
Proceedings of the AACR

Volume 59 | April 2018

Part A: Abstracts 1-3027

TABLE OF CONTENTS

TUMOR BIOLOGY:

Angiogenesis Inhibitors and Stimulators

Breast Cancer Metastasis

Dormancy and Aging: The Influential Microenvironment

Mechanisms Underlying Metastasis 1

Role of the Innate Immune System in Tumorigenesis

Targeting Cancer Stemness

The Mechanical Microenvironment in Tumorigenesis

Tumor Heterogeneity 1

EPIDEMIOLOGY:

Genetic Contributions to Cancer Epidemiology 1: GWAS and Pathway and Candidate Gene Studies

PREVENTION RESEARCH:

Chemoprevention of Cancer

BIOINFORMATICS AND SYSTEMS BIOLOGY:

Integrative Cancer Biology 1

MOLECULAR AND CELLULAR BIOLOGY / GENETICS:

BCL-2 Family and Mitochondrial Apoptosis

Cancer Predisposition and Synthetic Lethality

Chromatin Structure and Function

Emerging Concepts

Functional Genomics

High-Throughput Sequencing 1

Metabolism and Cellular Functions 1

MicroRNA Regulation in Cancer Biology

Noncoding RNAs as Oncogenes and Tumor Suppressors

Oncogenes and Tumor Suppressor Genes

CLINICAL RESEARCH:

Biomarker Discovery 1

Clinical Risk Factors and Outcomes in Solid Tumors / Survivorship Research

Immune Response to Therapies 1

Modifiers of the Tumor Microenvironment 1

Molecular Diagnostics 1: Cytogenetics, Clinical Molecular Genetics, and Clinical Imaging

Radiation Oncology

CANCER CHEMISTRY:

Chemical and Structural Biology

IMMUNOLOGY:

Inflammation, Immunity, and Cancer

Response and Resistance to Immune Checkpoint Blockade

Vaccines 1

EXPERIMENTAL AND MOLECULAR THERAPEUTICS:

Antibody-Drug Conjugates: Agents and Technology

Biomarker Identification and Novel Methods

Experimental Agents and Combinations for Hematologic Malignancies 1

Growth Factors and Protein Kinases as Targets

Modulators of Ionizing Radiation and Other Radiotherapeutics

New Therapy with New Mechanism of Action

Strategies to Reversing Drug Resistance

CLINICAL RESEARCH:

Pediatric Cancer: Poster Discussion

BIOINFORMATICS AND SYSTEMS BIOLOGY:

Computational Methods and Resources for Cancer Research

CANCER CHEMISTRY:

Molecules Addressing Challenges in Drug Discovery

CLINICAL RESEARCH:

Liquid Biopsy 1

ENDOCRINOLOGY:

Novel Roles of Steroid Hormone Receptors

EXPERIMENTAL AND MOLECULAR THERAPEUTICS:

Novel Therapeutic Agents and Combinations

IMMUNOLOGY:

Engineering Immunotherapies for Anticancer Activity

MOLECULAR AND CELLULAR BIOLOGY / GENETICS:

Cell Signaling Pathways

Epigenetic Mechanisms of Tumor Progression

MULTIDISCIPLINARY:

Radiation Science

TUMOR BIOLOGY:

Patient-Derived Models of Cancer: Present and Future

Building the Microenvironment through Crosstalk

MOLECULAR AND CELLULAR BIOLOGY / GENETICS:

Targeting the Cell Cycle: Mechanism and Therapy-- Poster Discussion

TUMOR BIOLOGY:

Adaptation and Checkpoints in Tumorigenesis

Advances in the Generation and Analysis of Patient-Derived Xenografts

Biomarkers of Metastasis

Mechanisms Underlying Metastasis 2

Regulation of Stemness in Cancer

Translational Therapeutics in Cancer Models 1

Tumor Heterogeneity 2

EPIDEMIOLOGY:

Descriptive Epidemiology of Cancer

Genetic Contributions to Cancer Epidemiology 2: Next-Generation Sequencing and Familial Cancers

PREVENTION RESEARCH:

Molecular Mechanisms and Targets for Cancer Prevention

BIOINFORMATICS AND SYSTEMS BIOLOGY:

Application of Bioinformatics to Cancer Biology 1

Integrative Cancer Biology 2

MOLECULAR AND CELLULAR BIOLOGY / GENETICS:

Autophagy

DNA Repair and Damage Response

Epigenetic Therapy

Genotype/Phenotype Correlations

High-Throughput Sequencing 2

Metabolic Pathways

Mitosis and Telomeres

Navigating the Cancer Chromatin Landscape

Novel Biomarkers and Drivers of the Cancer Transcriptome

Targeting the Cell Cycle: Mechanism and Therapy

CLINICAL RESEARCH:

Adoptive Cell Therapy 1

Biomarker Discovery 2

Liquid Biopsy 2

Molecular Diagnostics 2: Laboratory Correlates for Targeted Agents and Preclinical and Clinical Trials

Pediatric Cancer

Special Populations / Biostatistics in Clinical Trials

CANCER CHEMISTRY:

Target Based Drug Discovery

IMMUNOLOGY:

Immune Checkpoints 1

Immune Response to Therapies 2

Modifiers of the Tumor Microenvironment 2

Therapeutic Antibodies, Including Engineered Antibodies 1

ENDOCRINOLOGY:

Growth Factors and Nuclear Receptor Signaling

EXPERIMENTAL AND MOLECULAR THERAPEUTICS:

Acquired Resistance Against Molecularly Targeted Therapies

Cell Cycle, Drug Resistance, and Combinations

Experimental Agents and Combinations for Hematologic Malignancies 2

Identification of Molecular Drug Targets

New Targets 1

Therapeutic Resistance: New Targets and New Inhibitors

TUMOR BIOLOGY:

Epithelial Cancer Stem Cell Biology

Epithelial-to-Mesenchymal Transition and EMT and TGFbeta in Metastasis

Novel Mechanisms Regulating Angiogenesis

Pediatrics 1: Genomics, Epigenetics, and Biomarkers

Surveying the Tumorigenic Microenvironment

The Metastatic Microenvironment

Translational Therapeutics in Cancer Models 2

Tumor Heterogeneity 3

EPIDEMIOLOGY:

Screening, Early Detection, and Risk Prediction

PREVENTION RESEARCH:

Biomarkers, Intervention, and Early Detection for Cancer Prevention

BIOINFORMATICS AND SYSTEMS BIOLOGY:

Application of Bioinformatics to Cancer Biology 2

New Software for Data Analysis

MOLECULAR AND CELLULAR BIOLOGY / GENETICS:

CDK and CDK Inhibitors: Mechanism and Therapy

Ferroptosis, Metabolism, and Cancer Cell Death

Gene Discovery

Kinases and Phosphatases

Metabolic Regulation

Metabolism and Cellular Functions 2

Noncoding RNAs and Cancer

Nuclear Oncoproteins and Tumor Suppressor Genes

Receptors and Growth Factors

Transcription Factor Crosstalk and Aberrant Transcriptional Control

CLINICAL RESEARCH:

Adoptive Cell Therapy 2

Biomarker Discovery 3

Biomarkers of Therapeutic Response in Clinical Trials

Novel Immunotherapies and Laboratory Models in Pediatric Cancer

Prognostic Biomarkers

CANCER CHEMISTRY:

Antitumor Agents

Cancer Biology Insights Emerging from Proteomic Investigations

IMMUNOLOGY:

Immune Checkpoints 2

Immune Mechanisms Invoked by Therapies 1

Therapeutic Antibodies, Including Engineered Antibodies 2

EXPERIMENTAL AND MOLECULAR THERAPEUTICS:

Design, Structure/Activity, and Modeling

Modulation of DNA Damage and Repair

Monitoring the Evolution of Therapeutic Resistance

New Agents and New Targets

New Targets 2

Novel Experimental Combinations

BIOINFORMATICS AND SYSTEMS BIOLOGY:

Systems and Computational Biology: Poster Discussion

CLINICAL RESEARCH:

Molecular Predictors of Response, Mediators of Resistance, Mechanisms of Action, Pharmacodynamic Markers, and Novel Disease Subsets

Use of Liquid Biopsies in Clinical Trials

EPIDEMIOLOGY:

Genetic and Molecular Epidemiology of Cancer Risk and Prognosis

EXPERIMENTAL AND MOLECULAR THERAPEUTICS:

Identification of Biomarkers and Molecular Drug Targets

IMMUNOLOGY:

Defining New Immunotherapeutic Targets through Deep Molecular Characterization

MOLECULAR AND CELLULAR BIOLOGY / GENETICS:

Drugging the Undruggable: Using Synthetic Lethality and Other Approaches to Develop New Treatment Strategies

Cancer Epigenetics and Epigenetic Therapy

Sampling the Cancer Genome and the Epigenome: Opportunities and Exquisite Vulnerabilities

MULTIDISCIPLINARY:

Cancer Survivorship and Disparities

TUMOR BIOLOGY:

Deciphering Cancer Mechanisms in Animal Models

Organ-Specific Metastasis

# Sunday, April 15, 2018

## TUMOR BIOLOGY:

### Angiogenesis Inhibitors and Stimulators

#1

A kinome-wide CRISPR screen reveals BET inhibition-associated endothelial cell resistance to anti-angiogenic therapy.

Michael Y. He, Michael M. Halford, Marc G. Achen, Steven A. Stacker. _Peter MacCallum Cancer Centre, Melbourne, Australia_.

Angiogenesis inhibition is a useful strategy for treating cancer. However, the efficacy of anti-angiogenic therapy (AAT) in clinical oncology has been limited largely by highly variable patient response and the inevitable occurrence of resistance. The modest patient benefits have underscored a pressing need for qualified biomarkers and better knowledge of resistance mechanisms. Blood endothelial cells (ECs) are one of the main targets of AAT in the tumor microenvironment. Hence, understanding the EC response to AAT will provide insight into tumor response. To identify molecular modifiers of the EC response to AAT (in this study, bevacizumab, a humanized neutralizing anti-VEGF-A monoclonal antibody), we developed a high-throughput genetic screening platform. This involved a three-dimensional microcarrier-based culture system, CRISPR-Cas9-driven gene loss-of-function (LOF) and VEGF-A-dependent serum-free culture conditions for response modifiers. A pooled kinome-wide CRISPR-Cas9-based screen of 763 genes (with four single guide RNAs/sgRNAs targeting each gene) identified 18 candidate genes that upon LOF (represented by sgRNAs) were significantly enriched or depleted in the bevacizumab versus control treatment arm (P ≤ 0.005, FDR ≤ 0.3). Further candidate evaluation using siRNAs validated six genes whose knockdown conferred EC resistance or sensitization to bevacizumab (P < 0.05). Of these, knockdown of BRD2 or BRD3, which encode the epigenetic reader bromodomain-containing protein 2 or 3 (BRD2 or BRD3), respectively, conferred EC resistance to bevacizumab. The bromodomain and extraterminal domain (BET) inhibitors JQ1 and I-BET762, which selectively target the BET family of proteins (BRD2, BRD3, BRD4 and BRDT), reproduced the results of BRD2 or BRD3 LOF, with a more prominent phenotype (P < 0.05). Drug dose-response assessment indicated an anti-angiogenic effect of BET inhibitors regardless of the presence of bevacizumab. This inhibitory effect was unexpectedly attenuated when cells were co-treated with bevacizumab under VEGF-A-dependent conditions. Experiments to investigate the mechanistic basis for this phenotype of resistance are ongoing and include differential gene expression analysis using RNA-Seq and in vivo evaluation. Applying a non-biased approach to identify molecular modifiers of the EC response to AAT, we demonstrate in this study that BET inhibition is unexpectedly associated with resistance to bevacizumab, despite BET inhibition alone having an anti-angiogenic effect. These observations prompt further evaluation of epigenetic regulation in tumor angiogenesis, particularly in the context of interaction between BET inhibition and VEGF blockade. Clinically, these findings may facilitate development of potential predictive and/or response biomarkers and strategies to overcome resistance to AAT and/or BET inhibitors.

#2

CXCR2+ tumor cells mediate vascular mimicry driving anti-angiogenic therapy (AAT) resistance in glioblastoma (GBM).

Kartik P. Angara, Thaiz F. Borin, Mohammad H. Rashid, Roxan Ara, Bhagelu R. Achyut, Ali S. Arbab. _Augusta University, Augusta, GA_.

Glioblastoma (GBM) is a hypervascular and hypoxic neoplasia of the central nervous system with an extremely high rate of mortality. Owing to its hypervascularity, anti-angiogenic therapies (AAT) have been used as an adjuvant to the traditional surgical resection, chemotherapy, and radiation to normalize blood vessels and control abnormal vasculature. The benefits of AAT have been transient, and evidence of relapse exemplified in the progressive tumor growth following AAT reflects development of resistance and alternative neovascularization mechanisms in these resilient tumors to counter the AAT therapy insult. Vascular mimicry (VM) is the uncanny ability of tumor cells to acquire endothelial-like properties and lay down vascular patterned networks reminiscent of host endothelial blood vessels. The VM channels serve as an irrigation system for the tumors to meet with the increasing metabolic and nutrient demands of the tumor. In our current studies to understand the tumor-inherent mechanisms of AAT resistance, we identified a crucial pro-migratory and pro-angiogenic chemokine, CXCL8 or IL-8, to be highly upregulated in the GBM tumors treated with AAT. AAT-treated groups had significantly higher populations of CXCR2+ stem and endothelial-like subpopulations and these cells lined the VM-like vascular structures carrying functional RBCs in the tumors. These stem cell-like and endothelial-like populations were decreased following treatment with HET0016 or SB225002. Furthermore, knocking down CXCR2 led to smaller tumor size in the animals and improperly developed vascular structures without CXCR2+ GBM cells lining them. Also, HET0016 and SB225002 disrupt the tube-forming capability of U251 GBM cells in an in vitro Matrigel angiogenesis assay. This confirms our hypothesis that CXCR2+ GBM cells initiate VM and contribute to AAT resistance in GBM. Our present study suggests that tumor cell autonomous IL-8/CXCR2 pathway contributes to VM-mediated AAT resistance in GBM and that HET0016 and SB225002 have a great potential to target therapeutic resistance and can be combined with other chemotherapeutic agents in preclinical and clinical trials.

#3

Metronomic chemotherapy with cyclophosphamide (Cy) and the repositioned drug losartan (Los) for the treatment of M-234p triple negative murine mammary adenocarcinoma.

Julian Guercetti,1 Leandro E. Mainetti,1 M.Carolina Grillo,1 Antonela Del Giúdice,1 Maria V. Baglioni,1 Juan M. Cáceres,1 Ainelén Arboatti,2 Daniel Francés,2 Viviana R. Rozados,1 Maria J. Rico,1 O.Graciela Scharovsky1. 1 _Facultad de Ciencias Médicas, UNR, Rosario, Argentina;_ 2 _Facultad de Ciencias Bioquímicas y Farmacéuticas, UNR, Rosario, Argentina_.

Metronomic chemotherapy (MCT) refers to the chronic, equally spaced, delivery of low doses of chemotherapeutic drugs, without extended interruptions. Drug repositioning in oncology refers to the use of drugs formulated for other indications that showed antitumor potential. Los is an antagonist of angiotensin II receptor used to treat hypertension. We aim to study the combined effect of metronomic Cy+Los on M-234p tumor. Female BALB/c mice were orthotopically challenged with M-234p (day 0) and distributed, on day 8, into 4 groups (n=5-6/group) treated as follows, GI: Control, without treatment; GII: Cy 25mg/kg/day in the drinking water; GIII: Los 200mg/kg/day in the drinking water; GIV: Treated as GII+GIII. Mice were weighted, and tumor volume measured 3 times/week. When tumors were exponentially growing, mice were euthanized, tumors excised, and blood samples taken for immunohistochemistry, western blot and flow cytometry. GIV showed tumor growth inhibition compared to GI, GII and GIII and, as a consequence, higher survival (P<0.005). Also, GIV showed 60% (3/5) of complete tumor regressions, without relapses. No weight losses or sings of toxicity were observed. IHC analysis showed a lower N° of Ki67+ cells in GIV (P<0.05). Moreover, a significant increase of apoptosis in GIV vs Control was shown by TUNEL assay (P<0.05). No differences in circulating CD4+, CD8+ and Treg cells were shown by flow cytometry but, a marginally significant increase in Th17 cells in GII and GIV was seen. To share some light on the effect of the therapy on the cancer associated fibroblasts, the protein αSMA was measured by western blot; the expression of this marker was significantly lower in GIV vs GI (P<0.05). To evaluate the anti-metastatic effect of the therapy, mice were inoculated i.v. with 5x105 M-234p cells. On day 3, mice were distributed in the same experimental groups. When the first mouse showed signs of metastatic disease, all of them were euthanized, lungs excised and stained to highlight metastasis. The N° of lung metastasis was significantly lower in GII and GIV. No weight losses or any other sing of toxicity were observed. These results clearly show that metronomic chemotherapy with a combination of Cyclophosphamide and Losartan, administered as an intervention strategy, can inhibit, not only the growth of a triple negative mammary adenocarcinoma, causing permanent regressions, but also its metastasis, while being devoid of toxicity. Those effects would be achieved, at least partially, by inhibition of tumor proliferation, increase of apoptosis and modification of tumor microenvironment through tumor fibroblasts. The characteristics and the efficacy of the therapeutic schedule herein utilized suggest its implementation at the clinical setting in the near future.

#4

Antitumor activity of DLX1008, a single chain antibody fragment binding to VEGF-A, in in vivo preclinical models of Kaposi sarcoma and glioblastoma.

Anthony B. Eason,1 Sang-Hoon Sin,1 Emese Szabó,2 Douglas J. Phillips,3 Miriam Droste,3 Abdijapar Shamshiev,3 Dirk P. Dittmer,1 Michael Weller2. 1 _The University of North Carolina at Chapel Hill, Chapel Hill, NC;_ 2 _University Hospital Zurich and University of Zurich, Zurich, Switzerland;_ 3 _Cell Medica Switzerland AG, Schlieren, Switzerland_.

Background:

Angiogenesis mediated by vascular endothelial growth factor (VEGF) is a hallmark of several cancers. Efficacy of VEGF inhibitors could be increased by improving affinity or tumor penetration, for example by reducing the size of the drug. Recently, the exceptionally stable anti-VEGF single chain variable fragment (scFv) brolucizumab (RTH258, ESBA1008, Novartis) completed phase 3 clinical trials in ophthalmology and was shown to be more efficacious than the larger aflibercept. The same molecule, named DLX1008, is currently in pre-clinical development in gliobastoma and Kaposi sarcoma (KS) by Cell Medica. KS is the most angiogenic cancer known to date. Carcinogenesis is induced by KS-associated herpesvirus (KSHV) and compromised of endothelial cells expressing high levels of VEGFR. KS is unique because angiogenesis (growth of non-tumor endothelial cells) as well as proliferation of the primary tumor cells are dependent on VEGF. No VEGF antagonist has yet been demonstrated to unequivocally improve overall survival in either KS or glioblastoma, which may be due to limited tumor penetration.

Methods:

Binding affinity to VEGF-A was determined by kinetic exclusion assay (KinExA).

For in vivo studies, we used the L1T2 model of KS, whereby KSHV-positive human cells (L1T2/ ATCC® VR-1802™) are implanted subcutaneously onto NGS mice, and the U87 model of glioblastoma, where human glioblastoma cells (U87/ ATCC® HTB-14™) are implanted subcutaneously or intracranially onto SWISS nude mice. Tumor growth was followed by caliper measurements and pathology evaluated by histochemistry or imaging.

Results:

DLX1008 binds to VEGF-A165 with a KD of 1.05 pM. Bevacizumab binds with a KD of 32.4 pM. Hence, the affinity of DLX1008 to human VEGF is around 30-fold tighter than that of bevacizumab.

DLX1008 reduced growth of L1T2 cells in vivo (p ≤ 0.007 by mixed effects model, n = 10) compared to vehicle control at 15 mg/kg once daily 5x/week and (p≤0.001, n = 10) in a second biological repeat. Imaging showed that the tumor died from the inside out in response to DLX1008. DLX1008 reduced the growth of U87 cells subcutaneously implanted in vivo (p ≤ 0.0021 by ANOVA, n = 10) and improved median survival of mice intracranially implanted with U87 cells (p ≤ 0.00026) compared to control scFv at 50 mg/kg twice daily. Staining of intracranial U87 tumors showed decreased CD31-positive blood vessel density as well as decreased p-VEGFR1 immunoreactivity in vascular structures and glioma cells.

Conclusions:

The high affinity, highly stable scFv DLX1008 shows highly reproducible anti-tumor activity in in vivo preclinical models of KS and glioblastoma. Efficacy was attributable to reduced angiogenesis. Its small size offers advantages in tissue penetration and distribution as compared to bevacizumab. DLX1008 is an excellent antibody to advance into clinical trials.

#5

The histone acetyltransferase inhibitor CPTH6 impairs tumor angiogenesis acting on both endothelial and cancer cells.

Marta Di Martile,1 Marianna Desideri,1 Simonetta Buglioni,1 Carla Azzurra Amoreo,1 Daniela Trisciuoglio,2 Donatella Del Bufalo1. 1 _Regina Elena National Cancer Institute, Rome, Italy;_ 2 _National Research Council, Rome, Italy_.

Protein acetylation is typically catalyzed by enzymes with histone acetyltransferase (HATs) or histone deacetylase (HDACs) activity. To date, it has been identified the involvement of protein acetylation in different tumorigenic signaling events, including angiogenesis. The biological process of neo-vasculature formation from pre-existing blood vessels is widely considered to be an essential process to sustain tumor growth as well as to provide a route for tumor cell metastatization. In this context, the role exerted by HDACs in tumor angiogenesis is well known, whilst the role of HATs is largely unknown. Among molecules with HAT inhibitory activity, the thiazole derivative CPTH6 has been characterized by our group for its antitumor activity in different tumor models, including non-small cell lung cancer (NSCLC). In this study, we assessed the effect of CPTH6 on angiogenesis-related properties of both endothelial and NSCLC cells. The human umbilical vein endothelial cell (HUVEC) and H1299 NSCLC cell lines were used. HUVEC and H1299 morphogenesis was analyzed by plating cells on matrigel and evaluating their ability to organize capillary-like structures. The effect of CPTH6 on protein acetylation was assessed by WB analysis. Transwell supports were employed to evaluate HUVEC migration and invasion. Human Angiogenesis Antibody Array was used to test the conditioned media derived from CPTH6-treated H1299 cells. C57/BL6 and nude mice were used to perform matrigel plug assay and to evaluate tumor growth, respectively. IHC analysis of CD31 was employed to evaluate the number of intra-tumor vessels in tumor xenografts. The HAT inhibitor CPTH6 affected some endothelial cell functions in vitro. In particular, CPTH6 impaired HUVEC invasion, migration and differentiation abilities at doses that did not alter proliferation. Although CPTH6 did not affect histone H3 acetylation, it slightly reduced α-tubulin acetylation in HUVEC. In addition, CPTH6 decreased the neovascularization in vivo, as evidenced by the impairment of the VEGF-induced vascularization of matrigel plugs. Interestingly, CPTH6 affected also the angiogenesis-related properties of cancer cells. In particular, this compound reduced the ability of H1299 to organize capillary like structures and, conditioned media derived from CPTH6-treated H1299 cells, impaired HUVEC morphogenesis. Accordingly, CPTH6 reduced the secretion of some pro-angiogenic factors (VEGF, EGF, ANG, TIE-2, TNF-α) and, at the same time, increased the release of anti-angiogenic ones (Endostatin, PLG). Finally, in H1299-tumor xenografts, CPTH6 decreased significantly the number of intra-tumor vessels, even though it did not impair tumor growth. Overall, this study adds information to the role of HATs in tumor angiogenesis, and proposes HAT inhibition as an attractive target for antiangiogenic therapy of NSCLC.

#6

Role of Notch signaling in bevacizumab-induced vascular normalization in glioblastoma.

Norihiko Saito, Kazuya Aoki, Nozomi Hirai, Satoshi Fujita, Haruo Nakayama, Morito Hayashi, Takatoshi Sakurai, Satoshi Iwabuchi. _Toho University Ohashi Medical Center, Tokyo, Japan_.

Tumor angiogenesis occurs in the setting of a defective vasculature, which is associated with increased vascular permeability and enhanced tumor permeability. Bevacizumab is used to treat malignant glioma and was found to reduce microvascular density and prune abnormal tumor microvessels. Using electron microscopic observation of two autopsy cases, we investigated the effects of blood vessel normalization in glioblastomas treated with bevacizumab. Notch-1 and SMA immunostaining were used to compare initial surgical specimens with postmortem specimens obtained after bevacizumab treatment. Postmortem samples showed marked proliferation of SMA-positive cells (pericytes) in tumor vessels and marked proliferation of Notch-1–positive cells around vessels. Electron microscopic images confirmed the presence of pericytes surrounding the vascular endothelium. These findings suggest that bevacizumab treatment promotes vascular normalization by recruiting mature pericytes. Next, we investigated the effects of bevacizumab on VEGF inhibition in glioma stem cells. Bevacizumab treatment attenuated activation of VEGFR2 and increased Notch signaling expression. VEGF inhibition by bevacizumab treatment attenuated proliferation and self-renewal of glioma stem cells and induced endothelial and pericyte differentiation. In tumor angiogenesis, vascular endothelial growth factor induces sprouting angiogenesis and recruitment of vascular endothelial cells such as tip cells, stalk cells, and phalanx cells. Fully mature phalanx cells are in close contact with pericytes. Our results suggest that bevacizumab treatment induces glioma stem cells to differentiate to endothelium and pericytes. These mechanisms might be important in normalizing tumor vasculature after bevacizumab treatment and could be useful in improving the effectiveness of current glioma therapy.

#7

Platinum resistance in epithelial ovarian cancer is dependent on a PDGFR alpha-VEGF-A signalling mechanism that activates downstream angiogenesis pathways.

Aya El Helali,1 Nuala McCabe,1 Christopher Steele,2 Lara Dura Perez,2 Christina L. O'Neill,3 Naomi Dickson,2 Niamh McGivern,1 Caolan Harkin,1 Andrena McCavigan,2 Reinhold J. Medina,3 Laura A. Knight,2 Stephen McQuaid,4 Jacqueline A. James,4 Caroline O. Michie,5 Charlie Gourley,5 W Glenn McCluggage,6 Denis P. Harkin,2 Richard H. Wilson,1 Alan W. Stitt,3 Richard D. Kennedy1. 1 _Centre for Cancer Research and Cell Biology, Belfast, United Kingdom;_ 2 _Almac Diagnostics, Belfast, United Kingdom;_ 3 _Centre for Experimental Medicine, Belfast, United Kingdom;_ 4 _Northern Ireland Molecular Pathology Laboratory, Belfast, United Kingdom;_ 5 _Nicola Murray Ovarian Cancer Research Centre, Edinburgh, United Kingdom;_ 6 _Department of Pathology, Royal Group of Hospitals Trust, Belfast, United Kingdom_.

Introduction:

40-45% of patients with High Grade Serous Ovarian Cancer (HGSOC) will eventually relapse with platinum resistant disease. Tothill et al and TCGA are two independent gene expression datasets which have demonstrated the presence of a mesenchymal molecular subgroup, characterised by upregulation of angiogenesis regulating genes. Angiogenesis is known to be an integral pathological feature of HGSOC and anti-angiogenics have dominated the field of drug development in EOC. However, despite this, anti-angiogenic agents have failed to demonstrate a significant impact on overall survival (OS) benefit. In this study, we asked if platinum resistance could be associated with an improved response to anti-angiogenic agents and what the underlying biological rationale for this could be.

Methods:

A meta-analysis of 14 phase II and III clinical trials in EOC were used to investigate the association between platinum resistance and response to anti-angiogenic agents. In addition, we analysed gene expression in 12 matched pre- and post-chemotherapy EOC samples. Novel isogenic cisplatin-resistant HGSOC cell lines were established to study the development of an angiogenic phenotype. Further studies were performed in novel ascites-derived primary cell lines from HGSOC patients with known outcomes following platinum-based chemotherapy.

Result:

In the clinical trial meta-analysis, an OS benefit for antiangiogenics was observed in platinum-resistant disease (p=0.029), whilst platinum-sensitive EOC only derived progression free survival (PFS) (p=<0.0001) benefit and not OS (p=0.125). In the 12 matched pairs of patient samples, post-platinum samples had a higher micro-vessel density (MVD) relative to their paired treatment-naïve sample (p= 0.0001). Additionally, an in vivo angiogenesis matrigel plug assay demonstrated that cisplatin-resistant EOC cell lines were associated with an increase in MVD (p=<0.0001). MVD was reduced in the platinum-resistant cells following treatment with bevacizumab (p=0.001). Ascites-derived primary cells established from platinum-resistant patients demonstrated overexpression of VEGF-A, consistent with stimulating angiogenesis. Gene expression analysis of pre- and post-platinum paired samples identified that PDGFRα (p=0.007) and PDGFRβ (p=0.005) were differentially expressed in the post-platinum therapy samples. In vitro validation in the platinum-resistant cell lines demonstrated that VEGF-A expression was regulated by PDGFRα.

Discussion:

We have demonstrated that previous platinum therapy for EOC is associated with an increase in tumor PDGFα and VEGF-A expression, correlating with a response to anti-angiogenic therapies. This data suggests that platinum therapy resistance may inform the selection of EOC patients for novel antiangiogenic therapies in future clinical trials.

#9

Efficacy and functional study of tetraarsenic oxide as an anticancer drug in cervical cancer cell lines and cervical cancer patient-derived xenograft mouse.

Jeong-Won Lee, Jae Ryoung Hwang, Young-Jae Cho, Ji-Hye Kim, Ji Yoon Ryu, E-Sun Baik. _Samsung Medical Center, Seoul, Republic of Korea_.

Background: Arsenic compound has been used as a medicine in China and has been studied to treat acute promyelocytic leukemia, hepatocellular carcinoma, and melanoma. Tetraarsenic oxide (TAO; As4O6) is an inorganic arsenic compound that has been demonstrated to inhibit angiogenesis and cell growth in cervical cancer cells. In this study, we demonstrated anti-cancer effect of TAO in cervical cancer patient-derived xenograft (PDX) mouse model and the functional mechanism related with cervical cancer cell death.

Materials & Methods: We used SiHa, Caski, and HeLa cells for cervical cancer cells and HUVEC cells to study functional mechanism of TAO in an in vitro. MTT assay was performed to assess cell death and MMP-2-specific ELISA assay was used to detect MMP-2 expression. To establish the patient-derived xenograft (PDX) mouse model of cervical cancer, surgical patient tumor specimens were reduced to small pieces (less than 2-3 mm), implanted into the subrenal capsules of the left kidneys of BALB/C nude mice, and propagated by serial transplantation. The PDX model used in this study was generated with a tumor that was histologically defined as a FIGO stage Ib1 invasive squamous cell carcinoma. The patient was a 46-year-old woman who received primary debulking surgery followed by radiation therapy for 28 times.

Results: We measured IC50 of TAO in cervical cancer and HUVEC cells. IC50 of TAO in SiHa, Caski, and HUVEC was around 3 uM and it was about 0.6 uM in HeLa cells. Although TAO inhibited MMP-2 expression, an important protein for angiogenesis, in both cervical cancer cell lines and HUVEC cells, the functional mechanism was different in between cervical cancer cells and HUVEC cells. TAO inhibited Akt activation in SiHa, Caski, and HeLa. However, TAO had no effect on activation of Akt in HUVEC cells. TAO inhibited expression of VEGF receptor 2 in HUVEC cells but not in cervical cancer cells. We found that TAO inhibited autophagy determined by p62 expression level in cervical cancer cells but not in HUVEC cells. Cervical cancer PDX mouse study demonstrated that TAO inhibited tumor growth.

Conclusions: TAO inhibited cell growth of cervical cancer cells and HUVEC. The functional signaling pathway of TAO-induced cell death might different in between cervical cancer cells and endothelial cell, HUVEC. TAO inhibited tumor growth in cervical cancer PDX mouse probably through inhibition of cancer cell growth and inhibition of angiogenesis via inhibition of endothelial cell growth. These results suggest that TAO may be considered as a novel therapeutic compound for the treatment of cervical cancer.

#10

Evaluation of metronomic chemotherapy regimens in preclinical orthotopically implanted colon cancer models, and in patient-derived xenografts.

Jose Lopez,1 Paloma Valenzuela,1 Valerie Gallegos,1 Karla Parra,1 Valeria Rolih,1 Diana Gonzalez Garcia,1 Joel Martinez,1 Urban Emmenegger,2 Guido Bocci,3 Robert S. Kerbel,2 Giulio Francia1. 1 _UT El Paso, El Paso, TX;_ 2 _University of Toronto, Toronto, Ontario, Canada;_ 3 _University of Pisa, Pisa, Italy_.

Recent advances for the treatment of late stage colon cancer include a metronomic capecitabine plus bevacizumab maintenance regimen as reported for the CAIRO3 phase 3 clinical trial (Simkens et al, Lancet: 2015; 385: 1843-52) . We previously reported a phase 2 trial evaluating metronomic cyclophosphamide (CTX) plus UFT (a 5-fluorouracil prodrug) and celecoxib in gastrointestinal cancer patients (Allegrini et al, Angiogenesis: 2012; 15, 275-286). We also reported the preclinical evaluation of metronomic oral topotecan in mouse models of colon cancer (Hackl et al, Gut: 2013; 62, 259-71). Here we report our studies using orthotopic (intracecal) implantation of human HT29/luc/hCG colon cancer cells in SCID mice. Disease progression was noninvasively assessed using the transfected human chorionic gonadotropin (hCG), whose levels in the mouse urine correlate with tumor burden - and this was coupled with luciferase (luc) bioimaging of the mice. These implanted tumor models were used together with patient derived xenografts (PDX), to evaluate metronomic regimens of etoposide (80mg/kg/day, p.o.), gemcitabine (given i.p.; at either 120mg/kg every 3 days, or at the much lower 1mg/kg/day), or CTX given as a monotherapy (20mg/kg/day given via the drinking water) or combined with neutralizing antibodies against VEGF or EGFR. Our results indicate; 1) effective anti-tumor activity of daily etoposide, and of 2) daily gemcitabine (as well as when the gemcitabine was given every 3 days), and that 3) orthotopic HT29/luc/hCG colon tumors are weakly responsive to CTX or CTX plus targeted agents compared to the same tumors implanted subcutaneously or intraperitoneally. The effectiveness of metronomic etoposide was confirmed in a colon PDX model in NSG mice (n=5 mice/group; p<.05) - obtained from Jackson labs. Notably ovarian cancer and breast cancer PDX models did not respond to etoposide therapy (n=5/group), indicating the response may be tumor specific. Collectively, these results highlight the importance of advanced disease models to confirm sometime overly optimistic experimental therapeutic results from subcutaneously or intraperitoneally implanted tumor models. They also demonstrate the promising anti-tumor activity of metronomic etoposide and gemcitabine regimens for colon cancer, and they lead us to advocate the concerted use of both implanted xenografts and PDX for the testing of new therapeutic strategies for the treatment of colon cancer.

#11

Antitumor activity of lenvatinib in the renal cell carcinoma cell line RENCA model resistant to a VEGF specific inhibitor.

Kenji Ichikawa, Saori Watanabe Miyano, Yukinori Minoshima, Junji Matsui. _Tsukuba Research Laboratories, TSUKUBA-SHI, IBARAKI, Japan_.

Anti-vascular endothelial growth factor (VEGF) therapies have been in clinical use to treat patients with multiple types of cancers, especially in renal cell carcinoma (RCC) and hepatocellular carcinoma (HCC). However, benefits of anti-VEGF therapies are still limited because of the intrinsic/acquired resistance to the treatment. Lenvatinib mesilate (lenvatinib) is an oral multiple receptor tyrosine kinase (RTK) inhibitor that suppresses the kinase activities of VEGF receptors (VEGFR1-3), in addition to other proangiogenic and oncogenic pathway-related RTKs including fibroblast growth factor receptors (FGFR1-4), the platelet-derived growth factor receptor (PDGFR) α, KIT, and RET. Lenvatinib has been approved as a monotherapy for the treatment of radioiodine-refractory differentiated thyroid cancer and in combination with everolimus for the treatment of patients with RCC treated with one prior anti-VEGF therapy in the US and EU. In this study, we established an anti-VEGF treatment resistant preclinical mouse tumor model. We also examined whether lenvatinib, which targeted multiple receptor tyrosine kinases, showed anti-tumor activity in this model. To establish model resistant to anti-VEGF treatment, an expression vector encoding VEGFR-Fc protein (VEGF decoy) was introduced into a mouse RCC cell line RENCA. First, we confirmed the RENCAVEGFR-Fc transfectants secreted VEGF decoy, which selectively interacted with recombinant VEGF protein but not with ligands to other RTKs in in vitro. Tumor growth in vivo of RENCAVEGFR-Fc was significantly slower than that of RENCAMock. RENCAMock transfectants took 16 days to reach a mean tumor volume (TV) of 500 mm3 whereas RENCAVEGFR-Fc transfectants required 72 days to reach same TV. Furthermore, 72 days after inoculation of RENCAVEGFR-Fc transfectants, we resected tumor tissues and confirmed the expression of VEGF decoy protein. Tumor microvessel density was decreased in resected RENCAVEGFR-Fc tumor compared to the same size of RENCAMock tumor. From the above results, we characterized RENCAVEGFR-Fc tumor was resistant to VEGF decoy. Finally, anti-tumor activity of lenvatinib was challenged against RENCAMock or RENCAVEGFR-Fc tumors. When tumor volume reached approximately 300mm3, 10 mg/kg of lenvatinib was orally administered once daily for 14 days. Treatment of lenvatinib led to almost complete inhibition of tumor growth in both RENCAMock and RENCAVEGFR-Fc models. In conclusion, we developed a mouse RENCAVEGFR-Fc model resistant to anti-VEGF treatment, and lenvatinib showed anti-tumor activity in the resistant model as with RENCAMock model. Additional preclinical studies will be investigated to elucidate the signaling pathways associated with resistance to anti-VEGF treatment, in which lenvatinib may inhibit such as FGFR1-4, PDGFRα, KIT and RET.

#12

Antitumor activity of lenvatinib in combination with everolimus or an anti-PD1 antibody in preclinical RCC models.

Yusuke Adachi, Takayuki Kimura, Masahiro Matsuki, Junji Matsui, Yasuhiro Funahashi. _Eisai Co., Ltd., Tsukuba-shi, Ibaraki, Japan_.

Lenvatinib mesilate (lenvatinib) is an oral multiple receptor tyrosine kinase (RTK) inhibitor that selectively inhibits the kinase activities of VEGFR1-3, in addition to other proangiogenic and oncogenic pathway-related RTKs including FGFR1-4; PDGFRα; KIT; and RET. Lenvatinib plus everolimus for advanced renal cell carcinoma (RCC) after one prior anti-VEGF therapy was approved in the US and EU in 2016. Currently, Phase3 clinical study of lenvatinib in combination with everolimus or pembrolizumab for metastatic RCC patients is in progress. However, the detailed mechanisms underlying the efficacy of these combination treatments remain to be elucidated. In this study, we investigated the antitumor activities of lenvatinib in combination with everolimus or anti-PD1 mAb in preclinical RCC models. We examined antitumor activities in four human RCC (A-498, VMRC-RCW, Caki-1, and ACHN) xenograft models orally treated with lenvatinib, everolimus, and their combination. Expression of Ki67 and phosphorylation of S6 in RCC xenograft models were analyzed by immunofluorescence (IF) staining. Inhibitory effects of lenvatinib and everolimus on the phosphorylation of S6K (T389 and T421/S424), and S6 (S235/S236) were analyzed by western blot in vitro. We also examined antitumor activity of combination treatment of lenvatinib and anti-PD-1 mAb in RAG murine RCC model. The combination of lenvatinib with everolimus showed greater antitumor activity than that of either mono-treatment in all of 4 RCC xenograft models we examined and led to tumor regression in three out of 4 models (A-498, Caki-1, and ACHN). IF imaging showed expression of Ki67 was further suppressed with the combination treatment compared to lenvatinib and everolimus mono-treatments. Phospho-S6 (p-S6) signal were localized in perivascular regions of control group. Lenvatinib mono-treatment decreased p-S6 in the region apart from vessels, but some staining of p-S6 in perivascular regions were remained. The combination treatment further suppressed p-S6 staining regardless of the regions. In RAG syngeneic model, the combination treatment of lenvatinib at 10 mg/kg and anti-PD-1 mAb showed tumor shrinkage and tumors were regressed to nonpalpable sizes in 3 of 6 mice, although lenvatinib and anti-PD-1 mAb mono-treatments only slowed growths of RAG tumor. Our results indicate that combination of lenvatinib and everolimus enhances the inhibitory effects of mTOR signaling and tumor proliferation and showed greater antitumor activities. Combination of lenvatinib and anti-PD-1 mAb showed greater antitumor activity including tumor shrinkage, which was not demonstrated by each monotherapy in the murine RCC syngeneic model. These preclinical results provide one of the mechanisms of combinational effect of lenvatinib and everolimus, or PD-1 blockade in RCC.

#13

**Anti-angiogenic action of leukotriene-C** 4 **induced 15-hydroxyprostaglandin dehydrogenase in colon cancer cells is a TNF-α dependent phenomenon.**

Shakti R. Satapathy, Anita Sjölander. _Lund University, Malmo, Sweden_.

Colorectal cancer (CRC) is one of the leading causes of cancer-related deaths worldwide. Cyclooxygenase-2 (COX-2), which plays a key role in the biosynthesis of prostaglandin E2 (PGE2), is often up-regulated in CRC and in other types of cancer. PGE2 induces angiogenesis and tumor cell survival, proliferation and migration. The tumor suppressor 15-hydroxyprostaglandin dehydrogenase (15-PGDH) is a key enzyme in PGE2 catabolism, converting it into its inactive metabolite 15-keto-PGE2 and is often down-regulated in cancer. Interesting enough, CRC patients expressing high levels of Cysteinyl leukotriene receptor 2 (CysLTR2) have a good prognosis and therefore, we investigated a potential link between CysLTR2-signaling and the tumor suppressor 15-PGDH in colon cancer cells. TNF-α is considered as the main regulators of COX-2 and mPGES-1 that contribute to the increased synthesis of PGE2, which is inhibited by overexpressed 15-PGDH. Level of the pro-tumorigenic PGE2 are increased in CRC, previously attributed to increased production via TNF-α mediated COX-2 up-regulation but more recently attributed to decreased catabolism due to down-regulation of 15-PGDH. Elevation of 15-PGDH expression by leukotriene C4 (LTC4), a CysLTR2 ligand, exhibited anti-tumor activity in colon cancer cells with significant phosphorylation of β-catenin and down-regulation of anti-apoptotic marker Bcl-2 with concurrent activation of CASPASE-3 expression. We also observed a dramatic down-regulation of TNF-α on mRNA level and NF-κβ on both mRNA as well as protein level with LTC4 induced 15-PGDH in a TNF-α dependent manner. Moreover, TNF-α regulated anti-angiogenic action of 15-PGDH in HT-29 and Caco-2 colon cancer cells by depleting the mRNA level of MMP-2 and MMP-9 and protein level of VEGFR-1. Furthermore, our preliminary observation also suggested disrupted tube formation in HUVEC with LTC4 induced 15-PGDH which is mediated by TNF-α. Hence, restoration of 15-PGDH expression through CysLTR2-signaling promotes the anti-angiogenic action against colon cancer cells, indicating an anti-tumor as well as the anti-metastatic efficacy of CysLTR2-signaling.

#14

Preclinical evaluation of novel HIF-1α/P300 binding inhibitors.

Jonathan D. Strope,1 Emily M. Harris,1 Shaunna L. Beedie,1 Cindy H. Chau,1 Kristina M. Cook,2 Christopher J. Schofield,3 Kirk R. Gustafson,4 William D. Figg1. 1 _National Cancer Institute, Bethesda, MD;_ 2 _The University of Sydney, Sydney, Australia;_ 3 _University of Oxford, Oxford, United Kingdom;_ 4 _National Cancer Institute, Frederick, MD_.

A key component of tumor progression and metastasis is the hypoxic response. The hypoxic response regulates angiogenesis, tumor invasion, and metabolism. The Hypoxia Inducible Factor (HIF) is the transcriptional system responsible for the hypoxic response. The inhibition of the hypoxic response via inhibition of the HIF-1 pathway by disrupting its association with the transcriptional coactivator p300 presents a potential therapeutic target for multiple cancers where HIF is upregulated. To that end, we describe the preclinical development of previously described novel marine pyrroloiminoquinone alkaloids found using a HIF-1α/p300 assay in a high throughput screen of extracts from the National Cancer Institute's Natural Products Repository. Pyrroloiminoquinone alkaloids are a novel class of HIF-1α inhibitors, which interrupt the protein−protein interaction between HIF-1α and p300 and consequently reduce HIF-related transcription. The 2 lead candidates, discorhabdin L and H were assessed in inhibition of angiogenesis in an in vitro HUVEC assay and ex vivo rat aortic ring assay. The compounds were also evaluated for in vivo efficacy in prostate cancer cell tumor xenografts. Results show that development of these compounds for clinical use is warranted and may prevent the progression of multiple tumor types.

#15

PA28γ contributes to tumor angiogenesis through regulation of IL-6 and CCL2 in oral squamous cell carcinoma.

Xin Zeng, Jing Li, Jiajia Liu, Ning Ji, Hui Feng, Qianming Chen. _State Key Laboratory of Oral Diseases,West China Hospital of Stomatology, Sichuan University, Chengdu,Sichuan, China_.

Background: PA28γ has been shown to promote tumor development and progression. Although PA28γ has been suggested to play a role in tumor angiogenesis, direct evidence for the molecular mechanisms underlying this phenomenon have not been investigated.

Methods: Oral squamous cell carcinoma (OSCC) stable cell lines with varying levels of PA28γ expression were constructed. EA.hy926 human endothelial cells were co-cultured with these lines to evaluate the influence of PA28γ on EA.hy926 cell proliferation, migration, invasion, and tube formation. Using a subcutaneous xenotransplantation tumor model of under-expressing PA28γ HSC-3 cells, we explored effects on tumor angiogenesis in nude mice. We performed screens of the different angiogenesis-related genes and cytokines being expressed using PCR and cytokine chips to identify candidates with the potential to affect tumor angiogenesis. Then, candidates were verified using Western blot, and neutralization tests immunohistochemistry. Finally human OSCC clinical cohort was used to validate.

Results: PA28γ could enhance the ability of OSCC cells to interact with endothelial cells, thereby recruit endothelial cells and induce angiogenesis. Ablation of PA28γ inhibits tumor-induced angiogenesis in xenografts mice model in vivo. In vivo zebrafish model experiments showed that depleted PA28γ remarkably blocked the formation of intersegmental vessels in zebrafish embryos. Furthermore, Angiogenesis-related PCR, cytokine chips and Western blot analysis indicated that PA28γ through regulate IL-6 and CCL2 expression, thereby subsequent activation of the Stat3/VEGF and PI3K/MMP-1 axis to promote angiogenesis. Neutralization or block results IL-6 and CCL2 has an essential role in PA28γ-controlled promotes of angiogenesis. In addition, clinical OSCC cohort validate that PA28γ positively correlates with IL-6 or CCL2 expression.

Conclusion: Collectively, our data show that PA28γ contributes to the tumor angiogenesis, by regulating IL-6 and CCL2. PA28γ thus represents a novel therapeutic target for treating PA28γ-positive OSCC cancers.

#16

A wide-spectrum analysis of angiogenesis-related factors in ovarian cancer reveals a signature potentially for prognosis and therapeutic targeting.

Chih-Lin Kuo. _National Yang-Ming University, Taipei, Taiwan_.

Ovarian cancer is the first leading cause of death from woman gynecological malignancies worldwide. It is typically diagnosed at a late stage with a poor 5-year survival rate less than 30%. In the progress of ovarian cancer, angiogenesis-related factors not only induce neovascularization for supporting tumor growth but also accelerate ascites production, which helps the peritoneal metastasis and is associated with poor disease prognosis. Therefore, angiogenesis-related factors are thought to be particularly crucial in the persistence of ovarian cancer and have become key targets in clinical research. Although the expression of some angiogenesis-related factors in ovarian cancer has been sporadically reported, analysis of the profile of angiogenesis-related factors in a whole spectrum manner is needed to evaluate their cruciality and relationship. In our study, totally 104 angiogenesis-related factors were used to analyze their transcriptional changes along with ovarian cancer progression in the data of 601 patients obtained from TCGA datasets (AgilentG4502A_07_2 and AgilentG4502A_07_3). 33 candidates with significantly differential expression (p < 0.05) between normal and any stage were initially revealed. To narrow down the candidates, these factors were ranked by the significance of expressional changes between normal and all various stages and 13 factors with the most significant difference (p < 0.0001) were selected and their fold changes were further shown in a heat-map manner; these included MMP9, VEGFA, CXCL10, TIMP2, AGRP, MMP8, IGFBP-1, ENG, TEK, FGF7, CSF3, ANGPTL5, ANGPTL7. Moreover, Ingenuity Pathway Analysis (IPA) and DAVID database analysis were applied to analyze their functional categories in angiogenesis. In conclusion, we developed a signature of angiogenesis-related factors for patients with ovarian cancer. With appropriate validation, it can be applied as a prognostic index or used to select certain candidates for developing targeted therapies.

#17

Inhibition of LRG1 normalizes tumor vessels and improves efficacy of cancer therapeutics.

John Greenwood,1 Marie N. O'Connor,1 David Kallenberg,1 Rene-Filip Jackstadt,2 Angharad Watson,3 Julia Ohme,3 Laura Dowsett,1 Jestin George,1 Xiaomeng Wang,1 Ann Ager,3 Owen J. Sansom,2 Stephen E. Moss1. 1 _University College London, London, United Kingdom;_ 2 _Cancer Research UK Beatson Institute, Glasgow, United Kingdom;_ 3 _Cardiff University, Cardiff, United Kingdom_.

Introduction: The aim of this study was to determine whether deletion of the gene coding for the secreted glycoprotein, leucine-rich alpha-2-glycoprotein 1 (LRG1), or blockade of its action through function-blocking antibody treatment, improves tumor vascular function.

Experimental Procedures: The role of LRG1 was investigated in subcutaneous B16/F0 and LL2 mouse tumor models and in genetically engineered mouse models of intestinal (ApcMin) and pancreatic (KPC) cancers. Tumors were evaluated in wild type (WT) or Lrg1-/- mice or in WT mice treated with 15C4, a LRG1 blocking antibody. Tumor growth and survival were monitored and post-mortem analysis of vascular density, structure and function were undertaken. The effect of blocking LRG1 function on the efficacy of cisplatin or adoptive T cell therapy in B16/F0 tumor-bearing mice was determined.

Results: In Lrg1-/- mice or following functional blockade of LRG1 in WT animals there was a significant reduction in B16/F0 and LL2 tumor growth and improved survival in the ApcMin and KPC tumor-bearing mice. Vascular density was reduced in the B16/F0 and the KPC tumors but not in those of ApcMin. Most notably, we found that loss of LRG1 results in improved pericyte-endothelial cell association in the B16/F0 and ApcMin tumors. In the B16/F0 tumors we also observed an increase in the proportion of perfused vessels, and a reduction in vessel permeability and tumour hypoxia, consistent with our hypothesis that LRG1 is a vascular disrupting factor. Normalizing tumor vasculature to enhance vessel patency, reduce hypoxia and vascular leakage, and improve delivery of therapeutics has become a major objective. We therefore evaluated the effect of inhibiting LRG1 activity with the 15C4 antibody on the efficacy of cisplatin or adoptive CD8+ T cell therapy on B16/F0 tumor growth. Co-therapy revealed a highly significant reduction in tumor growth compared with monotherapy alone.

Conclusions: These data show that LRG1 subverts physiological angiogenesis by promoting dysfunctional vessel growth, and that therapeutic targeting of LRG1 reduces tumor neovascular growth and normalizes vascular function. We propose, therefore, that LRG1 is a potential therapeutic target in cancer, and that its inhibition may aid the delivery and efficacy of tumour therapeutics.

Funded by grants from the Medical Research Council UK, The Wellcome Trust, Rosetrees Trust, UCL Business and Moorfields Eye Hospital Special Trustees.

#18

MMP9 inhibition in mouse models of breast cancer: Therapeutic synergy with vinorelbine-based chemotherapy.

Francesca Reggiani,1 Loredana Vecchi,1 Valentina Labanca,1 Patrizia Mancuso,1 Amanda Mikels-Vigdal,2 Francesco Bertolini1. 1 _European Inst. of Oncology, Milan, Italy;_ 2 _Gilead Sciences, Inc., Foster City, CA_.

The combination of different therapeutic approaches, such as simultaneous targeting of tumor cells and stroma, may be a superior strategy to enhance anti-tumor responses in metastatic breast cancer (BC). Recently, the pro-tumorigenic activity of adipose tissue was further examined and suggested MMP9 promotes BC invasion, angiogenesis, and lung metastasis in mice (Reggiani et al., Cancer Res 2017). In the present work, MMP9 inhibition was combined with distinct therapeutic agents.

A monoclonal antibody that inhibits mouse MMP9 was administered to tumor-bearing (4T1) immune-competent BALB/c mice, alone or in combination with low-dose chemotherapy (vinorelbine, V or cyclophosphamide, CTX) or checkpoint inhibitors (anti-PD1 or anti-PDL1 monoclonal antibodies). MMP9 inhibition was also evaluated in diet-induced obese BC mice (Balb/c or FVB background).

The anti-tumor response of different combination treatments was assessed by tumor growth measurement, lung metastasis assessment, tumor angiogenesis evaluation through immunofluorescence, and immune system modulation by flow-cytometry.

The combination of V and MMP9 inhibition was superior to either agent alone. Tumor growth was reduced in mice administered with combined V and MMP9 inhibition compared to single treatment or control mice, administered with isotype control and saline (41% V+anti-MMP9 vs control, p=0.001). Lung metastases were significantly impaired (37% V+anti-MMP9 vs control, p=0.009). MMP9 inhibition exhibited the greatest reduction in intratumor angiogenesis among therapeutic agents tested (59% vs control, p=0.004). The combination of MMP9 inhibition with V compared to control, reduced B (1%, p=0.008), T cells (2.5%, p=0.01), and macrophages (35%, p=0.05) in the tumor.

Inhibiting MMP9 in diet-induced obese BALB/c mice was more effective in reducing tumor growth and metastatic progression, when compared to normal-weight mice (BC volume: 46%, p=0.01; lung metastasis: 53%, p=0.01). These data suggest that the increase of MMP9 release, observed in adipose progenitors when exposed to BC cells (Reggiani et al., Cancer Res 2017), was a mediator of the obesity-dependent pro-tumorigenic activity. Of note, when MMP9 inhibition was initiated in early phases of tumor growth in ErbB2+ BC FVB mice, the reduction of local tumor growth was more pronounced (started at 15 days vs 45 days: 18%, p=0.01; 15 days vs isotype control: 11%, p=0.01; 45 days vs isotype control: 64%, p=0.3 ns).

MMP9 inhibition is efficacious in mouse models of BC with concomitant obesity. In human BC patients, response to chemotherapy is reportedly lower, and the risk of relapse is higher in obese sub-population (Ewertz et al., JCO 2011). The additive effects of MMP9 inhibition with V may warrant further studies to characterize underlying molecular mechanisms in BC patients with high body mass index (BMI).

#19

Trastuzumab resistance accompanies vasculogenic mimicry in HER2-positive breast cancer cells.

Masafumi Shimoda, Ami Hori, Shinzaburo Noguchi. _Osaka University Graduate School of Medicine, Suita, Japan_.

Trastuzumab (Tzm) is a drug that targets HER2/ERBB2/Neu, and is essential for the treatment of HER2-positive breast cancer. However, resistance to the drug is a major obstacle in controlling the progression of this devastating disease. We hypothesized that Tzm load might induce a phenotypic change in HER2-positive breast cancer cells, enabling them to escape and survive Tzm activity. We conducted comprehensive immunophenotyping to detect the phenotypic changes in HER2-positive breast cancer cells loaded with Tzm, and compared the immunophenotype of Tzm-loaded cells with that of control cells based on the criteria determined a priori. Out of 242 cell surface antigens, 9 antigens were significantly upregulated and 3 were significantly downregulated. Surprisingly, all the antigens were related to endothelial and stem cell phenotypes, suggesting that Tzm load induced vasculogenic mimicry (VM). Moreover, we found VM markers like COX2, MMP2, MMP14, phospho-SMAD2/3, VEGFA, HIF1A, and TWIST1 to be upregulated in Tzm-loaded cells. However, Tzm-loaded cells did not exhibit tube formation on Matrigel, a hallmark of VM. Since the Tzm-loaded cells were still sensitive to Tzm, we used three Tzm-resistant cell lines to investigate if Tzm resistance accompanied VM. All Tzm-resistant cell lines exhibited tube formation on Matrigel. Importantly, several growth factors including EGF, FGF2, IGF1, and VEGF promoted VM in these cells, suggesting that single molecular-targeted drugs did not effectively inhibit VM as other growth factors could quickly compensate the VM pathways. We then examined if eribulin, a tubulin-binding chemotherapeutic drug that promotes vascular remodeling, inhibited VM in Tzm-resistant cells. Eribulin inhibited tube formation in Tzm-resistant cells at a clinically relevant concentration. In conclusion, Tzm load induces an incomplete vasculogenic phenotype in HER2-positive breast cancer cells. The cells exhibit VM after eventually acquiring Tzm resistance. Since VM drives metastasis, its regulation in Tzm-resistant HER2-positive breast cancer appears to be a promising approach in suppressing the progression of HER2-positive breast cancer.

#20

Inhibition of mutant EGFR in NSCLC promotes endothelin-1-mediated NSCLC disease progression and angiogenesis.

Stephen L. Ollosi,1 Margaret Soucheray,2 Jeffrey Becker,2 Ines Pulido,3 Annika Dalheim,2 Fatima Al-Shahrour,2 Wei Qui,2 Michael Nishimura,4 Julian Carretero,3 Simon Kaja,2 Takeshi Shimamura2. 1 _Loyola University, Lemont, IL;_ 2 _Loyola University, IL;_ 3 _University of Valencia, Spain;_ 4 _Loyola University, Maywood, IL_.

Despite recent advances in the treatment of NSCLC targeting of EGFR kinase domain mutations with tyrosine kinase inhibitors (TKIs), work needs to be done to reduce morbidity and improve survival for NSCLC patients. In NSCLC, tumor angiogenesis has been identified as important therapeutic target in combination with EGFR TKIs. However, only small advancements have been made for the use of angiogenesis inhibitors in NSCLC and it remains elusive why the inhibition of VEGF-mediated neovascularization is not therapeutically efficacious. We present evidence that a subpopulation of NSCLC cells with the EGFR TKI-induced epithelial to mesenchymal transition (EMT) contributes to the attenuation of response to anti-VEGF/VEGFR therapy. One of the hallmarks of cancer is heterogeneity and we have previously demonstrated that tumor heterogeneity within NSCLC cells lines harboring EGFR kinase domain mutations gives rise to divergent resistance mechanisms in response to treatment. In vivo admix models are instructive in studying intratumoral heterogeneity and in elucidating therapeutic responses. While NSCLC cell with acquired EGFR TKI resistance and EMT phenotype did not exhibit growth advantage in vivo, a 50% epithelial EGFR TKI sensitive and 50% mesenchymal EGFR TKI resistant admix provided significant growth advantage in vivo assessed by caliper measurement. Moreover, the admix tumors are resistant to EGFR TKI treatment. Interestingly, short-term in vitro co-culture of epithelial and mesenchymal cells did not provide a proliferative/growth advantage. The preliminary result led us to hypothesize that the epithelial-mesenchymal admix helps to create a tumor-host niche that is suitable for EGFR TKI resistance. To this end, we utilized the Luminex multiplex assay system to quantify secreted growth factors, cytokines, and chemokines. We have discovered that epithelial EGFR TKI sensitive cells secrete a significant amount of VEGF-A and cells with acquired/transient EGFR TKI resistance with an EMT phenotype secrete substantial amount of endothelin-1 (EDN1). Using an in vitro tube formation assay, we showed that secreted VEGF-A and EDN1 in admix conditions work synergistically to promote angiogenesis. Ectopic overexpression of EDN1 in EGFR-mutated HCC827 cells and predominantly secreting VEGF-A conferred significant resistance to gefitinib in vivo compared to control HCC827 cells. Importantly, the increased HUVEC cell tube formation in conditioned media from admix cells rich in both VEGF-A and EDN1 was significantly attenuated by the dual inhibition of VEGFR and EDNRA. Notably, the exposure of HCC4006 cells to 100nM gefitinib for 72 hours alone promoted transition from predominately VEGF-A secretion to EDN1 secretion. This suggests that the therapeutic efficacy of combining EGFR inhibitor with VEGFR inhibitors in the treatment of EGFR mutation positive NSCLC should be significantly tempered.

### Breast Cancer Metastasis

#21

p66ShcA is a contextual breast cancer metastasis promoter or suppressor depending on the tumor microenvironment.

Jesse Hudson,1 Kyle Lewis,1 Julien Senécal,2 Alexander Kiepas,2 Sébastien Tabariès,2 Valérie Sabourin,1 Ryuhjin Ahn,1 Rachel La Selva,1 Peter Siegel,2 Giuseppina Ursini-Siegel1. 1 _McGill University Lady Davis Institute for Medical Research, Montreal, Quebec, Canada;_ 2 _McGill University Goodman Cancer Research Centre, Montreal, Quebec, Canada_.

Introduction: Src homology and collagen A (ShcA) adaptor proteins are essential during breast cancer progression. However, the role of the largest isoform, p66ShcA, is conflicting and still poorly understood. Under high levels of stress p66ShcA is phosphorylated on serine36, within its CH2 domain, allowing it to translocate to the mitochondria and induce the formation of reactive oxygen species (ROS) to promote apoptosis. Previously, we provided the first in vivo evidence that p66ShcA can influence both pro and anti-tumorigenic functions in ErbB2+ luminal breast cancer. Stable overexpression of p66ShcA reduced tumor outgrowth while simultaneously elevating the expression of mesenchymal genes to promote tumor plasticity. In this study, we evaluated the role of p66ShcA in metastatic dissemination to the lung in a model of basal breast cancer, which typically is associated with poor outcome. Hypothesis: p66ShcA regulates basal breast cancer metastasis to the lung. Methods: Screening a panel of basal breast cancer cells in vivo selected to the lung, liver and bone, we found p66ShcA enriched in lung and liver metastatic variant cell lines relative to parental breast cancer cells and those in vivo selected through the mammary fatpad. Using CRISPR/Cas technology we genetically deleted p66ShcA and rescued with p66ShcA-WT or a p66ShcA-S36A mutant. Lung metastatic basal breast cancer cells were injected into the fourth gland of the mammary fatpad. Tumors were measured using calipers 3 times/week following first palpation (>50mm3) followed by surgical resection to monitor the lung metastatic burden. In addition, we performed tail vein injections to monitor lung metastatic burden following direct entry into the circulation. Results: Loss of p66ShcA significantly reduced the metastatic burden to the lung following surgical tumor resection and this reduction was partially rescued by stable overexpression of wild type (WT), but not S36A mutated p66ShcA from the primary site. These effects were not due to altered anti-oxidant expression levels, changes in oxidative DNA Damage or microvessel density. Intriguingly, we found that WT-rescue of p66ShcA significantly elevated the migratory speed of breast cancer cells in vitro and corroborates our in vivo metastatic burden data. However, this is in stark contrast to our tail vein data, where WT-rescue of p66ShcA significantly inhibited lung metastasis. Conclusion: p66ShcA is required for efficient metastasis to the lung in a mitrochondrial-ROS-dependent fashion from the primary site. Our data suggest that cues from the tumor microenvironement of the mammary fatpad are essential for successful colonization and outgrowth at oxygen rich sites, such as the lung, as breast cancer cells with elevated expression of p66ShcA directly entering the circulation suppressed lung metastatic burden.

#22

The Y537S ESR1 mutation is a dominant driver of distant ER-positive breast cancer metastasis.

Guowei Gu,1 Lin Tian,1 Meng Gao,2 Yassine Rechoum,1 Luca Gelsomino,1 Derek Dustin,1 Arnoldo Corona-Rodriguez,1 Amanda R. Beyer,1 Anna Tsimelzon,1 Xiang Zhang,1 Sebastiano Ando',3 Suzanne Fuqua1. 1 _Baylor College of Medicine, Houston, TX;_ 2 _MD Anderson Cancer Center, Houston, TX;_ 3 _University of Calabria, Cosenza, Italy_.

Background: Estrogen receptor (ESR1) mutations occur at a high frequency in metastatic breast tumors in patients treated with hormonal therapy in the metastatic setting. We do not know if these mutations are involved in metastasis. Experimental design and methods: We generated ESR1 Y537S homozygous mutations using CRISPR Casp-9 technology. Treatment synergy was evaluated using Compusyn. Athymic mice were used in tumor xenograft studies. ChIP-Seq and transcriptome analyses were performed. Results: We generated CRISPR ESR1 Y537S mutation homozygous knock-in clones and lentiviral stable pools in MCF-7 cells. Transcriptome profiling revealed elevated expression of Hallmark pathways, including epithelial mesenchymal transition (EMT) and estrogen-regulated gene expression. Mutant cell growth was resistant to tamoxifen, but responsive to fulvestrant treatment. Synergistic treatment effects were observed with fulvestrant and the mTOR inhibitor everolimus or the CDK4/6 inhibitor palbociclib. CRISPR Y537S mutant knock-in cells grown in the mammary fat-pad of athymic mice spontaneously metastasized to distant organs including the lung, intestine, and kidneys. In the presence of estrogen, there was no difference in the frequency of distant macrometastases between parental wild-type ER and CRISPR Y537S mutant ER mice. However, in the absence of estrogen, mimicking aromatase inhibitor treatment, 80% of CRISPR Y537S mutant ER mice displayed overt distant macrometastases, but none were observed in parental wild-type ER mice (p=0.04). Distant tumors retained ER expression and hormone sensitivity. Comparison of residual tamoxifen-treated metastatic tumors with tumors grown at the primary mammary fat-pad site using immunoblot analysis demonstrated significant reduction in estrogen-regulated gene expression, but no effect on the expression of biomarkers associated with EMT, suggesting a disconnect between EMT and distant metastasis in mutant cells. EMT genes were also identified as direct binding site targets in Y537S mutant cells compared with wide-type ER using ChIPSeq. We discovered that expression of the Y537S mutant was dominant, driving the growth of distant metastatic tumors when co-expressed with wild-type ER cells. A Y537S ER mutant-specific gene expression signature predicted poor disease-free survival of ER-positive patients using the METABRIC database, and lung-specific metastasis-free survival in a Memorial Sloan Kettering dataset. Conclusion: The Y537S ER mutation is a driver of distant metastasis in ER-positive breast cancer cells. A Y537S ER mutant-specific gene expression signature predicted poor disease-free, and distant lung metastasis in ER-positive patients. Mutation status is a potential new predictive factor for hormone therapy of metastatic breast cancer patients on maintenance hormonal therapy.

#23

Chemotherapy and tau interplay facilitates breast-to-brain metastasis.

Behnaz Saatian, Krutika Deshpande, Alex Julian, Brooke Naomi Nakamura, Ling Shao, Josh Neman. _University of Southern California, Los Angeles, CA_.

Breast cancer represents the second most common cause of brain metastasis with the rate of 10-16% after lung cancer. The overall survival in brain metastatic patients is very short ranging from 2 to 25 months. Considering the high incidence of brain metastasis and the limitation of available therapies, developing preventive strategies and early detection methods are required to improve the survival rate in the patients. Many primary and brain metastatic breast cancer patients represent cognitive deficits. Besides, chemo-treated breast cancer patients show memory problems before brain metastases diagnosis. Although the cause of cognitive impairments after chemotherapy have not been explored, a recent study revealed that chemotherapy promotes breast cancer metastasis. Correspondingly, our mRNA expression analysis showed downregulation of the tight junction markers in chemo-treated choroid plexus cells (CPs), suggesting that chemotherapy is likely to cause an increase in the blood-cerebral spinal fluid barrier (BCSFB) permeability. Chemotherapy also induces upregulation of tau expression in CP cells. Tau is known as a pathological hallmark of Alzheimer's disease (AD) and neurodegenerative disorders. The abnormally phosphorylated and aggregated form of tau play critical role in neurodegenerative diseases as of AD. Tau expression, both at the mRNA and protein level, is significantly elevated in breast to brain metastases (BBMs). Therefore, we hypothesized that chemotherapy upregulates tau expression in breast cancer cells leading to the BCSFB and blood-brain barrier (BBB) permeability, and consequently, breast to brain metastasis and neurodegeneration. To test our hypothesis, the effect of chemotherapy on the BCSFB and BBB permeability were investigated using the trans endothelial electrical resistance (TEER) and migration assays in the in vitro models. Our results show an increase in abnormal tau expression in chemo-treated primary breast cancer cells. BBM-derived tau is abnormal and forms paired helical filaments (PHFs), similar to Alzheimer's. We conclude that breast to brain metastasis is facilitated through an interplay between chemotherapy, and tau expression and release from breast cancer cells. Additionally, we suggest that tau plays a key role in neurodegeneration defects in brain metastatic patients. This study opens a new path towards prevention and early detection of breast cancer metastasis to the brain, cognitive decline and neurodegeneration in metastatic patients.

#24

Extracellular HMGA1 promotes tumor invasion and metastasis in breast cancer.

Olga Méndez,1 Vicente Peg,2 Candida Salvans,1 Mireia Pujals,1 Yolanda Fernández,3 Ibane Abasolo,3 Jose Perez,4 Ana Matres,1 Josep Tabernero,4 Javier Cortés,4 Joaquín Arribas,1 Josep Villanueva1. 1 _Vall D'Hebron Inst. of Oncology, Barcelona, Spain;_ 2 _Pathology department. Vall d´Hebron Institute de Recerca, Barcelona, Spain;_ 3 _Vall D'Hebron Institut de Recerca, Barcelona, Spain;_ 4 _Department of medical oncology. Vall D'Hebron University Hospital, Barcelona, Spain_.

The recent in-depth study of the cancer secretome suggests that a fraction of the intracellular proteome might be playing alternative roles in the extracellular space during tumorigenesis through its unconventional secretion. A proteomic study aimed at evaluating the role of unconventional protein secretion in the context of tumor invasion led us to investigate the high mobility group A1 (HMGA1) chromatin-binding protein whose overexpression has been correlated with high-grade carcinomas and reduced patient-survival. Here we show that HMGA1 is not only oversecreted in invasive breast tumor cells, but its blockage with specific antibodies in the extracellular space impairs tumor cell migration and invasion. The invasive phenotype caused by extracellular HMGA1 (eHMGA1) is dependent on ERK1/2 signaling and a blocking-Ab against eHMGA1 decreases pERK and re-localizes HMGA1 to the nucleus concomitantly to the loss of invasion capacity. Remarkably, pre-treatment of invasive breast tumor cells with an HMGA1-blocking Ab reduces their metastatic ability in an orthotopic breast cancer xenograft model. HMGA1 also shows a cytoplasmic localization in primary human triple-negative breast cancer (TNBC) tumors that developed distant metastasis while displaying a nuclear localization in TNBC tumors that did not progress to metastatic disease. Thus, eHMGA1 could be a novel drug target in aggressive breast cancer and a biomarker predicting the onset of distant metastasis.

#25

Neuroligin 4X: A neural cell adhesion molecule, in breast cancer.

Henry J. Henderson,1 Karanam Balasubramanyam,1 Rajeev Samant,2 Komal Vig,3 Shree R. Singh,3 Clayton Yates,1 Deepa Bedi1. 1 _Tuskegee University, Tuskegee, AL;_ 2 _University of Alabama at Birmingham, Birmingham, AL;_ 3 _Alabama State Univeristy, Montgomery, AL_.

Purpose: Neuroligins are neural cell adhesion molecules that are implicated in heterotopic cell adhesion. Our previous studies using a selection from a combinatorial random peptide library against breast and pancreatic cancer cell lines identified several peptides mimicking neuroligin (NLGN-1, 3 and NLGN4X respectively). In this study, we investigated the expression, relevance and functional significance of Neuroligin 4X in human breast cancer.

Methods: NLGN4X expression data for all breast cancer cell lines in the Cancer Cell Line Encyclopedia were analyzed. The correlation between NLGN4X levels and clinicopathologic parameters were established within Oncomine datasets. As a proof-of- principle, we evaluated survival by generating Kaplan–Meier plots using publicly available microarray datasets. To examine the expression of NLGN4X, immunohistochemistry of breast cancer tissue arrays was conducted. Additionally, flow cytometry and immunofluorescence staining were performed to investigate NLGN4X expression in MDA-MB-231, MCF-7, and MCF-7 treated with TGFβ. To observe the effect of NLGN4X gene knockdown, MDA-MB-231 cells were transfected with NLGN4X-specific siRNA. NLGN4X gene expression was analyzed by RT-PCR, western blot and flow cytometry. Post transfection, wound healing and cell viability assays were performed to determine the effect of NLGN4X knockdown on migration and proliferation. Apoptotic outcomes were examined through detection of caspase activation and Annexin V-FITC methods by using flow cytometry.

Results: NLGN4X showed abundant expression in breast cancer tissues. The evaluation of bioinfomatic datasets revealed that NLGN4X expression was higher in triple negative breast cancer (TNBC) and in metastatic tissues. Interestingly, high NLGN4X expression correlated with a decrease in relapse free-survival in TNBC. RT-PCR, flow cytometry and immunofluorescence validated that NLGN4X expression was high in MDA-MB-231 as well as MCF-7 TGFβ, which suggests that NLGN4X is associated with the mesenchymal phenotype. Knockdown of NLGN4X expression by siRNA significantly decreased cell proliferation(P<0.001) and migration(P<0.05) in MDA-MB-231 in conjunction with the induction of apoptosis as determined by annexin staining, elevated caspase 3/7 and cleaved PARP by flow cytometry.

Conclusion: Our findings suggest that NLGN4X could represent a novel biomarker and therapeutic target. This is the first study to link the expression of neuronal cell adhesion molecules, neuroligins, to breast cancer. We intend to continue the investigation of the role of NLGN4X to elucidate the mechanistic role of this adhesion protein in breast cancer progression and metastasis.

#26

MiR-205 targets integrin-α5 and inhibits triple-negative breast cancer metastasis.

Yajuan Xiao,1 Zhishan Wang,1 Yunfei Li,1 Jianjun Wu,1 Hua Tao,1 Aimin Li,2 Rongcheng Luo,2 Chengfeng Yang1. 1 _University of Kentucky, Lexington, KY;_ 2 _Southern Medical University, Guangzhou, China_.

Triple negative breast cancer (TNBC) is the most aggressive subtype of breast cancer, lacking effective target therapy compared to ER/PR+ or HER2+ breast cancer. Micro-RNAs are small non-coding RNAs playing a significant role in cancer stemness maintenance, tumor growth, and metastasis. Studies showed that miR-205 plays an important role in regulating EMT, cell proliferation and migration, tumor growth and metastasis, and drug-resistance of cancers, such as lung, renal, prostate and breast cancer. However, whether miR-205 plays a role in TNBC tumor growth and metastasis remains to be determined. We found that higher miR-205 expression level was strongly correlated with breast cancer patient better overall survival. The expression level of miR-205 was prominently downregulated in basal mesenchymal-like highly migratory and invasive TNBC cell lines, compared to basal epithelial-like TNBC and other breast cancer cell lines. Bioinformatics analysis identified that integrin- α5 (ITGA5) is a potential target of miR-205. Western blot analysis revealed that the expression level of integrin-α5 is significantly higher in basal mesenchymal-like TNBC cell lines than other breast cancer cell lines. Two miR-205 overexpression cell lines were established by transfecting TNBC LM2 and SUM-159 cell lines with miR-205 precursor expressing lentivirus. The expression level of ITGA5 was downregulated in miR-205 overexpressing LM2 and SUM-159 cells. Wound healing assay and transwell cell migration assay revealed that miR-205 overexpression reduced migration of TNBC cells, although no difference of cell proliferation was observed between miR-205 overexpression and vectors control cells as determined by the MTT assay. The cancer stem cell-like property of TNBC cells was inhibited by miR-205 overexpression as demonstrated by the mammary sphere formation assay. In in vivo experiments, mammary tumor growth and lung metastasis were inhibited by miR-205 overexpression using mammary fat pad xenograft tumor models. When ITGA5 was re-expressed in miR-205 overexpression cell lines, the migratory ability of TNBCs was rescued. The metastatic capability of TNBC mammary xenograft tumors was also restored. Further mechanistic studies proved that miR-205 can reduce the formation of lamellipodium, a hallmark of Rac1/cdc42 activation and cell migration, by targeting ITGA5. Rac1/cdc42 pull-down assay showed that miR-205 inhibited Rac1/cdc42 activation, while ITGA5 overexpression restored the activation of Rac1/cdc42. For uncovering the upstream regulator of Rac1/cdc42, several Rac inhibitors were scanned and it was found that inhibition of vav2 by EHop-016 reduced the downstream effects from ITGA5 signaling. CoIP revealed that Src/Vav2 pathway was activated by ITGA5. In conclusion, miR-205 suppresses TNBC cell migration and tumor metastasis by targeting ITGA5 and inhibits Src/vav-2/Rac1/cdc42 pathway.

#27

Regulating Rac1 signaling proteins influences migratory and protrusive behaviors of metastatic breast cancer cells in aligned, 3D collagen and collagen-like matrices.

Rachel Van Doorn, Joseph Szulczewski, Suzanne Ponik. _University of Wisconsin-Madison, Madison, WI_.

Breast cancer is the most commonly diagnosed cancer in women, with one out of every eight women being diagnosed in their lifetime. Our lab has previously shown that aligned collagen promotes tumor progression to metastatic disease, and predicts poor prognosis for breast cancer patients. Aligned collagen increases the persistence of cellular migration by limiting the number of protrusions from the cell body. The regulation of Rac1 and Rho proteins are important for cell motility through formation of lamelliepodia. Deregulation of these proteins and those upstream of Rac1, can result in cancer cell migration, invasion and metastases. Understanding how these proteins are regulated in different 3D collagen tumor microenvironments is important for determining phenotypes of invasive breast cancers. Inhibition of cell motility through the Rac1 pathway may limit cancer cell migration and prevent metastatic disease. While many studies have investigated cell migration in 2D, little is known regarding the cellular mechanisms that regulate both breast cancer cell migration and protrusive events in 3D aligned collagen. In vitro models will be used to recapitulate 3D aligned, and random collagen environments and assess the mechanisms of cell migration and protrusion dynamics by live-cell, time-lapse microscopy. Previous work from our group determined that MDA-MB-231 cells seeded into collagen environments had fewer protrusions, which localized in the directions of collagen alignment, when migrating along aligned fibers compared to MDA-MB-231 cells in the random collagen matrices. Preliminary data demonstrates that signaling proteins upstream of Rac1 such as, small GTPases, DOCK180 and FilGAP, dictate cellular protrusions and migratory persistence. MDA-MB-231 cell lines with doxycycline-induced knock-down Rac1 GTPase activating protein (GAP), FilGAP, and guanine nucleotide exchange factor (GEF), DOCK180, demonstrate that the regulation of Rac1 activity through these specific proteins plays a role in breast cancer cell migration and protrusion dynamics in an aligned collagen environment. Understanding the regulation of breast cancer cell migration in aligned and random 3D collagen environments will enhance the current understanding of progression to metastatic disease on a cellular level.

#28

Role of CSF in facilitating synaptic plasticity in breast-to-brain metastasis.

Krutika Deshpande, Alex Julian, Behnaz Saatian, Josh Neman. _University of Southern California, Los Angeles, CA_.

Breast cancer is the most common cancer in women worldwide, and 10-30% of all breast cancer patients eventually develop brain metastases. Breast-to-brain metastases (BBMs) in stage IV breast carcinoma are concurrent with poor prognosis, neurological degeneration, <10-month survival, and account for 10% of all breast cancer related deaths. Accumulating scientific evidence underlines the importance of the tumor microenvironment in the establishment and progression of metastatic cancers. Due to the inability of current therapies to cross the blood brain barrier it has become increasingly important to study the interaction of BBMs with the neuronal microenvironment, to develop novel therapeutic interventions. Although the role of the blood brain barrier (BBB) in breast to brain metastasis has been widely studied, the contribution of the cerebrospinal fluid as a microenvironmental niche has been largely unexplored. The cerebrospinal fluid (CSF) produced by the choroid plexus (CP) cells in the brain, bathes the brain and spine; and serves as an initial point of contact to the brain for circulating breast cancer cells that reach the central nervous system.

We treated EMT Her2\+ BBM cells with choroid plexus derived artificial CSF. Both mRNA and protein analysis showed enhanced expression of Reelin in treated BBMs, compared to controls. Reelin is an extracellular matrix protein that mediates maturation and synaptic communication (plasticity) in neurons. It has also been shown to be a negative regulator of invasive properties in tumor cells. mRNA expression analysis also showed that the increase in Reelin in the CSF-treated BBMs was correlated with a marked reduction in the expression of invasive EMT markers, and enhanced expression of genes involved in neuronal synaptic plasticity. Correspondingly, through immunofluorescence studies on surgically resected brain metastases from patients, we were able to show to for the first time, that Her2+ metastases in the brain parenchyma display enhanced expression of markers of neuronal synaptic plasticity (SNAP25, NLGN1/2, ENAH, Neurexin). We were also able to show that Her2+ BBMs express high levels of Reelin, its receptor ApoER2 (LRP8) and intracellular mediator Dab1. This suggests that metastatic breast cancer cells mimic their immediate microenvironment to establish themselves in the brain. Our data indicates that the CP/CSF microenvironment aids in establishing tumor synaptic plasticity by inducing Reelin expression, and altering EMT profiles in BBMs. The contribution of the choroid plexus/CSF in the establishment of brain metastases in breast cancer has not been singularly explored, and this study provides novel insights into understanding its role as a microenvironmental niche.

#29

RANK-RANKL signaling inhibition delays early breast cancer bone metastasis formation.

Sofia Sousa,1 Evelyne Gineyts,2 Sandra Geraci,1 Martine Croset,1 Philippe Clézardin1. 1 _Faculty of Medicine Laennec, University of Lyon 1, Lyon, France;_ 2 _National Institute of Health and Medical Research (INSERM) U1033, Lyon, France_.

Denosumab is a fully human monoclonal antibody to human RANKL, currently approved in the management of established bone metastases, mainly due to its anti-resorptive activity which blocks tumor-induced osteoclastogenesis and osteolysis.

Our aims are to determine the role of RANK expression in breast cancer (BC) bone metastasis and the efficacy of RANKL blocking by AMG161 (IgG1 equivalent to Denosumab). AMG161 does not recognize murine RANKL, so we use a BalB/c nude knock-in (KI) mouse model, which expresses a chimeric murine/human RANKL. Moreover, non-invasive in vivo imaging techniques such as bioluminescence and µCT were used to monitor metastatic tumor burden and bone degradation, respectively. To evaluate bone marrow early colonization by BC cells, DiD labelling and ex vivo laser scanning multiphoton confocal microscopy were used.

Long-term studies showed that intra-arterial (i.a.) injection of MDA-MB231 RANK versus MDA-MB231 leads to a poorer overall survival (OS), with the most striking differences seen in the earlier time-points. In this respect, the extent of radiographic osteolytic lesions was higher in the RANK overexpressing group than in the control group. Similar findings were observed following intra-tibial inoculation of RANK-expressing MCF-7 cells, when compared to that observed with parental MCF-7 cells. However, at later time points, bone and lung metastatic burden was similar between the two groups, suggesting that the poor OS of the RANK overexpressing group was due to a faster tumor progression owing to enhanced bone homing and colonization. Indeed, using short-term studies (5 and 14 days), i.a. injection of MDA-MB231 RANK led to higher incidence and extension of bone marrow micro-metastases than MDA-MB231, whereas there was no difference for lung micro-metastases. Daily subcutaneous injections of 1,5mg/kg AMG161 antibody to MDA-MB231RANK tumor-bearing animals, decreased bone micro-metastases and early bone marrow colonization without affecting lung micro-metastasis.

We therefore hypothesized that RANK overexpressing BC cells interact with RANKL produced by osteocytes, osteoblasts and other cells in the osteogenic niche, favoring survival and colonization of BC cells in bone. To test this hypothesis, we made use of heterotypic mammosphere assays in which BC cells were cultured with or without cells of the osteogenic niche (e.g. pre-osteoblasts, mesenchymal stem cells), recombinant RANKL and anti-RANK antibody or AMG161. Co-culture with recombinant RANKL or a cellular source of RANKL increased BC cell mammosphere formation, and blocking RANK/RANKL signaling impaired it.

In conclusion, RANK/RANKL signaling promotes the early engraftment of BC cells in the bone marrow. Blocking this signaling with AMG161 decreases bone marrow micro-metastasis formation in vivo, suggesting that adjuvant treatment of early-stage breast cancer patients with denosumab may prevent bone relapse.

#30

Effect of Wnt5a on aggressiveness of estrogen receptor-positive breast cancer and cancer cell migration through ALCAM pathway.

Yoshie Kobayashi,1 Takayuki Kadoya,1 Ai Amioka,1 Hideaki Hanaki,1 Shinsuke Sasada,1 Norio Masumoto,1 Koji Arihiro,2 Akira Kikuchi,3 Morihito Okada1. 1 _Hiroshima University, Hiroshima, Japan;_ 2 _Hiroshima University Hospital, Hiroshima, Japan;_ 3 _Osaka University, Osaka, Japan_.

Purpose: Wnt signaling occurs in β-catenin-dependent pathways, through β-catenin regulates the expression of many genes, and in the β-catenin-independent planar cell polarity (PCP) and Wnt/Ca2+ pathways. Wnt5a is a key ligand in activation of β-catenin-independent pathways, involved in cell motility and polarity through downstream signaling (e.g., JNK phosphorylation). Wnt5a expression correlates significantly with malignancy and stage of progression in malignant melanoma, gastric cancer, prostate cancer, lung cancer, and pancreatic cancer. In contrast, Wnt5a is a tumor suppressor in colorectal cancer, thyroid cancer, liver cancer, and malignant lymphoma. Thus, the effect of Wnt5a expression differs between organs. The role of Wnt5a expression in breast cancer remains elusive. In this study, we examined significance of Wnt5a expression in breast cancer.

Experimental design: The relationships between Wnt5a expression and clinicopathologic factors were assessed in 178 consecutive cases of invasive breast cancer resected in Hiroshima University Hospital between January 2011 and February 2014. In addition, altered gene expression following Wnt5a addition was mapped as possible Wnt5a-induced transformation pathways.

Results: Wnt5a was positively expressed in 69 of 178 cases (39%) of invasive breast cancer. Wnt5a expression was strongly correlated with estrogen receptor (ER)-positive (P < 0.001). Analysis of the relationship between Wnt5a expression and malignancy in 153 cases of ER-positive breast cancer revealed significant correlations with lymph node metastasis, nuclear grade, and lymphatic invasion. Relapse-free survival was shorter in cases of Wnt5a-positive breast cancer compared to Wnt5a-negative breast cancer cases (P = 0.024). Constitutive expression of Wnt5a in MCF7 cells enhanced migratory capacity, whereas knockdown of Wnt5a reduced this capacity. DNA microarray analysis identified activated leukocyte cell adhesion molecule (ALCAM) as the molecule which is primarily induced by Wnt5a. ALCAM was expressed in 69% of Wnt5a-positive breast cancers but only 27% of Wnt5a-negative breast cancers in 153 cases of ER-positive breast cancer, showing a statistical correlation between Wnt5a and ALCAM expressions. Inhibition of ALCAM in MCF7 constitutively expressing Wnt5a cells reverted the effect of Wnt5a, confirming the important of this molecule in migration of ER-positive breast cancer cells.

Conclusions: In ER-positive breast cancer, Wnt5a expression is related with high malignancy and poor prognosis. We speculate that Wnt5a expression increases malignancy in breast cancer by enhancing migratory capacity of cancer cells through induction of ALCAM expression. Wnt5a may be useful as a predictor of malignancy, a therapeutic target, and a prognostic indicator in ER-positive breast cancer.

#31

Targeted dissociation of PF1 from SIN3A chromatin regulator complex inhibits tumor growth and metastasis in triple-negative breast cancer.

Rama Kadamb, Nidhi Bansal, Boris A Leibovitch, Yeon-Jin Kwon, Ming-Ming Zhou, Eduardo Farias, Samuel Waxman. _Ichan School of Medicine at Mount Sinai, The Tisch Cancer Institute, New York, NY_.

Triple negative breast cancer (TNBC) is characterized by an aggressive, poorly differentiated phenotype associated with early recurrence. Chemotherapy is the only treatment option due to the lack of an effective molecular target. Targeting reversible epigenetic alterations in TNBC to reprogram the TNBC phenotype may increase chemosensitivity and identify effective targeted therapies. The SIN3 chromatin modifier complex is a multidomain scaffold protein which plays a key role in multiple cellular functions including epigenetic gene expression regulation, making SIN3 a potential therapeutic target. The PAH-2 domain of SIN3 binds with different affinities to a group of proteins that contain variations of an amino acid sequence known as the SIN3 interaction domain (SID). We previously reported the effects of blocking PAH-2 interaction with MAD-1, a SID sequence containing protein by using SID peptides and small molecule inhibitors (SMI) as decoys. We now report the effects of blocking the PAH-2 binding of PF1 and TGIF1, two other Sin3 interacting proteins with different amino acid sequence and PAH-2 binding affinities. Treatment with MAD-1 SID peptide and SMI revert the epithelial to mesenchymal transition (EMT) process, inhibit cancer stem cells expansion, cell invasion, tumor growth and metastasis development in human and mouse models of TNBC. TGIF1 interaction with SIN3 is associated with the invasive phenotype and EMT regulation by modulating wnt signaling and inhibiting beta catenin nuclear localization which is reverted by TGIF1 knockdown. PF1, which binds MRG15 and KDM5A/B, an epigenetic modifier, is involved in the regulation of cancer stem cell compartment expansion, DNA damage control, senescence, apoptosis and the expression of genes involved in metastatic progression. Interestingly, decrease in H3K4me3 is found in stem cell and EMT genes following 72 hour SID peptide treatment. PF1 dissociation from Sin3 by transfecting MDA-MB-231 and 4T1 cells with a PF1-SID expressing vector grown in 3D morphogenesis cultures display the formation of small organoids in the MDA-MB-231PF1-SID and tubular morphogenesis in the 4T1PF1-SID cells. There is increased activated caspase-3 and γH2AX, reduced Ki67, tenascin-C and marked cortical actin reorganization. This is accompanied by small organized colonies with loss of invasive and proliferative phenotype. In vivo, 4T1PF1-SID barely form tumors and have a 95% decrease in lung metastasis. These results show that decoys can be designed to block specific proteins that bind to the PAH-2 domain resulting in different outcomes which contribute to a common effect on inhibition of tumor progression and metastasis dissemination. Therefore, this strategy opens a potential therapeutic alternative for TNBC patients for whom there are no other therapeutic options besides chemotherapy.

#32

BCL11A is necessary for the expression of extracellular matrix genes and metastatic progression of triple-negative breast cancer.

Darcie D. Seachrist, Natasha N. Ingles, Molly M. Hannigan, Donny D. Licatalosi, Ruth A. Keri. _Case Western Reserve University, Cleveland, OH_.

Cancer stem cells (CSC) contribute to the high incidence of metastatic recurrence in triple-negative breast cancer (TNBC) and elucidating the mechanisms controlling CSC properties should provide novel targets for therapeutic development that improves patient outcomes. The transcription factor BCL11A is highly differentially expressed in TNBC versus all other subtypes and controls breast CSC phenotypes such as marker expression and tumor initiation. However, the genes that BCL11A targets to control CSC biology and whether BCL11A promotes metastatic progression are unknown. To address these questions, we assessed the impact of transiently silencing the expression of BCL11A in TNBC cells using siRNA transfections. Reducing BCL11A expression did not impact TNBC cell viability or in vitro migratory capacity. However, silencing BCL11A significantly decreased invasion of TNBC cell lines, in vitro. Furthermore, stable shRNA-mediated silencing of BCL11A in the highly metastatic MDA-MB-231 cell line reduced metastatic outgrowth when xenografted into immunocompromised mice, suggesting that BCL11A is critical for metastatic progression of TNBC. To identify the BCL11A-regulated transcriptome in TNBC, we performed RNA-seq analysis of cells transiently transfected with non-targeting or BCL11A-targeted siRNAs in the MDA-MB-231 cell line. Consistent with the impact of BCL11A on invasion, numerous genes involved in adhesion and extracellular matrix were reduced with BCL11A silencing. The gene encoding the matrix metalloproteinase, MMP1, was the most significantly differentially expressed gene, with a 10-fold decrease in RNA expression following BCL11A suppression. We confirmed this down-regulation by both qRT-PCR and western blots of independent samples, as well as in the HCC1143 TNBC cell line. Current studies are assessing the functional impact of MMP1 regulation by BCL11A on invasion and metastatic progression of TNBC.

#33

Calsequestrin 2 regulates proliferation, migration, and invasion in triple-negative breast cancer cells.

Ju Hee Kim, Bok sil Hong, Woohang Heo, Jong Min Han, Wonsik Han, Dong-Young Noh, Hyeong-Gon Moon. _Seoul National University Hospital, Seoul, Republic of Korea_.

Triple-negative breast cancer (TNBC) is the most heterogeneous and aggressive breast tumor subtype defined by absence of receptor for estrogen, progesterone, or HER2. However, the biologic mechanism for TNBC phenotype is still unclear. Here, we show that expression of Calsequestrin 2 (CASQ2), a Ca2+-binding protein, correlates with increase of proliferation, migration, and invasion, suggesting that intracellular Ca2+ may contribute to tumor growth and metastatic phenotype. CASQ2 is the main Ca2+-binding protein inside the sarcoplasmic reticulum of cardiomyocytes. CASQ2 forms a complex with ryodine receptor 2 (RyR2) luminal calcium release channel in cardiac muscle. Ca2+ is a sequestor and regulator of diverse cellular processes, and specific Ca2+ channels play important roles in cell proliferation and invasiveness of cancers. To know the role of CASQ2 TNBC cells, we established CASQ2-overexpressing stable cells in Hs578T (Hs578T-CASQ2) using retrovirus. Stimulation with caffeine triggered the remarkable increase of intracellular calcium in Hs578T-CASQ2 cells; in addition, the basal level of calcium in cells had much higher amount of Hs578T-CASQ2 than Hs578T. By contrast, calcium chelator BAPTM/AM blocked CASQ2-induced calcium release. Hs578T-CASQ2 cells showed higher level of proliferation, migration and invasion rate compared to Hs578T, which indicated that overexpression of CASQ2 related with cellular functions. We also found that CASQ2 overexpression elevates extracellular signal-related kinase (ERK) expression. In epidermal growth factor (EGF)-treated cells, Hs578T-CASQ2 cells had higher phosphorylated ERK compared to Hs578T, leading to the expression of epithelial-mesenchymal transition (EMT) marker, vimentin. These results indicate that CASQ2 overexpression increases the level of calcium and induces cell proliferation, migration and invasion through ERK signaling. Our findings from this study show a possible cause of migration and invasiveness in breast cancer cells. Taken together, these findings demonstrate that CASQ2 could be a new therapeutic target for breast cancer.

#34

Identification of a phosphorylation cascade triggering Wnt/planar cell polarity signaling in breast cancer cells.

Jean-Paul Borg. _CRCM Inserm-Institut Paoli-Calmettes, Marseille, France_.

The developmental Wnt/planar cell polarity (PCP) pathway is the most recently described branch of Wnt signaling strongly implicated in cancer development at early and late stages. Upregulation of Wnt/PCP components and signaling is indeed observed in many cancers, this event being mostly associated with cancer progression. However, how the function of this signaling is remains presently an open question. Recent work in our laboratory found that Prickle1 and Vangl2, two core Wnt/PCP components, are overexpressed in triple negative breast cancers and associated with poor prognosis. Prickle1 is a cytoplasmic PET/LIM-containing protein phosphorylated by the serine/threonine kinase Mink1 which triggers Prickle1 localization at the plasma membrane and regulates its activity. Activation of the Prickle1-Mink1 axis promotes breast cancer cell motility and metastatic spreading in the mouse by promoting β1-integrin turn-over, focal adhesion dynamics and cytoskeleton remodeling. However, Prickle1 is probably not the unique substrate of Mink1 in breast cancer cells. Through a combination of protein purification and mass spectrometry analysis, we identified novel partners of the Prickle1-Mink1 complex which includes LL5β, a PH domain containing protein involved in β1-integrin turn-over. In parallel, by an independent approach looking for Mink1 substrates in breast cancer cells, we identified LL5β as a potential substrate for this poorly described protein kinase. Using recombinant LL5β and Prickle1, we confirmed that Mink1 can directly phosphorylate both proteins in in vitro kinase assays. We also defined that, in breast cancer cells, Mink1 associates with and phosphorylates LL5β, controlling its subcellular localization, a key step for cancer cell motility. Our data provide novel insights on the regulation of the prometastatic Wnt/PCP pathway by characterizing an yet unknown multi-step phosphorylation cascade with promising targeting potential.

#35

Triple-negative breast cancer cells alter the pulmonary landscape to favor metastasis via exosome-mediated release of NM23.

Suzann Duan, Senny Nordmeier, Aidan E. Byrnes, Miles A. Brown, Iain L. Buxton. _University of Nevada, Reno School of Medicine, Reno, NV_.

There is strong evidence implicating exosomes, cell-derived nanoscale vesicles, and their protein cargo in facilitating dialogue between primary tumor cells and the premetastatic niche. Identified by our lab as one such protein, secreted nucleoside diphosphate kinase (NM23/NDPK) has been shown to activate endothelial remodeling in support of proangiogenic and prometastatic events. We have previously demonstrated that triple-negative breast cancer (MDA-MB-231) cells elaborate exosomes enriched with NDPK transphosphorylase activity and target the lung as a future site of metastasis. Using in vitro and in vivo approaches, we interrogate the functional role of NDPK in exosome-mediated lung remodeling. In cell-based assays, we demonstrate that MDA-MB-231 exosomes stimulate migration of pulmonary vascular endothelial cells and enhance monolayer permeability. These effects are reversed following treatment with an inhibitor of NDPK activity or an antagonist to the purinergic P2Y1 receptor. Further, we show that immune-compromised mice receiving intravenous injections of MDA-MB-231 exosomes exhibit enhanced pulmonary vascular leakage, whereas treatment with an inhibitor of NDPK attenuates this effect. Using tandem mass tag labeling and mass spectrometry, we profiled proteomic changes to the lung and identified the purinergic signaling pathway as among those significantly affected by exosome treatment. These results elucidate the functional role of exosomal NDPK in compromising vascular integrity and transforming the pulmonary landscape to support colonization by circulating breast cancer cells.

#36

Novel age-related tumor promoting and inhibiting genes in breast cancer.

Bingzhi Wang, Xiang Gu, Haiyan Zhu, Luzhe Sun. _UTHSCSA, San Antonio, TX_.

During aging, besides genetic alterations, genes with altered expression may promote breast cancer development. Using linear regression modeling, we identified 148 up-regulated and 189 down-regulated genes during aging, by analyzing the whole transcriptome profiling data of matched normal tissues from 82 female patients with age and menopausal status available from The Cancer Genome Atlas data. We also compared matched tumor and normal samples, 3356 up-regulated and 3124 down-regulated genes were identified to be affected by tumorigenesis. Overlapping of tumorigenesis-associated genes with age-dependent genes resulted in 14 up-regulated and 24 down-regulated genes with both age and tumorigenesis. These genes were significantly predictive in relapse free survival, indicative of their potential tumor promoting or suppressive functions respectively. To test this hypothesis, we selected two up-regulated genes-DYNLT3 and P4HA3, and two down-regulated genes-ALX4 and WDR86 to investigate their function in controlling breast cancer malignancy because they are novel genes to breast cancer. We found that knockdown of DYNLT3 or P4HA3, and overexpression of ALX4, significantly reduced breast cancer cell proliferation, migration and colony forming capacity in soft agar, while overexpression of WDR86 did not show any inhibitory activity in breast cancer cells. Consistent with the in vitro data, knockdown of DYNLT3, P4HA3 and overexpression of ALX4 inhibited tumor growth and lung metastasis when breast cancer cells were orthotopically implanted in mice, whereas such inhibition was not observed in mice when WDR86-overexpressing breast cancer cells were implanted. Taken together, our results support that some age-related genes harbor tumor promoting or inhibiting functions. Thus, proper modulation of their expression has potential to prevent or slow down the progression of breast cancer.

#37

Pulmonary pre-metastatic modifications induced by breast cancer.

Ruoqing Cai,1 Caitlin M. Tressler,1 Menglin Cheng,1 Kanchan Sonkar,1 Santosh K. Paidi,2 Ishan Barman,2 Kristine Glunde1. 1 _Johns Hopkins University School of Medicine, Baltimore, MD;_ 2 _Johns Hopkins University, Baltimore, MD_.

Breast cancer can be highly curable at early stages, while the survival rate plummets with the occurrence of metastasis. Thus, studies of molecular mechanisms that allow cancer cells to leave the primary tumor and ultimately establish colonies at distant sites are of crucial importance. First proposed by Stephen Paget in 1889, the "seed and soil" hypothesis suggests that the secondary organ microenvironment undergoes constant modification induced by the primary tumor through circulating chemokines. This leads to the migration of activated bone marrow derived cells to secondary organs, increased matrix metalloproteases (MMPs), and extracellular matrix (ECM) modifications in the secondary organ, collectively termed the pre-metastatic niche, which provides desirable docking sites for cancer cells. Our previous studies have shown persistent changes in the collagenous stroma and proteoglycans in pre-metastatic lungs of mice growing metastatic breast tumors, whose extent directly correlated with the malignancy of the primary tumor [1]. Elevated proteolytic MMP activity was also detected in these pre-metastatic lungs [2]. Based on these findings, we have continued our pre-metastatic niche studies by building an appropriate cell culture model, which consists of lung fibroblasts that were exposed to cell culture media conditioned by differentially malignant human breast epithelial cells. Cell lines used for fibroblast conditioning were nonmalignant MCF-12A breast epithelial cells (control), non-metastatic MCF-7 breast cancer cells, and highly metastatic, triple-negative MDA-MB-231 breast cancer cells. We focused our studies on ECM- and MMP-related changes, which we analyzed using qRT-PCR and immunoblotting. qRT-PCR analysis revealed that metastatic MDA-MB-231 cells induced an increase in the ECM protein Col14A1 as well as MMP-2 and MMP-14 in lung fibroblast as compared to induction by MCF-12A control. Immunoblotting analyses of lung fibroblasts showed that breast cancer cells induced increases in MMP expression in lung fibroblasts. Even higher MMP levels in fibroblasts were induced by metastatic MDA-MB-231 cells as compared to non-metastatic MCF-7 cells. Breast cancer cells also induced decreased expression levels of secreted MMPs in lung fibroblasts, most likely due to an enhanced release of these MMPs. This decrease was most significantly induced by the metastatic cell line. In conclusion, we have observed elevated levels in ECM molecules and MMPs in lung fibroblasts upon exposure of conditioned media from breast cancer cells. Our findings point towards an important role of lung fibroblasts in the evolution of the pre-metastatic niche in modifying the ECM, and their ability to directly respond to factors released from primary breast cancer cells with which they are not in direct contact. [1] Paidi SK et al. Cancer Res. 2017; 77:247-56. [2] Rizwan et al. Npj Breast Cancer 2015; 1: 15017.

#38

Mitophagy deficiency promotes osteolytic bone metastasis in breast cancer.

Rong Deng, Hai-Liang Zhang, Rui-Zhao Cai, Jun Tang, Xiao-Feng Zhu. _Sun Yat-sen Univ. Cancer Ctr., Guangzhou, China_.

Introduction: Metastasis of breast cancer to the bone is a common complication and occurs in 65-80% of late-stage patients. Specifically, the development of osteolytic bone lesions increases patient morbidity and mortality with debilitating skeletal complications. Mitophagy is a selective form of autophagy that removes damaged or excessive mitochondria in response to various metabolic stresses. However, the role of mitophagy in development of cancer remains largely unclear. The Janus-faced role of mitophagy during tumorigenesis leads to a novel challenge when targeting it for cancer therapies. This study focuses on the role of mitophagy deficiency in breast cancer osteolytic bone metastasis, and explores the potential of rescuing mitophagy deficiency as possible therapeutic targets for metastatic disease.

Methods: Specific single-guide RNAs (sgRNAs) were used to deplete ULK1 expression in breast cancer cell lines. Mitophagy was monitored by detecting Tom20 and Hsp60. In addition, mitochondrial DNA (mtDNA) was used to further confirm the reduction of mitochondrial mass. For bone metastasis studies, luciferase-tagged tumor cells were injected into the left cardiac ventricle of anesthetized female nude mice. Metastatic progression was monitored by bioluminescence imaging (BLI). Micro-CT imaging was used to detect bone volume, cortical wall thickness, trabecular thickness and number in mice of tibia. TRAP assay was used to detect osteoclast differentiation assays.

Results: We found that the core Atg protein ULK1 was not only expressed at low level in breast cancer, but also required for hypoxia-induced mitophagy. ULK1-related mitophagy deficiency promoted the formation of metastatic osteolytic lesions in experimental bone metastasis mouse model with MDA-MB-231 cells. TRAP staining revealed that mature osteoclasts significantly increased in RAW264.7 cells treated with CM from ULK1-knockout breast cancer cells. In addition, MEK inhibitor trametinib was sufficient to upregulate the expression of ULK1 and reduced the release of cytokines, which is key to osteoclast differentiation via triggering mitophagy, thereby effectively blocking breast cancer osteolytic bone metastasis.

Conclusion: In conclusion, we demonstrate the functional importance of mitophagy defect in breast cancer for promoting osteolytic bone metastasis. We also provide strong preclinical evidence for using MEK-ERK pathway inhibitors to disrupt ERK kinase-involved ULK1 degradation and induce mitophagy as a breast cancer bone metastasis therapy.

#39

LCN2, CD133 expressions in breast cancer spinal bone metastasis and CD151, TWIST1, EPHA2 in lung metastasis: Prognostic value and organ-specific relapse.

Aparna Maiti, Nitai C. Hait. _Roswell Park Cancer Institute, Buffalo, NY_.

Purpose: Breast cancer most often recurs and metastasizes to the distal organs that had their primary tumors surgically excised. Due to heterogeneous nature of the breast cancer, metastasis organotropism has poorly understood. In this study, we assessed the specific cancer-related gene expression changes occurring with metastatic breast cancer recurrence to distant organs comparing with non-metastatic breast cancer. Experimental design: We develop a clinically relevant metastatic syngeneic mouse model using two cell lines 4T1-luc2 that can only metastasize to lung and 4T1.2-luc2 metastasized to bone and lung. After the primary mammary tumor surgically excised from mice, we monitor mice using IVIS imaging for metastatic tumor lesion development in the distant organs followed by MRI scan to detect spinal bone metastases. Animals were sacrificed to collect spines, bones, and lung lesions for molecular analysis. We also validate the gene expression data using the RNA-seq cohort data from the Roswell Park Cancer Institute patient samples with advanced breast cancer bone metastases and matched non-metastatic breast cancer.

Results: Our cell lines, RNA-seq data analysis suggested that there are at least 50 genes which are statistically highly expressed in 4T1.2-luc2 compared to 4T1-luc2 cells. Out of which only a few genes are well established for cancer metastasis biology, include ANGPTL77, SERPINE2, TSPAN11, ESM1, LCN2 which are expressed more than 8 fold in 4T1.2-luc2 compared to 4T1-luc2 cells. To our surprise, we found that most of these genes are differentially expressed when metastasized to distant organ. Our data revealed that LCN2 (7 fold) and CD133 (above 20 fold) are overexpressed in the spine and bone compared to the primary or lung met lesions formed by 4T1.2-luc2. Conversely, LCN2 and CD133 genes are downregulated in breast cancer lung metastasis tissues, whereas CD151, EPHA2, and TWIST1 genes are highly overexpressed in lung metastatic lesion compared to primary, bone or spine. Further, the RNA-seq data of patient samples explained that LCN2, CD133 expressions are significantly higher (8 out of 10 patients) in advanced breast cancer bone metastases compared with matching non-metastatic breast cancer. Conclusion: Our data suggested that LCN2, CD133 would be a prognostic marker for breast cancer spinal bone metastasis, whereas CD151, TWIST1, EPHA2 would be a prognostic marker for lung metastasis and organ-specific relapse.

#40

The role of pressure-driven flow in invasion and chemoresistance of cancer cells in an engineered breast tumor model.

Allison K. Simi,1 Andreas P. Kourouklis,1 Alexandra S. Piotrowski-Daspit,1 Joe Tien,2 Celeste M. Nelson1. 1 _Princeton University, Princeton, NJ;_ 2 _Boston University, Boston, MA_.

Collapsed blood or lymphatic vessels in the tumor microenvironment often cause fluid buildup, leading to heterogeneous flow throughout the tissue. Here, we used a three-dimensional (3D) engineered tumor model to investigate how fluid flow specifically influences invasion and chemoresistance of breast cancer cells. To mimic breast tumors, we cultured aggregates of MDA-MB-231 human breast cancer cells embedded in 3D collagen channels. Collagen channels were flanked on the ends by two media reservoirs. By changing the relative heights of media in the reservoirs, we controlled the pressure-induced flow experienced by the tumor cell aggregate. We found that the direction of flow through the collagen channel determined the invasive phenotype of the engineered tumor. These analyses will be repeated with the addition of chemotherapy drugs taxol or 5-fluorouracil in the media to determine the effect of fluid flow on chemotherapeutic response. Our engineered tumor model provides insight into how physical forces influence the invasive phenotype of cancer cells.

#41

The role of epithelial-to-mesenchymal transition on breast tumorigenesis cancer associated to mesenchymal stem cells.

Fernanda Marques Rey,1 Carmen Lucia Pontes,1 Roberta Ribeiro Rosales,1 José Russo,2 Yanrong Su,2 Julia Santucci-Pereirab,2 Enilza Maria Espreafico,1 Daniel Guimarães Tiezzi1. 1 _FMRP-USP, Ribeirão Preto-Brasil, Brazil;_ 2 _FOX CHASE CANCER CENTER, Philadelphia, PA_.

The objective of this study is to identify and target the influence of tumor microenvironment with characteristics cancer stem cell (CSC) and clonal expansion on breast cancer population. For this purpose we are using the human breast epithelial cells (MCF-7) co-cultured with 1%, 10% or 30% mesenchymal stem cells (MSC) derived from Wharton's jelly. Flow cytometry: cell surface markers CD44+CD24−/low expression was determined by flow cytometry on MCF-7 cells after exposition for 5 days with MSC. Mammospheres formation was determined by using MCF-7 and MSC seeded in ultra-low-adhesion culture plates for ten days, dissociated and purified by FACS using the CD44+CD24−/low markers. Further cell characterization was performed using the LSM 780 multiphoton and Fiji software, for e-cadherin, β -catenin and n-cadherin expression. Time Lapse migration assay was done using eight well chamber and photographed at 10X magnifications at 20 min intervals for 72 hours. The sorted co-culture and MCF-7 cells were used for cytoplasmic extraction and Western blot analysis of the e-cadherin expression level. MSC and MCF-7 seeded in a collagen matrix, fixed and photographed at 10X and 20X magnifications using Fiji software were used for the 3D reconstruction. The ANOVA with Tukey's post-test were used for statistical analysis. Data were presented as mean ± standard deviation, p <0.05. Results The co-culture of MCF-7 with MSCs showed change on localization of e-cadherin from the membrane to the cytoplasm. For n-cadherin, the co-localization were predominantly in membrane after MCF7 has been exposed to MSC. MCF-7 cells showed cytoplasm and nucleus co-localization on β-catenin biomarker. This phenomenon is confirmed by WB showing increase levels of e-cadherin in the cytoplasm fraction of MCF-7. Amoeboid to mesenchymal transition morphology was showed on 3D assay, this interaction phenomena establish the cell-cell communication to modify the profile on cell migration and determine the epithelial to mesenchymal morphology by MSC interaction with MCF-7. The co-culture of MCF-7 with 30% MSCs increases the number of mammospheres and the expression of CD44+/CD24-. On time-lapse assay, the MCF-7 cells show migration on the direction to MSC cells, moving toward these cells as a single-cell migration, with collective migration in response to MSC interaction spindle-shaped cells by detaching from the rest of the cell culture These cells exhibit a differential invasive movement pattern in response to MSC interaction. Conclusion MSC boost aggressiveness of the breast cancer cell MCF-7 indicating that interaction of the tumor microenvironment is determinant of the clonal expansion making this process a relevant part of the mechanism in metastatic dissemination.

#42

Triple negative breast cancer cell heterogeneity in zebrafish-host microenvironment.

B.Ewa Snaar-Jagalska,1 Arwin Groenewoud,1 Letizia Astrologo,2 Lanpeng Chen,1 Marianna Kruithof-de Julio2. 1 _Institute of Biology, Leiden, Netherlands;_ 2 _University of Bern, Bern, Switzerland_.

Metastatic triple negative breast cancer (TNBC) can be considered as incurable endpoint in the progression of TNBC, prevention or treatment of metastasis therefore is of paramount importance. New insights into intra-tumoral heterogeneity have cast light on the existence of multiple functionally distinct cellular entities within one tumor, among others these cells can be divided based on their metastatic potential. The zebrafish provides us with the unique opportunity to study the first steps of metastatic initiation in great detail both microscopically and transcriptionally, on the side of both the host and cancer cells. Organotropic TNBC lines engrafted into this model recapitulate the tropism found in higher vertebrates. Using our zebrafish TNBC xenograft model we study these cellular sub-populations in vivo during metastatic initiation. These small metastatic lesions can be further analyzed ex vivo through the re-isolation of novel zebrafish specific organotropic cell lines. Re-engraftment of these greatly enhances the tumorigenic potential, and transcriptional analysis reveals increased EMT plasticity and an induction of stemness markers. Engraftment of these cells into murine tibia shows a significantly increased tumorigenic and osteolytic potential, further underscoring the critical role of this sub-population in cancer initiation. To determine the genetic drivers of this process we performed whole exome sequenced (NGS) of both, in vitro cultured cancer cells (maternal and re-isolated) and micro-dissected tissue samples derived from in situ zebrafish xenograft material. We are currently in the process of evaluating the candidate genes underpinning the enhanced tumorigenic potential in both the zebrafish and murine models used.

#43

Regulation of autophagy in bone metastatic breast cancer cells.

Ahmad H. Othman,1 Manish Tandon,2 Vivek Ashok,1 Marcus Winogradzki,1 Gary Stein,3 Jitesh Pratap1. 1 _Rush Univ. Medical Ctr., Chicago, IL;_ 2 _KBI Biopharma, TX;_ 3 _University of Vermont, Burlington, VT_.

Bone metastasis of breast cancer is a significant cause of patient mortality. Recent studies suggest that metastatic cancer cells induce autophagy to survive metabolic stress. During autophagy, cytoplasmic components and damaged organelles are captured by autophagosomes followed by lysosomal fusion and degradation, releasing metabolites as energy sources to meet metabolic demands. Although the components of the autophagy pathway have been well characterized, the regulatory mechanisms of autophagy in metastatic cancer cells remain unknown. Previously, we have shown that Runt-related transcription factor-2 (Runx2) promotes cell survival, bone metastasis, and osteolysis. Using a bone metastatic isogenic variant of breast cancer MDA-MB-231, we examined levels of the autophagosome specific marker LC3B to define the regulation of autophagy during bone metastasis. Additionally, we examined whether Runx2 regulates autophagy for increased cell survival in the bone microenvironment. Microscopic and biochemical studies showed elevated levels of autophagic flux among bone derived cells relative to parental breast cancer cells. Interestingly, we also observed that Runx2 enhanced the turnover of autophagic vesicles while Runx2 silencing resulted in accumulation of vesicles due to reduced turnover. Interestingly, Runx2 knockdown or inhibition of autophagy in bone derived cells increased AMPK levels suggesting higher levels of cellular stress as a consequence of impaired autophagy. In addition to AMPK activity, MAP kinase mutations have been demonstrated to result in constitutive activation of the autophagy pathway. Treatment with the MEK inhibitor PD184161 resulted in accumulation of LC3B-II in control cells. Furthermore, Runx2 knockdown in bone derived cells display lower levels of ERK activity relative to controls. These results suggest that Runx2/ERK signaling is critical for autophagy in metastatic breast cancer cells. Our mechanistic studies revealed that Runx2 promotes autophagy by increasing acetylation of α-tubulin sub-units of microtubules and enhancing trafficking of autophagic vesicles. Introduction of a mutant α-tubulin construct incapable of being acetylated resulted in the accumulation of autophagic vesicles in control cells, similar to silencing of Runx2. Inhibition of autophagy resulted in decreased adhesion, migration, and survival of Runx2 knockdown cells. Furthermore, analysis of LC3B protein in clinical breast cancer specimens and tumor xenografts revealed significant association between high Runx2 and low LC3B protein levels. Our studies reveal a novel regulatory mechanism of autophagy via Runx2 and provide insights into the role of autophagy in bone metastatic breast cancer cells.

#44

Characterization and targeting of a temporal micro-metastatic signature in human brain metastases.

Mohini Singh,1 Chitra Venugopal,1 Tomas Tokar,2 Nicole McFarlane,1 Minomi Subhapanditha,1 David Bakhshinyan,1 Maleeha Qazi,1 Parvez Vora,1 Neil Savage,1 Naresh K. Murty,1 Igor Jurisica,2 Sheila K. Singh1. 1 _McMaster University, Hamilton, Ontario, Canada;_ 2 _Princess Margaret Cancer Centre, Toronto, Ontario, Canada_.

Metastases to the brain (BM) are the most common neoplasms to affect the adult central nervous system, arising in 40% of cancer patients and at a rate 10 times greater than primary brain tumors. Despite the prevalence and poor survival rates, therapeutic strategies for BM remain limited, and a substantial cause is the lack of proper preclinical models available to interrogate the intricacies of BM development. Previous work in our lab utilized BM samples from patient-derived lung-to-brain metastases to successfully establish clinically relevant in vivo models of BM. Here we further characterized the cells responsible for BM, termed brain metastasis initiation cells (BMIC). Patient-derived BMICs were injected via intracranial (BT), intracardiac (IC) and intrathoracic (IT) routes into NOD/SCID mice and re-isolated at different stages of metastatic progression. We isolated cells from primary lung tumors (LT), cells injected via intra-thoracic route that crossed the blood brain barrier to seed the brain forming micro-metastases (BMIT), and cells injected via intracardiac route that seeded large macro-metastases (BMIC). Through RNA sequencing we determined cells from BMIT (micro-metastasis stage) to retain a vastly different genetic profile compared to BMICs isolated at other stages of metastasis. Several of these genes belonged to pathways implicated in autonomic central nervous system neoplasms and neural development. Through connectivity mapping of BMIT profiles we discovered drugs that could inhibit the micro-metatsasis signature, and further in vitro validation revealed apomorphine to reduce BMIC sphere formation and proliferation. In vivo treatment with apomorphine blocked both micro- and macro-metastatic stages of our BMIC model. Of 5 BMIT genes identified to be specifically targeted by apomorphine, KIF16B, TESK2 and SEPW1 were shown to have significant value when applied as an independent prognostic signature in a cohort of lung adenocarcinoma patients. Future work will further validate the efficacy of apomorphine in targeting BMICs in primary lung cancer patient samples. With this work we present a possible new avenue for therapeutic targeting toward the prevention of BM development, where it is anticipated to transform a uniformly fatal disease into one that is eminently more treatable.

#45

Snai2 is a new target to mediate glucocorticoid signaling on breast cancer cell migration.

Jun Ling, Adit Singhal, Zenaida P. Lopez-Dee, Brittany Porreca, Trinity Sprague. _Geisinger Commonwealth School of Medicine, Scranton, PA_.

Steroid hormone receptors such as estrogen and progesterone receptors are well studied in breast cancer pathology; they are also used as drug targets for breast cancer therapy in the clinic. In contrast, glucocorticoid (GC) as a ubiquitous stress activated steroid hormone is less investigated in breast cancer. However, GC is frequently used as a co-treatment for breast cancer chemotherapy that generates some controversial effects to even promote cancer progression or recurrence in certain subtypes of breast cancer. To address this clinical issue, we focused on investigating the specificity of GC signaling on breast cancer cell behaviors in this study. When breast cancer cells were treated by dexamethasone (Dex), they were more responsive to cell migration (measured by electric cell-substrate impedance sensing, ECIS) than the effects on cell proliferation and apoptosis, wherein MDA-231 (triple negative breast cancer) cell was more sensitive to Dex than MCF7 cell (luminal A subtype). Further gene expression analysis by qRT-PCR microarray revealed that the glucocorticoid receptor (GR) responsive gene patterns are different between these two subtypes of breast cancer cells. One of remarked changes was that Snai2 (a zinc finger transcriptional factor) was highly activated in both cell lines. Western blotting analysis confirmed that MDA-231 cells have much higher basal level of Snai2 but with less fold increase upon Dex treatment than MCF-7. GR response elements (GREs) were also identified in the promoter region of Snai2, thus confirming Snai2 as a new target gene of GR in breast cancer. When Snai2 was knocked out by CRISPR, it was found that the basal migration rate was decreased in both cell lines. More importantly, their response rates to Dex treatment were also decreased as compared to the wild type cells. These decreases in cell migration are similar to those treated by GR antagonist RU486, thus confirming that Snai2 is a mediator to regulate the effects of GC/GR signaling on breast cancer cell migration. In summary, this study identified Snai2 as a new target gene of GR to regulate cell migration and potentially metastasis in response to GC signaling. The differential regulation of different subtypes of breast cancer cells by GC signaling was also confirmed. (+ equal contribution; * Corresponding author).

#46

Endocytic recycling regulator EHD2 as a biomarker of poor prognosis and positive regulator of tumorigenesis in triple-negative breast cancer.

Timothy A. Bielecki,1 Insha Mushtaq,1 Bhopal C. Mohapatra,1 Sameer Mirza,1 Wei An,1 Fany Iseka,1 Alexandra Moffitt,1 Abir A. Muftah,2 Matthew D. Storck,1 Yuri Sheinin,1 Emad Rakha,2 Vimla Band,1 Hamid Band1. 1 _University of Nebraska Medical Center, Omaha, NE;_ 2 _University of Nottingham, Nottingham, United Kingdom_.

Triple-negative breast cancer (TNBC) subtype accounts for a disproportionate burden of breast cancer deaths due to higher propensity to metastasize and lack of targeted therapies. Oncogenesis is regulated by signals emanating from cell surface receptors whose surface expression is controlled by endocytic traffic. EPS15-Homolopgy Domain (EHD) 1-4 proteins are a family of membrane sculpting ATPases that regulate endocytic traffic. Our mouse knockout studies demonstrated key physiological roles of EHD family members, prompting studies of their roles in oncogenesis. EHD2 was selectively expressed in the basal/myoepithelial mouse mammary epithelium. Public gene expression database analysis revealed high EHD2 expression in TNBC, correlating with shorter survival. Q-PCR and immunoblot analyses confirmed EHD2 overexpression in basal B triple-negative but not in luminal ER+ cell lines. IHC analysis of 840 patient samples showed EHD2 overexpression in most triple-negative and a minority of other breast cancer subtypes, correlating with metastasis and shorter survival. EHD2 knockdown in Hs578t, MDAMB231 and BT549 TNBC cell lines reduced invasiveness, anchorage-independent growth, collective cell invasion in 3D and mammosphere growth. In an orthotopic mouse xenograft tumor model, TNBC cells with EHD2 knockdown exhibited a dramatic abrogation of tumor growth and expression of EMT markers. Studies of the MDAMB231 model demonstrated a requirement of EHD2 for tumor metastasis. These findings provide evidence that EHD2 is a novel biomarker of poor prognosis in TNBC, and suggest a previously uncharacterized role for EHD2 as a positive regulator of tumorigenicity in TNBC. Targeting EHD2 may provide a unique approach for treatment of TNBC.

#47

Brain metastatic versus non-metastatic breast cancer exosomes influence astrocyte response.

Megan Sayyad, Madhavi Puchalapalli, Bin Hu, Jennifer Koblinski. _Virginia Commonwealth Univ., Richmond, VA_.

Brain metastasis is a devastating, late-stage event affecting 10-30% of breast cancer patients, but it is unclear how breast cancer cells can cross the blood-brain barrier (BBB) and colonize the unique brain environment. Exosomes, endosomal-derived extracellular vesicles, have been reported to support premetastatic niche formation through recruitment of tumor growth-supporting cells, creating sites conducive to cancer development. Still, it is not known how exosomes might prime the distinctive brain environment for metastasis despite the ability of circulating exosomes to readily cross the BBB. Glial cells, namely astrocytes and microglia, are important contributors to brain metastasis, and astrocytes in particular secrete exosomes that promote tumor outgrowth in the brain. We therefore investigated the effects of breast cancer exosomes on astrocytes in promoting brain premetastatic niche formation. Using ultracentrifugation, we isolated exosomes from 4T1 metastatic and 67NR nonmetastatic mouse mammary carcinoma and MDA-MB-231 human triple-negative breast cancer cell-conditioned media. Exosomes were characterized using Western immunoblot, dynamic light scatter analysis, and transmission electron microscopy, and labeled with a fluorescent lipid dye. Mouse and human astrocytes were then treated with either 4T1 or MDA-MB-231 exosomes, respectively, and vesicle internalization was assessed using confocal microscopy. Astrocytes were indeed found to internalize cancer exosomes in vitro. Using qPCR analysis, we explored gene expression changes in mouse astrocytes treated with 4T1 exosomes compared to those treated with 67NR exosome nonmetastatic controls. 4T1 exosomes were found to significantly upregulate expression of tenascin C, an astrocyte-derived brain extracellular matrix protein, suggesting the brain matrix could be remodeled prior to cancer cell arrival. Further, astrocytes treated with 4T1 exosomes exhibited significant upregulation of inflammation-associated genes, namely COX-2, IL-6, CCL4 and PDGF-alpha, genes also associated with cancer and metastasis, specifically to the brain. Alternatively, 67NR exosome treated-astrocytes exhibited significant downregulation in these genes of interest, suggesting that there is a unique response profile associated with nonmetastatic versus metastatic breast cancer-derived exosomes. Taken together, these findings suggest that breast cancer exosomes could drive transformation of the brain environment towards a prometastatic site.

#48

Targeting HDAC6 to reduce the aggressiveness of metastatic breast cancer in immunotherapy.

Debarati Banik. _George Washington Univ., Hyattsville, MD_.

Histone modifiers are recognized to perform diverse functions above beyond their conventional roles in remodeling the chromatin landscape. These functionalities range from regulating the outcomes of cellular-health to systemic immune-diseases, e.g., autoimmunity and cancer, positioning the HDAC inhibitors at a crucial junction of immunotherapy. However, the toxicity from the broad-spectrum HDAC-inhibitors has deemed a better focus on inhibitors of individual HDAC-members associated with tumor progression. One such member is HDAC6 which was earlier reported to promote the pro-tumorigenic STAT3 pathway. By using pharmacological inhibitor of HDAC6, the downstream immune-modulatory pathways of STAT3 could be targeted, which directly link to T-cell mediated immunity through the co-stimulatory molecules PD-L1, PD-L2 and B7-H4. This relationship has been established in a wide variety of tumors, including melanoma and breast cancer. HDAC6 has been also involved in a number of structural functions related to cellular motility, shape and intracellular transport through the regulation of the acetylation of numerous targets, including tubulin and cortactin. This function is strongly suggestive of HDAC6 being a key player in metastatic cancer progression. In our initial studies, we observed that the selective HDAC6 inhibitor Nexturastat A (NextA) is capable of reducing the tumor growth in a highly aggressive murine mammary carcinoma that mimics certain features of the human triple negative breast cancer, under both orthotopic and subcutaneous conditions of implantation. NextA was also able to inhibit tumor cell invasion in presence of metastasis-promoting cytokine such as IL6, as investigated through in vitro invasion assays. Additionally, we observed that the size and number of secondary tumor nodules in lungs were significantly diminished after the HDAC6i treatment. In order to boost the anti-tumor T-cell response, we also tested check-point inhibitors, such as anti PD1 and CTLA4 antibodies against both metastatic breast cancer and melanoma. While both of the standalone treatments showed a certain degree of success in reducing tumor growth, we demonstrated that HDAC6i improves anti-tumor immune responses when combined with immune check-point blockade mediated by anti PD1 antibody. The enhancement in anti-tumor response was measured in terms of the composition of infiltrating immune cells as well as functionality of effector T cells in terms of intra-tumoral interferon gamma expression. In both respects we found that NextA alone and combined with check-point blockade improves the anti-tumor response.

#49

Stromal platelet derived growth factor receptor (PDGFRβ) signaling: A novel therapeutic target for breast cancer brain metastasis (BCBM).

Katie A. Thies,1 Anisha M. Hammer,2 B. Eason Hildreth,1 Luke O. Russell,2 Steven T. Sizemore,2 Anthony J. Trimboli,1 Raleigh D. Kladney,2 Chelsea M. Bolyard,2 Robert Pilarski,2 Lynn Schoenfield,2 Jose Otero,2 Arnab Chakravarti,2 Matthew Ringel,2 Balveen Kaur,3 Gustavo Leone,1 Michael C. Ostrowski,1 Gina M. Sizemore2. 1 _Medical University of South Carolina, Charleston, SC;_ 2 _The Ohio State University, Columbus, OH;_ 3 _University of Texas Health Science Center, Houston, TX_.

Stromal PDGFRβ has emerged as an actionable mediator of breast tumor-stromal communication. As a receptor tyrosine kinase, PDGFRβ is activated by its ligand, PDGFB, which is released by neighboring tumor epithelium and endothelium. However, how PDGF signaling mediates breast cancer initiation, progression, and metastasis remains unclear. To evaluate PDGFRβ in this disease, we developed a mouse model of stromal-specific PDGFRβ activation using the Fsp-cre transgene previously published by our group (PDGFRβ mutant). PDGFRβ mutant mammary glands exhibit increased tertiary side-branching and epithelial proliferation confirming a stromal-specific PDGFRβ effect on neighboring epithelium. To evaluate the functional relevance of PDGFRβ activation on metastatic progression, we performed tail vein injection of PDGFB expressing murine mammary tumor cells and, surprisingly, observed brain metastases in 50% of the PDGFRβ mutant mice while no brain lesions were seen in controls. There was no difference in the incidence of lung, liver or bone metastases. Mammary tumor cells expressing low PDGFB did not exhibit a similar increase in brain metastases in mutant mice. While there is no observable difference in blood brain barrier permeability in the mutant mice, we bypassed this variable by intracranially injecting mammary tumor cells and found that larger tumors formed in the brains of PDGFRβ mutant mice versus controls. To our knowledge, this is the first example where genetic manipulation of the stroma leads to an increased incidence of BCBM. Also, our pre-clinical data suggests that primary breast tumors that express high PDGFB could preferentially metastasize to the brain. To test this in patients, we analyzed PDGFB protein expression in a tissue microarray comprised of HER2-positive and triple negative breast cancer (TNBC) primary tumors. While high PDGFB did not correlate with site-independent metastatic recurrence, it was prognostic of brain metastasis, mirroring our mouse data. Evaluation of PDGFB in a small cohort of matched primary breast tumors with associated brain (n=5) and lung metastases (n=2) revealed intense PDGFB staining in 100% of the brain metastases, but only 50% of the lung metastases. Our findings suggest high primary tumor PDGFB expression defines a subset of breast cancer patients predisposed to brain metastases. These patients may benefit from therapeutic intervention of PDGFRβ signaling. To test this pre-clinically, we treated mice harboring intracranial tumors with the PDGFR specific inhibitor, Crenolanib. Excitingly, Crenolanib treatment significantly inhibited the brain tumor burden in these mice. Combined, our findings (1) advocate that primary tumor expression of PDGFB is a novel prognostic biomarker for the development of BCBM and (2) support clinical trial evaluation of PDGFR inhibitors for the prevention and treatment of BCBM.

#50

Elucidating the role of MUC5AC-MET axis in breast cancer brain metastasis.

Mohd W. Nasser,1 Shailendra K. Gautam,1 Ranjana Kanchan,1 Pranita Atri,1 Jawed Siddiqui,1 Rakesh Bhatia,1 Joseph Carmicheal,1 Dario Ghersi,2 Sukhwinder Kaur,1 Siddharth Mahapatra,1 Maneesh Jain,1 Surinder K. Batra1. 1 _University of Nebraska Medical Center, Omaha, NE;_ 2 _University of Nebraska, Omaha, NE_.

Metastasis of breast cancer (BC) is the leading cause of mortality in American women. In particular, brain metastasis is a significant clinical problem present in 30-40% of triple receptor negative (TN) and epidermal growth factor receptor 2 (HER2) positive BC patients. These patients also have the worst prognosis of all breast cancers in women. Due to advances in clinical management at both the diagnostic and therapeutic levels, the lifespan or overall survival of these patients has increased, but the manifestation of brain metastasis at later stages of the disease remains an unfortunate burden for these patients. In addition, the blood-brain barrier (BBB) poses an additional hurdle for treatment of BC brain metastases, because the majority of chemotherapeutic drugs fail to cross the BBB. Furthermore, there is no single targeted therapy available for TNBC patients with brain metastasis, and thus there is an urgent requirement to discover novel biomarkers and therapeutics to address BC brain metastasis. In this regard, using a bioinformatics approach, we observed that several mucins, including MUC5AC, are significantly up-regulated in brain metastases of BC compared to primary tumors. However, we found the secretory mucin MUC5AC protein to be substantially high in brain-seeking BC (BSBC) cell lines and metastatic brain tissues. We also observed that knockdown of MUC5AC reduced proliferation and metastatic potential in these cell lines. Furthermore, we observed that MUC5AC colocalizes with activated MET receptor. MUC5AC knockdown in BSBC cell lines reduced MET activation and its downstream targets FAK and paxillin. Interestingly, MET and FAK inhibition using pharmacological inhibitors led to substantial decrease in MUC5AC expression. Overall, these studies suggest that MUC5AC-MET axis may regulate breast cancer brain metastasis. Understanding the MUC5AC-MET axis could be a novel approach for the development of better prognosis and prevention of life-threatening and treatment-resistant breast cancer brain metastasis. 

### Dormancy and Aging: The Influential Microenvironment

#51

Role of hematopoietic cell kinase (HCK) in breast cancer dormancy.

Sachin Khiste,1 Elliot Eton,1 Kevin Hodgetts,1 Hrvoje Lusic,2 Kodaganur Gopinath,3 S Krishnamurthy,4 Shiladitya Sengupta,1 Aaron Goldman1. 1 _Harvard Medical School, Boston, MA;_ 2 _Brigham and Womens Hospital, Boston, MA;_ 3 _Bangalore Institute of Oncology, Banglore, India;_ 4 _Kidwai Memorial Institute of Oncology, Bangalore, India_.

Background: Triple negative breast cancer (TNBC) is an aggressive basal-like malignancy, which recurs more frequently than any other subtype of breast cancer. We recently discovered that TNBC can overcome drug pressure by switching to a hybrid basal-like (CD44Hi) and epithelial-like (CD24Hi) cell state, which is accompanied by a transient period of quiescence, or dormancy. These dormant, 'hybrid' cells highly express the phosphorylated SRC family kinase, hematopoietic cell kinase (HCK). Methods: Here, we used a panel of novel HCK inhibitors to study the effect of signaling-disruption in drug tolerance and tumor cell dormancy. Using flow cytometry and immunofluorescent microscopy we examined how HCK inhibition perturbs common features associated with dormancy such as proliferation and cell cycle status, intracellular levels of reactive oxygen species, and state of glucose metabolism. Finally, we validated the results using an ex-vivo human tumor model that predicts clinical response and recreates the native tumor microenvirnoment, CANscriptTM. Results: We determined that targeted inhibition of HCK down-regulates the features of tumor cell dormancy and drug tolerance, eliminating cells with a hybrid mesenchymal-epithelial phenotype, diminishing polyploidy and hyper-G2 endoreduplication, and suppressing features of a dysregulated glycolytic state. Conclusion: Expression of HCK drives cancer cells to a quiescent cancer cells subpopulation, resulting in tolerance to chemotherapy. Targeted disruption of HCK overcomes these features of dormancy. HCK inhibitors could emerge as a novel treatment for chemotherapy-induced breast cancer dormancy.

#52

A genomic editing approach for purification of viable quiescent cancer cells.

Ting La, Margaret Farrelly, Xu Guang Yan, Hamed Yari, Yuanyuan Zhang, Yuchen Feng, Seyed Hessamedin Tabatabaeehatambakhsh, Tao Liu, Lei Jin, Xu Dong Zhang. _The University of Newcastle, CALLAGHAN, Australia_.

Recurrent and metastatic cancer lesions often undergo a period of dormancy, which is closely associated with cancer cell quiescence, a state whereby cancer cells exit the cell cycle and are reversibly arrested in G0 phase. Quiescent cancer cells are inherently resistant to cell death and refractory to therapeutic drugs. However, the mechanisms responsible for the resistance remain largely undefined. This is closely associated with the lack of understanding of their biological properties as a consequence of technical hurdles in the isolation and analysis of viable quiescent cells. Nevertheless, it is known that quiescent cells are characteristically negative for the proliferation marker Ki67 and express high levels of the cyclin-dependent kinase (CDK) inhibitor p27. In this study, we developed a CRISPR/Cas9-based system to fuse a green fluorescent protein (EGFP) gene with endogenous CDKN1B, the gene encoding p27, and a red fluorescent protein (mCherry) gene with endogenous MKI67, the gene encoding Ki67 in the genome of human melanoma cells. By using this system, we have successfully isolated viable p27high/Ki67- melanoma cells using fluorescence-activated cell sorting (FACS). The quiescent state of these cells was confirmed by dual nucleic acid staining (DNA with Hoechst-33342, and RNA with Pyronin Y). This approach provides a unique research tool for further characterization of the biological properties of quiescent cells. Comparative RNA-seq and proteomics analyses are being currently carried out to reveal molecular networks involved in regulating cell quiescence, and to identify molecular targets for overcoming resistance of quiescent melanoma cells to treatment.

#53

Axl is sufficient but not necessary for disseminated prostate tumor cell dormancy.

Haley D. Axelrod, Kenneth C. Valkenburg, Jessica L. Hicks, Angelo M. DeMarzo, Kenneth J. Pienta. _Johns Hopkins School of Medicine, Baltimore, MD_.

Prostate cancer (PCa) remains the second most common cancer among men in the U.S., and kills nearly 30,000 men each year. While the majority of patients with localized disease are cured by surgery and/or radiation, 10-15% of these patients will develop metastasis most commonly in the bone marrow. The time from primary tumor removal to recurrence can range from years to decades, and constitutes a period of minimal residual disease in which cancer cells remain in the body but are clinically undetectable. These disseminated tumor cells (DTCs) are seeds for lethal bone metastases and are thought to maintain a long-term state of dormancy in the bone marrow. Very little is known about the mechanism(s) by which dormancy is regulated, but further investigation may lead to therapeutic strategies aimed at preventing recurrent metastasis altogether. In this effort, we have previously found the secreted bone marrow factor Gas6 and the expression of its receptor Axl to be associated with dormant bone marrow DTCs in xenograft mouse models. We hypothesize that bone marrow Gas6 signals through Axl to induce dormancy in prostate DTCs, and explored the functional consequences of altering Axl expression in these models. To determine if Axl is sufficient to induce dormancy, we overexpressed it in the Axl-negative human PCa C42B cell line and monitored tumor growth by BLI after intracardiac injection in NSG mice. By the time half of the control overexpression injected mice developed tumors, less than 10% of mice injected with Axl overexpression cells developed tumor signal. This delay in tumorigenesis indicates a prolonged period of dormancy that is mediated by Axl expression. To determine if Axl is necessary for dormancy, we knocked out Axl in the Axl-positive human PCa PC3 cells using CRISPR and monitored growth after intracardiac injection in the same manner as the overexpression cells. Knockout of Axl did not increase the time to tumor signal compared to control cells. Loss of Axl also did not decrease the fraction of EdU negative, non-proliferative cells present in the bone marrow at one week post-injection. These data indicate that Axl is likely not necessary for DTC dormancy. Altogether this study suggests that Axl overexpression is sufficient to induce dormancy, but endogenous Axl is not required for dormancy. This is further supported by negative immunohistochemical staining of Axl on cancer cells within growing primary and metastatic tumors from a PCa tissue microarray. Axl also correlated with decreased incidence of biochemical recurrence (Decipher GRID) and increased overall survival (TCGA). Ongoing work includes assessing the expression of Axl on patient CTCs and DTCs, and correlating it with proliferation status.

#54

Tumor dormancy in breast cancer cells: Identification of junctional adhesion molecule-A as a novel regulator.

Sri HariKrishna Vellanki, Rodrigo G. Cruz, Katherine M. Sheehan, Elaine W. Kay, Ann M. Hopkins. _RCSI, Dublin 9, Ireland_.

In tumor dormancy, a sub-population of tumor cells becomes quiescent but can re-enter the cell cycle upon certain environmental cues. The re-awakening of dormant populations has been associated with tumor cell proliferation and poor patient outcomes. Tumor dormancy can be modelled in vitro by exposing cancer cells on fibronectin-coated plates to bFGF-2, a mammary differentiation factor abundant in the bone marrow stroma, which causes partial re-differentiation, cell spreading and re-expression of integrin-α5β1. We have uncovered a novel potential role for the adhesion protein Junctional Adhesion Molecule-A (JAM-A) in tumor dormancy. JAM-A overexpression has previously been linked to increased risk of metastasis in breast cancer patients, and its expression regulates the angiogenic functions of bFGF-2. Furthermore, since loss of JAM-A downregulates the α5β1 downstream effector FAK, we hypothesized that JAM-A is required for maintenance of tumor dormancy. To investigate this, JAM-A was transiently silenced in MCF-7 breast cancer cells grown on fibronectin-coated plates and exposed to FGF-2. JAM-silenced cells exhibited expressional downregulations in integrin-α5β1 and FAK proteins, and failed to exhibit the cortical actin redistribution and morphological spreading which characterize dormant cells. in parallel, pharmacologically-induced loss of JAM-A (using an anti-tumor antibiotic) prevented tumor dormancy and was cytotoxic to dormant cells. We next investigated a potential relationship between JAM-A expression and pro-inflammatory cytokines linked with the re-awakening of dormant cells in post-menopausal women. Exposure of MCF-7 cells to IL-1β, TNF-α, IL-6 and IL-8 increased the protein expression of matrix metalloproteinase ADAM17, which in turn caused JAM-A cleavage and loss from the cell membrane. Analysis of bone metastasis sections from post-menopausal breast cancer patients revealed significant heterogeneity in JAM-A expression, with some regions strongly positive for JAM-A while other regions were negative. Nonetheless, 20/24 metastatic sections expressed either moderate or high JAM-A levels, suggesting that even if cells transiently lose JAM-A to reawaken from tumor dormancy, they may later acquire high JAM-A expression during disease progression. The fact that JAM-high dormant cells had high Aldefluor activity, a feature of stemness, might also support an important role for JAM-A in tumor evolution or progression. To conclude, our data support a model whereby JAM-A is important for the maintenance of dormancy in breast cancer cells in a simulated bone marrow microenvironment, and represents a novel therapeutic target worthy of investigation in breast cancer. This work was financially supported by Science Foundation Ireland (grant 13/IA/1994 to AMH).

#55

Intrinsic molecular characteristics of bone marrow-disseminated tumor cells.

Manabu Maeshiro, Satoru Shinriki, Takuya Nakamura, Hirofumi Jono, Hideki Nakayama, Yukio Ando, Hirotaka Matsui. _Kumamoto University, Kumamoto, Japan_.

Dormant or slow-cycling disseminated tumor cells (DTC) in bone marrow (BM) are known to persist in patients with various types of cancer including head and neck squamous cell carcinoma (HNSCC). BM-DTC has been thought to have possible metastatic stem cell traits although functional and clonal analysis of BM-DTC remain elusive. Recently, we reported that the intrinsic TGF-β2-triggered SDF-1-CXCR4 signaling axis was crucial for drug resistance dependent on a slow-cycling state in BM-DTC but not in lung metastatic cells. This study aimed to elucidate intrinsic molecular characteristics in BM-DTC. We used the human HNSCC cell line HEp3-originated sublines [i.e. parental line (P-HEp3), BM-DTC-derived (BM-HEp3), and lung metastases-derived sublines (Lu-HEp3)]. Our transcriptome analyses for these sublines revealed that slow-cycling and drug-resistant BM-HEp3 cells had unique gene expression signatures. In addition, our investigation for phosphorylation of 39 kinases associated with tumor progression showed enhanced activation of only Src in BM-HEp3 compared with P- and Lu-HEp3. Importantly, Src inhibition by dasatinib in these sublines led to a remarkable reduction in cell viability only in BM-HEp3. These data suggest that intrinsic molecular characteristics of BM-DTC are distinct from primary and lung metastatic tumor cells. Src activation may be important for survival of dormant or slow-cycling BM-DTC. Further studies should contribute to better understanding of biology of minimal residual disease in BM and metastatic initiation.

#56

CXCL5 is a master regulator of the dormancy switch to activate metastatic colonization of dormant breast cancer cells during bone metastasis.

Ricardo Romero-Moreno, Thomas Coughlin, Kimberly Curtis, Shourik Dutta, Glen Niebur, Laurie E. Littlepage. _University of Notre Dame, South Bend, IN_.

Bone is one of the most common and most dangerous sites for metastatic tumor growth across cancer types, including breast cancer. At death, roughly 73% of women with breast cancer have bone metastasis, most often growing in highly vascularized bones. These metastases are detrimental to the patient's quality of life, causing hypercalcemia, acid/base imbalance, aberrant hematopoiesis, immune response, and osteolysis (bone loss) resulting in severe pain and immobility. Current treatment options for patients with metastatic bone cancer include chemotherapy, palliative radiation, and surgical resection. However, bone metastasis is not considered curable with current therapies. Circulating tumor cells sometimes become arrested in blood vessels or within tissue, remaining in a quiescent state ("dormancy") until the right conditions induce the cancer cells to grow and thrive in the metastatic site ("colonization"). Switching cancer cells from dormancy to colonization is rate-limiting for bone metastasis, sometimes taking decades to induce metastatic tumor growth. With few experimental models available to study this last step of metastasis, the switch from cancer dormancy to colonization, or dormancy switch, has become one of the greatest challenges in cancer treatment and cancer research. Most of the experimental models that are used focus on either in vitro culture systems, lacking the heterogeneity of an intact bone, or in vivo animal models that are not amenable to higher-throughput experimentation. To model dormancy, we developed an ex vivo co-culture system of mouse bones and cancer cells. Using our culture system, we then identified distinct culture conditions for tumor cell dormancy and colonization in bone. In fact, conditioned media from our dormancy culture conditions induced dormancy of cancer cells. Profiling of a panel of cytokines, chemokines, and growth factors identified the chemokine CXCL5 as a candidate to induce the switch from dormancy to colonization. Interestingly, bones from mice that harbored tumor cells before collection support a higher cancer cell proliferation rate in co-culture and secrete more CXCL5 than bones from healthy mice. This suggests that bones primed with cancer cells form a niche that actively supports the proliferation of metastatic cells and is inducible by CXCL5. In culture, devitalized bones and conditioned media from devitalized cultures support cancer cell proliferation, which suggests that the bone and/or marrow express an inhibitor of cancer cell proliferation. Additional studies using CXCL5 recombinant protein further suggest that CXCL5 is sufficient to overcome breast cancer dormancy and promote proliferation in metastatic breast cancer. Together, this study supports the importance of the communication between the bone microenvironment and cancer cells to further promote metastatic colonization.

#57

Survival of dormant breast cancer cells and metastatic tumor recurrence is dependent upon the activation of autophagy.

Laura Vera Ramirez,1 Suman K. Vodnala,1 Ryan Nini,2 Kent W. Hunter,1 Jeffrey E. Green1. 1 _National Cancer Institute, US National Institutes of Health, Bethesda, MD;_ 2 _Department of Genitourinary Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX_.

Cancer recurrence resulting from the metastatic outbreak of dormant disseminated tumor cells following the apparent successful treatment of the primary tumor is a major cause of breast cancer mortality. However, little is known regarding the molecular mechanisms governing tumour cell dormancy and the dormant-to-proliferative switch, impeding the development of effective therapeutic strategies. We therefore investigated whether stress-induced autophagy may promote survival of dormant cancer cells and, consequently, inhibition of autophagy could prevent breast cancer recurrence. To address the functional role of autophagy in breast cancer progression and the potential therapeutic impact of its inhibition, we utilized mouse and human 3D in vitro and in vivo preclinical models of dormancy. The analysis of autophagy markers and use of an autophagic flux biosensor allowed direct visualization of autophagic vesicles and their evolution in breast cancer dormant cells over time. In agreement with our hypothesis, pharmacologic or genetic inhibition of autophagy in dormant breast cancer cells resulted in significantly decreased cell survival and metastatic burden in vitro and in vivo. Furthermore, the inhibition of autophagy prevented the spontaneous dormant-to-proliferative switch of highly metastatic cells. In contrast, proliferating disseminated cells were insensitive to autophagy blockade. Indeed, in vivo analysis of the autophagic flux over time confirmed that autophagy is a critical survival process activated and maintained in dormant breast cancer cells, which is shut down after the cells undergo the dormant-to-proliferative switch. Transcriptomic analysis and in vivo metastatic burden assays identified the autophagy-related 7 (ATG7) gene, but not Beclin1 (BECN1), to be essential for autophagy activation, indicating that a non-canonical autophagy pathway is activated in dormant breast cancer cells. Co-localization studies identified mitochondria as the predominant autophagosomal cargo in breast cancer dormant cells. Mechanistically, inhibition of the autophagic flux in dormant breast cancer cells led to the accumulation of depolarized mitochondria and reactive oxygen species (ROS), resulting in cell apoptosis. This study has important implications regarding the role of autophagy in breast cancer progression and suggests that inhibition of autophagy may be of therapeutic value in preventing breast cancer recurrence. Furthermore, it provides novel insights into the molecular mechanisms for survival of breast cancer dormant cells.

#58

Age-related elevated Th17 immune response contributes to prostate carcinogenesis.

Qiuyang Zhang,1 Sen Liu,1 Zongbing You,1 Brian G. Rowan,1 S M. Jazwinski,1 Alun R. Wang,1 Leann Myers2. 1 _Tulane University School of Medicine, New Orleans, LA;_ 2 _Tulane University School of Public Health and Tropical Medicine, New Orleans, LA_.

Prostate cancer is associated with advanced age, but how age contributes to prostate cancer remains unknown. Aging is characterized by a chronic, low-grade systemic inflammation, so called "inflamm-aging". Data supporting the role of inflammation in prostate cancer comes from a variety of fields, thus, inflamm-aging represents a strong candidate to connect age and prostate cancer. Recent studies have shown that T helper (Th) 17 immune responses are elevated in aging humans and mice. However, the contribution of age-related elevated Th17 immune responses to prostate cancer remains unclear. The aim of this study was to investigate the role of Th17 immune responses in the aging process in prostate carcinogenesis. Initial investigation of mouse prostate tissues from wild-type C57BL/6J mice showed that aged (>80-week-old) mouse prostates had significantly increased inflammatory cell infiltration, increased protein and mRNA levels of pro-inflammatory mediators and Th17 cytokines and activated NF-κB and ERK1/2 signaling compared to young (12-20-week-old) mouse prostates. To gain a mechanistic understanding of how the Th17 immune response promotes prostate carcinogenesis in the aging process, we isolated splenic T cells from young and aged mice. Naïve CD4+ T cells isolated from the young and aged mice were differentiated in Th17 polarization conditions. Th0 cells were cultured in the absence of cytokines to serve as control. Four days later cells were re-stimulated with anti-CD3 and CD28 with PMA and ionomycin in the presence of brefeldin A for 6 hours, then the cells were washed and re-cultured in serum free media for 24 hours. Cells were then collected and used for flow cytometry or qPCR. Conditioned media from both young and aged Th0 and Th17 cultures were collected and used in subsequent experiments. Results indicated that Th17 cells, Th17 cytokines and Th17/Treg ratio were significantly increased in aged mice compared to young mice. The human prostate cancer cell lines (LNCaP, DU-145 and PC3) and mouse prostate cancer cell line (PTEN-CaP8) was cultured in their suitable medium as in our previous study. These cell lines were treated with the above mentioned conditioned media (Th0 and Th17) for 48 and 72 hours and harvested for subsequent experiments. When human and mouse prostate cancer cell lines were exposed to the aged Th17 conditioned media, cell proliferation, migration and invasion were significantly increased, and the pro-inflammatory NF-κB pathway in PCa cell lines was activated compared to cells exposed to young ones. In summary, these results collectively indicate that Th17 immune responses are elevated in mice in the aging process, and this age-related elevated Th17 immune responses may play an important role in prostate carcinogenesis.

This work was supported by the NIH-National Institute of General Medical Sciences COBRE (2P20GM103629-06, PI: SMJ; Pilot Project PI: QZ).

#59

14-3-3zeta overexpressing breast cancer cells promote fibroblast senescence and CAF-like phenotype.

Hongzhong Li, Yi Xiao, Xiangliang Yuan, Wenling Kuo, Ping Li, Dihua Yu. _MD Anderson Cancer Center, Houston, TX_.

14-3-3ζ overexpression in breast cancer is associated with accelerated cancer progression and poor clinical outcome in patients. Our previous study revealed that 14-3-3ζ overexpression promotes epithelial mesenchymal transition (EMT) of breast cancer cells, which facilitates progression of ductal carcinoma in situ (DCIS) to invasive ductal carcinoma (IDC). However, the impact of 14-3-3ζ overexpressing breast cancer cells on tumor microenvironment (TME) and the underlying mechanisms have not been explored. We found dramatically increased cancer associated fibroblast (CAF) signatures in patients' matched IDC lesions compared to their DCIS lesions, suggesting that activated fibroblasts may be involved in DCIS to IDC transition. Global gene expression profiling uncovered increased expression of pro-inflammatory cytokines/chemokines, including IL6 and IL8, in 14-3-3ζ AND ErbB2 co-overexpressed 10A.ErbB2.1433ζ cells compared to ErbB2 overexpressed 10A.ErbB2 cells. This finding was further confirmed by ELISA and validated in other breast cancer cell lines. We tested whether increased pro-inflammatory cytokines in the conditional medium (CM) from 14-3-3ζ overexpressed cancer cells promotes CAF-like phenotype. Indeed, CM from 14-3-3ζ overexpressed cancer cells significantly increased SMA+ activated fibroblast cells compared to controls. Additionally, up-regulation of β-galactosidase and reduced cell proliferation, hallmarks of senescence cells, were detected in fibroblast cells cultured in CM from 14-3-3ζ overexpressed cancer cells. Next, we explored potential mechanism of how 14-3-3ζ overexpression in cancer cells leads to up-regulation of pro-inflammatory cytokines/chemokines. Since NF-kappaB is one of the master upstream regulator of IL-6 and IL-8, we next investigated the NF-kappaB signaling activation in 14-3-3ζ overexpressed vs control cancer cells. 14-3-3ζ overexpressing cancer cells had hyperactive canonical NF-kappaB responses, including accelerated IκBα degradation, p65 phosphorylation, as well as nuclear translocation compared to 14-3-3ζ low cancer cells. Prolonged NF-kappaB signaling activation upon pro-inflammatory cytokine stimulation was also detected in 14-3-3ζ overexpressing cancer cells. These results suggest that 14-3-3ζ overexpressing cancer cells may promote fibroblast cell senescence and CAF-like phenotype by creating by a chronic inflammatory microenvironment, which ultimately enhances DCIS to IDC transition.

#60

The association between cellular senescence of cancer associated fibroblasts and tumor progression in pancreatic cancer.

Takanobu Yamao, Yo-ichi Yamashita, Kensuke Yamamura, Naoki Umezaki, Masayo Tsukamoto, Yuki Kitano, Kota Arima, Tatsunori Miyata, Shigeki Nakagawa, Hirohisa Okabe, Katsunori Imai, Hidetoshi Nitta, Akira Chikamoto, Ishiko Takatoshi, Hideo Baba. _Kumamoto University, Kumamoto, Japan_.

Background: In recent days, cellular senescence of cancer associated fibroblasts (CAFs) is gaining increased attention, yet incompletely understood the role in the development of malignant diseases. Previous studies reported that Caveolin-1 plays a major role in cellular senescence, and its expression in CAFs. Therefore, we hypothesized that cellular senescence of CAFs introduces the tumor progression in pancreatic cancer (PC).

Methods: A total of 159 consecutive patients with PC who underwent curative resection between January 2004 and December 2016 were enrolled. The patients were divided into two groups according to the expression of Caveolin-1 in CAFs, which were analyzed by immunohistochemistry. First, we investigated the relationship between the expression of Caveolin-1 in CAFs and patients' clinicopathological factors including survival outcomes. Second, we established the CAFs-cell lines from the resected tissues of patient with PC. We stocked CAFs-cell line after 6 times passages at least. Third, we controlled the expression of Caveolin-1in CAFs, and underwent coculture of PC cell lines and CAFs-conditioned medium (CM) to evaluate the effect of cellular senescence in CAFs on invasion of PC cell lines.

Results: The high level of Caveolin-1 expression group counts of 49 patients (31%), and the low level of Caveolin-1 expression group counts of 110 patients (69%). As for patients' clinicopathological factors, the serum levels of CA19-9 (p=0.0008) and pathological T factor stage (p=0.014), defined by Japan Pancreas Society as the "General Rules for the Study of Pancreatic Cancer" were significantly higher in the high Caveolin-1 expression group than were those of the low expression group. The high Caveolin-1 expression group had significantly worse outcomes in overall (log-rank p = 0.022) and disease-free survivals (log-rank p = 0.011). We established 10 CAFs cell lines from clinical samples, and evaluated Caveolin-1 expressions. We identified the high Caveolin-1 expression CAFs (CAF-4 and 5), and suppressed its expression by transfection of short interfering (si) Caveolin-1 knockdown. We collected CAF-CM from CAF-4 and CAF-5 with siCaveolin-1suppressed the invasive ability of MiaPaCa-2 compared to that with si control (p<0.0001). Conclusion: The present result suggested that Caveolin-1 expression in CAFs in PC is associated with patients' poor prognosis. The downregulation of Caveolin-1 in CAFs would lead to less invasion of PC cells. Therefore, there is a possibility that Caveoin-1 of CAFs in PC is a new target for treatment.

#61

Obesity-induced T cell senescence contributes to prostate cancer progression.

Alejandra De Angulo, Peyton Travis, Christopher Jolly, Linda deGraffenried. _University of Texas at Austin, Austin, TX_.

Obese men are at greater risk of developing an aggressive form of prostate cancer. The T cell population from obese subjects have shown signatures of cell senescence including shortened telomeres and elevated production of proinflammatory cytokines. The objective of our studies is to better understand the cellular and molecular basis for altered T cell anti-prostate tumor immunity in obesity, and whether this altered immune function contributes to the worse outcomes observed in obese patients. To investigate whether obesity induces a pro-inflammatory senescent phenotype in CD4+ T cells, splenic CD4+ T-cells from 10-week-old male C57Bl/6 mice were isolated and, either pre-stimulated or co-stimulated with anti CD3/CD28 and conditioned media (CM) from macrophages stimulated under obese or normo-weight conditions. After 24 hours conditioned media from these CD4+ T cells was collected, and their relative cytokine expression profile was analyzed. The CD28 receptor is expressed on CD4+ T cells and provides co-stimulatory signals required for T cell activation. CD28 activation results in phosphorylation of Akt. Lack of Akt phosphorylation in CD4+ T cells after CD3+/CD28+ stimulation indicates CD4+ T cell dysfunction and senescence. Fresh murine splenic CD4+ T cells were isolated and stimulated with anti CD3/CD28 under obese or control conditions. Akt1 phosphorylation levels were assessed using Western blot analysis. To determine if obese conditions modify the impact of CD4+ T cell dysfunction on prostate cancer cell signal transduction pathways, prostate cancer epithelial cells were exposed to obese or control CD4+ T-cells CM for 30 minutes and western blot analysis was used to evaluate the modulation of key pro- and anti-tumorigenic pathways. Matrigel Invasion chambers were used to determine if obesity-modified CD4+ T cell CM impacts the invasive capacity of prostate epithelial cells. Obese conditions suppressed the expression of IFN-γ and IL-2 in CD4+ T-cells. Data also suggests that T cells exposed to obese conditions suppress STAT1 activation in prostate cancer cells. Additionally, obese conditions up-regulated the expression of IL-6 and IL-10 in CD4+ T-cells when compared to control. Importantly our data also suggests that T cells exposed to obese conditions induce STAT3 activation in prostate cancer cells. Preliminary data also suggests that obesity-modified CD4+ T cell CM impacts the invasive capacity of prostate cancer cells. Obesity may induce CD4+ T cell dysfunction, limiting their tumor suppressing activity. Findings form this study could substantially advance fundamental understanding of the ways in which obesity alters T cells, thereby promoting uncontrolled tumor growth and mortality from prostate cancer. This study could also provide a framework for future translational research aimed at improving the effectiveness of therapies in obese patients.

#62

Effect of cancer cell-derived exosome on energy metabolism associated with macrophage activation.

Yoonseok Kam, Brian P. Dranka. _Agilent Technologies, Lexington, MA_.

Macrophage is a critical microenvironmental component which can control tumor progression, and dynamic changes in cellular metabolism accompany the polarization. As well as other immune cells, the activation of macrophages can be modulated by cancer cells. Cancer-derived exosome is a newly emerging microenvironmental factor enables cancer cells to communicate with macrophages. In this study, examined were the activation-associated bioenergetic changes and the effect of cancer cell-derived exosome on the changes by a) monitoring the early metabolic kinetics and b) profiling bioenergetic phenotype resulted. In vitro activation of RAW264.7 macrophage cell line by injecting lipopolysaccharide (LPS) and interferon γ (IFNγ) in a Seahorse XF analyzer induced an immediate early increase in glycolytic rate. It also showed a delayed down-regulation of mitochondrial respiration, which resulted in a secondary elevation of glycolytic rate. This secondary glycolytic response was only obtained by LPS-IFNγ co-stimulation and mediated by induced nitric oxide synthase (iNOS) signaling. In contrast to the dynamic changes in metabolic phenotype, the total ATP production rate appeared to be stably maintained according to the XF data normalized by cell number. However, macrophage activation significantly increased glycolysis-dependent portion of ATP production. ATPs were generated almost solely by glycolysis after the delayed mitochondrial down-regulation by iNOS. Co-injection of cancer cell-derived exosomes with LPS slightly but significantly accelerated the immediate early glycolytic response as well as the delayed mitochondrial down-regulation. Furthermore, exosomes induced the immediate early glycolytic response even in the absence of LPS. These results imply that presence of cancer cell-derived exosomes may promote macrophage activation and accelerate pro-inflammatory immune response in a tumor microenvironment.

#63

**Microenvironment-derived mitochondria prime glioma chemoresistance by augmenting NAD** + **metabolism and PARP-dependent DNA repair.**

Yang Liu, Mark Gilbert, Chunzhang Yang. _Center for Cancer Research, National Cancer Institute, Bethesda, MD_.

Background: Mitochondria, the major organelles of cellular metabolism, can be released into the microenvironment and affect adjacent cells, supporting recipient cell metabolism and protecting them from unfavorable conditions. While our knowledge of intercellular mitochondria transfer under normal physiological conditions is expanding, a key question remains unanswered: do microenvironment-derived mitochondria influence the survival and malignant transformation of cancer cells?

Methods: In this study, we have established in vitro and in vivo models to aid in the understanding of mitochondrial transfer between astrocytes and glioma cells.

Results: We demonstrate that mitochondria are released from normal astrocytes through extracellular vesicles, and are adopted by neighboring glioma cells. Mitochondrial transfer not only improves oxidative metabolism in recipient glioma cells, but also supports the detoxification of chemotherapeutic agents by fueling PARP-dependent DNA repair with ATP and NAD+. Targeting astrocyte mitochondria-releasing pathways increased the cytotoxic effects of chemotherapy, reduced xenograft progression, and prolonged overall survival. Our findings suggest that microenvironment-derived mitochondria confer both metabolic capability and chemoresistance to cancer cells.

Conclusion: Targeting microenvironment-derived mitochondria may be important in reducing the capacity for cancer cells to recover from cancer treatment, thereby enhancing treatment efficacy.

#64

Macrophage perceive tumor-intrinsic glutamine metabolism and orchestrate immunosuppression through IL-23 inclear cell renal cell carcinoma.

Qiang Fu. _Fudan University, Shanghai, China_.

The signaling mechanisms between renal cancer cell metabolism and infiltrating immune cells may illuminate novel therapeutic approaches. Here, we showed the idiomatic glutamine metabolism of clear cell renal cell carcinoma (ccRCC) suppress immune surveillance through nonautonomous IL-23. Through analyzing multiple ccRCC clinical cohorts and human tumor samples, we uncovered SLC1A5 (solute carrier family 1 member 5) promotes ccRCC consumption of extrinsic glutamine, predicts patient prognostication, and associates with reduction of cytotoxic T cells and NK cells in the tumor microenvironment. Specifically, the immune surveillance suppression of tumor-intrinsic SLC1A5 is considerably conducted by IL-23, which could directly induce intratumoral accumulation of T helper 17 cells and regulatory T cells. Intriguingly, we identified macrophages as the major source of intratumoral IL-23, and macrophages secrete large amount of IL23 under low glutamine condition, as the consequence of tumor-driven glutamine consumption. Furthermore, blocking IL-23 using specific antibody in syngeneic mice tumor model prolonged their survival, and blocking IL-23 in an in vitro culture model of human tumors promote anti-tumoral T and NK cells reaction. Our results demonstrate that macrophage-secreting IL-23 is a key immune effector as the response to tumor-driving glutamine metabolism, and IL-23 profoundly facilitate an immunosuppressive microenvironment which suggest it as a promising target for cancer immunotherapy in ccRCC.

#65

Impact of tumor-infiltrating dendritic cells in esophageal squamous cell carcinoma treated with neoadjuvant chemotherapy.

Junya Nishimura, Hiroaki Tanaka, Yoshihito Yamakoshi, Soichiro Hiramatsu, Tatsuro Tamura, Takahiro Toyokawa, Kazuya Muguruma, Kosei Hirakawa, Masaichi Ohira. _Osaka City University, Osaka, Japan_.

Background: Tumor-infiltrating CD8⁺T cells have been found to be associated with favorable outcome of patients in many cancers including esophageal squamous cell carcinoma (ESCC). Dendritic cells (DCs) are the most potent antigen-presenting cells to induce CD8⁺T cells. Several investigators have shown that tumor infiltrating mature DCs (mDCs) were associated with favorable prognosis of cancer patients. We showed that mDCs were associated with favorable prognosis of ESCC patients who had not received preoperative treatment. Neoadjuvant chemotherapy (NAC) is the standard treatment for Stage II/ III thoracic esophageal carcinoma in Japan. The effect of chemotherapy on tumor infiltrating DCs remains unclear.

Purpose: The aim of this study was to examine the correlation of efficacy of NAC and locally infiltrating DCs in ESCC patients.

Object and Methods: Formalin-fixed, paraffin-embedded blocks of primary lesions were collected from 50 patients with ESCC who underwent surgical treatment after NAC at Osaka City University Hospital. Immunohistochemical analysis using anti-LAMP-3 antibody and CD8 antibody was used to evaluate tumor infiltrating DCs and CD8⁺T cells. The number of LAMP-3⁺DCs and CD8⁺T cells were counted at ×400 magnification. We divided the 50 cases into two groups according to the median number of tumor infiltrating DCs and CD8⁺T cells to examine the correlation of efficacy of NAC and locally infiltrating DCs.

Results: LAMP-3⁺DCs were predominantly observed in peritumoral area and sparsely found in intratumoral area, whereas CD8⁺T cells distributed between both peritumoral and intratumoral areas. LAMP-3⁺DCs infiltrated the peritumoral space from 0 to 19.0 (median 6.0) and the intratumoral space from 0 to 3.8 (median 0.0). Patients with histological effect (≧Grade 1a) had higher infiltration of mDCs than Grade 0. Patients with high infiltration of mDCs had better prognosis than those with low. The number of mDCs had positive correlation with the number of tumor infiltrating CD8⁺T cells.

Conclusions: Our results suggested that increase of tumor infiltrating mDCs was associated with histological effect of NAC and favorable prognosis in ESCC.

#66

MHC class I polypeptide related sequence A as contributing factor to chemotherapy-induced resistance.

Marcelo Jun Sakiyama,1 Ingrid Espinoza,1 Deepak Kumar,1 Amit Reddy,1 Eldrin Bhanat,1 Krista Syrigos,2 Roya Gordji,1 Christian Gomez1. 1 _Univ. of Mississippi Medical, Jackson, MS;_ 2 _Mississippi INBRE Research Scholar, MS_.

MHC class I polypeptide related sequence A (MICA) is a cell surface protein associated to tumor immunosurveillance activity, due to its ability to activate natural killer (NK) cells. Cleavage of MICA and generation of its soluble form (sMICA) has been described as an immunoevasion mechanism presented by aggressive tumors. In agreement with the role of MICA on tumor immunoevasion, recent studies correlated better prognosis for patients with higher levels of MICA expression in different types of cancer. Chemotherapeutic drugs are used to treat a great variety of malignant diseases. However, a fraction of patients under chemotherapy regimen stop responding to the treatment and develop recurrent tumors. We postulate that MICA can be a factor contributing to chemotherapy-induced resistance. To test our hypothesis, we tested the expression of MICA in prostate cancer-derived LNCaP and E006AA-hT cell lines were treated with bortezomib, a chemotherapeutic drug that inhibits proteasome activity. MTT cell viability assays showed that both cells had similar sensitivity to bortezomib, with IC50 of 10 nM and 8 nM for LNCaP and E006AA-hT, respectively. Flow cytometry analysis showed baseline difference in the cell fraction expressing surface MICA with 67% in LNCaP cells against 18% in E006AA-hT (p<000.1). Following treatment with bortezomib, the fraction of LNCaP cells expressing surface MICA increased 1.3-fold (p<0.01), while in the fraction of E006AA-hT cells expressing MICA yielded 2.3-fold increase (p<0.001). Baseline release rate of sMICA, assessed by ELISA analysis of culture supernatants, was 54 pg/ml/106cells for LNCaP and 5 pg/ml/106cells in E006AA-hT cells. Relative to baseline, sMICA release rate dropped 2-fold with bortezomib in LNCaP cells to 22 pg/ml/106cells (p<0.01). However, sMICA release rate in E006AA rose to 531 pg/ml/106cells (p<0.0001), more than 100-fold increase. Our findings show that, despite similar sensitivity to the killing effects of bortezomib in LNCaP and E006AA-hT, the release rate of sMICA in response to bortezomib is significantly distinct. These evidences lead us to propose that MICA is a contributing factor of resistance to chemotherapy, probably related to soluble MICA's immunoevasion properties. Further studies, testing cell specific ability of MICA's to affect cytotoxic activity in the context of chemotherapy-induced resistance will allow us to assess the value of this premise. This studies can open opportunities for new targeted immunotherapies, enhancing the innate immune system and avoiding the undesired effect of chemotherapy resistance.

#67

Chemotherapy induced pro-metastatic changes in the primary breast tumors of racially diverse patients.

George S. Karagiannis,1 Jessica M. Pastoriza,2 Sonali Lanjawar,1 Yarong Wang,1 David Entenberg,1 Esther Cheng,3 Timothy M. Dalfonso,4 Joan G. Jones,1 Jesus Anampa,2 Thomas E. Rohan,1 Joseph A. Sparano,2 John S. Condeelis,1 Maja H. Oktay1. 1 _Albert Einstein College of Medicine, Bronx, NY;_ 2 _Montefiore Medical Center, Bronx, NY;_ 3 _Weill Cornell Medical Center, New York, NY;_ 4 _Memorian Sloan Kettering Cancer Center, New York, NY_.

Neoadjuvant chemotherapy (NAC) induces influx of bone marrow-derived proangiogenic Tie2hi monocytes into the primary tumor, resulting in increased density of perivascular Tie2hi macrophages (1). Perivascular Tie2hi/Vegfhi macrophages in physical contact with Mena expressing cancer cells create micro-anatomic sites of transient vascular permeability called TMEM, which mediate cancer cell intravasation and dissemination (2). Cancer cells capable of intravasation via TMEM sites express high level of MenaINV, an isoform of Mena, induced by macrophage contact, which renders tumor cells intravasation-competent (3, 4). Consequently, breast cancers from mice and patients treated with NAC have increased density of TMEM sites and show increased expression of MenaINV (1) Moreover, in PyMT mouse mammary carcinoma and patient derived xenografts, NAC increases the number of circulating tumor cells and lung metastasis (1). The Tie2-inhibitor rebastinib, which inhibits TMEM function, can reverse chemotherapy-induced increases in the number of CTCs and lung metastasis in mouse mammary carcinoma (1, 5). Although NAC induces pro-metastatic changes in breast cancer microenvironment, large randomized prospective studies did not find significant differences in distant-recurrence free survival (DRFS) and overall survival (OS) between breast cancer patients treated with adjuvant and neoadjuvant chemotherapy in predominantly white populations. Since the breast cancer microenvironment in black women has higher microvascular density and density of Tie2hi macrophages, suggesting that black women may be more prone to develop TMEM-associated pro-metastatic changes in response to NAC, we questioned if there is a difference in DRFS in black women treated with NAC compared to AC. We evaluated DRFS in 1,211 racially diverse patients with localized or regionally advanced breast cancer treated with neoadjuvant or adjuvant chemotherapy between January 2000 and December 2016 and found that black patients with localized breast cancer treated with systemic neoadjuvant chemotherapy not only have inferior DRFS compared to white patients, but also worse DRFS when compared to black patients treated with adjuvant chemotherapy, after adjustment for clinical covariates in multivariate analysis. The biologic factors contributing to this finding, in particular TMEM-mediated pro-metastatic changes have been evaluated and will be discussed. 1. Karagiannis et al, Sci Transl Med. 2017;9:397. 2. Harney et al, Cancer discovery. 2015;5:932. 3. Pignatelli J et al, Sci Rep. 2016;6:37874. 4. Pignatelli J et al, Sci Signal. 2014;7:353. 5. Harney et al, Mol Cancer Ther. 2017;16:2486.

#68

Targeting macrophage that forms a chemotherapy-induced niche significantly reduces cancer stem cells and tumors.

Xi He,1 Paloma I. Giles,1 Edward H. Lin,2 Linheng Li1. 1 _Stowers Institute for Medical Research, Kansas City, MO;_ 2 _University of Washington, Seattle, WA_.

Failure in cancer therapy is often caused by relapse resulting largely from chemo-resistant cancer stem cells (CSCs). Most colorectal cancer patients, after receiving chemoradiotherapy, still harbor treatment-resistant residual tumor cells that are akin to colorectal adenoma. We therefore exploited the APCMin adenoma model that allowed us to study the natural and dynamic interaction between CSCs and their microenvironment in vivo following therapeutic stress. We observed that macrophages were directly recruited from blood vessel to the CSC niche in response to chemotherapy, playing a role in protecting and further promoting activation of CSCs. Reduction in the Ly6c-CD11bintCX3CR1\+ MHCII\+ population of tumor associated macrophages (TAMs) by celecoxib or clodrosome was correlated with a decline in slow-cycling drug-resistant CSCs and tumorigenesis. Molecularly, we found that COX-2 inhibitor celecoxib suppressed Akt and Wnt signaling, downstream of macrophage-dependent PGE2-EP pathway, thus reducing CSC survival and activation. Taken together, in the naturally formed adenoma reflecting human familial adenomatous polyposis, Chemotherapy-induced recruitment of TAMs to the CSC niche protected and promoted activation of surviving CSCs via PGE2-EP mediated Akt-Wnt signaling, leading to tumor regrowth. Targeting the TAMs will benefit clinical treatment of colorectal cancer.

#69

Chemotherapy-induced cancer cell death diminishes therapeutic efficacy through heme oxygenase-1-mediated inactivation of M1-like tumor associated macrophages.

Seung Hyeon Kim,1 Xiancai Zhong,1 Shin-Young Gwak,1 Ishrat Aklima Muna,1 Sin-Aye Park,1 Young-Nam Cha,2 Young-Joon Surh1. 1 _Tumor Microenvironment Global Core Research Center, College of Pharmacy, Seoul National University, Seoul, Republic of Korea;_ 2 _College of Medicine, Inha University, Incheon, Republic of Korea_.

One of the most common treatment options for cancer is a chemotherapy. After chemotherapy, however, unwanted host effects provoke tumor recurrence and aggressiveness of cancer cells, which often arise as a consequence of disruption in the patient's immune system. Lymphocytes, such as CD8+ cytotoxic T lymphocytes (CTLs), which have capability of suppressing cancer progression, are depleted following chemotherapy. Conventional chemotherapeutic agents kill cancer cells, which boosts recruitment of myeloid cells. Tumor-associated macrophages (TAMs), emerging as abundant tumor-infiltrating myeloid cells in tumor microenvironment, play an important role in immunosuppression caused by conventional chemotherapy. Chemotherapy-induced dying cancer cells could hijack accumulated TAMs, provoking tumor recurrence. Therefore, reprogramming of TAMs to maximize the chemotherapeutic efficacy is considered a promising novel anticancer strategy. In this study, we investigated whether dying cancer cells after chemotherapy could reduce therapeutic efficacy by modulating tumor-infiltrating macrophage activity. In a 4T1 syngeneic mouse breast cancer model, there was a marked decrease in the proportion of M1-like TAMs expressing CD86 with tumor-suppressive activity and reduced tumor infiltration of CTLs following treatment with paclitaxel. Paclitaxel treatment also resulted in an enhancement of heme oxygenase-1 (HO-1) mRNA levels in tumor-infiltrating myeloid cells engulfing dying cancer cells. Consistent with the in vivo profile of TAMs, bone marrow-derived macrophages (BMDMs) phagocytosing dying breast cancer cells exhibited significant upregulation of HO-1 expression. HO-1 induction in BMDMs engulfing dying breast cancer cells facilitated M2 polarization. In contrast, abrogation of HO-1 activity sustained M1 activation of BMDMs co-cultured with dying breast cancer cells. Pharmacologic inhibition of HO-1 activity with zinc protoporphyin IX augmented paclitaxel efficacy by stimulating tumor infiltration of CTLs in a 4T1 breast cancer model. Furthermore, blockade of HO-1 in breast tumor-bearing mice induced increased M1-like TAMs expressing CD86 that plays an important role in stimulating antitumor immune response. Taken together, HO-1 induction by the chemotherapy-derived dying cancer cells in TAMs dampens antitumor effects of chemotherapy by manipulating innate and adaptive immunity. Our results demonstrate that targeting overexpressed HO-1 in the breast tumor microenvironment can control the immune system to potentiate the efficacy of chemotherapy.

#70

Effect of aging on the antitumor activity of GITRL-Fc.

Angie Inkyung Park, Minu K. Srivastava, Rui Yun, Jenny Pokorny, Janice Yu, Fumiko Axelrod, Austin Gurney. _OncoMed Pharmaceuticals, Inc., Redwood City, CA_.

Mouse tumor models have been successfully used to generate preclinical data for numerous clinical programs including immunotherapy. Preclinical in vivo studies are typically carried out using young mice (often less than 2 months old) to generate efficacy data, predictive biomarker and pharmacodynamic markers. In notable contrast, the majority of human cancer occur in adult and older patients. It has become increasingly clear that the immune system of young and old mice is quite different with regards to the relative abundance and functionality of different cell populations. Data generated using young mice could provide a distorted assessment of the potential activity of immuno-oncology drugs in the clinic. Therefore, we tested the activity of GITRL-Fc protein in both young and old mice (>9 months). Previously using extensive young mice experiments, we have shown that GITRL-Fc promoted a robust antitumor immune response and enhanced tumor-specific T-cell responses, particularly of the Th1 type, and also led to a reduction in Treg-mediated immunosuppressive activity. Compared to young mice, tumors grew faster in older mice, and peripheral blood of older tumor-bearing mice has fewer T cells and NK cells. The total MDSC population was increased in the blood and spleen of old tumor-bearing mice, with a significantly higher number of G-MDSCs in the blood. On the other hand, old mice had reduced "antigen-presenting cells" (macrophages/dendritic cells expressing MHCII) in the blood. Furthermore, splenocytes from old mice had impaired production of IL-2. GITRL-Fc significantly inhibited tumor growth in both older and younger mice. However, efficacy was more pronounced in young mice, which frequently exhibited complete tumor regression. There were fewer tumor-infiltrating immune cells with less CD8 T and NK cells in older mice compared to young mice, consistent with faster tumor growth. Interestingly, GITR expression in CD8 T cells in old mice was lower compared with young mice at the tumor site. In old mice, GITRL-Fc (mIgG2a) was still able to deplete Tregs in tumor and increase Tregs in the spleen as has been previously shown with GITRL-Fc in young mice. On the other hand, GITRL-Fc deficient in effector function (mIgG2a (N297A)) did not deplete Tregs in the tumor but did retain some antitumor growth activity, indicating a role for GITR signaling in the mechanism of efficacy by GITLR-Fc. In conclusion, the results demonstrate the potential for the aging of the immune system to impact the efficacy observed with immunotherapy agents and highlight the potential benefits of conducting efficacy studies with both young and older mice.

#71

Caloric restriction promotes antitumor immunity through polarization of intratumoral macrophages to M1 phenotype via downregulation of miR-21.

Brittany A. Simone, Tiziana DeAngelis, Ajay Palagani, Kevin Ko, Larry Harshyne, D. Craig Hooper, Adam P. Dicker, Nicole L. Simone. _Thomas Jefferson Univ. Hospital, Philadelphia, PA_.

Tumor-associated macrophages (TAMs) are the most abundant inflammatory cells in the breast cancer (BC) tumor microenvironment (TME) and suppress antitumor immune function, contributing to treatment resistance. These M2-polarized macrophages are characterized by fatty acid oxidation as opposed to the glycolytic pathways associated with M1-polarization. MiR-21 is known to affect several metabolic pathways relevant to macrophage polarization. We have previously shown that caloric restriction (CR) can decrease miR-21 expression and increase response to radiation therapy (RT). We propose CR as a means of metabolically reprogramming TAMs toward M1 phenotype to increase efficacy of radiation therapy (RT).

To assess the effects of CR in vivo on the composition of the TME in breast cancer, 40 female Balb/c mice were orthotopically injected at 12 weeks old with 4T1 cells and assigned to one of four cohorts: ad lib (AL) fed, 8 Gy RT to the primary tumor (RT), 30% reduction in caloric intake (CR) or CR+RT. MiR-21 levels were significantly decreased in both the CR and CR+RT arms; however, miR-21 was increased in the RT arm. Increased expression of M2 markers was noted in tumors in the RT group as compared to the CR and CR+RT groups, which favored M1 markers. On microarray analysis, the top pathways affected by CR discovered by gene ontology pathway analysis were: inflammation mediated by chemokine and cytokine signaling (31/509 hits), Wnt signaling (21/509 hits) and apoptotic signaling (18/509 hits). Multiple genes related to macrophage polarization were altered by CR including Ccl2 and IL12A. IL-12 is a known target of miR-21, and CR decreases miR-21 expression, therefore we sought to determine if miR-21 may be the mechanism behind the shift in chemokine and cytokine expression in the TME. Cohorts of MMTV-PyMT mice with spontaneously developed breast tumors were bred with a miR-21-/- mouse to generate breast tumors that lack expression of miR-21. These tumors exhibited high levels of IL-12 as well as IL-23 and Cxcl10, all of which are associated with M1 phenotype. MiR-21+/+ tumors exhibited high levels of IL-4 and TGF-β, both associated with M2 phenotype. In addition, peripheral monocytes (PBMCs) were co-cultured with sera from each of the following mice: miR-21-/-, miR-21+/+, AL, RT, CR and CR+RT. M1-polarization was induced by sera from miR-21-/- mice as well as mice receiving CR alone and CR+RT. Ionizing radiation seemed to induce M2-polarization in this manner, as did the miR-21+/+ serum.

Taken together, these data suggest that CR can counteract the M2-polarization caused by RT in the TME. This is likely due, in part, to miR-21 downregulation leading to enhanced M1 response. By increasing M1-polarized macrophages and enhancing antitumor immunity, we believe CR is a viable adjunct to standard cytotoxic therapies that could help overcome resistance to chemotherapy and immunotherapy.

#72

Radiation induces LAP, latency-associated peptide of TGF-beta, on the surface of lymphoid cells in the tumor microenvironment.

Stavros Kopsiaftis, Xiufeng Song, Patricia E. Rao, Barbara S. Fox. _Tilos Therapeutics, Inc., Cambridge, MA_.

LAP, latency-associated peptide of TGFβ, is a protein that is co-synthesized with TGFβ and serves to effectively cage the cytokine, holding it in a latent state in the extracellular matrix and on the surface of immunosuppressive cell types. In the tumor microenvironment, activation of the LAP-TGFβ complex by integrins or proteases releases mature TGFβ, producing an immunosuppressive environment and fostering EMT. Anti-LAP antibodies have been shown to be effective at slowing tumor growth in mouse models (Gabriely et al., 2017) and offer a promising new approach to treating cancer. Anti-LAP antibodies are thought to mediate anti-tumor activity both by inhibiting the release of active TGFβ and through reducing the number of LAP+ inhibitory cells.

Radiation is used as a first line therapy for many forms of cancer and is known to be a potent inducer of TGFβ production. This study was designed to determine the effects of radiation on the expression of LAP in the tumor microenvironment. 30 Balb/c mice were inoculated sc in the right flank with 106 CT26 colorectal cancer cells. When the mean tumor volume was approximately 300 mm3, half of the mice received a single dose of 20 Gy of targeted ionizing radiation. Groups of 3 mice from each cohort were sacrificed 1, 2, 3, 7 and 14 days after radiation and spleen and TILs collected for analysis by flow cytometry. As expected, radiation reduced tumor growth, with mean tumor volumes of 250 mm3 at day 14 vs 3600 mm3 in the non-irradiated control group. This was accompanied by a marked increase in CD45+ cells in the tumor which started on day 2 post-radiation and peaked on day 7, when 84% of live cells recovered from tumors were CD45+ (vs 25% in the non-irradiated controls). Radiation induced a significant (p<0.001) increase in CD8+ T cell numbers in the tumor, and prevented the increase in M2 macrophage numbers that occurred in the non-irradiated tumors. Radiation increased LAP expression on multiple cell types. Most notably, expression of LAP on DC and M2 macrophages increased steadily in response to radiation, with 33% of DC and 64% of M2 macrophages expressing LAP, significantly more than in control animals (10%, p=0.007 and 25%, p=0.01, respectively). In addition, 30% of M-MDSC from irradiated tumors expressed LAP, compared to 6% of non-irradiated tumors (p=0.03); expression on M-MDSC peaked at day 7.

These data are the first demonstration that radiation increases cell-associated latent TGFβ in the tumor microenvironment. Radiation significantly altered both the distribution of immune cells in the tumor microenvironment and the expression of LAP on the surface of those cells. The increased LAP/TGFβ expression is anticipated to offset the effects of increased CD8+ effector cells. These observations suggest testing the use of an anti-LAP antibody in combination with radiation therapy to enhance effective immune responses against the tumor.

#73

Cancer stem cell driving immunomodulation in the tumor microenvironment.

Huei-Wen Chen,1 Ming-Fang Wu,1 Sheng-Fang Su,1 Hsuan-Yu Chen,2 Jeremy J. Chen3. 1 _National Taiwan University, Taipei, Taiwan;_ 2 _Academia Sinica, Taipei, Taiwan;_ 3 _National Chung-Hsing University, Taichung, Taiwan_.

Tumor microenvironment is crucial to support the tumor malignancy, chemo-resistance, metastasis and cancer stemness; as well as, a unique immune regulation. Such niche enriches with the stromal cells and immune cells having distinct communities can affect the heterogenic plasticity of cancer stem cells (CSCs) and immune modulation for cancer cell survive and evolution. Recently, we have identified that the cancer-associated fibroblasts (CAFs) can contribute on the tumor microenvironment to maintain the lung CSCs sub-population and we also established a lung CSCs/CAFs co-culture system to clarify the paracrine interactions in the CSC niche. Interestingly, we found that the crosstalk of lung CSCs with other stromal cells may form an immune-modulation microenvironment for tumor immune escape. Our data showed that the surrounding CAFs could provide the essential niche with paracrine networks (e.g., IGFII, HGF, LIF, ILs, BMPs, and CXCLs) for promoting the cancer stemness and may also controlling the regulation of immune checkpoints. Such signaling can trigger the up-regulation of the immune checkpoint molecules (PDL1) to suppress the anti-tumor immunity; also, play the role on education of the M2 macrophage and significantly increase the regulatory T cell (Treg) population. According to the transcriptomic/proteomic analysis of the CSCs and the niche, we have identified several important immune modulating signaling; affecting the Treg and Th17 cytokine profiling, which balance of the T cells population could correlate with lung cancer progression, metastasis, and immune escape. Our results may contribute on the knowledge that targeting on the cancer stemness niche may provide a potential novel therapeutic intervention to improve the efficacy of immune checkpoint blockade to benefit for the combinatory immune-therapy for cancer treatment.

#74

E2F6-mediated ceRNA and epigenetic silencing of miR193a lead to cancer stemness and anticancer immunity in ovarian cancer.

Frank Hsueh-Che Cheng,1 Hon-Yi Lin,2 Yin-Chen Chen,1 Tzy-Wei Hwang,3 Rui-Lan Huang,4 Chia-Bin Chang,1 Ru-Inn Lin,2 Ching-Wen Lin,1 Gary C.W. Chen,1 Jora M. J. Lin,1 Yu-Ming Chuang,1 Jian-Liang Chou,1 Chin Li,1 Alfred S.L. Cheng,5 Hung-Cheng Lai,4 Shu-Fen Wu,1 Je-Chiang Tsai,6 Michael W.Y. Chan1. 1 _Department of Biomedical Sciences, National Chung Cheng University, Min Hsiung, Chia Yi, Taiwan;_ 2 _Departments of Radiation Oncology, Buddhist, Dalin Tzu Chi General Hospital, Dalin, Chia Yi, Taiwan;_ 3 _Department of Mathematics, National Chung Cheng University, Min Hsiung, Chia Yi, Taiwan;_ 4 _Department of Obstetrics and Gynecology, School of Medicine, College of Medicine and Shuang Ho Hospital, Taipei, Taiwan;_ 5 _School of Biomedical Sciences, The Chinese University of Hong Kong, Hong Kong, China;_ 6 _Department of Mathematics, National Tsing Hua University, Hsin-Chu, Taiwan_.

Ovarian cancer is one of the most lethal cancers in the female reproductive system. Previous study suggested that long term treatment of estrogen such as hormonal replacement therapy (HRT) may increase the risk of ovarian cancer, however the role of estrogen in ovarian carcinogenesis is still controversial. To decipher this complicated process, we generated a mathematical model and found that estrogen-mediated up-regulation of E2F6 could upregulate the ovarian cancer stem/initiating marker, c-kit by two means one through epigenetic silencing of their co-targeted miR193a by binding of E2F6 which subsequently recruit EZH2 to miR-193a promoter; and second, by competing endogenous (ceRNA) mechanism. To confirm this model, treatment of E2 or environmental hormone, BPA resulted in upregulation of both E2F6 and c-kit but down-regulation of miR-193a in immortalized ovarian surface epithelial cells. Further bisulfite pyrosequencing, ChIP-qPCR and epigenetic treatment found that miR193a was epigenetically silenced by DNA methylation and H3K27me3 in CP70 but not HeyC2 ovarian cancer cells. Overexpression of miR193a inhibited tumor growth in vitro and in vivo. Depletion of EZH2 or E2F6 in CP70 restored miR-193a expression and decreased the number of "ovo" spheroid by reversing the repressive chromatin status of miR-193a promoter. To further explore the biological significance of this E2F6 ceRNA network, integrative RNA-Seq and computational analysis found that PBX1, a miR-193a target and transcriptional activator of the immunosuppressive cytokine IL-10, was down-regulated in E2F6 and EZH2 knockdown CP70 cells. Overexpression of E2F6 3'UTR containing miR-193a MRE but not MRE mutant increased the expression of PBX1 and IL10 in ovarian cancer cells. Importantly, co-culture of conditional media from E2F6 3'UTR overexpressing CP70 cells inhibited the differentiation of THP-1 monocytes into dendritic cell and the T-cell activating function of this THP-1 derived DC. This phenomenon can be rescued by incubation of anti-IL-10 antibody or pretreatment of CP70 cells with EZH2 inhibitor. Finally, clinical studies demonstrated that patients with higher promoter methylation of miR193a were associated with poor survival. Serum IL10 level was found to be higher in high staged ovarian cancer patients and patients with higher E2F6 mRNA level. Additional analysis from TCGA ovarian cancer expression microarray dataset demonstrated that ovarian cancer patients with low expression of EZH2, showed a positive correlation between E2F6, c-KIT and PBX1 resembling the ceRNA phenomenon between these mRNAs. Taken together, our results showed that estrogen-mediated E2F6 ceRNA network can regulate cancer stemness and anti-tumor immunity of DC through epigenetic silencing of miR-193a. Anti-estrogen therapy together with the EZH2 inhibitor may be a novel strategy against this deadly cancer.

#75

A phase I trial of mesenchymal stem cells transfected with a plasmid secreting interferon beta in advanced ovarian cancer.

Michael Andreeff,1 Frank C. Marini,2 Shannon N. Westin,1 Robert L. Coleman,1 Peter F. Thall,1 Vivian Aljahdami,1 Muzaffar H. Qazilbash,1 Katy Rezvani,1 Melissa Timmons,1 Lauren Heese,1 Rui-Yu Wang,1 Richard E. Champlin,1 Elizabeth J. Shpall,1 Amanda Olson1. 1 _UT MD Anderson Cancer Ctr., Houston, TX;_ 2 _Wake Forrest Baptist Medical Center, Winston-Salem, NC_.

Background: Standard treatment for ovarian cancer with a platinum and taxane is effective initially but associated with significant relapse. Our group was first to demonstrate genetically modified MSC preferentially engraft and secrete gene products such as interferon beta (IFNB) at tumor sites, resulting in complete responses in animal models (Studeny M et al. Cancer Res 2002;62:3603; JNCI 2004;96:1593).

Methods: We conducted a single-center phase 1 trial to evaluate the safety, feasibility tumor response of mesenchymal stem cells transfected with plasmid secreting IFNB administered via intraperitoneal infusion (IPI) for women with advanced refractory epithelial ovarian cancer. We electroporated MSC derived from male donors with a plasmid vector containing the IFNB gene (MSC-IFNB). MSC-IFNB were then administered on an outpatient basis via IPI. Pretreatment biopsy, peritoneal fluid sample, blood draw, and a CT were performed at enrollment.

Results: Three patients were enrolled at dose level 1 (105 MSC/kg) and received 4 weekly IPI of MSC-IFNB between 6-10/2016. After the 4th dose we performed a restaging CT and a biopsy, which was examined by FISH for the presence of male Y-chromosome positive MSCs, for production of IFNB assessed by multispectral multiplexed IHC, presence of injected MSCs, changes in immune cell phenotypes, and tumor cell death. We demonstrated detectable production of IFNB in the serum of patient 1, and at week 4 detection of IFNB in the peritoneal fluid, suggesting injected MSC-IFNB produce IFNB locally. We performed multispectral analysis to identify and quantitate the constituents of the ovarian tumor microenvironment in pre- and post-treatment biopsy specimens. We focused on ovarian tumor cells (CK3/5+,AE1+), as well as stromal cells (αSMA+), and IFNB. We observed in all 3 patients colocalization of IFNB (red) with αSMA-(green,) suggesting MSC homing and production of IFNB at tumor stroma interface. To confirm the presence of injected MSC (derived from male donors), we then analyzed the pre- and post-treatment tumor biopsies for presence of the "XY" chromosome by FISH. We identified "XY" chromosomes MSC only in the post-tumor biopsy, suggesting incorporation of injected MSC at the tumor site. There were no infusion- or MSC-related adverse events and 1 patient had stable disease on CT scan at 4 months after treatment.

Conclusion: We demonstrate, for the first time, proof of principle that MSC-IFNB home to and are incorporated into tumors and locally produce beta-interferon after IP injection in patients with ovarian cancer. We anticipate increased responses at higher MSC dose levels and envision MSC-IFNB may be used in combination with immunotherapies and chemotherapy, as we have shown in animal models that MSC-IFNB modulate the immune phenoptype of the tumor, recruit FOXp3 immune cells, and facilitate long-term control of tumor growth and metastases.

#76

Parasympathetic signaling via CHRM1 directly suppresses pancreatic carcinogenesis and cancer stemness through inhibition of EGFR/MAPK and PI3K/AKT pathway.

Bernhard W. Renz,1 Takayuki Tanaka,2 Ryota Takahashi,2 Marina Macchini,2 Yoku Hayakawa,2 Christoph B. Westphalen,1 Paul E. Oberstein,2 Alina C. Iuga,2 Richard A. Friedmann,2 Kenneth P. Olive,2 Timothy C. Wang2. 1 _Ludwig Maximilian University, Munich, Germany;_ 2 _Columbia University Medical Center, New York, NY_.

Background: In many solid tumors, parasympathetic input is provided by the vagus nerve, which has been shown to modulate growth through effects on both tumor and stroma. Whether parasympathetic signaling directly regulates progression of pancreatic cancer (PDAC) has not been defined.

Methods: KC mice were vagotomized and kept on regular water or water supplemented with a muscarinic agonist. Pancreatic tissue was collected and analyzed by IHC, and RT-PCR; cells were isolated and assayed for colony and sphere forming assays. Different human and murine PDAC cell lines were subjected to cholinergic and anticholinergic drugs and assayed by RT-PCR, Western blot and flow cytometry. CHRM1 was deleted in Pdx1-Cre/KRasG12D (KC) and Pdx1-Cre/KRasG12D/Trp53R172H (KPC) mice. A syngeneic model of metastatic PDAC was also utilized.

Results: In organoid cultures derived from pancreata harboring an oncogenic KRas mutation, cholinergic agonists suppressed sphere formation significantly. Pharmacologic inhibition or genetic knockout of the CHRM1 abolished this effect in vitro. When Panc-1 cells pretreated with direct parasympathetic agonists were assayed in a xenograft model, they formed fewer and smaller tumors than controls. Surgical vagotomy accelerated tumor development in KC, while treatment with the muscarinic agonist bethanechol rescued the phenotype, an effect also seen in KC-CHRM1 KO mice. In KPC mice, bethanechol significantly extended survival, while KO of the CHRM1 (KPM) shortened overall survival. These effects appeared to be mediated through the CHRM1, which inhibited the downstream MAPK/EGFR and PI3K/AKT pathways. Reduced cholinergic signaling led to an increase of pancreatic CSC, and accelerated metastatic growth in the liver.

Discussion: Taken together, these data suggest that parasympathetic signaling directly suppresses the growth of PDAC cells, and therapies directed at stimulating muscarinic receptors may be useful in inhibiting the progression of PDAC.

#77

Caloric restriction mimetics prevent tumor progression by tumor microenvironment remodeling.

Anirudh Goyal, Shakir M. Saud, Jessica D. Bedele, Mariana S. De Lorenzo. _Rutgers New Jersey Medical School, NJ_.

The only non-pharmacological intervention known to date to alleviate the age-related deleterious conditions is caloric restriction (CR). We have previously reported that CR induces metabolic and signaling changes that affect the tumor microenvironment preventing mammary tumors growth and metastases. Despite the beneficial effects of CR, it is not feasible to maintain long-term dietary restriction in humans. Based on this, investigators have been focused on the discovery of CR mimetics that evoke some of the benefits of CR without an actual reduction in the calorie intake. Metformin (MET) is an anti-diabetic and a well-known CR mimetic; while Orlistat (OR) is an anti-obesity drug which has also antitumor properties. In this study we used 4T1, a highly metastatic breast cancer, and the B16F10 melanoma mouse models. At first, we evaluated the effects of CR and MET alone. Then, we investigated whether the combination of MET+OR will enhance the CR mimetic properties of MET alone. Our overall hypothesis is that CR mimetics decrease tumor progression by affecting extracellular matrix, epithelial-to-mesenchymal transition (EMT) and inflammation. Briefly, 8-weeks old female mice were fed with normal diet (ND) or 40% CR and received vehicle, MET (in drinking water, 3mg/ml), OR i.p. injection (240mg/kg/d) or MET+OR. After 5 weeks on diets, 4T1 or B16F10 cells were injected into BALB/c or C57BL6 mice respectively; diets and treatments continued for the remainder of the experiment. CR and Mimetics treated-mice had a reduction in body weight (p<0.01) and adiposity (p<0.05) without any signs of toxicity. CR and mimetics treatments reduced tumor growth (p<0.01), microvessel density, total vessel length (p<0.01) and decreased the number and size of spontaneous lung metastases (p<0.01). Also, CR and mimetics tumor bearing mice had significantly lower levels of intra-tumor uPA, MMP-9 and TGF-β. Moreover, CR and mimetics-treated mice had lower levels of circulating insulin, leptin and increased levels of adiponectin and fibroblast growth factor 21 (FGF21) (p<0.01). In a cell invasion assay, we also showed that MET+OR resulted in a significantly reduction on invasion (p<0.01). It is known that the loss of E-Cadherin promotes metastasis. We observed that CR and treated-mice had greater expression of E-Cadherin compared to the controls; in particular MET+OR had higher expression compared to either agent alone. NFĸB activation, the linker between inflammation and cancer, induces the expression of genes that promote a mesenchymal phenotype. We found that the MET+OR decreased phosphorylated P65S536 in 4T1 tumors, which corresponded with a decreased expression in COX-2 and Bcl-2; and an increased expression of P53 and cleaved caspase-3. Our results suggest that CR and MET+OR reduce tumor growth and metastases may in part be due to NFĸB inhibition and by remodeling the ECM, preventing the loss of E-Cadherin and the local invasion.

#78

p38 kinase is a negative regulator of tumorigenesis and recurrence after surgery in a mammary carcinoma model.

Carla S. Capobianco,1 Johanna E. Sidabra,1 Maria F. Gottardo,1 Julio A. Aguirre-Ghiso,2 Daniel F. Alonso,1 Hernan G. Farina1. 1 _Quilmes National University, Bernal, Buenos Aires, Argentina;_ 2 _Icahn School of Medicine at Mount Sinai, New York, NY_.

The mitogen activated protein kinase (MAPK) p38 is a kinase involved in the response to different external stimuli, extremely diverse such as oxidative stress or growth factors. Its activation triggers different responses which include from promoting cell apoptosis or cell cycle arrest, to cell differentiation and the activation of survival pathways. Although the role of p38 in cancer is a well studied field, there are still multiple unanswered questions, given the large amount of evidence supporting both a tumorigenic but also a tumor suppressor function for p38 in cancer development and progression. In this study, the effects produced by p38 negative modulation using the chemical inhibitor SB203580 in different aspects of tumor biology are analyzed in vitro and in vivo. As a study model, F3II cell line was used, which is an aggressive murine mammary carcinoma. We found that p38 inhibition provokes contradictory effects in our mammary carcinoma model. On one hand, p38 inhibition provoked the reduction of cell viability and colony formation. On the other hand, SB203580 treatment increased cell adhesion and proliferation in matrix coated surfaces. In vivo we found that SB203580 treatment increases tumor aggressiveness. The shortened latency times, higher number of lung metastasis and larger local recurrences are a reflection of the aforementioned effect. Taking together, the results presented in this work position p38 as a tumor suppressor kinase, in the context of cells adapting to a new environment and developing the first stages of growth. P38 inhibition represented an advantage for cells newly inoculated in vivo. We consider that this phenotypic adaptation is probably a consequence of ERK activation and integrin α5 increased expression. Data presented here is of importance to show how determinant is the microenvironment in the responses elicited by the modulation of p38 kinase. We propose a differential role according to the environment conditions, which reflects the complexity of the pathway and the importance of the extracellular stimuli when evaluating the implications of treatment with p38 MAP kinase inhibitors.

#79

Stromal regulation of prostate cancer cell proliferation by mevalonate pathway enzymes HMGCS1 and HMGCR.

Shingo Ashida, Chiaki Kawada, Keiji Inoue. _Kochi Medical School, Nankoku, Japan_.

It has been suggested that the tumor microenvironment plays an important role in tumor progression, acquisition of androgen independence, and distant metastasis in prostate cancer (PC). However, little is known about the transcriptional basis of cellular interactions in the human PC microenvironment. To clarify the mechanism of PC progression and metastasis, we investigated the interaction of PC, epithelial, and stromal cells by analyzing genome-wide gene expression profiles. Our hypothesis is that PC cells could induce stromal cells to differentiate into so-called cancer-associated fibroblasts (CAFs), which might contribute to cancer invasion and metastasis. We identified the up-regulated genes in normal human prostate stromal cells (PrSC) co-cultured with human PC cells (LNCaP), which included the mevalonate pathway enzymes 3-hydroxy-3-methylglutaryl-CoA synthase 1 (HMGCS1) and 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR). Knockdown of endogenous HMGCS1 or HMGCR in PC cells by shRNA resulted in a significant reduction of PC cell viability. Importantly, exogenous overexpression of HMGCS1 or HMGCR in either PC cells or prostate stromal cells stimulated PC cell growth, suggesting a possible autocrine/paracrine regulation. Immunohistochemical analysis confirmed that HMGCS1 and HMGCR were overexpressed in PC stroma, especially in the early stage of PC. These results provide clues to the molecular mechanisms underlying PC invasion and metastasis, and suggest that HMGCS1 and HMGCR in PC, as well as in PC stroma, might serve as molecular targets for the treatment of PC.

#80

Glucocorticoids mediate DCIS transition to IDC through myoepithelial cell apoptosis.

Arantzazu Zubeldia-Plazaola,1 Leire Recalde-Percaz,1 Núria Moragas,1 Mario Mancino,1 Aleix Noguera,1 Miquel Prats de Puig,2 Flavia Guzman,3 Pedro Gascón,1 Paloma Bragado,1 Gemma Fuster1. 1 _IDIBAPS, Barcelona, Spain;_ 2 _Clínica Planas, Barcelona, Spain;_ 3 _Anatomical Pathology Service, Centro Médico Teknon, Barcelona, Spain_.

The most accepted theory in breast cancer evolution is the linear progression from ductal carcinoma in situ (DCIS) to invasive ductal carcinoma (IDC). DCIS is defined by a clonal proliferation of epithelial cells that accumulate within the lumen of a duct and surrounded by the myoepithelial cells layer and the basement membrane. The challenge is to predict which of the DCIS lesions will eventually progress to IDC and which will remain stable for a long time. Understanding the progression from DCIS to IDC is crucial to properly treat DCIS patients and to develop new and more approachable therapeutic strategies in this stage. The microenvironment, as well as stress factors like glucocorticoids, exerts strong influences in DCIS to IDC transition. These factors have been previously implicated in breast cancer progression however its role in the first stages of breast cancer and particularly in myoepithelial cells regulation remains elusive. In order to clarify glucocorticoids role in this breast cancer stage, we evaluated the effects of corticosterone and cortisol in epithelial cells and myoepithelial cells using in vitro 2D and 3D and in vivo techniques. Glucocorticoids induced a decreased in laminin levels and favour the disruption of the myoepithelial cell layer through myoepithelial cells apoptosis promotion. In addition, increased corticosterone levels, in an in vivo stress murine model, fostered the transition from DCIS to IDC via myoepithelial cell apoptosis and basement membrane vanishing. Furthermore, myoepithelial cell apoptosis is a more frequent event in DCIS+IDC than in DCIS breast cancer patients. Our findings show that stress, through glucocorticoids, plays a role in the progression of DCIS to IDC, and particularly by promoting apoptosis of myoepithelial cells and the apoptosis in myoepithelial cells seems to be a requirement to achieve invasive features in breast cancer patients.

### Mechanisms Underlying Metastasis 1

#81

Endogenous ADAMTS-13 regulates vasculogenic functions in glioblastoma cells.

Huiyuan Tang, Manfai Lee, Eun Ho Kim, Daniel Bishop, George M. Rodgers. _Univ. of Utah School of Medicine, Salt Lake City, UT_.

ADAMTS-13, a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13, is a zinc-containing metalloprotease that cleaves von Willebrand factor. Previous publications by our laboratory have shown that either endogenous or exogenous ADAMTS-13 may regulate angiogenesis in endothelial cells; for example, siRNA knockdown of endogenous endothelial cell ADAMTS-13 reduced angiogenic activities including cell migration, proliferation, and tube formation (Tang et al. Microvascular Res 2017; 113:85-70). Many tumor cells have the ability to develop a vascular phenotype, a process known as vasculogenic mimicry (VM). There is a multitude of evidence demonstrating that tumor cell-mediated VM may play a vital role in tumor development, independent of endothelial cell angiogenesis. For this study, we report that decreasing ADAMTS-13 levels inhibit vasculogenic functions in a glioblastoma cell line (LN-229), while increasing ADAMTS-13 levels had no effect. Incubating up to 30ng/ml of exogenous ADAMTS-13 did not promote cell proliferation and up to 200ng/ml did not change tube formation in LN-299 brain tumor cells. One possible explanation for this observation is that LN-299 brain tumor cells have almost twice the endogenous ADAMTS-13 expression than endothelial cells (~200pg/ml vs ~100pg/ml), so this cell line is not sensitive to exogenous ADAMTS-13. Using siRNA, we were able to successfully knockdown endogenous ADAMTS-13 in human brain tumor LN-229 cells. 50nM of ADAMTS-13 siRNA in a six-well plate inhibited HUVEC ADAMTS-13 expression levels by 75% after 48hr incubation, whereas control siRNA did not affect endogenous ADAMTS-13 levels. Additionally, expression of ADAMTS-1, a related family member of ADAMTS-13 was not affected by the knockdown. The effects of reduced endogenous ADAMTS-13 on glioblastoma vasculogenic functions were studied. Transfection of HUVEC with 50nM of ADAMTS-13 siRNA in a 24-well plate resulted in about 20% decrease in proliferation after 24hr and 48hr incubation. We demonstrated that decreased expression of endogenous ADAMTS-13 also affected vasculogenic mimicry as measured by glioblastoma cell tube formation using a Matrigel matrix method. The tube lengths, sizes and junction numbers of the ADAMTS-13 knockdown cells were all significantly lower compared to control cells by about 30-40%, respectively. Moreover, the expression of VEGF, a well-known regulator of angiogenesis, was significantly decreased by 25% after knockdown of endogenous LN-229 ADAMTS-13. Overall, these data suggest that endogenous ADAMTS-13 may regulate glioblastoma cell growth and function.

#82

Epigenetic reader ZMYND8 bridges BRD4 and hypoxia-inducible factors to mediate breast cancer progression and metastasis.

Yan Chen, Bo Zhang, Lei Bao, Lai Jin, Mingming Yang, Yan Peng, Jennifer Wang, Chenliang Wang, Xuan Zou, Yingfei Wang, Weibo Luo. _UT Southwestern Medical Center, Dallas, TX_.

Metastatic breast cancer has high rates of relapse and mortality. The absence of definitive prognostic biomarker and effective therapeutic target is the major obstacle to end this aggressive disease. Epigenetic dysregulation plays a crucial role in breast cancer metastasis. However, the mechanism by which epigenetic dysregulation stimulates the hypoxia-mediated breast cancer progression and metastasis remains unknown.

Based on luciferase reporter assays and analysis of expression changes of 720 epigenetic genes in three microarray datasets, we identified epigenetic reader ZMYND8 as a novel hypoxia-inducible factor (HIF) target gene. Human breast cancer tissue microarray data indicated that ZMYND8 is highly expressed in invasive breast tumors and this overexpression is significantly correlated with poor clinical outcomes in patients with breast cancer. ZMYND8 depletion dramatically attenuates the tumorigenic potential of breast cancer cells in colony formation, migration and invasion in vitro, and suppresses breast tumor growth and metastasis in mice. To elucidate the underlying mechanism, we performed co-immunoprecipitation, chromatin immunoprecipitation and quantitative PCR assays and found that ZMYND8 interacts with HIF complex and acetyl lysine 16 of histone H3 at the hypoxia inducible elements (HREs) to provoke the expression of HIF target genes LOX, AGR2, AQP1 and VEGFA. ZMYND8 controls breast cancer growth and metastasis through HIF in mice. We further found that p300 binds and acetylates ZMYND8 in breast cancer cells. Acetylated ZMYND8 interacts with and recruits BRD4 to the HREs to stimulate RNA polymerase II phosphorylation, thereby promoting transcriptional elongation of HIF target genes. ZMYND8 acetylation is necessary and sufficient for breast tumor growth and metastasis in vitro and in mice. Together, p300-ZMYND8-BRD4-HIF axis is critical for breast cancer progression and metastasis.

In summary, ZMYND8 represents a positive feedback mechanism that amplifies HIF-mediated breast cancer progression and metastasis, and provides a potential epigenetic target for the prognosis and treatment of breast cancer.

#83

The role of Nrf2-Keap1 pathway in the survival of circulating metastatic cancer cells.

Hashim M. Aljohani,1 John M. Furgason,2 Peter Amaya,3 Ayham Deeb,1 Jeffery J. Chalmers,3 El Mustapha Bahassi1. 1 _University of Cincinnati, Cincinnati, OH;_ 2 _Cincinnati Children's Hospital Medical Center, Cincinnati, OH;_ 3 _The Ohio State University, Columbus, OH_.

Approximately 90% of all cancer deaths arise from the metastatic spread of primary tumors. Of all the processes involved in carcinogenesis, local invasion and the formation of metastases are clinically the most relevant, but they are the least well understood at the molecular level. As a barrier to metastasis, cells normally undergo an apoptotic process known as "anoikis," in circulation. The recent technological advances in the isolation and characterization of rare circulating tumor cells (CTCs) will allow a better understanding of anoikis resistance. Detailed molecular and functional analyses of anoikis-resistant cells may provide insight into the biology of cancer metastasis and help identify novel targets for prevention of cancer dissemination. To uncover the molecular changes that govern the transition from a primary lung tumor to a secondary metastasis and specifically the mechanisms by which CTCs survive in circulation, we carried out whole-genome sequencing (WGS) of normal lung, primary tumors and the corresponding brain metastases from five patients with progressive metastatic NSCLC. We also isolated CTCs from patients with metastatic cancer and subjected them to whole-genome amplification and Sanger sequencing and quantitative digital PCR of genes of interest. While the primary tumors showed mutations in genes associated with cell adhesion and motility, brain metastases acquired mutations in adaptive and cytoprotective genes involved in the response to cellular stress such as Keap1, Nrf2 and E300, which are key players of the Keap1-Nrf2-ARE survival pathway. Nrf2 is a transcriptional factor that upon stress translocates into the nucleus, binds to the ARE (anti-oxidant response elements) and drives the expression of antioxidant genes. The identified mutations affect regulatory domains in all three proteins, suggesting a functional role in providing a survival advantage to CTCs in the peripheral blood, allowing their dissemination to distant organs.

#84

Identification of HNRNPLL as a novel metastasis suppressor of colorectal cancer.

Masahiro Aoki,1 Eiichi Sasaki,2 Kenya Kimura,2 Koji Komori,2 Yasuhiro Shimizu,2 Yasushi Yatabe,2 Keiichiro Sakuma1. 1 _Aichi Cancer Ctr. Research Inst., Nagoya, Japan;_ 2 _Aichi Cancer Ctr. Hosp., Nagoya, Japan_.

Despite the advances in surgery and chemotherapy for colorectal cancer patients, those with distant metastasis still suffer poor prognosis. Metastasis is a complex process regulated by a number of biomolecules, which can be conceptually divided into metastasis promoters and suppressors. To identify novel targets for prevention and/or therapy of colorectal cancer metastasis, we performed an in vivo shRNA library screen for colorectal cancer metastasis suppressor genes using an orthotopic transplantation model. CMT93 cells, a murine colorectal cancer cell line with poor metastasizing activity, were transduced with lentiviral shRNA library, and then transplanted into the rectum of C57/BL6 mice. Genomic DNA was collected from metastatic lesions, and the integrated shRNAs were retrieved by PCR for sequencing, followed by identification of the candidate genes targeted by the shRNAs. Among 47 candidate genes identified, we focused on Hnrnpll (heterogeneous nuclear ribonucleoprotein L-like) gene encoding a pre-mRNA binding protein involved in pre-mRNA splicing. Hnrnpll knockdown in CMT93 cells significantly increased their matrigel invasion in vitro and lung metastasis in vivo. An RNA-immunoprecipitation analysis identified Cd44 as a candidate target of Hnrnpll. The mRNA level of Cd44 variant 6 (Cd44v6), a splicing variant of Cd44 that had been shown to demarcate colon cancer stem cells driving colon cancer metastasis, was increased by knocking down Hnrnpll in colon cancer cells. Treatment with neutralizing anti-CD44v6 monoclonal antibodies suppressed matrigel invasion of colon cancer cells induced by Hnrnpll knockdown, suggesting that Hnrnpll may suppress invasion and metastasis, at least in part, by modulating the alternative splicing of Cd44. HNRNPLL expression level in colorectal cancer cells was drastically decreased upon induction of epithelial-mesenchymal transition (EMT), accompanied by increased expression of CD44v6. The link between HNRNPLL and EMT was further suggested by immunostaining analysis of clinical samples; colorectal cancer cells with low E-cadherin expression at the invasion front exhibited decreased HNRNPLL expression as compared with those in the tumor center. These results suggest that HNRNPLL is a novel metastasis suppressor in colorectal cancer. Possible mechanisms for regulation of HNRNPLL expression during EMT will also be discussed.

#85

Oleic acid promotes head and neck squamous cell carcinoma anoikis resistance and metastasis via ANGPTL4/fibronectin pathway.

Chih-Jie Shen,1 Shih-Hung Chan,2 Chung-Ta Lee,2 Wan-Chen Huang,3 Jhih-Peng Tsai,4 Kwang-Yu Chang,5 Wen-Chang Chang,1 Ben-Kuen Chen4. 1 _Taipei Medical University, Taipei, Taiwan;_ 2 _National Cheng Kung Univ. Hospital, Tainan, Taiwan;_ 3 _Academia Sinica, Taipei, Taiwan;_ 4 _National Cheng Kung Univ., Tainan, Taiwan;_ 5 _National Health Research Institutes, Tainan, Taiwan_.

Obese patients have higher levels of free fatty acids (FFAs) in their plasma and a higher risk of cancer than their non-obese counterparts. However, the mechanisms involved in the regulation of cancer metastasis by FFAs remain unclear. In this study, we found that oleic acid (OA) induced angiopoietin-like 4 (ANGPTL4) protein expression and secretion and conferred anoikis resistance to head and neck squamous cell carcinomas (HNSCCs). The autocrine production of OA-induced ANGPTL4 further promoted HNSCC migration and invasion. In addition, the expression of peroxisome proliferator-activated receptor (PPAR) was essential for the OA-induced ANGPTL4 expression and invasion. The levels of OA-induced epithelial-mesenchymal transition markers, such as vimentin, MMP-9, and fibronectin and its downstream effectors Rac1/Cdc42, were significantly reduced in ANGPTL4-depleted cells. Knocking down fibronectin not only inhibited the expression of MMP-9 but also repressed OA- and recombinant ANGPTL4-induced HNSCC invasion. On the other hand, ANGPTL4 siRNA inhibited OA-induced MMP-9 expression, which was reversed in fibronectin-overexpressing cells. Furthermore, the depletion of ANGPTL4 impeded the OA-primed metastatic seeding of tumor cells in the lungs. These results demonstrate that OA enhances HNSCC metastasis through the ANGPTL4/fibronectin/Rac1/Cdc42 and ANGPTL4/fibronectin/MMP-9 signaling axes. The inhibition of ANGPTL4 could be a potential strategy for the treatment of FFA-mediated HNSCC metastasis.

#86

Beta-arrestins regulate basal cell and cancer stem cell phenotype in muscle-invasive bladder cancer.

Georgios Kallifatidis, Diandra K. Smith, Jie Gao, Richard Pearce, Jiemin Li, Vinata Lokeshwar, Balakrishna L. Lokeshwar. _Augusta University, Augusta, GA_.

Background: The muscle-invasive bladder cancer (MIBC) is the most aggressive form of bladder cancer with a five-year survival of less than 15%. High metastatic potential, poor response to chemotherapy and a largely basal cell molecular signature characterize MIBC. We identified two cytoplasmic proteins, β-arrestin 1 and β-arrestin 2 (BARR1 and BARR2), that are unevenly expressed in bladder cancer cell lines. The function of BARR1 and BARR2 is unknown in bladder cancer although these proteins are well characterized in other systems, notably as regulators of G-protein coupled receptor activity in normal and abnormal cell physiology. We investigated the potential role of BARRs in established bladder cancer cells.

Methods: Manipulations of expression of individual arrestins were performed by establishing stable cell lines with forced overexpression by cDNA transfection, or depletion by shRNA or CRISPR-Cas9 mediated gene editing. The effect of altering BARRs in bladder cancer cells was investigated for changes in clonogenic survival, expression of basal cell markers, such as cytokeratins (CK), stem-cell associated markers, alteration in tumor cell motility and invasion.

Results: Established cell lines from MIBC origin (e.g., 253J and HT-1376) had either high-level expression of BARR1 and BARR2 or only high expression of BARR1. Normal bladder cells predominantly expressed BARR2. Depletion of BARR2 in 253J cells resulted in increased basal cell markers (e.g., CK5, CK14). Depletion of BARR2 elevated many cancer stem cell (CSC) markers (e.g., ALDH2). BARR2 overexpression reduced expression of stem cell markers, and increased sensitivity towards gemcitabine. Furthermore, when cells were grown as spheroids--a technique used to enrich CSCs--we observed decreased expression of BARR2, whereas expression of BARR1 increased. In 253J cells, BARR2 depletion significantly increased motility, chemotaxis, and invasive potential. CRISPR-Cas9 mediated gene knockout of BARR1 resulted in reversal of aggressive phenotype, including decrease in CD44, and phospho-STAT3.

Conclusions: This is the first report of the role of BARR1 and BARR2 in MIBC. The results presented here strongly suggest a critical role of BARRs in bladder cancer metastasis and resistance to chemotherapy drugs, two key factors that determine the poor survival of bladder cancer patients. Further exploration of the roles of β-arrestins in bladder cancer has potential to improve therapy for patients with muscle-invasive bladder cancer.

#87

Nuclear localization of p70 S6 kinase in ovarian cancer.

Jing ZHOU, Sally K. TO, Alice S. WONG. _School of Biological Sciences, HKU, Hong Kong, Hong Kong_.

Ovarian cancer is highly metastatic with a poor prognosis. The p70 S6 kinase (p70S6K) is a downstream effector of the phosphatidylinositol-3 kinase/Akt pathway that is frequently activated in ovarian cancer. Constitutive activation of p70S6K is significantly more often in malignant ovarian tumors than in normal or benign tissues, suggesting a role for p70S6K in tumor development and progression. In addition to its well-established role in cell growth and survival, we showed for the first time that p70S6K plays a pivotal role in other aspects of tumor progression such as metastasis. Here, we further showed that this was associated with the nuclear localization of p70S6K. Treatment of cells with the CRM1 inhibitor, leptomycin B, retained p70S6K in the nucleus. Growth factor stimuli led to p70S6K phosphorylation and triggered nuclear translocation of p70S6K. The phosphorylation was sensitive to p70S6K inhibitors. We further identified a potential region that was important in its nuclear localization. Expression of nuclear localization signal-p70S6K enhanced tumor cell migration and invasion when compared to non-targeted p70S6K. Our findings identify a region responsible for the nuclear entry of p70S6K and demonstrate a functional significance of nuclear p70S6K in tumor progression (This work is supported by RGC grant 17103417).

#88

Metastatic lymph nodes can serve as a source of systemic metastasis.

Kazu Takeda,1 Shiro Mori,2 Tetsuya Kodama1. 1 _Graduate school of Biomedical Engineering, Tohoku University, Sendai, Japan;_ 2 _Tohoku University Hospital, Sendai, Japan_.

Cancer metastasis is a major cause of death in cancer patients and metastatic lymph nodes (MLN) are associated with poor prognosis. In the field of breast cancer research, the role of MLN has been discussed for nearly a hundred years. Halsted formulated "Halsted theory", whereby breast cancer spread through the lymphatic system first to nearby lymph nodes and subsequently to other organs in the body. An alternative hypothesis, "Systemic theory" was put forward by Fisher who believed that breast cancer was a systemic disease, with tumors having the ability to spread to distant sites from the primary locus. Thu, MLN can hardly be the source of systemic metastasis. However, the actual role of MLN in routing cancer cells to distant organs remains incompletely characterized because of inadequate investigations in animal experiments. In the present study, we show that MLN can be the source of systemic metastasis using MXH10/Mo-lpr/lpr (MXH10/Mo/lpr) mice, which develop systemic swelling of lymph nodes up to 10 mm in diameter. Previously we developed a MLN model using MXH10/Mo/lpr by injection of tumor cells into its LN directory. In the present study, we first investigated the venous/lymphatic system around the subiliac LN (SiLN) where SiLN is one of the superficial LNs in the MXH10/Mo/lpr and the proper axially LN is the downstream LN of SiLN. The thoracoepigastric vein (TEV) runs along the lymphatic vessels between the SiLN and the PALN and connects to the inferior vena cava and subclavian vein via the SiLN and PALN. After intravenous injection of fluorescence liposomes, we demonstrated that intranodal venulas perforated the capsule of the SiLNs and anastomosed with the TEV. Next, we investigated the flow dynamics of a fluorescent solution that was injected into the SiLNs. We found that the fluorescent solution flowed into the efferent lymphatic vessels as well as the TEV. Moreover, the MLN model showed different flow dynamics compared to the control LN. Fluorescent solution tended to flow into the blood circulation more than the efferent lymphatic vessel. This finding suggested that the vasculature and lymphatic systems have a connection with the LN under specific mechanistic conditions, such as elevation of intranodal pressure by injection. This mechanical force could have facilitated the fluorescent solution to flow into the efferent lymphatic vessel along with the development of a connection between the two circulation systems in the LN. We previously reported that intranodal pressure increases in MLN during tumor cell proliferation and circulatory connection can be developed in the MLN. Therefore, tumor cells metastasized into the LN can route their metastatic pathways to the hematogenous system. Thus, the MLN has the potential to be a direct source of systemic metastasis. We anticipate that our results will facilitate the study of the progression of systemic metastasis via the MLN.

#89

Reduced kinase D-interacting substrate of 220kDa (Kidins220) in pancreatic cancer promotes EGFR/ERK signaling and disease progression.

Shuo Cai,1 Ping-Hui Sun,1 Xiangyu Gao,2 Ke Ji,2 Xiuyun Tian,2 Chunyi Hao,2 Bilal Al-Sarireh,1 Paul Griffiths,3 Steven Hiscox,4 Jared Tonkington,1 Jun Cai,1 Wen G. Jiang,1 Lin Ye1. 1 _Cardiff University School of Medicine, Cardiff, United Kingdom;_ 2 _Peking University Cancer Hospital & Institute, Beijing, China; _3 _Morriston Hospital, Swansea, United Kingdom;_ 4 _Cardiff University, Cardiff, United Kingdom_.

Kidins220 (Kinase D-Interacting Substrate, 220kDa), also known as ARMS (ankyrin repeat-rich membrane spanning) is a transmembrane scaffolding protein. Kidins220 acts as a platform for protein-protein interactions to coordinate cell plasticity, migration and neural growth receptor signalling. Present study aims to investigate the involvement of Kidins220 in the disease progression of pancreatic cancer. Pancreatic tumors (n=149) together with paired adjacent background tissues were collected at Peking University Cancer Hospital with written consent from the patients and also an ethical approval from the Peking University Cancer Hospital Research Ethics Committee. Kidins220 transcript levels were determined using real-time PCR. Relevance of Kidins220 transcript levels and pathologic/clinical features was analyzed. Knockdown of Kidins220 was carried out with a lentiviral Kidins220 shRNA. Immunohistochemical (IHC) staining of Kidins220 was performed on a pancreatic tissue microarray (TMA) (PA2081a, US Biomax, Inc., Rockville, MD, USA). A decreased expression of Kidins220 transcripts was observed in pancreatic cancer tissues in comparison with the paired background control (P=0.029). According to the TNM staging, early stage tumors (TNM 1&2) expressed higher levels of Kidins220 transcripts compared with its expression in more advanced tumors (TNM 3&4 stage, p=0.0034). In line with the finding from the Beijing pancreatic cancer cohort, a reduced expression of Kidins220 protein was seen in the pancreatic carcinomas of the TMA using IHC, p<0.001 compared with normal control. Moreover, based on the IHC results, primary tumors with distance metastases exhibited a decreased level of Kidins220 expression compared to those without metastasis. Knockdown of Kidins220 in pancreatic cancer cell lines PANC1 and MIA-PaCa-2 promoted migration and invasion of both cell lines. A protein array analysis (KinexTM) revealed an increased EGFR phosphorylation in the Kidins220 knockdown pancreatic cancer cell lines which was confirmed with Western blot. The Kidins220 knockdown-resulted increased invasion was eliminated by targeting EGFR using both gefitinib or with the irreversible pan-HER tyrosine kinase inhibitor, neratinib. Proliferation assays showed that neratinib was more effective to inhibit proliferation of PANC-1 and Mia-PaCa-2 cells at a range of concentration from 20nM to 2μM. In conclusion, the expression of Kidins220 was reduced in pancreatic cancer and the reduced expression in primary tumors was associated with distant metastases. Reduced Kidins220 expression promoted migration and invasion of pancreatic cancer cells through an upregulation of EGFR. The therapeutic potential of neratinib or gefitinib for reduced kidins220 relevant disease progression and metastasis in pancreatic cancers is yet to fully investigated.

#90

Metastatic melanoma and cervical tumor cell clusters can exit blood vessels through angiopellosis augmenting tumor formation ability.

Tyler Allen, Emmanuel Amu, Dana Asad, Ke Cheng. _NC State University, Raleigh, NC_.

Introduction Accumulating data suggests metastatic primary tumor cells can travel through the circulation and form secondary tumors as multicellular clusters. However, whether these clusters must first dissociate into single cells prior to exiting of blood vessels remains largely unknown. Of late, researchers have identified a mechanism of cell extravasation (exiting blood vessel), termed angiopellosis (Allen 2016). However, whether tumor cell clusters possess the ability to exit vessles using the angiopellosis method is unstudied. We hypothesized that circulating tumor cell (CTC) clusters could exit blood vessels while maintaining a multicellular phenotype, through angiopellosis.

Methodology To test this, we used an in vivo zebrafish larvae model using the tg(fli1a:egfp) line, in which blood vessels exclusively fluoresce. First, we infused fluorescent human melanoma (A375), mouse melanoma (B16F10), and human cervical (HeLa) cells into the circulation of the larvae. Next, we used lightsheet microscopy to observe the method the tumor cells used to exit blood vessels. Additionally, mouse melanoma (B16F10) cells were intravenously infused into the circulation of C57BL/6J mice as either tumor clusters or individual cells. The mice were imaged for metastatic lung foci, to determine cluster extravasation potential.

Experimental results The intravital lightsheet microscopy allowed us to determine that tumor cells can utilize angiopellosis to exit blood vessels in the zebrafish model, both as individual CTCs and CTC clusters. Extravasating CTCs exited blood vessels through angiopellosis at 94%, with only 6% exiting through the diapedesis method (n=30). CTCs which exited as a cluster through angiopellosis were observed to form tumor masses at an 88% higher rate compared to individual tumor cells in vivo, over the course of 96 hours. In the murine model, CTCs clusters which were infused via tail vein (n=4) showed significantly higher metastatic lung foci in comparison to individual CTCs that were infused (n=3). Following isolation and analysis of metastatic CTCs, multiple genes were shown to be differentially expressed in CTC clusters in both the zebrafish and murine models.

Discussion Here, we show circulating tumor cell clusters possess the ability to exit blood vessels through the recently identified angiopellosis. We determined tumor cell clusters exhibit an augmented ability to proliferate in vivo, while individually tumor cells are more prone to dormancy. Our results challenge the belief that circulating tumor cell clusters must first disassociate to exit the circulation, and posits an alternative model. We propose tumor cells both travel and exit the circulation while maintaining a multicellular phenotype, using angiopellosis, stimulating proliferation-augmenting genetic/molecular alterations. This has been termed the Cancer Exodus Hypothesis.

#91

RalA is frequently up-regulated in human hepatocellular carcinoma and promotes metastasis and cancer stemness.

Luqing Zhao, Lo-Kong Chan, Daniel Wai-Hung Ho, Goofy Yu-Man Tsui, Macrina Wai-Ling Lam, Charles Shing Kam, Karen Man-Fong Sze, Vanilla Xin Zhang, Abdullah Husain, Joyce Man-Fong Lee, Irene Oi-Lin Ng. _University of Hong Kong, Hong Kong, Hong Kong_.

RalA is a Ras-related small GTP binding protein A; however, its functional roles and regulatory mechanisms in hepatocellular carcinoma (HCC) are unclear. In this study, using the RNA-Seq data from TCGA database and our in-house HKU database, we observed that RalA expression was significantly up-regulated in human HCCs (P=0.001). This RalA over-expression was validated in a separate cohort of our HCC patients. Upon clinicopathological correlation of RalA in HCC, we found that over-expression of RalA was associated with more aggressive features of HCC patients, with more frequent tumor microsatellite formation (P=0.001), venous invasion (P=0.005) and absence of tumor encapsulation (P=0.005). The over-expression also correlated with poorer overall survival of HCC patients (P<0.001). Functionally, we established RalA stable knockdown (KD) in three HCC cell lines (Hep3B, BEL7402 and MHCC-97L-Luc) and demonstrated that KD of RalA significantly inhibited cell proliferation, colony formation, migration and invasion in vitro. Conversely, ectopic expression of RalA, by transfecting the RalA dominant active form G23V construct into HCC cells, promoted HCC metastasis using transwell invasion assays. Also, with immunofluorescence assay, RalA mainly located in the cytoplasm and cell membrane. Over-expression of RalA changed the HCC cell morphology from polygonal to spindled shape, suggestive of epithelial-mesenchymal transition. We conducted in vivo study using the orthotopic liver injection model of RalA stable-KD MHCC-97L cells in nude mice, and observed that KD of RalA suppressed HCC tumorigenicity and reduced lung metastasis (P<0.001). Moreover, KD of RalA also reduced sphere formation ability of HCC cells, as well as suppressed the expression of stemness markers, including CD24, NANOG, NOTCH1, NESTIN, and EpCAM, using qPCR. For the liver cancer stem cell surface markers, the expression level of CD24 was significantly decreased in RalA KD Hep3B and BEL7402 cells by flow cytometry. Additionally, with RNA-Seq analysis of Hep3B and BEL7402 shRalA clones, 12 genes were found to be significantly altered (among them 5 were up-regulated and 7 were down-regulated) for both cell lines, when compared with in-house cohort as well as TCGA cohort. Further analysis showed that RalA expression level was positively and negatively correlated with a small group of genes. Taken together, our findings have shown that RalA is significantly up-regulated in human HCCs and its over-expression enhances HCC metastasis and cancer stemness. Further investigation of the interplay between RalA and its downstream targets will derive novel mechanistic insight regarding the oncogenic role of RalA in HCC.

#92

CHIP/STUB1 ubiquitin ligase targets MZF1 and loss of its expression in breast cancer unleashes a MZF1-cathepsin pro-oncogenic program.

Bhopal C. Mohapatra,1 Haitao Luan,1 Timothy A. Bielecki,1 Insha Mushtaq,1 Sameer Mirza,1 Tameka A. Bailey,1 Robert Clubb,1 Wei An,1 Dena Ahmed,2 Rokaya El Ansari,2 Matthew D. Storck,1 Chittibabu Guda,1 Yuri Sheinin,1 Jane L. Meza,1 Srikumar Raja,3 Emad Rakha,2 Vimla Band,1 Hamid Band1. 1 _University of Nebraska Medical Center, Omaha, NE;_ 2 _University of Nottingham, Nottingham, United Kingdom;_ 3 _University of Northwestern, Chicago, IL_.

Triple-negative and ErbB2+ breast cancer subtypes carry inherently poorer prognosis. The overall therapeutic response and survival are markedly lower in metastatic disease. This identification of novel pathways that collide with driver oncogenes to promote more aggressive disease and metastasis could help identify biomarkers for selection of patients for more intensive therapy and provide avenues of targeted therapy for new combinatorial therapies. CHIP/STUB1 is a U-box containing ubiquitin ligase that has been established as a negative co-chaperone for HSP90/HSC70. Recent studies have shown its expression to be reduced or lost in several cancers, including breast cancer. However, the relationship of loss of CHIP with breast cancer subtypes and mechanisms by which loss of CHIP promotes oncogenesis in breast cancer are incompletely understood.

IHC analysis of an extensive breast cancer tissue collection revealed that loss of nuclear but not cytoplasmic CHIP predicted aggressive tumorigenesis and shorter survival, with loss of nuclear CHIP in two-thirds of TNBC/ErbB2+ and one-third of ER+ breast cancers. Reduced CHIP expression was confirmed in a panel of breast cancer patient-derived xenograft tumors and in ErbB2+ and TNBC cell lines. Ectopic CHIP expression in TNBC and ErbB2+ breast cancer cell lines suppressed in vitro oncogenic traits and in vivo xenograft tumor growth in nude mice. These analyses established CHIP as a barrier to tumorigenic progression and loss of CHIP as a key event in aggressive tumorigenesis in breast cancer.

To gain mechanistic insights into the linkage of the loss of nuclear CHIP expression with breast cancer aggressiveness, we carried out an unbiased screen for CHIP-regulated nuclear transcription factor-binding to their cognate DNA elements using an array methodology. This analysis identified many candidate transcription factors whose DNA-binding activity was up- or down-regulated upon re-expression of CHIP in CHIP-low ErbB2+ or TNBC cell line models. Western blotting of selected candidates confirmed the CHIP dependence of their expression levels. We focused our further efforts on the Myeloid Zinc Finger 1 (MZF1) protein as a CHIP target since it was recently identified as a positive regulator of cathepsin B/L (CTSB/L)-mediated tumor cell invasion in a model of oncogenic mutant ErbB2 overexpression in MCF-7 cells. We show that CHIP negatively regulates the expression of CTSB/L in TNBC and other breast cancer cell lines. Chemical inhibition of CTSB abrogated breast cancer cell invasion and matrix degradation in vitro and retarded xenograft tumor growth. We suggest that loss of CHIP remodels the cellular transcriptome to unleash critical pro-oncogenic pathways that can provide new therapeutic opportunities in breast cancer.

#93

**Tissue-specific CRISPR in zebrafish identifies** PVRL1 **as a novel metastasis suppressor gene in melanoma.**

Julien Ablain, Michelle Dang, Harriet Rothschild, Leonard I. Zon. _Boston Children's Hospital, Boston, MA_.

Defining factors that lead to the spread of cancer will have great impact on designing new treatment options. To search for such modifiers of melanoma, we developed a vector system based on the CRISPR/Cas9 technology of genome editing that targets genes specifically in the melanocytes of a zebrafish melanoma model. As a proof of principle for this system, we showed that pten inactivation in Tg(mitfa:BRAFV600E), p53-/- zebrafish significantly accelerated tumor onset. This system allows us to interrogate the functional relevance of recurrent genetic alterations found in cancer patients by next generation sequencing. Here, we identified a frequent focal deletion on chr11q23.3 by GISTIC analysis of the TCGA cohort of primary human melanomas. This region contains a gene called PVRL1 that is homozygously lost in 8% of tumors. PVRL1 encodes nectin-1, a transmembrane protein involved in adherens junctions. To assess the role of PVRL1 in melanoma in vivo, we injected Tg(mitfa:BRAFV600E), p53-/- zebrafish embryos with our melanocyte-specific CRISPR vector directed against pvrl1. We detected tumors earlier in fish expressing a vector targeting pvrl1 compared to fish expressing a control vector (median: 18 vs 20 weeks). Deep sequencing of the pvrl1 CRISPR sites in tumors confirmed gene inactivation through frameshift insertions or deletions. To further assess the in vivo phenotype of pvrl1-deficient cells, we designed a transplantation assay in which we injected 300,000 primary zebrafish melanoma cells subcutaneously in the back of irradiated, transparent recipients. In this assay, pvrl1 CRISPR melanoma cells consistently migrated away from the injection site, and eventually invaded internal tissues, whereas control cells remained localized in the dorsal region of transplanted fish. Thus pvrl1 loss favors the spreading of melanoma cells. To investigate the function of PVRL1 at the molecular level, we established pvrl1 knock-out zebrafish melanoma cell lines in culture and realized that pvrl1 was required for attachment and sustained growth in vitro in the absence of zebrafish serum. Nectin-1 was up-regulated upon serum stress in both zebrafish and human lines, presumably to support cell survival via cell-cell interactions. Additionally PVRL1 overexpression in human melanoma cell lines increased cell aggregation in vitro. Finally, we found that nectin-1 expression was higher in primary melanomas compared to benign nevi, but lower in metastases compared to primary melanoma, in human tissue-microarrays. Collectively, these data suggest a model whereby PVRL1 expression controls a balance between growth and invasion. Cancer cells deficient for PVRL1 are less fit but more prone to detach from the tumor under serum stress conditions. These results establish PVRL1 as a new metastasis suppressor gene in melanoma and cast light on the role of cancer cell adhesion in tumor spreading.

#94

DDX31 cooperates with mutant p53 and EGFR to promote the multistep progression of invasive bladder cancer.

Toyomasa Katagiri,1 Kei Daizumoto,1 Tetsuro Yoshimaru,1 Yosuke Matsushita,1 Tomoya Fukawa,2 Masaya Ono,3 Hiro-omi Kanayama4. 1 _Tokushima Univ., Tokushima, Japan;_ 2 _Institute of Biomedical Sciences, Tokushima University Graduate School, Tokushima, Japan;_ 3 _National Cancer Center Research Institute, Tokushima, Japan;_ 4 _Tokushima University Graduate School, Tokushima, Japan_.

Bladder cancer is the most common malignancy of the urinary tract worldwide. Approximately 70% of cases are diagnosed as non-muscle-invasive bladder cancer (NMIBC), while the remaining 30% of cases are classified as muscle-invasive bladder cancer (MIBC). Evidence based on molecular biology has highlighted that the aggressiveness of MIBC advances through a multistep mechanism due to many genomic alterations. Notably, alterations of TP53 and EGFR pathways frequently occur in bladder cancers and are associated with poor prognosis, respectively. However, the connection between the overexpression of EGFR and the p53 mutation in multistep carcinogenesis and the progression of MIBC remains unknown. Here we report the distinct critical roles of DEAD box polypeptide 31 (DDX31) in the multistep progression of muscle invasive bladder cancer (MIBC) through its sequential interaction with mutant p53 (mutp53) and EGFR. In early MIBC cells, nuclear DDX31 acts as a transcriptional co-activator that binds to mutp53/SP1 and enhances mutp53 transcriptional activation, thereby upregulating the EPB41L4B gene, which plays a critical role in metastatic behavior and promotes the invasion and migration of MIBC. Notably, in advanced MIBC, cytoplasmic DDX31, which is transported from the nucleus, functions as an adaptor scaffold protein that forms a complex with EGFR via its interaction with phosho-nucleolin (NCL), leading to constitutive activation of EGFR-Akt signaling. Significantly, high expression of both cytoplasmic DDX31 and p53 proteins is correlated with a poor prognosis in MIBC patients. More importantly, blocking the DDX31-NCL interaction via a dominant-negative peptide led to downregulation of EGFR-Akt signaling, resulting in a significant anti-tumoral effect of bladder cancer in vivo. Our findings reveal that DDX31 cooperates with mutp53 and EGFR to promote the multistep progression of MIBC and that inhibition of DDX31-NCL formation may lead to potential treatment strategies for advanced MIBC.

#95

Heparan sulfate and glypican-1 mediate renal carcinoma metastasis.

Henry Qazi,1 Heriberto Moran,1 Limary M. Cancel,1 Mariya Mayer,1 Sylvie Roberge,2 Peigen Huang,2 Lance L. Munn,2 John M. Tarbell1. 1 _The City College of The City University of New York, New York, NY;_ 2 _Edwin L. Steele Laboratory for Tumor Biology, Charlestown, MA_.

The surface proteoglycan/glycoprotein layer (glycocalyx) on tumor cells has been associated with cellular functions that can potentially enable invasion and metastasis. In addition, aggressive renal carcinoma cells (SN12L1) with high metastatic potential have enhanced invasion rates compared to low metastatic (SN12C) cells in response to interstitial flow stimuli in vitro. Our previous studies suggest that heparan sulfate (HS) and hyaluronic acid (HA) in the glycocalyx play an important role in this flow mediated mechanotransduction and upregulation of invasive and metastatic potential. In our recent study, SN12L1 cells were genetically modified to suppress HS production by knocking down its synthetic enzyme NDST1. Using modified Boyden chambers with defined interstitial flow, we showed that flow-enhanced invasion is suppressed in HS deficient cells. We also examined two prominent HSPGs on renal carcinoma cells, glypican-1 and syndecan-1, and one prominent HA receptor, CD44. We observed higher glypican-1 levels in flow dependent SN12L1 cells when compared to SN12C cells. Caki-1 (highly metastatic) cells did not display flow-dependent invasion in vitro and did not display elevated glypican-1 compared to low metastatic Caki-2 cells. However, we did observe significantly increased HS, HA, syndecan-1 and CD44 in Caki-1 compared to Caki-2 cells suggesting an alternative mechanism for reported higher metastatic rates in these cells. All of our data are consistent with the hypothesis that glypican-1 is the core protein responsible for flow sensing in metastatic cancer cells. This is also consistent with observations in endothelial cells. To assess the ability of tumor cells to metastasize in vivo, parental or HS knockdown SN12L1 cells expressing fluorescent reporters were injected into kidney capsules in SCID mice. Histological analysis confirmed that there was a large reduction (95%) in metastasis to distant organs by tumors formed from knockdown cells compared to control cells with intact HS. The reduction was even greater (98%) in lungs where most of the metastases from the control cells were observed. The ability of these knockdown cells to invade surrounding tissue was also impaired. The substantial inhibition of metastasis and invasion upon reduction of HS suggests an active role for the tumor cell glycocalyx and glypican-1 in tumor progression.

#96

Novel inverse functioning molecules, GSN as metastatic promoter and OAS2 as metastatic repressor in colorectal adenocarcinoma.

Jin Cheon Kim,1 Je Jin Ha,1 Ka Hee Tak,1 Seon Ae Roh,1 Chan Wook Kim,1 Yong Sik Yoon,1 Jong Lyul Lee,1 Seon-Kyu Kim,2 Ee Hong Kwon,1 Dong-Hyung Cho,3 Seon-Young Kim,2 Yong Sung Kim2. 1 _University of Ulsan College of Medicine, Seoul, Republic of Korea;_ 2 _KRIBB, Daejeon, Republic of Korea;_ 3 _Kyunghee University, Yongin, Republic of Korea_.

Background LVI and PNI, recognized as major histological traces of metastatic CRC, have been recognized as a category I prognostic factor representing aggressive CRC. The primary aim of our study was to find out molecules associated with LVI and PNI and to examine their biological behavior with their prognostic significance.

Methods LVI- and PNI-associated molecules were identified and verified by utilizing (1) RNA-seq and selecting 117 genes by differential expression values in primary cancer samples of 130 patients with and without systemic recurrence (n = 72 and 58) (2) association analysis to extract six genes associated with LVI or PNI (3) assessment of biological property measuring proliferation, invasion/migration, epithelial-mesenchymal transition, and autophagy flux (4) verification of disease-free survival using public data sets of gene

Results GSN-overexpressing LoVo cells showed more than 2-fold greater invasion compared with control cells 5 (p < 0.001 - 0.005), whereas OAS2-overexpressing RKO cells showed a significantly reduced invasion compared with control cells (p < 0.001 - 0.005). GSN repressed expressions of E-cadherin, β-catenin, claudin-1, and snail, whereas it enhanced N-cadherin and ZEB1 expressions. Reverse findings were noticeably identified in OAS2-overexpression. Interestingly, expressions of multiple autophagy-related proteins including ATG5-12, ATG6/BECN1, ATG7, and ATG101 were decreased in GSN-overexpressing Lovo cells. In addition, accumulation of p62, a well-known substrate of autophagic degradation, suggested that an autophagic flux was also reduced in the GSN-overexpressing Lovo cells. In contrast to GSN overexpression, reverse findings of autophagy flux was identified in OAS2-overexpressing RKO cells. The 5-year RFS rates were significantly greater in the GSN underexpression group than in the overexpression group (73.6% and 64.7%, p = 0.038), whereas patients with OAS2 overexpression presented a greater RFS rate than those with underexpression (73.4% and 63.7%, p = 0.01).

Conclusion GSN and OAS2, as a recurrence promoter and suppressor, respectively, suggest their potential value as recurrence predictor or therapeutic targets in future clinical validation studies.

#97

UBE3A regulates SIRT6 levels to promote tumorigenesis in an ANXA2 dependent manner.

Saishruti Kohli, Sanjeev Das. _National Institute of Immunology, New Delhi, India_.

Background: UBE3A is the founding member of the HECT family of E3 ubiquitin ligases, implicated in cervical, prostate and breast cancer. A recent study has reported that UBE3A promotes proliferation and invasiveness of cancer cells by inducing Annexin A2 (ANXA2) levels. We have delineated a cascade in which UBE3A brings about the proteasomal degradation of SIRT6. Furthermore, we have also established that SIRT6 transcriptionally regulates ANXA2 expression. Here, we have confirmed that UBE3A-mediated SIRT6 downregulation and consequent derepression of ANXA2 promotes tumor metastasis.

Methods: HepG2, a Hepatocellular carcinoma cell line, was used to study the effects of the UBE3A-SIRT6-AXA2 axis. HepG2 UBE3A knockdown, SIRT6 knockdown, UBE3A/SIRT6 double knockdown and SIRT6/ANXA2 double knockdown cells were used to examine in-vitro cell proliferation, invasion, plasmin generation and MMP activation. These cell lines were also used for obtaining subcutaneous and orthotopic xenografts in immunodeficient mice and multiple parameters were analyzed to determine tumor growth and metastases.

Results: We observed that upon UBE3A abrogation, cells exhibited reduced tumor-forming potential and resulted in smaller tumors with low metastasis. On the other hand, increased tumorigenesis, and much larger tumors with increased metastasis were observed upon SIRT6 knockdown, which was significantly reversed upon simultaneous knockdown of UBE3A. Furthermore, smaller tumors were observed in case of SIRT6/ANXA2 double knockdown cells as compared to UBE3A/SIRT6 double knockdown cells. These results suggest that UBE3A-mediated ANXA2 upregulation, via SIRT6 degradation, is critical for its oncogenic functions

Conclusion: UBE3A mediated SIRT6 degradation leads to ANXA2 upregulation and hence enhances tumorigenesis. It would be beneficial to identify drugs targeting UBE3A as a therapeutic approach in hepatocellular carcinoma. Alternatively, mechanisms which inhibit ANXA2 activity must be deciphered to identify druggable targets.

#98

Cell membrane cholesterol modulates lung cancer cell adhesion and rolling on E-selectin.

Amina Mohammadalipour, Christian Showalter, Harrison T. Muturi, Vishva Sharma, Amir M. Farnoud, Vishwajeet Puri, Sonia M. Najjar, Monica M. Burdick. _Ohio University, Athens, OH_.

Despite numerous advances in cancer research, lung cancer has remained the major cause of cancer-related mortality worldwide, mainly because of metastasis and the lack of curative systemic therapy. It has been well established that lipid rafts, the membrane microdomains containing cholesterol and glycosphingolipids, play an important role in cell adhesion and spreading. Notably, there are elevated levels of cholesterol-rich lipid rafts observed in cancer cells relative to normal ones. Thus, alterations in lipid rafts, such as through cholesterol depletion, have been considered as a strategy for cancer metastasis prevention. In this study three different treatments - methyl-beta-cyclodextrin (mβCD), sphingomyelinase (smase), and simvastatin (simva) - were used to investigate the effects of cholesterol depletion on the mechanics of adhesion and rolling velocities of multiple non-small cell lung cancer (NSCLC) cell lines (H1299, H23, H460, and A569) and a small cell lung cancer (SCLC) cell line (SHP-77) on E-selectin, a vascular endothelial molecule hypothesized to initiate recruitment of circulating tumor cells at metastatic sites. Under physiological conditions in a parallel plate chamber, it was observed that the number of adherent NSCLC cells significantly decreased through mβCD and simva treatments, while smase treatment did not show significant effect on those cells. In addition, only for NSCLC cells, significant increases in rolling velocities were detected after treatments with mβCD and simva, but not via smase. In contrast, cholesterol depletion did not affect the number of attached cells and rolling velocity of the SCLC cells. Our results show that cholesterol might regulate E-selectin-mediated adhesion of NSCLC cells to endothelium, most likely by disturbing functional E-selectin ligands expressed on the surface of the cancer cells. We have previously reported that CD44 expressed on colon cancer and breast cancer cells are functional E-selectin ligands that mediate cell adhesion and rolling on endothelium. Protein structure analysis by flow cytometry and western blot showed that all NSCLC cell lines strongly expressed CD44. After the cholesterol depletion through the various treatments, the molecular surface expression of CD44 was down regulated for all these cells. In contrast, the SCLC line did not show any expression of CD44. Although further studies must be performed to verify CD44 function as an E-selectin ligand, the data in total imply protein loss or absence due to the defection of the cell membrane integrity could impact cell adhesion and rolling, and consequently metastasis. Therefore, cholesterol depletion could have the potential to help prevent cancer metastasis through down regulation of relevant adhesion molecules.

#99

An oncogenic role of Deleted in Liver Cancer 1 (DLC1) in metastatic melanoma.

Xintao Yang,1 May Cheung,1 Hong-Lok Lung,2 Martin Cheung1. 1 _The University of Hong Kong, Hong Kong, Hong Kong;_ 2 _Hong Kong Baptist University, Hong Kong, Hong Kong_.

Melanoma is a type of skin malignancy originated from neural crest-derived melanocytes. Although the mortality rate has been significantly reduced due to early cancer diagnosis and treatment, distant metastasis still exhibits much inferior survival rates compared to the early stage of this disorder. Therefore, developing new target for therapy is of particular significance in combating metastatic melanoma. DLC1 is well known for its tumor suppressor role through activating small Rho-GTPases' (RHOA and CDC42) intrinsic capability to convert an active GTP to an inactive GDP-bound state, leading to inhibition of tumor growth and metastasis. So far, downregulation of DLC1 expression has been widely reported to associate with increased risk of various malignancies whereas elevation of its expression prevents tumor progression. In contrast to these studies, our pilot findings reveal that multiple melanoma cell lines exhibit higher levels of DLC1 than benign melanocytes and other cancer types, suggesting its unique expression profile in melanoma. Furthermore, in vitro functional assays demonstrate that silenced DLC1 inhibits proliferative, clonogenic, migratory and invasive capabilities of melanoma cells, whereas restoration of DLC1 rescue the oncogenic properties. More importantly, in lung metastasis model using nude mice, DLC1 knockdown suppresses melanoma cells colonizing to the lungs whereas their pulmonary metastases can be recovered with the reconstitution of DLC1. In addition, the manipulation of DLC1 expression does not alter the RHO-ROCK-MRLC signalling, indicating that its oncogenic effect is mediated through a RHO-independent manner. Thus, our results unravel an unprecedented paradigm-shift of DLC1 functioning as an oncogene instead of a tumor suppressor in metastatic melanoma, which may shed new light on disease pathogenesis and uncover novel therapeutic targets to treat this deadly cancer.

#100

Interleukin 1 receptor type II upregulates MMP1 expression and increases motility of keratinocytes cells.

Hsiu-Hua Wang, Te-Chang Lee. _Academia Sinica - Inst. of Biomedical Sci., Taipei, Taiwan_.

We have shown that long-term exposure to low doses of inorganic arsenite (iAs) induced malignant transformation of non-tumorigenic human keratinocytes, HaCaT cells. We further demonstrated that enhanced expression of a decoy type II receptor of interleukin-1 (IL1R2) in transformed keratinocytes mediated through IL-6 and VEGFR to increase the migration and invasion activities, indicating the oncogenic potential of IL1R2. In this present study, we further explored how IL1R2 enhances the cell motility in human keratinocytes. As described earlier, long-term and low dose iAs (0, 0.5 and 1 μM) exposed HaCaT cells were designated as HaCaT A0, A1 and A2 cells, respectively. The tumorigenic cell line, T4R2, was derived from the tumor in mice implanted with HaCaT A2 cells. By growing HaCaT A0, A1, A2 and T4R2 cells on an organotypic culture system using basal medium, we found that T4R2 cells formed multilayered morphology. By aid of siRNA and ectopic expression techniques, we further demonstrated that increased expression of IL1R2 in T4R2 cells was associated with increased matrix metalloproteinase-1 (MMP1) and enhanced cell motility. The roles of MMP1 on supporting the organotypic growth of T4R2 cells were confirmed by using the stably MMP1-silenced T4R2 cells (T4R2-shMMP1). The cell motility of T4R2-shMMP1 cells was also significantly reduced. Furthermore, IL1R2-induced MMP1 upregulation was remarkably inhibited by inhibitors of MAPK/ERK signaling. Our present results revealed that MMP1 was one of downstream targets of IL1R2 which plays a crucial role on the progression of malignancy. Therefore, IL1R2 may be a target for cancer intervention.

#101

Human bone marrow-derived endothelial progenitor cells promote the transformation of periostin-positive hepatocellular carcinoma cells into CD36-positive metastasis-initialing cells in pre-metastatic niche.

Xiaozai Xie,1 Pengyi Guo,1 Haitao Yu,1 Yi Wang,2 Gang Chen1. 1 _The First Affiliated Hospital of Wenzhou Medical Univ., Wenzhou, China;_ 2 _Department of Preventive Medicine, Wenzhou Medical University, Wenzhou, China_.

BACKGROUND: Recent studies have shown that metastasis-initiating cells (MICs) in the pre-metastatic niche play a key role in triggering tumor metastasis. In addition, bone marrow-derived endothelial progenitor cells (BM-EPCs), which are key components of the pre-metastatic microenvironment, can serve as "metastasis-initiators". Our previous studies have revealed that the periostin (POSTN) expression in HCC is positively correlated with the amount of EPCs and POSTNhigh hepatocarcinoma cells possess cancer stem cell-like properties. Meanwhile, CD36, a biomarker of MICs, is highly expressed in recurrent HCC. This study aims to clarify whether BM-EPCs in the pre-metastatic niche promote the generation of CD36-positive MICs and stimulate tumor metastasis through interacting with POSTNhigh hepatocarcinoma cells in HCC.

AIMS and METHODS: Cell co-culture experiments were conducted to investigate the effect of EPCs on malignant biological behaviors of POSTNhigh hepatocarcinoma cells. In addition, the change in the expression of CD36 antibody in co-cultured HCC cells was evaluated to investigate the generation of CD36-positive MICs. Furthermore, NF-E2-related factor 2 (Nrf2) was identified as a transcription factor that increases the expression of CD36 antigen and promotes stem cell-like properties of HCC cells. Finally, the changes in HCC invasion and metastasis by manipulating the POSTN/Nrf2/CD36 signal axis were evaluated.

RESULTS: POSTNhigh HCC cells co-cultured with EPCs showed enhanced proliferation, invasion and spheroid formation. Flow cytometry demonstrated that the expression level of CD36 on cell surface was significantly up-regulated in these co-cultured cells. ELISA tests showed that CCL2 secreted by EPCs was the key factor promoting the transformation of POSTNhigh HCC cells to a CD36-positive phenotype. Further experiments revealed that, after binding to the CCR2 receptor on HCC cell membrane, EPC-secreted CCL2 promoted the direct binding of Nrf2 to the promoter region of CD36 gene and upregulated CD36 expression when the αvβ3 pathway was activated, which eventually induced the transformation of POSTNHigh HCC cells into CD36-expressing MICs. The CCL2 monoclonal antibody and αvβ3 antagonist Cilengitide effectively blocked the POSTN/Nrf2 /CD36 axis, inhibiting the formation of CD36-positive MICs of HCC and suppressing tumor invasion and metastasis.

CONCLUSIONS: CCL2 protein secreted by BM-EPCs can promote the transformation of POSTNhigh HCC cells into CD36-positive MICs, thereby promoting formation of metastases.

#102

RUNX3 inhibits invasiveness and stemness through downregulation of Gli-1 in colon cancer cells.

Bo Ram Kim, Yoo Jin Na, Jung Lim Kim, Soyeon Jeong, Seong Hae Park, Min Jee Jo, YoonA Jeong, Dae-Hee Lee, Sang Cheul Oh. _Korea University, Seoul, Republic of Korea_.

Disabled tumor suppressor genes and hyperactive oncogenes contribute considerably to cell fates during cancer development via genetic alterations such as somatic mutations. To date, little is known about how tumor suppressor genes react to diverse oncogenes during tumor progress. Our previous report showed that RUNX3 inhibits metastasis by preventing vascular endothelial growth factor (VEGF) secretion and suppressed endothelial cell growth and tube formation in colorectal cancer. Hedgehog signaling is crucial for the physiological maintenance and self-renewal of stem cells, and its deregulation is responsible for their tumor development. The mechanisms that inhibit this pathway during the proliferation are poorly understood. Here, we show that the tumor suppressor RUNX3 modulates tumorigenesis in response to cancer cells induced by inhibition by oncogene Gli-1 ubiquitination. Moreover, we demonstrated that RUNX3 and Gli-1 expression had inverse correlation in colorectal cancer cells and tissues. We observed a direct interaction between RUNX3 and Gli-1, promoting ubiquitination of Gli-1 at the intracellular level. The increased ubiquitination of Gli-1 was induced through the E3 ligase β-TrCP. This novel RUNX3-dependent regulatory loop may limit the extent and duration of Hedgehog signaling during extension of tumor initiation capacity. Base on this research, a search for agents that can induce RUNX3 might result in new and effective therapies for colorectal cancer.

#103

Expression and regulation of MMP9 and RECK in human oral squamous cell carcinoma progression and invasion.

Kamdeo Kumar Pramanik,1 Basabi Rana,2 Ajay Rana,2 Rajakishore Mishra1. 1 _Central University of Jharkhand, Ranchi, India;_ 2 _The University of Illinois at Chicago, Chicago, IL_.

Purpose: Matrix metalloproteinase-9 (MMP-9), a Zn-dependent endopeptidase is known to play a central role in oral cancer pathogenesis. Recently we observed differential function of MMP-9 and reversion-inducing cysteine-rich protein with Kazal motifs (RECK) respectively in OSCC invasion. We hypothesize that the downregulation of MMP-9 via RECK promotes human oral cancer progression and invasion.

Experimental procedure: The mRNA expression, protein expression and activity of MMP-9/2, expression of transcription factors like Snail, c-Myc, β-catenin and protein-protein interaction of TIMP1/RECK with MMP9 and promoter methylation of RECK gene were assessed by using RT-PCR, immunohistochemistry (IHC), Western blotting, co-immunoprecipitation, gelatin zymography, and bisulphate modification-PCR analyses. Wnt5a and LPA mediated MMP-9 regulation as well as the MMP-9 regulation by the exogenously expression of RECK, GSK3β (WT) or non phosphoryable active GSK3β (S9A) was also performed in oral tongue SCC derived SCC-9 cells.

Summary: We observed up-regulation/activation of MMP-9 was associated with decreased expression of RECK and progression and invasion of OSCC. MMP-9 expression/ activity was positively correlated with inactivation of GSK3β signaling. Conversely, a link between inactivation of GSK3 signaling and the progressive silencing of RECK gene through promoter hypermethylation was observed. Finally, Wnt5a and LPA mediated increased MMP-9 and decreased RECK expressions were observed.

Conclusion: Taken together, these results demonstrated the influence of a novel signaling axis GSK3β-RECK-MMP-9 to drive OSCC progression and invasion. Thus, targeting this pathway may be exploited for treating OSCC.

#104

Identification of the molecular components required for the protumorigenic effect of MSLN in pancreatic cancer.

Leela Rani Avula,1 Michael Rudloff,1 Danielle Arons,1 Salma El-Behaedi,1 Xianyu Zhang,1 Theresa Guerin,2 Serguei Kozlov,2 Christine Alewine1. 1 _National Cancer Institute, National Institutes of Health, Bethesda, MD;_ 2 _Frederick National Lab for Cancer Research/Leidos Biomedical Research, Inc, Frederick, MD_.

Mesothelin (MSLN) is a GPI-linked protein expressed in >90% of pancreatic adenocarcinomas (PDAC) but not in healthy pancreas nor in the parenchyma of other vital organs. Due to this differential expression, multiple antibody-based therapies are targeting MSLN. The physiologic role of MSLN is yet unknown. The MSLN gene encodes for a precursor protein which is cleaved to produce mature MSLN protein and soluble Megakaryocyte Potentiating Factor (MPF), both of which can be secreted or shed. Previous reports suggested that overexpression of MSLN precursor protein increases tumorigenicity and metastatic potential of PDAC. Here, we have used CRISPR-Cas9 gene editing to delete MSLN from the KLM-1 pancreatic cancer cell line (ΔMSLN) and have examined the growth of these cells compared to mock deleted cells (Mock). The ΔMSLN and Mock cells grew at the same rate both in cell culture and as subcutaneous tumors in nude mice, but ΔMSLN had significantly impaired growth when inoculated into the mouse intraperitoneal (IP) cavity as well as when injected orthotopically into the pancreas. Histological analysis (H&E, Ki67 and Trichrome) showed no significant gross differences between ΔMSLN and Mock IP tumors. RNA deep sequencing analysis comparing ΔMSLN and Mock cell lines showed strong upregulation of MUC-4 in KO cells. Restoration of WT MSLN expression in ΔMSLN cells restored IP growth, however, transduction of a Y318A point mutant could not. Point mutation of Y318 diminished ability of shed MSLN to interact with KLM-1 cells, presumably by impairing association with MUC-16, the only known binding partner of MSLN. To determine the role of mature MSLN and MPF in tumorigenicity, we expressed these proteins separately in ΔMSLN cells and found that neither could restore IP growth, although mature MSLN was appropriately expressed on the cell surface and MPF secreted. This suggests either that both proteins are required for tumorigenicity or that when synthesized individually they are incorrectly processed by the cell. To examine the role of MSLN in tumorigenesis, MSLN KO mouse were bred into the KPC genetically engineered mouse model of PDAC, which spontaneously develops autochthonous tumors. Unlike PDAC patients, KPC typically die from complications of their primary tumors. Loss of MSLN had no effect on the survival of these mice. Effect of MSLN loss on metastasis in this model is currently being evaluated. In summary, we have demonstrated that MSLN enhances the IP growth of PDAC and defined key regions of MSLN important for this activity.

#105

CADM1 is a TWIST1-regulated suppressor of melanoma invasion and survival.

Edward J. Hartsough, Michele B. Weiss, Shea A. Heilman, Timothy J. Purwin, Curtis H. Kugel, Sheera R. Rosenbaum, Dan A. Erkes, Manoela Tiago, Inna Chervoneva, Andrew E. Aplin. _Thomas Jefferson University, Philadelphia, PA_.

Metastatic melanoma is the deadliest form of skin cancer; however, patients diagnosed and treated prior to metastatic dissemination have a good prognosis. The transcription factor, TWIST1 has been implicated in enhancing the migration and invasion of melanoma cells but the range of TWIST1-regulated targets is poorly described. In this study, we performed expression profiling to identify the TWIST1-regulated transcriptome. Gene ontology pathway analysis revealed that TWIST1 and epithelial to mesenchymal transition (EMT) were inversely correlated with levels of cell adhesion molecule 1 (CADM1). Chromatin immunoprecipitation (ChIP) studies and promoter assays demonstrated that TWIST1 physically interacts with the CADM1 promoter, suggesting TWIST1 directly represses CADM1 levels. Modulation of CADM1 resulted in significant effects on the migration and invasion of melanoma cells. In addition, elevated CADM1 elicited cell death in non-adherent conditions, an effect that could not be rescued with a pan-caspase inhibitor. Analyses suggest that CADM1 directed non-adherent cell death is associated with loss of mitochondrial membrane potential and subsequent failure of oxidative phosphorylation pathways. Furthermore, clinical data from TCGA indicates that CADM1 expression is correlated with better overall survival in patients. Together, these data suggest that CADM1 exerts tumor suppressive functions in melanoma by reducing invasive potential and may be a biomarker for improved survival of melanoma patients.

#106

Shock and ROR! Targeting ROR1 and ROR2 in a preclinical patient-derived model of ovarian cancer.

Claire E. Henry,1 Neville F. Hacker,2 Caroline E. Ford1. 1 _Lowy Cancer Research Centre, University of New South Wales, UNSW, Australia;_ 2 _Royal Hospital for Women, Randwick, Australia_.

BACKGROUND: New targets for ovarian cancer treatment are critically needed. The Wnt receptors ROR1 and ROR2 are overexpressed in all histotypes of ovarian cancer and appear to play a role in both the tumour and the surrounding microenvironment. We have previously conducted an extensive suite of in vitro experiments, exploring the functional role of ROR1 and ROR2 in ovarian cancer. Silencing either receptor using siRNA inhibited ovarian cancer cell migration and invasion, and silencing both receptors had an even stronger inhibitory effect on the metastatic potential of ovarian cancer cellS. We have also shown that silencing ROR1 and ROR2 sensitises chemoresistant ovarian cancer cells to chemotherapy. Combined, these studies have confirmed the importance of RORs in ovarian cancer, and provided a strong argument for these receptors potential as clinical targets.

However, these previous studies have utilised simple 2D in vitro models to investigate cancer cell growth and migration, which does not allow investigation of stromal involvement in ROR driven metastasis.

AIM: To investigate targeting ROR1 and ROR2 in a 3D primary co-culture model of epithelial ovarian cancer dissemination to the omentum.

METHODS: Primary fibroblasts (NOF) and mesothelial (HPMC) cells were isolated from fresh samples of omentum collected from women with benign or non-metastatic conditions and cultured with collagen to produce a organotypic 3D model. Stable shRNA knockdown of ROR1, ROR2 and double ROR1/ROR2 in OVCAR4 ovarian cancer cells were incorporated into the 3D model to measure adhesion, or using a transwell to measure invasion. Gene expression changes in primary cells upon OVCAR4 interaction was evaluated using indirect transwell co-culture.

RESULTS: Double knockdown of ROR1 and ROR2 strongly inhibited cell adhesion (p<0.05) and invasion (P<0.05) to the omentum model. ROR2, but not ROR1, was up- regulated in primary fibroblasts when cultured with OVCAR4 (P=0.05) and ectopic overexpression of ROR2 in NOFs increased cell migration.

CONCLUSION: The combination of ROR1 and ROR2 signalling influences ovarian cancer dissemination to the omentum, however ROR2 may also play a specific role in stromal activation during metastasis. Therefore, targeting both ROR1 and ROR2 may be a powerful approach to treating ovarian cancer. The development of a number of monoclonal antibodies targeting ROR1 currently in phase 1 trials for other tumour types makes this clinically feasible in the near future.

#107

Interaction of BIR domain of XIAP with Sp1/E2F1 activates MMP2 and bladder cancer cell invasion by inhibiting src protein translation.

Xiaohui Hua,1 Jiheng Xu,1 Honglei Jin,2 Chuanshu Huang1. 1 _New York Univ., Tuxedo Park, NY;_ 2 _School of Laboratory Medicine and Life Science, Wenzhou Medical University, Wenzhou, China_.

X-linked inhibitor of apoptosis protein (XIAP) is important in the regulation of cancer cell biological behaviors. However, little is known about the molecular mechanism by which the XIAP domains promote aggressive tumor behavior in human bladder cancer (BC). Herein, we discovered that ectopic expression of the BIR domains of XIAP rescued the MMP2 activation and human BC invasion in human BC cells with XIAP deletion. Interestingly, Src expression was upregulated in XIAP/BIR domain-deficient cells, whereas restoration of BIR domain in XIAP-deletion cells recused Src expression. Moreover, knockdown of Src reversed the defects of MMP2 activation and BC invasion in XIAP-deficient BC cells, revealing that Src acts as a XIAP downstream negative regulator for MMP2 activation and BC invasion. Mechanistic studies revealed that the regulatory effect of the BIR domains of XIAP on Src expression was due to directly inhibition of its protein translation by upregulation of miR-203 resulting from activation of the transcription factors Sp1 and E2F1, while the BIR domains of XIAP was found to interact with both Sp1 and E2F1 to activate these transcription factors. Overall, our studies demonstrate that BIR domain of XIAP is able to bind to Sp1 and E2F1 leading to their transcriptional activation, resulting their targeted miR-203 transcription, consequently directly targets the 3'-UTR of src mRNA and therefore attenuating SRC protein translation, and activating MMP2 and BC invasion.

#108

Abi1 positively regulates lung metastasis of aggressive breast cancer in PyMT mouse model.

Angelina Regua,1 Isabelle Bichindaritz,2 Tiffany Caza,1 Julie R. White,3 Alexander Nappi,1 Claudia Mondragon,1 Mira Krendel,1 Gennady Bratslavsky,1 Abirami Sivapiragasam,1 Leszek Kotula1. 1 _SUNY Upstate Medical Univ., Syracuse, NY;_ 2 _SUNY Oswego, Oswego, NY;_ 3 _MSKCC, New York, NY_.

Breast cancer is the most commonly diagnosed non-cutaneous cancer in American women and is estimated to cause 40,000 deaths this year. Despite standard of care, breast cancer patients often relapse after a few years of treatment thus highlighting the need for new molecular targets for improved management of metastatic disease. Abelson interactor 1 (Abi1) is an adaptor protein associated with the WAVE (Wiscott-Aldrich syndrome protein family verprolin homologous) regulatory complex and Arp2/3 (Actin-related proteins 2 and 3)-mediated actin cytoskeleton remodeling. Our analysis of human tumor data indicates that Abi1 is frequently upregulated in invasive breast cancers, is associated with poor survival, and may promote an aggressive breast tumor phenotype. Abi1 has been shown to positively regulate breast cancer cell proliferation, motility, division, and invasion in vitro however its exact role in vivo remains unknown. We therefore hypothesize that Abi1 promotes breast tumor progression and metastasis in a mouse breast cancer model. Using the well-characterized Polyoma Middle T (PyMT) breast cancer model, we conditionally deleted Abi1 from the mammary epithelium and determined the effects on tumor growth kinetics and WAVE complex protein levels. Interestingly, our studies show delayed tumor growth only in mice with heterozygous deletion of Abi1 while homozygote KO mice show relatively unchanged tumor growth. Western blot analyses of Abi1 KO breast tumors show concomitant loss of WAVE complex proteins supporting our previous findings that WAVE complex integrity is dependent on Abi1. Our data also show significant upregulation of Abi2 only in homozygous Abi1 KO mice, suggesting a potential compensatory role for Abi2 that may support primary tumor growth in absence of Abi1. Most interestingly, Abi1 null PyMT mice show significantly reduced incidence of lung metastasis, supporting our hypothesis that Abi1 promotes metastasis of breast cancer cells. In summary, Abi1 loss abrogates lung metastasis in PyMT mice however primary tumor growth remains largely unaffected, possibly due to a compensatory mechanism by a related protein, Abi2. This work will establish the biochemical dynamics between members of the Abi protein family and WAVE complex to improve targeted treatments in aggressive human breast cancer. [Supported by NCI grant R01-CA161018 and Carol M. Baldwin Breast Cancer Research Fund of CNY]

#110

PPARγ loss increases the metastatic potential of HER2+ breast cancer.

Elizabeth D. Lightbody, Kathleen MJ O'Connell, Hailey T. Newton, Rachel R. Rubino, Anthony J. Apostoli, Kevin Ren, Sandip K. SenGupta, Christopher J. Nicol. _Queens University, Kingston, Ontario, Canada_.

Breast tumors that overexpress human epidermal growth factor receptor 2 (HER2+) grow and spread faster than HER2-negative tumors, resulting in poor patient prognosis. Peroxisome proliferator-activated receptor (PPAR)γ is a nuclear transcription factor that controls the expression of genes essential for normal metabolism of fats and sugars in the body. Our laboratory previously showed that PPARγ expression suppresses environmental carcinogen DMBA-mediated breast tumor progression in vivo, and PPARγ-activating drugs further enhance this effect. However, the role of PPARγ and PPARγ agonists on HER2+ breast tumorigenesis and patient survival is unclear. We hypothesized that PPARγ loss enhances HER2+ breast tumor progression. To test this, this study generated a novel mouse model referred to as NIC:PPARγ-KO, which has targeted PPARγ deletion in the same HER2+ transformed mammary epithelial cells that drive breast tumorigenesis. Compared to NIC:PPARγ-WT mice, NIC:PPARγ-KO mice have increased mammary tumor incidences and lung metastases. Protein analysis of NIC:PPARγ-KO tumors shows PPARγ loss is inversely correlated with increased HER2 phosphorylation at tyrosine 877 (pY877HER2) in primary and metastatic tumorigenic tissue. Immunofluorescence also showed HER2 H-scores were significantly highest among tumors from NIC:PPARγ-KO mice, but also correlated with targeted PPARγ loss in DMBA-induced primary and metastatic mammary tumors among PPARγ-WT and PPARγ-KO mice (p<0.05). To further investigate the role of PPARγ in the metastatic process, we established cell lines from the freshly isolated lung metastatic tumors harvested from the NIC:PPARγ-KO model (NIC:PPARγ-KO-lmet). In vitro analysis of several human HER2+ breast cancer cells lines and our NIC:PPARγ-KO-lmet cells shows migration, invasion and tumorsphere-forming potential were significantly increased after epidermal growth factor (EGF, 20ng/mL) treatment and, more interestingly, that co-treatment with a PPARγ-activating drug (rosiglitazone, 10μM) significantly abrogated these effects (p<0.05). Together, these data provide the first evidence that PPARγ may be a useful prognostic/predictive biomarker for HER2+ breast tumors, and suggest that the novel inclusion of PPARγ-activating drugs may benefit a subset of HER2+ breast cancer patients.

### Role of the Innate Immune System in Tumorigenesis

#111

The CX3CR1-Fractalkine axis drives both circulating prostate cancer cells and macrophages to the bone metastatic microenvironment.

Ramanpreet Kaur,1 Chen Qian,1 Joseph Salvino,2 Olimpia Meucci,1 Alessandro Fatatis1. 1 _Drexel University, Philadelphia, PA;_ 2 _The Wistar Institute, Philadelphia, PA_.

Prostate cancer (PCa) is the second most frequently diagnosed cancer and its progression into metastatic disease reduces the 5-year survival rate to 29% in diagnosed men. A better understanding of the mechanisms underpinning metastatic dissemination and the role played by the tumor microenvironment at distant sites is essential. Our lab has shown that PCa cells express the chemokine receptor CX3CR1 and seed the skeleton when attracted by bone-derived Fractalkine (FKN) (a.k.a. CX3CL1). A crucial component of the bone microenvironment consists of heterogeneous populations of tumor-associated macrophages (TAMs), which can polarize into M1 or M2 phenotypes and are likely implicated in metastatic progression. TAMs migrate in close proximity of cancer cells and release growth factors, chemokines, and other inflammatory mediators that influence tumor growth. Using cell biology tools, we show that both PCa cells and macrophages express the CX3CR1 receptor, which signal to downstream targets when engaged by FKN and is inhibited by a novel small-molecule inhibitor. We also employed a mouse model of cancer cell dissemination and metastasis to show that our novel CX3CR1 antagonist impairs tumor seeding to the skeleton. Furthermore, as macrophages also express CX3CR1, targeting this receptor may potentially hinder TAMs' accumulation into the metastatic microenvironment, depriving it of crucial pro-metastatic conditioning. In conclusion, we propose that the CX3CR1-FKN axis drives both cancer cells and TAMs to metastatic sites and that the mitigation of the functional interactions between this receptor and its unique chemokine ligand by antagonists of CX3CR1 will impair the clinical progression of PCa metastasis.

#112

Tumor-associated macrophages promote enzalutamide resistance in microscale castrate-resistant prostate cancer models.

David Kosoff, Jiaquan Yu, Vikram Suresh, David J. Beebe, Joshua M. Lang. _University of Wisconsin, Madison, WI_.

Background: Despite recent advances in hormonal therapy for men with advanced prostate cancer, the development of therapeutic resistance continues to limit the efficacy of hormone-directed agents. This includes resistance to second-generation anti-androgens, such as enzalutamide, that can improve both morbidity and overall survival in men with castrate-resistant prostate cancer (CRPC). Tumor-associated macrophages (TAMs) are myeloid immune cells within the tumor microenvironment (TME), which have been shown to promote resistance to hormone therapies in prostate cancer. While they are known to have a role in the development of castrate resistance, their role in enzalutamide resistance is not yet understood.

Methods: We have developed a novel microscale cell culture platform that enables efficient multi-culture of >5 primary cell populations along with multiplexed analysis of cell viability, migration, mRNA and protein expression, and secretion of secretory factors. Utilizing this platform, we cultured androgen-sensitive prostate cancer cells (LNCaPs) with primary monocyte-derived macrophages (MDMs) as well as additional primary stromal and immune cell populations (T cells, fibroblasts). Cultures were treated with enzalutamide therapy and analyzed for cell viability, mRNA and protein expression, and cytokine secretion.

Results: After enzalutamide treatment, LNCaPs in co-culture with primary MDMs had an increase in viable cell number compared to LNCaPs in monoculture. CSF-1 expression was elevated in LNCaPs cultured with MDMs, and this expression was increased even further with enzalutamide treatment. In MDMs cultured with LNCaPs, there was a significant increase in expression of the CSF-1-responsive genes IL-10 and CCL18. This effect was observed consistently in MDMs from multiple patients.

Conclusions: Within microscale tumor models, we have demonstrated that primary, patient-derived MDMs promote resistance to enzalutamide therapy in androgen-sensitive prostate cancer cells. An increase in CSF-1 expression by LNCaPs in our tumor models was associated with an increase in CSF-1 responsive genes within the MDMs, including IL-10 and CCL18. Elevated expression of both of these cytokines has been associated with more aggressive prostate cancers in patients. IL-10 and CCL18 have also been shown to protect against apoptosis in vitro as well as promote immune suppression in vivo. Our findings suggest that CSF-1 secretion by tumor cells may promote hormone resistance in prostate cancer through TAM-mediated secretion of IL-10 and CCL18. These pathways represent potentially high-value targets to limit or prevent the development of castrate resistance in men with advanced prostate cancer. Further studies targeting the secretion of CSF-1, IL-10, and CCL18 in combination with enzalutamide treatment are ongoing.

#113

Gemcitabine treatment induces immunosuppressive microenvironment in pancreatic cancer by promoting the infiltration, growth, and polarization of macrophages.

Sachin Kumar Deshmukh,1 Arun Bhardwaj,2 Nikhil Tyagi,2 Mohammad Aslam Khan,2 Sanjeev K. Srivastava,2 Ahmed AL-Ghadhban,2 Kari Dugger,3 James E. Carter,4 Ajay P. Singh,2 Seema Singh2. 1 _Univ. of South Alabama Mitchell Cancer Institute, Mobile, AL;_ 2 _Univ. of South Alabama Mitchell Cancer Inst., Mobile, AL;_ 3 _University of Alabama at Birmingham, Birmingham, AL;_ 4 _College of Medicine, University of South Alabama, Mobile, AL_.

Resistance to chemotherapeutic drugs remains a major cause of therapeutic failure in pancreatic cancer (PC) patients. Efficacy of chemotherapy is further limited by its debilitating effects on immune system. Apart from genetic abnormalities in tumor cells, the tumor immune- microenvironment plays a crucial role in PC chemoresistance. However, it is unclear how chemotherapy affects the cancer immunity or how it affects the tumor microenvironment (TME). In the present study, we demonstrate gemcitabine induces an increased infiltration of CD68\+ M2 macrophages in pancreatic tumors isolated from an orthotopic xenograft murine model. Moreover, the conditioned media from PC cell lines cultured with gemcitabine (Gem-CM) promotes migration, invasion, and growth of RAW264.7 macrophage. Interestingly, Gem-CM induces an upregulation of tumor-associated or M2-polarized macrophage markers, arginase-1 and TGF-β1. Additionally, gemcitabine treatment of PC cell lines induces the expression of M2-polarization associated cytokines, growth factors and chemokines, including IL-8. IL-8 exhibits the greatest upregulation after culture with Gemcitabine. Further, IL-8 neutralization in Gem-CM diminished its ability to induce growth, migration, and invasion of RAW264.7 macrophages. Together, these findings suggest an indirect effect of gemcitabine on increasing macrophage trafficking and M2-polarization that potentially supports a pro-tumor microenvironment. These actions may contribute to the chemoresistance of PC. Prevention of gemcitabine-induced macrophage infiltration, growth and M2-polarization will offer a better strategy to counter chemotherapy-induced immunosuppression in PC.

#114

Basal-like breast cancer engages tumor-supportive macrophages via cytokines triggered by extracellular S100A4.

Lina Prasmickaite,1 Ellen M. Tenstad,1 Eivind Valen Egeland,1 Solveig Pettersen,1 Shakila Jabeen,2 Silje Nord,1 Mads Haugland Haugen,1 Siri Juell,1 Tove Øyjord,1 Olav Engebråten,1 Gunhild Mari Mælandsmo1. 1 _Oslo University Hospital, Oslo, Norway;_ 2 _Akershus Universitetssykehus, Oslo, Norway_.

Recently the biological and therapeutic perspective on cancer has evolved from focusing on tumor cells only, to include the complex impact of the tumor microenvironment (TME). The TME appears to have a strong influence on both tumor progression and response to treatment. In breast cancer, and in particular the triple-negative subgroup, patient outcome together with the therapeutic response are strongly linked to the tumor's inflammatory profile: presence of tumor-infiltrating leukocytes and their regulatory cytokine. Tumor-associated macrophages (TAMs) are among the most abundant immune cells in the TME, and expression of such cells has been strongly correlated with both poor outcome and development of resistance.

In this study we explored the effect of the pro-metastatic, inflammation-associated, TME factor S100A4 on breast cancer cells (BCCs) of different subtypes, and their further interactions with inflammatory cells. We show that S100A4 activates BCCs, stimulating secretion of pro-inflammatory cytokines, which, in turn, promotes monocyte conversion into TAM-like cells. This was in particular prominent for cytokines secreted from basal-like BCCs. The TAM-like cells possess pro-tumorigenic activities, including increased resistance and ability to migrate. In conclusion, we demonstrate that S100A4 instigate an inflammatory microenvironment, involving a network of cytokines and TAMs, which is particularly pronounced in basal-like BC and could facilitate aggressive phenotypes of this subtype.

#115

Crosstalk between breast cancer cells and macrophages in tumor microenvironment induces inflammasome activation.

Jeong-Hoon Jang, Do-Hee Kim, Sin-Aye Park, Su-Jung Kim, Young-Joon Surh. _Seoul National Univerisity, Seoul, Republic of Korea_.

The inflammasome is a multiprotein complex that is composed of NOD-like receptor, ASC and caspase-1. During the activation of the inflammasome, pro-interleukin (IL)-1β is cleaved by active caspase-1 to produce mature IL-1β. Although the inflammasome plays a controversial role in many types of tumor, emerging evidence indicates that the inflammasome functions as a negative regulator of breast cancer progression. Notably, high levels of IL-1β in breast cancer tissues are associated with a more virulent tumor phenotype and poor prognosis. However, it remains unresolved how IL-1β is accumulated in breast tumor microenvironment. In the present study, we have found that various human breast cancer cells (MDA-MB-231, MDA-MB-468, SKBr3, MCF-7 and T47D) retain cleaved-IL-1β whereas secretion of IL-1β into the culture medium was not observed. However, IL-1β was detected in the medium when human breast cancer cells were co-cultured with human monocytic (THP-1) cells. Among the various human breast cancer cells, triple-negative breast cancer cells (MDA-MB-231 and MDA-MB-468) effectively stimulated secretion of IL-1β. When co-cultured with MDA-MB-231 cells, THP-1 cells exhibited the elevated levels of pro- and cleaved-IL-1β. Consistent with this result, there was a marked increase in the secretion of IL-1β from THP-1 cells treated with the conditioned media of triple-negative breast cancer cells. Both triple-negative MDA-MB-231 and MDA-MB-468 breast cancer cells induced ASC oligomerization, which is a hallmark of inflammasome activation. Thus, it is likely that soluble factors from breast cancer cells promote IL-1β secretion from macrophages through activation of inflammasomes. In conclusion, breast cancer cells drive inflammasome activation in macrophages, thereby stimulating the accumulation of IL-1β in the tumor microenvironment. The secreted-IL-1β, in turn, promotes breast cancer progression.

#116

IGFBP2 promotes tumor progression by inducing alternative polarization of macrophages in pancreatic ductal adenocarcinoma.

Longhao Sun,1 Liang Liu,2 Weijun Tian,1 Zhixiang Zhang,1 Ya'an Kang,3 Huamin Wang,3 Jason B. Fleming,3 Boris C. Pasche,2 Wei Zhang2. 1 _Tianjin Medical University General Hospital, Tianjin, China;_ 2 _Wake Forest Baptist Medical Center, Winston-Salem, NC;_ 3 _The University of Texas MD Anderson Cancer Center, Houston, TX_.

Pancreatic ductal adenocarcinoma (PDAC) is the most lethal and aggressive malignancy. Tumor-associated macrophages (TAMs) mainly represent the M2-like phenotype, with potent immunosuppressive activity, and play a pro-tumor role in nearly all aspects of PDAC biology. Elevated infiltration of M2 TAMs is associated with poor prognosis in PDAC. Insulin-like growth factor binding protein 2 (IGFBP2) is a pleiotropic oncogene that induces PDAC progression and is involved in the regulation of immune responses. Our recent study demonstrated that IGFBP2 potentiates signal transducer and activator of transcription 3 (STAT3) transactivation activities in glioma. Considering the essential role of IGFBP2 in governing tumor progression and immune evasion of PDAC, we hypothesized that IGFBP2 promotes tumor progression by inducing alternatively activated macrophages in PDAC. In this study, immunohistochemical (IHC) staining for human PDAC clinical samples was used to reveal the relationship among IGFBP2 expression, M2 TAM accumulation and patient survival. The function of IGFBP2 in inducing alternative polarization of macrophages and promoting tumor progression in PDAC through the STAT3 pathway were evaluated using in vitro and in vivo assays. Ingenuity pathway and gene set enrichment analyses were used to identify the correlation of IGFBP2 gene expression with all other genes in the genome and enriched pathways associated with IGFBP2 gene expression. Our results provide evidence that IGFBP2 expression is associated with M2 macrophage accumulation in the tumor microenvironment and with poor prognosis in PDAC. IGFBP2 augments the production and secretion of IL-10 through activation of STAT3 in PDAC cells, thus reprogramming the TAMs polarization toward an M2 phenotype and promoting tumor progression. These findings established IGFBP2 as an immune-suppressor in the PDAC microenvironment and suggested a strategy for targeting IGFBP2 to improve PDAC immunotherapy.

#117

Lysyl oxidase secreted by PTEN-deficient glioblastoma cells recruits macrophages and promotes malignant growth.

Peiwen Chen, Alan Y. Wang, Ronald A. DePinho. _UT MD Anderson Cancer Center, Houston, TX_.

Glioblastoma (GBM) is the most lethal form of brain cancer in adults. The median survival of GBM patients is only about one year after initial diagnosis. Genomic profiling has stratified GBM into various subgroups, which are driven by specific genetic alternations of core signaling pathways, including RTK/RAS/PI3K, P53 and RB pathways. Loss of PTEN, TP53 and CDKN2A tumor suppressors, and amplification of EGFR are frequently observed in GBM. However, targeted therapies, such as therapy against EGFR, have failed in the clinic, and no effective therapeutic drugs available to target tumor suppressors. The main reason for this failure is tumor-cell genetic heterogeneity, which induces aberrant activation of multiple signaling pathways. Stromal/immune cells in the tumor microenvironment (TME) are genetically stable, which not only play a pivotal role in GBM progression by affecting multiple cancer hallmarks, but can also be educated by cancer cells. However, whether and how the behavior and function of specific stromal/immune cells in the TME are regulated by cancer cell genetic alternations in GBM remain relatively undefined. Here, we show that PTEN deficiency in GBM specifically triggers immune response by promoting macrophage recruitment, while without affecting macroglia and other immune cells. Using unbiased transcriptome profiling, we identified that lysyl oxidase (LOX) is preferentially secreted by PTEN-deficient cancer cells, and is a potent macrophage chemoattractant. Transcriptome profiling following Gene Set Enrichment Analysis (GSEA) and functional validation demonstrated that activation of SRC and AKT signaling pathways drives LOX upregulation in PTEN-deficient cancer cells via the YAP1-TEAD complex. Upon the secretion from PTEN-deficient cancer cells, LOX can be uptaken by macrophages via integrin β1 signaling. Using the phospho-kinase antibody array, we identified PYK2 as the main downstream of LOX that is responsible for macrophage migration. Genetic and pharmacological inhibition of LOX in PTEN-deficient cancer cells does not affect tumor cell proliferation in vitro, but markedly inhibits macrophage density and tumor growth in vivo. Bioinformatics analysis in clinical samples demonstrated that PTEN-YAP1-LOX-integrin β1 axis is enriched in GBM patients with higher macrophage density, and that these patients show lower survival. Together, our findings highlight the significance of PTEN-YAP1-LOX-integrin β1 axis in macrophage infiltration in GBM, and demonstrate a possibility of improving GBM treatment by targeting this axis-mediated macrophage recruitment.

#118

Immuno-modulation of macrophages by ovarian cancer stem cells in an in vitro 3D spheroid platform.

Shreya Raghavan, Geeta Mehta. _University of Michigan, Ann Arbor, MI_.

The ascites microenvironment of ovarian cancers is enriched in ovarian cancer stem cells (OvCSCs) and macrophages (MPs). The immune system is complicit in the progression, chemoresistance and metastasis of ovarian cancer, yet little is known about the exact nature of interactions between OvCSCs and MPs, especially in the malignant ascites microenvironment. Here, we describe the use of the 3D in vitro hanging drop spheroid platform to study the reciprocal interactions between OvCSCs and MPs. Hetero-spheroids contained: i) monocyte-derived Macrophages (MP); and ii) OvCSCs (ALDH+ CD133\+ OVCAR3). For immuno-modulation, we characterized MP polarization markers by flow cytometry and gene expression analysis. Using qPCR, we quantified changes in IL6, IL10, IL12, and Wnt7A, to understand signaling involved in OvCSC/MP hetero-spheroids. CD68 expression (~50%) was observed in OvCSC/MP hetero-spheroids, indicating the presence of MPs. OvCSCs drove higher CD206 expression in MPs (75%) when compared to unsorted ovarian cancer cells (OvCa; 40%). Even if MPs were pre-polarized to inflammatory M1 (HLA-DR+ CD206-), co-culture with OvCSCs reversed the M1 phenotype, and strongly induced CD206 expression. Pre-polarized M2 MPs resulted in 2-fold increase in ALDH+ cells in hetero-spheroids. Apart from CD206, MPs in hetero-spheroids also expressed iNOS, IFN-ϒ, and CD163. Gene expression analysis revealed increased immuno-suppressive IL10 in hetero-spheroids, with more IL10 in OvCSC/MP (3.5 fold) compared to OvCa/MP. OvCSC/MP and OvCa/MP spheroid also had consistently elevated (2-3 fold) increase in IL6 gene expression, consistent with the importance of the IL6/STAT3 axis in both cancer stem cell and macrophage signaling. Lastly, Wnt7A was significantly elevated in both differentiated MPs, and OvCSC/MP spheroids, indicating reciprocal trophic and immuno-suppressive signaling components at play. The 3D spheroid model can simulate biological interactions between OvCSCs and MPs within the malignant ascites. We describe that OvCSCs primarily polarize macrophages into a mixed phenotype that express both inflammatory and pro-tumoral markers. We believe that the putative involvement of Wnt7a in OvCSC/MP interactions drives this phenotype, and could have functional implications for tumorigenicity, response to chemotherapeutics and metastatic potential. This work was supported by the DOD OCRP W81XWH-13-1-0134, MIOCA, Rivkin Center for Ovarian Cancer and NIH T32DE00007057-41 and P30CA046592.

#119

Immunotherapy with dendritic cells inhibited tumor growth on an ID8 orthotropic mouse model of ovarian cancer.

Min-Je Kim,1 Joo-won Kim,1 Ju-hyun Kim,2 Young-Jae Lee,2 Shin-Wha Lee,3 Yong-Man Kim3. 1 _Asan Institute for Life Science, Seoul, Republic of Korea;_ 2 _Asan Medical Center, Seoul, Republic of Korea;_ 3 _College of Medicine, University of Ulsan, Asan Medical Center, Seoul, Republic of Korea_.

Background and Purpose: Ovarian cancer is the most frequent cause of death from gynecologic malignancy. Recently, immunotherapies have been suggested as ways to treat this malignancy. However, the ideal of immunotherapy has not been realized yet in ovarian cancer patients. Therefore, we investigated effects of monocyte-derived dendritic cells (mono DCs) and new-generation DCs subset (new DCs) on an ID8 orthotropic mouse model of ovarian cancer.

Methods: Mono DCs were prepared from bone marrow of C57BL/6 mice. We isolated monocyte from bone marrow by monocyte isolation kit. Isolated monocyte was incubated for 7 days with IL-4 and GM-CSF. In addition, monocytes were pulsed with LPS and ID8 tumor lysate while incubating. Likewise new DCs were isolated from bone marrow of C57BL/6 mice. We isolated lineage negative cells from bone marrow using lineage cell depletion kit. Isolated cells were incubated with IL-4 and GM-CSF; also the cells were pulsed by ID8 tumor lysate and Rg3, which is a ginsenoside. Orthotropic model of ID8 was made on C57BL/6 mice. We injected 5x106 cells into peritoneal cavity. 4 weeks after cell injection, mice were grouped into three groups, which were vehicle, mono DCs and new DCs. Mice were injected with mono DCs and new DCs. 5x106 cells of DCs were injected 1 time/week for three weeks. One week later, mice were sacrificed and organs were harvested such as peritoneum, phren, ovary and tumor mass for histologic analysis.

Results: DCs treated groups showed lower ascites formation than vehicle group. Ascites were fewest in new DCs group. Survival rate was lowest in vehicle group. Each survival rate is 0% in vehicle, 63% in new DCs and 80% in mono DCs. Also tumor growth at ovary was more decreased in mono DCs and new DCs group. In addition, mono DCs-treated mice showed low level of metastasis to other organs.

Conclusion: Immunotherapy with dendritic cells showed inhibition of tumor growth and metastasis on mouse ovarian cancer. These results indicated that DCs treatments are able to be a potential therapy for ovarian cancer. It also could have synergy with the combined treatment of existing anticancer drugs. However, there are several limitations such as a relatively high dose of DCs and the procedure of isolating DCs needed large amount of bone marrow. We will continue to study and improve these issues.

#120

Local immune cytolytic activity stratifies clinical outcome and is linked to the immune microenvironment in breast cancer.

Tstutomu Kawaguchi, Qianya Qi, Xuan Peng, Kerry-Ann McDonald, Sumana Narayanan, Jessica Young, Song Liu, Li Yan, Kazuaki Takabe. _Roswell Park Cancer Institute, Buffalo, NY_.

Purpose: Although a recent genomic landscape study identified molecular and genetic signatures of immune cytolytic activity (CYT) related to immune-mediated cancer elimination, less is known about the clinical relevance of CYT in the tumors linked to immune microenvironment in breast cancer. The aim of our study was to assess whether intratumor CYT associates with genetic alterations and tumor immune microenvironment, which impacts survival in breast cancer patients.

Experimental Design: We utilized genomic data from primary samples of 1,090 breast cancer patients with clinicopathologic information from The Cancer Genome Atlas (TCGA). We developed an immunogenomics pipeline that correlates CYT with clinical outcome and intratumor immune microenvironment, including infiltrating immune cell composition.

Results: Breast cancer displays a range of intratumor CYT. Patients with high CYT showed significantly better prognostic outcome, independent of age, hormonal status, and stage. CYT also has a strongly positive association with the expression of immune checkpoint genes such as PD1, PD-L1, and CTLA4, as well as regulatory T-cell (Treg) markers such as FOXP3 and CCR4. Enrichment of immune-response gene sets was observed in the high-CYT tumors. CYT was associated with higher composition of immune-elimination cells such as CD8+ T-cell, CD4+ T-cell, gamma delta T-cell, and M1 macrophage, as opposed to negatively associated with immunosuppressive cells, such as Treg and M2 macrophage. High CYT was also associated with improved survival in tumor-infiltrating lymphocyte (TIL)-positive breast cancer.

Conclusions: Patients with high CYT breast cancers show improved survival, likely due to high intratumor immunogenicity. CYT could also be used as a potential biomarker for immunologic response within tumors.

#121

Angiogenin and the mmp9-timp2 axis are strongly upregulated in pro-angigoenic dnk-like cells isolated from colorectal cancer patients.

Antonino Bruno,1 Giuseppe Pelosi,2 Luigi Boni,3 Lorenzo Dominioni,4 Lorenzo Mortara,5 Douglas Noonan,5 Adriana Albini6. 1 _IRCCS MultiMedica, Milano, Italy;_ 2 _University of Milan, Milano, Italy;_ 3 _IRCCS - Ca' Granda, Milano, Italy;_ 4 _University of Insubria, Milano, Italy;_ 5 _University of Insubria, Varese, Italy;_ 6 _Fondazione MultiMedica Onlus, Milano, Italy_.

Natural Killer (NK) cells are effector lymphocytes involved in tumor immunosurveillance. In solid malignancies, tumor associated (TANK; peripheral blood) and tumor infiltrating (TINK) NK cells have compromised functions. We functionally and molecularly characterize TINK and TANKs from blood and tissue samples of colorectal cancer (CRC) patients, a tumor type where inflammation and angiogenesis have clinical relevance, compared to NK isolated from control and non-oncologic inflammatory bowel disease patients.

NK subset distribution and cytokine profiling were performed by multicolor flow cytometry, using peripheral blood and tissue samples from CRC patients, for surface antigen and cytokine profiling characterization. Conditioned media (CM) from FACS-sorted NKs were used either for secretomic profiling using antibody membrane arrays or in functional in vitro angiogenesis assays.

CRC TINKs/TANKs showed decreased activation marker NKG2D, impaired degranulation activity, a decidual-like NK polarization toward the CD56brightCD16dim/- CD9+CD49\+ subset. TINKs and TANKs supernatants induced endothelial cell proliferation, migration, adhesion and formation of capillary-like structures in vitro. It has been reported that dNK release proangiogenic factors and metastasis-associated (MMP-9, TIMP1-2) proteins.

We describe for the first time the expression of angiogenin and MMP2, MMP9, TIMP1 and TIMP2 by CRC derived NK cells. This could be a phenotype relevant to their invasive capabilities and pro-angiogenic function. STAT-3/STAT-5 activation was observed in TANKs, and inhibition of the STAT5 pathway by pimozide, an antipsychotic drug, reduced proangiogenic factor VEGF and angiogenin production and capillary-like structure formation, but did not affect the levels of TIMP-1, TIMP-2 and MMP-9.

#122

Hypoxia-enhanced proliferation and effector function of NK cells.

Yunwon Moon,1 Seon Ah Lim,2 Kyung-Mi Lee,2 Hyunsung Park1. 1 _University of Seoul, Seoul, Republic of Korea;_ 2 _Korea University College of Medicine, Seoul, Republic of Korea_.

Hypoxia promotes tumor cell survival while providing immune cell shaping and resistance. Although NK cells function within hypoxic lymphatics and inflamed tissues, they were shown to mount impaired anti-tumor effector functions in ex-vivo hypoxic environment. This raises a question that additional cues exist in vivo to allow full NK cell activation. By varying the percentage and time of pO2 exposure ex-vivo, we were able to fine-tune a condition that reverses hypoxia-induced NK cell suppression to become highly proliferative and enhanced cytolytic phenotypes. We found that exposing NK cells to moderate hypoxia in the presence of IL-2 and feeder cells allowed HIF-1α stabilization and its target gene expression, metabolic changes toward glycolysis, specific upregulation of NKp44 receptor, and increased STAT3 phosphorylation with concomitant reduction of apoptosis and p16 senescence pathways. Therefore, the hypoxic exposure allows HIF-1α-mediated adaptation of mature NK cells toward highly proliferative and cytolytic NK phenotypes, which may occur in vivo at the time of tumor growth and inflammation. [This work was supported by the National Research Foundation of Korea (NRF), grants NRF-2013M3A9D3046248]

#123

Enhanced anti-tumor activity of the combination of entinostat and NKTR214 in renal and colon cancer tumor models.

Lei Wang,1 Fei Chen,2 Tingting Li,2 Peter Ordentlich,1 Jonathan Zalevsky3. 1 _Syndax Pharmaceuticals, Waltham, MA;_ 2 _Crown Bioscience (Taicang) Inc., China;_ 3 _Nektar Therapeutics, San Francisco, CA_.

Background: Combination strategies are required to improve outcomes for immune mediated anti-tumor therapies. Entinostat is a class 1 selective histone deacetylase inhibitor shown in preclinical studies to target immune suppressive mechanisms in the tumor microenvironment to improve the efficacy of immune checkpoint blockade, vaccines and cytokines. NKTR-214 is a novel CD122-biased agonist with promising safety, tolerability and anti-tumor efficacy. A previous phase 2 clinical trial of entinostat combined with high dose interleukin 2 demonstrated enhanced clinical efficacy and a well-tolerated safety profile in advanced renal cell cancer patients. Based on these data and potential for combinatorial anti-tumor activity, we investigated the antitumor efficacy and immune response of entinostat combined with NKTR-214 in CT26 colon cancer and RENCA renal cell cancer syngeneic tumor models.

Methods: CT26 and RENCA cells were subcutaneously inoculated into 6-8-week-old BALB/c mice (n=12 per arm) and when tumors reached ~140mm3 were randomized and treated with entinostat (p.o., 1 mg/kg or 5 mg/kg, QD x 28), NKTR-214 (i.v., 0.8mg/kg, Q9D x 4), the combination, or vehicle control. Phenotypic and functional analysis of tumor infiltrating cells in each group was conducted on tumors taken from 3 mice per arm, at day 7 post-treatment. Remaining mice (n=9 per arm) continued treatment until tumors reached 2,000mm3.

Results: In the CT26 model, the combination of entinostat (1mg/kg and 5mg/kg) and NKTR-214 was tolerated and demonstrated prolonged survival with a medium survival time not reached for the combination groups (7 of 9 animals still alive) as compared to NKTR-214 (44 days; 3 of 9 alive), entinostat or controls (16 days; none living). In RENCA, the treatments were less well tolerated and tumor growth inhibition is reported only for the entinostat 1mg/kg dose level with that combination demonstrating 60% tumor growth inhibition (n=8), compared to 34% in NKTR-214 alone (n=8), or 27% in entinostat alone (n=8) arms. Analysis of tumor infiltrating immune and myeloid cells after treatment indicated no significant phenotypic changes in any of the cell populations studied. However, measurement of IFNγ, TNFα and Granzyme B expression in T cell and NK cells revealed enhanced anti-tumor cytotoxicity. IFNγ was increased >15-fold in CT26 and >9-fold in RENCA treated with the entinostat (5mg/kg) and NKTR-214 combination along with increases in granzyme B (CT26 >17-fold; RENCA > 166-fold) and TNFα (CT26>3-fold; RENCA >2-fold).

Conclusions: These data demonstrate that entinostat combined with NKTR-214 results in enhanced cytotoxic Teff function which translates to significant anti-tumor effects. These results provide support for clinical testing of the combination with or without additional immune checkpoint blockade.

#124

The soluble factor SPINK1 is a biomarker of therapeutically damaged TME that confers acquired resistance and an exploitable molecular target in human cancer medicine.

Yu Sun. _Institute of Health Sciences, Shanghai Institutes for Biological Sciences, CAS, Shanghai, China_.

Clinical efficacy of most anticancer treatments is frequently compromised by the development of cancer resistance arising as either a consequence of innate adaptation of cancer cells, or acquired fitness conferred by extracellular factors generated by the surrounding tumor microenvironment (TME). Of note, stromal cells in the TME become overtly senescent upon genotoxic treatment and develop a senescence-associated secretory phenotype (SASP), the latter characterized by secretion of numerous soluble factors that are responsible for disease exacerbation. We recently disclosed significant upregulation of the serine protease inhibitor Kazal type I (SPINK1) in human prostate stroma after chemotherapy of prostate cancer (PCa) patients. SPINK1, originally reported as a pancreatic secretory trypsin inhibitor, prevents pancreatic tissues from protease degradation with expression normally restricted to gastrointestinal (GI) tract organs. However, the influence of SPINK1 as a TME-derived soluble factor on cancer progression remains unexplored. In this study, we defined the expression modality and biological roles of SPINK1 in a therapy-damaged and functionally activated TME under therapeutic pressure. Distinct from a recent finding that revealed the involvement of a regulatory circuit comprising HNF4G and HNF1A in the PCa-GI transcriptome, we found that SPINK1 expression in human stromal cells is regulated by alternative transcriptional factors including NF-κB and C/EBP in response to chemotherapy. TME-released SPINK1 can significantly enhance the aggressiveness of PCa cells including accelerated proliferation, increased migration, elevated invasion and more importantly, enhanced chemoresistance. SPINK1 triggers a typical epithelial-mesenchymal transition in cancer cells, a process mediated by EGFR/PI3K/Akt and MAPK/Erk signaling. Further, SPINK1 remarkably reprograms PCa cells by inducing a cancer stem cell (CSC)-like phenotype and promoting transdifferentiation of cancer cells toward neovasculature. As supporting evidence, our preclinical studies suggested that SPINK1 expression in the prostate TME substantially promoted cancer survival and disease progression. Clinical investigation revealed increased expression of SPINK1 in the stroma of multiple organ types including the prostate, lung and breast of cancer patients after chemotherapy, implying the systemic induction of SPINK1 by anticancer agents. Thus, our study demonstrates that SPINK1 is a soluble biomarker of therapeutically damaged TME and represents a potentially exploitable molecular target in human cancer clinics.

#125

Targeting autophagy inhibits B16-F10 melanoma growth by enhancing the infiltration of functional NK cells in a CCL5-dependent manner.

Tsolere Arakelian,1 Takouhie Mgrditchian,1 Jerome Paggetti,1 Muhammad Zaeem Noman,1 Elodie Viry,1 Etienne Moussay,1 Salem Chouaib,2 Guy Berchem,3 Bassam Janji1. 1 _Luxembourg Institute of Health (LIH), Luxembourg City, Luxembourg;_ 2 _Gustave Roussy Cancer Center, Villejuif, France;_ 3 _Luxembourg Institute of Health (LIH) and Centre Hospitalier du Luxembourg, Luxembourg City, Luxembourg_.

The failure in achieving a durable clinical immune response against cancer cells depends on the ability of cancer cells to establish a microenvironment that prevent cytotoxic immune cells to infiltrate tumors and kill cancer cells. Therefore, the key issue to achieve successful anti-tumor immune response is to harness strategies allowing the reorientation of immune cells to the tumor. While blocking tumor growth by inhibiting autophagy is experimentally well supported, its role on the infiltration of immune cells into tumors remains largely unknown. Here, the impact of targeting autophagy gene BECN1 on the infiltration of Natural Killer (NK) cells into melanomas was investigated using B16-F10 syngeneic melanoma mouse model. We showed that, in addition to decreasing tumor growth, targeting BECN1 increased the infiltration of functional NK cells into melanoma tumors. We provided evidence that driving NK cells to the tumor bed strikingly relied on the ability of BECN1-defective (BECN1-) tumors to transcriptionally overexpress chemokine gene CCL5 since such infiltration and tumor regression were abrogated by silencing CCL5 in BECN1- tumors. Mechanistically, we showed that the upregulated expression of CCL5 occurred through the activation of its transcription factor c-Jun by a mechanism involving the impairment of phosphatase PP2A catalytic activity and the subsequent activation of JNK. Similar to BECN1, targeting other autophagy genes such as ATG5, p62/SQSTM1, or inhibiting autophagy pharmacologically by chloroquine, also induced the expression of CCL5 in melanoma cells. Clinically, a positive correlation between CCL5 and NK cell marker NKp46 expression was found in melanoma patients and high expression level of CCL5 was correlated with a significant improvement of melanoma patients' survival. These results highlight the role of targeting autophagy in breaking the immunosuppressive tumor microenvironment barrier thus allowing the infiltration of NK cells into the tumor. We believe that this is the first study highlighting the benefit of targeting autophagy as a novel therapeutic approach to improve NK-based immunotherapy.

#126

MYC-driven lymphomas suppress NK surveillance by blocking maturation of early NK cells.

Line D. Heftdal, Srividya Swaminathan, Dean W. Felsher. _Stanford University, Stanford, CA_.

The MYC oncogene is commonly overexpressed in hematopoietic cancers. In a transgenic mouse model of MYC-induced T-cell lymphoma, we found that there was a systemic reduction in NK cell (CD3- NKp46High) numbers and activation in lymphoid organs such as spleen and bone marrow. MYC inactivation in lymphoma-bearing MYCON mice partially restored NK cell numbers and activation. Hence, the MYC oncogene appears to suppress NK cell-mediated immune surveillance of lymphomas. We evaluated whether reduction in mature and activated NK cells (CD3- NKp46High) in MYCON mice was due to increased death of NK cells in these mice when compared to normal and MYCOFF mice. Using flow cytometry, NK cells were measured in age matched spleens and bone marrows of normal (n = 8), MYCON (n = 8), and MYCOFF (n = 8) mice. Surprisingly, we observed a significant reduction in proportions of dead NK cells (NKp46+ PI+) in spleen and bone marrow of MYCON mice, in comparison to normal and MYCOFF cohorts. Hence, the reduction in activated NK cell numbers during MYC-driven lymphomagenesis does not occur because of increased death of NK cells. Next, we investigated whether MYC arrests early NK cell development in the bone marrow of lymphoma mice, leading to systemic NK suppression. NK cell lineage specification begins with CD122 expression, that is maintained throughout NK development starting at the precursor stage. NK precursors (NKP) transition to immature NK (iNK) cells by expressing the cytotoxicity marker NKp46. iNK cells leave the bone marrow to populate peripheral lymphoid organs. We measured NKP (CD122+ NKP46-), and iNK (CD122+ NKp46+) cells in bone marrow of normal (n = 8), MYCON (n = 8), and MYCOFF (n = 8) mice. We observed no significant changes in the percentages of NKP. However, the proportions of iNK cells were significantly reduced in MYCON mice, in comparison to MYCOFF and normal mice. Our results suggest that MYC may block transition from NKP to iNK stage during early NK cell development. The reduction of iNK cells in spleens of MYCON mice was concordant with the reduction of iNK cells in bone marrow from the same mice. We conclude that MYC-induced lymphomagenesis blocks early NK cell development, thereby suppressing NK-mediated immune surveillance.

#127

Exosomes derived from head and neck cancer enhance the cytotoxicities of NK cells by NAP1-IRF-3 pathway.

Wantao Chen, Yingnan Wang, Jianjun Zhang. _Ninth People's Hospital, Shanghai Jiao Tong Univ. School of Medicine, Shanghai, China_.

Objective: To examine the effect of tumor-derived exosomes (TEXs) from oral cancer on natural killer (NK) cells and to explore the underlying mechanism.

Materials and Methods: TEXs were isolated from the medium supernatant of oral cancer (OC) cells using ultrafiltration and affinity chromatography and were identified using electronic microscopy, nanoparticle tracking analysis and immunoblotting. The effects of TEXs on NK cells were analyzed using laser scanning confocal microscopy and several functional assays of NK cells. To explore the mechanism, antibody array, protein mass spectrometry and RNA interference were adopted.

Results: The particles isolated from the OC cells were identified as exosomes with satisfactory morphology, concentration and purity. The TEXs were internalized by the NK cells and then promoted the biologic functions of these cells, including proliferation, cytotoxicity and the release of perforin and granzyme M. Furthermore, we found that the TEXs increased the expression of interferon regulatory factor 3 (IRF-3) and phosphorylated IRF-3 by antibody array, which drove the expression of the type I interferon (IFN) gene and the chemokine (C-X-C motif) ligand (CXCL) genes, thereby promoting the function of NK cells. We also found that NF-κB-activating kinase-associated protein 1 (NAP1), an upstream activator of IRF-3, was enriched in TEXs, and treatment with TEXs increased NAP1 in NK cells. Importantly, NAP1-depleted TEXs obtained from OC cells dramatically weakened the influence of the TEXs on NK cells.

Conclusion: Our findings reveal a previously unrecognized function of exosome derived from oral cancer cells in enhancing the cytotoxicity of NK cells by NAP1- IRF-3 pathway.

This work was supported by the National Program on Key Research Project of China (2016YFC0902700).

#128

NKILA lncRNA promotes tumor immune evasion by mediating activation-induced cell death in tumor-specific CTLs.

Erwei Song. _Sun Yat-sen Memorial Hospital, Guangzhou, China_.

Activation-induced cell death (AICD) of T lymphocytes may be exploited by cancers to eliminate cytotoxic T cells (CTLs). However, the mechanisms underlying AICD in tumor specific CTLs remain unknown. Here, we demonstrated excessive apoptosis of tumor antigen-specific CTLs in breast and lung cancers. Interestingly, NKILA, an NFκB-interacting lncRNA, mediates AICD of CTLs by inhibiting NFκB activities after their activation. Mechanistically, NKILA promoter is closed for transcription by histone deacetylation in resting T cells, while calcium influx by TCR signaling activates calmodulin to remove the deacetylase from NKILA promoter and enhances its transcription. In vivo, administering CTLs with NKILA silencing to immunocompromised mice with breast cancer patient-derived xenografts (PDXs) effectively inhibits PDX growth by increasing CTL infiltration. Clinically, NKILA was overexpressed in the tumor-specific CTLs of breast and lung cancers, which was associated with less CTL infiltration in the tumors and shorter patient survival. Our findings present the first evidence that AICD in tumor-specific CTLs is crucial to cancer immune evasion, and targeting NKILA in CTLs emerges as a novel antitumor immunotherapy.

#129

Biobanking and feasibility considerations for prostate cancer gastrointestinal microbiome studies.

Sarah E. Ernst,1 Mark C. Markowski,2 Anuj Gupta,2 Sarah J. Wheelan,2 H. Ballentine Carter,3 Alan W. Partin,3 Cynthia L. Sears,4 Karen S. Sfanos1. 1 _Johns Hopkins University School of Medicine, Baltimore, MD;_ 2 _Sidney Kimmel Comprehensive Cancer Center, Baltimore, MD;_ 3 _James Buchanan Brady Urological Institute, Baltimore, MD;_ 4 _Bloomberg-Kimmel Institute of Immunotherapy, Baltimore, MD_.

The human microbiome may play a role in prostate health and disease as both direct and indirect interactions. Direct interactions between microbiota and prostate cancer include prostate infections, inflammation, prostatitis, and potentially interactions with the urinary microbiome. Indirectly, the gastrointestinal (GI) microbiota may influence prostate cancer via xenobiotic metabolism, augmentation of treatment response, and contributions to systemic inflammation and cytokines. In the present study, we are seeking to understand how different prostate cancer treatments may be affected by the microbial composition of the GI tract. We are currently developing a biorepository of fecal specimens from prostate cancer patients in different clinical states of disease. A key question during the planning phase of this biorepository was if samples can be collected and banked in the form of a rectal swab, instead of the more traditional method of a stool sample. Rectal swabs provide an advantage over collection of stool specimens in that they are easily collected (the patient can self-collect) in a standardized, "on demand" fashion during routine patient visits. Therefore, a pilot study was undertaken to compare the microbial profile of samples collected via both methods from the same individual. We concomitantly collected both stool samples and rectal swabs from 6 patients undergoing active surveillance for prostate cancer at the Johns Hopkins Hospital. All samples were stored at -80°C until we were ready to isolate bacterial DNA. DNA was extracted with a phenol:chloroform based protocol that we have optimized for microbiome studies that includes multiple enzyme digest and bead beating. We conducted Illumina amplicon sequencing of PCR products amplified with universal primers designed against the V6 hypervariable region of the bacterial 16S rRNA gene. We tested our sequencing strategy against Microbiome Reference Standards obtained from American Type Culture Collection (ATCC). The results of our analysis from prostate cancer patients indicated high similarity of bacterial profiles obtained for matched stool and swabs in 4 of the 6 patients. In both of the cases that were dissimilar, there was a greater representation of Enterobacteriaceae in the stool sample versus the swab. We conclude that collection of rectal swabs is a more feasible and convenient means of sampling the GI microbiota in prostate cancer patients, especially when aiming to conduct longitudinal studies with multiple sample collections to correlate microbiota profiles to treatments and/or treatment response. Due to the differences that were identified, we recommend deciding on the sample collection method at the onset of biobanking, and keeping the sampling method consistent throughout.

#130

Cancer teammate survives longer: Role of cancer-associated a2 V-ATPase in regulation of neutrophil survival.

Safaa A. Ibrahim,1 Arpita Kulshrestha,2 Gajendra K. Katara,2 Manoranjan Sahoo,2 Kenneth D. Beaman2. 1 _Faculty of Pharmacy, Cairo University, Cairo, Egypt;_ 2 _Rosalind Franklin University Of Medicine And Science, North Chicago, IL_.

Tumor associated neutrophils (TAN) represent a significant portion of tumor leukocytes and accumulate in many types of cancer. TAN can survive for weeks in cancer tissues and have been proposed as key mediator of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity. Identifying cancer associated factors that promote the survival and protumorigenic properties of neutrophils will provide new targets for cancer therapy. Cancer cells secrete a peptide from the Nterminal domain of a2isoform vacuolar ATPase (a2NTD) that promotes the protumorigenic properties of neutrophils that enhances angiogenesis and tumor cell invasiveness. The present study attempts to decipher the role of a2NTD in the regulation of neutrophil apoptosis. The treatment of human neutrophils with recombinant a2NTD led to 2.1 fold increase in the percentage survival of neutrophils (P < 0.001). a2NTD treatment activates NF- kBp65 by activating reactive oxygen species (ROS) generation in neutrophils. Activation of NF- kB pathway leads to upregulation of the gene expression of anti-apoptotic factors; Bcl2-A1, Bcl-xL, c-Flip, G-CSF and downregulates the pro-apoptotic factors such as BAX. Parthenolide; NF-kB inhibitor, negated the effect of a2NTD on enhancing neutrophil survival confirming the involvement of NF-kB pathway in the a2NTD mediated increase of neutrophil survival. Further analysis showed that a2NTD treatment stabilizes the neutrophil mitochondrial membrane potential and decreases Bax translocation to the mitochondria that was associated with a decrease in caspase 9 and caspase 3 activity. Since there is a direct link between the intrinsic and the extrinsic pathways of apoptosis, our results showed that there was no change in the Fas and Fas ligand expression upon a2NTD treatment. Further analysis would uncover the effect of a2NTD treatment on the extrinsic pathway of apoptosis in neutrophils. Together, our results demonstrate a novel role of the cancer associated a2-vacuolar ATPase on retarding the mitochondrial death pathway in neutrophils by the action of a2NTD.

#131

Neutrophil extracellular trap mediate mitochondrial biogenesis in cancer cells to promote growth of metastases.

Hamza Yazdani, Samer Tohme, Eva Roy, Patricia Loughran, Dirk Vander Windth, Hai Huang, Allan Tsung. _University of Pittsburgh, Pittsburgh, PA_.

Neutrophils through the release of neutrophil extracellular traps (NETs) in response to surgical stress were shown to be able to capture circulating cancer cells, and in doing so, support the development of metastatic disease. In addition, NETs persist within the tumor microenvironment long after the surgical stress happens. The exact nature of the NETs-tumor interaction and the role that NETs play in dictating the tumor microenvironment is unclear. In vivo, subcutaneous and liver metastasis models of colorectal and hepatocellular cancer were performed in wild type and PAD4 KO mice that lack the ability to form NETs. In vitro, bioenergetics profiles of both HCC and CRC cancer cells were studied after co-culture with NETs, isolated from human and murine neutrophils. We found a 3-fold decrease in the tumor volume of PAD4 KO mice compared to control mice 3 weeks after tumor injection in both subcutaneous and liver metastatic models. PAD4 KO tumors showed significantly decreased neutrophil infiltration and no NET formation within the tumor compared to WT mice. PAD4 KO tumors had decreased proliferation rates and increased apoptosis. In vitro, stressed cancer cells release stress signals such as cytokines, chemokines, and DAMPs (HMGB1 and histones), that enhanced neutrophil migration towards them and subsequently triggered NET formation. In turn, NETs resulted in a 2-fold increase in the expression of mitochondrial biogenesis regulators in cancer cells. Increase mitochondrial mass was observed when treated cancer cells with NETs. A significant increase mitochondrial oxygen consumption rate (OCR) and ATP levels were observed when compared with DNase treatment. Taken together, we suggest that cancer cells help in recruiting NETs which in turn regulates mitochondrial bioenergetics for tumor growth and metastasis.

#132

Role of IL-17-CXCR2 axis in neutrophil recruitment facilitating breast cancer metastasis and therapy resistance.

Lingyun Wu,1 Bhawna Sharma,2 Rakesh K. Singh1. 1 _University of Nebraska Medical Center, Omaha, NE;_ 2 _Frederick National Laboratory for Cancer research, Omaha, NE_.

The major challenges for breast cancer includes therapy resistance and metastasis to distant organs. Chronic inflammation has been intimately linked with these processes. We have observed increased level of CXCR2 ligands, inflammatory immunosuppressive microenvironment, and increased metastasis in chemotherapy resistant cells. CXCR2 and its ligands have been shown to be the primary mechanism for neutrophils recruitment to the sites of primary tumors and metastases. However, their precise roles in therapy resistance and metastasis remains unclear. The aim of this project is to investigate the role of neutrophils in chemotherapy resistance and metastasis in breast cancer. In this report, we investigated the mechanisms and putative role of neutrophils in chemotherapy resistance and metastasis. We evaluated the expression level of the factors critical for neutrophil recruitments, such as interleukin (IL)-17 and its receptors (IL-17R), granulocyte colony stimulating factor (GCSF), CXCR2 and its ligands in primary tumors and metastases established from parents and chemotherapy resistant cells. The frequency of neutrophils and T-helper 17 (Th17) cells were analyzed using immunohistochemistry. Chemotherapy-resistant cell lines express higher levels of GCSF, IL-17, and IL-17R compared to Cl66 parent cells. We observed higher expression of IL-17R, CXCR2, and CXCR2 ligands in metastatic lesions compared to primary tumors. Furthermore, there were more recruitments of neutrophils and Th17 cells in resistant tumors than parent tumors. In addition, treatment of tumor cells with IL-17 significantly increased cellular proliferation together with CXCL1 and CXCL5 expression in a concentration-dependent manner. Moreover, we observed higher expression of TGFβ 2 and Th17 cell priming factors, IL-6 and IL-23, in neutrophils co-cultured with therapy resistant cells compared to the control suggesting a feed forward loop. In addition, we observed that IL17 treatment of tumor cells did not increase their tolerance to chemotherapy drug, indicating its role in establishing immune suppressive microenvironments. Together our data demonstrate an IL17-CXCR2 ligands axis induces protumorigenic inflammation, facilitating therapy resistance and metastasis through recruited neutrophils.

#133

Synergistic effect between natural killer cells and anysomycin against hepatocellular carcinoma cells.

Chang Hoon Lee, Miok Kim, Kyung Won Lee. _Korea Research Institute of Chemical Technology, Daejeon, Republic of Korea_.

Although advances in the clinical management of hepatocellular carcinoma (HCC) have been made, HCC is the second leading cause of cancer-related death worldwide. Currently, there are limited treatment options for advanced HCC. Therefore, novel treatment strategies for HCC are required. Here, we described a promising anti-tumor effect of anisomycin, which not only had direct killing effects but also enhanced a natural killer cell-mediated immunotherapeutic effect in HCC. To better elucidate the anti-tumor mechanisms of anisomycin, we performed a genome-scale analysis of gene expression profiles. We found that anisomycin treatment of HCC differentially regulated a broad range of immune regulation-associated genes; among them, we found that CD58, which was significantly increased on anisomycin treated HCC cells was one of the critical components for natural killer cell-mediated immunotherapeutic effect enhanced by anisomycin. Notably, this is the first report for synergistic effect of anisomycin in combination with NK cell therapy as a potential therapeutic option for HCC.

#134

Dysbiosis of gut microbiota in chronic stressed patients with endometriosis.

Jing Xu, Yu Kang, Yinhua Yu. _Fudan University Affilicated Obstetric and Gynecology Hospital, Shanghai, China_.

Although endometriosis is a benign disease, it has malignant characters such as invasion and migration. Besides, there is an endometrial type of ovarian cancer, which means endometriosis is similar to malignant disease at some extent. Existing researches showed that patients with endometriosis often have high level of chronic stress. Furthermore, chronic stress can promote the growth of endometriosis by activating the β-adrenergic signaling pathway. Gut microbiota is reported to be related to the stress level through the gut-brain axis. So we wonder whether gut microbiota is associated with chronic stress level in endometriosis patients, which may provide a new clinical intervention method to improve prognosis. We grouped the endometriosis patients into stress group and control group with GAD-7 and PHQ-9 questionnaires and collected patients' fecal and tissues samples. Gut microbiota compositions were analyzed by the 16SrRNA gene sequencing-based method and we performed Immunohistochemistry (IHC) to detect the activation of inflammatory pathways in endometriosis tissues. We found that in patients with endometriosis, dysbiosis of gut microbiota is associated with patients' stress levels and Paraprevotella, Odoribacter, Veillonell and Ruminococcus show significant differences with reduced levels in chronic stressed endometriosis patients group, which represents a disease-specific change of gut microbiota on the genus level. Besides, compared to the control group, the expression level of inflammatory cytokines, NF-κB p65 and COX-2, increased in the chronic stressed patients group, which means that dysbiosis of gut microbiota may active the inflammatory pathway of gut-brain axis. In conclusion, we found new disease-specific changes of gut microbiota in chronic stressed patients with endometriosis, which can be a new examination target of chronic stress level and may provide an adjuvant therapy method for clinical psychological intervention, reducing the stress level and improving the prognosis of endometriosis patients.

#135

Immunosuppressive microenvironment in low-grade serous ovarian carcinoma.

Kwong-Kwok Wong, David M. Gershenson. _UT MD Anderson Cancer Ctr., Houston, TX_.

Low-grade serous ovarian carcinoma (LGSOC) accounts for a relatively large percentage of epithelial ovarian cancers in young women. Although LGSOC is considered to be an indolent cancer, with an estimated median overall survival of 80 months, an updated analysis of 350 women with LGSOC from our Low-Grade Serous Tumor Database indicated that over 85% developed recurrent disease, and almost all ultimately died of the disease. The overall response rate of recurrent LGSOC to various chemotherapy drugs is dismal - less than 4%. To explore the potential of immunotherapy in low-grade serous ovarian carcinoma, we investigate the immune profiles in low-grade serous ovarian carcinoma. In this study, we have generated gene expression profiles from microdissected epithelial tumor cells and the corresponding LGSOC bulk tumors. Such a novel comparison revealed an immune active tumor stromal microenvironment with highly expressed genes involved in immune response, antigen processing and presentation. The down-regulation of MHC-I gene expression or antigen-processing components of the MHC-I pathway in the tumor cells may lead to an escape from immune surveillance. A recent study has indicated that activation of the MAPK pathway in melanoma cancer cells could lead to internalization of MHC-I and subsequent sequestration within endolysosomal compartments. This sequestration may diminish antigen presentation and CD8+ T-cell recognition of tumor cells. This is further supported by our immunostaining analysis of CD8+ cells in LGSOC. The data indicated that the majority of LGSOC has less than five intratumoral CD8+ T-cells per high-power field, which is much less than the number of CD8+ T-cells observed in high-grade ovarian serous carcinoma. Thus, this low-number of intratumoral CD8+ cells and low level of MHC-I complex in LGSOC tumor cells would suggest a compromised antigen processing and presentation system. Furthermore, we also compared the number of CD45+ cells in LGSOC with or without BRAF/KRAS mutations. CD45 antigen (leukocyte common antigen) is a unique and ubiquitous membrane glycoprotein expressed on almost all hematopoietic cells, from which CD4+ helper T-lymphocyte, CD8+ cytotoxic T-lymphocyte and other lymphocytes are derived. Our data indicate that LGSOC with BRAF or KRAS mutant proteins had a high number of infiltrating CD45+ hematopoietic cells, which would suggest BRAF or KRAS mutant proteins are immunogenic and would partly explain our observation that patients with LGSOC carrying BRAF or KRAS mutation had longer overall survival. However, there were only a few or no CD8+ T-lymphocytes in the corresponding samples stained with CD45+ cells. In summary, our data suggest that an immune suppressive environment related to MAPK activation exists in LOSOC, which may prevent antigen presentation, maturation and activation of infiltrating cytotoxic T-cells.

#136

T-cell clonotype convergence in colorectal cancer driven by tumor-specific neoantigens.

Jose C. Machado,1 Joana Reis,1 Margarida Fernandes,2 Rafaela Silva,1 Luis Cirnes,1 Fatima Carneiro,3 Jose L. Costa1. 1 _IPATIMUP, Porto, Portugal;_ 2 _Centro Hospitalar São João, Porto, Portugal;_ 3 _Medical Faculty of Porto, Porto, Portugal_.

A cornerstone of the biological process which mediates response to immunotherapy in cancer patients is the expression of neoantigens resulting from somatic mutations in cancer cells. Little attention has been paid so far to the neoantigen's immune cell counterpart, the T-cell receptor (TCR). In this study, we aimed at characterizing the tumor infiltrating T-cell repertoire in colorectal cancer (CRC), and to evaluate evidence for neoantigen driven T-cell expansion within the tumor microenvironment. To characterize the T-cell repertoire, we used the Ion AmpliSeq Immune Repertoire Assay Plus with an Ion S5 XL system. This assay works with as little as 10ng of starting total RNA, allows 400bp read length, and offers complete characterization of the CDR1, CDR2 and CDR3 regions. A cohort of 20 CRC cases was used, including microsatellite instability (MSI) positive and MSI-negative cases. Our results show a higher T-cell richness in distal CRC samples compared with proximal samples (p=0.04), and lower T-cell richness in M1 as compared with M0 cases (p=0.0001). No association was observed with MSI status, inflammatory infiltrate, tumor grade or lymph node metastasis. We observed that the T-cell repertoire in CRC samples is highly convergent compared to the peripheral blood lymphocyte repertoire of healthy donors (p<0.0001). In CRC samples we observed evidence of selection of TCR clonotypes coding for the same CDR3 aminoacid sequence, suggesting neoantigen-driven TCR convergence. TCR clone overlap was observed only between pairs of samples (4 pairs of primary tumor and metastasis samples available) from the same patient, and not between samples from different patients, suggesting high tumor specificity of T-cell clonotype selection. A validation cohort of CRC cases, including additional pairs of primary tumor and metastasis samples, is currently being analyzed to validate our findings. In this study we provide evidence of T-cell clonotype selection in CRC driven by tumor-specific neoantigens. The specificity of this process is further reinforced by the T-cell clonotype overlap within patients. It remains to be seen whether it is possible to map convergent T-cell clonotypes to its respective tumor neoantigens.

#137

Pharmacologic activation of integrin CD11b as a novel therapeutic strategy against lung cancer.

Terese Geraghty,1 Anugraha Rajagopalan,1 Samia Khan,1 Judith Varner,2 Vineet Gupta1. 1 _Rush University Medical Center, Chicago, IL;_ 2 _University of California San Diego, Chicago, CA_.

Lung cancer is the leading cause of cancer related deaths in the United States. With a 5-year survival rate of 18%, lung cancer patients are in immediate need of new therapeutic options. A major problem facing immunotherapy for cancer is the active immune suppression by the tumor. Tumors show presence of large numbers of tumor associated macrophages (TAMs), which suppress the adaptive immune response, increase neo-vascularization to the tumor, and promote tumor survival. Integrin CD11b is highly expressed on TAMs and is essential for their recruitment and biological functions. Reasoning that CD11b activity is important for controlling tumor growth, we developed a novel small molecule agonist called leuhadherin-1 (LA1), which activates CD11b. We found that CD11b activation by LA1 significantly reduced tumor growth in wild type mice bearing Lewis Lung Carcinoma (LLC) tumors as early as one week post treatment. Conversely, LLC tumors grew at a faster rate in CD11b-/- mice, compared to wild type mice, showing that CD11b is important for controlling tumor growth. Importantly, LA1 treatment in tumor bearing CD11b-/- mice did not show any efficacy, demonstrating the specificity of LA1 to CD11b. These data suggest that CD11b activation via LA1 modulates TAMs in tumors and is a novel therapeutic strategy against cancer.

#138

PRMT6 promotes lung tumor growth via the modulation of macrophage M2 polarization.

Sreedevi Avasarala,1 Pei-Ying Wu,1 Samia Q. Khan,2 Michelle Van Scoyk,1 Yanlin Su,1 Odile David,1 Vineet Gupta,2 Mark T. Bedford,3 Robert A. Winn,1 Rama Kamesh Bikkavilli1. 1 _University of Illinois at Chicago, Chicago, IL;_ 2 _Rush University Medical Center, Chicago, IL;_ 3 _The University of Texas MD Anderson Cancer Center, Smithville, TX_.

Lung cancer kills more people in the United States than any other malignancy. High mortalities are in part due to the diagnosis at an advanced inoperable stage where the 5-year survival is only 4%. Therefore, improving survival will require increased knowledge of genes that drive the complex mechanisms of lung cancer initiation and progression. Emerging data implicate novel roles for protein arginine methylation in lung cancers. In protein arginine methylation, nitrogen/s of arginine can be post-translationally modified via the addition of a methyl group, catalyzed by a class of enzymes, protein arginine methyl transferases (PRMTs). Based on the computational analysis, tissue staining, and immunoblotting we have identified an increased expression of PRMT6 in lung tumors. To test the biologic relevance of PRMT6 in lung tumor development, we employed an inducible lung-targeted PRMT6 gain-of-function (GOF) mouse model. For the first time, we show that lung-targeted overexpression of PRMT6 results in spontaneous lung tumor development. We also observed a robust increase in lung tumors in the PRMT6 GOF mice in response to urethane, a KRas mutagen, and a prototypical model to study lung tumorigenesis. Excitingly, we detected an increase in the number of tumor-associated macrophages (TAMs) in the PRMT6 GOF mice. Further characterization of the TAMs from the PRMT6 GOF mice revealed that they were M2 polarized with immunosuppressive, angiogenesis-enhancing, and tumor-promoting activities. Taken together, these results demonstrate a novel role for PRMT6 in lung tumor development by way of macrophage M2 polarization. Therefore, defining the complete mechanism of PRMT6-mediated macrophage M2 polarization could lead to the development of novel therapeutic strategies to treat lung cancer.

#139

DNA damage and immunoprofiling with highly multiplexed tissue immunofluorescence (t-CycIF) in high-grade serous ovarian cancer.

Anniina Farkkila,1 Sameer S. Chopra,2 Jia-Ren Lin,2 Zoltan Maliga,2 Bose Koruchupakkal,1 Kyle C. Strickland,1 Brooke E. Howitt,1 Sandro Santagata,3 Ursula A. Matulonis,1 Kevin Elias,4 Elizabeth M. Swisher,5 Panagiotis A. Konstantinopoulos,1 Peter Sorger,2 Alan D. D'Andrea1. 1 _Dana Farber Cancer Institute, Brigham and Women's Hospital, Harvard Medical School, Boston, MA;_ 2 _Harvard Medical School, Boston, MA;_ 3 _Harvard Medical School, Brigham and Women's Hospital, Boston, MA;_ 4 _Brigham and Women's Hospital, Harvard Medical School, Boston, MA;_ 5 _Washington University, Seattle, WA_.

Immune checkpoint blockade (ICB) has emerged as a new promising therapeutic approach in multiple cancers, however, the responses to single-agent ICBs have been modest in high-grade serous ovarian cancer (HGSOC). Preclinical- and early clinical data show promising efficacy of combining DNA damaging agents with immunotherapy. An improved understanding of the interplay between DNA damage in cancer cells and anti-tumor immune responses may therefore accelerate the development of rational drug combinations and identify predictive biomarkers. The majority of HGSOC are deficient in homologous recombination (HR) DNA repair, and use alternative, error-prone DNA repair pathways, that have been shown to be associated with increased immune recognition (Strickland et al, 2016). Compelling evidence has shown that DNA damaging agents increase the expression of immune-regulatory genes, such as MCH class I antigens, and interferons in HR deficient tumors. Further, HR deficient tumor exhibit an increased response to ICBs, and DNA damage-driven activation of interferon signalling has been shown to overcome resistance to ICBs (Wang et al, 2016). To reveal the relationship between intrinsic and treatment-induced DNA damage and the HGSOC immune microenvironment we are employing a novel, high-multiplex tissue cyclic immunofluorescence (t-CycIF) platform (Lin et al, 2016& 2017) allowing for the simultaneous detection of up to 60 different antigens at single cell resolution. Utilizing t-CycIF we are in the process of profiling the DNA damage and immune responses in three unique HGSOC clinical cohorts including i) tumors with inherent DNA repair deficiencies ii) pre- and post DNA damaging therapy iii) tumors collected in an innovative clinical trial combining Poly- ADP Ribose Polymerase inhibitor (PARPi) Niraparib and ICB Pembrolizumab. Using this high-dimensional, quantitative data we are mapping the abundance, spatial arrangement and functional state of cancer cells, immune cells, and stroma in the HGSOC microenvironment. The highly multiplexed t-CycIF data are processed with established computational algorithms and correlated with clinical outcomes. Our preliminary data shows that t-CycIF sensitively captures immune cell subpopulations, tumor heterogeneity and DNA damage in HGSOC. We anticipate that t-CycIF could accelerate the development of rational strategies for combining DNA damaging agents with immunotherapy to ultimately improve the treatment and outcomes of patients with ovarian cancer.

#140

A novel signature of mesenchymal stromal cells in high-grade serous ovarian carcinoma.

Ali Hussain,1 Veronique Voisin,1 Stephanie Poon,1 Jalna Meens,2 Julia Dmytryshyn,2 Gary Bader,1 Benjamin Neel,3 Laurie Ailles2. 1 _University of Toronto, Toronto, Ontario, Canada;_ 2 _Princess Margaret Cancer Centre, Toronto, Ontario, Canada;_ 3 _Laura and Isaac Perlmutter Cancer Center, New York, NY_.

High grade serous ovarian cancer (HGSOC) is the most common and lethal subtype of ovarian cancer. Many cancers, including HGSOC, contain a "stromal" component comprised of cancer-associated fibroblasts (CAFs), immune cells, and blood vessels, as well as their secreted factors and extracellular matrix. This complex microenvironment plays a significant role in promoting tumor growth, therapy resistance, and invasion. The majority of studies of CAFs utilize cells cultured and passaged in vitro under crude conditions to maintain their viability. Such conditions alter the original phenotype of the cells and may select for specific subpopulations to grow out. To facilitate direct isolation of CAFs from primary tumor tissues, we screened a panel of antibodies to identify cell surface proteins that are uniquely expressed on CAFs. We discovered CD49e as a mesenchymal marker and then validated its specificity to CAFs through immunohistochemistry on tissue sections obtained from HGSOC patients. The discovery of CD49e as a CAF-specific cell surface marker facilitated fluorescence-activated cells sorting (FACS) to isolate CAFs directly from primary tumors, allowing us to avoid in vitro manipulation and to characterize their transcriptional and functional profiles in the primary setting. We next performed transcriptional profiling of primary CAFs isolated from 12 HGSOC patients and found that CAFs fall into two subgroups with unique gene expression signatures. A cell surface protein, fibroblast activation protein (FAP) is the defining marker for separating the two subgroups (FAP-Hi and FAP-Lo). The FAP-Hi subgroup possesses the classical gene signature of CAFs that is reported in the literature. When we isolated FAP-Hi and FAP-Lo cells and placed them into classical CAF growth conditions in vitro, both cell types had mesenchymal features, but FAP-Hi cells grow faster. Thus under the classical conditions for growing CAFs, FAP-Hi cells have a growth advantage, outcompeting FAP-Lo cells and becoming the dominant cells used for experimentation. Flow cytometry for FAP indicates that both CAF subtypes co-exist in every tumor, but their ratio varies from one patient to another. Patients whose tumors are dominated by FAP-Hi CAFs have worse clinical outcome than patients whose tumors are dominated by FAP-Lo CAFs. Thus, we have functionally characterized the role of FAP-Hi and FAP-Lo fibroblasts in the context of HGSOC. We have shown that FAP-Hi fibroblasts support tumor proliferation and invasion of ovarian cancer cells in vitro and in vivo. On the other hand, FAP-Lo cells suppress cancer cell proliferation and invasion in vitro and in vivo. 

### Targeting Cancer Stemness

#141

HEY1-mediated inhibition of glioma stem cell proliferation is associated with restoration of glioma stem cell division asymmetry and transcriptional repression of PDGFRA.

Damian A. Almiron Bonnin, Jaclyn Sullivan, Mark A. Israel, Matthew Havrda C. Havrda. _Geisel School of Medicine at Dartmouth, Lebanon, NH_.

Emerging evidence suggests that deregulation of cell-fate regulatory mechanisms in subpopulations of stem-like tumor cells can contribute to the initiation, progression, and recurrence of glioma. In this study, we report that Hey1 expression suppresses the expansion of oligodendroglial progenitor (OPC)-like glioma stem cells, which are a putative cell of origin for high-grade gliomas. We found that Hey1 expression reduced the proliferation of primary neural progenitor cells isolated from mice without the tumor suppressor Tp53, and OPC-like glioma stem cell cultures isolated from a mouse model of glioma driven by an EGFR gain-of-function mutation and Tp53 loss (v-ErbB/p53-/-). Further analysis of these cultures revealed that Hey1-mediated reductions in proliferation were associated with suppression of self-renewal and a reduced percentage of symmetric cell divisions. Mechanistically, we found that HEY1 directly bound to novel promoter elements and repressed the mRNA expression of a key marker and mitogenic driver of OPCs, the PDGF Receptor A (Pdgfra). Furthermore, Hey1-mediated of Pdgfra expression was associated with inactivation of the canonical PDGF/PDGFRA downstream target, extracellular signal-regulated kinases (ERK), in v-ErbB/p53-/- glioma stem-like cells. Key findings made in the murine system were validated in patient-derived glioma stem-like cells. Moreover, analyzing publicly available databases, we found that the subset of glioma patients with high levels of HEY1 expression had improved survival compared to patients with tumors expressing low levels of HEY1. Further supporting a role for HEY1 in the regulation of the oligodendroglial lineage, ontologic analysis revealed that the subset of patients with high levels of HEY1 expression were also characterized by the expression of gene sets associated with oligodendroglial differentiation. These cellular, molecular and genetic findings provide support for a model in which HEY1 functions to suppress glioma progression by inhibiting the expansion of glioma stem cells.

#142

cdk5 inhibition promotes neurogenesis while reducing self-renewal of brain tumor stem cells.

Subhas Mukherjee, Cheryl Olson, Daniel Brat. _Northwestern University, Chicago, IL_.

Cancer stem cells are biologically potent and support tumor growth, in part by deregulating asymmetric stem cell division. Asymmetric cell division of stem cells is a type of mitotic division where a stem cell generates one stem and one differentiated cell. Cancer stem cells, however, tend to generate more stem-like dividing cells to promote neoplasm. To uncover mechanisms and potential therapeutic targets relevant to self-renewal in human glioma stem cells (GSCs), we performed a genetic screen for kinases that reverse the tumor phenotype of our novel adult Drosophila brain tumor model and found that knockdown of the brain-specific dCdk5 was highly effective in suppressing the self-renewing stem cell components. A highly specific CDK5 inhibitor is promoting neurogenesis among these fly brain cancer stem cells. These cancer stem cells, although they express glial markers in tumors, do not differentiate into glia upon CDK5 suppression. Additionally, while the tumor generation in adult flies completely disrupts the mushroom body, the main cognitive center in adult fly brain, the CDK5 inhibition partially rescues the mushroom body and extends life span of these flies. dCdk5 and its human ortholog CDK5 (79% identity) are aberrantly activated in some forms of cancer. We demonstrate that GBMs highly express CDK5 mRNA and protein, likely related to copy number gains of chromosome 7, with strong positive correlation with stem cell markers and marked effect on tumor-propagating properties. Using human GSCs isolated from tumor spheres and mouse xenografts, we found that CDK5 directly phosphorylates CREB1 independent of cAMP and stops stem cell renewal. These data together show that CDK5 inhibition not only can attenuate self-renewal properties of brain tumor stem cells, but also can promote neurogenesis, which could potentially become a better treatment strategy for patients and improve their cognitive function as well.

#143

**MAPK pathway blockade effects on glioma stem cells and immunotherapy in BRAF** V600E **mutant gliomas.**

Stefan Grossauer,1 Katharina Koeck,1 Malek Chouchane,2 Joanna J. Phillips,2 Claudia K. Petritsch,2 Theodore Nicolaides2. 1 _Knappschaftsspital Bochum, Bochum, Germany;_ 2 _UCSF, San Francisco, CA_.

BRAFV600E mutations are frequent in pediatric gliomas. We previously showed antitumor activity when using BRAF and/or MEK inhibitors against BRAFV600E glioma syngeneic grafts (Grossauer et al., Oncotarget 2016;7:775839). While early clinical trial reports suggest an encouraging response rate to BRAFV600E inhibitors alone (Burger et al., Oncol Rep 2017;38:3291) and in combination with MEK inhibition (Brown et al., CNS Oncol 2017:6:291), resistance has been reported in BRAFV600E mutant melanoma. Moreover, we reported that resistance to BRAFV600E inhibitor monotherapy occurs in mouse models and is driven by stem-like cells (Lerner et al., Cancer Res 2015;75:5355). Rapid reactivation of MEK pathway and prosurvival signaling via EGFR have been implicated as mechanisms for acquired resistance (Yao et al., Oncotarget 2017;8:583). It is just unclear why stem-like cells survive treatment better than non-stem like tumor cells and how they respond to additional therapies, such as MEK inhibition but also immune checkpoint inhibition. Immunocompetent, syngeneic FVB/N mice were intracranial-engrafted with luciferase-modified BRAFV600E mutant CDKN2A-deficient murine glioma cell line 2341luc and treated with BRAFV600E and/or MEK inhibitors, either alone or in combination, and either with or without PD-L1 (Clone 10F.9G2) and CTLA-4 (Clone 9D9) mAbs. While the combination of BRAF and MEK inhibitors significantly increased the median survival from 46 to 70 days (P <0.05), additional treatment with PD-L1 and CTLA-4 mAbs further increased survival to a median of 150 days (P <0.001). Notably, PD-1 and CTLA-4 blockade, alone, only marginally increased survival to 60.5 days (P <0.05). To understand the mechanism underlying the impact on survival, we examined the tumor microenvironment using CyToF as well as T cell and CD133+ cancer stem cell composition using flow cytometry in treated tumors. Our preliminary findings showed that the survival benefit of quadruple treatment associates with an increased frequency of CD8+ cells and a decrease in T regulatory cells and cancer stem cells. In summary, we report that in BRAFV600E mutant gliomas when MAPK pathway and immune checkpoint blockades are combined, an immune profile indicative of productive tumor immunity is observed. CyToF analyses of treated and untreated tumors and RNA sequencing analyses of stem and non-stem like tumor cells from untreated and BRAFV600E inhibitor treated mice are on the way. These data provide rationale for further understanding this therapeutic approach, as well as the potential for clinical consideration in the near future.

#144

Dasatinib enhances the effects of paclitaxel on chemotherapy-resistant breast cancer through targeting breast cancer stem cells.

Jun Tian, Chieh Lo, Fatmah Al Raffa, Meiou Dai, Jean-Jacques Lebrun. _McGill University, Montreal, Quebec, Canada_.

Many patients with triple negative breast cancer (TNBC) exhibit poor prognosis and are at high risk of tumor relapse and metastasis due to the resistance to conventional chemotherapy. These tumor recurrences have been attributed to the presence of breast cancer stem cells (BCSCs), a subpopulation of breast cancer cells that possesses stem-like properties and displays resistance to chemotherapy. Therefore, targeting BCSCs is a priority to overcoming chemotherapy failure in TNBCs. We generated chemotherapy-resistant TNBC cells through cyclic treatments with paclitaxel (pac). The pac-resistant cells displayed increased self-renewal potential compared to the parental TNBC cells, higher percentage BCSCs and increased phosphorylation of Src kinase at Tyr416. After performing a kinase library screening on BCSCs in chemotherapy-resistant cells, we identified dasatinib, a Src kinase family inhibitor, potently reduced BCSC self-renewal and two BCSC populations (ALDH+ and CD24lowCD44high) as well as phosphor-Src. We also demonstrated dasatinib to block pac-induced BCSC enrichment and Src activation in the parental TNBC cells. Interestingly, we found that dasatinib induced an epithelial differentiation of pac-resistant cells, resulting in their enhanced sensitivity to paclitaxel. The combination treatment of dasatinib and paclitaxel not only decreased the proportion of BCSCs and their self-renewal capacity but also synergistically reduced cell viability of pac-resistant cells. In vivo studies further demonstrated the effectiveness of dasatinib/paclitaxel combination treatment in inhibiting breast tumor growth. All these data suggest that dasatinib is a promising anti-BCSC agent and can be used in combination with paclitaxel to overcome chemotherapy resistance in TNBCs.

#145

Pharmacologic inhibition of SIAH2 stabilizes DYRK2 and inhibits survival and self-renewal in chronic myeloid leukemia (CML) leukemic stem cells.

Chun Shik Park,1 Ye Shen,1 Andrew Lewis,1 Koramit Suppipat,1 Monica Puppi,1 Julie Tomolonis,1 Taylor Chen,1 Paul Pang,1 Toni-Ann Mistretta,1 Leyuan Ma,2 Michael Green,3 Rachel Rau,1 Daniel Lacorazza1. 1 _Baylor College of Medicine, Houston, TX;_ 2 _MIT, Boston, MA;_ 3 _University of Massachusetts Medical School, Worcester, MA_.

Leukemia stem cells (LSCs) are a rare population able to sustain and recapitulate leukemia through a poorly understood mechanism of self-renewal. Because more than half of patients relapse after the cessation of TKI therapy, it is clear that a cure is not possible with TKIs alone, and LSC-specific drugs are urgently needed to simultaneously eliminate bulk leukemia with TKIs and LSCs. Here we report that KLF4 promotes disease progression in chronic myeloid leukemia (CML) by repressing an inhibitory mechanism in LSCs that can be activated with small molecules. Deletion of the Klf4 gene severely abrogated maintenance of BCR-ABL(p210)-induced CML by impairing survival and self-renewal in LSCs whereas increased self-renewal was observed in hematopoietic stem cells during serial transplantation. Mechanistically, KLF4 represses the Dyrk2 gene and thus loss-of-KLF4 resulted in elevated levels of the DYRK2 kinase in LSCs, which was associated with p53-mediated apoptosis and inhibition of self-renewal through depletion of c-Myc protein. Supporting this model, stabilization of DYRK2 protein, by inhibiting the ubiquitin E3 ligase SIAH2 with vitamin K3, promoted apoptosis in a panel of CML cell lines (K562, KU-812, and KCL-22) by inducing DYRK2 expression. Knocking out the DYRK2 gene in K562 cells by Cas9/CRISPR abrogated cytotoxicity induced by vitamin K3 and the presence of p53 significantly lowered IC50. In vivo treatment of CML mice diminished the number of LSCs evaluated in secondary transplants. In humans, vitamin K3 induced apoptosis in bone marrow cells from CML patients but not in healthy individuals by inducing DYRK2, p53 phosphorylation, and c-Myc depletion; furthermore, vitamin K3 abrogated capacity of CD34+ cells to generate colonies in methylcellulose only in CML. Altogether, our results suggest that DYRK2 is a molecular checkpoint controlling both p53 and c-Myc mediated regulation of survival and self-renewal in CML LSCs that can be activated pharmacologically.

#146

KDM1A inhibition is effective in reducing stemness and treating triple-negative breast cancer.

Mei Zhou,1 Suryavathi Viswanadhapalli,1 Mengxing Li,1 Xiaonan Li,1 Takayoshi Suzuki,2 Rajeshwar R. Tekmal,1 Andrew Brenner,1 Ratna K. Vadlamudi,1 Gangadhara R. Sareddy1. 1 _University of Texas Health, San Antonio, TX;_ 2 _Kyoto Prefectural University of Medicine, Kyoto, Japan_.

Background: Triple-negative breast cancer (TNBC) lacks targeted therapy, exhibits an aggressive clinical course and contributes to a disproportional share of the overall mortality from breast cancer. TNBC is characterized by significant heterogeneity, a high proportion of cancer stem cells (CSCs), and resistance to conventional therapies. CSCs are highly tumorigenic, spared by chemotherapy, sustain the tumor growth and enhance the tumor recurrence after conventional therapies. Therefore, eradication of CSCs is critical for the development of efficient therapeutic strategies for TNBC. The lysine-specific histone demethylase 1A (KDM1A) is highly expressed in TNBC and is essential for maintenance of CSCs. However, the role of KDM1A in CSC function remains unknown. The objective of this study is to examine the role of KDM1A in CSCs and test the utility of KDM1A inhibitors using in vivo models.

Methods: We have generated KDM1A knockout cells using the CRISPR/Cas9 system and knockdown cells using KDM1A shRNA lentiviral particles. The effect of KDM1A inhibitors on the cell viability was examined by MTT assays. The effect of KDM1A knockdown and KDM1A inhibition using novel inhibitor NCD-38 on stemness and self-renewal of CSCs was examined using mammosphere formation and extreme limiting dilution assays. Global transcriptional changes modulated by KDM1A inhibition were examined by RNA-sequencing. Mechanistic studies were conducted using Western blot, RT-qPCR, and immunoprecipitation (IP) analysis. Mouse xenograft and patient-derived xenograft models were used for preclinical evaluation of the KDM1A inhibitor.

Results: Western blot and RT-qPCR analysis demonstrated that KDM1A is highly expressed in CSCs compared to non-CSCs. Cell viability assays using various KDM1A inhibitors revealed that novel KDM1A inhibitor NCD-38 is highly potent in reducing cell viability of TNBC cells compared to other inhibitors. NCD-38 effectively reduced the cell viability of therapy-sensitive and therapy-resistant TNBC cells. Flow cytometry analysis revealed that KDM1A knockout or KDM1A inhibitor treatment reduced the CSC population on TNBC cells. Further, KDM1A inhibition reduced the cell proliferation, mammosphere formation and self-renewal ability of CSCs. RNA-seq analysis revealed that KDM1A inhibition modulated pathways that are involved in the stemness of TNBC. IP analysis and GST pulldown assays revealed that KDM1A interacts with TLX and KDM1A inhibition modulated TLX target genes. Knockout of KDM1A reduced the in vivo tumor growth in xenograft models and KDM1A inhibitor NCD-38 treatment significantly reduced the tumor growth in patient-derived xenograft (PDX) models.

Conclusions: Our results establish KDM1A inhibitors as a novel class of drugs for treating TNBC and contribute to the development of a highly promising therapy that can significantly extend patient survival with minimal toxicity.

#147

Targeting USP22 suppresses tumorigenicity and enhances sensitivity to cisplatin in cancer-initiating cells from lung adenocarcinoma through downregulating ALDH1A3.

Keqiang Zhang, Xinwei Yun, Jinhui Wang, Melisa Bonner, Lu Yang, Jun Wu, Dan J. Raz. _City of Hope, Duarte, CA_.

We previously found that loss of monoubiquitination of histone H2B (H2Bub1) was associated with poor differentiation and enhanced malignancy of lung adenocarcinoma. Herein, we investigated the expression and roles of USP22, an H2Bub1 deubiquitinase, in cancer-initiating cells (CICs) with stem cell-like characteristics from primary lung adenocarcinoma. CICs from patient-derived cancer tissues were isolated and characterized, and the association of USP22 with CICs was investigated using flow cytometry, aldehyde dehydrogenase (ALDH) activity, Western blot, and fluorescence microscope assays. The impact of USP22 on stem cell-like characteristics and cisplatin resistance in CICs was examined by in vitro tumorsphere formation, in vivo transplantation, and apoptosis assays. We identified that USP22 is predominantly expressed in CICs, a subpopulation of cells with high expression of CD133 stem cell biomarker. The expression of USP22 in CICs is dramatically reduced upon serum-induced differentiation. Moreover, knockdown of USP22 significantly suppressed in vitro tumorsphere formation and in vivo xenograft growth in NOD-SCID mice. Notably, USP22 is dramatically elevated in tumorsphere cells that survived cisplatin treatment, while knockdown of USP22 significantly sensitizes tumorsphere cells to cisplatin. Interestingly, ALDH1A3, a dominant isoform of ALDH implicated in enhancing cisplatin resistance in lung adenocarcinoma, is significantly downregulated upon knockdown of USP22 in tumorsphere cells. Furthermore, knockdown of ALDH1A3 significantly sensitizes tumorsphere cells to cisplatin. Therefore, our data indicate that USP22 plays critical roles in tumorigenicity and cisplatin resistance in lung adenocarcinoma, and targeting USP22 may represent a potential therapeutic approach to eliminate CICs in lung adenocarcinoma through downregulation of ALDH1A3 expression.

#148

Canonical Wnt activation as a therapeutic strategy in pediatric medulloblastoma.

Sheila Kumari Singh, Branavan Manoranjan, Anna Dvorkin-Gheva, Chitra Venugopal, Steven Moreira, Michelle Kameda-Smith, Minomi Subapanditha, Ashley Adile, David Bakhshinyan, Neil Savage, Blake Yarascavitch, Olufemi Ajani, Adam Fleming, Bradley Doble. _McMaster University, Hamilton, Ontario, Canada_.

Brain tumors represent the leading cause of childhood cancer mortality, of which medulloblastoma (MB) is the most frequent malignant pediatric brain tumor. Current molecular subgroups of MB recognize distinct disease entities of which activated Wnt signaling (monosomy 6, exon 3 mutations in CTNNB1, and Wnt gene signature) is associated with a distinct subgroup and the best overall outcome. In contrast, only non-Wnt MBs are characterized by metastatic disease, increased rate of recurrence, and poor overall survivorship. Given the excellent clinical outcome in patients with Wnt-driven MB, we aimed to convert treatment-resistant MB subgroups into an ostensibly benign tumor through selective activation of the canonical Wnt pathway. Initial characterization of patient-derived Wnt and non-Wnt MB lines demonstrated a significant reduction in in vitro self-renewal and proliferative capacity of Wnt MBs. This was further validated by RNA-seq, which identified a marked reduction in the expression of stem cell self-renewal genes Bmi1 and Sox2 in Wnt MBs compared to non-Wnt MBs. Further, Wnt MB-derived xenografts maintained a significant increase in overall survival compared to non-Wnt MB xengrafts, further highlighting the protective nature of activated Wnt signaling in MB. Activated Wnt signaling by way of small molecule Wnt agonists in treatment-refractory MBs resulted in decreased in vitro self-renewal and expression of self-renewal genes, Bmi1 and Sox2. In order to validate the therapy-sensitive nature of Wnt-activated cells, we developed stable patient-derived lines containing a 7XTOPFlash reporter for endogenous Wnt signaling. Rare subclonal Wnt-active cells demonstrated a reduced self-renewal and tumor-initiating capacity through in vivo limiting dilution assays when compared to bulk Wnt-inactive cells. The therapeutic relevance of these findings were demonstrated with an in vivo survival advantage in those mice with orthotopic injections of cells with endogenous Wnt activity when compared to xenografts generated from Wnt-inactive cells. To develop a rationale clinical therapeutic, we used a novel substrate-competitive peptide inhibitor for GSK. Treatment with our peptide inhibitor showed a significant reduction in tumor burden with a corresponding increase in survival of patient-derived tumors that were otherwise treatment-resistant. The clinical utility of our findings is further supported by our analysis of integrated genomics data from 763 primary MBs, in which a validated Wnt gene signature was found to predict improved survivorship among children with poor-outcome and metastatic MBs. Our work establishes activated Wnt signaling as a novel treatment paradigm in childhood MB, identifies a rationale therapeutic approach for recurrent MB, and provides evidence for the context-specific tumor suppressive function of the canonical Wnt pathway.

#149

Tumor suppression by regulator of asymmetric cell division in glioma.

Mathieu Daynac, Malek Chouchane, Alex Valenzuela, Claudia K. Petritsch. _UCSF, San Francisco, CA_.

We previously discovered that oligodendrocyte progenitor cells (OPC) undergo asymmetric cell division and symmetric, self-renewing divisions at an equal rate, whereby they may establish homeostasis. The proteoglycan and OPC marker NG2 plays a critical role in establishing asymmetric cell division (1). Studies in genetically engineered mouse models identified OPC as a putative cellular origin of human glioma (2,3). Studies also showed that premalignant OPC (aka glioma precursor) increase rates of symmetric, self-renewing divisions at the expense of asymmetric division, suggesting that tipping the balance towards symmetric divisions might disrupt homeostasis and lead to cancer. The objective of this project is to gain functional insights into asymmetric division to assess its role in maintaining tissue homeostasis and tumor suppression. Using our novel FACS-based approach we were able to identify that several conserved regulators of asymmetric division, including the WD40 protein Lethal giant larvae1 (Lgl1) protein, are upregulated in asymmetric versus symmetric dividing OPC. We genetically enforced deletion of Lgl1 in OPC conditionally by injecting tamoxifen into triple transgenic mice obtained by breeding Lgl1fl/fl NG2CreERT2 and red fluorescence Cre reporter mice. Cre-conditional deleted Lgl1 OPC were analyzed for changes in cell division mode, proliferation rate and differentiation in vivo and ex vivo. Whole-transcriptome analyses of Lgl1-deleted OPC followed by Ingenuity Pathway Analyses revealed significant changes in several pathways, including in receptor-mediated endocytosis. Functional analyses were conducted to test if Lgl1 regulates endocytosis in OPC. Our results show that Lgl1 deletion cooperates with CDKN2A homozygous deletion to form glioma from OPC, albeit with long latency. Lgl1-deleted OPC show increased rates of symmetric, self-renewing division and proliferative rate at the expense of asymmetric divisions and differentiation. Lgl1 regulates endocytosis of NG2 and by routing NG2 complex to the lysosome for degradation, Lgl1 may establish the asymmetric NG2 distribution, which is critical for asymmetric division. Taken together, our data identified a conserved regulator of asymmetric OPC division and showed that loss of such a regulator not only disrupts asymmetric divisions but also leads to premalignant changes and neoplastic transformation and the accumulation of glioma precursor. Investigations into the mechanistic details of asymmetric division and the potential role for endocytosis in cell fate determination of OPC and gliomagenesis are ongoing.

References:

1. Sugiarto et al., Cancer Cell 2011;20:328.

2. Persson et al., Cancer Cell 2010:18:229.

3. Liu et al., Cell 2011:146:209.

#150

Elucidating the molecular mechanisms of cutaneous melanoma development from melanocyte stem cells.

Hyeongsun Moon, Leanne Donahue, Jerry Zhu, Andrew White. _Cornell University, Ithaca, NY_.

Cutaneous melanoma is the most lethal form among all skin cancers. Multiple genetic etiologies have been identified in primary and metastatic tumors, however, the cellular origin for cutaneous melanoma and their related molecular mechanisms required for early steps of melanoma initiation have not been well defined. Although great progress has been made in the development of targeted therapies for malignant melanoma, knowledge on the factors that can cause tumor formation would provide valuable insight for melanoma prevention and control. Here, our study aims to determine potential means of prevention by identifying the extrinsic environmental factors and cellular conditions responsible for melanoma initiation particularly from tumor-competent melanocyte stem cells. In the study, adult stem cells have been discovered as a compelling candidate for melanoma formation. We also found that quiescent stem cells are relatively unable to develop melanocytic tumors. On the other hand, environmental and physiological stresses can significantly alter the status of melanocyte stem cells which in turn causes melanoma development. Screening the genetic changes that occur in the melanoma-competent stem cells in tumor initiation stages can help to elucidate which molecular pathways may cause the formation of cutaneous melanoma. Our data will provide a new framework for the early initiation steps of cutaneous melanoma from quiescent but tumor-prone stem cells, with the ultimate goal to find better preventative measurements and targeted therapy for cutaneous melanoma.

#151

Phenotypic selection screening reveals Annexin A2 as a pancreatic cancer stem cell target.

Elaine M. Hurt, Matt Flynn, Paul Hynes, M. Carla Cabrera, Suneetha B. Thomas, Lilian van Vlerken-Ysla, Louise Slater, Alan Sandercock, Steven Rust, Ralph Minter, Ronald Herbst. _MedImmune, LLC, Gaithersburg, MD_.

Cancer stem cells (CSCs) represent an important clinical entity given their role in tumor initiation, metastasis and patient relapse. Phenotypic selection allows for identification of novel antigens important in biological processes. Therefore, we undertook a phenotypic selection campaign using a designed ankyrin-repeat (DARPin) library to identify new cancer stem cell (CSC) targets. CSCs generated from late-stage pancreatic cancer patient-derived xenograft (PDX) models were used to pan a designed Ankyrin repeat (DARPin) library. The most prevalent target identified in this screen was Annexin A2 (ANXA2). ANXA2 is a member of the Annexin family that has diverse roles in cancer including proliferation and invasion and is widely expressed in cancer. Furthermore, expression of ANXA2 is linked to poor prognosis in pancreatic cancer. Targeting of Annexin A2 on the surface of pancreatic cancer cells using DARPins resulted in decreased frequency of CSCs both in vitro and in vivo in several pancreatic patient-derived xenografts. ANXA2 DARPins also reduced proliferation and tumor growth of HPAC cells both in vitro and in vivo. Mechanistically, we found that blocking ANXA2 resulted in decreased IL6 and IL8 secretion, two cytokines that have been linked to CSC expansion and survival. Additionally, we found that pharmacological blockade of ANXA2 resulted in decreased plasmin activation, a pathway that has been linked to dissemination of cancer cells. Together our data suggests that targeting of cell surface ANXA2 is effective at reducing CSCs and could be a novel target for the treatment of pancreatic cancer.

#152

An epigenetic mechanism of p53 inactivation in colorectal cancer stem cells as a potential therapeutic target.

Veronica Veschi, Alice Turdo, Matilde Todaro, Giorgio Stassi. _University of Palermo, Palermo, Italy_.

Cancer is one of the most complex biological systems, in which genetic, epigenetics microenvironment and heterogeneity are the key words to decipher the "cancer code". The existence of sub-clonal tumor populations and the development of resistance mechanisms led to a failure of cutting-edge diagnostic and therapeutic approaches. Recently it has become increasingly clear that the assumption one mutation-one gene -one drug of targeted therapy is very limited. How to overcome the limitations of the precision medicine? We need to consider tumor complexity beyond the single-gene version. Complexity is growing exponentially, and the targeted therapy generated resistance mutations. In particular, the epigenetic regulation in colon cancer and other cancer is probably one of the principal causes of this complexity. Cancer stem cells (CSCs) are a dynamic population that undergoes genetic and epigenetic changes leading to phenotypic switching (i.e. from an epithelial to a mesenchymal status), thus rendering even more difficult to treat tumors. The increasing number of recently discovered mutations in epigenetic regulators shed new light on the importance of epigenetic dysregulation in tumor initiation and in the biology of CSCs. SETD8 is a histone methyltransferase, an epigenetic enzyme that besides other non-histone targets, monomethylates p53 on lysine 382 (p53K382me1), attenuating the pro-apoptotic and growth arrest functions of p53, a pivotal tumor suppressor critical for maintaining genomic stability and preventing cancer development. Recently, we identified SETD8, the H4K20me1 methyltransferase, as a crucial epigenetic regulator of growth and differentiation in neuroblastomas (NBs). Moreover, we found that SETD8 mRNA expression levels are higher in samples derived from colorectal cancer (CRC) patients compared with healthy patients. Interestingly, p53K382me1 is highly expressed in freshly isolated CSCs derived from CRC patients and, in particular, at the tumor front of invasion, indicating that this epigenetic inactivation may be a specific event occurring in CR-CSCs. Western blot analysis showed that 8/11 (72%) p53 WT CR-CSCs express p53K382me1 and at least 50% of these show a high p53K382me1/p53 ratio, as determined by densitometry. Interestingly, in two commercially established cell lines that present a differentiated status, p53K382me1 is nearly undetectable, despite the high levels of total p53 protein. Moreover, p53K382me1 is highly expressed in tumor leading edge CR-CSCs CD44v6+, a CSC marker required for metastatic potential. Immunofluorescence analysis demonstrates that 80% of CSCs in the tumor leading edge express p53K382me1 compared with 15% of cells in intra-tumor tissue. This suggests that p53 methylation may play a synergistic role in the tumorigenic and metastatic capabilities of CSCs.

#153

A genome-wide CRISPR screen in hESC uncovers JNK pathway mediated inhibition of human definitive endoderm formation.

Qing Li, Danwei Huangfu. _Memorial Sloan Kettering Cancer Center, New York, NY_.

Human embryonic stem cells (hESCs) are uniquely suitable for interrogating human development and birth defects with high-throughput genetic manipulation. Using CRISPR/Cas, we have conducted a forward genetic screen for genes that regulate the formation of human definitive endoderm (DE), the first step towards the generation of cells in respiratory and gastrointestinal organs. Our screen identified both known and unknown regulators of endoderm differentiation, including a previously unrecognized inhibitory mechanism mediated by the JNK/JUN pathway. JNK inhibitor treatment significantly improves the efficiency of hESC differentiation to endoderm, leading to more efficient creation of endoderm-derived pancreatic and lung lineage cells. The AP1 transcription factor JUN co-occupies ESC enhancers with SMAD2/3 and OCT4, and impedes the re-configuration of the SMAD2/3 chromatin-binding landscape during the ESC-to-DE transition. Thus the hESC-based CRISPR screen enables unbiased interrogation of the functional genome during human development, which can inform hESC directed differentiation for regenerative medicine and disease modeling.

#154

Inhibition of WEE1 expression reduces lipid metabolism and stemness in hepatocellular carcinoma.

Keon Uk Park,1 Min Ji Ko,2 Ilseon Hwang,2 Hun-Mo Ryoo,3 Yun-Han Lee2. 1 _Keimyung Univ. Dongsan Medical Ctr., Daegu, Republic of Korea;_ 2 _Keimyung Univ. School of Medicine, Daegu, Republic of Korea;_ 3 _Daegu Catholic University, Daegu, Republic of Korea_.

Background: Classical therapeutic regimens predominantly target proliferating cells, which are unlikely to be cancer stem cells (CSCs). Similarly, new generation therapies, e.g. sorafenib for hepatocellular carcinoma (HCC), do not seem to target CSCs as evidenced by frequent tumor relapse and resistance after therapy. Thus, identification and characterization of signaling pathways and biomarkers associated with CSC biology are therefore priorities for developing new paradigms of molecular cancer therapeutics. Increase of WEE1 kinase activity through an epigenetic regulation plays an important role in the development of HCC. However, the functional role of WEE1 in HCC progression remains to be clarified.

Methods: Human HCC cell lines were transfected with WEE1 siRNA and tested for growth inhibition, apoptotic induction, molecular changes in both RNA and protein levels, and changes in CSC phenotype using various methods such as MTS, FACS, microscopic analysis, Real-time PCR, Western blotting, sphere forming assay. To obtain insights into the molecular changes in response to WEE1 knockdown, global changes in gene expression were examined using RNA sequencing.

Results: Here we demonstrated that WEE1 siRNA silencing caused inhibition of HCC cell growth through blockade of cell cycle progression and induction of apoptosis. The anti-proliferative effects were driven by a subset of molecular alterations including the upregulation of cdk inhibitor p21 and the downregulation of AKT1, CDK2, cyclin B1 (CCNB1), PARP1 and GPAM which are functionally involved in control of cell cycle, apoptosis and lipid metabolism. Wee1 silencing in tumor cells also resulted in a strong inhibition of lipogenesis (SREBP1C, FAS) and caused a marked decrease in fat accumulation. Of importance, knockdown of WEE1 dramatically reduced the portion of liver CSC population through co-downregulation of cancer stemness genes and weakened the capacity of sphere formation and the ability of cancer cell migration. Systemic delivery of a modified WEE1 siRNA encapsulated in lipid nanoparticles significantly inhibited human HCC growth in murine xenograft models, and increased mice survival. Our findings suggest that the epigenetic modifier WEE1 functionally involve to HCC lipid metabolism and CSC-like phenotype maintenance and that molecular targeting of WEE1 may be an effective systemic therapy for prevention of tumor recurrence via elimination of CSCs in liver tumor microenvironment.

#155

KDM4 inhibition targets breast cancer stem-like cells.

Jochen Maurer. _University Hospital Aachen/RWTH, Aachen, Germany_.

Traditional treatments for breast cancer fail to address therapy-resistant cancer stem-like cells that have been characterized by changes in epigenetic regulators such as the lysine demethylase KDM4. Here we describe an orally available, selective and potent KDM4 inhibitor (QC6352) with unique preclinical characteristics. To assess the antitumor properties of QC6352, we established a method to isolate and propagate breast cancer stem-like cells (BCSC) from individual triple-negative tumors resected from patients after neoadjuvant chemotherapy. Limiting-dilution orthotopic xenografts of these BCSC regenerated original patient tumor histology and gene expression. QC6352 blocked BCSC proliferation, sphere formation and xenograft tumor formation. QC6352 also abrogated expression of EGFR, which drives the growth of therapy-resistant triple-negative breast cancer cells. Our findings validate a unique BCSC culture system for drug screening and offer preclinical proof of concept for KDM4 inhibition as a new strategy to treat triple-negative breast cancer.

#156

Identifying novel therapeutics to inhibit the WNT self-renewal pathway in leukemia stem cells.

Meghan Green Haney, Chunming Liu, Jessica Blackburn. _University of Kentucky, Lexington, KY_.

The relapse rate of pediatric Acute Lymphoblastic Leukemia (ALL) is 15-20%, and these patients have limited treatment options and a poor prognosis. Relapse rate is likely due to a small population of cells known as leukemia stem cells (LSCs), which have the ability to self-renew and can re-form a leukemia from a single cell. Additionally, a major clinical concern is whether these LSCs are effectively killed by conventional cytotoxic chemotherapies. Current efforts to study LSCs have faced serious limitations, which have impeded our understanding of this important population of cells.

Prior work in our lab has established a zebrafish Myc-induced T-cell acute lymphoblastic leukemia (T-ALL) model that mimics the most aggressive and treatment resistant form of human T-ALL. Using this system, we isolated single LSCs through a novel transplantation strategy. Limiting dilution analysis showed significant differences in the rate of self-renewal between different LSCs, suggesting heterogeneity in LSC relapse potential. We generated a library of zebrafish T-ALL with high self-renewal rates (about 1 in 10 leukemia cells is a LSC) for use in bulk RNAseq, as well as single cell qPCR and single cell RNAseq to identify a self-renewal signature unique to LSCs. Single cell analyses showed a population of cells that expressed known self-renewal genes and clustered separately from the rest of the leukemia cells in the population. Ongoing work in our laboratory is focused on functionally testing whether this gene expression profile is linked with self-renewal. Additionally from this analysis, the Wnt pathway, more specifically ß-catenin, was enriched in the putative LSC population. These data support previous findings in mammalian models of ALL.

We hypothesize that inhibitors of the Wnt pathway will inhibit self-renewal of LSCs and force them to terminally differentiate. However, there are no WNT inhibitors clinically available due to undesirable side-effects. We are currently using a 6xTCF/LEF:GFP zebrafish Wnt reporter model for high-throughput screens to identify new, less toxic Wnt pathway inhibitors. Thus far, several compounds have showed significantly decreased TCF/LEF activity after drug treatment, with no effects on animal health. Future studies will test these inhibitors in our zebrafish high LSC leukemia samples and patient derived xenograft models. Ultimately, these inhibitors may represent a potential therapeutic strategy for targeting treatment-resistant LSCs and preventing ALL relapse.

#157

Targeting cancer stem-like cells in triple negative breast cancer.

Fokhrul Hossain,1 Ayse D Ucar Bilyeu,1 Claudia Sorrentino,1 Judy Crabtree,1 Antonio Pannuti,1 Margarite Matossian,2 Matthew Burow,2 Todd Golde,3 Barbara Osborne,4 Lucio Miele1. 1 _LSUHSC, New Orleans, LA;_ 2 _Tulane University, New Orleans, LA;_ 3 _University of Florida, Gainesville, FL;_ 4 _University of Massachusetts at Amherst, MA_.

Triple Negative Breast Cancer (TNBC), an aggressive, heterogeneous subtype of breast cancer, and immunohistochemically negative for estrogen receptor α (ER-), progesterone receptor (PR-), and lacking amplification of the human epidermal growth factor receptor 2 locus (HER2-). TNBC patients have high risk of recurrence and metastasis, and current treatment options remain limited. Cancer stem-like cells (CSCs) have been linked to cancer initiation, progression and chemotherapy resistance. Therefore CSC-targeted therapies are keenly sought. There is strong evidence for the involvement of Notch signaling in TNBC. Notch1 is highly expressed in Basal-like 1 (BL1) and especially Mesenchymal-Stem-Like (MSL) TNBCs. Expression of Notch1 and its ligand Jagged1 correlates with poor prognosis. Moreover, strong evidence supports key roles of different Notch paralogs in breast CSCs. Moreover the role of non-canonical Notch signaling in TNBC remains unknown. Here, we demonstrate that Notch1 promotes cell survival in MDA-MB-231 cells, representative of MSL TNBC, in part by activating NF-κB. Notch activation by Jagged1-expressing stromal cells enhances transcription of the anti-apoptotic gene cIAP-2 (BIRC3), a known NF-κB target. This event is dependent on recruitment of NF-κB subunits, IKKα and Notch1 to the cIAP-2 promoter. Short term exposure of MDA-MB-231 cells (MSL, PTEN wild-type), but not MDA-MB-468 cells (BL1, PTEN-null) to recombinant Jagged1 leads to AKT phosphorylation. This is suppressed by dual mTORC1/2 inhibitors, AKT inhibitors and IKKα inhibitors but not Everolimus (mTORC1-selective inhibitor). These observations support a model where canonical and non-canonical mechanisms downstream of Notch1 trigger AKT phosphorylation and NF-κB activation in PTEN wild type TNBC cells. This suggests a bidirectional crosstalk between the IKKα and AKT arms of this Jagged1-activated pathway. We also demonstrate that recombinant Jagged1 increases the cellular metabolism of TNBC cells and knockdown of Notch1 or IKKα by siRNA decreases mitochondrial respiration and glycolysis. We have found that CSCs derived from MDA-MB-231 cells have increased Notch1, p-AKT, and mitochondrial respiration. AKT inhibition or NF-κB inhibition decreases both mitochondrial respiration and glycolysis of TNBC derived CSCs. Pharmacological inhibition of Notch cleavage by gamma secretase inhibitor (PF-03084014) in combination with AKT inhibitor (MK-2206) or NF-κB inhibitor (Bay11-7082) blocks CD90hi or CD44+CD24low sorted secondary mammospheres formation. These data suggest that combination treatments affecting Notch, NF-kB or AKT pathways have potential therapeutic importance in targeting CSCs of TNBC.

#158

GSKJ4, a selective jumonji histone H3 lysine27 demethylase inhibitor, targets ovarian cancer stem cells.

Hirotsugu Sakaki. _Yamagata Univ., Yamagata, Japan_.

Background/Aim: Global increase in the trimethylation of histone H3 at lysine 27 (H3K27me3) has been associated with the differentiation of normal stem cells and cancer cells, however, the role of H3K27me3 in the control of cancer stem cells (CSCs) remains poorly understood. We investigated the impact of increased H3K27me3 on ovarian CSCs using GSKJ4, a selective jumonji H3K27 demethylase inhibitor.

Materials and Methods: The effect of GSKJ4 on the viability as well as on the self-renewal and tumor-initiating capacity of CSCs derived from A2780 and TOV21G human ovarian cancer cell line, was examined.

Results: GSKJ4 induced cell death in A2780 CSC and TOV21G CSC at a concentration non-toxic to normal human fibroblasts. Furthermore, GSKJ4 induced differentiation and inhibited sphere formation as well as stem cell marker expression of A2780 CSC and TOV21G CSC that survived GSKJ4 treatment.

Conclusion: Using ovarian CSCs as a model, we have demonstrated for the first time that GSKJ4 can target CSCs, suggesting a critical role for H3K27 methylation in their maintenance and survival. Our findings thus provide an initial clue to explore the role of GSKJ4 as a potent CSC-targeting agent for ovarian cancer and other types of human cancer.

#159

**Frankincense essential oil and doxorubicin treatment inhibited cell proliferation and induced apoptosis in CD133** \+ **and CD90** + **subpopulation hepatocellular carcinoma cancer stem cells.**

Amira S. Fyala, Ahmed S. Sultan. _Alexandria University., Alexandria, Egypt_.

Frankincense essential oil has been known in traditional medicine of many countries as wealth of health for the treatment of inflammatory diseases, anti-bacterial, anti-fungal activities, and might possess anti-cancer activities, based on their anti-proliferative and pro-apoptotic activities, but its anticancer role against cancer stem cells (CSC) in hepatocellular carcinoma (HCC) cell lines and the underlying molecular mechanisms remain uninvestigated. Doxorubicin is one of the efficient factors for HCC treatment, but the resistance to it is presenting a major obstacle for HCC treatment. The main aim of this study, isolated CD133+/CD90\+ subpopulation from HCC cell line by flow cytometry, and evaluated the effects of Frankincense essential oil and Doxorubicin treatment on cell viability, colony formation, cellular proliferation, cell cycle distribution and apoptosis induction through DNA fragmentation of HCC cells and its isolated CD133+/CD90+ CSCs. Cell viability was monitored using an MTT assay. In addition, apoptosis induction was labeled by Annexin V-fluorescein isothiocyanate/propidium iodide and measured using flow cytometry. Western blotting was used to examine the protein expression of p53, Bcl-2, cyclin D1, and cleaved-caspases-3 and -9. Our results demonstrated that Frankincense essential oil or Doxorubicin suppressed the cell viability of CD133+/CD90+cells in a concentration-dependent manner after 24hrs of treatment. In addition, Frankincense essential oil or Doxorubicin induced a significant apoptosis induction in CD133+/CD90\+ subpopulation cells through DNA fragmentation, caspase-9 and caspase-3 activation, increased the expression of pro-apoptotic proteins, including p53, decreased the protein level of anti-apoptotic protein Bcl-2, and regulated the expression of cell cycle regulators as cyclin D1. Levels of cyclin D1 expression were gradually suppressed by Frankincense essential oil in tested cells. Our data revealed that Frankincense essential oil has been shown to arrest cancer cells at the G1-phase of cell cycle, suppresses cyclin D1, and E, cdk 2, as well as increases expression of p21 through a p53-independent pathway. Furthermore, the Frankincense essential oil treatment strongly inhibited stemness characteristics of CSC subpopulation, colony formation and dramatically inhibited CD133+/CD90\+ tumor growth in vitro. In conclusion, our results suggested that Frankincense essential oil and Doxorubicin may be a potential treatment for apoptosis induction in CSC and may provide an attractive therapeutic strategy against HCC.

#160

**Novel strategy targeting breast cancer stem cells CD44** + **/CD24** -low **responsible for breast cancer progression.**

Fatma A. Abouelnazar,1 Ahmed S. Sultan2. 1 _Faculty of Science-Alexandria University and Faculty of Allied Medical Sciences-Pharos University, Alexandria, Egypt;_ 2 _Faculty of Science-Alexandria University, Alexandria, Egypt_.

The present study has been designed to isolate the highly tumorigenic sub-population of cells from the breast cancer cell line (T47-D). The effect of Manuka honey (MH), Frankincense essential oil (F), Metformin (M) and combined treatment on eradicating breast cancer stem cells (BCSCs) that are responsible for cancer progression was investigated.

The isolated (BCSCs) were subjected to different concentrations of (MH), (F), (M) and combined treatment to visualize their effects on BCSCs viability, morphological changes, caspase-3 activity, apoptosis induction, clonogenic survival, β-catenin, ALDH6A1, Notch-1, Notch-4 and Nonog protein expression, using western blot analysis and protein localization by immunocytochemical technique.

Inhibited viability of isolated breast cancer stem cells, which showed apoptotic morphological changes were observed beside increased caspase-3 activity and inhibited clonogenic survival compared to control. At the protein level, our data showed that (MH), (F), (M) and combined synergistic treatment decreased the expression of β-catenin levels, ALDH6A1 and Notch-1 compared to control. In addition, (MH) showed no effect on Notch-4 expression, but significantly decreased the expression level of Nanog. In contrast, (F) treatment enhanced the protein expression of Nanog level in a dose dependent manner compared to control. Furthermore, (MH), (F), (M), and combined treatment showed conspicuously decrease in the nuclear and cytoplasmic level of β-catenin and ALDH6A1 of CD44+/CD24-low (BCSCs) compared to control.

Taken together, natural compounds of Manuka honey and Frankincense essential oils and their combination with Metformin have been emerged as a potential strategy to target and eradicate (BCSCs).

#161

Adult myogenic stem cells inhibit prostate cancer cell proliferation and increase docetaxel-mediated cell toxicity.

Krishna M. Sinha,1 Rozita Yarmand,2 Reid Andrew,1 Johnny Huard1. 1 _UT Health Science Center, Houston, TX;_ 2 _UT MD Anderson Cancer Center, Houston, TX_.

Current treatments for prostate cancer (PCa) are primarily based on use of anti-androgen agents to prevent disease progression and bone lesions. However, metastatic PCa (mPCa) continues to progress in an androgen-independent manner, and consequently, bone and muscle tissues deteriorate, often resulting in severe pain and fragility. Adult stem cells have been used in regenerative medicine approaches for many years for treating human diseases through gene therapy. We have demonstrated for nearly two decades that a population of slow-adhering myogenic progenitor cells, called muscle progenitor cells (MPCs), has tremendous ability to regenerate bone, skeletal muscle, cartilage, nerve, and cardiac muscle after injury. However, to date, the potential therapeutic use of MPCs in cancers has not yet been investigated. We hypothesized that MPCs can inhibit proliferation and survival of PCa cells and increase docetaxel-induced cytotoxicity of the PCa cells. Moreover, MPCs may be effective in decreasing PCa-induced skeletal lesions and promote bone and muscle tissue repair. The objective of this study was to evaluate the paracrine roles of secreted factors from MPCs with respect to proliferation and survival of PCa cells. Here we showed that co-culture of PC3 and C42B cells with MPCs in transwell inserts strongly decreased PCa cell proliferation and survival by activating CDK inhibitors (p16 and p27) and apoptotic BBC3 and NOXA genes. Analysis of factors secreted by MPCs using ELISA protein array and mass spectrometry identified several proteins with known antiproliferative functions, including IGFBP6/7, pigment epithelium-derived factor, HPCAL1, NO, and SPARC. Additionally, conditioned media (CM) from MPCs increased the docetaxel-induced cytotoxicity of both androgen-dependent and -independent PCa cells. Molecular analysis also showed decreased expression of AR, MLL, FOXA1, TWF1, and TGIF1, which are known oncogenic regulators of PCa progression. Since metastatic PCa causes both osteolytic and osteoblastic lesions through the secretion of RANKL and BMPs, respectively, we then showed that treatment of PCa cells with OPG and noggin, antagonists of RANKL and BMPs, respectively, inhibited their cell proliferation. Further, CM from PCa cells pretreated with noggin and OPG resulted in decreased osteoblast activity in primary osteoblasts and decreased osteoclast activity in macrophage cells, respectively, when compared to CM from untreated PCa cells. These results suggest that noggin and OPG could be used as therapeutic proteins for reducing PCa-induced skeletal lesions through MPC gene delivery. Taken together, our data suggest that use of adult stem cell-based therapy along with current chemotherapy agents (e.g., docetaxel) is a promising effective strategy for treatment of PCa and preventing metastatic skeletal lesions.

#162

UBE2T: A molecular regulator for cancer stemness and drug resistance in hepatocellular carcinoma.

Pui Yu Ho, Kin Wah Lee. _The Hong Kong Polytechnic University, Hong Kong, Hong Kong_.

Hepatocellular carcinoma (HCC) is one of the common cancers worldwide. Poor prognosis of HCC patients is attributed to high frequency of tumor relapse and chemoresistant nature of this disease. Increasing evidence showed the critical role of cancer stem cells (CSCs) on tumor relapse and therapeutic resistance. Since normal stem cells and CSCs share high similarity, we would like to identify the molecules/pathways crucial in liver CSCs by determining what molecules involved in normal liver stem cells during liver regeneration. Comparison of expression profiles between early regenerating liver and intact liver revealed the upregulation of DNA Damage Response pathways in the self-renewing liver, in which Ubiquitin Conjugating Enzyme E2 T (UBE2T) was the most significantly upregulated. This, together with the publicly available dataset (GSE5975) shows upregulation of UBE2T in EpCAM-enriched liver CSC populations, suggest the potential role of UBE2T on regulation of cancer stemness. By qPCR analysis UBE2T mRNA level was overexpressed in 91% of the clinical HCC specimens (n=56) and associated with aggressive tumor behavior and poorer patients' survival. The overexpression of UBE2T was further confirmed by western blot and immunohistochemical analysis. By lentiviral based knock-down approach, we demonstrated the role of UBE2T in regulation of liver CSC properties, including self-renewal, tumorigenicity, drug resistance and expression of liver CSC markers. In orthotopic HCC model, UBE2T was found to play pivotal role in lung metastasis in vivo. Mechanistically, UBE2T is found to interact directly with E3 ligase Mule and regulate its protein expression via ubiquitination. Since Mule is found to directly degrade β-catenin protein, we are examining whether UBE2T mediates liver T-IC function through direct regulation of Mule-mediated β-catenin degradation. In conclusion, we have uncovered the novel role of UBE2T signaling cascade in regulation liver CSCs, which provides attractive therapeutic target for potential treatment of HCC.

#163

Glioblastoma, cancer stem cells, and reactive species balances: A case for GTP cyclohydrolase 1.

Anh N. Tran,1 Kiera Walker,1 David G. Harrison,2 Wei Chen,2 James Mobley,1 Lauren Hocevar,1 James R. Hackney,1 Randee Sedaka,1 Jennifer Pollock,1 Matthew S. Goldberg,1 Dolores Hambardzumyan,3 Sara J. Cooper,4 G Yancey Gillespie,1 Anita B. Hjelmeland1. 1 _University of Alabama at Birmingham, Birmingham, AL;_ 2 _Vanderbilt University, Nashville, TN;_ 3 _Emory University, Atlanta, GA;_ 4 _HudsonAlpha Institute for Biotechnology, Huntsville, AL_.

Glioblastoma (GBM), or grade IV astrocytoma, is a deadly disease due in part to the high degree of intratumoral heterogeneity that contributes to treatment failures. Previous studies have shown the importance of reactive species balances, partially controlled by the coupling of nitric oxide synthases (NOS) with their cofactor, in maintenance of glioma stem cell (GSC) phenotype as well as survival of cancer cells in general. In this study, we investigated the roles of GTP cyclohydrolase 1 (GCH1), which is the first and rate-limiting enzyme of the pathway producing of NOS cofactor producing pathway, in GBM stem cell phenotypes via redox balances. We found that GCH1 RNA and protein expression were increased in GSCs in comparison to non-GSCs, but that GCH1 was not exclusive to the GSC fraction. Indeed, GCH1 was elevated in GBM in comparison to normal brain. Overexpression of GCH1 in GBM cells increased cell growth in vitro and neurosphere-forming capability and decreased survival in an intracranial GBM mouse model. In contrast, GCH1 knockdown with short hairpin RNA in GBM cells led to growth inhibition in vitro as well as increased survival in animal models. GCH1 increased CD44 expression and was upregulated in the detrimental mesenchymal GBM subtype in which CD44 served as a marker. Mechanistically, we found that the expression of GCH1 increased BH4 production, as well as augmented multiple antioxidant pathways, including the expression of PARK7, was critical for controlling reactive species balance, including suppressing reactive oxygen species production. In silico analyses demonstrated that higher GCH1 levels in glioma patients correlate with higher glioma grade, recurrence and worse survival. Together, our data suggest that upregulation of GCH1 in GSCs promotes tumor maintenance and is a key regulator of reactive oxygen species in GBM, and GCH1 pathway is a potential target for therapy.

#164

Ston2 knockdown promotes stem-like properties via dnmt1-mediated muc1 upregulation in ovarian cancer.

Shanshan Xu. _Zhejiang University, Hangzhou, China_.

Cancer stem cells possess the ability of self-renewal, unlimited proliferation,and non-sensitivity to physical and chemical factors, which is regarded to be a cause of ovarian cancer recurrence, but the mechanism is currently unclear. We found that the expression of STON2 was significantly lower in ovarian cancer-stem like cells by LC-MS/MS-based label free quantitative proteomics approach and western blot validation. STON2 knockdown upregulated the expression of the CSC-related markers in protein level, increased the spheroids number and CD44+CD24−proportion. Besides, in vivo xenograft experiments, the volume of tumors from the shSton2 group were much bigger compared with the shNC group. Moreover, STON2 overexpression decreased the CSC-related markers and inhibited the spheroids number compared with the control group. Transcriptome sequencing data showed that MUC1 is negative correlation with STON2, which were verificated by RT-PCR and western blot analysis. Subsequently, we found that MUC1 mediated the role of STON2 in regulation of stem-like properties in ovarian cancer. MUC1 knockdown resulted in a significant decrease in the sphere-forming ability, CSC-related markers and CD44+CD24−proportion. In addition, MUC1 overexpression caused an increment of CSC-related markers expression and CD44+CD24−proportion. Furthermore, we found that the reducing of sphere-forming ability caused by STON2 overexpression was rescued by MUC1 overexpression. As MUC1 can be regulated through epigenetic mechanisms, we found that DNA methylation inhibitor Azacitidine was able to rescue the upregulation of MUC1 by STON2. The DNA methylation levels of MUC1 in one CpG-rich region were then evaluated with pyrosequencing, and the data displayed that the siSton2 group is lower than the control group. Furthermore, we found that the DNMT1 as a member of the DNA methyltransferase family decreased following with STON2 knockdown in protein level, rather than in mRNA level. We thus knock down DNMT1 using specific siRNA, and found that MUC1 overexpression both in mRNA level and protein level. In this study, we focused primarily on STON2 repression leading to DNMT1 downregulation and MUC1 activation is a main mechanism underlying the maintenance of stem-like properties in ovarian cancer.

#165

Cancer stem cell phenotypes determine mechanisms of resistance to targeted therapies.

Joshy George,1 Yaohui Chen,2 Keiko Yamamoto,3 Scott Adamson,4 Jeff Chuang,1 Kyuson Yun2. 1 _The Jackson Laboratory, Farmington, CT;_ 2 _Houston Methodist Research Institute/Weill Cornell Medical Collge, Houston, TX;_ 3 _The Jackson Laboratory, Bar Harbor, ME;_ 4 _The Jackson Laboratory, Houston, CT_.

Despite promising, and sometimes dramatic, efficacy of targeted therapies, primary and acquired resistance to targeted therapies limits their broad and long-term use in the clinic. Therefore, the ability to rationally design anticipatory or combination therapies as early in the treatment process as possible could significantly improve the utility of targeted therapies and cancer outcome. Here, we report that one can predict a priori the types of mutations that will occur in SHH pathway inhibitor treated medulloblastomas based on different cancer stem cell (CSC) phenotypes in each tumor. CSCs are a subset of cancer cells that are more resistant to cytotoxic therapies (chemo and radiation) than bulk tumor cells, and are responsible for tumor recurrence. Previously, we reported that cell-of-origin is a major driver of CSC phenotypes. In particular, we showed that CSCs retain epigenetic memory of their cells-of-origin and the mitogenic pathways that drive CSC proliferation and survival are reflective of their cells-of-origin. We also demonstrated that CSCs and bulk tumor cells do not necessarily depend on the same mitogenic/survival signaling pathways. An important implication of this finding is that targeted therapies selected based on bulk tumor analyses may not be effective in ablating CSCs in some tumors. Furthermore, the selective pressure imposed on CSCs in individual patient tumors by a targeted therapy will vary greatly, depending on whether the selected drug targets a critical pathway in CSCs and not just bulk tumor cells. Another clinically significant implication is that based on the resident CSC phenotype, we may able to predict whether individual SHH tumors will or will not acquire treatment-induced mutations in the SHH pathway upon SHH pathway inhibitors (SMOi) treatment. To test this hypothesis, we treated allografts of spontaneous Ptch;p53 mouse medulloblastomas arising from different cells-of-origins with SMOi's (LDE225 and GDC0449). While all tumors responded to SMOi's initially, most acquired resistance over time. We took an integrated genomics approach to identify molecular mechanisms of therapy resistance in these SMOi-resistant tumors. We report that acquired mutations in SHH pathway genes occurred only in tumors that contained CSCs that depended on the SHH pathway. In tumors where only the bulk tumor cells, but not CSCs, depended on SHH signaling, no acquired mutations in SHH pathway genes were detected. In sum, we report that CSCs drive mechanisms of therapy resistance and also provide the first proof-of-principle evidence that it is possible to predict the types of mutations that will occur in therapy-resistant tumors in advance of treatment, which can be exploited to design anticipatory therapies in the future.

#166

Sam68 modulates the response to PARP inhibitors in breast cancers regardless of BRCA mutational status.

Alice Turdo, Miriam Gaggianesi, Aurora Chinnici, Elisa Lipari, Tiziana Apuzzo, Veronica Veschi, Emanuela Scavo, Annalisa Nicotra, Giorgio Stassi, Matilde Todaro. _Univ. of Palermo, Palermo, Italy_.

Background: Breast cancer (BC) is the most common cancer among women worldwide. The high mortality rate is based on metastatic disease that is largely incurable and resistant to common therapeutic agents. Sam68 is an RNA/DNA binding protein involved in a plethora of biological processes and plays a crucial role in breast cancer onset and progression. Most recently, a novel function of Sam68 in DNA damage response has been unveiled in mouse embryonic fibroblasts and colon cancer cells. Sam68 is recruited to the DNA and, together with PARP1, orchestrates the formation of polymers of ADP-ribose (PAR) and the consequent activation of NF-kB and its anti-apoptotic target genes. On the other hand, Sam68 is a transcriptional co-activator of TP53 wt following DNA damage, suggesting a tumor suppressive role of Sam68 in TP53 wt cancers. The use of PARP inhibitors in breast cancers, regardless of BRCA1/2 mutations, has been proposed in those cancers with defects in the homologous recombination machinery showing considerable cytotoxicity. However, biomarkers of therapeutic response to predict the efficacy of PARP inhibitors are far from being used the clinical settings.

Material and methods: BC stem cells (BCSCs) were obtained from mechanic and enzymatic digestion of human breast cancer specimens. Sam68 knockdown (shSam68) was generated by cells lentiviral transduction followed by puromycin selection. BC cells (3 x 105) were suspended in 1:6 matrigel and injected in the mammary fat pad of NOD/SCID mice.

Results: Tissue microarray (TMAs) analysis showed that high expression levels of Sam68 are associated with a reduced distant relapse free survival in the most aggressive subtypes of BCs (p=0.008) namely Luminal B Her2+, Her2+ and Triple negative (TN) BCs, harboring the highest frequency of TP53 mutations. Stable knockdown of Sam68 reduced the proliferation, invasiveness and capability to form colonies in vitro of both BC cell lines and patient-derived BCSCs. Orthotopic injection of shSam68 TNBC cell lines in immunocompromised mice did not result in primary tumor formation and metastatic spreading in liver and lungs, as compared to the control. Additionally, the depletion of Sam68 sensitized BC cells to the administration of genotoxic compounds. The combination of genotoxic stress and a PARP inhibitor showed enhanced toxicity in TP53 mut/BRCA wt cells. Interestingly, we observed similar results in terms of cell viability in TP53 wt BC cells upon knockdown of Sam68.

Conclusions: The possibility to interfere with DNA repair mechanism is a promising strategy to counteract the growth of BCs harboring DNA-repair defects. Herein, we showed that Sam68 fosters proliferation and invasive behavior of BC cell lines as well as patient-derived BCSCs and is implicated in the recovery from DNA damaging agents.

#167

Targeting the NF-kappaB pathway with bardoxolone methyl to inhibit ovarian cancer spheroid formation.

Michelle K. Ozaki,1 Carrie D. House,1 Krystyna Mazan-Mamczarz,2 Madhu Lal-Nag,2 Craig Thomas,2 Christina M. Annunziata1. 1 _NCI, Bethesda, MD;_ 2 _NCATS, Rockville, MD_.

Over 70% of patients with advanced stage ovarian cancer relapse within 2 years, with long term overall survival being only 25%. Tumor-initiating cells (TICs) are thought to contribute to this high rate of recurrence as they can resist killing by platinum-based chemotherapy. In this study we sought to identify pathways unique to TICs and to disrupt these pathways in order to more effectively target this population of cells. We conducted an siRNA screen which identified NF-kappaB signaling as an important pathway in the maintenance of ovarian cancer cells grown in non-adherent spheroid cultures that mimic TICs. Downregulation of several known NF-kappaB target genes, including NXF1, RAC2, CDC42, BCL2L1 and SERPINB3, significantly decreased growth of TIC cultures but not the corresponding adherent cultures, thereby indicating that expression of these genes is necessary for growth in TIC cultures. These findings are consistent with previous studies which showed that higher NF-kappaB activity is linked with drug resistance in patients. In parallel, we conducted a drug screen to identify compounds that target TICs. One clear candidate was bardoxolone methyl, which has been shown to inhibit IKK, an upstream NF-kappaB signaling protein. We confirmed the results of the drug screen in a panel of ovarian cancer cell lines, showing that bardoxolone methyl decreased known TIC markers CD133 and ALDH by flow cytometry, and decreased NF-kappaB proteins by western blot. To further elucidate the mechanism of bardoxolone methyl on TICs we conducted an RNAi screen +/- bardoxolone methyl. Interestingly, the knockdown of genes including BRAF, MAP3K8, LMNA and SMAD6 acted in a synthetically lethal manner with bardoxolone methyl in the TICs. To begin translating to a clinically relevant model, we proceeded to test whether bardoxolone methyl could eliminate TICs that remained after treatment with platinum-based chemotherapy. We confirmed in vitro that bardoxolone methyl in combination with chemotherapy reduced TIC populations. Our ongoing studies will address whether this drug is effective in preventing relapse in vivo after chemotherapy treatment of ovarian cancer in mouse models.

#168

Cancer stem-like properties and drug resistance are dependent on purine synthetic metabolism mediated by the mitochondrial enzyme MTHFD2.

Noriko Gotoh,1 Tatsunori Nishimura,1 Asuka Nakata,1 Shin-ichi Horike,1 Susumu Kohno,1 Chiaki Takahashi,1 Tomoyoshi Soga,2 Arinobu Tojo3. 1 _Cancer Research Institute, Kanazawa University, Kanazawa, Japan;_ 2 _Institute for Advanced Biosciences, Keio University, Tsuruoka, Japan;_ 3 _Institute of Medical Science, The University of Tokyo, Tokyo, Japan_.

Tumor recurrence is attributable to cancer stem-like cells (CSCs), the metabolic mechanisms of which currently remain obscure. Here, we uncovered the critical role of folate-mediated one-carbon (1C) metabolism involving mitochondrial methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) and its downstream purine synthesis pathway. MTHFD2 knockdown greatly reduced tumorigenesis and stem-like properties, which were associated with purine nucleotide deficiency, and caused marked accumulation of 5-aminoimidazole carboxamide ribonucleotide (AICAR)—the final intermediate of the purine synthesis pathway. Lung cancer cells with acquired resistance to the targeted drug gefitinib exhibited increased stem-like properties and enhanced expression of MTHFD2. MTHFD2 knockdown or treatment with AICAR reduced the stem-like properties and restored gefitinib sensitivity in gefitinib-resistant cancer cells. Thus, MTHFD2-mediated mitochondrial 1C metabolism appears critical for cancer stem-like properties and resistance to drugs including gefitinib through consumption of AICAR, leading to depletion of the intracellular pool of AICAR. Because CSCs are dependent on MTHFD2, therapies targeting MTHFD2 may eradicate tumors and prevent recurrence.

#169

Identification of RNA aptamers selectively recognizing and affecting glioblastoma stem cells.

Alessandra Affinito,1 Cristina Quintavalle,1 Maurizio Albero,1 Claudia Vilardo,1 Francesco Palma,1 Giuseppina Roscigno,1 Lucia Ricci Vitiani,2 Roberto Pallini,3 Carla Lucia Esposito,4 Vittorio de Franciscis,4 Gerolama Condorelli1. 1 _Federico II University of Naples, Napoli, Italy;_ 2 _Istituto Superiore di Sanità, Rome, Italy;_ 3 _Università Cattolica del Sacro Cuore, Rome, Italy;_ 4 _CNR, Napoli, Italy_.

Glioblastoma (GBM) is the most frequent and aggressive primary brain tumour in adults. Despite advances in surgical and medical neuro oncology, prognosis for GBM patients remains dismal, with a median survival of about 15 months. It has been demonstrated that the modest benefit of conventional therapies depends on a small population of cancer stem cells within the tumor, named Glioma Stem Cells (GSCs) that cause tumor relapse and chemoresistance and therefore could play a key role in GBM recurrence. Thus, the identification of new specific ligands for GSCs could be a fundamental challenge for the development of effective glioma therapies. Here, we developed an in vitro evolution based approach, named differential whole cellSELEX; it is used to generate nucleic acid ligands, named aptamers, with high affinity and specificity for GSCs. Aptamers, were obtained through the iterative evolution of a random pool of sequences using human primary GSCs as target. Among different potential candidates we focused on one sequence, named 40L. The 40L aptamer and its truncated form, 40S, were selective for human GSCs distinguishing them from tumor differentiated cells, obtained from the stem cells induced to differentiate. 40L revealed to be functionally active on target cells and able to inhibit stemness, cell growth and migration. 40s preserves binding ability of 40L sequence and it has further proven to strongly reduce tumor proliferation in in vivo experiment. Moreover, both 40L and 40s were able to rapidly internalize upon target binding and therefore may serve as selective vehicle for therapeutics.In conclusion, our results indicate that 40L and its short form 40s can selectively target GSCs both in vitro and in vivo. Given the crucial role of these cells in GBM recurrence and therapy resistance, 40L and 40s represent innovative drug candidates with a great potential in the GBM treatment.

#170

Targeting colorectal cancer stem cells with the anticancer molecule thymoquinone.

Farah R. Ballout,1 Maamoun Fatfat,2 Rana Abdel-Samad,2 Nadine Darwiche,2 Regine Schneider-Stock,3 Wassim Abou-Kheir,2 Hala-Gali Muhtasib2. 1 _American University of Beirut, Beirut, Lebanon;_ 2 _American University of Beirut, beirut, Lebanon;_ 3 _Friedrich-Alexander University of Erlangen-Nürnberg, Erlangen, Germany_.

Introduction: 5-fluorouracil (5-Fu) remains the standard chemotherapy for metastatic colorectal cancer (CRC), but drug resistance and unpredictable cardiotoxicity limit its effectiveness. The high recurrence rates and the common resistance are thought to be due to a population of self-renewing cancer stem cells (CSCs). The black seed extract Thymoquinone (TQ) is a promising anticancer molecule known to inhibit cancer cell growth and progression in numerous cancer systems both in vitro and in vivo. This project aims to investigate the effect and mechanism of action of TQ on colon cancer stem/progenitor cells using two isogenic HCT116 colon cancer cell lines that differ in their 5-Fu drug sensitivity.

Methods: Sphere-formation and propagation assays were used to assess the efficacy of TQ on targeting self-renewal capacity of colon CSCs enriched from the sensitive and resistant cell lines in 3D cultures over several generations in comparison to 2D monolayers. In addition, xenotransplantation experiments were used to assess for the tumor initiation of 2D vs. 3D cells.

Results: Our results of TQ efficacy in 2D cell culture system showed that it reduced the viability of both cell lines. Importantly, our 3D results showed that TQ inhibits HCT116 colonosphere growth at 10-fold lower concentrations than those required to inhibit the growth of 2D monolayer cells. Interestingly, the injection of 100 spheres derived from HCT116 sensitive cell line and not the 2D equivalent cell density into NOD-SCID immunocompromised mice resulted in tumor development, suggesting that spheres are rich in cells with stem-like properties.

Conclusion: In summary, our data suggests that TQ might be an effective treatment strategy and may prevent colorectal cancer recurrence by targeting CSCs.

### The Mechanical Microenvironment in Tumorigenesis

#171

Acid-induced collagen remodeling promotes cancer progress as a result of niche engineering.

Ava Niazi, Johnson J. Joseph, Robert J. Gillies, Mehdi Damaghi. _Moffitt Cancer Ctr. Research Inst., Tampa, FL_.

Growing tumors are dynamic and nonlinear ecosystems, wherein cancer cells in tumors adapt to their local microenvironment, and these adaptations further modify the environment, inducing more changes. From nascent intraductal neoplasms to a disseminated metastatic disease, several levels of evolutionary adaptations and selections occur. Here, we focus on one example of such an adaptation mechanism, namely, niche construction and remodeling promoted by adaptation to acidosis, which itself is a metabolic adaptation to the early harsh environment in intraductal neoplasms. Early carcinogenesis is an avascular and hypoxic disease leading to extracellular acidosis. Hence, we investigated how acid-adapted tumor cells engineer their niche to support their growth, proliferation, and invasion. We show that relatively rare collagens are produced by acid-adapted cancer cells to create an environment that contributes to their survival. We found collagen and other fibrillar structure in the periluminal region of DCIS lesions using Second Harmonic Generation Microscopy (SHG). We compared the microarray of DCIS samples to normal tissue and observed that Col10a1 and Col11a1 were significantly increased. We have shown previously that DCIS lesions are acidic. To test if these were related, we adapted low grade MCF7 and DCIS-10AT breast cancer cells to growth in acidic conditions and probed them for collagen production using qPCR, western blotting and Immunofluorescence. We found acid adaptation increases number of collagens including Col10a1, Col11a1, and different subtypes of Col4. Further, we developed a window chamber model to study the collagen remodeling real time through intra-vital microscopy. SNARF and collagen imaging simultaneously showed the collagen structural modifications in acidic regions. Further proteomic and secretome analysis of acid adapted cells versus non-adapted cells identified additional enzymes involved in collagen remodeling such as TGM2 and LOXL2 that have been shown to play major role in matrix remodeling. These findings were also validated by western blot and ICC. To test the role of collagens in viability of cancer cells we grew acid adapted and non-adapted MCF7 cells as 3D spheroids and treat them with FAK inhibitors. FAK inhibitors dramatically affected the acid adapted spheroids. The specific role of each of collagen genes in survival and invasion is still under study. We discuss that adaptation to acidosis induces cancer cells to adopt strategies that assist them to be more independent, particularly in harsh conditions such periluminal areas of DCIS that they don't have access to stroma cells; they produce the collagen they need and will remodel it by the enzymes produced by themselves to be able to survive, grow, and invade. Finally these adaptation mechanisms may present a vulnerability to design new therapeutics against the emerging aggressive phenotypes during cancer cell evolution.

#172

**Collagen increases tumorigenic characteristics of papillary thyroid cancer cells harboring BRAF** V600E **mutations.**

Anna Sharabura, Jonathan Jenkins, Laura MacDonald. _Hendrix College, Conway, AR_.

Thyroid cancer is the most common endocrine cancer, and incidence is increasing worldwide. Thyroid cancer can be classified as either well differentiated or poorly differentiated. Of well-differentiated thyroid cancers, papillary thyroid cancer is most common, and is associated with activating BRAF mutations. While our understanding of the genetic basis for thyroid cancer is fairly extensive, less is known about how the tumor microenvironment alters characteristics of tumor cells. Recently, Jolly and others reported that papillary thyroid tumors derived from cells harboring BRAFV600E mutations and PTEN deletions are enriched with fibrillar collagen. Notably, overexpression of collagen was also associated with decreased survival and worse overall prognosis in patient samples. In this study, we investigated whether growth on collagen enhanced tumorigenic characteristics of papillary thyroid cancer cell lines with BRAFV600E mutations. Three distinct cell lines derived from mouse papillary thyroid cancer tumors were grown in the presence and absence of collagen and assessed for changes in morphology, proliferation, and drug resistance. Interestingly, our results suggest that growth on collagen contributes to a more mesenchymal morphology, increased proliferation, and decreased sensitivity to chemotherapy drugs. These and other results implicate an important role for collagen in the progression of thyroid cancer.

#173

Adipocytes-derived collagen reorganization in microenvironment promotes breast cancer progression.

Wei Xiaohui, He Jinyong, Li Sijing, Yuan Shengtao, Sun Li. _China Pharmaceutical University, Nanjing, China_.

Purposes

Breast cancer cells recruit surrounding stromal cells, such as cancer-associated fibroblasts (CAFs), to reorganize collagen and promote tumor metastasis. Adipocytes are the most abundant stromal partners in breast tissue, whether cancer-associated adipocytes (CAAs) involve in the collagen reorganization and ultimately promoting metastasis of breast cancer is still unknown. In this study, we seek to understand whether adipocytes involve in the extracellular matrix collagen reorganization in breast cancer and its underline mechanism.

Methodology

In vitro mature adipocytes and breast cancer transwell co-culture model and orthotopic xenograft mouse model of human breast tumor formation in vivo were used to study the crosstalk between adipocytes and breast cancer cells, and then using a combination of proteomics, database analysis and clinical breast cancer samples analysis to study the mechanism mediated adipocytes-derived collagen reorganization.

Results

Here, we found that CAAs co-cultured with breast cancer cells increased the linearly aligned type I collagen fibers deposition in vitro and in vivo, which accompanied with increasing collagen lysyl hydroxylation 2 (LH2) and lysyl oxidase (LOX) expression. Breast cancer cells migrated orderly along the CAAs-derived collagen, while randomly migrated through the mature adipocyte-derived collagen. Simultaneously, clinical breast cancer samples analysis showed LH2 was high expression in the areas where cancer cells infiltrated into fatty tissue, which accompanied with poor prognosis. It is well-known that interaction between adipocytes and cancer altered the secretion of cytokines. In the present study, proteomics analysis showed that IL-6, MIF-1, TECK, MCP-1, PAI-1, IGFBP-1, TIMP1 and TIMP2 are highly secreted in adipocytes-breast cancer system. And using agonist or antagonist of these cytokines showed that these cytokines might involve in the reorganization of adipocyte-derived collagen. Mechanistic investigations demonstrated PI3K/AKT pathway and JAK/STAT3 signaling pathway involved in the rearrangement of adipocyte-derived collagen, which mediated by promoting forkhead-box(FOX) and phosphorylation STAT3(p-STAT3) translocate to the nucleus, respectively. Moreover, FOX and STAT3-dependent induction of LH2 expression further potentiated the adipocytes-derived collagen reorganization, which thus promoting breast cancer metastasis.

Conclusion

Collectively, our findings reveal new insights underlying increased breast cancer progression and offer new opportunities for stromal-targeted therapies in breast cancer.

#174

Collagen fiber orientation influences tumor cell growth and Hedgehog signaling in the breast tumor microenvironment.

Ana M. Reyes, Jorge Almodovar, Maribella Domenech. _University of Puerto Rico-Mayaguez, Mayaguez, PR_.

Collagen matrix architecture is an important player in tumor transition to therapeutic resistance. Several studies have shown that high level of fiber orientation and density of collagen type I matrix enhances tumor cell invasion, decrease drug response and influence tumor metabolism, but the mechanisms of action that support transition to therapeutic resistance are not fully understood. We developed a 3D culture platform to evaluate the effects of collagen matrix architecture in the sensitivity of tumor and stromal cells to tumor promoting ligands and pharmacological inhibitors. Estrogen (E2) and Hedgehog (Hh) signaling pathways were selected for evaluation due to their relevance in breast cancer prognosis and tumor progression. In this 3D culture platform, electrospun collagen fiber sheets of random or aligned orientation are bound to double-sided medical grade tape to generate collagen fiber stickers. Automated cutting plotter allows razor-printing for precisely cutting shapes from collagen fiber sticker sheets. Round shapes were applied like a sticker to the bottom of culture well plates prior to cell seeding. The percentage of matrix porosity fiber orientation and diameter was quantified by Scanning Electron Microscopy (SEM) and Image J analysis. For estrogen signaling studies, MCF-7 cells were seeded in collagen fibers of random or aligned orientation using hormone free-culture conditions. Cells were treated +/- E2 (10nM) ligand and +/- estrogen receptor signaling inhibitors (Fulvestrant and Tamoxifen). Tumor cell proliferation was quantified at 48hrs via Image Cytometry. For Hh signaling studies, expression levels of GLI1, PTCH1 and SMO Hh target genes were monitored in triple negative breast cancer (MDA-MB-231 and MDA-MB-468) and mesenchymal cells. Cells were seeded on collagen fibers and treated with sonic hedgehog ligand for 24hrs. Tumor and mesenchymal cells were harvest for viability and qRT-PCR analysis respectively. Evaluation of cell growth in MCF-7 cultures shows that a fibrillar structure of collagen matrix in combination with high degree of fiber orientation can significantly increase cell proliferation as compared to gelatin or tissue culture plastic substrates. Collagen fibers significantly supported hormone-independent tumor growth at similar levels to those observed in E2 treatment. Increased proliferation levels were maintained in ER inhibition groups particularly when cultured in aligned collagen fibers. In Hh signaling studies, overexpression of GLI1 and SMO genes were observed for collagen fibers matrixes as compared to gelatin and tissue culture plastic. Overall our results show for the first time that collagen fiber architecture can promote hormone-independent growth dependence in breast cancer cells and activation of hedgehog signaling in tumor and mesenchymal cells.

#175

Mechanical transduction mediated by Integrin-ILK dependent actin dynamics drives stem-plasticity leading experimental metastatic colonization of prostate cancer leading experimental metastatic colonization of prostate cancer.

Lanpeng Chen, Stefano Coppola, Nick Landman, Arwin Groenewoud, Thomas Schmidt, B.Ewa Snaar-Jagalska. _Leiden University, Leiden, Netherlands_.

Incurable bone metastasis is a main cause of death in prostate cancer. Metastasis is believed to be initiated by a small subpopulation of cancer cells termed tumor initiating cells (TICs) or cancer stem-like cells (CSCs). One of the key steps of the metastatic cascade is metastatic colonization of circulating tumor cells/clusters (CTCs) in a distant niche from circulation. From clinical database analysis, a significant up regulation of genes related to cell-ECM interaction was observed in prostate cancer patients derived CSCs, implying that the enhanced focal adhesion ability and its correlated cytoskeleton remodeling of CSCs plays a critical role in regulating metastatic colonization. However, due to a lack of tools to visualize the low amount of disseminated CSCs at high imaging resolution, it remains unknown how those cells behave when they interact with the stromal compartment at the inception of metastatic colonization. Hereby, we intravenously injected osteotropic prostate cancer cells with fluorescently-labeled F-actin into transparent zebrafish embryos to monitor single cell dynamics during metastatic colonization. In this model we observed a highly dynamic actin-based cytoskeletal remodeling in ALDHhi CSCs that controls extravasation and metastatic colonization. Transcriptome analysis revealed that this cytoskeleton remodeling was regulated by the focal adhesion factors: Integrinβ1 and integrin linked kinase (ILK). Genetic targeting of Integrinβ1 and ILK significantly inhibited expression of pluripotency genes, ALDH activity, metastatic colonization and outgrowth, suggesting the Integrinβ1-ILK axis as a key metastatic regulator that drives stem-like properties of CSCs during metastasis. Further in vitro and in vivo analysis showed that the Integrinβ1-ILK axis controls prostate cancer stem plasticity by generating contractile force through small GTPase-CDC42 and invadopodia regulator-N-wasp when the cells physically interact with the ECM. This mechanical transduction further activates mechanical sensor YAP/TAZ in a hippo-independent manner and promotes the expression of pluripotency genes that support metastatic initiation. Interference with this process by blocking CDC42-N-wasp signaling significantly attenuated mechanical transduction, impaired YAP/TAZ nuclear translocation and eventually inhibited metastatic initiation. Taken together, this study indicates a fundamental role of the mechanical transduction between ECMs and CSCs mediated by integrin/ILK dependent cytoskeleton remodeling at metastatic onset and outgrowth. Pharmacological targeting of this process may be a potent approach to attenuate the formation of prostate cancer metastasis on clinic.

#176

DDR2-depleted mesenchymal stem cells attenuate the tumor-promoting effect of breast cancer cell engulfment.

Maria E. Gonzalez,1 Yu-Chi Chen,2 Celina G. kleer2. 1 _Univ. of Michigan Comp. Cancer Ctr., Ann Arbor, MI;_ 2 _University of Michigan, Ann Arbor, MI_.

Background: Mesenchymal stem cells (MSCs) are recruited to the tumor microenvironment and promote tumor progression. We have demonstrated that MSC expressing DDR2, a collagen receptor, mediates stromal-breast cancer direct interactions and promotes metastatic growth. Tumor microenvironment studies have shown that transfer of cellular constituents from stromal cells to cancer cells stimulate malignant behavior but the mechanisms need further study. Pathologists have noticed that metastatic carcinoma cells in tissues display cell "cannibalism", and MDA-MB-231 cells were recently found to cannibalize MSC, but the in vivo consequences and mechanisms are unclear. We hypothesize that breast cancer cells engulf MSC which enables metastatic dissemination, and that DDR2 inhibition in MSC may block this effect.

Methods: We used GFP labeled breast cancer cells (BCC) of high metastatic potential (MDA-MB-231 and -436), non-metastatic (MCF7), and non-tumorigenic (HME and MCF10A), and Ds-RED labeled MSC controls (MSC-shC) and with DDR2 shRNA knockdown (MSC-shDDR2). 3D-Co-culturing MSC-Ds-Red and BCC-GFP and Image Stream flow cytometry single cell system were used to quantify cell engulfing. Live Imaging Delta Vision was used to visualize engulfing. We developed a microfluidic high-throughput cell paring and retrieval platform to study BCC-MSC-shC and shDDR2 engulfing clones which allowed selective retrieval of single cells. RNA sequencing of engulfing MSC-shC or MSC-shDDR2 BCCs clones was compared to non-engulfing BCCs. The in vitro and in vivo relevance of engulfment of MSC-shC or MSC-shDDR2 by BCCs was assessed by WB, invasion, migration, mammosphere assays, and xenografts.

Results: Subpopulations of metastatic MDA-MB-231 and -436 cells engulf MSCs. MSC engulfment was not detected in non-metastatic and non-tumorigenic breast cells. Using our high-throughput cellular pairing platform we visualized MSC engulfment, and retrieved engulfing and non-engulfing BCCs. RNA sequencing revealed a 7-gene engulfing signature by comparing BCC engulfing MSC-shC, and BCC engulfing MSC-shDDR2, BCC non-engulfing. Functionally, MSC engulfment enhances EMT, mammospheres, migration and invasion of BCCs.

Conclusions: We developed a high-throughput cellular pairing platform to study MSC engulfment by BCC, and demonstrated that engulfment of MSC by BCC is a key mechanism enabling tumor progression. Our study suggests that DDR2 knockdown in MSC reduces their ability to enhance tumorigenic functions after engulfment by BCC. We identify an MSC engulfment gene signature with potential for developing of new tissue-based biomarkers of metastasis.

#177

Increased stiffness of the tumor microenvironment in colon cancer leads to an increase in activin and metastatic potential.

Jessica Bauer,1 Jonas J. Staudacher,2 Georgina Mancinelli,1 Nancy Krett,1 Emon Bashar,3 Paul Grippo,1 M Taher A. Saif,3 Barbara Jung1. 1 _University of Illinois at Chicago, Chicago, IL;_ 2 _Charite, Berlin, Germany;_ 3 _University of Illinois at Urbana-Champaign, Urbana, IL_.

Colorectal cancer (CRC) is the second deadliest cancer in the US and understanding the switch to metastatic behavior and developing therapeutic strategies to target this process are clinical challenges. Stromal cells and cancer associated fibroblasts (CAF) play a critical biophysical role in cancer progression. CAFs are contractile and mechanosensitive which remodel the tumor microenvironment (TME) by cross linking, deforming and stiffening the matrix and release growth factors, which offers a unique niche for the cancer cells to become metastatic. We hypothesize that CAF-generated mechanical forces lead to metastatic spread directly or indirectly via release of growth factors including a member of the TGFb superfamily, namely activin. We will explore the mechanistic link between fibroblast force, activin release, and the induction of EMT and compare it with the activin serum levels of a CRC patient cohort. We measured activin serum levels by ELISA assay from a retrospective cohort of twenty-eight colorectal cases and twenty-eight polyp free controls. Conditioned media from co-culture of human CRC epithelial cells (FET) and stromal cells (CCD18) was examined by ELISA assay for secreted activin and was also used to culture CRC FET cells to measure induction of metastatic behavior through increase in proteins involved in epithelial-to-mesenchymal transition (EMT). CCD18 were seeded on substrates with increasing stiffness (2kPa, 10kPa, 40kPa) and treated for 72 hours with or without TGFb to generate conditioned media. FET cells were incubated with this media and subjected to trans-well migration and metabolic activity assays.Activin levels were up regulated in serum from stage IV CRC patients. High activin levels may be a predictive marker for metastasis in this cohort, with an Area Under the Curve of 0.8163. CCD18 cells secreted a 10-fold higher level of activin than FET cells alone and co-culture of CCD18 and FET cells resulted in an additional 2-fold increase in activin. Treatment of conditional media from TGFb activated stromal cells with the activin ligand trap, follistatin, leads to a decrease in FET cell migration and reversal of EMT indicating that the activin increase is functionally relevant and critical to the pro-oncogenic stromal response by TGFb. We plated CCD18 cells 2, 10 and 40 kPa polyacrylamide (PA) substrates functionalized by fibronectin with and without TGFb. We found that, with increasing gel substrate stiffness, (a) FET became more migratory, (b) CCD18 contractility increased with increasing stiffness, (c) activin in the condition media increased with increasing force. Inhibiting activin with follistatin decreased the TGFb induced activin secretion and migration in FETs. We suggest that increased TME stiffness leads to stromal cells amplification of TGFb pro-oncogenic function through thus far unrecognized induction and utilization of activin signaling.

#178

Quantification of matrix remodeling during H1299 lung cancer cell migration in microfluidic devices.

Maria Anguiano, Xabier Morales, Mikel Ariz, Martín Martínez Villar, Carlos Ortiz-de-Solorzano. _Foundation for Applied Medical Research, Pamplona, Spain_.

Introduction

Metastasis is a hallmark of cancer and represents a major clinical challenge. Since cell migration is a key process for metastasis, it is of great importance to understand the mechanisms of cancer cell migration. In the last decade, novel microfluidic devices, containing extracellular matrix (ECM) biomimetic hydrogels, have become attractive tools to recapitulate the tumor microenvironment, thus permitting the study of cancer cell migration in a more physiological 3D environment.

Previously, we have used microfluidic platforms, filled with hydrogels of mixed collagen-Matrigel composition, to investigate the effect of the microenvironment on H1299 NSCLC migration, and demonstrated how the biomechanical properties of the hydrogels determined the phenotype and migration speed of the cells. We concluded that a balanced composition of collagen and Matrigel favors cell migration, due to an increased matrix stiffness and pore size of the hydrogels.

Objectives

To further examine the impact of the ECM on cell migration, we now quantify matrix remodeling due to metalloproteinase (MMP) activity during H1299 migration, in mixed collagen-Matrigel hydrogels. Our second goal is to study the relationship between H1299 cancer cell migration and H1299 integrin expression levels in the same hydrogels, to understand how integrin profile is modulated by hydrogel composition.

Methods

We used three different hydrogels with the same collagen concentration of 2 mg/ml and 0 (C), 2 (CM) and 4 (CM+) mg/ml of Matrigel respectively. Proteolytic degradation of hydrogel caused during H1299 cell migration was quantified as the volume of a dye-quenched protein substrate (DQ collagen I) at 25 µg/ml, imaged by confocal microscopy (40X) and measured using in-house developed image analysis tools.

β1 and β3 integrin expression at cell surface was quantified by flow cytometry after stimulating cell migration inside the hydrogels for 24 h.

Results

The DQ volume per cell ratio revealed the proteolytic degradation was ~5 times higher in Matrigel-containing hydrogels CM and CM+ than in collagen-only hydrogel C. Cytometry assays showed β1 integrin is expressed by H1299 cells significantly more than β3 integrin in all three hydrogels. Moreover, we found β1 integrin expression at cell surface was higher in CM and CM+ hydrogels than in C hydrogels.

Conclusions

Hydrogel composition modifies H1299 integrin profile at cell surface. Accordingly, β1 integrin expression is higher than β3 integrin expression in all hydrogels studied as a result of collagen type I being the main binding ligand to β1 integrin.

Matrigel, at an intermediate, balanced concentration, enhances cell migration by stimulating both integrin expression at cell surface and matrix degradation due to MMP activity, thus compensating the higher degree of cell confinement compared to collagen-only hydrogels.

#179

Cellular anisotropies and phenotypic diversity regulate the contact guidance response to aligned matrix architectures in the tumor microenvironment.

Arja Ray,1 Rachel Morford,1 Zaw Win,1 Rachel Edwards,1 Oscar Lee,2 Deok-Ho Kim,2 Patrick Alford,1 Paolo P. Provenzano1. 1 _Univ. of Minnesota, Minneapolis, MN;_ 2 _Univ. of Washington, Seattle, WA_.

Directed carcinoma cell migration along aligned stromal collagen due to contact guidance is known to promote disease progression and leads to poor outcomes in human patients. In order to elucidate the physical and molecular mechanisms governing contact guidance of carcinoma cells, intravital imaging, as well as a number of powerful engineered 2D and 3D platforms, have been employed. We demonstrate that for single cells, and particularly those that have undergone epithelial-to-mesenchymal transition, aligned extracellular matrix architectures providing contact guidance cues induce constrained focal adhesion maturation and associated F-actin alignment. This consequently orchestrates anisotropic traction stresses that drive cell orientation and directional migration along aligned collagen architectures. Thus, alignment of mature force bearing adhesions allow single mesenchymal-like cells to spontaneously 'sense' and follow matrix alignment. Consistent with this understanding, relaxing spatial constraints to adhesion maturation either through a pharmacologically-induced reduction in adhesion size or reduction in substrate alignment density diminishes the contact guidance response. Furthermore, the response to extracellular matrix guidance cues is diminished when the complexity of the mechanical interaction is increased through the addition of forces across cell-cell adhesions. Indeed, intercellular interactions within epithelial clusters temper anisotropic cell-substratum forces, resulting in substantially lower directional response along guidance cues. These results demonstrate that the response to contact guidance is regulated by a balance of cell-substratum and cell-cell interactions that is further modulated by cell phenotype-specific cytoskeletal arrangements. Accordingly, our findings elucidate how phenotypically diverse cells perceive ECM alignment at the molecular and cellular levels and elucidate why phenotypically distinct cells respond to guidance cues to differing degrees that depend on combinations of cell phenotype, matrix architecture, and the length scale of the guidance cues. Indeed, rapid phenotypic switching, particularly in cancer stem cell subpopulations, can profoundly enhance directed migration through aligned matrices, consistent with observations during live tumor imaging. Thus our findings suggest that a complementary approach of targeting carcinoma cell adhesion dynamics in concert with stroma targeting approaches in order to re-engineer the tumor microenvironment to disrupt direct migration cues may be a robust strategy to disrupt disease progression.

#180

OPNa variant expression is associated with matrix mineralization in thyroid cancer cell lines.

Luciana B. Ferreira. _INCA, Rio de Janeiro, Brazil_.

Osteopontin (OPN) and its three spliced variants (OPN-SV: OPNa, OPNb and OPNc) are overexpressed in several tumors and frequently associated with cancer progression. This holds true for papillary thyroid carcinoma (PTC), which is the most common variety of thyroid cancer (TC), being the histologic type which often presents desmoplasia (collagen deposition) and dystrophic calcification, including a fairly typical feature, the psammoma bodies (PB). This study aims to investigate the OPN-SV expression in classical variant of PTC (cPTC) and the role of OPN in calcification and collagen deposition into the extracellular matrix of thyroid cancer cell lines. Total OPN and OPN-SV expression was analyzed by immunohistochemistry and real time PCR in a series of 48 cPTC cases and three diffuse sclerosing PTCs. The association of OPN expression and the presence of PB as well as between PB in cPTC and the clinicopathological features of the tumors were evaluated. TPC-1 and c643 TC cell lines overexpressing OPN-SV were tested for the ability to promote calcification and to synthesize collagen in vitro. We also evaluated the expression of calcification markers, including osteocalcin and collagen I. Overexpression of OPNa transcripts was significantly associated with the presence of PB in cPTC samples. The presence of PB in cPTC was associated with younger patients and lymph node metastasis. Moreover, OPNa overexpression displayed a strong capacity to promote calcification and substantial collagen deposition in thyroid cancer cell lines. Upon OPN knockdown, the mRNA expression levels of calcification markers (osteocalcin and collagen I) were downregulated, further evidencing the involvement of OPN on activating these processes. Our data suggest that OPNa plays a role in the extracellular matrix calcification associated with cPTC. Basic research on the interactions between OPNa overexpression by tumor cells and the surrounding microenvironment can give clues for a better understanding of cPTC biology and phenotype.

#181

Biomimetic, 3D hydrogels to investigate effects of microenvironment biophysical properties on patient-derived glioblastoma (GBM) and endothelial cells (ECs).

Alireza Sohrabi, Jesse Liang, Deepthi Muthukrishnan, Itay Solomon, Carolyn Kim, Amelia Lao, Harley Kornblum, Stephanie Seidlits. _UCLA, Los Angeles, CA_.

Introduction: This study investigated how the mechanical microenvironment of glioblastoma (GBM) tumors may affect morphology and phenotype of patient-derived GBM and brain endothelial cells (ECs)-- both critical components in the perivascular invasive niche.

Materials and Methods: Hydrogels were fabricated from thiolated hyaluronic acid (HA-SH) (0.5 w/v%) and 4-arm PEG-SH (1.5 w/v%), 8-arm PEG-norbornene (1.125 w/v%). Gelation occurred upon exposure to UV light (365 nm, 3-5.75 mw/cm2) for 15 s in the presence of a cytocompatible photoinitiator LAP (Lithium Pehnyl (2, 4, 6-trimethylbenzoyl) phosphinate, 0.025 w/v%). Patient-derived gliomaspheres or single dissociated ECs were mixed with hydrogel precursors prior to gelation. Phase contrast images were acquired every 3 days to monitor cell migration. After 9 days, 3D cultures were fixed, and cells visualized with CellMaskTM Green (ThermoFisher) and Hoescht (nuclei). Cell survival following encapsulation was evaluated using a Live/Dead Assay (Life Technologies). A Leica LSP5 confocal microscope was used to image 3D cultures. Storage moduli (G') of hydrogels were measured using shear rheometer (TA DHR-2) with an 8-mm flat plate geometry.

Results and Discussion: Hydrogel stiffness (G') was controlled by varying UV intensity to achieve a range of 150-1500 Pa, which is representative of the normal brain and tumor microenvironments, respectively. Gliomaspheres and ECs cultured showed comparable survival 7 days post-encapsulation in both soft and stiff gels. Gliomaspheres cultured in stiff hydrogels remained did not migrate away from spheroids, while cells encapsulated in soft hydrogels exhibited robust migration. Our previous results showed that ECs residing in GBM perivasculature differentially express high amount of integrin-binding sialoprotein (IBSP). Survival of ECs in hydrogels baring different biophysical properties is an appropriate platform to study the effect of stiffness on ECs protein expression.

Conclusion: Patient-derived GBM and ECs were successfully cultured in 3D HA hydrogels mimicking biophysical properties of brain or GBM tumor tissue. GBM cells cultured in stiffer environments were unable to migrate, but upon culture in soft environments were found to migrate up to 200 µm away from seeded spheroids within 9 days of cultures.

Acknowledgment: This project is funded by NIH 1R21NS0931099-01A1 grant and University of California Cancer Research Coordinating Committee Research Award. NIH Training Grant in Genomic Analysis and Interpretation T32HG002536.

#182

Superior targeting of tumor-stromal interactions and endothelial migration with a bispecific antibody to α5 and αv integrins.

Raghav Joshi, Wenying Ren, Paul Mathew. _Tufts Medical Ctr., Boston, MA_.

Introduction: The integrin α5β1 has been implicated in the adhesive and migratory response of prostate cancer cells induced by fibronectin fragments secreted by human bone-marrow derived mesenchymal stromal cells (hBM-MSCs) [Joshi, Cell Adh and Migr 2016). Genetic inactivation of integrin α5 in PC-3 cells downregulated the bcl-2 family of proteins and induced programmed cell death (Ren, Mol Cell Res 2017) - linking hBM-MSCs to a putative bone marrow niche survival program for prostate cancer cells. We hypothesized that the fibronectin-binding integrins α5β1 and integrin αv collaborate in broader tumor-stromal interactions and sought to examine the impact of combinatorial inactivation of these integrins.

Methods: Monospecific antibodies to α5 (or its obligate α5β1 heterodimer) and to integrin alpha v (or the αvβ3 heterodimer) were utilized to assess the efficacy of a combinatorial neutralization approach over either single agent in α5/αv co-expressing cells from diverse tumor types including prostate, breast, glioma, cervix and uterine cancer. Assays included adhesion, migration, cell survival and the induction of endothelial migration in co-culture with stromal cells. To address the hypothesis that a bispecific integrin targeting antibody would be superior to a combinatorial approach with monospecific integrin antibodies, a bispecific antibody prototype that simultaneously targets the α5β1 and αv integrins [Bsα5β1/αvAb] was constructed and deployed in comparative assays.

Results: Combined α5 and av neutralization with dual monospecific antibodies was superior to individual single agents in blocking adhesion, migration and the induction of endothelial chemotaxis across diverse tumor types that co-expressed α5 and αv integrins. However, a significant superiority of the Bsα5β1/αvAb was noted over the combinatorial approach with monospecific antibodies in these assays. In addition, a significant reduction in cell survival was noted in selected tumor types with the Bsα5β1/αvAb. Strikingly, the Bsα5β1/αvAb was significantly more potent than bevacizumab in the inhibition of endothelial migration induced by tumor-stromal cell interactions

Conclusions: Combinatorial α5 and av integrin targeting in tumor cells that co-express α5 and av integrins effectively abrogates tumor-stromal interactions and the resultant endothelial migratory response. A Bsα5β1/αvAb demonstrates a significant improvement over combinatorial monospecific antibodies likely through diverse mechanisms including cross-priming. Targeting tumor, stromal and endothelial cells simultaneously with a Bsα5β1/αvAb approach represents a potentially effective therapeutic strategy for targeting diverse mechanisms of progressive disease in the tumor microenvironment.

#183

HERG1 potassium channels perturb the β1 integrins mediated force transduction machinery in pancreatic cancer.

Stefano Coppola,1 Claudia Duranti,2 Annarosa Arcangeli,2 Thomas Schmidt1. 1 _Physics of Life Processes, Huygens-Kamerlingh Onnes Laboratory, Leiden University, Leiden, Netherlands;_ 2 _Department of Experimental and Clinical Medicine, University of Florence, Florence, Italy_.

Ion channels regulate cell proliferation, differentiation, and migration in normal and neoplastic cells through cell-cell and cell-extracellular matrix (ECM) transmembrane receptors called integrins. K+ flux through the human ether-à-gogo-related gene 1 (hERG1) channel shapes action potential firing in excitable cells such as cardiomyocytes. Its abundance is often aberrantly high in human tumors, including the pancreatic ductal adenocarcinoma (PDAC). We recently demonstrated that the direct interaction of β1 integrins with hERG1 channels in cancer cells stimulated distinct signaling pathways that depended on the conformational state of hERG1 and affected different aspects of tumor progression [1].

We hypothesized that hERG1 channels compromise the PDAC mechano-reciprocity, the ability to dynamically respond to externally applied forces by exerting forces, which enhances invasion and compromises treatment.

Using elastic micropillar arrays of varying stiffness (20-150 kPa) [2], we quantified the β1-integrin mediated forces exerted by PANC-1 cells. Micropillars coating with collagen and/or fibronectin derived peptides allowed us to direct cell surface receptor binding specificity (i.e. α2β1 and α5β1 integrins). Independently of the substrate coating, PANC-1 cells exerted higher forces as a function of substrate stiffness, as already demonstrated for other cell types. Remarkably, the disruption of the β1/hERG1 interaction through E4031 inhibition of hERG1 channels resulted in a significant increase in the detected cellular forces by ~20%, accompanied by an increase of focal adhesion areas as demonstrated by staining with vinculin.

Our results suggest that, in addition to alter distinct signaling pathways and tumor progression [1], the direct interaction of β1 integrins and hERG1 channels perturbs the force transduction machinery. These findings encouraged us to develop bispecific antibodies (scDb, single-chain diabody) binding to the β1/hERG1 complex that are now being validated in PDAC cell lines. In particular, we have developed a bispecific antibody (scDb-β1/hERG1) [3], which is composed by the variable domains (VH and VL chains) of monoclonal antibodies binding two different antigens, hERG1 and β1 integrin. Data obtained from in vitro studies demonstrates that the scDb antibody impacts cell viability and migratory behavior in Mia PaCa-2 cell lines, holding promises for translational impact after a further validation in vivo as a novel candidate in cancer therapeutics.

[1] Becchetti et al. Science Signaling (2017), 10 (473), eaaf3236.

[2] van Hoorn et al. Nano Lett (2014), 14, 4257-4262.

[3] Patent: "Novel antibodies". Inventors: Annarosa Arcangeli, Claudia Duranti, Silvia Crescioli, Laura Carraresi. Patent Deposit: July 2017. Patent Ref: 102017000083637 (University of Florence).

#184

Three-dimensional density model promotes invasive characteristics in normal mammary epithelial cells.

Shayan Nazari, Pinku Mukherjee. _UNC Charlotte, Charlotte, NC_.

Background: Breast cancer (BC) is the most diagnosed cancer and the fourth leading cause of cancer related mortality among women. One of the highest risk factors for developing BC is dense breast tissue. Mammographic density (MD) is defined by the amount of stromal and epithelial tissues present in the breast. Approximately, 50% of women in the US have high MD and are 4-6 times more likely to develop BC in their lifetime. The stromal microenvironment is characterized by extracellular matrix (ECM) reorganization and stiffness. One of the most abundant molecules in ECM is collagen type I (Col1). High Col1 expression is seen in women with high MD and is also correlated with cell proliferation, survival, and invasion. Unfortunately, the underlying cellular and molecular mechanisms for the association between MD and BC are poorly understood. Therefore, understanding the molecular pathways involved in the unique transformation of normal mammary epithelial cells (nMECs) to cancer cells in a dense microenvironment is an important step in finding diagnostic tools and therapeutic targets for prevention and treatment of BC.

My hypothesis is that 3-dimensional (3D) collagen-dense matrix promotes an invasive phenotype and activates oncogenic cell signaling pathways in nMECs, and hTERT-HME1 cell line, contributing to their transformation.

Methods: I conducted an in vitro study using hTERT cells cultured in 3D COL1-dense gels todetermine the phenotypical changes and cellular signaling alterations caused by high collagen density.

Results: I have observed that hTERT cells in high 3D COL1-dense gels form invasive phenotypes with protrusive structure compared to cells in low density gels. My previous data shows that, hTERT cells cultured in 2D-COL1 coated plates will express mesenchymal markers and over-express ERK1/2 and PI3K pathways which promote cancer cell proliferation and tumor cell survival. My plan is to further analyze the oncogenic cell signaling pathways that drive the transformation of hTERT cells into invasive cells, using my 3D COL1-dense gels. These pro-oncogenic pathways include, MAPK pathway, PI3 Kinase pathway, the activation of Epithelial to Mesenchymal Transition (EMT) markers as well as the expression level of tumor-associated MUC1 (tMUC1) . Unlike MUC1, tMUC1 is hypo-glycosylated and is over expressed in more than 90% of invasive breast carcinomas. I plan to investigate the pathway of association between inflammation caused by dense tissue, MUC1 signaling and cell transformation that could drive carcinogenesis.

Conclusion: A collagen-stiff mammary microenvironment can induce carcinogenic phenotypes and cell signaling behavior in nMECs, which highlights the possible first steps in the transformation of nMECs into cancer cells. Given that high MD is one of the major risk factors for BC development, it is critical to decipher the molecular mechanisms associated with the high risk.

#185

p300 and STAT3 drive YAP-independent mechanotransduction during breast cancer invasion.

Joanna Y. Lee, Jessica Chang, Sungmin Nam, Hong-pyo Lee, Antonia A. Dominguez, Sushama Varma, Lei S. Qi, Robert B. West, Ovijit Chaudhuri. _Stanford University, Stanford, CA_.

83% of non-invasive breast cancers are diagnosed as ductal carcinoma in situ (DCIS). While some DCIS tumors remain confined in the mammary duct, about half progress into invasive ductal carcinoma (IDC), when carcinoma cells break through the basement membrane (BM) into the type-1 collagen (col-1) rich stroma, a key first step towards metastasis. As 90% of cancer-related deaths are due to metastasis, preventing progression to invasive disease could significantly reduce cancer mortality. However there are no established biomarkers for invasive potential and current diagnostic methods cannot predict which DCIS cases will progress to IDC. Interestingly, increased tissue stiffness correlates with invasion and the transcriptional regulator YAP has been implicated as a mechanotransducer, largely based on 2D culture studies. To identify the drivers of DCIS progression, we encapsulated mammary epithelial cells (MECs) in 3D hydrogels with a range of stiffness and that presented either BM-ligands or col-1 containing microenvironments to the cells. RNA-seq identified the global gene expression changes induced by increased 3D culture stiffness in BM-like environments. 3SEQ analysis of breast cancer patient samples revealed that genes regulated by 3D culture stiffness were upregulated in breast cancer patients, demonstrating the relevance of 3D culture models and suggesting expression of S100A7 as a potential biomarker of breast cancer progression. Interestingly gene expression changes induced by increased 3D stiffness in BM-like environments were distinct from col-1 like environments. Col-1 exposure in stiff gels promotes expression of genes whose protein products remodel the col-1 network including FN1 and LOX. As col-1 remodeling promotes cell dissemination, this suggests that col-1 exposure following BM invasion induces pro-metastatic changes in carcinoma phenotype. Surprisingly, enhanced stiffness induced invasion in MECs independently of YAP activation in both BM-like and col-1 rich 3D hydrogels. Instead, bioinformatic analysis identified transcriptional regulators p300 and STAT3 as mediators of 3D mechanosensing. Inhibition of p300 and STAT3 in stiff 3D BM-like environments and, conversely, overexpression in soft 3D BM-like environments confirm a role during stiffness-induced proliferation. This suggests p300 and STAT3 as possible targets for preventing progression to invasive disease. While genetic alterations initiate transformation, these results reveal the miroenvironment events that initiate breast cancer invasion.

#186

Small-molecule inhibition of cathepsins L and K as potential therapeutics for macrophage-driven breast cancer.

Samantha S. Dykes, Dietmar W. Siemann. _University of Florida, Gainesville, FL_.

Tumor-associated macrophages secrete many factors including proteases and growth factors, which ultimately promote the metastatic phenotype of mammary tumors. Due to their numerous pro-tumor functions, tumor-associated macrophages represent an attractive cell population for stromal-targeted anti-cancer therapies. Interleukin-4 stimulation promotes macrophage differentiation from a baseline M0 state to a more pro-tumorigenic M2 phenotype, which is often associated with increased expression of proteases including the lysosomal cathepsin L. Cathepsin L is important in both macrophage and tumor cell invasion, whereby secreted cathepsin L degrades the extracellular matrix allowing for cell infiltration. M0 to M2 differentiation was not accompanied by an overall increase in the protein expression of cathepsin L, however, secreted cathepsin L was increased in the M2 macrophage population. The present study examined the role of cathepsin L in M0 to M2 differentiation and macrophage-mediated tumor cell invasion using the novel cathepsin L/K inhibitors KGP94 and KGP207 [Dr. Kevin Pinney, Baylor University]. Boyden chamber assays revealed that KGP94 and KGP207 prevented in vitro M2 macrophage invasion and reduced macrophage-stimulated invasion of 4T1 murine breast cancer cells. KGP94 and KGP207 treatment also partially prevented IL-4-stimulated M0 to M2 differentiation of macrophages as determined by a decrease in the IL-4-induced expression of the M2 marker Arginase-1 upon drug treatment. Furthermore, exogenous recombinant cathepsin L partially stimulated the expression of Arginase-1 in M0 macrophages. Together, these data suggest that cathepsin L may play a role in macrophage M0 to M2 differentiation. In summary, the novel cathepsin L/K inhibitors KGP94 and KGP207 altered M0 to M2 differentiation, reduced macrophage invasion, and reduced macrophage-stimulated invasion of breast cancer cells. These data highlight the importance of cathepsin L in macrophage functions and suggest that cathepsin inhibition strategies may be therapeutically beneficial by impairing the progression of tumors, particularly those with high recruitment of M2 macrophages.

#187

Breast cancer cells exhibit enhanced proliferation, invasion, PLAU expression and chemoresistance when exposed to fluid shear stress in an innovative 3D bioreactor.

Caymen Novak, Eric Horst, Shreya Raghavan, Geeta Mehta. _University of Michigan, Ann Arbor, MI_.

The cells within the mammary tumor microenvironment experience a range of shear stresses from both interstitial fluid flow, as well as, vascular blood flow, and this shear stress is increased as the tumor expands. Many in vitro models recapitulate the 3D microenvironment but fail to include physiological shear stress. To address this issue, a bioreactor capable of applying 3D shear stress to cells encapsulated within a 3D environment was designed, characterized, and utilized to investigate the impact of shear stimulation on breast cancer cell morphology, proliferation, gene expression, and chemoresistance. A shear bioreactor capable of applying variable 3D shear stress to hydrogel constructs was designed and fabricated in house. Shear stress of 4.92 dynes/cm2 was applied for 72 hours to MDA-MB-231, MDA-MB-468, and MCF7 breast cancer cells housed within an interpenetrating agarose-collagen type I hydrogel (IPN). The IPN hydrogel was characterized using SEM imaging and rheometric testing, and the viscoelastic modulus was determined to be 10 kPa, within the range of aggressive tumor microenvironments. Histological stains were evaluated for morphology. Immunohistochemistry staining was used for proliferation and protein evaluation. Changes in gene expression were monitored through qPCR analysis. The chemotherapeutic drug paclitaxel was investigated to observe changes in chemoresistance while under shear stress stimulation. Finally, the PLAU inhibitor amiloride was investigated for impact on cellular response to shear stress stimulus. All cell lines were found to have a significant increase in cellular area and a decrease in circularity when exposed to shear stress. Shear stimulated cells also displayed an increase in proliferation as quantified by Ki67 expression. qRTPCR analysis revealed a greater than 2-fold increase in the expression of PLAU, a common factor in breast cancer progression and metastasis. Preliminary results also show enhanced chemoresistance. Overall, shear stress stimulus was found to enhance the expression of PLAU, contribute to breast cancer proliferation, morphological changes, and chemoresistance. This bioreactor provides an ideal in vitro 3D platform for understanding the influence of shear stress stimulus on a variety of cell types and co-culture investigations.

Acknowledgements: This material is based upon work supported by the DOD OCRP Early Career Investigator Award W81XWH-13-1-0134, MIOCA and Rivkin Center for Ovarian Cancer grants to GM. This research was supported by the National Cancer Institute of the National Institutes of Health under award number P30CA046592. CMN is supported by the National Science Foundation Graduate Research Fellowship under Grant No. 1256260.

#188

Regulation of breast cancer progression by stromal DDR1.

Xiujie Sun. _UT Health Science Center at San Antonio, San Antonio, TX_.

Adipose stromal cells (ASC) constitute a significant component of breast stroma. Emerging evidence demonstrates a functional interaction between ASC and breast cancer (BC) cells. Here, we report that stromal discoidin domain receptor 1 (DDR1) is important for ASC-BC communication. Using a DDR1 knockout mouse model, we find that syngeneic mammary tumors grow more slowly in KO mice than wild-type littermate controls. In vitro, loss of DDR1 in stromal vascular fraction (SVF) impairs its ability to stimulate migration and invasion of tumor cells. Furthermore, we show that DDR1-dependent IL-6 production in SVF is responsible for the SVF effect on tumor cells. These results uncover a previously unrecognized role of stromal DDR1 in BC progression and a potential therapeutic target for BC treatment.

#189

Examining how the extracellular environment alters gene expression in breast cancer.

Joanne Nolan,1 Maeve Kiely,1 Colum P. Dunne,1 Aoife J. Lowery,2 Patrick A. Kiely1. 1 _University of Limerick, Limerick, Ireland;_ 2 _National University of Ireland, Galway, Galway, Ireland_.

Breast cancer represents 25% of all cancers diagnosed in women globally with almost 1.7 million new cases annually. The average survival rate for patients with early stage breast cancer has increased, however the survival rate for those with metastatic breast cancer has not improved. Metastasis is the number one cause of death in breast cancer patients; it remains a scientific enigma, with no available early detection screen or targeted therapies available to patients. Therefore, understanding how and why cancer cells migrate is of significant biological and clinical interest. Accumulating evidence suggests the metastatic potential of breast cancer is influenced by the composition of the extracellular matrix (ECM). The ECM is composed of fibrous proteins and other non-cellular biomolecules and has a well-documented role in influencing tumour structure and behaviour. We are examining genes that code for several categories of proteins involved in regulating the composition of the ECM. These genes code for proteins that are involved in signal transduction from the cell membrane or for proteins that are secreted into the ECM. We are also examining genes that code for fibrous proteins, glycoproteins, transmembrane proteins, growth factors and proteases. To refine our panel, we used STRING [1], an online database, to examine the relationships between the proteins coded by our genes based on the experimental determination of protein co-expression and protein homology. Using qRT-PCR, we are examining the expression of our refined gene set in three different cell lines, (representing the major sub-categories of breast cancer) which have been cultured on collagen, fibronectin, laminin and stimulated by various growth factors. We extended our study to examine the expression pattern in cells that were maintained in 3-Dimensional culture over a period of 10 days in an attempt to mimic more closely the physiological process. We have determined that there is significant differential gene expression of integrins and growth factor binding proteins as we change the composition of the cells culture environment. This work is helping us to refine a signature set by identifying genes whose expression is dysregulated in response to changes in the stromal environment. We are moving now to determining whether dysregulation of the expression of our gene panel correlates with disease progression by examining matched normal and diseased patient tissue. This could provide the opportunity to generate a novel gene signature that may be used as a prognostic tool to predict invasive cancers. It may also provide information on the design of novel treatment therapies for patients with metastatic cancers.

1. Szklarczyk, D., et al., The STRING database in 2017: quality-controlled protein-protein association networks, made broadly accessible. Nucleic Acids Res, 2017. 45(D1): p. D362-d368.

#190

Tumor endothelial cells as a targetable gateway that modulates access of drugs to cancer cells.

Yue Wu, Michael J. Greene, Jianmin Wang, Gary J. Smith. _Roswell Park Cancer Inst., Buffalo, NY_.

Introduction: Human endothelial cells (EC) lining the tumor vasculature of human prostate adenocarcinoma are preserved in primary xenografts of intact surgical remnant tissue. Androgen deprivation (AD) causes de-endothelialization of primary xenografts that is followed by re-endothelialization with human endothelial cells in the absence of testosterone (T). This study determined the consequences of targeted perturbation of prostate endothelium on access of chemotherapeutic agents to prostate cancer (CaP) cells. Methods: SCID mice were castrated and implanted with a device for sustained delivery of T to establish "humanized" mice. Fresh surgical specimens of human CaP or benign human prostate were transplanted onto the "humanized" mice and allowed 30 days to engraft before the integrity of the human microvascular EC barrier was perturbed by removal of the T delivery device, T-deprivation (T-D). Cisplatin (Cis-Pt) was investigated because it is excluded effectively by the membrane pumps/transporters in human prostate ECs, and it effectively kills slowly/non-proliferating cancer cells. Fresh prostate cancer tissue, benign prostate tissue, or xenografts after various times/treatments were enzymatically disaggregated and EC and CaP cells isolated using cell type-specific antibody-conjugated magnetic beads. Transcriptomes of isolated cells were acquired using RNASeq. Cis-Pt-DNA adducts in the xenografts or isolated cells were analyzed using immunohistochemistry (IHC) or Cytof, respectively. Results: Enhanced access to the xenograft tissue of circulating imaging markers during the interval of the compromised EC barrier, and after re-endothelialization, was demonstrated by: MRI (contrast dyes); MS (nanoparticles); 3) IHC (lectins); and Photo-Acoustic Imaging (hemoglobin). Cis-Pt-DNA adducts in xenografts treated during the T-D induced "window" were at greater levels than xenografts treated in intact mice. Genes classically associated with epithelial or endothelial cells demonstrated the effectiveness of the enrichment of cell types. Global transcriptome analyses revealed comparable gene expression profiles of the same cell type isolated from fresh tissue or pre-T-D xenografts. T-D induced dynamic changes in gene transcriptomes of endothelial cells, epithelial cells and stromal cells, and the transcriptomes evolved over the time after T-D. Transcriptomes of endothelial cells before T-D and after re-endothelialization were significantly different. The profile of uptake transporters and efflux pumps varies between epithelial, endothelial and stromal cells, and the expression pattern within the individual populations also evolved in response to T-D. Conclusions: Primary xenografts provide a unique tool for analysis of the roles of targeted microvascular damage in organ-specific therapies, and of the evolution of the EC compartment in response to iatrogenic intervention.

#191

Paracrine role of endothelial cells in HER3-mediated colon cancer cell survival.

Rui Wang, Rajat Bhattacharya, Fan Fan, Xiangcang Ye, Delphine Boulbes, Lee M. Ellis. _UT MD Anderson Cancer Ctr., Houston, TX_.

INTRODUCTION: Colorectal cancer (CRC) remains the second-leading cause of cancer-related death in the United States. Median survival of patients with metastatic CRC (mCRC) is ~2.5 years and patients often become resistance to systemic therapy within 1 year of treatment. Therefore, a better understanding of the regulation of CRC cell survival pathways is necessary in the development of new therapeutic strategies that will improve outcomes for patients with mCRC. We have previously demonstrated that endothelial cells (ECs) from the liver, the most common site of CRC metastases, secrete soluble factors in the conditioned medium (CM) that, in turn, increase the cancer stem cell phenotype in CRC cells.

OBJECTIVES: In this study, we sought to (1) elucidate the paracrine role of liver ECs in mediating CRC cell growth and chemoresistance, and (2) determine the mechanism(s) involved.

METHODS: Primary ECs from non-malignant liver parenchyma were isolated and confirmed. CRC cells were incubated with either their own CM (control) or CM from ECs. The effect of CM on CRC cell growth was determined by the MTT assay. The effect on chemoresistance was determined by fluorescence-activated cell sorting (FACS)-based assay for apoptosis after treating CRC cells with 5-fluorouracil (5-FU) either in control CM or EC CM. Effects of CM on CRC tumor growth in vivo was determined using a subcutaneous (subQ) xenograft tumor model. CRC cells were pretreated with control or EC CM and then injected subQ in an inoculation mixture of Matrigel and concentrated CM. Tumor burden was assessed over time by bioluminescence with in vitro imaging system (IVIS) and by measuring tumor volume with calipers.

RESULTS: CM from liver ECs significantly increased cell growth and chemoresistance in CRC cells in vitro via activating the AKT pathway. We identified human epidermal growth factor receptor 3 (HER3, also known as ERBB3) being the only receptor tyrosine kinase (RTK) activated by EC CM. Furthermore, we found that inhibition of HER3, either by a HER3 inhibitor or by HER3 siRNAs, blocked EC CM-induced AKT activation, cell growth, and chemoresistance in CRC cells. In our in vivo tumor model, CRC cells treated with EC CM formed larger tumors with higher growth rates than that with control CM.

CONCLUSIONS: Our results demonstrated a paracrine role of liver ECs in promoting cell growth and chemoresistance via activating HER3-AKT in CRC cells. Identification of the liver EC-secreted factor(s) for activating CRC-associated HER3 is currently under study. This study suggested a potential strategy of treating mCRC patients with HER3 antibodies/inhibitors that are already being assessed in clinical trials for various cancer types.

#192

Preclinical evaluation of TQBWX220, a small-molecule inhibitor of IDO1.

Shilan Liu,1 Guibai Liang,1 Dahai Wang,1 Shuhui Chen,1 wenyuan Qian,1 Chi-Chung Chan,1 Xiquan Zhang,2 Lin Yang,2 Xin Tian,2 Yong Gao,2 Tiantian Dong,2 Mei Liu,2 Lihua He2. 1 _WuXi App Tec (Shanghai) Co., Ltd., Shanghai, China;_ 2 _Chia Tai Tianqing Pharmaceutical Group Co., Ltd., Nanjing, China_.

Objective: Indoleamine-2, 3-dioxygenase-1 (IDO1) catalyzes conversion of tryptophan to kynurenine (Kyn), which triggers signaling through GCN2, mTOR and AHR and affects differentiation and proliferation of T cells. Expression of IDO1 by tumor cells or host APCs can inhibit tumor-specific effector CD8+ T cells and enhance the immunosuppressing activity of regulatory T cells. Targeting the IDO pathway via inhibition of IDO1 or blocking its downstream signaling pathways is recognized as one of the prime immunomodulation approaches to unleash antitumor immunity in the tumor microenvironment. As reported, inhibition of IDO1 pathway may also synergize with inhibitory Abs of immune checkpoint proteins, PD-1 and CTLA-4, etc. We report herein TQBWX220, a potent small-molecule inhibitor of IDO.

Method: Potency of TQBWX220 was determined by corresponding IDO1 biochemical and cell assays. Antitumor efficacy of TQBWX220 was evaluated in the syngeneic CT26 xenograft colon cancer model. CT26 cells were engrafted onto the back of BALB/c mice. BID oral administration of vehicle, positive control (INCB024360, 100 mg/kg) and TQBWX220 at different doses were performed. Tumor volumes and mouse body weight were measured at time points after the start of the treatments. Other assays were carried out by using standard methods.

Result: TQBWX220 displayed good IDO1 enzymatic (IDO1 IC50=22 nM) and cellular (Hela EC50=117 nM) potency. It has excellent solubility (>500uM pH7.4) and low inhibition on CYPs and hERG. TQBWX220 significantly inhibited tumor growth at tested doses (60% TGI at 50 mpk/PO BID and 73% TGI at 100 mpk /PO BID) (P<0.05) in the CT-26 syngeneic model. As a result of IDO1 inhibition, reduction of Kyn level in the tumor was also observed.

Conclusion: The promising preclinical antitumor results and its favorable DMPK properties indicate that TQBWX220 has high potential in the clinic as a cancer immunotherapy in combination with checkpoint inhibitors such as anti-PD1 antibodies.

#193

**The tumor microenvironment protects against ibrutinib but not rituximab-mediated control of Waldenström macroglobulinemia (WM)** in vivo **.**

Weiguo Han,1 Brandon L. Sklavanitis,2 David A. Jackson,2 Stephan J. Matissek,1 Sherine F. Elsawa1. 1 _University of New Hampshire, Durham, NH;_ 2 _Northern Illinois University, DeKalb, IL_.

The tumor microenvironment (TME) plays an important role in the development and progression of cancer. It has also been shown to play a protective role in resistance to therapy. Therefore, understanding the role of the TME is a vital component of our understanding of malignant cell biology. In Waldenström macroglobulinemia (WM), a B cell malignancy characterized by the overproduction of a monoclonal IgM protein, several studies have shown that the TME plays an important role in malignant disease. Therefore, the goal of this study was to investigate the efficacy of combined targeting of the TME with either Ibrutinib or Rituximab therapy in WM in vivo. SCID mice were injected with RPCI-WM1 + HS-5 stromal cells (5:1 ratio) and upon tumor development, mice were treatment with Ibrutinib (αBTK), Actemra (αIL-6), or a combination of Ibrutinib+Actemra or vehicle controls. An additional group of mice were injected with BCWM.1 + HS-5 cells (5:1 ratio) subcutaneously followed by treatment with Rituxmab (αCD20), Actemra (αIL-6) or both Rituximab+Actemra or controls. There was no improvement in survival of mice treated with Ibrutinib or Actemra as single therapy and combined treatment did not improve survival compared to controls. We also found a reduction in tumor growth rate in mice treated with Ibrutinib + Actemra compared with mice treated with Ibrutinib alone. There was no difference in human IgM secretion among different groups when examining serum samples by ELISA. In the cohort of mice treated with Rituximab, we found that the TME did not provide a protective effect on Rituximab therapy as Rituximab significantly enhanced the survival of mice (p<0.0001). Combined treatment with Rituximab and Actemra did not significantly improve survival compared with Rituximab alone. We also found that mice treated with Rituximab alone had a significant reduction in tumor growth rate and combined therapy did not reduce tumor growth rate beyond that of Rituximab alone. Furthermore, we found a significant reduction in human IgM secretion in mice serum in mice treated with Rituximab alone (p=0.005) or combined therapy (p=0.0007) compared with control mice. However, there was no difference between Rituximab alone and combined therapy. There was no significant change in mice weight over the course of the treatment suggesting that none of these therapies had toxic effects. Taken together, our data suggests that the TME provided a protective effect against Ibrutinib, but not Rituximab therapy and inhibition of IL-6, a cytokine known to modulate the TME in WM did not enhance either therapy.

#194

Prognostic implication of stromal hyaluronic acid protein expression in resected oropharynx and oral cavity cancer.

Yoon Ho Ko,1 Hye Sung Won,1 Der Sheng Sun,1 Soon Auck Hong,2 Ji Hyung Hong3. 1 _Catholic Univ. of Korea, Uijeongbu-Si, Republic of Korea;_ 2 _Soonchunhyang University Cheonan Hospital, Cheonan, Republic of Korea;_ 3 _Catholic Univ. of Korea, Incheon, Republic of Korea_.

Background: Hyaluronic acid (HA) is a well-characterized component of extracellular matrix (ECM), which plays a critical role in a variety of cellular processes and regulates cell adhesion, migration, and proliferation associated with prognosis of various tumor types. However, the clinical prognostic significance in resected head and neck squamous cell carcinoma (HNSCC) is not well identified.

Methods: The resected tissues from oropharynx or oral cavity cancer patients undergoing surgery were made for tissue microarray (TMA) divided into stroma and cancer panels. Including HA protein expression, HA synthases (HAS) and hyaluronidases (HYAL1) was also assessed by immunohistochemistry.

Results: A total of 160 resected oropharynx or oral cavity cancer tissues between 1994 and 2012 were analyzed in this study. Stromal HA expression, positive in 31 (19.4%) of patients, showed a trend favoring for HPV negative tumor. Other clinicopathological characteristics did not differ according to stromal HA protein expression. However, the HA expression in stroma were statistically associated with clinical outcomes. In the multivariate analysis for clinical outcomes, stromal HA expression was found to be an independent indicator for disease recurrence. (HR, 2.716; 95 % CI, 1.323 - 5.575, P = 0.007), and the combined analysis of tumoral and stromal expression of HA showed that patients with a cancer and stroma-positive HA expression showed a significantly worse outcome than those with other combinations (RFS, P = 0.023, OS, P = 0.082). In addition, when using stratification of HPV stastus, the stromal HA expression remained an independent indicator for disease recurrence and survival(HR, 2.712; 95% CI, 1.323 - 5.56; P = 0.006).

Conclusions: The HA expression in stroma could be related with poor prognosis independently in surgically resected HNSCC. Thus, the finding suggested that HA in stroma may serve not only as a prognostic marker but also as a supposed target for HNSCC treatment.

#195

Electrochemical treatment produces pH changes in the tumor microenvironment that are toxic to cancer cells.

Nicholas Perkons, Elliot Stein, Chike Nwaezeapu, Joseph Wildenberg, Daniel Ackerman, Gregory Nadolski, Stephen Hunt, Terence Gade. _University of Pennsylvania, Philadelphia, PA_.

Background: Locoregional therapy is playing an increasingly central role in the management of hepatocellular carcinoma (HCC), where ablation is distinguished among these techniques by its capacity to effect cure. Electrochemical treatment (EChT), a technique that facilitates necrosis via the application of direct current at low voltages shows promise; however, widespread use has been limited as studies of the underlying biology and the mechanism of action have been limited.

Methods: A novel assay for the study of EChT mechanism was designed, wherein HCC cells (2.5e5 cells/mL) were embedded in low-melting temperature agarose (1.5%). EChT was performed on these assays between nitinol cathodes (d = 0.5 mm) and platinum anodes (d = 0.5 mm) in a variety of geometries. Buffering capacity of the encapsulation assay was varied via the addition of HEPES buffer at final concentrations of 10mM, 50mM, or 200mM. Either during or immediately following EChT, the following measurements were recorded: cell viability, pH, ROS burden, transmembrane potential, and temperature.

Results: Following EChT, acidic pH was appreciated surrounding the anodes and basic pH surrounding the cathodes in regions where cell death was observed by fluorescence microscopy. Upon increasing the buffering capacity of the assay, the total area of cell death decreased (p < 10-6). There was not sufficient heat generation nor transmembrane potential to account for the observed cell death. A pH-threshold was identified, where a pH > 10.8 or < 4.4 was found to cause cell necrosis in the three different buffer conditions studied. By varying the number of electrodes, their spacing, and length of ablation, it was possible to shape the zone of cell death observed to an arbitrary geometry.

Conclusions: The mechanism of EChT-induced cancer cell death is through the spread of acidic and basic species generated by hydrolysis of water upon electron transfer at the electrodes. The diffusion of these species leads to cell death in a predictable, pH dependent manner. These results suggest that an electrochemical ablation device could treat complex HCC tumor geometries with appropriate electrode placement and charge delivery.

#196

The effect of modulated electro-hyperthermia on B16-F10 melanoma tumor growth.

Balázs Besztercei, Enikő Major, Anett Benedek, Zoltán Benyó, Andrea Balogh. _Institute of Clinical Experimental Research, Budapest, Hungary_.

The objective of the present study was to examine the effect of modulated electro-hyperthermia (mEHT) on B16-F10 mouse melanoma cell line in vitro and in vivo. mEHT induces tumor-selective heat shock at ~42°C and is used as adjuvant to conventional cancer therapy. For in vitro comparison of the effect of mEHT to conventional hyperthermia (convHT), cells grown on coverslips were treated using mEHT or convHT in water-bath at 42°C for 1x60 min. Changes in gene expression, viability and invasiveness were measured. For in vivo studies, B16-F10 cells mixed with Matrigel were injected subcutaneously into C57Bl/6 mice as the tumors were exposed to 42°C for 30 minutes with mEHT every second day for three times. Tumors were removed 48 hours post treatment, weighed and digested to single-cell suspension. The supernatant of tumor cells and the plasma of tumor-bearing mice were used to screen a membrane-based antibody array representing a panel of 111 cytokines. The tumor cells were stained for flow cytometry to determine the changes induced on melanoma cells and tumor-infiltrating leukocytes. As a marker of in vitro treatment we measured HSPA2 expression with qPCR. Similar expression patterns were induced both by mEHT and convHT. Three hours after treatment the peak levels of HSPA2 were 3 times higher as compared to the untreated control in a time-course experiment. Next, we showed that in parallel to the upregulation of proapoptotic genes Puma, Bak-1 and Bax, downregulation of prosurvival genes XIAP, Bcl-2, Bcl-XL was induced both by convHT and mEHT with similar kinetics and magnitude. Furthermore, cell viability decreased and the invasivness was impaired as measured by scratch assay by both treatment types. The in vivo mEHT of primary melanoma tumors resulted in considerable tumor size reduction. Flow cytometry analysis revealed the increase of MHC class I on the surface of melanoma cells and concomitant activation of CD8+ cytotoxic T lymphocytes in response to treatment. Increased number of CD4+ T cells and Gr1+ granulocytes was detected, although changes in the various subpopulations need further analysis. F4/80+CD11b+ macrophages and mature DC-s were slightly elevated in the treated tumors. The cytokine array analysis shows that several cytokines responsible for leukocyte recruitment were enriched in the treated tumor (SDF-1, osteopontin, IL-1). Complement activation was induced by mEHT both in the tumor microenvironment and the plasma, indicating an acute inflammatory response to treatment. Furthermore, along the short pentraxins, CRP and SAP, the long pentraxin PTX3 was elevated in the tumor and plasma of the treated mice, suggesting that mEHT induced the tumor clearance. Taken together, our results demonstrate that in vitro mEHT and convHT produce comparable effects, whereas the in vivo mEHT treatment was effective in reducing the tumor size by enhancing immune recognition of B16-F10.

This study was supported by NVKP 16-1- 2016-0042 grant.

#197

Astragalus polysaccharides (APS) effects on betel-nuts related head and neck squamous cell carcinoma (HNSCC) in Taiwan.

Jo-Pai Chen,1 Jui-Ying Chang,1 Sung-Hsin Kuo,2 Ruey-Long Hong2. 1 _National Taiwan Univ. Hospital, Yun-Lin Branch, Taipei, Taiwan;_ 2 _National Taiwan Univ. Hospital, Taipei, Taiwan_.

Backgrounds: The treatment of HNSCC in Taiwan is still very challenging and might be related to betel-nuts use. Betel-nuts chewing might contribute to (1)strong inflammation, invasion, and angiogenesis; (2)poor response to chemotherapy , radiation, and epidermal growth factor receptor inhibitors. In our previous studies, betel-nuts related TW2.6(p53 defective mutation, p16 loss, BCL2+) was resistant to traditional chemotherapies, radiation, and EGFR inhibition. In addition to PI3K/mTOR dual inhibition, polo-like kinase inhibitor with radiation, CDK4/6 inhibitor, WEE1 inhibitor, ALK/IGF-1R inhibitor, Bcl2 inhibitor, and eribulin, FGFR inhibitor & VEGFR2/FGFR/PDGFR or VEGFR2/c-MET/Axl triple blockage might be effective on TW2.6 and reverse treatment refractoriness, maybe through the inhibition of mesenchymal transformation & PI3K/Akt/mTOR signaling and the modulation of stemness & PD1/PDL1 pathway. PI3Kalpha inhibitor, Bcl2 inhibitor, and ALK/IGF-1R inhibitor also have roles to reverse treatment refractoriness. Astragalus polysaccharides is a kind of traditional Chinese medicine with immuno-modulatory effects(enhancing mature DC, NK cells, & effector CD8 cytolytic activity; adjusting chronic inflammation in tumor microenvironment; reversing M2 polarization; suppressing Treg/MDSC) and maybe anti-tumor efficacy. APS has also been manufactured to treat cancer-related fatigue in Taiwan.

Purpose: We try to find out whether APS has anti-tumor effects on betel-nuts related HNSCC in Taiwan.

Methods: APS was tested on TW2.6 to evaluate (1)the in vitro drug sensitivity; (2)synergistic effects with some target therapies by MTT assay, colon formation assay, flow cytometry, and western blot assay; (3) HUVEC response and invasion capacity by wound healing.

Results: APS has minimal cytotoxic effect on TW2.6; however, it could inhibit tumor migration, suppress signals of epithelial-mesenchymal transformation & PI3K/AKT/mTOR pathway, and induce PDL1 drop. APS re-sensitizes TW2.6 to respond to afatinib & volasertib and could enhance further response of TW2.6 to BYL719, foretinib, BGJ398, and eribulin. APS and eribulin combinations might drop BMI-1.

Conclusions: APS could possibly reverse TW2.6 treatment refractoriness to EGFR inhibitor & polo-like kinase inhibitor and further enhance PI3Kalpha inhibitor, VEGFR/c-MET/Axl triple inhibitor, FGFR inhibitor, & eribulin effects on TW2.6. APS can be further investigated to be combined with these targeted therapies and even immunotherapy in betel-nuts related HNSCC in Taiwan in the future, by modulating EMT, PI3K/AKT/mTOR pathway, immune escape, & cancer stemness.

#198

MUC13 promotes pancreatic tumor-stromal interactions by influencing tumor microenvironment.

Sheema S. Khan, Kyle Doxtater, Sonam Kumari, Saini Setua, Mohammed Sikander, Shabnam Malik, Murali Yallapu, Stephen Behrman, Subhash Chauhan, Meena Jaggi. _Univ. of Tennessee Health Science Ctr., Memphis, TN_.

Objective: Pancreatic cancer (PanCa) is the third leading cause of cancer-related deaths in the U.S. MUC13, mucin, is aberrantly expressed in PanCa and its overexpression leads to tumorigenic and metastatic behavior of cancer cells. Herein, we investigate the role of MUC13 in tumor microenvironment (TME) that favors PanCa progression/metastasis and drug resistance.

Methods: MUC13 expressing (Panc-1) and knockdown PanCa cells (HPAF-II) and pancreatic stromal cells (PSCs), derived from freshly collected human pancreatic tissues, were utilized for the study. Using immunoblotting, ELISA, qPCR and RT-PCR techniques, we assessed the MUC13-induced alterations in the expression of important molecules that are involved in promotion of pancreatic tumor stromal interactions and drug resistance. Cytation 3 live cell imaging was performed for 3D tumor-stromal cell co-culture and invasion assays. Xenograft mice tumor tissues generated using ectopically expressing MUC13 and null cells were used for the study.

Results: Our results demonstrate that MUC13 activates SHH signaling pathway in PanCa cells via upregulation of SHH and its related important downstream targets such as CXCR-4, Gli-1, SMO, PTCH1/2, NFκB and p-AKT in PanCa cells using immunoblotting and PCR. We also observed increased expression of SHH, Gli-1, CXCR-4 and α-SMA in MUC13-expressing xenograft tumors using Immunohistochemistry. Ectopic MUC13-expressing PanCa cells grown in co-culture with PSCs showed enhanced secretion of IL-6 and CXCL-12 (ELISA), increase in invasion (Matrigel invasion assay) and migratory (Boyden chamber assay) potential of both the cultured PanCa as well as stromal cells. Additionally, ectopically MUC13-expressing cells were resistant to treatment with gemcitabine as depicted by proliferation assay and qPCR results showed enhanced expression of ribonucleoside-diphosphate reductase (RRM1/2) and miR-21, which are known to be involved in gemcitabine resistance. Overall, our results suggest that MUC13 modulates tumor-stromal crosstalk, promoting tumor growth, invasion and drug resistance in TME.

Conclusion: Our results suggest that MUC13 expression might influence signaling supportive of interactive TME, leading to increased tumor growth and drug resistance. These findings illustrate mechanisms by which MUC13 promotes tumorigenic TME and contributes to PanCa cell survival, metastasis and drug resistance.

#199

Host CYP27A1 expression is essential for ovarian cancer progression.

Sisi He,1 Amy E. Baek,1 Liqian Ma,1 Joanna Burdette,2 Erik R. Nelson1. 1 _University of Illinois at Urbana-Champaign, Champaign, IL;_ 2 _University of Illinois at Chicago, Chicago, IL_.

Ovarian cancer continues to have a high mortality and recurrence rate. Hence, there is an urgent need to develop new therapeutic or lifestyle strategies. In this regard, epidemiological studies have implicated elevated cholesterol as a negative prognostic factor. Conversely, ovarian cancer patients prescribed cholesterol lowering drugs (HMGCoA-R inhibitors; statins) exhibit significantly increased progression free survival (PFS). Therefore, cholesterol appears to be clinically important for the progression of ovarian cancer. Considering the potential mechanisms by which cholesterol impacts ovarian cancer progression, it was noteworthy that CYP27A1 encodes the first enzyme in the alternative pathway of cholesterol catabolism, producing 27-hydroxycholesterol (27HC), a primary metabolite of cholesterol that has recently been shown to have the capacity to activate the estrogen receptors and liver x receptors. Our bioinformatics analysis of human tumoral mRNA expression indicated that low CYP27A1 expression was associated with increased PFS. Conversely, high CYP7B1 expression, the enzyme that metabolites 27HC, was associated with increased PFS. Therefore, we hypothesized that CYP27A1 is involved in ovarian cancer pathophysiology via 27HC signaling. To directly investigate the role of CYP27A1 in ovarian cancer progression, we monitored the growth of tumors grafted into the ovarian bursa of wildtype and CYP27A1 knockout (KO) mice. Strikingly, we found that ovarian tumors failed to thrive in CYP27A1 KO mice, eventually completely regressing. Importantly, treatment with exogenous 27HC was able to sustain tumor growth in CYP27A1 KO mice. In exploring the potential mechanisms by which this occurs, we found 27HC had very little effect on ovarian cancer cell proliferation in vitro, however, was associated with the enrichment of certain myeloid populations within tumors. Specifically, treatment with exogenous 27HC significantly enhanced the infiltration of M-MDSCs, while significantly fewer M-MDSCs were found in tumors from CYP27A KO mice. M-MDSCs are a myeloid-immune cell type known to be pro-tumorigenic, at least in part through their suppression of cytotoxic CD8+ T cells. This would suggest that inhibition of CYP27A1 might improve the efficacy of immune checkpoint inhibition. Therefore, we tested the therapeutic utility of combining a small molecule inhibitor of CYP27A1 with anti-PD-L1. Indeed, combining these two approaches significantly decreased ovarian tumor growth in a preclinical model. Collectively, our preliminary data strongly indicate that CY27A1 expression is critical for sustaining ovarian cancer progression and is a viable target in combination with checkpoint inhibition.

This work was supported by the NIH R00CA172357 (E.R.N.), a Cancer Scholars for Translational and Applied Research (C*STAR) Award (S.H.) and NIH T32EB019944 (S.H.).

#200

FOXD1 promotes stromal investment in clear cell renal cell carcinoma.

Kyle H. Bond,1 Jennifer Fetting,2 Ashwani Gupta,2 Christine W. Duarte,2 Michele Karolak,2 Clare B. Congdon,3 Ivette Emery,2 Eoghainín Ó hAinmhire,4 Benjamin D. Humphreys,4 Leif Oxburgh2. 1 _University of Maine, Orono, ME;_ 2 _Maine Medical Center Research Institute, Scarborough, ME;_ 3 _Bowdoin College, Brunswick, ME;_ 4 _Washington University School of Medicine, St. Louis, MO_.

Forkhead transcription factors (FOX) play a role in the development of many cancers, including clear cell renal cell carcinoma (ccRCC). FOXD1, essential for angiogenesis in the developing kidney, has not previously been implicated in ccRCC. Tumor microarrays (TMAs) containing 41 patient-derived ccRCC tumors and 26 healthy cortex samples were stained for FOXD1, with 65% of tumor samples staining positively. Due to the effect of FOXD1 on angiogenesis, staining for stromal markers PECAM, αSMA, PDGFRβ and NG2 was also performed. A direct correlation was found only between FOXD1 and PDGFRβ (p<4.5x10-6), suggesting interstitial fibroblast recruitment by FOXD1+ cancer cells. Increased expression of FOXD1 was found to correlate with increased stage (p=9.1x10-5) and reduced survival (FOXD1 low= 2830 days; FOXD1 high= 1913 days) from a Kaplan-Meier analysis of RNA-seq data from The Cancer Genome Atlas (TCGA). Potential FOXD1 target binding sites were determined using the TRANSFAC FOXD1 binding site matrix. SLIT2, a factor known to inhibit migration of pericytes, was uncovered from the analysis and was further found to be downregulated in response to FOXD1 overexpression in renal proximal tubule cells (RPTECs). To test the influence of SLIT2 on interstitialfibroblast migration, scratch assays on NRK-49F (rat kidney fibroblast) and Gli-1 (cardiac fibroblast) cells were performed and showed that SLIT2 reduced PDGFBB-induced cell migration (p<0.027). A multiplex proximity ligation assay for multiple signaling pathways showed that SLIT2 treatment reduced the STAT signaling response to PGDFBB. To model the influence of SLIT2 on ccRCC stromal invasion in vitro, we devised a novel 3D invasion assay. In summary, 786-O ccRCC cancer cells were seeded into a 200um thick silk scaffold and cultured for 3 days to allow deposition of ECM. Scaffolds were then placed on top of a monolayer of interstitial fibroblasts and invasion into the cancer cell-filled scaffold was measured using confocal microscopy. The results showed a significant reduction of cell invasion into the cancer-filled matrix with the addition of recombinant SLIT2 to media (5µm p=1.34x10-4; 50µm p=1.64x10-2). In conclusion, we show that FOXD1 expression has prognostic relevance to patient survival in ccRCC, and that this may be due to modulation of SLIT2 expression. 

### Tumor Heterogeneity 1

#201

Multiregion joint somatic genotyping in ovarian cancer proves optimal tumor sampling for clinical sequencing.

Lorinc Pongor,1 Gyongyi Munkacsy,2 Ildiko Vereczkey,3 Imre Pete,3 Balazs Gyorffy1. 1 _MTA TTK, Budapest, Hungary;_ 2 _Semmelweis University, Budapest, Hungary;_ 3 _National Institute of Oncology, Budapest, Hungary_.

Introduction. Genetic heterogeneity of ovarian tumors is caused by the accumulation of novel mutations through time. A result of this process is a spatially heterogeneous tumor with a fractal-like architecture, comprising of a mixture of spatially separated clones, sub-clones, and single cells. Spatial heterogeneity renders clinical sequencing a challenging task because clinically relevant mutations can go undetected. Here, we compare the genetic composition of single tumors with multiple DNA samples extracted from different regions of the tumor in diverse combinations to examine the effects of spatial separation on tumor heterogeneity.

Methods. We derived tumor samples from five patients with ovarian cancer. Three tumor tissue samples were collected from each patient, in each of these, DNA was extracted from a total of eight segments of different sizes (5x3x8 total segments). Exome sequencing was performed and we examined DNA mutations identified from a biopsy sample, from three merged tissue segments with immediate vicinity to the biopsy, and from segments from spatially distant tissue samples merged into one. We compared identified mutations and copy-number variations called from the exome-seq data.

Results. Three patients had heterozygous germline alterations affecting the BRCA pathway, paired with somatic TP53 mutations, one patient had multiple somatic mutations affecting the PI3K pathway, the last patient had the most common activating mutations in the PIK3CA and KRAS genes. When comparing the different sequencing runs, increasing the size of the tumor sample did not affect the overall quantity of mutations identified. Clonal mutations were identifiable in all samples, while sub-clonal mutations shifted. In case of a hypermutating phenotype, increasing the sequenced sample size strongly decreased the number of identified somatic mutations, most probably due to dilution of sub-clones. These observations are confirmed by an in silico model, in which we shuffled regions from publicly available multi-region sequencing data into known compositions.

Conclusion. In hypermutating tumors the number of detected mutations increases as the size of the sequenced sample decreases. Generally, increasing the tumor sample size did not affect the number of identifiable mutations. Our findings suggest that clinical sequencing using small biopsy samples can generate adequate mutation calling results.

#202

Functional heterogeneity in glioblastoma: cell-cell communication regulates temporospatial events in tumor recurrence.

Kevin D. Woolard,1 Patrick Huang,1 Thomas K. Sears,1 Matthew Settles2. 1 _UC Davis School of Vet Med, Davis, CA;_ 2 _UC Davis, Davis, CA_.

Patients with glioblastoma frequently develop progressive disease following treatment. This is believed to occur because of an inherent or acquired resistance to chemo- and radiotherapy within residual tumor burden. However, how discrete tumor subpopulations coordinate tumor recurrence and progression is still poorly understood. Here, we describe the isolation of glioma stem cells (GSCs) from a patient prior to treatment (0203), and then from multiple sites in the brain following tumor recurrence. These sites include the recurrent tumor at the primary site (T3/T4), as well as disseminated tumor cells in the contralateral hemisphere (T6/T7). Whole exome sequencing identifies loss of all unique genomic signatures within the original tumor cells (0203) following treatment, suggesting eradication of the original tumor bulk. Moreover, genomic signatures of the recurrent tumor (T3/T4) clusters alongside disseminated subpopulations of tumor cells isolated from the contralateral hemisphere (T6/T7), suggesting that tumor recurrence is driven by repopulation of the original tumor location. Copy number variants (CNVs) between the original tumor (0203), the recurrent tumor (T3/T4), and distant tumor sites (T6/T7), identify a more primordial genome in distant tumor sites (T6/T7), which lack canonical CNVs found in the original tumor and in other subpopulations (deletion of PTEN or amplification of EGFR). Thus, tumor dissemination likely occurs early in disease development. We are able to model the patient's disease progression by using co-culture techniques and orthotopic xenograft studies. Our data indicates that 0203 cells actively suppress migration of disseminated tumor sub-populations (T6/T7) in vitro and in vivo, which is replicated by using cell-free conditioned media. In stark contrast, the induction of apoptosis in 0203 cells (or treating with conditioned media from apoptotic 0203 cells) rapidly recruits migration of disseminated cells (T6/T7) in a co-culture system. Co-culture of disseminated GSCs (T6/T7) with viable 0203 cells also results in suppression of pathways implicated in glioma migration, such as AKT, CXCR4, TrkA, and CLCN3; suppression that is reversed upon induction of apoptosis in 0203 cells. These data indicate that while widespread tumor dissemination occurs early in glioblastoma, a dominant subpopulation may actively suppress proliferation of separate, disseminated subpopulations. Moreover, abrogating this suppression following therapy triggers migration and proliferation of specific, distant tumor sub-populations, resulting in tumor recurrence. This suggests that glioblastoma recurrence may not be as simple as selection of radioresistant cells, rather it may result from the active recruitment of disseminated subpopulations, which migrate to the original tumor microenvironment.

#203

Regenerable altruism drives chemotolerance in clonal cancer cells.

Kee Wah Lee,1 Muhammad Sufyan Masroni,1 Karen Meiling Tan,2 Mo-Huang Li,3 Lihan Zhou,4 Steven Tucker,5 Lynette Su Mien Ngo,6 Chan Fong Chang,1 Boon Huat Bay,1 Soo Yong Tan,1 Mikael Hartman,1 Huiwen Chua,2 Tze Ping Loh,2 Thomas Putti,1 Sai Mun Leong,1 Evelyn Siew-Chuan Koay1. 1 _National University of Singapore, Singapore;_ 2 _National University Hospital, Singapore;_ 3 _CellSievo Private Limited, Singapore;_ 4 _Mirxes Private Limited, Singapore;_ 5 _Tucker Medical Center, Singapore;_ 6 _Raffles Hospital, Singapore_.

Introduction: Cancer is thought to represent a breakdown of multicellular cooperation and reversion to Darwinian dynamics characterized principally by self-interested competition. However, there is increasing evidence that cancer cells can behave as communities, manifesting social behaviors that influence cancer progression.

Methods: Circulating tumor cells enrichment, microRNA extraction, qRT-PCR, MTS assays, RNA in situ hybridization, immunohistochemical staining, SILAC-based mass spectrometry, flow cytometry, ChIP-sequencing, and western blotting.

Results: We report here evidences of altruistic cooperation in clonal breast cancer cells. Heterogeneity in expression of a microRNA, miR-125b, leads to sectoring of clonal breast cancer cell populations into minority miR-125b-high and majority miR-125b-low subpopulations. Enhanced population-wide tolerance to taxane-based chemotherapy is mediated by the miR-125b-high minority, in part through increased secretion of extracellular public goods such as insulin-like growth factor binding protein 2 (IGFBP2) and chemokine (C-C motif) ligand 28 (CCL28). Cost-benefit analysis established this helping behavior to be altruistic, as survival benefits conferred to the miR-125b-low cells, via public goods sharing, occurred at a fitness cost to the miR-125b-high cells. Notwithstanding this fitness cost, miR-125b-high altruists regenerate readily from isolated populations of miR-125b-low defectors, via Kruppel-like factor 2-mediated epigenetic mechanism of histone deacetylation. As therapeutic proof-of-principle, we demonstrated that targeting extracellular IGFBP2 and CCL28, through ligand neutralization, markedly blunted the tolerance response of cancer cells towards taxane chemotherapy.

Conclusions: Our results demonstrate how positive social engagement such as altruism may be employed by heterogeneous clonal cancer communities to drive chemotolerance, and understanding of such social behaviors could help formulate more effective treatment strategies.

#204

Reconstructing testicular germ cell cancer progression: Primary tumor heterogeneity and early metastatic clone selection.

Lambert C. Dorssers,1 Ad J. Gillis,1 Hans Stoop,1 Ronald Van Marion,1 Marleen M. Nieboer,2 Job Van Riet,1 Harmen J. Van de Werken,1 J Wolter Oosterhuis,1 Jeroen de Ridder,2 Leendert H. Looijenga1. 1 _Erasmus Medical Center, Cancer Institute, Rotterdam, Netherlands;_ 2 _University Medical Center Utrecht, Center for Molecular Medicine, Utrecht, Netherlands_.

Background. Testicular germ cell cancer (TGCC) is initiated from a primordial germ cell during early life and the most frequent malignancy in young Caucasian males. Although treatment of TGCC is generally curative, rare cases of therapy resistance occur. Little information is available on the presence of heterogeneity of the complex primary tumor and derived metastases after systemic treatment. The goals of this study are to determine the intratumor heterogeneity, and to unravel tumor progression from initiation till therapy-resistant metastases.

Methods. In this study, we have investigated 42 purified samples of four cases of nonseminoma with intrinsic resistance to chemotherapy including different histological elements (embryonal carcinoma, yolk sac tumor and teratoma), metastatic specimens and precursor lesions (germ cell neoplasia in situ, GCNIS) using whole genome-, targeted-, and RNA sequencing, as well as methylation profiling. Enriched tumor samples were prepared using laser-assisted micro-dissection (PALM) or macro dissection. Sequencing data were used to reconstruct the evolutionary history of these rare cases.

Results. A low frequency of somatic mutations (~0.1 per Mb) was found in these TGCC cases with a BRCA-like mutational signature without evidence for direct involvement of BRCA1 and BRCA2 genes. Intratumor molecular heterogeneity was observed and did not correspond to the supposed histological evolution of the primary tumor. Metastases after systemic treatment were derived from precursors frequently not identified in the primary cancer. GCNIS mostly lacked the molecular marks of the primary TGCC (including gain of chromosome 12p) and comprised dominant subclones that had failed to progress into a manifest malignancy. The low read frequencies of somatic variants in the enriched samples indicated early genome duplication.

Conclusions. Our data strongly support the hypothesis that TGCC is initiated by whole genome duplication, followed by copy number alterations, dynamic acquisition of chromosome 12p overrepresentation, and accumulation of low numbers of somatic mutations resembling a BRCA-like mutational signature. Moreover, metastatic clones may originate early in tumor development and follow an independent path of genetic evolution. The observations of heterogeneity at all stages of tumorigenesis should be considered when treating patients with clinically overt TGCC, or with GCNIS-only disease.

#205

Reliance upon ancestral mutations is maintained in colorectal cancers that heterogeneously evolve during targeted therapies.

Mariangela Russo,1 Simona Lamba,2 Annalisa Lorenzato,3 Alberto Sogari,3 Giorgio Corti,2 Giuseppe Rospo,2 Monica Montone,2 Luca Lazzari,3 Sabrina Arena,3 Daniele Oddo,3 Michael Linnebacher,4 Andrea Sartore-Bianchi,5 Filippo Pietrantonio,6 Salvatore Siena,5 Federica Di Nicolantonio,3 Alberto Bardelli3. 1 _University of Turin, Department of Computer Science, Candiolo (TO), Italy;_ 2 _Candiolo Cancer Institute - IRCCS, FPO, Candiolo (TO), Italy;_ 3 _University of Turin, Dept. of Oncology, Candiolo (TO), Italy;_ 4 _University of Rostock, Germany, D-18057 Rostock, Germany, Germany;_ 5 _Niguarda Cancer Center, Grande Ospedale Metropolitano Niguarda, Milano, Italy;_ 6 _Fondazione IRCCS Istituto Nazionale dei Tumouri, Milano, Italy_.

Clinical effectiveness of targeted therapies is often transitory and virtually all patients develop secondary resistance. The heterogeneous mechanisms of resistance and the sub-clonal evolution pattern of cell populations that emerge upon pharmacological selection limit disease control with subsequent lines of therapy. We used colorectal cancer (CRC) to test the hypothesis that ancestral oncogenic event (such as WNT pathway alterations) shared by every cell sub-clone might be better suited as therapeutic targets than heterogeneous events in the branches, as the former remain present in each drug-resistant cell independently from its genetic drift.To test our assumption, we first generated a panel of CRC cells resistant to commonly used targeted agents, including EGFR and BRAF inhibitors. Treatment with targeted therapies, although initially effective, fuels clonal evolution and further amplifies molecular diversity. Exome sequencing and phylogenetic tracking unveiled a complex sub-clonal architecture in CRC resistance cell populations, indicating parallel evolution of multiple independent cellular lineages, associated with distinct genomic landscapes.Functional and pharmacological modulation of WNT signaling induces cell death in CRC preclinical models from patients that relapsed during treatment, regardless of drug type or resistance mechanisms. Pharmacological blockade of WNT ligands secretion resulted in translocation of β-catenin from the cytoplasm and nucleus to the plasma membrane, decreased of β-catenin dependent Tcf/LEF transcriptional activity, and cell growth impairment despite massive molecular drift of resistant derivatives. Concomitant blockade of WNT and MAPK signalling restrained evolution of drug-resistant clones.In summary, reliance of CRC on the WNT pathway is preserved in later phases of colorectal carcinogenesis when tumors face genomic bottlenecks and sub-clonal evolution driven by administration of target therapies; thus offering the possibility of a common therapeutic strategy to overcome secondary resistance to treatments.

#206

The Consensus Molecular Classification (CMS) of primary colorectal tumors and their matched liver metastasis: Investigating the concordance.

Loredana Vecchione,1 Ines Beumer,2 Mireille Snel,2 Ramon Salazar,3 Filippo Pietrantonio,4 Fotios Loupakis,5 Cristina Santos Vivas,3 Maria Mercedes Martinez-Villacampa,3 Xavier Sanjuan,6 Antonia Martinetti,4 Giovanni Fuca',4 Marta Schirripa,5 Matteo Fassan,7 Rene' Bernards8. 1 _Charité - Universitätsmedizin Berlin, Berlin, Germany;_ 2 _Agendia, Amsterdam, Netherlands;_ 3 _Catalan Institute of Oncology, IDIBELL, Barcelona, Spain;_ 4 _Fondazione IRCCS, Istituto Nazionale dei Tumori, Milan, Milan, Italy;_ 5 _IOV- IRCCS, Padua, Italy;_ 6 _, L'Hospitalet de Llobregat, Barcelona, Spain;_ 7 _University of Padua, Padua, Italy;_ 8 _Agendia and NKI/AvL, Amsterdam, Netherlands_.

Background The Consensus Molecular Subtypes (CMS) identified four different molecular groups in colorectal cancer (1). Coding mutations are concordant between primary tumors (PT) and matched metastasis (MM) while gene copy numbers seem highly discordant (2). This could somehow influence the molecular classification of PT and MM. We therefore aimed to investigate the concordance of the CMS between PT and MM.

Material and methods We retrospectively collected 25 fresh matched samples of primary (P) and synchronous liver metastasis (LM) (discovery cohort) and 50 formalin-fixed-paraffin embedded (FFPE) samples of P and synchronous LM (validation cohort) of chemonaive metastatic colorectal cancer patients. All samples contained at least 30% of tumor cells. RNA was extracted from four sections of 5-μm by using the RNeasy FFPE kit (Qiagen). RNA yield was quantified using a NanoDrop spectrophotometer. Extracted RNA was amplified using a TransPLEX C-WTA whole-transcriptome amplification kit. Amplified cDNA was labeled using the Genomic DNA Enzymatic Labeling Kit and hybridized onto Agendia's full genome arrays both according to the manufacturer's instructions. For FFPE samples, no reference channel was used. Gene expression intensities were normalized using Lowess normalization method implemented in Matlab software version R2012a, and the CMS classification was applied as in Guinney et al (1).

Results and conclusion Clinical data were currently available for 66% of patients. Median age at the diagnosis was 64 (35-76), 30% of patients were female and 70% were male. 42% of PT were right side located and 58% were left side located. 42% of tumors were well differentiated, 40% moderately differentiated, 15% poorly differentiated and 3% were not classified. 3% of tumors were MSI, 45% were MSS. No data on the MMR status was available (NA) for 52% of the tumors. Mutations in KRAS were detected in 18% of the tumors while 30% were wild type (WT) and 52% NA. BRAF(V600E) mutation was detected in 3% of tumors, 39% were WT and 58 NA. 73% of patients received surgery of both P and LM simultaneously and before starting a systemic treatment. 72% of patients received a first line treatment. The results of the molecular classification will be presented at the conference.

References 1. Guinney J et al. The consensus molecular subtypes of colorectal cancer Nat Med. 2015 Nov;21(11):1350-62. 2.Mamlouk S et al. DNA copy number changes define spatial patterns of heterogeneity in colorectal cancer. Nat Commun. 2017 Jan 25;8:14093

#207

CML as a prototypical malignancy.

Dmitriy Sonkin, Richard Simon. _NCI, Rockville, MD_.

Chronic myelogenous leukemia (CML) was the first malignancy for which clinical outcome was drastically improved by kinase inhibitor therapy. Kinase inhibitors targeting other well-known oncogenes have been introduced into clinical practice, but none have shown the same magnitude of clinical benefit as ABL1 inhibition in CML. CML is sometimes described as an atypical malignancy or a one-hit malignancy. However careful examination of accumulation of additional oncogenic alterations in genes such as TP53, CDKN2A/B, IKZF1 and RUNX1 during CML progression from chronic phase to blast phase indicates that this is not the case. Instead CML provides a window into how many types of cancer may look and behave at an early stage, prior to diagnosis and the development of additional genomic alterations. The remarkable clinical benefits of ABL1 inhibition is likely due to early detection of CML at a stage in which the tumor is driven by single oncogenic alteration which can be successfully controlled by the inhibitor. The majority of CML patients in chronic phase of the disease treated with ABL1 inhibitor achieve a major molecular response (MMR) which corresponds to 1000 times decrease in BCR-ABL fusion levels. As a result, there is drastic reduction in number of cells which may accumulate additional alterations in genes like TP53, CDKN2A/B, IKZF1, RUNX1 leading to greatly decreased chance of progression to blast crisis. Thinking of CML as a prototype for effective systemic treatment based on early cancer detection may help to provide strategies for improving treatment for other types of cancer.

#208

Multi-omics analysis in primary cell cultures reveals the genomic basis of phenotypic diversity within tumors.

Sixue Liu,1 Zuyu Yang,1 Guanghao Li,1 Yanting Luo,1 Qiang Gong,1 Xin Wu,1 Tao Li,1 Zhiqian Zhang,2 Baocai Xing,2 Xiaolan Xu,3 Xuemei Lu1. 1 _Beijing Institute of Genomics,Chinese Academy of Sciences, Beijing, China;_ 2 _Peking University Cancer Hospital and Institute, Beijing, China;_ 3 _Institute of Biophysics,Chinese Academy of Sciences, Beijing, China_.

To uncover the functionally essential variations related to tumorigenesis and tumor progression from large amount of cancer genomics data is still challenging due to the genetic diversity between patients, and extensive inter- and intra-tumor heterogeneity at the genomic, epigenomic, and transcription levels. Multiple primary cultures derived from phenotypically distinct subpopulations of a single patient may not only represent the diversity and heterogeneity of tumor cell types and states (such as metastasis and drug-resistant), but also narrow the analysis in genomic basis of phenotypic diversity down to the relatively small

number of variations between subpopulations with the same genetic background. Performing multi-omics sequencing, we identified variations in single nucleotide, copy number, DNA methylation and gene expression among four primary cell cultures from primary and recurrent tumors in a hepatocellular carcinoma patient. We observed the discrepancy between the phylogenetic relationships revealed by single nucleotide variations (SNVs) and transcriptional profiles in the four cell cultures. The differences in gene expression, which indicated the distinct cellular characteristics and tumorigenic capability in one of the two recurrent cell clones, were correlated to the copy number alterations (CNAs) and DNA methylation variation in gene body and confirmed by the characterization of cellular phenotype. Our multi-omics analysis in heterogeneous clones derived from an individual revealed that CNAs and epigenomic changes, rather than SNVs, dominantly contribute to the phenotypic diversity among subpopulations in tumors, which could shed light on developing new cancer diagnosis and treatment strategies based on gene dosage and epigenetic modification

#209

Laser capture microdissection of metastatic colon adenocarcinoma samples enables RNA sequencing analysis of tumor and stromal compartments.

Stephanie Littlewood, Rebekka Krumbach, Gemma Corr, Laura Collins, Adriana Gambardella, Emma Hickman. _Immunocore, Abingdon, United Kingdom_.

Gene expression data from whole tumor samples represent averaged expression of all the tumor cells, stromal cells and normal adjacent cells present in a given sample. Identification of colon tumor-specific genes may be hampered by contamination from surrounding normal adjacent tissue or stroma. In order to better understand gene expression derived specifically from colon tumor cells we performed laser-capture microdissection to obtain separate samples of tumor and stromal/normal adjacent tissue. We generated data from 12 samples of well-differentiated colon adenocarcinoma metastasized to the liver, lung or ovary. Snap-frozen samples were sectioned, stained with an RNA-preserving haematoxylin and eosin staining procedure and tumor cell and stromal cell containing areas were defined with the support of a pathologist. Tumor and non-tumor samples (containing both stroma and normal adjacent tissue) were captured separately and total RNA isolated from these alongside whole (non-dissected) sections. RNA-sequencing was performed with Kapa HyperPrep Riboerase library preparation and Illumina sequencing at UCL Genomics, London, UK and FPKM values were calculated. For tumors metastasized to liver, analysis of liver-specific genes (including ALB, HP and ORM1) showed that in 6/9 samples these genes were present in the whole section, but absent in the microdissected tumor cells. Some samples (3/9) showed very low expression of liver-specific markers in either the whole section or the dissected non-tumor compartment, indicating low levels of normal adjacent tissue contamination, confirmed by pathology analysis. Similarly in tumors metastasized to the lung, we found absence of lung-specific genes including surfactants in the microdissected tumor cells (2/2 samples), despite high expression in whole sections and the non-tumor compartment. We were able to demonstrate localization of known and novel colon tumor genes in the tumor cell compartment. For example, a cancer testis antigen expressed in 13% of colon adenocarcinoma samples in the TCGA dataset was localized specifically to the tumor compartment in 3/12 microdissected samples and was absent from the non-tumor fraction. We were also able to identify genes derived from immune infiltrates and cancer-associated fibroblasts in the non-tumor compartment. This unique dataset can be used to identify genes expressed in metastatic colon adenocarcinoma without interference from stromal cell, immune infiltrate or normal adjacent tissue.

#210

Uncovering a novel layer of complexity in the architecture of pancreatic cancer.

Matteo Ligorio,1 Srinjoy Sil,1 Jose Malagon-Lopez,1 Sandra Misale,2 Murat Karabacak,1 Linda Nieman,1 Shyamala Maheswaran,1 Daniel A. Haber,1 Andrew L. Warshaw,1 Carlos Fernandez-Del Castillo,1 Cristina R. Ferrone,1 Wilhelm Haas,1 Martin Aryee,1 David T. Ting1. 1 _Massachusetts General Hospital, Boston, MA;_ 2 _Memorial Sloan Kettering, New York City, NY_.

Single cell technologies have described heterogeneity across tissues, but the spatial distribution and forces that drive single cell phenotypes has not been well defined. We integrated single cell RNA-seq (scRNA-Seq) and phospho mass cytometry (CyTOF) with high-content digital imaging to investigate tumor heterogeneity of human pancreatic cancer (PDAC) using a novel tumor-architecture approach.

Patient-derived PDAC cells co-cultured at different ratios with cancer associated fibroblasts (CAFs) were found to heterogeneously acquire proliferative (PRO) and invasive (EMT) phenotypes. scRNA-Seq enabled the identification of a novel cell phenotype with upregulation of both PRO and EMT programs, which we named the double positive (DP) phenotype. Functional studies confirmed the heterogeneous activation of PRO and EMT programs with different patterns of tumor growth and metastasis in mice orthotopically xenografted with distinct PDAC:CAF ratios.

In a time-course experiment of PDAC cells with CAF conditioned media (CM), mass spectrometry-based phosphoproteomics demonstrated selective enrichment of MAPK and STAT3 pathways. This was validated with mass cytometry (CyTOF) at the single cell level in our cell line model as well as from primary PDAC tumors, which identified the dual enrichment of MAPK and STAT3 signaling in the DP cell population. Functional validation of MAPK and STAT3 signaling was performed with small molecule inhibitors of MAPK (Trametinib) and STAT3 (pyrimethamine) in our cell line models.

To evaluate the generalizability of these cell subpopulations, we performed RNA-ISH for EMT (FN1) and PRO (MKI67) across 195 human PDAC primary tumors and scored 319,626 cancer cells revealing significant heterogeneity of PRO, EMT, and DP cells among patients. Single-cell analysis within the context of tissue architecture

revealed these cells were grouped together in discrete tumor glands. Using each of the 3 cell types (PRO, EMT, and DP), we classified 8 distinct types of tumor glands in these primary tumors. This provided the ability to define inter and intra-tumor heterogeneity at the tumor gland level, which provides an extra layer of tumor cell organization not appreciable with single cell analysis alone. We showed that tumor glands are independent functional units in human PDACs carrying distinct prognostic information. In fact, by comparing their prognostic power we noticed that some tumor glands are positively linked with worsened survival (Type I p=0.003, Type II p=0.04, Type II p=0.001 and Type VII p=0.02), while Type III glands are associated with a favorable patient prognosis (p=0.01, log-rank test).

In conclusion, our study showed for the first time at this scale level that integrating single-cell technologies with tissue architectural information provides a more comprehensive landscape of intra-tumoral cancer cell heterogeneity that has implications on PDAC cell behavior and patient outcomes.

#211

Spin coated peripheral blood film reveals tumor heterogeneity in circulation.

Andy Cheng,1 Leo Xu,2 Bin Hong1. 1 _TeloVISION LLC, West Lafayette, IN;_ 2 _Adventist Healthcare Medical Center, MD_.

Peripheral blood film (PBF) is an important laboratory workup for clinical characterization of haematological malignancies that involves blood smear on microscope slide and cytology of blood cells. PBF preparation, typically manually operated, requires technical training and empirical experience and is not without human errors. Automated blood smearing by traditional spin coating has been an alternative. However, with intended use originally for semiconductor industries, it utilizes volatile organic solvents, employs powerful centrifugation at high speed, which frequently alters morphology and damages cells, and inherently creates radius dependent pattern. In addition, manual blood dispensing could bring contamination and spin-off of excess blood as aerosol can be hazardous. While improvement of traditional spin coating technology to overcome intrinsic defects and compensate operational variations is possible, low-speed spin coating is more predicable and controllable for uniform PBF thickness. An innovative automatic PBF coating system has been developed to smear monolayer of dispersed blood cells for cytopathology and thick PBF of multilayered blood cells as well for identification, enumeration, and examination of rare tumor cells in peripheral blood. Explicitly, whole blood is dispensed through nozzle directly from the blood collection tube that slides radially on rails above substrate, and coated on the spinning substrate at low speed in non-contact mode circle-by-circle with no intra/inter- circular overlaps and negligible centrifugal force. Consequently, there is no spin-off, saving blood samples and preventing potential biohazard. External contamination is also eliminated by dispensing blood directly from the tube. Temperature control of substrate is further enabled to preserve cellular viability and morphology. At speed of 60 rpm, contact angle of 10°, stepper of 0.5 mm, and room temperature, monolayer PBF with intact cellular morphology across the entire substrate was created using healthy fresh whole blood directly from the BD Vacutainer K2-EDTA tube. To study tumor cell heterogeneity in circulation, thick PBFs have been evenly coated on 100 mm diameter discs using 2 mL of pre-stained, cancer patient blood samples at speed of 30 rpm, contact angle of 45°, stepper of 1 mm and elevated temperature of 37°C and fully dried. PBFs were then scanned by our innovative Spinning Disc Imaging system (iSDI) to pinpoint fluorescent nucleated cells of epithelial origin. Images of suspects were automatically displayed with identities of location, area, orientation, width, height, and signal intensity. Results demonstrated tumor cells with variable sizes, shapes and expression levels of EpCAM, in addition to clusters and fragments. Despite advances in genetic and molecular diagnostics, PBF remains an indispensable part of diagnostic jigsaw in unraveling heterogeneity and evolution of tumor in liquid phase.

#212

High-resolution barcoding in patient-derived xenografts of triple-negative breast cancer reveals reversible chemoresistance conferred by non-mutational mechanisms.

Gloria Vittone Echeverria,1 Sahil Seth,1 Zhongqi Ge,1 Alessandro Carugo,1 Christopher Bristow,1 Prabjhot Mundi,2 Sabrina Jeter-Jones,1 Xiaomei Zhang,1 Xinhui Zhou,1 Aaron McCoy,1 Shirong Cai,1 Yizheng Tu,1 Yuting Sun,1 Joseph Marszalek,1 Andrea Califano,2 William F. Symmans,1 Stacy L. Moulder,1 Jeffery T. Chang,3 Timothy P. Heffernan,1 Helen Piwnica-Worms1. 1 _UT MD Anderson Cancer Ctr., Houston, TX;_ 2 _Columbia University Medical Center, New York, NY;_ 3 _UT Health Science Center, Houston, TX_.

Fifty percent of patients with localized triple negative breast cancer (TNBC) have substantial residual cancer burden following treatment with neoadjuvant chemotherapy (NACT), resulting in a 40-80% risk of recurrence which leads to distant metastases and death for most patients. Intra-tumor heterogeneity (ITH) is a pervasive feature of TNBCs but the relative contributions of heterogeneous tumor cell populations to chemoresistance are not understood. To investigate the clonal dynamics that accompany chemotherapy treatment, we employed orthotopic patient-derived xenograft (PDX) models of treatment-naïve TNBC, thus enabling experimentation with heterogeneous populations of human tumor cells that have undergone minimal manipulation.

To monitor the fates of PDX tumor cell lineages undergoing treatment with front-line NACT, we treated multiple treatment-naïve PDX models with Adriamycin combined with Cytoxan (AC). Some PDXs initially exhibited partial sensitivity followed by maintenance of residual tumors that were resistant to chemotherapy. Residual tumors re-grew to regain partial chemo-sensitivity. To conduct barcode-mediated clonal tracking, we introduced a pooled lentiviral barcode library (Cellecta; 50M unique barcodes) into freshly dissociated PDX tumor cells which were orthotopically engrafted into recipient mice. Strikingly, residual tumors maintained the same clonal architecture as untreated tumors. In contrast, only 20% of residual tumor clones repopulated tumors after discontinuation of treatment. Whole-exome sequencing revealed conservation of mutant allele frequencies throughout treatment. Together, these studies demonstrate that re-growth of residual tumors is accompanied by a non-random selection of subclones, that residual tumors surviving AC maintain the same levels of ITH as untreated tumors, and that selection of genomic subclones did not confer the observed chemoresistance.

Transcriptomic, proteomic, and histologic profiling revealed that residual tumors exist in a distinct state characterized by alterations in EMT, metabolic, and cell adhesion programs. This state was reverted as tumors re-grew after discontinuation of AC treatment, indicating that the residual state may be a unique therapeutic window. In silico prediction of drug sensitivity revealed candidate drivers of resistance in the residual tumor state, and pharmacologic targeting identified multiple existing therapies that significantly delayed the regrowth of residual tumors. These data suggest that sequential administration of AC followed by these targeted agents could prolong TNBC responses, which may delay time to recurrence for patients with this highly aggressive disease.

#213

Exome sequencing of paired primary and relapsed small cell lung cancers reveals increased copy number aberration complexity to be associated with disease relapse.

Marcelo V. Negrao,1 Ming Tang,1 Ying Jin,2 Yamei Chen,2 Xiao Hu,2 Huarong Tang,2 Haimiao Xu,2 Kelly Quek,1 Jianhua Zhang,1 Xizeng Mao,1 Xingzhi Song,1 John V. Heymach,1 Ignacio Wistuba,1 Lauren A. Byers,1 Bonnie S. Glisson,1 Andrew Futreal,1 Ming Chen,2 Jianjun Zhang1. 1 _MD Anderson Cancer Center, Houston, TX;_ 2 _Zhejiang Cancer Hospital, Hangzhou, China_.

Introduction: Although small-cell lung cancer (SCLC) is sensitive to first-line radiation and chemotherapy, nearly all patients recur often with treatment-resistant disease. Therefore, delineating subclonal architecture and molecular evolution of SCLC after treatment by comparing genomic profiles of recurrent and paired treatment-naïve tumors may provide new insights into mechanisms underlying recurrence and susceptibility to further treatment.

Methods: Treatment-naïve and paired recurrent tumor samples from 9 patients with limited-stage SCLC treated with concurrent chemoradiation (CCRT) underwent whole exome sequencing (WES) and data is currently available for 6 pairs. All 6 patients had disease recurrence more than 3 months after finishing treatment. Genomic landscape including somatic mutations, somatic copy number aberrations (SCNA), subclonal architecture and transversion rate (percentage of base changes from pyrimidine to purine and vice-versa) were compared between treatment-naïve and paired recurrent tumor samples. Circulating cell-free DNA (cfDNA) collected at the time of recurrence was also subject to deep sequencing of 461 cancer genes.

Results: SCNA analyses revealed significantly more complex chromosomal gains in relapsed SCLCs compared to pre-treatment primary tumors (7.7% vs 13.2% genes with copy number gain in primary and relapsed SCLC respectively, p=0.034). As expected, TP53 and RB1 were the most commonly mutated genes (83% and 67% respectively) and tobacco exposure related signatures were predominant in all samples. Primary and relapsed tumor pairs showed very similar genomic landscape including tumor mutational burden (11.8/MB in primary SCLC vs 9.7/MB in relapsed tumors), intra-tumor heterogeneity complexity (MATH score 38.2 in primary vs 39.2 in relapse) and transversion rates, that have been reported to be associated with alkylating-agent treatment (62% in primary vs 65% in relapse). An average of 72.25% (range: 53-86%) somatic mutations, including all canonical cancer gene mutations, were shared between primary SCLC and relapsed tumors. In addition, the subclonal architecture was also very similar between treatment-naïve primary SCLC and paired relapsed tumors. Of note, 5 out of 6 patients achieved disease control on retreatment with chemotherapy.

Conclusion: Copy number gains may play a role in post-CCRT relapse of SCLC. Paired pre-treatment and relapse samples show genomic similarities which could account for benefit to subsequent treatment. Additional sample analysis and ctDNA assessment is ongoing to better address these findings, especially in patients with chemoresistant relapse. These findings also suggest that other molecular changes involving gene expression (eg. methylation) may play a role in SCLC relapse and resistance.

#214

Genetic and transcriptional instability alters cell line drug response.

Uri Ben-David,1 Ben Siranosian,1 Gavin Ha,1 Helen Tang,1 Nicholas J. Lyons,1 Robert Burns,2 Anwesha Nag,2 Beth Cimini,1 Peter Tsvetkov,3 Andrew A. Tubelli,1 Bang Wong,1 Aaron R. Thorner,2 Joshua Bittker,1 Matthew Meyerson,1 Rameen Beroukhim,1 Todd R. Golub1. 1 _Broad Inst. of MIT and Harvard, Cambridge, MA;_ 2 _Dana Farber Cancer Institute, Boston, MA;_ 3 _Whitehead Institute, Cambridge, MA_.

Inconsistencies in cell line-based studies jeopardize reproducibility of cancer research. Natural evolution leading to genetic and transcriptional heterogeneity within cancer cell lines may contribute to such inconsistencies. To systematically test this hypothesis, we performed comprehensive genetic and transcriptomic characterization of 27 strains of the common breast cancer cell line MCF7, and then assessed the response of those strains to 321 anti-cancer compounds. We found extensive variation in genomic profiles and responses to small molecules across strains. Genetic variation occurred at all levels – point mutations, rearrangements, and copy number changes – and affected multiple oncogenes and tumor suppressor genes. Similar findings were obtained across 23 strains of the lung cancer cell line A549, indicating that such variation is a general property of cancer cell lines. These changes resulted in substantial differences in gene expression programs, cell morphology and proliferation, and strikingly high variability in drug response. Over 75% of the compounds that exhibited strong activity in some of the strains, were completely inactive in other strains. These results indicate that genomic variation within commonly used cancer cell lines is extensive, resulting in disparate drug responses. Such variation may contribute to difficulties in conducting reproducible cell line-based studies.

#215

Reconstructing the Barrett's-dysplasia-adenocarcinoma sequence from patient-matched stem cells.

Wa Xian,1 Yanting Zhang,2 Marcin Duleba,1 Shan Wang,1 Frank McKeon1. 1 _University of Texas, Houston, TX;_ 2 _University of Houston, Houston, TX_.

Introduction: Both esophageal and gastric adenocarcinoma (EAC; GAC) evolve from intestinal metaplasia (Barrett's and gastric IM, respectively) in a multi-decade and multi-step process involving transitions through low- and high-grade dysplasia often referred to as the "Correa Sequence". In early stage EAC, each of these lesions can be seen co-existing in the 2-D topology of the distal esophagus. We have adapted methods we originally developed for cloning normal stem cells of the gastrointestinal tract to capturing stem cells of each of these lesions. We anticipate that a clonal analysis of these stem cells will reveal key steps in the evolution of highly lethal EAC.

Methods: Libraries of stem cell clones were generated from a series of patient-specific endoscopic biopsies derived from normal, Barrett's, dysplasia, and EAC. Single cell-derived clones were analyzed by in vitro differentiation, whole genome expression analysis, and SNP-based copy number variation.

Results: Highly undifferentiated, ground state stem cells were cloned from multiple endoscopic biopsies of patients with early EAC. Based on sampling site and adjacent biopsy histology, tissue corresponding to normal esophagus, gastric epithelia, Barrett's, and EAC were taken in these biopsies dissociated for stem cell cloning. All the clones derived showed unlimited proliferative potential and the ability to differentiate to normal or diseased epithelia in vitro. Thus the normal esophageal and gastric stem cells gave rise to squamous and gastric epithelia, the Barrett's stem cells yielded intestinal metaplasia, and dysplastic and EAC clones yielded highly proliferative and disordered epithelia marked by a loss of cell polarity. In contrast to the germline genotypes of the normal stem cells, those from Barrett's, dysplasia, and EAC sustained increasingly large numbers of copy number variation events, single nucleotide variations, and chromosomal aneuploidies. Significantly, all single cell-derived clones of EAC yielded "intestinal" adenocarcinoma following xenografting to immunodeficient mice. We are presently exploiting the genomic alterations present in hundreds of sample clones from each patient to reconstruct the evolutionary events that accompanied the progression to a highly lethal cancer.

Conclusions: Stem cells from all stages of lesions along to the path to epithelial cancers can be cloned and independently propagated to yield insights into the evolution of cancer. In addition, these cloned cells should enable insights into intratumor heterogeneity, therapy resistance, and drug discovery.

#216

Functional stability, progression and evolution of targeted drug sensitivity of HER-2-positive breast cancer patient-derived xenografts.

Lorena Landuzzi,1 Marianna L. Ianzano,2 Claudio Ceccarelli,2 Enrico Di Oto,2 Giordano Nicoletti,1 Veronica Giusti,2 Roberta Laranga,2 Tania Balboni,2 Carla De Giovanni,2 Massimiliano Dall'Ora,2 Sofia Asioli,2 Arianna Palladini,2 Donatella Santini,3 Maria Pia Foschini,2 Mario Taffurelli,2 Pier-Luigi Lollini,2 Patrizia Nanni2. 1 _Rizzoli Orthopedic Institute, Bologna, Italy;_ 2 _University of Bologna, Bologna, Italy;_ 3 _Policlinico S. Orsola-Malpighi University Hospital, Bologna, Italy_.

Human tumors are dynamic entities that undergo variation, selection and progression within the patient. How well patient-derived xenografts (PDX) recapitulate tumor dynamics? To investigate these aspects we established a collection of breast cancer PDX, representative of the main intrinsic subtypes. From 66 primary breast cancer specimens, we obtained 5 transplantable PDXs (8%). The highest rate of PDX stabilization was obtained for HER2-positive (40%) followed by triple negative (17%) and luminal B (14%) subtypes. In 3/66 cases a human lymphoma developed without any further evidence of breast cancer. Two HER-2-positive and one non-amplified, HER-2++, luminal B PDXs were serially transplanted in mice for 7-25 passages over a period of 2-4 years. Morphological and immunohistochemical (ER, PR, Ki67, p53, HER2, HER1, HER3, IGFR) features were highly stable over serial passages of PDXs, which retained the histology and the expression pattern of the tumor of origin. After the second passage, one HER-2++ amplified PDX, named BO-HAT4, was split in six different sublines, which were then studied separately to analyze random and selective events in long-term evolution. Two sublines progressively acquired a marked acceleration of tumor growth rate, whereas the remaining four did not. Metastatic spread was absent in early passages and appeared sporadically in late passages. In vitro cultures derived from early in vivo passages showed a rapid onset of cell senescence, whereas late in vivo passages gave rise to long-term in vitro cultures. However, we have not yet been able to obtain continuous cell lines from breast cancer PDX. To study the onset of resistance to HER-2 targeted therapies, sequential in vivo passages of BO-HAT4 were treated with trastuzumab, leading to a progressive loss of sensitivity. After one year of treatment BO-HAT4 was completely resistant to trastuzumab. We are currently investigating the molecular changes underlying trastuzumab resistance. Interestingly, both trastuzumab-sensitive and -resistant tumors were highly inhibited by neratinib. In conclusion, the low take rate as PDX prevents the generalized analysis of human breast cancer patients. Individual PDX allow the analysis of target therapy response and onset of resistance, however long-term study of transplantable breast cancer PDX show that tumor progression in these model systems is a late and random event. Supported by grants from the Italian Association for Cancer Research (AIRC) and the University of Bologna, Italy.

#217

The role of exosomes and exosomal miRNA in mediating EGFR-TKI sensitivity in heterogeneous NSCLC.

Chien-Chung Lin,1 Wu-Chou Su,1 Chin-You Wu2. 1 _National Cheng Kung Univ. Hospital, Tainan, Taiwan;_ 2 _National Cheng Kung University, Tainan, Taiwan_.

Tumor heterogeneity has impacts on tumor biopsy strategy, characterization of actionable targets and drug resistance. At lung cancer, recent study demonstrated that the discordance rates of EGFR mutation implying tumor heterogeneity in metachronous and synchronous settings were 14.3% and 9.1% respectively. And other study further showed intratumoral EGFR mutational heterogeneity was correlated with the response and prognosis of EGFR-TKIs. Extracellular vesicles (EVs) such as exosomes serve as the transporter of bioactive molecules between cells and become one of the major mechanisms contributing intratumoral heterogeneity via intercellular transference of genetic information. Since most patients harboring EGFR mutation showed excellent response, we hypothesized that EVs such as exosome mediating the crosstalk between EGFR mutant cell and EGFR wild type cell contributing the change of sensitivity of EGFR wild type cell to EGFR-TKI in heterogeneous NSCLC. We used ultrafiltration (UF) method to isolate the exosomes. The conditioned media were centrifuged and then the supernatants were passed through 0.22 μm filters and then Vivaspin nanomembrane concentrators step by step. We first incubated EGFR wild type lung cancer cell (CL1-5) with medium containing PKH26 labeled exosomes derived from PC9 cell (EGFR Del 19) for 24 h. The uptake of exosomes from PC9 in CL1-5 cell can be observed under immunofluorescence microscope and recorded by time-lapse microscope. And the EGFR Del19 DNA and specific protein can be detected in recipient CL1-5 cells by digital PCR and Western blotting respectively. Then EGFR wild type lung cancer cell (CL1-0, CL1-5 and H1299) were first treated with exosome from themselves or PC9 cell respectively for 48h and then subjected to gefitinib (0.1 μM) for 72h. We found only PC9 cell derived EV sensitized EGFR wild type cell to gefitinib. To mimic tumor heterogeneity, we next tested the significance of exosome on EGFR-TKI sensitivity in co-culture system with pretreatment with or without GW4869, which blocks exosome secretion by inhibiting neutral sphingomyelinase. We first verified that treating lung cancer cell with GW4869 at concentrations of 2.5, 5 and 10μM inhibited exosome secretion without cytotoxicity. We demonstrated that EGFR wild type lung cancer cell became sensitive to EGFR-TKI after co-culture with PC9 cell for 48h and then subjected to gefitinib for 72h. However, the pretreatment with GW4869 for 48 hours reversed the sensitivity to EGFR-TKI in co-culture system with PC9. We further compared the exosomal miRNAs from PC9 to those from CL1-5 and identified a panel of discriminative miRNAs. Some miRNA among the panel such as miR-200 family have been identified associated with resistance to EGFR-TKI. Our study, at least in part, suggests that exosome release from EGFR mutant cell might be a possible mechanism for excellent EGFR-TKI sensitivity even in heterogeneous NSCLC.

#218

The evolutionary history of 2,658 cancers.

Clemency Jolly,1 Moritz Gerstung,2 Ignaty Leshchiner,3 Stefan C. Dentro,4 Santiago Gonzalez,2 Thomas J. Mitchell,4 Yulia Rubanova,5 Pavana Anur,6 Daniel Rosebrock,3 Kaixian Yu,7 Maxime Tarabichi,1 Amit Deshwar,5 Jeff Wintersinger,5 Kortine Kleinheinz,8 Ignacio Vásquez-García,4 Kerstin Haase,1 Subhajit Sengupta,9 Geoff Macintyre,10 Salem Malikic,11 Nilgun Donmez,11 Dimitri G. Livitz,3 Mark Cmero,12 Jonas Demeulemeester,1 Steve Schumacher,3 Yu Fan,7 Xiaotong Yao,13 Juhee Lee,14 Matthias Schlesner,8 Paul C. Boutros,5 David D. Bowtell,15 Hongtu Zhu,7 Gad Getz,3 Marcin Imielinski,13 Rameen Beroukhim,3 S Cenk Sahinalp,16 Yuan Ji,9 Martin Peifer,17 Florian Markowetz,10 Ville Mustonen,18 Ke Juan,10 Wenyi Wang,7 Quaid D. Morris,5 Paul T. Spellman,6 David C. Wedge,19 Peter Van Loo,1 PCAWG Evolution and Heterogeneity Working Group. 1 _The Francis Crick Institute, London, United Kingdom;_ 2 _European Molecular Biology Laboratory, European Bioinformatics Institute (EMBL-EBI), Cambridge, United Kingdom;_ 3 _Broad Institute of MIT and Harvard, Cambridge, MA;_ 4 _Wellcome Trust Sanger Institute, Cambridge, United Kingdom;_ 5 _University of Toronto, Toronto, Ontario, Canada;_ 6 _Oregon Health and Science University, Portland, OR;_ 7 _The University of Texas MD Anderson Cancer Center, Houston, TX;_ 8 _German Cancer Research Center (DKFZ), Heidelberg, Germany;_ 9 _NorthShore University HealthSystem, Evanston, IL;_ 10 _Cancer Research UK Cambridge Institute, University of Cambridge, Cambridge, United Kingdom;_ 11 _Simon Fraser University, Vancouver, British Columbia, Canada;_ 12 _University of Melbourne, Melbourne, Australia;_ 13 _Weill Cornell Medicine, New York, NY;_ 14 _University of California Santa Cruz, Santa Cruz, CA;_ 15 _Peter MacCallum Cancer Centre, Melbourne, Australia;_ 16 _Indiana University, Bloomington, IN;_ 17 _University of Cologne, Cologne, Germany;_ 18 _University of Helsinki, Helsinki, Finland;_ 19 _Big Data Institute, Oxford, United Kingdom_.

Cancer develops through a continuous process of somatic evolution. Whole genome sequencing provides a snapshot of the tumor genome at the point of sampling, however, the data can contain information that permits the reconstruction of a tumor's evolutionary past.

Here, we apply such life history analyses on an unprecedented scale, to a set of 2,658 tumors spanning 39 cancer types. We estimated the timing of large chromosomal gains during tumor evolution, by comparing the rates of doubled to non-doubled point mutations within gained regions. Although we find that such events typically occur in the second half of clonal evolution, we also observe distinctive and early chromosomal gains in some cancer types, such as gains of chromosomes 7, 19 and 20 in glioblastoma, and isochromosome 17q in medulloblastoma. By integrating these results with the qualitative timing of individual driver mutations, we obtained an overall ranking, from early to late, of frequent somatic events per cancer type, which both identified novel patterns of tumor evolution, and incorporated additional detail into known models, such as the progression of APC-KRAS-TP53 in colorectal cancer proposed by Vogelstein and Fearon.

To estimate how mutational processes acting on the tumor genome change over time, we classified mutations in each sample according to three broad time periods (early clonal, late clonal, and subclonal), and quantified the activity of mutational signatures in each period. Most mutational processes appear to remain remarkably constant, however, certain signatures show clear and consistent changes during clonal evolution. Particularly, mutational signatures associated with exposure to carcinogens, such as smoking and UV light, tend to decrease over time. In contrast, signatures associated with defective endogenous processes, such as APOBEC mutagenesis and defective double strand break repair, show an increase between early and late phases of tumor evolution.

Making use of clock-like mutational signatures, we converted mutational time estimates for large events, such as whole genome duplication (WGD), and the emergence of the most recent common ancestor (MRCA), into real time estimates, which allowed us to combine our analyses into overall timelines of cancer evolution, per tumor type. For example, the typical timeline of ovarian adenocarcinoma development shows that early tumor evolution is characterized by mutations in TP53, and widespread genome instability, with WGD events taking place on average 8 years prior to diagnosis. In later stages of evolution, signatures of defective repair processes increase, and the MRCA emerges on average 1 year before diagnosis.

Taken together, these data reveal the common and divergent evolutionary trajectories available to a cancer, which might be crucial in understanding specific tumor biology, and in providing new opportunities for early detection and cancer prevention.

#219

Development of TEAPOT algorithm to reconstruct individual ovarian tumors' evolutionary history based upon bulk and single cell whole exome sequencing data.

Jianshu Zhang,1 Helaman Escobar,2 Harshmi Shah,1 Mickey Miller,2 Yang Wei,3 Kristen Schneider,3 Michelle Knirr,3 Kenny Day,2 Christopher Johnson,2 Baoli Yang,3 Eric Devor,3 Kristina Thiel,3 Lincoln Nadauld,2 Kimberly Leslie,3 Donghai Dai3. 1 _Immortagen Inc., Coralville, IA;_ 2 _Intermountain Precision Genomics, St George, UT;_ 3 _Univ. of Iowa, Iowa City, IA_.

Background. Mutation detection through genetic testing is playing an increasingly important role in personalized precision medicine in cancer. However, current tests identifying driver mutations as therapeutic targets are based on detection of common mutations in cancer genes. These tests are not patient specific and do not address intra-tumor heterogeneity. Ubiquitous intra-tumor genetic heterogeneity is a mechanism of drug resistance and cancer recurrence.

Methods. Approximately 16-24 microsamples are acquired to represent the entire cancer cell population for every ovarian tumor. Each microsample consists of a few cells within a clone and is selected to substitute for a single cell and overcome the large allele dropout rate commonly seen in single genome amplification and sequencing. TEAPOT (Tumor Evolution Assay for Personalized Oncology Therapy) algorithm has been developed to reconstruct a tumor's evolutionary history through integration of whole exome sequencing data from the bulk primary tumor and 16-24 microsamples taken from the bulk tumor. The evolutionary history for an individual tumor is expressed as a rooted and binary tumor developmental tree representing the mitotic process starting from an ancestral cancer cell. Individual mutations are assigned to the cells where they originally occur. The offspring size carrying a mutation was estimated based on tumor purity, variant allele frequency and the variant's copy number.

Results. TEAPOT algorithm builds a tumor's evolutionary history with the following features: 1) a tumor's evolutionary history is unique for each ovarian cancer patient; 2) the size of a tree is proportional to the number of microsamples selected; 3) 16-24 microsamples builds a tree with 5 or more generations; 4) TEAPOT detects a driver mutation's occurrence at a specific developmental stage such as 1-cell, 2-cell, 4-cell, etc; 5) The size of offspring carrying a mutation thus the intra-tumor prevalence of the mutation can be estimated; 6) multiple driver mutations can be located separately in different clones. Therefore, TEAPOT provides a quantitative description of intra-tumor genetic heterogeneity and identifies sub-clonal driver mutations in a tumor.

Conclusion. TEAPOT reconstructs a tumor's developmental process thus providing a patient-specific evolutionary history. Quantitation of intra-tumor prevalence of driver mutations may inform selection of an effective targeted agent and may provide rationale for cocktail treatment targeting multiple driver mutations simultaneously. TEAPOT can be also used for other solid and liquid cancers. A driver mutation's role in a patient may be functionally defined and quantitated based upon the growth advantage (fitness) it confers on its host cells in the reconstructed tumor evolutionary history.

#220

Intra-tumor immune heterogeneity is associated with prognosis of oropharyngeal head and neck squamous cell carcinoma.

Takahiro Tsujikawa,1 Guillaume Thibault,2 Young Hwan Chang,2 Edward El Rassi,2 Daniel R. Clayburgh,2 David Sauer,2 Akihito Arai,1 Molly F. Kulesz-Martin,2 Motomi Mori,2 Shigeru Hirano,1 Paul W. Flint,2 Lisa M. Coussens2. 1 _Kyoto Prefectural University of Medicine, Kyoto City, Kyoto, Japan;_ 2 _Oregon Health & Science University, Portland, OR_.

As cell-cell interactions among tumor and immune cells are known to contribute to tumor progression, in depth evaluation of tumor-immune heterogeneity will enable biomarker-guided patient stratification and improvement of treatment response. Here we examined head and neck squamous cell carcinomas (HNSCC) to reveal the prognostic significance of leukocytes in tumors with regards to their complexity, effector status and spatial characteristics via multiplex immunohistochemistry and image cytometry. To accomplish this, we examined oropharyngeal HNSCC (N = 38), where lymphoid, myeloid, and hypo-inflamed leukocyte complexity correlated with HPV-status (Tsujikawa et al. Cell Reports, 2017). In the present study, quantitative analysis of cell density and distribution of 14-distinct immune cell lineages (e.g., CD8+ T cells, regulatory T cells, Th1, Th2, Th17, Th0 lymphocytes, B cells, NK cells, CD163\+ and CD163- macrophages, mature and immature dendritic cells, mast cells, granulocytes) was analyzed via immune cell density mapping and tissue segmentation algorithms. We revealed tropism of polarized Th1-type cells within tumor nests versus stroma in HPV-associated HNSCC. In addition, Cox regression analysis of cell density and distribution of the 14 immune populations revealed that CD66b+ granulocyte infiltration within tumor nests reflected a negative prognostic indicator for HNSCC outcome. Furthermore, cell-cell proximity analysis in HPV-associated HNSCC further revealed a correlation between PD-L1 positive immune cells, and micro-regionally polarized immune characteristics biased towards Th1, coincident with high density of CD8+ T cells. These results reveal intra-tumor immune heterogeneity is associated with micro-regional immune complexity profiles, and provide insight into in situ immune characteristics that may aid patient stratification for immune therapy going forward. Acknowledgement: This project was supported by the Japan Society for the Promotion of Science Grant-in-Aid for Young Scientists (Start-up, 17H07016), Oregon Clinical and Translational Research Institute (OCTRI), grant number (UL1TR000128) from the National Center for Advancing Translational Sciences (NCATS) at the National Institutes of Health (NIH), and P30 CA069533-17 OHSU Knight Cancer Institute. LMC acknowledges support from the NIH/NCI, DOD BCRP Era of Hope Scholar Expansion Award, Susan G. Komen Foundation, Stand Up To Cancer - Lustgarten Foundation Pancreatic Cancer Convergence Dream Team Translational Research Grant, Breast Cancer Research Foundation, and the Brenden-Colson Center for Pancreatic Health.

## EPIDEMIOLOGY:

### Genetic Contributions to Cancer Epidemiology 1: GWAS and Pathway and Candidate Gene Studies

#221

Inherited variants at 3q13.33 and 3p24.1 influences risk of diffuse large B-cell lymphoma.

Geffen Kleinstern,1 Michelle Hildebrand,2 Vijai Joseph,3 Sonja I. Berndt,4 Hervé Ghesquières,5 James McKay,6 Sophia S. Wang,7 Alexandra Nieters,8 David Cox,9 Alain Monnereau,10 Angela R. Brooks-Wilson,11 Qing Lan,4 Mads Melbye,12 Rebecca D. Jackson,13 Lauren R. Teras,14 Mark P. Purdue,4 Claire M. Vajdic,15 Demetrius Albanes,4 Anne Zeleniuch-Jacquotte,16 Simon Crouch,17 Yawei Zhang,18 Susan L. Slager,1 Xifeng Wu,2 Karin E. Smedby,19 Gilles Salles,20 Christine F. Skibola,21 Nathaniel Rothman,4 Stephen J. Chanock,4 James R. Cerhan1. 1 _Mayo Clinic, Rochester, MN;_ 2 _MD Anderson Cancer Center, Houston, TX;_ 3 _Memorial Sloan-Kettering Cancer Center, New York, NY;_ 4 _National Cancer Institute, Bethesda, MD;_ 5 _Centre Léon Bérard, Lyon, France;_ 6 _International Agency for Research on Cancer, Lyon, France;_ 7 _City of Hope Beckman Research Institute, Duarte, CA;_ 8 _Center for Chronic Immunodeficiency, Freiburg, Germany;_ 9 _Cancer Research Center of Lyon, Lyon, France;_ 10 _Centre for Research in Epidemiology and Population Health (CESP), Villejuif, France;_ 11 _BC Cancer Agency, Vancouver, British Columbia, Canada;_ 12 _Statens Serum Institut, Copenhagen, Denmark;_ 13 _The Ohio State University, Columbus, OH;_ 14 _American Cancer Society, Atlanta, GA;_ 15 _University of New South Wales, Sydney, Australia;_ 16 _NYU School of Medicine, New York, NY;_ 17 _University of York, York, United Kingdom;_ 18 _Yale School of Public Health, New Haven, CT;_ 19 _Karolinska Institutet, Stockholm, Sweden;_ 20 _Hospices Civils de Lyon, Lyon, France;_ 21 _Emory University, Atlanta, GA_.

Background: We previously identified 5 SNPs at 4 susceptibility loci for diffuse large B-cell lymphoma (DLBCL) in individuals of European ancestry through a large genome-wide association study (Cerhan et al, Nat Gen 2014;46:1233-8); however, much of the heritability remains unexplained. To further elucidate genetic susceptibility to DLBCL, we sought to validate 2 loci at 3q13.33 and 3p24.1 that were suggestive in the original report.

Methods: We selected two SNPs rs9831894 (3q13.33) and rs6773363 (3p24.1) for de novo replication from studies at the Mayo Clinic, MD Anderson, and Memorial Sloan Kettering. Logistic regression was used to estimate odds ratios (ORs), using a log-additive model, and adjusting for age, gender and significant eigenvectors for each genotyping center separately. For both SNPs, we then conducted a meta-analysis of all replication studies (n=3) and our original GWAS studies (n=4), encompassing in total 5662 cases and 9237 controls for rs9831894, and 5510 cases and 12,817 controls for rs6773363. Meta-analysis was conducted using the fixed-effects inverse variance method based on the β estimates and standard errors from each study.

Results: In a meta-analysis of the 4 original GWAS scans (3856 cases and 7666 controls), rs9831894 (MAF=0.40) was associated with DLBCL risk (OR=0.84, P=4.52 x 10-9) and this replicated in a meta-analysis of the 3 studies with de novo genotyping (OR=0.80, P=4.17 x 10-5); the overall meta-analysis showed a strong association with DLBCL risk (OR=0.83, P=3.62 x 10-13). This locus maps near a plausible candidate gene, CD86, a protein coding gene and a member of the immunoglobulin superfamily that encodes a type I membrane protein. Binding of CD86 with cytotoxic T-lymphocyte-associated protein 4 negatively regulates T-cell activation and diminishes the immune response, while binding of CD86 with CD28 antigen is a costimulatory signal for activation of the T-cell. In a meta-analysis of the same 4 GWAS scans, rs6773363 (MAF=0.45) was tentatively associated with DLBCL risk (OR=1.17, P= 3.68 x 10-7) and this replicated in a meta-analysis of the studies with de novo genotyping (OR=1.27, P=3.78 x 10-7); the overall meta-analysis showed a strong association with DLBCL risk (OR=1.20, P=2.31 x 10-12). This locus also maps near a plausible candidate gene, eomesodermin (EOMES), a transcription factor crucial for embryonic development of the central nervous system and also putatively involved in T-cell differentiation in viral infection defense.

Conclusion: In this follow-up analysis of our initial GWAS, we have identified two additional loci associated with risk of DLBCL, the most common lymphoma subtype. These loci provide additional evidence for the role of immune function in the etiology of DLBCL.

#222

Genome-wide association study of acute lymphoblastic leukemia in children with Down syndrome.

Austin L. Brown,1 Adam J. de Smith,2 Michael E. Scheurer,1 Noah A. Kallsen,3 Shanna A. Peyton,3 Gareth E. Davies,3 Erik A. Ehli,3 Michael E. Zwick,4 Naomi Winick,5 Kelly Maloney,6 Anne L. Angiolillo,7 Reuven Schore,7 MIchael M. Burke,8 Wanda L. Salzer,9 Nyla A. Heerema,10 Andrew J. Carroll,11 Michael J. Borowitz,12 Brent L. Wood,13 William L. Carroll,14 Elizabeth A. Raetz,15 Elanor Feingold,16 Stephanie L. Sherman,17 Wenjian Yang,18 Meenakshi Devidas,19 Kyle Walsh,20 Andrew T. DeWan,21 Maria S. Pombo-de-Oliveira,22 Jeffrey W. Taub,23 Daniel Sinnett,24 Jasmine Healy,24 Jillian M. Birch,25 Lisa F. Barcellos,26 Helen Hansen,2 Ivan Smirnov,2 Charles G. Mullighan,18 Stephen P. Hunger,27 Ching-Hon Pui,18 Mignon Loh,2 Joe L. Wiemels,2 Xiaomei Ma,21 Catherine Metayer,28 Beth A. Mueller,29 Mary V. Relling,18 Jun J. Yang,18 Philip J. Lupo,1 Karen R. Rabin1. 1 _Baylor College of Medicine, Houston, TX;_ 2 _University of California San Francisco, San Francisco, CA;_ 3 _Avera Institute for Human Genetics, Sioux Falls, SD;_ 4 _Emory University, Atlanta, GA;_ 5 _University of Texas Southwestern Medical Center, Dallas, TX;_ 6 _Children's Hospital Colorado, Aurora, CO;_ 7 _Children's National Medical Center, Washington, DC;_ 8 _Medical College of Wisconsin, Milwaukee, WI;_ 9 _U.S. Air Force Medical Service, Fort Detrick, MD;_ 10 _Ohio State University, Columbus, OH;_ 11 _University of Alabama Birmingham, Birmingham, AL;_ 12 _Johns Hopkins Hospital, Baltimore, MD;_ 13 _University of Washington Medical Center, Seattle, WA;_ 14 _New York University, New York, NY;_ 15 _University of Utah Huntsman Cancer Institute, Salt Lake City, UT;_ 16 _University of Pittsburgh, Pittsburgh, PA;_ 17 _Emory University School of Medicine, Atlanta, GA;_ 18 _St. Jude Children's Research Hospital, Memphis, TN;_ 19 _University of Florida, Gainesville, FL;_ 20 _Duke University, Durham, NC;_ 21 _Yale School of Public Health, New Haven, CT;_ 22 _Instituto Nacional do Cancer, Rio de Janeiro, Brazil;_ 23 _Wayne State University, Detroit, MI;_ 24 _University of Montreal, Montreal, Quebec, Canada;_ 25 _University of Manchester, Manchester, United Kingdom;_ 26 _University of California Berkeley, Berkeley, CA;_ 27 _Children's Hospital of Philadelphia, Philadelphia, PA;_ 28 _University of California Berkeley School of Public Health, Berkeley, CA;_ 29 _Fred Hutchinson Cancer Research Center, Seattle, WA_.

Purpose: Children with Down syndrome (DS) have a 20-fold increased risk of acute lymphoblastic leukemia (ALL) compared to children without DS. While genome-wide association studies (GWAS) have identified several susceptibility loci in childhood ALL, studies of ALL in children with DS are lacking. Therefore, we conducted the first GWAS of DS-ALL.

Methods: We analyzed independent cohorts of: 1) 226 newly diagnosed DS-ALL cases from Children's Oncology Group (COG) ALL trials (2000-2013) and 436 DS controls from the National Down Syndrome Project (NDSP), 2) 124 additional COG ALL cases (2011-2015) and 336 additional NDSP DS controls, 3) 20 DS-ALL cases and 275 DS controls from Michigan neonatal bloodspots, and 4) 157 DS-ALL cases and 145 DS controls largely from neonatal bloodspots from California and Washington. Genotyping was performed with Affymetrix or Illumina single nucleotide polymorphism (SNP) arrays. STRUCTURE software was used to define European (372 cases, 1,056 controls), Hispanic (140 cases, 136 controls), and African (15 cases, 62 controls) genetic ancestry. After genome-wide imputation and quality control, ancestry- and cohort-specific associations were evaluated at >6,000,000 autosomal SNPs with minor allele frequency ≥1%. Associations were meta-analyzed across cohort and ancestry groups, assuming additive allelic effects.

Results: Genome-wide significant (p<5x10-8) association signals were identified for known ALL susceptibility loci, including rs58923657 near IKZF1 (Odds Ratio [OR]=2.02, p=5.32x10-15), CDKN2A missense mutation rs3731249 (OR=3.63, p=3.91x10-10), rs3781093 near GATA3 (OR=1.73, p=2.89x10-8), and rs7090445 near ARID5B (OR=1.57, p=2.93x10-8). A novel potential risk locus was identified at chromosome 20q11.21 (rs78019519, OR=3.17, p=5.11x10-7) with consistent effects observed across each cohort and ancestry group. This SNP is in the promoter region of the oncogene TPX2 and is also associated with expression of HM13 in whole blood in the Genotype-Tissue Expression (GTEx) Portal.

Conclusion: We confirmed that known ALL susceptibility loci in children without DS, including IKZF1, CDKN2A, GATA3, PIP4K2A and ARID5B, also confer risk of ALL in children with DS, with CDKN2A showing the largest effect size. We also identified a potentially novel locus associated with ALL susceptibility in DS at chromosome 20q11.21. Additional investigation of these loci is ongoing and may advance our understanding of DS-ALL etiology and biology.

#223

A meta-analysis of genome-wide association studies of multiple myeloma among African Americans.

Zhaohui Du,1 Chi Song,1 Kristin Rand,2 Niels Weinhold,3 David Van Den Berg,2 Amie Hwang,2 Xin Sheng,2 Victor Hom,2 Sikander Ailawadhi,4 Ajay K. Nooka,5 Seema Singhal,6 Karen Pawlish,7 Edward S. Peters,8 Cathryn Bock,9 Ann Mohrbacher,2 Alexander Stram,10 Sonja I. Berndt,11 William Blot,12 John David Carpten,2 Antoinette Stroup,13 Andrew Olshan,14 Wei Zhang,15 African Ancestry Breast & Prostate Consortium, Stephen Chanock,16 Jayesh Mehta,6 Graham A. Colditz,17 Jeffrey Wolf,18 Thomas G. Martin,18 Michael Tomasson,19 Mark A. Fiala,17 Howard Terebelo,20 Nalini Janakiraman,21 Laurence Kolonel,22 Loic LeMarchand,22 Elad Ziv,18 Daniel Stram,2 Ravi Vij,17 Leon Bernal-Mizrachi,5 Gareth J. Morgan,23 Jeffrey A. Zonder,24 Carol Ann Huff,25 Sagar Lonial,5 Robert Z. Orlowski,26 David V. Conti,2 Christopher A. Haiman,2 Wendy Cozen2. 1 _USC Norris Comprehensive Cancer Ctr., Los Angeles, CA;_ 2 _University of Southern California/Keck School of Medicine, Los Angeles, CA;_ 3 _Myeloma Institute, AR;_ 4 _Mayo Clinic, MN;_ 5 _Emory University, GA;_ 6 _North Western University Medical School, IL;_ 7 _New Jersey Department of Health, NJ;_ 8 _Louisiana State University School of Public Health, LA;_ 9 _Wayne State University, Karmanos Cancer Center, MI;_ 10 _Genomic Health Inc., CA;_ 11 _National Cancer Institute, NIH, DHHS, Bethesda, MD;_ 12 _International Epidemiology Institute, MD;_ 13 _Rutgers University, NJ;_ 14 _UNC Gillings School of Public Health, NC;_ 15 _Vanderbilt University, TN;_ 16 _National Cancer Institute, NIH, DHHS, MD;_ 17 _Washington University School of Medicine, MO;_ 18 _University of California at San Francisco, CA;_ 19 _University at Iowa, IA;_ 20 _Providence Hospital Southfield, MI;_ 21 _Henry Ford Hospital, MI;_ 22 _University of Hawaii, HI;_ 23 _Myeloma Institute, University of Arkansas, AR;_ 24 _Karmanos Cancer Institute, MI;_ 25 _John Hopkins University, MD;_ 26 _University of Texas MD Anderson Cancer Center, TX_.

Multiple myeloma (MM) is twice as common in African Americans (AA) compared to European Americans (EA). The reported familial clustering and the elevated MM risk among first-degree relatives of cases implicate genetic susceptibility. Previous genome-wide association studies (GWAS) in EA have identified 16 novel risk loci. In this study, we tested the generalizability of the established risk alleles to AA and conducted a meta-GWAS analysis using two sets of AA to identify additional novel common MM risk variants. In the first study, we genotyped 1,305 incident AA MM cases from the African American Multiple Myeloma Study (AAMMS) using the Illumina HumanCore GWAS array and compared them to 7,078 AA controls from the African Ancestry Prostate Cancer Consortium (AAPC) and African Ancestry Breast Cancer Consortium (AABC) using the Illumina 1M-Duo. In the second study, 95 additional AAMMS cases and 435 AA MM cases from the University of Arkansas for Medical Sciences (UAMS) were genotyped using the Illumina MegaBead Chip and compared to 2,390 AA controls from the Multiethnic Cohort. ‎The Haplotype Reference Consortium (HRC) was used to impute the overlapping typed SNPS from each GWAS case and control set together. Per-allele risk associations were tested for 8,715,278 overlapping genotyped and imputed variants with >1% frequency and >0.8 imputation score using unconditional logistic regression in both sets, and the combined effects were estimated using a fixed-effect meta-analysis. Of the 16 reported risk loci discovered in EA, directional consistency was present for 15 variants; eight of these replicated at nominal significance p<0.05, with the most statistically significant variant being rs4487645 at 7p15.3 (OR=1.38, p=3.56×10-6). AA individuals with polygenic risk scores from these 16 variants (PRS) in the top 10% stratum had a 1.44-fold increased MM risk compared to those with a PRS in the 25th -75th percentiles. Additionally, we identified three suggestive novel loci located at 12q12, 9p24.3 and 9p13.1 at p<1×10-6, with ORs ranging from 1.25-1.55, but none reached genome-wide significance. The variant at 9p24.3 is located in an intron in the KANK1 gene and a correlated SNP in EAs (r2=0.5) is strongly associated with gene expression in neoplastic plasma cells (unpublished, Weinhold and Morgan). Our study replicated most of the reported risk loci discovered among EA, demonstrated that a PRS constructed using the 16 reported risk alleles was associated with MM risk, and provides suggestive evidence for additional loci associated with MM risk in AAs.

#224

Evidence for a shared genetic basis between breast cancer and mammographic density phenotypes.

Hongjie Chen,1 Julie Douglas,2 William Gordon,1 Deborah Thompson,3 Fergus Couch,4 Peter Fasching,5 Per Hall,6 John Hopper,7 Gertraud Maskarenic,8 Marina Pollan,9 Jennifer Stone,10 Rulla Tamimi,11 Sara Lindstroem1. 1 _University of Washington, Seattle, WA;_ 2 _University of Michigan, Ann Arbor, MI;_ 3 _University of Cambridge, Cambridge, United Kingdom;_ 4 _Mayo Clinic, Rochester, MN;_ 5 _Friedrich Alexander University of Erlangen-Nuremberg, Comprehensive Cancer Center Erlangen-EMN, Erlangen, Germany;_ 6 _Karolinska Institutet, Stockholm, Sweden;_ 7 _Cancer Council Victoria, Melbourne, Australia;_ 8 _University of Hawaii Cancer Center, Honolulu, HI;_ 9 _Carlos III Institute of Health, Madrid, Spain;_ 10 _University of Western Australia, Perth, Australia;_ 11 _Harvard T.H. Chan School of Public Health, Boston, MA_.

Background: Radiologically dense area (DA) on a mammogram represents the stromal and epithelial tissue in the breast, while non-dense area (NDA) represents adipose tissue. Quantitative measurements of DA, NDA as well as the proportion of dense area in the breast (percent density, PD) adjusted for age and body mass index (BMI) have all been associated with breast cancer (BC) risk. Previous genome-wide association studies (GWAS) have identified genetic loci that show association with both mammographic density (MD) phenotypes and BC, but little is known about the genome-wide and local genetic correlation between MD phenotypes and BC.

Methods: We estimated the genome-wide and local genetic correlation between MD phenotypes adjusted for age and BMI and BC, leveraging GWAS summary statistics for MD phenotypes based on 12,866 women (MODE consortium) and for BC based on 122,977 cases and 105,974 controls (BCAC consortium). We also conducted partitioned heritability analysis to identify specific functional annotations that contribute to MD heritability.

Results: We observed a negative genetic correlation between DA and NDA (-0.62, p=0.002). Both DA and PD showed positive genetic correlations with overall BC (DA: rg=0.24, p=0.004; PD: rg=0.27, p=0.0001), estrogen receptor-positive (ER+) BC (DA: rg=0.23, p=0.005; PD: rg=0.23, p=0.001), and ER- BC (DA: rg=0.26, p=0.01; PD: rg=0.32, p=0.0006). In contrast, NDA showed a nonsignificant negative genetic correlation with BC (rg=-0.11, p=0.21). Local genetic correlation analyses between MD phenotypes and BC reflected known findings including positive correlation at 10q21 region between both PD and DA and BC (both p<5x10-7), negative correlation at 22q13.1 between DA and BC (p=4.6x10-6), and positive correlation at 8p11.23 between NDA and BC (p=2.8x10-6). Two known BC regions that have not previously been associated with MD showed suggestive genetic correlations between BC and DA: 1q21.2 (negative genetic correlation, p=1.3x10-4) and 12q21.31 (positive genetic correlation, p=6.7x10-4). In exploratory analyses estimating the genome-wide genetic correlation between MD and hormone-related phenotypes in UK Biobank (n=337,199), we observed nominally significant positive genetic correlations between PD and benign mammary dysplasia (rg=0.45, p=0.01), NDA and endometriosis (rg=0.45, p=0.02), and negative genetic correlation between NDA and menstrual cycle length (rg=-0.30, p=0.05). We identified multiple functional annotations that contribute to MD heritability including histone mark H3K27ac that marks active enhancers (~2,5-fold enrichment in heritability for both DA and NDA) and DNAsel Hypersensitive Sites (DHS, 9.1-fold enrichment in heritability for DA).

Conclusion: Our results provide evidence of shared genetic basis between mammographic density and breast cancer.

#225

Tumor-infiltrating CD8-positive T-lymphocytes in tubo-ovarian high-grade serous cancer are associated with multiple germline variants in 22q12.1 in a genome-wide association analysis.

Yanina Natanzon,1 Martin Köbel,2 Stacey J. Winham,1 Sebastian M. Armasu,1 Bryan M. McCauley,1 Robert A. Vierkant,1 Julie M. Cunningham,1 David Bowtell,3 Ian G. Campbell,3 Jenny Chang-Claude,4 Anna deFazio,5 Peter A. Fasching,6 Mark T. Goodman,7 Beth Y. Karlan,7 Francesmary Modugno,8 Kirsten B. Moysich,9 Roberta B. Ness,10 Weiva Sieh,11 Paul D. Pharoah,12 Susan J. Ramus,13 Ellen L. Goode1. 1 _Mayo Clinic, Rochester, MN;_ 2 _University of Calgary, Calgary, Alberta, Canada;_ 3 _Peter MacCallum Cancer Centre, Melbourne, Australia;_ 4 _German Cancer Research Center (DKFZ), Heidelberg, Germany;_ 5 _The University of Sydney, Sydney, Australia;_ 6 _Friedrich-Alexander University, Erlangen, Germany;_ 7 _Cedars-Sinai Medical Center, Los Angeles, CA;_ 8 _University of Pittsburgh School of Medicine, Pittsburgh, PA;_ 9 _Roswell Park Cancer Institute, Buffalo, NY;_ 10 _The University of Texas, Houston, TX;_ 11 _Icahn School of Medicine at Mount Sinai, New York, NY;_ 12 _University of Cambridge, Cambridge, United Kingdom;_ 13 _University of New South Wales, Sydney, Australia_.

Tubo-ovarian high-grade serous cancer (HGSC) is the most common histotype of epithelial ovarian cancer (70%-80%) and the leading cause of death from gynecological malignancy. Novel monoclonal antibody and adoptive cell transfer immunotherapies have found success in cancer in the past five years; however success in HGSC treatment has been elusive thus far. We have recently shown that CD8+ T-lymphocytes infiltrating the tumor proper (TILs) associate in a dose response manner with longer patient survival time regardless of age, stage or residual disease. As understanding host factors associated with this clinically relevant tumor phenotype will contribute to the development of targeted therapies, we sought to identify germline genetic factors associated with CD8+ TIL levels in HGSC. We performed a germline genome-wide association study (GWAS) of CD8+ TILs using 1204 HGSC patients of European ancestry from twelve studies with centralized genotyping, immunohistochemistry, and harmonization of clinical data. Germline genotypes were assayed using the Illumina Infinium OncoArray Beadchip and were imputed to over 11.4 million variants based on the 1000 Genomes Project reference panel. CD8 immunohistochemistry was performed on tissue microarrays using the Leica Bond Rx stainer and scored into four levels of CD8+ TILs per x400 magnification of a 0.55-mm diameter field for each tumor hotspot: none (no CD8+ TILs), low (1-2 CD8+ TILs), moderate (3-19 CD8\+ TILs), high (20 or more CD8+ TILs). GWAS used multinomial regression modelling of these four CD8+ TIL levels adjusted for study site, age, and European ancestry principal components. On the average, CD8+ TIL levels were 18%, 19%, 44% and 18% for none, low, moderate and high levels, respectively. As expected, CD8+ TIL levels associated with overall survival time (p < 2.6x10-8), and CD8+ TIL levels and age at diagnosis were similarly distributed across study sites. We identified a region on chromosome 22 with multiple, correlated variants (MAF > 20%) associated with CD8+ TIL levels at p-value < 2.0x10-5. The most statistically significant variant was an indel (rs557925408) located in the an intron of the MYO18B gene (Myosin XVIIIB, p-value < 8.7x10-6). Each copy of this variant associated with a reduced extent of CD8+ TIL infiltration (OR low v none = 0.92; OR moderate v none = 0.61). An independent set of 1200 OncoArray genotyped HGSC cases are undergoing CD8+ TIL scoring in December 2017 and will be included in final genome-wide analysis. This will provide improved statistical power and enable consideration of additional genetic models (e.g., linear trend tests). Comprehensive integrative analysis of germline, tumor, and clinical features provides a model for clinical molecular epidemiology studies and will be key to increasing our understanding of this important HGSC immunophenotype.

#226

Single variant and gene-based replication analysis of reproductive aging in African American women in the AMBER Consortium.

Marie V. Coignet,1 Qianqian Zhu,1 David G. Cox,2 Kathryn Lunetta,3 Elisa V. Bandera,4 Christopher Haiman,5 Andrew Olshan,6 Julie Palmer,3 Christine Ambrosone,1 Song Yao1. 1 _Roswell Park Cancer Inst., Buffalo, NY;_ 2 _INSERM, Lyon, France;_ 3 _Boston University, Boston, MA;_ 4 _Rutgers Cancer Institute of New Jersey, New Brunswick, NJ;_ 5 _University of Southern California, Los Angeles, CA;_ 6 _University of North Carolina, Chapel Hill, NC_.

The two main hormonal events of a woman's life, menarche and menopause, have a paramount impact on the duration of exposure to estrogen. Reproductive aging phenotypes, including age at menarche (AM) and age at natural menopause (ANM) have been consistently associated with breast cancer risk. Despite an estimated strong genetic component, genome-wide association studies (GWAS) for AM and ANM found that common variants identified to date account for only 7.4% for SNPs related to AM and 2.5-4.1% for ANM. As most previous GWAS on AM and ANM were conducted in women of European ancestry (EA), studies examining genetic components of reproductive aging in African-American (AA) women are needed. We hypothesize that although the index GWAS variants discovered in EA women may differ from those in AA women, rare and low-frequency causal variants may reside in the same genetic regions.

A candidate analysis of previously identified GWAS variants and genes in association with AM, ANM was conducted in the African American Breast Cancer Epidemiology and Risk (AMBER) Consortium. All SNPs within a 500kb window of previously discovered GWAS SNPs for AM and ANM were extracted from the Illumina Human Exome Beadchip v1.1, leading to 1,505 candidate SNPs from 125 genes for AM and 1,198 candidate SNPs from 35 genes for ANM in a total of 7,886 AA subjects. Single SNP association tests were run in PLINK using linear regressions for the continuous trend test for AM/ANM and logistic regression for the extreme AM phenotype (<11 v. >=15 years). The SKAT-O test for the gene-based analyses was performed using the SKAT R package to aggregate variants with an MAF upper bound of 5%.

The top variants related to AM were two SNPs, rs314277 (β=0.11, MAF= 0.45, p=6.24E-05) and rs4742314 (β= -0.11, MAF= 0.39, p=6.44E-05), located in LIN28B and KDM4C respectively. rs974828 (RORA, MAF= 0.23, OR=0.71, p=0.0003) and rs314277 (p=0.0007) were found to be the top variants in association with the extreme AM phenotype. For ANM, rs16991615, located in MCM8, was the most significant variant associated with increased ANM ((β=2.06, MAF= 0.01, p=0.0005). rs314277 (LIN28B) has been previously associated with AM, and rs16991615 (MCM8) had also been related to ANM in previous GWAS in EAs. In gene-based analysis for AM, SLC38A3 (p=0.0007) and WDR6 (p= 0.003) were

nominally significant; and EPS8L1 (p= 0.005) and RBM6 (p= 0.01) were associated with the AM phenotype. For ANM, RBMS2 (p= 0.006) was nominally significant in gene-based analysis.

This is to date the largest study in AA women for reproductive life events to interrogate rare and low-frequency variants, which are beyond the spectrum of common variants in previous GWAS. Although the overall replication success rate is low, our analyses identified several rare and low-frequency variants in regions from previous GWAS. Our data contributed to the literature on genetic variation for reproductive aging in AA women.

#227

Germline variation at 8q24 and prostate cancer risk in men of European ancestry.

Marco Matejcic,1 Edward J. Saunders,2 Tokhir Dadaev,2 Mark Brook,2 Ali Amin Al Olama,3 Fredrick R. Schumacher,4 Sonja I. Berndt,5 Sara Benlloch,2 Kenneth Muir,6 Koveela Govindasami,2 Victoria L. Stevens,7 Susan M. Gapstur,7 Catherine M. Tangen,8 Jyotsna Batra,9 Judith Clements,9 APCB (Australian Prostate Cancer Bio Resource), Henrik Gronberg,10 Nora Pashayan,3 Johanna Schleutker,11 Demetrius Albanes,5 Alicja Wolk,12 Catharine West,6 Lorelei Mucci,13 Peter Kraft,14 Géraldine Cancel-Tassin,15 Stella Koutros,5 Karina Dalsgaard Sorensen,16 Lovise Maehle,17 Eli Marie Grindedal,17 Sara Strom,18 David E. Neal,3 Freddie C. Hamdy,19 Jenny L. Donovan,20 Ruth C. Travis,19 Robert J. Hamilton,21 Sue Ann Ingles,22 Barry Rosenstein,23 Yong-Jie Lu,24 Graham G. Giles,25 Adam S. Kibel,26 Ana Vega,27 Jeanette Bensen,28 Manolis Kogevinas,29 Fredrik Wiklund,30 Stephen Chanock,5 Douglas F. Easton,3 Rosalind A. Eeles,2 Zsofia Kote-Jarai,2 David V. Conti,1 Christopher A. Haiman1. 1 _USC Norris Comprehensive Cancer Center, Los Angeles, CA;_ 2 _The Institute of Cancer Research, London, United Kingdom;_ 3 _University of Cambridge, Cambridge, United Kingdom;_ 4 _Case Western Reserve University, Cleveland, OH;_ 5 _National Cancer Institute, Bethesda, MD;_ 6 _University of Manchester, Manchester, United Kingdom;_ 7 _American Cancer Society, Atlanta, GA;_ 8 _Fred Hutchinson Cancer Research Center, Seattle, WA;_ 9 _Queensland University of Technology, Brisbane, Australia;_ 10 _Department of Medical Epidemiology and Biostatistics, Stockholm, Sweden;_ 11 _University of Turku, Turku, Finland;_ 12 _Karolinska Institutet, Stockholm, Sweden;_ 13 _Harvard School of Public Health, Boston, MA;_ 14 _Harvard T.H. Chan School of Public Health, Boston, MA;_ 15 _Tenon Hospital, Paris, France;_ 16 _Aarhus University Hospital, Aarhus, Denmark;_ 17 _Oslo University Hospital, Oslo, Norway;_ 18 _University of Texas, Austin, TX;_ 19 _University of Oxford, Oxford, United Kingdom;_ 20 _University of Bristol, Bristol, United Kingdom;_ 21 _Princess Margaret Cancer Centre, Toronto, Ontario, Canada;_ 22 _University of Southern California, Los Angeles, CA;_ 23 _Icahn School of Medicine at Mount Sinai, New York, NY;_ 24 _Queen Mary University of London, London, United Kingdom;_ 25 _Cancer Council Victoria, Melbourne, Australia;_ 26 _Brigham and Womens Hospital, Boston, MA;_ 27 _CIBERER, IDIS, Santiago de Compostela, Spain;_ 28 _University of North Carolina, Chapel Hill, NC;_ 29 _Barcelona Institute for Global Health, Barcelona, Spain;_ 30 _Karolinska Institute, Stockholm, Sweden_.

We performed an in-depth and well-powered investigation of genetic variation across the cancer susceptibility region at chromosome 8q24 (127.6-129.0 Mb) to search for novel risk variants associated with prostate cancer (PCa) risk in the European ancestry population. We combined genotyped and imputed data from the PRACTICAL/ELLIPSE OncoArray and iCOGS consortia consisting of 71,535 PrCa cases and 52,935 controls of European ancestry. Variants with high imputation quality score (>0.8) were retained for a total of 5,600 overlapping variants between the two datasets. Associations of genetic variants with PCa risk were evaluated using unconditional logistic regression with adjustment for country and ten principal components. The marginal risk estimates for the 5,600 variants that passed quality control were combined by a fixed effects meta-analysis. A meta-stepwise selection was performed on variants marginally associated with PCa risk from the meta results (P<0.05). A polygenic risk score and the contribution to the familial relative risk of PCa were estimated for variants from the final model. Of the 5,600 variants at 8q24 retained for analysis, 1,268 (23%) were associated with PCa risk at P<5x10-8 while 2,772 (49%) were marginally associated at P<0.05. In the stepwise model, 12 variants remained statistically significantly associated with PCa risk with conditional meta p-values between 2.93x10-137 and 4.28x10-15. The independent stepwise signals were confirmed by Joint Analysis of Marginal (JAM) summary statistics, which defined the credible sets of variants driving those signals. Three of the variants (rs1914295, rs190257175, rs12549761) were weakly correlated (r2≤0.17) with any known PCa risk marker, and may define novel association signals. Men in the top 1% of the polygenic risk score distribution had a 3.97-fold relative risk (95%CI=3.87-4.07) compared to men with "average risk" (25th-75th percentiles). The 12 independent signals at 8q24 capture 11.54% (95%CI=9.86-13.65) of the familial relative risk of PCa, which is approximately one quarter of the total PCa familial relative risk explained by known genetic risk factors. Most of the independently associated signals have good evidence for biologic functionality; in particular, many reside within putative transcriptional enhancers and/or binding sites for AR and FOXA1 transcription factors in prostate cell lines. In summary, we defined 12 independent association signals among men of European ancestry, with three of the risk variants representing putative novel association signals. Whereas the individual associations of these variants with PCa risk are relatively modest (ORs<2.0), their cumulative effects are substantial, and their contribution to the overall familial relative risk of PCa is substantially greater than any other known prostate cancer risk locus.

#228

Association analysis across different populations identifies 26 new cutaneous melanoma risk loci.

Cristina Pellegrini,1 Matthew Law,2 Mark Iles,3 Mitch Machiela,4 Alex Stratigos,5 Tongwu Zhang,4 Kevin Brown,4 Julia Newton-Bishop,3 Nick Hayward,2 Nick Martin,2 Alisa Goldstein,4 Rose Yang,4 Stephen Chanock,4 Florence Demenais,6 Susana Puig,7 Eduardo Nagore,8 Jianxin Shi,4 Tim Bishop,3 Stuart McGregor,9 Maria Teresa Landi,4 GenoMEL Consortium,3 MelaNostrum Consortium4. 1 _University of L'Aquila, L'Aquila, Italy;_ 2 _QIMR Institute, Australia;_ 3 _University of Leeds, United Kingdom;_ 4 _NCI, NIH, MD;_ 5 _Athens Medical School, Greece;_ 6 _INSERM, France;_ 7 _University of Barcelona, Spain;_ 8 _Istituto Valenciano de Oncologia, Spain;_ 9 _QIMR, Australia_.

Heritability of cutaneous melanoma is among the highest across cancer types and is mostly determined by common, noncoding genetic variants. Previous genome-wide association studies (GWAS), mostly including subjects from Australia and Northern European countries, had identified twenty genetic loci associated with melanoma risk. To characterize the genetic landscape of cutaneous melanoma across different populations, we conducted a new association study in Southern European countries including 6,043 melanoma cases and 10,383 controls from the MelaNostrum Consortium. Moreover, we largely expanded the analyses of melanoma cases and controls from other populations, for a total of 27,450 histologically confirmed melanoma cases and 49,888 controls from Northern Europe, Southern Europe, USA and Australia. Imputation was done using the Haplotype Reference Consortium as a reference panel. We assessed the association between cutaneous melanoma risk and single-nucleotide polymorphisms (SNPs) adjusting for country and ancestry-informative principal components. We identified 26 new genetic loci associated with melanoma risk achieving genome-wide significance. The pigmentation pathway, with TYRP1 and MITF, and the telomere-related pathway with RTEL1, TERC, and POT1 among the candidate genes, continued to show an important role for melanoma susceptibility. Additional candidate pathways emerged, including signaling (with GPRC5A and DOCK8), cell-cell junction (with CDH1), immune-related functions (with DCST2, involved in antigen processing), transcriptional regulation (with FOXD3, which represses MITF expression), or tumor-suppressor genes (TP53). Adding genotype data from 6,130 self-reported melanoma cases in a sensitivity analysis identified eight additional novel genetic loci involved in the HLA region, signaling transduction and transcription factor functions. A combined analysis of the melanoma GWAS with the GWAS of pigmentation characteristics from the UK Biobank including 500,000 UK persons, and with a GWAS of nevus density including over 52,000 persons, revealed substantial overlap across phenotypes. Although the effect size of the SNPs on melanoma risk was consistent across populations, substantial differences in minor allele frequency (MAF) were observed in pigmentation-related genes between Northern and Southern European populations; for example, MC1R, SLC45A2, and ASIP SNP MAF was 8.2, 3.1 and 14.2 in Northern and 2.6, 8.8, and 5.5 in Southern European subjects, respectively. Additional analyses based on melanoma histologic subtypes and body site distribution are ongoing. These results provide further insight into genetic susceptibility to cutaneous melanoma and provide the opportunity for genetic risk scores in risk-prediction models for early detection and targeted prevention of the disease.

#229

Genome-wide association study by colorectal carcinoma subtype.

Tabitha A. Harrison,1 Yiwen Lu,1 Chenjie Zeng,1 Flora Qu,1 Kristin Anderson,2 Hermann Brenner,3 Daniel D. Buchanan,4 Peter T. Campbell,5 Andrew T. Chan,6 Jenny Chang-Claude,3 Graham G. Giles,7 Bethany Van Guelpen,8 Michael Hoffmeister,3 Mark A. Jenkins,4 Noralane M. Lindor,9 Roger L. Milne,7 Polly A. Newcomb,1 Reiko Nishihara,10 Michael O. Woods,11 Shuji Ogino,12 John D. Potter,1 Martha L. Slattery,13 Wei Sun,1 Stephen N. Thibodeau,9 Li Hsu,1 Ulrike Peters1. 1 _Fred Hutchinson Cancer Research Center, Seattle, WA;_ 2 _University of Minnesota School of Public Health, Minneapolis, MN;_ 3 _German Cancer Research Center, Heidelberg, Germany;_ 4 _University of Melbourne, Melbourne, Australia;_ 5 _American Cancer Society, Atlanta, GA;_ 6 _Massachusetts General Hospital, Boston, MA;_ 7 _Cancer Council Victoria, Melbourne, Australia;_ 8 _Umea University, Umea, Sweden;_ 9 _Mayo Clinic, Rochester, MN;_ 10 _Dana-Farber Cancer Institute, Boston, MA;_ 11 _Memorial University of Newfoundland, St. John's, Newfoundland and Labrador, Canada;_ 12 _Harvard Medical School, Boston, MA;_ 13 _University of Utah Health Sciences Center, Salt Lake City, UT_.

Over 50 genetic variants have been associated with colorectal cancer (CRC) risk through genome-wide association studies (GWAS), yet these variants represent only a fraction of the total estimated heritability. CRC is a heterogenous disease with diverse tumor etiology. Assessing genetic risk in molecular subtypes may help to identify novel loci and characterize genetic risk among tumor subtypes. We used microsatellite instability (MSI), an established CRC classifier with etiological and therapeutic relevance, to define CRC subtypes for GWAS analyses. We conducted a case-case analysis to estimate odds ratios (OR) and 95% confidence intervals (CI) for association of genome-wide variants with microsatellite stable (MSS) versus unstable (MSI) carcinomas. We ran an inverse-variance weighted fixed-effects meta-analysis across GWAS in a discovery set of 4,163 population-based CRC cases with harmonized microsatellite instability (MSI) marker and imputed genotype data. For each analysis, we used log-additive logistic regression, adjusting for age, sex, and principal components to account for population substructure. We then followed up with replication of 102 SNPs that reached p-values less than 5x10-6 in 1,698 cases. A total of 845 (20.3%) cancer cases were microsatellite unstable in the discovery population and 174 (10.2%) were unstable in the replication population. No variants reached the genome-wide significance level of 5x10-8 in the discovery set. However, we identified two variants that reached a Bonferroni corrected p-value of 4.0x10-4 in the replication set. This included one variant in MLH1 (Replication: OR=1.74, 95% CI=1.53-1.98, p=1.63x10-5; Discovery+Replication: OR=1.45, 95% CI=1.37-1.54, p=9.76x10-11) and one variant in LOC105377645 (Replication: OR=1.70, 95% CI=1.49-1.94, p=5.13x10-5; Discovery+Replication: OR=1.45, 95% CI=1.37-1.54, p=9.76 x 10-11). The MLH1 gene is a DNA mismatch repair gene implicated in Lynch Syndrome, the hallmark of which is microsatellite instability. This is the first genome-wide scan to identify a common variant in MLH1 that is associated with CRC. This variant (minor allele frequency, MAF = 23% in this all European ancestry population) is located in the 5'-untranslated region of MLH1 and is thought to act as a long-range regulator of DCLK3, a potential tumor driver gene. The second variant, located in LOC105377645 with an MAF of 22%, is in an uncharacterized region of the genome and has not previously been implicated in cancer development. These findings suggest that accounting for molecular heterogeneity is important for discovery and characterization of genetic variants associated with CRC risk. We plan to run polytomous regression analyses, increase our sample size, and further investigate CRC subtypes by CIMP, BRAF mutation, KRAS mutation status.

#230

Joint genome-wide association study of endometrial cancer and ovarian cancer identifies a novel genetic risk region at 14q23.3.

Tracy A. O'Mara,1 Dylan M. Glubb,1 Daniel D. Buchanan,2 Diether Lambrechts,3 Per Hall,4 Emma Tham,4 Jone Trovik,5 Ellen L. Goode,6 Peter Fasching,7 Thilo Dörk,8 Rodney J. Scott,9 Paul L. Auer,10 Roger L. Milne,11 Graham G. Giles,11 John Perry,12 Immaculata de Vivo,13 Ian Tomlinson,14 Douglas F. Easton,12 Deborah J. Thompson,12 Amanda B. Spurdle,1 E2C2, BCAC, OCAC, ECAC. 1 _QIMR Berghofer Medical Research Institute, Herston, Australia;_ 2 _The University of Melbourne, Parkville, Australia;_ 3 _University of Leuven, Leuven, Belgium;_ 4 _Karolinska Intitutet, Stockholm, Sweden;_ 5 _University of Bergen, Bergen, Norway;_ 6 _Mayo Clinic, Rochester, MN;_ 7 _University Hospital Erlangen, Erlangen, Germany;_ 8 _Hannover Medical School, Hannover, Germany;_ 9 _University of Newcastle, Newcastle, Australia;_ 10 _University of Wisconsin-Milwaukee, Milwaukee, WI;_ 11 _Cancer Council Victoria, Melbourne, Australia;_ 12 _University of Cambridge, Cambridge, United Kingdom;_ 13 _Harvard Medical School, Boston, MA;_ 14 _University of Birmingham, Birmingham, United Kingdom_.

Ovarian cancer is the most lethal gynecologic malignancy and the sixth most common cause of death from all cancer in women, with an estimated 140,000 deaths per year worldwide. Endometrial cancer (cancer of the uterine lining) is the most commonly diagnosed gynecologic cancer, ranking sixth in incident cancer in women. Ovarian and endometrial cancer share many epidemiologic, histopathologic, and tumor genetic characteristics. Meta-analyses of genome-wide association study (GWAS) datasets across etiologically related diseases have successfully been used to increase statistical power and identify novel genetic risk regions. We hypothesised that joint meta-analysis of ovarian and endometrial cancer GWAS datasets would identify novel genetic loci for both cancers. Following quality control, summary statistics for >11 million genetic variants were available from the largest genome-wide association studies performed by the Ovarian Cancer Association Consortium (OCAC, Phelan et al., Nature Genetics 2017) and the Endometrial Cancer Association Consortium (ECAC, unpublished). A total of 35,312 cancer cases from all histologic subtypes (22,406 ovarian and 12,906 endometrial cancer cases) and 149,920 controls were included in the analysis. Summary statistics were combined using an inverse-variance, fixed-effects model in METAL and identified ten loci at genome-wide significance (P < 5 x 10-8), of which one locus at 14q23.3 (rs4072776 combined OR 0.94; 95% CI 0.92-0.96; P = 8.0 x 10-9) had not been previously associated with the risk of either cancer. Integration with chromatin conformation data (RNAPolII HiChIP) from the Ishikawa endometrial cancer cell line suggests interaction between the risk signal and the promoter of FUT8. FUT8 encodes a fucosyltransferase and its expression has been implicated in breast, stomach, and melanoma and lung cancer. Interestingly, FUT8 is a downstream target regulated by the loss of PAX2 and mutated p53, which is the earliest known molecular aberration in the progression of the fallopian tissue epithelial to serous ovarian cancer. Further functional evaluation of the 14q23.3 risk region is required to determine the regulatory effect of genetic risk variants on FUT8. Future meta-analyses of datasets, stratifying results by histologic subtypes, will be undertaken.

#231

Identification of susceptible loci and enriched pathways for post-radiotherapy pain in breast cancer patients.

Eunkyung Lee,1 Christina Takita,2 Jean L. Wright,3 Eden Martin,2 Susan Slifer,2 Jennifer Hu2. 1 _Univ. of Central Florida, Orlando, FL;_ 2 _Univ. of Miami, Miami, FL;_ 3 _Johns Hopkins University, MA_.

Pain is one of the most prevalent symptoms among breast cancer survivors, which negatively impact on their quality of life (QOL). Adjuvant radiotherapy (RT) can increase the risk of development or persistence of pain; however, little is known about the mechanisms of RT-related pain or genetic susceptibility. The current study aimed to identify susceptible loci and enriched pathways for clinically relevant pain, which is defined as pain score ≥ 4, at the completion of RT (post-RT pain). We conducted a genome-wide association study (GWAS) of 1,112 breast cancer patients with 1,344,832 tagging SNPs, gene-based analysis using PLINK set-based tests for 19,621 genes, and functional enrichment analysis of a gene list (875 genes having p < 0.05) using NIH DAVID functional annotation module with KEGG pathways and GO terms (n=380). About 29% of patients reported post-RT pain, and 4 SNPs showed suggestive associations; rs16970540 in RFFL or near to LIG3 gene (p=1.7x10-6), rs4584690 and rs7335912 in ABCC4/MPR4 gene (p=5.5x10-6 and p=7.8x10-6, respectively), and rs73633565 in EGFL6 gene (p=8.1x10-6). Gene-based analysis suggested the importance (involvement) of neurotransmitter, olfactory receptor, and cytochrome P450 in post-RT pain, and functional analysis suggested glucuronidation and olfactory receptor activity as the most enriched biological processes/features. With a limited size of sample, this is the first GWA study showing (confirming) that post-RT pain is a complex polygenic trait influenced by many biological processes and functions. If validated in a larger population, the results can provide biological targets for pain management to improve cancer survivor's QOL. Additionally, these findings may be used as predictive biomarkers of personalized pain management.

#232

Genome-wide association study (GWAS) of total nicotine equivalents: A composite biomarker of nicotine uptake.

Linda M. Polfus,1 S. Lani Park,1 Yesha M. Patel,1 Sharon E. Murphy,2 David Conti,1 Christopher Haiman,1 Daniel O. Stram,1 Loïc Le Marchand3. 1 _University of California, Los Angeles, CA;_ 2 _University of Minnesota, Minneapolis, MN;_ 3 _University of California, Honolulu, HI_.

The primary risk factor for lung cancer (second most common cancer in men and women in the U.S.) is tobacco smoking. Prior GWAS have identified variants in the CHRNA5-CHRNA3-CHRNB4 cluster (chr 15q25) to be associated with smoking. Total nicotine equivalents (TNE) measured from urine represents the sum of nicotine, cotinine, and trans-3′-hydroxycotinine and its glucuronides, as well as nicotine N-oxide, and is a biomarker of nicotine uptake and, when adjusted for cigarettes smoked per day (CPD), a biomarker of smoking intensity. In addition to smoking status, genetic variation is likely to influence smoking behavior (e.g., number of cigarettes, number of puffs, depth of inhalation) and, thus, one's internal exposure to nicotine and tobacco carcinogens. We conducted a genome-wide association study of TNE (i.e. smoking dose) among 2,239 cancer-free, current smokers from the Multiethnic Cohort, comprised of 5 ethnic groups. TNE was measured using liquid chromatography-tandem mass spectrometry analysis in overnight or first morning urine samples. Genotyping was conducted utilizing the Illumina 1M array and the data were imputed to the 1000 Genomes reference panel, for a total of 11.9 million SNPs and indels. Linear regression models were conducted in all subjects combined and each ethnic group using the log transformed TNE measurement, adjusted for age, BMI, sex, and the first ten principle components (Model 1). A separate model additionally adjusted for log transformed cigarettes per day (Model 2). Genome-wide significant associations with TNE for Model 1 included the chr 15q25 region, specifically, intronic common SNPs in CHRNB4 (rs17487223) and CHRNA3 (rs55676755) with P=7.37x10-9 (Beta (SE) = 0.21 (0.04)) and P=2.22x10-8 (Beta (SE) = 0.19 (0.03)), respectively. Ethnic specific analyses pointed to European American, African American, and Hispanic individuals mainly contributing to the signal. Nicotinic receptors, as ligand-gated channels consist of five subunits and are the primary targets for nicotine and initiate the brain responses to smoking. Our findings with nicotine uptake confirm previous reports that these biologically plausible genes influence smoking behavior. For Model 2 in all pooled ethnicities combined, three intronic SNPs were near genome-wide significance (with lowest P=7.13x10-8 for rs55934265) in the beta-1,3-galactosyltransferase (B3GALT1) gene, Beta (SE) = -0.57 (0.10). The B3GALT1 signal was primarily driven by the African American data (N=364) with ethnic specific results at P=5.3x10-4, Beta (SE) = -0.47 (0.13). B3GALT1 is expressed exclusively in the brain and is involved in the biosynthesis of glycolipids. Next steps include constructing a genetic risk score (GRS) of TNE, and evaluating the GRS in association with lung cancer risk independent of self-reported levels of smoking.

#233

Evaluating glioma risk associated with extent of European admixture in African-Americans and Latinos.

Quinn T. Ostrom,1 Kathleen M. Egan,2 L. Burt Nabors,3 Travis Gerke,2 Reid C. Thompson,4 Jeffrey J. Olson,5 Renato LaRocca,6 Sajeel Chowdhary,7 Jeanette E. Eckel-Passow,8 Georgina Armstrong,9 John K. Wiencke,10 Christopher I. Amos,9 Jonine L. Bernstein,11 Elizabeth B. Claus,12 Dora Il'yasova,13 Christoffer Johansen,14 Daniel H. Lachance,15 Rose Lai,16 Ryan T. Merrell,17 Sara H. Olson,11 Siegal Sadetzki,18 Joellen Schildkraut,19 Sanjay Shete,20 Richard S. Houlston,21 Robert B. Jenkins,15 Beatrice Melin,22 Melissa L. Bondy,9 Jill S. Barnholtz-Sloan1. 1 _Case Western Reserve Univ. School of Med., Cleveland, OH;_ 2 _H. Lee Moffitt Cancer Center & Research Institute, Tampa, FL; _3 _University of Alabama at Birmingham, Birmingham, AL;_ 4 _Vanderbilt University Medical Center, Nashville, TN;_ 5 _Emory University School of Medicine, Atlanta, GA;_ 6 _Norton Cancer Institute, Louisville, KY;_ 7 _Lynn Cancer Institute, Boca Raton, FL;_ 8 _Mayo Clinic College of Medicine, Rochester, MN;_ 9 _Dan L. Duncan Comprehensive Cancer Center, Baylor College of Medicine, Houston, TX;_ 10 _University of California, San Francisco, San Francisco, CA;_ 11 _Memorial Sloan Kettering Cancer Center, New York, NY;_ 12 _Yale University, New Haven, CT;_ 13 _Georgia State University School of Public Health, Atlanta, GA;_ 14 _Danish Cancer Society, Copenhagen, Denmark;_ 15 _Mayo Clinic Comprehensive Cancer Center, Mayo Clinic,, Rochester, MN;_ 16 _University of Southern California Keck School of Medicine, Los Angeles, CA;_ 17 _NorthShore University HealthSystem, Evanston, ID;_ 18 _Tel-Aviv University, Sackler Faculty of Medicine, Tel-Aviv, Israel;_ 19 _University of Virginia School of Medicine, Charlottesville, VA;_ 20 _University of Texas MD Anderson Cancer Center, Houston, TX;_ 21 _The Institute of Cancer Research, Sutton, Surrey, United Kingdom;_ 22 _Umeå University Faculty of Medicine, Umeå, Sweden_.

Glioma incidence is highest in non-Hispanic Whites, where it occurs ~2x as frequently compared with other race/ethnicity groups. Glioma GWAS to date have included European ancestry populations only, and it is unknown whether variants identified by these analyses are associated with glioma in non- European ancestry populations. African Americans and Hispanics are admixed populations with varying proportions of European ancestry. While global ancestry may be similar within admixed groups, the proportion of European ancestry at each allele can vary across the genome. As glioma is more common in European ancestry populations, the presence of increased local European ancestry in these admixed populations could be used to identify glioma risk loci. Here we assessed whether excess European ancestry at established risk loci (Melin et al, Nature Genetics, 2017) was associated with glioma risk in non-European ancestry populations. Global ancestry was estimated using fastStructure, and local ancestry was estimated using RFMix. Both methods used 1,000 genomes project reference populations (African: YRI; European: CEU; East Asian: CHB/JPT; and Native American: CLM/PEL/MXL). We evaluated differences in local European ancestry between cases and controls using logistic regression conditioned on global European ancestry within 500kb of 25 previously identified risk variants among individuals with ≥50% African ancestry, and ≥30% Native American ancestry for all gliomas, and for grade IV glioblastoma (GBM) and grade II-III non-GBM. There were 347 individuals (184 cases and 163 controls) with ≥50% global African ancestry, and 277 individuals (153 cases and 124 controls) with ≥30% global American ancestry. There was no significant difference in proportion of global European ancestry between cases and controls with ≥50% global African ancestry (cases: 18.2%, controls: 17.7%, p=0.6834), and no significant difference in proportion of global European ancestry between cases and controls with ≥30% global American ancestry (cases: 51.1%, controls: 49.0%, p=0.2123). Among individuals with >50% African ancestry, we observed a nominally significant association between all glioma and increased local European ancestry at 7p11.2 (EGFR, pmin=0.0070) and between GBM and increased local European ancestry at 22q13.1 (CSNK1E, pmin=0.0098), both near SNPs previously associated with glioblastoma in majority European-ancestry populations. The dataset used for this analysis represents the largest collection of genotyped non-European glioma cases. These results suggest that glioma risk in African Americans may be associated with an increased local European ancestry variants at glioma risk loci previously identified in majority European ancestry populations (7p11.2 and 22q13.1).

#234

Understanding melanoma susceptibility through GWAS of risk phenotypes.

David T. Bishop,1 Mark M. Iles,1 Julia A. Newton-Bishop,1 Jennifer H. Barrett,1 Adam Trouwer,1 Matthew Law,2 Stuart MacGregor,2 David Duffy,2 Nicholas G. Martin,2 Nicholas Hayward,2 Anne Cust,3 Maria T. Landi,4 Alisa Goldstein,4 Jianxin Shi,4 Mitch Machiela,4 Florence Demenais,5 Peter Kanetsky,6 GenoMEL & MELANOSTRUM. 1 _University of Leeds, Leeds, United Kingdom;_ 2 _QIMR Berghofer, Brisbane, Australia;_ 3 _University of Sydney, Sydney, Australia;_ 4 _National Cancer Institute, Washington, DC;_ 5 _INSERM, Paris, France;_ 6 _Moffitt Cancer Center, Tampa, FL_.

Melanoma susceptibility SNPs can be categorized by their impact on known susceptibility phenotypes (pigmentation, nevus count, telomere length, or other). In an ongoing meta-analysis of melanoma, at this stage involving 17,800 cases but soon expanding to more than 30,000 cases, 26 SNPs have achieved genome-wide significance. UK Biobank is a population-based cohort of 500,000 UK persons aged at recruitment 40-69 years; participants reported pigmentation characteristics including ease of tanning, natural hair color, skin color (in the absence of tan), and number of childhood sunburns. To conduct analyses, we separately analyzed the red hair phenotype while ordering the other hair colors from black to blond. Among the 26 SNPs from the melanoma GWAS, 15 were associated with at least one pigmentation factor assessed in UK Biobank (each at p <10-5) with 5 being associated with all pigmentation factors (e.g., SLC45A2, ASIP, MC1R) while others were associated with a subset of factors (e.g., RAD23B/TAL2 was strongly associated with hair color alone). 13 of the melanoma SNPs were associated with "ease of tanning." Detailed analysis of "ease of tanning" SNPs found 29 genome-wide significant in a subset of UK Biobank, of which 13 were associated with melanoma risk (p <0.001) but notably 4 of the SNPs showed no evidence of melanoma risk even when being among the strongest effects for "ease of tanning" and the remainder showing weaker evidence of association. For all pigmentation factors (except having red hair), the effect sizes for a SNP on melanoma risk and pigmentation were highly correlated. We examined the 26 melanoma SNPs in recent GWAS of nevus count and telomere length, and similarly observed overlaps with these phenotypes. While the telomere, nevus and pigmentation instruments were largely distinct, we found evidence of some overlap in genetic susceptibility (for instance, the PLA2G6 SNP rs2092180 is strongly associated with nevus count, ease of tanning and skin color [all p <10-12]). Combined analysis of the melanoma GWAS results with the GWAS of each of these phenotypes provides overwhelming evidence of further susceptibility loci, indicating that GWAS of intermediate phenotypes can benefit identification of the underlying genetic profile.

Supported by Cancer Research UK (C588/A19167) & NIH (CA083115). This research has been conducted using the UK Biobank Resource.

#235

Type 2 diabetes and glycemic traits in relation to colorectal cancer risk: A Mendelian randomization study.

Mingyang Song,1 Yiwen Lu,2 Marc Gunter,3 Neil Murphy,3 Barbara L. Banbury,2 Wenjie Ma,1 Jennifer Prescott,4 Graham Casey,5 Stephen B. Gruber,6 Edward L. Giovannucci,7 Ulrike Peters,2 Andrew T. Chan1. 1 _Massachusetts General Hospital and Harvard Medical School, Boston, MA;_ 2 _Fred Hutchinson Cancer Research Center, Seattle, WA;_ 3 _International Agency for Research on Cancer (IARC), Lyon, France;_ 4 _Booz Allen Hamilton, Boston, MA;_ 5 _University of Virginia School of Medicine, Charlottesville, VA;_ 6 _University of Southern California Norris Comprehensive Cancer Center, Los Angeles, CA;_ 7 _Harvard T.H. Chan School of Public Health, Boston, MA_.

Background: Although type 2 diabetes (T2D) has been linked to an increased risk of colorectal cancer (CRC), a causal relationship remains to be established. Also, the influence of hyperglycemia and impaired beta-cell function on CRC remains to be determined.

Methods: We performed a Mendelian randomization (MR) study using common single nucleotide polymorphisms (SNPs) identified from prior genome-wide association studies for T2D (78 SNPs), fasting glucose (36 SNPs), glycosylated hemoglobin (HbA1c, 11 SNPs), and homeostatic model assessment for beta-cell function (HOMA-B, 13 SNPs) (all SNPs reached genome-wide significance at 5 x10-8). We first assessed the association of each SNP with CRC risk using logistic regression in 26,357 CRC cases and 20,505 control participants in the Colon Cancer Family Registry, Colorectal Transdisciplinary Study and the Genetics and Epidemiology of Colorectal Cancer Consortium. The MR estimates for the association of each risk factor with CRC were calculated using the inverse-variance weighted method that combines summary data on the association of the SNPs with each trait derived from previous genome-wide association studies. Given the potential sex difference in the relationship of metabolic factors with CRC, all analyses were performed in men and women separately.

Results: We did not find any statistically significant association between each of the genetically predicted risk factors and CRC incidence for sex-stratified or combined analyses. The odds ratios (ORs) of CRC associated with genetic risk of T2D were 1.02 (95% confidence interval [CI], 0.95-1.08, P=0.62) in women and 0.96 (95% CI, 0.90-1.03, P=0.25) in men. For fasting glucose, the ORs of CRC associated with one mmol/L increment was 0.91 (95% CI, 0.68-1.20, P=0.49) in women and 1.20 (95% CI, 0.90-1.59, P=0.22) in men, respectively. For HbA1c, the ORs of CRC associated with one percent increment was 0.83 (95% CI, 0.57-1.22, P=0.35) in women and 0.99 (95% CI, 0.67-1.45, P=0.95) in men. For HOMA-B, the ORs of CRC associated with a unit increase in log-transformed measurement was 0.92 (95% CI, 0.53-1.60, P=0.76) in women and 0.77 (95% CI, 0.44-1.35, P=0.36) in men.

Conclusion: Our findings do not support a causal association of T2D, impaired beta-cell function, and hyperglycemia with CRC risk. However, the null findings may be due to weak instrument bias since these common SNPs account for only a limited proportion of the inter-individual variation in these glycemic traits.

#236

Identification of novel cancer target genes by combining data from the cancer genome-wide association studies (GWAS), regulatory DNA elements and The Cancer Genome Atlas (TCGA).

Diptee A. Kulkarni, Karl Guo, Junping Jing, Mugdha Khaladkar, Kijoung Song, Coco Dong, David Cooper, Benjamin Schwartz. _GlaxoSmithKline, Collegeville, PA_.

Unlike tumors that develop because of somatic genetic alterations such as frequent coding mutations or chromosomal deletions, amplifications or translocations, some tumors are addicted to expression of certain genes for their sustained proliferation and survival - for example, LIM domain only 1 (LMO1) expression in neuroblastoma (NB). Numerous genome-wide association studies (GWAS) have identified significant associations between germline single-nucleotide polymorphisms (SNPs), many in the non-coding genome, and cancer. A robust genome-wide association has been reported previously between an intronic LMO1 SNP and NB susceptibility. By further sequencing and epigenetic fine mapping of the SNP locus, it was demonstrated that the causal SNP is part of a transcription factor binding site within an enhancer element, regulates expression of LMO1 and in turn increases the tumorigenic potential. To identify additional examples of regulatory SNPs as cancer drivers, we overlaid published genome-wide significant cancer associations with active chromatin marks from Encyclopedia of DNA Elements and searched for SNPs that resided within gene regulatory elements. To map these SNPs to candidate genes and determine direction of effect, we co-localized GWAS signals with expression quantitative trait (eQTL) signals from the Genotype-Tissue Expression (GTEx) Consortium database. Lastly, we checked for gene amplification and/or overexpression of the mapped genes in the Cancer Genome Atlas (TCGA) data to identify a set of target genes that not only exhibit significant cancer association in GWAS, but also have evidence for epigenetic regulation and propensity for amplification and/or overexpression in tumors. We identified more than 25 novel cancer-target pairs with strong germline, regulatory and somatic evidence. A look up through synthetic lethality screen data available in-house suggested that several of these targets are self-lethal, further underscoring their importance for cancer cell proliferation and survival. Additional in vitro experiments are being planned to further validate the targets.

#237

The genetic architecture of African esophageal cancer.

Christopher G. Mathew,1 Wenlong C. Chen,1 Hannah Bye,2 Natalie J. Prescott,2 Marco Matejcic,3 Robyn Kerr,1 Elvira Singh,1 Cathryn M. Lewis,2 Chantal Babb de Villiers,4 Mohamed I. Parker3. 1 _University of the Witwatersrand, Johannesburg, South Africa;_ 2 _King's College London, London, United Kingdom;_ 3 _University of Cape Town, Cape Town, South Africa;_ 4 _University of Cambridge, Cambridge, United Kingdom_.

Esophageal squamous cell carcinoma (ESCC) is common in many Black populations of sub-Saharan Africa, with high incidence regions in Eastern and Southern Africa. Clinical presentation in Africa is late, and treatment is mainly palliative with a very poor prognosis. Various environmental risk factors have been identified, but the possible contribution of inherited genetic variants to disease risk is unresolved. Genetic association studies of African ESCC have been limited to the analysis of small numbers of single nucleotide polymorphisms (SNPs) in candidate genes, and carried out only in the Black and Mixed Ancestry populations of the Western Cape of South Africa. Thus far no genetic associations with ESCC have been replicated in African populations.

We tested SNPs which have been associated with ESCC in genome-wide association studies (GWAS) from Asian and European populations for association with ESCC in the South African Black population. Black cases with a histologically confirmed diagnosis of ESCC and matching population controls were recruited, after informed consent and institutional ethical approval, from the Western Cape and Gauteng provinces of South Africa. SNPs were genotyped either by individual TaqMan assays (Applied Biosystems) or in a multiplex MassARRAY (Agena Bioscience) and genotypes were tested for association. We genotyped 26 SNPs from OSCC risk loci and 10 ancestry informative markers in 880 Black South African (SAB) OSCC cases and 939 controls recruited by the Johannesburg Cancer Study. No significant associations were detected at the CASP8, ALS2CR12, ADH1C TMEM173, PLCE1, ALDH2, ATP1B2/TP53 or CHEK2 loci, although some SNPs showed the same direction of effect observed in other populations. 8 SNPs from these loci were genotyped in a further 493 cases and 820 controls from the Western Cape, and a combined test for association done in a total of 1373 cases and 1759 controls. The only SNP that was significantly associated with ESCC in the combined analysis after correction for multiple testing was rs1033667 in CHEK2 (OR = 1.18, P = 0.002).

The lack of transference of most loci from Asian and European populations in an African population could result from smaller or absent effects of these loci in Africa. Alternatively, since we are likely to be genotyping tagging SNPs rather than the actual causal variants in these studies and linkage disequilibrium (LD) is lower in African genomes, our power to detect association with tagging SNPs will be reduced. This question could be resolved by carrying out a well-powered GWAS in African ESCC using a SNP array with sufficient African content to identify known or novel associations. We are currently conducting a GWAS for ESCC by genotyping 1700 ESCC cases and 6000 controls using a 2.4M SNP array designed by the H3 Africa Consortium.

#238

Interaction analysis between germline susceptibility loci and somatic alterations in lung cancer.

Yuzhuo Wang, Cheng Wang, Meng Zhu, Guangfu Jin, Hongbing Shen. _Nanjing Medical University, Nanjing, China_.

Emerging evidence indicates that germline variations may interact with somatic events in carcinogenesis. However, the germline-somatic interaction in lung cancer remains largely unknown. In the present study, we investigated whether lung cancer driver genes (CDGs) were more likely to locate within cancer susceptibility regions. Additional pathway enrichment analysis was performed to identify common pathways underlying CDGs and cancer susceptibility genes (CSGs). Next, we analyzed the associations between lung cancer risk SNPs and somatic alterations, including mutations and copy number alterations, in the level of genes, pathways, and overall burden of alterations. Enrichment analysis showed that lung CDGs are more likely to locate within cancer susceptibility regions (P = 8.40×10-3). Both of lung CSGs and CDGs showed significant enrichment in pathways such as cell cycle and p53 signaling pathway. Gene-based analysis showed that rs36600 (22q12.2) was associated with somatic mutations within ARID1A (OR = 2.45, 95%CI: 1.47-4.08, P = 5.78×10-4). Pathway-based analysis identified rs2395185 and rs3817963 at 6p22.1 were associated with somatic truncation mutations in cell cycle pathway (OR = 1.56, P = 3.61×10-4 for rs2395185; OR = 1.58, P = 4.15×10-4 for rs3817963), and rs3817963 was also associated with somatic truncation mutations in MAPK signaling pathway (OR = 1.54, P = 8.58×10-4). Further analysis associated rs2395185 at 6p22.1 (HLA class II genes) with increased APOBEC3A expression (P = 9.50×10-3) and elevated APOBEC mutagenesis (P = 3.58×10-3). These results provide evidence for the germline-somatic interactions on lung tumorigenesis. This can help to uncover the molecular mechanisms by which risk SNPs influence lung cancer risk.

#239

Development of DNA chip for detection of predictive SNPs for gemcitabine-induced adverse events.

Noriaki Nakamura,1 Koichi Hirayama,1 Hirofumi Yamano,1 Hiroshi Okamura,1 Hitoshi Zembutsu2. 1 _Toyo Kohan Co., Ltd., Yamaguchi, Japan;_ 2 _Japanese Foundation for Cancer Research, Tokyo, Japan_.

[Introduction] Anticancer agent gemcitabine is one of the effective chemotherapies and has been widely used for the treatment of patients with various tumors. However, 13-35% of patients receiving gemcitabine experience severe leukopenia/neutropenia. In our previous studies, we identified four novel SNPs (rs11141915, rs1901440, rs11719165 and rs12046844) associated with this toxicity. To predict the risk of gemcitabine-induced myelosuppression, we developed a high accuracy DNA chip system for genotyping of these four SNPs.

[Methods] We used 17 and 170 germline DNA samples of patients who received gemcitabine therapy in the first stage and second stage respectively. For SNP genotyping using the DNA chip, we amplified target regions by PCR with 10 ng of DNA extracted from blood samples. The PCR product was hybridized with the probe on the DNA chip. Genotype was detected by genotyping score, which was calculated the ratio of risk allele fluorescence intensity to total fluorescence intensity. To confirm the accuracy and reliability of this method, TaqMan genotyping assay was performed for 187 samples.

[Results and Discussion] In the first stage, genotypes of 17 samples were clearly divided into 3 types (non-risk type homo, hetero type, risk type homo). We defined an appropriate cut off value of genotyping score for detection of genotype according to the results of first stage. In the second stage, 170 samples, whose genotype were unknown, were genotyped using the DNA chip. The genotype results were completely concordant (100%) with those using TaqMan genotyping assay. Our DNA chip system is extremely useful as a simple method that is capable of genotyping using DNAs extracted from small amount of blood samples in general hospitals. The development of our DNA chip system provides new insights into personalized anti-cancer drug therapy including gemcitabine for the patients with cancer.

#240

SNP-mediated repression of DCBLD1 by YY1 may explain a GWAS finding on lung cancer.

Yu Wang,1 Rongna Ma,1 Hongyan Lin,1 Xiao Yu,1 Herbert Yu,2 Biyun Qian1. 1 _Shanghai Jiao Tong University School of Medcine, Shanghai, China;_ 2 _University of Hawaii Cancer Center, Honolulu, HI_.

Genome-wide association studies (GWAS) have identified many cancer-associated single nucleotide polymorphisms (SNP) with limited understanding of their real implication in cancer. To study the biological relevance of a GWAS-discovered SNP, we performed linkage disequilibrium (LD) analysis on rs9387478 and identified four potentially functional SNPs. We found that SNP rs17079281 C>T was associated with lung cancer in 766 Chinese cases and 773 matched controls, and the association was validated by another independent study (558 cases and 534 controls). Luciferase assay and CRISP/Cas9-generated genotype experiments showed that transcription factor YY1 suppressed DCBLD1 expression in an allele-specific manner, higher inhibition to T than to C alleles. In vitro and in vivo experiments on gain and loss of function further demonstrated that DCBLD1 behaved like an oncogene for lung cancer. These findings suggest that rs17079281 genotype may differentially affect YY1's suppression on DCBLD1 which influences the risk of lung cancer as an oncogene.

#241

Contribution of female breast cancer predisposition SNPs to risk of male breast cancer.

Nick Orr,1 Michael Jones,2 Sarah Maguire,1 Anthony Swerdlow2. 1 _Queen's University Belfast, Belfast, United Kingdom;_ 2 _Institute of Cancer Research, London, United Kingdom_.

Genome-wide association studies have identified more than 170 loci that confer susceptibility to breast cancer. Male breast cancer accounts for approximately 1% off all newly diagnosed cases of breast cancer and several reports have implicated germline genetic factors as significant in the aetiology of the disease. Inherited germline mutations in the BRCA2 gene are thought to account for up to 10% of cases. Here we demonstrate that there are substantial overlaps between polygenic susceptibility to male and female breast cancer and that a subset of shared predisposition loci confer significantly larger risk effects in males than in females. Men in the highest quintile of the distribution of risk allele count have a 2.7-fold (2.17-3.30) increased risk of breast cancer compared to those in the lowest quintile. Furthermore, comparisons of the risk allele distributions in male and female cases suggests that the overall impact of genetic predisposition to breast cancer may be greater in a susceptible minority of males from the general population than in females.

#242

Large pathway and gene set analysis of GWAS data identifies novel associations for pancreatic cancer.

Naomi Walsh,1 Han Zhang,2 Paula L. Hyland,3 Qi Yang,2 Evelina Mocci,4 Mingfeng Zhang,2 Erica J. Childs,4 Zhaoming Wang,2 Stephen Chanock,2 Patricia Hartge,2 Robert Hoover,2 Peter Kraft,5 Donghui Li,6 Eric J. Jacobs,7 Gloria M. Petersen,8 Brian M. Wolpin,9 Harvey A. Risch,10 Laufey T. Amundadottir,2 Kai Yu,2 Alison P. Klein,4 Rachael Z. Stolzenberg-Solomon2. 1 _Dublin City University, Dublin, Ireland;_ 2 _National Cancer Institute, Bethesda, MD;_ 3 _Food and Drug Administration, Silver Spring, MD;_ 4 _Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins School of Medicine, Baltimore, MD;_ 5 _Harvard School of Public Health, Boston, MA;_ 6 _University of Texas MD Anderson Cancer Center, Houston, TX;_ 7 _American Cancer Society, Atlanta, GA;_ 8 _Mayo Clinic College of Medicine, Rochester, MN;_ 9 _Dana-Farber Cancer Institute,, Boston, MA;_ 10 _Yale School of Public Health, New Haven, CT_.

Objective Genome-wide association studies (GWAS) identify associations of individual SNPs with cancer risk but usually only explain a fraction of the inherited variability. Pathway analysis of genetic variants has been shown for many disorders to be a powerful tool in discovering novel networks of susceptibility genes. Design We have conducted a large agnostic pathway-based meta-analysis of GWAS data using the summary-based adaptive rank truncated product (sARTP) method to identify novel gene sets and pathways associated with pancreatic ductal adenocarcinoma (PDA) in 9,040 cases and 12,495 controls. We performed expression quantitative trait loci (eQTL) analysis and functional annotation of the top SNPs in genes identified in PDA-associated gene sets and pathways. Results We identified 14 gene sets and pathways associated with PDA at FDR < 0.05. Five of the strongest signals (P-value ≤ 1.3x10-5 Bonferroni corrected) were in genes from KEGG maturity onset diabetes of the young, Reactome regulation of beta cell development, Biocarta role of epidermal growth factor (EGF) receptor transactivation by G-protein-coupled receptors in cardiac hypertrophy pathway, Nikolsky breast cancer chr17q11-q21 amplicon gene set, and Pujana ATM Pearson correlation coefficient (PCC) network gene set. We identified and validated rs876493 and three correlated SNPs (PGAP3) and rs3124737 (CASP7) as eQTLs in two normal derived pancreas tissue datasets. Conclusion Our agnostic gene set and pathway analysis integrated with functional annotation and eQTL analysis provides novel insight into genes and pathways that may be biologically relevant for risk of PDA.

#243

Genetic polymorphisms of diabetes-related genes, their interaction with diabetes status, and breast cancer incidence and mortality: The Long Island Breast Cancer Study Project.

Humberto Parada,1 Rebecca J. Cleveland,2 Kari E. North,2 June Stevens,2 Susan L. Teitelbaum,3 Alfred I. Neugut,4 Regina M. Santella,4 Maria E. Martinez,5 Marilie D. Gammon2. 1 _San Diego State University, San Diego, CA;_ 2 _University of North Carolina at Chapel Hill, Chapel Hill, NC;_ 3 _Icahn School of Medicine at Mount Sinai, New York, NY;_ 4 _Columbia University, New York, NY;_ 5 _University of California, San Diego, La Jolla, CA_.

Background: Diabetes is associated with increased risk of breast cancer and subsequent mortality; however, whether genetic variants that increase diabetes risk also influence breast cancer has received little attention. Herein, we examined the associations between 143 single nucleotide polymorphisms (SNPs) identified from genome-wide association studies of diabetes risk, and breast cancer incidence and subsequent mortality. For SNPs significantly associated with breast cancer incidence or mortality, we evaluated effect modification by diabetes status.

Methods: Caucasian women diagnosed with first primary invasive breast cancer (n=817) and controls (n=1,021) from a population-based case-control study were interviewed to assess diabetes status. Using the National Death Index, women with breast cancer were followed for more than 18 years during which we identified 340 deaths including 139 from breast cancer. Genomic DNA was extracted from blood samples and genotyping was done using high-throughput matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. We used logistic regression to estimate age-adjusted odds ratios (ORs) and 95% confidence intervals (CIs) for breast cancer incidence and used Cox regression to estimate age-adjusted hazard ratios (HRs) and 95% CIs for all-cause and breast cancer-specific mortality.

Results: Twelve SNPs were associated with incident breast cancer in additive models, at an alpha of 0.05. The top three most significantly associated SNPs included SLC30A8 rs4876369 (P=0.0034), HHEX rs11187146 (P=0.0086), and CDKN2A/CDKN2B rs1333049 (P=0.0086). Diabetes status modified two SNP-breast cancer incidence associations: SLC30A8 rs4876369 was associated with a 25% increase (OR=1.25, 95% CI=1.02-1.53) in odds of breast cancer among women without diabetes, and with a 330% increase (OR=4.30, 95% CI=1.66-11.17) in odds of breast cancer among women with diabetes (PInteraction=0.0150). IRS2 rs2241745 was inversely associated (OR=0.76, 95% CI=0.61-0.94) with breast cancer among women without diabetes, and associated with a 76% increase (OR=1.76, 95% CI=0.86-3.58) in odds of breast cancer among women with diabetes (PInteraction=0.0283). Three SNPs were associated with all-cause (CDKAL1 rs981042, P=0.0032; HHEX rs1111875, P=0.0361; and INSR rs919275, P=0.0488) and three with breast cancer-specific (CDKN2A/CDKN2B rs3218020, P=0.0225; CDKAL1 rs981042, P=0.0246; and TCF2/HNF1B rs3094508, P=0.0344) mortality in additive models, at an alpha of 0.05.

Conclusions: SNPs identified in GWAS studies of diabetes risk were associated with breast cancer incidence and mortality among a population-based sample of women. These associations may highlight important biological mechanisms of breast carcinogenesis and progression.

#244

Pathway analysis of OncoArray data identifies biological pathways involved in lung cancer development.

Zhihui Wang,1 Ruyang Zhang,2 Li Su,2 Rayjean Hung,3 Christopher Amos,4 David C. Christiani2. 1 _Harvard University, Cambridge, MA;_ 2 _Harvard T.H. Chan School of Public Health, Boston, MA;_ 3 _Samuel Lunenfeld Research Institute of Mount Sinai Hospital, Ontario, Canada;_ 4 _Dartmouth Geisel School of Medicine, Hanover, NH_.

Background:Genome-wide association studies (GWAS) have identified susceptible loci associated with lung cancer development. However, these variants only account for a small proportion of lung cancer heritability. With the aim to identify the missing heritability, we propose to conduct pathway analysis to test the joint effects of rare and common variants using the OncoArray data. By grouping SNPs into genes and pathways, we may shed light into the underlying mechanisms for lung cancer development and identify novel candidate genes and pathways.Methods:We applied sequence kernel association test (SKAT) to the OncoArray data of the Transdisciplinary Research of Cancer in lung of the International Lung Cancer Consortium (TRICL-ILCCO) that includes 34,432 individuals (19,028 cases and 15,404 controls) across over twenty lung cancer studies. A total of 403 KEGG and Biocarta pathways containing 5,555 genes and 58,717 SNPs were included in the study. As a score-based variance-component test, SKAT calculated p values for each pathway set by fitting the null model containing only age, gender, smoking, and first three PCAs. Results:KEGG neuroactive ligand receptor interaction (p=1.18×10-4, FDR=0.0285) and KEGG pancreatic cancer pathways (p=1.41×10-4, FDR=0.0285) were significantly associated with lung cancer. Gene-based analyses found that the most significant genes on the KEGG neuroactive ligand receptor interaction pathway to be CHRNA5 (p=2.33×10-8, FDR=0.0003), CHRNA3 (p=2.85×10-7, FDR=0.0019), and CHRNB4 (p=7.49×10-7, FDR=0.0034), while the most significant genes for KEGG pancreatic cancer pathway to be BRCA2 (p=2.23×10-5, FDR=0.0505). Stratified analyses highlighted five pathways (KEGG intestinal immune network for IGA production, KEGG leishmania infection, KEGG bladder cancer, KEGG pancreatic cancer, and KEGG axon guidance) for squamous cell carcinoma and one pathway (Biocarta ACH pathway) for adenocarcinoma with FWER<0.05. Noteworthy, the most significant gene on ACH pathway for adenocarcinoma was TERT gene, whereas the top two significant pathways for squamous carcinoma shared the most significant HLA-DQA1 gene, along with 11 other HLA genes at 6p21.Conclusion:The results suggest that the underlying pathways differ by cancer cell types and further research should be conducted to investigate the effects of immune pathways and HLA genes on squamous cell carcinoma etiology.

#245

Genetic variation related to innate immunity and colorectal cancer risk.

Jessica Citronberg,1 Barbara Banbury,1 Andrew T. Chan,2 Peter T. Campbell,3 Graham Casey,4 Jenny Chang-Claude,5 Steven J. Gallinger,6 Tabitha Harrison,1 Michael Hoffmeister,5 Mark A. Jenkins,7 Loic Le Marchand,8 Hongmei Nan,9 Hongmei Nan,9 Li Jiao,10 Robert E. Schoen,11 Hermann Brenner,5 Emily White,1 Ulrike Peters,1 Polly A. Newcomb1. 1 _Fred Hutchinson Cancer Research Center, Seattle, WA;_ 2 _Massachusetts General Hospital, Boston, MA;_ 3 _American Cancer Society, Atlanta, GA;_ 4 _University of Virginia, Charlottesville, VA;_ 5 _German Cancer Research Center, Heidelberg, Germany;_ 6 _Mount Sinai Hospital, Toronto, Ontario, Canada;_ 7 _University of Melbourne, Melbourne, Australia;_ 8 _University of Hawaii Cancer Center, Honolulu, HI;_ 9 _Indiana University, Indianapolis, IN;_ 10 _Baylor College of Medicine, Houston, TX;_ 11 _University of Pittsburgh Medical Center, Pittsburgh, PA_.

Introduction: Previous studies have shown that polymorphisms in the Toll-like receptor 4 (TLR4) gene may be associated with obesity, inflammatory bowel disease, and various cancers, including prostate, breast, and gastric cancer. However, the data regarding the associations between various TLR4 single-nucleotide polymorphisms (SNPs) and colorectal cancer (CRC) risk are inconsistent. In addition, the effect of interactions between TLR4 SNPs and obesity on the risk of CRC remains unclear.

Methods: We selected candidate SNPs involved in the TLR4 pathway that were previously associated with the risk of CRC or other cancer (N=31). SNPs with low minor allele frequency (MAF < 0.05) were excluded (N=2), leaving 29 SNPs in the main analysis. We examined the associations of these SNPs with CRC risk using logistic regression on 10,998 cases of colorectal cancer and 10,691 controls drawn from 14 studies within the Genetics and Epidemiology of Colorectal Cancer Consortium (GECCO) and the Colon Cancer Family Registry (CCFR). BMI was dichotomized into non-obese (BMI ≤25) and obese (BMI >25) categories. Overall and BMI-stratified analyses were performed across studies, with regression models adjusting for sex, age, study site, and the first three principal components from EIGENSTRAT to account for potential population substructure. A false discovery rate at 0.2 was applied to correct for multiple testing.

Results: Before adjustment for multiple comparisons, TLR4 SNPs rs10116253, rs11536891, rs7873784, rs1927911, rs4986791, and rs4986790 were associated with CRC risk. Additionally, for both rs4986791 and rs4986790, this association was more pronounced in those with a BMI ≤25 (rs4986791 - OR: 1.17; 95% CI: 1.02-1.34; rs4986790 - OR: 1.20; 95% CI: 1.04-1.38) compared to those who had a BMI >25 (rs4986791 - OR: 1.03; 95% CI: 0.93-1.15; rs4986790 - OR: 1.03; 95% CI: 0.92-1.15). However, after accounting for multiple comparisons, there were no statistically significant associations between candidate SNPs and CRC nor any statistically significant SNP interactions with BMI.

Conclusion: This large study provides evidence that neither these identified TLR4 SNPs nor their interaction with obesity are associated with CRC risk.

#246

An integrative genetic epidemiologic approach to identify susceptibility genes for post-menopausal breast cancer.

Roxana Moslehi,1 Nur Zeinomar,2 Cristy Stagnar,3 Hui-Shien Tsao,1 Amiran Dzutsev4. 1 _University at Albany, Albany, NY;_ 2 _Columbia University, New York City, NY;_ 3 _New York University, New York City, NY;_ 4 _National Institutes of Health, Bethesda, MD_.

Background: Up to 30% of common (peri- and post-menopausal) breast cancer cases in the general population are believed to be due to segregation of mutations and variations with low to medium risk [i.e., 1.5< relative risk <20] for breast cancer. The majority of these genes remain to be identified. We used a hypothesis-driven integrative genetic epidemiologic approach to test the association of post-menopausal breast cancer with Nucleotide Excision Repair (NER) genes using several gene expression microarray and genome-wide association studies (GWAS) datasets. Methods: As per inclusion criteria, all datasets contained Caucasian cases with invasive ductal breast carcinoma diagnosed post-menopause (≥50 years of age) and healthy controls (with no personal history of breast cancer). Raw data from four gene expression microarray datasets (GSE5764, GSE10797, GSE10780, E-TABM-276) were used for pathway and meta-analysis. Raw data from three GWAS datasets, the Cancer Genetic Markers of Susceptibility (CGEMS) (1089 cases, 1093 controls), a nested case-control (351 cases, 884 controls) within National Surgical Adjuvant Breast and Bowel Project (NSABP), and a nested case-control (465 cases and 1394 controls) within the Women's Health Initiative (WHI) Hormone Therapy Trials, were analyzed using logistic regression models adjusting for covariates. Linkage disequilibrium patterns and deviations from Hardy-Weinberg Equilibrium (HWE) were assessed for single nucleotide polymorphisms (SNPs) within all candidate genes. Individual haplotypes and diplotypes were determined for all subjects. Odd ratios (OR) and 95% confidence intervals (CI) were calculated. Results: Individual analysis of gene expression microarray datasets and meta-analysis of GSE10780 and E-TABM-276 identified significant up-regulation of several NER genes including ERCC6 (P=7.95*10-6) and ERCC8 (P=4.67*10-6). Increased risk of breast cancer was found in association with variations in ERCC6 in all GWAS datasets. ERCC6 SNPs were in strong LD in all datasets with no deviations from HWE. In CGEMS, increased risk of breast cancer was found with ERCC6 rs4253082 (OR=1.32, 95%CI: 1.09-1.61, P=0.005), rs3750749 (OR=1.40, 95%CI: 1.07-1.8, P=0.01), and rs3750751 (OR=2.91, 95%CI: 1.05-8.06, P=0.04). In NSABP, increased risk of breast cancer was found with ERCC6 rs2229760 (OR=1.37, 95%CI: 1.02-1.83, P=0.03). In WHI, increased risk of breast cancer was found with ERCC6 rs1012553 (OR=1.35, 95%CI: 1.07-1.71, P=0.01) and rs2228528 (OR=1.29, 95%CI: 1.01-1.66, P=0.04). Gene-gene interaction analysis was significant for interactions between ERCC6 and several NER genes, including between specific diplotypes in ERCC6 and ERCC8 (Pinteraction=0.0009 in CGEMS). Conclusion: Genes involved in the NER pathway, including ERCC6 and ERCC8, may be associated with low to medium risk for susceptibility to post-menopausal breast cancer.

#247

Molecular markers of the vitamin D pathway and esophageal adenocarcinoma survival.

Elizabeth Loehrer,1 Rebecca Betensky,1 Ruyang Zhang,2 Li Su,1 Edward Giovannucci,1 David Christiani1. 1 _Harvard T.H. Chan School of Public Health, Boston, MA;_ 2 _Nanjing Medical University School of Public Health, Nanjing, China_.

We investigated the potential prognostic association between vitamin D levels (25-Hydroxyvitamin D [25(OH)D]) and vitamin D pathway single nucleotide polymorphisms (SNPs) with overall survival (OS) in esophageal adenocarcinoma (EA) patients. EA patients were recruited from Massachusetts General Hospital near the time of their diagnosis between 1999 and 2016. At the time of recruitment, patients completed a baseline questionnaire and provided blood and serum samples. Serum 25(OH)D levels were measured using radioimmunoassay and adjusted for month of blood draw. Genotypes for 48 tag SNPs from seven candidate genes in the vitamin D pathway (GC, CYP2R1, CYP27A1, CYP27B1, CYP24A1, VDR and RXRA) were determined from whole blood DNA. For survival outcomes, we used log-rank test and a multivariable extended Cox model to estimate adjusted hazard ratio (HR) of death. HR models for each SNP were restricted to patients who identified as White and adjusted for age, sex, smoking status, diagnosis year, and treatment, stratifying baseline hazard by clinical stage and modeling surgical resection as a time-dependent covariate. HR models by quartiles 25(OH)D, additionally adjusted for body mass index (BMI), and timing of blood draw. Our analyses included EA patients with complete information on relevant confounders and predictors of OS. The 25(OH)D analysis included 463 EA patients and 337 deaths, with median follow-up of 23.1 months. The mean 25(OH)D level at diagnosis was 20.7 ng/mL. We found no evidence that OS curves differed across quartiles of vitamin D (log rank p=0.83). In the adjusted extended cox model, we found no difference in OS among the highest quartile and quartiles 2-4 (respectively, HR (95% confidence interval), 0.95 (0.70, 1.31), 1.03 (0.76,1.39), 1.01 (0.73, 1.38), global p=0.97). We did not find evidence of interaction between 25(OH)D and clinical stage (p=0.88) or BMI (p=0.43) on OS, and this relationship did not differ by timing of blood draw. Preliminary SNP analysis included 424 EA patients and 305 deaths, with median follow-up of 24.1 months. Two SNPs in CYP24A1 (SNP, HR (95% confidence interval) per minor allele: rs1570669, 1.23 (1.04-1.45); rs927650, 1.22 (1.04-1.44)), two SNPs in GC (rs4588, 1.25(1.04-1.49); rs7041, 0.84(0.71-0.99)), and one SNP in RXRA (rs7039190, 1.57(1.06, 2.31)) were marginally associated with OS in the multivariable survival model. SNPs were not statistically significant after adjusting p-value for multiple testing. Based on the available evidence from our analyses, markers in the vitamin D pathway at the time of diagnosis are not clinically relevant markers of EA survival.

#248

**Association of vitamin D related polymorphisms with hereditary breast cancer in 431 patients wild type for** BRCA1 **and** BRCA2 **mutations.**

Valentina Aristarco,1 Davide Serrano,1 Monica Barile,1 Davide Bondavalli,1 Mariarosaria Calvello,1 Debora Macis,1 Harriet Johansson,1 Aliana Guerrieri Gonzaga,1 Irene Feroce,1 Valeria Pensotti,2 Sara Gandini,1 Bernardo Bonanni1. 1 _European Inst. of Oncology, Milano, Italy;_ 2 _Cogentech, IFOM, Milano, Italy_.

Background: A high proportion of Breast Cancer (BC) patients with a positive family history is not explained by mutations in BRCA1 and BRCA2 genes and the biology and BC outcome are different compared to patients with BRCA mutations. Low serum 25-hydroxyvitamin D (25(OH)D) has been found to be associated with an increased cancer incidence and poorer prognosis. Single nucleotide polymorphisms (SNPs) of vitamin D receptor (VDR) and vitamin D binding protein (VDBP) genes (also called GC (Globulin Complex)), may contribute to final vitamin D activity. The aim of this study is to assess the role of 25(OH)D and VDBP serum levels, VDR and GC SNPs on BC recurrence and survival in a cohort of patients with positive family history, wild type (WT) for BRCA1 and BRCA2.

Methods: VDR (ApaI, FokI, TaqI, BsmI) and GC (rs2282679, rs4588, rs7041) SNPs were analyzed, by TaqMan SNP Genotyping Assays (Thermo Fisher Scientific), for 431 WT patients. Serum levels of 25(OH)D and VDBP were measured using an electro-chemiluminescence immunoassay (Abbott) and Human Vitamin D BP Quantikine ELISA kit (R&D) respectively.

Results: A cohort of 431 BC patients was followed from 2001 to 2015 (median follow-up 9 years). Women with the GC rs228679 variant had significantly lower levels of circulating VDBP and 25(OH)D compared to carriers of the major allele, whereas the GC rs7041 variant was associated with a greater level of VDBP and 25(OH)D.

Multivariate Cox models showed that BsmI is significantly associated with BC outcome: patients with the major allele of BsmI (C) have half the risk of recurrence (CC vs. TT HR=0.43; 95%CI: 0.27-0.68; and CC vs. CT HR=0.66; 95%CI: 0.45-0.99). Finally, low serum VDBP concentrations are significantly associated with a higher risk of BC recurrence (VDBP<187 vs >321: HR=1.79; 95%CI: 1.03-3.13) and 25(OH)D>30 ng/ml in late summer/autumn vs <20 ng/ml in winter/spring is associated with a significant decrease risk of mortality (HR=0.44; 95%CI: 0.22-0.89).

Conclusions: 25(OH)D and VDBP serum levels were related with survival and breast cancer recurrence and influenced by genetic variants of GC SNPs. An allele VDR dose-related decrease of breast cancer recurrence was achieved with the increasing number of allele C of BsmI rs1544410(C>T). Our study provides evidence for the pivotal role of vitamin D metabolism in breast cancer outcome of high risk patients with a positive family history, wild type for BRCA1 or BRCA2 genes.

#249

Genome-scale analysis of aberrant DNA methylation for emerging, high incidence of colorectal cancer in Taiwan.

KuoHsing Chen,1 Liang-Chuan Lai,2 Hsien-Fang Chang,3 Yu-Liang Chao,1 Been-Ren Lin,1 Jin-Tung Liang,1 Ann-Lii Cheng,1 Eric Y. Chuang,4 Kun-Huei Yeh1. 1 _National Taiwan University Hospital, Taipei, Taiwan;_ 2 _Graduate Institute of Physiology, National Taiwan University, Taipei, Taiwan;_ 3 _Bioinformatics and Biostatistics Core, Center of Genomic Medicine, National Taiwan University, Taipei, Taiwan;_ 4 _Graduate Institute of Biomedical Electronics and Bioinformatics, National Taiwan University, Taipei, Taiwan_.

Background:

The incidence of colorectal cancer (CRC) in Taiwan is increasing in decades, and the definite etiology remains elusive. This study aimed to explore the association of aberrant DNA methylations and the development of colorectal cancer in Taiwan.

Materials and methods: We prospectively collected 27 CRC patients' tumors, the adjacent normal colons (frozen specimens), and clinical characteristics, including age, sex, body mass index (BMI), stage at diagnosis and medical history of diabetes mellitus and hyperlipidemia. We conducted a genome-scale analysis of DNA methylation in these 27 CRC and 5 adjacent normal colon samples by using Illumina Methylation EPIC Beadchips. We used Wilcoxon rank-sum test (p < 0.05) and β value difference (≧ 0.25 or ≦ -0.25) to evaluate the methylation differences between CRC and adjacent normal colon samples.

Results: Overall, 7,672 differentially methylated genes (DMGs) were found between 27 CRCs and 5 adjacent normal colons. Principle component analysis demonstrated that a distinct cluster of adjacent normal colon and CRC tissue samples. Pathway analysis (β value difference ≧ 0.4 or ≦ -0.4) showed that ERK/MAPK signaling, GP6 signaling, CREB signaling in neurons, CDK5 and IGF-1 pathways were the top 5 pathways that were involved by DMGs. Subgroup analysis revealed most DMGs (N = 5,228) are shared by all 3 subgroups, however, patients who had metabolic syndrome had fewest subgroup-specific DMGs between tumors and adjacent normal colons, compared to patients who had no metabolic syndrome with normal weight or overweight (BMI < 25 or ≧ 25).

Conclusion: We demonstrated a genome-scale difference of aberrant DNA methylation in colorectal cancer in an East Asian population. These data will be validated further in a larger cohort.

#250

**Multiple lines of evidence make** ZDHHC3 **a possible marker for breast cancer in African American women.**

Nicholas A. Kinney,1 Robin T. Varghese,1 Ramu Anandakrishnan,1 Harold R. Garner2. 1 _The Edward Via College of Osteopathic Medicine, Blacksburg, VA;_ 2 _Gibbs Cancer Center & Research Institute, Spartanburg, SC_.

Rates of breast cancer mortality continue to be divided among race and ethnicity without a definitive explanation. Between the years 2005-2009 the racial disparity in breast cancer mortality caused about 9 extra death per 100 breast cancer cases in African American women compared to Caucasian women. In addition, young African Americans tend to have more aggressive forms of breast cancer. The source of this health disparity likely stems from a complex combination of mammography rate and frequency; segregation; socioeconomic status; and genetics. We contribute to what is known about genetics by investigating microsatellites - a type of repetitive DNA - and possible links to the breast cancer mortality gap. Microsatellites are understudied compared to single nucleotide polymorphisms and have the capacity to affect gene expression. We screen 33,854 microsatellites in germline DNA of African American women with and without breast cancer: four are statistically significant. We preform further analysis of genes harboring these microsatellites; the results reveal that ZDHHC3 is particularly interesting. Its location (3p21) is already linked to early invasive breast cancer. Kaplan-Meier analysis shows that ZDHHC3 alterations may be associated with poor breast cancer survival. Data from cBioPortal suggests that ZDHHC3 mRNA expression is lower in African Americans compared to Caucasians. These independent lines of evidence make ZDHHC3 a candidate for further investigation. 

## PREVENTION RESEARCH:

### Chemoprevention of Cancer

#251

Luteolin-mediated increase in miR-26a inhibits prostate cancer cell growth and induces cell cycle arrest targeting EZH2.

Rajnee Kanwal,1 Stephen Moreton,2 Daniel Franco,1 Sanjay Gupta1. 1 _Case Western Reserve Univ., Cleveland, OH;_ 2 _Louis Stokes VA Medical Center, Cleveland, OH_.

Luteolin (3',4',5,7-tetrahydroxyflavone), a flavonoid found in several vegetables and fruits, has been reported to possess anticancer and apoptosis inducing properties. However, the molecular targets of luteolin action are presently unknown. The enhancer of zeste homolog 2 (EZH2) is a catalytic subunit of polycomb repressive complex 2 (PRC2), which catalyzes the trimethylation of histone H3 on Lysine 27 (H3K27) and is involved in chromatin remodeling and gene silencing. EZH2-mediated epigenetic gene silencing plays an important role in several human cancers including prostate neoplasm. Emerging studies demonstrate that miR-26a, a non-coding microRNA, regulates EZH2 expression and is frequently lost during cancer progression. Here we investigated the potential involvement of luteolin-mediated miR-26a upregulation and EZH2 suppression leading to cell cycle arrest and apoptosis in cancer cells. Human prostate cancer DU145 and PC-3 cells, which possess high constitutive EZH2 expression, were treated with 5-20 µM luteolin at various times significantly inhibited EZH2 and H3K27 trimethylation in dose and time dependent manner. Luteolin treatment further resulted in increased expression of miR-26a in both cell lines, as exhibited by EZH2 inhibitor GSK126. Mechanistic investigations revealed that miR-26a overexpression suppressed cell cycle regulatory molecules such as cyclin D and E, cyclin dependent kinases CDK4 and CDK6, and CDK inhibitors p14/ARF, p16/INK4A and p21/WAF1 in EZH2-dependent manner. Interestingly, similar observations were noted in prostate cancer cells with a marked decrease in cyclins and CDKs along with concomitant increase in CDK inhibitors after luteolin treatment resulting in G0/G1 phase cell cycle arrest. In plasma specimens, obtained from healthy individuals, high-risk subjects and prostate cancer patients exhibit a progressive decrease in miR-26a expression. Taken together, our results indicate that miR-26a functions as a growth suppressive miRNA lost in prostate cancer, whereas luteolin is a promising agent which demonstrate anti-proliferative effects mediated by upregulation of miR-26 repressing EZH2 in prostate cancer cells.

#252

Prostate carcinogenesis inhibition through AKT-PKCα signaling pathway by diosmetin.

Rebecca Pakradooni, Ahmad Khalifa, Ilaha Isali, Sanjeev Shukla. _Case Western Reserve Univ., Cleveland, OH_.

Mammalian target of rapamycin (mTOR) integrates nutrient and mitogen signals to regulate cell growth (increased cell mass and cell size) and cell division. mTOR protein kinase have two different complexes; complex-I contains major component raptor, whereas complex II (insensitive to rapamycin), has major component rictor. Rictor plays a critical role in AKT (Ser-473 phosphorylation), which facilitates phosphorylation of AKT on Thr-308 by PDK1 for full activation. When we genetically disrupted Rictor, it decreased AKT phosphorylation (Ser-473) and impaired PKCα (Ser-657) expressions. Growth factors viz., IGF-1 and IL-6 are the major player in prostate cancers, these increases Rictor expression. We need to have an agent that can modulate kinase activity of AKT and PKCα to inhibit prostate cancer cell growth. Diosmetin (5, 7-Trihydroxy-4′-methoxyflavone) could be such an agent. Diosmetin, a natural flavone present in plant legumes, has anti-mutagenic and anti-allergic properties. Diosmetin inhibited IGF-1 and IL-6 induced rictor expression in LNCaP and PC-3 prostate cancer cells. Diosmetin dose response treatment to these cells inhibited cell growth and induced apoptosis with no significant growth inhibition in normal prostate epithelial cells (RWPE1). Diosmetin treatment to prostate cancer cells modulated cell survival machinery by down regulating key molecules viz., c-Myc, Survivin and XIAP (X-Linked Inhibitor of Apoptosis). Similarly diosmetin (20 and 50μg/animal/day) feeding to nude mice models, which were orthotopically implanted with luciferase tag PC-3 cells in ventral prostate represented significant decrease in tumor volume than control mice. Diosmetin fed tumor bearing mice reduced phosphorylation of Rictor, (Thr-1135), AKT (Ser-473) and PKCα (Ser-657) expressions to inhibit tumor growth significantly. Moreover, luciferase activity of implanted tumor decreased after diosmetin dose response feeding than control mice. Additionally bone radiographic images suggested that diosmetin treatment inhibited homing of PC-3 cells in the bone. These effects were associated with decrease in cyclin D1 expression and their activating partner, cyclin-dependent kinase (cdk) 2 and 4 with concomitant upregulation of p27/KIP1. Further diosmetin treatment induced apoptosis which was evident by increased expression of cleaved caspase-3. We are documenting these evidences for the first time in animal model system that diosmetin acts against potential molecular targets to alter cellular events to elicit anticancer effects in prostate cancer.

#253

Gnetin C as a candidate for targeted chemopreventive and therapeutic measures in prostate cancer.

Urvi Kolhatkar,1 Kshiti Dholakia,1 Gabriela Sikorska,1 Avinash Kumar,1 Luis A. Martinez,2 Anait S. Levenson1. 1 _Long Island University, College of Pharmacy and Health Sciences, Brooklyn, NY;_ 2 _Stony Brook School of Medicine, Stony Brook, NY_.

Overexpression of chromatin modifier protein, metastasis-associated protein 1 (MTA1) in prostate cancer contributes to tumor aggressiveness and metastasis. MTA1 ChiP-Seq analysis identified downstream targets that are transcriptionally regulated by MTA1 and suggested a link between MTA1 and ETS2. The effects of ETS2 in cancer are context-dependent and both oncogenic and tumor suppressive functions have been described. We have shown that there is a positive correlation between MTA1 and ETS2 using loss of function studies and various preclinical models of prostate cancer. Our conclusion that MTA1 functions as a co-activator for regulating ETS2 expression in prostate cancer leading to promotion of prostate cancer progression, prompted us to look for pharmacological inhibitors of MTA1/ETS2 axis. In our previous studies, we reported inhibition of MTA1 by resveratrol and its potent analog pterostilbene in vitro and in vivo. We have demonstrated that pterostilbene treatment blocks the progression of PIN and adenocarcinoma in xenografts and transgenic mouse models by inhibiting MTA1 expression and signaling. Here, we found that Gnetin C, double-resveratrol, has more potent inhibition of MTA1/ETS2 axis than other stilbenes. Gnetin C, a resveratrol dimer, is found abundantly in melinjo plant widely cultivated in Southeast Asia; its seeds and fruits are common ingredients in Indonesian culinary. As resveratrol and pterostilbene, Gnetin C has been reported to possess anti-inflammatory and anticancer activity, however it has not been considered as chemopreventive agent in prostate cancer. Using two prostate cancer cell lines, namely DU145 and PC3M cells, we found that Gnetin C shows significant inhibitory effect on cell proliferation, more potent effects in colony formation and wound healing assays than resveratrol or pterostilbene. Importantly, Gnetin C specifically and strongly downregulates MTA1 and ETS2 expression in prostate cancer cells. Taken together, our findings implicate the potential of Gnetin C in MTA1/ETS2-mediated chemoprevention and therapy in prostate cancer.

#254

Chemopreventive effects of angiotensin II receptor type 2 agonist on prostate carcinogenesis by the downregulation of the androgen receptor.

Hiroji Uemura,1 Yusuke Ito,2 Aya Naiki-Ito,3 Hiroyuki Kato,3 Shugo Suzuki,3 Toshiya Kuno,3 Satoru Takahashi3. 1 _Yokohama City Univ. Medical Center, Yokohama, Japan;_ 2 _Yokohama City Univ. Graduate School of Medicine, Yokohama, Japan;_ 3 _Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan_.

[Background] We previously reported that angiotensin II receptor blockers (ARB), anti-hypertensive drug, have chemopreventive and chemotherapeutic potential against prostate cancer via the reduction of androgen receptor (AR) expression. It is generally known that angiotensin II binds to two kinds of receptors, AT1R and AT2R, and the biological function of AT2R signaling has the opposite effect to that of AT1R signaling in many aspects. We therefore hypothesized that AT2R agonism might attenuate the proliferative activity in prostate cancer cells. In this study, we investigated the effects of the AT2R agonist Compound 21 (C21) designed by Vicore Pharma (Sweden), which is expected to play similar roles to an ARB, on prostate carcinogenesis using the transgenic rat for adenocarcinoma of prostate (TRAP) model previously established in our laboratory.

[Materials and Methods] In vitro analyses of the cell growth, Western blotting and PSA-reporter gene assays were performed using LNCaP and 22RV1 cells. TRAP rats at 6 weeks of age were randomly divided into 3 groups of 12 animals each and treated with C21 at 1 or 2 mg/kg/day in drinking water for 12 weeks. They were pathologically examined the classification as prostatic intraepithelial neoplasia and non-invasive adenocarcinoma. The proteins extracted from TRAP prostate glands were examined for Western blotting.

[Results] C21 reduced the proliferation activity of prostate cancer cells and down-regulated the PSA promoter activity, medium PSA levels and the AR protein expression. We discovered that C21 inhibited the progression of prostate carcinogenesis in TRAP rats and decreased the incidence of adenocarcinoma in the lateral prostate. A significant increase in the apoptotic index with activation of caspase 3 and 7 were observed by immunohistochemistry and Western blotting analyses. C21 also down-regulated the expression of AR significantly in TRAP rat prostate. C21 decreased the expression of AR and AR-relevant genes reduced the proliferation activity effectively in prostate cancer cells and TRAP rat prostate.

[Conclusion] The present study demonstrated that the epoch-making AT2R agonist C21 down-regulated the proliferation activity and the expression of AR both in vitro and in vivo. C21 is a promising drug not only for hypertension but also for human prostate cancer chemoprevention.

#255

Gross thymic extract induces cell cycle arrest and apoptosis in Ehrlich ascites carcinoma in vivo.

Nariman K. Badr El-Din,1 Azza I. Othman,1 Maggie E. Amer,1 Mamdooh Ghoneum2. 1 _Mansoura Univ., Mansoura, Egypt;_ 2 _Charles R. Drew University of Medicine and Science, Los Angeles, CA_.

Objective. Gross thymic extract (GTE) obtained from pathogen-free pigs is composed of thymosin, thymomodulin, and other peptides. We have previously demonstrated that GTE induces apoptosis in human breast cancer cells in vitro. The present study was undertaken to examine the chemopreventive effect of GTE in mice bearing Ehrlich ascites mammary carcinoma (EAC) and to elucidate the mechanisms underlying its effect.

Materials and Methods. Swiss albino mice were inoculated with EAC cells in the thigh to develop solid mass. Oral treatment with GTE (5.45mg/kg body weight) was started either 14 days prior to tumor cell inoculation or 9 days post inoculation. Tumor incidence and growth were monitored for 30 days. Cell cycle progression, apoptosis, and the expression of their related proteins were analyzed by flow cytometry and western blot. Mitochondrial membrane potential (MMP) and natural killer (NK) cell cytotoxicity were examined by flow cytometry. Molecular analysis of DNA fragmentation was analyzed by agarose gel electrophoresis. GTE was provided by YS Nature Company, Tokyo, Japan.

Results. Pretreatment of mice with GTE markedly delayed tumor growth and reduced tumor incidence by 38.9% as recorded on day 30. Significant tumor growth inhibition reached 90.5% and 55.0% for pre-inoculation and post-inoculation cases, respectively. GTE treatment prior to tumor inoculation decreased tumor cell proliferation as indicated by marked suppression in the expression of PCNA, Ki-67, and Cyclin D1 in cancer cells with concomitant upregulation of p53 , p21 , and p27 expression relative to the untreated control. This was associated with significant G0/G1 phase arrest of the cell cycle accompanied by induction of apoptosis as indicated by the appearance of a significant sub-G0/G1 peak and confirmed by Annexin V/PI assay and DNA laddering pattern. Additionally, GTE decreased MMP, activated caspase-3, and increased Bax/Bcl-2 ratio by 2.4 fold in neoplastic cells, indicating that GTE induces apoptosis via an intrinsic pathway. Treatment with GTE markedly enhanced NK cell cytotoxicity by 5.8 fold relative to the untreated control. Data also showed the effectiveness of GTE treatment in post-inoculation cases but to a lesser extent than for pre-inoculation.

Conclusion. Our data demonstrate that GTE intake results in profound tumor growth inhibition by a mechanism involving cell cycle arrest, apoptosis via a mitochondrial dependent pathway, and modulating the immune system via increased NK cell activity. GTE may represent a new class of adjuvants for the treatment of breast cancer.

#257

Dual targeting of PI3K/AKT and AR pathway by combination therapy with phloretin in pre-clinical and patient derived xenografts of enzalutamide-resistant prostate cancer.

Sarita Saraswati,1 Abdulqader A. Alhaider,1 Abdelgalil M. Abdelgadir2. 1 _King Saud University, Riyadh, Saudi Arabia;_ 2 _King Saud bin Abdulaziz University for Health Sciences, King Abdulaziz Medical City, Riyadh, Saudi Arabia_.

Background:

With the emergence of resistance to FDA-approved inhibitors of androgen receptor (AR) signaling in prostate cancer, AR remains a viable target, engaged in the regulation of processes of pathological importance. CRPC tumors adapt the PI3K/AKT survival pathway to escape ADT. Thus, co-targeting AR and PI3K/AKT signaling is more effective therapeutic means for CRPC patients. Phloretin (PH), is a polyphenolic compound, found in apples, cider etc. It is known to possess anticancer and anti-angiogenetic activity. However, it`s effect against CRPC cells and the underlying mechanism are still unknown.

Experimental procedure:

Ten human PCa cell lines were tested in vitro for sensitivity to the PI3K/AKT inhibitor LY294002; enzalutamide (ENZ); PH; LY294002 + PH; ENZ + PH and LY294002 + ENZ + PH. In vivo studies were conducted using VCaP, 22Rv1, MR49F xenografts and LuCaP and 136CR PCa PDX tumors. Molecular docking was done with AutoDock Vina program. CSF Chimera and Ligplot programs were used for analysis of ligand and proteins interactions.

Results:

LY294002; ENZ; PH; LY294002 + PH; ENZ + PH and LY294002 + ENZ + PH inhibited in vitro growth of 7 of 10; 9 of 10; 9 of 10; 9 of 10; 9 of 10; 10 of 10 PCa cell lines, respectively, with increased sensitivity under androgen depletion. PH inhibited tumor growth in VCaP, 22Rv1, MR49F, MR49C xenografts and LuCaP and 13CR PCa PDX in a dose-dependent manner. Further, dual combination of ENZ + PH was more effective than LY294002 + PH in inhibiting tumor growth, whereas LY294002 + ENZ + PH diminished the tumor growth (P<0.005) and demonstrated a longer disease specific survival. PH alone inhibited the AR-DNA interaction directly or by interfering with AR dimerization and inhibited AR-V7 expression and transcriptional activity. Further, it blocked the R1881 induced expression of AR-target genes kallikrein-related peptidase 3 (KLK3, also known as PSA); transmembrane protease, serine 2 (TMPRSS2); and FK506 binding protein 5 (FKBP5), glucocorticoid receptor (GR). In LuCaP PCa PDX tumors, PH administration decreased TV (P=0.021), proliferation (p=0.0024), PSA (p<0.005) and decreased AR signaling and nuclear glucocorticoid receptor (nGR) localization. Computer aided molecular docking data shows the binding affinity and ligand efficiency scores of PH in following chronological order: AR > AKT1 > GR > PI3K.

Conclusion:

Our studies suggested triple combination improved the efficacy of ENZ and PH. PH could be promising candidates for prevention of CRPC. This pre-clinical synergy provides a strong rationale for clinical evaluation of this combination.

#258

Targeting Nanog in 3D explant models for the evaluation of cancer prevention agents.

Sam Khan,1 Ankur Karmokar,1 Zahirah Sidat,2 Nalini Foreman,1 David Moore,1 Jennifer Higgins,1 Emma Parrott,1 Despoina Theofanous,1 Dominic Hobbs,1 Lynne Howells,1 Anne Thomas,1 Karen Brown1. 1 _University of Leicester, Leicester, United Kingdom;_ 2 _Hope Against Cancer Clinical Trials Facility, Leicester, United Kingdom_.

Background: The transcription factor Nanog is crucial for the self-renewal of cancer stem-like cells (CSCs). Nanog expression in colorectal cancer (CRC) tissue correlates with lymph node metastasis and poor prognosis. Since Nanog is not expressed in most tissues, including normal adult stem-cells, it represents a therapeutic target specific to cancer cells. Curcumin inhibits proliferation and expansion of CSCs derived from CRC and adenomas. In NOD/SCID mice bearing xenografts from patient-derived CRC CSCs, curcumin significantly inhibited tumour growth and improved survival. In addition, curcumin binds directly to Nanog recombinant protein (quantified using microscale thermophoresis). We have developed 3D in-vitro primary human explant models to further characterise the effects of curcumin on Nanog. In this work the hypothesis is tested that Nanog may be an early marker of response for CRC prevention agents.

Methods: Patient-derived CRC and adenoma tissue was cubed (2x2x2mm) and treated for 24 hours with curcumin. Following treatment, explant tissues were processed for analysis by immunohistochemistry (IHC) (n=6) and flow cytometry (n=17). The effect of curcumin on CSCs (defined by expression of aldehyde dehydrogenase (ALDH) or Nanog) and differentiation (via Mucin 2 expression) was analysed using IHC. Additionally, cells expressing Nanog (Nanog+) or Nanog plus proliferation marker Ki67 (Nanog+Ki67+) were assessed using flow cytometry.

Results: A range of adenoma (n=5) and Dukes stage A-C CRC (n=18) samples were studied. Following exposure to curcumin, a 30% reduction was observed in Nanog+ and Nanog+Ki67+ cells. Nanog+ cell number was decreased in a curcumin concentration-dependent fashion in 6 samples and concentration-independently in a further 8. No response was observed in 3 samples. A reduction in Nanog and ALDH with concurrent increase in differentiation was observed via IHC in one sample.

Conclusion: Our data suggest Nanog is targeted by curcumin in adenoma and CRC tissues. Nanog may serve as a biomarker in clinical trials to identify individuals most amenable to treatment with curcumin alone or in combination treatment. Crucially, this will help select those who are likely to benefit from curcumin as a cancer prevention agent. Ultimately, this concept may be applicable to the evaluation of novel CRC prevention agents.

#259

Integrated multi-omics analyses reveal ɑ-lipoic acid-regulated cell proliferation via Grb2-mediated signaling in colorectal cancer cells.

Lan Yang,1 Chunlei Hu,2 Guoqing LV,2 Bei Li,2 Shaowei Xiong2. 1 _Shenzhen Second People's Hospital, Shenzhen, China;_ 2 _Peking University Shenzhen Hospital, Shenzhen, China_.

Background: The use of antioxidants as cancer prevention and treatment agents has become a focus of research in recent years due to their limited adverse effects. Alpha lipoic acid (ɑ-LA), a naturally occurring antioxidant and metabolic enzyme co-factor, has been reported to inhibit the growth of various cancer cells, but the precise signaling pathways that mediate the effects of ɑ-LA on colorectal cancer cells development remain unclear.

Methods: In this study, we combined the use of an RNA sequencing (RNA-Seq) database and isobaric tag for relative and absolute quantitation (iTRAQ)-based quantitative proteomics data to investigate the dynamic genomic/proteomic response of human colorectal cancer cells (HT29) to α-LA stress. The CCK-8 assay was used to assess cell proliferation in CRC cell lines after ɑ-LA treatment. The expression of proteins were evaluated by western blotting. Grb2 levels were restored in α-LA-treated cells by transfection of a plasmid carrying Grb2 and were reduced in CRC cells via specific siRNA-mediated knockdown.

Results: A total of 4,107 differentially expressed genes (2,259 downregulated and 1,848 upregulated) were identified via RNA-Seq in HT29 after exposure to α-LA for 24 h. Moreover, GO and KEGG pathway analyses showed that cancer-relevant cell membrane proteins were significantly affected. An interaction network analysis predicted that Grb2 may mediate the key target pathways activated by exposure to ɑ-LA. In verifying the multi-omics results, Grb2 was confirmed to mediate the ɑ-LA-induced inhibition of cell proliferation in vitro. Moreover, additional experiments indicated that Grb2 inhibition partially abolished EGF-induced phospho-EGFR and phospho-ERK1/2 activity. Furthermore, the analysis of human colorectal cancer specimens obtained from the GEO database showed that the expression of EGFR correlated with that of Grb2.

Conclusion: These findings provide a novel mechanism by which ɑ-LA regulates cell colorectal cancer proliferation via the down-regulation of growth factor-stimulated Grb2 signaling.

#260

**Apigenin nanoparticle suppresses sphere formation in CD133** + **/ALDH1** high **prostate cancer stem cells through downregulation of stem cell markers.**

Rajnee Kanwal,1 Sanjeev Shukla,1 Agata A. Exner,1 Andreas G. Tzakos,2 Sanjay Gupta1. 1 _Case Western Reserve Univ., Cleveland, OH;_ 2 _University of Ioannina, Ioannina, Greece_.

The recurrence of prostate cancer after definitive treatment(s) is thought to be due to the potential of rare cancer stem cells (CSCs) to withstand therapy and initiate tumor formation. CSCs are a subpopulation of cancer cells capable of self-renewal and plastic adaptation mediating tumorigenesis, metastasis, and therapy resistance. CSCs are associated with cancer progression and clinical outcome in cancer patients. In the present study, we isolated CSCs from various human prostate cancer 22Rv1, DU145 and PC-3 cells by applying selection of cluster of differentiation 133 cell surface marker and high ALDH1 activity (CD133+/ALDH1high). Next we aimed to identify CD133+/ALDH1high CSCs isolated from human prostate cancer cells and compare their profiles with non-CSCs as bulk counterparts of the population. Subsequently, the CSC population continued to grow in the three-dimensional multicellular spheroids. Differentiation investigated with stem cell-related genomic characteristics exhibited significant increase in genes viz. CD44, Nanog, β-catenin, c-Myc, SOX2 and Oct4 in CSCs at the message level. In further studies we determine the effect of natural plant flavone apigenin on CSC population. Apigenin is abundantly present in common fruits and vegetables and possess remarkable anticancer, anti-angiogenic and anti-metastatic properties. Apigenin loaded polyethylene glycol nanoparticles (Ap-NP) was characterized for particle size, morphology, zeta potential, drug release and encapsulation. Cellular entry and intracellular localization of Ap-NP were assessed by transmission electron microscopy. Treatment of CD133+/ALDH1high CSCs with Ap-NP inhibited cell viability and sphere formation at higher extent in time dependent manner, compared to the non-CSCs counterparts. Furthermore, Ap-NP exposure resulted in the downregulation of Nanog, β-catenin, c-Myc, SOX2 and Oct4 in the CSCs. Taken together, our study demonstrate overexpression of potential CSC markers and the ability to target these CSCs by the use of non-toxic agent apigenin killing this rare population that may lead to the total abolition of tumor recurrence and/or metastasis.

#261

Chemopreventive effects of flavonoids on pancreatic carcinogenesis.

Hiroyuki Kato, Aya Naiki, Shugo Suzuki, Yoriko Yamashita, Satoru Takahashi. _Nagoya City University Graduating School of Medical Science, Nagoya, Japan_.

Although clinical therapies are being developed for combating pancreatic cancer, its prognosis remains dismal; consequently, chemopreventive strategies may be needed to manage this disease. Luteolin (Lut), a flavone contained in perilla, is one of the most effective antioxidant flavonoids and has demonstrated potential for the treatment of inflammatory and neoplastic disease. Hesperetin (Hes) is a flavone contained in citrus, and the Ohsaki cohort study showed that citrus consumption was correlated with reduced pancreatic cancer (RR=0.62, 95%CI=0.38-1.00) (Int.J.Cancer, 2010: Li.W). However, there are no studies dealing with their antineoplastic effects against pancreatic cancer. In the present study, to assess the chemopreventive effects of Lut and Hes for pancreatic carcinogenesis, 6 week-old syrian golden hamsters were subcutaneously injected with N-nitrsobis(2-oxopropyl)amine four times and then fed Quick Fat Diet (control, n=14) or Quick Fat Diets with Lut 100 ppm (n=13) or Hes 100 ppm, 1000 ppm (n=13,13) for 13 weeks. There were no differences in body weights or liver or kidney weights among the groups. Serum cholesterol was significantly decreased in the Lut group. The incidence and multiplicity of pancreatic cancer was significantly suppressed in the Lut group (Incidence/ Multiplicity: 23%/ 0.23±0.44; P<0.05), but not the Hes groups (100ppm: 54%/ 0.69±0.75, 1000ppm: 62%/ 0.77±0.73), compared to the controls (71%/ 0.93±0.73). The Ki67 labeling index in pancreastic intraepithelial neoplasias (PanINs) was significantly decreased by Lut, but not Hes. Therefore, we focused further investigation on Lut. In vitro, cell proliferation of both hamster (HPD1NR) and human (MiaPaCa, PANC1) pancreatic cancer cell lines was inhibited by Lut in a dose-dependent manner. Multi-blot analysis for phosphorylated proteins related to cell signaling revealed that phosphorylated forms of Stat3 (tyr705) were down-regulated by Lut in MiaPaCa and PANC1 cells. We confirmed them in a dose-dependent manner in the three pancreatic cell lines and also found that Lut down-regulated cyclin D1. In vivo, the nuclear positive proportion of pStat3 (tyr705) in both PanINs and adenocarcinomas was significantly decreased by Lut. Because pStat3 (tyr705) is regulated by cytokines such as Il6, we examined mRNA levels of several cytokines including Il6 in hamster pancreatic tissues. Lut decreased the mRNA expression of Tgfb, Tnfa, Il10, and Il6. Immunohistochemistry of alpha smooth muscle actin revealed that the positive area of activated pancreatic stellate cells, which can produce these cytokines in cancerous tissue, tended to be decreased by Lut. In conclusion, these results indicate that Lut, but not Hes, has potential as a chemopreventive agent for pancreatic cancer and inhibition of pStat3 signaling may be involved in its mechanism of action.

#263

Effects of the sodium glucose cotransporter 2 inhibitor tofogliflozin on hepatoma cell lines and liver tumorigenesis.

Yohei Shirakami, Koki Obara, Masaya Kubota, Hiroyasu Sakai, Takuji Tanaka, Masahito Shimizu, Mitsuru Seishima. _Gifu Univ. Graduate School of Medicine, Gifu, Japan_.

Obesity, diabetes mellitus, and their related metabolic abnormalities are associated with increased risk of hepatocellular carcinoma (HCC). Anti-diabetic agents sodium glucose cotransporter (SGLT)-2 inhibitors are expected to ameliorate the abnormalities associated with metabolic syndrome including non-alcoholic fatty liver disease (NAFLD). In this study, we investigated the effects of the SGLT2 inhibitor tofogliflozin on the development of NAFLD-related liver tumorigenesis in C57BL/KsJ-+Leprdb/+Leprdb (db/db) obese and diabetic mice. The direct effects of tofogliflozin on human hepatoma cell lines were also evaluated. Male db/db mice were administered diethylnitrosamine-containing water for two weeks and were then treated with tofogliflozin throughout the experiment. Tofogliflozin was kindly provided by Kowa Co., Ltd. and the chemical structure will be disclosed at the time of presentation at the meeting. In mice treated with tofogliflozin, the development of hepatic pre-neoplastic lesions, foci of cellular alterations, was markedly suppressed, and hepatic steatosis and inflammation significantly reduced, as evaluated using the NAFLD activity score (NAS), in comparison to those in the control mice. Serum levels of glucose and free fatty acid and mRNA expression levels of pro-inflammatory markers in the liver were reduced by tofogliflozin treatment. Conversely, the proliferation of SGLT2 protein-expressing hepatoma cells was not inhibited and the insulin signaling in those cells was not altered by this agent. These findings suggest that tofogliflozin suppressed the early phase of obesity- and NAFLD-related liver carcinogenesis by attenuating chronic inflammation and steatosis in the liver, while the agent had no significant direct effect on the proliferation of hepatoma cells. Therefore, SGLT2 inhibitors may have a chemopreventive effect on obesity-related HCC.

#264

Vaccination against neoantigens induced in future tumors in the setting of recurrence and premalignancy.

Greta Garrido Hidalgo,1 Brett Schrand,1 Ailem Rabasa,1 Agata Levay,1 Alexey Berezhnoy,1 Shrey Modi,1 Giri Bhuwan Bhuwan,1 Vikas Dudeja,1 Elien Doorduijn,2 Thorbald van Hall,2 Eli Gilboa1. 1 _Univ. of Miami, Miami, FL;_ 2 _Leiden University Medical Centre, Netherlands_.

Development of therapeutic strategies to prevent recurrence in cancer patients, or tumor progression in at risk individuals has been challenging given the long and often unpredictable time to the emergence of the malignant tumors. Neoantigens represent the most potent antigens to induce antitumor immunity, yet the ability to predict which neoantigens will be expressed in future tumors is at present not an option. We have developed a simple and broadly applicable approach to induce neoantigens in tumor cells in situ by reducing the expression of antigen processing mediators like TAP, ERAAP, or invariant chain (Ii), using siRNAs that were targeted to tumor cells by conjugation to a nucleolin-binding aptamer. Exploiting the ability to induce neoantigens in situ, we describe a novel vaccination strategy targeting potent neoantigens to control the growth of the future tumors whereby patients in remission or individuals at risk are first vaccinated against the antigen processing downregulation-induced neoantigens and when or if tumor develops the same antigens are induced in the tumor, termed prorapeutic vaccination (prophylactic + therapeutic). Mice were vaccinated against the TAP downregulation-induced neoantigens by targeting the corresponding siRNAs to dendritic cells (DC) in situ via conjugation to a short CpG oligonucleotide (ODN); the CpG ODN also activated the targeted DC to avoid induction of tolerance against the neoantigens. Vaccination with CpG ODN-TAP siRNA conjugates inhibited subsequently implanted or autochthonous tumors in several tumor models, provided the said neoantigens were also induced in the tumor by nucleolin aptamer-targeted siRNA inhibition of TAP. The CpG-siRNA and nucleolin aptamer-siRNA conjugates are simple, chemically- synthesized, off-the-shelf, and broadly applicable reagents to vaccinate against neoantigens and control the growth of future tumors irrespective of their origin.

#265

Epi-magnolin suppresses EGF-induced neoplastic cell transformation by inhibition of Akt/mTOR signaling pathway in JBC Cl41 cells.

Cheol-Jung Lee, Sun-Mi Yoo, Seung-Min Kim, Seon-Yeon Cho, Juhee Park, Yu-Mi Shin, Yong-Yeon Cho, Hye Suk Lee. _The Catholic University of Korea, Bucheon-si, Republic of Korea_.

The phosphatidylinositol-3-kinase (PI3K)/Akt/the mammalian target of rapamycin (mTOR) signaling pathway is frequently activated in human cancer and plays a pivotal role in cell proliferation and transformation. In this study, we reveal epi-magnolin, a phytochemical found in Magnolia flos, as an inhibitor of Akt/mTOR signaling pathway. Epi-magnolin inhibits EGF-induced Akt phosphorylation at Thr308 and Ser473, but not extracellular signal-regulated kinases (ERKs)/ribosomal S6 kinase 2 (RSK2) signaling pathway. Moreover, we demonstrate that epi-magnolin suppresses JB6 cell proliferation by impairing EGF-induced G1/S cell cycle transition in a dose-dependent manner. Notably, epi-magnolin abrogates phosphorylation of c-Jun at Ser63 and 73 as well as total c-Jun protein level, which leads to downregulation of AP-1 transactivation activity. The inhibition effect of Akt/mTOR signaling pathway and AP-1 transactivation activity by epi-magnolin suppressed EGF-induced anchorage-independent cell transformation. Based on these results, epi-magnolin might be a chemopreventive agent by targeting Akt/mTOR signaling pathway.

Key words: chemoprevention, cell transformation, Akt/mTOR signaling pathway

#266

Disulfiram overcomes docetaxel resistance in prostate cancer by targeting the neddylation pathway and down-regulation of Skp2 expression.

Liankun Song,1 Bin Yu,2 Hong-Min Liu,2 Xiaolin Zi1. 1 _University of California, Irvine, Orange, CA;_ 2 _Zhengzhou University, Zhengzhou, China_.

Disulfiram (DSF), an inhibitor of aldehyde dehydrogenase and a dithiocarbamate chelator of copper, has been clinically used for treatment of alcohol dependence for decades. A population-based case control study has suggested that the use of DSF is associated with reduced risk of prostate cancer. In addition, copper is accumulated in prostate cancer and can act synergistically with DSF to inhibit the growth of prostate cancer cells. These accumulating data suggested that DSF is a very promising agent to be repurposed for prostate cancer treatment and prevention. However, the anti-cancer mechanisms of DSF currently remain largely unknown. Here we have shown that DSF treatment decreases the neddylation levels of NEDD8-conjugating enzyme UBE2M and Cullin-1 leading to down-regulation of Skp2 expression in prostate cancer DU145 cells. Further co-immunoprecipitation experiment revealed that DSF disrupted the formation of the defective in Cul neddylation 1 protein (DCN1) and UBE2M complex. DSF synergistically enhanced the growth inhibitory effect of docetaxel on prostate cancer cell lines and overcame the drug resistance. Taken together, these data may provide proof of concept for rationally designed repurpose of DSF to treat docetaxel-resistant prostate cancer by involvement of the neddylation pathway.

#267

Nonsteroidal anti-inflammatory drugs induce ER stress- and BID-dependent immunogenic cell death to suppress colorectal tumorigenesis.

Rochelle Elayne Fletcher, Brian Leibowitz, Yijun Wang, Fernando Concha-Benavente, Robert Ferris, Robert Schoen, Jian Yu, Lin Zhang. _University of Pittsburgh School of Medicine, Pittsburgh, PA_.

Prevention or early detection represents a key approach for reducing the mortality and morbidity of CRC. Use of non-steroidal anti-inflammatory drugs (NSAIDs) has been associated with decreased risk of CRC in numerous epidemiological studies, clinical trials, and animal model studies. However, the mechanism by which NSAIDs suppress colorectal tumorigenesis has remained unclear. We previously demonstrated that the chemopreventive effect of NSAIDs is mediated by a synthetic lethal interaction between death receptor signaling and loss of the gatekeeper APC tumor suppressor, which is dependent on the BH3-only Bcl-2 family member BID. In this study, we found a critical role of endoplasmic reticulum (ER) stress upstream of death receptor signaling and BID activation. Elevated levels of ER stress and cell death markers were detected in advanced adenomas from patients taking NSAIDs including aspirin. Inhibiting ER stress using a pharmacological approach abolished the apoptotic effect of NSAIDs in CRC cells and normal colonic epithelial cells with APC loss and also suppressed the chemopreventive activity of the NSAID sulindac in APCMin/+ mice. Importantly, our results unveiled a critical role of ER stress and BID-dependent cell death in triggering immune-mediated elimination of nascent cells that lose APC. Markers of immunogenic cell death (ICD), including plasma membrane translocation of calreticulin and phagocytosis by dendritic cells, were suppressed in cells with BID knockout or ER stress inhibition. BID knockout or ER stress inhibition also reduced tumor infiltrating lymphocytes (TILs) in sulindac-treated APCMin/+ mice. Collectively, our results suggest that NSAIDs suppress colorectal tumorigenesis by enhancing immunosurveillance through ER stress and BID-dependent ICD. These results provide novel insight into the chemopreventive mechanism of NSAIDs, which may help design more effective chemopreventive strategies and agents.

#268

Combined inhibition of cyclooxygenase with docosahexaenoic acid augments apoptosis in KRAS mutant non-small cell lung cancer cells.

Felix Amissah,1 Kylee J. Jones,1 Rosemary A. Poku2. 1 _Ferris State University, Big Rapids, MI;_ 2 _Central Michigan University, Mount Pleasant, MI_.

Cyclooxygenase (COX) inhibitors are widely used in the treatment of various inflammatory disorders. Their antiproliferative effects have also been widely reported. This health benefit of COX inhibitors is limited by some potential adverse effects, including gastrointestinal irritation. Docosahexaenoic (DHA), commonly derived from fish oils, is also known to possess anticancer effects. We examined whether COX inhibitors (aspirin and diclofenac) combined with DHA would synergize their antiproliferative activity. This would allow for a decrease in the amounts of each compound used for chemoprevention, which may likely decrease the risk of adverse effects. While treatment with DHA resulted in concentration-dependent cell death in K-Ras mutant (A549 and NCI-H1573) lung cancer cells (EC50 of 5.5 µM and 9.5 µM), combination with diclofenac resulted in relatively more significant reduction in cell viability (EC50 of 3.7 µM and 3.2 µM) and cell proliferation (p<0.0001). In addition, the combination treatment was more effective in inhibiting clonogenic cell growth and inducing apoptosis with a significant increase in proapoptotic proteins (p<0.001). Furthermore, the combination treatment yielded more marked inhibition of K-Ras expression as well as altered localization in the cell membrane. These data point to the promising chemopreventive potential of combining low doses of COX inhibitors with DHA with a possible decrease in their adverse effects.

#269

Reactivation of mutant p53 in human prostate cancer cells as a critical mechanism for inhibition of tumor growth by phenethyl isothiocyanate.

Monika Aggarwal,1 Rahul Saxena,2 Nasir Asif,1 Elizabeth Sinclair,1 Judy Tan,2 Idalia Cruz,1 Deborah Berry,1 Bhaskar Kallakury,1 Quynhchi Pham,3 Thomas Ty Wang,3 Fung-Lung Chung1. 1 _Lombardi Comprehensive Cancer Center, Georgetown University, Washington, DC;_ 2 _Georgetown University, Washington, DC;_ 3 _USDA, Beltsville, MD_.

Mutations in the p53 gene occur in a wide variety of human cancers at remarkably high frequencies. Prostate cancer (PCa), the most commonly diagnosed cancer and the second leading cause of cancer-related death in men in the United States, show a significantly high frequency of p53 mutations (~30%-70%). However, the role of mutant p53 as a target for the treatment or prevention of PCa remained to be investigated. In this study, we showed that cruciferous vegetable-derived phenethyl isothiocyanate (PEITC) can reactivate p53 mutants, irrespective of its site of mutation, in PCa cells in vitro. Importantly, this study showed that dietary supplementation of PEITC can significantly inhibit DU145 p53 mutant xenograft tumor in a nu/nu Balb/c mice. Interestingly, the animals in the PEITC-treated group showed a significant decrease in the mutant p53 expression levels as compared to the animals in the control group. DU145 cells harboring p53V274F/P223L mutant display maximum sensitivity to PEITC-induced growth inhibition as compared to the wild-type p53 LNCaP and p53 null PC-3 cells. PEITC treatment also caused a significant decrease in the p53 expression level in DU145 cells. No significant inhibition of cell proliferation was detected for the normal prostate epithelial RWPE1 cells treated with PEITC. Mechanistic studies revealed that PEITC induces a conformation change and restores the transactivation functions to the p53 mutant. Accordingly, in PEITC-treated cells the reactivated p53 mutant(s) induces apoptosis by activating canonical wild-type p53 targets, inducing a G1 and S phase delay and by phosphorylating ATM. PEITC treatment of isogenic PC-3 p53 null cells revealed that PC-3 cells expressing p53P223L mutant exhibit maximum inhibition of cell proliferation, suggesting that PEITC acts in a p53P223L mutant-dependent manner. Furthermore, PEITC treatment restores the transactivation functions to p53P223L mutant as shown by the activation of p53 regulated downstream target genes. Interestingly p73, a member of the p53 gene family that shares high functional homology and regulate cell-growth/ cell-death pathways in a manner similar to p53, does not play a role in p53-mediated pathway activation in PEITC treated cells. We further showed that PEITC inhibits cell proliferation and induces apoptosis in prostate cancer cells with hotspot mutants R248W (VCaP) and R175H (LAPC-4). Importantly, PEITC can induce a conformation change, reactivates p53 mutants and induces G1 and S-phase delays in these cells. These results demonstrate that PEITC can reactivate mutant p53, irrespective of the site of p53 mutation, in human prostate cancer cells. These studies provide the first example of targeting mutant p53 in prostate cancer cells by a dietary-related compound that may have impact on the prevention and treatment strategies of this disease.

#270

Effect of dietary methylseleninic acid and Se-methylselenocysteine on carcinogen-induced, androgen-promoted prostate carcinogenesis in rats.

Maarten C. Bosland,1 Michael J. Schlicht,1 Yibin Deng,2 Junxuan Lu3. 1 _Univ. of Illinois College of Med. at Chicago, Chicago, IL;_ 2 _University of Minnesota Hormel Institute, Austin, MN;_ 3 _College of Medicine, Pennsylvania State Univ., Hershey, PA_.

Selenomethionine (SeMet) did not inhibit development of prostate cancer in the SELECT trial and in preclinical rat models. However, we and others have shown that oral intake of next-generation selenium (Se) forms, especially methylseleninic acid (MSeA) and Se-methylselenocysteine (MSeC), inhibits prostate carcinogenesis in the TRAMP mouse model and growth of human prostate cancer cells xenografted in immunodeficient mice. We recently showed that MSeA suppresses progression of high-grade prostatic intraepithelial neoplasia to adenocarcinoma in pten-deficient mice (Cancer Prev Res 2016;9:35-42). To determine whether next-generation Se forms inhibit prostate carcinogenesis in models driven by a different etiology, we tested the in vivo effect of different Se forms fed to rats undergoing chemically induced androgen-promoted prostate carcinogenesis. We previously established that SeMet was not inhibitory in this model (Cancer Prev Res 2010;3:381-92). Wistar-Unilever (WU) rats (10-12 wks) were sequentially treated with androgen receptor blocker flutamide for 21 days (10 mg/kg/day by gavage), followed by testosterone propionate (TP, 10 mg/kg, s.c) on day 22. Three days after TP, the carcinogen methylnitrosourea (MNU, 30 mg/kg/bw) was administered by i.p. injection. One week later, the rats received slow-release Silastic implants containing testosterone. Rats were then randomized to one of three groups fed the AIN-93M diet supplemented with 3 ppm of Se as MSeA or MSeC or control AIN-93M diet. Moribund animals were euthanized and the study was terminated 400 days after MNU injection. Mean survival (days ± SEM) after MNU was 344±11 in controls (n=31), 354±8 in MSeA fed rats (n=30), and 363±10 in rats given MSeC (n=29). These differences were not statistically significant, which was confirmed by survival analysis using the logrank test. Overall tumor incidence in all accessory sex glands combined (dorsolateral and anterior prostate plus seminal vesicle) was 68% in controls, 83% in MSeA rats, and 72% in MSeC rats. The incidence of large tumors of uncertain origin was 42% in controls, 40% in MSeA rats, and 41% in MSeC rats. The incidence of smaller tumors clearly confined to dorsolateral and/or anterior prostate was 19% in controls, 20% in MSeA rats, and 38% in MSeC rats (some rats had more than one tumor). None of these differences was statistically significant and there were no shifts from large to small tumor or small to large tumors. Thus, MSeA and MSeC were not preventive in this rat prostate carcinogenesis model. The contrast with the inhibitory effects of MSeA in the TRAMP and pten-deficient mouse models may be due to differences in administered dose or fundamental rat-mouse differences in Se metabolism that impact chemoprevention by Se compounds.

Supported by NIH grant CA172169.

#271

Diterpenoid pseudolaric acid B induces M-phase arrest and apoptosis primarily through JNK stress signaling in LNCaP prostate cancer cells.

Kartick C. Pramanik,1 Nehal Gupta,2 Min Ye,3 Junxuan Lu,1 Cheng Jiang1. 1 _Pennsylvania State University College of Medicine, Hershey, PA;_ 2 _Texas Tech University Health Sciences Center, Amarillo, TX;_ 3 _Peking University, Beijing, China_.

Pseudolaric acid B (PAB) is a major diterpenoid component isolated from the root bark of the traditional Chinese medicinal plant Pseudolarix kaempferi Gordon (Pinaceae). PAB has shown potential as an anticancer agent via inducing cell cycle arrest, apoptosis and DNA damage in several cancer models. To explore potential use of PAB for prostate cancer chemoprevention, we used human LNCaP prostate cancer cells that harbor wild-type p53, functional androgen receptor (AR), and defective PTEN that are characteristic of early stage prostate adenocarcinomas to study cell fates and signaling mechanisms upon PAB exposure in cell culture. Our results show that PAB inhibited LNCaP cell proliferation in a dose- and time-dependent manner (IC50 ~5.4 µM at 24 h and ~ 0.2 µM at 72 h). PAB induced M phase cell cycle arrest as confirmed by increased phosphorylation of histone H3 (p-H3 Ser10). PAB also induced apoptosis evidenced by Annexin V staining and cleavage of the canonical caspase substrate PARP. PAB at lethal exposure concentration decreased AR protein and its best-known target prostate-specific antigen (PSA). Timewise, PAB treatment increased p-H3 (mitosis marker) and the expression of another mitosis/prosurvival protein survivin several hours ahead of PARP cleavage. PAB induced p-H2A.X, which would mark DNA double-strand breaks, and the best-known DNA damage response protein P53 and its target P21Cip1 preceding cPARP. PAB stimulated phosphorylative activation of c-Jun N-terminal kinase (JNK), and the JNK inhibitor SP600125 decreased PAB-induced p-H2A.X, P53/P21Cip1 and cleavage of PARP as well as M phase arrest. Furthermore, PAB significantly increased 2',7'-dichlorofluorescin diacetate (DCF)-reactive oxygen species (DCF-ROS) in LNCaP cells and addition of antioxidant N-acetyl-L-cysteine (NAC) attenuated P53/P21Cip1 cascade and cPARP/apoptosis, but did not affect M phase arrest. Knocking-down of P53 or P21Cip1 by siRNA attenuated cPARP and apoptosis without affecting p-H2A.X. Taken together, these results suggest a rapid induction of M phase arrest by PAB that was primarily mediated by JNK stress signaling, either parallel to or upstream of ROS-DNA damage-driven P53-P21Cip1 apoptosis cascade in LNCaP cells.

#272

Effect of inositol hexaphosphate on cancer stem cell pool of prostate tumors.

Komal Raina,1 Anil K. Jain,1 Dileep Kumar,1 Vijay Mohan,1 Paul Maroni,2 Rajesh Agarwal1. 1 _Univ. of Colorado Denver School of Pharmacy, Aurora, CO;_ 2 _Univ. of Colorado Hospital, Aurora, CO_.

Prostate cancer (PCa) is the most frequently diagnosed invasive malignancy and second leading cause of cancer-associated deaths in the males in the United States. Inositol hexaphosphate (IP6) a nutrient constituting 6.4% (w/w) or even higher levels in most cereals, legumes, nuts, oil seeds and soybean has been shown to protect against growth and progression of PCa in pre-clinical animal models. Our completed studies in a spontaneous transgenic mouse model of PCa (TRAMP) have shown that IP6 feeding suppresses growth and progression of PCa via its ability to alter tumor vascularity and the energy generating metabolic events in the tumor cells. We also employed quantitative high-resolution nuclear magnetic resonance spectroscopy (1H-, 13C- and 31P-NMR) to assess the metabolic profile and energy state of the IP6-treated human PCa PC3 cell line which validated these in vivo findings. Next, we assessed stage-specific efficacy of IP6 feeding on PCa initiation and growth, progression and angiogenesis, and elucidated the molecular events involved in IP6 effects during these stages. Different groups of male TRAMP mice starting at 4, 12, 20 and 30 weeks of age were fed with regular drinking water or 2% IP6 in regular drinking water for 8-10 weeks. Study end point assessments showed that IP6 treatment results in arrest of tumor grade at earlier stages and prevents its progression to more advanced forms of the disease. In more recent studies, to determine whether these protective effects are mediated via the effect of IP6 on expansion of cancer stem cells (CSCs) pool; we next assessed whether IP6 had the potential to affect the CSC pool in TRAMP prostate tumors. We observed that the anti-PCa effects of IP6 are associated with its potential to eradicate PCa CSC pool, which is recognized to be involved in the initiation, progression, relapse, and therapy-resistance of PCa. IP6 feeding also showed inhibitory effects on the CSC associated transcription factors and signaling pathways. Next, we performed in vitro sphere cluster assays to determine IP6 effect on the self-renewal capacity of the CSCs of PCa cell lines PC3 and HMVP-2. The % of floating spheroids (prostatospheres) generated in the presence of macrophage U-937 conditioned media (with and without IP6 pre-treatment) after 1-2 weeks were determined. The results indicated an inhibitory effect of IP6 on both number and size (volume) of prostatospheres. Since formation of spheroids under specific in vitro conditions is a measure of stemness, it is evident that IP6 has the potential to target the self-renewal of CSCs in PCa cell lines. Since these mechanistic events eventually result in arrest of tumor grade at neoplastic stages, this protective effect of IP6 against PCa might have clinical implications in controlling the malignancy at an early stage. Together, these findings suggest the practical and translational potential of IP6 treatment in suppressing growth and progression of PCa in humans.

#273

**Upregulation of 15-hydroxyprostaglandin dehydrogenase expression by 15-Deoxy-Δ** 12,14 **-prostaglandin J** 2 **through inactivation of DNA methyltransferase 1.**

Hye-Ok Jang,1 Hye-Rim Kim Kim,1 Ha-Na Lee,1 Do-Hee Kim,1 Young-Joon Surh,1 Hye-Kyung Na2. 1 _Seoul National University, Seoul, Republic of Korea;_ 2 _Sungshin Women's University, Seoul, Republic of Korea_.

15-Hydroxyprostaglandin dehydrogenase (15-PGDH) catalyzes the oxidation of prostaglandin E2 (PGE2) to a biologically inactive keto metabolite 15-keto-PGE2. However, the molecular mechanisms underlying regulation of 15-PGDH expression remain largely elusive. 15-Deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2), an endogenous ligand of peroxisome proliferator-activated receptor γ, has been reported to have anti-inflammatory and anti-carcinogenic activities. In the present study, we have found that 15-PGJ2 upregulates expression and catalytic activity of 15-PGDH in human breast cancer (MDA-MB-231) cells. 15d-PGJ2 treatment decreased the CpG methylation in the 15-PGDH promoter in MDA-MB-231 cells as determined by the bisulfite genome sequencing and methyl-specific PCR. 15d-PGJ2 inhibited catalytic activity of methyltransferase 1 (DNMT1) but did not influence DNMT expression. 15d-PGJ2 increased the expression of c-Fos which is a functional subunit of AP-1. Chromatin-immunoprecipitation analysis revealed that 15d-PGJ2 significantly attenuated DNMT1 binding to the site for AP-1 transcription factor present in the 15-PGDH promoter regions, while increasing c-Fos binding. Biotin-tagged 15d-PGJ2 directly bound to DNMT1, and reduced its catalytic activity. A non-electrophilic analogue 9,10-dihydro-15d-PGJ2 failed to suppress the methylation of CpG islands present in 15-PGDH promoter and did not affect both DNMT1 activity and 15-PGDH expression. This findings suggests that the α,β-unsaturated carbonyl group of 15d-PGJ2 is essential for its inactivation of DNMT1 and expression of 15-PGDH. In conclusion, 15d-PGJ2 directly interacts with DNMT1 and consequently suppresses DNMT1-mediated hypermethylation of 15-PGDH promoter, leading to upregulation of 15-PGDH expression.

#274

**Selective chemopreventive efficacy of 1,4-phenylenebis(methylene)seleno-aspirin (** p **-XS-Asp) towards lung cancer.**

Daniel Plano,1 Srinivasa Ramisetti,1 Sang-Yub Kim,1 Manoj Pandey,2 Cesar Aliaga,1 Deepkamal N. Karelia,1 Arthur Berg,1 Carmen Sanmartin,3 Junxuan Lu,1 Shantu Amin,1 Arun K. Sharma1. 1 _Penn State Univ. College of Medicine, Hershey, PA;_ 2 _Cooper Medical School of Rowan University, Camden, NJ;_ 3 _University of Navarra, Pamplona, Spain_.

1,4-Phenylenebis(methylene)selenocyanate (p-XSC) has been shown to inhibit tobacco carcinogen NNK induced lung tumor development in several animal models. This had placed p-XSC on the National Cancer Institute´s (NCI) list of chemopreventive agents for clinical development, but there were systemic toxicity issues. p-XSC metabolizes through the formation of active bis-selenol (p-XSeH) along with the release of poisonous hydrogen cyanide (HCN). We recently developed p-XS-Asp, with a rationale that it would cleave in vivo to release the active p-XSeH and aspirin, thus making the compound less toxic and possibly more potent than p-XSC. Indeed, we previously presented (AACR Annual Meeting 2014) that p-XS-Asp inhibited NNK-induced lung tumorigenesis in A/J mice more effectively than p-XSC, and was also more tolerable. At doses of 15 ppm and 7.5 ppm Se, p-XS-Asp showed a significantly marked decrease in the percentage of lung cancer incidence in vivo with only 50% and 87% of tumor incidence, as compared to p-XSC (79% and 100%), respectively. NNK-control showed an 100% tumor incidence. Likewise, the tumor multiplicity for p-XS-Asp group was 0.87 and 1.93 tumors/mouse as compared to the NNK-control (11.53) and p-XSC (1.66 and 4.10 tumors/mouse, respectively) at the two doses tested. Notably, blood chemistry and tissue analyses did not show systemic toxicity for the p-XS-Asp fed group.

We have now evaluated the underlying mechanisms of lung cancer preventive action of p-XS-Asp and its efficacy for inhibiting azoxymethane (AOM)- and dimethyl hydrazine (DMH)-induced Aberrant Crypt Foci (ACF) in Fischer F344 rats. At a dose of 7.5 ppm Se, p-XS-Asp was able to restore the expression of several genes (MMP9, COX-2, Myc, SphK1 and RELA), that were over-expressed in the NNK group, to control or even lower levels. The AKT1 gene expression was much lower in the lung tissue of p-XS-Asp treated mice at this dose compared to both negative and NNK control groups. Therefore, p-XS-Asp might be exerting its chemopreventive effect on NNK-induce lung tumorigenesis via inhibiting COX-2 mediated PI3K/Akt signaling pathway.

Interestingly, contrary to the striking inhibition of lung tumorigenesis, p-XS-Asp failed to significantly inhibit AOM- or DMH-induced formation of ACF in Fischer F344 rats. In both AOM and DMH models, aspirin (positive control) significantly reduced the number of ACF and large ACF per area (cm2) by 37.4% and 33.8%, respectively. On the other hand, both p-XSC and p-XS-Asp showed no significant inhibitory effect on the formation of ACF and large ACF. Taken together, our results have shown p-XS-Asp to selectively prevent the lung, but not colon, tumorigenesis, and thus is a promising candidate for further development as a lung cancer preventive agent.

#275

Flavokawain B, a kava chalcone, acts synergistically with proteasome inhibitors (i.e., bortezomib and MG132) to reduce the growth of prostate cancer cells via downregulation of Skp2.

Xuesen Li,1 Victor Pham,1 Matthew Tippin,1 Shan Xu,1 Dongjun Fu,1 Liankun Song,1 Bang H. Hoang,2 Xiaolin Zi1. 1 _UC Irvine, Orange, CA;_ 2 _The University Hospital for Albert Einstein College of Medicine, Orange, CA_.

Flavokawain B, a chalcone isolated from kava root extracts, has been shown to inhibit the in vitro and in vivo growth of various cancer cell lines via induction of apoptosis. However, the mechanism of flavokawain B's action remains largely unknown. Here we have demonstrated that flavokawain B caused Skp2 degradation in an ubiquitin and proteasome dependent manner. In addition, flavokawain B inhibited NEDD8 conjugations to both Cullin1 and Ubc12 in PC3 cells and Ubc12 NEDDylation in an in vitro assay. Overexpression of dominant-negative cullin1 (1-452) or K720R mutant cullin1 rescued the effect of flavokawain B on Skp2 degradation, but further increased the expression of p27. However, siRNA knock-down of Skp1, CSN5, and UBC12 expression resulted in down-regulation of Skp2 expression and further enhanced the effect of flavokawain B on Skp2 degradation. siRNA knock-down of Cdh1, a known E3 ligase of Skp2 for targeted degradation, also didn't rescue the effect of flavokawain B on Skp2 degradation, but overexpression of F-box deleted Skp2 can reverse the effect of flavokawain B on Skp2 degradation. These results suggest that degradation of Skp2 by flavokawain B is involved in Cullin1. Furthermore, flavokawain B selectively inhibited the growth of pRb deficient cell lines and PC3 cells with Skp2 overexpression. Flavokawain B also acts synergistically with Bortezomib and MG132 to inhibit the growth of prostate cancer cell lines via down-regulation of Skp2 and up-regulation of p27 and p21 expression. These finding provide rationale for combination of flavokawain B and Bortezomib in treatment of prostate cancer.

#276

Targeting cancer stem cells with chemopreventive agents for breast cancer prevention.

Naing Lin Shan, Jeffrey Yang, Min Ji Bak, Joseph Wahler, Nanjoo Suh. _Rutgers University, Piscataway, NJ_.

Breast cancer accounts for one of the most common causes of cancer-related deaths in women. Despite the available treatment modalities to cure breast cancer, therapeutic resistance and recurrence remain major challenges, owing to the culminating evidence of the presence of cancer stem cell subpopulations in breast tumors. The purpose of the study is to identify chemopreventive and dietary compounds that effectively target cancer stem cells to inhibit breast cancer development and progression. The mammosphere culture system has been proven to enrich cancer stem-like cells, and cells from mammospheres are shown to be more tumorigenic in vivo. By culturing mammospheres from breast cancer cell lines, we have shown that 1,25 vitamin D3, a vitamin D3 analog, BXL0124, pterostilbene (trans-3,5-dimethoxy-4-hydroxystilbene), tocopherols and triterpenoids (CDDO-Im) decreased the mammosphere forming efficiency (MFE) in a dose-dependent manner. Vitamin D compounds (1,25D3 and BXL0124) decreased the MFE in SUM159, MCF10DCIS and MCF-7 breast cancer cell lines (dose range 1-100 nM). Pterostilbene, a bioactive component of blueberries, effectively decreased the MFE in the MCF-7 cell line. The size of the mammospheres was found to be smaller with increasing concentrations (10-100 μM). Tocopherols significantly decreased estrogen-stimulated MFE in MCF-7 cells (1-10 μM). CDDO-Im, a synthetic triterpenoid derived from natural oleanolic acid, has also been shown to decrease MFE and sphere size in MCF-7 cells (50-100 nM). The effect on the shape of the spheres was also observed, suggesting the differentiation-inducing properties of these compounds. Taken together, we demonstrate that naturally occurring compounds as well as synthetic analogs derived from natural and dietary compounds significantly inhibit sphere forming efficiency and cancer stemness in breast cancer cells. Our study suggests that chemopreventive compounds, 1,25D3, BXL0124, pterostilbene, tocopherols, and CDDO-Im, are agents with the potential to target cancer stem-like cells to prevent breast cancer.

#277

Gaining insights into pancreatic cancer intervention with bitter melon, a natural agent, in combination with gemcitabine using patient derived xenografts.

Deepanshi Dhar,1 Dileep Kumar,1 Stacey Bagby,2 Todd M. Pitts,2 Wells A. Messersmith,2 Komal Raina,1 Rajesh Agarwal1. 1 _Univ. of Colorado Denver, Skaggs School of Pharmacy, Aurora, CO;_ 2 _Univ. of Colorado Denver, Division of Medical Oncology, Aurora, CO_.

Pancreatic cancer (PanC) remains the 4th leading cause of cancer related-deaths in U.S. resulting in a dismal survival rate of <5%. This generates a critical need for identifying novel non-toxic agents aimed at effective PanC management with minimal patient distress. Bitter melon (Momordica charantia), a dietary agent, is actively being examined for its anti-cancer efficacy against a panel of malignancies. Recent data from our group highlights the anti-PanC potential of bitter melon juice (BMJ); its role in PanC-cancer stem cell (CSC) self-renewal/ kinetics and gemcitabine (GEM) resistance in PanC cells. GEM is the standard of care drug used in PanC therapeutics but there has been a continual increase in GEM resistant PanC patients lately. In our current study, we determined the nature of combinations for BMJ (0.5- 2% v/v) and GEM (2- 25 μM) in vitro using 3 human PanC cell lines. Combination indices (CI) determined using Chou-Talalay method for BMJ and GEM were: PANC1 (no observed interaction), AsPC1 (CI=0.5-1, leaning towards synergism) and BxPC3 (CI=0.1-0.8, synergistic). Patient-derived xenografts (PDX) are gaining importance as representatives of the genomic complexities and interactions with tumor microenvironment like primary patient tumors, thereby contributing to accelerated therapeutic approaches. Subsequently, we focused on assessing the efficacy of BMJ alone, or in addition to GEM using the PanC-PDX mouse model. Treatment regimen had 4 cohorts: Controls (untreated); BMJ alone- 200mg/Kg; GEM alone- 50mg/Kg; and Combo (BMJ+GEM). BMJ efficacy was examined in 3 human PanC-PDX explants in nude mice, denoted by PanC-PDX 272, 266 and 271, alongside GEM, and Combo. Tumor volumes recorded post first part of study (~day 35; first sacrifice time-point) showed significant tumor growth inhibition in all treated groups. GEM and Combo displayed comparable efficacies by decrease in tumor volume (~70-80% decrease), followed by BMJ (~50-60% decrease). Interestingly, tumor regrowth determination with subsequent treatment washout period (~30 days), showed maximum tumor regrowth in GEM-treated mice, nearing the untreated controls. BMJ consistently showed a prolonged effect by containment of tumors until the study end (~day 64; second sacrifice time-point). Combo tumors remained close to BMJ, suggesting no apparent antagonistic interference. Notably, at study completion (day 64), BMJ or Combo displayed the most significant and sustained effect on tumor volumes (~60-80% decrease for BMJ, 80-90% decrease for Combo) compared to GEM (~40-50% decrease), after treatments were stopped. Together, these results point to the chemopreventive efficacy of BMJ as a monotherapeutic as well a perfect combinatorial candidate in studies with chemotherapeutic agents like GEM aimed at PanC management utilizing the resourceful PanC-PDX mouse model.

#278

Inhibition of skin tumor promotion by TPA using a combination of topically applied ursolic acid and curcumin.

Lisa Tremmel, Okkyung Rho, John DiGiovanni. _UT Austin College of Pharmacy, Austin, TX_.

Despite the many advances that have been made in understanding cancer development and the discovery of new therapies, cancer still remains the second leading cause of death in the US. In addition, approximately two out of five people are expected to be diagnosed with an invasive cancer in their lifetime. Prevention remains an important strategy to reduce the burden of cancer. One approach to prevent cancer is the use of phytochemicals in various combinations as safe and effective cancer preventive agents. The purpose of this study was to examine the effects of the combination of ursolic acid (UA) and curcumin (Curc) for potential synergistic chemopreventive activity to inhibit tumor promotion using the mouse two-stage skin carcinogenesis model. UA and Curc alone have been shown to inhibit tumorigenesis in several mouse models, and both have been shown to inhibit mouse skin tumor promotion by 12-O-tetradecanoylphorbol-13-acetate (TPA). The anti-cancer activity of these phytochemicals is due primarily to their anti-inflammatory, anti-oxidant, and anti-proliferative effects. These effects are accomplished by acting on a broad spectrum of signaling pathways. In initial short-term experiments (mice treated twice weekly for two weeks with TPA), the combination of UA and Curc pretreated topically inhibited TPA-induced activation of epidermal NF-κB, Cox-2, epidermal growth factor receptor (EGFR) and Stat3 to a greater extent than either compound alone. Additionally, these compounds increased levels of programmed cell death protein 4 (Pdcd4). The alterations in these signaling pathways by the combination of UA and Curc were associated with decreased epidermal proliferation and skin inflammation as assessed by measuring BrdU incorporation and inflammatory gene expression, respectively. Furthermore, results from skin tumor experiments (mice initiated with DMBA and promoted twice weekly with TPA) indicate that the combination of UA and Curc given topically significantly inhibited mouse skin tumor promotion by TPA to a greater extent than the individual compounds given alone. The greatest effects were seen on tumor free survival and on tumor size. These results suggests the potential for the combination of UA and Curc to be used topically for chemopreventive strategies in non-melanoma skin cancer.

#279

Cranberry proanthocyanidins mitigate reflux-induced transporter dysregulation in esophageal adenocarcinoma.

Katherine M. Weh,1 Bridget A. Tripp,2 Jennifer L. Clarke,2 Amy B. Howell,3 Jules Lin,1 David G. Beer,1 Andrew C. Chang,1 Laura A. Kresty1. 1 _University of Michigan, Ann Arbor, MI;_ 2 _University of Nebraska - Lincoln, Lincoln, NE;_ 3 _Rutgers University, Chatsworth, NJ_.

Esophageal adenocarcinoma (EAC) incidence rates have increased sharply (>500%) throughout the Westernized world since the 1980s, despite the widespread use of endoscopy and anti-reflux medications. Reasons for the rapid increase in EAC are still being unraveled; however, persistent, symptomatic reflux of gastric and duodenal contents, known as gastroesophageal disease (GERD), is considered the strongest risk factor. Our laboratory utilizes the rat esophagogastroduodenal anastomosis (EGDA) surgical model of reflux-induced EAC to evaluate mechanisms by which cranberry proanthocyanidins (C-PAC) delivered in the drinking water inhibit reflux-induced EAC. Findings show that C-PAC inhibits EAC formation by 83% with concomitant restoration of the gut microbial profile and significant reduction of primary and secondary bile acid metabolites in the esophagus of animals with reflux-inducing surgery, but through unknown mechanisms. Herein, we investigated whether transporters, important in bile acid transport, buffering capacity, excretion, and even resistance to anticancer agents, were restored by C-PAC in the context of reflux. Additionally, we utilized a publicly available GEO dataset (GSE37203) to assess transporter expression levels in patients with metaplastic Barrett's esophagus (BE) versus those with high grade dysplasia (HGD) or EAC for translational relevance. Methods included esophageal RNA isolation, transcriptome sequencing using the Illumina HiSeq platform, followed by validation utilizing the PrimePCR Drug Transporter array plate and database mining. Results show significant changes in solute carriers (SLC), ATP-binding cassette (ABC) and aquaporin transporters in the rat reflux-induced model for EAC with C-PAC mitigating alterations. EGDA altered one or more members in 71% of the SLC families with frequent changes observed in SLC25, SLC4, SLC35, SLC2 and SLC6. C-PAC favorably impacted >40% of the EGDA-induced SLC changes. A number of ABC transporters involved in glutathione transport and also implicated in drug resistance were altered by EGDA including Abcc1, Abcc3, Abcc4, Abcc5 and Abcg2 and C-PAC mitigated these changes. Aquaporins, Aqp3 and Aqp4, were significantly modulated in EGDA and restored with C-PAC. Significant differences in ABCC9, ABCC10, AQP6 and AQP9, in addition to several members of the SLC25, SLC4, SLC35, SLC2 and SLC6 families, were observed in patients with HGD/EAC compared to those with BE metaplasia. Therefore, altered expression of transporters following reflux-inducing surgery or GERD is likely a defensive mechanism by which cells attempt to adapt or protect from injurious bile acid exposure. Further research is warranted to investigate agents that restore normal transporter function, which may serve to concomitantly improve epithelial barrier function and mucosal integrity in the context of esophageal cancer progression. 

## BIOINFORMATICS AND SYSTEMS BIOLOGY:

### Integrative Cancer Biology 1

#281

Identifying genetic variation and cellular heterogeneity with a comprehensive cancer analysis toolkit.

Sarah Garcia, Rajiv Bharadwaj, Stéphane Boutet, Claudia Catalanotti, Valeria Giangerra, Josephine Lee, Jessica Terry, Stephen Williams, Grace X. Zheng, Tarjei Mikkelsen, Michael Schnall-Levin, Ben Hindson, Deanna M. Church. _10x genomics, Inc., Pleasanton, CA_.

Understanding disease development, progression and response to treatment remains elusive for most types of cancer. This is due both to the genetic complexity seen in tumors as well as differences in how the surrounding cells and immune system respond to the developing tumor. To fully understand all aspects of disease progression and response to treatment, a systems level approach is needed. We have developed a platform that allows for high resolution multi-omics analysis of the tumor and its microenvironment. This platform is built upon a microfluidic system that enables the production of hundreds of thousands to millions of partitioned barcoded reactions. On this platform we generate Linked-Reads that allow for the detailed resolution of complex structural variant events. To obtain more detailed information about tumor heterogeneity and clonal structure, we also perform single cell DNA analysis that allows for the detection of sub-megabase copy number variants at the single cell level.The platform also provides detailed resolution of the cells occupying the microenvironment surrounding the tumor. Using a combination of single cell gene expression and enriched V(D)J B and T cell repertoire analysis, we identify cells constituting a tumor, as well as the immune and nonimmune cells surrounding the tumor. We also provide single cell profiling of the paired antigen receptor chains for both B and T cells. As a demonstration, we have performed this analysis on three types of tumor samples: metastatic melanoma (MM), primary colorectal cancer (CRC) and primary clear cell renal carcinoma (CCRC). We are able to identify a clonal expansion in the CCRC sample and determine there is no expansion in the CRC sample. The combination of these approaches allows for unparalleled characterization of disease development, progression and response.

#282

Inferring transcriptional regulatory programs in gynecological cancers.

Hatice U. Osmanbeyoglu,1 Petar Jelinic,2 Douglas Douglas,2 Christina Leslie1. 1 _Mem. Sloan Kettering Cancer Ctr., New York, NY;_ 2 _New York University Langone Medical Center, New York, NY_.

Cancer cells acquire genetic and epigenetic alterations that often lead to dysregulation of oncogenic signal transduction pathways, which in turn alter downstream transcriptional programs. Transcription factors (TFs) are the main link between signaling pathways and the transcriptional regulatory programs. The Cancer Genome Atlas (TCGA) has studied several of the most common and aggressive gynecologic tumors including high-grade serous ovarian carcinomas (HGSOC), uterine carcinosarcoma (UCS), and the serous-like subset of endometrial cancer (UCEC), together with basal breast cancer, which shares many genomic features with serous ovarian tumors. TFs impacts on gene regulation have not been well characterized in gynecological and basal breast cancers. The majority of these tumors lack accurate predictors of response and resistance and share an unmet need for adequate treatment of recurrent disease. We developed a multitask learning framework for integrating regulatory sequence from ATAC-mapped promoters and enhancers from cell line models with RNA-seq data from patient tumors in order to infer transcription factor (TF) regulatory activities and explore similarities and differences between uterine, ovarian, and basal breast tumors. We showed that our multitask learning framework enables us to selectively share the information across tumors and strongly improves the accuracy of gene expression prediction models for gynecological and basal breast tumors. Our analysis identified histologic type specific and common TF regulators of gene expression as well as predicted distinct dysregulated transcriptional regulators downstream of somatic alterations in these different cancers. Moreover, many of the identified TF regulators were significantly associated with survival outcome within the histological subtype. Computationally dissecting the role of TFs in these cancers may ultimately lead to new therapeutics tailored to groups of subtypes or individuals.

#283

Identification of a high-risk subgroup in primary prostate cancers presenting with targetable immune biology.

Emma Reilly,1 Andrena McCavigan,1 Steven M. Walker,1 Nuala McCabe,1 Eileen Parkes,2 Denis P. Harkin,1 Richard D. Kennedy,1 Laura A. Knight1. 1 _Almac Diagnostics, Craigavon, United Kingdom;_ 2 _Queen's University Belfast, Belfast, United Kingdom_.

Background Recent studies have demonstrated limited success of immune checkpoint therapies in unselected prostate cancer. We therefore assessed an immune based DNA Damage Repair Deficiency (DDRD) assay, that we have previously reported represents activation of the cGAS STING pathway in the TCGA dataset of primary prostate cancers, to investigate the presence of targetable immune biology in prostate cancer. In addition we applied a second assay (the prostate cancer metastatic signature-PCM) that predicts risk of metastatic recurrence for early prostate cancer to assess if immune therapy could have a role in treating high risk disease. Methods 498 samples in the TCGA dataset with RNA sequencing data were scored with the PCM and DDRD assays. Integrative analysis was performed on 488 of those samples with matched RNA sequencing, promoter site methylation, somatic mutation and somatic copy number variation. Gene expression of n=6 immune checkpoint targets was investigated with the subgroups identified using T-tests. The prevalence of immune infiltration in each subgroup was tested by applying a cut off to the leukocyte fraction. The viability of reproducing those subgroups with RNA sequencing alone was tested in the TCGA dataset and an independent validation dataset of 321 resected primary prostate cancers. Cox proportional hazards regression analysis was performed for biochemical recurrence and metastatic events in both datasets. Results Integrative analysis of the TCGA dataset identified four patient subgroups characterised primarily by variances in copy number and genomic mutation. One of these subgroups 'Metastatic-like DDRD' had significantly higher PCM scores and DDRD immune scores compared to the other subgroups (p < 2E-12). This subgroup of patients showed elevated leukocyte fraction and expression of immune checkpoint genes: CD274 (PDL1), CTLA4, ICOS, IDO1, HAVCR2 (TIM3) & LAG3 (p < 2E-6). Genomic instability with amplification of 8q and a larger prevalence of somatic mutations including that of TP53 was also detected in this subgroup. The 'Metastatic-like DDRD' subgroup was found to have a significant association with poor survival outcome in TCGA (multivariable: p < 0.008), a result that has also been replicated in an independent dataset in both univariate (p < 0.001) and multivariable analysis (p < 0.01). Conclusions We have identified and validated a poor prognostic subgroup, representing 10-20% of early prostate cancer patients that are at increased risk of developing metastatic disease and present with targetable immune biology. These patients may represent a viable target population for immune checkpoint and DNA damaging therapies in prostate cancer.

#284

Integrated proteogenomic analysis of laser microdissected primary breast tumors define proteome clusters.

Praveen-Kumar Raj-Kumar,1 Tao Liu,2 Lori A. Sturtz,1 Albert J. Kovatich,3 Marina A. Gritsenko,2 Vladislav A. Petyuk,2 Brenda Deyarmin,1 Viswanadham Sridhara,1 James Craig,1 Jason E. McDermott,2 Anil K. Shukla,2 Ronald J. Moore,2 Matthew E. Monroe,2 Bobbie-Jo M. Webb-Robertson,2 Jeffrey A. Hooke,3 J.Leigh Fantacone-Campbell,3 Leonid Kvecher,1 Jianfang Liu,1 Jennifer Kane,1 Jennifer Melley,1 Stella Somiari,1 Stephen C. Benz,4 Justin Golovato,4 Shahrooz Rabizadeh,5 Patrick Soon-Shiong,5 Richard D. Smith,2 Richard J. Mural,1 Karin D. Rodland,2 Craig D. Shriver,6 Hai Hu1. 1 _Chan Soon-Shiong Institute of Molecular Medicine at Windber, Johnstown, PA;_ 2 _Pacific Northwest National Laboratory, Richland, WA;_ 3 _Clinical Breast Care Project, Murtha Cancer Center, Uniformed Services University/Walter Reed NMMC, Bethesda, MD;_ 4 _NantOmics, Culver City, CA;_ 5 _NantWorks, Culver City, CA;_ 6 _Murtha Cancer Center, Uniformed Services University/Walter Reed NMMC, Bethesda, MD_.

Introduction: Breast tumors have 4 well-established intrinsic subtypes based on transcriptome profiling. However, clusters defined by proteomics are often in disagreement with those defined by transcriptomics. Here, we report the findings of proteogenomic profiling of 118 laser microdissected (LMD) breast tumors using RNA-Seq and mass-spectrometry (MS)-based proteomic technologies.

Methods: Cases used in this study were drawn from the Clinical Breast Care Project, with patients consented using an IRB-approved protocol. A total of 118 primary breast tumors embedded in OCT were selected and processed by LMD. Total RNA and protein were extracted using the Illustra triplePrep kit. Paired-end RNA sequencing of 118 cases was performed using the Illumina HiSeq platform, and the reads were preprocessed using a PERL-based pipeline involving the preprocessing tool PRINSEQ, splice-aligner GSNAP and HTSeq for quantifying expression. Quantitative global proteomics analyses were performed on 113 cases using isobaric TMT 6-plex labeling with the "universal reference" strategy. MS data were acquired using a Q-Exactive instrument and analyzed using Proteome Discoverer with Byonic node. Sample-to-sample normalization was conducted to remove pipetting errors and ComBat was used to remove batch effect. K-means clustering was done using Bioconductor package Consensusclustering.

Results: The number of preprocessed RNA sequencing reads for the 118 cases ranged from over 43 to 295 million. An average of 83% of reads was mapped, and 24,518 genes with a mean expression of ≥ 10 counts across 118 tumor samples were identified. The PAM50 algorithm was used for intrinsic subtyping, yielding 37 Basal-like, 16 HER2-enriched, 39 Luminal A and 26 Luminal B calls. Unsupervised clustering of 3,000 highly varying genes reflected 4 intrinsic subtypes. In the global proteomics data, 840 proteins were identified across all 113 cases. Unsupervised K-means consensus clustering on all 840 or just using the top 210 highly varying proteins indicated the optimal number of clusters to be 3. These 3 clusters were identified as Basal-enriched, Luminal A-enriched and Luminal B-enriched. HER2-enriched cases were distributed among these clusters.

We did not observe a stromal-enriched cluster in this analysis of LMD-prepared samples that selected against stromal components of the tumor.

Conclusion: Analysis of LMD breast tumors using proteogenomic technologies resulted in 3 clusters for proteome data: basal-enriched, luminal A-enriched and luminal B-enriched. Unlike a recent report on proteomics clustering using bulk processing of tumors, a stromal-enriched cluster was not observed in this analysis which excluded stromal components of the samples.

The views expressed in this abstract are those of the author and do not reflect the official policy of the Department of Army/Navy/Air Force, Department of Defense, or U.S. Government.

#285

Uncovering the disorder of breast invasive carcinoma homeostasis proteins.

Julia V. Castro, Evan O. Paull, Jose I. Garzon, Diana Murray, Andrea Califano, Barry Honig. _Columbia University Medical Center, New York, NY_.

Breast cancer is one of the most common cancers among women worldwide. Recent studies suggest that cancer cell homeostatic stable phenotypes may be implemented, controlled and maintained by Master Regulators (MR) proteins. In essence, all MR proteins are Transcription Factors, which frequently are intrinsically (IDP) or partially disordered proteins, depending on the extent of disordered regions (IDRs). IDPs/IDRs are known to be promiscuous binders that commonly function as central hubs in protein networks. Furthermore, they contain Molecular Recognition Features (MoRFs) and/or Short Linear Motifs (SLiMs), regions that occupy a unique structural and functional niche in which function is a direct consequence of intrinsic disorder. Gathering information on MR protein interactome has significant implications for understanding the molecular basis of the mechanisms responsible for the stability of tumor cell states. In this work, we focused on a subset of 128 MR proteins predicted from RNA-Seq profiles to be responsible for the Breast Invasive Carcinoma (BRCA) tumor homeostatic control across five distinct cancer subtypes and analyzed their disorder prevalence, functional importance, and potential roles in protein association networks. The disorder profile annotation was performed using the MobiDB-lite disorder meta-predictor. The proteins were classified according to the percentage of intrinsically disordered residues (PIDR) as highly ordered (PIDR < 10%), moderately disordered (10% ≤ PIDR < 30%), and highly disordered (PIDR ≥ 30%). We also evaluated target proteins using the CH-CDF plot, which is a combination of two binary disorder predictor methods: the charge-hydropathy plot (CH), that considers charge and hydrophobicity of amino acids, and the cumulative distribution function (CDF) plot, which is based on the distribution of per-residue disorder predictor scores. Disorder functional annotation was performed by mapping MoRFs and SLiMs using experimentally curated databases. The PrePPI database was used to predict interactivity of the BRCA MR proteins by constructing five cancer-specific protein-protein interaction (PPI) networks. Statistical significance of connectivity for the subtype interactomes was measured by the network-based distance method. Annotation of abundant/ housekeeping genes and network topology (hubs and bottlenecks) was introduced to correlate disorder, gene essentiality, and promiscuous binding activity. The result of this work is a comprehensive, functionally annotated inventory of the intrinsically disordered status of BRCA MR proteins. Our work revealed that the master regulator candidates are enriched in moderate (44%) and high (28%) intrinsic disorder and provide the groundwork for experimental studies that may uncover novel pathways associated with the aberrant protein activity that determine stable tumor states and predict IDPs as novel drug targets.

#286

Transcriptomic features predicting drug sensitivity and resistance in acute myeloid leukemia.

Ashwini Kumar,1 Disha Malani,1 Bhagwan Yadav,2 Mika Kontro,2 Matti Kankainen,1 Swapnil Potdar,1 Simon Anders,3 Kimmo Porkka,2 Olli Kallioniemi Kallioniemi,1 Caroline Heckman1. 1 _Institute for Molecular Medicine Finland (FIMM), University of Helsinki, Helsinki, Finland;_ 2 _Haematology Research Unit Helsinki, University of Helsinki, Helsinki, Finland;_ 3 _Center for Molecular Biology of University of Heidelberg (ZMBH), Heidelberg, Germany_.

Acute myeloid leukemia (AML) is an aggressive, heterogeneous disease with poor survival after disease recurrence. Although new targeted therapies have recently been approved for specific AML subtypes, the majority is treated with conventional cytotoxic therapy with variable outcome. To identify novel therapies, we performed comprehensive ex vivo drug sensitivity testing with 515 drugs and RNA sequencing on 127 AML patient samples, allowing us to identify associations between transcriptomic profiles and drug responses. Bone marrow or peripheral blood mononuclear cells (MNCs) were collected from diagnostic (n=66), relapsed (n=39) and refractory (n=22) AML patients. RNA was prepared, sequenced and analyzed as described previously (PMID:28818039). Drug sensitivity and resistance testing was performed on the MNCs with 515 approved and investigational oncology chemical compounds (PMID:24056683). To identify expression profiles associated with drug response, generalized linear regression and elastic net regression models were applied. In the analysis, confounding factors including the patient's gender, RNA extraction and library preparation methods were taken into account. Elastic net regression analysis resulted in significant (FDR<0.1) positive or negative associations between 1110 genes and 105 drugs. Clustering of the genes depicted 4 major hubs where drugs with the same mode of action grouped together, e.g. chemotherapeutics, BCL-2, and FLT3 inhibitors. Functional enrichment analysis of each hub using DAVID tool revealed genes involved in regulation of cell proliferation (43 genes, FDR=9.19E-18), which can be explained by the cytotoxic chemotherapy drugs. Genes involved in cell death (68 genes, FDR=1.67E-5) were associated with BCL-2 inhibitor response, while genes involved in cell surface receptor linked signal transduction (51 genes, FDR=2.33E-11) were associated with FLT3 inhibitors. The fourth hub was enriched with cell adhesion (45 genes, FDR=1.11E-21) and focal adhesion (50 genes, FDR=5.11E-28), specifically integrin family (29 genes, FDR=3.17E-18) genes, that play an important role in drug resistance and were negatively correlated with cytotoxic drugs. Finally, the linear regression analysis revealed significant positive correlation between tyrosine kinase inhibitors (sorafeninb, sunitinib, tivozanib) and FLT3LG and KITLG and negative correlation with BEX2 and BEX5 genes. Regression analysis for MEK inhibitors resulted in expected positive correlation with RRAS and JAK2 gene expression. In conclusion, identifying associations between transcriptomic profiles and drug responses may reveal clinically actionable drugs for AML patients characterized by specific molecular features. Our results indicate potential gene expression biomarkers for key targeted drugs, which can be used to identify AML patients likely to benefit from these therapies.

#287

Germline variants and somatic mutation signatures in breast cancer across populations of African and European ancestry.

Shengfeng Wang,1 Jason J. Pitt,2 Yonglan Zheng,2 Toshio F. Yoshimatsu,2 Guimin Gao,2 Markus Riester,3 Ayodele Sanni,4 Olayiwola Oluwasola,5 Dominic Fitzgerald,2 Temidayo Ogundiran,5 Chinedum Babalola,5 Abiodun Popoola,6 Adeyinka Falusi,5 Wendy Winckler,3 John Obafunwa,4 Oladosu Ojengbede,5 Nasiru Ibrahim,4 WABCS Working Group, Jordi Barretina,3 Kevin P. White,2 Dezheng Huo,2 Olufunmilayo I. Olopade2. 1 _Peking University, Beijing, China;_ 2 _University of Chicago, IL;_ 3 _Novartis Institutes for BioMedical Research, MA;_ 4 _Lagos State University Teaching Hospital, Nigeria;_ 5 _University of Ibadan, Nigeria;_ 6 _Lagos State University, Nigeria_.

Background: Somatic mutation signatures may represent the footprints of genetic and environmental exposures that cause different types of cancer. However, few studies have comprehensively examined the association between germline variants and somatic mutation signatures, and none in African ancestry populations. Methods: Study samples consist of 1,011 breast cancer patients from The Cancer Genome Atlas (TCGA; 168 Black, 737 White, and 106 other ancestries, discovery set) and 170 Nigerian women with breast cancer (validation set). SomaticSignatures was employed to extract mutation signatures from whole-genome or whole-exome sequenicng data, and to estimate contributions of signatures on each sample. SNPtest and SKAT-O were used to conduct association tests for the association between somatic signatures and common single nucleotide polymorphisms (SNPs) or rare deleterious variants. SNP array-based heritability was calculated by GCTA. Results: Nine stable signatures were inferred in total. Four of them -- APOBEC C>T, APOBEC C>G, aging, and homologous recombination deficiency (HRD) -- were highly similar to known COSMIC signatures. These together explained the vast majority (60%~85%) of mutation signature contributions regardless of race/ethnicity or tumor subtype. The heritability estimates displayed significantly heritable components for APOBEC C>T signature (h2 = 0.575, P = 0.010) and combined APOBEC signature (h2 = 0.432, P = 0.042). In TCGA dataset, seven common SNPs on/near GNB5 were significantly associated with an increased proportion (beta = 0.33, 95% CI = 0.21~0.45) of APOBEC signature contribution at genome-wide significance, while rare deleterious variants in MTCL1 and HIV-EP1 were also significantly associated with this signature contribution (P =1.9×10-6, 2.2×10-5, respectively). The combined rare deleterious variants in BRCA1 and BRCA2 were significantly correlated with the contribution of HRD signature (P =1.0×10-8, 1.1×10-5, respectively). In the Nigerian cohort, only the associations in HIV-EP1 and BRCA2 were replicated with relatively higher mutation frequencies (N = 9, 8; P = 0.05, 0.01, respectively). In addition, the combined rare deleterious variants in HRD pathway genes was significantly associated with the contribution of HRD signature in both TCGA (P = 2.8×10-5) and Nigerian samples (P = 0.01). After excluding BRCA1/2, this association turned to be nonsignificant. Conclusion: Our study identified associations between germline variants and mutational patterns in breast cancer across different ethnicities, especially in African women for the first time. This finding has important public health implications as it provides evidence to substantiate causal links between germline genetic variants and carcinogenesis process. Further replications in larger and diverse populations are needed.

#288

Genomic profiling of metastatic gastric adenocarcinoma.

Kazuto Harada, Shumei Song, Yan Xu, Brain D. Bagewell, Melissa P. Pizzi, Jiankang Jin, Ying Wang, Ailing W. Scott, Lang Ma, Fatemeh G. Amlashi, Makoto Kobayashi, Jody V. Vykoukal, Cristina Ivan, Jeannelyn S. Estrella, Sinchita R. Chowdhuri, George A. Calin, Samir Hanash, Ju-Seog Lee, Bin Liu, Jaffer A. Ajani. _UT MD Anderson Cancer Ctr., Houston, TX_.

Objectives: Metastatic gastric adenocarcinoma (mGAC) is highly resistant to therapy and is incurable. Particularly, peritoneal carcinomatosis leads to severe morbidities and short survival. The genome wide approaches have been conducted in primary GAC and have uncovered useful information. However, detailed understanding of mGAC is limited. Thus, we aimed to characterize the genomic landscape of mGAC.

Methods: Thirty-four malignant ascites specimens were prospectively collected from at The University of Texas MD Anderson Cancer Center (Houston, USA) from February 2016. 25 specimens had whole exome sequencing and 30 specimens had RNA sequencing.

Results: The top highly mutated genes in mGAC were TP53, ATM, SYNE2, TTN, CDH11, FAT4/3, KMT2C, FGF23, NCOR1, RP1, CYP2A, and ADAMTS8/9. Compared with mutations in primary GAC (TCGA and TMUCIH), mutations in mGAC shared only 13.7% with TCGA and 10.9% with TMUCIH data. The commonly shared mutations in all three sets were TP53, ATM, FAT4, CDH1, SYNE1, PLEC, PRKDC, and ZFHX3, while unique mutations in mGAC were TTN, SYNE2, ZNF503, PUS7L, PPP2R5C, NBPF12, MROH1, IFNL1, and COL18A1. Furthermore, RNA- sequencing revealed two frequent fusions; 36.7% of samples with C15orf57-CBX3 (t(15;7)) fusion and 10% with SLC35A1-TSHZ2 (t(6;20)). We explored gene signatures in C15orf57-CBX3 fusion subgroup and C15orf57-CBX3 non-fusion subgroup. Gene set enrichment analysis revealed that C15orf57-CBX3 fusion group had enrichment of immune-response related genes, while non-fusion subgroup had enrichment of genes related to the segregation of chromatid, DNA replication, and histone exchange. The clustering analysis of RNA expression using the top 2000 genes with most median absolute deviation identified two subgroups that correlated either with C15orf57-CBX3 fusion or mutations.

Conclusions: We have identified a novel genomic landscape of mGAC, such as unique mutation and fusions. Further studies are needed to gain insights into the mechanism of progression and resistance phenotype of mGAC.

#289

Probing the non-enhancing component of glioblastoma: Targeting what is left behind.

Sen Peng,1 Rebecca Halperin,1 Harshil Dhruv,1 Sara Byron,1 Christophe Legendre,1 Joanna Phillips,2 Michael Prados,2 Michael Berens,1 Nhan Tran3. 1 _Translational Genomics Research Institute (TGen), Phoenix, AZ;_ 2 _University of California San Francisco, San Francisco, CA;_ 3 _Mayo Clinic Arizona, Scottsdale, AZ_.

While genomic profiling and therapeutic selection support individualized GBM treatment, such therapeutic decision-making is usually made with reference to tumor obtained from the enhancing core region. GBM is known to be heterogeneous and exhibits a high resistance to standard therapies. To address whether non-enhancing tumor (representing the majority of tumor left behind after surgery) shows distinct genomic characteristics and therapeutic targets compared to the enhancing tumor core, we performed genome-wide exome-sequencing and RNA-sequencing for 12 patients with matched enhancing region and at least one non-enhancing region. Non-enhancing biopsies show a surprisingly high level of tumor content, with a median of 28% tumor cells and 6 of the 22 samples having greater than 50% tumor cells. Cognate non-enhancing and enhancing specimens demonstrated overall concordance in therapeutically actionable alterations (single nucleotide variants) and copy number alterations. However, non-enhancing regions were not genetically identical and did reveal additional and distinct variants compared to enhancing cores. For example, the non-enhancing region of patient 1 showed two nonsense NF1 mutations (R1534X; R2517X) while the enhancing region showed an NF1 frameshift mutation (F1247fs). Clonality analysis by LumosVar also indicated that 7 out of 12 patients harbored dissimilar cellular prevalence patterns between enhancing and non-enhancing regions. In addition, comparison of alternative polyadenylation between enhancing and non-enhancing regions uncovered distinct 3' UTR usage: e.g. SGMS2 and TOB1 tended to have longer 3' UTR in enhancing regions whereas longer 3' UTR of SYNPO and NOS1AP were

prevalent in non-enhancing regions. We posit that the enhancing component of glioblastoma probably underrepresents the genomic alterations in patients' tumors. Given non-enhancing tumor is left behind after surgical debulking, genomic profiling of this region would potentially reveal more accurate tumor vulnerabilities and lead to more effective therapy.

Supported by a grant from the Ben & Catherine Ivy Foundation.

#290

Integrative analytics: A framework for precision medicine.

Darragh G. McArt, Seedevi Senevirathne, Aideen Roddy, Jessica Black, Alan Gilmore, Suneil Jain, Philip Dunne, David Waugh. _Queen's University Belfast, Belfast, United Kingdom_.

Technology advancements have enhanced our abilities to gain greater insight to the tumor environment. However, such emergent methods have brought new considerations in data storage, access and analysis. Modern large data projects and clinical trial materials could be explored to a greater degree if appropriate infrastructure could be built to support efforts. Such a scaffold would be an integrated and dynamic framework where novel hypotheses could be investigated in modern big data collections. Placing discovery back in the hands of the researcher through a reactive and supportive framework will enhance our understanding of cancer aetiology. The Cancer Integromics Research Application Framework (CIRAFm) was created to emulate current platforms that have a rigid analytical interface. We designed a robust architecture that supports a reactive user-friendly interface blended to several cross-platform coding technologies. It has been developed to accommodate an individualised framework where we can create an 'app-store' of key software to fit research questions. In efforts to encapsulate and accelerate differing data types it sits astride of two NoSQL based database management systems, minimising data redundancy. Initial requirements have begun to create algorithms to investigate alignment-free applications on next generation sequencing (NGS) data enhancing analysis of spatial and temporal heterogeneity in cancer. Key drivers can be explored by assisting software to build correlative marker associations using Darwinian approaches such as genetic algorithms. Information requirements from external platforms are assisted through a novel domain specific language (DSL) to enable a singular interface. The modularised architecture of the platform is enabled through an Angular framework supported by interactive and dynamic data visualisation software, D3.js. Data analytics can be explored through the 'apps' created, investigating new markers in large data and enhancing our understanding of tumor heterogeneity. Alignment-free phylogenetics of NGS data harnesses our capabilities to display fully sequence evolution in patient data. This highlights possible sub-types, markers of interest and maps to therapeutic compounds via the DSL to our externally developed drug discovery software, QUADrATiC. Future scientific endeavours further defining new tumor subtypes are paramount in efforts to help uncover treatment strategies. Unburdened by legacy pipelines and data, flexible and robust models of architecture provide an effective and efficient framework for research on our ever increasing search for precision medicine.

#291

Building a program in cancer bioinformatics and computational biology: A balancing act.

John N. Weinstein, and the Department of Bioinformatics and Computational Biology. _The University of Texas MD Anderson Cancer Center, Houston, TX_.

Bioinformatics is a transdisciplinary field based on statistics, machine learning, computer science, biology, and medicine. Computational biology can be considered a subfield of bioinformatics in which computation is analogous to experiments in laboratory science; the emphasis is on the biologist's endpoint (1). There is no standard blueprint for a program in bioinformatics, but perhaps our experience in building the Dept. of Bioinformatics and Computational Biology (DBCB) at UT MD Anderson Cancer Center (MDACC) can offer some instructive ideas, particularly a set of balances that must be struck.

Current Personnel (106):

• Faculty (15) plus visiting professors

• Statistical analysts (17); BioAnalysts (a new job description) (2)

• Software engineers in-house (6); on contract (4)

• IT staff (4)

• Wet laboratory staff (14)

• Graduate students (15), postdoctoral fellows (22)

• Administrators (7)

Essential goals and metrics:

• Excellence in academic research: Faculty averaging 9.2 authorships each (in 2016), including 14 overall with IF > 20.

• Excellence in Bioinformatics support for projects and programs: In 2016, analysts working on 376 projects for 122 PIs in 50 MDACC departments

• Excellence in Education: Teaching in the UT Graduate School of Biomedical Science, etc.

• Development of cutting edge bioinformatics software tuned for biologists and clinical researchers (e.g., NGCHMs, MBatch, TCPA, Tanric, MuSE, TransVar, SpliceSeq, VirusSeq, SoS)

• Excellence in career development for all: Intensive mentoring

Balances to be struck:

• Academic research vs. collaborative support: Faculty supervise support, but analysts (17 of them PhDs) do most of it, sharing expertise (and authoring 4.1 authorships each/yr). Faculty's academic projects often flow from collaborative support interactions.

• Centralized vs. distributed system for support: We would wish for (a) total immersion of bioinformatics personnel in the basic, translational, or clinical domain being supported; (b) total immersion in the relevant technology and its analytical tools; and (c) total immersion in the bioinformatics community for sharing of expertise, continuing career development, and morale. Clearly, a compromise solution is necessary. Our (imperfect) answer is a hybrid system that emphasizes longitudinal association of faculty and analysts with the biomedical domain department or program supported.

• Pure computational vs. combined computational/laboratory research: 5 of 14 faculty have combined programs. There is career benefit but also a cost in time and focus.

• Investigator-initiated projects vs. projects that originate from interactions within the institution vs. national/international projects: Our answer has been a self-chosen mix, with deep participation and leadership in NIH projects, notably TCGA.

Reference:

1. Weinstein JN. Cancer bioinformatics. In Holland-Frei Cancer Medicine, 9th edition (R. Bast et al., Eds.), 2016.

#293

Heterogeneous nucleocytoplasmic regulation of an incompletely penetrant ErbB2 onco-phenotype.

Lixin Wang, Kevin A. Janes. _University of Virginia, Charlottesville, VA_.

Perturbation of cancer cells often leads to heterogeneous outcomes, in that most cells exhibit a dominant phenotype, but the rest appear resistant or hypersensitive to the perturbation. If the penetrance of such a phenotype is heritably incomplete, then it becomes extremely difficult to decipher the upstream molecular events that heterogenize the population and cause response variability. By combining quantitative measurements with dynamical models, systems approaches should be useful if provided with a core network of important biomolecules. The daunting hurdle lies in identifying phenotype-relevant regulatory heterogeneities that define the network for penetrance at the single-cell level. Here, we exploit a new approach, called stochastic frequency matching (SFM), which elaborates the molecular networks upstream of incompletely penetrant phenotypes. SFM identifies and parameterizes single-cell heterogeneities—which emerge after a uniform perturbation but before the appearance of a variable phenotype—to hone in on regulatory states corresponding to future penetrance. For a multi-acinar onco-phenotype incompletely triggered by ErbB receptor tyrosine kinase signaling in 3D cultured breast epithelia, we implemented SFM using microarrays to uncover a network of critical nucleocytoplasmic regulators, including the inner-ring nucleoporin NUP37 and the exportin CSE1L. Gain- and loss-of-function perturbations of NUP37 and CSE1L alter the frequency of ErbB2-triggered multiacinus formation, supporting that SFM can identify mechanisms of incompletely penetrant phenotypes. In addition, population-level transcriptomics revealed other ErbB2-induced changes in nucleocytoplasmic shuttling proteins: KPNA2, RANBP1, and XPO1. The net result of these alterations on shuttling is complicated, and the specific cargo relocalized by ErbB2 signaling is not known. We hypothesize that ErbB signaling heterogeneously reconfigures the nucleocytoplasmic shuttling state of cells to determine incomplete penetrance of the onco-phenotype. Using proximity labeling, we seek to identify novel ErbB2-induced NUP37 and CSE1L interactors that may serve as effectors for the multiacinar phenotype. Systems models of the nucleocytoplasmic shuttling network will evaluate the impact of observed abundance changes and identify characteristics of cargo predicted to be most sensitive to network reconfiguration. The importance of key effectors will ultimately be tested in vivo with murine cells engineered for spontaneous Erbb2 amplification and in patient samples with HER2 amplification.

## MOLECULAR AND CELLULAR BIOLOGY / GENETICS:

### BCL-2 Family and Mitochondrial Apoptosis

#294

BCL-2 family compensation regulates T cell homeostasis and reveals a BIM:BCL-W axis in T-ALL.

Lindsey M. Ludwig,1 Lauren E. Roach,1 Samuel G. Katz,2 Jill K. Fisher,3 Melissa Burns,3 Matthew R. Schnorenberg,1 Riyue Bao,1 Makda Zewde,1 Yusuke Nakamura,1 Alejandro Gutierrez,3 Loren D. Walensky,3 James L. LaBelle1. 1 _University of Chicago, Chicago, IL;_ 2 _Yale School of Medicine, New Haven, CT;_ 3 _Dana-Farber Cancer Institute, Boston Children's Hospital, Harvard Medical School, Boston, MA_.

BIM is the master BH3-only BCL-2 family regulator of lymphocyte survival. To understand how long-term loss of BIM affects apoptotic resistance in T and B cells we examined peripheral blood counts in mice with T or B cell-specific deletion of Bim. In contrast to CD19CRE Bimfl/fl animals, LCKCRE Bimfl/fl mice had pronounced early lymphocytosis followed by lymphocyte count normalization as they aged. To explore BCL-2 family specific mechanism(s) leading to this normalization we compared cell death sensitivity and BCL-2 expression patterns of young versus old LCKCRE Bimfl/fl T cells. Young (6-12 weeks) and old (>40 weeks) LCKCRE Bimfl/fl mice had consistently abnormal thymocyte development hallmarked by a decrease in CD4+8+ double positive (DP) cells and accumulation of double negative (DN) and single positive (SP) cells as previously described in global Bim knockout mice. Thymocytes maintained cell death resistance to a number of apoptotic stimuli throughout all ages, reflecting a consistent loss of BIM. In contrast to thymocytes, mature splenic-derived LCKCRE Bimfl/fl T cells from young mice were equally sensitive to apoptotic stimuli as WT controls, indicating BCL-2 family compensation. Expression profiling in these cells revealed upregulation of other BH3-only proteins, including Noxa = Puma << Bmf, suggesting that these proteins functionally compensated for the loss of BIM. To determine if this was a T cell-intrinsic process and if the presence of other BH3-only proteins with pan-BCL-2 family binding affinities were required for lymphocyte number and apoptotic normalization, we analyzed peripheral blood from mice globally deleted in Bim, Bid, and Puma (TKO). TKO, like LCKCRE Bimfl/fl mice, developed profound early lymphocytosis followed by normalization as they aged. A subset of TKO mice developed T-ALL hallmarked by upregulation of Bmf and Bcl-w, an under characterized anti-apoptotic BCL-2 family protein, suggesting that BCL-W allowed for malignant T cell survival despite the presence of BMF. To determine if such a relationship exists in human disease, expression profiling of pediatric T-ALL patient samples revealed a significant negative correlation between BIM and BCL-W expression in developmentally immature and mature T-ALL. Our findings suggest a model in which the loss of BIM in T cells can be compensated for through upregulation of BMF, a BH3-only protein mechanistically similar to BIM. Loss of other 'direct activating' BH3-only proteins increases the chances of malignant cell escape despite BMF upregulation, particularly when BCL-W is overexpressed. This work provides a developmental framework between BIM and BMF in T cells and for a BIM:BCL-W axis in T-ALL. Overexpression of BCL-W in BIMlow-expressing T-ALL may be an additional target responsible for early immature T-ALL therapeutic resistance.

#295

Efficacy of MCL1 inhibitor S63845 in small cell lung cancer.

Yuto Yasuda, Hiroaki Ozasa, Takahiro Tsuji, Takashi Nomizo, Tomoko Funazo, Hironori Yoshida, Yuichi Sakamori, Toyohiro Hirai, Young Hak Kim. _Kyoto University, Kyoto, Japan_.

Lung cancer is the leading cause of cancer death. Small cell lung cancer (SCLC) is a histologic subtype of lung cancer and the proportion of SCLC is approximately 15%. New molecular targeted drugs and immune checkpoint inhibitors are successfully effective for non-small cell lung cancer. However, treatment of SCLC has not been improved over recent decades. It is important to explore new treatment strategies of SCLC. MCL1 is a member of the BCL-2 family, which regulates apoptosis. Targeting MCL1 represents a potential breakthrough of cancer treatment. We tested S63845, a MCL1 inhibitor, in four SCLC cell lines (DMS114, DMS53, SW1271, and NCI-H69) and, in addition, one patient derived SCLC cell (KTOR201). S63845 had greater efficacy in two of five SCLC cells (DMS114 and KTOR201). These two SCLC cells had higher expression of MCL1 and lower expression of BCL-XL, which is another member of the BCL-2 family. The other three SCLC cells (DMS53, SW1271 and NCI-H69) were resistant to S63845 and had a higher expression of BCL-XL or lower expression of MCL1. These data suggested that S63845 might be a powerful treatment of SCLC as a new therapeutic strategy. It is possible that the expressions of MCL1 and other members of the BCL-2 family predict the sensitivity of S63845.

#296

Loss of Romo1 enhances TRAIL-induced apoptosis through Bax upregulation in colon cancer.

Minjee Jo, Jung Lim Kim, So Yeon Jeong, Bo Ram Kim, Yoo Jin Na, Seong Hye Park, Yoon A Jeong, Dae-Hee Lee, Sang Cheul Oh. _Korea University Guro Hospital, Seoul, Republic of Korea_.

Tumor necrosis factor-related apoptosis inducing ligand (TRAIL) is a protein which is induces cell apoptosis in various cancer. To identify novel sensitizer of TRAIL, we knocked down Reactive Oxygen Species modulator 1 (Romo1). Romo1 is a mitochondrial membrane protein that is in charge of increasing ROS. Romo1 was highly upregulated in various cancers including colorectal cancer. Inhibition of Romo1 significantly increased TRAIL-induced apoptosis in colorectal cancer cells, not colon normal cells. Silencing of Romo1 has resulted in upregulation of Bax protein expression. It has been reported that Bax degraded by ubiquitination. We identify interaction between Bax and Parkin decreased in knockdown of Romo1 condition. Also, suppression of Romo1 lead to mitochondrial dysfunction. Our study suggests that inhibition of Romo1 enhanced TRAIL-mediated apoptosis by regulating Bax ubiquitination.

#297

AZD4573: Mechanistic PKPD model linking CDK9 inhibition to Mcl1 depletion and induction of apoptosis in preclinical AML model.

Douglas Ferguson, Theresa Proia, Justin Cidado, Scott Boiko, Maryann San Martin, Steven Criscione, Wenlin Shao, Lisa Drew. _Astrazeneca, Waltham, MA_.

Cyclin-dependent kinase 9 (CDK9) regulates elongation of transcription through phosphorylation of RNA polymerase II (pSer2-RNAPII), and its short-term inhibition results in the selective downregulation of genes with short-lived transcripts and labile proteins - including the anti-apoptotic protein Mcl1. AZD4573 is a selective inhibitor of CDK9 with short pharmacokinetic (PK) half-life. Intermittent dosing of AZD4573 in mouse MV411 (AML cell line) xenograft models results in progressive reduction in tumor volume with the mechanism of action believed to be via induction of apoptosis following depletion of Mcl1. The aim of this work was to derive a quantitative understanding of the relationships between extent and duration of CDK9 inhibition, depletion of Mcl1 and rate of induction of apoptosis in MV411 tumor cells.

A mechanistic model has been established that quantitatively and dynamically connects AZD4573 plasma and tumor PK to the rate and extent of modulation of pSer2-RNAPII and Mcl1 in the tumor and rate of induction of cell death (as measured by reduction in tumor volume). Tumor pSer2-RNAPII and Mcl1 pharmacodynamics were modeled using a series of linked indirect response models. Production rate of pSer2-RNAPII was modeled as being directly inhibited by AZD4573 concentration in the tumor. Production rate of Mcl1 was linked to pSer2-RNAPII via a series of transit compartments to capture the transcription/translation driven delay in onset of response. Induction of intrinsic apoptosis in the MV411 tumor cells was modelled as being inhibited by Mcl1.

Tumor pSer2-RNAPII exhibited a rapid, dose-dependent decrease following IP dosing of AZD4573 in mice. The free concentration of AZD4573 that resulted in half-maximal inhibition of pSer2-RNAPII production rate was estimated to be in the range 11-21 nM. Following a brief delay, tumor Mcl1 also exhibited a relatively rapid decrease that was proportional to the pSer2-RNAPII response. Mcl1 protein half-life was estimated to be 0.3 hr. Rate of induction of apoptosis could be decribed as a saturable first-order process (Kmax ~ 0.2 hr-1) and appeared to exhibit a steep response to the depletion of Mcl1, with reduction of Mcl1 to 25% (of the baseline value) being estimated to result in half-maximal rate of induction of apoptosis in the MV411 cells.

The described MV411 PKPD/efficacy model has been assumed to be representative of AML in human patients and was used to derive preliminary predictions of clinical efficacy at a range of possible IV dosing regimens.

#298

Identifying mechanisms of anti-apoptotic adaptation to enhance BAX activation and overcome resistance to apoptosis.

Andrea G. Lopez-Arroyo, Evripidis Gavathiotis. _Albert Einstein College of Medicine, New York, NY_.

The BCL-2 protein family includes both pro- and anti-apoptotic proteins, which have a central role in regulating apoptosis during tumor formation and in response to chemotherapy. Cancer cells can evade apoptosis through upregulation of the BCL-2 anti-apoptotic proteins and/or the downregulation or inhibition of the BCL-2 pro-apoptotic proteins. BAX is a crucial pro-apoptotic BCL-2 family protein that upon activation translocates from the cytosol to the mitochondria to execute mitochondrial outer membrane permeabilization, the "point of no return" for mitochondrial apoptosis. BH3 mimetics, small molecules that have been developed to bind specific anti-apoptotic BCL-2 proteins, such as Venetoclax, have shown efficacy in hematologic cancers but generally are less effective in solid tumors. Here we explored a panel of non-small cell lung cancer (NSCLC) cell lines for elucidating their mechanism of anti-apoptotic adaptation and identify novel therapeutic strategies to overcome resistance to apoptosis. We used the BH3 profiling approach in NSCLC cell lines, as a tool to understand addiction to specific BCL-2 family members, and identified cells that depend on one or more anti-apoptotic proteins for their survival. Furthermore, these cell lines have low expression levels of the activators BH3-only proteins suggesting that downregulation of BH3-only proteins could be a second apoptotic block. Using size-exclusion chromatography of cytosolic cellular extracts we found that NSCLC cell lines contain BAX in dimeric conformation, which is previously characterized to provide resistance to BAX activation, suggesting that this could be a third factor increasing the apoptotic threshold. Our laboratory recently developed a lead BAX activator small molecule, BTSA1, that mimics BIM BH3 helix and interacts with the BAX activation site inducing apoptosis in vitro and in vivo in hematological cancers. We hypothesized that availability of increased amounts of cytosolic BAX monomers in addition to direct BAX activation by BTSA1 would be sufficient to broadly overcome resistance to apoptosis in NSCLC cells. To systematically identify such mechanisms, we have performed combinatorial high-throughput screens using a library of FDA-approved and investigational chemotherapeutic drugs. We identified drugs that significantly enhanced apoptosis when are combined with direct BAX activation. Our work highlights three different mechanisms of anti-apoptotic adaptation and resistance in NSCLC and provides novel pharmacological strategies that may lead to effective therapies for cancer patients.

#299

**A susceptible-quiescent model can describe** **biphasic** **cell-kill by MCL1 inhibitor AZD5991** in vitro **.**

Francis D. Gibbons, Matthew Belmonte, Paul Secrist, Alwin Schuller. _AstraZeneca, Boston, MA_.

AZD5991 is a potent and selective inhibitor of Mcl-1, now in phase 1 clinical development. Mcl-1 is an anti-apoptotic Bcl2-family protein that is up-regulated by cancer cells in order to avoid apoptosis. Disruption of binding of Mcl-1 to pro-apoptotic BH3-only proteins such as Bax and Bak results in loss in cellular viability of several cell-line models of multiple myeloma (MM). As a function of time, many cell lines display a biphasic response, with an initial period of rapid cell kill, followed by a period of slower kill. Such biphasic kill curves are not uncommonly seen in bacteria. As a function of concentration, cell lines display a range of efficacy (extent of cell kill) and potency (concentration of half-maximal cell kill). Here we apply a model originally developed for bacterial cell-kill to parameterize the in vitro cell kill over time and across several orders of magnitude in concentration. Cells from 10 MM cell lines were grown in culture, then plated at uniform density in microtiter plates where they were incubated at concentrations of AZD5991 ranging from 0.3nM to 30µM (with DMSO control), each plate for a duration of 0.5h to 72h. After the desired incubation period, Cell-Titer Glo® was used to assess cellular viability by luminescence. Plates were run in duplicate. A model of cell survival was implemented in MATLAB, in which cells can be in one of two states: a proliferating state 'S' in which they are susceptible to a saturable concentration-dependent drug-induced cell death, or a quiescent state 'Q' in which they don't proliferate and have only a constitutive death rate independent of drug concentration. Transition between the two states is possible in either direction. By fitting the model to the data across time and over a wide range of concentrations, we can succinctly and precisely describe the rate of growth and drug-induced cell death, as well as transition between the susceptible and quiescent states. To our knowledge, this represents the first application of such a model to a BH3 mimetic such as AZD5991. We find that the cell lines show much greater variation in efficacy (i.e., maximum inducible cell kill rate) than in potency, suggesting that the main difference between these lines lies in their dependence on Mcl-1 for survival.

#300

Developing a methodological platform for the exploration of MCL1 inhibitors.

Jan Erik Ehlert, Daniel Müller, Sarah Umber, Carolin Heidemann-Dinger, Constance Ketterer, Alokta Chakrabarti, Daniel Feger, Oliver Siedentopf, Marianne Birkle, Christoph Schächtele, Michael H. Kubbutat. _ProQinase GmbH, Freiburg, Germany_.

Many cancer cells succeed to survive by neutralizing apoptotic triggers. A frequently found evasive mechanism is to keep proapoptotic BCL2 family proteins (e.g. BAK; BIM) in check. To avoid apoptosis, cancer cells overexpress anti-apoptotic BCL2 family proteins (e.g. BCL2, BCLXL or MCL1). Latter proteins block pro-apoptotic effectors via binding to the so called BH3 binding domain and thus act as oncogenic survival factors. Therapeutic strategies aim at the disruption of this protein-protein interaction to reactivate apoptosis. Small molecular weight compounds targeting BCL2 and BCLXL such as Navitoclax (ABT-263) or Venetoclax (ABT-199) have been successfully promoted into clinics. However, there is still an urgent need for inhibitors to MCL1, which would be especially useful for MCL1 addicted cancers as well as for cancers that turn resistant to the previously mentioned drugs.

To facilitate discovery and optimization of compounds targeting MCL-1, we established a methodological platform that allows the differentiated analysis of MCL1 inhibitors. We report on the establishment of FRET-based biochemical assays measuring the interaction of MCL1 as well as of BCLXL with either BAK or BIM derived peptides. Cellular phenotypic assays were set up suitable to analyze the impact of potential inhibitors on proliferation and Caspase 3/7-related apoptosis in MCL1-high expressing NCI-H929 cells and on K562 control cells. In addition, a combination treatment assay was set up to analyze the synergistic effect of MCL1 inhibitors on cell lines poorly responsive to BCL2/BCLXL-inhibitor Navitoclax such as OPM2 cells. Finally, mechanistic assays were established focusing on the impact of MCL1-inhibition on the MCL1 stability in e.g. HCT116 cells.

Applying these assay systems, we here report for the first time on the side-by-side comparison of benchmark MCL1-inhibitors A1210477 and S63845 in identical biochemical and cellular assay systems. Our data support the suitability of our methodological platform for the development of MCL1 inhibitors. With synergistic studies of these compounds in combination with Navitoclax on a large panel of ~ 100 cell lines we furthermore provide novel data on the relevance of BCLXL/MCL1 co-inhibition in several entities.

#301

Targeting the anti-apoptotic BCL-2 family protein provides an effective and precise therapeutic strategy for NHL.

Qiu-Yun Luo,1 Han-Jie Yi,1 Xiang-Lei Yan,1 Miaozhen Qiu,1 Bao-Xia Li,1 Lin Zhang,1 Wen-Tao Pan,1 Lu-Ping Yuan,1 Zhen-Yi Liu,1 Douglas D. Fang,2 Yifan Zhai,2 Jian Sun,1 Da-Jun Yang1. 1 _Sun Yat-sen University Cancer Center, Guangzhou, China;_ 2 _Ascentage Pharma Group Corp. Limited, Suzhou, China_.

Background: Despite advances in the treatment of non-Hodgkin's lymphoma (NHL), refractory NHL remains a major clinical challenge. The B-cell lymphoma protein-2 (BCL-2) family is frequently overexpressed in NHL, which is associated with poor prognosis by promoting cell growth and resistance to antitumorigenic agents. In this study, by using different BCL-2 family protein inhibitors, we aim to further classify the survival dependency of NHL for anti-apoptotic BCL-2 family members and explore novel approaches to rationally develop combination therapies by completely blocking the activity of the Bcl-2 family proteins.

Methods: Cell viability was determined by CCK-8 assay. Apoptotic activity was confirmed by Annexin V-propidium iodide (PI) apoptosis detection, Western blot analysis and mitochondrial membrane potentials detection analysis. In vivo, NHL xenograft models were established to analyze the antitumor effect of the BCL-2 family protein inhibitors.

Results: We evaluated the inhibitory effect of of Bcl-2 targeted inhibitors and synergy in combination with other molecular targeted agents in NHL. While those NHL cell lines with only high levels of BCL-2 protein are sensitive to Bcl-2 selective inhibitor, the dual Bcl-2/Bcl-xL inhibitor APG-1252 and its active metabolite APG-1252-M1 potently induced mitochondria-dependent apoptosis in NHL cell lines that possess high expression of both BCL-2 and BCL-XL proteins. After the treatment of APG-1252-M1, apoptosis including caspase-3 activation, PARP cleavage, and mitochondrial membrane potentials change was observed rapidly within four hours. We further demonstrated that APG-1252-M1 showed synergistic effects with Bruton's tyrosine kinase (BTK) inhibitor ibrutinib in those sensitive cell lines by modulating STAT3 signaling pathways. However, cell lines harboring high expression of anti-apoptotic protein MCL-1 were resistant to APG-1252-M1. Furthermore, we observed that APG-1252-M1 exhibited strong synergistic effect with dual PI3K/mTOR inhibitors NVP-BEZ235 in those resistant cell lines with high levels of Mcl-2 protein.

Conclusions: Taken together, our data provide an effective and precise therapeutic strategy for NHL through determining NHL addiction to anti-apoptotic BCL-2 family proteins, and then target-guided application of BCL-2 family inhibitors as a monotherapy or in combination with other molecular targeted agents to completely block the Bcl-2 family proteins, thus to restore the apoptotic cell death.

#302

Selective Mcl-1 inhibition by AZD5991 induces on-target cell death and achieves antitumor activity in multiple myeloma and acute myeloid leukemia.

Adriana E. Tron,1 Matthew A. Belmonte,1 Steven Criscione,1 Edwin A. Clark,1 Eric Gangl,1 Francis D. Gibbons,1 Jeffrey W. Tyner,2 Stephen E. Kurtz,2 Qing Ye,1 Alexander W. Hird,1 Alwin Schuller,1 J. Paul Secrist1. 1 _AstraZeneca, Boston, MA;_ 2 _Oregon Health & Science University, Portland, OR_.

Mcl-1 is a member of the Bcl/Mcl family of proteins that promotes cell survival by preventing induction of apoptosis in a broad range of cancers. High expression of Mcl-1 has been linked to tumor development and resistance to anticancer therapies, underscoring the potential of Mcl-1 inhibitors as anticancer drugs. We have previously shown that AZD5991, a rationally designed macrocycle with sub-nanomolar affinity for Mcl-1 and high selectivity, induces rapid and irreversible commitment to apoptosis in Mcl-1-dependent cancer cells in a manner dependent on proapoptotic BAK. Here, we demonstrate that AZD5991 exhibits cytotoxic activity (GI50<100nM) in various MM and AML cell lines in vitro with an activity profile distinct from the selective Bcl2 inhibitor venetoclax. In vivo, AZD5991 shows potent antitumor activity with complete (100%) tumor regression in several mouse MM and AML xenograft models after a single tolerated dose. AZD5991 shows enhanced efficacy in vivo when combined with standard-of-care agents. Pharmacodynamic studies confirmed that AZD5991 kills cancer cells by activation of the mitochondrial apoptotic pathway. Ex vivo analysis indicates that AZD5991 has single agent activity in primary AML patient samples with LC50 values in the low nanomolar range. Consistent with our findings in AML cell lines, the activity profile for AZD5991 in AML primary samples was distinct from venetoclax, highlighting the unique therapeutic opportunity for AZD5991. Based on these data a phase I clinical trial has been launched for evaluation of AZD5991 in patients with MM and other hematologic malignancies (NCT03218683).

#303

Novel role of nuclear BH3-only protein BNIP3 in regulation of cellular proliferation.

Amandeep Singh, Meghan Azad, Spencer Gibson. _University of Manitoba, Winnipeg, Manitoba, Canada_.

The objective of this study is to investigate the role of Bcl-2 nineteen kilodalton interacting protein 3 (BNIP3) in regulating cellular proliferation and identifying the proliferation pathways mediated by BNIP3. BNIP3 is a BH3-only proapoptotic member of Bcl-2 family of cell death regulating proteins. The function of BNIP3 is dependent on its localization and expression levels. Under normal conditions, low levels of BNIP3 are present in the nucleus of cells. Low oxygen or hypoxic conditions lead to increased expression of BNIP3, and high levels of BNIP3 are found in the cytoplasm. Cytoplasmic BNIP3 is associated with caspase-independent apoptotic cell death through mitochondria. If, however, BNIP3 levels are increased in the nucleus, it promotes survival of cells by transcriptionally inhibiting several apoptosis-inducing factors. High levels of BNIP3 in nucleus are seen in majority of glioblastoma tumors, where hypoxic environment of tumor core causes an increase in expression of BNIP3 but high amounts of BNIP3 localize to the nucleus, possibly aiding in the survival of the tumor cells. In this study, we found that BNIP3 knockout mice brains have increased cellularity compared to wild-type brains. We also found that both mouse primary astrocytes and embryonic fibroblasts (MEFs) lacking BNIP3 expression have an increased capacity for proliferation compared to wild-type MEFs. Overexpressing BNIP3 in the nucleus of HEK 293 cells also resulted in reduced proliferation. In contrast, both astrocytes and MEFs lacking BNIP3 failed to show differences in cell death compared to wild-type cells. This reveals a novel function for BNIP3 in regulating cellular proliferation that may lead to new targets for cancer therapy. In future studies, we will identity genes regulated by nuclear BNIP3 that may lead to suppression of cellular proliferation in cancer cells.

#304

**Dual PI3K/BCL-2 family inhibition in colorectal cancers with** Apc **and** Pik3ca **mutations.**

Devon D. Miller, Christopher P. Babiarz, Susan N. Payne, Cheri A. Pasch, Linda Clipson, Kristina A. Matkowskyj, Dustin A. Deming. _Univ. of Wisconsin, Madison, WI_.

Background: Colorectal cancer (CRC) is a leading cause of cancer-related death in the United States. Approximately 20% of human CRCs possess mutations in the PIK3CA gene, resulting in a constitutively active form of phosphoinositide-3 kinase (PI3K). These PIK3CA mutations most commonly occur concomitantly with the loss of adenomatous polyposis coli (APC). Copanlisib is a beta-isoform sparing PI3K inhibitor. Here we examine the response of CRCs with Apc and Pik3ca mutations to copanlisib alone and in combination with navitoclax, a BCL-2, BCL-xL, and BCL-w inhibitor.

Methods: Murine-derived organotypic cancer spheroids (MDOCS) with Apc and Pik3ca mutations generated from transgenic mice were cultured in Matrigel and treated by exchanging feeding media containing desired concentration of each agent over the spheroids. Spheroids were treated with feeding media (control), 200nM ABT263, 200nM copanlisib, or the combination of both treatments. The spheroid culture response was quantified as the median relative change in the sphere diameter, comparing pre- and posttreatment 4x optical microscopic images to those obtained 48 hours post-treatment.

Results: Untreated MDOCS had a median growth in sphere diameter of 137%. ABT263 did not significantly affect growth; however, copanlisib significantly reduced median sphere size by 24.6% (p<0.001) and the combination treatment reduced median sphere size by 33% which was significant to both control and copanlisib only treated spheres (p<0.001 and p=0.014, respectively). Phosphorylation of ribosomal protein S6 and 4EBP1 were suppressed with copanlisib treatment. Induction of apoptosis in these spheres treated with the combination regimen was confirmed with immunofluorescence for cleaved caspase 3 with minimal apoptosis observed in those spheres treated with copanlisib alone.

Conclusion: In this study, we demonstrated the ability of copanlisib alone and combined with ABT263 to cause a marked decrease in sphere size of CRC MDOCS with Apc and Pik3ca mutations. Future tests will examine this novel regimen in vivo and potentially in future clinical trials.

#305

Cerdulatinib induces Bim expression and synergistic cell kill in combination with venetoclax in follicular lymphoma cell lines.

Andrew J. Steele,1 Greg Coffey,2 JiaJia Feng,2 Matthew D. Blunt,1 Francis DeGuzman,2 Deogracias Canivel,2 Jack Rose,2 Kenneth Der,2 Janet Leeds,2 Anjali Pandey,2 Pamela Conley2. 1 _University of Southampton, Southampton, United Kingdom;_ 2 _Portola Pharmaceuticals, South San Francisco, CA_.

Follicular lymphoma (FL) is the most frequently occurring indolent B-cell non-Hodgkin lymphoma. Treatment typically involves rituximab in combination with bendamustine, CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone), or CVP (cyclophosphamide, vincristine, and prednisone). Disease relapse is high, and current treatment options for these patients are limited. BTK inhibition has limited clinical activity in FL patients, and while the PI3Kδ inhibitor idelalisib was recently approved, tolerability has been an issue. Limited clinical activity was observed with the Bcl-2 inhibitor venetoclax, due to a compensatory upregulation of Mcl-1 by the tumor cell. Consequently, novel treatment strategies are required for patients with relapsed/refractory FL. Cerdulatinib is a dual SYK/JAK inhibitor that targets the BCR and cytokine signaling pathways. We previously demonstrated in chronic lymphocytic leukemia (CLL) cells that cerdulatinib inhibited signaling via the BCR and IL-4 signaling pathways and induced apoptosis in a dose- and time-dependent manner. However, the mechanism behind cerdulatinib-induced tumor killing has not previously been investigated. Herein we explore the function of cerdulatinib alone and in combination with venetoclax for the treatment of FL. To confirm cerdulatinib activity in FL derived cells lines WSU-FSCCL, DOHH2 and SU-DHL6, cells were incubated with cerdulatinib prior to BCR or cytokine receptor engagement. Cerdulatinib (0.1-1µM) significantly inhibited anti-Ig-induced pAKTS473, pERKThr202/Tyr204, pS6 ribosomal subunit Ser235/236 and cytokine-induced STAT signaling. The Bcl-2 protein was highly expressed in all cell lines consistent with a t(14:18) translocation, whereas basal Mcl-1 was expressed at lower levels. Bcl-2 levels remained unchanged following cerdulatinib treatment in all three cell lines. However, in contrast to CLL where cerdulatinib inhibited Mcl-1 expression, modulation of Mcl-1 protein expression by cerdulatinib in FL cell lines was subtle and inconsistent. Cerdulatinib alone induced 12%-44% cell death in all cell lines tested in a time dependent manner (24-72h) and this correlated with a robust increase in Bim expression at the RNA and protein level. We subsequently treated DOHH2, WSU-FSCCL and DHL6 cells with cerdulatinib in combination with 10-100nM venetoclax. Venetoclax synergized with cerdulatinib to induce significantly greater levels of apoptosis in all cell lines investigated compared to either agent alone and appeared to function by displacing Bim from Bcl-2. To demonstrate in vivo capability, we xenografted SU-DHL6 subcutaneously in nude mice. Cerdulatinib in combination with venetoclax led to greater antitumor activity when compared to vehicle or either drug alone. Together these data highlight a rationale for combining cerdulatinib with venetoclax for the treatment of FL.

#306

A mechanistic rationale for combining acalabrutinib with CDK9 inhibitor, AZD4573, in ABC-DLBCL.

Scott Boiko, Theresa Proia, Maryann San Martin, Justin Cidado, Wenlin Shao, Lisa Drew. _AstraZeneca, Boston, MA_.

Cyclin-dependent kinase 9 (CDK9) regulates transcription elongation through phosphorylation of RNA polymerase II at serine 2 (pSer2-RNAPII), and its short-term inhibition downregulates genes with short-lived transcripts and labile proteins, thus providing a mechanism to inhibit key survival proteins like Mcl1 and Myc. We developed a selective CDK9 inhibitor, AZD4573, that exhibits anticancer activity across a diverse set of hematologic cell lines and is being evaluated in patients with hematologic malignancies. Through efforts to identify combinations with AZD4573, we observed that combined treatment with acalabrutinib, our Bruton tyrosine kinase (BTK) inhibitor, led to more robust and durable responses in preclinical ABC-DLBCL models. We demonstrate BTK inhibition with acalabrutinib upregulates the proapoptotic BH3-only protein Bim in BTKi sensitive ABC-DLBCL cell lines, priming cells for apoptosis induction and further sensitizing them to CDK9 inhibition with AZD4573. Specifically, acalabrutinib upregulated proapoptotic BH3-only proteins Bim and Bmf (>3-fold, 24h) in two BTK-dependent ABC-DLBCL cell lines, OCILy10 and TMD8 but not in a BTK-independent GCB-DLBCL cell line Karpas-422. In each model, treatment with 100nM AZD4573 resulted in rapid induction of cleaved caspase-3 (CC3), and subsequent combination with acalabrutinib accelerated induction of CC3 and increased the magnitude of cell death in the BTK-dependent cell lines. Apoptotic priming by acalabrutinib was validated in vitro in OCILy10 cells by siRNA-mediated knockdown of BH3-only proteins followed by a phenotypic comparison of combination dosing schedules. Both inhibitors dosed concurrently (unprimed) resulted in a minimal increase in the percentage of apoptotic cells compared to treatment with AZD4573 alone (49% of population). In contrast, when dosed sequentially with a pretreatment of acalabrutinib (primed) before the addition of AZD4573, nearly all cells were apoptotic (>90%). Consistent with the in vitro results, the combination of AZD4573 with acalabrutinib resulted in rapid and complete tumor growth inhibition in both BTK-dependent ABC-DLBCL xenograft models. In the OCILy10 model, the combination led to complete tumor regressions and 100% of animals treated in the combination arm (8/8) demonstrated a 60-day delay to regrowth off treatment compared to single-agent AZD4573. Similarly in TMD8, the combination resulted in complete regressions in 100% of treated animals with a highly durable response (17/19 animals tumor-free at 100+ days) whereas the combination yielded no additional benefit in Karpas-422. Acalabrutinib was recently approved for relapsed/refractory mantle cell lymphoma and is being evaluated in patients with additional subtypes of lymphomas. Based on our findings, the combination of acalabrutinib with AZD4573 could be an effective treatment option for patients with ABC-DLBCL.

#307

Targeting BCL-2 and BCL-xL with a novel dual inhibitor APG-1252 triggers cell death and inhibits tumor growth in small cell lung cancer models.

GuangFeng Wang,1 Ping Min,1 MiaoYi Wu,1 Shuo Dang,1 ChuanYan Tang,1 Fei Zhang,1 Ming Guo,1 Shaomeng Wang,2 Jing Deng,1 Douglas D. Fang,1 DaJun Yang,3 YiFan Zhai1. 1 _Ascentage Pharma, China;_ 2 _School of Medicine, University of Michigan, Ann Arbor, MI;_ 3 _Sun Yat-Sen University Cancer Center, Guangzhou, China_.

Small cell lung cancer (SCLC) is one of the most deadly diseases with a dismal five year survival rate less than 7%. Even though most SCLC patients respond to the initial platinum-based cytotoxic or radiation therapies, they inevitably relapse and succumb to the disease. Anti-apoptotic proteins BCL-2 and BCL-xL, which are highly expressed in 40-60% of SCLCs and play a critical role in tumorigenesis and drug resistance, have been emerging as a promising target for therapeutic intervention. We have recently developed a novel dual BCL-2/BCL-xL inhibitor APG-1252 for cancer therapy. In this study, the effect of APG-1252 was evaluated in a panel of SCLC cell lines for discovery of indications and predictive biomarkers. The results show that the sensitivity of SCLC cell lines with sub-µM or nM IC50 values are correlated with the higher expression levels of BCL-2/BCL-xL, BIM and/or PUMA but lower levels of MCL-1. Conversely, the resistant cell lines either lack of BCL-2/BCL-xL protein, or exhibit higher level of MCL-1 protein. In xenograft tumor models, consistent with in vitro results, APG-1252 exhibits antitumor activities in the models derived from the sensitive cells but not in those from the resistant cells. Interestingly, while ABT-263 failed to inhibit H146 xenograft tumor growth, despite of its similar in vitro killing ability as APG-1252, APG-1252 showed potent antitumor activity in the xenograft model. To overcome the drug resistance conferred by MCL-1 in the resistant cells, we explore the combination therapy with other targeted agents. We found that our novel MDM2 inhibitor APG-115 was able to overcome the intrinsic resistance and sensitize those cells to APG-1252 in vitro, suggesting that reducing the apoptotic threshold by inhibiting other anti-death proteins like MCL-1 or increasing apoptotic function through p53 can enhance SCLC sensitivity to APG-1252. Collectively, APG-1252 represents a novel opportunity that can neutralize the protection from BCL-2/BCL-xL and trigger cell death and inhibit tumor growth in SCLC models. With the significance of these preclinical data, APG-1252 has been granted for phase 1 clinical trials in USA (NCT03080311) and China.

#308

**Yeast Bax Inhibitor,** BXI1 **, is involved in calcium homeostasis of the ER in** Saccharomyces cerevisiae **.**

Nicholas Andrews, Nicholas Mello, Liam McDonough, Joseph Alisch, David Eagan, James Mullin, Alfredo Gonzalez, Nicanor Austriaco. _Providence College, Providence, RI_.

Yeast Bax inhibitor-1 (BXI1/YBH3) encodes a protein that belongs to the Bax Inhibitor (TMBIM) family of proteins that all contain a transmembrane BAX inhibitor motif. The crystal structure of a prokaryotic member of the family, BsYetJ, has revealed that the Bax inhibitor proteins are pH sensitive calcium leaks. In mammals, the Bax inhibitor family of proteins has cytoprotective properties that are most evident in paradigms of endoplasmic reticulum (ER) stress. Our published studies have shown that yeast Bxi1p is localized to the endoplasmic reticulum and is involved in the unfolded protein response (UPR) that is triggered by ER stress. BXI1 is thought to act via a mechanism involving altered calcium dynamics. We now show that cells lacking BXI1 accumulate higher levels of calcium in their ER as compared to their wildtype counterparts. We have also over expressed Bxi1p in E.coli and have used a fura-2 based calcium assay to show that the protein facilitates the influx of extracellular calcium into the cell. Our preliminary data with Δbxi1 Δpmr1 double mutants suggest that Bxi1p and Pmr1p, the Golgi-ER calcium pump, function in an antagonistic manner. [Our laboratory is supported by grant NIGMS R15 GM110578, awarded to N. Austriaco.]

#309

Targeting anti-apoptotic reprogramming to counteract drug tolerance in EGFR-inhibited colorectal tumors.

Simonetta M. Leto, Irene Catalano, Valentina Vurchio, Francesca Cottino, Giorgia Migliardi, Livio Trusolino, Andrea Bertotti. _Candiolo Cancer Institute FPO – IRCCS, Candiolo (TO), Italy_.

Treatment with EGFR monoclonal antibodies, such as cetuximab, has improved the outcome of patients with metastatic colorectal cancer (mCRC). However, EGFR inhibition - even in cases that respond with tumor shrinkage - is cytostatic rather than cytotoxic, with persistence of drug-tolerant cells that appear to be less prone to undergo apoptosis and poised to foster residual disease, which preludes tumor relapse.

Our preliminary data, obtained in mCRC patient-derived xenografts (PDXs), indicate that CRC residual cells surviving EGFR blockade display increased expression of the anti-apoptotic proteins BCL-2 and BCL-XL. Inhibition of such proteins together with EGFR prompts apoptosis of PDX-derived organoids, suggesting a pro-survival adaptation triggered by cetuximab and neutralized by pro-apoptotic drugs. On these premises, we plan to investigate how the entire apoptotic machinery is modulated in response to cetuximab in mCRC, with the final goals to: (i) identify and validate new mechanisms sustaining the persistence of drug-tolerant cells, and (ii) find new therapeutic strategies to eradicate residual disease.

We first performed a gene expression analysis of the main BCL-2 family members in cetuximab-sensitive PDXs after prolonged EGFR inhibition and found a pronounced reprogramming whereby anti-apoptotic outcomes prevail over cell death signals and apoptosis gets aborted. In order to generate a list of candidates potentially involved in sustaining this anti-apoptotic adaptation, we plan to combine gene expression analysis with "Dynamic BH3 profiling" (DBP), a novel functional approach that enables assessing (i) how cetuximab treatment changes CRC cell propensity to undergo apoptosis and (ii) which anti-apoptotic proteins prevent such priming from switching into overt apoptosis. We performed DBP in two PDX-derived organoid lines and found that exposure to cetuximab induced a strong dependence on BCL-XL in one model. We next examined the effect of combining cetuximab with a specific BCL-XL inhibitor in both lines and found that while either drug alone was insufficient to cause cell death, the combination therapy induced massive apoptosis only in the case featuring cetuximab-induced BCL-XL dependence, as predicted by DBP. This analysis will be extended to a larger number of PDX-derived organoids; then, therapeutic strategies with significant activity in vitro will be validated in vivo in matched PDXs and the underlying mechanistic underpinnings will be further investigated.

These preliminary results illustrate how perturbations of the apoptotic machinery can influence the response to EGFR blockade in mCRCs. This information will guide the identification of critical functional nodes involved in sustaining the drug-tolerant state, thus providing new hints to design rational ways to convert the cytostatic effect of cetuximab into a cytotoxic, fully apoptotic outcome.

#310

AZD4573, a novel CDK9 inhibitor, rapidly induces cell death in hematological tumor models through depletion of Mcl1.

Justin Cidado, Theresa Proia, Scott Boiko, Maryann San Martin, Steven Criscione, Douglas Ferguson, Wenlin Shao, Lisa Drew. _AstraZeneca, Waltham, MA_.

Cyclin-dependent kinase 9 (CDK9) regulates elongation of transcription through phosphorylation of RNA polymerase II (pSer2-RNAPII), and its short-term inhibition downregulates genes with short-lived transcripts and labile proteins. We developed a novel and selective CDK9 inhibitor, AZD4573, with nanomolar potency and physicochemical properties suitable for IV administration and short exposure.

Initial transcriptomic and proteomic analyses were performed on MCF7 breast cancer cells treated with AZD4573 for 4h or 8h to determine which genes were most rapidly modulated by CDK9 inhibition. Of the 1,290 genetically-linked, cancer-associated genes analyzed on these platforms, MCL1 ranked amongst the top as most robustly and sustainably suppressed at both the mRNA and protein level with AZD4573 treatment. MCL1 is an anti-apoptotic member of the BCL2 gene family and is frequently amplified and/or over-expressed in various cancers, conferring a survival advantage to the tumor cell. Mcl1 has also been notoriously difficult to drug with small molecules, although inhibitors are now in early clinical development. Transient inhibition of CDK9 with AZD4573, however, provides a means to indirectly target Mcl1 and induce apoptosis in Mcl1-addicted tumors.

In human cancer cell line panel screens, AZD4573 demonstrated the ability to induce rapid caspase activation (6h) and loss of viability (24h) across a diverse set of hematological cancers (median caspase EC50=30nM, GI50=11nM) but with minimal effect on solid tumors (median EC50 & GI50 >30μM). The activity of AZD4573 in the cell line panels displayed a strong correlation with that of the Mcl1 inhibitor, AZD5991 (r2 = 0.837), suggesting transient CDK9 inhibition induces cell death through an Mcl1-dependent mechanism. Furthermore, knockdown of BAK and/or BAX in sensitive cell lines rescued the cells from AZD4573-mediated caspase induction and cell death, underscoring the mechanistic involvement of the intrinsic apoptosis pathway. In the AML cell line, MV411, AZD4573 led to a rapid dose- and time-dependent decrease in pSer2-RNAPII with concomitant loss of Mcl1 mRNA and protein, resulting in caspase induction and loss of cell viability. In contrast, protein levels of the other anti-apoptotic proteins, Bcl2 and BclxL, remained unchanged out to 6h post-AZD4573 treatment of MV411 cells.

With intermittent dosing of AZD4573, in vivo efficacy was observed in multiple hematological tumor xenograft models. AZD4573 also exhibited the ability to drive deeper and more durable regressions in combination with multiple targeted agents. Based on our findings, AZD4573 could be an effective treatment option, either as single agent or in combination, for patients with hematological malignancies. Thus, we have initiated a first-time-in-human study with AZD4573 in October 2017 (NCT03263637).

#311

**AZD4320 is a potent, dual Bcl-2/x** L **inhibitor that rapidly induces apoptosis in preclinical hematologic tumor models.**

Justin Cidado, J Paul Secrist, Francis D. Gibbons, Edward J. Hennessy, Stephanos Ioannidis, Edwin A. Clark. _AstraZeneca, Waltham, MA_.

Apoptosis is a normal cellular process that is regulated by the dynamic interaction of pro- and anti-apoptotic proteins of the B-cell lymphoma 2 (Bcl-2) family. Cancers, however, have evolved mechanisms to hijack this process and tip the balance in favor of anti-apoptotic proteins, conferring a survival advantage for tumor cells as well as a means of resistance to anti-cancer therapies. Indeed, the Bcl-2 family are some of the most frequently amplified genes and over-expressed proteins across various tumor types. As a result, tumor cells can become addicted to Bcl-2 family members and, hence, vulnerable to targeted BH3 mimetics. Clinical validation of this concept has been demonstrated by venetoclax with its approval for treatment of R/R CLL patients with 17p deletion. Given the great potential that directly targeting the apoptotic machinery holds in treating cancer, developing BH3 mimetics is an attractive proposition.

To that end, we have developed a potent small molecule, AZD4320,1 that has nanomolar affinity for Bcl-2 and Bcl-xL, similar to navitoclax, but has physicochemical properties suitable for IV administration. This will help mitigate toxicities observed with oral administration of navitoclax (e.g. allow recovery of platelets), thus improving therapeutic index. AZD4320 also displays the hallmarks of a bona fide BH3 mimetic, most notably the ability to disrupt the complex formation of Bcl-2 with BH3-only proteins and the necessity for intact BAK and BAX to propagate the apoptotic cascade. A kinetic study was also conducted to explore apoptosis induction in the Bcl-2-addicted B-ALL cell line, RS4;11, which revealed both a dose- and time-dependent increase in cleaved caspase-3 (CC3) and corresponding reduction in cell viability. In an expanded panel of human cancer cell lines, AZD4320 rapidly induced CC3 (6h) and loss of viability (24h) in a diverse set of hematological lines with a median EC50 of 182nM. Solid tumor cell lines, however, were much less responsive (median EC50 >30μM). A comparison to venetoclax from the same cell line panel screen revealed that many more hematological tumor cell lines were sensitive to AZD4320, highlighting the utility and promise of a dual Bcl-2/xL inhibitor. Furthermore, in a venetoclax-resistant derived ABC-DLBCL cell line, AZD4320 was equally potent when compared to the parental cell line whereas venetoclax exhibited a >20-fold reduction in activity. Lastly, for in vivo efficacy studies with RS4;11 xenograft tumors, regressions with corresponding induction of CC3 were observed following a single dose of AZD4320.

Together, these results highlight the therapeutic potential of a dual Bcl-2/xL inhibitor to be used as a foundation therapy across a broad range of hematological tumor types as well as combat resistance to other BH3 mimetics and targeted therapies.

1Hennessy, E; et al. ACS National Meeting 24 (2015).

#312

Emodin inhibits colon cancer by modulating apoptotic and cell survival signals.

Ian T. Saunders, Neeraj Kapur, Hina Mir, Shailesh Singh. _Morehouse School of Medicine, Atlanta, GA_.

Colon cancer remains a formidable health care concern and is the third most common cancer among men and women. Late diagnosis and development of resistance to chemotherapeutic regimens significantly impact overall outcome and survival. Various phytochemicals have been shown to have promising anti-cancer properties. Consequently, such natural agents are emerging as an alternative treatment modality for various cancers, including colon cancer. In this study, we have tested the effect of Emodin, an anthraquinone derivative of rhubarb, on human colon cancer cell lines. Our antibody microarray data and pathway analysis indicated that Emodin induced apoptosis in colon cancer cells by triggering both intrinsic and extrinsic apoptotic pathways. These effects of Emodin were associated with modulation of death receptor expression, changes in mitochondrial membrane integrity, and DNA degradation. Further, western blot analysis confirmed that Emodin significantly increased pro-apoptotic proteins (Bak, Bax, Bad) as well as activated caspase-3 and -8. Additionally, Emodin reduced ERK1/2, NF-κB, and STATs at transcript and protein levels, suggesting it also interferes with cell survival. Hence, the ability of Emodin to induce apoptosis and inhibit cell survival by altering molecular pathways underscore its potential as chemo-preventive as well as -therapeutic agent in combination with conventional chemotherapy.

#313

An Akt3 splice variant lacking the serine 472 phosphorylation site promotes apoptosis and suppresses mammary tumorigenesis.

Rachel B. Hazan. _Albert Einstein College of Medicine, Bronx, NY_.

The Akt pathway is a well-known promoter of tumor malignancy. Akt3 is expressed as two alternatively spliced variants, one of which lacks the key regulatory serine 472 phosphorylation site. Whereas the function of full-length Akt3 isoform (Akt3/+S472) is well-characterized, that of Akt3/-S472 isoform remains unknown. Despite being expressed at a substantially lower level than Akt3/+S472 in triple negative breast cancer cells, specific ablation of Akt3/-S472, enhanced, whereas overexpression, suppressed mammary tumor growth-consistent with a significant association with patient survival duration relative to Akt3/+S472. These effects were due to striking induction of apoptosis, which was mediated by Bim upregulation, leading to conformational activation of Bax and caspase-3 processing. Bim accumulation was caused by marked endocytosis of EGF receptors with concomitant ERK attenuation, which stabilizes BIM. These findings demonstrate an unexpected function of an endogenously expressed Akt isoform in promoting - as opposed to suppressing - apoptosis, underscoring that Akt isoforms may exert dissonant functions in malignancy.<!--EndFragment-->

#314

A novel MDM2-p53 antagonist APG-115 induces p53-mediated apoptosis and enhances radiosensitivity in colorectal cancer.

Han-Jie Yi,1 Xiang-Lei Yan,1 Qiu-Yun Luo,1 Luping Yuan,1 Baoxia Li,1 Wentao Pan,1 Lin Zhang,1 Miao-Zhen Qiu,1 Guangfeng Wang,2 Yifan Zhai,2 Da-Jun Yang1. 1 _Sun Yat-sen University Cancer Center, Guangzhou, China;_ 2 _Ascentage Pharma Group Corp. Limited, Suzhou, China_.

Background: Colorectal carcinoma is the third leading cause of cancer-related mortality in China. MDM2-p53 inhibitor APG-115 is a novel potential anticancer agent. We aim to investigate the anticancer effect and radiosensitivity enhancement of APG-115 in colorectal cancer.

Materials and methods: Human colorectal cancer cell lines (LOVO, HCT116, RKO, HT29, SW480 and SW620) were treated with the APG-115 alone or with radiation (0-8Gy). Transwell, clonogenic survival assays, CCK-8, flow cytometry, RT-qPCR, Western blot were used to assay cell invasion, cell survival, cell cycle progression, quantification of mRNA expression and DNA damage repair. In vivo, xenograft mouse models (RKO Cell line) were established to analyze the anticancer effect and radiosensitivity enhancement of APG-115.

Results: In vitro, APG-115 not only inhibited invasion and proliferation of colorectal cell lines, but also induced cell-cycle arrest in G1/S phase. Meanwhile, APG-115 increased the mRNA and protein expression of p53 downstream targets dependent on MDM2-p53 signal pathway. The combination of APG-115 and radiation caused cells to accumulate in G2/M and resulted in the accumulation of H2AX-related DNA damage, indicating an increasing of radiosensitivity. In xenograft mouse models, single therapy with either radiation or APG-115 was equivalent in delaying tumor growth, although APG-115 slightly outperforming therapy with radiation alone. Combination therapy delayed tumor growth more significantly compared with either single agent therapy. By analyzing proliferation marker Ki67 in xenograft tumor specimens, we found that the number of proliferating cells significantly decreased in tumors receiving combination treatment of APG-115 and radiation compared with tumors receiving single agent treatment. These results strongly confirmed the enhanced antitumor capacity of APG-115 when combined with radiation.

Conclusions: Our data strongly suggest that MDM2-p53 inhibitor APG-115 may serve not only as a stand-alone therapy, but also as an effective adjunct to current radiotherapeutic regimens for treating colorectal cancer dependent on MDM2-p53 axis.

#315

Cell death of AML blasts induced by cytarabine and enhanced by the Vitamin D2/Carnosic acid cell differentiating combination involves apoptosis signal-regulating kinase 1 (ASK1) activation.

Xuening Wang,1 Jonathan S. Harrison,2 George P. Studzinski1. 1 _Rutgers New Jersey Medical School, Newark, NJ;_ 2 _University of Connecticut, Farmington, CT_.

Acute Myeloid Leukemia (AML) is a disease with a grim prognosis and limited curative treatment options. The principal agent used to induce disease remissions is Cytarabine (AraC), typically combined with an anthracycline, though multiple other combinations have been explored. We have previously reported that the exposure of AML blasts, in which the DNA is already damaged by AraC, to the Vitamin D2 analog Doxercalciferol combined with Carnosic Acid (D2/CA) enhances AraC cytotoxicity, resulting in an increased cell kill of the blasts. To obtain an understanding of the underlying mechanisms, and thus facilitate the translation of this finding to the clinic, we conducted in vitro studies on HL60, U937 cell lines and on AML blasts in primary culture. The initial findings were that the kinases BRAF and JNK1 and the pro-apoptotic protein BIM participate in the enhancement of AraC action. We now report that the kinase activity of ASK1 (MAP3K5) is upregulated by both AraC and D2/CA, and this appears to be additive thus increasing the apoptosis by this sequential combination regimen. Interestingly, we also noted that BRAF directly binds to ASK1, and since it is a kinase, this raises the possibility that it activates ASK1 in this experimental system. This possibility is strengthened by the report that JNK activation which leads to apoptosis is dependent on BRAF. The ongoing investigation of additional details of the molecular events responsible for enhanced cell death may provide clues for the improvement of the therapy of AML. (Supported by NIH grant R01CA044722-26 from NCI).

#316

Cancer cell apoptosis is induced by soluble factors secreted from normal mammary fibroblasts.

Matyas Abel Tsegaye, Mati Nemera, Zachary T. Schafer. _University of Notre Dame, Notre Dame, IN_.

The tumor microenvironment (TME), comprised of a heterogeneous population of transformed and non-transformed cells, plays a crucial role in cancer growth. Studies have shown that there is continuous bidirectional communication between cancer cells and the different cell populations within the TME. This communication can either work to promote tumor growth, as seen with carcinoma associated fibroblasts (CAFs), or inhibit tumor growth, as observed with tumor infiltrating lymphoid cells. To date, the majority of research focusing on understanding the role of fibroblasts in the TME has been on CAFs and their ability to enhance tumorigenesis. Although the majority of studies have focused on CAFs, some studies have shown that certain fibroblast populations can antagonize tumor growth. Despite this evidence for an inhibitory role of normal fibroblasts, the precise molecular mechanism by which these cells modulate cancer cell growth has yet to be elucidated. Here, we show that normal mammary fibroblasts (fibroblasts isolated from normal breast tissue) secrete soluble factors that induce caspase mediated cell death in a variety of cancer cell lines. This phenotype was observed in MDA-MB-231s as well as T47D, and BT474 cell lines. Further verification of this phenotype was performed in MDA-MB-231s using Annexin V staining and flow cytometry. When examining the mechanism of apoptotic induction, we observed a substantive increase in the cleavage of caspase 8, evidence that the extrinsic apoptotic pathway is activated. We are currently working to further elucidate the signaling pathways activated in the aforementioned cancer cell lines resulting in the induction of apoptosis by normal mammary fibroblasts. Our research reveals a novel and potentially tumor suppressive function of normal mammary fibroblasts in the tumor microenvironment through secretion of soluble factors that induce apoptosis in cancer cells.

#317

Role of KLLN in DNA damage-induced apoptosis is associated with the regulation of p53 phosphorylation and acetylation in breast cancer cells.

Madhav Sankunny, Charis Eng. _Cleveland Clinic Foundation, Cleveland, OH_.

KLLN was initially discovered as a target of p53 involved in S-phase checkpoint regulation. Germline hypermethylation in the promoter of KLLN is known to increase the predisposition for breast and clear cell renal cell carcinomas in PTEN-wildtype Cowden syndrome (CS) patients. KLLN is a known tumor suppressor protein and required for p53-mediated apoptosis. In this study, we explore the underlying mechanism by which KLLN regulates DNA damage response (DDR), specifically apoptosis. Our DDR studies were conducted on one breast normal and four breast cancer cell lines using doxorubicin as the DNA damaging agent. We initially showed that doxorubicin-induced DNA damage increases KLLN expression at 16h and 24h post-damage which could be abrogated by RNAi-mediated knockdown of KLLN expression. Response to DNA damage as determined by the presence of γH2AX foci formation and expression of γH2AX protein were both decreased after KLLN knockdown. Using TUNEL assay and cell viability studies, we demonstrated that a lack of KLLN expression decreases apoptosis and increases cell viability at 16h and 24h post-damage. Definitive markers of apoptosis such as cleavage of caspase 3 and PARP were also decreased. Since KLLN is a known transcription factor and regulates the expression of DDR genes such as TP53 and CHK1, we assessed the regulation of these genes in response to DNA damage and found that lack of KLLN expression did not affect the expression of either TP53 or CHK1. Cell cycle analysis results using flow cytometry also suggested that KLLN does not play a role in S-phase cell cycle regulation in response to DNA damage as was previously thought, since KLLN knockdown did not change accumulation of cells in S-phase after doxorubicin-induced DNA damage. DDR in cells is immediate and therefore we assessed the abrogation of DDR after KLLN knockdown at early timepoints (1h and 2h) post-damage. We found that lack of KLLN expression decreased Ser15 phosphorylation of p53 and Thr68 phosphorylation of CHK2 at 1h and 2h post-damage. KLLN regulation of p53-mediated apoptosis could also be associated with its interaction with deleted in breast cancer (DBC1). DBC1 is a known inhibitor of SIRT1, a deacetylase that is known for its inhibitory deacetylation of p53. Phosphorylated DBC1 binds to SIRT1 and inhibits its deacetylation of p53. We found that lack of KLLN expression decreased the phosphorylation of DBC1 on Thr454 in response to DNA damage. Therefore, KLLN displays a 2-pronged regulation of the activation of p53 and the downstream cascade of proteins involved in apoptosis. SIGNIFICANCE: Our study demonstrates a clear role for KLLN in DNA damage-induced apoptosis and elucidates the underlying mechanism through the regulation of p53 phosphorylation and deacetylation in response to DNA damage. Our observations add to the basis of KLLN as a tumor suppressor and as a susceptibility gene for inherited cancers.

#318

Inhibition of FASN induces apoptosis and senescence in breast cancer.

Travis Vander Steen, Ruth Lupu. _Mayo Clinic, Rochester, MN_.

Fatty Acid Synthase (FASN), the key multienzyme in de novo lipogenesis, is upregulated and activated in numerous carcinomas, including breast cancer. Fatty acids play a fundamental role in cellular structure, energy production and storage and are intermediates in the biosynthesis of hormones and other biologic molecules. Therefore, FASN is a novel therapeutic target for treatment in cancer cells that have upregulated FASN protein. BT474 breast cancer cells, which overexpress FASN, were treated with a pharmacologic FASN inhibitor and resulted in apoptosis in a dose-dependent manner. Inhibition of FASN simultaneously upregulates the pro-apoptotic BCL2 family members Bim, Puma, and Noxa; additionally, FASN inhibition alters the NDAPH/NADP+ balance and induces reactive oxygen species (ROS) production. Most significantly, FASN inhibition induces sensitization of breast cancer cells to the BH3 mimetic navitoclax, a potential opportunity to use BH3 mimetics for the treatment of breast carcinomas. These results support further preclinical and potentially clinical development of a dual FASN and Bcl-2 treatment for breast cancer tumors that express FASN. In addition to apoptosis, there is an increase in cellular senescence in breast cancer cells overexpressing FASN when treated with FASN inhibitors. The increase in ROS, a known effect of FASN inhibition, is known to induce cellular stress, resulting in both apoptosis and senescence. Increased senescence was initially observed by x-gal staining of BT474 cells treated with FASN inhibitors via microscopy and FLOW. We also observed an increased expression of several pro-senescent genes both at the protein and mRNA levels, including p16/INK4a, p14ARF/p19ARF, p15, and p21. A hallmark of age-induced cellular senescence is the shortening of telomeres. However, telomere shortening was not observed, suggesting that senescence was a result of FASN inhibition and activation of senescence-induced genes. Therefore, inhibition of FASN appears to be an ideal target for therapeutic treatment of breast cancers that overexpress FASN through both apoptosis and senescence.

#319

Syntenin regulates multiple myeloma cell survival.

Tyler Moser-Katz, Catherine M. Gavile, Benjamin G. Barwick, Sagar Lonial, Lawrence H. Boise. _Emory University, Atlanta, GA_.

Multiple myeloma is the second most common hematological malignancy with an estimated 30,000 new diagnoses in 2017. Myeloma cells retain numerous characteristics of normal plasma cells including reliance on survival signals in the bone marrow for long term viability. However, they have gained the capacity to proliferate, causing harmful bone lesions in patients, and in advanced stages become extramedullary. Therefore, we are investigating molecular mechanisms of myeloma cells that allow them to survive independently of the bone marrow microenvironment. We have identified syntenin as a protein involved in myeloma cell survival and a potential therapeutic target. Syntenin has been reported to bind to and regulate levels of the plasma cell marker, CD138. We used the CoMMpass dataset, a longitudinal study of myeloma patients following their transcriptomic expression throughout treatment, and discovered that patients expressing in the top quartile for syntenin trend toward lower overall survival, progression-free survival, and shorter response duration than those in the bottom quartile. To determine whether it is involved in myeloma cell survival, we used short hairpin RNA to knock down syntenin (shsyn) in RPMI 8226 and MM1.s myeloma cell lines and determined cell death using Annexin-V staining four days following lentiviral infection. We observed increased death in syntenin-silenced cells compared to our empty vector control in both RPMI 8226 (control=57.83% viable, shsyn=29.47% viable, p=0.04) and MM1.s cell lines (control=91.43% viable, shsyn=70.1% viable, p=0.04) suggesting that syntenin is important for myeloma cell survival. Syntenin is a PDZ-domain containing protein that has been shown to regulate expression of several transmembrane proteins. We, therefore, wanted to look at correlation of syntenin expression with CD138 and CD86, two PDZ-binding domain containing proteins expressed on the surface of myeloma cells. Using the CoMMpass dataset, we found patients with high expression of syntenin had increased expression of CD86 while syntenin-low patients had decreased CD86 expression. This correlation does not exist with CD138 as syntenin-low patients express higher levels of CD138. The correlation with CD86 but not CD138 suggests a previously undescribed role for syntenin in myeloma cells. Our lab has shown that expression of CD86 is necessary for myeloma cell survival, and signals to confer drug resistance. When we silenced syntenin, we observed decreased CD86 expression. Moreover, knockdown of CD86 showed increased expression of syntenin. Taken together, these data suggest that syntenin may regulate CD86 expression on the cell surface. We now show a novel role for syntenin in myeloma cell viability and as a potential regulator of CD86 expression. Syntenin has not previously been explored in multiple myeloma and determining its molecular function is warranted as it may be an effective target for therapeutic treatment of the disease. 

### Cancer Predisposition and Synthetic Lethality

#320

Frequency and patient characteristics of homologous recombination deficiency in metastatic cutaneous melanoma.

Kevin B. Kim,1 Sherri Z. Millis,2 Jeffrey Ross,3 Laurie M. Gay,2 Elham Vosoughi,1 John Moretto,1 Stanley P. Leong,1 Mark I. Singer,1 Brian M. Parrett,1 David R. Minor,1 Mohammed Kashani-Sabet1. 1 _California Pacific Medical Center, San Francisco, CA;_ 2 _Foundation Medicine, Cambridge, MA;_ 3 _Albany Medical College, Albany, NY_.

Purpose: In various cancer types, cells with a homologous recombination (HR) deficiency have been shown to be sensitive to PARP inhibitors. We investigated the frequency of HR pathway gene mutations in melanoma.Experimental Design: Next-generation sequencing analysis that included the entire coding sequence of 315 cancer-related genes was performed on formalin-fixed, paraffin-embedded melanoma samples. Clinical and pathologic characteristics of 62 patients with advanced melanoma were reviewed, and compared with a dataset of 1,986 melanoma patients with available molecular profiling data.Results: Nineteen (30.6%) patients had ≥1 functional HR mutation. The characteristics of patients with HR-deficient melanoma were: median age 69, male predominance (79%) and primary head and neck melanoma (53%). TMB was high in 12 (67%) patients and low in 1 (6%) patient. The most commonly mutated HR-associated gene was ARID2 (13%), followed by ARID1A, ATM, BRCA1, and FANCA (3% each). Among the 19 patients with HR mutation(s), concurrent NF1, NRAS, V600 BRAF, and KIT mutations were found in 7 (37%), 6 (32%), 5 (26%) and 1 (5%) patients, respectively. Presence of HR pathway mutation was associated with absence of ulceration at the primary site, high TMB and clinical response to checkpoint immunotherapy. A larger dataset analyzed by Foundation Medicine, Inc. (n=1,986) showed a similar frequency and pattern of HR mutations. Conclusions: HR pathway gene mutations are frequently observed in advanced melanoma. Melanomas with these alterations may represent a unique subset of patients who are more likely to benefit from checkpoint blockade, and also may be targeted with PARP inhibitors.

Clinical and pathologic characteristics of patients with genetic HR mutations

---

|

HR mutation (n=19) | Non HR mutation (n=43) | p-value

Ulceration status | n=16* | n=33* | 0.0050

Present | 2 (12.5%) | 18 (54.5%) | 0.0114

Absent | 14 (87.5%) | 15 (45.5%) | 0.0479

Tumor mutation burden | n=18* | n=38* | 0.0114

Low | 1 (5.6%) | 17 (44.7%)

|

Intermediate | 5 (27.8%) | 8 (21.1%)

|

High | 12 (66.7%) | 13 (34.2%)

|

Response to anti-PD-1 antibody inhibitors (+/- ipilimumab) | n=12* | n=23* | 0.0479

Response | 10 (83.3%) | 10 (43.5%)

|

Early Progression | 1 (8.3%) | 10 (43.5%)

|

Stable disease | 1 (8.3%) | 3 (13.0%)

|

#321

**Synergistic activity of the ATR inhibitor BAY 1895344 in combination with DNA damage inducing and DNA repair compromising therapies in preclinical tumor models** **.**

Antje Margret Wengner, Gerhard Siemeister, Ulrich Luecking, Julien Lefranc, Kirstin Meyer, Eleni Lagkadinou, Bernard Haendler, Pascale Lejeune, Dominik Mumberg. _Bayer AG, Berlin, Germany_.

The DNA damage response (DDR) system consists of complex signalling pathways that secure the integrity of the genome in eukaryotic cells. DDR pathway activation follows recognition of DNA damage and results in cell cycle arrest, suppression of general translation, induction of DNA repair, cell survival or even cell death. Proteins that directly recognize aberrant DNA structures recruit and activate kinases of the DDR, such as ATR (ataxia telangiectasia and Rad3-related). ATR responds to a broad spectrum of DNA damages, including double-strand breaks (DSB) and lesions derived from interference with DNA replication as well as increased replication stress. Therefore, inhibition of ATR kinase activity could be the basis for a novel anti-cancer therapy in tumors with increased DNA damage, deficiency in DDR or replication stress. The potential of combining ATR kinase inhibitor with DNA damage inducing or DNA repair compromising anti-cancer therapeutics was studied in preclinical tumor models. We assessed the novel ATR kinase inhibitor (ATRi) BAY 1895344 in combination with external beam radiation therapy (EBRT), poly ADP ribose polymerase (PARP) inhibition or anti-androgen (AA) therapy. In cellular anti-proliferation assays as well as in tumor xenograft studies we could demonstrate synergistic activity of BAY 1895344 in combination treatment with the PARP inhibitor AZD-2281 in the homologous recombination (HR) defective breast cancer model MDA-MB-436, and with the non-steroidal AA darolutamide in the hormone-dependent prostate cancer model LAPC-4. Strong synergistic anti-tumor activity of BAY 1895344 could be further demonstrated in combination with EBRT inducing long-lasting tumor growth inhibition in the colorectal cancer xenograft model LOVO. The mechanism-based potential of combining DNA damage induction by EBRT with ATRi BAY 1895344 suggests a potential new treatment option for radiation therapy-resistant patients. Furthermore, the inhibition of parallel DDR pathways, as a combination of ATRi BAY 1895344 with a PARP inhibitor, indicates novel treatment opportunities in breast cancer patients with homologous recombination deficiencies, as does the synergism of BAY 1895344 and AA darolutamide therapy in hormone-dependent prostate cancer patients. BAY 1895344 is currently under clinical investigation in patients with advanced solid tumors and lymphomas (NCT03188965).

#322

High EMSY expression defines a BRCA-like subgroup of high-grade serous ovarian carcinoma with superior survival and platinum sensitivity.

Robert L. Hollis,1 Michael Churchman,1 Tzyvia Rye,1 Andrena M. McCavigan,2 Laura A. Knight,2 Richard Kennedy,2 C. Simon Herrington,1 Charlie Gourley1. 1 _Nicola Murray Centre for Ovarian Cancer Research, Edinburgh CRUK Centre, MRC IGMM, University of Edinburgh, Edinburgh, United Kingdom;_ 2 _Almac Diagnostics, Craigavon, United Kingdom_.

EMSY, encoding a BRCA2-binding protein, is reportedly amplified in 6-18% of high grade serous ovarian carcinomas (HGSOCs). As a negative regulator of BRCA2, HGSOCs with EMSY overexpression may mimic BRCA-mutant patients, who experience superior clinical outcome and hypersensitivity to platinum chemotherapy and PARP inhibition by virtue of homologous recombination deficiency (HRD).

Here we investigate the impact of EMSY expression, extracted from local and publicly available transcriptomic data, on the outcome and platinum sensitivity of HGSOCs. Platinum response data were collected from the Edinburgh Ovarian Cancer Database. Overall survival (OS) and progression-free survival (PFS) differences were assessed using multivariate cox regression accounting for age, stage at diagnosis and debulking status.

Within the Edinburgh cohort, high EMSY expression was associated with superior OS and PFS (table 1). Datasets from the MRC ICON7 trial control arm, Tothill et al, Mateescu et al, Pils et al, and TCGA cohorts demonstrated similar benefit for high-EMSY patients. Within the Edinburgh cohort, high-EMSY patients displayed significantly superior complete response (CR) rate to platinum at second (radio CR 44.4% vs 12.5%, P = 0.035; CA125 CR 88.0% vs 55.0%, P = 0.002) and third exposure (radio CR 50.0% vs 5.9%, P = 0.080; CA125 CR 53.3% vs 21.3%, P = 0.021), with prolonged time to progression (median 127 vs 83.5 days from second platinum, P = 0.084; median 151.5 vs 60.5 days from third platinum, P = 0.004). Within high-risk (advanced stage suboptimally debulked) HGSOCs, more high-EMSY patients remained recurrence-free 5 and 10 years from diagnosis (17.6% vs 2.7%, P = 0.031; 12.5% vs 0.9%, P = 0.041).

Together these data demonstrate a subgroup of HGSOCs defined by high EMSY expression experience superior clinical outcome and platinum sensitivity, consistent with HRD. This subgroup may therefore represent HGSOCs that are also sensitive to PARP inhibition. | |  | |

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

Dataset | Event | Multi HR | 95% CI | P

Edinburgh | OS | 0.59 | 0.39 – 0.90 | 0.013

|

PFS | 0.60 | 0.38 – 0.93 | 0.022

MRC ICON7 control arm | OS | 0.21 | 0.07 – 0.68 | 0.009

Tothill (primary tumour masses) | OS | 0.28 | 0.09 – 0.90 | 0.032

Mateescu | OS | 0.43 | 0.18 – 0.99 | 0.048

Pils | OS | 0.27 | 0.08 – 0.87 | 0.028

TCGA advanced stage patients | PFS | 0.69 | 0.45 – 1.05 | 0.081

#323

DNA damage response (DDR)-targeting strategy by targeting WEE1 and or ATM/ATR works in biliary tract cancer.

Ah Rong Nam,1 Ji Eun Park,1 Ju-Hee Bang,1 Mei Hua Jin,1 Yung-Jue Bang,2 Do-Youn Oh2. 1 _Cancer Research Institute, Seoul National Univ. College of Medicine, Seoul, Republic of Korea;_ 2 _Department of Internal Medicine, Seoul National University Hospital, Seoul, Republic of Korea_.

Background: Currently, targeting DNA damage response (DDR) is one strategy for new cancer drug development. DDR pathway involves DNA damage repair, cell cycle progression, apoptosis, and regulate the immune system. Biliary tract cancer (BTC) has a poor prognosis with a huge unmet medical need. In BTC, DNA repair pathway, which includes BAP1, MSH6, BRCA1, ATM, MLH1, and MSH2, is altered in about 20 % of cases. TP53 module is observed in 33.9% of cases (Nat Genet 2015). The purpose of this study is to evaluate DNA damage response (DDR)-targeting strategy in BTC.

Methods: Using 10 kinds of BTC cell lines (SNU245, SNU308, SNU478, SNU869, SNU1079, SNU1196, HuCCT-1, TFK-1, SNU2670, and SNU2773). AZD1775 (Wee1 inhibitor), AZD6738 (ATR inhibitor) and AZD0156 (ATM inhibitor) were tested.

Results: Among 10 cell lines, SNU308 and HuCCT1 were very sensitive to AZD1775, and SNU2670 and SNU2773 were relatively resistant to AZD1775. AZD1775 blocked phosphorylation of CDK1 (Y15) and CDK2 (Y15) in sensitive cells and but increased rH2AX in all cells. AZD1775 significantly increased apoptosis (cleavage of PARP and caspase-7) and G2/M arrest. Interestingly, Wee1 inhibitor increased pATR and pATM levels in resistant cells. AZD1775 in combination with AZD6738 or AZD0156 showed more potent antitumor effect than monotherapy of each drug.

Conclusion: Inhibition of Wee1 has an antitumor effect in some BTC cells. In combination with ATM/ATR inhibitors, the resistance to Wee1 inhibition could be overcome. Taken together, this study supports the further clinical development of DDR-targeting strategy in BTC as monotherapy or in combination.

#324

SIK2 inhibitors regulate DNA repair pathway and sensitize ovarian cancer to PARP1 inhibitors.

Zhen Lu,1 Wequn Mao,1 Lan Pang,1 Janice M. Santiago-O'Farrill,1 Haling Yang,1 Ahmed Ahmed,2 Hariprasad Vankayalapati,3 Robert C. Bast1. 1 _UT MD Anderson Cancer Center, Houston, TX;_ 2 _Oxford University, Oxford, United Kingdom;_ 3 _Arrien Pharmaceuticals, Salt Lake City, UT_.

Genomic instability is a recognized hallmark of cancer. Germline mutations in critical DNA-repair and DNA-damage response genes, including BRCA1and BRCA2, predispose to cancer development, but also create vulnerabilities that can be exploited for cancer therapy. The successful development of PARP inhibitors for cancers with BRCA1/2 mutations and a deficiency in homologous recombination DNA repair has provided proof of concept. Inhibition of kinases that promote transcription of DNA repair genes might provide targets that enhance the activity of PARP-inhibitors. One novel candidate, salt-inducible kinase 2 (SIK2), is overexpressed in approximately 30% of high grade serous ovarian cancers. Inhibition of SIK2 induces tetraploidy, triggers apoptotic cell death, prevents metastasis, reduces AKT/survivin signaling and decreases phosphorylation of class-IIa histone deacetylases (HDACs) enhancing inhibition of gene expression. In contrast to other HDACs, class-IIa HDACs function as signal-responsive transcriptional corepressors for the myocyte enhancer factor-2 (MEF2) family of transcription factors. MEF2s play important roles in regulating growth factor responses, neuronal survival, T-cell apoptosis and, importantly, B-cell DNA double strand break (DSB) repair. Here we ask whether inhibition of SIK2 can enhance HDAC class-IIa inhibition of MEF activity, decrease DNA repair and enhance sensitivity to the PARP inhibitor Olaparib in ovarian cancer cells.

Treatment with SIK2 inhibitors (ARN3236 or ARN3261) enhanced sensitivity to Olaparib in each of 8 ovarian cancer cell lines without BRCA1/2 mutations (OC316, OVCAR8, IGROV1, A2780, HCC5030, HCC5032, SKOv3 and OVCAR5) and significantly reduced the IC50 of Olaparib in two PARP inhibitor-resistant ovarian cancer cell lines (A2780CP-R and UWB1-289-10R). Synergistic cytotoxicity, judged with a Chou-Talaylay combination index (CI), was observed in all 8 cell lines. Treatment with a SIK2 inhibitor decreased the phosphorylation of class-IIa HDAC4/5/7, abolished class-IIa HDAC4/5/7-associated transcriptional activity of MEF2 and decreased MEF2 binding to regulatory regions with high-chromatin accessibility in DNA repair genes, resulting in repression of critical gene expression in DNA repair pathways. DNA damage, judged by rH2AX expression was enhanced by SIK2 inhibition. These observations not only provide new insights into the transcriptional regulation of DNA repair gene expression, but also have important implications for enhancing sensitivity of high grade serous ovarian cancers to PARP inhibition, with or without BRCA1 or BRCA2 germline mutations.

#325

A functional homologous recombination assay predicts primary chemotherapy response and long-term survival in ovarian cancer patients.

Manuela Tumiati,1 Sakari Hietanen,2 Johanna Hynninen,2 Elina Pietilä,1 Anniina Färkkilä,3 Katja Kaipio,4 Amjad Alkodsi,1 Minna Tuominen,1 Sampsa Hautaniemi,1 Olli Carpén,1 Liisa Kauppi1. 1 _University of Helsinki, Helsinki, Finland;_ 2 _Turku University Hospital, Turku, Finland;_ 3 _Dana-Farber Cancer Institute, Boston, MA;_ 4 _University of Turku and Turku University Hospital, Turku, Finland_.

Background:

High-grade serous ovarian cancer (HGSOC) is the most common and aggressive type of ovarian cancer (OC) and the fifth most common malignancy occurring in women, meaning that 1 in 70 will develop OC during her lifetime. The current first-line treatment for HGSOC patients consists of cytoreductive surgery and platinum/taxanes-based chemotherapy. Homologous recombination deficiency (HRD) correlates with platinum sensitivity in OC patients, which clinically is the most useful predictor of sensitivity to PARPi. To date, there are no reliable diagnostic tools to anticipate response to platinum-based chemotherapy, thus we aimed to develop an ex vivo functional HRD detection test that could predict both platinum-sensitivity and patient eligibility to targeted drug treatments.

Methods:

We obtained a functional HR score by quantifying homologous recombination (HR) repair after ionizing radiation-induced DNA damage in primary ovarian cancer samples (n=24). Samples spontaneously clustered in 3 categories: HR-deficient, HR-low and HR-proficient. We analysed the HR score association with platinum sensitivity and treatment response, platinum-free interval (PFI) and overall survival (OS), and compared it with other clinical parameters (treatment strategy, FIGO stage, age). In parallel, we performed DNA sequencing of HR genes to assess if functional HRD can be predicted by currently offered genetic screening.

Results:

Low HR scores predicted primary platinum sensitivity with high statistical significance (p=0.001) and associated with longer PFI (HR-deficient vs HR-proficient: 531 vs 113 days). The HR score was the only factor that significantly correlated with improved OS (HR score <35 vs ≥35, hazard ratio=0.12, 95% CI=0.02 to 0.79, p=0.026). At the genomic level, we identified a few mutations of unclear significance in HR genes but, overall, DNA sequencing failed to predict functional HRD.

Conclusions:

We developed an ex vivo assay that detects tumor functional HRD and an HR score able to predict platinum sensitivity, which holds the clinically relevant potential to become the routine companion diagnostic in the management of ovarian cancer patients.

#326

A novel antimetabolite, FF-10502-01 exhibits potent antitumor activity via inhibition of both DNA replication and DNA damage repair in solid tumor cells.

Kazunori Saeki,1 Rena Uematsu,1 Kaoru Morimura,1 Takayuki Yamada,1 Hiroyuki Iwamura,1 Shinichi Watanabe,1 Timothy Madden,2 Shinji Hagiwara1. 1 _Fujifilm Corporation, Tokyo, Japan;_ 2 _Fujifilm Pharma US (FPHU), Cambridge, MA_.

Introduction FF10502-01 is a synthetic pyrimidine nucleoside analogue that is structurally similar to gemcitabine (GEM) with a substitution of sulfur for oxygen in the pentose ring. FF-10502-01 demonstrated potent anti-tumor activity in a number of preclinical studies. In both Capan-1 and SUIT-2 pancreatic cancer xenograft models, FF-10502-01 achieved superior tumor growth suppression and survival prolongation, respectively, compared to GEM, with less toxicity at clinically relevant doses. In GEM-resistant pancreatic PDX models, FF-10502-01 showed greater efficacy and tolerability than GEM. A Phase 1 trial of FF-10502-01 in patients with advanced cancer has been conducted in United States with positive results, and a Phase 2a trial is scheduled for 2018.

Methods To determine the putative differences between FF-10502-01 and GEM activity the following studies were undertaken. The inhibitory activity for polymerase alpha and beta was measured using a DNA synthesis assay in vitro. Purified human polymerases and tri-phosphates of test drugs were incubated for 30 minutes at 37 °C in the presence of [methyl-3H]dTTP, and radioactivity was measured by liquid scintillation counting. SUIT-2 cells were cultured in serum-free medium for 72 hours to establish a dormant cell model. The test drugs, with DNA damage inducers, were then added as combination treatment. After 72 hours, the cell growth inhibition was evaluated using an ATP cell viability assay. DNA damage in the dormant cell model was evaluated by alkaline comet assay.

Results To explain why FF-10502-01 was more potent than GEM in vivo, we investigated possible differences in their mechanisms of action (MOA). Both FF-10502-01 and GEM inhibited DNA replication in vitro and in vivo. On the other hand, only FF-10502-01 showed potent cytotoxic activity against serum starvation-induced dormant SUIT-2 cells in combination with DNA damage inducers (DDIs: H2O2, cisplatin, or temozolomide). Similar results were obtained in cholangiocarcinoma and ovarian cancer cell lines. In vitro enzymatic assays showed that FF-10502-01 was far more potent than GEM in inhibiting DNA polymerase-beta, which plays an essential role in repairing oxidative DNA damage via the base-excision repair pathway. The inhibitory activity of DNA repair may explain the differential effect of FF-10502-01 in solid tumors in which reactive oxygen species are generated by tumor specific microenvironment.

Conclusions Unlike GEM, FF-10502 is unique in preventing DNA repair via polymerase-beta inhibition when combined with DDIs, suggesting a dual MOA for FF-10502 in solid tumors compared to GEM.

#327

**Functional characterization of** RAD50 **germline deleterious variants identified from deep targeted sequencing study of familial esophageal squamous cell carcinoma.**

Shiu Yeung Lam, Lvwen Ning, Hoi Yan Ng, Maria Li Lung, Josephine Mun-Yee Ko. _The University of Hong Kong, Hong Kong, Hong Kong_.

Esophageal squamous cell carcinoma (ESCC), a histological subtype of esophageal cancer, is a highly prevalent malignancy in Southeast Asia with poor prognosis and high mortality rate. From previous Next-Generation Sequencing (NGS) studies that sampled familial ESCC cases from Henan, a hotspot of ESCC cases in China, rare germline variants of RAD50 homolog, double strand break repair protein (RAD50) were identified and predicted to be deleterious by in silico models. We hypothesized that ESCC patients carrying dominant-negative germline or somatic RAD50 alterations with disrupted MRN (MRE11-RAD50-NBS1) functions may show improved prognosis, as tumor cells are sensitized to genotoxic agents such as cisplatin and ionizing radiation(IR). The current study aims to characterize if these identified germline loss-of-function (LOF) mutants of RAD50 exert dominant-negative impacts on double-strand break (DSB) repair and ATM/ATR signalling. A stop-gain mutation at the zinc-hook domain (RAD50-SG1), and a missense mutation at the C-terminal ATP-binding cassette (RAD50-MS1) were chosen for functional characterization. These mutations were introduced into RAD50 ORF by site-directed mutagenesis. The wildtype (RAD50-WT), RAD50-SG1 and RAD50-MS1 were expressed alongside a vector-alone control (VA) in two cell lines, U2OS and KYSE70(ESCC), using a lentiviral system. DSB repair efficiency was assessed by γH2AX foci recovery. Expression of RAD50-WT reduced the number of γH2AX foci-(+) cells compared to VA at 6 hours post-IR, hinting of an improved DSB repair efficiency while RAD50-MS1 showed no reduction in γH2AX foci-(+) cells. Intriguingly, the number of γH2AX foci-(+) cells was significantly elevated in RAD50-SG1, indicating a reduced DSB repair efficiency in a dominant-negative manner as U2OS expresses wildtype RAD50 endogenously. RAD50-SG1 expressed U2OS was sensitized to Chk-inhibitor AZD7762, when compared to RAD50-WT, RAD50-MS1 and VA control, as measured by MTT assay. Details about the downstream ATM/ATR signalling for reduced phosphorylation of ATM/ATR substrates for impaired checkpoint signalling, data of dominant-negative functional impacts of the RAD50-SG1 mutant regarding cell proliferation in the presence of DSB stimuli (cisplatin treatment/γ irradiation), DSB repair efficiency and sensitivity towards PARP inhibitor will be presented. We expect our study to provide insight into the potential therapeutic impacts of RAD50 LOF mutants with PARP inhibition or Chk inhibition. It should provide a strategy for design of new therapeutic regimens for combined targeted disruption of MRN function with PARPi or Chki, which is expected to benefit ESCC patients, who show poor responses to conventional chemotherapy and radiotheapy.

#328

Targeting Mcl-1 enhances DNA replication stress sensitivity for cancer therapy.

Guo Chen,1 Andrew Magis,2 Ke Xu,1 Dongkyoo Park,1 David Yu,1 Taofeek Owonikoko,1 Gabriel Sica,1 Sarah Satola,1 Suresh Ramalingam,1 Walter Curran,1 Paul Doetsch,1 Xingming Deng1. 1 _Emory University, Atlanta, GA;_ 2 _Institute for Systems Biology, Seattle, WA_.

DNA double-strand breaks (DSBs) are mainly repaired by homologous recombination (HR) and non-homologous end-joining (NHEJ) pathways. Here we report that Mcl-1 acts as a functional switch in pathway choice between HR and NHEJ. Mcl-1 was cell cycle-regulated, with expression peaking in S/G2 phase when HR occurred. Endogenous Mcl-1 depletion reduced HR and enhanced NHEJ. Mcl-1 overexpression resulted in a net increase in HR over NHEJ. Mcl-1 directly interacted with Ku via its BH1 and BH3 domains, which are required for Mcl-1 to inhibit Ku-mediated NHEJ and promote Mre11 complex-mediated DNA resection and HR-dependent DSB repair. Using the Mcl-1 BH1 domain (aa256-265) as docking site, we identified a novel small molecule, MI-223, that directly bound to BH1 and blocked Mcl-1-stimulated HR DNA repair leading to a significant sensitization of cancer cells to hydroxyurea- or olaparib-induced DNA replication stress. Combined treatment with MI-223 and hydroxyurea or olaparib exhibited a strong synergy against lung cancer in vivo. This mechanism-driven combination of agents provides a highly attractive therapeutic strategy to improve lung cancer outcome.

#329

Highly potent and selective ATM kinase inhibitor M3541: A clinical candidate drug with strong antitumor activity in combination with radiotherapy.

Thomas Fuchss, Werner W. Mederski, Frank T. Zenke, Heike Dahmen, Astrid Zimmermann, Andree Blaukat. _Merck KGaA, Darmstadt, Germany_.

M3541 is an ATP-competitive inhibitor of the Ataxia telangiectasia mutated (ATM) kinase (IC50 < 1 nM), which targets tumor cell survival and growth by inhibiting double-strand break (DSB) repair as well as checkpoint control. DSB repair is crucial for survival of malignant tumor cells, especially under treatment with DNA damaging chemo- and radiotherapy. As such, the rationale of pharmacological inhibition of ATM is to increase and maintain the extent of unrepaired DNA damage generated by radio-, chemotherapy, and targeted therapies to drive tumor cells into cell death. We have developed an orally administered, sub-nanomolar potent & selective kinase inhibitor of ATM, M3541, for cancer therapy in combination with DNA damaging modalities. Here, we present the optimization of initial hit matter & its structure-activity relationships leading to our clinical candidate M3541, its chemical structure (first-time disclosure) & preclinical characterization using biochemical, cellular & human tumor xenograft models. M3541 sensitizes tumor cell lines to radiation therapy in vitro and strongly enhances the anti-tumor activity of ionizing radiation in vivo. These effects are due to the inhibition of ATM kinase activity as demonstrated by the levels of the phosphorylation of its primary downstream target CHK2 in human tumor cell lines. M3541 is currently being investigated in a multicenter Ph I clinical trial.

#330

BRCA1 and BLM helicase compensate for replication fork defects in the absence of 53BP1 protein.

Erin H. Sybouts, Sonal S. Tonapi, Alexander J. Bishop. _UT Health San Antonio, San Antonio, TX_.

53BP1 is well-known for its involvement in DNA double strand break (DSB) repair, binding DNA and influencing choice of DSB repair. In S/G2 phases of the cell cycle, 53BP1 accumulates at DSBs, but can be removed by BRCA1, allowing end-resection and homology-directed repair (HDR) to occur. Without BRCA1, HDR is defective unless 53BP1 is also lost, in which case HDR can proceed. Such secondary mutation is relevant as a mechanism of acquired chemoresistance in BRCA1 deficient tumors. BRCA1 is also important for maintaining replication fork stability, particular for blocking end resection by MRE11a at a collapsed fork. We therefore set out to ask whether 53BP1 also plays a role in maintenance of fork stability and how this role relates to the role of BRCA1 at a collapsed fork. 53BP1 has been indicated to co-localize with collapsed forks and indeed we find that in its absence fork stability is compromised in a DNA2, but not MRE11a, dependent manner. With decreased fork stability in the absence of 53BP1 we observe increased HDR, indicating a role for BRCA1 to compensate for the lack of 53BP1 by promoting HDR. However, if both 53BP1 and BRCA1 are both necessary for fork stability, then there must be additional compensating mechanisms providing fork stability when both are absent. In fact, absence of both 53BP1 and BRCA1 results in better replication progression than when either 53BP1 or BRCA1 alone are absent. A third mechanism of fork stabilization was identified involving BLM; a RecQ helicase involved in dissolving homologous recombination intermediates, can facilitate fork restart and reverse fork regression. Additionally, BLM has been shown to interact with both 53BP1 and BRCA1, is found at stalled replication forks, and promotes BRCA1 recruitment to sites of DNA damage. Targeting BLM in cells with impaired 53BP1 and BRCA1 increases chemosensitivity to DNA damaging agents, supporting the redundant nature of these three proteins at stalled replication forks. Our findings indicate that targeting BLM may be an effective treatment for HDR defective cancers that have developed chemoresistance; with applicability to BRCA1 and homologous recombination deficient cancers, such as breast, ovarian, and Ewing sarcoma.

#331

Therapeutic co-targeting of WEE1 and ATM has synergistic effects and contributes to downregulation of PD-L1 expression in pancreatic cancer.

Meihua Jin, Ah-Rong Nam, Ji Eun Park, Ju Hee Bang, Yung-Jue Bang, Do-Youn Oh. _Seoul National Univ. College of Medicine, Seoul, Republic of Korea_.

Background: Currently, targeting DNA damage response (DDR) is one of strategies for new cancer drug development. DDR pathway involves DNA damage repair, cell cycle progression, apoptosis, and regulate the immune system. In pancreatic cancer (PC), one of the most fatal disease, TP53 mutation was found in approximately 78% of cases and BRCA pathway alteration including BRCA1, BRCA2, the ataxia telangiectasia mutated (ATM) and PALB2 was observed in 5% (germline) and 12% (somatic) of patients. The purpose of this study is to evaluate the DDR-targeting strategy using Wee1 inhibitor and ATM inhibitor in PC.

Methods: Using a total of 11 kinds of PC cell lines (AsPC-1, Capan-1, Capan-2, MIA PaCa-2, PANC-1, SNU213, SNU324, SNU410, SNU2822, SNU2913, and SNU2918), AZD1775 (Wee1 inhibitor) and AZD0156 (ATM inhibitor) were tested.

Results: AZD1775 significantly inhibited cell proliferation in all of the PC cell lines. AZD1775 monotherapy induced apoptosis and S phase arrest, and decreased Wee1/ p-wee1/ p-CDC2 expression. An increase of rH2AX and caspase-7 cleavage were occurred by AZD1775. When the cells were treated with AZD1775, upregulation of p-ATM was observed.

The combination of AZD1775 and AZD0156, the synergism was found. P-wee1 and p-CDC2 were downregulated more obviously in co-treated cells compared with monotherapy.

In some cells, PD-L1 was increased after AZD1775 treatment. Interestingly the combination of AZD1775 and AZD0156 synergistically reduced PD-L1 protein level in the cancer cells, especially in SNU213 and SNU2913 cells.

Conclusion: Therapeutic WEE1 and ATM co-targeting strategy demonstrated promising anti-cancer effect in pancreatic cancer cells. Moreover, this co-treatment blocked PD-L1 expression. Taken together, this supports further clinical development of DDR targeting strategy in pancreatic cancer.

#332

The BET bromodomain inhibitor JQ1 synergizes with WEE1 inhibitor AZD1775 by impairing non-homologous end joining and enhancing DNA damages in non-small cell lung cancer.

Yuta Takashima,1 Eiki Kikuchi,1 Junko Kikuchi,1 Tetsuaki Shoji,1 Megumi Furuta,1 Hajime Kikuchi,1 Jun Sakakibara-Konishi,1 Ichiro Kinoshita,2 Hirotoshi Dosaka-Akita,2 Masaharu Nishimura1. 1 _First Department of Medicine, Hokkaido University School of Medicine, Sapporo, Japan;_ 2 _Department of Medical Oncology, Hokkaido University Graduate School of Medicine, Sapporo, Japan_.

Background: The bromodomain and extraterminal domain (BET) inhibitors are broadly active in different cancer types, including non-small cell lung cancer (NSCLC). Although their activity on oncogene expression such as c-Myc has been addressed in many studies, the mechanism of BET inhibition on the cytotoxicity remain unknown. BET proteins have been also reported to interact with some DNA damage repair-related genes. AZD1775, a selective WEE1 G2 checkpoint kinase inhibitor, induces DNA damage and consequent apoptosis by abrogating G2 cell cycle arrest and inducing premature mitotic entry. We hypothesized that repression of BET activity would increase WEE1 inhibitor-induced cytotoxicity by impairing DNA damage repair. Here, we evaluate the efficacy and mechanistic rationale for combining AZD1775 and JQ1 as a potential therapy for NSCLC.

Methods: NSCLC cell lines (A549, H1299, H1975) and human embryonic kidney cells line (293T) were used in the present study. Anti-tumor activities of JQ1, AZD1775, or the combination were analyzed using MTT survival assay. Changes in protein expression were analyzed by Western Blot analysis. mRNA expressions were evaluated by quantitative rt-PCR. Cell cycle was analyzed by flow cytometry using PI staining. Activity of non-homologous end joining (NHEJ) was evaluated using NHEJ reporter plasmid.

Results: The combination of AZD1775 and JQ1 showed synergistic effects for NSCLC cell lines in vitro with combination indices of 0.1-0.5. The JQ1 monotherapy did not induce gamma-H2AX expression, but JQ1 increased and prolonged AZD1775-induced gamma-H2AX expression. The mRNA analysis showed that JQ1 significantly repressed NHEJ-related genes, such as XRCC4 and LIG4. Moreover, NHEJ reporter assay revealed JQ1 diminished NHEJ activity.

Conclusions: Our data demonstrate that the combination of JQ1 and AZD1775 has a synergistic effect against NSCLC cell lines via a mechanism of compromised DNA damage repair by JQ1. This combination therapy can be a novel therapeutic strategy for NSCLC.

#333

USP1 is required for replication fork stability in BRCA1-deficient tumors.

Kah Suan Lim,1 Heng Li,2 Emma A. Roberts,1 Emily F. Gaudiano,1 Connor Clairmont,1 Karthikeyan Ponnienselvan,1 Jessica C. Liu,1 Kalindi Parmar,1 Ning Zheng,2 Alan D'Andrea1. 1 _Dana-Farber Cancer Inst., Boston, MA;_ 2 _University of Washington, Seattle, WA_.

Homologous-recombination (HR) deficient tumors with BRCA1 and BRCA2 mutations exhibit replication fork stability defects. To date, PARP inhibitors are the only targeted therapy available in the clinic against HR deficient tumors, and alternative therapies are needed. In this study, we found a deubiquitinase, USP1, to be significantly upregulated in tumors with mutations in BRCA1. SiRNA mediated silencing or small molecule inhibition of USP1 activity resulted in replication fork destabilization and decreased viability of BRCA1 deficient cells, revealing a synthetic lethal relationship between USP1 and BRCA1. The cofactor of USP1, UAF1, had previously been shown to have DNA-binding activity. We observed that USP1 independently binds to and is stimulated by fork DNA. It is therefore the first known deubiquitinase (DUB) to be directly regulated by DNA binding. A truncated form of USP1, lacking its DNA binding region, was not stimulated by DNA and failed to localize and protect the replication fork. Persistence of monoubiquitinated PCNA at the replication fork was the mechanism of fork destabilization and cell death in the absence of USP1. Loss of monoubiquitinated PCNA, resulting from RAD18 knockdown, rescued the sensitivity and replication fork instability induced by USP1 inhibition. USP1 therefore is the first DUB enzyme exhibiting DNA-mediated activation at the replication fork, and is required for fork protection in BRCA1 deficient cells. We propose small molecule inhibitors against USP1 as a therapeutic option for BRCA1 deficient cancers.

#334

Detection of BRCAness by a functional biomarker.

Chih-Ying Lee, Min-Yu Ko, Kai-Hang Lei, Peter Chi. _Institute of Biochemical Sciences, National Taiwan University, Taipei, Taiwan_.

Homologous recombination (HR) is an error-free repair pathway to eliminate DNA double-strand breaks and cells with deficiency of HR repair are highly sensitive to poly(ADP-ribose) polymerase (PARP) inhibitors. Thus, targeting HR-defective cells becomes a new therapeutic approach for cancer patients. HR deficiency can be identified as BRCAness which may be due to mutations or epigenetic silencing in genes involved in HR, such as BRCA1 and BRCA2. Recent studies have developed several approaches to analyze HR status of cancer cells including RAD51 focus formation assay, large-scale mutational signatures analysis, and genome-wide transcriptome profiling. However, these assays provide an indirect estimation of HR status. We wish to directly measure HR rate in cancer cells. We have successfully developed two approaches, plasmid-based and virus-based functional assays, to directly quantify HR activity in cells and serve as a functional biomarker. Our results show that the signal of the functional biomarker specifically depends on the presence of the key enzyme, RAD51 recombinase, in HR. Finally, these functional assays can be utilized in different cancer cell types to determine the HR activity and can be a functional biomarker to evaluate the "BRCAness" status for the treatment of PARP inhibitors.

#335

Variable off-target effects of clinically advanced PARP inhibitors.

Monica E. Wielgos, Jay Patibandla, Michelle Firlit, Elke Van Oudenhove, Paulina Cybulska, Petar Jelinic, Douglas A. Levine. _NYU Langone Health, New York, NY_.

High-grade serous ovarian carcinoma (HGSOC) is the most common subtype of ovarian cancer with low 5-year survival rates. Inhibitors of poly (ADP-ribose) polymerase (PARP) are promising novel agents that uniquely target HGSOCs with DNA repair deficiencies. The majority of patients with DNA repair proficient HGSOCs do not respond to PARP inhibitor monotherapy, highlighting the need for novel treatment approaches for these women. The goal of this study was to expand the patient population that could benefit from PARP inhibition by identifying the off-target effects that are favorable in combination with CHK1 inhibitors among the five most clinically advanced PARP inhibitors.

Cell cycle and western blot analysis revealed that the four most potent PARP trappers (rucaparib, olaparib, niraparib, and talazoparib) have a strong effect on the cell cycle. The PARP trappers in particular activated CHK1 through phosphorylation at the serine 345 residue, decreased total protein levels of CDC25C, and arrested cells in the S/G2 phase of the cell cycle. These results were not observed after treatment with veliparib, the least potent PARP trapper. We created phospo-CHK1 mutants to confirm CHK1 activation at serine 345 and to further investigate whether potent PARP trappers activate CHK1 on serine 317, another residue that is also phosphorylated in response to replication stress. The data suggest that PARP trappers activate CHK1 at both the serine 317 and 345 phosphorylation sites.

Next, we examined whether PARP inhibition-induced activation of CHK1 is a determinant of synergy between PARP and CHK1 inhibitors. We exposed one of our established BRCA wild-type, TP53 mutant HGSOC patient-derived xenograft models to a PARP1 inhibitor (veliparib or talazoparib) or to the CHK1 inhibitor (MK-8776) or a combination of PARP and CHK1 inhibitors. CHK1 inhibition slightly increased sensitivity to talazoparib compared to single agent therapy. However, the tumor volume was significantly reduced by the combination of CHK1 inhibition and veliparib treatment compared to either single agent alone. This study suggests that there are off-target cell cycle effects that vary among the five most clinically-advanced PARP inhibitors that likely influence the response to combinatorial treatment. We propose the use of PARP and CHK1 inhibition as a strategy for HGSOC patients who would otherwise have minimal benefit to PARP inhibitor monotherapy.

#336

Anti-tumor activity of the Chk1 inhibitor prexasertib (LY2606368) as a single agent in triple negative breast cancer models.

Wenjuan Wu, Constance King, Gregory Donoho, Philip Iversen, Andrew Capen, Mark Castanares, Jennifer Stephens, Yan Ding, Susan Pratt, Ricardo Martinez, Sean Buchanan, Christoph Reinhard, Richard Beckmann, Aimee Lin. _Eli Lilly and Company, Indianapolis, IN_.

Triple-negative breast cancer (TNBC), the most aggressive subtype of breast cancer, is diagnosed in approximately 15% of all breast cancer patients and characterized by high level of genomic instability, defects in DNA damage response (DDR) and increased replication stress (RS). Currently the treatment options for TNBC are limited, and new approaches are needed. Checkpoint kinase 1 (Chk1) is a key kinase that regulates the cell cycle, DDR and RS through the modulation of cell cycle checkpoints and replication fork licensing; and has emerged as an attractive target for anti-cancer therapy. Prexasertib (LY2606368), an ATP-competitive inhibitor of Chk1 has demonstrated single agent activity in vitro and in vivo. Prexasertib is being evaluated in an ongoing TNBC Phase 2 trial sponsored by NCI; and preliminary results suggest modest single-agent activity in sporadic TNBC patients (https://academic.oup.com/annonc/article/27/suppl_6/231PD/2798970). To understand the biology and mechanisms underlying prexasertib's activity and/or identify the potential biomarkers that may predict response or identify novel combinations that could improve prexasertib's efficacy, we evaluated the anti-tumor activity of prexasertib as a single agent in TNBC cancer cell lines in vitro and in vivo, including xenograft, orthotopic and patient-derived xenograft (PDX) tumor models. Prexasertib inhibited cell proliferation in 12 TNBC cell lines with IC50 values ranging from 0.32 nM to 117.3 nM; and demonstrated inhibition of Chk1 auto-phosphorylation and activation of γH2AX, RPA32 and DNAPKc in TNBC cell lines including HCC1187, HCC1806, MX-1 and MDA-MB-231. Treatment with prexasertib inhibited tumor growth by 83.8%, 85.5% and 94.2% in HCC1187, MX-1 and HCC1806 xenograft models, respectively. Prexasertib also inhibited primary tumor growth by 74.4% and lung metastasis by 97.48% in a MDA-MB-231 mammary fat pad orthotopic model. The anti-tumor effect of prexasertib was further assessed in 40 TNBC PDX models. Prexasertib treatment resulted in 10% (4/40) of models achieving complete tumor regression, 40% (16/40) achieving partial tumor regression/stasis and 50% (20/40) not deriving benefit. Potential biomarkers of prexasertib were investigated. TNBC cells with higher phosphorylation levels of DNAPKc and RPA32 demonstrated higher sensitivity to prexasertib treatment. TNBC PDX tumors with higher RNA message levels of CyclinE1, Cyclin D1 and c-Myc showed better prexasertib response. Together these findings will inform subsets of TNBC and/or individual patient characteristics that should be considered for future clinical assessments and development of potential combination therapy strategies.

#337

The PARP inhibitor olaparib is synergistic with the ATR inhibitor AZD6738 in ATM deficient cancer cells.

Rebecca Lloyd, Katarynza Falenta, Paul W. Wijnhoven, Christophe Chabbert, Jonathan Stott, James Yates, Alan Y. Lau, Lucy A. Young, Simon J. Hollingsworth. _AstraZeneca, Cambridge, United Kingdom_.

The poly(ADP-ribose polymerase) (PARP) inhibitor olaparib is FDA approved for the treatment of BRCA-mutated ovarian cancers. Olaparib inhibits PARP function during DNA single-strand-break repair and also by trapping of PARP on DNA to create lesions. These complexes cause replication-associated DNA damage including stalling and collapse of DNA replication forks. Cells which have lost BRCA1/2-dependent homologous recombination repair are highly sensitive to olaparib. In addition, other DNA damage response (DDR) pathways mediated by the ataxia telangiectasia mutated (ATM) and ataxia telangiectasia mutated and Rad3-related (ATR) kinases are hypothesised to be important survival pathways in response to PARP inhibitor treatment. Here we show that olaparib combines synergistically with the orally bioavailable ATR inhibitor AZD6738, in vitro, leading to cell death in ATM-deficient backgrounds. The growth inhibitory and cell kill effect of a range of concentrations of AZD6738 (0.01-1 µM) and olaparib (0.03-3 µM) were assessed using the Loewe model of additivity in the isogenic ATM knockout (KO) and wildtype (WT) FaDu head and neck cancer cell lines. The combination of AZD6738 and olaparib induced cell death in the ATM KO cells across a wide range of concentrations, with 3 µM olaparib plus 1 µM AZD6738 inducing 84% cell kill, in contrast to only 37% growth inhibition in the ATM WT cells. The observed combination synergy in the ATM KO cells is a result of increased DNA replication-associated DNA damage together with abrogation of the G2-M checkpoint activated by olaparib. Olaparib treatment led to a 10 and 20% increase in G2 cells at 24 hours in the ATM KO and ATM WT FaDu cell lines respectively, however co-administration of AZD6738 (0.1-0.3 µM) completely released the olaparib treated cells from the G2 arrest. DNA metaphase spread analysis showed that combination treatment with olaparib (1 µM) plus AZD6738 (0.1 µM) in the ATM KO cells caused 12.8 chromosomal DNA aberrations per cell compared to 4.4 or 3.3 aberrations per cell when treated with olaparib or AZD6738 alone respectively (n = 2). In contrast, in the ATM WT cells the olaparib plus AZD6738 combination treatment caused only 1.5 chromosomal aberrations per cell. Collectively, these data show that olaparib-induced DNA damage activates an ATR-dependent G2-M checkpoint allowing time to repair damaged DNA, and that AZD6738 abrogates this checkpoint, permitting cells to undergo mitosis in the presence of DNA damage leading to increased chromosomal aberrations. These data provide a mechanistic understanding of the combination and supports the clinical line of sight for the development of AZD6738 in combination with olaparib.

#338

A new investigational ATM Inhibitor, M3541, synergistically potentiates fractionated radiotherapy and chemotherapy in cancer cells and animal models.

Astrid Zimmermann,1 Frank Zenke,1 Heike Dahmen,1 Christian Sirrenberg,1 Thomas Grombacher,1 Lyubomir T. Vassilev,2 Thomas Fuchss,1 Andree Blaukat1. 1 _Merck KGaA, Darmstadt, Germany;_ 2 _EMD Serono Research and Development Institute, Billerica, MA_.

Physical or chemical DNA damaging agents are among the most widely used classes of cancer therapeutics today. Radiotherapy and topoisomerase inhibitors induce double strand breaks (DSB) considered most dangerous if left unrepaired because they can induce growth arrest, chromosomal abnormality and/or cell death. ATM is an essential kinase which orchestrates one of the two major pathways for repair of DSBs via the high fidelity homologous recombination (HR) mechanism. In addition, ATM acts as an upstream signaling kinase, regulating cell cycle checkpoint control in the response to DSB damage. Inhibitors of ATM kinase are expected to suppress DSB DNA repair, block checkpoint controls and enhance the therapeutic effect of radiation therapy and other DSB-inducing treatment modalities. We have developed a novel, highly potent and selective inhibitor of ATM, M3541, which modifies cellular response to DSB damage leading to potentiation of radiation therapy and DSB-inducing chemotherapy. Here, we present data from the preclinical evaluation of M3541 therapeutic potential in cellular and xenograft models of cancer. ATM inhibitor sensitized multiple tumor cell lines to ionizing radiation and a topoisomerase inhibitor in vitro. Oral administration of M3541 to nude mice bearing human tumor xenografts in a clinically relevant radiation regimen strongly enhanced the antitumor activity, leading to complete tumor regression. These activities resulted from inhibition of ATM kinase activity and modulation of its downstream effects in the xenograft tissues. M3541 is currently under clinical investigation in a Phase I trial.

#339

**Heterozygous germline mutation in** ABRAXAS **causes BRCA1 mislocalization and DNA damage response defects.**

Robert Winqvist,1 Muthiah Bose,1 Juliane Sachsenweger,2 Niina Laurila,1 Ann Christin Parplys,3 Jonas Willmann,4 Leila Eshraghi,1 Thomas W. Dunlop,5 Marco Groth,6 Katrin Rapakko,7 Pentti Nieminen,8 Thomas W. Friedl,9 Lisa Heiserich,10 Felix Meyer,3 Hanna Tuppurainen,1 Ganapathy Raman Devarajan,1 Hellevi Peltoketo,1 Heli Nevanlinna,11 Katri Pylkäs,1 Kerstin Borgmann,3 Lisa Wiesmuller,9 Helmut Pospiech12. 1 _University of Oulu / NordLab Oulu, Oulu, Finland;_ 2 _Ulm University / University of Oulu, Ulm / Oulu, Germany;_ 3 _University Cancer Center Hamburg (UCCH) / University Medical Center Hamburg-Eppendorf, Hamburg, Germany;_ 4 _University Cancer Center Hamburg (UCCH), Hamburg, Germany;_ 5 _University of Eastern Finland, Kuopio, Finland;_ 6 _Leibniz Institute of Aging - Fritz Lipmann Institute, Jena, Germany;_ 7 _Northern Finland Laboratory Centre NordLab Oulu, Oulu, Finland;_ 8 _University of Oulu, Oulu, Finland;_ 9 _Ulm University, Ulm, Germany;_ 10 _Medipan GmbH, Berlin, Germany;_ 11 _University of Helsinki / Helsinki University Central Hospital, Helsinki, Finland;_ 12 _University of Oulu / Leibniz Institute of Aging - Fritz Lipmann Institute, Oulu / Jena, Finland_.

Whilst heterozygous germline mutations in the ABRAXAS gene have been associated with hereditary breast cancer predisposition, their initial effect on promoting tumorigenesis at the cellular level has not been explored. Here, we demonstrate in patient-derived cells that the Finnish ABRAXAS founder mutation (c.1082G>A, Arg361Gln), even in the heterozygous state leads to decreased BRCA1 protein levels as well as reduced nuclear localization and foci formation of BRCA1 and CtIP. This causes disturbances in basal BRCA1-A complex localization, which is reflected by a restraint in error-prone DNA double-strand break (DSB) repair pathway usage, attenuated DNA damage response, deregulated G2-M checkpoint control and apoptosis. Most importantly, mutation carrier cells display a change in their transcriptional profile, which we attribute to the reduced nuclear levels of BRCA1. The current study clearly demonstrates how the Finnish ABRAXAS founder mutation acts in a dominant-negative manner on BRCA1 to promote genome destabilisation in heterozygous carrier cells.

#340

CTDP1 regulates FANCI activation and DNA repair.

Kimiko L. Krieger,1 Wen-Feng Hu,2 Dragana Lagundzin,1 Nicholas T. Woods1. 1 _University of Nebraska Medical Center, Omaha, NE;_ 2 _Ziangya Hospital, Central South University, Changsha, China_.

Objective: The modular BRCA1 C-terminal (BRCT) domain plays a central role in the regulation of DNA damage repair (DDR). RNA Pol II C-terminal domain phosphatase 1 (CTDP1), the only phosphatase encoding a BRCT domain, is one of the few BRCT domain-containing proteins without a formal role ascribed to DDR. Thus, the goal of this study is to characterize the DDR-specific role of CTDP1 through interrogation of the BRCT domain-protein interaction with FANCI and the repair of DNA interstrand cross-links (ICL) through the Fanconi anemia pathway.

Methods: Novel CTDP1 BRCT domain-protein interactions were identified with mass spectrometry and analyzed with gene ontology enrichment. Molecular biology experiments were conducted to examine the effects of CTDP1 knockdown or overexpression on cell growth, survival, apoptosis, regulation of FANCI activation and localization, FANCD2 foci formation and ubiquitination, and homologous recombination repair. Mass spectrometry was used to determine both CTDP1-regulated FANCI phosphorylation sites and FANCI protein interactions.

Results: The BRCT domain of CTDP1 was found to interact with three Fanconi anemia proteins-FANCA, FANCD2, and FANCI. This interaction remains unchanged upon mitomycin C treatment. Loss of CTDP1 induces a four-fold decrease in homologous recombination repair efficiency. CTDP1 prevents ICL-induced apoptosis. Knockdown of CTDP1 negatively affects plating efficiency and decreases the rate of cellular growth in breast cancer cell lines, but not the untransformed MCF-10A breast cell line. CTDP1 prevents sensitivity to DNA-damaging agents repaired by homologous recombination, but not UV damage or treatment with paclitaxel. CTDP1 knockdown negatively impacts FANCD2 activation measured by DNA damage-induced foci formation and monoubiquitination, which is critical for the onset of ICL repair by the ID2 complex and downstream effector proteins of the Fanconi anemia pathway. CTDP1 regulates 15 phosphorylation sites in FANCI determined by mass spectrometry profiling of cells overexpressing wild-type CTDP1 compared to phosphatase-dead D302K mutant. Paradoxically, CTDP1 phosphatase significantly increases SQ motif phosphorylation of S559 and S556, which is important for dormant origin firing and replication fork restart. This suggests that phosphorylation regulation of FANCI at non-SQ motif residues could regulate FANCI activation potential, resulting in increased phosphorylation at the SQ motif.

Significance: This is the first demonstration of a formal role of CTDP1 in DDR. CTDP1 is implicated in the regulation of the specific ICL-repair Fanconi anemia pathway, and regulates breast cancer cell survival in vitro and in vivo. Delineating the mechanism by which CTDP1 regulates FANCI activation can provide the basis for preclinical studies targeting CTDP1 in breast cancer to promote sensitivity to DNA-damaging therapies.

#341

The role of telomere protective protein TPP1 in hepatocellular carcinoma.

Jiang Zou, Ru Sun, Jingruo Xia, Dan Xiao, Chang Liu, Hebin Liao, Lei Xu, Qiang Ma, Yan Cai, Jingdong Li, Xiaolan Guo. _North Sichuan Medical College, Nanchong, Sichuan, China_.

Hepatocellular carcinoma (HCC) is a worldwide malignancy. To date, surgery is still the most effective treatment with curative potential, but only about 20% of patients with HCC are eligible for surgical intervention. Therefore exploring the pathogenesis of liver cancer, looking for molecules targeting therapy, is very important. Telomeres, the chromosome extremities, are protected by specific proteins, such as POT1, RAP1, TIN2, TRF1, TRF2,and TPP1. More and more findings have discovered that TPP1 plays an important role in the tumorigenesis. However, the mechanism of TPP1 in HCC remains unknown. The purpose of this study is to explore the role of TPP1 in HCC. Currently 42 cases of HCC patients were recruited in this study. First, the patients' tumor tissue, adjacent tissue and normal tissue were analyzed by Western blot. The results showed that TPP1 expression in patients' adjacent tissue and normal tissue was lower than that in tumor tissue, respectively (P<0.05; P<0.05). Furthermore, investigation on the protein expression levels of TPP1 with clinicopathologic characteristics discovered that the higher TPP1 in patients' tumor tissue was correlated with the tumor clinical stages, demonstrating that those patients who were in earlier stages (I + II) showed lower TPP1 protein levels than those in later or advanced stages (III + IV) (P<0.05). More importantly, the survival analysis showed that the overall survival was significantly shorter in patients with high TPP1 expression than in others (P<0.05). To further study the role of telomere protective protein TPP1 in HCC, we used lentiviral shRNA embedding TPP1 to knock down the expression of TPP1 in HepG2 and SK-Hep1 cells. The cell growth curve results showed that knockdown of TPP1 could inhibit the growth and proliferation of the above cells. Meanwhile we found that the migration and invasion abilities of the cells were significantly reduced by knocking down the expression of TPP1 compared with the cells transfected with the control lentiviral shRNA. Through scratch test and cell invasion transwell chamber test, TPP1 depletion dramatically suppressed the colony formation on soft agar, which indicated that TPP1 loss could reduce the transformation ability of HCC. More interestingly, we found that ATM pathway was activated after knockdown of TPP1, resulting in γ-H2AX and phosphorylated ATM(p-ATM) upregulation. On the other hand, removal of TPP1 initiated mTOR pathway activation, which resulted in phosphorylated mTOR (p-mTOR) augmentation, followed by the increase of p70S6K and 4EBP1 phosphorylation. Eventually the autophagy marker LC3 I/II conversion and p62 declined obviously by depletion of TPP1 in HepG2 and SK-Hep1 cells. Taken together, TPP1 could be a potential predictor of hepatocellular carcinoma staging and overall survival, and targeting TPP1 might be a therapeutic approach for HCC.

#342

Strigolactone analogues show potential as new combination therapy agents against pancreatic cancer.

Matthew D. Park,1 Jefferson M. Haake,1 Erika Parasido,2 Christopher Albanese,1 Ronit I. Yarden1. 1 _Georgetown University, Washington, DC;_ 2 _Georgetown University Medical School, Washington, DC_.

There is an increasing appreciation for combination therapy of antineoplastic drugs that target key pathways often through a synergistic effect, reducing the chance for tumor growth, self-renewal of cancer stem cell (CSC), spread of metastasis and drug resistance. The combination of DNA repair impairment and DNA damage was shown to be a successful approach. Previously, we had shown that strigolactone (SLs; synthetic analogues, SLAs), a novel class of phytohormones, causes DNA double strand breaks (DSBs) and inhibits breast and osteosarcoma cancer cells and CSC viability. Here, we tested whether SLAs inhibit the growth of pancreatic ductal adenocarcinoma (PDAC) cells and stem cells and whether SLAs sensitize the effects of Gemcitabine (Gemzar), the most commonly prescribed drug for PDAC patients. We show that SLAs induce DSBs in different PDAC cells and the combination of SLAs with Gemcitabine significantly increases apoptosis when compared to PDAC cells treated with Gemcitabine alone. This additive effect is independent of BRCA2 expression, which suggests that SLAs are effective at impairing multiple repair mechanisms and is suitable in combination with multiple DNA damaging agents. Interestingly, we show that SLAs sensitize patient-derived Gemcitabine non-responsive, PDAC Conditionally-Reprogrammed Cells (CRCs) to Gemcitabine, reducing their overall viability by more than 30%. Accordingly, the SLA- Gemcitabine combination has a significant impact on tumorsphere (CSCs) growth outcome. These findings demonstrate the potential for a novel drug combination of SLAs and Gemcitabine and underscore the potential of SLAs for multiple highly valuable translational applications regarding alternative therapies to combat cancer.

#343

Targeting DNA replication as a therapeutic strategy for acute myeloid leukemia with DNMT3A mutations.

Kartika Venugopal, Daphné Dupéré-Richer, Jonathan Licht, Olga Guryanova. _University of Florida, Gainesville, FL_.

Acute Myeloid Leukemia (AML) is an aggressive malignancy of the blood system, wherein somatic mutations in epigenetic modifier and chromatin remodeling genes are common. Recurrent somatic mutations in the DNA methyltransferase 3 alpha (DNMT3A) gene are detected in about 30% of AML cases, and predict poor outcomes in patients treated with anthracycline-based induction chemotherapy, due to suboptimal response to therapy and subsequent early disease relapse. DNA methylation profiling in both primary AML samples and in animal models carrying a DNMT3A mutation detected modest DNA hypomethylation that did not fully explain how mutant DNMT3A contributed to AML pathogenesis.

Recent studies have uncovered disordered nucleosome remodeling in response to DNA topological stress in DNMT3A-mutant cells, which was most pronounced during DNA replication. These changes were accompanied by negative enrichment of the cell cycle-associated gene expression signatures and attenuated CHK1 signaling, implicated in DNA damage response and replication fork integrity. Therefore, we hypothesized that targeting the S-phase in leukemic cells with DNMT3A mutations by pharmacologic agents that stall replication may offer enhanced therapeutic benefit. Indeed, our studies show increased sensitivity to replication stalling pharmacological treatments such as nucleoside analogues cytarabine and fludarabine in different cellular systems harboring DNMT3A mutations. This was accompanied by increased apoptosis as measured by PARP cleavage and annexin V binding.Analysis of the DNA damage signaling revealed elevated CHK1 phosphorylation signaling and accumulation of the DNA damage marker gammaH2A.X. A similar drug sensitivity trend was observed in murine Dnmt3a-mutant bone marrow cells treated with cytarabine, using clonogenic survival in MethoCult as readout.

In order to examine this, we are developing isogenic cellular systems with differing DNMT3A mutational status via the CRISPR-Cas9 gene-editing approach. Further, these isogenic cell lines will be used as a tool to characterize the effect of mutant DNMT3A on replication progression, arrest, and resolution under pharmacologically induced replication stress. This will include analysis of the chromatin remodeling at the replication forks, proficiency of DNA damage repair, and dynamic localization of wild-type and mutant DNMT3A within the cells throughout the cell cycle. Finally, we will test the efficacy of replication stress inducing therapies in vivo in a genetically accurate mouse model of Dnmt3a-mutant AML. These studies will shed light on the mechanism of a potential vulnerability in DNMT3A-mutant leukemia cells and may lead to a more effective treatment strategy for this common subtype of AML.

#344

A high-throughput functional screen reveals synthetic lethal interactions associated with replication stress in HORMAD1-expressing triple-negative breast cancers.

Dalia Tarantino,1 Callum Walker,2 Daniel Weekes,1 Helen Pemberton,2 Jessica Frankum,2 Rachel Brough,2 Christopher J. Lord,2 Anita Grigoriadis,1 Andrew Tutt1. 1 _Breast Cancer Now Research Unit, King's College London, London, United Kingdom;_ 2 _Breast Cancer Now Research Centre, The Institute of Cancer Research, London, United Kingdom_.

HORMAD1, expression of which is usually restricted to germline cells, is expressed in ~60% of triple-negative breast cancers (TNBCs) where it is associated with higher levels of allelic imbalance. In experimental systems, HORMAD1 also drives Homologous Recombination (HR) deficiency, PARP inhibitor and cisplatin sensitivity. To provide further insights into the role of HORMAD1 in TNBC, we generated a doxycycline-inducible HORMAD1 expression system in the copy number stable, HORMAD1-negative, TNBC cell line SUM159. Clonal, HORMAD1-positive, populations were established which displayed increased levels of genomic instability and reduced levels of homologous recombination. To identify synthetic lethal therapeutic targets associated with HORMAD1 expression in TNBC, we performed an RNAi library screen, in which the viability of HORMAD1-expressing SUM159 clonal cell populations was assessed following the targeted depletion of 1743 genes. Candidate synthetic lethal (SL) genes were validated in a secondary screen using 3 additional HORMAD1-expressing SUM159 clones. The primary siRNA screen identified 63 genes which were SL with elevated HORMAD1 expression, including XRCC1, TDP1, Pol η, BRIP1 and ATR - genes with a known function in mitigating replication stress. DNA polymerase η (POLH) is a Y-family DNA polymerase involved in translesion synthesis (TLS), a DNA damage tolerance pathway employed by proliferating cells to bypass replication fork stalling DNA lesions and to prevent replication fork collapse. We hypothesised that HORMAD1 expression leads to Pol η localisation to replication factories, to mediate lesion bypass. Consistent with this, we found an enrichment in Pol η and monoubiquitylated-PCNA in the chromatin fraction of HORMAD1-expressing SUM159 cells, suggesting that Pol η and TLS might buffer the effect of elevated HORMAD1 expression in TNBC. Our data suggest that HORMAD1 expression in mitotic cells leads to a dependency on pathways implicated in preventing or resolving replication stress, such as TLS, and suggests that vulnerabilities associated with specific DNA repair processes could be used to target HORMAD1-positive TNBCs.

#345

**Characterizing and therapeutically targeting G-quadruplex DNA in** ATRX **-mutant glioma.**

Yuxiang Wang,1 Carla Danussi,2 Kasthuri Kannan,3 Timothy A. Chan,1 Jason T. Huse2. 1 _Memorial Sloan Kettering Cancer Center, New York, NY;_ 2 _MD Anderson Cancer Center, Houston, TX;_ 3 _New York University Medical Center, New York, NY_.

Inactivating ATRX mutations are defining molecular alterations in several cancer variants, including large subsets of malignant glioma. ATRX encodes a chromatin binding protein widely implicated in epigenetic and transcriptional regulation. However, ATRX is also thought to guard against genomic instability, in part by mitigating the formation of deleterious G-quadruplex (G4) DNA secondary structures. G4s have been shown to stall DNA replication leading to single-strand breaks and, potentially, DNA damage and genomic instability. To study this biology in a glioma-relevant context, we generated an isogenic astrocyte model featuring intact or deficient ATRX. While ATRX deficiency had no effect on baseline proliferation, cell cycle progression, and apoptosis, it led to markedly increased G4 formation, DNA damage, and replication stress signaling. These effects were reversed by ATRX re-expression. Moreover, ATRX-deficient astrocytes acquired copy number alterations over time, unlike ATRX-intact counterparts. Given these findings, we hypothesized that therapeutically targeting G4s might serve to exploit the inherent genomic vulnerabilities of ATRX-deficient cancer. Remarkably, treatment with a G4 stabilizing compound (CX3543) selectively inhibited growth and colony formation in ATRX-deficient astrocytes at nanomolar concentrations, and synergistically potentiated the effects of ionizing radiation and hydroxyurea. Finally, CX3543 significantly inhibited the growth of patient-derived, ATRX-deficient glioma xenografts in mice, with only minimal effects on ATRX-intact glioma xenografts. These results confirm and characterize a novel functionality for ATRX in the mitigation of genomic instability and point towards a viable strategy for therapeutically targeting ATRX deficiency in cancer.

#346

Targeting DNA repair with the combination of p97 and HDAC6 inhibitors in mantle cell lymphoma.

Pratikkumar Vekaria, Anusha Vallurupalli, Dharmalingam Subramaniam, Frank Schoenen, Joseph McGuirk, Rekha M. Rao. _Univ. of Kansas Medical Ctr., Kansas City, KS_.

p97 is an ATPase which is involved in remodeling multi-subunit protein complexes. It plays a pivotal role in the retrotranslocation and proteasomal degradation of misfolded ubiquitinated proteins in the endoplasmic reticulum. In association with cytosolic histone deacetylase 6 (HDAC6), p97 promotes the autophagic degradation of polyubiquitylated proteins and damaged organelles. More recently p97 has been implicated in regulating cell division and in facilitating DNA repair by promoting the recruitment of DNA repair proteins to the site of DNA damage. In this study, we determined the molecular mechanisms through which HDAC6 promotes p97 function in mantle cell lymphoma (MCL) cells. We report that treatment of MCL cells with the p97 inhibitors, DBeQ, ML240, NMS-873 and CB-5083 induces ER stress markers GRP78 and CHOP and resulted in dose-dependent apoptosis. Treatment of Z138C xenografts in NSG mice with CB-5083, the first in-class p97 inhibitor, resulted in increased accumulation of polyubiquitylated proteins and improved survival in vivo compared to control mice. Co-treatment with CB-5083 and the HDAC6-selective inhibitor ACY-1215, resulted in accumulation of polyubiquitylated proteins, impaired DNA double-stranded break repair and resulted in the partial depletion of BRCA1 and Cyclin D1 in MCL cells compared to either agent alone. We also demonstrate that shRNA-mediated knockdown of HDAC6 induced enhanced accumulation of H2AX-γ under basal conditions and following treatment with CB-5083 in cultured and primary MCL cells. Finally, treatment of MCL cells with p97 inhibitors and ACY-1215 results in synergistic apoptotic cell death in MCL cells. Collectively our studies create a strong rationale to test efficacy of the combination of p97 inhibitors in combination with HDAC6 inhibitors in MCL.

#347

Targeting the AICDA/RAD51 axis: A novel gain-of-function synthetic lethal therapy for the treatment of AICDA-expressing cancers.

Muneer Hasham,1 Kin-hoe Chow,1 Tyler Maclay,2 Amber Cyr,2 Darryl Patrick,2 Melinda Day,2 Kevin D. Mills2. 1 _The Jackson Laboratory, Bar Harbor, ME;_ 2 _Cyteir Therapeutics, Inc, Cambridge, MA_.

A key characteristic of many cancers is genomic instability, and is often associated with poor prognosis. While genomic instability can promote tumorigenesis, it also provides a therapeutic opportunity for synthetic lethality. The induction of DNA damage creates stress that necessitates highly active DNA repair as a critical survival system for many transformed cells. Recently, therapeutics that target PARP1 have been approved by the FDA as synthetic lethal therapeutics for cancers with deficiencies in BRCA1/2 and associated pathways. Here we present the early development of a new synthetic lethal therapy that leverages gain-of-function abnormalities to selectively target cancer cells. Activation Induced Cytidine Deaminase (AICDA or AID) is a DNA-directed cytidine deaminase that is normally expressed exclusively and transiently in activated B-lymphocytes, where it plays critical roles in somatic hypermutation and immunoglobulin class switching. AICDA is a DNA damaging enzyme, producing DNA base pair mismatches which can subsequently be converted into mutations, DNA single strand breaks (SSB), or DNA double strand breaks (DSB). Numerous cancers show constitutive overexpression of AICDA, leading to hypermutation, genomic instability, and tumor evolution. We have previously demonstrated that cells expressing AICDA are critically dependent upon the DNA repair factor RAD51. Here we present data illustrating the effectiveness of a novel small molecule, CYT02A, in targeting the AICDA/RAD51 axis. Cell culture assays showed CYT02A to be a potent DNA repair inhibitor that targets RAD51 subcellular localization and filament formation. Although CYT02A was stable when incubated with liver microsomes. Although CYT-02A showed limited oral bioavailability, it was stable in liver microsome assyas assays and the pharmacokinetic profile following intravenous injection revealed dose proportionality and acceptable in vivo exposures. CYT02A was efficacious in a human-to-mouse xenograft model of chronic lymphocytic leukemia (CLL). CYT02A was administered intravenously at a concentration of 50 mg/kg to AID+ CLL xenograft mice daily for up to 9 days. Treated mice showed a significant reduction in CLL burden in the bone marrow compared to vehicle control animals. CYT02A was well tolerated by the animals, with no observable change in behavior or complete blood counts (CBC). Taken together, these data validate a novel gain of function synthetic lethal approach targeting the RAD51/AICDA axis. Preclinical efficacy data support this strategy for the treatment of AICDA-expressing cancers. We continue to build upon this foundation to produce a new therapeutic paradigm that may be effective in a wide range of both hematologic malignancies and solid tumors.

#348

Targeting histone chaperone FACT complex and APE1 sensitizes colon cancer cells to chemotherapy.

Heyu Song, Shrabasti Roychoudhury, Pranjal Biswas, Jiping Zeng, Kishor Bhakat. _University of Nebraska Medical Center, Omaha, NE_.

Apurinic/apyrimidinic (AP) sites are one of the most common types of DNA damage that occur spontaneously and following chemotherapy. Human AP-endonuclease (APE1) is primarily responsible for initiating the repair of AP sites via the highly coordinated DNA base excision repair (BER) pathway. Although the repair of AP sites by APE1 utilizing naked DNA or nucleosomal DNA substrate has been extensively investigated in vitro, till to date, how APE1 repairs AP sites in chromatin in cells via BER pathway remains largely unknown. Chromatin remodeling histone chaperone FACT (facilitates chromatin transcription) complex has been previously shown to mediate nucleosome disruption which allows DNA repair proteins to gain access to damage sites. Its involvement in nucleotide excision repair (NER) pathway has been demonstrated. However, the role of FACT in BER remains unclear. Our lab has recently found that APE1 is acetylated at AP sites damage in chromatin and acetylation enhances the DNA repair activity of APE1. Here, we show that APE1 interacts with histone chaperone complex FACT. Subsequently, we confirmed the interaction of APE1 with FACT complex by Co-IP and immunofluorescence (IF) and found that both subunit of FACT complex, SPT16 and SSRP1, interact with APE1 in the nucleus and in chromatin. By Western blot (WB), IF and co-IP, we found enhanced interaction upon induction of DNA damage. To understand the functional importance of FACT, we downregulated its levels using siRNA and noted increased cell sensitivity upon damage by using MTT and colony formation. The number of AP sites in the genome increased significantly upon FACT knockdown. FRAP data demonstrated increased APE1 turnover time in damage sites after FACT knock-down suggesting the role of FACT in nucleosome disruption in facilitating the APE1 function in BER pathway. Decreasing FACT level led to reduced acetylation level of APE1 at damage sites and impaired DNA repair as evident by comet assay. Additionally, we found elevated levels of AcAPE1 and FACT in colon cancer tissues and multiple cell lines as compared to normal adjacent tissues and cells. We used curaxin which blocks the function of FACT and found increased cell sensitivity to various chemotherapeutic agents in multiple colon cancer cell lines. In nude mice xenograft, combination of curaxin and 5-FU group slowed the growth of tumor to the largest extent as compared to 5-FU and curaxin groups. Together our study suggests that FACT is required for APE1 to gain access to AP sites in the context of chromatin. Interruption of APE1 and FACT interaction exhibits synergistic effect on cell viability and proliferation upon DNA damage with chemotherapeutic drugs. We demonstrate targeting both FACT and BER pathway together as a novel strategy for chemosensitization of colon cancer cells.

### Chromatin Structure and Function

#349

Dynamic 3d chromosomal landscapes in acute leukemia.

Palaniraja Thandapani,1 Andreas Kloetgen,1 Charalampos Lazaris,1 Xufeng Chen,1 Panagiotis Ntziachristos,2 Aristotelis Tsirigos,1 Iannis Aifantis1. 1 _New York Univ. School of Medicine, New York, NY;_ 2 _Northwestern University, New York, NY_.

T-cell acute lymphoblastic leukemia (T-ALL) accounts for approximately 10-15% of pediatric and 25% of adult ALL cases. Despite improved prognosis driven by the significant advances in the molecular understanding of T-ALL, the outcome of T-ALL patients with primary resistant and relapsed leukemia remains poor. Recent evidences have highlighted a role for changes in 3D chromatin architecture in cancer progression. Herein, for the first time we explored genomewide changes in chromatin organization in primary T-ALL samples relative to T cells from healthy donors. We show how the organization of topologically associated domains (TADs) influences key oncogenic and tumor-suppressive loci by modifying the promoter-enhancer landscape, deregulating the transcriptional program and ultimately causing disease progression.

To comprehensively characterize the chromatin landscape of clinically relevant loci in T-ALL, we sought to identify changes in intra-TAD activity and TAD disruptions on a genome-wide scale. We developed and implemented new computational approaches for Hi-C data analysis with a focus on Hi-C data integration with ChIP-Seq and RNA-Seq. We found significant correlations of TAD activity with CTCF occupancy, super-enhancer activity and gene expression of leukemia relevant loci. Furthermore, among the dozens of TAD disruptions we identified, we found a novel TAD "fusion" event around the MYC locus, which allows for chromatin interactions between a recently reported Notch-dependent MYC super-enhancer and promoter to drive overexpression of MYC. This particular TAD fusion is accompanied by a complete CTCF loss in the TAD boundary and increased MYC expression in T-ALL samples. Overall, this study sheds new light into how changes in chromatin architecture influence disease progression by restructuring oncogene and tumor suppressor landscapes.

#350

Che-1/aatf-induced transcriptionally active chromatin promotes cell growth in multiple myeloma.

Tiziana Bruno,1 Francesca De Nicola,1 Frauke Goeman,1 Matteo Pallocca,1 Cristina Sorino,1 Valeria Catena,1 Gianluca Bossi,1 Bruno Amadio,1 Giovanni Cigliana,1 Enrico Spugnini,1 Maria Rosaria Ricciardi,2 Maria Teresa Petrucci,2 Alfonso Baldi,3 Mario Cioce,1 Giancarlo Cortese,1 Elisabetta Mattei,4 Roberta Merola,1 Umberto Gianelli,5 Francesco Pisani,1 Svitlana Gumenyuk,1 Andrea Mengarelli,1 Katja Hopker,6 Thomas Benzing,6 Aristide Floridi,1 Claudio Passananti,7 Giovanni Blandino,1 Simona Iezzi,1 Maurizio Fanciulli1. 1 _Regina Elena National Cancer Inst., Rome, Italy;_ 2 _Sapienza University of Rome, Rome, Italy;_ 3 _Campania University, Caserta, Italy;_ 4 _CNR, Rome, Italy;_ 5 _University of Milan, Rome, Italy;_ 6 _University Hospital of Cologne, Cologne, Germany;_ 7 _CNR-Institute of Molecular Biology and Pathology, Rome, Italy_.

Tumor transformation is the result of genetic and epigenetic modifications that alter gene transcription, consequently producing a specific oncogenic program. Multiple myeloma (MM) is a neoplasia characterized by the accumulation of proliferating antibodies producing plasma cells in the bone marrow. This disease is the second most frequent haematological malignancy in the US and Europe. In this study we demonstrate that Che-1 plays a crucial role in the control of transcription and cellular proliferation by regulating the state of the chromatin and by increasing its accessibility, highlighting Che-1 as an essential component of the transcription machinery in MM. Che-1 loss induces a global transcription shut-off, by reducing RNA Pol II recruitment onto the DNA. Most importantly, transcriptional inhibition in Che-1 depleted cells could already be detected at pre-mRNA levels, leading to the hypothesis that Che-1 could regulate RNA pol II activity directly. Our study shows that Che-1 is required for the maintenance of open chromatin in MM cells, and is involved in general histone acetylation. Morever, we found that Che-1 downregulation further sensitizes MM cells to bromodomain and extra-terminal (BET) inhibitors. Indeed Che-1 silencing increased JQ1 sensitivity of MM cells. In summary, our findings identify Che-1 as a key player for maintaining the open chromatin structure required for sustaining MM growth. These findings support Che-1 as a possible target for MM therapy, alone or in combination with BET inhibitors.

#351

Epigenetic mechanisms regulate DNA amplification and drug-resistant gene selection.

Sweta Mishra, Capucine Van Rechem, Damayanti Chakraborty, Johnathan R. Whetstine. _Massachusetts General Hospital & Harvard Medical School, Charlestown, MA_.

DNA copy gains and amplifications are often associated with tumorigenesis, poor prognosis and drug resistance. Little is known about how these regions of the genome are selected for amplification. Our group discovered that modulation of epigenetic factors and their associated chromatin states control DNA copy gains of drug-resistant regions. In fact, transient site-specific copy number gains (TSSGs) occur in both normal and cancer cells upon manipulation of a histone H3 lysine 9/36 (H3K9/36) tri-demethylase KDM4A. However, the mechanism by which KDM4A is targeted to specific genomic regions and whether additional chromatin regulators are involved in this process were unknown. To begin addressing these questions, we performed a series of unbiased genetic screens against all the histone lysine-modifying enzymes and followed their ability to generate copy gains of regions within chromosome 1. Our screen uncovered insights into both KDM4A targeting and the identification of other epigenetic factors modulating DNA amplification of drug-resistant regions of the genome. We have uncovered KDM4A-independent DNA amplifications, which established that additional regions are undergoing epigenetic control for amplification. These currently unpublished data will be discussed at the meeting.

#352

DNA replication licensing factor MCM5 is a target for SETD8 mediated methylation.

Fabio Pittella, Gil Blum, Yongxia Zhu, Chamara Senevirathne, Minkui Luo. _Memorial Sloan Kettering Cancer Center, New York, NY_.

SETD8 methyltransferase activity is implicated in several fundamental cellular processes such as transcriptional regulation and heterochromatin formation as well as processes that ensures genomic stability including DNA replication and the DNA damage response. It has been proposed that SETD8 affects DNA replication as a positive regulator of origin licensing through H4K20 methylation and by supporting Okazaki fragment processing through PCNA methylation. However, there is no evidence whether other key element in the replication machinery is directly modified by SETD8. To address this question, we used a combination of SILAC-based BPPM (Bioorthogonal Profiling of Protein Methylation) to profile new substrates for SETD8. We genetically engineered SETD8 and identified mutants amenable to accommodate non-native SAM analogues containing a terminal alkyne moiety for click chemistry. The engineered SETD8 can transfer this unique chemical moiety into target proteins for subsequent identification of the modified substrates. Stable cell lines expressing catalytically active or catalytically dead SETD8 mutants were systematically cultured with distinct stable isotope labeled amino acids allowing quantitative proteomic analysis of the identified substrates. With this method we could validate previously known substrates of SETD8 and identify novel targets, including MCM5, a subunit of the hexameric minichromosome maintenance (MCM) DNA helicase complex. MCM5 directly interacts with MCM2 and 3 to form the MCM2-7 hexamer which associates with the origins of DNA replication to form part of the pre-replicative complex (preRC), playing a major role during replication initiation and elongation. We found that SETD8 mediated methylation of MCM5 directly affects its binding affinity to MCM2 and 3. Amino acid substitutions at the methylated lysine further evidenced a stronger binding to its interacting partners contributing to the formation of the MCM hexamer. In addition, CRISPR based modifications on MCM5 methylated lysine affected the replication process resulting in defective cell cycle progression. Taken together our results indicate that MCM5 methylation contributes to the assembly of the MCM complex revealing a novel role for SETD8 in replication initiation. Our findings brings new perspectives on the biological importance of SETD8 during DNA replication.

#353

Using cancer-associated variants to dissect functions of SETD8.

Yongxia Zhu, Lihui Wang, Chamara Senevirathne, Shi Chen, Fabio Pittella Silva, Minkui Luo. _Memorial Sloan Kettering Cancer Center, New York, NY_.

Protein lysine methyltransferases (PKMTs) play important roles in carcinogenesis through methylation of histone and non-histone substrates. SETD8 is the sole human methyltransferase for the monomethylation of histone H4 Lys20 (H4K20me1). Besides histone substrates, SETD8 modifies multiple non-histone substrates including proliferating cell nuclear antigen (PCNA), Numb, and p53. The biological functions related to the methyltransferase activity of SETD8 have been implicated in many essential biological processes including DNA replication, DNA damage response, cell cycle regulation, and transcription modulation. The SET domain of SETD8 is required to catalyze the methylation in substrates using the cofactor S-adenosyl-L-methionine (SAM). The C-terminal region post the SET domain (cSET) contains both the cofactor and protein substrate-binding sites. Recent genomics data from samples of cancer patients have revealed distinct variants in the SET domain of SETD8. Missense mutations, such as, E289Q, R292L, V315I, and H347Q were found in multiple types of cancers. To help dissect the roles of SETD8, we proposed the identification and characterization of SETD8 variants with partial loss-of-function phenotypes. Our data showed that overexpression of the SETD8 mutants increased the migration and tumor sphere formation of breast cancer cells without affecting H4K20me1. In vitro biochemical assay also confirmed that the SETD8 mutants lose the methyltransferase activity on H4K20 substrate. The RNA-seq data in the context of SETD8 mutant indicated that the c-Myc pathway was upregulated without affecting c-Myc expression. With the technology of Bioorthogonal Profiling of Protein Methylation (BPPM), we have found that SETD8 can methylate cancer-associated transcription factors in a cellular context. Collectively, these findings suggest that native SETD8 can act on cancer driving-associated transcription factors and its mutant maintains this methyltransferase activity but spare H4K20 methylation for cancer malignancy.

Acknowledgements: This project was supported by 1R01GM120570.

#354

Role of KAT5 in lung tumorigenesis.

Gilbert Pan,1 Eunyoung Heo,1 Mariko Ando,1 Masanori Fujii,1 Ikei Kobayashi,1 Daniel G. Tenen,2 Hideo Watanabe,3 Susumu S. Kobayashi1. 1 _Beth-Israel Deaconess Medical Center, Boston, MA;_ 2 _Cancer Science Institute of Singapore, Singapore;_ 3 _Icahn School of Medicine at Mount Sinai, New York, NY_.

Epigenetic modifications in oncogenesis have long been an area of interest in cancer research. Histone modifications are crucial in activities such as transcriptional activation, gene silencing, and epigenetic cellular memory. In particular, lysine acetylation via lysine (K) acetyltransferases (KATs) have been implicated in cancer development. Interestingly, KAT5, also known as Tip60, has been reported to possess both tumor promoting and tumor suppressing properties depending on the context of malignancy. Herein we report that KAT5 contributes to tumorigenesis in EGFR-mutated lung cancer, and Kat5-knockout mice models demonstrate significantly reduced lung tumor burden. To probe the aberrant modification of KAT5, we demonstrated that KAT5 binds to and is phosphorylated by oncogenic EGFR in co-immunoprecipitation experiments. Next, to investigate whether KAT5 is involved in cell proliferation and survival, H1975 cells harboring L858R-T790M double-activating mutations were transfected with doxycycline inducible shRNA targeting KAT5 (shKAT5). Following treatment, shKAT5 cells were observed to have suppressed proliferation rates. Pharmacological inhibition using TH1834, a known KAT5 inhibitor, also suppressed proliferation rates in shKAT5 cells; in contrast BEAS-2B cells, an immortalized normal bronchial cell line, surprisingly exhibited increased viability compared to the transformed H1975 cells, supporting KAT5's context-dependent role in normal and abnormal cell homeostasis. To further investigate KAT5 in lung tumorigenesis in vivo, we generated EGFR-mutant conditional Kat5 knockout mice using a tetracycline-induced Cre/loxP system. Following doxycycline treatment for 10 weeks, isolated mice lungs for EGFRTL/CCSP-rtTA/Cre/Kat5F/F possessed significantly lower tumor volume compared to EGFRTL/CCSP-rtTA/Cre/Kat5W/F and EGFRTL/CCSP-rtTA/Cre/Kat5W/W mice lungs. Hemotoxylin and eosin staining showed no evident hyperproliferation in lungs isolated from EGFRTL/CCSP-rtTA/Cre/Kat5F/F mice whereas lungs isolated from EGFRTL/CCSP-rtTA/Cre/Kat5W/W and EGFRTL/CCSP-rtTA/Cre/Kat5W/F did, signifying that KAT5 has a potential regulatory role in cellular proliferation. RNA-Seq and ChIP-Seq analysis of shKAT5 H1975 cells identified downstream targets involved in tumorigenic pathways. Further studies are in progress to validate the mRNA and protein expression levels of reported targets. Taken together, these data offer insight into a KAT5 mediated oncogenic pathway that can provide novel therapeutic approaches in treating lung cancer.

#355

Histone acetyltransferase KAT6A upregulates PI3K/AKT signaling through TRIM24 binding.

Deguan Lv,1 Feng Jia,1 Yanli Hou,1 Youzhou Sang,1 Angel A. Alvarez,2 Weiwei Zhang,1 Bo Hu,2 Shi-Yuan Cheng,2 Jianwei Ge,1 Yanxin Li,1 Haizhong Feng1. 1 _Shanghai Jiao University School of Medicine, Shanghai, China;_ 2 _Northwestern University Feinberg School of Medicine, Chicago, IL_.

Lysine acetyltransferase KAT6A is a chromatin regulator that contributes to histone modification and cancer, but the basis of its actions are not well understood. Here we identify a KAT6A signaling pathway that facilitates glioblastoma (GBM) where it is upregulated. KAT6A expression was associated with GBM patient survival. KAT6A silencing suppressed cell proliferation, cell migration, colony formation and tumor development in an orthotopic mouse xenograft model system. Mechanistic investigations demonstrated that KAT6A acetylates lysine 23 of histone H3 (H3K23), which recruits the nuclear receptor binding protein TRIM24 to activate PIK3CA transcription, thereby enhancing PI3K/AKT signaling and tumorigenesis. Overexpressing activated AKT or PIK3CA rescued the growth inhibition due to KAT6A silencing. Conversely, the pan-PI3K inhibitor LY294002 abrogated the growth-promoting effect of KAT6A. Overexpression of KAT6A or TRIM24, but not KAT6A acetyltransferase activity- deficient mutants or TRIM24 mutants lacking H3K23ac binding sites promoted PIK3CA expression, AKT phosphorylation and cell proliferation. Taken together, our results define an essential role of KAT6A in glioma formation, rationalizing its candidacy as a therapeutic target for GBM treatment.

#356

Polyadenyaltion of canonical histone mRNA: A potential mechanism of arsenic-induced carcinogenesis.

Qiao Yi Chen,1 Danqi Chen,1 Thomas Kluz,1 Feng Wu,1 Yusha Zhu,1 Thomas Des Marais,1 Jinquan Li,2 Xiaoru Zhang,1 Ashley Jordan,1 Hong Sun,1 Chunyuan Jin,1 Max Costa3. 1 _New York University School of Medicine, New York, NY;_ 2 _Wuhan University of Science and Technology, Wuhan, China;_ 3 _New York University School of Medicine, Tuxedo, NY_.

Naturally occurring inorganic arsenic has been identified as a prominent causal agent in lung, bladder, liver, and prostate cancers. Although arsenic (As) has long been known to induce carcinogenicity via epigenetic mechanisms, alteration in histone gene expression has not been explored. The replication-dependent histone genes (also known as canonical histone genes) are the only genes found in multicellular organisms whose messenger RNA (mRNA) does not terminate with a poly(A) tail at the 3' end. Instead, they contain a conserved 26-nucleotide sequence that forms a stem-loop structure, and serves as the binding site for the Stem-loop binding protein (SLBP). SLBP is critical for canonical histone pre-mRNA processing and histone mRNA translation. Our previous studies demonstrated that arsenic exposure increases polyadenylation of histone H3.1 mRNA (a canonical histone H3) and its presence outside of S phase of the cell cycle due to the loss of SLBP protein and mRNA. Here we report that ectopic expression of polyadenylated H3.1 mRNA induces cell transformation and that the transformed cells are able to form tumors in athymic nude mice. These carcinogenic effects are dependent on the level of polyadenylation of canonical H3.1 mRNA. Notably, transfection of H3.1 cDNA with a stem-loop sequence in the front of the poly(A) signal generates less polyadenylated H3.1 mRNA and thereby attenuates cell transformation. Furthermore, the increase in polyadenylated H3.1 mRNA is able to antagonize histone H3.3 (a non-canonical variant of H3) in the promoter of active genes and perturb expression of cancer-related genes. In addition transfection of H3.1 with a poly(A) tail causes mitotic block in the cell cycle as well as chromosome instability. Furthermore, acute arsenic exposure by inhalation in mice results in the loss of SLBP and gain of polyadenylated H3.1 mRNA in lung tissues. These results suggest that the polyadenylation of canonical histone mRNAs induce by Arsenic is very disruptive to cellular genomic integrity and contributes to development of human cancers.

#357

Phosphorylated histone H3 mediates epigenetic regulation of Brf1 and Pol III genes in alcohol-associated breast cancer.

Lei Lu,1 Zeng Fang,2 Wen Li,2 Zhimin He,1 Shuping Zhong3. 1 _Guangzhou Medical University, Guangzhou, China;_ 2 _Sun Yat-sen University, Guangzhou, China;_ 3 _University of Southern California, Los Angeles, CA_.

Approximately 3.6% human cancers worldwide derive from chronic alcohol drinking, including cancers of breast and multiple other organs. Alcohol intake is consistently associated with an increased risk of BC (breast cancer). Alcohol consumption is more pronounced in ER+ BC cases than in ER- cases. However, the molecular mechanism remains to be determined. RNA polymerase (Pol) III genes, such as 5S rRNA and tRNAs, are elevated in both transformed and tumor cells. Brf1 (TFIIIB-related factor 1), a subunit of TFIIIB complex, specifically regulates Pol III gene transcription. Our studies have demonstrated that Brf1 expression and Pol III gene transcription are increased in alcohol-induced mice liver tumor. Alcohol-induced deregulation of Pol III gene transcription may be fundamental to the development of BC. We have found that alcohol-increased Pol III gene transcription in ER+ BC cells is significantly higher than in ER- normal breast cells and BC cells. The induction is ER dependent. The cellular levels of Brf1 protein and mRNA are increased in ethanol-treated MCF-7 cells. Ethanol markedly stimulates phosphorylation of MSK1 (MSK1ph) and histone H3 (H3ph). Our previous studies demonstrated that MSK1 mediates H3ph. MSK1 mediates ERα expression. Inhibition of MSK1 signal reduces alcohol-induced ERα expression to decrease cellular level of Brf1 and Pol III genes. Repression of Brf1 expression decreases alcohol-induced anchorage-independent cell growth and tumor formation. Furthermore, our analysis indicates that ethanol treatment increases occupancy of ERα to Brf1 promoter. The results by using Co-IP and ChIP assays indicate that MSK1, ERα and Brf1 form a complex to mediate Pol III gene transcription. Studies of human biopsies of BC patients indicate that Brf1 expression is increased in human cases of BC. High expression of Brf1 displays longer overall survival period after Tamoxifen treatment. Co-localization analysis indicate that MSK1ph and H3S10ph localizes in nuclear with Brf1. These results support the idea that alcohol activates MSK1 and induces epigenetic modification of H3ph to enhance ERα and Brf1 expression and Pol III gene transcription, resulting greater phenotypic changes. Together, our studies indicate that ethanol-induced epigenetic modification of H3ph may play a critical role in alcohol-induced cell transformation and alcohol-associated ER+ BC. The project is supported by NIH grants: AA017288, AA021114, AA02324 and AA024169

#358

miRNAs/Ago2/YY1/PcG protein/DNMT complex mediates epigenetic silencing of locus genes and associates with bortezomib-induced apoptosis of leukemia.

Ju-Ming Wang,1 Yu-Yi Chu,1 Chiung-Yuan Ko,2 Shao-Ming Wang,3 Pin-I Lin,1 Han-Ying Wang,1 Wen-Chi Lin,1 Dong-Yu Wu1. 1 _Institute of Bioinformatics and Biosignal Transduction, Tainan, Taiwan;_ 2 _The Ph.D. Program for Neural Regenerative Medicine, Taipei, Taiwan;_ 3 _The Ph.D. Program for Neural Regenerative Medicine, Taipei, Taiwan, Tainan, Taiwan_.

miRNAs are a class of non-coding RNAs, which bind to the 3'-untranslated regions of their target mRNAs to repress translation. However, the details of miRNA-mediated epigenetic regulation targeting genomic DNA remained largely unknown. Here, we demonstrated that transcription factor CCAAT/enhancer binding protein delta (CEBPD) was responsive to the anticancer drug bortezomib, is a highly selective reversible inhibitor for leukemia treatment, and resulting in caspase activation and apoptosis. Interestingly, following the identification of CEBPD-induced miRNAs, we found that miR-744, miR-3154 and miR-3162 feedback to suppress the transcription of CEBPD itself and genes adjacent to its 5'-flanking region, including protein kinase DNA-activated catalytic polypeptide (PRKDC), minichromosome maintenance-deficient 4 (MCM4) and ubiquitin-conjugating enzyme E2 variant 2 (UBE2V2), upon bortezomib treatment. Additionally, we previously demonstrated that the Yin Yang 1 (YY1)/polycomb group (PcG) protein/DNA methyltransferase (DNMT) complex is important for mediating epigenetic silencing of the CEBPD gene; we further found that Argonaute 2 (Ago2) interacts with YY1 and binds to the CEBPD promoter. Moreover, we revealed that miRNA binding plays a key role for YY1/PcG group protein/DNMT complex-mediated epigenetic gene silencing and is associated with bortezomib-induced methylation on genomic DNA. Altogether, we provided new insights for miRNA-mediated epigenetic regulation in the case of bortezomib-induced cell death in leukemia.

#359

IWS1 is essential for mouse development.

Arturo Orlacchio,1 Claudia Foray,1 Tyler Sheetz,1 Erika Reese,1 Foued Amari,1 Aaron E. Stark,1 Krista La Perle,2 Ioannis Sanidas,3 Philip N. Tsichlis,3 Dario Palmieri,1 Vincenzo Coppola1. 1 _The Ohio State University and Arthur G. James Comprehensive Cancer Center, Columbus, OH;_ 2 _The Ohio State University and Arthur G. James Comprehensive Cancer Center,College of Veterinary Medicine, Columbus, OH;_ 3 _Tufts Medical Center, Boston, MA_.

Iws1 (Interacts with Spt6) was originally identified in Saccharomyces Cerevisiae as a binding partner of Spt6, a histone H3/H4 chaperone.As a transcription factor, Iws1 plays a key role in defining the composition of the RNA polymerase II (RNAPII) elongation complex and in modulating the production of mature mRNA transcripts.Iws1 is phosphorylated, following stimulation with growth factors, by Akt3 and Akt1 at Ser720/Thr721 in human cells (Ser667/Thr668 in the mouse).Preliminary evidence supports a critical protumorigenic role of Iws1 and its phosphorylation in several types of malignancies such as lung cancer (NSCLC), mammary adenocarcinomas and melanomas.For example, it has been shown that, while IWS1 expression in normal human lung is very low, dramatically increases in many NSCLCs. Interestingly the level of IWS1 phosphorylation is variable between tumors and correlates with Akt3 expression.Moreover data from the TCGA database show that Iws1 is expressed at higher levels in tumors than in normal mammary gland specimens and also that basal and HER-2-enriched tumors express the highest levels. The genetic depletion of Iws1 in human mammary adenocarcinoma cell lines inhibits anchorage-independent growth in soft agar and mammosphere formation and growth. Similar experiment revealed that human melanoma cells were extraordinarily sensitive to the knockdown of Iws1. These experiments identify mammary adenocarcinomas and melanomas as tumors potentially addicted to Iws1. However, Iws1 biological functions are poorly characterized and there is no reported in vivo investigation about this RNA-processing factor.To begin to shed some light on the function of this protein at organismal level, we performed a thorough analysis of the expression of Iws1 and generated Iws1-deficient mice.The systematic analysis of expression in mouse tissues shows that Iws1 is ubiquitously expressed. We also report that ablation of Iws1 causes lethality at very early stages of mouse development. In fact, Iws1-deficient mouse embryos die at pre-implantation stage. Overall, these results show that Iws1 is an essential gene for mouse development and suggest a critical function in mammary adenocarcinomas as well as in melanoma.

#360

The role of polybromo1 in stress response.

Elizabeth G. Porter, Emily C. Dykhuizen. _Purdue University, West Lafayette, IN_.

Polybromo1 (PBRM1) is mutated in 3.5% of human cancers with the highest incident in renal clear cell carcinoma (40%). PBRM1 is a member of the PBRM1-Brg-Associating Factors (PBAF) chromatin remodeling complex and is characterized by six tandem bromodomains. PBRM1 acts as a tumor suppressor and when deleted along with VHL, the most frequently mutated gene in RCCC, tumors develop in mice. We have previously shown that PBRM1 regulates the expression of genes involved in cell adhesion, apoptosis, and negative cell proliferation in a RCCC cell line. Additionally, we have demonstrated that four of the six bromodomains work together to regulate binding of the complex to chromatin, gene expression, and tumor suppression in RCCC, which correlates with pattern of PBRM1 missense mutations found in RCCC patient tumors. Through this work we identified histone 3 lysine 14 acetylation (H3K14Ac) as the chromatin mark essential for PBRM1 binding. While H3K14Ac is generally found at active promoters, it is also induced at the promoters of stress genes when cells experience stressors such as metabolic stress, osmotic stress, ER stress, or reactive oxygen species (ROS) stress. Knockdown of PBRM1 in normal epithelial cells leads to an increase in proliferation and a partial epithelial to mesenchymal transition (EMT), consistent with low grade ROS. RNA-seq analysis further confirmed a role for PBRM1 in regulating stress response genes. Although loss of PBRM1 increases proliferation in epithelial cells, it also increases cell vulnerability to high levels of ROS. Together this suggests that PBRM1 can be growth promoting or growth inhibiting to cells depending on the cells environment and stress response needs.

#361

Identification of a novel dependence on the SWI/SNF chromatin remodeling complex in uveal melanoma.

Florencia Rago,1 Xiaoqin Xiang,1 Aurore Desplat,2 Emilie Niemezyk,2 David Ruddy,1 Grainne Kerr,2 Eusebio Manchado Robles,2 Deborah Castelletti,2 Zainab Jagani1. 1 _Novartis Institutes for Biomedical Research, Cambridge, MA;_ 2 _Novartis Institutes for Biomedical Research, Basel, Switzerland_.

Uveal melanoma is a rare and aggressive cancer that originates in the pigmented tissues of the eye, which include the iris, the choroid and the ciliary body. To date, no targeted therapies have been approved for this cancer, and much remains to be described about the underlying biology of the disease. The most common driver mutation is an activating mutation in either the GNAQ or GNA11 GTPase, which in turns leads to hyperactivation of the MAPK pathway, but the precise downstream effectors driving tumorigenesis are poorly understood. Using pooled shRNA screening, we discovered that the SWI/SNF complex represents a novel genetic vulnerability in uveal melanoma, and further interrogated this dependency by a more targeted analysis across various uveal melanoma cell lines. While we observed the dropout of multiple SWI/SNF complex subunits in the screens, we defined the ATPase activity of SWI/SNF as central to the observed phenotype, implicating the chromatin remodeling function of the SWI/SNF complex in this phenotype. The regulation of gene expression by transcription factors can involve coordinated activity of SWI/SNF directed chromatin remodeling activity, so we began to investigate mechanistic links between SWI/SNF and other transcription factors such as MITF, which is known to play important roles in the differentiation of melanocytes. We observed that depletion of SWI/SNF affected transcription of certain MITF target genes, and are further investigating the precise pathways that are altered with SWI/SNF modulation. Taken together, these data suggest that the SWI/SNF complex plays an important role in maintaining tumor cell survival in uveal melanoma, and may represent a novel therapeutic target.

#362

EGFR signaling activates S100 gene expression in HER2-positive breast cancer cells.

Miguel Nava,1 Nathan R. Zemke,2 Arnie Berk,2 Robin Farias-Eisner,2 Jay Vadgama,1 Yanyuan Wu1. 1 _Charles Drew Univ. of Medicine & Science, Los Angeles, CA; _2 _University of California at Los Angeles, Los Angeles, CA_.

Introduction: Human Epidermal Growth Factor Receptor-2-positive (HER2+) breast cancer is one of the four major molecular types of breast cancer. HER2 is a classical receptor tyrosine kinase (RTK) and its kinase activity is stimulated by heterodimerization with other ligand bound HER family members, such as EGFR/HER1. Treatment for HER2+ breast cancer includes the use of Trastuzumab, a monoclonal antibody that binds to the HER2 extracellular domain and inhibits downstream signaling. Resistance to Trastuzumab involves several cellular and molecular alterations, including gain of function mutations in proteins that are downstream of HER2 activation. The purpose of this study is to examine EGFR signaling in HER2+ breast cancer cells to discover novel putative drug targets.

Methods: HER2-positive breast cancer cell line (SKBR3) was used before and after EGF treatment. In order to determine whether EGFR signaling was activated, the levels of several proteins of interest were determined by western blot analysis. RNA-seq and ChIP-seq for H3K18ac and H3K27ac was conducted following an EGF time course in SKBR3 cells.

Results: Treatment of SKBR3 cells with EGF resulted in a transient increase in pERK1/2 and pAKT, which returned to basal levels at 1h and 2h post-EGF treatment, respectively. This was surprising because pEGFR remained higher in treated cells compared to untreated cells throughout the 24h time course. Immediate early genes (IEGs) became activated in SKBR3 cells, as previously reported in other cell types, during the early phase of EGFR stimulation (<4h). Using mRNA-seq we determined expression patterns during a 24h period and found that SKBR3 cells treated with EGF resulted in the activation and repression by 2-fold or more of over 4000 transcripts. We examined the chromatin landscape following EGFR stimulation with chromatin immunoprecipitation for H3K18ac and H3K27ac followed by next generation sequencing (ChIP-seq). We determined that, regardless of transcript levels, H3K18ac and H3K27ac oscillated at all regulated genes and globally. A closer look at the genes that peaked in expression at 24h post-EGF treatment revealed that S100 genes steadily increased in expression. Moreover, the chromatin surrounding the locus that contains the majority of S100 genes increased in H3K18ac and H3K27ac 1h post-EGF treatment, suggesting that S100 genes are a direct target of EGFR/HER2 signaling. Summary: We hypothesize that S100 proteins, which act as Ca2+ sensors, could play a role in EGF induced tumor cell growth and metastasis, contribute to Herceptin resistance and are likely drug targets in HER2+ breast cancer.

#363

Role of the SAGA histone acetylation module in lung cancer.

Lisa Maria Mustachio, Jason Roszik, Sharon YR Dent. _MD Anderson Cancer Center, Houston, TX_.

Lung cancer causes the highest mortality in cancer-related deaths. Definition of novel molecular targets is needed since these cancers often become resistant to existing therapies. Epigenetic modifications may provide such targets. Recent reports suggest that the histone acetyltransferase (HAT) module within the transcriptional coactivator SAGA complex plays a role in cancer, creating a new link between epigenetic regulators and this disease. We are working to define the role of the SAGA HAT protein GCN5 in the regulation and function of oncogenes in lung cancers. Since our prior work indicates that GCN5 serves as a coactivator for MYC target genes, we hypothesize that inhibition of GCN5 will reduce lung oncogenesis by suppressing MYC functions. We also propose that inhibition of GCN5 will reduce lung cancer formation and progression in mouse models of lung cancer. Our preliminary data indicate that both GCN5 and MYC expression levels are upregulated and positively associated in mouse and human lung cancers. Both proteins are also associated with MYC target genes in lung cancer tissues. Inhibition of the GCN5 active site or bromodomain significantly (p < 0.05) reduces cell proliferation compared to normal lung epithelium cells. Studies investigating how GCN5 inhibition affects other lung cancer phenotypes are underway. Specific genes and pathways affected by GCN5 loss will be determined using RNA-Seq and RPPA assays to identify mechanisms behind observed phenotypes. In addition, we will determine whether knockout of Gcn5 reduces lung formation and progression in the KrasLA2 lung cancer mouse model. Our ongoing studies will determine whether targeting GCN5 represses multiple oncogenic pathways in cancer and whether it inhibits oncoproteins difficult to directly target, such as MYC. We expect that our findings will highlight the importance of epigenetic regulators in carcinogenesis and extend beyond our current research focus to other cancer types.

#364

Differentially methylated super-enhancers regulate gene expression in human papillomavirus-related head and neck squamous cell carcinoma.

Emily L. Flam,1 Dylan Z. Kelley,1 Elena Stavrovskaya,2 Ludmila Danilova,1 Theresa Guo,1 Michael Considine,1 Jiang Qian,1 Joseph A. Califano,3 Alexander V. Favorov,1 Elana J. Fertig,1 Daria A. Gaykalova1. 1 _Johns Hopkins Univesity, Baltimore, MD;_ 2 _Moscow State University, Moscow, Russian Federation;_ 3 _University of California, San Diego, San Diego, CA_.

Human papillomavirus-related head and neck squamous cell carcinoma (HPV+ HNSCC) is characterized by abundant gene expression changes, which cannot be explained by limited genetic alterations alone. We hypothesized that epigenetically regulated super-enhancers (SEs) are the drivers of aberrant gene expression in HPV+ HNSCC. We used integrated analysis of DNA methylation and gene expression data for cancer and noncancer oropharyngeal tissues to discover actionable SEs that regulate expression of target genes in HPV+ HNSCC. Since no SEs are currently defined for any HNSCC samples; we investigated 6196 SE elements that were obtained from the public domain for closely related tissues, including normal and tumor lung, and cervical cancer cell lines. We analyzed the methylation of these elements and gene expression of their nearby genes for 47 HPV+ HNSCC and their 25 noncancer controls. Overall, we found 122 differentially methylated SEs that had putative target (nearby) genes whose expression was correlated with enhancer methylation in HPV+ HNSCC. Of these, 107 were hypermethylated, and 15 were hypomethylated in tumors relative to normal samples. The pathway analysis revealed that the inferred SE-regulated genes were associated with pathways known to regulate carcinogenesis. Our data demonstrate that gene expression in HPV+ HNSCC may be regulated by epigenetic alterations in SE elements of related tissues.

#365

The impact of the RASSF1C-PIWIL1-piRNA pathway on DNA methylation.

Yousef G. Amaar, Mark E. Reeves. _VA Loma Linda Healthcare Systems and Loma Linda University School of Medicine, Loma Linda, CA_.

Introduction: Epigenomic regulation is becoming critically important in human cancers as epigenetically regulated genes can be used as biomarkers for diagnosis, prognosis, molecular classification of tumors, and predicting response to therapies. Hence, identification of epigenetic biomarkers for specific cancers is highly desirable for development of precision medicine tools. Recently we have identified a novel RASSF1C-PIWIL1-piRNA pathway that appears to promote lung cell proliferation and migration. PIWI-like proteins interact with piRNAs to form complexes that regulate gene expression at the transcriptional (epigenetic control) and translational (mRNA silencing) levels, leading to stimulation of cell stem renewal and proliferation. We have shown that RASSF1C modulates the PIWIL1-piRNA gene axis, suggesting the hypothesis that the RASSF1C-PIWI-piRNA pathway could potentially contribute to lung cancer stem cell development and progression, in part, through modulation of gene methylation of both oncogenic and tumor suppressor genes. Thus, we wanted to validate this hypothesis using a non-small cell lung cancer (NSCLC) cell model to determine if we can identify candidate genes targeted by the RASSF1C-PIWIL1-piRNA pathway through a gene methylation mechanism.

Methods: We conducted a pilot study on the impact of over-expressing RASSF1C and knocking down RASSF1C and P1WIL1 expression on global gene DNA methylation in the NSCLC cell line H1299. DNA from cells over-expressing RASSF1C, RASSF1C-knockdown, PIWIL1-knockdwn, and control cells was used for the methylation study using the Reduced Representation Bisulfite Sequencing (RRBS) method.

Results: Candidate Differentially Methylated Regions (DMR) were identified by comparing DNA methylation profiles of experimental and control cells. We found that over-expression of RASSF1C and knocking down RASSF1C and PIWIL1 modulated DNA methylation of genomic regions; and statistically significant candidate genes residing in hypo- and hyper-methylated regions in lung cancer cells were identified, including the Dual-specificity phosphatase 4 gene (DUSP4) that functions as a tumor suppressor.

Conclusion: The RASSF1C-PIWI-piRNA pathway may play a critical role in epigenome regulation and genesis of lung cancer stem cells through modulation of key genes such as DUSP4. We found that RASSSF1C over-expression increases, while knock-down of RASSF1C or PIWIL1 decreases methylation/expression of the DUSP4 gene perhaps providing a potential mechanism through which the RASSF1C-PIWIL1-piRNA pathway could promote lung cancer stem cell development and progression. DUSP4 is an interesting target gene because knockdown of DUSP4 has recently been shown to enhance cancer stem cell formation.

#366

**Association of** MLL2/KMT2D **and** MLL3/KMT2C **with** **chronic myeloid leukemia.**

Doralina do Amaral Rabello Ramos,1 Vivian D'Afonseca da Silva Ferreira,1 Maria Gabriela Berzoti-Coelho,2 Sandra Mara Burin,2 Cíntia Leticia Magro,2 Maira da Costa Cacemiro,2 Belinda Pinto Simões,2 Felipe Saldanha-Araujo,1 Fabíola Attié Castro,2 Fábio Pittella-Silva1. 1 _University of Brasilia, Brasilia, Brazil;_ 2 _University of São Paulo, São Paulo, Brazil_.

Chronic myeloid leukemia (CML) is a clonal myeloproliferative neoplasm linked to the Philadelphia chromosome presence that generates the BCR-ABL1 fusion oncogene. Tyrosine kinase inhibitors (TKI) such as imatinib mesylate (IM) dramatically improved the treatment efficiency and survival of CML patients by targeting BCR-ABL tyrosine kinase. Although patients in the chronic phase respond well to treatment, patients in the accelerated phase or blast crisis usually show therapy resistance and CML relapse. It is crucial, therefore, to identify biomarkers to predict CML genetic evolution and resistance to TKI therapy, considering not only the effects of genetic aberrations but also the role of epigenetic alterations during the disease. Although dysregulations in epigenetic modulators such as histone methyltrasnferases have already been described for some hematologic malignancies, to date very limited data is available for CML, especially when considering the MLL2/KMT2D and MLL3/KMT2C genes of histone methyltrasnferases. Here we investigated the expression profile of both genes in CML patients in different stages of the disease, in patients showing different responses to therapy with IM and in non-neoplastic control samples. Imatinib sensitive and resistant CML cell lines were also used to investigate whether treatment with other tyrosine kinase inhibitors interfered in their expression. Both genes were either upregulated or with basal expression level during the chronic phase compared to controls. Interestingly, MLL3/KMT2C and specially MLL2/KMT2D levels decreased during disease progression correlating with distinct clinical stages. Furthermore, MLL2/KMT2D was decreased in patients resistant to IM treatment. Our results established a new association between MLL2/KMT2D and MLL3/KMT2C genes with CML and suggest that MLL2/KMT2D is associated with disease evolution and may be a potential marker to predict the development of therapy resistance.

#367

Methods to enhance the optical resolution for 3D-SIM for the study of Hodgkin's lymphoma nuclei.

Aleksander T. Szczurek,1 Fabio Contu,1 Roberta Vanni,2 Hans Knecht,3 Nathalie Johnson,3 Tina Haliotis,3 Sabine Mai1. 1 _University of Manitoba, Winnipeg, Manitoba, Canada;_ 2 _University of Cagliari, Cagliari, Italy;_ 3 _McGill University, Montreal, Quebec, Canada_.

In spite of a great deal of knowledge already available, the precise higher order organization of chromatin is still a subject of an debate. Moreover, cancerous cells have an altered chromatin organization in comparison to their healthy counterparts. This is also the case for Hodgkin's lymphoma (HL) - a lymphoid malignancy of B cell origin. HL comprises two cell types, the Hodgkin cells and the diagnostic bi- or multinucleated Reed-Sternberg (RS) cells readily distinguishable from healthy lymphocytes. Patient stratitifcation has been proposed through the quantitative analysis of 3D nuclear telomere organization. With the advent of super resolution microscopy (SRM), further insights into the spatial organization of the cancer genome become feasible. This includes the study of chromatin arrangement with SRM. However, its application to HL remains limited due to technical difficulties: HL cells display thickness of >10µm resulting in light scattering and decreased signal-to-noise ratio.

Structured Illumination Microscopy (SIM, a SRM type) in particular has advanced studies of the sub-cellular structures. It relies on projection of excitation pattern on to a fluorescently labeled sample. Sample's information is then shifted to a detectable band doubling the 3D resolution. Sample's refractive index homogeneity and counteracting photo-bleaching are both crucial for maintenance of the sharp illumination pattern and high-quality SIM, particularly in thick samples. Nevertheless, sample mounting media for SIM have not undergone a significant evolution since almost a decade.

By identification and systematic comparison of non-hazardous components of mounting media we demonstrate increased sample's transparency and consequently improved SIM-quality. Using these novel sample clearing protocols we demonstrate high-resolution chromatin organization at ~120nm level in large RS cells of HL cell lines and in patient samples compared to lymphocytes. This work is of relevance of identification of new diagnostic structural biomarkers for HL.

### Emerging Concepts

#368

B-lymphoid transcriptional repression of the pentose phosphate pathway reveals a unique therapeutic vulnerability of B cell malignancies.

Gang Xiao,1 Zhengshan Chen,1 Lai N. Chan,1 Daniel Braas,2 Thomas G. Graeber,2 Huimin Geng,3 Hassan Jumaa,4 Xiaoyan Jiang,5 Markus Müschen1. 1 _City of Hope Comprehensive Cancer Center, Duarte, CA;_ 2 _University of California Los Angeles, Los Angeles, CA;_ 3 _University of California San Francisco, San Francisco, CA;_ 4 _University Medical Center Ulm, Ulm, Germany;_ 5 _University of British Columbia, Vancouver, British Columbia, Canada_.

Introduction Activation during normal immune responses and oncogenic transformation impose increased metabolic demands on B-cells and their ability to retain redox homeostasis. While the serine/threonine-protein phosphatase 2A (PP2A) was identified as tumor suppressor in multiple types of cancer, our genetic studies revealed an unexpected lineage-specific dependency on PP2A in a broad range of B-cell malignancies.

Results PP2A regulates glycolysis rate and balances energy supply against anti-oxidant protection of B-cells through the pentose-phosphate pathway (PPP). In contrast to robust PPP-activity in other hematopoietic lineages, B-lymphoid transcription factors (PAX5, IKZF1) restrict PPP-activity by transcriptional repression of G6PD and other rate-limiting PPP enzymes. Constitutively low PPP-activity and limited capacity to balance redox fluctuations cause a unique dependency on PP2A in B-cell tumors. Pharmacological ablation of PP2A and PPP-activity identify this pathway as a novel lineage-specific therapeutic target in a broad range of B-cell malignancies.

Highlights • Conditional deletion of Ppp2r1a, the central scaffold assembling the PP2A holoenzyme, induces acute cell death of early and mature B-cells but does not affect other hematopoietic lineages. • PP2A redirects glucose carbon utilization from glycolysis to the PPP to mitigate oxidative stress. • B-cell malignancies exhibit constitutively low PPP activity, owing to B-cell-specific transcriptional repression of G6PD and other rate-limiting PPP enzymes. • Transcriptional repression of PPP activity in B-cells represents the mechanistic basis for the unique dependency of B-cell malignancies on PP2A. • Small molecule inhibitors of PP2A and G6PD act synergistically and overcome conventional drug-resistance in B-cell tumors.

Summary Genetic lesions of PP2A are frequent in solid tumors and myeloid leukemia, but not in B-cell malignancies. Unlike other types of cancer, our genetic studies revealed an essential role of PP2A in B-cell tumors. Thereby, PP2A redirects glucose carbon utilization from glycolysis to PPP to salvage oxidative stress. This unique vulnerability reflects constitutively low PPP activity in B-cells and transcriptional repression of G6PD and other key PPP enzymes by the B-cell transcription factors PAX5 and IKZF1. Reflecting B-cell-specific transcriptional repression of PPP activity, glucose carbon utilization in B-cells is heavily skewed in favor of glycolysis resulting in lack of PPP-dependent antioxidant protection. These findings reveal a novel gatekeeper function of the PPP in a broad range of B-cell malignancies that can be efficiently targeted by small molecule inhibition of PP2A and G6PD.

#369

The retinoblastoma protein regulates glucose metabolism in lung cancer.

Lindsey Reynolds,1 Susan Dougherty,2 Traci Kruer,2 Brian Clem1. 1 _University of Louisville, Louisville, KY;_ 2 _James Graham Brown Cancer Center, Louisville, KY_.

Lung cancer is among the most frequently diagnosed cancers and is the leading cause of cancer-related deaths in the United States. Of its subtypes, non-small cell lung cancer (NSCLC) accounts for 85% of all lung cancer diagnoses. The retinoblastoma protein (Rb) is a tumor suppressor which functions to regulate cell cycle progression by altering the transcriptional activity of the E2F family of transcription factors. In most NSCLC cases, Rb inactivation occurs via hyperphosphorylation due to loss of upstream regulatory mechanisms including overexpression of cyclin D1 or loss of p16INK4A. Although the canonical fucntions of Rb have been well established, advances in our understanding of Rb have highlighted additional cellular processes regulated by this tumor suppressor in both normal and transformed cells. Increased glucose utilization has become a hallmark of transformed cells and is driven in part by specific genetic alterations in various oncogenes or tumor suppressors. In preliminary studies, we demonstrate a role for Rb in regulating glucose metabolism in both in vitro and in vivo models of lung cancer. Using A549 lung adenocarcinoma cells, which harbor a p16 deletion that allows CDK4/6 inactivation of Rb, we performed in vitro metabolic assays, including glucose uptake and glycolysis as well as measured the expression of key enzymes within glycolysis and downstream metabolic pathways. Each assay was performed following treatment with vehicle control or Palbociclib, a CDK4/6 inhibitor which inhibits the phosphorylation and subsequent inactivation of Rb. Following treatment with Palbociclib, A549 cells show decreased glycolysis and expression of glycolytic genes, without altering glucose uptake. To define the metabolic fates of glucose in vitro, we performed stable isotope resolved metabolomics (SIRM) analysis of 13C-glucose in A549 cells. Consistent with in vitro metabolic assays and gene expression data, SIRM analysis revealed treatment with Palbociclib decreases glucose-derived carbons into glycolytic intermediates and other metabolic pathways. To determine if Rb1 loss results in an altered metabolic phenotype in an in vivo model of lung cancer, we have crossed Rb1lox/lox mice with Kras+/LSLG12D mice to generate Kras+/LSLG12D/Rb1lox/lox and Kras+/LSLG12D/Rb1+/+ mice. Rb1 KO mice exhibit increased tumor burden as well as decreased overall survival compared to Rb1 WT mice. Additionally, SIRM analysis of 13C-glucose incorporation into lung tumors of Kras+/LSLG12D/Rb1+/+ and Kras+/LSLG12D/Rb1lox/lox mice revealed increased glycolysis in lung tumors of Rb1 KO mice compared to Rb1 WT mice. Lastly, immunohistochemistry was performed to determine the metabolic transporter/enzyme expression profile in these tumors. We have observed increased levels of glycolytic genes in Rb1 KO lung tumors compared to Rb1 WT. Taken together, these data indicate that Rb may regulate the metabolic reprogramming of lung cancer.

#370

Hyperpolarized magnetic resonance metabolic imaging in pancreatic cancer research: Early detection, assessing aggressiveness and real-time monitoring treatment response.

Prasanta Dutta, Erick Riquelme Sanchez, Yu Zhang, Travis Salzillo,, Priyank Raj, Jaehyuk Lee, Niki Millward, Anirban Maitra, Florencia McAllister, Pratip Bhattacharya. _UT MD Anderson Cancer Center, Houston, TX_.

Pancreatic cancer is on pace to become the second leading cause of cancer-related death. The high mortality rate results from a lack of methods for early detection and the inability to successfully treat patients once diagnosed. Pancreatic cancer cells have extensively reprogrammed metabolism, which is driven by oncogene-mediated pathways and the unique physiology of the tumor microenvironment. In our research, we are interrogating reprogrammed metabolism in pancreatic cancer. We have employed hyperpolarized metabolic imaging to measure noninvasively the metabolic plasticity as the diseases initiate, evolve and respond to the drugs. Genetically engineered mouse (GEM) model with progression of pancreatic intraepithelial neoplasm (PanIN) lesions has been used for early detection, patient-derived xenografts (PDX) model to assess aggressiveness, and orthotopic pancreatic cancer model for monitoring metabolically targeted therapeutic response. Hyperpolarization of pyruvate and in-vivo 13C MRS were performed using Hypersense (Oxford Instruments) and 7T MRI scanner with a dual tuned 1H/13C volume coil (Bruker), respectively. Tissue alanine and lactate concentrations were determined using a Bruker 500 MHz NMR spectrometer coupled with cryoprobe. Histology and immunohistochemistry were performed on excised tissue samples. Hyperpolarized pyruvate metabolism in PDX tumor was well captured in real time. Pyruvate was readily metabolized to lactate and alanine in vivo. The quantitative flux ratios lactate-to-pyruvate (Lac/Pyr) and alanine-to-lactate were determined. The most aggressive tumors showed the highest value of Lac/Pyr ratio. GEM model such as P48Cre;LSLKrasG12D mice were used for detection of early and advanced PanIN, which usually develop in these mice between 20 and 25 weeks. The imaging experiments were performed at the different stages of the disease. Progression of disease from tissue containing predominantly low-grade PanIN to tissue with high-grade PanIN showed a decreasing alanine/lactate concentration ratio as measured by 1H-NMR metabolomics. These results demonstrate that there are significant alterations of alanine transaminase (ALT) and lactate dehydrogenase (LDH) activities that favor the transformation of aggressive pancreatic cancer from PanIN lesion. The efficacy of a metabolically targeted drug was monitored in orthotopic pancreatic cancer mice by measuring flux ratio of Lac/Pyr after injecting hyperpolarized pyruvate before the tumor shrinkage. Metabolic imaging with hyperpolarized pyruvate and NMR metabolomics enabled detection and monitoring of the progression of pancreatic cancer lesions. Translation of this HP-MRI technique to the clinic has the potential to improve early detection and the management of patients' care.

#371

Utilizing metabolic pathways to improve diagnosis and risk stratification of prostate cancer.

Aymen Alqazzaz, Dexue Fu, Arman Karimi, Gustavo Ferreira, Mary McKenna, Mohummad M. Siddiqui. _University of Maryland School of Medicine, Baltimore, MD_.

Prostate cancer has well-characterized changes that take place metabolically in the citric acid cycle (CAC) such as increased lactate formation, reversal of physiologic buildup of citrate and breakdown into downstream CAC products. Metabolic MR spectroscopy utilizing 13C-labeled pyruvate now allows for clinical characterization of metabolic pathways in tissue. However, the traditionally utilized substrate of [1-13C]pyruvate has limitations because as the pyruvate is broken down, the labeled carbon does not enter the CAC and instead is lost as CO2. We propose that [2-13C]pyruvate, is better to characterize changes in the CAC. Specifically, [2-13]C-pyruvate can be converted to 13C-labeled acetyl CoA and subsequently metabolized in the CAC as 13C-labeled intermediate compounds (such as glutamate which rapidly pools alpha-ketogluterate). Alternatively in altered bioenergetic states, [2-13C]pyruvate is reduced to 13C-labeled lactate (Warburg effect) or transaminated to 13C-labeled alanine. By exposing prostate cancer cells to [2-13C]pyruvate and determining the relative labeling of lactate, glutamate, and alanine, we can elucidate which metabolic pathways (CAC, Warburg, transamination) are utilized in prostate cancer metabolism. The aim of this study was to determine how the metabolism of [2-13C]pyruvate via the CAC, reduction to lactate, and transamination differed in high and low malignancy prostate cancer cells.Methods: Tumorigenic prostate cells LNCaP and PC3 were exposed a medium containing [2-13C]pyruvate for 4 hours. PCA extraction was then performed to obtain the metabolites. 13C labeled compounds formed intracellularly and those released into the medium were analyzed using NMR chemical shifts with focus on peaks associated with pyruvate, lactate, alanine, glutamate, and other compounds related to the CAC. Dioxane was used as an internal standard for NMR characterization. Results: The labeled carbon from the [2-13C]pyruvate can be identified in the downstream metabolites of the citric acid cycle, after reduction to lactate, and/or transamination by determining labeled metabolites by 13C-NMR analysis. This allows for the ability to calculate the relative metabolism of pyruvate via the three pathways; transamination (74%), lactate formation (16%), and CAC (10%).

Conclusions: [2-13C]pyruvate can be successfully utilized as a tool to explore the alterations in pathways of metabolism, including the citric acid cycle, in prostate cancer cells.

#372

Obesity matters: Prostate cancers that overexpress fatty acid binding protein 5 are more common in men who are overweight or obese.

Sandra M. Gaston,1 Soroush Rais-Bahrami,2 James Kearns,3 Dennis Otali,2 Denise Oelschlager,2 Mark S. DeGuenther,4 Jeffrey W. Nix,2 Peter N. Kolettis,2 James E. Bryant,2 Robert A. Oster,2 William E. Grizzle5. 1 _Tufts Medical Center, Tufts Univ School of Medicine and New England Baptist Hospital, Boston, MA;_ 2 _University of Alabama at Birmingham, Birmingham, AL;_ 3 _Tufts Medical Center, Boston, MA;_ 4 _Urology Centers of Alabama, Birmingham, AL;_ 5 _University of Alabama at Birmingham, Boston, MA_.

BACKGROUND: One of the hallmarks of the malignant phenotype is an increase in the cell's demand for fatty acids (FA). Many cancers, including prostate cancer, show increased expression of fatty acid synthase (FASN), a multi-component enzyme that catalyzes the de novo synthesis of the fatty acid palmitate. However recent observations suggest that alternate mechanisms for FA acquisition are also important, including the utilization of exogenous palmitate when it's available. Recently, through a biopsy-based molecular analysis of high risk prostate cancer, we identified a tumor subtype with very high (>10 fold) "outlier" overexpression of fatty acid binding protein 5 (FABP5). FABP5 is involved in lipid transport and signaling, and it facilitates the utilization of palmitate and other FAs from outside of the cell. De novo palmitate synthesis by the FASN pathway is energetically expensive compared to utilization of exogenous fatty acids.

STUDY DESIGN: We proposed that prostate cancers that can efficiently utilize exogenous palmitate are at a selective advantage when dietary FAs are abundant. In this study, to test that hypothesis, we compared FABP5 and FASN expression levels in prostate cancers obtained from diagnostic biopsies from normal weight, overweight and obese study subjects. A total of 201 consecutive eligible prostate biopsy patients were prospectively enrolled; 151 (75%) of these patients were cancer positive. Prostate cancer FABP5 and FASN mRNA expression were measured by qrtPCR and FABP5/FASN and FASN/FABP5 expression ratios were determined for each cancer positive core. BMI and self-identified race were obtained from hospital records.

RESULTS: The expression of FABP5 and the FABP5/FASN expression ratio were significantly higher in overweight and obese individuals compared to normal weight individuals (mean FABP5/FASN overexpression ratio of 20.2 in overweight/obese subjects vs 4.0 in normal weight subjects; p < 0.05). The highest levels of prostate cancer FABP5 outlier overexpression (FABP5/FASN > 100) were observed in African American subjects with BMI > 30, and high levels of FABP5 outlier overexpression were more strongly associated with overall Gleason sum in African American than in European American subjects.

CONCLUSIONS: In our study population, prostate cancer subtypes with high levels of FABP5 overexpression were more common in biopsy patients who were overweight or obese compared to normal weight patients. The highest levels of FABP5 overexpression in were observed in prostate cancers from obese African American subjects. Such FABP5 dominant prostate cancers may utilize exogenous fatty acids more efficiently and be more sensitive to dietary interventions than the more widely studied FASN dominant prostate cancers.

Funding: DOD Prostate Cancer Research Program Awards W8XWH-10-1-0543 (Grizzle, PI) and W81XWH-10-1-0544 (Gaston, PI)

#373

Bioluminescent non-lytic metabolite and cytotoxicity assays for cancer and immunotherapy research.

Donna M. Leippe, Natasha Karassina, Michael P. Valley, James J. Cali, Jolanta Vidugiriene. _Promega Corporation, Madison, WI_.

The growing interest in understanding metabolic adaptations that occur in cancer and immune cells in the tumor microenvironment and the advances made towards developing effective immunotherapies have increased the need for sensitive and efficient methods for studying cell metabolism and viability. We have developed bioluminescent assays for monitoring cell metabolism and cytotoxicity during cell growth and in response to environmental conditions and treatments. The plate-based assays employ coupled enzyme reactions and bioluminescent NADH detection chemistry to detect metabolites and enzymes. We used the assays in a non-lytic format, analyzing samples of cell culture medium and leaving the cells intact for additional measurements or multiplexing with other assays. In this way, changes occurring in a single well of cells were followed over time by collecting medium at multiple time points. We used assays for glucose, lactate, glutamine and glutamate detection to obtain information about the key metabolic pathways, glycolysis and glutaminolysis, and metabolically profile cancer cell lines. When we compared the metabolism of two ovarian cancer cell lines, OVCAR-3 and SKOV-3, the rates for glucose consumption and lactate secretion were similar, but the SKOV-3 cells consumed more glutamine and secreted 20-fold more glutamate. Glutamate secretion by the OVCAR-3 cells was low, only 5-fold higher than background controls. Analysis of the metabolites in cell lysates further supported the conclusion that the two cell lines differed in glutamine metabolism. The metabolite detection assays were also used to monitor metabolic pathway reprogramming in response to the environment. The induction of glycolysis is an early, critical step in the activation of T cells, and results in increased lactate production. We investigated the requirements for T-cell activation by measuring lactate levels over time and observed robust activation only in the presence of glutamine and anti-CD3/anti-CD28 costimulatory signals. In addition to metabolite levels, cell culture medium was used to assess cytotoxicity by following lactate dehydrogenase enzyme (LDH) release into cell culture medium. We used this assay to study cancer cell cytotoxicity induced by immunotherapeutic treatments, including antibody-drug conjugates (ADC) and antibody-dependent cell-mediated cytotoxicity (ADCC). The high sensitivity of the assay permitted the use of fewer cells, in 2D and 3D cultures, and the earlier detection of small changes (10 lysed cells in medium lacking serum). These sensitive bioluminescent assays require small amounts of sample and minimal sample processing to facilitate (1) identification of unique cancer cell metabolic signatures and potential therapeutic targets, (2) studies of connections between metabolism and effective immune cell function, and (3) the evaluation of cytotoxic immunotherapies.

#374

Direct measurements of cellular ATP levels in tumor cell lines using real-time, quantitative live-cell analysis.

Cicely L. Schramm,1 Grigory S. Filonov,1 Michael L. Bowe,1 Yong X. Chen,1 Laura A. Skerlos,1 Dyke P. McEwen,2 Daniel M. Appledorn1. 1 _Essen BioScience, Ann Arbor, MI;_ 2 _University of Michigan, Ann Arbor, MI_.

Metabolic reprogramming is a hallmark of tumor cells, owing to the increased energetic and biosynthetic demands associated with maintaining high rates of proliferation. Therapeutic strategies targeting unique, critical metabolic pathways of tumor cells are under investigation and in clinical trials. Standard approaches to monitoring drug induced metabolic perturbations are limited to endpoint assays that provide population-based measurements and limited kinetic information. We developed a genetically encoded ATP sensor to enable direct, automated analysis of cellular ATP levels using IncuCyte® S3. In this study, our live cell imaging approach was utilized to evaluate the effect of cancer therapeutics on ATP levels in tumor cell lines, focusing primarily on compounds that target metabolic vulnerabilities. Cell lines stably expressing a genetically encoded, fluorescent ATP sensor or a control (non-ATP binding) sensor were generated. ATP levels were monitored and analyzed using an IncuCyte® S3 equipped with a specialized filter set and data acquisition module. Cellular ATP levels were measured over the course of hours to days following compound treatment. Reductions in ATP levels could be observed within an hour of administration, highlighting the sensitivity and temporal resolution of the IncuCyte® S3 ATP sensor assay. Transient reductions in ATP followed by recovery, which would have been missed by typical endpoint assays, were noted using our live cell analysis approach. Sustained decreases in ATP were associated with enhanced antiproliferative efficacy compared to conditions under which recovery of ATP levels were observed. For example, triple-negative breast cancer (TNBC) cell lines have been shown to be more dependent on activity of glutaminase 1 (GLS1), which catalyzes the first step in utilization of glutamine to fuel mitochondrial metabolism, than their receptor-positive counterparts. Using our live cell analysis approach, we observed a rapid drop in ATP upon glutamine deprivation or inhibition of GLS1 by CB-839 in the TNBC cell line MDA-MB-231. ATP levels remained below that of vehicle-treated cells for the duration of the three-day time course. In contrast, the estrogen receptor-positive cell line MCF-7 displayed a more modest decrease in ATP and full recovery within 48 hours. Quantification of phase confluence confirmed that CB-839 had a stronger antiproliferative effect in MDA-MB-231 cells compared to MCF-7 cells. Visualization of morphology in tandem with automated ATP sensor analysis provide additional insight into the heterogeneity of cellular responses to compound addition. Overall, these data highlight the ability of IncuCyte® S3 to provide direct, kinetic measurement of ATP by live-cell analysis.

#375

Origins of fractional control in regulated cell death.

Luis C. Santos,1 Robert Vogel,2 Jerry E. Chipuk,1 Marc Birtwistle,3 Gustavo Stolovitzky,2 Pablo Meyer2. 1 _Icahn School of Medicine at Mount Sinai, New York, NY;_ 2 _IBM T. J. Watson Research Center, Yorktown Heights, NY;_ 3 _Clemson University, Clemson, SC_.

Fractional killing of a population of cancer cells is often linked to cell-to-cell variability due to stochastic gene expression and fluctuations in protein levels. We found that mitochondria abundance is a major source of cell-to-cell variability that determines the fraction of cells that live or die in response to TNF-related apoptosis-inducing ligand (TRAIL). We show that the IC50 of TRAIL of clonogenic cancer cell populations correlates with mitochondria abundance, and cell-to-cell variability in mitochondria abundance controls up to 40% of the response to TRAIL. Our coarse-grained quantitative model of apoptosis reveals that increasing the mitochondrial surface area decreases the density of proapoptotic Bax pores, which in turn decreases the sensitivity of cells to TRAIL. We demonstrate that the clinically relevant small-molecule inhibitor of antiapoptotic Bcl-2 proteins ABT-263 reduces the IC50 of TRAIL by reducing the contribution of mitochondria to variability in apoptosis.

#376

γ-Tocotrienol negative modulation of the Warburg effect is mediated by the AMPK activation in malignant breast cancer cells.

Venkatesh Dronamraju, Ibrahim G. Algayadh, Paul William Sylvester. _University of Louisiana at Monroe, Monroe, LA_.

Previous studies showed that γ-tocotrienol, a natural vitamin E derivative is associated with decrease in the rate of glycolysis in human mammary tumor cells. Therefore, studies were conducted to investigate the effects of γ-tocotrienol on key regulatory pathways of glucose metabolism in MCF-7 and MDA-MB-231 breast cancer cells. Unregulated proliferative capacity of cancer cells places huge metabolic load on cancer cells, which ultimately promotes the dysregulation of normal metabolic pathways. This dysregulation of energy metabolism manifests as a characteristic phenomenon called as the "Warburg effect". The Warburg effect is an over-reliance on aerobic glycolysis by cancer cells to meet energy demand of proliferation. Akt has been reported as key regulator of Warburg effect in several tumors and is a known promoter of glycolysis. Additionally, 5'-AMP activated protein kinase (AMPK) is a known metabolic sensor that is activated when energy balance is disrupted in cells. In addition to their role in regulating cellular energy metabolism, Akt and AMPK also share several downstream targets like FoxO3a, a transcription factor. In the present study, protein expression using western blot and immunocytochemistry revealed that in human breast cancer cell lines MCF-7 and MDA-MB-231, γ-tocotrienol induced dose-dependent inhibition of Akt. This was associated with a rescue of Akt-induced inhibition of FoxO3a. In addition, γ-tocotrienol also induced a decrease in aerobic glycolysis as evidenced by a decrease in consumption of glucose and a decrease in glycolytic enzyme expression. Furthermore, results showed that γ-tocotrienol treatment induced activation of AMPK in breast cancer cells. Immunoprecipitation analysis also revealed a possible interaction between AMPK and FoxO3a in γ-tocotrienol treated cells. To study the effect of γ-tocotrienol-induced activation of AMPK on its downstream targets, microarray analysis was done using RT2-Profiler PCR array. The microarray revealed that γ-tocotrienol-induced AMPK activation resulted in downregulation of several genes that are critical regulators of aerobic glycolysis. In summary, results show that anticancer effects of γ-tocotrienol treatment are associated with significant inhibition in pro-Warburg signaling such as Akt activation, and a corresponding increase in activity of AMPK in human breast cancer cells. In addition, γ-tocotrienol reversal of Warburg effect is directly related to a reduction in the rate of glycolysis in these breast cancer cells. Finally, γ-tocotrienol-induced activation of AMPK signaling was found to significantly alter gene expression of various downstream targets of AMPK. These findings highlight the potential of vitamin E derivatives in targeting the metabolism of breast cancer cells. This work was supported in part by funds provided by the Louisiana Cancer Foundation.

#377

Time-restricted feeding: A dietary intervention to treat breast cancer in postmenopausal obese mice.

Manasi Das, Emilie Gross, Deepak Kumar, Consuelo Sauceda, Hyun-Tae Park, Dorothy D. Sears, Lesley Ellies, Nicholas Webster. _University of California, San Diego, La Jolla, CA_.

Background: Abundant evidence indicates that obesity increases risk for breast cancer and the incidence further increases by 40% in obese postmenopausal women. Therefore it is envisioned by diverse research groups that the risk of breast cancer in obese postmenopausal women is an ever-increasing menace that needs to be curbed soon with effective practical strategies that could have a far-reaching impact. In this setting, time-restricted feeding (TRF), the practice of consuming ad libitum energy during the normal active phase, has been demonstrated to reinforce normal metabolic regulation, thereby attenuating obesity-driven

metabolic deregulation. Although studies have assessed the effect of TRF on metabolism, no investigations have been carried out in human or mouse exploring TRF in cancer remission. Therefore, the present work is to understand the efficacy of TRF for improved breast cancer remission in postmenopausal obese female mice. Further, the work explores the mechanistic link of TRF for reduced tumor growth.

Methods: Ovariectomized and 4-vinylcyclohexene diepoxide (VCD) treated mice were used as a postmenopausal model. Both ovariectomized and VCD mice were made obese by feeding them with 60% high-fat diet (HFD) and then grouped into ad libitum group (24 h access to food) and TRF group (8 hr access to food at night) to assess metabolic and tumor growth effect of TRF. To develop breast tumor, mice were injected with py230 breast cancer cell line into the mammary fat pad. The metabolic effect of TRF was assessed by glucose tolerance test, insulin tolerance test, body weight measurement, food intake, lipid accumulation in liver by HE staining and measurement of different tissue weight. Measuring the tumor volume over time and tumor weight assessed TRF effect on tumor growth. Performing a tumor growth study in mice fed with HFD and HFD containing diazoxide assessed insulin dependency of tumor growth in ad libitum group. Insulin-dependent tumor growth was validated by tumor growth study in normal chow-fed mice implanted with insulin pump or without pump (control).

Results: The preliminary studies in ovariectomized and VCD-treated postmenopausal mice suggest that restricting access to Western-style HFD in active night phase improves insulin resistance, glucose tolerance, and hepatic steatosis. Further, TRF exhibited reduced tumor growth compared to ad libitum group. More importantly, the results from tumor growth study in mice fed with HFD with/without diazoxide or normal chow mice with/without insulin pump, suggest that the tumor growth is insulin dependent and TRF may be acting through attenuating insulin signaling.

Conclusion: Experimental and animal model data corroborate that TRF improves metabolic deregulation and reduces breast tumor growth in HFD-fed obese mice. The results suggest putative application of such therapeutic intervention for breast cancer therapy.

#378

Stat3 promotes ovarian cancer by modulating the energy metabolism and develops drug resistance in patients.

Vaishnavi Raja,1 Shailendra Giri,2 Sharif Sakr,1 Miriana Hijaz,1 Raymond Quiles,1 Adnan R. Munkarah,1 Ramandeep Rattan1. 1 _Henry Ford Health System, Detroit, MI;_ 2 _Henry Ford Hospital, Detroit, MI_.

Signal Transducers and Activators of Transcription (STAT) are a group of transcription factors that are known to play a major role in cancer progression. In ovarian cancer, increased STAT3 leads to cancer proliferation in response to cytokines and confers resistance to chemotherapy-induced apoptosis in epithelial malignancies. STAT3 is constitutively activated in patient derived ovarian cancer cells, and increased STAT3 expression is a predictor of poor prognosis. Apart from its function as a transcription factor, recently STAT3 has been shown to modulate mitochondrial function to promote carcinogenesis. The aim of our study was to investigate if STAT3 activation can modulate cellular metabolism of ovarian cancer cells. Stable clones expressing STAT3 were generated in A2780 ovarian cancer cells, along with empty vector clones. Ectopic expression of STAT3 in A2780 ovarian cancer cell line resulted in increased proliferation (p<0.01) and colony formation ability (p<0.001) in vitro and led to large ovarian tumors (p<0.01) compared to parental and vector controls in xenograft mouse model. Bioenergetics profiling showed higher mitochondrial respiration (OCR) and glycolysis (ECAR) in STAT3 clones compared to parental and vector clones. Ratio of ECAR/OCR in the STAT3 overexpressing cells placed them in the 'metabolically active' phenotype, while parental A2780 and vector clones were in 'metabolically less active' phenotype. A selective inhibitor of STAT3, STATTIC, inhibited the STAT3 mediated growth of A2780 cells both in vitro (p<0.01) and in vivo (p<0.01). In addition, STATTIC treatments reversed the 'metabolically active' state of STAT3 overexpressing clones to a 'lower metabolic state', placing them in the same category as the control cells. In addition, STATTIC inhibited the cell proliferation and modulated bioenergetic phenotype of other ovarian cancer cells lines (PEO4, C200 and OVCAR3) that display a 'metabolically active' phenotype. Overall, STAT3 can induce metabolic changes in ovarian cancer cells, maybe as survival mechanism and enhances the cellular fitness of the ovarian cancer cell resulting in increased oncogenic abilities.

#379

Calorie restriction reduces PI3K/Akt signaling and tumorigenic potential in HER2-overexpressing breast cancer.

Laura A. Smith, Magdalena A. Rainey, Nishita T. Sheth, Ciara H. O'Flanagan, Laura W. Bowers, Stephen D. Hursting. _University of North Carolina at Chapel Hill, Chapel Hill, NC_.

Purpose: Approximately 20% of breast cancers (BC) among US women overexpress HER2, an oncoprotein that stimulates increased cellular proliferation and survival. HER2 acts through two major signaling pathways, PI3K/Akt and Raf/MAPK, and its overexpression is associated with aggressive disease, resistance to therapy, and poor prognosis. Our laboratory has previously demonstrated in preclinical models that calorie restriction (CR) modulates HER2 BC pathogenesis, decreasing incidence and increasing latency in transgenic MMTV-HER2/neu mice. Our current study aims to determine the impact of CR on HER2 signaling and cancer cell phenotypes, including proliferation, cell cycle progression, and apoptosis. Methods: In vitro experiments were performed using a BC cell line derived from the MMTV-HER2/neu mouse model. To mimic CR, cells were treated with nutrient restricted media containing either reduced glucose (1mM), reduced serum (1%) or both (1mM/1%). The impact of CR on proliferation and survival was measured using growth curve experiments, and flow cytometric analysis of cell cycle and apoptosis. HER2 signaling proteins were assessed by western blotting. Results: Relative to cells grown in control media (25mM glucose/10% BCS): a) cells grown in glucose restricted (1mM) + serum restricted (1%) media grew 87% slower (p<0.01); b) cells grown in serum-restricted media (1%) with control glucose levels (25mm) grew 86% slower (p<0.01); and c) cells grown in glucose restricted media (1mM) with control serum levels (10%) grew 67% slower (p<0.05). Serum restricted groups, regardless of glucose levels, also exhibited increased G1 cell cycle arrest following 24 hours of treatment and increased percentage of apoptotic cells following 72 hours of treatment compared to control. In contrast, glucose restriction alone did not significantly affect apoptosis compared to control; however, it induced a G2/M arrest, possibly explaining reduction in growth. Western blotting analysis revealed that serum restricted cells had reduced levels of pAkt (Ser473) but no differences in Akt, pERK, or ERK, compared to the control and 1mM groups following 24 hours of CR treatment. Conclusions: In vitro models of CR, specifically reduced serum proteins alone or in combination with glucose, reduced activation of PI3K/Akt in HER2 BC cells. This altered signaling was associated with reduced tumorigenic potential, as evidenced by decreased growth and increased apoptosis. These findings suggest that combining a nutrient restriction regimen or pharmacologic mimetic of CR with HER2-targeted therapy, which also inhibits PI3K/Akt signaling, may act synergistically. Future experiments will test the hypothesis that CR modulates the efficacy of HER2 targeted therapies through in vitro and in vivo models of HER2 BC.

#380

The association of sleep with metabolic pathways and metabolites: Evidence from the Dietary Approaches to Stop Hypertension (DASH)-Sodium Feeding Study.

Vanessa L. Gordon-Dseagu,1 Andriy Derkach,1 Qian Xiao,2 Ishmael Williams,3 Joshua Sampson,1 Rachael Stolzenberg-Solomon1. 1 _NCI, Rockville, MD;_ 2 _University of Iowa, Iowa City, IA;_ 3 _Howard University, Washington, DC_.

Background

Sleep is increasingly being viewed as an important determinant of health, and appears to be associated with cardiovascular disease, diabetes and several site-specific cancers. Few epidemiologic studies have used data from a feeding study to explore the impact of sleep habits up on metabolomic profile.

Methods

The Dietary Approaches to Stop Hypertension (DASH)-Sodium feeding trial randomized individuals to either the DASH (low fat, high protein, low-fat dairy and high fruits and vegetables) or control diet (12 weeks) and three levels of sodium intake (30 days each). In a subgroup of 97 participants, we measured the levels of 531 metabolites in serial fasting plasma samples at two time points using liquid chromatography and gas chromatography mass spectrometry. We then assessed the association between each metabolite and sleep using a linear random effects model adjusted for age, sex, race, dietary pattern and sodium level. The resulting p-values were combined using Fisher's method to estimate the association with 38 metabolic pathways.

Data about sleep were collected at the end of two dietary intervention phases. Participants recorded times for going-to-sleep and waking while wearing 24-hour ambulatory blood pressure monitors. From this we produced two sleep variables - sleep mid-point (the median time between going to sleep and waking) and sleep duration. We undertook further analysis to explore the associations between bedtime, wake-time and metabolic pathways and metabolites.

Results

Nineteen pathways were significantly associated (p <0.05) with sleep mid-point, but only the γ-glutamyl amino acid metabolism pathway was associated with sleep mid-point and wake-time at Bonferroni-corrected threshold of 0.0013 (7 individual peptide metabolites contributed and showed positive associations). Fifty-six metabolites were associated with sleep mid-point (FDR<0.20), although none of them reached Bonferroni-corrected significance (p-value <10-5). Notable top metabolites (pathways given in brackets) associated with sleep mid-point and wake-time were CMPF (fatty acid, decarboxylate), erythrulose (an advanced glycation end-product), isovalerate (fatty acid metabolism) and HWESASXX (polypeptide). Neither sleep duration nor bedtime were associated with metabolite pathways or metabolites and there was no significant interaction between sex, race, BMI or dietary intervention and sleep.

Conclusion

Within our study we found multiple metabolites associated with sleep timing. In particular, the γ-glutamyl pathway metabolites were associated with sleep mid-point and wake-time. These metabolites are formed using the enzyme γ-glutamyltransferase (GGT), which is expressed in response to oxidative stress and glutathione synthesis - a process that may play a role in carcinogenesis, diabetes, and cardiovascular disease.

#381

Uptake of biguanides in mitochondria appears to be related to increased glucose uptake and medium acidification but not necessarily related to inhibition of growth of bladder and colon cancer cells.

Michael A. Lea, Haneulsol Kim, Charles desBordes. _Rutgers New Jersey Medical School, Newark, NJ_.

Biguanides including metformin have been used in the treatment of Type 2 diabetes and they are under investigation as anti-cancer agents. Investigation on the mechanism of action of biguanides has largely focused on inhibition of complex 1 in the mitochondrial electron transport chain with subsequent activation of AMP-dependent protein kinase. Studies by Bridges et al. (BMC Biology 14:65, 2016) indicate that inhibitory effects on complex 1 in cells require uptake of biguanides into the mitochondria. In the present work the action of two biguanides that enter the mitochondria (buformin and phenformin) were compared with the action of two biguanides with poor uptake (phenyl biguanide and proguanil). Effects on growth, glucose uptake and medium acidification were studied with two human colon cancer cells (Caco-2 and HT29) and seven bladder cancer cell lines (5637, HT1197, HT1376, RT4, T24, TCCSUP and UM-UC-3). After incubation for 72 hours with 25 µM and 50 µM concentrations of the biguanides, growth inhibition was greatest with proguanil followed by phenformin and with little or no growth inhibition at these concentrations with buformin and phenylbiguanide. In contrast, increased glucose uptake and acidification of the medium was observed with buformin and phenformin with no change or decreased medium acidification with phenyl biguanide and proguanil. Studies with buformin over a concentration range from 10 µM to 500 µM indicated that there was generally growth inhibition at concentrations greater than 50 µM. Studies on medium acidification and glucose uptake revealed biphasic responses with increases at lower concentrations that were reversed at higher concentrations of buformin where growth inhibition was observed. The data suggested that the effect of biguanides on glucose metabolism requires mitochondrial uptake while the mechanism for growth inhibition by biguanides remains to be established.

#382

Inhibition of mitochondrial spare respiratory capacity by new securinine derivatives as a novel differentiation therapy for acute myeloid leukemia.

Sheela Karunanithi, Ruifu Liu, Anne Roe, Stephen Moreton, Yongchun Hou, Natasha Oldford, Juan Valentin Goyco, David Wald. _Case Western Reserve University, Wolstein Research Building, Cleveland, OH_.

Acute myeloid leukemia (AML) is characterized by a differentiation block in myeloid progenitors resulting in aberrant proliferation of immature myeloid cells. Therapeutics that induce differentiation of leukemic cells would overcome the high toxicity and low potency of conventional chemotherapy. Securinine, a plant-derived alkaloid from Securinega suffruticosa, has been shown to be a promising AML differentiation agent through an unclear mechanism, but is limited by its low potency (~20μM) and toxicity. We performed lead optimization chemistry on securinine that led to derivatives 250 and 317, with nanomolar efficacy and reduced toxicity. These derivatives effectively inhibit growth of AML cell lines and patient samples. Treatment of AML cell lines with compounds 250 and 317 induces the expression of differentiation markers CD11b and CD14c. Compounds 250 and 317 significantly reduced leukemic burden in a circulating model of AML in NSG mice. Additionally, these compounds were effective in inhibiting cell growth of HL60 cells that are resistant to a conventional chemotherapeutic, doxorubicin. By using the drug-affinity responsive target sensitivity (DARTS) assay, we identified that the redox mediator thioredoxin reductase (TrxR) is the primary target of these derivatives. 250 and 317 inhibit TrxR activity in vitro and in cells, and the resultant alterations in redox status are overcome by addition of a scavenger of reactive oxygen species (ROS), N-acetylcysteine. The ensuing oxidative metabolic stress rapidly alters the metabolite profile in AML cells including depletion of malate. Securinine derivatives decreased basal oxygen consumption rate and spare respiratory capacity, resulting in differentiation and cell death. Downregulating TrxR1 expression increased the sensitivity of OCI-AML3 cells to compounds 250 and 317. Together, we show here that these new derivatives of securinine exhibit preclinical efficacy in AML by decreasing mitochondrial spare respiratory capacity through the inhibition of TrxR.

#383

Metabolomic analysis of uterine serous carcinoma with acquired resistance to paclitaxel.

Manabu Seino, Tsuyoshi Ohta, Hirotsugu Sakaki, Takeshi Sudo, Satoru Nagase. _Yamagata University, Yamagata, Japan_.

Introduction: Uterine serous carcinoma (USC) is more aggressive than other subtypes of endometrial carcinoma and is associated with a poor prognosis. We analyzed the metabolomic profile of USC with acquired resistance to paclitaxel.

Method: We compared metabolic profiles and reactions to paclitaxel in both a wild-type USC cell line (USPC-1) and PTX-1, a cell line derived from USPC-1 that acquired paclitaxel resistance, using a capillary electrophoresis CE-MS/MS system.

Results: Glutathione (GSH) concentration in PTX-1 cells was higher than in USPC-1 cells. In addition, GSH concentration in the USPC-1 cells increased after treatment with paclitaxel but was unchanged in PTX-1 cells. Glucose-6-phosphate (G6P) and ribose-5-phosphate (R5P) concentrations in PTX-1 cells were higher than those in USPC-1 cells. G6P concentration in the USPC-1 cells was unchanged after treatment with paclitaxel, while it decreased in PTX-1 cells.

Conclusion: Our results indicate that increased GSH and glucose metabolism may be related to acquiring resistance to paclitaxel in USC and thus may be targets for anti-USC therapy.

#384

Role of PD-L1 immunoregulatory protein in breast cancer cells metabolic reprogramming.

Julie Berthe,1 Jérôme Kluza,1 Hassiba El Bouazzati,1 Isabelle Briche,1 Xavier Thuru,1 Sylvie Aliegue-Zouitina,1 Bruno Quesnel2. 1 _Univ. Lille, Inserm UMR-S 1172 - JPARC (Centre de Recherche Jean-Pierre AUBERT, Neurosciences et Cancer), Lille, France;_ 2 _CHU Lille (Service des Maladies du Sang), Univ. Lille, Inserm UMR-S 1172 - JPARC (Centre de Recherche Jean-Pierre AUBERT, Neurosciences et Cancer), Lille, France_.

When evolving to a neoplastic state, normal cells acquire many characteristics; indeed, tumor cells follow abnormal metabolic pathways and exhibit the ability to avoid immune destruction, partly by exploiting immune checkpoints. Many of these are currently under clinical investigation for new cancer treatments, notably the PD-1/PD-L1 axis.

Programmed Death-Ligand 1 (PD-L1) molecule belongs to the B7 immunoregulatory proteins family and was originally described as mediating tumor immuno-escape through interaction with its receptor PD-1 on T cells. Associated with poor cancer outcome, aberrant PD-L1 expression has been observed in hematologic malignancies and in multiple solid tumor types. Interestingly, some reports highlight PD-L1 intrinsic effects in cancer cells. Actually, this protein has been shown to regulate tumor cell proliferation and resistance to chemotherapy through apoptosis inhibition, without interacting with PD-1. However, cellular mechanisms modulated by PD-L1 and involved in these functions are still unclear. Abnormal metabolic pathways are known for contributing to tumor growth and therapy resistance; therefore, in this study, we investigated the role of PD-L1 in cancer cell metabolic reprogramming.

By using genome editing, we knocked-out the CD274 gene encoding PD-L1 in breast cancer cell line MDA-MB-231 and investigated metabolic functions after PD-L1 overexpression in the same cells. We observed that PD-L1 induces a shift from oxidative phosphorylation to glycolysis, indicating this molecule promotes the Warburg effect in these tumor cells. Moreover, in agreement with an increasing mitochondrial reactive oxygen species (ROS) production, transcriptomic study suggested that PD-L1 represses Nrf2-mediated oxidative stress response pathway, especially NQO2, GSTM3 and ABCC2 genes. Additionally, in silico analysis of breast cancer patients databases highlighted a correlation between CD274 gene and oxidative stress gene signature (GSTM3; CYBB) or glucose transporters genes (SLC2A1; SLC2A3) expressions. Besides, glucose is mostly used by cancer cells to favor biosynthesis of diverse biomolecules required for cellular proliferation; assuming that PD-L1 overexpression modulates glycolytic capacities of MDA-MB-231 cells, we are performing a metabolomic study to determine the impact of this protein on the diverse biosynthetic outputs linked to glucose metabolism. The above results could explain our human breast cancer cells xenograft experiments in NUDE mice demonstrating that PD-L1 increases tumoreginicity. Furthermore, to identify which protein domain is implicated in these metabolic functions, experiments on PD-L1 mutant forms are ongoing.

Thus, our study evidences novel PD-L1 intrinsic tumor-promoting functions, suggesting that therapeutic agents inhibiting these mechanisms would be promising for breast cancer treatment.

#385

HIF1A can regulate Wnt signaling in human colon cancer cells.

Yung Lyou,1 George Chen,2 Marian Waterman2. 1 _UCI Medical Center, Orange, CA;_ 2 _University of California Irvine, Irvine, CA_.

Standard-of-care treatment for KRAS mutant metastatic colorectal cancer (mCRC) combines a cytotoxic chemotherapy backbone (FOLFOX) with an antiangiogenesis biologic agent (i.e., bevacizumab). Despite significant efforts to improve treatment, the prognosis for this type of cancer remains poor with an average 5-year overall survival of ~11%. Oncogenic KRAS mutations lead to the overactivation of RAS/ERK/MAPK pathwa,y driving many hallmarks of cancer such as cell proliferation, growth, and altered metabolism. In particular, several studies have shown that oncogenic activation of KRAS leads to normoxic stabilization of HIF1A protein, which can then reprogram cellular metabolism towards aerobic glycolysis. Our studies have discovered that overactive Wnt signaling can also reprogram cancer cell metabolism towards aerobic glycolysis. Of note, KRAS (~40%) and Wnt signaling (~90%) are very frequently coactivated in colorectal cancer. Interestingly, an important, key metabolic gene targeted by Wnt signaling, Pyruvate Dehydrogenase Kinase I (PDK1), is also directly regulated by HIF1A, suggesting there may be crosstalk between the HIF1A and Wnt pathways. Indeed, several published studies support the idea that HIF1A can influence Wnt signaling, and our data show this may occur through regulation of the Wnt mediating transcription factors LEF1 and TCF1 (TCF7 gene). Using luciferase reporters driven by LEF/TCF promoters, we observed significantly increased LEF1/TCF7 promoter activity in hypoxic colon cancer cells but not for other TCF family members (TCF7L1/TCF7L2). We identified putative hypoxia responsive elements (HREs) in the human LEF1 promoter, and deletion of these HREs reduced responsiveness to hypoxia. Furthermore, we have found that targeting glycolysis and Wnt can be synergistic in inhibiting colon cancer cell colony growth. We find that HIF1A and LEF/TCF/beta-catenin complexes coregulate gene expression of metabolic targets as well as Wnt signaling components (e.g., LEF1 and TCF7). These data suggest that cross-regulation between the HIF1A and Wnt signaling pathway is important and may be a potential Achilles heel for novel therapeutics in targeting KRAS mutant colon cancer. Current studies are under way to further investigate the mechanisms of crosstalk and joint regulation of metabolic reprogramming in KRAS mutant colon cancer cells.

#386

Heme function in promoting tumorigenic potential of lung cancer cells.

Chantal Vidal, Sagar Sohoni, Li Zhang. _UT Dallas, Richardson, TX_.

Heme, also known as iron protoporphyrin IX, is an essential molecule for living organisms. Heme serves as a prosthetic group in enzymes, and acts as a signaling molecule in diverse cellular processes. Mammalian cells can synthesize heme endogenously, or they can import heme from circulation via heme transporters. The synthesis of heme is carried out by eight enzymes, the first of which is the rate-limiting enzyme 5-aminolevulinate (ALA) synthase. Heme can also be transported intracellularly by transporters, such as HCP1 (heme carrier protein 1) and HRG1 (heme responsive gene 1). Finally, levels of heme are also maintained by the degrading enzyme, heme oxygenase-1 (HO-1). HO-1 is localized to the endoplasmic reticulum and is the rate-limiting enzyme in the catabolism of heme. HO-1 cleaves heme to biliverdin and carbon monoxide. Biliverdin is converted to bilirubin, which is an antioxidant and scavenger of ROS. Heme also serves as a catalytic site for various proteins that are involved in aerobic metabolism, such as respiratory chain cytochromes. For example, complex II, III, and IV of the mitochondria require heme for proper function. Importantly, epidemiologic studies have shown the association of dietary heme intake, mainly from red meat, with different cancers, including lung cancer. Therefore, we have analyzed the levels of heme synthesis, uptake, degradation, oxygen consumption and mitochondrial proteins in an immortalized, nontumorigenic human bronchial epithelial cell line (NL20) compared to the tumorigenic counterpart (NL20-TA). These studies showed that the tumorigenic cell line expressed significantly higher levels of heme flux compared to the nontumorigenic cell line. This led us to alter heme availability by overexpressing the rate-limiting enzyme of heme synthesis as well as the rate-limiting enzyme of heme degradation. Experiments are under way to characterize their effects on tumor growth and metastasis.

#387

Glutamine metabolism reprogramming in adipocyte-tumor microenvironment promotes triple-negative breast cancer growth.

Yang Liu, Tingli Zhao, Zhengzheng Li, Lai Wang, Mei Yang, Shengtao Yuan, Li Sun. _China Pharmaceutical University, NANJING, China_.

OBJECTIVE

Adipocytes are the most abundant stromal partners in breast tissue. However, the crosstalk between breast cancer cells and adipocytes has been given less attention compared to cancer-associated fibroblasts. We try to explain whether cancer associate adipocytes (CAAs) in microenvironment can lead to metabolic remodeling of triple-negative breast cancer, promoted cancer proliferation, migration and the potential mechanism.

METHOD

A Transwell co-culture model in vitro and orthotopic mouse model of human breast tumor formation in vivo were used to study the crosstalk between adipocytes and breast cancer cells, and then using clinical breast cancer sample analysis to study the mechanism of glutamine reprograming.

RESULTS

We find that while cocultivated adipocytes with breast cancer cells, it exhibited enhanced oxygen consumption rate (OCR) and ATP, accompanied by increasing glucose and glutamine uptake. Interestingly, the metabolic way is significantly different between phenotype-specific breast cancer cells. Compared with non-triple-negative breast cancer cells, triple-negative breast cancer cells (MDA-MB-231, MDA-MB-468) are more dependent on glutamine. We show that cancer associate adipocytes (CAAs) promote growth of breast cancer cells through key enzymes such as GLUL/GLS/GDH and transporters such as SLC1A5/SLC38A1/SLC38A2 related to glutamine metabolism in vitro. Using cytokine antibody microarrays, IL-6/IL-8/ leptin/TIPM2 is secreted by CAAs and is required to enhance breast cancer cells malignancy in vitro. Consistently, the correlation of inflammatory cytokines and adipokines with prognosis is analyzed by the Kaplan-Meier plotter, the data suggest that inflammatory cytokines and adipokines is associated with glutamine metabolism in triple-negative breast cancer microenvironment.

CONCLUSIONS

These results suggest that adipocytes promote proliferation of triple-negative breast cancer via metabolism reprogramming potentially by inducing inflammatory cytokines and adipokines upregulation of cancer associate adipocytes (CAAs).

#388

p16/MTAP status as a model for investigating gene-environment interactions in metabolism.

Sydney M. Sanderson,1 Peter Mikhael,2 Ziwei Dai,1 Jason W. Locasale1. 1 _Duke University School of Medicine, Durham, NC;_ 2 _Duke University, Durham, NC_.

While the consequences of tumor suppressor gene deletions have been extensively characterized, little is known about how concurrent deletions of neighboring genes can influence the interaction of cancer cells with the environment. Using a panel of tissue-matched cancer cell lines exhibiting either wild-type or homozygous deletions of the p16 locus, metabolomics analysis showed that concurrent deletion of the proximal gene encoding the methionine salvage enzyme methylthioadenosine phosphorylase (MTAP) is associated with a subtle but reproducible global shift in metabolite levels. However, MTAP status does not appear to be predictive of metabolic responsiveness to restriction of key nutrients that feed into and out of the methionine cycle. Of particular interest is the finding that cell lines exhibit remarkable heterogeneity in responsiveness to nutrient availability, seemingly independent of MTAP status or tissue of origin. Similarly, MTAP-deleted cell lines exhibit variable global metabolic reprogramming in response to acute ectopic expression of MTAP, and maintain their heterogeneous responsiveness to nutrient restriction. These findings illustrate the exceptional metabolic diversity across cancer cell lines despite similar culture conditions, demonstrating the usefulness of examining a variety of cell lines in studies of cancer cell metabolism. 

### Functional Genomics

#389

**Integrative genomic analyses of mutation signatures, immunogenicity and germline copy number variation of the** APOBEC **genes in multiple cancer types.**

Xingyi Guo, Xiao-ou Shu, Jiandong Bao, Wanqing Wen, Qiuyin Cai, Jirong Long, Wei Zheng. _Vanderbilt University Medical Center, Nashville, TN_.

The APOBEC-signature mutation has been frequently observed in multiple cancer types, including bladder, breast, and lung adenocarcinoma. Increased expression of the APOBEC3A and APOBEC3B genes is known to induce APOBEC-signature mutations in these cancer types. Limited studies have investigated whether the mutation patterns are associated with individual isoforms of the APOBEC3A and APOBEC3B genes, particularly the isoform uc011aoc from an APOBEC3A/3B chimera that is primarily generated by a common germline deletion. Previous studies have shown that somatic missense mutations contribute to neoantigen load and consequently may affect cancer immunogenic abilities such as attracting tumor infiltration lymphocytes (TILs). However, the influence of APOBEC-signature mutations on tumor immunology remains largely unknown. In this study, we systematically analyzed the APOBEC isoform expression and mutation signature, neoantigen load, and copy number variations in ~4000 tumor samples across 10 cancer types using data from The Cancer Genome Atlas. Using multiple regression analyses that included all isoforms of both APOBEC3A and APOBEC3B, we found that the isoforms uc003awn (APOBEC3A) and uc011awo (APOBEC3B) were significantly associated with APOBEC-signature mutations (defined as TCW change to either TTW or TGW mutations) in multiple cancer types, while the association for uc011aoc was observed only in breast cancer. We also observed that APOBEC-signature mutations were positively associated with neoantigen load in all cancer types except lung adenocarcinoma and kidney carcinoma (P < 0.05). To further investigate the effects of these mutations on TILs, we used gene expression data and the tool CIBERSORT to predict the relative compositions of immune cells for each cancer type. We found that APOBEC-signature mutations were significantly associated with the compositions of CD8+ and CD4 memory-activated T cells only in breast and bladder cancer types. Furthermore, we found that neoantigen load was significantly associated with these cell compositions in multiple cancer types, but was not observed in breast cancer. Lastly, our results showed that a germline common deletion in APOBEC3A/B was associated with a significantly increased expression level of uc011aoc isoform, but decreased expression levels of both uc003awn and uc011awo across cancer types. In addition, we found that the deletion was associated with the evaluated neoantigen load and the composition of T cells (CD8+) in breast cancer. The results suggest potential mechanisms for the association of APOBEC3A/B gene deletion with increased breast cancer risk reported from previous genome wide association studies. Our study provides novel insights into understanding the biological and immunological mechanisms through which APOBEC genes may be involved in carcinogenesis.

#390

Genome-wide CRISPR screens in brain tumor initiating cells (BTICs) identify potent sensitizers and resistors of conventional chemoradiotherapy.

Chirayu Chokshi,1 David Tieu,2 Kevin Brown,2 Chitra Venugopal,1 Parvez Vora,1 Katherine Chan,2 Amy Tong,2 Maleeha Qazi,1 Mohini Singh,1 Neil Savage,1 Andrea Habsid,2 Jason Moffat,2 Sheila Singh1. 1 _McMaster University, Hamilton, Ontario, Canada;_ 2 _University of Toronto, Toronto, Ontario, Canada_.

Glioblastoma (GBM) is a highly aggressive and most common form of malignant primary brain tumors in adults (WHO grade IV). Despite surgical and therapeutic interventions, including chemotherapy with the alkylating agent Temozolomide (TMZ) and cranial irradiation, GBM relapse is inevitable with a median survival of <15 months. Extensive intratumoral heterogeneity in GBM is believed to be the leading cause of therapy resistance and disease relapse, suggesting that therapy acts as a bottleneck for tumor evolution. Recently, the advent of CRISPR-Cas9 technology has led to the development of genome-wide libraries of sgRNAs capable of introducing insertion-deletion (indels) within exonic regions of genes, leading to a frameshift mutation two-thirds of the time. Here, we present the first genome-wide CRISPR-Cas9 knockout screen in patient-derived GBM BTICs aimed to discover synthetic lethal sensitizers of conventional chemoradiotherapy. Briefly, we performed genome-wide CRISPR-Cas9 screens in treatment-naïve GBM BTICs subjected to in vitro chemotherapy with TMZ and irradiation. By comparing sgRNA dynamics at each doubling period, we were able to identify potent sensitizer genes exclusive to combined chemoradiotherapy, and not TMZ or irradiation alone. Candidate sensitizer genes were validated in an arrayed format to evaluate impact on GBM BTIC self renewal, proliferation, and sensitivity to TMZ and radiation. We aim to further validate these sensitizers of conventional chemoradiotherapy by performing a focused CRISPR-Cas9 genetic screen in our patient-derived xenograft model of treatment-refractory GBM. Ultimately, adjuvants targeting sensitizer genes could greatly enhance the impact of conventional chemoradiotherapy in GBM patients, leading to an increase in patient survival.

#391

**Evolutionary trajectories and** KRAS **gene dosage define pancreatic cancer phenotypes.**

Sebastian Mueller,1 Thomas Engleitner,1 Roman Maresch,1 Magdalena Zukowska,1 Sebastian Lange,1 Thorsten Kaltenbacher,1 Björn Konukiewitz,2 Rupert Öllinger,1 Maximilian Zwiebel,1 Alex Strong,3 Hsi-Yu Yen,4 Ruby Banerjee,3 Sandra Louzada,3 Beiyuan Fu,3 Barbara Seidler,1 Juliana Götzfried,1 Kathleen Schuck,1 Zonera Hassan,1 Nina Schönhuber,1 Sabine Klein,1 Christian Veltkamp,1 Mathias Friedrich,3 Lena Rad,1 Maxim Barenboim,1 Christoph Ziegenhain,5 Julia Hess,6 Oliver M. Dovey,3 Stefan Eser,1 Swati Parekh,5 Fernando Constantino-Casas,7 Jorge de la Rosa,3 Marta I. Sierra,8 Mario Fraga,8 Julia Mayerle,9 Günter Klöppel,2 Roland M. Schmid,1 Juan Cadiñanos,3 Pentao Liu,3 George Vassiliou,3 Wilko Weichert,2 Katja Steiger,2 Wolfgang Enard,5 Fengtang Yang,3 Kristian Unger,6 Günter Schneider,1 Ignacio Varela,10 Allan Bradley,3 Dieter Saur,1 Roland Rad1. 1 _University Hospital TU Munich, Munich, Germany;_ 2 _Institute of Pathology, Technische Universität München, Germany;_ 3 _The Wellcome Trust Sanger Institute, Genome Campus, United Kingdom;_ 4 _German Cancer Consortium (DKTK), German Cancer Research Center (DKFZ), Munich, Germany;_ 5 _Anthropology & Human Genomics, Department of Biology II, Ludwig-Maximilians Universität, Munich, Germany; _6 _Helmholtz Zentrum München, Research Unit Radiation Cytogenetics, Germany;_ 7 _Department of Veterinary Medicine, University of Cambridge, United Kingdom;_ 8 _Institute of Oncology of Asturias (IUOPA), HUCA, Universidad de Oviedo, Spain;_ 9 _Medizinische Klinik und Poliklinik II, Klinikum der LMU München-Grosshadern, Germany;_ 10 _Instituto de Biomedicina y Biotecnología de Cantabria (UC-CSIC), Munich, Spain_.

Pancreatic ductal adenocarcinoma (PDAC) has frequent alterations in few genes (KRAS, CDKN2A/TP53/SMAD4) and extensive heterogeneity of cancer drivers beyond. The expectation that mutational landscapes of rare drivers could explain phenotypic diversity has -with few exceptions- not come true. Likewise, PDAC metastasis is not understood, and comparisons of primary/metastasis pairs did not find recurrently mutated "metastasis genes". Here we show that key aspects of PDAC biology are defined by gene-dosage variation of PDAC signature genes, evolving along distinct evolutionary routes. We found increased gene dosage of the initiating KRAS mutation (KRASMUT-iGD) in human PDAC precursors. Mouse models revealed the importance of KrasMUT-iGD for both, early progression and metastasis, rationalizing the high frequency of PDAC dissemination at diagnosis. To overcome limitations posed to gene dosage studies by PDAC´s stroma-richness, we developed murine cell culture resources comprising 135 primaries/metastases. Integrative analyses of their genomes, transcriptomes and tumor phenotypes, combined with human studies and functional analyses revealed a series of additional KrasMUT-dosage effects: different KrasMUT-levels define distinct cellular morphologies, histopathologies and clinical outcomes, with highest KrasMUT-expression underlying the most aggressive undifferentiated phenotypes. We also observed KrasMUT-dosage-associated cellular plasticity, including epithelial-to-mesenchymal transition. Mechanistically, oncogenic dosage-variation is linked to distinct evolutionary routes, characterized by defined types/states of tumor-suppressor alterations: Phylogenetic tracking studies revealed convergent evolution of KrasMUT-iGD-gains, with dependence on prior homozygous Cdkn2a- or Trp53-loss. By contrast, in Cdkn2aHET cancers, amplifications of known and novel oncogenes (Myc, Yap1, Nfkb2) collaborate with KrasMUT-HET to drive progression, yet with lower metastatic potential. These results also reveal oncogene-selective/context-dependent Cdkn2a-haploinsufficiency, for which Tgfβ pathway alterations provide permissiveness. Our study uncovers universal principles underlying PDAC biology and phenotypic diversification. It describes evolutionary trajectories, identifies their genetic hallmarks and shows how oncogenic dosage-variation is differentially licensed along individual routes to control critical disease characteristics, including early progression, histopathology, metastasis, cellular plasticity and clinical aggressiveness.

#392

Reverse functional genomics: Identifying differentially regulated functional networks as predictive biomarkers of pivotal vulnerabilities in pancreatic cancer.

Johnathon L. Rose, Alessandro Carugo, Wantong Yao, Sahil Seth, Sanjana Srinivasan, Michael Peoples, Traver Hart, Timothy Heffernan, Giulio Draetta. _MD Anderson Cancer Center, Houston, TX_.

Pancreatic Ductal Adenocarcinoma (PDAC) is an incurable disease characterized by poor survival, a dense desmoplastic stroma, and frequent activating mutations in KRAS (<90%) with a relatively flat mutation landscape. Despite recent advances in RNA sequencing analysis that enabled the characterization of PDAC into at least two tumor subtypes, this alone is insufficient to define more specific patterns of oncogenic dependency. As a result, there is currently no approach for stratifying these KRAS-driven tumors for targeted therapy. To inform on the more nuanced oncogenic dependencies we investigated how the deregulation of proteins localized to the extracellular face of the cell, or the "surfaceome", plays a role in tumor maintenance and progression by utilizing an in vivo pooled screening platform in patient-derived models. A PDAC prioritized surfaceome gene set was established based on aberrant transcription in microdissected and bulk tumor datasets, functional amplification observations via the TCGA dataset, and KRAS dependency via SILAC screening in KRASG12D inducible mouse lines. Refining this initial gene set for functional relevance, through parallel loss of function shRNA screens in orthotopic and 2D conditions, we were able to successfully define an in vivo enriched PDAC functional surfaceome. To interconnect each protein of the functional surface gene set, we utilized a protein-protein interaction dataset (STRING) and built a custom CRISPR sgRNA library for the purpose of informing the downstream oncogenic PDAC dependencies. Starting from oncogenic signaling at the cell surface, we established a set of connected oncogenic modules to functionally fingerprint each PDAC model both in vivo and in vitro. Our custom algorithm leveraging essentiality to define distinct oncogenic routes has allowed insight into the shared core oncogenic dependencies within PDAC (KRAS, MYC, etc), as well as model-specific central dependencies. Notably, many of the more central model-specific nodes, like AKT, EGFR, and UBC, were druggable targets. These unique centralities have been confirmed through differential sensitivity to MK-2206, Gifitinib/Erlotinib and AUY922 respectively. Importantly, by driving the defined network from periphery to center, we associated the unique pivotal dependencies to larger functional module sets to be leveraged as potential biomarkers. The functional modules suggested by the network and the associated vulnerability match the transcriptionally defined PDAC subtypes. To validate these module-based biomarker sets, we are currently leveraging a larger PDAC xenograft cohort, as well as patient data, to quantify the predictive capacity of the differentially regulated modules in the context of variable drug dependencies.

#393

Dynamic networks of transcriptome, DNA methylome, and DNA hydroxymethylome during T-cell lineage commitment.

Byoung-Ha Yoon. _KRIBB (Korea Research Institute of Bioscience & Biotechnology), Daejeon, Republic of Korea_.

Immunotherapy is a treatment that uses a part of a personal immune system to overcome diseases such as cancer. T cell therapy has been used in a variety of cancer types, including lymphoma, melanoma and colon cancer. There are several strategies used to take advantage of cell-mediated anti-tumor properties of T cells. Cellular immune responses to prevent tumors are typically caused by CD4 and CD8 T cells. The stepwise development of T cells from a multipotent precursor is guided by diverse mechanisms including interactions of lineage specific transcription factors and epigenetic changes and acquires lineage-specific trait. However, in this T-cell development process, a greater understanding of the regulation of genes involved in immunotherapy is needed.

To elucidate the transcriptional networks and epigenetic mechanisms underlying T-cell therapy and T cell lineage commitment, we investigated genome-wide changes in gene expression, DNA methylation and hydroxymethylation among five successive populations of T cell development (DN3, DN4, DP, CD4+, and CD8+) using RNA-seq, MBD-seq and hMeDIP-seq, respectively. And investigated interactive networks of transcription factors, chromatin modifiers, and DNA methylation. We found stage specific differentially methylated regions and some of them occurred near the specific differentially expressed genes related to immune cells or cancer immunotherapy such as Cd4, Ccr9 and Pdcd1. Dynamic changes of DNA methylation and hydroxymethylation is associated with the recruitment of the stage specific transcription factors.

This study may provide a frame work for understanding the complexity of T cell lineage commitment and immunotherapy-related genes were regulated during T-cell differentiation.

#394

Elucidating tissue-specific effects of the 8q24 multicancer risk locus via CRISPR/Cas9 scarless genome editing.

Nicole B. Coggins, Henriette O'Geen, David J. Segal, Luis G. Carvajal-Carmona. _Univ. of California, Davis, Davis, CA_.

In the past decade, genome-wide association studies have identified a large number of genomic variants, specifically single-nucleotide polymorphisms (SNPs), associated with increased cancer risk. The molecular mechanisms underlying the vast majority of these associations remain largely unknown. Precise cellular models containing the variant of interest could be a crucial tool for establishing disease-relevant function in order to translate these discoveries into clinical application. CRISPR/Cas9 gene editing has provided a convenient and flexible method for creating isogenic cell lines via the homology-directed repair (HDR) pathway; however, methods for efficient generation and subsequent isolation of precisely edited cells have proven both expensive and time consuming. In response to this need, we have combined CRISPR/Cas9 HDR gene editing with an innovative high-throughput genotyping pipeline utilizing KASP (Kompetitive Allele-Specific PCR) technology to create scarless isogenic cell models of cancer risk variants in ~1 month without selectable markers or specialized methods such as digital droplet PCR or NGS. Utilizing this technology, we have been able to create cell lines differing by only a single base to model risk-associated SNPs located in the 8q24 risk locus in multiple cell types. The 8q24 risk locus is associated with increased risk for colorectal, breast, prostate, and, more recently, thyroid cancer. In this study, we use engineered isogenic cell lines modeling functional risk SNP rs6983267 in each of these cancer types to identify tissue-specific mechanisms of this multicancer risk locus. Rs6983267 is located within a known c-Myc enhancer and has been shown to exert risk-allele specific increase in enhancer activity. Oncogenic c-Myc is known to play a significant role in the pathogenesis of many cancers. However, how or if this mechanism varies between the different cancers this locus has been associated with remains uncertain. From preliminary studies, we do observe cell type variation in the regulatory effect of rs6983267. Not only does there seem to be a varying effect of the risk allele on c-Myc expression across the different cell types, but also risk allele enhancement of binding of the TCF7L2 transcription factor to the c-Myc enhancer rs6983267 is located within. Interestingly, rs6983267's regulatory effect seems most significant between homozygous reference cells and heterozygous cells within the same tissue type. We plan to delve further into these differences using RNA-seq and 4C-seq to assess whole transcriptomic and chromosomal interaction changes with changes in risk status. This study is one of first to compare risk mechanisms across different cancer tissue types; using precisely edited cellular models, we are able to detect cell-type specific variations in functional risk SNP effects on cancer development.

#395

Identifying the functional drivers of noncoding somatic mutations in ovarian cancer.

Rosario I. Corona,1 Ji-Heui Seo,2 Dennis J. Hazelett,1 Xianzhi Lin,1 Paulette Y. Mhawech-Fauceglia,3 Jenny Lester,1 Sohrab Shah,4 David G. Huntsman,4 Beth Y. Karlan,1 Benjamin P. Berman,1 Matthew L. Freedman,2 Simon A. Gayther,1 Kate Lawrenson1. 1 _Cedars-Sinai Medical Center, Los Angeles, CA;_ 2 _Dana-Farber Cancer Institute, Boston, MA;_ 3 _University of Southern California, Los Angeles, CA;_ 4 _The University of British Columbia, Vancouver, British Columbia, Canada_.

Epithelial ovarian cancer (OC) is a heterogeneous disease that has been stratified into different histologic subtypes: high-grade serous (HGSOC), clear cell (CCOC), endometrioid (EnOC) and mucinous (MOC). HGSOC is the most common, but every histotype is characterized by largely distinct germline genetics, somatic alterations and clinical biomarkers. Recently, whole-genome sequencing (WGS) studies have catalogued genome-wide somatic variation for most OC histotypes. These data demonstrate that OC histotypes harbor thousands of noncoding somatic mutations and our next major challenge is to distinguish the few important noncoding somatic drivers from the thousands of passenger mutations. We hypothesized that driver noncoding somatic mutations impact disease development and progression through altering the sequence of regulatory elements (REs), such as enhancers and promoters, eventually resulting in perturbation of the expression of target genes. To systematically address this hypothesis, we established genome-wide H3K27ac epigenomic profiles, annotating active REs for the different ovarian cancer histotypes using chromatin immunoprecipitation sequencing (ChIP-seq) in 20 fresh frozen primary OC tissue samples—five tumors for each major histotype. In parallel, we performed transcriptional profiling using RNA sequencing (RNA-seq). Together, these two datasets enabled us to evaluate epigenetic alterations and the transcriptome. We identified histotype-specific active REs, and common active REs across all histotypes. We used the RNA-seq data to assess the effect of the histotype-specific REs in gene expression, and to find target genes of cis-REs and novel histotype-specific biomarkers. Next, we integrated these unique profiles with WGS data from 232 OCs (169 HGSOCs, 35 CCOCs and 28 EnOCs). The number of somatic single-nucleotide variants per sample range from 481 to 40,764 (mean=7199, sd = 5751). Of these, 9.4% were noncoding and overlapped active REs in OC. Using a Poisson binomial distribution, we tested the significance of the observed number of mutated samples for any given active RE. Importantly, the method adjusted for interpatient mutation rate heterogeneity. Using the Benjamini-Hochberg procedure for p-value correction, we identified several significantly mutated active REs, including the promoters of POLR3E and WDR74. In conclusion, we have used an integrative method to identify functional, driver noncoding somatic mutations for ovarian cancer based on their interaction with disease-specific regulatory elements, and their putative target genes. This represents a powerful way to distinguish important noncoding somatic drivers from a much larger number of passenger mutations that accumulate during tumor development. The approach can be applied to other cancer types if sufficient depth and quality of genetic, transcriptomic and epigenomic datasets are available.

#396

Sex-specific genomic architecture and regulatory mechanisms underlying hepatocellular carcinoma.

Heini M. Natri, Melissa Wilson Sayres, Kenneth Buetow. _Arizona State University, Tempe, AZ_.

The occurrence and mortality rates of most types of cancer are significantly higher in males than in females. Environmental risk factors may explain some portion of the sex-bias, but cellular and molecular differences are also likely important. The sexes differ in their endocrinological and immunological functions, genetic makeup, and in their response to some forms of chemotherapy and immunotherapy. However, sex differences are rarely considered in the development of cancer therapies. Targeted approaches to the treatment of male and female tumors will require characterizing and understanding the fundamental biological mechanisms that differentiate them.

Hepatocellular carcinoma (HCC) is the second leading cause of cancer death worldwide. HCC is influenced by genetic susceptibility, environmental factors such as oncogenic viral infections, metabolic syndrome, and alcohol use, and shaped by numerous biological processes, resulting in a high degree of genetic and transcriptional heterogeneity. HCC incidence in the US has doubled in the last 3 decades, attributable to increased rates of obesity. HCC exhibits sex bias in occurrence with 2.1-fold higher rate in HBV+ males, 1.9-fold in HCV+ females.

While sex-bias in HCC is partly attributed to sex-specific differences in risk behaviors and environmental exposure, the relationship of these factors and sex as a biological variable has not been systematically investigated. Moreover, most GWAS and eQTL studies as yet have failed to explore the role of sex-specific impacts of regulatory variants.

Here, we utilize an integrative systems genetic approach to investigate the mechanisms involved in HCC. By sex-specific analyses of genotypic, transcriptomic, and phenotypic data from GTEx, TCGA, and ICGC datasets, we have produced a detailed characterization of regulatory networks underlying HCC occurrence and susceptibility. As mis-mapping of sequence reads along the sex chromosomes may increase the rate of false negatives when accounting for sex-biased gene expression, we have utilized custom sex-specific reference genome sequence alignments. We have carried out gene and isoform level analyses of differential expression, pathway enrichment analyses, cis- and trans-eQTL mapping, and de novo network analyses to identify the mechanisms underlying HCC oncogenesis in each sex. Substantial sex-biased gene expression was detected in normal liver tissue, as well as tumor-adjacent and tumor tissues. Analyses of paired tumor and tumor-adjacent samples imply that distinct oncogenic pathways shape HCC in males and females. We found considerable differences in significant eQTLs between sexes. Constructed networks further elucidate the sex-specific regulatory mechanisms of HCC oncogenesis. Our results illustrate the highly sex-dependent genomic architecture of HCC, with implications for future studies and targeted therapies.

#397

Systematic functional annotation of somatic mutations in cancer.

Han Liang, Patrick Kwok-Shing Ng, Jun Li, Kang Jin Jeong, Yiling Lu, Song Yi, Nidhi Sahni, Gordon Mills. _UT MD Anderson Cancer Center, Houston, TX_.

Recent sequencing studies have identified thousands of unique somatic mutations across patient tumors. However, the functional impact of the vast majority of these mutations remains unknown, representing a critical knowledge gap for implementing precision oncology. Here, we report the development of a moderate-throughput functional genomic platform consisting of efficient mutant open reading frame generation, sensitive viability assays using two growth factor-dependent cell models, and functional proteomic profiling of downstream signaling effects for select aberrations. We apply the platform to annotate >1,000 genomic aberrations, including gene amplifications, point mutations, indels, and gene fusions, potentially doubling the number of driver mutations characterized in clinically actionable genes. We show that our platform has higher sensitivity for characterizing weak drivers than pooled screening. Our data are accessible through the user-friendly, open-access data portal we created. Our study will facilitate the discovery of novel biomarkers, improvement of existing prediction algorithms, and development of new therapeutic approaches.

#398

Functional coding and noncoding drivers of EMT-mediated acquired drug resistance.

Russell O. Bainer,1 Catherine Wilson,1 Marinella Callow,1 Siyu Feng,2 Michael Costa,1 Colin Watanabe,1 Oleg Mayba,1 Eva Lin,1 Scott Martin,1 Bob Yauch,1 Richard Bourgon,1 Christiaan Klijn1. 1 _Genentech, Inc., South San Francisco, CA;_ 2 _The University of California at San Francisco, San Francisco, CA_.

In response to diverse stimuli, tumor cells can undergo a process resembling the epithelial-mesenchymal transition (EMT) observed during development. During this transition, epithelial-like tumor cells acquire mesenchymal-like physiologic features, become more motile and invasive, and acquire insensitivity to many therapeutic agents. While the importance of this phenomenon to cancer treatment and patient outcomes is well known, considerable heterogeneity exists across experimental models of EMT, and diverse mechanisms have been proposed to explain its various associated malignant phenotypes. Consequently, the extent to which these processes are shared uniformly across instances of EMT, as well as their relative importance in treatment contexts, remains unclear. We have used RNAseq, ATACseq, and parallelized high-throughput cell viability screening to identify differences in gene expression levels, chromatin accessibility, and drug sensitivity that correlate with EMT in a series of cell line models. By contrasting these changes across different genetic backgrounds and mechanisms of EMT induction, we are able to identify sets of elements in the transcribed and noncoding genome that are characteristic of different modes of EMT induction. Then, by comparing these candidate sets with observed differences in acquired drug sensitivity in the respective models, we were able to define a set of genetic elements whose biologic activity is likely to influence the efficacy of these compounds. Finally, we used a series of targeted CRISPR-based knockout screens to assess the relative importance of these elements to the viability of isogenic epithelial-like and mesenchymal-like cells, alone and in the presence of multiple therapeutic drugs. Using this approach, we were able to validate known mechanisms of acquired drug resistance and identify new candidate effectors. Notably, we identify multiple candidate noncoding elements containing CTCF binding sites that appear to meaningfully influence cell viability in a drug-specific manner, potentially implicating genome conformational changes in acquired therapeutic resistance.

#399

NCI Office of Cancer Genomics: Supporting structural and functional genomics and development of bioinformatic approaches to advance precision oncology.

Pamela C. Birriel,1 Caitlyn W. Barrett,1 Tanja M. Davidsen,2 Martin L. Ferguson,1 Patee Gesuwan,2 Nicholas B. Griner,1 Jaime M. Guidry Auvil,1 Yiwen He,2 Subhashini Jagu,1 Freddie L. Pruitt,1 Daniela S. Gerhard1. 1 _National Cancer Institute, Bethesda, MD;_ 2 _National Cancer Institute, Rockville, MD_.

The National Cancer Institute's Office of Cancer Genomics (OCG) aims to advance the molecular understanding of cancer, with the goal of improving clinical outcomes. OCG supports large-scale cancer genomics and translational research programs that share data and resources with the research community, thereby accelerating discoveries into the clinic and contributing to precision oncology. The OCG initiatives promote: i) generation and dissemination of molecular and clinical data via programmatic databases and the Genomic Data Commons, ii) advances in bio- and chemi-informatic methodologies, and iii) creation of valuable experimental reagents, resources, models, and standard operating procedures.

OCG currently supports four innovative and collaborative programs which conjointly generate, analyze, and translate genomic and other datasets into biologically and clinically-relevant information for the scientific and medical communities. The Cancer Genome Characterization Initiative (CGCI) and the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) initiatives use comprehensive genomic, transcriptomic, and epigenomic approaches to analyze tumor and patient matched normal tissue samples with the goal of identifying therapeutic targets and biomarkers. CGCI characterizes both adult and pediatric cancers, including those prevalent in HIV-positive individuals, while TARGET focuses predominantly on high-risk cancers affecting children and young adults. The Cancer Target Discovery and Development (CTD2) Network advances cancer research by bridging the knowledge gap between cancer genomics and precision oncology for the development of effective combinatorial cancer treatments to minimize treatment resistance. The Network develops bioinformatics and analysis tools, generates diverse datasets, and further validates subsets of these data. The Human Cancer Models Initiative (HCMI) is an international consortium which will develop next generation patient-derived cancer models that are representative of the biology and complexity of human tumors. Models will be molecularly characterized and clinically annotated, and models along with the data will be available as a community resource.

Data, analytical tools, and resources generated by OCG programs are made available through the OCG website (https://ocg.cancer.gov/) to the research community to accelerate the discovery of efficient treatment strategies for cancer. This poster will include an overview of the programs and emphasize the usability and functionality of the OCG databases and resources. Presenters will also provide data access information and examples of past data use highlighting the benefit of the OCG programs to the scientific community.

#400

**Noncanonical role of** SEC23B **in thyroid carcinogenesis: Anemia gene meets cancer.**

Lamis Yehia,1 Ying Ni,1 Supriya Jindal,2 Ata Abbas,1 Anton A. Komar,2 Charis Eng1. 1 _Cleveland Clinic, Cleveland, OH;_ 2 _Cleveland State University, Cleveland, OH_.

Hereditary cancer syndromes serve as powerful models to uncover cancer-relevant genes. Cowden syndrome (CS), typically caused by germline PTEN mutations, is a heritable cancer syndrome with high risks of breast and thyroid cancers. However, there is a growing subset of CS without PTEN mutations. By exome sequencing cum family-studies, we identified heterozygous SEC23B variants in CS-associated and apparently sporadic thyroid cancer. Functional characterization revealed mutant CS-associated SEC23B led to endoplasmic reticulum (ER)-stress-mediated cell-colony formation and survival, growth and invasion, reflecting major hallmarks of cancer.

Classically, SEC23B is a component of coat protein complex II (COPII) vesicles that transport secretory proteins from the ER to the Golgi apparatus. Germline homozygous or compound heterozygous loss-of-function SEC23B mutations cause Congenital Dyserythropoietic Anemia Type II (CDA II), resulting in decreased SEC23B levels. Intriguingly, our in vitro data does not recapitulate the CDA II-like decreased SEC23B protein expression, suggesting possible change-of-function effects in a cancer context. Here, we show mutant SEC23B exists within nucleoli, besides classical distribution at the ER/Golgi. This occurs in the absence of nucleolar localization of other COPII protein components and does not compromise canonical secretory function. Global transcriptomic and polysome profiling analyses reveal increased expression of ribosomal protein and translation-related genes, accompanied by enhanced translational capacity in mutant cells in response to ER stress. We also show mutant SEC23B directly binds to Upstream Binding Transcription Factor (UBF), concomitantly with increased UBF binding at the ribosomal DNA (rDNA) promoter region with ER stress. Unbiased proteomic analyses further show SEC23B, both wildtype and mutant, can interact with proteins in the ER stress and EIF2 signaling pathways. We validate through immunoprecipitation the binding interaction between SEC23B and UBA52, with notably increased binding with mutant SEC23B. UBA52 is ribosomal protein RPL40, known to regulate selective translation of stress-related transcripts, protein synthesis, and the cell cycle. Knockdown of SEC23B in vitro resulted in a concomitant decrease in protein levels of UBA52, further supporting that SEC23B and UBA52 are potentially co-regulated. All in all, our data suggest that SEC23B has non-canonical COPII-independent function, particularly within the ribosome biogenesis pathway, that may contribute to the pathogenesis of cancer-predisposition.

#401

Functional characterization of the 14q24 renal cancer susceptibility locus implicates SWI/SNF complex member DPF3 via inhibition of apoptosis.

Leandro Machado Colli, Lea Jessop, Timothy A. Myers, Mitchell J. Machiela, Jiyeon Choi, Mark Purdue, Kevin Brown, Stephen J. Chanock. _NCI, Rockville, MD_.

To identify promising regulatory regions under the peaks of Renal Cell Carcinoma (RCC) GWAS signals, we deployed the Massively Parallel Reporter Assay (MPRA), a powerful tool to dissect putative regulatory regions in parallel. Using MPRA, we identified rs4903064 within the 14q24 RCC risk region as a potential transcriptional enhancer with allele-preferential activity. By MPRA, the risk allele C leads to higher expression, an effect confirmed by luciferase assay in three RCC cell lines. Using electromobility shift assays, we observed preferential protein binding to the T allele of rs4903064, one of the strongest signals in the RCC GWAS at 14q24. Motif analysis shows that this SNP resides in a binding site for the transcriptional repressors IRX2 and IRX5 and the C allele is predicted to disrupt the binding site. These results suggest that the higher expression seen in MPRA and luciferase assays when the C allele is present is likely due to reduced binding of transcriptional repressor proteins. Consistent with this, an eQTL analysis of RCC TCGA data showed that C allele is associated with higher expression of the double PHD Fingers 3 gene (DPF3) (p=5.5e-8). DPF3, a plausible candidate gene influenced by the RCC susceptibility allele is a member of the nucleosome remodeling SWI/SNF complex, components of which are frequently mutated in multiple cancers. PBRM1, a member of SWI/SNF complex, is the second most common mutated gene in RCC (40%), demonstrating the importance of SWI/SNF complex for RCC oncogenesis. We generated three kidney cells lines (two RCC- ACHN and UOK-121 and one normal kidney cell line- HK2) with DOX-inducible expression of both DPF3 isoforms (a or b), separately. ATAC-seq analysis of HK2 cells overexpressing DPF3a or DPF3b showed reduced chromatin accessibility compared to controls, suggesting that DPF3 overexpression is disrupting SWI/SNF activity. RNA-seq analysis of DPF3a or DPF3b over expressing cell lines showed deregulation of apoptotic genes, especially overexpression of CEMIP. Cell lines over expressing DPF3a or DPF3b had a higher growth rate, suggesting an oncogenic effect of both DPF3 isoforms. Knockdown of CEMIP by si-RNA blocked the effect of DPF3a and DPF3b on growth rate, suggesting that part of the DPF3 effect on cell growth can be attributed to overexpression of the apoptosis inhibitor CEMIP. We treated cells overexpression of either DPF3a or DPF3b with cisplatin or camptothecin to induce apoptosis and observed cleavage of Caspase 3 and PARP by Western Blot, both markers of apoptosis. Using AnexinV and 7-AAD, flow cytometery analysis confirmed that DPF3a or DPF3b overexpression reduces apoptosis in RCC cell lines. In summary, our results suggest that the RCC risk C-rs4903064 at the 14q24 locus increases expression of DPF3, which disrupts the normal function of the SWI/SNF complex leading to dysregulation of CEMIP and a reduction of apoptosis.

#402

Gene editing for functional analysis of Ewing sarcoma target genes.

Timothy J. Triche,1 Sheetal A. Mitra,2 Hyung G. Kang,2 Jonathan D. Buckley1. 1 _USC/Children's Hospital Los Angeles, Los Angeles, CA;_ 2 _Children's Hospital Los Angeles, Los Angeles, CA_.

Gene editing tools like CRISPR, using Cas9 protein and selected guide RNAs, are now the preferred method to study many coding gene functions, but historically have been hampered by low efficiency (typically <20% of cells), which limits their utility in suppressing highly expressed oncogenic drivers of cancer like fusion genes. Ewing sarcoma (ES) is driven by the EWS-FLI1 or equivalent fusion gene and expresses CD99 on its cell surface. CD99 itself is a direct target of the fusion gene, making it a universal target potentially exploited by CD99 targeted methods. We have utilized CD99 targeted nanoparticles containing plasmid constructs (Cas9-EGFP-guide RNA) targeting the EWS, FLI1, or ETS domains of the fusion gene to achieve up to ~70% knockdown after one transfection in TC32 ES cells. We show that targeting the 5' EWSR1 region of the fusion gene results in dramatically lower expression of the fusion gene (30% of control), with lesser effect when the 3' ETS region is targeted (40% of control). We then compared the genes targeted by the CRISPR EWS-FLI1 KO in TC32 cells with those whose expression was altered by knock down of the fusion gene using shRNA directed against the EWS-FLI1 3'UTR in CHLA9 ES cells and found >2,000 targets in common based on total RNA Seq data with a 2-fold or greater delta in expression. Most targets were down regulated after fusion gene suppression (~1,300 vs ~1,000), indicating they are up regulated by the oncoprotein. Importantly, over half of the affected transcripts were non-coding lncRNAs (Up regulated targets: 421 coding, 539 ncRNA; Down regulated targets: 299 coding, 448 ncRNA) Nearly 600 pseudogenes were also dysregulated. The most statistically significant targets were sense and anti-sense intragenic (regulatory) ncRNAs, documenting the likely importance of ncRNAs in the genesis of Ewing sarcoma. Further, while both coding and non-coding targets were enriched for multimeric GGAA repeats within 1 kb of their TSS when compared to a randomly selected control population, the vast majority of both classes of transcripts lacked multimeric GGAA repeats in their promoters or even GGAA repeats in enhancers within 50kb of the targeted transcripts, indicating that regulation of Ewing sarcoma target gene expression is mediated proximally by factors other than direct or indirect binding of the oncoprotein in cis with the target gene. Our results demonstrate that use of Ewing-specific, CD99 targeted nanoparticles allows high-efficiency (~70%) permanent editing and knockdown of the fusion gene. This enables analysis of fusion gene targets in a native, unperturbed tumor setting, free of artifacts introduced by transfection methods or induced fusion gene expression. It also allows functional studies of the fusion gene RNA targets themselves, using the same gene editing methods, which could enable pre-clinical therapeutic proof-of-concept studies directed against the fusion gene, its validated coding and non-coding targets, or both.

#403

Analysis of the functional consequences of single nucleotide polymorphisms in CYP3A4 gene to prostate cancer in men of African ancestry.

Solomon O. Rotimi,1 Chidiebere Ndukwe Ogo,2 Olubanke Olujoke Ogunlana,1 Shalom Nwodo Chinedu,1 Titi Akinremi,3 Omolara Fatiregun,4 Funlayo Buraimoh,4 Adewunmi Alabi,5 Kehinde Tijani,5 Ayo Salako,6 Emmanuel Omonisi,7 Ruth Agaba,8 Folakemi Odedina9. 1 _Covenant University, Ota, Nigeria;_ 2 _Federal Medical Centre, Abeokuta, Ogun State, Abeokuta, Nigeria;_ 3 _Federal Medical Centre, Abeokuta, Nigeria;_ 4 _Lagos State University Teaching Hospital and College of Medicine, Ikeja, Nigeria;_ 5 _University of Lagos Teaching Hospital, Lagos, Nigeria;_ 6 _Obafemi Awolowo University Teaching Hospital, Ile Ife, Nigeria;_ 7 _Ekiti State University Teaching Hospital, Ado Ekiti, Nigeria;_ 8 _Prostate Cancer Transatlantic Consortium, Ota, Nigeria;_ 9 _University of Florida, Gainesville, FL_.

Prostate cancer (CaP) is the leading cause of cancer death among Black men and its pathogenesis is linked to testosterone metabolism. CYP3A4 deactivates testosterone and the polymorphisms of its gene are known to alter testosterone metabolism. In this study, computational techniques were used to investigate the effects of nonsynonymous single nucleotide polymorphisms (nsSNPs) in the CYP3A4 gene in African Americans exome sequence. A case-control study was also carried out on 100 prostate cancer patients and 100 controls, among Yoruba men in Nigeria, to investigate the allelic variants of CYP3A4*1B (rs2740574 A>G) using polymerase chain reaction restriction fragment length polymorphism. A total of 22 nsSNPs were found in CYP3A4 gene of African American; out of which, 13 mutations (Q472R, M445T, K421R, R403C, I396T, L373F, R372I, I335T, T309I, V191A, D174H, R162W and S134C) were predicted to have deleterious effects on the CYP3A4 protein. The protein structural analysis of these amino acid variants revealed that M445T mutation could result in an increased free energy with a significant (p<0.05) loss of allosteric site at M445, altered metal binding, and loss of catalytic site at R446. T309I mutation which also resulted in increase free energy was found to be associated with a loss of catalytic site at E308. All the Nigerian samples were homozygous for the variant CYP3A4*1B allele. Hence, the presence of GG homologous variant in the Yoruba population of Nigeria, coupled with the computationally predicted deleterious mutations, could contribute to the high prevalence and aggression of CaP in black men. These genetic variants identified through in silico analysis can provide a better understanding of the wide range of disease susceptibility associated with this gene in Black men and aid the development of new molecular diagnostic markers for CaP particularly in men of West African ancestry.

#404

**Amplification of** SOX4 **promotes PI3K/Akt signaling in human breast cancer.**

Gaurav Mehta,1 Michael Gatza,1 Steve Angus,2 Gary Johnson2. 1 _Rutgers Cancer Institute of New Jersey, New Brunswick, NJ;_ 2 _Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC_.

Basal-like or triple-negative breast cancer, if considered its own disease, would rank as the 5th leading cause of cancer deaths for women in the USA each year with ~10,000 deaths annually. Given the lack of drug-able targets expressed by TNBC tumors, few therapeutic options exist beyond currently utilized cytotoxic therapies, and the overall prognosis for these patients remains poor. The oncogenic PI3K/Akt signaling axis contributes to the dysregulation of many dominant features in breast cancer including cell proliferation, survival, metabolism, motility, and genomic instability. While multiple studies have demonstrated that basal-like or triple-negative breast tumors have uniformly high PI3K/Akt activity, genomic alterations that mediate dysregulation of this pathway in this subset of highly aggressive breast tumors remain to be determined. In this study, we present an integrated genomic analysis based on the use of a PI3K gene expression signature as a framework to analyze orthogonal genomic data in human breast tumors from TCGA (The Cancer Genome Atlas) including RNA expression, DNA copy number alterations, and protein expression. These analyses identified copy number alterations associated with PI3K activity in human breast tumors (n=1,031), including known and potentially novel drivers of PI3K signaling. Data from a genome-wide RNA interference (RNAi) screen were used to discriminate between essential and non-essential genes within identified amplicons. Integrating DNA copy number and RNAi analyses identified amplified SOX4 as a novel and essential regulator of PI3K/Akt signaling. Importantly, by analyzing protein expression data (n=733), we determined that SOX4 amplified tumors have increased activation of pAkt substrates. Moreover, depleting SOX4 levels in HCC38 and HCC1143, basal-like breast cancer cell lines with high PI3K activity and high SOX4 expression, demonstrated significant abrogation of p-Akt levels. This validated our in silico findings and confirmed the role of SOX4 in regulating PI3K pathway activity. To identify the potential mechanism(s) by which SOX4 mediates PI3K pathway activity, we performed genome-wide analyses of these cell lines following SOX4 inhibition. Transcriptomic profiling by RNA-sequencing, protein profiling by Reverse Phase Protein Array (RPPA) and Kinome profiling through multiplexed kinase inhibitor beads and mass spectrometry (MIB-MS) based approach identified TGFBR2, as a potential regulator of SOX4-mediated PI3K/Akt signaling. Further studies will elucidate the mechanism by which SOX4 modulates TGFBR2 expression and drives the oncogenic PI3K/Akt signaling pathway in basal-like breast cancer.

#405

**PBRM1 re-introduction in** PBRM1 **-mutant kidney cancer cell lines drives an Interferon-γ expression signature.**

Rahmat K. Sikder, Wafik S. El-Deiry, Philip H. Abbosh. _Fox Chase Cancer Center, Philadelphia, PA_.

Introduction

Clear cell renal cell carcinoma (ccRCC) is the most common type of kidney cancer and is driven by loss of chromosome 3p. The 3p tumor suppressor PBRM1 is mutated in about 1/3 of tumors. VHL loss is well-known to drive the hypoxic response, but the role of PBRM1 loss is less well understood. PBRM1 is part of the SWI-SNF complex, an ancient chromatin modifying machine, so we hypothesized that its loss would alter chromatin profiles and subsequently expression of SWI-SNF target genes, and would result therapeutic susceptibility to histone H3-K27 methyltransferase inhibitors. Therefore we sought to reintroduce PBRM1 back into ccRCC cell lines which carry PBRM1 truncations in conjunction with 3p LOH and to characterize these cells in detail.

Methods

All cell lines harbored chromosome 3p loss with truncation of PBRM1 in the remaining allele. Tet-inducible PBRM1 or GFP was introduced into RCC-ER (D251fs), RCC-FG2 (Q445*), and RCC-MF (N496fs). Western blot and immunohistochemistry were used to confirm BAF180 (the protein encoded by PBRM1) and p21. CellTiter Glo was used to measure cell viability to drug treatments. RNA seq was used followed by gene-set enrichment analysis (GSEA) and Ingenuity Pathway Analysis (IPA) for gene expression profiling.

Results

BAF180 and p21 (a BAF180 target gene) were re-expressed in a time- and dose-dependent manner in all 3 cell lines. BAF180 was localized to the nucleus. EZH2 inhibitor UNC1999 completely ablated clonogenic formation in all 3 cell lines but there was not differential sensitivity between PBRM1-induced and uninduced cells. All cell lines were resistant to EPZ11011989, a different EZH2 inhibitor. We also characterized sensitivity to everolimus, an MTOR inhibitor, and sunitinib, an antiangiogenic multi-tyrosine kinase inhibitor. BAF180 expression did not alter sensitivity to these drugs either. Therefore, we performed RNA sequencing to evaluate the numerous signaling or epigenetic pathways which may have been altered in cells re-expressing BAF180. Using IPA, we saw that several immune signaling pathways were increased in cells upon reintroduction of PBRM1 including Interferon-γ signaling (RCC-ER and RCC-FG2), JAK/STAT signaling, and IL6 pathway. GSEA confirmed the finding that Interferon-γ pathway is activated in each cell line after 7 days of BAF180 induction. These findings were confirmed using Western blot for STAT1/2/3 and their phosphoepitopes, JAK1 and phospo-JAK1, IRF1 and IRF9.

Conclusions

Our hypothesis that PBRM1 loss would lead to a targeted vulnerability with EZH2 inhibitors was not supported. However, our finding that Interferon-γ signaling is increased when PBRM1 is reintroduced is significant. Given the prominence of Interferon signaling in the response to PD1 and PDL1 inhibitors, somatic PBRM1 inactivation in ccRCC may have significant biological and clinical implications for triaging patients into immune checkpoint therapy.

#406

A proteome-transcriptome-miRnome integrated analysis identifies similarity between UV-exposed skin and wounding skin.

Tran N. Nguyen,1 Kimal Rajapakshe,2 Stanislav Avdieiev,1 Courtney Nicholas,3 Vida Chitsazzadeh,4 Eric Welsh,1 Bin Fang,1 John Koomen,1 Cristian Coarfa,2 Janine Einspahr,5 Kenneth Y. Tsai1. 1 _Moffitt Cancer Center, Tampa, FL;_ 2 _Baylor College of Medicine, Houston, TX;_ 3 _MD Anderson Cancer Center, Houston, TX;_ 4 _University of Texas Health Science Center at Houston, Houston, TX;_ 5 _The University of Arizona Cancer Center, Tucson, AZ_.

Cutaneous squamous cell carcinoma (cuSCC) is the second most common skin cancer, for which long term UV exposure and chronic wounding are the dominant risk factors. Despite these clinically established connections, little is understood about the early molecular response of human skin to UV exposure and its connection to acute wounding and cuSCC. Thus, our goal is to find common and specific signatures driven by UV-exposure and wounding as a means of developing new approaches for treating and preventing cuSCC. Here, we perform integrated analyses of proteomic, RNA-seq and miR-seq on 3 datasets: (1) UV-unexposed and acute UV-exposed human skin, (2) public dataset on acute wound healing and (3) our previously published dataset on normal skin and cuSCC from humans. We find that biological signatures and processes regulated by acute UV exposure and wounding has profound similarity. miR-seq data shows that miR-223, miR-132 and miR-142 are significantly upregulated in both acute events. Combined gene set enrichment analysis shows that G-protein-coupled-receptors (GPCRs) pathways are upregulated, possibly through Gαi activation. While ECM remodeling is significantly enriched in all three datasets, gene expression regulated by PPARα is suppressed. Interestingly, upregulation of matrisome components is observed among all three datasets. This suggests that these changes are important early events that regulated by both UV-exposure and wounding which eventually can promote cuSCC initiation. Thus, our findings suggest that these common signatures can be potentially validated as chemopreventive targets for cuSCC.

#407

Novel targets in metastatic HNSCC.

Maria Kondratyev,1 Aleksandra Pesic,1 Troy Ketela,1 Azin Sayad,1 Stephano Marastoni,1 Jason Mofat,2 Carl Virtanen,1 Natalie Stickle,1 Reider Grenman,3 Brad Wouters1. 1 _University Health Network, Toronto, Ontario, Canada;_ 2 _University of Toronto, Toronto, Ontario, Canada;_ 3 _Turku University and Turku University Hospital, Turku, Finland_.

Head and neck carcinoma (HNSCC) is 6th most common malignancy in the world. Despite advances in diagnosis and treatment, the survival rates remain low due in large part to metastatic disease. The underlying biology associated with metastatic disease and poor outcome in HNSCC remains unclear. Importantly, metastatic cells acquire new properties that permit them to invade surrounding tissues and seed metastasis at distant sites. While these acquired properties contribute to aggressiveness of metastatic cancer and interfere with success of therapies, they can also potentially be exploited to target metastatic cells selectively, sparing toxicity in normal tissues. The idea behind this selective targeting is based on discovering molecular pathways that became essential in metastatic cells, and then exploiting this vulnerability through targeted agents. We used functional genomic technologies to identify new potential therapeutic targets for advanced disease in HNSCC. These targets were identified by conducting whole genome shRNA screens in matched sets of cell lines derived from primary tumors and their respective metastatic sites, with the goal of identifying genes that become essential for cell survival only following metastasis. Since hypoxia is an important attribute of aggressive and therapy resistant subpopulations of HNSCC tumor cells, we also aimed to identify genes that became essential when cells are exposed to hypoxia. To complement the functional screens data, we performed targeted sequencing of the most commonly altered genes in HNSCC as reported by the TCGA profiled gene expression in the HNSCC cell lines cultured under normoxia and hypoxia using Illumina microarrays. These analyses led to the discovery of genes that are differentially essential in metastatic cells as well as in cells exposed to hypoxic conditions. Utilizing CRISPR technology, we assembled a library of guide RNAs targeting the discovered hits and are currently validating the top identified genes using both in vitro and in vivo systems. To validate hits that are essential in metastatic cells in vivo, we engineered selected "matched" pairs of HNSCC cell lines to express CAS9 protein upon induction with doxycycline. The cells are then transduced with the library of guides and injected subcutaneously into mice; tumors from control and doxycycline treated animals are compared. The difference in genes that are essential for growth of tumors seeded by primary tumor derived and metastasis derived HNSCC cell lines is then assessed in a quantitative manner.

#408

Evidence of DLL4, NNAT, and SLC35C2 in suppression of breast cancer initiation, growth, and metastasis.

Cody Plasterer, Angela Lemke, Dana Murphy, Carmen Bergom, Amit Joshi, Hallgeir Rui, Michael J. Flister. _Medical College of Wisconsin, Milwaukee, WI_.

Breast cancer affects 1 in 8 women, resulting in 40,000 deaths annually. In most cases, a single cause of breast cancer cannot be found, but rather multiple environmental and genetic factors contribute to overall disease susceptibility. This, combined with complex gene interaction in both malignant tumor cells and nonmalignant tumor microenvironment (TME) cells, poses significant challenges in sifting through the many variants that contribute to the ~31% of breast cancer risk that is heritable. Here, we narrowed the regions associated with breast cancer risk on rat chromosome 3 (RNO3) by introgressing portions of RNO3 derived from the BN rat (protective strain) onto the genomic background of the SS rat (susceptible strain). These SS.BN3 congenics were then phenotyped for DMBA-induced mammary tumor incidence, latency, and multiplicity, which revealed two loci in close proximity that contribute to mammary tumor risk: chr3:95-130Mb (QTL1) and chr3: 154-177Mb (QTL2). By comparing these data with a previous study (Flister et al. Breast Cancer Res Treat. 2017 Aug;165(1):53-64.), we concluded that QTL1 is dependent on the host TME, whereas QTL2 directly modifies breast tumorigenesis and cancer cell proliferation. By combining the congenic mapping studies with genomic and transcriptomic sequencing, and functional analysis, we have now localized the top three candidate modifiers on RNO3: DLL4 (QTL1; TME modifier), NNAT (QTL2; cancer cell modifier), and SLC35C2 (QTL2; cancer cell modifier). Collectively, these data demonstrate the effects of several novel breast cancer modifiers, as well as highlight the potential interactions between modifiers of the malignant cancer cells and the nonmalignant host TME.

#409

Dual-direction CRISPR transcriptional regulation screening uncovers gene networks driving drug resistance.

Carlos le Sage, Steffen Lawo, Prince Panckier, Tim M. Scales, Syed Asad Rahman, Annette S. Little, Nicola J. McCarthy, Jonathan D. Moore, Benedict C. Cross. _Horizon Discovery, Cambridge, United Kingdom_.

Functional genomic screening with CRISPR-Cas9 has provided a powerful and precise new way to interrogate the phenotypic consequences of gene manipulation in high-throughput, unbiased analyses. Rapid development of pooled lentivirus and deep-sequencing-led approaches have allowed us and others to exploit this technology in target ID, target validation, drug MOA analysis and patient stratification.

Many screens have been conducted using loss-of-function driven by CRISPR-Cas9 knock-out. Whilst powerful, this approach is not suitable for the examination of hypomorphic phenotypes, which include essential genes. In addition, CRISPR-Cas9 knock-out does not allow the analysis of gene activation, leaving a significant hole in the functional genomic analysis. To address this, we have developed two new paired pooled screening platforms using inactivated Cas9 (dCas9) to control gene expression in both loss- and gain-of-function screens. Our CRISPRi platform uses a streamlined and high-performance approach with an improved tracrRNA sequence incorporated into a single-shot transduction protocol. Our CRISPRa platform is based on the SAM system and both platforms have been adapted to use next generation, highly optimised whole-genome libraries to enact maximum gene expression modulation. Validation analysis of both tools revealed outstanding performance and sensitivity, with greater than ten-fold improvement in detection rates compared to existing platforms, including RNAi.

We used these paired tools to explore the development of resistance mechanisms to the BRAF V600E inhibitor, vemurafenib. Both platforms allowed for the unambiguous discovery of expected hits, additional complex members not identified previously, as well as multiple novel genes involved in drug resistance. Most interestingly, simultaneous evaluation of both activating and inhibiting perturbations revealed direct and opposing phenotypic effects within complex gene networks, switching the response of affected cells to either sensitisation or resistance. In addition to providing a compelling strategy for the validation of drug resistance targets, the parallel analysis of CRISPRi and CRISPRa data sets also allows the identification of pathway modulators that affect both drug sensitivity and resistance, but in which sgRNAs are not reciprocally lost or increased in each screen.

Taken together, our data indicate that CRISPRa and CRISPRi screens in parallel provide an exceptionally powerful dataset for target identification. Dual directional approaches such as ours provide the new opportunity to achieve increased depth of hit finding in the analysis and offer novel discovery pathways by exploring opposing function and more complete gene network perturbation.

#410

**Genome-wide and focused CRISPR screens to study PARP inhibitor resistance mediated by mutations in** PARP1 **.**

Stephen J. Pettitt,1 Dragomir B. Krastev,1 Inger Brandsma,1 Amy Dréan,1 Feifei Song,1 Maria I. Harrell,2 Rachel Brough,1 Jessica Frankum,1 Jung-Min Lee,3 Elizabeth M. Swisher,2 Kosuke Yusa,4 Alan Ashworth,5 Christopher J. Lord1. 1 _Institute of Cancer Research, London, United Kingdom;_ 2 _University of Washington, Seattle, WA;_ 3 _National Cancer Institute, Bethesda, MD;_ 4 _Wellcome Trust Sanger Institute, Hinxton, United Kingdom;_ 5 _UCSF, San Francisco, CA_.

To investigate mechanisms of PARP inhibitor (PARPi) cytotoxicity, we carried out genome wide CRISPR-Cas9 screens for mutants resistant to the potent PARPi talazoparib (BMN 673) in mouse ES cells and human breast cancer cells. Many talazoparib resistant clones had loss of function Parp1 mutations as expected from to the proposed role of trapped PARP1 in cytotoxicity. However, we also isolated a point mutation affecting a single amino acid in the Parp1 DNA binding domain. This mutant encodes a stable Parp1 protein that cannot bind DNA and does not become trapped in the presence of inhibitors. Thus the CRISPR screen implicated PARP1 DNA binding directly in determination of PARPi cytotoxicity and was extremely informative about the mechanism of action compared to conventional loss-of-function mutagenesis.

We extended this approach by synthesising a high-density focused sgRNA library targeting only PARP1. We developed a reporter cell line that allows us to selectively isolate in-frame mutations that preserve PARP1 protein expression. By deep sequencing mutagenised and appropriately selected cells we identified a series of subtle mutations in PARP1 that result in PARP inhibitor resistance, giving us a detailed insight into structure-function relationships in PARP1. Among these, we found mutants that display trapping despite conferring PARPi resistance, suggesting that PARP trapping is not sufficient for cytotoxicity. Mutations that confer resistance are restricted to DNA binding domains and a network of residues in the WGR and helical domains of PARP1 that may be involved in intramolecular activation of PARP1 upon DNA binding and thus affect trapping.

We also established that PARP1 loss can be tolerated in some BRCA1 mutant breast and ovarian tumour cell lines, despite the well-described synthetic lethality of PARP1 and BRCA1 loss. Further investigation indicated that these cell lines that tolerate PARP1 loss of function mutations have exon 11 BRCA1 mutations, and may retain some function due to a splice variant that skips exon 11. siRNA knockdown of remaining BRCA1 function in SUM149 or COV362 cells with PARP1 mutations elicited the expected synthetic lethality. A PARP1 mutation identified in a PARPi resistant patient is localised to the WGR domain cluster identified in our screens and abolishes PARP1 trapping in a microirradiation assay. PARP1 mutation also caused talazoparib resistance in a SUM149 xenograft model. These results raise the possibility that PARP1 loss may be a clinical mechanism of PARP inhibitor resistance. Importantly, BRCA1 mutant cells with PARP1 mutations had distinct drug sensitivities to other known mechanisms of PARPi resistance - for example PARP1 mutation did not result in cross resistance to cisplatin or camptothecin, unlike BRCA1 reversion or 53BP1 loss. This suggests that studying the mechanism of acquired PARPi resistance may be important to inform subsequent therapy choice.

#411

Risk-dependent enhancer transcription defines oncogenic gene regulatory networks for high-risk neuroblastoma.

Zhezhen Wang, Ivan P. Moskowitz, John M. Cunningham, Xinan Holly Yang. _The University of Chicago, Chicago, IL_.

Neuroblastoma is the most frequent extracranial solid tumor in childhood. High-risk neuroblastoma (HR-NB) with less than 50% five-year event-free survival rates (EFSR) is distinct from non-high-risk neuroblastoma (NHR-NB) with above 90% EFSR. Investigating risk-dependence is therefore essential, and that of noncoding regulation is extra essential because to date only few coding mutations have been found in pediatric tumors. Among noncoding regulators, active enhancer plays the most crucial role to precisely control gene expression. However, given surrogate marks of enhancers, identifying functional enhancer is challenging. We have recently developed a noncoding transcriptome-based computational approach to define functional enhancers, given that some long noncoding RNAs (lncRNAs) transcribed from enhancers are required for enhancer activity. Here, we apply risk-dependence of lncRNA expression to identify oncogenic enhancers and enhancer-associated lncRNAs, determining an oncogenic regulatory network in HR-NB.

LncRNAs are often unstably expressed thus have a higher proportion of risk-dependence than coding genes. We re-analyzed the noncoding transcriptome of sequenced primary HR-NB (n=176) versus NHR-NB (n=322) samples (tumors cell content >60%) and identified 8.3k lncRNAs (3.4k up-regulated and 4.9k down-regulated) for the high-risk patients (FC>2, FDR<0.05). Similarly, from the coding transcriptome profiling of the same patients, we identified 694 up-regulated and 925 down-regulated genes.

Noncoding transcriptomic risk-group dependence is a robust metric for enhancer activity. Comparing risk-dependent distal lncRNAs with that of adjacent risk-dependent genes there exists a significant correlation in their direction and magnitude (coefficient=0.70, P<2e-16). Furthermore, risk-dependent lncRNAs reside at active enhancers specific to the neural tube (VISTA, 37 out of 257 validated enhancers, empirical P<0.005). Importantly, we observed three strong oncogenic associations for these 37 "risk-dependent and tissue-specific enhancers." 1) 70 human transcription factors (TFs) showed significant binding potency that indicates enhancer function, exampled by the tumorigenic FOXM1. These TFs significantly over-represent known oncogenes (FET P=0.01, odds=5) and tumor suppressors (P=0.0003, odds=7.5). 2) Risk-dependent target genes that remarkably over-represent the proliferation of neuroblastoma cell lines (P=0.0001, Ingenuity Pathway Analysis) indicate a directly tumorigenic impact by these risk-dependent enhancers. 3) Risk-dependent enhancer lncRNAs reside at neuroblastoma susceptibility loci more often than by chance.

In summary, risk-dependence of noncoding transcripts illuminate oncogenic enhancers with tissue-specific activity and neuroblastoma susceptibility, therefore is a general facet of oncogenic enhancer activity.

#412

8p12 amplification pattern dictates FGFR1 dependency in squamous cell lung cancer.

Florian Malchers, Martijn Henricus van Attekum, Martin Peifer, Roman Kurt Thomas. _University of Cologne, Cologne, Germany_.

Squamous cell lung cancer (SQLC) is the second most common lung cancer subtype after adenocarcinoma and accounts for 30% of all lung cancer cases. However, in contrast to adenocarcinoma, SQLC lacks therapeutic targets like activating EGFR mutations, ALK or ROS translocations. Besides immune-checkpoint-inhibition a promising treatment option in SQLC is the recurrent amplification of the tyrosine kinase fibroblast growth factor receptor 1 (FGFR1) within the 8p12-p11 region. Thus, small molecules inhibiting FGFRs have been employed to treat FGFR1-amplified SQLC. However, only about 10% of such FGFR1-amplified tumors respond to single agent inhibitor therapy. To investigate the mechanism of FGFR inhibitor resistance in 8p12-p11 amplified SQLC we performed deep genomic and transcriptome sequencing on 53 FGFR1 amplified samples including primary tumors (n=33), patient derived xenografts (n=13) and cell lines (n=7). For 22 of these samples the response to FGFR inhibition was known. We detected frequent breaks within NSD3 (n=3), also known as WHSC1L1, favoring the expression of NSD3-short, which is known to enhance MYC expression. Furthermore, the amplification pattern for all samples was compatible with a breakage-fusion-bridge (BFB) mechanism, showing chromosomal telomeric losses, copy number, and frequent intrachromosomal head to head and tail to tail breaks. Genomic reconstruction of one sample suggests a tandem duplication followed by a BFB mechanism, implying that the BFB mechanism is a later event in tumor genesis. Intrachromosomal tail to tail fusions within a 400kb region close to the FGFR1 open reading frame, have been detected in 75% of patients with a partial response to FGFR inhibitor therapy (3 of 4 patients). A similar break was detected in the FGFR inhibitor sensitive cell line H1581. All samples, which responded to FGFR inhibition (n=9), demonstrated a centered amplification pattern on NSD3 / FGFR1 and excluded amplification of the adjacent disintigrin and metalloproteinase family members (ADAM) genes. In contrast, the main amplification peak of the non-responding samples (n = 13) was centered on ADAM family members, corresponding to an increase in ADAM expression. These data suggest strong relevance of these genes for tumor development and growth, and warrant further investigation.

#413

Emerging principles in synthetic lethality in glioblastoma.

Pia Hoellerbauer, Sonali Arora, Megan Kufeld, Lucas Carter, Emily J. Girard, Heather Feldman, Philip Corrin, James M. Olson, Patrick J. Paddison. _Fred Hutchinson Cancer Research Ctr., Seattle, WA_.

Synthetic lethality occurs when mutations in two otherwise nonessential genes are combined to cause lethality. Because cancer is a disease of genetic alteration, synthetic lethality has been heralded as a method to identity candidate therapeutic targets, e.g., where a target gene could be "synthetic lethal" with a cancer driver mutation. To define synthetic lethal relationships in glioblastoma (GBM), we have performed multiple focused-set and genome-wide CRISPR-Cas9 lethality screens in patient-derived GBM stem-like cells (GSCs) and nontransformed human neural progenitor cells. Because GSCs isolates likely represent a sub-clone of the original tumor and we can determine GSCs' genetic and epigenetic makeup, it is possible to address the concept of synthetic lethality for GBM. To this end, we recently performed comprehensive CRISPR-Cas9 retests of all scoring GBM lethal genes (>900) from screens in three patient isolates with different and overlapping genetic drivers. We then performed secondary retests of high-priority gene targets in 13 GSC harboring various alterations commonly found in GBMs, e.g., EGFRamp, NF1mut, PIK3CAmut, PTENloss/mut, TP53mut, etc. The results were surprising, first in what we did not find. We failed to find synthetic lethal targets for TP53loss/mut, RB1mut, or TERT expression, suggesting that synthetic lethal relationships for these alterations may not exist for GBM. Second, NF1mut interactors defined a broader class of synthetic lethal targets with general overactivity of the RTK/Ras pathway, which can arise from various activating lesions. Third, candidate synthetic lethal relationships can be observed, but, so far, only with EGFRamp, MYC/MYCNamp, and PTEN/PIK3CA alterations. Thus, in general our results suggest that the majority of synthetic lethal relationships in GBM arise from oncogenic activation of the RTK/Ras and PI-3 kinase pathway or amplification of MYC/MYCN. (Synthetic lethal targets will be revealed and discussed at the meeting.)

#414

Broad range chromosomal abnormality detection through Bionano genome mapping.

Alex R. Hastie. _BioNano Genomics, San Diego, CA_.

Current methods for detection of balanced structural variation can be broken down into two categories: traditional cytogenetics and molecular methods. Cytogenetics may include chromosomal karyotyping, fluorescence in situ hybridization (FISH), chromosomal microarray and adaption of them. Molecular methods primarily include NGS sequencing based methods. Bionano genome mapping, an optical mapping approach in a NanoChannel array system, is a method that combines the advantages of different categories while solving many of the limitations. Compared to cytogenetics, Bionano mapping is high throughput and removes manual interpretation, it also has much higher resolution, detecting balanced events as small as about 30 kbp compared to multi-megabases needed for cytogenetic approaches, and unbalanced events starting at 1 kbp. NGS based methods often are limited by read lengths that cannot provide unambiguous information across repeat elements longer than individual reads. This limitation results in significantly reduced sensitivity for balanced variation and abnormalities as well as for insertions and even some categories of larger deletions. This is particularly true in highly medically relevant locations where segmental duplications mediate chromosomal abnormalities. Genome mapping using Bionano Genomics' Saphyr System offers high-throughput, genome-wide visualization of extremely long DNA molecules in their native form. It allows researchers to interrogate genomic structural variations (SVs) in the range of one kilobase pair and above. We present several in silico and biological validation experiments that demonstrate the sensitivity and specificity of Bionano mapping for the detection of insertions, deletions, inversions and translocations compared to benchmark studies using short read and long read sequencing. We also show a new direct labeling method that dramatically improves assembly contiguity such that chromosome arms can be assembled into single maps. Bionano mapping is a fast and cost effective method for the detection of a broad range of traditionally refractory SVs across the human genome.

#415

A single molecule detection system for comprehensive analysis of cancer related cellular pathways.

Manohar R. Furtado,1 Rixun Fang,1 Norman Burns,1 Rebekah Burich,2 Catherine Le,1 Soumya Ivaturi,1 Rob Hartlage,1 Ed Zizminskas,1 Jim Jahncke,1 Philip C. Mack,2 Bryan P. Staker1. 1 _Apton Biosystem Inc., Pleasanton, CA;_ 2 _UC Davis Comprehensive Cancer Center, Sacramento, CA_.

The purpose of this study was to evaluate the system capabilities of a new single-molecule detection platform capable of both genomic and proteomic analysis of cellular pathways using very small amounts of tumor material. The system has 4-color optics, single-fluorophore detection capability, localization of molecules to within a 20nm area, a flow cell with an area of 940 mm2 and the ability to detect > 109 molecules on the surface. NSCLC cell lines were cultured and untreated or treated with the EGFR tyrosine kinase inhibitor erlotinib. Cells were harvested and used to generate protein lysates, isolate mRNA and gDNA. Cellular molecules were covalently attached to an epoxysilane-coated surface on the flow cell and then repeatedly probed 10-15 times with labeled antibodies and oligonucleotides to detect, count and quantitate proteins, protein phosphorylation levels, mRNAs, fusion transcripts, and DNA mutations. We detected protein phosphorylation changes for EGFR, ERK, MET and MEK comparable to that observed by traditional western blots; however, the system required only 0.5 to 2 cell equivalents of protein lysate, containing sub pico-Molar levels of protein. We demonstrated robust and statistically significant detection of 2-fold changes in protein levels across 3 slides, in triplicate lanes and performed over 3 non-consecutive days by 3 operators (Tukey-Kramer tests). We also detected 8 mRNAs, with no PCR amplification required, in a multiplexed format with relative levels similar to that observed with TaqMan® qPCR tests. We demonstrated protein detection at ~ 100% recovery compared to an ELISA test and nucleic acid recovery at ~ 30% with no amplification. We also detected exon 19 deletions in EGFR, point mutations at L858R, T790M in EGFR and V600E in BRAF, at 0.5% minor allele levels, by using an oligonucleotide ligation assay off-chip and then attaching the ligated product to the surface. mRNA levels were detected using 10-20 cell equivalents of RNA. We believe the system will enable comprehensive analysis of cancer-related pathways, requiring only a few cells, to help decipher cellular pathway activity induced by driver mutations and consequently help with selection of efficacious drugs tailored for specific individuals based on comprehensive molecular analysis.

#415A

**Regulation of** NCAPG **by** miR-99a-3p **(passenger strand) inhibits cancer cell aggressiveness and is involved in CRPC.**

Takayuki Arai,1 Atsushi Okato,1 Yasutaka Yamada,1 Sho Sugawara,1 Akira Kurozumi,1 Satoko Kojima,2 Yukio Naya,2 Tomohiko Ichikawa,1 Naohiko Seki1. 1 _Graduate School of Medicine, Chiba University, Chiba, Japan;_ 2 _Teikyo University Chiba Medical Center, Ichihara, Japan_.

In developed countries, prostate cancer (PCa) is one of the most commonly diagnosed cancers, identified by prostate-specific antigen (PSA) screening; PCa is also the third leading cause of cancer-related death among men. Most naïve PCa initially responds well to androgen-deprivation therapy (ADT). However, during ADT treatment, PCa cells acquire ADT treatment resistance and progress to a lethal pathology known as castration-resistant prostate cancer (CRPC). Cancer cells that have reached CRPC can cause distant metastasis, and effective treatments for patients with CRPC have not yet been established. Identification of the molecular pathogenesis underlying acquisition of androgen-independent and metastatic signaling pathways based on advanced genomic approaches is essential for further understanding of this disease.MicroRNAs (miRNAs) are endogenous small RNA (molecules 18-23 bases in length) that act as central players regulating the expression control of protein-coding and protein-noncoding RNAs. Interestingly, a single miRNA can directly regulate a vast number of RNAs in human cells. Therefore, aberrant expression of miRNAs can disrupt normal control of RNA expression in cancer cells. Furthermore, dysregulation of miRNAs is contributed to cancer cell malignancies, such as progression, metastasis, and treatment resistance. Analyses of RNA-sequencing of microRNA (miRNA) expression revealed that miR-99a-3p (passenger strand) is significantly downregulated in several types of cancers. In this study, we aimed to identify novel miR-99a-3p regulatory networks and therapeutic targets for CRPC. The expression levels of miR-99a-3p was significantly lower in PCa and CRPC tissues than in normal tissues (P = 0.0047 and P = 0.0001). Ectopic expression of miR-99a-3p significantly inhibited cancer cell proliferation, migration, and invasion in PCa cells (P < 0.0001). Non-SMC condensin I complex subunit G (NCAPG) was a direct target of miR-99a-3p in PCa cells. Overexpression of NCAPG was detected in CRPC clinical specimens and TCGA data showed that high NCAPG expression was significantly associated with shorter disease-free survival (P = 0.0009) and advanced clinical stage in PCa. Knockdown of NCAPG markedly inhibited cancer cell proliferation, migration, and invasion (P < 0.0001). Moreover, the proliferation, migration, and invasion abilities were recovered by cotransfection with NCAPG and miR-99a-3p. These results indicated that NCAPG affected the aggressiveness in PCa. The passenger strand miR-99a-3p acted as an antitumor miRNA in naïve PCa and CRPC. NCAPG was regulated by miR-99a-3p, and its overexpression was involved in CRPC pathogenesis. Involvement of passenger strand of miRNA in cancer pathogenesis is novel concept and identification of antitumor miRNA regulatory networks in CRPC might be provided novel prognostic markers and therapeutic targets for this disease.

### High-Throughput Sequencing 1

#416

Impact of virus integration into the genomes of hepatocellular carcinoma patients with prior hepatitis B virus infection.

Yutaka Midorikawa,1 Kenji Tatsuno,2 Shogo Yamamoto,2 Genta Nagae,2 Kazuhiko Koike,2 Mitsuhiko Moriyama,1 Tadatoshi Takayama,1 Kyoji Moriya,2 Hiroyuki Aburatani2. 1 _Nihon Univ., Tokyo, Japan;_ 2 _Univ. of Tokyo, Tokyo, Japan_.

It has been elucidated that hepatitis B virus (HBV) DNA is frequently integrated to some cancer related genes such as TERT or KMT2B in patients with chronic HBV infection. On the other hand, it has not yet become clear of the integration of HBV to the genome of patients with prior infection with HBV. To identify the integration sites of HBV, we performed next generation of target capture technology for hepatocellular carcinoma samples obtained from 73 HBc antibody positive patients but negative for HBs antigen nor hepatitis C antibody (HCV) (Group 1; prior infection), 78 HBs antigen positive patients (Group 2; positive control), and 33 patients without any antigens nor antibodies for HBV (Group 3; negative control). Among these patients, overall HBV integration in Group 2 (54 patients, 69.2%) was significantly frequent compared with that in Group 1 (12, 16.4%) (P < 0.001), and was not detected in any patients of Group 3. On the other hand, HBV integration occurred in the known driver genes; TERT in seven (9.5%) and 12 (15.3%) patients (P = 0.332), MLL4 in two (3.5%) and four (5.1%) patients (P = 682), and CCNE1 in 0 and one (1.2%) patient (P = 1.000) in Group 1 and 2, respectively. Notably, integration of any driver genes was not observed in 56 patients with prior infection with HBV who were positive for HCV infection.Taken together, despite the disappearance of HBV, the frequency of driver events by HBV integration in patients with prior HBV infection is similar to those with chronic HBV infection, and patients with prior HBV infection are still at the risk of hepatocellular carcinoma.

#417

Comparative analysis of clinically validated NGS-based assays reveals high concordance across short variants.

Ninad P. Dewal,1 Joel Skoletsky,1 Wai-Ki Yip,1 Caitlin Patriquin,1 Yuting He,1 Jeffrey S. Ross,1 Vincent A. Miller,1 Philip J. Stephens,1 Christine Burns,1 Christine Vietz,1 Yali Li,1 Colin Pritchard,2 James X. Sun1. 1 _Foundation Medicine, Inc., Cambridge, MA;_ 2 _University of Washington, Seattle, WA_.

Background: Traditional FDA-approved assays employing PCR, FISH, or IHC report presence or absence of single markers for therapeutic guidance. Comprehensive Genomic Profiling (CGP) via hybrid-capture NGS-based assays, however, can simultaneously interrogate hundreds of markers to offer a broader picture for clinical actionability. To support the reliability of using hybrid-capture NGS as a clinical platform, we present a comparative analysis on the variant calls detected between two clinically validated assays - FoundationOne CDx (F1CDx), which is the first FDA-approved NGS-based platform that indicates personalized therapies; and a clinically-validated NGS tumor panel assay, performed in an experienced CLIA-certified and CAP-accredited lab in the academic setting. The assays capture 324 and 262 genes, respectively.

Methods: DNA from 188 clinical cases across an array of tumor types was selected to be sequenced by both assays, performed at the respective labs. For F1CDx, CGP was performed using a hybrid-capture, adaptor ligation-based NGS assay to a mean coverage depth of over 500X. Short variants - base substitutions, small insertions and small deletions - were identified and curated. A similar procedure was followed for the academic lab. Resulting variant calls were compared at FMI.

Results: Across the 157 genes that overlap between the assays, 550 clinically reportable short variants that met filtering criteria were identified. Of these, 491 were called by both, 15 were unique to the academic assay, and 44 to F1CDx. This resulted in a bi-directional agreement of ~94% (97.0% with the academic NGS assay as the reference method, and 91.0% using F1CDx as the reference method). The bi-directional agreement for base substitutions alone is (97.4% (discordances due to low allele fraction (AF)), 92.5%, respectively), while for indels in non-homopolymer regions is (96.1%, 89.7%, respectively). Negative agreement between the assays was greater than 99.8%.

Conclusions: Overall concordance among short variant calls between the two NGS assays is high, comparable to reproducibility results at low AF in qPCR. A further advantage over traditional assays is that variant calling is mostly automated but with some curation, whereas with use of IHC or FISH, the call is wholly dependent on pathologic review and interpretation. This supports the ability of robustly validated hybrid-capture based NGS to subsume sequential testing using other technologies commonly employed in oncology in the clinical diagnostics space.

#418

Highly efficient duplex DNA tagging strategy improves accuracy of detecting ultra-low-frequency mutations through consensus read reconstruction.

Jiashi Wang, Kevin Lai, Madelyn Light, Layla Katiraee, Kristina Giorda, Mirna Jarosz, Yun Bao, Criss Walworth, David Kupec, Caifu Chen. _Integrated DNA Technologies, Redwood City, CA_.

Introduction: Molecular diagnostics and precise personalized care continue to increase the sensitivity and specificity requirements for detecting low (~5%) to ultra-low (<1%) frequency actionable mutations by next generation sequencing (NGS). These low-frequency variants often occur below the reliable limit of detection of standard NGS as they are confounded by errors introduced during the NGS workflow. We have developed adapters containing unique molecular identifiers (UMIs) that permit tagging of double-stranded DNA and statistical reconstruction of reads sequenced as duplicates. These novel adapters are compatible with standard library preparation and enrichment methods and reagents, yet provide significantly enhanced error correction. Methods: Libraries were prepared from the 3 most commonly used oncology sample types: genomic, FFPE, or plasma-derived, cell-free DNA, using a standard, commercially available kit combined with standard adapters or the novel duplex UMI adapters. The libraries were enriched with a custom xGen Lockdown Panel targeting a 75 kb polymorphic region, and then deep sequencing and variant calling were performed. A consensus read building tool was developed to collapse PCR duplicates based on molecular barcodes, and the tool was used to evaluate the utility of UMIs in error correction. Results: Compared to standard adapters, the duplex adapters presented comparable or better library yield and mean deduplicated sequencing depth for all sample types and input masses tested. To evaluate variant-calling accuracy, we established mixtures of DNA of known SNP genotype to mimic ultra-low-frequency variant samples. For genomic DNA samples, DNA from NA12878 and NA24385 were mixed to generate minor allele frequency (MAF) down to 0.1%, and variant calls were evaluated against annotations in Genome in a Bottle. Commercial FFPE and cell-free DNA samples were genotyped and mixed to present MAF at 0.5%. When standard adapters were used, variants present at the MAF were detected with 90% sensitivity for all sample types, but only under conditions that also called >4,000 false positives, resulting in a positive predictive value (PPV) of <3%. Using the duplex UMI adapters in conjunction with consensus read construction, >90% detection sensitivity was achieved for genomic and cell-free DNA samples with 0 false positives, resulting in 100% PPV, a >40-fold error suppression. For FFPE DNA samples, the duplex adapters provided >30-fold PPV improvement compared to standard adapters. Conclusions: The duplex UMI adapters eliminate background NGS errors by collapsing duplicated reads using their unique tags. This leads to unprecedented accuracy in detecting true low-frequency variants, regardless of the input DNA source. Such advances are key to refining diagnostics and improving precision cancer care.

#419

Reproducibility assessment of mutations calls in exome- and whole-genome sequencing using consensus calling from TCGA and ICGC.

Matthew H. Bailey,1 Liang-Bo Wang,1 Wen-Wei Liang,1 Steven Foltz,1 Guanlan Dong,1 Michael C. Wendl,1 Michael McLellan,1 Angela C. Hirbe,1 Jared Simpson,2 Mark Gerstein,3 Li Ding1. 1 _Washington University in St. Louis, St. Louis, MO;_ 2 _Ontario Institute of Cancer Research, Ontario, Ontario, Canada;_ 3 _Yale University, New Haven, CT_.

Two large cancer genomic consortia recently published the largest and highest-quality consensus mutations calls for both whole-exome sequencing (WES) and whole-genome sequencing (WGS) in cancer: The Cancer Genome Atlas (TCGA), and the International Cancer Genetics Consortia (ICGC), respectively. Together these datasets encompass more than 60M mutations from ~13,000 samples (~10,000 WES and ~3,000 WGS). An intersecting set of 742 samples, from 22 cancer types, was sequenced using both platforms and mutations were identified using a combined 13 variant calling tools (7 WES and 5 WGS). These samples represent an ideal dataset to compare and contrast WES with WGS performance, reliability, and reproducibility of mutation calling in exons, and provide the community with key regions flanking exons that play a role in carcinogenesis. MAF files were collected using strict filtering criteria for initial file release, including the elimination of germline contaminants, 8-oxo-guanine artifacts, depth filtering and repeat masking. Additional filtering included minimum coverage requirements and restriction of both WES and WGS to variants detected within targeted exons. Finally, we restricted our data to known cancer genes. This final step suggests that these 742 samples have anywhere between 11.5K to 12.3K mutations from covered exons in potential cancer driver genes—WES and WGS, respectively. Preliminary results found that ~70% of samples had had >80% congruent mutations between both platforms; ~25% of samples had had >80% congruent mutations calls in one or the other platform; and the remaining samples had poor performance in replicating identical mutations. We observed that a majority of the variants unique to a sequencing platform were primarily from mutations with low VAF. We also sought to explore regions of the genome that are captured by both technologies despite the knowledge that WES did not target these regions. This is made possible by obtaining access to the primary data resources, and relaxing filtering criteria to include other regions such as 3' and 5' UTR, exon flanking regions, and intronic regions. We identified many recurrent mutations from non-exonic regions that were corroborated using both platforms that have not been previously reported in pan-cancer efforts. At this historic junction in time, as preliminary results from whole-genome sequencing efforts emerge and large exome sequencing efforts taper, 742 samples spanning both efforts can provide insights into the lessons learned from exome sequencing, and provide a solid foundation stepping forward into whole-genome analysis. We will continue to glean insights into the etiology of human disease by using both technologies; however, these mutation calls highlight the challenges that still exist in somatic variant calling, and provide grounds for more critical evaluation of genomic findings in cancer.

#420

Targeted deep sequencing of mitochondrial DNA in epithelial ovarian carcinomas.

Nicholas B. Larson,1 Stacey J. Winham,1 Chen Wang,1 Jared M. Evans,1 Sebastian M. Armasu,1 Bryan M. McCauley,1 Sean J. Yoder,2 Lan Min Zhang,2 Catherine M. Phelan,2 Ellen L. Goode1. 1 _Mayo Clinic, Rochester, MN;_ 2 _Moffit Cancer Center, Tampa, FL_.

Introduction: Mitochondria are intracellular organelles that play an important role in energy metabolism, reactive oxygen species generation, and apoptosis. They possess their own haploid genome distinct from nuclear DNA, and mutations in mitochondrial DNA (mtDNA) have previously been linked to cancer risk, disease progression, and treatment response for multiple tumor types. However, the spectrum of mtDNA variation in epithelial ovarian carcinoma (EOC) and associations with clinical characteristics have not been fully explored.

Methods: We extracted mtDNA from fresh frozen EOC tumors of women diagnosed with primary invasive epithelial ovarian, fallopian tube, or peritoneal carcinoma, and performed targeted sequencing using the Illumina MiSeq Reporter mtDNA workflow. Homoplasmic and heteroplasmic SNVs and indels were called using an in-house bioinformatics pipeline. We estimated mtDNA haplogroup using Haplogrep and compared to results from available germline mtDNA SNP genotyping. We derived regional weighted variant burden scores based on mtDNA functional domains, accounting for underlying haplogroup, heteroplasmic fraction, and predicted functional impact. To assess potential differences in variant burden by histotype, we compared burden scores using Kruskal-Wallis testing. Among high-grade serous tubo-ovarian cases, we explored associations with overall survival (OS) using Cox proportional hazards regression. After quality control filtering, 389 mitochondrial genomes were analyzed, including those from 320 tubo-ovarian high-grade serous tumors, 42 endometrioid tumors, and 18 clear cell tumors.

Results: Accounting for predicted haplogroup, we identified a median count of two global private heteroplasmies per tumor (range: 0-9). Elevated mutation rates were identified in the regulatory control region as well as among the various mt-tRNAs (>3 per 10kb per sample) relative to the remainder of the mtDNA. The majority of recurrent variants across tumors were also identified in the mtDNA control region. We did not observe an association with overall mtDNA variant burden and patient age at diagnosis. Regional mtDNA burden scores revealed a significant association with histotype (P<0.001) for the oxidative phosphorylation complex IV genes, with higher burden in tubo-ovarian high-grade serous and clear-cell tumors relative to endometrioid tumors. For patients with tubo-ovarian high-grade serous tumors, increased variant burden in MT-CO1 (cytochrome C oxidase I) was associated with improved OS (HR = 0.32; 95% CI = [0.14,0.70], LRT P = 0.001) after adjustment for age, stage, and debulking status.

Conclusions: We demonstrate a wide range of tumor mtDNA variant patterns in the largest known collection of EOC patients. This work provides the first extensive evaluation of mtDNA variation in these diseases and highlights the potential prognostic relevance of mitochondrial dysfunction.

#421

Improved detection and identification of microsatellite instability features in colorectal cancer: Implications for immunotherapy.

Giwon Shin, HoJoon Lee, Susan M. Grimes, Matthew A. Kubit, Hanlee P. Ji. _Stanford University, Stanford, CA_.

Colorectal cancers with loss of DNA mismatch repair display microsatellite instability (MSI). These tumors have a better prognosis and an improved response rate to immune check point therapies compared to non-MSI tumors. MSI-positive tumors demonstrate a surprising range of somatic alterations in repetitive sequence tracts. For example, elevated microsatellite alterations at selected tetranucleotide repeats (EMAST) that may be related to functional loss of MSH3. Current MSI tests are based on PCR and capillary electrophoresis. This conventional approach is suboptimal because i) only a small number of microsatellite markers are interrogated per assay, and ii) sensitivity is insufficient for low allelic fraction alleles in heterogeneous tumors. Next generation sequencing permits the inclusion of more microsatellite markers, but these NGS method still rely on PCR methods, which is the major source of artifacts that obscure the true somatic alterations.

In this study, we analyzed a series of colorectal cancers using a new sequencing technology that sequences in parallel more than 200 microsatellite regions - we evaluate mono-, di- , tri- and tetranucleotide repeats to provide a broader spectrum of MSI events. This deep sequencing technology provides greater than 2,000X coverage, uses in vitro CRISPR-Cas9 segmentation, and eliminates the need for PCR amplification. Because every read corresponded to a single DNA molecule, we eliminated the artifacts introduced by PCR that obscured true MSI events. Moreover, we improved the sensitivity of detecting minor allele fractions to as low as 0.1% by measuring microsatellite haplotypes. Included in our analysis was new method for somatic copy number changes in 82 cancer genes, by which we can infer chromosomal instability (CIN) status.

Among our cases, 10 cancers had MSI when considering both mononucleotide and tetranucleotide repeats. Interestingly, diagnostic testing of two of these tumors were false negatives and were demonstrated to be MSI-high. There were three major types among the 10 MSI cancers: MSI-high, EMAST, and the cancers having both. However, such types were distinguishable only by microsatellites with intermediate length (10 - 20 bp). MSI-high tumors (n = 3) showed no sign of CIN while all the EMAST tumors (n = 4) also showed CIN phenotype. Our results suggest that the current gold standard MSI diagnostic test may have issues with both false negatives and false positives, indicating that NGS approaches are better suited for evaluating MSI status of tumors and consideration of immunotherapy.

#422

Single-stranded library preparation enables unbiased sequencing of damaged, FFPE-derived DNA for both targeted gene capture and WGS applications.

Anna Vilborg. _TOMA Biosciences, Foster City, CA_.

Next Generation Sequencing is increasingly used in translational cancer research and as a diagnostic test. Most tumor specimens available for testing are formalin-fixed, paraffin-embedded (FFPE) blocks. FFPE-derived DNA is typically damaged, causing difficulties for standard library preparation methods. Frayed ends prevent blunt-ended double-stranded adapter ligation, used for whole genome sequencing (WGS) and targeted bait hybridization library preparation. Fragmented DNA and modified bases interfere with PCR, which underlies amplification based targeted sequencing.

We present a single-stranded library preparation method especially forgiving for low quality FFPE-derived DNA. By excising damaged bases and using single-stranded adapter ligation, we omit repair aimed at generating blunt-ended double-stranded DNA, and eliminate the need for whole genome PCR. After ligation of the first adapter, libraries can either undergo second adapter ligation to generate WGS libraries, or be used as input to oligo-selective sequencing (OS-Seq) target capture.

We show that our FFPE-derived PCR-free WGS libraries exhibit coverage depth and uniformity highly similar to double-stranded PCR-free libraries of matched cell line (non-FFPE) materials. Additionally, we present performance of our WGS library preparation on clinical FFEP samples.

We further demonstrate reproducible high and uniform coverage for reference standards and clinical FFPE samples in targeted capture. We show a lack of correlation between DNA quality and sequencing metrics such as on-target rate (R2=0.005), uniformity (R2=0.0002), and coverage (R2=0.06), with successful removal of C>T FFPE artifacts comparable to standard repair methods. We demonstrate sensitive copy number alteration detection through analysis of coverage depth, feasible due to the absence of pre-capture PCR.

In conclusion, our single-stranded library preparation method is forgiving for sample quality and provides uniform and high coverage as the basis for somatic variant calling.

#423

Investigating the germline deleterious rare variants for genetic susceptibility of esophagus squamous cell carcinomas by target sequencing.

Lvwen Ning,1 Josephine Mun Yee Ko,1 Lisa Chan Lei,1 Li Dong Wang,2 Maria Li Lung1. 1 _The University of Hong Kong, Pokfulam, Hong Kong, Hong Kong;_ 2 _Zhengzhou University, Henan, China_.

Purpose:

The genetic basis of esophageal squamous cell carcinoma (ESCC) still remains unclear. Several genome-wide association studies identified some common variants associated with small effects. Here we aimed to identify some rare deleterious variants in cancer predisposition genes, which may contribute to the genetic susceptibility of ESCC with a large effect, using family history-positive (FH+) samples from the Mainland China Henan high-risk region by targeted sequencing.

Methods:

A two-phase study was conducted, the discovery phase and the validation phase. In the discovery phase, we sequenced the blood DNAs of 214 independent family history ESCC cases and 123 non-ESCC hospital controls by Illumina HiSeq platform using a custom capture kit. The capture kit included more than 1000 genes, which were cancer-related genes, DNA repair genes, and other genes reported in some ESCC functional studies. For each gene, the exonic region plus 3'-UTR, 5'-UTR, upstream and downstream regions were designed for targeted capture with a total capture size of ~11 Mb. The sequencing reads were aligned to the human reference genome (GRCh37/hg19) using burrows-wheeler aligner (bwa). PCR duplication was marked by Picards. Local realignment was performed with genome analysis toolkit (GAKT). Annovar was used to do the annotation. Loss-of-function (LoF) variants, including splicing, stopgain, and frameshift InDel variants in the cases and controls were identified. The damaging effect of the missense variants was predicted by the combination of 4 different bioinformatics tools (CADD score, MutationTaster, SIFT, Polyphen2). We also used the 1000genomes or ExAC dataset as public controls. All the identified LoF variants in BRCA2 were validated by Sanger sequencing.

Results:

There was a total of 110 LoF genes identified. Pathway enrichment analysis of these genes found that they were enriched in the Fanconi Anemia (FA) pathway after the background correction of all captured genes. BRCA2 is in the FA pathway and was found to have the most LoF variants; this included 6 individuals from the 214 FH+ cases, and only 1 in the control cohort. We did not find many somatic mutations in ESCC tissues in BRCA2. BRCA2 expression was also significantly upregulated in ESCC tumor tissues, as compared to adjacent normal tissues from in-house and 3 other publicly available RNA-seq datasets. The variant spectra of candidate genes were compared with the public controls to filter out some genes.

Conclusions:

In the discovery study, we identified some LoF variants in BRCA2 and other genes in the FA pathway that may contribute to ESCC genetic susceptibility. Further validation with a larger sample size is now underway.

Acknowledgements: This work was supported by the Research Grants Council Collaborative Research Fund grant C7031-15G and the Asian Fund for Cancer Research to MLL.

#424

Defining prostate cancer subtypes by genomic features associated with defects in DNA repair processes.

Navonil De Sarkar, Emily Kohlbrenner, Ilsa Coleman, Peter S. Nelson. _Fred Hutchinson Cancer Research Ctr., Seattle, WA_.

Introduction: Prostate cancer (PC) exhibits a spectrum of genomic aberrations, a subset of which associate with responses to specific therapeutics. In particular, mutations in genes involved in DNA repair processes expose therapeutic vulnerabilities that include PARP inhibitors and DNA-damaging chemotherapy for homology-directed repair(HR) defects and immune checkpoint blockade for mismatch repair (MMR) defects. Deploying appropriate therapy is dependent on accurate tumor classification.

Experimental Procedures: We evaluated whole-exome sequences derived from localized (n = 465) and metastatic (n = 208) PCs to identify mutations and structural aberrations predicted to disrupt or alter the function of genes involved in DNA repair processes. We annotated all tumors for trinucleotide (COSMIC) mutation signatures by comparing somatic alterations in tumor versus paired normal exomes. We compared the type and frequency of presumed pathogenic mutations, tumor ploidy, copy number aberrations, and mutational signatures between primary and metastatic tumors and evaluated associations between specific DNA repair gene aberrations and mutation signatures.

Results: Overall, 54% of localized PCs and 24.5% of metastatic PCs had a component of their somatic mutation burden associated with a mutation signature indicative of a DNA repair defect. Metastatic PCs had significantly higher numbers of somatic mutations (n=223) compared to primary PCs (n=93) (p<0.008). Aneuploid and hypermutated tumors are also more common in metastatic PCs compared to primary PCs. Tumors with high LOH (>0.1) scores were enriched for pathogenic mutations in BRCA1/2 and lowest LOH scores were observed in tumors with mutation/loss of CDK12, which were near-diploid. In addition to relatively frequent aberrations in BRCA2, BRCA1 and ATM, we found CDK12 mutations in ~1% of primary PCs and 5.3% of metastatic PCs. Tumors with BRCA1/2 loss or ATM and RecQ-Helicase pathway aberration exhibited larger amplifications and loss events, whereas PCs with CDK12 aberrations demonstrated a high number (n>73 to 261) of focal (500b-1mb range) amplifications with rare focal losses.

Conclusions: 102 individual metastatic cases with some fraction of HR-defect signature and 41 (~40%) of them are without any bona fide HR pathway mutations. DNA repair gene (DRG) aberrations are common in metastatic PC and associate with distinct mutational signatures and structural aberrations that partition PCs into distinct subtypes. Epigenetic processes may contribute to HR signatures in tumors where DRG mutations were not identified. Preclinical and clinical studies are required to determine if mutation signatures are accurate predictors of responses to therapeutics. Thus, with these signatures, we have proposed a few molecular subcategories of prostate tumors and such information can be harnessed for future precision cancer therapies.

#425

Linked Target Capture: Rapid and high-performance NGS target enrichment for clinical sequencing applications.

Joel Pel,1 Wendy Choi,1 Milenko Despotovic,1 Laura Gelinas,1 Amy Leung,1 Lloyd Ung,1 Andre Marziali2. 1 _Boreal Genomics, Vancouver, British Columbia, Canada;_ 2 _Univ. of British Columbia, Vancouver, British Columbia, Canada_.

With the continued decrease of DNA sequencing costs, targeted next-generation sequencing (NGS) is seeing broad adoption in many research, commercial and clinical settings. However, current target capture methods typically have long, multiday workflows that can limit their clinical utility. In addition, small panels for applications such as liquid biopsy typically have high off-target rates or low uniformity, resulting in an increased cost of sequencing required to reach the desired coverage for variant detection. We have developed a novel library preparation and enrichment method named "Linked Target Capture" that replaces typical multiday "PCR-capture-PCR" workflows with a single, linked "PCR/capture" step. This approach, which uses physically linked capture probes and universal PCR primers, enables users to work from extracted DNA to loaded sequencer in less than a single day with minimal hands-on time. The linked PCR and capture step is cycled multiple times to reduce off-target DNA and increase uniformity, ultimately reducing the cost of sequencing. Additionally, the universal primers and captures probes can be combined to enable detection of DNA fusions with unknown partners. Using this method, we evaluated the ability to detect multiple mutation types from both tissue and cell free DNA, as a function of sequencing usage. Unique Molecular Identifiers (UMIs) were integrated and enabled the detection of variants below 0.1%, represented on both senses of the starting DNA (duplex sequencing). Fusion detection was also demonstrated without a priori knowledge of the fusion partner. Using a 30-gene pan-cancer panel, on-target fraction was measured to be above 95%, and the fraction of the panel covered at greater than 25% of the mean depth was greater than 99%. In summary, we have developed a method that enables rapid and cost-effective detection of clinically relevant cancer variants, such as SNVs and gene fusions. This method holds promise for many NGS areas, including clinical applications requiring rapid and simple workflows, or groups new to NGS where simple workflows and repeatable performance help remove barriers to technology adoption.

#426

Variant allele frequency changes and treatment responses in advanced prostate cancer.

Panagiotis J. Vlachostergios, Ana M. Molina, David M. Nanus, Himisha Beltran, Scott T. Tagawa. _Weill Cornell Medical College and Meyer Cancer Center, New York, NY_.

Introduction: Serial monitoring of genomic alterations in tumors is gaining interest as a research tool to better understand the biology of disease progression in different tumor types, including prostate cancer (PC). We hypothesized that intra-patient enrichment of mutations shared among different tumor samples from patients with advanced PC (castration-resistant or neuroendocrine) may parallel their clinical behavior in response to systemic therapy.

Methods: We examined patients with PC enrolled in our prospective Precision Medicine cohort, with at least 2 consecutive tissue or/and liquid biopsies available. By whole exome sequencing (WES) we assessed the variant allele frequencies (VAF) in gene alterations which were shared between spatiotemporally different samples from the same patients. We investigated associations between VAF changes and radiologic responses using the Chi-square test.

Results: Six patients with paired WES from samples 1-15 months apart with advanced PC were studied. Distribution of VAF in shared gene alterations and treatment outcomes (response vs. progression of disease) are presented in Table 1. In progressing patients (N=5), 20/25 (80%) of VAFs of shared genes were increased while 5/25 (20%) were reduced. In the one responding patient, 6/6 (100%) VAF of shared genes were decreased between samples. A significant association was found between VAF and radiologic responses (p = 0.0006).

Conclusions: Serial monitoring of VAF of shared genes between spatiotemporally distinct tumors within the same patient may assist in predicting radiologic responses to systemic therapies in advanced PC. Testing of these hypothesis-generating analysis results in larger, prospective studies is suggested.

Table 1. | |  | |  | |

|

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

|

Patient 1 | Patient 2 | Patient 3 | Patient 4 | Patient 5 | Patient 6

Sample A | site | bone | bone | prostate | lymph node | lymph node | prostate

|

Gene – VAF (%) | APC: 45 | GRIN3B: 20 | TP53: 26.8 | TP53: 75.8 | TP53: 71.4 | TP53: 86.7

|  | CAPN9: 51.6 | BRCA2: 38.1 | BRCA2: 31.2 | CSMD1: 67.1 | APC: 73.7 | TRIM27: 39.2

|  | RCOR3: 57.1 | ZFHX3: 29.8 | ANK2: 39.1 | ARHGAP4: 88.6 | |

AICF: 65.4

|  | ARHGAP36: 49.2 | DNA2: 14.4 | CPS1: 100 | DUSP12: 51.3 | |

SCN3A: 62.8

|  | LAMA4: 36 | PDL1M1: 19.4 | |

ZNF335: 65.8 | |

SLCO3A1: 76.7

|  | RPTN: 27.5 | LRIG3: 19.8 | |

SEC14L1: 75 | |

SIRT2: 53,4

|  | ROR2: 31.1 | |  | |

|

Sample B | site | bone | bone | lymph node | liver | blood | liver

|

Gene – VAF (%) | APC: 75 | GRIN3B: 58.7 | TP53: 44.7 | TP53: 88.7 | TP53: 6.4% | TP53: 60.6

|  | CAPN9: 89.4 | ZFHX3: 85 | BRCA2: 40 | CSMD1: 73.3 | APC: 0.8% | TRIM27: 36.1

|  | RCOR3: 64 | DNA2: 50 | ANK2: 85.7 | ARHGAP4: 92.7 | |

AICF: 51.9

|  | ARHGAP36: 88.2 | PDL1M1: 53.7 | CPS1: 98.4 | DUSP12: 58.8 | |

SCN3A: 41

|  | LAMA4: 66.7 | LRIG3: 35.5 | |

ZNF335: 53.1 | |

SLCO3A1: 27.9

|  | RPTN: 58.2 | |  | SEC14L1: 67.1 | |

SIRT2: 35.7

|  | ROR2: 52.5 | |  | |

|

Sample C | site | |

blood | |  | |

|

Gene – VAF (%) | |

BRCA2: 71.6 | |  | |

Treatment | Leuprolide | Enzalutamide | Cisplatin/

Docetaxel, Etoposide | Enzalutamide | Docetaxel | Carboplatin/

Etoposide

Radiologic assessment | progression | progression | progression | progression | progression | response

#427

High-quality whole-genome sequencing of FFPE samples.

Shannon T. Bailey, Jim Lund, Hao Wang, Weiren Cui, Chen Hao, Hongye Sun, Jeffrey R. Gulcher. _WuXi NextCODE Genomics, Cambridge, MA_.

Next-generation sequencing analysis of formalin-fixed, paraffin-embedded (FFPE) samples has the potential to lead to major advances in cancer treatment and prevention. However, whole-genome sequencing of these samples has remained challenging due to the difficult process of isolating high-quality DNA and distinguishing true variant calls from artifacts. Previous studies have attempted to overcome these challenges by using whole-exome sequencing or smaller exon panels at high coverage depths to analyze FFPE samples. While these approaches have produced useful results, they have had varying degrees of success, tend have coverage biases due to probe capture issues, and are limited to only a fraction of the genome. Thus, there has not been widespread adoption of using FFPE samples for next-generation sequencing.

In an attempt to improve on the process of next-generation sequencing of FFPE samples, we first developed an efficient DNA extraction method that produces high quantities of high-quality DNA. Whole-genome sequencing of DNA generated by this method demonstrated uniform high coverage that was comparable to DNA prepared from fresh frozen samples at the same sequencing depth. This result is in contrast with data generated from DNA isolated by a traditional FFPE extraction method, which had lesser sequencing coverage even with twice the sequencing depth. Further analysis of the data generated using our method demonstrated similar numbers of mutations called and mutational signatures without artifact contamination.

We extended this analysis to a large cancer cohort consisting of over 1,000 FFPE samples in tumor normal pairs that were up to 6 years old. Whole-genome sequencing of these samples demonstrate that there was an average of 98% of the genome covered at 20X or greater using our method with no drop off for the older samples. Mutational analyses of these samples demonstrate similar findings as that previously published using TCGA datasets.

In summary, our study of greater than 1,000 samples is the largest report of whole-genome sequencing of FFPE samples to date. Our results demonstrate the feasibility of using our method to assay tissues stored in archival tissue biobanks, analyze patient samples for clinical trial recruitment, or examining sections as part of normal clinical workflows, all of which hold the potential for greatly benefiting human health.

#429

Optimization of an RNA sequencing method for low-quantity degraded samples.

Dan Su, Jason G. Powers, Thomas A. Halsey, Patrick Hurban. _Q2 Solutions- EA Genomics, Morrisville, NC_.

RNA-sequencing (RNA-seq) is an effective tool for gene expression analysis, promising to be a diagnostic tool for patient stratification and individualized therapy. However, it has been challenging to apply RNA-seq to low-quantity and degraded RNA derived from formalin fixed paraffin embedded clinical samples. The Illumina TruSeq® RNA Access approach, utilizing capture probes targeting known exons to enrich for coding RNAs, has shown high performance to profile poor quality RNA samples at input amounts at or above 20 ng. Here, we have further optimized the Illumina TruSeq® RNA Access method for samples with low quality and low quantity, and have compared results to those obtained with libraries prepared with SMARTer® Stranded Total RNA-Seq- Pico Input Kit. Libraries were prepared using 1 ng and 10 ng of degraded RNA from Banked FFPE liver specimens, and were compared to libraries prepared using the standard RNA Access method with an input of 100 ng. With the optimized low-input RNA Access method, the alignment rate was consistently high (95-97%) across both input amounts, with 82-84% of aligned reads mapped to the transcriptome. This is comparable to results obtained with the standard 100 ng input. Gene detection however, changed as a function of input, with ~ 15,900 genes detected at an input of 10 ng, and ~ 13,500 detected at 1 ng of input. In contrast, the SMARTer method detected a relatively constant number of genes (~ 14,500 genes) at both input amounts, albeit with a lower % aligned reads to transcriptome (46-47%), and an increased % reads aligned to Ribosomal RNA when compared to RNA Access. Thus, while both methods are capable of comparable levels of performance, the RNA Access method is more cost-effective from a sequencing standpoint. These characteristics were maintained across a group of additional FFPE-derived samples tested by both methods.

Taken together, these results demonstrate that an optimized RNA Access method has consistent mapping performance and a high gene detection rate, and is thus suitable for the analysis of low-quantity degraded RNA.

#430

Detection of low-frequency variants from highly degraded DNA samples using a novel library preparation method.

Ushati Das Chakravarty,1 Zac Zwirko,1 Yu Zheng,1 Madelyn Light,1 Kevin Lai,1 Keith Bryan,2 Scott Rose,2 Yun Bao,1 Mirna Jarosz,1 Caifu Chen1. 1 _Integrated DNA Technologies, Inc., Redwood City, CA;_ 2 _Integrated DNA Technologies, Inc., Coralville, IA_.

Diagnostic tools based on next generation sequencing are fundamentally transforming clinical oncology. However, there is a lack of adequate library preparation strategies for highly degraded, clinically relevant samples, such as cell-free DNA (cfDNA) and FFPE DNA. Furthermore, clinical samples are often available in limited quantity, making the detection of pathologic variants particularly challenging. Due to the extreme heterogeneity of these sample types, targeted sequencing is often used to achieve deep coverage of genomic loci and enable detection of low-frequency variants. Commercially available protocols for library preparation require stringent size-selection to remove adapter-dimers, which reduces library complexity. Achieving high specificity can be challenging because low-frequency artifacts arise from a variety of sources, including DNA extraction, library construction, PCR, hybrid selection, and sequencing. These artifacts can be identified by "duplex sequencing", where strand-specific unique molecular identifiers (UMIs) are used to confirm the presence of an alteration on both strands of an input molecule. However, duplex sequencing typically delivers low conversion rates with degraded samples due to poor ligation efficiency and template loss during size-selection. We present a novel library construction chemistry that eliminates adapter dimers and utilizes duplexed UMIs to increase sensitivity. The method employs a two-step ligation procedure that does not require size-selection or adjustment of AMPure bead ratios during cleanup. We achieve maximal library conversion using a unique, mutant DNA ligase and proprietary sequencing adapters that increase ligation efficiency and suppress chimera formation. We demonstrate performance using three sample types: sheared genomic DNA, cfDNA, and FFPE DNA, across a wide input range (1-1000 ng). To mimic performance with degraded DNA, we created libraries from mixtures of NA12878 and NA24385 cell line DNA sheared to 150 bp with inputs of 1-25 ng and mutant allele fractions (MAFs) down to 0.25%. Libraries were enriched using a 75 kb panel of xGen Lockdown Probes, followed by ultra-deep sequencing and variant calling. When compared to commercially available methods, our approach yielded a 50-100% increase in library complexity with significantly improved sensitivity to <1% variants. We also obtained 100% specificity using duplexed UMI correction, which removed all false-positive calls. To highlight clinical utility, we extended our study to cfDNA samples with inputs of 5-25 ng and MAFs down to 1%, and FFPE DNA samples with inputs of 25-100 ng and MAFs down to 1%.

#431

Mitochondrial-nuclear crosstalk influences accumulation of mitochondrial DNA mutations in mammary tumor progression.

Takae M. Brewer, Amanda E. Brinker, Sharon Manley, Carolyn J. Vivian, Danny R. Welch. _University of Kansas Medical Center, Kansas City, KS_.

Introduction: Previous studies demonstrated that mitochondrial inheritance may contribute to aggressiveness of metastatic disease. Accumulating evidence suggests the mitochondrial genetic background may influence how certain cancers behave. We utilized Mitochondrial-Nuclear eXchange (MNX) female mice crossed with transgenic mice over-expressing the Her-2 gene and showed mitochondrial DNA (mtDNA)-dependent differences in tumor latency, lung metastasis number and lung metastasis size. We hypothesized that mtDNA mutations accumulate as mammary tumors progress and that the evolution is associated with mtDNA-nuclear DNA cross-talk. To test this hypothesis, we conducted next generation sequencing analyses to examine the spectra of mutations in the mitochondrial genome.

Methods: Normal mammary gland, primary tumor and lung metastases [n=5 each] were obtained from FVB/NJ mice with FVB/NJ (designated FF), C57BL/6J (designated FC) or BALB/cJ mtDNA (designated FB). Epithelial cells from mammary gland or tumor cells were carefully isolated by laser capture microdissection in order to minimize contamination from surrounding stromal cell mtDNA. mtDNA was deep sequenced using three pools totaling 182 overlapping primers spanning the whole mitochondrial genome using the Ion TorrentTM PGM System. Sequences were compared to an FVB mtDNA reference sequence to detect variants.

Results: Significant differences in the total number of mtDNA mutations were observed between the wild-type and MNX mice. Both FB and FC MNX cohorts exhibited increased mtDNA mutations compared to the wild-type (FF). As tumor progressed, the numbers of and distribution of mutations across the mitochondrial genome increased. 'Hotspots' were observed in FB mice (S12 rRNA, COX I, ND4, CYTB) that were distinct from common mutations in the FC mice (16S rRNA, COX I).

Discussion: As predicted, tumor cells accumulated more mutations in mtDNA as neoplastic cells from the primary tumor progressed to metastasis. Surprisingly, wild-type (FF) mice, even though more clinically aggressive (i.e., more metastases), accumulated fewer mtDNA mutations than tumors arising in the MNX mice. The mutations appear to occur in different sites, depending upon the nuclear-mitochondrial combination. Whether the mtDNA mutations function as contributors to metastatic efficiency has not yet been determined. Nonetheless, the data imply that nuclear-mitochondrial cross-talk influences mtDNA mutational spectra and metastasis and that defining the critical mtDNA genes most commonly involved may eventually be used to predict patient prognosis.

#432

Establishment of reference samples for the detection of somatic variants in cancer.

Li Tai Fang,1 Wenming Xiao,2 Somatic Mutation Working Group, SEQC2 Consortium. 1 _Roche Sequencing Solutions, Belmont, CA;_ 2 _FDA/NCTR, Jefferson, AR_.

The FDA is well aware of the crucial role that next generation sequencing (NGS) plays in precision medicine, and this technology is already leveraged in regulatory decision-making. However, lack of well-characterized and community-validated reference samples and data benchmarks creates a potential challenge for development and review of NGS applications.

To address this challenge, 60+ institutions and 160+ scientists have come together to form a somatic mutation working group within the FDA-led Sequencing Quality Control Phase II (SEQC2) consortium. We present genomic samples with high-confidence germline and somatic mutation data in order to establish a community standard against which new tools and platforms can be evaluated and benchmarked. Our initial reference samples are a triple-negative breast cancer cell line: HCC1395, and its B lymphocyte derived normal cell line: HCC1395BL. To minimize bias, we sequenced these two cell lines at up to 10 sequencing centers using multiple sequencing platforms. In totality, we have performed whole genome sequencing of the two cell lines to coverage depths of 2400X on Illumina HiSeq, 450X on NovaSeq, 600X on 10X Genomics and 50X on PacBio. Additionally, we performed whole exome sequencing at 1200X on HiSeq and 100X on Ion Torrent. Lastly, we sequenced (RNAseq) >5000 single cells for cellular heterogeneity studies. We applied machine learning algorithms to define high confidence mutation calls with datasets from 8 NGS platforms combined with 9 bioinformatics analysis pipelines. Each mutation in the "gold standard" dataset was annotated with confidence scores and supporting evidence. To independently evaluate accuracy of our call set, we selected a subset of mutation calls for additional validation using orthogonal methods, including ddPCR.

To summarize, we present the first release of high-confidence germline variants and somatic mutations on a pair of well characterized cell lines. We plan to continuously advance accuracy and completeness of our reference sample data, in order to serve as a standard for the community for years to come.

#433

Diagnostic targeted sequencing panel for hepatocellular carcinoma genomic screening.

Viola Paradiso,1 Andrea Garofoli,1 Nadia Tosti,1 Valeria Perrina,1 Manuela Lanzafame,1 Luca Quagliata,1 Matthias S. Matter,1 Stefan Wieland,2 Markus H. Heim,3 Salvatore Piscuoglio,1 Charlotte K. Ng,1 Luigi M. Terracciano1. 1 _Institute of Pathology, Basel, Switzerland;_ 2 _Department of Biomedicine, Basel, Switzerland;_ 3 _Department of Gastroenterology and Hepatology, Basel, Switzerland_.

Commercially available targeted or exome panels miss some genes and regions frequently mutated in hepatocellular carcinoma (HCC); we aim to design and benchmark an HCC-specific panel as a robust high-throughput and cost-effective panel for genomic screening in HCC.

We designed an Ion AmpliSeq panel targeting all exons of 33 liver cancer protein-coding genes, recurrently mutated long non-coding RNA genes MALAT1 and NEAT1, the recurrently mutated promoter regions of TERT, WDR74, MED16 and TFPI2, as well as mutation hotspots in 7 additional cancer genes. We profiled DNA from fresh frozen tumor (FFT, n=10, median depth 1495x) and/or formalin-fixed paraffin-embedded (FFPE) tumor with low input DNA (n=36, median depth 530x), and their non-tumoral counterparts from 39 HCCs using the custom panel. We benchmarked the somatic mutations and copy number alterations (CNAs) identified against those from Illumina whole-exome sequencing (WES) of the equivalent FFT samples (median depth 112x).

At least one somatic mutation was identified in 35/39 cases using the custom panel. Median of 2.5 (0-74) and 3 (0-76) mutations were identified in FFT and FFPE samples, respectively. 98% (61/62) of the mutations identified from WES of the 10 FFT samples were successfully recovered using the custom panel, with the identification of an additional 6 and 32 mutations in coding and non-coding regions, respectively. Similarly, all 104 mutations identified from WES were also found based on the analysis of our custom panel of the 36 FFPE biopsies. We identified an additional 18 and 70 somatic mutations in coding and non-coding genes, respectively, using our custom panel, including 2 CTNNB1 activating mutations. Furthermore, copy number analysis revealed high correlation (r = 0.8) in the identification of gains/ amplifications and losses/ homozygous deletions in the genes included in the custom-panel between our custom panel and the WES.

Here, we develop a robust high-throughput and cost-effective tool for HCC genomic screening that allows the identification with high sensitivity and specificity of somatic mutations as well as copy number alterations in routine diagnostic tissue specimens.

#434

100,000 Genomes Project: Cancer program.

Alona Sosinsky,1 Pavlos Antoniou,1 John Ambrose,1 Martina Mijuskovic,1 Antonio Rueda-Martin,1 Daniel Perez-Gil1,1 Enric Serra,1 Shirley Henderson,1 Angela Hamblin,1 Anna Schuh,2 Nirupa Murugaesu,1 Clare Turnbull,1 Augusto Rendon,1 Mark Caulfield1. 1 _Genomics England, London, United Kingdom;_ 2 _University of Oxford, Oxford, United Kingdom_.

The 100,000 Genomes Project aims to improve cancer care for patients in the UK's National Health Service by applying personalized medicine paradigms based on whole-genome sequencing (WGS). The program has been designed to enable return of WGS-based results to clinicians in a clinically meaningful timeframe in order to enable diagnosis and treatment decisions for patient.

Validation of WGS analysis results against standard-of-care tests: WGS analysis of tumors has a potential for replacing multiple standard-of-care tests, such as gene panels, genotyping arrays, FISH and karyotyping. For structural variants, WGS also provides much higher genomic resolution in comparison to FISH, the current standard of care. An important challenge when validating the analytical performance of WGS against existing technologies is to be able to systematically compare across a wide range of structural variant classes, low resolution events with WGS ascertained variants, which have often base pair resolution. We present here the results of successful validation for WGS analysis against targeted sequencing, genotyping array and FISH for a cohort of patients with chronic lymphocytic leukaemia (CLL).

Quality control of sequencing data: To ensure high sensitivity and specificity of reported variants we developed a protocol for DNA quality control based on PCA analysis of six metrics derived from sequencing data: AT and CG dropout, unevenness of local genome coverage, percentage of mapped reads, percentage of chimeric DNA fragments and average fragment size. For each new sample, these six metrics are projected into the transformed space and outliers are determined by comparing with a cohort of 672 fresh frozen samples. Samples having a p below 10-4 (p: probability density after multivariate normal fitting) are manually reviewed. This procedure allowed us to quickly spot the sets of samples that were extracted with the violation of DNA-extraction protocol and to prevent polluting results with high rates of false positive variant calls.

Reporting outcomes: Currently our bioinformatics analysis of WGS includes somatic small variants and somatic structural variants, germline pertinent findings, mutational signatures and mutational burden. Somatic small variants are linked to approved therapies, UK clinical trials and prognosis guidelines. We are working on implementing recently derived genomic signatures for predicting therapeutic response for specific tumor types. We are presenting here an overview of clinical utility for our reported outcomes. Bioinformatics reports for WGS with links to potentially relevant therapies and clinical trials have been already delivered for hundreds of cancer patients in the UK. We are collecting feedback from clinicians and working on improving our analyses to provide better benefits for cancer patients.

#435

Accurate measurement of tumor mutation burden through tumor-only sequencing using a 500-gene panel.

Alex Steven So, Shannon Kaplan, Chen Zhao, Shile Zhang, Li Liu, Phillip Le, Raakhee Vijayaraghavan, Tingting Jiang, Sven Bilke, Traci Pawlowski, Karen Gutekunst. _Illumina, Inc., San Diego, CA_.

Introduction: Recently, it has been shown by various studies that tumor mutation burden (TMB) can correlate with a cancer patient's responsiveness to checkpoint inhibitor immunotherapy. These findings have stimulated a widespread interest in the development of cost-effective assays that accurately measure TMB. Here we assess whether TMB measured through next-generation sequencing (NGS) of a 2-megabase 500-gene panel correlates with that determined through whole exome sequencing (WES). We further investigate whether sequencing tumor samples alone combined with a germline filtering and background noise removal algorithm can measure TMB accurately compared with sequencing of tumor-normal pairs.

Experimental Method: WES data of tumor and subject-matched normal DNA were either downloaded from The Cancer Genome Atlas (TCGA) or generated in-house. A 500-gene targeted DNA sequencing panel, which includes various cancer-relevant genes and covers two megabases, was also developed in-house. TMB was calculated as mutations (single nucleotide variants, insertions, and deletions) per megabase.

Data Summary: Using downloaded TCGA data from 2,385 subjects across colon, lung and melanoma cancer types, TMB was measured using tumor-normal WES or the in-silico-filtered tumor-only 500-gene panel and the two approaches correlated linearly (R2 = 0.96). Furthermore, we demonstrated that the targeted panel achieved >90% sensitivity and >85% specificity when classifying TMB-high and low tumor samples at the cutoff of 15 compared to the same TCGA data. TMB as determined from tumor-only WES was also analyzed using computational germline-filtering and background noise-removal to demonstrate excellent concordance with WES of tumor-normal pairs. To further validate these findings, by analyzing WES data and 500-gene panel targeted NGS data of >45 subjects generated in house. Through interrogating the 500-gene NGS data, we found that TMB determined using tumor-only sequencing was also similar from that ascertained through tumor-normal pairs. Of interest, further analysis showed that TMB determined from the 500-gene panel data using tumor-only sequencing along with germline filtering and background noise removing was comparable to that calculated from WES of tumor-normal pairs.

Conclusion: Collectively, our data demonstrates that TMB determined from NGS of a two megabase DNA region utilizing a 500 gene panel correlates strongly with TMB measured through WES, with a sensitivity of >90% and a specificity of >85%. In addition, sequencing tumor samples alone combined with bioinformatic germline-filtering and background noise-removal was similar to sequencing subject-matched normal DNA. These findings support the use of a 500 gene panel for accurate measurement of TMB while the addition of bioinformatics enables tumor-only sequencing.

#436

Comparative analysis of PCR-free and bead-linked transposome libraries for somatic mutation detection by whole genome sequencing.

Coralie Viollet, Xijun Zhang, Harvey B. Pollard, Matthew D. Wilkerson, Clifton L. Dalgard. _Uniformed Services University, Bethesda, MD_.

PCR-free DNA whole genome sequencing libraries are a standard in population-scale genomics, with high uniformity of coverage, reproducible addressable space and minimal amplification bias. However, high input requirements (>1µg) can be challenging for small volume biopsy, thin scroll, or low-weight tumor sources. Nextera DNA Flex is a newly developed, bead-linked transposome-based DNA library workflow that requires minimal amplification. It features a simplified single-tube technical process and could be an alternative for low input (~100ng) DNA samples.

Here, we assessed library and sequencing metrics for both the TruSeq DNA PCR-free and the Nextera DNA Flex workflows using a reference genomic DNA (NA12878), and human normal-tumor lung adenocarcinoma pairs. PCR-free and Flex libraries were profiled equally on the Illumina HiSeq X, either at >30x or >90x depth for germline and tumor, respectively. Read sequences were aligned to the human reference genome using the Isaac aligner. Genomic alterations were identified by Starling, Strelka, and Canvas algorithms.

The PCR-free workflow at 1µg input produced libraries with a >10nM yield and 550bp peak size, while the Flex workflow using a 10-fold reduced input of 100ng resulted in >25nM, 600bp libraries, with a wider fragment size distribution. Single lane output on HiSeq X yielded equivalent Picard coverage for both workflows (>30x). In contrast, tumor libraries generated 122x and 108x coverage for PCR-free and Flex workflows respectively. This 11% decrease in coverage was associated with a higher duplicate rate from smaller insert size observed in the Flex workflow. Furthermore, there was high variant (SNP and INDEL) concordance of >98% and >97% for germline and tumor, respectively. Additionally, CNV coverage was highly correlated between Flex and PCR-free samples (average r=0.952 per chromosome). Across tumor-normal pairs, somatic mutations were detected in both workflows with a >79% concordance using a 0.15 allele fraction cutoff. Moreover, somatic mutation calls are observed in PCR-free only and Flex only groups at equal ratios (~10% of total calls). In addition, tumor purity and mutant allele fractions between matched samples were in high agreement, providing another measure of genomic concordance between workflows. Finally, review of canonical mutations identified COSMIC annotated KRAS mutations in both workflows.

In summary, germline and somatic alterations identified by the Flex workflow achieved overall high agreement on matched assays of the PCR-free workflow. Nextera Flex may be a promising alternative for low-input tissue scenarios.

#437

Leveraging new methods in single-cell copy number analysis and clonotype detection to uncover and characterize hidden subclones within standard cell lines.

Enrique I. Velazquez Villarreal,1 Vijay Kumar,2 Yifeng Yin,2 John D. Carpten,1 David W. Craig1. 1 _USC, Los Angeles, CA;_ 2 _10x Genomics, Inc., Pleasanton, CA_.

Single-cell sequencing is an important and powerful tool for improving our understanding of cancer heterogeneity. While previous reports have largely focused on single-cell RNA-seq, we examined the utility of novel approaches for single-cell copy-number analysis and clonotype detection using 10x Chromium™ Technology capable of processing thousands of single cells. In contrast to bulk sequencing, this technology is able to call cell clonotypes down to 1% of 1,000 cell inputs, while providing detection of CNVs down to 100 Kb events on clones consisting of 10 or more cells. As a pilot study, we processed 300 single cells from COLO829 through the system, generating a barcoded short-read library, which was sequenced to a raw depth of approximately 600 million reads. Previous efforts had characterized COLO829 by bulk whole-genome sequencing of tumor/normal cell-line lineages grown and sequenced across four laboratories to help establish tumor/normal reference standards. We used barcode-aware bioinformatic analysis to recover reads derived from single cells, analyzing them in 20 Kb cassettes of reads providing both regional copy number and genome-wide ploidy estimates. This high resolution of CNV detection is enabled by aggregation of reads across cells within the same clone. Somewhat surprisingly, we find the presence of multiple clones within a single cell line growth evident by large-scale copy number changes, in some cases spanning entire chromosome arms. We show an ability to better resolve absolute copy number, whereas previous analysis approaches on bulk sequencing have lagged due to a reliance on indirect inferences. Consideration of prior publications on this cell line are consistent with the undescribed existence of clonal heterogeneity in these, and when considered with these data provide greater insight into copy number-driven events. Overall, our results suggest that single-cell copy number provides unique and important insight into larger-scale events, providing unique and distinct insight into cancer heterogeneity.

#438

High-quality CNV segments from low-coverage whole genome sequencing from FFPE cancer biopsies based on an evaluation of multiple CNV tools.

HoJoon Lee,1 Li Charlie Xia,1 Stephanie Greer,1 John Bell,1 Sue M. Grimes,1 Christina Wood Bouwens,1 Giwon Shin,1 Billy TC Lau,1 Lucas Johnson,1 Noemi Andor,1 Kenneth Day,2 Mickey Miller,2 Helaman Escobar,2 Lincoln Nadauld,2 Hanlee P. Ji,1 Paul Van Hummelen1. 1 _Stanford University, Palo Alto, CA;_ 2 _Intermountain Precision Genomics, St George, UT_.

Changes in DNA copy number, i.e., somatic CNVs, are common genetic aberrations in cancers. The effects of CNV include alteration in gene dosage across large segments of the cancer genome affecting the expression of cancer driver genes by amplifications, or cancer suppressor genes by deletions. In addition, CNVs are markers of underlying rearrangements within or between chromosomes and there is increasing evidence supporting a greater role for CNVs in developing and maintaining neoplastic cell population diversity.

Copy number aberrations can be estimated from next generation sequencing data, with high sensitivity and genomic resolution by sequencing the whole genome (WGS). For this study, we demonstrated that high quality CNV calls can be extracted in a fast and cost-effective way from low-coverage whole genome sequencing. Novaseq S2 flowcells (Illumina Inc) enables to obtain an average coverage of 3-4x per sample after pooling up to 96 samples per flowcell.

We examined three different copy number detection tools (CNVkit, BicSeq, and seqCBS) from paired tumor and normal WGS using microarray data as a reference. Pearson correlations were computed between the reference and CNVs from the WGS in two fashion; i) segment based and ii) gene based. The segment based comparison used sliding window of 100 K bp while gene based comparison used segments at the gene level.

We found high correlations between microarray and WGS segments. The highest correlations were obtained by CNVkit, ranging from 0.964 to 0.985 (SD: 0.973 - 0.007) and BicSeq, ranging from 0.963 to 0.986 (SD: 0.975 - 0.008). These results open the prospect of assessing large cancer cohorts of hundreds of samples at a reasonable cost. We are planning to apply this method to a large cohort of Stage III colon cancer patients and determine the clinical relevance of CNVs for survival.

#439

Elucidation of genetic susceptibility in nasopharyngeal carcinoma (NPC) by next-generation sequencing.

Wei Dai, Josephine Mun Yee Ko, Bianca Hoi Yan Ng, Hong Zheng, Maria Li Lung. _Univ. of Hong Kong, Hong Kong, Hong Kong_.

Background: NPC has a striking geographical distribution. Hong Kong is one of the endemic regions in Southern China with incidence of 20 per 100,000 in males, 10 times higher than the rest of the world. NPC diagnosis usually occurs 20 years earlier than the other common cancers. Family clustering is often observed in both high- and low-risk regions. The relative risk in first degree relatives of NPC patients is increased from 6-10-fold compared to the general population, suggesting the important role of genetics in NPC development. Previously, we applied a whole-exome sequencing (WES) approach to characterize the germline variants in NPC and identified a novel genetic susceptibility gene MST1R associated with NPC cases having an early onset. This study also discovered several potential candidates involved in DNA repair, immune response, and epigenetic regulation.

Aim: We aim to 1) validate MST1R as a genetic susceptibility gene and evaluate the clinical significance of its germline variants in association with survival and 2) to examine the association of the rare deleterious variants of fourteen candidate genes in a larger NPC case-control cohort.

Methods: We examined the germline variants in 1224 cases (FH: 271 and sporadic 953) and 1256 age/gender matched Hong Kong (HK) controls by the targeted sequencing approach using Roche NimbleGen SeqCap EZ custom platform.

Results: The MST1R rare deleterious variants were significantly associated with NPC, especially in the FH+ cases (OR=2.37, p=0.032). Combined analysis including both discovery (WES) and validation cohorts showed that the MST1R variants clustered at the Sema and tyrosine kinase (TK) domains were associated with NPC, respectively. The controls are intolerant to the rare deleterious variants in the TK domain, suggesting the more severe functional impact of those variants to its function. The sporadic cases with MST1R deleterious variants had disease progression (relapse or metastasis) much earlier than those without variants (n=756, HR=3.0, 95% CI 1.2-7.5, p=0.017). Among another fourteen candidates, multiple loss-of-function (LOF) germline mutations of a critical DNA repair gene, ATM, were identified in the FH+ cases, but not in any of sporadic cases and HK controls.

Conclusions: This study confirmed MST1R as a genetic susceptibility gene in NPC. It encodes macrophage-stimulating 1 receptor, which has an important function in innate immunity for maintaining the macrophage hemostasis. The function of this gene and impact of its germline variants in NPC development and progression are under investigation. Our study also highlights the role of a DNA repair gene, ATM, in NPC susceptibility.

Acknowledgements: NPC AoE funding was provided by the Hong Kong Research Grants Council (AoE/M-06/08) to MLL

#440

Single-cell whole-genome sequencing reveals somatic mutation signatures in normal somatic cells predictive of cancer later in life.

Xiao Dong, Lei Zhang, Moonsook Lee, Alexander Y. Maslov, Jan Vijg. _Albert Einstein College of Medicine, Bronx, NY_.

Single-cell sequencing for analyzing DNA mutations across the genome in somatic tissues is critically important for studying development, cancer and aging. However, current procedures are prone to artifacts and to date a reliable protocol for single-cell somatic mutation analysis remains to be developed. We address the two largest sources of artifacts, i.e., DNA denaturation-related cytosine deamination and allelic bias-driven whole genome amplification errors. We first reconfigured multiple displacement amplification (MDA) into an efficient protocol for whole genome amplification of single cells without cytosine deamination artifacts, i.e., Single Cell MDA (SCMDA). We then developed a new single-cell SNV caller (SCcaller) that distinguishes real somatic mutations and amplification errors by utilizing a SNP-based localized estimate of allelic amplification bias. The procedure was validated by comparing SCMDA-amplified single cells with unamplified clones derived from single cells from the same population.

Using this highly accurate single-cell whole-genome sequencing method we analyzed human B lymphocytes from donors varying in age from birth to over 100 year, studying both genome distribution and functional impact of base substitution mutations. Mutations per cell were found to increase with age from less than 500 in cord blood to over 3,000 in cells from individuals over 100. While overall mutations were randomly distributed across the genome with all chromosomes equally affected, 24 hotspot regions were identified, five of which were part of immunoglobulin variable regions subject to somatic hypermutation. Age-related mutation accumulation was found to be significantly slower in genomic sequences directly involved in cellular function, such as exons and gene regulatory sequences. Still, on average, B lymphocytes from aged individuals contained 3-15 damaging mutations in the transcribed part of the exome identified by RNA-seq, plus 15-50 mutations in transcription factor binding sites identified by ATAC-seq. Analysis of the spontaneous mutation spectra in these normal cells revealed signatures similar to those previously found associated with B cell leukemia and other cancers. These results indicate that cancer risk is already part of age-related mutation accumulation in normal cells. Taken together, our single-cell sequencing method provides a firm foundation for analyzing cellular genetic heterogeneity in normal human tissues. 

### Metabolism and Cellular Functions 1

#441

**NOX2 inhibition reduces oxidative stress and prolongs survival of mice with** Kras **induced myeloproliferative disease.**

Ebru Aydin, Alexander Hallner, Hanna Grauers Wiktorin, Anna Staffas, Kristoffer Hellstrand, Anna Martner. _University of Gothenburg, Gothenburg, Sweden_.

0 0 1 300 1716 University of Gothenburg 14 4 2012 14.0 Normal 0 false false false SV JA X-NONE /* Style Definitions */ table.MsoNormalTable {mso-style-name:"Table Normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-parent:""; mso-padding-alt:0cm 5.4pt 0cm 5.4pt; mso-para-margin-top:0cm; mso-para-margin-right:0cm; mso-para-margin-bottom:8.0pt; mso-para-margin-left:0cm; line-height:107%; mso-pagination:widow-orphan; font-size:11.0pt; font-family:Calibri; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-ansi-language:SV;} Mutations leading to Ras gene activation are important contributors to myeloid leukemogenesis. Kras is one of three homologues of the Ras family, and oncogenic Kras is frequently found in monocytic forms of acute myeloid leukemia (AML), chronic myeloid leukemia (CML) and myeloproliferative diseases (MPD). Earlier studies show that excessive amounts of reactive oxygen species (ROS) are produced in Kras-mutated myeloid malignancies. Myeloid cells, including Kras-mutated leukemic cells, express the ROS-generating enzyme NOX2. Relatively little is known about the impact of ROS formation, in particular NOX2-derived ROS formation, on leukemogenesis. Mice where oncogenic Kras was induced in hematopoietic cells of LSL-KrasG12D and Mx1-Cre double transgenic mice with ensuing myeloproliferation were treated with a histamine H2-receptor selective NOX2 inhibitor, Nα -methyl histamine (NMH) to assess whether NOX2 is a feasible therapeutic target in Kras-driven leukemia. Spleens of diseased Kras mice were infiltrated with mature CD11b+Gr1+ myeloid cells that expressed NOX2 and produced ROS. The NOX2-dependent formation of ROS in CD11b+Gr1+ cells was inhibited by NMH. In vivo administration of NMH delayed the development of myeloproliferative disease and significantly prolonged survival of Kras mice (p=0.003). In contrast, treatment with NMH did not alter the survival of mice when Kras expression was induced in hematopoietic cells of NOX2-deficient mice that did not produce ROS (Nox2-/-, LSL-KrasG12D and Mx1-Cre triple transgenic mice). In Kras-mutated Nox+/+ mice, but not in Kras-mutated Nox-/- mice, the in vivo administration of NMH entailed diminished ROS levels and reduced oxidative stress as reflected by reduced DCFDA and anti-8 Hydroxyguanosine staining. We propose that NOX2-derived ROS in Kras-mutated hematopoetic cells mediate genomic instability that may contribute to the progression of leukemia. Strategies to target NOX2 should be further evaluated in Ras-mutated hematopoietic cancer. <!--EndFragment-->

#442

Serine protease inhibitor Kazal type 1 (SPINK1) drives anoikis resistance in ovarian clear cell carcinoma.

Christine Mehner, Mathew A. Coban, Alexandra Hockla, Derek C. Radisky, Evette S. Radisky. _Mayo Clinic Cancer Center Jacksonville, Jacksonville, FL_.

Patients diagnosed with ovarian cancer face an average 5 year survival rate of 46%; low survival is driven by early and ongoing intraperitoneal dissemination and metastasis of the tumor. Among the different histotypes of ovarian cancer, ovarian clear cell carcinoma (OCCC) has a particularly poor prognosis when diagnosed in late stage, as these tumors tend to be chemoresistant, leaving no effective therapeutic options. Intraperitoneal metastasis requires cells to become anoikis resistant, where apoptosis normally induced by loss of attachment is suppressed. We seek to better understand the mechanisms involved in anoikis resistance in OCCC, which in the future could lead to new therapeutic strategies for these patients.

We have found that Serine Protease Inhibitor Kazal type 1 (SPINK1), an endogenous inhibitor of trypsin like serine proteases, is also an important regulator of anoikis resistance in some ovarian cancers. In this study, we aimed to dissect the role of SPINK1 specifically in OCCC, defining its contribution to anoikis resistance using cultured OCCC cell lines and elucidating its mechanism of action. We compared anoikis resistance of cells with endogenous SPINK1 expression versus cells in which SPINK1 was silenced using lentiviral shRNA constructs, when culturing cells on ultra-low attachment plates to mimic cell detachment from the extracellular matrix. We found that knockdown of SPINK1 reduced survival and stimulated apoptotic pathways in OCCC cells grown under detached conditions, implicating SPINK1 in anoikis resistance of OCCC cells. As a secreted protease inhibitor, SPINK1 may be expected to confer anoikis resistance by inhibiting an extracellular serine protease involved in triggering anoikis. Because many proteases represent potential targets of SPINK1, we have designed a strategy using Activity Based Protein Profiling (ABPP) to discover the targets of SPINK1 from among this large pool of candidates. In pilot studies, we have successfully used a novel activity-based probe to covalently label the active sites of serine proteases secreted from OCCC cells. In ongoing efforts, we are optimizing methods to identify labeled proteases with high sensitivity using tandem mass spectrometry. We will then identify those proteases regulated by SPINK1 through comparison of labeled proteomes from OCCC cells with and without SPINK1 treatment,, and candidate proteases will be further analyzed for their role in triggering anoikis. The identification of SPINK1 regulated proteases that are responsible for mediating anoikis in our models will give insight into important mechanisms whereby tumor cells acquire resistance to anoikis in OCCC. In-depth understanding of anoikis resistance, a critical component of ovarian cancer progression and metastasis, may lead to novel biomarkers of disease progression as well as important therapeutic targets.

#443

The anion channel GPHR/GPR89 regulates protein-folding homeostasis by regulating the IRE1α pathway in breast cancer.

Riccardo Ferro,1 Pierfrancesco Marra,1 Ana Mendes Pereira,1 Virinder Reen,1 Cynthia Prince,1 Sumi Mathew,1 Alessandra Facchetti,1 Priyanka Ghongane-Salpe,1 Sedigeh Kareemaghay,1 Patrycja Gazinska,1 David Robertson,2 Farzana Noor,1 Fara Braso' Maristany,1 Daniel Weekes,1 Dragomir Krastev,2 Tencho Tenev,2 Hasan Mirza,1 Nirmesh Patel,1 Anita Grigoriadis,1 Andrew Tutt1. 1 _Breast Cancer Now, king's College London, London, United Kingdom;_ 2 _Breast Cancer Now, Institute of Cancer Research, London, United Kingdom_.

In order to identify novel tumor drivers and gene addictions in breast cancer we have recently completed an integrated in silico/in vitro analysis of gene copy-number alterations and gene-expression profiles in a Triple Negative Breast Cancer-enriched cohort. An amplified and overexpressed gene that appeared to be a driver required for breast cancer cell proliferation is GPHR/GPR89. Several lines of evidence have recently suggested GPHR to be an anion channel, resident in the Golgi, where it controls intraluminal pH. Perturbation of GPHR function leads to altered secretory protein modification and impairment of intracellular transport. Additional data obtained in non-mammalian systems suggest that the localization of GPHR is wider and includes the endoplasmic reticulum (ER). Here, we show that GPHR is amplified and highly expressed across the breast cancer subtypes in our cohort, which we validate in the publicly available TCGA and METABRIC datasets. Using orthogonal characterization by both siRNA-shRNA and CRISPR/Cas9 gene editing, we demonstrate that GPHR down regulation impairs breast cancer cell proliferation and clonogenic ability. To further exclude the possibility that the observed phenotype was due to an "off target" effect, we also performed a rescue of function experiment overexpressing a GPHR protein resistant to GPHR shRNA that confirmed specificity. Importantly, breast cancer spheroids inducibly overexpressing GPHR are shown to have an increased area compared to control spheroids, demonstrating an important role of GPHR in breast cancer cell proliferation. Furthermore, by gradient sucrose subcellular fractionations and immunofluorescence analysis, we show that GPHR is localized in the ER of breast cancer cells. Unfolded protein response (UPR) signatures based on first-degree protein-protein interactions indicate that GPHR is positively correlated with IRE1α and negatively correlated with ATF4/CHOP networks. GPHR is also positively correlated with the XBP1 gene signature. We then further demonstrate that over expression or down regulation of GPHR are able to modulate the IRE1α/XBP1 network of proteins. In summary, in order to sustain uncontrolled growth, breast cancer cells undergo significant stress due to accelerated metabolism, deficient nutrient provision and oxidative stress. UPR signaling and GPHR expression are then essential to protect cells and to allow tumor growth by reducing protein synthesis and favoring chaperonine functions. Here we demonstrate that GPHR is over expressed across the breast cancer subtypes where it controls IRE1α/XBP1 network, and, therefore, can be a novel target for breast cancer therapy as malignant breast cells overexpressing GPHR are selectively dependent on GPHR expression.

#444

**ER Stress marker GRP78 is** not **expressed on ER** - **/PR** - **/Her2** - **human breast cancer cell surface** or **secreted.**

Jesus E. Serrano,1 Andrea Rivera-Ruiz,1 Krishna Baksi,2 Dipak K. Banerjee1. 1 _Univ. of Puerto Rico School of Medicine, San Juan, PR;_ 2 _Universidad Central del Caribe, Bayamon, PR_.

GRP78 (a Mr 78kDa calcium dependent glucose binding protein) located in ER lumen. It functions as ER chaperone and translocates proteins for glycosylation at the asparagine residue present in the sequon Asn-X-Ser/Thr (i.e., N-linked glycosylation). This important biochemical event is essential for glycoprotein folding and function. When targeted in ER-/PR-/HER2+ breast tumor in athymic nude mice with the N-glycosylation inhibitor tunicamycin, the cancer growth was reduced ~55% in three weeks. Paraffin sections of the tumor exhibited reduced N-glycan level, and reduced angiogenesis but increased GRP78 expression in microvasculature as well as in the tumor tissue (J. Biol. Chem. 286, 29127–29138, 2011). This supported the presence of ER stress. We have now evaluated the effect of tunicamycin on the proliferation of metastatic ER-/PR-/HER2\- (MDA-MB-231) and non-metastatic ER+ (MCF-7) human breast cancer cells. Tunicamycin inhibited proliferation of both cells types in a time and dose-dependent manner. Interestingly, GRP78 expression (protein and mRNA) was higher in tunicamycin (1.0 µg/ml) treated MDA-MB-231 and in MCF-7 cells and supported by both Western blotting and qPCR. Since GRP78 is an ER stress marker, so we have followed its intracellular localization by immunofluorescence microscopy after treating the cancer cells with tunicamycin as well as after subjecting them to various stress conditions. We have used unfixed cells and stained with either FITC-conjugated Concanavalin A (Con A), or Texas-red conjugated wheat germ agglutinin (WGA) or with anti-GRP antibody. There were expression of N-glycans but not GRP78. GRP78 however became detectable after permeabilization of cells with a brief exposure to ice-cold methanol. GRP78 was not detected in the conditioned media of the cancer cell but a high level of MMP-1 did. We have therefore, concluded that GRP78 is expressed neither on the outer-leaflet of the (ER-/PR-/HER2-) human breast cancer cells nor it is secreted into the culture media during tunicamycin-induced ER stress. This is obviously, contradicted the current dogma that GRP78 expression on the tumor cell surface interferes with the cancer therapeutic(s) and make them as tumor promoters and not tumor suppressors. Our study, thus, suggests strongly that anti-angiogenic and anti-tumorigenic action of tunicamycin can be modeled to develop next generation cancer therapy, i.e., glycotherapy for treating breast tumor and all other sold tumors. Supported in part by the university funds and NIH/NIMHD G12MD007583 (KB).

#445

The unfolded protein response sensor PERK mediates extrinsic ER stress-induced inhibition of glioblastoma stem cell self-renewal.

Frank A. Kruyt, Natalia M. Peñaranda-Fajardo, Coby Meijer. _University Medical Center Groningen, Groningen, Netherlands_.

Introduction: Glioblastoma (GBM) is the most prevalent and aggressive brain tumor in adults and has a very poor prognosis. GBM cancer stem cells (GSCs) rather than differentiated offspring are thought to be critical for tumor progression and therapy resistance. Hence, targeting GSCs is essential for effective therapy. In this study we aimed to investigate whether ER-stress aggravation is an effective approach for targeting GSCs. ER stress is known to impair protein production leading to activation of the unfolded protein response (UPR). This adaptive response attempts to restore protein homeostasis and cell survival, but induces cell death when damage is overwhelming.

Methods: Patient-derived GBM neuropsphere cell lines enriched for GSCs were used. Differentiated counterparts were obtained by serum exposure. Thapsigargin (Tg) was used to induce extrinsic ER-stress. Cytotoxic effects and GSC targeting were determined by MTS and neurosphere formation assays; apoptosis was studied by caspase activation and use of pan-caspase inhibitor ZVAD; necrostatin-1 was applied for demonstrating necroptosis. Various known chemical modulators of the PERK and IRE1 branches of the UPR were employed. ATF6 and PERK expression was blocked by RNAi and CRIPSR/Cas9, respectively. Expression of genes was studied by RT-qPCR and western blotting. RNA sequencing was used for UPR gene transcriptional profiling.

Results: Tg effectively reduced viability of the GBM neurospheres that was accompanied by induction of both apoptosis and necroptosis. Modulation of ATF6, IRE1 and PERK activity showed that the PERK branch is mediating cytotoxicity. This was confirmed by genetic ablation of PERK expression. Interestingly, Tg potently reduced neurosphere formation indicating targeting of GSCs, which coincided with down-regulation of the stem cell transcription factor SOX2. The PERK branch was found to be instrumental for this effect. Serum-differentiated neurospheres appeared to be more resistant for Tg. Transcriptional profiling of GBM neurospheres and differentiated counterparts overall revealed enhanced expression of key UPR branch genes in neuropspheres that may be linked to enhanced sensitivity for ER stress. Genes displaying a more than 2-fold differential expression included sterol regulatory element-binding proteins (SREBPs) known to modulate for example ATF6 activity.

Conclusions: ER stress induced by Tg effectively reduces viability of GBM neurospheres and targets the self-renewal capacity of GSCs. The PERK branch of the UPR is instrumental in these cytotoxic effects. ER stress aggravation as well as PERK modulation appear to be promising approaches for further exploration as possible therapeutic strategies in GBM.

#446

HSP90 inhibitors disrupt a transient HSP90-HSF1 interaction and identify a noncanonical model of HSP90-mediated HSF1 regulation.

Megan L. Tigue,1 Toshiki Kijima,1 Thomas L. Prince,2 Kendrick H. Yim,1 Harvey Schwartz,1 Kristin Beebe,1 Sunmin Lee,1 Marek A. Budzynski,3 Heinric Williams,2 Jane B. Trepel,1 Lea Sistonen,3 Stuart Calderwood,4 Len Neckers1. 1 _NIH, Bethesda, MD;_ 2 _Geisinger Clinic, Danville, PA;_ 3 _Åbo Akademi University, Turku, Finland;_ 4 _Harvard Medical School, Boston, MA_.

Heat shock factor 1 (HSF1) is an evolutionarily conserved transcription factor that initiates the cytoprotective heat shock response (HSR) and mediates a cancer-specific transcriptional program, which is comprised of over 500 genes and has been associated with poor clinical outcomes. HSF1 is thought to be regulated by molecular chaperones, including Heat Shock Protein 90 (HSP90). The purpose of our study was to describe the interactions between molecular chaperones, such as HSP90 and HSP70, and HSF1 and to determine the mechanism(s) by which HSP90 inhibitors promote the HSR in mammalian cells. Using a combination of techniques including IP-WB, ChIP, and RT-qPCR analysis, we demonstrated that HSP90 binding to HSF1 depends on HSP90 conformation and is only readily visualized for the ATP-dependent, N-domain dimerized chaperone, a conformation rarely sampled by mammalian HSP90. We also showed that ATP-competitive, N-domain targeted HSP90 inhibitors disrupt this interaction and enhance HSF1 phosphorylation, resulting in the increased duration of HSF1 occupancy of the hsp70 promoter and significant prolongation of both the constitutive and heat-induced HSF1 transcriptional activity. While our data do not support a role for HSP90 in sequestering HSF1 monomers to suppress HSF1 transcriptional activity, as has been previously proposed, our findings do identify a noncanonical role for HSP90 in providing dynamic modulation of HSF1 activity by participating in removal of HSF1 trimers from heat shock elements in DNA, thus terminating the heat shock response.

#447

A novel proteotoxic combination therapy for EGFR+ and HER2+ cancers.

Mengxiong Wang, Renan Ferreira, Mary Law, Elham Yaaghubi, Coy Heldermon, Ronald Castellano, Brian Law. _University of Florida, Gainesville, FL_.

EGFR and/or HER2 are frequently overexpressed oncoproteins in HER2+ breast cancer, Triple Negative Breast Cancer (TNBC), and Inflammatory Breast Cancer (IBC). However, EGFR and/or HER2 targeting agents have failed to make major advances in improving survival for these cancer patients, largely because these cancers frequently develop resistance to these drugs. The identification of novel treatment strategies is critically needed. Overexpression of EGFR and/or HER2 presents a severe burden to the secretary pathway, as HER-family proteins EGFR, HER2, HER3 share conserved extracellular cysteine-rich and proline-rich repeats that form numerous disulfide bonds and control cis-trans isomerization in the protein folding processes. We demonstrated that the disulfide bond disrupting Agents (DDAs) selectively killed EGFR+ and/or HER2+ breast cancer cells in vitro and decreased tumor growth of HER2+ cancer in a xenograft mouse model. DDA-dependent anti-cancer actions resulted from downregulation of EGFR/HER2/HER3, induction of AKT dephosphorylation, and activation of Endoplasmic Reticulum (ER) stress. We also investigated a novel proteotoxic combination therapy employing DDAs and Proline Isomerase Inhibitors (PIIs) in treating breast cancers. PIIs potentiated the efficacy of DDAs in downregulating EGFR/HER2/HER3 levels and initiating ER stress, and the combination was highly synergistic in reducing EGFR+ and/or HER2+ breast cancer cell viability. Importantly, the combination of DDAs and PIIs overcame single drug resistance, significantly decreased tumor volumes and increased the survival of xenograft mice. Evidence that the drug combination was effective against metastatic breast cancer cells is also provided.

#448

**The novel role of ecdysoneless (ECD) in maintain ER Ca** 2+ **homeostasis .**

Aniruddha Sarkar,1 Irfana Saleem,1 Sameer Mirza,1 Keshore Bidasee,1 Rajini Rao,2 Hamid Band,1 Vimla Band1. 1 _University of nebraska medial center, omaha, NE;_ 2 _The Johns Hopkins University School of Medicine, Baltimore, MD_.

Background and Significance: Calcium (Ca2+) is an essential signal transduction element involved in the regulation of several cellular activities such as gene transcription, proliferation and apoptosis. Endoplasmic Reticulum (ER) is the Ca2+ reservoir inside the cell that strictly orchestrate Ca2+ gradient across the Plasma Membrane(PM). Importantly, intracellular Ca2+ flux, is a key to orderly regulate cell proliferation, if altered can promote uncontrolled proliferation leading to cancer. Ca2+ entry through the [Ca2+]PM channels during [Ca2+]ER depletion is called the Store Operated Ca2+ Entry (SOCE). Impairment of endogenous SOCE leads to sustained [Ca2+]ER depletion and results in accumulation of unfolded proteins in the ER (known as ER stress). Understanding the basic molecular mechanism regulating PM to ER Ca2+ flow and developing novel strategies to target ER Ca2+ signaling could be beneficial from cancer therapeutic perspective. We have identified Ecdysoneless (ECD), an evolutionary conserved novel cell cycle regulator protein. ECD is overexpressed in several cancers, including breast and pancreatic cancers and its overexpression in breast cancer correlates with poor prognosis and short survival of patients. Recently our lab demonstrates that ECD is a negative regulator of ER stress; however, there is no mechanistic insights of how ECD senses and acts to maintain ER homeostasis.

Experimental Design: We have utilized mouse embryonic fibroblasts (MEFs) from Tet(O)-FhECD-IRES-eGFP-rtTA and Ecdflox/flox mice transgenic mice to regulate ECD levels in cells to assess the role of ECD in Ca2+ signaling in a highly controlled system. Structured illumination microscopy(SIM) and biochemical fractionation techniques were performed to identify localization of ECD. Live cell imaging of cells stained with Ca2+ binding dyes like Fluo4-AM and Fluo5N-AM were used to access [Ca2+]Cyto and [Ca2+]ER levels respectively. Fluorescence resonance energy transfer (FRET) and immunoprecipitation methods were conducted to analyze SOCE pathway. Western blotting was used to assess the levels of various proteins involved in Ca2+ signaling.

Results: We demonstrate several novel findings: (i) ECD is a smooth ER associated protein; (ii) ECD regulates assembly of ER-PM protein complex to drive Ca2+ entry through SOCE into the ER, maintaining [Ca2+]ER homeostasis; iii) ECD is overexpressed upon Ca2+ depletion and provides survival benefit to cells; v) ECD regulates Ca2+ mediated cell proliferation via CaMKII phosphorylation.

Conclusion: Our results demonstrate ECD is a sensor of ER stress, induced by Ca2+deprivation, and overexpressed ECD promotes cell proliferation and survival under ER stress. These findings suggest ECD could be a potential target of Cancer therapy.

#449

Targeting PKR-like endoplasmic reticulum kinase modulates metabolism to promote T-cell effector function and PD1 immunotherapy responsiveness.

David R. Soto-Pantoja, Yismeilin R. Feliz-Mosquea, Kenysha YJ Clear, Adam S. Wilson, Katherine L. Cook. _Wake Forest University, Winston Salem, NC_.

The development of immunotherapy is a major recent advance in clinical oncology. However the majority of patients do not respond and those who do, experience resistance and relapse. Therefore, understanding the molecular mechanisms of immune checkpoint inhibitor resistance is critical to develop combinatorial drug strategies to potentiate therapeutic responsiveness to reduce mortality. Highly secretory cells, such as T-cells, have a larger endoplasmic reticulum (ER) to handle the increased protein translation capacity required by the cell. Therefore, T-cells may be highly sensitive to ER stress. The high nutrient needs of the tumor deplete resources in the microenvironment subjugating infiltrating T-cells to reduced nutrient availability resulting in stress. Our data indicate that co-culturing melanoma cells with T-cells increase T-cell specific unfolded protein response (UPR) signaling. Stimulating ER stress decreased cytolytic T-cell function in TALL-104 T-cells, enabling Mun2b melanoma survival. Knockdown of PERK restored T-cell killing capacity in ER stress induced cells. In an ex-vivo model of antigen-specific mediated T-cell death, Pmel-1 T-cells stimulated with gp100 displayed increased B16 melanoma killing when treated with GSK2606414 (a small molecule PERK inhibitor). PERK inhibition also elevated glycolysis proteins and mitochondrial bioenergetics in T-cells, suggesting PERK inhibition increases T-cell metabolism. As proof-of-concept, we determined the effect of PERK inhibition in a syngeneic B16 melanoma model. Male C57/Bl6 mice were inoculated with B16 melanoma cells. At 5 days post injection, mice were treated with a control IgG, antisense morpholino targeting PERK, PD1 antibody, or a combination of PERK morpholino and PD1 antibody. PERK morpholino treatment alone was sufficient to reduce tumor volume by 46.5%. PD1 antibody therapy alone reduced tumor volume by 47.4%. The combination of PD1 antibody and PERK targeting therapy significantly reduced B16 melanoma tumor volume by 64.5% demonstrating that targeting PERK in vivo enhanced immunocheckpoint therapy efficacy. Treated B16 tumors were homogenized and infiltrating T-cells isolated by flow cytometry. Gated CD3+ infiltrating leukocytes were then counted for CD8 and PD1 expression. Tumor infiltrating CD8+ T-cells doubled with PERK inhibition alone, giving further proof indicating that PERK may be a novel immune checkpoint target. Small molecule PERK inhibitors were previously shown to cause β-islet damage and insulin resistance; therefore we tested the effect of our PERK morpholino on glucose tolerance in the B16 melanoma model. PERK morpholino treatment had no overall impact on glucose tolerance and therefore may be a novel therapeutic to induce T-cell metabolism and potentiate immune checkpoint therapy efficacy with reduced off-target toxicities.

#450

Obesity favours leukemogenesis through enhanced preleukemic stem cell self renewal via polyunsaturated fatty acid-dependent ER stress relief.

Luca Mazzarella, Paolo Falvo, Annagiulia Sanarico, Elena Gatti, Piergiuseppe Pelicci. _European Inst. of Oncology, Milan, Italy_.

Obesity increases risk for multiple tumors, but impact on leukemia and its underlying molecular mechanism are poorly understood. Studies from our group and others showed that risk and outcome of Acute Promyelocytic Leukemia (APL) are more strongly associated with obesity than other hematological neoplasms. Intriguingly, in 2 independent cohorts (Italian and TCGA), obesity was associated with ~4 fold higher incidence of internal tandem duplications of the leukemogenic kinase FLT3 (FLT3-ITD), the first example of obesity-associated genetic alterations in cancer (unpublished). Here we propose a molecular mechanisms underlying obesity-associated leukemogenesis through our studies in murine models. Exposure to High fat Diet (HFD), an established model of obesity-related pathology, strongly accelerated disease onset and mortality in 3 murine models of hematological cancer: PML-RARa knockin (PRKI), developing an APL-mimicking disease; FLT3ITD knockin (FIKI) developing myeloproliferation; a new double PRKI-FIKI knockin mouse, developing APL-like disease but, surprisingly, with delayed latency compared to PRKI, presumably due to the known exhaustion of hematopoietic stem cells (HSC) associated with FLT3ITD. Preleukemic HSCs from HFD PRKI mice showed increased DNA damage in Comet assays, but surprisingly this was not associated with increased mutational load as revealed by a novel whole genome sequencing-based method. Instead, HSCs derived from HFD PRKI mice showed enhanced self-renewal in in vitro replating assays. Similarly, in the FIKI model we observed enhanced engraftment in serial competitive transplantation experiments in HFD-fed recipients. Delving more deeply in the molecular mechanisms, through bioinformatic analysis of RNAseq data from the TCGA we found upregulation of the linoleic acid (LA) pathway in obese APL patients. LA is the richest fatty acid in HFD and in many western diets, thus a likely candidate for mediating HFD activity. LA significantly enhanced PRKI serial replating efficiency; this was abolished by pharmacologically blocking LA metabolism through a 5-lipoxygenase inhibitor or its transcriptional effects through a PPARd inhibitor. In the FIKI model and in a FLT3ITD-inducible cell line, we revealed a previously unappreciated induction of ER stress by FLT3ITD, likely to underlie FLT3ITD-induced HSC exhaustion. HFD (in vivo) and LA (in vitro) relieved FLT3ITD-induced ER stress, as revealed by the attenuation of markers of adaptive response to ER stress (Unfolded Protein Response and RNA IRE1-dependent RNA decay). In conclusion, our study suggests a novel and somewhat counterinuitive model for obesity-associated cancerogenesis: rather than inducing cellular stress, HFD-induced obesity enhances the self-renewal of preleukemic stem cells through LA-mediated alleviation of oncogene-induced proteotoxic stress

#451

Proteomic profiling of the unfolded protein response identifies patients benefiting from bortezomib in pediatric acute myeloid leukemia.

Fieke W. Hoff,1 Yihua Qiu,2 Wendy Hu,3 Amina A. Qutub,3 Alon S. Gamis,4 Richard Aplenc,5 E Anders Kolb,6 Todd A. Alonzo,7 Eveline SJM de Bont,1 Steven M. Kornblau,2 Terzah Horton8. 1 _University Medical Center Groningen, Netherlands;_ 2 _UT MD Anderson Cancer Ctr., Houston, TX;_ 3 _Rice University, Houston, TX;_ 4 _Children's Mercy Hospitals and Clinics, Kansas City, MO;_ 5 _Children's Hospital of Philadelphia, Philadelphia, PA;_ 6 _Nemours Center for Cancer and Blood Disorders, Wilmington, DE;_ 7 _University of Southern California, Los Angeles, CA;_ 8 _Texas Children's Cancer Center, Houston, TX_.

Background: The unfolded protein response (UPR) is a cellular stress response triggered by accumulation of misfolded proteins in the endoplasmatic reticulum. Bortezomib is a proteasome inhibitor that triggers the UPR. Our goal was to globally assess the expression and activation of cell stress proteins, including the UPR, and to determine if proteins in the UPR pathway are prognostic of clinical response or predictive of bortezomib resistance in pediatric acute myeloid leukemia (AML).

Methods: We analyzed 5 UPR proteins (CAV1, EIF2S1, EIF2S1.pS51, ERN1, and GRP78) involved in the UPR by reverse phase protein arrays (RPPA) in ''bulk'' (CD3-/19-) AML cells from 505 de novo pediatric AML patients who participated in the Children's Oncology Group AAML1031 phase 3 clinical trial. Progeny clustering was used to identify subgroups of patients (protein clusters) based on similar protein expression. Clusters were correlated with outcome, as well as patient characteristics.

Results: Four UPR protein clusters (C1-4) were recognized based on relative protein expression levels. UPR clusters were correlated with cytogenetics (high risk underrepresented in C3+C4, P<0.001), MLL-rearrangement (low in C3, p<0.001), t(8;21) (enriched in C3, p<0.001) and the CEPBA (low in C3+C4, p= 0.023). In patients treated with standard therapy, cytarabine/daunorubicin/etopside (ADE), protein clusters were prognostic for overall survival (OS) (p=0.024) and event-free survival (EFS) (p=0.003), with C2 having an unfavorable prognosis (OS estimate C2: 55% vs. 73-80% for C1, C3+C4 at 4yr). Multivariate Cox regression analysis identified C2 as independent prognostic variable for EFS (p=0.009). Adding bortezomib (ADE+B) did not show an outcome difference overall (p=0.65). However, the response of patients in C2 improved with the addition of bortezomib (ADE: 46% 4yr-OS vs. 65% with ADE+B, Fisher's exact: p=0.014). This cluster was characterized by relative low levels of CAV1 and ERN1, in combination with slightly elevated expression of EIF2S1, EIFS2S1.pS51 and reduced GRP78 compared to normal CD34+ cells.

Conclusion: We analyzed UPR in pediatric AML and identified four protein clusters that were prognostic of OS and EFS. We were able to identify a subgroup of patients that benefited from the addition of bortezomib to ADE chemotherapy. We hypothesize that patients with low UPR activation are more susceptible to protein cell stress, and protein cell stress susceptibility is amplified by the addition of bortezomib with ADE. This suggests that certain subsets of pediatric AML patients benefit from the ADE+B therapy. The use of UPR screening could identify patients who would benefit from ADE+B therapy.

#452

Direct delivery of a synthetic ATP binding protein reduces ATP and induces apoptosis in Hela cells.

Selina M. Martinez,1 Shanika Kingston,1 Arlene Ngor,2 John C. Chaput,2 Nagaraj J. Vinay,3 Shaleen B. Korch1. 1 _Midwestern Univ. - Glendale Campus, Glendale, AZ;_ 2 _University of California Irvine, Irvine, CA;_ 3 _Arizona State University, Tempe, AZ_.

Introduction: Reprogramming of cellular metabolism is a hallmark characteristic of cancer cells. As opposed to normal cells, which generate most of their adenine triphosphate (ATP) through oxidative phosphorylation, cancer cells generate ATP less efficiently by aerobic glycolysis (Warburg effect). In addition to deciphering metabolic switches, there is growing interest in understanding the multifaceted role ATP plays in cancer development, progression and chemotherapeutic resistance. One approach to untangling these roles is to significantly reduce intracellular ATP and investigate the cellular response. With that, we designed and created a man-made protein that chelates ATP with high specificity and affinity. This synthetic protein, called DX, has been genetically expressed in a living organism, Escherichia coli, where it reduced intracellular ATP to below the levels of detection. Hypothesis: Delivery of the DX protein into HeLa cancer cells will reduce intracellular bioavailable ATP affecting energy metabolism, viability and drug susceptibility. Methods: A cationic lipid mixture was complexed with active, purified DX protein to generate a DX/lipid complex. The integrity of the complexed protein was verified by western blot analysis. HeLa cells were incubated with the DX/lipid complex to allow efficient delivery of DX into the cytoplasm of the HeLa cells. The impact of DX on cell viability was determined using a tetrazolium-based colorimetric cell viability assay and a caspase 3/7 assay. To correlate phenotypic/viability change with DX activity, bioavailable ATP levels were measured at specific time points following DX delivery. To establish whether DX impacts drug export, the retention of calcein-AM, a substrate of the ATP-dependent p-glycoprotein pump, was measured post DX-delivery. Results: Western blot analysis confirmed the stability of DX prior to- and post- delivery to HeLa cells. In a time- and dose- dependent manner, DX negatively impacted cell growth and induced cell death via apoptosis, at a time concomitant with a decrease in bioavailable ATP. Significantly, HeLa cells treated with DX retained calcein-AM, suggesting reduced p-glycoprotein activity. Conclusion: Advances in protein engineering have made it possible to create artificial proteins with specific functions. In addition to direct clinical applications, synthetic proteins are powerful tools that have the potential to reveal something new about biology. Direct delivery of a synthetic ATP-binding protein potentiated apoptosis in HeLa cells contemporaneous with reduced intracellular ATP. Having established DX activity in a living system, future studies will examine the cancer cell metabolome in response to significant ATP stress. Finally, a reduction in p-glycoprotein activity in DX-treated HeLa cells advocates examination of concurrent DX/chemotherapeutic treatment, principally in drug resistant scenarios.

#453

The role of centrosome amplification in HCC.

Sai Fung Yeung, Yick Pang Ching. _The University of Hong Kong, Hong Kong, Hong Kong_.

Centrosome is the major microtubule organizing center (MTOC) in mammalian cells which maintain cell polarity, organization of microtubules and regulate cell cycle progression. In normal cells, centrosome duplication cycle is tightly coupled with cell cycle, DNA synthesis, mitosis as these event were controlled by parallel pathways. Centrosomal defect is reported in numerous type of solid and hematologic malignancy, both structurally (enlarged PCM, string-like etc) and numerically (<2 or >=3). Centrosome amplification (CA, n>=3) is the most commonly reported. CA is casually linked to chromosomal instability (CIN), tumor-initiation, advanced tumor stage and chemo-resistance. CA arises through multiple distinct but non-redundant mechanisms in tumor, for example, aberrant cell cycle, hypoxia, and chemotherapy. Recently studies suggest CA might not be responsible for HCC initiation. Yet, around 90% of HCC cells carry certain degree of CA. It is associated with p53 mutation and tumor-aneuploidy but no other clinicopathological features. The role of CA in HCC progression deserve a more comprehensive study. To investigate the role of CA in HCC, first, we use CA20, an expression signature-score includes centrosomal structural genes and genes results in CA when dyregulated. We use CA20 as surrogate marker to evaluate the clinical outcome of CA in HCC from GEO datasets. CA carrying HCC was found to have poor overall survival and larger tumor size. Next, we developed several inducible PLK4 overexpressing HCC cell line. Preliminary data shown that PLK4 overexpression concomitantly leaded to centrosome over-duplication in HCC cell lines. Functionally, PLK4 overexpression resulted in increased cell migration, but retarded growth rate in p53+/+ HCC cells. Furthermore, we identified another cell cycle-related kinase - Aurora A, as a novel interacting partner of PLK4 suggest that these kinases work together to promote malignant transformation.

#454

The role of autophagy in multiple myeloma disease progression.

Ioanna Savvidou,1 Sahan Chandrasekara,2 Tiffany Khong,1 Andrew Spencer1. 1 _Australian Center for Blood Diseases, Alfred Hospital, Melbourne, Australia;_ 2 _Monash University, Melbourne, Australia_.

Multiple Myeloma (MM) is an incurable hematologic neoplasm characterized by accumulation of malignant plasma cells in the bone marrow (BM). MM cells establish close ties with the BM, however at late stages they utilize as yet unknown mechanisms to acquire independence from the microenvironment and survive in extramedullary/nutrient deprived sites. Autophagy, a process responsible for degradation of proteins and organelles under stress conditions, has been suggested as a mechanism whereby tumor cells can withstand metabolic stress.

Using two newly acquired human myeloma cell lines (HMCL), TK1 and TK2, from the medullary and extramedullary sites of a MM patient respectively, we investigated the ability of autophagy to confer stress/glutamine deprivation tolerance.

Results

HMCLs require exogenous L-Glutamine (Gln) for sustained proliferation in vitro. However, when TK1 and TK2 were grown under Gln deprivation conditions (8mM, 4mM, 2mM, 0mM) we observed striking differences in their growth response. The TK2 (peripheral blood) cell line continued proliferating until day 14 (p=0.0046), whereas proliferation of the TK1 cell line significantly decreased in the absence of Gln (p=0.0028 between 8mM and 0mM). Furthermore, the proliferative advantage of TK2 was abrogated at 2mM and 0mM of Gln by the use of an autophagy inhibitor (chloroquine [CQ] (p=0.000235 and p=0.000069 respectively), implicating this process in Gln deprivation tolerance of TK2.

Additionally, Gln concentration was inversely correlated with TK1 survival, but not TK2, as measured by annexin-V/PI staining. Cell death in TK1 increased at 24h, from 36.022% at Gln 8mM to 56.2% at Gln 0mM, irrespective of autophagy inhibition. In TK2 cells, Gln deprivation did not cause a significant increase in cell death, whereas inhibition of autophagy increased cell death by two-fold. Accordingly, apoptosis (as measured by PARP cleavage) was induced by the addition of CQ only in TK2 under Gln deprivation. Resistance of TK2 cells to metabolic stress also correlated with up-regulation of the autophagic pathway upon CQ exposure, as demonstrated by a higher LC3BII/I turnover and the presence of autophagic vacuoles in TK2 (but not TK1) cells.

In addition to upregulation of autophagy, Gln deprivation also induced the expression of glutamine synthetase in TK2 but not TK1 cells. Chemical inhibition of this enzyme by methionine-sulfoximine (5mM) under Gln deprivation conditions was able to induce significant cell death in TK2 and not TK1 (32.57% and 2.53% increase respectively). These data demonstrate that Gln synthesis is an additional mechanism whereby TK2 cells tolerate Gln starvation.

In conclusion we have shown that MM survival outside the BM can be attributed to the ability of cells to overcome "glutamine addiction" due to up-regulation of autophagy and ability to synthesise Gln. Thus, autophagy inhibition is an attractive alternative approach to target late-stage, Gln independent, metastatic cells.

#455

Cellular response to oxaliplatin treatment shows altered DNA damage, nucleolar and ribosomal stresses as main altered pathways.

Tomáš Oždian,1 Dušan Holub,1 Zuzana Macečková,1 Lakshman Varanasi,1 Gabriela Rylova,1 Jiří Rehulka,1 Jana Vaclavkova,1 Hanuš Slavík,1 Pavel Moudry,1 Pawel Znojek,1 Jarmila Stanková,1 Juan Bautista de Sanctis,2 Marian Hajduch,1 Petr Džubák1. 1 _Institute of Molecular and Translational Medicine, Olomouc, Czech Republic;_ 2 _National Center of Clinical Immunology and FOCIS Center of Excellence, Caracas, Bolivarian Republic of Venezuela_.

Oxaliplatin is a platinum-based drug playing a critical role in the treatment of colorectal carcinoma and various Hodgkin and non-Hodgkin lymphomas. The primary mechanism of action is reported to be DNA damage and double strand breaks response. However, platinum-based drugs are usually very reactive and covalently binds not only to DNA but also to RNA, proteins or glutathione. Concerning very high reactivity of platinum compounds, we have performed whole-cell proteomic profiling on highly chemosensitive CCRF-CEM cell line to identify all possible cellular response mechanisms.

For modeling of actual drug response, CCRF-CEM cell line was treated with 5 x IC50 (29.3 µM) for half-time to caspase activation (240 min). Such cells were analyzed using current high-resolution mass spectrometry. Totally, 4049 proteins were identified over three biological replicates. Among these proteins, 76 were significantly downregulated and 31 significantly upregulated in the whole experiment. Bioinformatic analysis showed nucleolar and ribosomal stress, changes in secretory, mitotic and DNA damage related proteins. These response pathways were confirmed by functional analyses and targeted experiments aimed to expression changes of selected proteins.

Results observed in this experiment suggests nucleolar and ribosomal stress as the most abundant stress responses to oxaliplatin treatment at a halftime to apoptosis. Down-regulation of ribosomal proteins indicates stop of ribosome synthesis, which is one kind of response to stress. In such a case, energy saved by stopping ribosome biogenesis could be invested in solving stress conditions. Dysregulated ribosomal proteins are reported activators of p53 as well. In conclusion, oxaliplatin-induced cellular stress caused an acute response at an early stage of the experiment. Acute response stopped ribosomal biosynthesis and reduced the nucleolar size by the currently unknown mechanism. Nucleolar and ribosomal stress then triggers apoptosis in both p53 dependent and independent pathways.

This work was supported by Internal Grant of Palacky University in Olomouc (LF_2016_019), Ministry of School, Education and Youth of the Czech Republic (LO1304 and LM2015064) and Technological Agency of the Czech Republic (TE02000058).

#456

WR1065, the active metabolite of amifostine modulates chemistry and biology of cisplatin.

Tatjana Paunesku,1 Jelena Popovic,2 Andrijana Klajn,2 Natasa Kovacevic-Grujicic,2 Qing Ma,1 Milena Stevanovic,2 Gayle E. Woloschak1. 1 _Northwestern University, Chicago, IL;_ 2 _Institute of Molecular Genetics and Genetic Engineering, Belgrade, Serbia_.

WR-1065 [2-((aminopropyl)amino)ethanethiol] is metabolically active form of amifostine (also known as Ethyol, an agent used to mitigate side effects of chemo-radiotherapy in head and neck cancer). In vitro, WR-1065 was also found to decrease DNA damage caused by cisplatin. In some human studies, amifostine was found to be effective against chemotherapy-induced peripheral neuropathy (CIPN). While this effect was not dramatic in all such studies, it was notable in patients who received cisplatin alone. Because CIPN is the most common and longest lasting side effect of chemotherapy we decided to explore possible biological and chemical basis of amifostine mediated protection against platinum (II). Two human cell lines were used in this study: SK-N-SH neuroblastoma cells and NT2/D1 embryonal carcinoma cells. Importantly, their response to cisplatin treatment corresponded to their differentiation. In both cases, differentiated cells survived exposure to cisplatin better than their non-differentiated counterparts; cisplatin IC50 doses in differentiated cells were regularly two times higher. Addition of WR-1065 to cisplatin treated differentiated cells increased their viability regardless of whether that treatment was done, before, during or after cisplatin treatment. Similar approach was used to prepare samples for chemical studies using X-ray absorption near edge structure (XANES) spectroscopy. XANES can be used to investigate oxidation-reduction changes of Pt in biological samples. This was used by others to follow gradual reduction of Pt(IV) into Pt(II) inside cells. In this work, XANES was used to estimate Pt (II) to Pt (IV) ratio in samples of cisplatin treated cells and cisplatin mixed with cellular components such as DNA. Comparisons were done between such samples and their counterparts additionally exposed to WR-1065. In all cases, presence of WR-1065 changed the ratio of two forms of Pt, suggesting that chemistry of this interaction may be a possible mechanism of WR-1065 protection against cisplatin. At the moment, amifostine is not used in clinical practice in combination with platinum based chemotherapy drugs. Reasons for that may lay in the fact that there remains a concern that Amifostine could possibly protect cancer cells from therapy and not only normal tissues. This work shows that the mechanisms underlying ability of amifostine to decrease detrimental effects of platinum (II) are compatible with delayed CIPN treatment. This means that amifostine could be used to reduce CIPN symptoms significantly after cancer treatment, in patients who are cured from cancer but suffer treatment complications.

#457

Protein and chemotherapy profiling of extracellular vesicles harvested from therapeutic-induced senescent triple-negative breast cancer cells.

Emma L. Kavanagh,1 Sinéad Lindsay,1 Melinda Halasz,1 Luke C. Gubbins,1 Karolina Weiner-Gorzel,1 Matthew Ho Zhi Guang,1 Aloysius McGoldrick,1 Eoin Collins,1 Michael Henry,2 Peter O'Gorman,3 Patricia Fitzpatrick,1 Michaela J. Higgins,3 Paul Dowling,4 Amanda McCann1. 1 _University College Dublin, Dublin, Ireland;_ 2 _Dublin City University, Dublin, Ireland;_ 3 _Mater Misericordiae University Hospital, Dublin, Dublin, Ireland;_ 4 _National University of Ireland Maynooth, Kildare, Ireland_.

Triple negative breast cancer (TNBC) is an aggressive subtype with relatively poor clinical outcomes and limited treatment options. Chemotherapy, while killing cancer cells, can result in the generation of highly chemoresistant therapeutic induced senescent (TIS) cells that potentially form stem cell niches resulting in metastases. Intriguingly, senescent cells release significantly more extracellular vesicles (EVs) than non-senescent cells. Our aim was to profile EVs harvested from TIS TNBC cells compared with control cells to identify a potential mechanism by which TIS TNBC cells maintain survival in the face of chemotherapy. TIS was induced and confirmed in Cal51 TNBC cells using the chemotherapeutic paclitaxel (PTX) (Taxol). Mass spectrometry (MS) analysis of EVs harvested from TIS compared with control Cal51 cells was performed using Ingenuity Pathway Analysis and InnateDB programs. We demonstrate that TIS Cal51 cells treated with 75 nM PTX for 7 days became senescent (senescence-associated β-galactosidase (SA-β-Gal) positive, Ki67-negative, increased p21 and p16, G2/M cell cycle arrest) and released significantly more EVs (P=0.0002) and exosomes (P=0.0007) than non-senescent control cells. Moreover, TIS cells displayed an increased expression of the multidrug resistance protein 1/p-glycoprotein. MS analysis demonstrated that EVs derived from senescent Cal51 cells contained 142 proteins with a significant increased fold change compared with control EVs. Key proteins included ATPases, annexins, tubulins, integrins, Rabs and insoluble senescence-associated secretory phenotype (SASP) factors. A fluorescent analogue of PTX (Flutax-2) allowed appreciation of the removal of chemotherapy in EVs from senescent cells. Treatment of TIS cells with the exosome biogenesis inhibitor GW4869 resulted in reduced SA-β-Gal staining (P=0.04). In summary, this study demonstrates that TIS cells release significantly more EVs compared with control cells, containing chemotherapy and key proteins involved in cell proliferation, ATP depletion, apoptosis and the SASP. These findings may partially explain why cancer senescent cells remain viable despite chemotherapeutic challenge.

#458

Relationship between autophagy, activation and senescence in normal and cancer-associated human oral fibroblasts.

May Leng Tan,1 Eric K. Parkinson,2 Lee Fah Yap,1 Ian C. Paterson1. 1 _University of Malaya, Kuala Lumpur, Malaysia;_ 2 _Queen Mary University of London, London, United Kingdom_.

Cancer-associated fibroblasts (CAFs) isolated from oral squamous cell carcinoma (OSCC) display an activated phenotype in that they express α-smooth muscle actin (αSMA) and are frequently prematurely senescent. OSCC cell lines can induce senescence in normal fibroblasts in vitro by a mechanism that involves TGF-β and reactive oxygen species. Another phenotype closely associated with senescence is autophagy, but the relationship between autophagy, activation and senescence in CAFs has not been examined in detail. We first examined autophagosome accumulation in normal human oral fibroblasts (NHOFs; n=3) and in a panel of CAFs (n=8) that exhibited different degrees of senescence. Autophagosome accumulation was observed in senescent CAFs, as shown by the increased expression of the LC3B-II protein and by the number of LC3B cytoplasmic puncta, which were quantified using immunofluorescence. The number of LC3B puncta was significantly greater (p<0.01) in senescent CAFs compared to NHOFs, and senescent CAFs contained a higher fraction of autophagic fibroblasts than NHOFs, as indicated by increased number of LC3B puncta per cell. Further, the number of LC3B puncta correlated positively with the degree of senescence, as demonstrated by senescence-associated β-galactosidase (SA β-Gal) activity. To investigate whether autophagy, activation and senescence develop simultaneously when fibroblasts are stimulated to undergo senescence, we examined the temporal induction of these phenotypes in NHOFs in response to TGF-β1. NHOFs were treated with TGF-β1 (4 ng/ml) for up to 10 days to induce autophagy, αSMA expression and SA β-Gal activity. The results showed that autophagic flux preceded fibroblast activation, which in turn was followed by senescence. In summary, our results demonstrate that CAFs from OSCC concomitantly display autophagic, activated and senescent phenotypes and that the induction of autophagy occurs before fibroblast activation and sensecence. Taken together, these data suggest that autophagy, activation and sensecence in fibroblasts may be part of a unified programme leading to the development of CAFs.

#459

Therapy-induced senescence is reversible in vitro and in vivo.

Tareq Saleh,1 Moureq R. Alotaibi,2 Sarah L. Kyte,1 Emmanuel K. Cudjoe,1 Ajinkya Kawale,1 Jingwen Xu,3 Zeinab Elsayed,1 Joseph W. Landry,1 Scott C. Henderson,4 Vasily Yakovlev,1 Lynne W. Elmore,5 David A. Gewirtz1. 1 _Virginia Commonwealth Univ., Richmond, VA;_ 2 _King Saud University, Riyadh, Saudi Arabia;_ 3 _Shenyang Pharmaceutical University, Richmond, China;_ 4 _The Scripps Research Institute, La Jolla, CA;_ 5 _American Cancer Society, Atalanta, GA_.

While cellular senescence has long been recognized as an irreversible form of growth arrest, evidence in the literature has suggested that subpopulations of senescent tumor cells may retain proliferative capacity. To directly address this question, H460 non-small cell lung cancer cells induced into senescence by exposure to etoposide, and enriched based on β-galactosidase staining and size, were shown to recover reproductive capacity, which was accompanied by resolution of the DNA-damage-response (downregulation of p53 and p21Waf1/Cip1 induction), attenuation of the senescence-associated secretory phenotype (SASP) as well as downregulation of miRNA34 expression and an increase in c-myc. The senescence-enriched lung cancer cells were also capable of tumor growth upon transplantation into immunodeficient mice. Re-emergence from etoposide-induced senescence was also observed using HCT116 colon cancer cells, as evidenced by concomitant downregulation of a senescence-associated reporter (BTG1-RFP). Collectively, these findings indicate that therapy-induced senescence (TIS) may ultimately be a transient process in that at least a subpopulation of tumor cells can resume proliferation. We propose that some forms of tumor dormancy that lead to disease recurrence may reflect cells that enter into and ultimately escape from senescence.

#460

Reversibility of chemotherapy-induced senescence in breast tumor cells in culture and in vivo.

Liliya Tyutyunyk, Joseph Landry, Tareq Saleh, David Gewirtz. _Virginia Commonwealth University, Richmond, VA_.

Despite the ability of chemotherapy (and/or radiation) to eliminate the majority of tumor cells, some residual surviving cells may be able to escape cell death by entering into a prolonged growth arrest from which they can ultimately re-emerge and proliferative . In this study, we evaluated two mechanisms, autophagy and senescence, that in theory could allow tumor cells to escape apoptotic cell death, and which therefore might be responsible for cancer recurrence. 4T1 murine breast cancer cells exposed to the chemotherapeutic agent, Adriamycin (doxorubicin), underwent both autophagy and senescence. Senescence induced by anticancer drugs or radiation is a state of prolonged growth arrest that is thought to be irreversible and is considered an alternative (but nevertheless desirable) response to apoptosis. Our studies, however, suggest that Therapy Induced Senescence (TIS) is reversible and serves as a mechanism of evading cell death. When subjected to acute treatment with Adriamycin, some of the 4T1 cells underwent apoptosis; however, a large population entered into a state of prolonged growth arrest that lasted for several days, after which time the cells recovered proliferative capacity and formed colonies. At the time points when cells were arrested, prominent Senescence Associated β-Galactosidase staining and polyploidy were detected, markers indicative of senescence. Inhibition of autophagy did not prevent the tumor cells from undergoing senescence and later escaping growth arrest. To further establish the reversibility of senescence, senescent cells that were isolated by Flow Cytometry (based on their enlarged morphology and detection of a fluorescent β-Gal substrate (C12FDG)), plated and monitored over time; these cells were also shown to recover, as was the case from with the non-sorted senescent cells. Sorted and isolated senescent cells implanted into either NSG (immunodeficient) or syngeneic (immunocompetent) mice also recovered and developed into tumors; recovery was accelerated in the NSG mice, suggesting that the immune system may (at least transiently) recognize and eliminate the tumor cells either at senescence or during recovery from senescence. Senescent cells are characterized by a specific (though often heterogeneous) secretory profile (Senescence Associated Secretory Phenotype or SASP), some components of which have been shown to promote tumorigenesis and may assist in neoplastic escape after therapy. Furthermore, senescence and escape from senescence may represent a form of tumor dormancy and disease recurrence, respectively. Therefore, it is important to treat Therapy Induced Senescence as a deleterious outcome of treatment and identify strategies that might prevent senescent cells from neoplastic escape and induce cell death to completely eliminate residual tumors.

#461

**An essential role of the iron-sulfur cluster assembly enzyme isu-1 in the aging and stress responses of** C. elegans **.**

Rui Xiao, Yi Sheng. _University of Florida, Gainesville, FL_.

Iron is essential for animal cellular homeostasis by acting in the biogenesis of two important redox-reactive prosthetic groups of enzymes: iron sulfur clusters (ISC) and heme. Previous studies have indicated that the misregulated iron metabolism is associated with multiple aging-related neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease. However, how iron metabolism modulates the aging process remains largely elusive.In the current study, we aim to reveal the roles and mechanisms of iron metabolism in the aging process of C. elegans, a well-established genetic model organism for aging research. Through our preliminary studies, we have found that the iron-sulfur cluster assembly gene isu-1 plays an important role in the lifespan modulation and stress resistance of C. elegans. Specifically, RNAi knocking-down of isu-1 causes a significantly upregulated mitochondrial unfolded protein response (mitoUPR). Furthermore, the isu-1 RNAi-treated worms are significantly more resistant to heat shock and oxidative stress. Lastly, isu-1 RNAi significantly extends lifespan (~25.7%).Currently, we are examining the transcription factors downstream of isu-1 in regulating aging and stress responses. Surprisingly, the isu-1 RNAi-triggered lifespan extension is independent of insulin and IGF pathway, an evolutionarily conserved master regulator of animal growth and aging. Next, we will characterize the mechanisms underlying the effects of iron-sulfur cluster in animal aging and stress response using various genetic and biochemistry approaches.

#462

Hyperactivation of ERK/MAPK pathway reduces pancreatic cancer cells' proliferation and tumor progression through phosphoproteome reprogramming.

Marie-Camille Rowell,1 Xavier Deschênes-Simard,2 Benjamin Le Calvé,3 Stéphane Lopes-Paciência,1 Ana Fernandez Ruiz,1 Filippos Kottakis,4 Nabeel Bardeesy,4 Gerardo Ferbeyre1. 1 _University of Montreal, Montreal, Quebec, Canada;_ 2 _McGill University, Montreal, Quebec, Canada;_ 3 _University of Namur, Namur, Belgium;_ 4 _Massachusetts General Hospital Cancer Center, Harvard University, Boston, MA_.

This study aims to better understand the loss of tumor -uppression functions of ERK in pancreatic cancer progression. Pancreatic cancer is the 4th leading cause of death by cancer in Canada and USA with a 5-year survival rate below 7%. We have identified a high level of phosphorylation of the MAP kinase ERK in benign pancreatic intraepithelial neoplasms (PanIN), followed by a reduction in phospho-ERK levels in pancreatic ductal adenocarcinoma (PDAC) both in human patient samples and KRas-driven mouse models for pancreatic cancer. Since PanINs are considered senescent lesions, we hypothesized that phospho-ERK levels were reduced in PDAC to bypass oncogene-induced senescence and allow tumor initiation. We propose that reactivating high ERK in PDAC could restore tumor-suppression mechanisms lost during progression. To do so, we use a tamoxifen-inducible constitutive allele of RAF1 kinase, which acts upstream of ERK (∆RAF1-ERT). In human (PANC-1, KP-4) and murine PDAC cell lines, activated RAF1 slows cell proliferation and induces cellular senescence in vitro in an ERK-dependent and p53/p16-independent manner. Activation of ERK kinases by this system in subcutaneous PDAC xenografts slows tumor growth in nude mice. To characterize key mediators of ERK-dependent tumor suppression, we did phosphoproteomics by mass spectrometry and identified a reprogramming of nuclear vs. cytoplasmic phosphoproteome, involving key ribosome biogenesis factors. Depletion of these nucleolar targets by shRNAs and nucleolar stress inducers such as RNA polymerase I inhibitor CX-5461 recapitulates ERK-dependent tumor suppression. At the same time, we are also investigating autophagy-related processes involved in RAF/MEK signaling blockade in PDAC cells. In summary, we suggest that high levels of p-ERK1/2 in early PanIN stage act as a tumor-suppressor mechanism, which is lost with cancer progression. Our future work aims to better understand the mechanism by which high ERK signaling exerts tumor suppression in order to find downstream effectors that could be used as therapeutic targets in pancreatic cancer.

#463

Glucose-regulated protein 94 modulates the response of osteosarcoma to chemotherapy.

Huang Chien-Yu,1 Po-Li Wei,2 Yu-Jia Chang2. 1 _Shuang Ho Hospital, Taipei Medical University, Taipei, Taipei, Taiwan;_ 2 _Taipei Medical University and Hospital, Taipei, Taiwan_.

Background: Osteosarcoma (OS) is the most common and most aggressive primary solid malignant bone tumor in children and young adults and has high rates of recurrence and metastasis. The endoplasmic reticulum (ER) stress pathway is important in regulating chemo-responsiveness in cancer. However, the role of glucose-regulated protein 94 (GRP94) in regulating the chemotherapy response of OS has never been explored.

Method: In this study, GRP94-knockdown (GRP94-KD) MG63 OS cells were generated using short hairpin RNAs, and the response to chemotherapy was assessed by an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay.

Results: GRP94-KD cells were shown to be more resistant to chemotherapy than scrambled control cells, and the scrambled control cells had higher levels of apoptosis after chemotherapy than GRP94-KD cells.

Conclusion: Therefore, silencing GRP94 may increase resistance to chemotherapy treatment in OS cells by inhibiting the activation of apoptosis. Thus, GRP94 may be a key biomarker for the chemotherapeutic response of OS.

#464

Cell death and senescence: Role of protein kinase C in promoting senescence.

Etta Livneh, Assaf Ben Ari, Udi Zurgil. _Ben-Gurion Univ. of the Negev, Beer-Sheva, Israel_.

Senescence is characterized by permanent cell cycle arrest and loss of proliferative capacity, despite continued viability and metabolic activity. Senescent cells undergo massive genome modulation including induction of the senescence-associated secretory phenotype (SAPS). Components of SASP can implement cell cycle arrest or recruit the immune system to clear senescent cells, thus contributing to tumor suppression. However, some secreted pro-inflammation molecules, act as pro-tumorigenic agents contributing to tumor progression, suggesting that elimination of senescent cells could be beneficial. Very few studies showed a role for PKC in senescence. Here we show that PKCeta, an epithelial specific and anti-apoptotic kinase (epithelia is the origin of about 90% of human tumors), promotes senescence induced by oxidative stress and DNA damage. Using PKCeta-knockdown breast adenocarcinoma MCF-7 cells we show that PKCeta promotes senescence induced by oxidative stress and DNA damage via its ability to upregulate the expression of the cell cycle inhibitors p21Cip1 and p27Kip1 and to modulate transcription of major components of SASP such as IL-6 and IL-8. Moreover, we demonstrate that PKCeta creates a positive loop for reinforcing senescence by increasing the transcription of both IL-6 and IL-6 receptor. Thus, the expression of PKCeta modulates major components of SASP. Furthermore, our studies demonstrate that PKCeta interferes with γH2AX phosphorylation, which mark DNA double-strand breaks for repair. The phosphorylation of γH2AX may reflect the extent of DNA breaks but also the cellular response to DNA damage. Our experiments suggest that the response to DNA damage (repair processes) was more efficient in PKCeta-knockdown cells. The phosphorylation on ATM and Chk2 was also lower in PKCeta expressing cells. Revealing the molecular regulators of senescence will improve our ability to develop new therapeutic strategies for clearing tumor cells. Our studies showing a role for PKCeta in senescence could identify PKCeta and/or its activated signaling cascades as targets for intervention senescence induction. Since PKCeta is highly expressed in epithelia that are the origin of about 90% of human tumors, this may provide critical inhibitors as therapeutic adjuvants for different types of carcinomas.

#465

Regulation of HMGB1 release protects chemoradiotherapy-associated mucositis.

Keon-Il Im,1 Young-Sun Nam,1 Nayoun Kim,1 YuneJin Song,1 Young-Woo Jeon,2 Jung-Yeon Lim,1 Seok-Goo Cho2. 1 _The Catholic University of Korea, Seoul, Republic of Korea;_ 2 _Department of Hematology, Catholic Blood and Marrow Transplantation Center, Seoul St. Mary's Hospital, The Catholic University of Korea, Seoul, Republic of Korea_.

Oral mucositis (OM) is a common complication in cancer patients undergoing anticancer treatment. Despite the clinical and economic consequences of OM, there are no drugs available for its fundamental control. Here, we show that high-mobility group box 1 (HMGB1), a "danger signal" that acts as a potent innate immune mediator, plays a critical role in the pathogenesis of OM. In addition, we investigated treatment of OM through HMGB1 blockade using NecroX-7 (tetrahydropyran-4-yl)-[2-phenyl-5-(1,1-dioxo-thiomorpholin-4-yl)methyl-1Hindole-7-yl]amine). NecroX-7 ameliorated basal-layer epithelial cell death and ulcer size in OM induced by chemotherapy or radiotherapy. This protective effect of NecroX-7 was mediated by inhibition of HMGB1 release and downregulation of mitochondrial oxidative stress. Additionally, NecroX-7 inhibited the HMGB1-induced release of TNF-α, IL-1β and MIP-1β, as well as the expression of p53-upregulated modulator of apoptosis (PUMA) and the excessive inflammatory microenvironment, including nuclear factor kB (NF-kB) pathways. In conclusion, our findings suggest that HMGB1 plays a key role in the pathogenesis of OM; therefore, blockade of HMGB1 by NecroX-7 may be a novel therapeutic strategy for OM.

#466

Cellular senescence drives skin carcinogenesis.

Fatouma Alimirah, Alexis Valdovinos, Emily Chang, Sena Alptekin, Tanya Pulido, Elijah Jones, Chandani Limbad, Michael Velarde, Marco Demaria, Albert Davalos, Judith Campisi. _Buck Institute for Research on Aging, Novato, CA_.

Squamous cell carcinoma (SCC) is the second most common non-melanoma skin cancer, and has a high rate of recurrence and metastasis. Systemic genotoxic chemotherapeutic drugs such as doxorubicin (Doxo) can be used to treat SCC, but these agents can have deleterious long-term side effects, including fueling the development of more aggressive cancers. Some of the adverse effects of genotoxic chemotherapies might be due to their ability to induce cellular senescence. Cellular senescence is a tumor suppressive mechanism that entails a permanent cell growth arrest accompanied by several metabolic changes. Senescent cells increase during aging, and accumulating evidence suggests they can contribute to a variety of age related pathologies, including cancer, by secreting numerous pro-inflammatory molecules, growth factors and proteases, a feature termed the senescence-associated secretory phenotype (SASP). We recently showed that genotoxic and cytotoxic chemotherapeutic agents induce senescence and a SASP in vivo. However, a direct role for senescent cells in skin carcinogenesis in vivo remains unexplored. To investigate the role of senescent cells and the SASP in squamous cell skin carcinogenesis, we first induced senescence with Doxo in p16-3MR transgenic mice. These mice are designed to permit the selective elimination of senescent cells expressing the established senescence marker p16INK4a (p16) by administrating ganciclovir (GCV) which has a high affinity for the herpes simplex viral thymidine kinase (HSV-TK). Phosphorylated GCV is a DNA chain terminator that kills HSV-TK expressing cells by apoptosis. After Doxo treatment, we initiated skin SCC in p16-3MR mice with 7, 12-dimethylbenz[a]anthracene (DMBA), followed by treatment with 12-O-Tetradecanoylphorbol-13-acetate (TPA) to promote skin carcinogenesis. 25 weeks after Doxo treatment, we observed a significant increase in p16 mRNA levels as well as an increase in expression of the SASP factors IL-1α, MMP-9, and IL-17 in the skin of Doxo- compared to PBS- treated mice. Further, the presence of senescent cells resulted in significantly larger and more proliferative tumors compared to PBS-treated mice. Importantly, systemic elimination of Doxo-induced senescent cells using GCV reduced the expression of SASP factors and significantly reduced tumor size, suggesting that senescent cells fuel skin tumor progression. These findings will help our understanding of the role of chemotherapy-induced cellular senescence in skin carcinogenesis and will pave the way for developing novel therapeutics against squamous cell carcinoma and the deleterious side effects of genotoxic chemotherapies.

#468

Antioxidant supplements may accelerate the formation and progression of hepatocellular carcinoma in vitro and in vivo.

Xin Zhang, Eva Lee, Yu-Man Tsui, Man-Fong Sze, Yung-Tuen Chiu, Lo-Kong Chan, Daniel Wai-Hung Ho, Lu-Qing Zhao, Macrina Wai-Ling Lam, Lu Tian, Joyce Man-Fong Lee, Derek Lee, Carmen Chak-Lui Wong, Irene Oi-Lin Ng. _The University of Hong Kong, Hong Kong, Hong Kong_.

Antioxidants are electron donors that can neutralize and inhibit reactive oxygen species (ROS). Some evidence has shown that not only normal cells but cancer cells also require antioxidants, as cancer cells produce even more ROS as a result of their rapid growth. N-acetylcysteine (NAC) and glutathione (GSH) are popular antioxidant supplements for humans. A recent study done by Martin O' team has revealed that NAC could accelerate lung cancer via reducing the ROS-related DNA damage in mice. Tak Mak's group has reported that the GSH is essential for breast cancer initiation in mice. Liver cancer (hepatocellular carcinoma) is the fifth leading cause of cancer-related death worldwide. Whether NAC and GSH would aggravate HCC remains unknown. In this study, we aimed to investigate the effects of popular antioxidant supplements including NAC and GSH on HCC both in vitro and in vivo. We first screened the cell viability and colony formation effects of NAC and GSH on 5 HCC cell lines, MHCC97L cells showed the most promoting effects upon treatments. To further evaluate the effects of NAC and GSH on HCC initiation, growth and progression in vitro, we performed functional tests such as sphere formation, cell proliferation, migration and invasion assays in MHCC97L cells. Subcutaneous implantation and orthotopic liver injection mouse models were employed to further investigate the effects of NAC on tumorigenicity and tumor progression in vivo. To reveal the underlying mechanism, we also tested the expression levels of proteins involved in the ROS defence system. From the in vitro experiments, NAC-treated HCC cells promoted colony formation, suggesting that NAC might facilitate tumor cell growth. GSH increased the sphere formation, which is a test for self-renewal ability in vitro, and cell proliferation in MHCC97L cells. Significant enhancement in cell migratory and invasive abilities was observed in both NAC- and GSH-treated HCC cells. For the in vivo tumorigenicity assay, 120 mg/kg/day of NAC (based on conversion from the human equivalent dosage) was given to mice immediately after the tumor cell injection subcutaneously. Toxic side effects were observed in NAC-treated mice such as distended abdomen and weight loss despite a reduction in tumor incidence. In the orthotopic liver injection model, mice were treated with 60 mg/kg/day of NAC after tumor onset. NAC administration significantly increased the tumor masses and incidence of lung metastases when compared to the control group. Furthermore, NAC reduced the expression of p21 by western blotting while GSH showed no significant effect on protein markers involved in ROS defence pathways, such as AMT, P53 and H2AX. To conclude, our data show that antioxidants supplements could exacerbate HCC growth and progression in HCC cells in vitro and mice in vivo. The role of antioxidant supplements such as NAC and GSH in cancer therapy deserves more extensive studies.

#469

Therapy-induced senescence associated secretory phenotype enhances breast cancer cell invasion and stemness via CXCR1/2-CXCR1/2 ligands axis.

Yong Won Choi, Hyun-Young Cha, Tae Jun Park, Mi Sun Ahn, Hyun Woo Lee, Seong Hyun Jeong, Seok Yun Kang, Joon Seong Park, Jin-Hyuk Choi. _School of Medicine, Ajou University, Suwon, Republic of Korea_.

Various chemotherapeutic agents have been demonstrated to induce senescence of cancer cells in vitro and in vivo. This therapy-induced senescence (TIS) has been regard as a favorable therapeutic response, although it is not a cell death but a permanent cell cycle arrest. However, adverse aspects of TIS have been also suggested since senescence-associated secretory phenotype (SASP) enables to affect microenvironment of senescent cancer cells. Nonetheless, when senescent cancer cells are intermingled with non-senescent cells after chemotherapy, the effects of SASP from senescent cancer cell to non-senescent cell have not yet been fully elucidated. Therefore, TIS, confirmed by measuring senescence associated β-galactosidase activity and cell cycle arrest, was induced by the treatment with adriamycin and cisplatin in variable breast cancer cell lines in vitro. To evaluate the effects of SASP from senescent cancer cell to non-senescent cell, conditioned media (CM) from TIS of MCF7 (poorly-aggressive and non-invasive cell line) was treated in parent non-senescent MCF7 and their invasiveness and stemness were measured by invasion assay and mammosphere forming assay, respectively. In MCF7 treated with CM from TIS, invasiveness and mammosphere forming ability were significantly increased. To discover the mediators of the observed effects, cytokine array was performed by collecting CM from non-senescent control and therapy-induced senescent cells. In the secretome of TIS, CXCR2 ligands, including growth-related protein-α (CXCL1) and interleukin-8 (CXCL8) were focused on for further analysis. Increased mRNA of CXCR2 ligands (CXCL1, 2, 3 and CXCL8) in TIS and increased secretion of CXCL8 protein in CM of TIS were confirmed by real time PCR and ELISA, respectively. To investigate the contribution of CXCR1/2-CXCR1/2 ligands axis in enhanced invasion and stemness ability by CM of TIS, a CXCR1/2 specific inhibitor, SCH-527123, was applied. The pretreatment of SCH-527123 in the concentration without affecting cell proliferation and survival (25, 50μM), significantly inhibited the enhanced invasion and mammosphere forming ability by CM of TIS. The potential mechanisms of cancer cell invasion and stemness enhancement by CXCR1/2-CXCR1/2 ligands axis, are currently under investigated. In summary, SASP of TIS can be harmful by augmenting invasion and stemness of surrounding non-senescent cancer cell thorough the CXCR1/2-CXCR1/2 ligands axis and CXCR1/2 inhibitors can be used as therapeutic agents, by antagonizing the adverse effects of TIS.

#470

p53 isoform delta133p53 in tumor senescence.

Natalia Von Muhlinen, Jessica Beck, Curtis C. Harris. _National Cancer Institute, Bethesda, MD_.

The tumor suppressor gene TP53 expresses at least 12 isoforms due to alternative splicing, alternative initiation of translation, and alternative promoter usage. These isoforms include p53β and Δ133p53, which can modulate p53 transcriptional activity and apoptosis. The p53 isoform Δ133p53 is expressed in normal tissue, but is abnormally expressed in several cancer types. However, its role in tumor progression is still unclear. Normal somatic cells undergo a limited number of cell divisions, eventually leading to an irreversible proliferative growth arrest known as replicative senescence. Replicative senescence can act as a tumor suppressor mechanism, thus constituting a critical barrier to tumor progression in vivo. Previous studies showed that, p53β and Δ133p53 are endogenous regulators of replicative senescence in normal human fibroblasts. Interestingly, expression of Δ133p53 is upregulated in several cancer types. For instance, enhanced levels of Δ133p53 are associated with inhibition of cellular senescence in colon carcinoma as well as with progression of premalignant colon adenomas to colon carcinomas. However, little is known about the role of Δ133p53 in cancer. We are studying the roles of Δ133p53 in proliferation and senescence of cancer cell lines. We hypothesized that depletion of otherwise enhanced Δ133p53 isoform induces cellular growth arrest and increases the secretion of senescence-associated secretory phenotype (SASP) pro-inflammatory cytokines such as IL-6 and IL-8 of cancer cell lines, and thus Δ133p53 may be a therapeutic target to repress tumor cell growth.

### MicroRNA Regulation in Cancer Biology

#471

High expression of miR-182 promotes prostate cancer aggressiveness in African-Americans compared to Caucasians.

Marisa Shiina, Yutaka Hashimoto, Guoren Deng, Varahram Shahryari, Majid Shahana, Soichiro Yamamura, Priyanka Kulkarni, Pritha Dasgupta, Divya Bhagirath, Sharanjot Sharan, Yuichiro Tanaka, Rajvir Dahiya. _UCSF/VA Medical Center, San Francisco, CA_.

Background: African-Americans (AfA) have higher risk for developing prostate cancer and when diagnosed, the cancer is more aggressive with worse survival compared to Caucasians (CA). To clarify the mechanisms involved in this disparity, we analyzed the role of miR-182 in AfA and CA prostate cancer tissues and cells. Materials and Methods: We analyzed miR-182 expression in AfA and CA prostate cancer tissue samples by Real Time PCR. We selected two cell lines, DU-145 (CA) and MDA-PCa-2b (AfA), which express different levels of miR-182, to mimic the tissue samples and help identify the mechanisms related to racial disparity. We generated lentivirus stable miR-182 knockdown cells lines and performed cell viability assays using a CellTiter-Glo luminescent assay. For cell cycle analysis, cells were stained with PI/RNase staining buffer and analyzed for DNA content by BD FACSVerse. Cancer pathway-focused gene expression profiling was done using a human RT2 Profiler Cancer Pathway Finder and Oncogenes and Tumor Suppressor Genes PCR Array. Results: We found miR-182 expression to be higher in AfA prostate cancer tissues compared to CA patients with localized disease. Also, expression of miR-182 in AfA cell line, MDA-PCa-2b, was significantly higher compared to CA cell line, DU-145. To determine the role of miR-182 in the differences between AfA and CA, miR-182 was knocked down in MDA-PCa-2b and DU- 145 cells. miR-182 knockdown in MDA-PCa-2b cells effectively inhibited cell migration, invasion and colony formation compared to control cells, while there was no difference in DU-145 cells. Knockdown of miR-182 caused a decrease in MDA-PCa-2b cell viability compared with negative control. In addition, the cell cycle profile showed an increase in the G1 phase of MDA-PCa-2b cells (control 7.3% compared to miR-182 knockdown cells 18.6%), whereas no change was observed in DU-145 cells. Similar to these results, wound healing was significantly slower in the miR-182 knockdown MDA-PCa-2b cells compared to controls. PCR array analysis was performed to determine the molecular effects of miR-182 knockdown in MDA-PCa-2b and DU-145 cells. We found from our PCR array data some potential targets genes regulated by miR-182. Conclusion: We conclude that high levels of miR-182 is associated with prostate cancer aggressiveness in African-Americans.

#472

Tumor DNA methylation and microRNA expression in breast cancer among American women of African or European ancestry.

Zhihong Gong,1 Dan Wang,2 Jie Wang,3 Matthew F. Buas,1 Song Liu,1 Steven A. Belinsky,4 Michael J. Higgins,1 Christine B. Ambrosone1. 1 _Roswell Park Cancer Inst., Buffalo, NY;_ 2 _Eli Lilly and Company, Indianapolis, IN;_ 3 _University at Buffalo, Buffalo, NY;_ 4 _Lovelace Respiratory Research Institute, Albuquerque, NM_.

American women of African ancestry (AA) are more likely than those of European ancestry (EA) to be diagnosed with aggressive, estrogen receptor (ER) negative breast cancer, and to die of this disease. The reasons remain largely unknown. Epigenetic mechanisms, particularly differential DNA methylation and altered microRNA expression, may contribute to this racial disparity. We previously conducted genome-wide DNA methylation profiling of fresh-frozen breast tumor samples from 58 AA and 80 EA women, with subsequent analyses restricted to CpGs within coding genes. In this study, we focused on CpGs within loci encoding microRNAs, and sought to identify differentially methylated loci (DML) by ER status and by race. We identified a total of 297 DML (Δβ >0.1; FDR<0.05) between ER negative and ER positive breast tumors. Of these, 113 were differentially methylated regardless of race; 65 were specific to EAs; and 119 were specific to AAs. The top DML common to both races were located within miR-155HG, -365-1, -135b, -196b, and -190b, while the top race-specific DML were located within miR-1256, -1224, -1249, -9-3, -657/-338, and miR-25 (EA), or within miR-2053, -526a1, -518B, -489, and -125b1 (AA). We also identified 25 DML (Δβ >0.1; P<0.05) between AA and EA tumors. Among these 25 DML, all of which were hypomethylated in AAs versus EAs, three were specific to ER+ tumors (miR-548G/FILIP1L,-572, -661/PLEC1), 21 were specific to ER- tumors (e.g., miR-2053, -1323, -125b1), and one was common to both tumor subtypes (miR-662). In order to assess how DNA methylation may impact gene expression, we performed miRSeq on tumors from a subset of included women (29 AA and 29 EA). We observed that altered methylation at certain DML was inversely associated with microRNA expression; for example, three DML in the promoter region of miR-190b (Spearman's rho=-0.65, -0.68 and 0.55, respectively; all P<1.2x10-6), and two in miR-135b (rho=-0.56 and -0.59, respectively; both P<1.2x10-07). Interestingly, multiple loci within the promoter region of miR-190b were hypermethylated in ER- compared to ER+ tumors, and we further observed that miR-190b expression was significantly downregulated in ER- versus ER+ tumors (log2FC=-2.45 and -4.92 in EAs and AAs, respectively; FDR<0.05). Lower expression of miR-190 has been linked previously to reduced breast cancer survival, potentially reflecting effects on IGF-related pathways. In summary, based on genome-wide profiling of tumor DNA methylation and microRNA expression, our results suggest that DNA methylation patterns differ by tumor subtype and by race, and that this altered methylation may affect gene expression. These findings will provide the basis for further functional analyses, which are likely to improve our understanding of race-related tumor biological differences and lead to specific targeted preventative and therapeutic strategies.

#473

miR-135b mediates gemcitabine sensitivity in breast cancer cells by modulating epithelial-to-mesenchymal transition and mTOR-signaling.

Anna Tessari, Meghan Pawlikowski, Kareesma Parbhoo, Olivia Willetts, Marianna Hernandez, Giovanni Nigita, Ashley Braddom, Joseph J. Mills, Dario Palmieri, Carlo M. Croce. _The Ohio State University, Columbus, OH_.

Background: We previously identified miR-135b as a possible biomarker for gemcitabine sensitivity in metastatic breast cancer patients. Here we aimed to better understand the role of this microRNA in breast cancer cells in order to identify new clinically relevant approaches to revert gemcitabine resistance.

Methods: miR-135b KO MDA-MB-231 breast cancer cells were generated through CRISPR-Cas9 technology and tested for gemcitabine sensitivity by cell survival and apoptosis activation assays. Gene expression profile was performed by GeneChip® Human Transcriptome Array 2.0 (HTA 2.0 - Affymetrix). The One-Way between-subject ANOVA algorithm was used to calculate statistical significance of pairwise comparisons. The validation of different gene expression was assessed by qRT-PCR. The mesenchymal phenotype of miR-135b KO cells was tested by wound-healing and migration assay. mTOR pathway has been tested by Western blot analysis.

Results: miR-135b KO MDA-MB-231 showed increased resistance to gemcitabine than WT cells. Gene expression profiling of miR-135b WT and KO cells revealed the upregulation of several genes, including miR-21 and gene involved in the epithelial-to-mesenchymal transition (EMT) in the absence of the microRNA. Functional enrichment analysis indicated the deregulation of multiple tumor-associated pathways, including focal adhesion, senescence and autophagy. Accordingly, miR-135b KO cells showed higher migrating properties and expression of mesenchymal genes. Moreover, miR-135b KO cells showed deregulation of the mTOR-signaling pathway upon starvation or treatment with gemcitabine. Based on these observations, we assessed whether inhibition of mTOR activation using a clinically approved inhibitor (everolimus) could enhance gemcitabine sensitivity in the absence of miR-135b. Our data indicate that blocking mTOR pathway could represent a valid approach to revert resistance to gemcitabine even in cancer cells with low levels of miR-135b.

Conclusions: We identified several deregulated genes and pathways in the absence of miR-135b in breast cancer cells, in particular related to the EMT and mTOR pathway. These results confirm the role of miR-135b in breast cancer and pave the way for new studies for the combination of gemcitabine and mTOR inhibitors in a specific subset of metastatic breast cancer patients that could largely benefit from it.

#474

Alterations of micro-RNAs are associated with melanoma resistance to BRAF inhibitors: Role of miR-126.

Simona Caporali,1 Lauretta Levati,1 Ester Alvino,2 Adriana Amaro,3 Pedro Miguel Lacal,1 Laura Bonmassar,1 Cristian Bassi,4 Laura Lupini,4 Gian Carlo Antonini Cappellini,1 Ulrich Pfeffer,3 Massimo Negrini,4 Nadia Felli,5 Alessandra Carè,5 Giandomenico Russo,1 Stefania D'Atri1. 1 _Istituto Dermopatico dell'Immacolata-IRCCS, Rome, Italy;_ 2 _National Council of Research, Rome, Italy;_ 3 _IRCCS-AUO San Martino, National Cancer Research Institute, Genoa, Italy;_ 4 _University of Ferrara, Ferrara, Italy;_ 5 _Istituto Superiore di Sanità, Rome, Italy_.

Altered expression of miRNAs has been demonstrated in tumor tissue and plasma/serum of cancer patients. miRNAs have been shown to have both diagnostic and prognostic significance and to potentially constitute novel targets and therapeutic agents for cancer treatment. Experimental evidences also support an involvement of miRNAs in tumor cell response to therapy. In this study, we explored the role of miRNAs in melanoma resistance to BRAF inhibitors (BRAFi). The melanoma cell line A375 and its dabrafenib-resistant subline A375R, which displays increased invasiveness and VEGF secretion, were analyzed for miRNA expression using Affymetrix GeneChip® miRNA 3.1 microarrays. Differential expression of selected miRNAs was confirmed in the two cell lines using qRT-PCR. miR-126, previously shown to act as a tumor suppressor gene in melanoma, was chosen for additional studies that were performed also in a second pair of matched melanoma cell lines, sensitive or resistant to dabrafenib (i.e SK-Mel28 and SK-Mel28R). The resistant sublines were transfected with 50 nM Pre-miR hsa-miR-126 miRNA Precursor (pre-miRNA-126) or Pre-miR miRNA Precursor Negative Control#1 (Ambion®) and analyzed for proliferation 6 days later using the MTT assay. Seventy-two hours after transfection, the cells were also assayed for invasion of the extracellular matrix and VEGF-A secretion using Boyden Chamber and ELISA assays, respectively. In 33 melanoma patients treated with BRAFi, alone or in combination with MEKi, plasma samples were collected before the beginning of therapy (T0), and two months later (T2) and subjected to miRNA expression profiling by small RNA-seq. A375R cells displayed 13 up-regulated miRNAs and 32 down-regulated miRNAs with respect to A375 cells (SAM analysis; fold change ≥ 2). Down-regulation of miR-126 was confirmed by qRT-PCR analysis in both A375R and SK-Mel28R cells. Restoration of miR-126 expression in A375R and SK-Mel28R cells by pre-miRNA-126 transfection impaired proliferation, invasion and secretion of VEGF-A, a validated target of miR-126. Fifthy and 28 circulating miRNAs were differentially expressed at T0 and at T2, respectively, between patients who responded to therapy (n=28) and patients who did not (n=5). Our results demonstrate that down-regulation of miR-126 expression is associated with acquired resistance to dabrafenib in melanoma cells and suggest that restoration of this miRNA in dabrafenib-resistant melanomas might restrain tumor growth and metastasis. They also show that in melanoma patients with primary resistance to BRAFi, a set of circulating miRNAs is differentially expressed with respect to patients responding to therapy, suggesting a potential role of circulating miRNAs as biomarkers for early prediction of drug response. Supported by the Italian Ministry of Health, grant 5PerMille-2010 and AIRC, IGP 17585

#475

Characterization of miR-21 expression in budding colon cancer cells using digital images of multiplex fluorescence stained slides obtained by confocal scanning microscopy.

Boye Schnack Nielsen,1 Kirsten Nguyen Knudsen,2 Jan Lindebjerg,2 Alexandra Kalmár,3 Béla Molnár,3 Flemming Sorensen,2 Torben Hansen2. 1 _Bioneer A/S, Horsholm, Denmark;_ 2 _Vejle Hospital, Vejle, Denmark;_ 3 _Semmelweis University, Budapest, Hungary_.

This study was undertaken to analyze the presence and prevalence of microRNA-21 (miR-21) positive budding cancer cells in colon cancer using confocal scanning microscopy. Budding cancer cells are locally invasive cancer cells with increased metastatic properties and characteristics of epithelial to mesenchymal transition (EMT). Expression of miR-21 in stromal fibroblastic cells in colorectal cancers is well documented (1,2), whereas miR-21 expression in budding cells is poorly described. In order to characterize miR-21 positive budding cells, we first developed a multiplex fluorescence staining method by combining miR-21 in situ hybridization with immunohistochemical staining for cytokeratin and budding cell marker laminin-γ2, and stained 16 colon cancer cases (stage II, n=5, stage III, n=11). We then employed a confocal scanning microscope to obtain 15-55 GB digital images covering areas of 10-40 mm2 of the invasive front. The high resolution of the confocal digital images allowed detailed examination of the 4-fluorophore-stained slides using extended focus and z-stack images, e.g. in the discrimination of epithelial cells from adjacent stromal cells. All cytokeratin-positive budding cells were evaluated for the presence of miR-21 and laminin-γ2. Five out of the 16 cases had more than 10% miR-21 positive budding cells, a few of which were also laminin-γ2 positive. All five cases were stage III cancers. The presence of miR-21 in the budding cancer cells was not associated with the level of tumor budding. These observations suggest that the miR-21 expression in tumor budding cells increases with the progression of the cancer and is independent of laminin-γ2. References: 1. Nielsen BS et al, Clin Exp Mets 2011, 28:27-38. 2. Kjaer-Frifeldt S et al, Br J Cancer 2012, 107:1169-1174.

#476

A human scFv as a tool to understand the biogenesis of a subset of oncogenic microRNAs.

Valerio Embrione,1 Michael Scarpati,2 Zachary D. Uzzel,1 Rahimah Ahmad,2 Fortuna A. De Sury,1 Tyler J. Sheetz,1 Vincenzo Coppola,1 Shaneen M. Singh,2 Anjana D. Saxena,2 Carlo M. Croce,1 Dario Palmieri1. 1 _The Ohio State University, Columbus, OH;_ 2 _Brooklyn College, Brooklyn, NY_.

Trans-acting factors such as RNA-binding proteins (RBPs) and microRNAs (miRNA) have recently been recognized as prime regulators of gene expression during tumorigenesis. Nucleolin (NCL) is the most abundant nucleolar RNA-binding protein, controlling critical cellular processes such as chromatin remodeling, ribosome biogenesis and miRNA biogenesis. In the cytoplasm, NCL regulates gene expression post-transcriptionally by controlling mRNA stability and/or translation via ARE- and miRNA-mediated pathways. In contrast, NCL at the cell surface plays pivotal roles in cancer progression and metastasis in many types of tumors. Therefore, surface-NCL is an appealing target for cancer therapy. Earlier our group described 4LB5, a human-derived single chain fragment variable (scFv) that specifically targets surface-NCL exerting cytotoxic effects, both in vitro and in vivo. We have identified the central RNA-binding domains (RBD1-4) of NCL as 4LB5-interacting sites. 4LB5-mediated blocking of NCL-RBDs significantly impairs the biogenesis of a subset of oncogenic miRNAs, such as miR-21, -221 and -222, and their binding to the microprocessor complex. The structural fold of all the 4 RBDs is highly conserved, but there are significant differences in their sequences. RBD1-2 have been well studied in their mechanism and capability to form high-affinity RNA-binding complexes, but RBD 3-4 have not been explored to the same extent. We hypothesize that consecutive or selective combinations of RBDs in NCL might have distinct roles in RNA biology. NCL RBD1-2 domains are well documented in the literature for their high affinity for mRNAs and rRNAs. However, RNA-specificity on NCL-RBDs has yet to be elucidated. In this study, we used protein-antibody and protein-RNA docking analyses to identify potential differential binding affinities between multiple NCL modules (RDB1-2, RBD3-4 and RBD1-4) and either 4LB5 or miRNAs. In silico results were also evaluated by multiple biochemical assays (ELISA, Surface Plasmon Resonance, Pull-Down and Electrophoretic Mobility Shift Assays) using NCL Modules, 4LB5 and RNA molecules. Finally, competition assays were performed to assess ability of our scFv to impair miRNA binding to NCL. Our results indicate the importance of the NCL region targeted by 4LB5 in microRNA maturation. Functional studies will help us to better elucidate the biologic role of NCL RBDs, and their involvement in cancer pathogenesis.

#477

Targeting of ErbB3 with functional cooperative miRNAs enhances efficacy of trastuzumab in ErbB2-overexpressing breast cancer cells.

Hui Lyu, Jingcao Huang, Bolun Wang, Amy Han, Bolin Liu. _Univ. of Colorado Anschutz Medical Campus, Englewood, CO_.

Introduction: ErbB3 serves as a critical co-receptor of erbB2 and plays a vital role in the development of erbB2-overexpressing (erbB2+) breast cancer. It is thought to be an important compensatory target for combinatorial strategy to improve the treatments for erbB2+ breast cancer patients. MiRNAs regulate gene expression by the sequence-specific targeting of mRNAs, leading to translational repression or mRNA degradation. ErbB3 is a direct target of both miR-125a and miR-205. Here, we study the potential activity of miRNA based erbB3-targeted therapy in erbB2+ breast cancer cells.

Methods: Cell growth assays were used to determine cell viability. Western blot analyses were performed to assess the expression and activation of proteins. Flow cytometry analysis was carried out to examine cell cycle progression. Lentiviral vector containing one or two miRNAs was used to ectopically express miR-125a and/or miR-205.

Results: Co-expression of miR-125a and miR-205 showed a potent activity to downregulate erbB3 while single miRNA had no or little effect on erbB3 when miR-125a or miR-205 was only increased below 10-fold in BT474 cells. Combination of the two miRNAs not only resulted in a dramatic reduction of phosphorylated erbB3 (P-erbB3) and the downstream signaling kinases Akt (P-Akt) and Src (P-Scr), it also inhibited cell proliferation and increased the cells at G1 phase. More importantly, concomitant expression of the two miRNAs significantly enhanced trastuzumab-mediated growth inhibition and cell cycle G1 arrest in BT474 cells. Interestingly, the expression levels of miR-125a, but not miR-205 in the trastuzumab-resistant BT474-HR20 cells were much lower than that in the parental BT474 cells. Ectopic expression of miR-125a alone profoundly inhibited proliferation of BT474-HR20 cells. These data suggested that reduced miR-125a might be a novel mechanism leading to trastuzumab resistance; and upregulation of miR-125a could be a useful tool to abrogate the resistance.

Conclusions: Co-expression of miR-125a and miR-205 is a new approach to inhibit erbB3. Specific targeting of erbB3 via the functional cooperative miRNAs enhances efficacy of trastuzumab against erbB2+ breast cancer cells. Our data support further exploration of the possible role of miR-125a in the development of trastuzumab resistance.

Keywords: ErbB3, ErbB2, miRNA, Trastuzumab, Breast Cancer

#478

**Regulation of** MUC13 **by microRNA in colorectal cancer.**

Linda Bartu, Alena Opattova, Pavel Vodicka, Veronika Vymetalkova. _The Czech Academy of Sciences, Prague, Czech Republic_.

Colorectal cancer (CRC) represents a serious health problem with high incidence and mortality worldwide. The main reason of high mortality is the late diagnosis. For the Duke stage I, the average 5-years survival rate is 90%. In comparison to patients diagnosed at IV stage, the 5-years survival rate decreases to 3%. Mucinous CRC is generally defined as having greater than 50 % of the tumor area with a mucinous differentiation by histological examination. The increased incidence occurs in proximal colon and among younger patients, as compared to nonmucinous adenocarcinoma. Mucinous CRCs have been found to have a higher Duke stage at diagnosis, and, consequently a poorer prognosis. Mucines are surface or secreted glycoproteins with high molecular weight. Dysregulation of their expression may lead to malignant transformation. MUC13 overexpression influences colon cancer tumorigenesis and metastasis via multiple oncogenic proteins. Recently, we have observed that rs1532602 polymorphism in microRNA (miRNA) binding sites in MUC13 gene was associated with significantly decreased risk of CRC. The aim of the present study is to investigate regulation in vitro by miRNAs as a potential mechanism for increased MUC13 expression in CRC. Two different miRNAs were selected in this investigation. MiRNA-4647 is predicted to bind to the rs1532602 in MUC13. MiRNA-137 functions as a tumour suppressor in colon. Additionally, we investigated the impact of miRNAs mentioned above on regulation of MUC13 in a cohort of 113 patients with sporadic CRC. The present work provides important insights into role of miRNAs which bind to MUC13. Definition of functions, mechanisms of MUC13 regulation and its impact on CRC may contribute to better prediction and individual treatment of CRC. Acknowledgement: Grant Agency of the Ministry of Health of the Czech Republic (AZV MZ 15-26535A) and Grant Agency of the Czech Republic GACR 17-16857S.

#479

miR-372 **enhances tumorigenesis and drug resistance in oral carcinoma by targeting ZBTB7A transcription factor.**

Li-Yin Yeh,1 Chung-Hsien Chou,1 Chung-Ji Liu,2 Shu-Chun Lin,1 Kuo-Wei Chang2. 1 _National Yang-Ming Univ. Institute of Oral Biology, Taipei, Taiwan;_ 2 _National Yang-Ming Univ. School of Dentistry, Taipei, Taiwan_.

miRNA-mediated post-transcriptional regulation of targeted genes plays crucial roles in neoplastic process. miR-372 has been shown an oncogenic miRNA which is hypoxia-inducible and is conspicuously up-regulated in oral squamous cell carcinoma (OSCC). Zinc finger and BTB domain containing 7A (ZBTB7A) is a transcriptional repressor modulating a great diversities of physiological or oncogenic regulation. This study identified that ZBTB7A was a direct target of miR-372. ZBTB7A was down-regulated in 75% of OSCC tumors relative to adjacent oral mucosa. Reverse correlation across miR-372 expression and ZBTB7A expression was found in tumor samples. In OSCC cells with the stable knockdown of ZBTB7A, the oncogenic potential and drug resistance increased. Whereas, such increase was attenuated by ZBTB7A expression. High throughput screening and validation assay confirmed that ZBTB7A was able to repress multiple oncogenic factors and activate the expression of Trail-R1, Trail-R2 and Fas to increase the drug sensitivity in OSCC cells. miR-372 induction drastically repressed the expression of apoptosis genes by inhibiting ZBTB7A. This was accompany with the enrichment of oncogenicity and the increased tolerance to the toxicity of cisplatin and taxol. This study signifies the importance of miR-372-ZBTB7A-downstream effectors in mediating pathogenesis and drug resistance of OSCC. Interception of this pathogenic cascade would confer therapeutic benefits against oral carcinoma.

#480

Regulation of crosstalk between AR and Wnt/Beta-catenin pathways by miR-644a in prostate cancer.

Alexis R. Plaga, Girish C. Shukla. _Cleveland State University, Cleveland, OH_.

Prostate Cancer (PCa) is the most commonly diagnosed cancer in males with 161,360 people diagnosed in the United States and is predicted to cause 26,730 deaths in 2017. The current treatment of androgen deprivation therapy (ADT) initially depletes circulating androgens; however, intratumoral androgens rescue androgen receptor (AR) signaling and promotes the development of castration-resistant prostate cancer (CRPC). The dysregulation of the Wnt/Β-catenin signaling pathway has been implicated in the development of many cancers including PCa. The disruption of this signaling leads to the stabilization of Β-catenin which upregulates many genes involved in tumorigenesis. Additionally, Β-catenin acts as AR cofactor. Our previous studies show that AR is a direct target of miR-644a. We hypothesized that targeting the expression of both the Β-catenin pathway and AR by conventional drugs and/or tumor suppressor miR-644a would have synergistic therapeutic benefits. In this study, we are investigating miR-644a mediated posttranscriptional downregulation of GSK3-Β and Β-catenin in the wnt/Β-catenin signaling pathway. The effect of miR-644a in combination with inhibitors (Β-catenin or GSK3-Β) was also assessed using viability assays and a significant downregulation was observed. We will further study the posttranscriptional effects of tumor suppressor miR-644a on the wnt/Β-catenin pathway as well as assess if miR-644a can inhibit the cross-talk of these pathways.

#481

QKI, a direct target of miRNA-200, inhibits epithelial-to-mesenchymal transition.

Eun Ju KIM,1 Der Sheng Sun,2 Hye Sung Won,2 Yoon Ho Ko,2 Young-Ho Ahn1. 1 _College of Medicine, Ewha Womans University, Seoul, Republic of Korea;_ 2 _College of Medicine, The Catholic University, Seoul, Republic of Korea_.

MicroRNA (miR)-200 family members are well-known regulators of epithelial-to-mesenchymal transition (EMT) in epithelial tumors. To identify novel miR-200 targets, we analyzed TargetScan and TCGA data sets. Among 20 candidate genes, we selected QKI gene and studied its role during EMT process in human head and neck squamous cell carcinoma (HNSCC) and non-small cell lung carcinoma (NSCLC) cells. The expression of QKI was proved to negatively correlate with those of miR-200 members in TCGA data sets. miR-200 suppressed QKI expression directly, which was confirmed by 3'-UTR reporter assays. Interestingly, shRNA-mediated knockdown of QKI promoted migration, invasion, and EMT in cancer cells and increased tumor growth in a xenograft mouse model. These results indicate that QKI inhibits EMT and tumorigenesis in HNSCC and NSCLC cells. Even though further studies are necessary to elucidate the underlying mechanism, we suggest that QKI and miR-200 form a feedback loop to maintain homeostatic responses to EMT-inducing signals.

#482

Epstein-Barr virus-encoded miRNAs target ATM-mediated response in nasopharyngeal carcinoma.

Raymond W. Lung,1 Po-Man Hau,1 Joanna H. Tong,1 Ka-Hei Lam,1 Anthony W. Chan,1 Kevin Y. Yip,1 George S. Tsao,2 Kwok-Wai Lo,1 Ka-Fai To1. 1 _The Chinese University of Hong Kong, Hong Kong, Hong Kong;_ 2 _The University of Hong Kong, Hong Kong, Hong Kong_.

Nasopharyngeal carcinoma (NPC) is a highly invasive epithelial malignancy that is prevalence in southern China and Southeast Asia. It is well-known to be associated with latent Epstein-Barr virus (EBV) infection. By using small RNA sequencing, we previously established a comprehensive miRNA profile in a panel of NPC samples and revealed that the EBV-encoded miRNAs derived from BamHI-A rightward transcripts (miR-BARTs) are abundantly expressed. The important roles of miR-BARTs in cancer development have been reported extensively. In the present study, we identified multiple putative binding sites of miR-BART5-5p, BART7-3p, BART9-3p and BART14-3p on the 3'-UTR of a critical DNA double-strand break (DSB) repair gene, Ataxia-Telangiectasia-Mutant (ATM). Notably, the expression of these 4 miR-BARTs represented more than 10% of all EBV-encoded miRNAs in NPC cells while downregulation of ATM expression was observed in our local primary NPC samples in both qRT-PCR and immunohistochemical (IHC) staining analysis. In addition, the abilities of miR-BARTs to downregulate ATM expression were demonstrated in the transient transfection experiments. By manipulating the miR-BARTs expression in epithelial cell lines, we further revealed that these four viral miRNAs could inhibit both ionizing radiation (IR)-induced ATM kinase activity and BZLF1-induced viral lytic reactivation. In conclusion, our findings suggest that miR-BART5-5p, BART7-3p, BART9-3p and BART14-3p work co-operatively to modulate DNA damage response and to maintain viral latency, contributing to the NPC tumorigenesis. Acknowledgement: Theme-based Research Scheme (T12-401/13-R) and GRF (14104415 and 14138016), Research Grant Council, Hong Kong.

#483

BRCA1 protein expression is downregulated by miR-185, miR-93 and miR-107 in breast cancer tumors.

Valentina A. Zavala, Patricia Gajardo, Paola Faundez, Carolina Alvarez, Pilar Carvallo. _Pontificia Universidad Católica de Chile, Santiago, Chile_.

BRCA1 is a tumor-suppressor gene whose mutations confer a high risk to breast cancer. Fifty percent of hereditary breast cancer tumors without germline mutation have impaired BRCA1 expression, as well as 30% of sporadic cases. Somatic events that silence BRCA1 expression have been described in breast cancer tumors, such as promoter hypermethylation and BRCA1 gene deletion; however, these mechanisms do not completely explain the loss of BRCA1 expression in breast tumors. Our aim is to identify miRNAs differentially expressed in tumors with negative or moderate BRCA1 protein, which could regulate BRCA1 expression. Total RNA from 50 fresh-frozen breast cancer tumors was isolated and miRNA profiling was performed using miRNA microarrays. Sixteen miRNAs were found exclusively upregulated in BRCA1-negative, and ten in BRCA1-moderate tumors. Among them, in silico analyses predicted eleven miRNAs that could regulate BRCA1 expression. The effect of five of these microRNAs was assessed using a luciferase reporter assay in HEK293T cells, where the complete BRCA1 3'UTR, or a segment of its coding region, was cloned within the 3' end of the Luciferase gene. Cells were co-transfected with each of the selected microRNAs. MiR-185, miR-93 and miR-107 significantly downregulated luciferase activity by binding to the 3'UTR. Also, we transfected these microRNAs into a nontumorigenic breast cell line (MCF10A), where miR-93 diminished endogenous BRCA1 expression in 48%, miR-185 in 39% and miR-107 in 38%, in relation to non-transfected cells. These results strongly support that these microRNAs are potential regulators of BRCA1 expression in breast cancer tumors.

FONDECYT1120200, CONICYT21120269.

#484

Tumor suppressor miRNA-101 modulates leukemogenesis by targeting the EZH2/Wnt/β-catenin signaling pathways.

Estrella Gonzales-Aloy, Dylan G. Grebert-Wade, Jenny Y. Wang. _Children's Cancer Inst., Randwick, Australia_.

The survival rate of acute myeloid leukemia (AML) patients undergoing standard chemotherapy remains poor due to disease relapse. The main cause of relapse is the failure of chemotherapeutic drugs to eradicate self-renewing leukemic stem cells (LSCs) (Cancers 2017;9:74-97). Because of their intrinsic drug resistance, LSCs are extremely difficult to target by single-gene therapies. MicroRNAs (miRNAs), a class of small noncoding RNAs capable of regulating multiple target genes, represent a promising therapeutic target for elimination of LSCs. While miRNA-101 (miR-101) has been identified as a tumor suppressor in several types of cancers including prostate (Science 2008;322:1695-99) and liver cancer (Cancer Res 2009;69:2623-29), its expression and functional role in AML remains unknown. In this study, we uncover that miR-101 plays a crucial role in leukemogenesis through modulation of key epigenetic and signaling pathways activated in LSCs of mixed-lineage leukemia (MLL)-rearranged AML. Our data showed that overexpression of miR-101 in murine pre-LSCs inhibited cell proliferation in vitro and delayed leukemogenesis in vivo, confirming a tumor-suppressor function of miR-101 in MLL-AF9-induced AML, a highly aggressive leukemia. To assess the therapeutic value of miR-101, we next examined the antitumor function of miR-101 in a xenograft model of human MLL-AF9 AML. Briefly, miR-101 was overexpressed in human MLL-AF9 AML cell line, MOLM-13, that was then transplanted into NOD-scid IL2Rgammanull (NSG) mice followed by in vivo bioluminescence imaging to monitor engraftment of human leukemic cells. Our result revealed a significantly lower engraftment in the xenograft mouse model carrying miR-101-overexpressing leukemic cells compared to control mice. This suggests that inhibition of tumor suppressor miR-101 is essential in the initiation of an aggressive form of human AML. To investigate the potential mechanism by which miR-101 impairs leukemogenesis, we examined key oncogenic pathways regulated by miR-101 by performing luciferase assays and Western blotting. Our data showed that miR-101 inhibited leukemogenesis via directly targeting Enhancer of zeste homolog 2 (EZH2), a crucial epigenetic regulator in leukemia maintenance, as well as key components of Wnt/β-catenin signaling, an oncogenic pathway necessary for LSC self-renewal in AML. Furthermore, immunofluorescence analysis revealed a significant reduction in active β-catenin in the nucleus of miR-101-overexpressing leukemic cells, indicating a direct regulation of miR-101 in β-catenin activity. Collectively, these findings reveal a novel role for miR-101 in leukemia progression that may be used to develop a targeted therapy for AML treatment.

#485

Modulation of microRNA expression in EGFR-tyrosine kinase inhibitor-resistant non-small cell lung cancer cell lines.

Inamul Haque,1 Hannah Motes,2 Mukut Sharma,2 Emma Borrego-Diaz Reyes,2 Andrew K. Godwin,1 Chao H. Huang2. 1 _University of Kansas Medical Center, Kansas City, KS;_ 2 _Kansas City VA Medical Center, Kansas City, MO_.

Objectives: Epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) are now standard of care in lung cancer patients with EGFR mutation. Invariably, these patients develop resistance and require treatment with another EGFR-TKI or chemotherapy. The gatekeeper mutation T790M is responsible for about 50% of resistance. Other mechanisms may also confer resistance in these patients. High expression of microRNA-10b (miRNA-10b) is associated with worse prognosis in resected lung cancer patients with EGFR mutation. Both miRNA-27a and -27b are associated with increased expression of c-met, which is a potential mechanism of resistance to EGFR-TKI. The goal of this study is to determine the optimal treatment and miRNA characteristic of EGFR mutation lung cancer cells resistant to EGFR-TKI. We hypothesized that miRNA-10b, -27a and -27b modulated Erlotinib (ERL) resistance by different EGFR-TKIs and chemotherapeutic agents in EGFR mutant NSCLC cell lines.

Materials and Methods: Lung cancer cell lines with EGFR mutation HCC827 and HCC4006 resistant to ERL were generated. ERL-resistant cells were then treated with 10 nM afatinib (Afa), 0.5 mg/mL pemetrexed (Pem), and combination of 10 nM Afa/0.5mg/mL Pem for 72h. The cells were harvested and microRNA assay was performed by real-time PCR. NCI-H460 cell line without EGFR mutation was used as control.

Results: We observed increased expression of miRNA-10b, miRNA-27a and miRNA-27b in ERL-resistant HCC4006 compared to control cells NCI-H460. There was an upregulation of miRNA-27a and -27b but not of miRNA-10b in ERL-resistant HCC827. Treatment of ERL-resistant cells with Afa, Pem, and combined treatment changed the expression of miRNA-10b, miRNA-27a and miRNA-27b. These miRNAs were downregulated by 2nd-generation EGFR-TKI therapy alone or in combination with Pem. Different treatments had different effects on these miRNAs. There might be differences in miRNA regulation in different cell lines when treated with EGFR-TKI or chemotherapy. Our results suggest that microRNA may be used as targets for developing novel approach to treating patients with EGFR-TKI resistance.

Conclusion: This study may have implications in the management of patients resistant to EGFR TKI. MicroRNA analysis showed upregulation of several microRNAs that should be confirmed in clinical setting.

#486

MicroRNA-30 family members inhibit breast cancer invasion, osteomimicry, and bone destruction by directly targeting multiple bone metastasis-associated genes.

Martine Croset,1 Francesco Pantano,1 Casina Kan,1 Edith Bonnelye,1 Francoise Descotes,2 Catherine Alix-Panabières,3 Charles Lecellier,4 Richard Bachelier,1 Nathalie Allioli,5 Saw See Hong,6 Kai Bartkowiak,7 Klaus Pantel,7 Philippe Clézardin1. 1 _INSERM, Lyon, France;_ 2 _Hospices Civils de Lyon, Lyon, France;_ 3 _University Medical Centre, Montpellier, France;_ 4 _Université Montpellier 1, Montpellier, France;_ 5 _Institut des Sciences Pharmaceutiques et Biologiques, Lyon, France;_ 6 _University Lyon 1, Lyon, France;_ 7 _University Medical Centre Hamburg-Eppendorf, Hamburg, Germany_.

MicroRNAs (miRNAs), which are master regulators of gene expression, have emerged as key players in cancer metastasis. Here, we established miR-30 family members (miR-30a, miR-30b, miR-30c, miR-30d and miR-30e) as breast cancer bone metastasis suppressor genes. Low expression levels of miR-30s in primary tumors of breast cancer patients were associated with poor relapse-free survival. Additionally, miR-30s levels were significantly lower in estrogen receptor (ER)-negative/progesterone receptor (PR)-negative tumors than in their hormone-responsive counterparts. Overexpression of miR-30s in ER/PR-negative breast cancer cells resulted in the reduction of bone metastasis burden in vivo. MiR-30s overexpression also reduced tumor outgrowth, when tumor cells were implanted subcutaneously in animals. In vitro, miR-30s did not affect tumor cell proliferation, but inhibited tumor cell invasion. Furthermore, miR-30s restored bone homeostasis by reversing the in vitro effects of the tumor cell conditioned medium on stimulation of osteoclastogenesis and inhibition of osteoblastogenesis. We identified a number of genes associated with osteoclastogenesis stimulation (IL-8, IL-11), osteoblastogenesis inhibition (DKK-1), tumor cell osteomimicry (RUNX2, CDH11) and invasiveness (CTGF, ITGA5, ITGB3) that were direct and/or indirect targets for repression by miR-30s. Among these genes, integrin ITGA5 was a previously unknown miR-30 target. By silencing ITGA5 in ER-/PR-negative breast cancer cells, colonization of the bone marrow by tumor cells was drastically reduced in vivo. Overall, our data indicate that miR-30s employ multiple mechanisms to impede breast cancer bone metastasis. These findings may pave the way to a new field of therapeutic interventions in breast cancer patients with bone metastasis.

#487

MicroRNA regulation of oral squamous cell carcinoma.

Gary Guishan Xiao, Min Cao. _Dalian Univ. of Technology, Dalian, China_.

Oral squamous cell carcinoma (OSCC) remains the sixth most common human cancer with high mortality rate worldwide. Understanding the molecular mechanisms is important for development of an effective treatment for the disease. MicroRNA is a short, noncoded nucleotide playing an important role in tumorigenesis and metastasis. This study is to identify novel microRNAs and their targets in order to understand the molecular mechanisms underlying OSCC and metastasis using label-free proteomics and microRNA microarray techniques. Human oral tissue biopsies from three groups of subjects who had benign, localized carcinoma and distal carcinoma were collected from the archived tissue bank at Pathology in Creighton University. Total RNA and proteins were extracted from the core biopsy of the same subject. MicroRNA identification was performed by using Affymetrix GeneChip miRNA arrays and comprehensive data analysis. Protein was analyzed by using label-free LTQ orbit trap mass spectrometry. Forty-four miRNAs were upregulated significantly, including let-7b, miR-720, miR-21, and miR-1274b, while 41 miRNAs were remarkably downregulated, including miR-195, and miR-932, at localized and distal carcinoma compared to benign tissues. Additionally, as compared to benign, 38 miRNAs upregulated at local carcinoma were downregulated significantly at distal carcinoma, while other 17 miRNAs downregulated significantly at localized carcinoma were upregulated gigantically at distal carcinoma. Those miRNAs were further validated by qRT-PCR and FISH. Among those upregulated at both local and distal, miR-720 was further studied, and tryptase alpha-1 as its target was identified. MicroRNA-720 mimics suppressed significantly the expression of tryptase alpha-1 in cultured human OSCC stem cell lines. Results from miR-720 tissue microarray analysis showed that 98.8% of sensitivity and 100% of specificity for OSCC detection were achieved, suggesting that miR-720 may be developed as a novel biomarker for detection of human OSCC.

#488

Epigenetic regulation of colorectal cancer stem cells by the miR-221/QKI5 axis.

Junko Mukohyama,1 Yohei Shimono,1 Piero Dalerba,2 Taichi Isobe,3 Qingjiang Hu,4 Debashis Sahoo,5 Naoki Shibuya,1 Hironobu Minami,1 Koshi Mimori,6 Yoshihiro Kakeji,1 Akira Suzuki1. 1 _Kobe University Graduate School of Medicine, Kobe, Japan;_ 2 _Columbia University, New York, NY;_ 3 _Stanford University, Stanford, CA;_ 4 _Kyushu University Hospital, Fukuoka, Japan;_ 5 _University of California San Diego, San Diego, CA;_ 6 _Kyushu University Beppu Hospital, Beppu, Japan_.

Colorectal cancer stem cells (CSCs) have extensive abilities to initiate tumor formation, and are responsible for the progression and metastasis of colorectal cancers. MicroRNAs (miRNAs) are non-coding RNAs of 18-24 nucleotides that post-transcriptionally regulate expression of multiple genes by targeting their 3'untranslated regions (UTRs). miRNAs regulate various biological processes, and function as important regulators of CSC properties.

miR-221 is one of the oncogenic miRNAs frequently upregulated in human cancers including colorectal cancer. Previous studies have shown that miR-221 enhances proliferation, migration, and invasion of cancer cells. However, the role of miR-221 in human colorectal CSCs is not fully elucidated. In this study, we analyzed the colorectal CSCs directly isolated from the surgical specimens of colorectal cancer patients to explore the role of miR-221 in CSCs.

Expression profile of 754 miRNAs in the CD44+/EpCAM+ CSC and non-tumorigenic cancer cell (NTC) populations was analyzed by multiplex semi-quantitative PCR. Prognostic impact of miR-221 expression in CSCs was analyzed using the TCGA database. Target genes of miR-221 were validated by luciferase reporter assays and Western blot experiments. The roles of miR-221 and its target gene QKI-5 in colorectal CSCs were evaluated by organoid and xenotransplantation assays.

Comparison of the expression levels of 754 miRNAs in the CSCs and NTCs resulted in the identification of 10 miRNAs upregulated or downregulated in CSCs compared to NTCs. Among them, miR-221 was most highly expressed in CSCs and its expression level was very low in NTCs and human normal colon epithelial cells of human colorectal cancer specimens (n=6, p<0.05). The estimated 5-year overall survival rate for miR-221high patients (n=177) was significantly lower than miR-221low ones (n=116) in TCGA dataset (54.6% vs. 73.6%; p<0.001). The TNM stages at diagnosis were not significantly associated with the expression level of miR-221. In a multivariate analysis, miR-221 expression were significantly associated with overall survival (p=0.009). miR-221 targeted an RNA binding protein (RBP) QKI-5 and suppressed its expression. Knockdown of miR-221 in human colorectal cancer patient-derived xenograft cells suppressed the organoid formation in vitro in a QKI-5-suppression-dependent manner, and tumor formation initiated by colorectal CSCs in vivo (n=6, p=0.015 ).

Our results suggest that the miR-221/QKI5 axis plays important roles in the regulation of CSC properties in human colorectal cancers. Because miR-221 was preferentially and highly upregulated in human colorectal CSCs, but not in normal stem/progenitor cells, it is possible that miR-221 will be a biomarker that reflects amount and/or activity of CSCs in colorectal cancers and an attractive target to selectively attach colorectal CSCs by avoiding damages on their normal counterparts.

#489

Up-regulation of miR-130b is involved in prostate cancer racial disparity thorough FHIT- β-catenin pathway inactivation.

Yutaka Hashimoto,1 Masrisa Shiina,1 Taku Kato,1 Soichiro Yamamura,1 Yuichiro Tanaka,1 Shahana Majid,1 Sharanjot Saini,1 Varahram Shahryari,1 Priyanka Kulkarni,1 Pritha Dasgupta,1 Divya Bhagiratha,1 Nadeem Bhat,1 Guoren Deng,1 Laura Tabatabai,2 Deepak Kumar,3 Rajvir Dahiya1. 1 _UCSF, San Francisco, CA;_ 2 _VAMCSF/ UCSF, San Francisco, CA;_ 3 _North Carolina Central University, Durham, NC_.

Purpose: Prostate cancer (PCa) is a common cancer in men. African-American (AfA) men have higher incidence and significantly higher prostate cancer mortality rates than Caucasian-American (CaA) men. In this study, we investigated the biochemical role of miR-130b in this disparity.

Methods: We used meta-analyses and data from Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA). PCa cell lines from CaA (DU145, LNCaP, PC3), AfA (MDA-PCa-2b, E006AA-hT) and a normal epithelial cell line (PWR1E) were used for examining miR-130b expression levels and/or establishing stable miR-130b knock-down clones. Total RNA was extracted from clinical samples and cell lines. miR-130b and mRNA levels of its target genes were determined by quantitative real-time PCR (qPCR). qPCR based gene expression arrays were used to identify miR-130b target genes. Cell proliferation, apoptosis and cell cycle were monitored after stable miR-130b knock down. Western blotting was performed to detect miR-130b target protein expression levels.

Results: TCGA data showed up-regulation of miR-130b in AfA PCa tissue samples compared with CaA samples. miR-130b was significantly up-regulated in AfA prostate cancer tissues and cell lines compared to CaA cells and tissues. Utilizing VAMCSF and NDRI patient cohorts, we confirmed that miR-130b expression was linked to a racial difference between AfA/CaA PCa patients. Knock down of miR-130b showed decreased growth and cell cycle arrest, though the effect was less in CaA compared to AfA cells. We found unique changes in biological pathways associated with miR-130b knock down in AfA cells by qPCR array. Evaluation of the altered pathways showed that Fragile Histone Triad (FHIT) - β-catenin (CTNNB1) axis was markedly decreased in the AfA compared with CaA cells.

Conclusion: These results demonstrate that miR-130b may be a central regulator of key events that contribute racial differences in prostate cancer. FHIT-CTNNB1 may also be a new AfA specific oncogenic pathway that may be of therapeutic importance in AfA prostate cancer patients.

#490

Single nucleotide variations within and around microRNA-binding sites.

Amber Budmark*,1 Michael Catalano*,1 Tyrel Haley*,1 Brady Hicks*,1 Maria Koenen*,1 Thea Patrick*,1 Tyler Larson*,1 Tyler Wagner*,1 Clark Butler,1 Joshua Feiner,1 Rebecca Frick,1 Sierra Haage,1 James Miller,1 Mackayla Nohr,1 Dillon Stadlman,1 Dillon Turner,1 Sara Husher,1 Nicholas Woslum,1 Nathan Stadem,1 John Dosch,1 Tyler Fortuna,1 Chandler Fredrich,1 Elise Hadley,1 Brooklynn Oehlerking,1 Delayna Paulson,1 Cal Wiese,1 Paula Mazzer,1 Tim Mullican,1 Cynthia Anderson,2 Mark Larson,3 Elena Paryiskaya,4 Alexandra Kharazova,4 Paola Vermeer,5 Samuel Milanovich,5 Alexei Savinov,5 John Collins,6 Alexander Kofman1. 1 _Dakota Wesleyan University, Mitchell, SD;_ 2 _Black Hills State University, Spearfish, SD;_ 3 _Augustana College, Sioux Falls, SD;_ 4 _St. Petersburg State University, St. Petersburg, Russian Federation;_ 5 _Sanford Research, Sioux Falls, SD;_ 6 _University of Tennessee at Martin, Martin, TN_.

* equally contributed

Grant #5P20GM103443 (NIGMS).

Single nucleotide polymorphisms (SNPs) can either create or destroy microRNA binding sites. We analyzed 200 previously reported cancer associated SNPs within microRNA binding sites and found that more than 90% of them were surrounded by single and multiple low-frequency SNPs. The low-frequency SNPs were positioned within the expected microRNAs seed matching areas (58%), within the whole microRNA matching regions (71% incidence), and within the distance where they potentially can affect microRNA-mRNA interaction (36% incidence). We further analyzed the presence of SNPs within microRNA-binding sites in the 3'UTRs of mRNAs encoding the human V-set domain containing T-cell activation inhibitor 1 (VTCN1) and an AT-rich interaction domain 5B (ARID5B). In VTCN1 single SNPs were present in 36% of microRNAs seed matching areas, and two and more SNPs in 8% of microRNA seed matching sites. For ARID5B, single SNPs were present in 42%, and two and more SNPs in 26% of microRNA seed matching sites. In both VTCN1 and ARID5B, some microRNA seed matching areas harbored as many as 4 SNPs. The predicted binding site for microRNA-6870-5p (miR-6870) within the VTCN1 3'UTR consists of 11 uninterrupted and 2 additional Watson-Crick pairs. The length of mature miR-6870 is 19 bases, and the corresponding VTCN1 mRNA fragment harbors 6 nucleotide variations: rs758251859, rs1001277215, rs551576201, rs539444165, rs949692788, and indel rs9055595515. Hypothetically, the rs1001277215 minor allele (MA) eliminates the miR-6870-mRNA complementarity between the corresponding nucleotides. The rs551576201 MA weakens miR-6870-mRNA complementarity by creating a wobble G-U pair, the rs949692788 MA enhances miR-6870-mRNA complementarity by switching from a non-canonical G-U to canonical Watson-Crick A-U pair, and for indel rs9055595515, the absence of deletion preserves miR-6870-mRNA complementarity between CA and GU nucleotides. The probabilities of the "best" and "worst" matches between miR-6870 and VTCN1 mRNA are 0.009995% and 0.000006% correspondingly. The binding between miR-6870 and VTCN1mRNA may also be affected by variations between A and G within rs758251859 (MA frequency is unknown). As microRNA-binding efficacy depends on the mRNA sequences outside the target region, the indels rs35182629 (located 5 nucleotides upstream and covers 2 bases) and rs896747700 (located 1 nucleotide downstream and covers 1 base) may also impact miR-6870-mediated regulation of VTCN1 expression. The possible coincidence of hyper-functional or completely disabled microRNA-binding sites may result in significant phenotypic variations and predisposition to cancers.

#491

Understanding the roles of miRNAs in melanoma susceptibility through miQTL study using miRNA transcriptomes from 106 individuals.

Jiyeon Choi,1 Tongwu Zhang,1 Michael Kovacs,1 Mai Xu,1 Andrew Vu,1 NISC Comparative Sequencing Program, Stacie Loftus,2 William Pavan,2 Kevin Brown1. 1 _NCI, Bethesda, MD;_ 2 _NHGRI, Bethesda, MD_.

microRNAs (miRNA) are important mediators of post-transcriptional gene regulation and play critical roles in tumorigenesis. In melanoma, miRNAs are involved in proliferation, migration, invasion and drug resistance modulating RAS/MAPK and MITF pathways and others. Melanoma arises in melanocytes, and understanding heritable regulation of miRNAs in primary melanocytes could enhance our knowledge of genes and pathways contributing to melanoma risk. While a handful of studies in limited tissue types have identified miRNA transcripts with expression levels that are correlated with common genotypes using an expression quantitative loci approach (miQTL), expression levels of miRNAs are highly tissue-specific, further correlating with tissue-specific mRNA expression. Given the lack of cell-type specific miQTL data, we established a miQTL dataset using primary cultures of human melanocytes from 106 newborn males mainly of European decent. To assess comprehensive miRNA transcript levels from these samples, we performed RNA sequencing of the small RNA population (single-end, stranded, 51bp). An average of 63M reads per sample were obtained, and subsequently processed following miRMaster pipeline (ccb-compute.cs.uni-saarland.de/mirmaster) to detect known and novel miRNAs. A total of 961 known and 1262 novel miRNA transcripts were expressed above the background noise (> 0.05 Read Per Million and >5 reads per transcript in >=10 samples). Genotypes of 106 individuals were also assessed by direct genotyping using Illumina OmniExpress arrays followed by imputation using Michigan Imputation Server to produce a total of 10,687,550 genotypes. Subsequent QTL analyses using FastQTL (fastqtl.sourceforge.net) and QTLtools (qtltools.github.io/qtltools) identified 33 known and 34 novel miRNA transcripts as cis-miQTLs (q-value < 0.05) and 2 known and 6 novel miRNAs as trans-miQTLs (FDR < 0.1), respectively. We also observed a subset of significant cis-miQTLs overlapping with marginally significant mRNA trans-eQTLs from the same RNA samples, suggesting miRNA-mediated trans regulation. Further identification of melanocyte-specific miQTLs and their target genes will provide better understanding of melanocyte-specific miRNA regulation network. To identify candidate melanoma susceptibility genes functioning through heritable miRNA expression, melanocyte miQTLs will also be overlaid with melanoma genome-wide association data. In addition to the results from these analyses, further analyses of differentially expressed miRNAs and target genes between our primary melanocytes and melanoma tumors (available through The Cancer Genome Atlas) will shed light on the altered gene networks in melanomagenesis that might have therapeutic relevance through miRNA-mediated intervention.

#492

Downregulation of microRNA-17-5p promotes EMT by targeting vimentin in colorectal cancer.

Tea Won Kim,1 Yeo Song Lee,2 Hye Kyung Hong,3 Su Jeong Song,3 Nak Hyeon Yun,1 Yong Beom Cho4. 1 _SAIHST, Sungkyunkwan University, Seoul, Republic of Korea;_ 2 _Sungkyunkwan University School of Medicine, Seoul, Republic of Korea;_ 3 _Samsung Biomedical Research Institute, Seoul, Republic of Korea;_ 4 _Department of Surgery, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Republic of Korea_.

Background: Colorectal cancer (CRC) is one of the most frequent forms of cancer worldwide. Cells with abnormal growth have the ability to invade or spread to other parts of the body (metastasis). Metastasis is the most common cause of death in CRC patients and activation of epithelial-mesenchymal transition (EMT) triggers metastasis. Thus, it is important to understand the mechanisms of EMT to increase the survival rate of CRC. MicroRNA-17 (miRNA-17) has been proven to be significantly higher in CRC tissues than normal tissues. However, there are few papers about the role of miRNA-17 in CRC metastasis. Therefore, in this study, we evaluated the mechanism underlying miRNA-17-5p related EMT in colon cancer cells.

Methods: Expression level of miRNA-17-5p as well as target mRNA were analyzed in HCT-15, HT29, LoVo, SW480 and COLO205 cell lines. In order to evaluate the correlation between miRNA-17-5p and EMT phenomenon, selected cell lines were transiently transfected either miRNA-17-5p mimic or miRNA-17-5p inhibitor and then phenotyping studies were conducted. Moreover, to find out the direct target of miRNA-17-5p, we performed AGO2 immunoprecipitation in each colon cancer cell lines.

Results: Real-time PCR revealed that miRNA-17-5p expression reversely correlated with vimentin expression in five colon cancer cell lines. Overexpression of miRNA-17-5p inhibited vimentin expression, cell migration and cell invasion in both LoVo and HT29 cells. On the other hand, inhibition of miRNA-17-5p promoted vimentin expression, cell migration and cell invasion in the same parallel cell lines. Through AGO2 immunoprecipitation analysis, we observed that miRNA-17-5p directly bind to vimentin 3'UTR to influence its stability and expression at the transcriptional level.

Conclusion: Our data demonstrated that down-regulated miRNA-17-5p promotes colon cancer cell migration and invasion by regulating vimentin expression. These findings propose that miRNA-17-5p may serve as a potential prognostic biomarker in colorectal cancer.

#493

Circulating biliary tract microRNA signature discriminates cholangiocarcinoma from pancreatic cancer.

Jisce R. Puik,1 Laura L. Meijer,1 Tessa Y. Le Large,1 Michal Heger,2 Frederike Dijk,2 Niccola Funel,3 Ingrid Garajová,4 Elisa Giovannetti,1 Geert Kazemier1. 1 _VU University Medical Center, Amsterdam, Netherlands;_ 2 _Amsterdam Medical Center, Amsterdam, Netherlands;_ 3 _University of Pisa, Pisa, Italy;_ 4 _University of Bologna, Bologna, Italy_.

Accurate diagnosis of lesions in the pancreatic head is a challenge with substantial clinical consequences in daily practice. Located in the pancreatic head, both distal cholangiocarcinoma (CCA) and pancreatic ductal adenocarcinoma (PDAC) are highly lethal malignancies with limited but distinct therapeutic options. Given these different therapeutic considerations, discovery of novel biomarkers that can ensure differential diagnostic specificity is an unmet clinical need. This study aims to identify a circulating microRNA (miRNA) signature to accurately diagnose and discriminate distal CCA from PDAC. In the initial discovery phase, microarray profiling of a panel of 752 miRNAs was performed on seven distal CCA and seven age- and sex-matched healthy control plasma samples. Raw data were normalized to the global mean. Based on probability value and fold change, candidate miRNAs were selected for further validation. In the validation phase, selected candidate miRNAs were analyzed in an independent cohort of distal CCA (n = 22) and healthy controls (n = 32) using RT-qPCR. To further assess the origin of lesions, candidate miRNA expression profiles were analyzed in malignant disease (n = 90) compared to benign disease (n = 15), and distal CCA (n = 22) compared to PDAC (n = 30). Following an empirically based selection of potential reference genes, raw data were normalized to a combination of two normalizing reference miRNAs. In the discovery phase, microarray analysis revealed nineteen miRNAs that were significantly deregulated in patients with distal CCA compared to healthy controls. Thirteen miRNAs were selected for further validation. In the validation phase, using logistic regression analysis, a three-miRNA panel was identified as the most robust diagnostic signature to discriminate malignant from benign disease (AUC = 0.881). Remarkably, two miRNAs of this panel were validated as specific classifier for distal CCA, with an AUC of 0.814 in differentiating distal CCA from PDAC. In addition, a combination of miR-93 and miR-101 was used as bona fide normalizing reference gene for these analyses. In conclusion, a combined panel of plasma miRNAs shows promising diagnostic capability to serve as unique minimally invasive distal CCA biomarker and has the potential to help clinical decision making for patients affected by pancreatobiliary disease in the pancreatic head.

#494

Intravital discovery of miRNA drivers of human cancer cell directional invasion.

Konstantin V. Stoletov, Lian Willetts, Juan Jovel, Emma Woolner, John D. Lewis. _Univ. of Alberta, Edmonton, Alberta, Canada_.

Metastatic cancer cells use directional ECM cues such as blood vessels or collagen fibers when invading through live tissue. Oncogenic miRNAs have been implicated as key regulators of cancer progression, yet the systemic discovery of miRNAs that drive directional cancer cell invasion has not been achieved. Here we describe the first in vivo quantitative whole human miRNAome screen for miRNA drivers of directional cancer cell invasion that is based on high-resolution intravital imaging. We identified several novel miRNAs that promote cancer cell invasion during the key rate-limiting step of cancer metastasis: the initiation of overt metastatic lesions. In vivo 4D cancer cell tracking revealed that these prometastatic miRNAs are required for successful invasion into collagen-rich tissue and for attachment to the outer surface of the vascular wall. Deregulation of these miRNAs led to formation of loose contacts with the vasculature and chaotic, nondirectional cancer cell invasion patterns in living tissue. Intravital SHG microscopy showed that inhibition of the expression of these miRNAs blocked the ability of cancer cells to rearrange the disorganized collagen network into collagen fiber bundles and stably protrude along these bundles. Further imaging analysis revealed that this miRNA expression specifically blocks vesicular transport of key cell invasion machinery proteins such as MT1-MMP to the cancer cell invadopodia. Human cancer gene expression database analysis showed that our top miRNA candidates are specifically deregulated in invasive pancreatic cancer. Indeed, intravital imaging analysis showed that blocking of metastatic miRNA function inhibited MT1-MMP secretion and ECM degradation by human pancreatic cancer cells. In summary, we identified a novel panel of human miRNAs that are functionally involved in the regulation of directional invasion and metastasis. This work establishes these miRNAs as promising therapeutic targets to block the metastatic spread of lethal cancers.

#495

Leptin facilitates ovarian cancer metastasis through miR-28-3p and matrix metalloproteinase-1 in the omental tumor microenvironment.

Chi Lam Au Yeung, Ngai Na Co, Tsz-Lun Yeung, Samuel C. Mok. _UT MD Anderson Cancer Center, Houston, TX_.

Advanced stage serous ovarian cancers spread beyond the pelvis and preferentially metastasize to the omentum, which is mainly composed of adipose tissue, suggesting that the omental microenvironment is a favorable niche for ovarian cancer cells. By comparing the transcriptome profiles of microdissected ovarian cancer tissues from the primary ovarian site and from the omental site, we identified matrix metalloproteinase-1 (MMP-1) as one of the most significant genes that was up-regulated in the omental site compared with that in the primary site. To identify mediators secreted by the omental microenvironment that up-regulated MMP-1 in the omental site tumors, transcriptome profiling was performed on microdissected ovarian tumor associated adipose tissue in the omental site and the ovarian stromal tissue in the primary ovarian tumor site. We identified leptin as one of the top differentially expressed genes in ovarian tumor associated adipose tissue in the omental site, suggesting that it might up-regulate MMP-1 in the omental ovarian cancer. To further delineate the role of MMP-1 in mediating the effect of leptin on ovarian cancer cell metastasis, ovarian cancer SKOV3 and OVCA433 cells were treated with leptin. The results showed that leptin increased MMP-1 mRNA and protein expression as assessed by quantitative RT-PCR and Western blot analyses, respectively. Furthermore, we demonstrated that leptin induced both the cell migration and invasion potential of SKOV3 and OVCA433 cells, and the effects were abrogated by transfecting cells with MMP-1 siRNAs. In addition, using Ion Torrent next generation sequencing, we found that miR-28-3p is down-regulated in leptin-treated SKOV3 and OVCA433 cells compared to the untreated cells, suggesting that it may play a role in mediating the effect of leptin on MMP-1 expression and the subsequent ovarian cancer cell motility and invasion potential. In conclusion, leptin secreted by the adipocytes in ovarian cancer associated omental microenvironment may be important in facilitating the ovarian cancer cell metastasis through the miR-28-3p/MMP-1 pathway.

#496

The synergistic role of miR-150-5p and miR-661 in regulating epithelial mesenchymal transition in triple negative breast cancer.

Patricia MM Ozawa,1 Bruna M. Sugita,1 Aline S. Fonseca,2 Yara Rodriguez,2 Yuriy Gusev,2 Paolo Fadda,3 Deepak Kumar,4 Luciane R. Cavalli2. 1 _Universidade Federal do Paraná, Curitiba, Brazil;_ 2 _Georgetown Lombardi Comp. Cancer Ctr., Washington, DC;_ 3 _The Ohio State University, Columbus, OH;_ 4 _North Carolina Central University, Durham, NC_.

MiRNA dysregulation has been linked to cancer initiation and progression, through their control in multiple cancer associated signaling pathways. MiRNA low specificity of target interaction confer them the ability to cooperatively regulate a single biological process. Epithelial mesenchymal transition (EMT) plays pivotal roles in cancer progression and metastasis, through the regulation of transcription factors and proteins, such as E-cadherin, Slug, Snail, Twist and Zeb1. This process is also controlled post-transcriptionally by a complex and regulated network of miRNAs. We have shown that miR-150-5p and miR-661, up-regulated in triple negative breast cancer (TNBC) when compared to normal tissue and other breast cancer subtypes, exert an oncogenic action and modulate cell proliferation, clonogenicity, migration and cytotoxic response in in vitro TNBC models. The main objective of this study was to determine whether the modulation of these phenotypes were due to the regulation of their corresponding gene targets associated with EMT. By conducting an integrated computational and experimental analysis, in which miR-150-5p and miR-661 were suppressed or ectopically expressed in transfected MDA-MB-231 cells, we showed that they present common regulatory roles in cell adhesion and motility signaling networks, including those involved in EMT. Gene expression analysis in a targeted panel of 770 genes with primary functional annotations in EMT, cell adhesion and motility and metastasis, revealed that: i. the suppression of miR-150-5p and miR-661, affected the expression of 160 and 123 genes, respectively; 18% of these genes were commonly affected by both miRNAs, including ACVR1C (ALK-7), CHAD, GIMAP6, SFRCL1, SPARCL1 and TBX1; ii. the ectopic expression of miR-150-5p and miR-661, affected the expression of 283 and 267 genes, respectively; 27% of which commonly affected by both miRNAs, including AKT1, DCL1, MAPK2, MMP1, SFRP1 and TPM2. iii. 26 genes were inversely affected by the suppression and ectopic expression of miR-150-5p and miR-661 (consistent with the general post-transcriptional regulation of miRNA/mRNA targets); 73% of these genes were predicted to be miRs150-5p and 661 direct or indirect targets. In summary, based on the computational and experimental data presented, we provide evidences that strongly suggest that miRNA-150-5p and miR-661 modulate the aggressive tumor phenotype in TNBC cells by regulating common gene targets associated with the EMT process. Further functional studies in TNBC experimental models will allow us to dissect the mechanisms by which they contribute to the TNBC aggressive phenotypes by synergistically regulating EMT.

Funding: Partially funded by Georgetown University Center of Excellence in Regulatory Science and Innovation (CERSI U01FD004319), CNPq, Capes-Brazil (scholarship to PMMO, BMS, ASF).

#497

Identification of master microRNA regulatory networks for the mesenchymal glioblastoma subtype.

Lan Zhao,1 William Cho,2 Hu Zhu,1 Bing Shi,1 Hong Yan1. 1 _City University of Hong Kong, Hong Kong, Hong Kong;_ 2 _Queen Elizabeth Hospital, Hong Kong, Hong Kong_.

Introduction. Glioblastoma (GBM) is one of the most aggressive cancer types, accounting for more than 60% of malignant brain tumors in adults. Genomic profiling defined four subtypes of GBM termed proneural, neural, classical, and mesenchymal. The mesenchymal subtype is characterized by epithelial-mesenchymal-transition (EMT) and is associated with poor prognosis. Our aim in this study is to identify the mesenchymal subtype-specific biomarkers that may drive EMT, resulting in worse clinical outcome.

Materials and methods. We investigated 433 primary GBM patients from TCGA that have both gene and microRNA (miRNA) expression profiles. A prediction analysis for microarrays (PAM) classifier was built based on the predefined 141 patient labels and 738 gene signatures. The classifier was then used to classify the remaining unlabeled patients into one of the four subtypes. Comparisons between the mesenchymal and non-mesenchymal subtypes were performed using two-sided t-test statistics. Anti-correlated miRNA-mRNA interactions were used for network inferred by ARACNe (Algorithm for the Reconstruction of Accurate Cellular Networks), and the network were visualized by RedeR package. We tested the statistically significant over-representation of EMT signature genes in the regulon of each miRNA. MiRNAs of top significance (false discovery rate, FDR < 0.05) were selected as master regulators.

Results and Discussion. We classified the remaining unlabeled 292 GBM patients by using the PAM classifier. Gene set enrichment analysis (GSEA) and survival analysis were performed to do molecular and clinical characterization of the four subtypes, respectively, which all align with previous characteristics. Such as, mesenchymal subtype is enriched for EMT signatures, and proneural subtype has the best clinical outcomes. We also classified the 43 GBM cell lines from the Cancer Cell Line Encyclopedia (CCLE) into one of the four subtypes. In total, we identified 54 miRNAs downregulated, and 2,520 genes upregulated in mesenchymal compared with the non-mesenchymal subtypes. ARACNe predicted 28,586 miRNA-mRNA interactions, 2,784 of which were stable and therefore used for network construction. A miRNA regulatory network was subsequently built to describe the regulatory relations among miRNAs and target genes. Master regulator analysis identified 6 miRNAs, namely miR-9, miR-488, miR-95, miR-551b, miR-30c and miR-101, which may functioning as master regulators of the EMT program.

Conclusion. We used a relatively large GBM cohort to infer a miRNA-mRNA regulatory network associated with EMT. Further in vitro and in vivo experiments are ongoing to access the 6 miRNAs role in the EMT program.

#498

Dual functions of miR-200b in triple-negative breast cancer metastasis and chemoimmuno-resistance.

Xiaohui Tan,1 Shuchang Ren,1 Woojin Lee,1 Xiaoling Wu,2 Katayoon Rezaei,1 Yan-gao Man,3 Patricia Latham,1 Robert S. Siegel1,1 Rachel F. Brem,1 Zhongwu Li,4 Xiaofeng Chang,5 Sidney W. Fu1. 1 _George Washington Univ., Washington, DC;_ 2 _Chengdu Military General Hospital, China;_ 3 _Henry Jackson Foundation, Bethesda, MD;_ 4 _Peking University Health Science Center, Beijing, China;_ 5 _Xian Jiaotong University College of Stomatology, Xi'an, China_.

Triple-negative breast cancer (TNBC) is a subtype of breast cancer that is negative for estrogen and progesterone receptors (ER/PR) and human epidermal growth factor receptor 2 (HER2). It is typically associated with high rate of metastasis and limited targeted treatment options. Chemotherapy is the standard treatment for metastatic TNBC. However, the development of chemoresistance limits its clinical application. Elevated expression of immune-related genes in TNBC suggests that immunotherapy strategies may provide new therapeutic options for TNBC. Programmed death 1 (PD-1) and programmed death-ligand 1 (PD-L1) immune checkpoint inhibitors have been approved by the FDA for TNBC treatment. However, tumor immune evasion is considered an important obstacle. BRCA1 is a nuclear-cytoplasmic shuttling protein that plays a key role in preventing the development of a malignant phenotype. BRCA1 dysregulation and nuclear export are an important mechanism in cancer development and chemoresistance, especially in TNBC. Blocking BRCA1 nuclear export could be used as a strategy to prevent resistance. BRCA1 nuclear export has been reported to be mediated by several proteins such as BRCA1-binding protein 2 (BRAP2) and chromosomal maintenance 1 (CRM1, also known as exportin 1, XPO1). CRM1-mediated events have been implicated in breast cancer and involved in chemoimmunotherapy. microRNA-200b (miR-200b) is a cell-autonomous suppressor of EMT (epithelial-mesenchymal transition) and involved in tumor metastasis. In our present work, we discovered that miR-200b overexpression resulted in significant BRCA1 nuclear retention accompanied by downregulated expression of CRM1 and STAT1 (signal transducer and activator of transcription 1). Bioinformatics analysis indicated that miR-200b directly targets STAT1, which was confirmed by luciferase assay. We demonstrated that STAT1 is a transcription factor (TF) of CRM1, by both Transfac analysis and chromatin immunoprecipitation (ChIP)-qPCR assay. In patient tissue samples, we found that miR-200b expression was relatively lower in TNBC compared to non-TNBCs. Furthermore, we demonstrated that miR-200b-mediated BRCA1 nuclear retention is associated with significant PD-L1 downregulation, and sensitizes the TNBC cells to chemotherapeutic agents. In addition, high level PD-L1 expression is associated with not only chemoimmunoresistance but also tumor metastasis. These data provide strong evidence that miR-200b-mediated regulation of BRCA1 nucleus retention is through transcriptional regulation of CRM1 by STAT1, and miR-200b regulates PD-L1 expression in TNBC. In conclusion, this novel dual role of miR-200b may serve as a strategy in metastatic TNBC therapy by repressing STAT1-mediated CRM1 transcription regulation, and reversing the chemoimmunoresistance via PD-L1 inhibition.

#499

MicroRNA-411 downregulation promotes tumorigenesis and inhibits chemotherapy sensitivity by upregulating MLLT11 expression in human bladder cancer.

Honglei Jin,1 Haishan Huang,1 Chuanshu Huang,2 Xiaohui Hua,2 Junlan Zhu1. 1 _School of Laboratory Medicine and Life Science, Wenzhou Medical University, China;_ 2 _Nelson Institute of Environmental Medicine, New York University, NY_.

Although several previous studies have reported the implication of various microRNAs (miRNAs) in regulation of human bladder cancer (BC) development, alterations and function of many miRNAs in BC carcinogenesis and anti-cancer therapy are not explored yet at present. Here, we screened 1900 known miRNAs and first discovered that miR-411 was one of major miRNAs that was downregulated in a n-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN)-induced BC mouse model. This miR-411 downregulation was also observed in human BC tissues and cell lines. The results from evaluating the relationship between miR-411 and patient survival in BC using the TCGA database also indicated that miR-411 was positively correlated with survival (>3 years). Our studies also showed that miR-411 inhibited tumorigenesis and increased chemotherapy sensitivity in human BC. Mechanistic studies revealed that overexpression of miR-411 repressed expression of ALL1-fused gene from the chromosome 1q (MLLT11) by binding to the 3′-untranslated region of MLLT11 mRNA, induced p21 expression, and caused cell cycle arrest at the G2/M phase, in turn inhibiting BC tumorigenic growth. Ectopic expression of miR-411 also increased the sensitivity of BC cells to the chemotherapeutic agents, whereas MLLT11 restoration in miR-411-overexpressing cells increased resistance to the chemotherapeutic agents.

#500

A p53/miR-30a/ZEB2 axis controls basal-like/triple-negative breast cancer aggressiveness.

Valentina Damiano,1 Alessandra di Gennaro,1 Giulia Brisotto,1 Michela Armellin,1 Tiziana Perin,1 Antonella Zucchetto,1 Michela Guardascione,1 Herman P. Spaink,2 Claudio Doglioni,3 Ewa Snaar-Jagalska,2 Manuela Santarosa,1 Roberta Maestro1. 1 _CRO Aviano National Cancer Institute, Aviano, Italy;_ 2 _Leiden University, Leiden, Netherlands;_ 3 _San Raffaele Scientific Institute, Milano, Italy_.

Inactivation of p53 plays a major role in the poor prognosis of basal-like /triple-negative breast cancers (TNBC). However, how p53 inactivation impinges upon TNBC aggressive phenotype is only partially defined. Here we report the identification of a novel axis involving p53, miR30a and ZEB2. We provide evidence that p53 affects the expression of ZEB2, a transcription factor involved in epithelial-mesenchymal transition (EMT), by relying on miR-30a. In particular, we found that p53 regulates miR-30a expression by direct promoter binding and that miR30a targets ZEB2. Finally, we provide evidence that the new p53/miR-30a/ZEB2 axis controls tumor cell invasion and migration and that reduced miR-30a expression correlates with p53 inactivation and dismal prognosis in human breast cancers. Overall, this study highlights the existence of a novel axis linking p53 to EMT via miR-30a, and adds a novel element to the complex network whereby p53 inactivation impinge upon tumor aggressiveness. 

### Noncoding RNAs as Oncogenes and Tumor Suppressors

#501

A RET-related microRNA, miR-153-3p, acts as a tumor suppressor in medullary thyroid carcinoma (MTC) via S6K signaling.

Lauren Jin Suk Joo,1 Jocelyn Weiss,2 Anthony J. Gill,3 Roderick Clifton-Bligh,1 Himanshu Brahmbhatt,2 Jennifer A. MacDiarmid,2 Matti L. Gild,3 Bruce G. Robinson,1 Stan B. Sidhu,1 Jing Ting Zhao1. 1 _Kolling Institute of Medical Research, The University of Sydney, St Leonards, NSW, Australia;_ 2 _EnGeneIC Ltd., Lane Cove West, Sydney, NSW, Australia;_ 3 _Royal North Shore Hospital, St Leonards, NSW, Australia_.

Background

Medullary thyroid carcinoma (MTC), which comprises 3-5% of all thyroid cancers, originates from a small population of neuroendocrine calcitonin C-cells of thyroid gland. Gain-of-function mutations of the human Rearranged during Transfection (RET) proto-oncogene have been recognized as the key driver of MTC tumorigenesis. RET has been targeted by tyrosine kinase inhibitors (TKIs), however with modest efficacy. Many of the microRNAs (miRNAs) identified in human genome have been associated with cancer and have prompted interest in the establishment of miRNA-based therapeutics. However miRNA studies in MTC has been hampered due to the lack of normal C-cell tissue as a differential expression comparator.

Aims

We aimed to characterize differentially expressed miRNAs, which play fundamental roles in MTC tumorigenesis and in modulation of TKI responses.

Methods

RET of human MTC cells were silenced using TKI, Cabozantinib, or siRNA. Small RNA sequencing was performed (AGRF) to identify differentially expressed miRNAs before and after RET inhibition. MiRNA effects on cell proliferation (MTS assay), cell cycle and apoptosis (flow cytometry, Western blot) were investigated with gain-of-function studies. For in vivo study, MTC xenografts were established and miRNAs were intravenously delivered using nanoparticle delivery vehicles invented by biotechnology company - EnGeneIC Ltd.

Results

miR-153-3p was identified to be under-expressed in MTC following RET inhibition. Restoration of miR-153-3p significantly reduced cell proliferation, induced G2 cell arrest and increased cell apoptosis. Furthermore, combined treatment of miR-153-3p and Cabozantinib caused greater cell proliferation inhibition compared to individual treatment. miR-153-3p targets ribosomal protein S6 kinase B1 (RPS6KB1) and reduced phosphorylation of Bcl-2 associated death promoter (BAD) protein. In vivo delivery of miR-153-3p significantly impeded tumor growth compared to scrambled sequence. Combination of miR-153-3p delivery and oral administration of Cabozantinib resulted in further tumor stabilization effect.

Conclusion

miR-153-3p reveals a significant tumor suppressive role in MTC via S6K signaling, demonstrating its important therapeutic potential for MTC patients.

#502

Circulating exosomal miR-203 is associated with metastasis via inducing tumor-associated macrophages in colorectal cancer.

Dai Shimizu,1 Takaaki Masuda,1 Yuki Takano,1 Hisae Imamura,2 Rui Yamaguchi,3 Kuniaki Sato,1 Taro Tobo,1 Hidenari Hirata,1 Yosuke Kuroda,1 Sho Nambara,1 Naoki Hayashi,1 Tomohiro Iguchi,1 Shuhei Ito,1 Hidetoshi Eguchi,1 Takahiro Ochiya,4 Katsuhiko Yanaga,5 Satoru Miyano,3 Koshi Mimori1. 1 _Kyushu University Beppu Hospital, Beppu, Japan;_ 2 _Teikyo University, Tokyo, Japan;_ 3 _University of Tokyo, Tokyo, Japan;_ 4 _National Cancer Center Research Institute, Tokyo, Japan;_ 5 _Jikei University School of Medicine, Tokyo, Japan_.

Distant metastasis is the leading cause of mortality in patients with malignant tumor including colorectal cancer (CRC). A primary tumor can create a premetastatic niche in distant organs to facilitate the development of metastasis by communicating with host cells. However, little is known about the communicating mechanism is known. Understanding this mechanism should uncover a new field of cancer therapy through the targeting of the interaction between tumor and host. In this study, we focused on circulating microRNAs (miRs) as the communication tool between host and tumor cells that were mainly carried by exosomes in circulation. As a result of gene expression microarray and gene set enrichment analysis, we identified miR-203 as a putative signaling molecule between tumors and monocytes in metastatic CRC patients. The level of miR-203 expression was significantly upregulated in a TNM stage-dependent manner. High exosomal miR-203 expression in serum was significantly associated with poor DFS and OS (P < 0.01), and was an independent poor prognostic factor. Additionally, high exosomal miR-203 was associated with distant metastasis (P < 0.01). We also found that exosomes carrying miR-203 from CRC cells were incorporated into monocytes and miR-203 promoted the expression of M2 markers such as CD163 and STAT3 in vitro. It suggested that serum exosomal miR-203 released from CRC cells promoted the differentiation of monocytes to M2-tumor-associated macrophages (TAMs) which facilitated premetastatic niche and distant metastasis. Co-injection of miR-203-transfected CRC cells and monocyte increased a ratio of liver metastasis in vivo compared with only control CRC cells or only miR-203-transfected CRC cells injection. On the other hand, overexpression of miR-203 did not increase the proliferation, invasion and migration capacity of CRC cells. This results indicated that serum exosomal miR-203 might create a premetastatic niche whereas did not affect to malignant phenotype of CRC cells. Our study indicated that serum exosomal miR-203 expression could be a novel and feasible biomarker for predicting metastasis, possibly via promoting the differentiation of monocytes to M2-TAMs in CRC.

#503

MicroRNA-1205 as a tumor suppressor in castration resistant prostate cancer.

Michelle K. Naidoo,1 Dibash K. Das,1 Adeodat Ilboudo,2 Akintunde Orunmuyi,3 Gabriel O. Ogun,3 S. A. Adebayo,3 E. O. Olapade-Olaopa,4 Olorunseun Ogunwobi1. 1 _Hunter College and The Graduate Center of the City University of New York, New York, NY;_ 2 _Hunter College of the City University of New York, New York, NY;_ 3 _University of Ibadan, Ibadan, Nigeria;_ 4 _University of Ibadan, Ibandan, Nigeria_.

Prostate cancer (PCa) is the second most common cancer and third deadliest cancer in American men, yet the only established risk factors are familial genetics, age and race. Men of African Ancestry (moAA) have higher PCa incidence rates when compared to Caucasian men in the United States, which are due to differences in genetic susceptibility variants. Moreover, high mortality rates of PCa are associated with castration-resistant prostate cancer (CRPC) due to maintenance of androgen receptor (AR) signaling in PCa cells following androgen ablation therapy. The 8q24 chromosomal locus is a highly susceptible PCa region that has frequent amplifications of the c-MYC oncogene and downstream PVT1 gene. PVT1 is a long non-protein coding gene that encodes six annotated microRNAs (miRNAs), including microRNA-1205 (miR-1205), yet the function of these miRNAs are poorly understood. To elucidate the role of miR-1205 in PCa, we examined miR-1205 mRNA expression in a cohort of normal (n=22), benign (n=42), and malignant (n=26) histologically confirmed prostatic tissues obtained from prostatectomy or transrectal biopsies of moAA men in Ibadan, Nigeria. One-way ANOVA analysis determined changes in the relative expression of miR-1205 between groups (F(2,87) = 1.153) and a Tukey post hoc test revealed decreased miR-1205 expression in benign (4.61 ± 7.5) and malignant tumors (3.39 ± 3.53) when compared to normal tissues (6.55 ± 9.5). These data suggest that miR-1205 may function as a miRNA tumor suppressor and is characteristic to moAA-associated PCa. To elucidate the molecular mechanism of miR-1205, we examined AR and c-MYC mRNA expression using androgen-dependent LNCaP and CRPC C4-2B and 22RV1 cells. We observed a two-fold decrease of miR-1205 expression and overexpression of AR and c-MYC in C4-2B and 22RV1 cells when compared to LNCaP, suggesting that miR-1205 may regulate AR and c-MYC signaling in CRPC. Next, we identified Fry-like (FRYL) as a putative target using a miSVR computer algorithm and subsequently performed whole transcriptome analysis on prostate tumors and adjacent normal tissue from fourteen PCa patients using the Galaxy web platform. FRYL and AR overexpression was observed in diseased patients suggesting that FRYL may function as an oncogene. Moreover, FRYL was overexpressed in C4-2B and 22RV1 cells when compared to LNCaP, further suggesting a role in CRPC development. C4-2B cells transfected with miR-1205 and the 3'UTR of FRYL in a luciferase expressing vector revealed a significant decrease in luciferase activity when compared to control cells, indicating direct binding of miR-1205 to the 3'UTR of FRYL. These observations strongly suggest that miR-1205 acts as a tumor suppressor that may regulate AR and c-MYC expression, and directly targets the 3'UTR of FRYL in PCa cells. Further understanding the role of miR-1205 regulation of FRYL, AR, and c-MYC signaling may provide novel insights into the molecular mechanisms of CRPC.

#504

miR-484 acts as an "oncomiR" in triple negative breast cancer cells to promote tumor growth and progression by targeting HOX5A.

Nashwa N. Kabil, Recep Bayraktar, Cristina Ivan, Nermin Kahraman, Hamada A. Mokhlis, George A. Calin, Gabriel Lopez-Berestein, Bulent Ozpolat. _UT MD Anderson Cancer Ctr., Houston, TX_.

Breast cancer (BC) is the most common malignancy and the second leading cause of cancer-related deaths in women worldwide. Triple negative breast cancer (TNBC) is the most aggressive and chemo-resistant subtype of BC that represents an unmet therapeutic challenge. Lack of therapeutic targets and significant tumor heterogeneity are the major reasons contributing to poor prognosis and high mortality rates. To meet these challenges, we extensively analyzed BC patient databases and discovered that the high expression of a novel non coding RNA- microRNA 484- was correlated with poor survival and prognosis of BC patients. Moreover, miR-484 expression was elevated in all molecular subtypes of BC patients, especially in TNBC, compared to healthy controls. Thus, our preliminary data suggests that miR-484 could be a possible oncogenic driver, with clinical and prognostic significance in BC patients, and may thus serve as a potential molecular target. However, the role and mechanism of action of miR-484 in TNBC has not been previously elucidated. Thus, the aim of this study is to elucidate the functional role and mechanism of action of miR-484 in TNBC. To identify potential targets for miR-484, we used four different algorithms that predict the mRNA targets of miRNAs: TargetScan, miRDB, microrna.org, and Dianna microT, and based on their target prediction strategy, HOXA5 was the common target for miR-484 in all four databases. The homeobox protein (HOXA5) is a transcription factor that has been shown to play important roles in cell differentiation, hematopoiesis, and tumorigenesis. Additionally, previous studies have shown that HOXA5 may function as tumor suppressor in BC. Our results showed that ectopic inhibition of miR-484 reduced cell proliferation, colony formation, invasion/migration, and induced apoptosis in TNBC cells. Furthermore, ectopic overexpression of miR-484 reduced HOXA5 mRNA and protein expression levels in TNBC cells, suggesting that miR-484 may function in TNBC cells by inhibition of HOXA5. We further confirmed that HOXA5 is a direct target of miR-484 by luciferase reporter assay, and showed reduced luciferase activity in TNBC and HEK293 cells expressing the 3'UTR region of HOXA5 and treated with miR-484 mimic. Furthermore, in vivo therapeutic targeting of miR-484 significantly reduced tumor growth and progression in orthotopic xenograft TNBC mouse models. Overall, our findings suggest that miR-484 promotes tumor growth and progression by targeting HOXA5 in TNBC.

#505

Low miR-522 expression is correlated with paclitaxel resistance in ovarian cancer cells.

Mayuko Miyamoto, Kenjiro Sawada, Akihiko Yoshimura, Erika Nakatsuka, Michiko Kodama, Kae Hashimoto, Seiji Mabuchi, Tadashi Kimura. _Osaka University, Suita city, Japan_.

Background: Platinum-paclitaxel combination chemotherapy is standard postoperative treatment of ovarian cancer. Overcoming paclitaxel resistance is a critical issue in ovarian cancer treatment. Objectives: The aim of this study is to identify key miRNAs which regulate paclitaxel resistance and to pursue those potential as therapeutic targets. Methods: Using two serous ovarian cancer cell lines, SKVO3ip1 and HeyA8, paclitaxel resistant cell lines were established by a continuous exposure to paclitaxel. Taqman miRNA PCR array was performed and several miRNAs which were down-regulated in paclitaxel resistant cell lines were picked up. After the validation by miRNA PCR assay, miR-522 was found to be one of those. The clinical impact of miR-522 expression in ovarian cancer tissues was examined using a public database (PROGmiRV2). From a patient who had Stage IC clear cell carcinoma, RNA was extracted from formalin fixed paraffin-embedded (FFPE) tissue by laser microdissection. Thereafter, the miR-522 expression was compared with that from a normal contralateral ovary. Further, the miR-522 expression was analyzed among 4 ovarian cancer cell lines (RMG-1, RMG-2, HAC2 and KOC7C) and each IC50 value of paclitaxel was analyzed. The effect of miR-522 expression was assessed by transducing the precursor miRNA into paclitaxel resistant cells. Results: While the IC50 values of parental SKOV3ip1 and HeyA8 were 6.2 and 1.3 nM respectively, SKOV3ip1-PR and HeyA8-PR were 194.8 and 135.5 nM. MiRNA PCR array revealed 5 miRNAs including miR-522 were down-regulated in both paclitaxel resistant cell lines. Among those, the validation assay by miRNA PCR showed miR-522 was significantly down-regulated. In a public database analyzing 549 patients with serous adenocarcinomas, patients with the low expression of miR-522 had significantly correlated with worse relapse free survival than those with the high expression. In clinical paclitaxel resistant ovarian cancer tissue, the miR-522 expression was significantly down-regulated compared with that of a normal contralateral ovary. Each IC50 value of ovarian cancer cell lines (RMG-1, RMG-2, HAC2 and KOC7C) was 4.8, 222.1, 75.6, 66.9nM, respectively. Among those, IC50 values of 3 cell lines (RMG-2, HAC2 and KOC7C) were more than 50nM. In these cell lines, the expression level of miR-522 was significantly lower than that of paclitaxel sensitive SKOV3ip1. Transduction of miR-522 into paclitaxel resistant cells significantly sensitized to paclitaxel. Conclusion: MiR-522 is down-regulated in paclitaxel resistant cell lines and the low expression of miR-522 is correlated with shorter relapse free survival, indicating that the dysregulation of miR-522 is associated with the acquisition of paclitaxel resistance. The transduction of miR-522 into paclitaxel resistant cells sensitized these to paclitaxel. MiR-522 can be considered a therapeutic target to overcome paclitaxel resistance.

#506

miR-203a-3p may play a tumor suppressor role in esophageal cancer by targeting GATA Binding Protein 6.

Caiqiao Yin,1 Xiaohui Tan,1 Qingfeng Tan,2 Jingjing Wang,2 Jiqiao Zhang,2 Xiaoling Wu,3 Tao Chen,3 Hongmei Jiao,4 Sidney W. Fu1. 1 _George Washington University, Washington, DC;_ 2 _Affiliated Minda Hospital of Hubei University of Nationalities, Enshi, China;_ 3 _Chengdu Military General Hospital, Chengdu, China;_ 4 _Peking University First Hospital, Beijing, China_.

Esophageal cancer (EC) is the eighth most common cancer and the sixth most common cause of cancer death worldwide. MicroRNAs (miRNAs) are small 19-22nt non-coding single-strand RNAs that regulate diverse cellular processes and are dysregulated in a variety of cancers including EC. Using bioinformatics analysis, we identified a list of dysregulated miRNAs from most recent studies on miRNA expression profiling in EC, including miR-203a-3p, which was reported as a tumor suppressor miRNA and dysregulated in many malignancies. GATA Binding Protein 6 (GATA6) is a member of zinc finger transcription factors that is amplified or overexpressed in many tumors, such as breast cancer, pancreatic cancer, esophageal cancer, etc. Our bioinformatics analysis showed that GATA6 is a potential target of miR-203a-3p. Therefore, we hypothesize that miR-203a-3p functions as a tumor suppressor miRNA in esophageal cancer by targeting GATA6. Four esophageal cancer cell lines, including esophageal squamous cell carcinoma (ESCC) cell lines (KYSE70, KYSE180) and adenocarcinoma (EAC) cell lines (JHU-ad1,FLO-1), along with one normal esophageal cell line (HET-1A) were cultured. Six of ESCC FFPE samples were obtained and microdissected into normal, dysplasia and cancer tissues. The expression levels of miR-203a-3p and GATA6 were determined by TaqMan and CYBR green qRT-PCR assays, respectively from the cell lines and patient tissues after total RNA isolation by the Trizol reagent. The miR-203a-3p inhibitor or mimic was transfected to the above cell lines by the Lipofectamine® RNAiMAX reagent. MTT assays were used to determine the cell proliferation. The t-test was used for statistical analysis. After successful transfection of miR-203a-3p mimic, we found that cell proliferation was decreased accompanied by significantly lower GATA6 expression compared to the mock groups (p<0.05) in four cell lines (KYSE70, Het-1A, JHU-ad1 and FLO-1). However, when cells were transfected with miR-203a-3p inhibitor in KYSE70, KYSE180, JHU-ad1 and FLO-1, cell proliferation rate and GATA6 expression were both increased compared to the inhibitor mock controls (p<0.05). In clinical samples, we found that miR-203a-3p expression was downregulated during the progression of EC (100%). To determine the specificity of the functional relationship between miR-203a-3p and GATA6 in EC, we performed the luciferase array, invasion assay and other functional assays, and in the process of obtaining more clinical samples to determine if miR-203a-3p functions differently in ESCC and EAC. Our data has shown an inverse correlation between miR-203a-3p and GATA6 expression in EC, which, along with our bioinformatics analysis, indicates that miR-203a-3p downregulates GATA6 expression by directly targeting its 3'-UTR in EC. Therefore, miR-203a-3p, as a tumor suppressor, may be involved in EC development, and it may serve as a novel marker for EC management.

#507

MiR-873 functions as a potential tumor suppressor in pancreatic cancer by targeting KRAS.

Hamada A. Mokhlis,1 Recep Bayraktar,1 Nashwa N. Kabil,1 Nermin Kahraman,1 Richard Bouchard,1 Tamer Abdelghany,2 Ahmed Ashour,2 Abdel-Aziz H. Abdel-Aziz,2 George A. Calin,1 Gabriel Lopez-Berestein,1 Bulent Ozpolat1. 1 _The University of Texas MD Anderson Cancer Ctr., Houston, TX;_ 2 _Al Azhar University-Faculty of Pharmacy, Cairo, Egypt_.

Pancreatic ductal adenocarcinoma (PDAC) is an incurable cancer with a median survival of 6 months with current therapeutic strategies. The majority (~85%) of patients present with locally advanced or metastatic disease, rendering the 5-year survival rate to be only around 1-4%. The mutated K-RAS oncogene is amont the most common oncogenes in human cancer and is present in about 90% of PDAC cases. However, current treatment options targeting KRAS are still very limited, thus warranting the need for development of effective targeted therapeutics in order to improve PDAC patient outcome. miRNAs are a class of small noncoding RNAs that negatively regulate gene expression at the post-transcriptional level. Dysregulation of miRNAs is involved in the pathogenesis of a variety of diseases, including cancer, by regulating cell signaling pathways including apoptosis, cell cycle, migration/invasion, and metastasis. Oncogenic miRNAs have become major targets for novel therapies, while tumor suppressor miRNAs are being developed as therapeutic tools to inhibit expression of oncogenes in various cancers. Thus, inhibition of KRAS using miRNAs represents an appealing strategy to suppress PDAC progression. To identify potential regulators of KRAS we used various predictive algorithms including TargetScan, Diana tools and microRNA.org, and identified miR-873 as a potential regulator of KRAS. We analyzed basal miR-873 expression in PDAC cells and found it to be significantly downregulated compared with normal pancreatic epithelial cells. Using luciferase-gene reporter assay, we demonstrated that miR-873 directly binds to the 3′-UTR of KRAS mRNA and suppresses its expression. In vitro delivery of miR-873 into mutated KRAS-driven PDAC cells (PANC 1 and Mia-Paca-2) inhibited their proliferation, colony formation, migration and invasion. Furthermore, ectopic delivery of miR-873 decreased the expression of KRAS downstream signaling pathways (e.g., Akt, ERK). To target KRAS in PDAC models we recently developed single-lipid based nanoparticles (SLNP) loaded with KRAS siRNA or microRNA, which provide robust and sustained silencing of target genes, including KRAS. We also found that in vivo targeting of mutated KRAS significantly enhances in vivo perfusion of tumors detected by photoacoustic imaging. Remarkably, in vivo intravenous systemic administration of SLNP-MiR-873 nanotherapeutics (0.3mg/kg miR-873) suppressed tumor growth in PDAC xenograft models (PANC 1 and Mia-Paca-2) and silenced mutated KRAS expression. Overall, our results suggest that targeted delivery and restoration of miR-873 may be a potential therapeutic approach against PDAC.

#509

Upregulation of miR-424 promotes cell cycle progression and cell proliferation in esophageal squamous cell carcinoma.

Jing Wen, Xiuying Xie, Jianhua Fu. _Sun Yat-sen Univ. Cancer Ctr., Guangzhou, China_.

Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive malignancies of the gastrointestinal tract. miR-424 was one of the most significantly upregulated miRNAs in the ESCCs compared with normal esophageal tissues (NEs) identified by our previous miRNA arrays. The effects of miR-424 in tumor development and progression are conflicting according to previous reports till now. Here, we characterized the role of miR-424 in ESCC cell cycle progression and proliferation regulation. Expression of miR-424 was determined using qRT-PCR analysis. Cell proliferation assay by CCK-8 and the xenograft tumor model were used to determine ESCC proliferation in vitro and in vivo. Cell cycle distribution was analyzed by flow cytometry after synchronizing and releasing cells from G0/G1 phase, the onset of S-phase, or mitosis. Targets of miR-424 were validated using luciferase assays. The presence of E2F1-binding sites in the putative promoter of miR-424 gene was investigated using Chromatin immunoprecipitation (ChIP). As a result, miR-424 expression was elevated in ESCC specimens than in their normal counterparts. High miR-424 expression was a predictor of poor survival for Stage II-III ESCC patients undergoing surgery. Consistent with clinical data, knockdown miR-424 function in ESCC cells remarkably suppressed ESCC proliferation in vitro and tumor formation in mice model, while opposite results were observed after overexpressing miR-424. Cell cycle analyses revealed that miR-424 knockdown negatively regulates both G1/S and G2/M transition in ESCC cells, which were mediated by two newly identified miR-424 targets PRKCD and WEE1, respectively. During G1/S transition, increased level of PRKCD protein in KYSE410-miRZip-424 cells resulted in increased activation of PRKCD and downstream p38MAPK and JNK, which enhanced the stability of p21Cip1 protein. p21Cip1 proceeded to block CDK2 activation during G1/S transition. On the other hand, level of CDC2 phosphorylation at Tyr15, the inactive state of CDC2, was increased as a result of increased WEE1 expression by miR-424 knockdown when cells were at the G2/M border. Further evaluation of miR-424 expression in various cell cycle phases showed that miR-424 expression increased along with pri-miR-424 and pre-miR-424 when cell cycle progressed from G0/G1- to S-phase in ESCC cells. ChIP results demonstrated that G1/S transcriptional activating regulator E2F1 was enriched in regions of the putative miR-424 promoter containing the predicted E2F1-binding sites. Here, our results proved that miR-424 functions as an onco-miRNA in ESCCs. Activation of E2F1 when ESCC cells at the G1/S border induced the transcription of miR-424, which in turn targeted the expression of key genes to promote cell cycle progression and cell proliferation of ESCCs.

#510

MicroRNA-8073 as a tumor suppressor and a potential diagnostic and therapeutic target.

Atsuko Mizoguchi, Aiko Takayama, Taiga Arai, Yuho Kida, Junpei Kawauchi, Hiroko Sudo. _Toray Industries, Inc., Kanagawa, Japan_.

MicroRNAs (miRNAs) generated inside cells are also present in peripheral blood. MiR-8073 has been studied in various disease model, but its molecular function had been unknown. In this study, we demonstrated that miR-8073 regulates target genes that participate in cell survival and works as a tumor suppressor controlling only cancer cells.

First, we observed that miR-8073 expression was evidently decreased in multiple types of cancer cells compared to normal cells, but was increased in cancer exosome secreted into cultured supernatant. We then transfected synthetic miR-8073 mimic into various cancer cell lines and found dramatic reduction in cell proliferation, which was not detected in normal cells. Additionally, in tumor xenograft mice model, three-time subcutaneous injections of miR-8073 mimic at 2nM suppressed 57% growth of colorectal tumor weight.

Through mRNA expression analysis and in silico sequence analysis, we further identified five potential miR-8073 targets that plays critical roles in regulation of cell proliferation, cycle and apoptosis, and demonstrated that miR-8073 indeed bound with the 3'-UTR of these target mRNAs and reduced both mRNA and protein expression.

These results suggest that miR-8073 is a potential tumor suppressor, possibly discarded from cancer cells for their survival out to extracellular space, and circulates in bloodstream in the form of exosome. Our findings promise miR-8073 as a novel cancer diagnostic and therapeutic target.

#511

HPV mediates HNSCC carcinogenesis by exosomal exclusion of tumor suppressive miRNAs.

Madhu Khullar, Varinder Singh, Nilambra Dogra, Malay Sannigrahi, Naresh Panda. _PGIMER, Chandigarh, India_.

Background: Exosomes are small vesicles between 40-150 nm in diameter that are secreted by various cell types, including tumor cells. Exosomes of cancer cells are known to be involved in tumor pathology and have altered expression of microRNAs (miRNAs). However, the effect of HPV-16 (Human Papilloma Virus-16) infection on miRNA content of exosomes released by infected HNSCC cells has not yet been explored.

Materials & Methods: Exosomes were isolated from media of cultured HPV positive 93VU-147T and HPV negative UPCI:SCC 116 HNSCC cells and were characterized by Transmission Electron Microscopy. Presence of exosomal marker CD63 was confirmed using western blotting. Small RNA seq of exoRNA of HPV positive and HPV negative cells was carried out. For validation of identified miRNAs, HPV16 E6E7 was cloned into pcDNA3.1(-ve) vector and was transfected into SCC116 cells and stable cells were selected using G418. Differentially expressed miRNAs were validated in cells and exosomes using Real Time PCR.

Results: Seven miRNAs 6724-5p, 6087, 4497, 4792, 4508, 199a-3p and 199b-3p were found to be significantly upregulated in HPV positive HNSCC exosomes as compared to HPV negative HNSCC exosomes from NGS data. Upregulation of these miRNAs was further confirmed in exosomes derived from HPV positive 93VU-147T cells and SCC116 E6E7 cells as compared to HPV negative cells (SCC 116 (3.1-ve) (vector only transfected SCC 116) cells. Expression of these miRNAs was significantly downregulated in 93VU14T and SCC116 E6E7 (HPV +ve) cells as compared to HPV negative cells. Three of these miRNAS (4792, 199a-3p and 199b-3p) are known tumor suppressor miRNAs. miRNA 6724-5p, a novel miRNA was found to be down regulated the most in HPV +ve cells. We identified CBX6, PDK2 and FOXK1 which are known to be involved in carcinogenesis, as its target genes through Targetscan. Expression of these genes was found to be significantly upregulated in HPV positive 93VU-147T cells and SCC116 E6E7 cells as compared to HPV negative cells indicating tumor suppressive role of miRNA 6724-5p in carcinogenesis.

Conclusion: Our results suggest that HPV may mediate HNSCC carcinogenesis by exosomal exclusion of tumor suppressive miRNAs.

#512

miR-29 expression regulates B7-H3 mediated angiogenesis in medullablastoma.

Ian Purvis,1 Maheedhara R. Guda,1 Sujatha Venkataraman,2 Rajeev Vibhakar,2 Kiran K. Velpula,1 Swapna Asuthkar1. 1 _University of Illinois College of Medicine at Peoria, Peoria, IL;_ 2 _University of Colorado School of Medicine, Aurora, CO_.

B7-H3 (CD276), an immune checkpoint member of the B7/CD28 family, plays a key role in the repression of the immune response by cancer cells. B7-H3 is overexpressed exclusively on tumor and tumor-associated cells, making it an interesting therapeutic target. B7-H3 also appears to play a role outside of immune evasion, contributing to the progression of cancer via invasion, migration, and angiogenesis. Our preliminary data have confirmed the presence of B7-H3 in highly aggressive MYC+ medulloblastoma (MB) tumors. Data-mining studies revealed that B7-H3 is highly expressed across the subgroups of MB, and is correlated with MYC expression in Group 3 and 4 MBs. Recently, miR-29, a tumor-suppressor miRNA, was shown downregulating B7-H3 expression by targeting the 3' UTR of B7-H3 mRNA. Immunoblot analysis of D283 and D458 cells demonstrated reduced expression of both cellular and soluble form of B7-H3 when transfected with miR-29 plasmid. Our preliminary data have shown a novel method of targeting B7-H3 expression by miR-29 in MB cells, inhibiting the ability of MB cells to induce angiogenesis. Because both miR-29 and B7-H3 form an inversely related axis that plays a role in tumor angiogenesis, inhibition of B7-H3 by miR-29 may have potential value in developing novel antiangiogenic treatments in MB.

#513

**Antitumor** miR-451a **regulatory network is associated with poor prognosis of renal cell carcinoma.**

Yasutaka Yamada,1 Sho Sugawara,1 Takayuki Arai,1 Atsushi Okato,1 Satoko Kojima,2 Yukio Naya,2 Tomohiko Ichikawa,1 Naohiko Seki1. 1 _Graduate School of Medicine, Chiba University, Chiba-shi, Japan;_ 2 _Teikyo University Chiba Medical Center, Ichihara-shi, Japan_.

Renal cell carcinoma (RCC) is the most common neoplasm of the kidney, and approximately 70-80% of RCCs are classified as clear cell-type (ccRCC). Approximately 350,000 new cases are diagnosed worldwide, with over 140,000 deaths, every year. Although surgical resection remains the only known curative treatment for localized RCC, a significant percentage of these patients develop relapses or metastatic disease, which are associated with a poor prognosis. Recently developed targeted molecular therapies and immunomodulatory agents are currently being widely used for patients with metastatic or recurrent RCC. However, those advanced treatments are restricted to RCC patients in advanced stages of disease and the 5-year survival rate of the patients is only 5-10%.

MicroRNAs (miRNAs) belong to a class of noncoding RNA molecules that fine-tune the expression of protein coding/noncoding RNAs by repressing translation or cleaving RNA transcripts in a sequence-dependent manner. Aberrantly expressed miRNAs contribute to the initiation, development, and metastasis of several types of cancers, including RCC. Current advanced technologies can be used to identify dysregulated miRNAs in HNSCC clinical specimens. Identification of differentially expressed miRNAs is the first step to elucidating novel miRNA-mediated RNA networks in cancer cell.

Analyses of miRNA expression signature by RNA sequencing revealed that miR-451a is significantly downregulated in RCC tissues. Here, we aimed to identify novel RNA networks for miR-451a regulation in RCC cells. Ectopic expression of miR-451a significantly inhibited cancer cell aggressiveness in RCC cells. The Cancer Genome Atlas (TCGA) showed that low expression of miR-451a was significantly associated with poor survival of patients with RCC (p=0.00305). Genome-wide gene expression analyses were applied to identify miR-451a target genes. A total of 16 genes were identified as putative targets of miR-451a in

RCC cells. Among these targets, high expression of 8 genes (F2, CRELD2, MSC, LENG8, CLEC2D, SPC25, PMM2, BST2 and NSMAF) were significantly associated with poor prognosis of the patients with RCC by large numbers of cohort analyses.

Downregulated miR-451a acted as an antitumor miRNA in RCC cells. Antitumor miR-451a regulatory network was contributed to RCC aggressiveness. The identification of antitumor-mediated oncogenic networks may lead to a better understanding of RCC pathogenesis.

#514

**Lncrna** uca1 **interacts directly with angiomotin to activate Hippo-YAP signaling in epithelial ovarian cancer.**

Xianzhi Lin, Tassja J. Spindler, Simon A. Gayther, Kate Lawrenson. _Cedars-Sinai Medical Center, Los Angeles, CA_.

The long noncoding RNA (lncRNA) urothelial cancer associated 1 (UCA1) is upregulated in multiple cancers, including epithelial ovarian cancer (EOC). Our present study aims to dissect the functional and mechanistic role of UCA1 in EOC. We first explored the effects of stable UCA1 knockout mediated by CRISPR/Cas9 genome editing. The effects of UCA1 loss were more profound in vivo, as UCA1 knockout significantly impaired tumor growth in a mouse xenograft model. Pathways downstream of UCA1 were catalogued using reverse phase protein arrays, which revealed that YAP signaling is activated in UCA1-overexpressing ovarian cancer cells. To characterize the UCA1 interactome, in vivo RNA antisense purification (UCA1-iRAP) experiments were performed using a modified ChIRP and RAP protocol to isolate in vivo RNA direct interactomes based on hybridization of short (20 base) biotinylated probes at a physiologically relevant temperature (37°C). UCA1-iRAP was performed using an EOC cell model, and proteins extracted were profiled by mass spectrometry (MS). Altogether, 19 unique proteins were reproducibly detected by UCA1-iRAP-MS. Interestingly 16 of these UCA1-associated proteins were not previously known RNA-binding proteins. Integration of UCA1-iRAP-MS data with multilayered functional characteristics of UCA1 knockout/knockdown identified angiomotin (AMOT), a known Hippo-YAP signaling modulator, as potential interacting partner for UCA1. The interaction between UCA1 and AMOT was confirmed using both Western blotting for AMOT using UCA1-iRAP proteins, and RT-qPCR for UCA1 RNA after AMOT immunoprecipitation in 3 independent EOC cell lines. Knockdown experiments showed that AMOT is required for UCA1 to activate the expression of YAP target genes, including CYR61 and AXL. Our results indicate that UCA1 interacts directly with AMOT to promote cell growth by activation of Hippo-YAP signaling in EOC, and for the first time identify UCA1 as a novel upstream regulator of YAP signaling. Moreover, the optimized iRAP method we developed in this study can be readily applied to characterizing the protein, DNA, and RNA interactomes of noncoding or coding transcripts implicated other complex traits.

#515

**Integrative analysis of LncRNA-drug interactions in cancer identified** EPIC1 **as an oncogenic lncRNA that regulates breast cancer tumorigenesis and iBET resistance through regulating MYC transcriptional activity.**

Yue Wang, Zehua Wang, Bo Yang, Min Zhang, Da Yang. _Univ. of Pittsburgh, Pittsburgh, PA_.

Emerging evidence suggests that lncRNAs can serve as promising biomarkers and therapeutic targets in cancer. Here, by integrating multiple dimensional pharmacogenomic data of 11,950 lncRNA in 5,605 tumors and 1,005 cancer cell lines, we build lncRNA drug response models for 265 anti-cancer compounds across 27 cancer types. Our study reveals the cancer cell lines can realistically recapitulate patient tumors in IncRNA genomic and epigenetic alterations. Using Elastic Net (EN) regression model combined with bootstrap cross validation, our analysis identified 162,327 unique lncRNA-drug interactions. The robustness of lncRNA based EN-models is validated by independent cancer cell line drug response data. By applying lncRNA EN-models of 49 FDA approved drugs to TCGA pan-cancer patient samples across 21 caner types, we have shown that the cancer cell line based EN-models could readily predict chemotherapy responses in patients with breast, stomach, thyroid, and colon rectal cancer. Further lncRNA-protein coding gene co-expression analysis reveals that drug-predictive lncRNAs regulated drug-metabolism and drug-target pathways, which provide underlying mechanism for lncRNAs' regulation of drug response. This analysis has identified a novel intergenic lncRNA, EPIC1, regulates breast cancer tumorigenesis and iBET (Bromodomain Inhibitors) resistance through directly interacting with MYC and regulating MYC transcriptional activity. Knockdown of EPIC1 in breast cancer cells leads to inhibition of colony formation, cell cycle arrest, and suppression of tumor growth in vitro and in vivo. Overexpression of EPIC1 increased MYC target expression, breast tumorigenesis in vitro and in vivo, and promote iBET resistance in breast cancer. Mechanistically, EPIC1 directly interacts with MYC through EPIC1's 129-283 nt region. EPIC1 knockdown reduces the occupancy of MYC protein to the promoters of its target genes (e.g., p21, CCNA2, CDC20, and CDC45) without influencing MYC expression. The oncogenic effect of EPIC1 can be abolished by MYC knockdown. To our best knowledge, this is the first study to link noncoding genotypes with drug response phenotypes in both cancer cell lines and patient tumors.

#516

Long noncoding RNA NEAT1 promotes lung metastasis of soft tissue sarcoma.

Jianguo Huang,1 Eric Xu,1 Mohit Sachdeva,1 Timothy Robinson,2 Xiaodi Qin,1 Dadong Zhang,1 Kouros Owzar,1 Nalan Gokgoz,3 Andrew Seto,3 Irene Andrulis,3 Jay Wunder,3 Tomoyo Okada,4 Samuel Singer,4 Alexander Lazar,5 Brian Rubin,6 David G. Kirsch1. 1 _Duke University Medical Center, Durham, NC;_ 2 _Moffitt Cancer Center, Tampa, FL;_ 3 _Lunenfeld-Tanenbaun Research Institute, Toronto, Ontario, Canada;_ 4 _Memorial Sloan Kettering Cancer Center, New York, NY;_ 5 _MD Anderson Cancer Center, Houston, TX;_ 6 _Cleveland Clinic Lerner Research Institute, Cleveland, OH_.

Soft tissue sarcomas (STS) are malignant tumors from diverse mesenchymal tissues. About 40% STS patients develop fatal lung metastasis with a median survival of 15 months. The mechanisms driving the development of lung metastasis in sarcoma patients are poorly understood. Therefore, our lab has developed a genetically engineered mouse model (GEMM) of high-grade primary STS with conditional mutations in Kras and Trp53 (KP) where 40% of mice tumors develop lung metastasis. This KP model recapitulates human patients with Undifferentiated Pleomorphic Sarcoma (UPS), one of the most common subtypes of STS diagnosed in adults. RNA sequencing (RNA-Seq) was performed on paired primary and lung metastases in KP mouse sarcomas and determined that the expression of the long non-coding RNA (lncRNA) Neat1 is increased in lung metastases compared to paired primary tumors. Real time PCR (qPCR) in 22 paired KP mouse primary sarcomas and lung metastases further confirmed that Neat1 is significantly upregulated in lung metastases. In addition, RNA-Seq data from 5 pairs of human primary sarcomas and matched lung metastases also showed that NEAT1 levels are increased in lung metastases. Furthermore, NEAT1 RNA in situ hybridization (ISH) on tissue microarrays (TMAs) of human primary UPS and lung metastases determined that the expression of NEAT1 is upregulated in lung metastases. Next, CRISPR/Cas9 technology was applied to delete Neat1 in primary mouse sarcoma cells and loss of expression of Neat1 was confirmed by qPCR and northern blot in knockout (KO) clones. In addition, loss of Neat1 significantly reduced lung metastasis in vivo following tail vein injection of these modified cells into nude mice. To further test that loss of Neat1 reduces lung metastasis in primary sarcoma mouse model, primary tumors with Neat1 deletion are generated in GEMMs using in vivo CRISPR/Cas9 technology and KrasLSL-G12D/+; Trp53Flox/Flox; Neat1-/- (KPN) mouse model. Finally, RNA-Seq and capture hybridization analysis of RNA targets (CHART) are performed to determine mechanisms by which Neat1 regulates lung metastasis. Overall, these results suggest that upregulation of Neat1 promotes lung metastasis of soft tissue sarcoma and NEAT1 is a potential target to prevent or treat lung metastasis in sarcoma patients.

#517

LncRNA expression at the different site of high-grades serous carcinoma.

Natalie Filippov-Levy,1 Yoav Smith,1 Claes Gøran Trope,2 Ben Davidson,3 Reuven Reich1. 1 _Hebrew University of Jerusalem, jerusalem, Israel;_ 2 _Oslo University Hospital, Norwegian Radium Hospital, Norway;_ 3 _University of Oslo, Oslo University Hospital, Norwegian Radium Hospital, oslo, Norway_.

Background: High-grades serous carcinoma (HGSC) is the most aggressive subtype of ovary cancer. The disease appears at three anatomic sites: ovary tumor, solid metastasis and effusions. Not much is known about the regulatory pathways involved in cancer progression. Long non coding RNA (lncRNA) is non protein coding RNA with size >200bp. Exosomes are 30-100nm endosomal-derived vesicles containing mRNA, miRNA, lncRNA, transcription factors, proteins and lipids. They are secreted from various cells and their substance and amount alter between healthy and diseases state.

Objective: Identification of lncRNA effecting cancer progression and patient survival.

Methods: Custom made lncRNA microarray (composed off ̴17,000 lncRNAs and ̴22,000 mRNAs) followed by validation series of 180 specimens from three anatomic sites, including 77 effusions, 40 ovarian HGSC specimens, 21 solid metastases and exosomes from 42 effusion supernatants. 10 selected lncRNA were subjected to clinicopathologic parameters and survival association analysis.

Results: out of 10 lncRNA chosen for validation, 5 lncRNA- ESRG, Link-A, MEG3, GATS and PVT1 - were differently expressed between the anatomic sites and exosomes (all p<0.001). In univariate survival analysis, higher ESRG levels were significantly related to longer overall survival (OS) in the entire cohort (p=0.023), Higher Link-A levels in post-chemotherapy specimens was significantly associated with longer OS (p=0.015). MEG3 levels were significantly higher in pre-chemotherapy effusions tapped at diagnosis (n=40) compared to post-chemotherapy specimens (p=37; p=0.017). Higher GATS expression was seen in specimens from patients who were sub-optimally debulked (p=0.046). Moreover, H19 which did not change significantly between the anatomic sites, was higher in effusions from patients whose tumors showed primary resistance to chemotherapy (PFS≤6 months).

Conclusion: lncRNAs are differently express at different anatomic site and exosomes in HGSC and can provide new therapeutic targets. Furthermore, their level in effusion fluids can serve as a prognostic marker.

#518

Dual strands of pre-miR-223 (miR-223-5p and miR-223-3p) possess antitumor roles and these are involved in bladder cancer pathogenesis.

Sho Sugawara, Yasutaka Yamada, Takayuki Arai, Atsushi Okato, Mayuko Kato, Tomohiko Ichikawa, Naohiko Seki. _Chiba University, Chiba City, Japan_.

Bladder cancer (BC) is a common urologic cancer and the ninth most common cause of death worldwide. In 2015, it was estimated that approximately 76,000 new cases were diagnosed as BC and 1600 patients died in United States of America. Approximately 70-80% of patients are diagnosed with non-muscle-invasive BC (NMIBC) at the first visit and high recurrence rates (50%-70%) are observed in this patients. Moreover, 15% of recurrent BC progress to muscle-invasive BC (MIBC) and 5-year survival rate for patients with MIBC is only approximately 60%. At present, there are no effective treatments for patients with advanced BC or failure of first-line treatment. MicroRNAs (miRNAs) are endogenous small non-coding RNA molecules (19-22 bases in length) that act as fine-tuners for RNA expression in a sequence-specific manner. Unique nature of miRNA is a single miRNAs might be controlled a large number of protein-coding or protein noncoding RNAs in cells. Abnormal expression of miRNAs will be caused disruption of intracellular RNA networks and these events are involved in human diseases. In this study, we focused on both strands of pre-miR-223 (miR-223-5p and miR-223-3p) based on miRNA signature of bladder cancer (BC) and to investigate the functional significance of these miRNA in BC cells. Ectopic expression of these miRNAs showed that as in miR-223-3p (guide strand), miR-223-5p (passenger strand) inhibited the abilities of cancer cell migration and invasion in BC cells. Combination of gene expression and in silico database analyses demonstrated that a total of 33 putative targets were identified as miR-223-5p regulation in BC cells. Among these target genes, high expression of 5 genes (ANLN, HMMR, CDC20, GJB2 and BUB1B) were significantly associated with poor prognosis of the patients with BC by The Cancer Genome Atlas (TCGA) database analyses. Moreover, knockdown of ANLN significantly inhibited cell proliferation, migration, and invasion (P < 0.0001). The ANLN was highly expressed in BC clinical specimens. These results indicated that ANLN acts as an oncogene in BC. Aberrant expressed ANLN was detected in BC clinical specimens and its high expression was deeply involved in BC pathogenesis. Involvement of passenger strands of miRNAs in cancer pathogenesis is a novel concept of cancer research and novel miRNAs-mediated molecular networks may lead to a better understanding of BC pathogenesis and the development of new treatment protocols.

#519

**The long non-coding** **RNA (lncRNA)** HOXB-AS3 **regulates transcription of ribosomal RNA (rRNA) in** NPM1 **-mutated (** NPM1 **mut) acute myeloid leukemia (AML).**

Dimitrios Papaioannou,1 Andreas Petri,2 Sara Terreri,3 Charlotte A. Thrue,2 Deedra Nicolet,1 Frances A. Collins,1 Lauren A. Woodward,1 Prasanthi Kumchala,1 Malith Karunasiri,1 Felice Pepe,1 Marius Bill,1 Nina Zitzer,1 Guramrit Singh,1 Sakari Kaupinnen,2 Clara D. Bloomfield,1 Adrienne M. Dorrance,1 Ramiro Garzon1. 1 _The Ohio State University, Columbus, OH;_ 2 _Aalborg University, Copenhagen, Denmark;_ 3 _National Council of Research, Naples, Italy_.

Background: A HOXB-locus-embedded lncRNA, named HOXB-AS3 significantly associates with NPM1 mutations in AML. Herein, we evaluate the functional role of HOXB-AS3 expression in NPM1mut AML.

Methods: HOXB-AS3 expression was measured by real-time PCR. Knock-down (KD) of HOXB-AS3 was performed in vitro and in vivo with locked nucleic acid-modified gapmers. RNA antisense purification (RAP), RNA-immunoprecipitation (RIP), and Chromatin-immunoprecipitation (ChIP) experiments were performed according to published protocols.

Results: Among 7 AML cell lines tested, only OCI-AML3 cells, which harbor NPM1mut, showed detectable HOXB-AS3 expression. HOXB-AS3 was more abundant in NPM1mut AML patient (pt) blasts than blasts of AML pts with wild-type NPM1 (P=.001) and bone marrow samples from healthy donors(P=.001). HOXB-AS3 localized in the nucleus and did not associate with isolated polysomes of OCI-AML3 cells.

In vitro HOXB-AS3 KD in OCI-AML3 cells decreased the cells in S phase (P<.001) and increased those in G2/M phase (P=.006). HOXB-AS3 KD reduced the number of formed colonies by OCI-AML3 cells (P=.02). In contrast, overexpression of HOXB-AS3 in K562 cells increased the cells in S phase (P=.02) and decreased those in G0/G1 phase (P=.008). HOXB-AS3 KD in blasts of 3 NPM1mut AML pts decreased the number of formed colonies (P=.03, P=.02, and P<.001). In vivo HOXB-AS3 KD in murine patient-derived xenografts of 2 NPM1mut AML pts prolonged their overall survival (P<.001 and P=.03). RAP-based isolation of HOXB-AS3 and comparative proteomic analyses identified 23 candidate HOXB-AS3-binding proteins. EBP1 was validated as the most avid HOXB-AS3 interactor (P<.001) by RIP experiments. Manipulations of HOXB-AS3 impacted on the (previously reported) EBP1 interaction with NPM1; HOXB-AS3 KD reduced, whereas overexpression of HOXB-AS3 increased the EBP1-NPM1 complex formation. Consequently, HOXB-AS3 KD reduced transcription of rRNA and de novo protein synthesis in OCI-AML3 cells (P<.001 and P=.002) and AML pt blasts (P<.001 and P=.03, respectively). Overexpression of HOXB-AS3 increased rRNA transcription (P<.001), de novo protein synthesis (P=.001). ribosomal DNA (rDNA) promoter occupancy by RNA-Polymerase I (P=.001), and activity of an rDNA promoter-containing luciferase reporter (P=.002) in K562 cells. We hypothesized that HOXB-AS3 guides EBP1 to the rDNA locus. RAP-DNA experiments validated the interaction of HOXB-AS3 with rDNA chromatin (P=.001) and HOXB-AS3 KD decreased the occupancy of the rDNA promoter by EBP1 (P=.002), as shown by ChIP assays.

Conclusions: We describe the function of the HOXB-AS3 lncRNA as a compensatory mechanism, which mediates increased rRNA transcription and adequate protein production, in

NPM1mut AML. From a therapeutic standpoint, we show that HOXB-AS3-targeting yields anti-leukemic activity in pre-clinical models.

#520

circEPSTI1 as a prognostic marker and mediator of triple-negative breast cancer progression.

Bo Chen, Xiaojia Huang, Weidong Wei, Hailin Tang, Xiaoming Xie. _Sun Yat-Sen University Cancer Center, State Key Laboratory of Oncolog, Guangzhou, China_.

Purpose: Circular RNAs (circRNAs) represent a class of non-coding RNAs which play a vital role in modulating gene expression and several pathological responses. However, it is largely unknown about the expression profile and function of circRNAs in triple negative breast cancer (TNBC). In the current study, we investigated the expression profile of human circRNAs in TNBC tissues and identified circEPSTI1 (hsa_ circRNA_000479) as one circRNA significantly upregulated.

Experimental Design: We performed circular RNAs microarray assay to screen circular RNAs expression profiles of TNBC and further investigated circEPSTI1. We observed the effect of circEPSTI1 on proliferation, clonal formation and apoptosis of TNBC by knockdown of circEPSTI1 in three TNBC cell lines. According to the MREs analysis and luciferase reporter assay, we found circEPSTI1 could bind to miRNAs as a miRNA sponge and the co-target gene of miRNAs and performed xenografts in mice to confirm our findings. We evaluated circEPSTI1 levels of 240 TNBC patients by ISH.

Results: We found that knockdown of circEPSTI1 could inhibit TNBC cell proliferation and induce apoptosis. In vitro and in vivo experiments implicated that circEPSTI1 could bind to miR-4753 and miR-6809 as miRNAs sponge to regulate BCL11A expression and affect TNBC proliferation and apoptosis. High level of circEPSTI1 was correlate with shorter survival in TNBC patients.

Conclusions: The circEPSTI1-miR-4753/6809-BCL11A axes can affect the proliferation and apoptosis of triple negative breast cancer through the mechanism of competing endogenous RNAs. Meanwhile, our results provide circEPSTI1 as an independent prognostic marker for survival in patients with TNBC.

#521

Dynamic measurements of circulating microRNAs reflect different biological effects of radiofrequency ablation and transarterial chemoembolisation in liver cancer patients.

Jaroslav Juracek,1 Tomas Andrasina,2 Barbora Cechova,1 Petra Vesela,1 Jan Zavadil,2 Tana Machackova,1 Jiri Sana,1 Marek Vecera,1 Natalia Gablo,1 Marek Svoboda,3 Nahum Goldberg,4 Ondrej Slaby1. 1 _CEITEC - Central European Institute of Technology, Brno, Czech Republic;_ 2 _University Hospital Brno, Brno, Czech Republic;_ 3 _Masaryk Memorial Cancer Institute, Brno, Czech Republic;_ 4 _Beth Israel Deaconess Medical Center/Harvard Medical School, Boston, MA_.

Introduction: The majority of primary or metastatic liver tumors are unresectable (because of tumor size, location, poor performance status or multifocality), therefore other therapeutic modalities as radiofrequency ablation (RFA) and transarterial chemoembolization (TACE) are applied. RFA is a localized thermal treatment technique designed to produce tumor destruction by heating tumor tissue, while TACE combines cytotoxic effect of particle based tumor ischemia and locoregional chemotherapy. Both methods cause characteristic changes in liver tissue (inflammation, hypoxia, elevated temperature, tissue destruction) accompanied by targeted systemic secretion of microRNA into the bloodstream. Since RFA and TACE differ in the dynamics with which they affects the tumor tissue, we aimed to investigate whether the expression level of circulating microRNAs related to hypoxia (miR-21 and miR-210), liver injury (miR-122) and epithelial-mesenchymal transition (miR-200a) could reflect such changes.

Material and methods: This study consisted of 14 patients diagnosed with primary hepatocellular carcinoma (HCC) (median age 73; TACE) and 20 patients diagnosed with liver metastases of colorectal cancer (median age 63; 17 patients - RFA, 3 patients - TACE). RFA was performed using the rf/mw generator (AngioDynamics). For TACE drug eluting beads (Biocompatibles Ltd.) loaded with irinotecan for mCRC patients and doxorubicin for HCC patients were used. The concentrations of miRNA were determined for all patients in series of blood plasma from 4 time points (before intervention, immediately after intervention, 24 hours after intervention, 1 week after intervention) using miRNA-specific TaqMan assays and qRT-PCR method.

Results: In RFA cases we observed significant increase of investigated miRNA concentrations immediately after intervention (miR-122, FC = 15, P = 0.0002; miR-200a, FC = 1.9, P = 0.015). In TACE we observed delayed increase in circulating miRNA concentrations at time point 24 hours after intervention (miR-21, FC = 10.4, P < 0.0001; miR-210, FC = 9.0, P = 0.03; miR-122, FC = 27, P = 0.0004; miR-200a, FC = 4.0, P = 0.0098). In both methods, the initial increase was followed by a steady decline of miRNA levels. Identified dynamic changes in circulating miRNA levels were in accordance with the nature of RFA and TACE biologic effects. In selected cases, we observed specific dynamic miRNA patterns to be linked to the course of the disease (e.g. necessity of additional intervention).

Conclusions: Our preliminary data indicates potential usage of circulating miRNAs for monitoring of the systemic effects of RFA and TACE therapy and their ability to reflect efficacy of intervention procedures. This work was supported by Ministry of Health of the Czech Republic, grant nr. 15-33158A, 15-34553A, 15-31627A, 16-31314A, and 15-34678A.

#522

Histone deacetylase 6-let-7i-5p-thrombospondin-1 signaling axis suppresses CD47-dependent tumorigenic behavior of liver cancer.

Hee Doo Yang, Jung Woo Eun, Qingyu Shen, Hyung Seok Kim, Sang Yean Kim, Suk Woo Nam. _Lab of Oncogenomics and Functional RNomics Research Center, Department of Pathology, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea_.

Histone deacetylase 6 (HDAC6) uniquely endows as tumor suppressor in liver tumorigenesis, but the underlying mechanisms leading to liver cancer are not fully understood. To identify the large-scale microRNA (miRNA) expression changes by HDAC6, we performed miRNA expression analysis in HDAC6-overexpressing Hep3B cells. From this, let-7i-5p was suggested as most highly associated miRNA with HDAC6 in liver cancer cells. We also found that let-7i-5p expression was up-regulated in a large cohort of HCC patients, and high expression of let-7i-5p was significantly associated with poor prognosis of HCC patients. Ectopic expression of antisense let-7i-5p (AS-let-7i-5p) caused cancer cell growth inhibition mediated by caspase-dependent cell death processing, and inhibited epithelial-mesenchymal transition (EMT) of liver cancer cells. Computational analysis suggested that thrombospondin-1 (TSP-1) is a specific target for let-7i-5p in liver cancer cells. Increased secretion of TSP1 was detected in AS-let-7i-5p transfected HCC cells. We found that partially purified TSP-1 from culture media (ppTSP-1) induced cellular death processing and simultaneously inhibited tumor angiogenesis and metastatic behavior of liver cancer cells. In addition, we observed that anti-TSP-1 antibody containing CD47 receptor binding motif (3F352) attenuated HDAC6, AS-let-7i-5p and TSP-1 effects on liver cancer cells. Furthermore, TSP-1 and ppTSP-1 treatments significantly enhanced macrophage phagocytosis through blocking CD47-dependent "Don't eat me" signal in liver cancer cells. Our findings suggest that HDAC6-let-7i-5p-TSP-1 signaling axis regulates CD47-dependent neoplastic and phagocytic behaviors of hepatocytes providing a novel target for the molecular treatment of liver malignancies.

#523

miR-206 inhibits pleural mesothelioma by targeting an active KRAS/CDK4/CCND1 pathway.

Anand Singh, Roma Pahwa, Li Zhang, Nathanael Pruett, R Taylor Ripley, David S. Schrump, Chuong D. Hoang. _National Cancer Institute, NIH, Bethesda, MD_.

Introduction: Since there remains no effective, durable chemotherapy against malignant pleural mesothelioma (MPM), we propose to leverage microRNA(miR)-based approaches that recently reached phase I trial testing. We posit there are many miR with therapeutic potential yet to be identified in MPM. Methods/ Results: Our prior miRNA expression profiling results of MPM (GSE40345) were cross-referenced with a novel p-value minimization analytic technique to identify prognostic miRNA. Both MPM cell lines and tissues (tumors and normal pleura) were assessed using quantitative and functional biologic assays. In silico algorithms identified gene targets that were verified by 3'-UTR luciferase assay.In silico analyses showed underexpression of miR-206 by 12.1-fold in MPM tumors compared to normal pleura. Using our p-value minimization technique, we assessed the clinical impact of this underexpressed miR in TCGA data (n=73 MPM) and noted a significant association to worse survival for patients with lower miR-206 expression (p=0.024). We confirmed that miR-206 was significantly underexpressed in tumor tissues by qRT-PCR analysis of a new, randomly selected MPM cohort (n=41) versus normal pleura (n=14). In a set of established MPM cell lines with low endogenous miR-206 level, ectopic re-expression of miR-206 dramatically suppressed cell proliferation, invasiveness, colony foci formation, and growth in soft-agar. Treatment of non-malignant mesothelial cells MeT-5a and LP9 with ectopic miR-206 showed no untoward effects. In silico gene target predictions for miR-206 were summated across 3 databases and screened by prognostic effect based on Kaplan-Meier results from TCGA data (n=87 MPM). Interestingly, we noted several overexpressed MPM-prognostic genes (p<0.05) regulated by miR-206: KRAS, CDK4, and CCND1. qRT-PCR analysis of tissues confirmed elevated transcripts of KRAS, CDK4, and CCND1 in MPM. This signaling axis of KRAS/CDK4/CCND1 is important in MPM as it summates well-known dysregulated tyrosine kinase receptors (EGF, IGF-1, VEGF, MET, etc) that are upstream. The KRAS/CDK4/CCND1 axis is associated with cell cycle progression and survival of cancer cells, but is not an easily druggable target. miR-206 treatment significantly downregulated KRAS, CDK4 and CCND1 genes in MPM cell lines. Conclusion: In vitro, miR-206 exerts tumor suppressive effects in MPM via inhibition of KRAS/CDK4/CCND1 signaling. miR-206 restoration in MPM mimics simultaneous blocking of multiple tyrosine kinases. Loss of miR-206 and concomitant overexpression of KRAS, CDK4 and CCND1 formed a poor prognostic signature of MPM. Our results identify miR-206 as a rational therapeutic agent to studied further in preclinical MPM models.

#524

Impact of age, sex, smoking, body mass and physical activity on circulating small non-coding RNA expression profiles.

Trine Ballestad Rounge,1 Sinan Ugur Umu,1 Andreas Keller,2 Eckart Meese,2 Giske Ursin,1 Steinar Tretli,1 Robert Lyle,3 Hilde Langseth1. 1 _Cancer Registry of Norway, Oslo, Norway;_ 2 _Saarland University, Saar, Germany;_ 3 _Oslo University Hospital, Oslo, Norway_.

Non-coding RNAs (ncRNA) are regulators of cell functions and circulating ncRNAs from the majority of the RNA classes, such as miRNA, tRNA, piRNAs, lncRNA, snoRNA, snRNA and miscRNAs, are potential non-invasive cancer biomarkers. Understanding how non-disease traits influence ncRNA expression is essential for assessing their biomarker potential. The aim of the study was to investigate associations between sex, age, smoking, body mass, physical activity, technical factors such as sample storage and processing, and serum ncRNA expression profiles. Serum samples from 526 healthy individuals in the Janus Serum Bank Cohort were included in the study. The samples were collected in the time-period 1972-2004 with varying collection procedures, and stored at - 25º C. Information on smoking habits, body mass and physical activity was linked from health examination survey data. RNA was extracted from 400 µl serum using phenol-chloroform separation and the miRNA Neasy Serum/Plasma kit (Qiagen). Small RNAseq was performed using NEBNext Small RNA Library Prep Set for Illumina with an average sequencing depth of 18 million reads per sample. The RNAseq reads were initially trimmed for adapters using Adapter Removal (v2.1.7). We then mapped the collapsed reads (generated by FASTX v0.14) to the human genome (hg38) using Bowtie2 (10 alignments per read were allowed). We compiled a comprehensive annotation set from miRBase (v21) for miRNAs, pirBAse (v1.0) for piRNAs, GENCODE (v26) for other RNAs and tRNAs. We used SeqBuster (v3.1) to get isomiR and miRNA profiles. To count the mapped reads, HTSeq (v0.7.2) was used. Differential gene expression analyses based on the negative binomial distribution and Wald significance tests were performed for each trait using the R package DESeq2 version 1.14.1. We identified associations between all RNA classes and traits. Ageing showed the strongest association with ncRNA expression, both in terms of statistical significance and number of RNAs, regardless of RNA class. Serum processing modifications and storage times significantly altered expression levels of a number of ncRNAs. Smoking cessation generally restored RNA expression to non-smoking levels, although for some isomiRs, mRNA fragments and tRNAs smoking-related expression levels persisted. sncRNA expression levels in serum are considerably age-dependent and age should be adjusted for in studies of circulating sncRNA expression. Certain biomarkers are also influenced by body mass, smoking, physical activity, serum processing and storage conditions.

#525

Role of a novel microRNA gene at frequently deleted chromosome 8p region in prostate cancer.

Divya Bhagirath, Thao Yang, Shahana Majid, Varahram Shahryari, Soichiro Yamamura, Z. Laura Tabatabai, Rajvir Dahiya, Guoren Deng, Sharanjot Saini. _UCSF and SFVAMC, San Francisco, CA_.

A frequent alteration in the prostate oncogenome is the loss of chromosome (chr) 8p21-22 region that has been traditionally associated with the loss of homeodomain protein, NKX3.1, that plays important roles in prostate cancer (PCa) initiation. Genomic deletions of this region increase significantly with tumor grade and are associated with poor prognosis in prostate cancer suggesting a link of this region with PCa progression. We proposed and validated a novel, paradigm shifting hypothesis that this frequently deleted locus is associated with a cluster of miRNA genes - miR-3622a/b and miR-383- that are lost in PCa and play an important mechanistic role in PCa progression and metastasis by regulating Epithelial-mesenchymal transition (EMT) and stemness. Extending our hypothesis, in this study, we evaluated the role of another miRNA gene located within this region- miR-4288- in prostate cancer. To understand the role of miR-4288 in prostate cancer, we performed miRNA expression profiling in laser capture microdissected (LCM) PCa tissues (n=64) and matched adjacent normal regions by real-time PCR. miR-4288 expression was down regulated in ~66% of tissue samples. miR-4288 expression was not altered significantly in 14% cases and high miR-4288 expression was observed in 20% cases. We performed ROC (Receiver Operating Characteristic) analyses to test the diagnostic potential of miR-4288 expression. Our analyses showed that miR-4288 expression can be a significant parameter to discriminate between normal and tumor tissues with an area under the ROC curve (AUC) of 0.711 (95% CI: 0.625-0.787, P<0.0001). We found that miR-4288 exhibits 81.54% sensitivity and 51% specificity as a diagnostic parameter. Association with clinicopathological parameters of the disease showed that miR-4288 expression was not significantly correlated with age, tumor stage, Gleason grade, biochemical recurrence or serum PSA levels. However, significant correlation was observed between miR-4288 expression and race. Analyses of miR-4288 expression in prostate cell lines showed that its expression is specifically attenuated in PCa cell lines compared to normal or immortalized prostate epithelial cells. To evaluate the functional role of miR-4288 in prostate cancer, miR-4288 precursor was overexpressed in PCa cell lines followed by functional assays. miR-4288 overexpression in PC3 and LNCaP cell lines led to decreased cellular viabilities as compared to control cells. Our preliminary data suggests that miR-4288 overexpression lead to morphological changes in these cell lines consistent with mesenchymal to epithelial transition (MET). In conclusion, our data suggests that miR-4288 is commonly downregulated in prostate cancer and plays an important regulatory role.

#526

Mechanistic studies of TAp63 regulated oncogenic long noncoding RNAs in breast cancer metastasis.

Xiaobo Li. _Moffitt Cancer Center, Tampa, FL_.

Advanced breast cancer metastasis is the major cause of relapse. However, no effective treatment exists for the metastatic stage of breast cancer. Recently, regulatory long non-coding RNAs (lncRNAs) have been highlighted in p53 regulated genome protection in cancer metastasis; therefore approaches targeting lncRNAs is a promising strategy to treat breast cancer metastasis. Our laboratory aims to design such approaches through focusing on the p53 family member, TAp63 and its regulated lncRNAs. TAp63 has critical tumor suppressor function in breast cancer. Loss of function transforms mammary epithelial cells (MECs) into tumor initiating cells.

Our lab showed that loss of TAp63 in mouse MECs induces the expression of several lncRNAs. A locus conservation strategy was applied to identify the homologous lncRNAs relevant in human breast cancer progression. Targeting these lncRNAs with CRISPR/CAS9 system in human breast cancer cell line in mouse xenograft model demonstrated that they are required for the tumorigenic and metastatic potential of human breast cancer cells in vivo. The high-throughput protein microarray screening method was used to identify proteins that interact with these lncRNAs and facilitate their oncogenic functions. Importantly, the expression of both protein and lncRNA is negatively correlated with patient survival in breast cancer dataset, and positively correlated with breast cancer progression in human tissue microarrays.

We demonstrate the oncogenic effects of these lncRNAs in promoting breast cancer metastasis. Our data indicates that targeting lncRNAs directly or their effectors indirectly will provide innovative and effective therapeutics aiming the metastatic stage of breast cancer.

#527

Identification of novel target mRNAs of microRNA-143 in glioblastoma cells.

Eunice Lozada-Delgado,1 Fatma Valiyeva,2 Maria Marcos,3 Pablo Vivas3. 1 _University of Puerto Rico- Rio Piedras, PR;_ 2 _University of Puerto Rico- Comprehensive Cancer Center, PR;_ 3 _University of Puerto Rico- Medical Sciences Campus, PR_.

The purpose of this study is to assess the biological role of microRNA-143 (miR-143) in Glioblastoma multiforme (GBM). In the United States, the incidence of GBM is about 17% of all primary brain tumors and about 60-75% of all Astrocytomas. Many patients recur even after therapy with a median survival rate of 15 months. MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression post-transcriptionally. Deregulated miRNAs have been identified in cancer including GBM. In this study, we aim to thoroughly uncover the role of miR-143 by using GBM cell lines, mouse models, and patient samples. Previous qPCR data from FFPE samples of Puerto Rican GBM patients showed a higher relative expression of miR-143 in GBM patients compared to control individuals and paired surrounding non-cancerous tissue suggesting an oncogenic role. Moreover, in vitro data of transient transfections of a miR-143 oligonucleotide inhibitor in GBM cells showed reduced cell proliferation (clonogenic assay), increased apoptosis and cell cycle arrest in the G0/G1-S phase (flow cytometry and western blot analysis). Furthermore, we want to identify novel target mRNAs of miR-143 in GBM cells that may explain these results. Using in silico analysis tools and a qPCR analysis we identified 5 candidate genes as potential miR-143 targets. Western blot analysis of the proteins encoded by these genes using miR-143 overexpressing cells showed that the Integral membrane protein 2B (ITM2B) and Zinc transporter 8 (SLC30A8) protein levels were reduced and when miR-143 was transiently inhibited their protein levels where increased. These results suggest that miR-143 targets these mRNAs. Ongoing luciferase reporter assays will determine direct binding of miR-143 to the 3'UTR of these mRNAs in GBM cells.

#528

Identification of MYC as a novel repressor of miR-29 in pancreatic ductal adenocarcinoma.

Jason Kwon, Kayla Quirin, Tricia Factora, Janaiah Kota. _Indiana Univ. School of Medicine, Indianapolis, IN_.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignant disease with a dismal 5-year survival rate of 8%. The lack of effective therapies reinforces the need to better understand molecular mechanisms in PDAC. In our previous work, we found that downregulation of miR-29 is a common phenomenon of PDAC, and its restored expression had a potent tumor-suppressive effect in pancreatic cancer cells. However, the mechanism associated with the loss of miR-29 in PDAC has yet to be elucidated. In our initial work, using PDAC cell lines, we found that miR-29 is downregulated independent of KRAS. In an effort to understand the transcriptional factors mediating miR-29 repression, we selected several transcription factors that have a role in PDAC pathogenesis and binding sites in the miR-29 promoter region. siRNA mediated knockdown (KD) of three candidate transcription factors, SMAD4, MYC, and Gli3 revealed that the KD of MYC resulted in the highest gain of miR-29 expression (2-fold increase). Furthermore, MYC nuclear localization negatively correlates with miR-29 expression in various pancreatic cancer cell lines, and treatment of pancreatic cancer cells with small-molecule MYC inhibitor resulted in a dosage-dependent increase of miR-29 expression. miR-29a/b1 locus located on human chromosome 7 has two canonical MYC binding motifs upstream of the miR-29 transcription start site, which were functionally validated via luciferase assay. Finally, autophagy has been shown to be upregulated by MYC and is known to mitigate stressors of the integrated stress response (ISR) pathway in cancer. In our previous work, we showed that miR-29 blocks autophagy in pancreatic cancer cells. Consistently, we found miR-29 overexpression led to decreased cancer cell viability in combination with gemcitabine and induced the ISR as indicated by increased phospho-eIF2α. Taken together, our results demonstrate that MYC plays a key role in the regulation of miR-29 expression in pancreatic cancer. Furthermore, our data indicate the potential use of miR-29 as a novel therapeutic agent for PDAC, and these results could help form the basis for future studies to develop a more targeted approach of miR-29 therapeutic administration.

#529

PTEN status regulates let-7a-3p mediated therapeutic response in glioma stem cells.

Rajbir Singh, Kamalakannan Palanichamy, Saikh Jaharul Haque, Arnab Chakravarti. _The Ohio State Univ., Columbus, OH_.

Cancers of the brain and central nervous system are among the leading cause of deaths in patients less than 40 years of age and second most common cancer types in children and adolescents. Glioblastoma (GBM) is the most common primary tumor of the central nervous system, and is highly invasive, proliferative and vascularized. A key player in the GBM pathogenesis is a distinct subpopulation of tumor cells which are capable of self-renewal, highly angiogenic and possess the potential for extensive proliferation and multi-lineage differentiation. This cell population termed Glioma Stem Cells (GSCs) is highly resistant to the chemo- and radiotherapies and plays an important role in tumor initiation, progression, and recurrence. In response to radiation therapy, GSCs can trigger the activation of multiple signaling pathways including Wnt, Notch, and Hedgehog, and their DNA damage repair system is proactive. The miRNA network adds a dimension of regulatory control, which serves to maintain pluripotency and reprograms multiple stemness and radioresistance promoting pathways. However, not much information is available that could provide mechanistic insights regarding how specific miRNAs modulate radioresistance pathways. Our initial efforts involving a global screening of microRNA profiles in different GSCs after radiation treatment resulted in identification of multiple microRNAs that were significantly altered in response to radiation. Based on our preliminary data, we selected let-7a-3p as one of the potential candidates. We used specific lentiviral constructs to manipulate let-7a-3p in GSCs, and carried out multiple in vitro studies to analyze the effects of the respective alteration in relation to radioresistance and proliferation. We also carried out an extensive in silico analysis to identify the potential targets of these microRNAs, followed by analyzing various downstream components of the target genes. Additionally, we carried out an assessment of the overall effects to probe if there is an integration of one or more pathways that could lead to radiation sensitivity. Our in vitro findings revealed a specific roles of let-7a-3p in regulating the radioresistance and proliferation of glioma cells, which was contingent upon the PTEN status of the cell type. Furthermore, we analyzed miR-seq data from TCGA for functional validation of our study and discovered a defined correlation of let-7a-3p and its downstream effectors. In conclusion, our study provides a basis for understanding the role of miRNAs in radioresistance phenotype and furthermore, it may lead towards another tool to inhibit GSC proliferation.

### Oncogenes and Tumor Suppressor Genes

#531

**Heterozygous** IDH1R132H/WT **created by "single base editing" inhibits human astroglial cell growth by downregulating YAP.**

Shuang Wei,1 Jie Wang,2 Olutobi Oyinlade,1 Ding Ma Ma,1 Shuyan Wang,1 Shuli Xia1. 1 _Kennedy Krieger Research Inst., Baltimore, MD;_ 2 _Johns Hopkins School of Medicine, Baltimore, MD_.

Mutations in the isocitrate dehydrogenase 1 (IDH1) gene have been identified in a number of cancer types, including brain cancer. The Cancer Genome Atlas project has revealed that IDH1 mutations occur in 70-80% of grade II and grade III gliomas. Until recently, most of the functional studies of IDH1 mutations in cellular models have been conducted in overexpression systems with the IDH1 wild type background. In this study, we employed a modified CRISPR/Cas9 genome editing technique called "single base editing", and efficiently introduced heterozygous IDH1 R132H mutation in human astroglial cells (~20%). Compared with astroglial cells with IDH1 wild type, monoallelic IDH1 R132H mutation (IDH1R132H/WT) inhibited cell proliferation and promoted cell migration. Global gene expression analysis identified molecular targets and pathways altered by IDH1R132H/WT, including cell proliferation, extracellular matrix, and cell migration, consistent with our phenotype characterization. Global DNA methylation profiling showed hypermethylation as well as hypomethylation induced by IDH1R132H/WT. Using our mutated IDH1 models generated by genome editing, we identified novel molecular targets of IDH1R132H/WT, namely Yes-associated protein (YAP) and its downstream signaling pathway Notch, to mediate the cell growth-inhibiting effect of IDH1R132H/WT. Our findings are in line with the clinical observations that brain tumor patients with mutated IDH1 have a better prognosis. In summary, the "single base editing" strategy has successfully created heterozygous IDH1 R132H mutation that recapitulate the naturally occurring IDH1 mutant. Our isogenic cellular systems that differ in a single nucleotide in one allele of the IDH1 gene provide a valuable model for novel discoveries of IDH1R132H/WT-driven biological events.

#532

PELP1 and AIB1 (SRC-3) complexes promote cancer stem cell-associated phenotypes in ER+ breast cancer models.

Thu H. Truong, Hsiangyu Hu, Nuri A. Temiz, Kyla M. Hagen, Brian J. Girard, Nicholas J. Brady, Kathryn L. Schwertfeger, Carol A. Lange, Julie H. Ostrander. _University of Minnesota Masonic Cancer Center, Minneapolis, MN_.

Proline, glutamic acid, and leucine rich protein 1 (PELP1) is overexpressed in approximately 80% of invasive breast tumors. PELP1 dynamically shuttles between the nucleus and cytoplasm, but is primarily nuclear in normal breast tissue. However, altered localization of PELP1 to the cytoplasm is an oncogenic event that promotes breast cancer initiation and progression. Herein, we sought to identify interacting partners unique to cytoplasmic PELP1 and determine the mechanisms by which these interactions promote oncogenic PELP1 signaling. We identified AIB1 (amplified in breast cancer 1; also known as SRC-3 or NCOA3) as a novel binding partner of cytoplasmic PELP1 in both estrogen receptor-positive (ER+) and ER-negative cell lines. Cytoplasmic PELP1 expression elevated basal phosphorylation levels (i.e., activation) of AIB1, enhanced ALDH+ tumorsphere formation, and upregulated specific target genes independently of hormone stimulation. Direct manipulation of AIB1 levels using shRNA abrogated cytoplasmic PELP1-induced tumorsphere formation and downregulated cytoplasmic PELP1-specific target genes. SI-2, an AIB1 inhibitor, inhibited the PELP1/AIB1 interaction and decreased cytoplasmic PELP1-induced tumorsphere formation. Similar results were observed in a murine-derived MMTV-AIB1 tumor cell line (J110). Furthermore, in vivo syngeneic tumor studies showed that PELP1 knockdown in J110 cells resulted in increased survival of tumor-bearing mice as compared to mice injected with control cells. These studies suggest that manipulating PELP1 location or levels has the potential to mitigate tumor progression, particularly in the context of AIB1-mediated tumorigenesis. Taken together, our data demonstrate that cytoplasmic PELP1/AIB1-containing complexes function to promote advanced cancer phenotypes, including outgrowth of stem-like cells, associated with estrogen-independent breast cancer progression.

#533

OTULIN promotes resistance and metastasis upon chemotherapy in TNBC by activating Wnt/β-Catenin signaling.

Wei Wang, Jixiao Niu, Bo Zhang, Zhaohui Wu. _Univ. of Tennessee Health Science Ctr., Memphis, TN_.

Triple-negative breast cancer (TNBC) or Basal-like breast cancer is characterized by worse prognosis than other breast cancer subtypes due to the rapid-arising therapeutic resistance accompanied by aggressive metastasis. The therapy-resistant tumor cells, including cancer stem-like cells (CSCs) with intrinsic resistance or tumor cells acquired resistance during treatments, could repopulate the primary tumors and serve as seeds for recurrent metastatic lesions. However, the molecular events orchestrating the innate and acquired therapeutic resistance are not completely understood. We found that the linear ubiquitin-specific deubiquitinase OTULIN is overexpressed in breast tumors, especially in the basal-like subtype, compared with normal breast tissues. Moreover, OTULIN overexpression significantly increased TNBC cells resistance to chemotherapy and is associated with shorter DFS in breast cancer patients. Mechanistically, we found genotoxic treatments activate OTLULIN, which promotes epithelial-to-mesenchymal transition (EMT) in TNBC cells through increased activity of Wnt/β-Catenin signaling. OTULIN overexpression significantly enhanced TNBC cells resistance to chemodrugs while inhibiting Wnt/β-Catenin signaling or OTULIN sensitized TNBC xenografts to Doxorubicin treatment in vivo. Our findings suggest that OTULIN-mediated Wnt/β-Catenin activation may increase TNBC therapeutic resistance and aggressiveness by promoting EMT. Targeting OTULIN and the chemotherapy-induced Wnt/β-Catenin activation may serves as promising strategy to mitigate drug resistance and reduce metastasis in TNBC patients receiving chemotherapy.

#534

Oncogenes alter mammary epithelial cell lineage identity prior to tumor formation.

Jerrica Breindel,1 Felix Nampanya,2 Aditya Datye,1 Daniel H. Miller,3 Piyush Gupta,3 Charlotte Kuperwasser1. 1 _Tufts University, Boston, MA;_ 2 _University of Massachusetts Boston, Boston, MA;_ 3 _Massachusetts Institute of Technology, Cambridge, MA_.

Background: Breast cancer is a heterogeneous disease encompassing multiple subtypes that differ in their molecular profiles, clinical course of progression, and response to therapeutics. Interestingly, breast cancers fall broadly into luminal and basal subtypes, which resemble the normal luminal and basal epithelial cells of the breast. This correlation suggests that the molecular mechanisms controlling epithelial cell lineage identity during normal mammary development may also influence tumor subtype during cancer formation. Since many breast cancers are driven by oncogenes that are important for normal mammary development, we hypothesize that dysregulated oncogene signaling may alter mammary epithelial cell lineage identity at an early step of cancer initiation, priming the breast tumor subtype that will be formed upon full tumorigenesis.

Methods: Human breast cell lines, MCF10a and MCF10f, and primary human breast tissues were infected with virus expressing single breast cancer oncogenes. Flow cytometry and quantitative RT-PCR were performed on breast cell lines to determine changes in the balance of luminal to basal cells and expression of luminal or basal gene sets. Primary tissues were grown in hydrogels and stained for luminal and basal markers to determine the effect of oncogene expression on the growth of mammary ductal structures. Mouse tumor models, MMTV-Myc and MMTV-Wnt1, were investigated for changes in their mammary epithelial architecture by histology and flow cytometry at ages prior to tumor formation.

Results: Oncogene overexpression in human breast cell lines causes shifts in basal and luminal cell balance that correlate with changes seen in mammary ductal growth in hydrogel cultures of primary human breast epithelial cells. In mouse models, individual oncogenes alter mammary epithelial architecture and cell balance at early stages prior to tumor formation. In both models, individual oncogenes are able to regulate mammary epithelial cell lineage identity in the absence of other tumor-initiating signals, indicating that oncogenes may influence breast cancer subtype through mammary epithelial cell lineage regulation at early stages of cancer progression.

#535

The impact on transcriptome diversity after oncogenic transformation of human mammary epithelial cells.

Anthony Tran. _Dana Farber Cancer Institute, Philadelphia, PA_.

Breast cancer is a leading cause of cancer-related morbidity and mortality worldwide. In the USA, one out of eight women will be diagnosed with breast cancer over the course of their lifetime. A major obstacle hindering the treatment of breast cancers is intratumor heterogeneity since cytotoxic drug sensitivities among different clonal populations may vary. Previously, we have shown that cancer cells display an increased repertoire and abundance of all transcripts within a cell (termed transcriptome diversity) compared to normal cells. The driving mechanism(s) of cellular intratumor heterogeneity has yet to be fully elucidated. In this study, we utilize immortalized human mammary epithelial cells (MCF10A) transformed with an array of doxycycline-inducible lentiviral vectors encoding known genetic drivers of breast tumorigenesis (TP53 mutant, BRCA1 mutant, PIK3CA mutant, shPTEN and c-MYC) alone and in combination to determine their impact on transcriptome diversity based on RNA-seq. Our strategy enables strict temporal control and reversibility of gene expression alterations via the doxycycline-inducible promoter. Transcriptome diversity will also be interrogated using single-cell RNA sequencing to determine how individual cellular transcriptional profiles contribute to the overall population-based transcriptional landscape. These results will be compared to patient tumor samples' transcriptome diversity and correlated with their respective clinicopathologic characteristics. Advancing our understanding of cellular transcriptome diversity will enable new avenues of treatment for breast cancers resistant to current clinical interventions such as metastatic and triple negative breast cancers, both of which are characterized by high transcriptome diversity.

#536

Targeting PRPK and TOPK for skin cancer prevention and therapy.

Eunmiri Roh,1 Mee-Hyun Lee,2 Tatyana A. Zykova,1 Feng Zhu,3 Hong-Gyum Kim,2 Ki Beom Bae,1 Yong Yeon Cho,4 Ann M. Bode,1 Zigang Dong1. 1 _The Hormel Institute, University of Minnesota, Austin, MN;_ 2 _China-US (Henan) Hormel Cancer Institute, Zhengzhou, China;_ 3 _Huazhong University of Science and Technology, Wuhan, China;_ 4 _The Catholic University of Korea, Bucheon, Republic of Korea_.

Solar ultraviolet (sUV) irradiation is a major environmental carcinogen that can cause inflammation and skin cancer. The costs and morbidity associated with skin cancer are increasing, thus identifying molecules that can help prevent skin carcinogenesis is important. In this study, we identified the p53-related protein kinase (PRPK) as a novel oncogenic protein that is phosphorylated by the T-LAK cell-originated protein kinase (TOPK). Knockdown of TOPK inhibited PRPK phosphorylation and conferred resistance to solar simulated light (SSL)-induced skin carcinogenesis in mouse models. In the clinic, acute SSL irradiation significantly increased epidermal thickness as well as total protein and phosphorylation levels of TOPK and PRPK in human skin tissues. We identified two PRPK inhibitors, FDA-approved rocuronium bromide (Zemuron®) or betamethasone 17-valerate (Betaderm®) that could attenuate TOPK-dependent PRPK signaling. Importantly, topical application of either rocuronium or betamethasone attenuated SSL-induced epidermal hyperplasia, neovascularization and cutaneous squamous cell carcinoma (cSCC) development in SKH1 (Crl: SKH1-Hrhr) hairless mice by inhibiting PRPK activation, and also reduced expression of the proliferation and oncogenesis markers COX-2, cyclin D1 and MMP-9. This study is the first to demonstrate that PRPK- targeted therapy could be useful against sUV-induced cSCC.

#537

CK1 delta as a therapeutic target in bladder cancer.

Chun-Han Chen. _Taipei Medical Univ., Taipei, Taiwan_.

Bladder cancer is a common malignancy worldwide, but targeted therapy is currently unavailable. Therefore, there is an unmet medical need for bladder cancer patients. Oncomine database revealed that gene expression of CK1 delta is higher in bladder cancer compared to normal tissue. The purpose of this study is to investigate the role of CK1 delta in the progression of bladder cancer, and the feasibility of CK1 delta as a therapeutic target. Lentiviral delivery of shRNA and CK1 delta inhibitors was used to modulate gene expression or kinase activity of CK1 delta in bladder cancer cell lines. Cell viability, cell cycle progression, apoptosis, migration and downstream signaling pathway were analyzed. The results showed that knocking down CK1 delta or treatment of CK1 delta inhibitors decreased cell viability and inhibited wnt/β-catenin pathway in bladder cancer cell lines. CK1 delta inhibitors increased the accumulation of sub-G1 phase by flow cytometry analysis, as well as the activation of caspase-3, 8, 9 and PARP cleavage by Western blot, indicating the promotion of cell apoptosis. Knocking down of CK1 delta and the treatment of CK1 delta inhibitors impaired cell migration activity, and altered protein expression of EMT markers, such as E-cadherin, vimentin, snail and slug. In conclusion, this study demonstrates that CK1 delta is a potential target for bladder cancer therapy.

#538

**ASPM promotes prostate cancer tumorigenesis by bolstering cancer stemness through Wnt-Dvl-3-** b **-catenin signaling.**

Kelvin K. Tsai, Vincent C. Pai, Chung-Chi Hsu, Tze-Sian Chan. _Taipei Medical University, Taipei City, Taiwan_.

Metastasis is the major cause of cancer mortality in androgen-independent prostate cancer (PCA) and understanding it is crucial to improving the outcome of patients. Recent evidence suggests that activation of Wnt signaling in cancer stem cells (CSCs) contributes to cancer progression in malignant tumors. Here we report that a novel Wnt activator ASPM (Abnormal spindle-like microcephaly associated) maintains the prostate CSC subpopulation through augmenting the Wnt-β-catenin signaling in PCA. Functional studies showed that downregulation of ASPM expression attenuated the proliferation and invasive of PCA cells and reduced the number of prostate CSCs and inhibited cancer stemness and tumorigenesis. Mechanistically, ASPM interacts with dishevelled-3 (Dvl-3) and inhibits its proteasome-dependent degradation, thereby increasing the protein stability of Dvl-3 and enabling the β-catenin transcriptional activity in PCA cells. The clinical significance of the above findings was credentialed by the profound up-regulation of ASPM in metastatic PCA and the strong correlation of cytoplasmic ASPM expression with poor metastasis-free survival of patients with resected PCA. Collectively, our data points to ASPM as a novel oncoprotein and an essential regulator of cancer stemness in PCA, which has important clinical and therapeutic significance (supported by Ministry of Science and Technology grants MOST 104-2314-B-400-022 and MOST 105-2314-B-400-018 and National Health Research Institutes NHRIs grant CA-106-PP-09 to K.K.T).

#539

Pro-proliferative function of the histone deacetylase SIRT3 in prostate cancer.

Chandra K. Singh,1 Gagan Chhabra,1 Minakshi Nihal,1 Kenneth A. Iczkowski,2 Nihal Ahmad1. 1 _University of Wisconsin-Madison, Madison, WI;_ 2 _Medical College of Wisconsin, Milwaukee, Madison, WI_.

Worldwide, prostate cancer (PCa) is the second most frequently diagnosed cancer and fifth leading cause of cancer death in males. The existing treatments, as well as surgical approaches, have not been fully effective either for prevention or treatment of PCa. This necessitates a need to intensify our efforts towards the understanding of genetics and mechanism(s) of PCa. This may lead to the identification of the novel molecular target(s) and mechanism-based approaches for the management of this neoplasm. Sirtuin-3 (SIRT3) is a nicotinamide adenine dinucleotide (NAD)-dependent deacetylase primarily located in the mitochondria and known to play important roles in the regulation of a variety of cellular processes, including transcription and programmed cell death. The fact that SIRT3 can regulate several cellular processes those are critical in cancer cell proliferation, makes it a potential therapeutic target for cancer management. Moreover, SIRT3 is a central regulator of mitochondrial adaptive responses that relate to metabolic reprogramming of cancer cells. However, the role of SIRT3 in cancer, including PCa, is not well understood and it has been shown to act both as a tumor suppressor as well as a tumor promoter. In this study, we determined the role of SIRT3 in PCa, employing in vitro and ex vivo approaches. The first step in our efforts to understand the role of SIRT3 in PCa was to check the expression profile of SIRT3 in a panel of human PCa cell line (DU145, 22Rν1, PC3, LNCaP, C4-2, MDA PCa 2b, E006AA-Par and E006AA-hT) by RT-qPCR and immunoblot analyses. Compared to normal human prostate epithelial cells (NrPEC), PCa cells showed higher expression of SIRT3, both at mRNA and protein levels. Further, we determined the expression profile of SIRT3 by immunostaining of a tissue microarray (TMA) containing paraffin-embedded sections of 40 cases of prostate adenocarcinoma and 8 normal prostate tissues. Our data demonstrated a significant upregulation of SIRT3 in cancerous prostatic tissues compared to the normal tissues. We next determined the effect of chemical inhibition of SIRT3 using a recently described SIRT3 inhibitor viz. 4'-Bromo-Resveratrol (4BR), in human PCa cells (DU145 and 22Rν1). 4BR treatment at 10, 20 and 40 μM concentrations for 48 and 72 h resulted in a dose- and time-dependent decrease in cell growth and proliferation. Further, 4BR treatment resulted in a marked decrease in clonogenic survival of DU145 and 22Rν1 PCa cells. Furthermore, 4BR treatment resulted in a marked cleavage of PARP, an indicator of apoptosis induction; and a decrease in the level of PCNA, a marker of cellular proliferation, in human PCa cells. Overall, our data suggest a possible pro-proliferative function of SIRT3 in PCa. Further studies are underway to unravel the role and functional significance of SIRT3 during PCa development and progression.

#540

β-Catenin/LIN28B promotes cell proliferation of human trophoblast cells via let7a repression.

Jing Wu, Baoxin Luan, Huandi Yu, Yinhua Yu, Congjian Xu, Hongbo Zhao. _OB/GYN Hospital of Fudan University, Shanghai, China_.

LIN28 is a highly conserved RNA-binding proteins, have been implicated in key biological functions such as development, glucose metabolism and pluripotency. LIN28 overexpression induces the reprogramming of pluripotent stem cells (iPSCs) and supports the proliferative and metabolic capacities of iPSCs. Therefore LIN28 is crucial for the regulation of the cell fate and human embryonic development. In addition, LIN28B is frequently up-regulated in a variety of cancers and correlates with poor prognosis and cancer recurrence, indicating the crucial role of LIN28B in tumorigenesis. Aberrantly expression of LIN28B is responsible for the decrease in the let-7 microRNA family observed in many cancers and Let-7 is frequently down-regulated in numerous different tumors and lower levels of let-7 correlates with poor prognosis. Therefore, LIN28/Let7 may be crucial for the regulation of cell cycle and cell proliferation. In our present study, We first detected the expression of LIN28B in normal placental villi and choriocarcinoma tissue. Our data showed LIN28B was highly expressed in human choriocarcinoma tissues and most LIN28B staining was localized in the nucleus. Comparatively, LIN28B staining was faint in normal placental villi. Our data then demonstrated that LIN28B was highly expressed in human choriocarcinoma cell lines JEG-3 and JAR. However, LIN28B expression was undetectable in HTR-8/Svneo normal trophoblast cells. Our data then demonstrated that LIN28B knockdown by small interfering RNA (siRNA) caused an increase in Let-7a expression. Further, silencing of LIN28 inhibited IGF2BP1 expression and suppressed cell proliferation capacity, which can be markedly restored by Le7a siRNA. These findings reveal that LIN28 may promote IGF2BP1 expression and cell proliferation by Let-7a repression. Moreover, β-catenin knockdown resulted in reduced LIN28 expression and Let-7a up-regulation. β-catenin is the major mediator of canonical Wnt signaling pathway, and activation of canonical Wnt signaling has been reported to play a critical role in the proliferation, invasion, and differentiation of various tumors. Collectively, our present data indicated that β-catenin/LIN28B/Let7a pathway may be crucial for cell proliferation regulation in human choriocarcinoma cells.

#541

Involvement of miRNA expression may contribute for FoxO3a oncogenic role in uterine leiomyosarcoma.

Natalia Garcia,1 Anamaria Ritti Ricci,2 Bruna Cristine de Almeida,2 Thais Gomes Almeida,1 Isabela Werneck Cunha,3 Edmund Chada Baracat,1 Kátia Candido Carvalho1. 1 _Univ. of São Paulo Faculty of Medicine, Brazil;_ 2 _Univ. of São Paulo Faculty of Medicine, São Pulo, Brazil;_ 3 _AC Camargo Cancer Center, São Paulo, Brazil_.

Introduction: Leiomyoma (LM) and leiomyosarcoma (LMS) are uterine mesenchymal tumors that present variable clinical behavior. LM are extremely common estrogen and progesterone dependent benign tumors. On the other hand, LMS are rare and aggressive malignant tumors with limited available treatment options. FoxO3a seems to have a tumor suppressor role and its loss of function may determine deregulation in the cell proliferation, with DNA damage accumulation. Several studies demonstrated FoxO3a with an important role in several tumors development; however, its regulation or function is unknown in uterine mesenchymal tumors. Objective: Here, our goals were to assess the FoxO3a expression profile in uterine LM and LMS, to evaluate the mechanisms involved on FoxO3a regulation, and to assess its use as a marker for diagnosis, prognosis and malignancy risk prediction. Methods: Formalin-Fixed Paraffin-Embedded tissues of 56 leiomyosarcomas (LMS), 80 leiomyomas (LM) and 20 myometrium (MM) from patients, were used for FOXO3a protein evaluation by immunohistochemistry (IHQ). Epigenetic regulation of FoxO3a expression was assessed by 84 miRNAs gene analysis, using Real Time PCR method (MIHS 109Z, Qiagen). MM, LM and LMS cells were evaluated for FoxO3a gene expression by Real Time PCR. Results: FOXO3a protein showed an increased expression profile in MM, LM and LMS. Higher protein levels were observed in the comparison between LMS and MM (p<0,05). Gene expression analysis corroborate the protein data, with higher FoxO3a expression in LMS compared to MM and LM cells. Concerning the miRNAs analysis, we found oncogenic has-96-5p and has-155-5p overexpressed in LMS samples (p=0,008 and p=0,0007; respectively), while the tumor suppressor miR-let7c-5p was down expressed (p=0.0026). Conclusion: FoxO3a seems to display an oncogenic role in uterine tumors and could participate in the tumors risk of malignancy. miRNAs expression profile corroborates the FoxO3a negative regulation in these tumors. However, further studies are necessary for the understanding of the FoxO3a role in LM and LMS biology.

#542

A disintergrin and metalloprotease (ADAM) 15 promotes non-small cell lung cancer (NSCLC) proliferation through cytosolic domain, not extra-cytosolic metalloprotease activity.

Hsin-Han Hou. _National Taiwan University Hospital, Taipei, Taiwan_.

Emerging evidence indicates that proteins of a disintergrin and metalloprotease (ADAM) family contribute to cancer progression and metastasis. ADAM15 is up-regulated in multiple cancers and the enzymatic activities of its extracellular metalloprotrease domain promote cancer proliferation and migration by mediating the ErbB signaling pathway. In lung cancer, the enzymatic activity of ADAM15 extracellular domain is reported to be independent of ErbB signaling, indicating an alternative mechanism in promoting lung cancer progression. However, the role of ADAM15 in non-small cell lung cancer (NSCLC) and its correlation to patient prognosis is not well understood. This study demonstrated the longest ADAM15 isoforms, isoform 6 (i6), which contained the most cytoplasmic Src homology 3 (SH3) binding motifs, was significantly up-regulated in progressive NSCLC cell. Comparison of i6 and isoform 1 (i1) revealed that the cytoplasmic domain was essential for NSCLC cell proliferation and aggressiveness. ADAM15 i6, but not i1, mediated the activation the activation of the Src homolog 2 domain containing (Shc) through physical interaction with growth factor receptor-bound protein 2 (Grb2). ADAM15 i6 overexpression promoted NSCLC cell growth in a xenograft mouse model, while ADAM15 or Grb2 knockdown resulted in tumor shrinkage. ADAM15 was overexpressed in primary NSCLC tissues compared to adjacent normal tissues. Patients with ADAM15 i6 high-expressing lung tumors had shorter survival times. Thus, a novel mechanism of the ADAM15 cytoplasmic domain is identified in NSCLC tumor progression. This may help shed light on the molecular mechanisms of ADAM proteins and facilitate development of novel therapy for NSCLC.

#543

IL-11, a novel diagnostic biomarker for lung adenocarcinoma, is involved in lung cancer tumorigenesis.

Laura Ojeda Márquez,1 Patricia Yagüe Sanz,2 Sonia Molina Pinelo,3 Amancio Carnero Moya,3 Alejandro Sweet-Cordero,4 Luis Paz-Ares Rodríguez,2 Irene Ferrer Sánchez2. 1 _UCM-CNIO, Madrid, Spain;_ 2 _H12O-CNIO, Madrid, Spain;_ 3 _IBiS, Sevilla, Spain;_ 4 _UCSF, San Francisco, CA_.

Introduction

Interleukin-11 (IL-11) belongs to the IL6-family of cytokines, which are related to tumor development and progression in several types of cancer. These cytokines activate downstream pathways such as JAK/STAT, PI3K/AKT and RAS/ERK, triggered by their common receptor subunit, GP130. We have recently described that IL-11 is overexpressed in bronchoalveolar lavage fluid from patients with lung adenocarcinoma compared to non-cancer or other histological subtype's patients, thereby being useful as a diagnostic biomarker for this disease. So far, the role of IL-11 in lung cancer has been poorly studied. We aim to unravel the role of IL-11 in lung adenocarcinoma tumorigenesis, and its possible role as a prognostic biomarker.

Material and Methods

We have characterized a broad panel of human lung adenocarcinoma cell lines in terms of their expression levels of IL-11 and IL-11RA. We have studied the activation status of JAK/STAT, MEK/ERK and PI3K/AKT signaling pathways in these cell lines, after IL-11 stimulation. Additionally, several lung adenocarcinoma cell lines were alternatively transfected to overexpress IL-11RA or infected to silence IL-11 using CRISPR/Cas9 technology. We performed several different functional assays in these generated cell lines, such as growth curves, colony formation assays, soft-agar assays and xenografts models, in order to determine the tumorigenic effects of IL-11. Furthermore, we analyzed overall survival and progression-free survival of lung adenocarcinoma patients expressing high levels of IL-11 versus patients expressing low levels, using a public database.

Results

Different pathways are activated in adenocarcinoma cell lines after IL-11 stimulation. However, only the STAT1/3 pathway is differentially activated in IL-11RA-overexpressing cell lines subsequently stimulated with IL-11, or inhibited in IL-11-silenced cell lines. These molecular effects were translated into a slight increase or decrease in tumorigenic properties in the in vitro and in vivo assays when overexpressing IL-11RA and stimulating with rhIL-11 or when silencing IL-11, respectively. Furthermore, high levels of IL-11 correlated to poorer Overall-Survival and Progression-Free-Survival in patients with lung adenocarcinoma.

Conclusions

1. IL-11 activates STAT1/3 pathways in lung adenocarcinoma.

2. IL-11 exerts oncogenic effects in vitro and in vivo.

3. High levels of IL-11 correlate with poorer outcomes in lung adenocarcinoma patients.

#544

Characterization of serine/threonine kinase LASK2 as a biomarker and therapeutic target for lung cancer.

Yataro Daigo,1 Atsushi Takano,1 Yusuke Nakamura2. 1 _Institute of Medical Science, The University of Tokyo, Tokyo, Japan;_ 2 _University of Chicago, Chicago, IL_.

As the number of lung cancer patients responding well to standard therapies is still small, further development of new anti-cancer drugs with minimum risk of adverse event and highly sensitive cancer biomarkers is urgently awaited. We have been developing new molecular therapies targeting oncoproteins with their companion biomarkers by screening system as follows; i) To identify up-regulated genes in 120 lung cancers by the gene expression microarray representing 27,648 genes, ii) To verify the candidate genes for their low expression in 23 normal tissues, iii) To validate the clinicopathological significance of their protein expression by tissue microarray covering hundreds of non-small cell lung cancers (NSCLCs), iv) To verify whether they are essential for the growth/invasion of cancer cells by siRNAs. During this process, we selected dozens of druggable oncoproteins with various enzymatic activities, and identified activation of serine/threonine kinase LASK2 (lung cancer-associated kinase 2) in the majority of various histological types of lung cancers, but not in normal tissues except testis. Immunohistochemical analysis showed that strong LASK2 positivity was an independent prognostic factor for patients with NSCLC (P<0.0001). Suppression of LASK2 expression with its siRNAs inhibited growth of lung cancer cells. The enhanced cellular proliferation by introduction of LASK2 in mammalian cells also supported its oncogenic function in vitro and in mice model. Induction of LASK2 appeared to increase the levels of phosphorylation of oncogenic signal proteins for lung cancer such as MAPK. The data suggest that LASK2 is a candidate prognostic biomarker and possible therapeutic target for lung cancers.

#545

NOTCH1 co-expression analysis reveals novel insights underlying its opposing effects as an oncogenic and tumor suppressor in lung cancer.

Sara L. Sinicropi-Yao, David P. Carbone, Kevin R. Coombes, Joseph M. Amann, David Lopez Lopez. _The Ohio State University, Columbus, OH_.

Background: Aberrations in the family of Notch receptors (1, 2, 3, and 4) have been implicated in a range of solid tumors, including lung cancer. NOTCH1 biology is complex where Notch plays an oncogenic and tumor suppressor role in lung adenocarcinoma and lung squamous cell carcinoma, respectively. Although the role of Notch in cancer development and progression has received increased appreciation in recent years, there is still a lack of understanding of the mechanisms underlying these opposing activities in lung cancer.

Methods: The Cancer Genome Atlas datasets were used to examine gene co-expression patterns of Notch1 in lung adenocarcinoma and lung squamous cell carcinoma. Biological pathways implicated by gene families were assessed using functional annotation tools (DAVID, ToppGene, and IPA). In vitro and in vivo knockdown studies assessed the functional role of Notch1.

Results: This novel co-expression analysis supports the hypothesis that NOTCH1 is co-expressed with different genes in lung adenocarcinoma and squamous cell carcinoma. Knockdown of Notch1 in vitro and in vivo support our in silico finding of opposing effects of NOTCH1. Our analysis implicates genes associated with metabolic pathways, immune pathways, angiogenesis and cell cycle that may underlie the differential role of NOTCH1 in lung adenocarcinoma and squamous cell carcinoma. In vitro and in vivo studies support our bioinformatics analysis.

Conclusion: These results demonstrate different NOTCH1 gene co-expression patterns in lung adenocarcinoma and squamous cell carcinoma. These findings provide novel insights underlying the context-dependent role of Notch as an oncogene and tumor suppressor in subtypes of lung cancer. Understanding the similarities and differences in co-expression patterns reveal novel insights, suggesting that tumor intrinsic and extrinsic pathways may underlie the dual role of NOTCH1 in lung cancer. Recognition of the mechanisms underlying NOTCH1 opposing roles in cancer may help direct development of Notch-targeted therapies as a monotherapy or in combination with approaches focusing on the tumor microenvironment.

#546

The nuclear sirtuin SIRT6 promotes epithelial-mesenchymal transition in human melanoma cells.

Liz M. Garcia, Gagan Chhabra, Mary A. Ndiaye, Nihal Ahmad. _University of Wisconsin-Madison, Madison, WI_.

According to the recent statistics, the incidence as well as mortality from malignant melanoma has risen steadily for the past two decades in the United States. In 2017, an estimated 87,110 new cases of melanoma will be diagnosed and 9,730 people will die due to invasive melanoma. Therefore, intense research is needed to enhance our understanding of the molecular mechanisms of melanoma development, progression and metastasis. We have previously shown that the sirtuin SIRT6 was significantly overexpressed in melanoma, and a transient short hairpin RNA (shRNA)-mediated knockdown of SIRT6 resulted in a marked anti-proliferative response in melanoma cells (Genes & Cancer; In Press). In this study, we tested a hypothesis that the nuclear sirtuin SIRT6 is involved in the regulation of epithelial-mesenchymal transition (EMT) in melanoma. Indeed, EMT is an important and well-choreographed process where cells transition from a non-mobile epithelial cell type to a more motile and invasive mesenchymal state, thereby enhancing the migratory capacity of cancer cells. Activation of the EMT process allows cancer cells to propagate from the primary tumor site to adjacent tissues and subsequently metastasize by spreading through the lymphatic system and/or bloodstream. The role of SIRT6 in EMT appears to be cell- and context- dependent, since it has been shown to either promote or inhibit EMT depending on cancer type. The role of SIRT6 in melanoma has not yet been explored. In this study, we determined the effect of SIRT6 manipulation on EMT-associated markers, employing lentiviral short hairpin RNA (shRNA) mediated stably transfected SIRT6 knockdown A375 melanoma cells. Our data demonstrated that SIRT6 knockdown resulted in a significant downregulation of the mesenchymal markers N-cadherin, β-catenin, and vimentin, with a concomitant upregulation of the epithelial marker E-cadherin, both at mRNA and protein levels. Further, a wound healing scratch assay showed a marked inhibition of cell migration potential following SIRT6 knockdown in melanoma cells. Interestingly, 72 h after performing the scratch, the wound was completely closed in control cells (stably transfected with nonsense shRNA), whereas SIRT6 knockdown cells showed limited migration and the wound was still visible. Similarly, compared to control cells, the A375 shSIRT6 cells showed a significant inhibition in invasive potential, as assessed by a Matrigel invasion assay. Taken together, our data suggests that SIRT6 may be a contributing factor in the EMT process and its downregulation may inhibit mesenchymal properties in melanoma. Further detailed studies are underway to understand the complete mechanism and the ways that SIRT6-associated signaling molecules affect the EMT process in melanoma.

#547

The potential role of polo-like kinase 4 in non-melanoma skin cancers.

Debra R. Garvey, Mary A. Ndiaye, Chandra K. Singh, Ambria Noll, Nihal Ahmad. _University of Wisconsin-Madison, Madison, WI_.

Nonmelanoma skin cancers (NMSC), including basal cell carcinoma (BCC) and squamous cell carcinoma (SCC), are the most common malignancies in the United States, with over 5.4 million cases treated in more than 3.3 million people each year. The existing preventive and therapeutic strategies have not been fully effective in NMSC management. Therefore, an enhanced knowledge of the molecular mechanisms of NMSC may provide novel targets for prevention and treatment of these cancers. This study was undertaken to determine the potential role of the serine/threonine kinase, polo-like kinase 4 (PLK4) in NMSC. PLK4 plays an important role in cell division by regulating centriole duplication during the cell cycle. Essentially, PLK4 is a low abundance suicidal kinase capable of auto-phosphorylating itself to cause its own destruction to limit centriole duplication once per cell cycle phase. Since centrosome aberrations are frequently seen in cancer, the central role of PLK4 in centriole duplication suggests its significance as a potential target for cancer management. In this study, employing in vitro and ex vivo approaches, we tested the hypothesis that PLK4 is differentially expressed in NMSC and may be used as a potential new target for the management of these neoplasms. Employing real time quantitative polymerase chain reaction as well as immunoblot analyses, we found that compared to normal human epidermal keratinocytes (NHEKs), PLK4 was significantly overexpressed in human epidermoid carcinoma A431 cells and basal cell carcinoma UW-BCC1 cell lines at both mRNA and protein levels. Further, we determined the expression profile of PLK4 in NMSC using immunohistochemical analysis of multiple tissue microarrays containing cores of normal skin, BCC and SCC. Our data demonstrated a marked overexpression of PLK4 in cancerous tissues, compared to normal skin. To further assess the role of PLK4 overexpression in NMSC, we determined the effects of small molecule inhibitors of PLK4, centrinone-B and CFI-400945 in vitro. We found that treatment with centrinone-B or CFI-400945 resulted in dose- as well as time-dependent decreases in the growth and viability. Further, centrinone-B and CFI-400945 treatments also resulted in a marked reduction in the clonogenic survival of skin cancer cells. Taken together, our study suggests that PLK4 has a pro-proliferative function in NMSC. Additional detailed studies are ongoing in our laboratory to determine the functional significance of PLK4 in NMSC. Based on our data, we suggest that PLK4 should be further evaluated as a potential target and prognostic biomarker for the management of NMSC.

#548

**The effects of Sirtuin 6 inhibition on autophagy-related pathways in melanoma** **.**

Liz M. Garcia-Peterson, Mary A. Ndiaye, Chandra K. Singh, Gagan Chhabra, Nihal Ahmad. _University of Wisconsin Madison, Madison, WI_.

Melanoma is an aggressive skin cancer that can rapidly metastasize to become fatal, if not diagnosed early. Despite therapeutic advances, clinical management of melanoma remains challenging. The existing chemotherapeutic drugs either fail to achieve greater than 25% response or ultimately develop resistance to therapy. Therefore, novel molecular targets and treatments are required for an effective management of melanoma. In order to design novel and more effective approaches for lasting cure of melanoma, a deeper understanding of the mechanisms involved in melanomagenesis are needed. In our laboratory, we are assessing the role and functional and therapeutic significance of sirtuin proteins in melanoma. We have previously demonstrated that Sirtuin 6 (SIRT6) is significantly overexpressed in human melanoma cells and clinical tissues, and its genetic knockdown resulted in a marked anti-proliferative response in human melanoma cells (Genes & Cancer; In Press). A limited number of studies have implicated SIRT6 in autophagy regulation. Autophagy forms part of a basic cellular process that copes with cellular stress and is considered to be an important metabolic mechanistic essential for degradation and recycling of unnecessary intracellular components. In melanoma autophagy is believed to be a tumor suppressing process in early stages of cancer, but tumor-promoting in established tumors. This study was designed to determine the connection between SIRT6 and autophagy in melanoma. Employing lentiviral short hairpin RNA-mediated knockdown of SIRT6 in A375 melanoma cells, we determined the modulation in autophagy related pathways. We employed a PCR array containing 84 genes that are involved in the regulation of autophagy. Our data demonstrated that 17 of the 84 genes were significantly modulated (two-fold or more) upon SIRT6 knockdown. Of these 17 genes, 2 were upregulated (ATG10 and GAA) and 15 were downregulated (AKT1, ATG12, ATG3, ATG7, BAK1, BCL2L1, CLN3, CTSB, CTSS, DRAM2, HSP90AA1, IRGM, NPC1, SQSTM1, and TNF). Modulated genes were analyzed using Ingenuity Pathway Analysis (IPA) for predicted gene-gene interactions and functional networks. Many of the network genes were found to have definitive links to cancer. These genes were found to be associated with cell transformation and tumor invasion. Further validation at mRNA and protein levels, confirmed the significant modulations in autophagy markers BECN1, SQSTM1, ATG3, ATG7, ATG10 and GAA, upon SIRT6 knockdown. Moreover, SIRT6 knockdown was found to cause a marked decrease in the LC3 II protein level, which is a central protein in the autophagy pathway. Taken together, our data suggest that SIRT6 knockdown significantly alters pathways related to autophagy in melanoma cells. Future studies are required to carefully study the role of SIRT6 and autophagy in early versus late melanomas.

#549

The CUG-translated WT1, not AUG-WT1, is an oncogene.

Kun Yeong Lee, Hong-Gyum Kim, Joohyun Ryu, Do Young Lim, Hanyong Chen, Ann M. Bode, Zigang Dong. _The Hormel Institute, Austin, MN_.

The Wilms' tumor 1 (WT1) gene is believed to act as a canonical tumor suppressor. However, it has also been reported to function as an oncogene. Germline WT1 deletion is associated with Wilms' tumor, and exogenous WT1 cDNA introduction into cells induces the transcriptional suppression of its oncogenic target genes. In contrast, high WT1 expression is associated with poor prognosis in patients with various cancers. Why WT1 acts as a tumor suppressor under certain conditions but as an oncogene under other conditions is unknown. Here, we report that CUG initiation site for WT1 protein synthesis (CUG)-translated WT1 (cugWT1), an N-terminally extended form of canonical AUG initiation site for WT1 protein synthesis (AUG)-translated WT1 (augWT1), was overexpressed in most cancer cell lines and cancer tissues and functioned as an oncogene, whereas the classical augWT1 acted as a tumor suppressor as reported previously and inhibited the function of cugWT1. Translation of cugWT1 is initiated from a CUG codon upstream and in-frame with the coding region of augWT1. cugWT1 induced cell transformation and increased the gene expression of c-myc, bcl-2 and egfr, whereas overexpression of augWT1 repressed colony formation of cancer cells and inhibited the expression of the same target genes by recruiting histone deacetylase 1 (HDAC1). In addition, we found that protein kinase B (AKT)-phosphorylated cugWT1 on Ser62 and protected cugWT1 from proteasomal degradation induced by the F-box/WD repeat-containing protein 8 (FBXW8). These results provide an important breakthrough in the field of cancer biology and contribute significantly to the resolution of the chameleon function of WT1.

#550

One enhancer stimulates cancer cells proliferation and metastasis via the regulation of PDL1 expression.

Chunyan Li,1 Han Chen,2 Han Liang2. 1 _Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, China;_ 2 _The University of Texas MD Anderson Cancer Center, Houston, TX_.

Cancer cells employ multiple mechanisms to evade the recognition by the host immune system, including the expression of the negative T cell regulatory molecule PD-L1. PD-1/PD-L1-mediated immune escape plays key role during cancer development. It is crucial to unravel the regulatory mechanism of PD-1/PD-L1. In this study, we seek one enhancer (we named "enh9") that regulates the transcription of PD-L1 by systematic exploration. First, we found that PD-L1 and enh9 were co-expressed in multiple cancer types, including both lung adenocarcinoma and lung squamous cell carcinoma, which was further validated in a cohort of 130 lung cancer cell lines from the Cancer Cell Line Encyclopedia database. Then the direct interaction between PD-L1 and enh9 was further confirmed by Hi-C dataset. To validate the transcriptional regulation of PD-L1 by enh9, we knocked out enh9 using CRISPRi (CRISPR/ clustered regularly interspaced short palindromic repeats interference) in lung adenocarcinoma cell line A549, and single cell clones with double-copy deletion of enh9 were screened out. The expression of PD-L1 was dramatically reduced at both mRNA and protein level in enh9 knockout cells. Furthermore, we found that the cell proliferation and metastasis of enh9 knockout cells were significantly decreased compared to the control cells. Based on the previous studies that PD-L1 intrinsically regulated cancer cells' proliferation in ovarian and melanoma cancer cells, ongoing work is to determine whether enh9 regulates the cancer cell hallmarks by direct regulation of the expression of PD-L1 in lung cancer cells. The regulatory mechanisms will be deeply studied. In conclusion, we found an enhancer regulated the expression of PD-L1, which was one famous immune checkpoint, and it may play key roles in the cancer cell hallmarks.

#551

**Discovering the co-drivers of mutant** TP53 **that promote breast cancer progression.**

Anasuya Pal, Jin Park, Joshua LaBaer. _Arizona State University, Tempe, AZ_.

Breast cancer is a heterogeneous disease and has different subtypes. Genetically 30% of all breast tumors and 80% of basal-like breast tumors harbor mutations in TP53 and numerous somatic mutations in other genes. This molecular heterogeneity has posed a challenge in developing effective therapeutic regimens and targeted therapies. Different TP53 missense mutations co-exist with other mutations in tumors and we hypothesize that tumor heterogeneity results from combinatorial effects of neo-morphic functions of specific TP53 driver mutations and a distinct subset of functionally important co-mutations (or 'co-drivers'). We ectopically expressed the ten most prevalent missense TP53 mutants found in breast cancer tumors in non-transformed mammary epithelial cells and examined their phenotypes associated with the hallmarks of cancer. The results showed wide spectrum of phenotypic changes in cell survival, resistance to apoptosis and anoikis, cell invasion and cell polarity. As a proof of concept for the 'co-driver', we knocked-out PTEN in a non-invasive TP53 Y234C mutant using the CRISPR system and observed increased cell invasion. A subsequent genome-wide CRISPR library-based screen on non-invasive TP53 Y234C expressing cells identified co-driver candidates that were highly enriched in the invasive population of Y234C cells. The top candidates included several known mutated genes in breast cancer patients with TP53 mutations, and were associated with signaling and cytoskeletal pathways that play crucial role in regulating cell invasion. These results demonstrate that this approach can pinpoint the key loss-of-function mutations in clinically important TP53 mutant backgrounds that can promote cancer-like behavior, and identify the combinations of 'TP53 mutant and co-driver pathways' that can be targeted by specific inhibitors.

#552

PEPD is an essential regulator of p53 tumor suppressor.

Lu Yang, Yun Li, Arup Bhattacharya, Yuesheng Zhang. _Roswell Park Cancer Inst., Buffalo, NY_.

Peptidase D (PEPD), also known as prolidase among other names, is an enzyme that hydrolyzes dipeptides with proline or hydroxyproline at the carboxy terminus and is believed to be important for collagen metabolism, as proline and hydroxyproline are very abundant in collagen. Interestingly, we recently found that eliminating cellular PEPD causes cell death and tumor regression due to p53 activation. Here, we show that PEPD binds to and suppresses over half of nuclear and cytoplasmic p53 under normal conditions, independent of its enzymatic activity. PEPD binds to the proline-rich domain in p53, which inhibits phosphorylation of nuclear p53 and MDM2-mediated mitochondrial translocation of nuclear and cytoplasmic p53. Indeed, PEPD competes with MDM2 for p53 binding. However, the PEPD-p53 complex is critical for p53 response to stress, as stress signals doxorubicin (DOX) and hydrogen peroxide each must free p53 from PEPD in order to achieve robust p53 activation, which is mediated by reactive oxygen species. Thus, PEPD stores p53 for stress response, but this also renders cells dependent on PEPD for survival as it suppresses p53. Our results reveal a major p53 regulatory mechanism and a critical physiological function of PEPD. The p53-PEPD system likely operates in most if not all cells, since both p53 and PEPD are expressed ubiquitously. Disrupting PEPD suppression of p53 may be an important therapeutic strategy in cancer, as our data show that PEPD knockdown by RNA interference in tumors in mice causes p53 activation in the tumor

tissues and tumor regression. Our study also reveals a previously unrecognized anticancer mechanism of DOX. We show that the key step in DOX-induced p53 activation is the disruption of p53 association with PEPD via reactive oxygen species. This finding also raises the intriguing question of whether other stress-inducing anticancer agents also disrupt the PEPD-p53 complex for p53 activation and suggests that antioxidants may inhibit the anticancer activity of DOX and other agents by inhibiting p53 separation from PEPD. This work is supported by NCI grants and Roswell Park Alliance Foundation Grants.

#553

p53 target BRMS1A is a metastatic suppressor.

Takashi Tokino, Ryota Koyama, Masashi Idogawa, Yasushi Sasaki. _Sapporo Medical Univ., Sapporo, Japan_.

Tumor suppressor p53 plays an important role in the cell fate determination in response to diverse upstream signals. As a transcription factor, p53 regulates a number of genes that are involved in cell-cycle arrest, apoptosis, senescence, and maintenance of genomic stability. Recent studies revealed that p53 is critical for the regulation of cell invasion and migration. Microarray analysis showed that breast cancer metastasis-suppressor 1-like (BRMS1L) is upregulated by p53. We identified two responsive sequences of p53 in the first intron and upstream of the BRMS1L gene. These two response elements are well conserved among mammals. Functional analysis showed that ectopic expression of BRMS1L inhibited cancer cell invasion and migration. In contrast, knockdown of BRMS1L by siRNA induced the opposite effect. Notably, clinical databases revealed that reduced BRMS1L expression correlated with poor prognosis in patients with breast and brain cancer. Altogether, these results strongly indicate that BRMS1L is one of the mediators downstream of the p53 pathway, and inhibits cancer cell invasion and migration, which are essential steps in cancer metastasis. Collectively, our results demonstrate that BRMS1L is involved in cancer cell invasion and migration, and may be a therapeutic target for cancer.

#554

APC loss alters DNA damage repair in breast cancer cells.

Stephanie M. McClintock Batista. _University of Notre Dame, Notre Dame, IN_.

Breast cancer is the second leading cause of cancer deaths in U.S. women. Many of these deaths have been attributed to the development of chemotherapeutic resistance. Thus, understanding the process of chemotherapeutic resistance will help develop new approaches to combat breast cancer and ultimately cause a decrease in breast cancer related deaths. The Adenomatous Polyposis Coli (APC) tumor suppressor is silenced or mutated in up to 70% of sporadic breast cancer; however, the possible effects of APC loss on chemoresistance has not been elucidated. Using the ApcMin/+ mouse crossed to the Polyoma middle T antigen (PyMT) transgenic model, we previously showed that APC loss decreased cisplatin and doxorubicin-induced apoptosis. We also have shown that APC loss increased mRNA expression of the multidrug resistance protein 1 (MDR1), which was augmented by doxorubicin treatment. Treatment with doxorubicin and A69, a STAT3 inhibitor, normalized MDR1 gene expression in MMTV-PyMT;ApcMin/+ cells. Here, we hypothesize that loss of APC causes an increase in doxorubicin efflux and a decrease in DNA damage. First, we show that APC loss increased MDR1 activity as measured by calcein flux. To determine whether MDR1 inhibition would restore doxorubicin induced DNA damage, a combination treatment of doxorubicin and the MDR1 inhibitor, Valspodar, was able to restore doxorubicin sensitivity in MMTV-PyMT;ApcMin/+ cells compared to the control MMTV-PyMT;Apc+/+ cells. In addition, we have shown a decrease of γH2AX, a marker of damaged DNA, in MMTV-PyMT;ApcMin/+ cells treated with doxorubicin suggesting that the MMTV-PyMT;ApcMin/+ cells either have less damage due to increased drug efflux via MDR1 or due to increased DNA damage repair. Due to APC binding to Topoisomerase IIa, we also examined the degree of Topoisomerase IIa inhibition (via doxorubicin and etoposide) in MMTV-PyMT;ApcMin/+ compared to MMTV-PyMT;Apc+/+ cells. Topoisomerase IIa activity will also be measured to determine the effect of APC loss on Topoisomerase IIa. To test the hypothesis that loss of APC affected DNA damage repair, the expression of phosphorylated H2AX was measured in MMTV-PyMT;ApcMin/+ and MMTV-PyMT;Apc+/+ cells treated with the following: doxorubicin, etoposide, Valspodar, or a combination treatment. The longevity of doxorubicin-induced double stranded breaks was observed. Future studies will measure DNA damage repair pathway efficiency via reporter plasmids. We also will measure downstream signaling of yH2AX. Overall with an understanding of the role of APC in DNA damage repair, the use of a combination therapy could be advantageous to overcome chemotherapeutic resistance.

#555

Loss of function Cbl-c mutations in solid tumors.

Silvano R. Daniels, Stephen C. Kales, Mariya Liyasova, Marion M. Nau, Phil E. Ryan, Jeffrey E. Green, Stanley Lipkowitz. _National Institutes of Health, Bethesda, MD_.

Receptor Tyrosine Kinase (RTK) signaling, which is essential for cellular growth and proliferation, can lead to malignant transformation and tumorigenesis when aberrantly activated. Cbl proteins (Cbl, Cbl-b, and Cbl-c) are a conserved family of RING finger (RF) ubiquitin ligases (E3) that negatively regulate the activity of several tyrosine kinases such as the EGFR. Cbl mutations have recently been identified in 5% of human myeloid neoplasms, with a majority of mutations clustered within the RF and linker domains. Cbl-c is the most recently identified Cbl protein and is exclusively expressed in epithelial cells. We recently identified a novel cDNA isolated from a mouse mammary tumor in the C3(1) Large T Antigen transgenic mouse model. This mutant cDNA encodes a protein that has a deletion in the RF domain of Cbl-c, thereby resembling known Cbl family mutations associated with myeloid neoplasias. Genomic analyses of both parental and transgenic lines show no evidence of germline mutation, suggesting that this mutation is most likely somatic. The mutant Cbl-c protein enhances transformation of NIH 3T3 cells when co-transfected with SV40 Large T Antigen. In overexpression studies, this mutant Cbl-c fails to mediate ubiquitination of activated EGFR and acts in a dominant negative manner to prevent ubiquitination of activated EGFR by wildtype Cbl proteins. Datamining also reveals Cbl-c mutations associated with human solid tumors. Cell-based analyses demonstrate a similar loss of E3 function in some of these human mutations. These data collectively suggest that, like Cbl mutations in myeloid neoplasms, loss of function Cbl-c mutants may also contribute to the pathogenesis of solid tumors in murine models and in humans.

#556

Oncogenic properties and response to HDAC inhibitor treatment of H1299 cells expressing GOF p53 mutants.

Brianna Flores, Elizabeth E. Hull. _Midwestern Univ. - Glendale Campus, Glendale, AZ_.

The tumor suppressor p53 protein is mutated in over 50% of cancers. The most frequent mutations in p53, termed "hotspot" mutations, occur in the DNA binding domain of the p53 protein. These mutations not only remove normal p53 function but also convey additional oncogenic properties greater than loss of tumor suppressor activities associated with loss of wild-type (WT) p53 activities. With newly acquired oncogenic properties, mutations in the DNA binding domain of p53 are classified as gain of function (GOF) mutations. GOF p53 mutants can be subdivided into conformational mutations (e.g. R175H, H193Y) involving residues necessary for maintenance of structure and contact mutations (e.g. R248Q, R273H) involving resides necessary for DNA interaction. Cancer cells bearing a GOF p53 mutation increase metastasis with application of histone deacetylase inhibitors (HDACi). This uncharacterized pro-oncogenic GOF p53 property is of clinical relevance as HDACi are FDA approved for hematological cancers but have failed in clinical trials for solid tumors. To explore this hypothesis, metastatic capacity and proliferation rates of common GOF p53 mutations were assessed with and without HDACi treatment and in the presence or absence of specific inhibitors of target GOF p53. The p53-null, H1299 non-small cell lung carcinoma line was transfected with GOF p53 mutants and WT p53 using a tetracycline inducible expression system. Inducible expression of stable subclones was varified by immunofluorescence and western blot. Proliferation rate was measured by incorporation of EdU and metastatic capacity was assessed by measuring MMP activity and migration assays with and without HDACi treatment. Specific GOF p53 mutations possess a subset of the oncogenic properties of the entire GOF p53 class. Data suggest that the GOF p53 mutants display differing rates of proliferation with the R175H mutation exhibiting the highest rate. Application of NSC319726 significantly decreases the proliferation rate and appears to successfully refold the R175H mutant to a WT conformation. The R248Q mutation exhibited morphological change suggestive of the epithelial to mesenchymal transition associated with increased metastatic capacity. Preliminary data suggest that HDACi affect among these cell lines in conjunction with NSC 319726 and SCH 529074 treatment. In conclusion, HDACi treatment may be contraindicated while treatment with HDACi may have great benefit in tumors which are WT or null for p53 protein expression.

#557

Aberrant methylation of tumor-suppressor genes in pediatric myelodysplastic syndrome/acute myeloid leukemia.

Akira Shimada, Hirotaka Kanzaki, Hisashi Ishida, Takehiro Matsubara, Michinori Aoe. _Okayama University Hospital, Okayama, Japan_.

Introduction: Not only somatic mutations, but also epigenetic modifications in acute myeloid leukemia (AML) are estimated to have a pivotal role in leukemogenesis and it could be a therapeutic target. Especially, aberrant methylation of the promotor region of tumor suppressor genes (TSGs) were the causative events in leukemogenesis, however, there was only limited information in pediatric myelodysplastic syndrome (MDS) and AML. Materials and Methods: We analyzed the aberrant methylation of promotor region of 25 TSGs in pediatric 7 low grade MDS, 4 advanced MDS and 22 AML patients by FAB classification by Methylation-Specific Multiplex Ligation-dependent Probe Amplification (MS-MLPA) method (10 Male, 12 female, 0-14 years old, median 6 years ols). Simultaneously, detection of SNVs, In/del, and copy number variants were also analyzed by target sequence of more than 150 leukemia related genes. Especially, specific chimeras were analyzed in each patient. Results: Total 12/22(54.5%) patients had more than one methylated TSGs (number of methylated TSGs was 0-5 in each patient, median 1). More than 20% of patients had methylated TSGs including CDKN2B, CADM1, CDH13 and ESR1. Especially, there was a specific methylation pattern of TSGs, for example, all (4/4) t(8;21) patients had CDH13 methylation, 3/4 (75%) patients with inv(16) had ESR1 mutation and normal karyotype did not. Normal control samples, complete remission samples, and low grade MDS samples were not completely methylated. All advanced MDS patients had 1 or 2 methylated TSGs. Simultaneously, several leukemogenic genes were mutated such as GATA1, IKZF1, KRAS, KIT, FLT3, SH2B3, PIK3CD, PU.1, SMARC1, ALK, etc. Several genes were deleted such as CTCF, RB1, SRP72, ATM, NF1, U2AF1 etc. Discussion: In adult MDS, the increased number of methylated TSGs correlated to the development of AML and the poor prognosis. In this study, a few TSGs were methylated as the adult study (Hess CJ., Leuk Lymphoma 2008), furthermore, many genes related to epigenetic modification such as ASXL1, TET2 and EZH2 were not mutated in this study. Recently, anti-methylation drug were used in adult MDS/AML, larger study will be needed for adjusted use of epigenetic modifier in pediatric MDS/AML.

#557A

The clinicopathoogical significance of FBXW7 expression in GIST.

Yuki Koga, Masaaki Iwatsuki, Kouhei Yamashita, Yuki Kiyozumi, Hiroshi Sawayama, Yukiharu Hiyoshi, Yoshihfumi Baba, Yuji Miyamoto, Naoya Yoshida, Hideo Baba. _Department of Gastroenterological Surgery, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan_.

Objective: Gastrointestinal stromal tumor (GIST) is a rare mesenchymal tumor originated from the interstitial cells of Cajal in the gastrointestinal tract. A significant breakthrough was achieved with the identification of the expression of the c-kit (CD117) antigen in GISTs. However, alternate oncogenic pathways have not sufficiently been understood. FBXW7 is a ubiquitin ligase that mediates ubiquitylation of oncoproteins, which is known as tumor-suppressor gene in various malignancies. Previous reports showed that FBXW7 is associated with tumor progression by regulating some oncoprotein such as c-myc and c-jun. The aim of this study is to elucidate the clinicopathological significance of FBXW7 in GIST.

Methodology: (i) We examined the FBXW7 expression in GIST cells by immunostaining and correlation clinicopathological characteristics. (ii) We examined cell proliferation by Cytotoxicity assays in GIST-T1 cells suppressed by FBXW7-specific siRNA.

Results: (i) In 90 cases of GIST samples with surgical resection, high FBXW7 and observed are 47 cases and low expression are 43 cases. There are significantly more large size, located small intestine, high mitotic index in low FBXW7 cases (P=0.05, P=0.01, P=0.04). There are significantly more high risk cases in modified-Fletcher classification in low FBXW7 expression cases (P=0.0008). There are significantly higher value of SUV max in high risk cases of modified-Fletcher

classification and low FBXW7 expression cases (P=0.007, P=0.02). In GIST cases with distant metastasis, there are significantly more low FBXW7 expression cases than GIST cases without distant metastasis (P=0.009). FBXW7 expression is significantly inversely correlated with C-MYC expression (P=0.0004). (ii) The GIST cells suppressed FBXW7 expression by siRNA have higher proliferation rate compared to control GIST cells (P=0.02).

Conclusion: We demonstrated that FBXW7 expression is correlated with modified-Fletcher classification, SUV max value, c-myc expression and cell proliferation in GIST. These results are suggesting that FBXW7 may be associated with tumor progression. These findings suggest that it is possible that FBXW7 can be a promising biomarker for disease progression of GIST. 

## CLINICAL RESEARCH:

### Biomarker Discovery 1

#558

Prognostic gene signature use in checkpoint inhibitor monotherapy for melanoma.

Mariaelena Capone,1 Gabriele Madonna,1 Paolo Ascierto,1 Patrick Danaher,2 SuFey Ong,2 Sarah Warren,2 Joseph M. Beechem,2 Alessandra Cesano2. 1 _Unit of Melanoma, Cancer Immunotherapy and Development Therapeutics at the Istituto Nazionale Tumori IRCCS Fondazione G. Pascale, Napoli, MD;_ 2 _NanoString Technologies, Seattle, WA_.

Introduction: The successful deployment of checkpoint inhibitors in cancer immunotherapy relies on the responsiveness of an individual's immune system for relief of that particular blockade in the cancer immunity cycle. As most patients fail to respond to immunotherapy, there is a need for biomarkers that can predict clinical benefit of the therapeutic by identifying the patient population most likely to respond. Gene expression signatures characterizing basal immune state within tumor have shown utility in retrospective analysis of clinical trials. This study utilizes tumor and PBMCs from patients with metastatic melanoma receiving either anti-CTLA-4 or anti-PD-1/PD-L1 to characterize local and peripheral patterns of gene expression associated with clinical benefit of therapy. The goal of this study is to evaluate existing gene signatures and develop novel signatures that predict response to checkpoint inhibitors in melanoma.

Methods: Pretreatment biopsies from metastatic lesions of melanoma stage IV patients treated with ipilimumab (anti-CTLA4) or pembrolizumab (anti-PD-L1) were retrieved from the Department of Pathology archives. Cohorts were assembled of 30 patients receiving each drug, equally stratified between responders and nonresponders. RNA expression in tumor biopsies will be profiled with a pilot version of the NanoString® IO360 gene expression panel. In parallel, pre- and post-treatment PBMC from independent cohorts receiving either ipilimumab (anti-CTLA4) or pembrolizumab (anti-PD-1) were collected and profiled with the NanoString PanCancer Immune Panel (gene expression) and Immune Profiling Panel (protein expression).

Results: Patterns of gene expression in the tumor biopsies and gene and protein expression in the PBMCs will be assessed for correlation to clinical outcome (PFS, OS, ORR). Specifically, the Tumor Inflammation Signature described by Ayers et al. (1), an investigational 18-gene signature of suppressed adaptive immune response that enriches for clinical response to pembrolizumab, will be assessed in these cohorts. Other patterns of gene and protein expression that correlate with response to immunotherapy or lack thereof will also be evaluated.

Conclusion: Correlating patterns of gene expression from tumor with clinical response can lead to the development of biomarkers to better select patients who will respond to immunotherapy. It can also lead to identifying immune evasion. Gene and protein profiling from a readily accessed sample such as PBMC may give insights into early on-treatment signatures of efficacy. Utilization of a clinical grade platform such as the NanoString nCounter® may speed the development of diagnostic assays that can be used to predict and monitor patient response to immunotherapy.

Reference: 1. Ayers et al., J Clin Invest 2017;127:2930.

#559

Development of a novel approach to identify cancer drivers for hepatitis B virus-associated hepatocellular carcinoma.

SHIH-CHUN SHEN,1 Amy Jiaen Lu,2 John Caleb Shieh,2 Yih-Ping Su,1 Bilal Nasir,3 Ning Ling,3 Daryl T. Lau,3 Jamin D. Steffen,2 Fwu-Shan Shieh,4 Wei Song,4 Ying-Hsiu Su1. 1 _The Baruch S Blumberg Institute, Doylestown, PA;_ 2 _JBS Science, Inc., Doylestown, PA;_ 3 _Beth Israel Deaconess Medical Center, Boston, MA;_ 4 _U-Screen Dx Inc., Doylestown, PA_.

Infection with Hepatitis B virus (HBV) is a major etiology of hepatocellular carcinoma (HCC), the third leading cause of cancer-related deaths worldwide. Studies have shown that some cells infected with HBV contain integrated viral DNA, and integrated viral DNA has been observed more frequently in HCC than in non-HCC infected liver. Although integration occurs randomly throughout host chromosomes, recurrent integration sites have been reported. Interestingly, these integration sites are located in genes known to be involved in carcinogenesis. During HCC carcinogenesis, tumor cells undergo uncontrolled clonal expansion in which a particular integration junction will become dominant (or a major junction). We have adapted a method known as primer extension capture (PEC) to enrich integrated HBV DNA for next-generation sequencing (NGS) to detect these integration junctions that occur at low frequency. We next developed a novel program, "HccDriverFinder," to identify major junctions, incorporate PubMed data mining for driver identification, detect HBV mutations (revealing a striking 98.8% HCC-linked mutations identified in cancer), and visualize integration events. By analyzing NGS data generated from 13 matched HCC and adjacent non-HCC tissue samples, we found specific integrations in known targets such as TERT and CCNE1, and uncovered new recurrent HBV integration sites in genes including CSAD and ABCC13 (both of which are reportedly linked with tumorigenesis). We have further optimized the PEC approach to enrich for the whole HBV genome and have applied it to urine DNA from 31 hepatitis, 27 cirrhosis, and 29 HCC patients for the potential of identifying HBV junctions in circulation. Our approach holds the promise of building a driver identification kit for HCC detection, drug development and precision medicine.

#560

**Identification of a novel** MUTYH **mutation in non-small cell lung cancer.**

Alvin Hong-Wai Fong,1 Eunice Yuen-Ting Lau,1 William Ka-Chun Cheung,2 William Chi-Shing Cho1. 1 _Queen Elizabeth Hospital, Hong Kong;_ 2 _DiagCor Bioscience Incorporation Limited, Hong Kong_.

Introduction: Lung cancer is the leading cause of death from cancer in Hong Kong and worldwide. Nearly 80-85% of lung cancers are diagnosed with non-small cell lung cancer (NSCLC). Treatment strategies can be refined according to the distinct difference of NSCLC molecular profiling among different ethnic populations. Moreover, oncogenic driver mutations are closely linked to NSCLC in non-smokers and young people. Therefore, the urge of finding pathogenic mutations is inevitable. This study aims to discover novel mutations in NSCLC patients in Hong Kong by next-generation sequencing and characterize the clinical significance of these mutations. Methods: In order to identify mutations, DNA was purified from 6 pairs of lung tumor-normal tissues from 2 stage I adenocarcinoma (LUAD), 2 stage III LUAD and 2 stage I squamous cell carcinoma (LUSC) patients. Samples were sequenced by targeted sequencing with the TOMA OS-Seq 130 cancer gene panel. Sequence alignment and variant calling were performed by combining TOMA Stratus and in-house pipelines. Results: The mean aligned reads exceeded 14 million and the mean coverage of target region was over 1000X. MUTYH deletion was found in 66.7% (4/6) of the patients. Data from The Cancer Genome Atlas (TCGA) revealed that MUTYH deletion is significantly associated with poorer disease-free survival (DFS) (p=0.0063) and overall survival (p=0.022) in LUAD patients. TCGA data also showed that MUTYH copy number correlates with gene expression (r=0.473) and LUAD patients with MUTYH mRNA overexpression have better DFS (p=0.0205). However, such correlations were not observed in LUSC patients. MUTYH is a DNA glycosylase which involves in the base excision repair and it repairs oxidative DNA damage by preventing G:C to T:A transversion. Numerous studies indicated that biallelic mutations in MUTYH cause MUTYH-associated polyposis and increase the lifetime risk of colorectal cancer. MUTYH mutations could be associated with increased risk of endometrial and breast cancer. Conclusion: To elucidate the clinical significance of MUTYH in LUAD, an independent cohort will be sequenced to validate the primary result. Immunohistochemistry, RT-qPCR and functional assays will be performed to examine the protein expression, the gene expression and the functional role. All these findings suggested that MUTYH deletion may play a crucial role in LUAD and potentially lead to the development of biomarker and therapeutic target.

#561

Circulating DNA clean up and enrichment from liquid biological samples for rare biomarker detection.

Samantha R. Lewis, Charles Cowles, Kathryn Oostdik, Curtis Knox, Brad A. Hook. _Promega Corporation, Madison, WI_.

A major roadblock to using circulating cell free DNA (ccfDNA) for cancer biomarker detection is the difficulty of obtaining a pure sample. Contaminating genomic DNA (gDNA) can overwhelm already rare events within a CCF sample and prevent sensitive detection of these key mutations. Unfortunately, liquid biological samples often are not collected and processed in an ideal manner, resulting in decreased quality and purity. Many studies report difficulties in detecting mutations in ccfDNA which may originate from variable levels of gDNA background based how samples are processed. To this end, we present an effective method for purifying relevant small nucleic acids from larger genomic species after DNA purification from plasma.

Using a simple magnetic bead based technique, we demonstrate efficient removal of gDNA even in highly contaminated samples. This method can be used to enrich liquid biological samples for improved detection of rare genetic events such as microsatellite instability and mutations in key oncogenes. Using plasma from three individuals with colorectal cancer tumors harboring known mismatch repair gene mutations, we compare detection of somatic mutations in ccfDNA before and after this gDNA clean up method. Microsatellite instability profiles were obtained from ccfDNA and compared to normal and tumor profiles to determine if evidence of mismatch repair abnormalities could be detected. Targeted amplicon sequencing was then performed to detect tumor associated mutations. DNA isolated from matched adjacent normal and tumor tissues were used as controls in these experiments to confirm mutations were of tumor not germline mutations.

Effectively removing gDNA contamination from ccfDNA samples can improve biomarker detection consistency from liquid biological samples. We demonstrate a magnetic bead-based method for ccfDNA clean-up, which can be used prior to microsatellite instability detection by fragment analysis or rare variant detection by next generation sequencing.

#562

HPRT surface localization on prostate cancer cells as a biomarker for immunotherapy.

Michelle H. Townsend, Abigail M. Felsted, Taylor P. Cox, Zachary E. Ence, Stephen R. Piccolo, Richard A. Robison, Kim O'Neill. _Brigham Young University, Provo, UT_.

The purpose of this study is to evaluate the expression and possible upregulation of the salvage pathway enzyme hypoxanthine guanine phosphoribosyltransferase (HPRT) in prostate cancer cells to determine if it could serve as a biomarker for prostate cancer diagnosis and treatment. Prostate cancer is the second most lethal cancer in men, and an estimated 26,730 men will die from the disease in 2017. We chose to evaluate the HPRT enzyme due to its involvement in nucleotide synthesis and cell cycle progression. Two prostate cancer cell lines were used for this analysis (PC3 and DU145) along with malignant tissue from 35 patients with prostate carcinoma. The surface localization of HPRT was determined utilizing flow cytometry and scanning electron microscopy, while upregulation within tissue was assessed using immunohistochemistry. Additionally, RNA-seq data from TCGA was used to evaluate general HPRT upregulation in patients with prostate cancer (n = 502) when compared to healthy individuals (n = 52). Throughout our investigation, we found a significant association between HPRT and the plasma membrane of DU145 cells, but found no presence on PC3 cells. Flow cytometry showed insignificant (p = 0.14) changes in fluorescence when PC3 cells were exposed to HPRT antibodies, while there was a significant increase in fluorescence when DU145 cells were treated with fluorescent HPRT antibody (p = 0.0004). To determine the distribution of HPRT across the membrane and ensure the observed expression was not due to cytoplasmic HPRT, gold conjugated antibodies were used for analysis with an electron microscope. The distribution of the gold on the cell surface showed random HPRT binding across the membrane with no patterns of localization. This analysis aided in confirming HPRT surface presence as the gold weight % of DU145 cells increased significantly when exposed to HPRT antibodies (p < 0.0001). In addition to being presented on the surface of DU145 cells, tissue samples revealed variable HPRT expression as approximately 53% of prostate cancer patients evaluated had elevated levels of HPRT compared to normal controls, while 47% of patients had no upregulation. TCGA data revealed a significant (p = 1.53x10-4) increase in HPRT levels upon malignancy. While some patients had levels consistent with healthy controls, there was a significant number of patients with increased protein expression upon cancer development. The control of HPRT expression within these cancer cells has been linked preliminarily to p53 functionality. While PC3 cells are null for p53, DU145 cells have a gain of function (GOF) p53 mutation. GOF p53 mutations are known to influence salvage pathway enzyme expression and is an influential gene in HPRT expression in these cancer cells. These results strongly indicate a unique relationship between prostate cancer cells and HPRT and suggest HPRT as a possible biomarker for the detection and treatment of patients with prostate cancer.

#563

Identification of a novel biomarker to predict pathologic complete response for neoadjuvant chemoradiotherapy in locally advanced rectal cancer.

Jong Gwang Kim, In Hee Lee, Soo Jung Lee, Yee Soo Chae, Byung Woog Kang. _Kyungpook National University Hospital, Daegu, Republic of Korea_.

Neoadjuvant chemoradiotherapy (CRT) followed by total mesorectal excision (TME) is the standard treatment for locally advanced rectal cancer (LARC). Good pathologic response to CRT is associated with better outcome. However, there is a lack of effective methods to select rectal cancer patients who would or would not have a benefit from CRT. Therefore, we aimed to perform whole-exome sequencing (WES) for rectal cancer to identify the novel genetic variations predicting CRT benefit in LARC. Two independent sets were used to evaluate the genetic differences between the good response group and poor response group to CRT. Fifteen patients who achieved pathologic complete response were divided into the good response group and 15 patients with pathologic stage III were classified as poor response group. Among these 30 patients, WES was examined. To validate discovery set, additional samples (n=67) were genotyped for candidate variants using TaqMan SNP genotyping assays or Sanger sequencing. Overall, the current study included a total of 97 LARC patients who were treated with neoadjuvant CRT followed by curative surgery at Kyungpook National University Medical Center. We found 5 candidate variants significantly associated with pathologic complete response (BCL2L10 rs2231292, DLC1 rs3816748, DNAH14 rs3105571, ITIH5 rs3824658, RAET1L rs912565) using WES. In the dominant model, patients with GC/CC genotype of DLC1 rs3816748 (p=0.032), AC/CC genotype of DNAH14 rs3105571 (p=0.009) and TT genotype of RAET1 rs912565 (p<0.0001) showed higher rate of pCR compared to those of other genotype patients. In the recessive model, the BCL2L10 rs2231292 (p=0.036) and ITIH5 rs3824658 (p=0.003) were significantly associated with pathologic CR. Four SNPs (DLC1 rs3816748, DNAH14 rs3105571, ITIH5 rs3824658, RAET1L rs912565) found to be significantly correlated with pCR in codominant model. However, none of the SNPs was associated with the relapse-free survival or overall survival. The present results suggest that the genetic variation of the BCL2L10 rs2231292, DLC1 rs3816748, DNAH14 rs3105571, ITIH5 rs3824658, and RAET1L rs912565 genes can be used as biomarkers predicting CRT response for patients with LARC.

#564

Identification of biomarkers predictive of response to panitumumab combined with neoadjuvant chemotherapy for primary inflammatory breast cancer.

Xiaoping Wang, Naoko Matsuda, Bora Lim, Savitri Krishnamurthy, Juhee Song, Yu Shen, Wenhui Wu, Jianhua Hu, Wendy A. Woodward, James M. Reuben, Anthony Lucci, Debu Tripathy, Vicente Valero, Naoto T. Ueno. _UT MD Anderson Cancer Center, Houston, TX_.

Inflammatory breast cancer (IBC) is the most lethal and aggressive form of breast cancer, and there are no FDA-approved targeted therapies specific for IBC. We recently completed a single-arm phase II study of neoadjuvant therapy with the humanized anti-EGFR monoclonal antibody panitumumab (PmAb), nab-paclitaxel, and carboplatin followed by 5-fluorouracil, epirubicin, and cyclophosphamide in patients with newly diagnosed HER2-negative IBC (N = 40), which produced the highest pathologic complete response (pCR) rate (47%) ever observed in patients with triple-negative IBC (TN-IBC; negative for estrogen receptor, progesterone receptor, and HER2; N = 19). In the work described here, we aimed to identify biomarkers predictive of response of IBC to this combination therapy. We used immunohistochemical staining to examine the expression of key molecules involved in EGFR signaling, which regulates cancer stem cells and epithelial-mesenchymal transition in IBC, including EGFR, phosphorylated EGFR (pEGFR), E-cadherin, vimentin, COX-2, and Nodal, in paired tumor samples obtained before and after the first dose of PmAb in our phase II trial. Our data showed that the expression of pEGFR (P = .05) and COX-2 (P = .05) in pre-PmAb tissues marginally correlated with pCR; the change of pEGFR expression (P = .09) between pre-PmAb and post-PmAb samples showed a trend toward correlation with pCR. We conducted RNA-seq on 13 pairs of tumor biopsy specimens obtained before and after the first dose of PmAb. Analysis of all samples did not identify any candidate genes with significant changes after PmAb treatment. In the TN-IBC samples, we identified 2 genes (POU3F3 and EGR1) that were significantly downregulated and 4 genes (BBOX1, GLYATL2, MUCL1, and LCN2) that were significantly upregulated after PmAb treatment, but we did not identify any gene whose change in expression predicted pCR status, possibly because the small sample size of the non-pCR group (N = 2) limits the statistical power. We validated the change in expression of EGR1, MUCL1, and LCN2 after PmAb treatment in SUM149 IBC cells. In the hormone-receptor-positive, HER2-negative tumor samples, we identified 19 genes that were significantly downregulated and 10 genes that were significantly upregulated after PmAb treatment. These genes function in cellular movement, cell morphology, cell-to-cell signaling and interaction, molecular transport, and the cell cycle. Our results suggest potential roles of pEGFR and COX-2 expression at baseline and PmAb-modulated pEGFR, EGR1, MUCL1, and LCN2 expression in predicting PmAb response. Validation of these biomarker candidates and studies of the mechanisms by which they affect IBC response are ongoing.

#565

CDX2 targets, GPA33 and LI-cadherin, are novel biomarkers with prognostic value in gastric cancer.

Nair Lopes,1 Christian Bergsland,2 Merete Bjornslett,2 Jarle Bruun,2 Ragnhild Lothe,2 Raquel Almeida,1 Leonor David1. 1 _Ipatimup/i3S - University of Porto, Porto, Portugal;_ 2 _Institute for Cancer Research - University of Oslo, Oslo, Norway_.

Gastric cancer is one of the most common types of cancer and the third cause of cancer-related death worldwide. The aggressiveness of the disease is related to, among other factors, tumour differentiation. CDX2 is a known intestinal differentiation marker with prognostic value in gastric cancer and two of its targets are expressed at the cell surface: GPA33 (glycoprotein A33) and LI-cadherin (liver intestine cadherin). Whether these membrane proteins are good biomarkers in gastric cancer and could refine the significance of CDX2 expression remains unknown. In order to answer these questions, we evaluated the expression of CDX2, GPA33 and LI-cadherin using immunohistochemistry in 350 gastric cancer samples arranged in TMAs (tissue microarrays) and evaluated the co-localization of the biomarkers using fluorescent multiplex immunohistochemistry.

CDX2 was expressed in 36% of the cases, while GPA33 and LI-cadherin were positive in 55% and 66%, respectively. All markers were significantly correlated with each other and are more often expressed in early stage (I and II) cancers (p < 0.05). Overall survival analysis showed that both GPA33 and LI-cadherin predict better outcome. When stratifying the series in early (I and II) and late (III and IV) stages, both proteins significantly associate with better outcome for late stages of disease progression. Overall, these data indicate that the presence of an intestinal differentiation program in gastric cancer is a marker of good prognosis.

The concordance rate between CDX2 and GPA33 and CDX2 and LI-cadherin expression was 68% and 64%, respectively. Since CDX2 displays a heterogeneous pattern of expression and TMA sampling can, by chance, select a negative area in a positive tumour, we also compared CDX2 expression between whole-tissue sections and TMAs. In our series we obtained 68 discrepant cases (positive for CDX2 in whole-tissue sections and negative in the TMAs). For the majority of these cases (50, corresponding to 74%) the combined expression of CDX2 targets, GPA33 and LI-cadherin, can rescue the underrepresentation of CDX2 expression in the TMAs and identifies CDX2-dependent intestinal differentiation.

We conclude that CDX2, GPA33 and LI-cadherin are significantly associated with each other and are commonly expressed in early stages of gastric cancer. The expression of GPA33 and LI-cadherin is associated with better overall survival, particularly in late-stage disease patients. Finally, GPA33 and LI-cadherin are good cell surface surrogate markers for CDX2 expression, not only because of the good concordance of expression, but also because the combined expression of GPA33 and LI-cadherin rescues false negative CDX2 cases in TMAs versus whole-tissue samples.

#566

High dimensional single cell analysis predicts response to anti-PD-1 immunotherapy.

Carsten Krieg,1 Malgorzata Nowicka,2 Silvia Guglietta,3 Sabrina Schindler,4 Felix J. Hartmann,2 Lukas M. Weber,2 Reinhard Dummer,4 Mark D. Robinson,2 Mitchell P. Levesque,4 Burkhard Becher2. 1 _Medical University of South Carolina (MUSC), Charleston, SC;_ 2 _University Zurich, Switzerland;_ 3 _European Institute of Oncology, Italy;_ 4 _University Hospital Zurich, Switzerland_.

Checkpoint inhibition of programmed cell death protein 1 (PD-1) has revolutionized cancer therapy. As more and more patients with metastatic melanoma but also other cancers show a dramatic increase in progression-free survival, still a large proportion of patients do not show durable response. As options for monotherapies, as well as combination immunotherapies, increase in the clinics, predictive biomarkers of clinical response are urgently needed. We employed high-dimensional single cell mass cytometry and a machine-learning bioinformatics pipeline for the in-depth characterization of all immune cells in the peripheral blood of stage IV melanoma patients before and at 12 weeks of anti-PD-1 immunotherapy. We observed a clear treatment response of patients under immunotherapy. However, a strong predictor of progression free and overall survival in response to anti-PD-1 immunotherapy before therapy was the frequency of CD14+CD16-HLA-DRhi monocytes. We could confirm this by conventional flow cytometry in an independent, blinded validation cohort and propose this as a novel predictive biomarker for therapy decisions in the clinic. We further present a high dimensional analysis workflow to analyze predictive and prognostic immune biomarkers during future clinical trials.

#567

The lysosomal transporter SLC46A3 is a potential predictive biomarker for antibody-drug conjugates bearing the non-cleavable warheads SG3376 and DM1.

Krista Kinneer,1 Yu-Tzu Tai,2 Sriram Sridhar,1 John Meekin,1 Sandrina Phipps,1 Ben Ruddle,1 Ryan Fleming,1 Nazzareno Dimasi,1 Ronald Herbst,1 Arnaud Tiberghien,3 Luke Masterson,3 Philip Howard,3 Kenneth Anderson,2 David Tice1. 1 _MedImmune, Gaithersburg, MD;_ 2 _Dana Farber Cancer Institute, Boston, MA;_ 3 _Spirogen, London, United Kingdom_.

Antibody drug conjugates (ADCs) combine a monoclonal antibody with a potent cytotoxic drug to preferentially eliminate antigen-positive cells for the treatment of cancer. ADCs bearing non-cleavable drug-linkers such as the pyrrolobenzodiazepine (PBD) SG3376 or the maytansinoid DM1 rely upon lysosomal degradation of the antibody for release of the cytotoxic warheads, which must then escape the lysosome and bind to their intracellular targets. We sought to identify the mechanism of lysosomal transport of these membrane-insoluble warheads and have recently reported that the lysosomal transporter SLC46A3 is required for cytotoxic activity of ADCs bearing SG3376 and DM1, and that loss of SLC46A3 is a mechanism of acquired resistance to Trastuzumab-DM1 (T-DM1). Here we explore the potential translational relevance of SLC46A3. Analysis of SLC46A3 mRNA expression across several tumors showed that breast cancer cell lines and primary tumor samples have relatively high levels of SLC46A3 compared to samples derived from multiple myeloma (MM), esophageal, and upper aerodigestive tract malignancies, all of which are being targeted in clinical trials with non-cleavable DM1 ADCs. Deeper analysis indicates that levels of SLC46A3 are decreased in MM patients compared to healthy donors, and levels are further decreased in the relapsed/refractory setting compared to samples taken at diagnosis. We measured the expression of SLC46A3 in a panel of MM cell lines by RT-PCR and found that 5 of 9 (56%) had undetectable levels of SLC46A3. In vitro cytotoxicity assays demonstrate that SLC46A3-negative cells are resistant to B-cell maturation antigen (BCMA)-targeted ADCs prepared with non-cleavable DM1 and SG3376 warheads, but remain sensitive to the cleavable payload PBD SG3249 (Tesirine). Taken together, our results suggest that SLC46A3 may be a predictive response marker for ADCs bearing the non-cleavable warheads SG3376 and DM1, and this marker may be particularly useful in trials involving MM.

#568

Recurrent drug sensitivity patterns in myelodysplastic syndrome patients are recapitulated by ex vivo drug response profiling.

Alexey Aleshin,1 Bruno C. Medeiros,1 Savita Kamble,1 Diane Heiser,2 Marianne Santaguida,2 Sacha Prashad,2 Peter L. Greenberg1. 1 _Stanford University, Stanford, CA;_ 2 _Notable Labs, San Francisco, CA_.

Myelodysplastic syndromes (MDS) are a collection of clonal diseases of dysfunctional hematopoietic stem cells, characterized by ineffective hematopoiesis, cytopenias, and dysplasia. Limited conventional treatment options exist for these patients, with hypomethylating agents remaining the standard of care for higher-risk MDS patients. Drug sensitivity and resistance testing on myelodysplastic syndromes (MDS) samples should provide important functional information to guide actionable target and biomarker discovery. We provide proof-of-concept data by profiling the effects of 50 common oncology drugs on 14 myelodysplastic (MDS) samples from both treatment-naïve and refractory cases. Ex vivo high-throughput functional screening was performed on lysed peripheral blood and/or bone marrow aspirate patient samples. This set of samples was analyzed 72 hours post incubation and blasts were quantified by flow cytometry. Unsupervised hierarchical clustering was performed to determine similar responses between patient samples, and produced two distinct groups with differing sensitivity to tested drugs. We defined these groups as "sensitive" and "resistant" clusters based on the prevailing drug sensitivity patterns these groups displayed, the latter being strongly enriched for HMA refractory samples. Ex vivo testing recapitulated known patient drug sensitivity patterns, with 12/14 samples from known HMA refractory vs. sensitive patients showing similar ex vivo phenotypes. Interestingly, sensitive and resistant clusters strongly correlated with known features associated with therapy resistance and poor prognosis in MDS patients, including number of somatic driver mutations (average 2.38 vs. 4.5, P <0.05), absence vs. presence of RUNX1 or ASXL1 mutations (P 0.015 and 0.0256, respectively), and HMA sensitive vs. refractory status (P <0.05). Interestingly, percent bone marrow blasts, cytogenetic risk groups and other somatic mutations had no effect on sensitivity patterns. Furthermore, while the resistant cluster showed increased resistance to most drugs, some drugs seemed to have increased activity in this cluster, including calcitriol and poly ADP ribose polymerase (PARP) inhibitor rucaparib. This unique platform, applied to predict ex vivo therapeutic response of MDS patient samples to various classes of drugs, recapitulates known clinical and molecular predictors of therapeutic response, and possible new therapeutic targets. These data suggest the possible utility of using this methodology to aid decision making for therapeutic selection in the management of MDS patients.

#569

A comprehensive immunoscore to predict immunotherapy responses based on multivariate genomic/transcriptomic features.

Mengchi Wang, Aaron Wise, Han Kang, Vitor F. Onuchic, Ali Kuraishy, Sven Bilke, Kristina M. Kruglyak, Shile Zhang. _Illumina, Inc., San Diego, CA_.

In recent years, immune checkpoint inhibitors have shown great promise in treating various cancer types; however, only a fraction of patients respond to this type of immunotherapy. Currently, PD-L1 ligand expression and microsatellite-instability (MSI) status are FDA-approved biomarkers to guide selected checkpoint-based immunotherapy; however, due to the complexity of tumor-immune interactions, it is unlikely that any single biomarker will be able to comprehensively predict clinical outcomes across the gamut of immunotherapeutics. Many genomic and cellular features have been shown to contribute to the effectiveness of immunotherapy, including tumor mutational burden (TMB), tumor T cell infiltrate, HLA gene expression, and Treg /myeloid-derived suppressor cell (MDSC) infiltrates. Here we have built a machine learning model trained on multiple features derived from whole exome sequencing (WES) and whole transcriptome sequencing (WTS) data. The resulting patient-specific "immunoscore" describes the likelihood that a patient will respond to a specified immunotherapy. Additionally, we have developed a novel visualization schema, which summarizes the full model immunoscore, as well as the weight and impact of each genomic and transcriptomic feature.

In this study, we assessed WES and WTS data from two melanoma cohorts, the first treated with anti-PD1 immunotherapy and the second with anti-CTLA4 immunotherapy. First, we extracted genomic/transcriptomic features around five functional groups: antigen presentation, tumor lymphocyte infiltration, checkpoint gene signatures, interferon-gamma gene signatures, and Treg/MDSC gene signatures. Next, random forest classification was performed to identify significant features and weight the relative importance of each. A final immunoscore was calculated as the patient-specific probability of immunotherapy response, scaled from zero (0% likelihood of response) to ten (100% likelihood of response). We noted that the highest-weighted feature for anti-PD1 response came from the antigen presentation feature group, while the highest-weighted feature for anti-CTLA4 response came from the tumor lymphocyte infiltration feature group, which is consistent with the underlying mechanistic difference between the two checkpoint inhibitors. Finally, our immunoscore has shown significantly better performance compared to any single feature based on 3-fold cross validation (p<0.05). In summary, we have built a machine learning model to predict patient responses to immunotherapies based on WES and WTS data and show that integrating multiple established biomarkers delivers superior performance compared to any individual biomarker. Moreover, our framework can be extended to include novel genomic/transcriptomic features that are identified as mediating immunotherapy response.

#570

High-resolution microbiome profiling of 16SrRNA sequencing data, Microbiome Arrays, and expression arrays identifies differential bacterial communities in head and neck cancer patients treated with surgery, chemo-radiation, and PD-1 checkpoint blockade therapy.

Rafael E. Guerrero-Preston,1 Kelvin Navarro,2 Filipa Godoy-Vitorino,2 James White,3 Anne Jedlicka,4 Gustavo Rivera-Alvarez,1 Barbara Mora-Lagos,5 Winston Timp,6 William Westra,5 Wayne Koch,5 Luigi Marchionni,5 Young Kim,7 David Sidransky5. 1 _University of Puerto Rico School of Medicine, San Juan, PR;_ 2 _Inter American University of Puerto Rico, San Juan, PR;_ 3 _Resphera Biosciences, Baltimore, MD;_ 4 _Johns Hopkins School of Public Health, Baltimore, MD;_ 5 _Johns Hopkins School of Medicine, Baltimore, MD;_ 6 _Johns Hopkins University, Baltimore, MD;_ 7 _Vanderbilt University School of Medicine, Nashville, TN_.

We have previously shown altered microbiota in saliva from head and neck squamous cell carcinoma (HNSCC) patients, when compared to controls. We now applied high-resolution microbiome profiling (Resphera Insight) to analyze 16S rRNA sequencing and Axiom Microbiome Array (Affymetrix) data in tissue samples from HNSCC patients and healthy controls. DNA from HNSCC (n=43) and controls (n=12) tissue samples was processed for 16SrRNA sequencing and microarray analyses. Raw sequences were processed for quality and length, screened for chimeras and filtered for contaminant human and chloroplast DNA. High-quality passing sequences were submitted to for species-level taxonomic assignments, followed by differential abundance analysis. Compositional arrangement was obtained with the R microbiome package, using a 25% compositional filtration of differentially significant Resphera OTUs, obtained with the Negative Binomial Wald test (p value <0.05). DESeq2 Variance Stabilizing Transformation algorithm was used for normalization after samples underwent the 25% prevalence threshold filtration. We found five assigned phyla dominating across the samples after 16S rRNA sequencing: Firmicutes, Proteobacteria, Bacteroidetes, Fusobacteria and Actinobacteria. We found three assigned phyla dominating across the samples hybridized to the microarray: Enterobacteria, Fusobacteria, and Prevotella. Alpha Rarefaction curves using the Chao1 index revealed that species richness did not differ between tumor and normal oral mucosa. However, there were differences by anatomic site and tumor staging when comparing HNSCC tissue samples. Differential abundance analysis between tumors and normals at the species level using the negative binomial test revealed that Lactobacillus salivarius, Lactobacillus gasseri, Lactobacillus johnsonii and Prevotella nanceiencis had the highest tumor/normal ratio (FDR-adjusted p values <0.05). We also found that Fusobacterium nucleatum (F.nucleatum), an oral cavity flora commensal bacterium linked to colon cancer, is enriched 51x higher in tissue in a subset of HNSCC patients with advanced tumors (T3 or above). F. nucleatum was detected in samples obtained before and after treatment with chemo-radiation, but not with surgery alone. Interestingly, we identified upregulation of the oncogenic Wnt/beta catenin pathway (Wnt7B, FZD6, SFRP4) and downregulation of immune system pathways (TLR10, IRF8) with genome-wide mRNA arrays (Affymetrix) in HNSCC tissue samples enriched for F. nucleatum. Together these data suggest the existence of complex interactions between the tissue microbiome in oral cavity mucosa and HNSCC tumor tissue, which may be modulating.

#571

Training and validation of a gene expression signature for microsatellite instability.

Alessandra Cesano,1 Patrick Danaher,2 SuFey Ong,2 Nathan Elliott,2 Sarah Warren2. 1 _NanoString Technologies, Inc., Redwood City, CA;_ 2 _NanoString Technologies, Inc., Seattle, WA_.

Introduction: Clinical response to cancer immunotherapy can be predicted from two biologically distinct variables: tumor foreignness, manifested in some tumors as deficient DNA mismatch repair (dMMR)/microsatellite instability (MSI), and antitumor immunity, typically measured with gene expression signatures or immunohistochemistry. Clinical benefit should be more accurately predicted by a synthesis of these two variables than by either alone. However, measuring both variables requires multiple clinical assays to be run in parallel. Here, we investigate the ability of gene expression alone to provide a surrogate measure of both tumor foreignness and immunogenicity, empowering a single assay to measure both axes of predictive biology. In addition, we explore the relationships between tumor foreignness and immunogenicity among both tumors of different dMMR-statuses and within tumors themselves.

Methods: Using TCGA datasets from colon, esophageal, stomach and uterine cancers, we trained two algorithms predicting hypermutation: the first detecting loss of expression of mismatch repair genes (MLH1, MSH2, MSH6 and PMS2) and the second identifying expression patterns shared by hypermutated tumors across these four cancer types. A final algorithm synthesizes the two above algorithms into a single score. To independently validate our TCGA discoveries, we collected a cohort of 60 colorectal cancer (CRC) tumor FFPE samples, divided into 30 dMMR and 30 MMR-proficient (pMMR) (previously identified by immunohistochemistry), and a cohort of 10 MSI and 5 MSS endometrial and neuroendocrine tumors. We analyzed all samples using the NanoString nCounter® platform and a smaller subset using the NanoString® Digital Spatial Profiling (DSP) Technology.

Results: We show that our algorithms successfully predicted hypermutation phenotypes and MMR status in TCGA training data and in the independent CRC and endometrial datasets analyzed with NanoString. In CRC samples, the algorithms showed 85% sensitivity and 100% specificity; in endometrial samples, they predicted dMMR with 90% sensitivity and 100% specificity. Additionally, we demonstrate that higher mutational burden is linked to a heightened tumor immune environment as shown by gene expression and protein signatures specific to the adaptive immune response.

Discussion: Gene expression proves to be a powerful predictor of microsatellite instability and hypermutation in cancers where dMMR subtypes are known to exist. This discovery raises the possibility that a gene expression algorithm measuring both hypermutation and immune activity can achieve unprecedented accuracy in predicting response to checkpoint inhibition in these cancer types.

#572

Genome-scale CRISPR/Cas9 screen identifies factors required for sensitivity to pyrimidine nucleoside analogs.

Awa SARR,1 Jennifer Bré,1 Peter Mullen,1 Sarah Blagden,2 In Hwa Um,1 David J. Harrison,1 Paul A. Reynolds1. 1 _University of St Andrews, St Andrews, United Kingdom;_ 2 _University of Oxford, Oxford, United Kingdom_.

NUC-1031 (NuCana plc), is a phosphoramidate transformation of gemcitabine, which bypasses cancer cell resistance mechanisms to gemcitabine. This compound showed promising results in clinical studies (phase I/II), demonstrating tumor reduction and durable disease control in patients who were refractory to or had relapsed on gemcitabine. Here, we adopted an unbiased approach to uncover genes and pathways involved in gemcitabine and NUC-1031 resistance. We performed a genome-wide knockdown screen using CRISPR/Cas9 technology in the pancreatic cancer cell line MiaPaCa2. Potential candidates from the screen were validated by gene inactivation using individual or combined single guide (sg)RNA targeting sequences. The effect of gene knockdown on NUC-1031 and gemcitabine resistance was assessed by monitoring cell survival after drug treatment using Celigo cytometry to assess cell number. EC50 values were determined using the resulting survival curves. This screen generated a list of 4 candidate genes potentially involved in NUC-1031 resistance. Two candidates were investigated further: DCK and DCTPP1, both involved in pyrimidine metabolism, especially in the maintenance of the dCMP/dCTP pool. DCK knockdown induced significant resistance to gemcitabine (EC50 350 times higher than in control cells) and a degree of resistance to NUC-1031 (EC50 7 times higher than in control cells). A less robust effect was observed when DCTPP1 was inactivated. DCTPP1 knockdown induced a small decrease in sensitivity to NUC-1031 (EC50 1.5 times higher than in control cells).

These results highlight the potential role of dCMP/dCTP pool regulation by DCK and DCTPP1 in sensitivity to pyrimidine nucleoside analogs and may provide a useful biomarker for patient selection.

#573

BRAF, KRAS, and NRAS mutations in archival tumor samples and samples of cell-free DNA from serum and bone marrow aspirates from patients with multiple myeloma.

Filip Janku,1 Qian Li,2 Helen J. Huang,1 Yafei Wang,2 Zeng Cao,2 George Karlin-Neumann,3 Zhiqiang Liu4. 1 _MD Anderson Cancer Center, Houston, TX;_ 2 _Tianjin Medical University Cancer Institute and Hospital, Tianjin, China;_ 3 _Bio-Rad Laboratories, Pleasanton, CA;_ 4 _Tianjin Medical University, Collaborative Innovation Center of Tianjin for Medical Epigenetics, Tianjin, China_.

Background: Oncogenic mutations in BRAF, KRAS and NRAS genes have been reported in multiple myeloma and can be explored as biomarkers for selection of targeted therapies. Methods: Unamplified DNA from archival tumor tissue samples and cell-free DNA (cfDNA) from serum and bone marrow aspirates from patients with multiple myeloma was tested for BRAF V600, KRAS G12/G13, NRAS G12/G13 and NRAS Q61 mutations using multiplex assays for droplet digital PCR (Bio-Rad). Agreement among methods was evaluated and results were compared to clinical outcomes. Results: Of 88 patients with multiple myeloma (stage I, 13 [15%]; stage II, 26 [30%]; stage III, 34 [39%]; stage unknown, 15 [17%]), 4 (5%) patients were found to have BRAF V600 mutation (all confirmed V600E), 15 (17%) KRAS G12/G13 mutation, 3 (3%) NRAS G12/G13 mutation and 16 (18%) NRAS Q61 mutation in archival tumor tissue. Most mutations were subclonal with a median variant allelic frequency (VAF) 1.55% (range, 0.13%-34%). Survival data were available for 47 patients and patients with high mutation VAF had a trend to shorter survival than patients with low mutation VAF (25.3 months vs. not reached, P=0.087). Of 88 patients, 22 (25%) had simultaneous serum cfDNA collection, which demonstrated BRAF V600 mutation in 7 (32%; 3 confirmed as V600E), KRAS G12/G13 mutation in 2 (9%), NRAS G12/G13 mutation in 0 (0%) and NRAS Q61 mutation in 4/22 (18%) of samples and median VAF was 2.9% (range, 0.34%-18.8%). Agreement rates for serum compared to tissue were 73% for BRAF V600, 95% for KRAS G12/G13, 100% for NRAS G12/G13 and 95% for NRAS Q61. In addition, 13 (15%) patients had simultaneous bone marrow aspirate cfDNA collection, which demonstrated BRAF V600 mutation in 1 (8%; confirmed as V600E), KRAS G12/G13 mutation in 0 (0%), NRAS G12/G13 mutation in 0 (0%) and NRAS Q61 mutation in 1 (8%) of samples. Agreement rates for bone marrow aspirates compared to tissue were 100% for BRAF V600, 92% for KRAS G12/G13, 100% for NRAS G12/G13 and 100% for NRAS Q61. Conclusion: Common oncogenic mutations in BRAF, KRAS and NRAS genes are prevalent with relatively low VAF in multiple myeloma and can be detected with sensitive techniques such as ddPCR. Their prognostic significance and therapeutic utility needs to be further investigated.

#574

BRAFV600E **mutation and loss of CDX2 synergize in serrated colorectal tumorigenesis.**

Naoya Sakamoto,1 Ying Feng,2 Takuya Hattori,1 Masayuki Shimoda,3 Akira Ishikawa,1 Ririno Honma,1 Takao Hinoi,4 Hiroyuki Egi,1 Hideki Ohdan,1 Yasunori Okada,5 Eric Fearon,2 Wataru Yasui1. 1 _Institute of Biomedical & Health Sciences, Hiroshima University, Hiroshima, Japan; _2 _University of Michigan, Ann Arbor, MI;_ 3 _Keio University School of Medicine, Tokyo, Japan;_ 4 _National Hospital Organization Kure Medical Center and Chugoku Cancer Center, Kure, Japan;_ 5 _Juntendo University Graduate School of Medicine, Tokyo, Japan_.

While many CRCs arise from adenomatous polyps through what we call "adenoma-carcinoma sequence," it has been estimated that up to 20% of CRCs likely evolve from an alternative pathway, so-called ''serrated pathway.' However, only 8-10% of CRCs display definitive serrated morphology at diagnosis. We previously examined expression of differentiation and molecular markers that are most likely to be involved in serrated tumor development by using 36 serrated CRCs and found CDX2 loss or BRAF mutations often together. We then generated a mouse model that can be induced concurrent biallelic inactivation of Cdx2 (Cdx2-/-) and expression of mutant BRAFV600E in adult mouse colon epithelium, and the Cdx2-/-/BrafV600E tumors showed a quite similar phenotype to that of CDX2-negative serrated CRCs. Through the validation of global gene expression profile including the mouse models and human CRCs, we focused on ADAM28 as a candidate because ADAM28 was significantly upregulated in Cdx2-/-/BrafV600E tumor and CRCs with low CDX2 expression and BRAFV600E mutation. We examined ADAM28 expression using 12 CRC cases with BRAFV600E mutation (8 of 12 displayed serrated feature) and 12 CRC cases with KRAS codon 12/13 mutation and found that ADAM28 was specifically upregulated in BRAF mutant CRC cases. Among 10 CRC cell lines (DLD-1, Lovo, SW48, SW480, SW837, SW1116, Caco2, RKO, HT-29, HCT-116), only HT-29, which has many phenotypic and genetic similarities to serrated CRC, showed a robust expression of membranous type ADAM28, and 9 of 10 CRC cells, except for RKO, showed weak to modest expression of active-form ADAM28. Further in-depth studies are ongoing to clarify the solid evidences, especially the utility of ADAM28 as a diagnostic marker for serrated CRCs and the direct effect of ADAM28 onto cell growth of CRC cells using ADAM28-specific antibody. We present a novel transgenic model of human serrated CRC to highlight the suitability of centering on CDX2 loss and BRAFV600E in the pathogenesis. This model has a great potential to figure out the mechanisms underlying in serrated tumor progression and identify the candidates of new biomarkers and therapeutic targets of CRCs with serrated morphology.

#575

Prediction of genomic instability in triple-negative breast cancer patients utilizing a CTC phenotype classifier.

Adam Jendrisak, Nadia Ebrahim, Angel Rodriguez, Jerry Lee, Ramsay Sutton, Yipeng Wang, Mark Landers, Ryan Dittamore. _Epic Sciences, San Diego, CA_.

Background: Genomic scars and HRD gene mutations are biomarkers for PARP inhibitor (PARPi) and platinum agent therapy response in breast cancer. Tests for these markers are usually performed by next-generation sequencing (NGS) of tumor tissue or circulating tumor DNA (ctDNA) and have limitations such as sample accessibility/availability and under-sampling due to inter/intratumor heterogeneity. Previously we predicted genomic instability (GI) using CTC phenotypic features without the use of NGS in metastatic castration-resistant prostate cancer (mCRPC) pts with high accuracy (>76%) (ASCO 2016). In addition, patients with phenotypically predicted GI (pGI) CTCs had significantly better PSA responses on abiraterone + veliparib (92%) vs. abiraterone alone (23%) (ESMO 2016), and also responded better to platinum agents vs. taxane agents (ESMO 2017). Here, we sought to develop and analytically validate an algorithm for predicting CTC pGI in TNBC.

Methods: Training set: 521 CTCs from 26 mCRPC pts were detected with the Epic Sciences CTC platform and analyzed for 19 phenotypic digital pathology features, including protein expression and cell morphology. The same CTCs were single-cell sequenced for the number of large-scale transitions (LSTs) as an indicator of GI. A linear regression algorithm to predict GI by CTC phenotype was developed, cross validated, and utilized to generate a CTC pGI score. Test set: 114 CTCs from 8 TNBC blood samples, median of 8 CTCs/pt, were sequenced for GI and phenotypically predicted for pGI.

Results: Analytical cutoff for GI (8.74) was determined in the mCRPC training set by separation of a binomial distribution low vs. high GI scores. pGI cutoff (8.74) was set the same as GI. The training set performance was: sensitivity = 69%, specificity = 83%, accuracy = 77%. In the TNBC test set, pGI vs. GI demonstrated: sensitivity = 86%, specificity = 92%, accuracy = 88%.

Conclusions: Previous studies showed that pGI was an analytically validated biomarker with clinical utility to predict PARPi or platinum therapy response in mCRPC pts. Here we show the same test concept can be applied to TNBC. Further analytical validation in a larger cohort is ongoing. The ability to identify PARPi/platinum sensitivity using an IF CTC staining method for CTC phenotype without the use of NGS will help to stratify patients more rapidly, at reduced cost, and aid in the acceleration of drug development.

#576

A cancer drug atlas enables prediction of parallel drug vulnerabilities.

Ravi Narayan,1 Piet Molenaar,2 Jian Teng,3 Bakhos Tannous,3 Tom Wurdinger,1 Jan Koster,2 Bart Westerman1. 1 _VU Medical Center, Amsterdam, Netherlands;_ 2 _Academic Medical Center Amsterdam, Amsterdam, Netherlands;_ 3 _Experimental Therapeutics and Molecular Imaging Lab, Neuroscience Center, Neuro-Oncology Unit, Charlestown, MA, Massachusetts General Hospital and Harvard Medical School, Boston, MA_.

Personalized cancer treatments using synergistic combinations of drugs is attractive but proves to be highly challenging. The combinatorial nature of such problems results in an enormous parameter space that cannot be resolved by empirical research, i.e. testing all combinations for all molecularly defined tumors. In addition, effective drug synergy is hard to predict. Here we present an approach to map data of drug-response encyclopedias and represent these as a drug atlas. This atlas consists of a framework of chemotherapeutic responses that represents a drug vulnerability landscape of cancer. Based on data from the literature we found that many synergistic drug combinations show distinct inter therapy responses and drug sensitivities. We confirmed this by performing a drug combination screen against glioblastoma where we used 270 combination experiments. From the identified dual therapies we were able to predict and validate a triple drug synergy which was validated in vivo. This new and generalizable strategy opens the door to unforeseen personalized multidrug combination approaches.

#577

Oral microbiome biomarker panel to detect oral and oropharyngeal cancers in a clinical setting.

Yenkai Lim,1 Naoki Fukuma,2 Makrina Totsika,1 Liz Kenny,3 Mark Morrison,3 Chamindie Punyadeera1. 1 _Queensland University of Technology, Brisbane, Australia;_ 2 _Obihiro University of Agriculture and Veterinary Medicine, Hokkaido, Japan;_ 3 _University of Queensland, Brisbane, Australia_.

Oral microbiome plays a role in the instigation of oral diseases that affect other systemic conditions. These associations encourage the exploration of oral dysbiosis leading to the pathogenesis of cancers. In this study, saliva was used to characterise the oral microbiome fluctuation associated with oral and oropharyngeal cancers. The study cohort consists of normal healthy controls (n = 10, between 20 to 30 years of age; n = 10, above 50 years of age), high-risk individuals (n = 11, above 50 years of age with bad oral hygiene and/or oral diseases) and oral and oropharyngeal cancer patients (n = 31, HPV-positive; n = 21, HPV-negative). Saliva samples were analysed using 16S rRNA gene amplicon sequencing on the MiSeq platform. Kruskal-Wallis rank test was used to identify genera associated with oral and oropharyngeal cancers. A logistic regression analysis was carried out to determine the performance of these genera as a biomarker panel to detect oral and oropharyngeal cancers. In addition, a two-fold cross-validation with a bootstrap procedure was carried out on R to investigate how well the panel would perform in a real clinical scenario. Our data indicates that the oral microbiome is able to predict the presence of oral and oropharyngeal cancers with sensitivity and specificity of 100% and 90% respectively. With further validation, the panel could potentially be implemented into clinical workflow as oral and oropharyngeal cancers diagnostic and prognostic biomonitors.

#578

Loss of MED12 predicts for a DNA damage repair deficiency phenotype and activates immune signalling via the STING pathway.

Folake A. Orafidiya,1 Catherine Davidson,1 Richard D. Wilkinson,1 Steve M. Walker,2 Laura A. Knight,2 Paul D. Harkin,2 Nuala McCabe,1 Richard D. Kennedy1. 1 _Queen's University, Belfast, United Kingdom;_ 2 _Almac Diagnostics, Craigavon, United Kingdom_.

Introduction: Genome integrity is maintained by a number of pathways which recognize and repair both exogenous and endogenous DNA damage. Deficiency in these pathways results in mutations that accelerate tumorigenesis. A subgroup of cancers such as those with mutations in the BRCA/ Fanconi anemia (FA) pathway have been reported to benefit from DNA damaging chemotherapy. Previously, we identified a molecular subgroup in breast cancer characterised by upregulation of immune genes. This group referred to as the DNA damage repair deficient (DDRD) group also showed enhanced response to DNA damaging chemotherapy. Here, we identify the same subgroup in prostate cancer and investigate the mutations associated with the subgroup in prostate cancer. Experimental procedures: Unsupervised hierarchical clustering in primary prostate cancer identified a subset of patients which demonstrated activation of immune signalling genes and enrichment for BRCA mutations, representing the DDRD group. Using data from The Cancer Genome Atlas (TCGA), we show that mutation of the MED12 gene is associated with the DDRD group. In order to study the role of MED12 in prostate cancer, isogenic cell lines with knockdown of MED12 were generated using RNAi technology. We evaluated the expression of immune genes in these cell lines using RT-qPCR. Immunofluorescence, DNA-RNA immunoprecipitation and western blot analyses were also used in the characterization of these cell lines. Results: Our data reveals that the loss of MED12 leads to a significant upregulation of the immune genes associated with DNA repair deficiency; CXCL10, CCL5, MX1 and the immune check-pointing gene PD-L1 (p<0.05). Accumulation of ɣH2AX foci was identified in cells depleted in MED12 (p<0.001). A significant increase in DNA-RNA hybrids (Rloops) was also observed in MED12- deficient cells (p<0.0001) indicating an endogenous source of genomic instability. Furthermore, we show the presence of cytosolic nucleic acid which triggers immune response via the STING/TBK1/IRF3 pathway which is characteristic of the DDRD-like phenotype in MED12-deficient cells.Conclusion: Mutations in the MED12 gene have previously been reported in 5 % of prostate cancer. Our findings demonstrate that the loss of MED12 in prostate cancer results in a DDRD-like phenotype. Upon further characterization, MED12 may be used as a predictor of response to stratify patients for chemotherapy or immune checkpoint targeted therapy in prostate cancer

#579

Magnetic resonance imaging of the breast and radiomics analysis for an improved early prediction of the response to neoadjuvant chemotherapy in breast cancer patients.

Katja Pinker-Domenig,1 Amirhessam Tahmassebi,2 Georg Wengert,3 Thomas H. Helbich,3 Zsuzsanna Bago-Horvath,3 Elizabeth A. Morris,1 Anke Meyer-Baese4. 1 _Memorial Sloan Kettering Cancer Center, New York, NY;_ 2 _Floriada State University, Tallahassee, FL;_ 3 _Medical University of Vienna, Vienna, Austria;_ 4 _Florida State University, Tallahassee, FL_.

Background and Aim

In patients undergoing neoadjuvant chemotherapy for breast cancer the achievement of a pathological complete response (pCR) is associated with a significantly improved disease-free and overall survival. Therefore, accurate means to predict treatment response as early as possible are desirable to identify women who don't benefit from this cytotoxic therapy. Several studies have demonstrated that dynamic contrast-enhanced MRI is the most sensitive method for the assessment and prediction of treatment response. In the past decade, the field of medical image analysis has grown exponentially, with an increased number of pattern recognition tools and an increase in data set sizes. These advances have facilitated the development of processes for high-throughput extraction of quantitative features that result in the conversion of images into mineable data and the subsequent analysis of these data for decision support. This emerging field in medical research is termed radiomics. The aim of this study was to assess radiomics with dynamic contrast-enhanced (DCE) MRI for the early prediction of pathological complete response (pCR) in breast cancer patients undergoing neoadjuvant chemotherapy.

Methods and materials

In this IRB approved prospective study 41 women with breast cancer scheduled for NAC were included and underwent MRI of the breast at 3T with DCE and T2-weighted imaging prior to and after two cycles of NAC. For each lesion a total of 14 features were extracted ranging from morphological and kinetic MRI ADC parameters. A recursive feature elimination method along with five different classifiers was performed including: linear support vector machine (SVM), linear discriminant analysis (LDA), logistic regression (LR), random forest (RF), and stochastic gradient descent (SGD) was employed to rank the features. Histopathology using the Residual Cancer Burden (RCB) score and class calculated from post-treatment surgical specimen and patient outcomes were used as the standard of reference.

Results

Classification accuracy was assessed for pCR as defined by the RCB score, metastases and disease specific death. Radiomics analysis of MRI data achieved AUCs for RCB score (AUC 0.85), metastases (AUC 0.87) based on RF and death (AUC 0.92) based on SVM. The most relevant parameters for prediction of RCB score were mass internal enhancement characteristics, shape and margins with DCE, for metastasis peri-tumoral edema on T2-weighted imaging, mass margins and internal enhancement characteristics, and for death high signal intensity on T2-weighted imaging, mass margins, and internal enhancement characteristics.

Conclusion

Radiomics with MRI of the breast using DCE and T2-weighted imaging enables prediction of response to NAC with high accuracy and thus can provide predictive information to guide treatment decisions.

#580

A method for estimating mutation load from tumor research samples using a targeted next-generation sequencing panel.

Ruchi Chaudhary, Dinesh Cyanam, Vinay Mittal, Warren Tom, Janice Au-Young, Seth Sadis, Fiona Hyland. _Thermo Fisher Scientific, South San Francisco, CA_.

Introduction: Tumor mutation load predicts durable benefit from immune checkpoint inhibitors in several cancer types. Existing methods to estimate tumor mutation load have large input DNA and extensive infrastructure requirements and are associated with delays due to shipping precious biopsy samples to central laboratories. Herein, we demonstrate the ability of a targeted panel with fast turn-around time to estimate mutation load from tumor research samples using low input. Methods: We developed a single sample analysis workflow to estimate mutation load (mutation count per megabase) from FFPE and fresh frozen tumor research samples. The assay utilizes a PCR-based target enrichment panel that covers 409 genes and 1.7 Mb of genomic regions. The workflow requires only 10 ng of input DNA, and enables a 2.5-day turn-around time from sample to the final report. The workflow enables < 60 minutes of hands-on time for automated library preparation and templating on a batch of 8 samples. Sequencing is performed on high throughput semiconductor sequencing platform to achieve sufficient depth (~500x coverage) and accuracy. Our analysis pipeline calls variants at ≥5% allelic frequency with parameters optimized for high accuracy. The workflow is tumor sample only, with no matched normal sample required, and germ-line variants, along with background noise, are removed through application of filters based on variants prevalent in population databases. Results: In-silico analysis demonstrated that the predicted mutation counts associated with the covered regions of the targeted panel could effectively stratify responders and non-responders to immune checkpoint inhibitors in 3 separate cohorts with high statistical significance. The assay was applied to colorectal specimens previously typed for microsatellite instability (MSI) and distinguished samples that were MSI-H (median mutation count 94.5) and MSS (median mutation count 17.8). Matched tumor-normal analyses on 7 colorectal tumor samples showed that the single tumor analysis workflow detected somatic mutations with strong correlation (r = 0.97) with tumor-normal analysis. To assess reproducibility, we compared somatic mutation count in 6 library replicates of breast cancer cell line HCC1143 and observed low relative variability (CV=9.57%) with 19.17 average mutation count. The analysis report characterizes substitution type and context of somatic mutations, and highlights mutation signatures consistent with UV damage and spontaneous deamination of 5-methyl-cytosine, as well as FFPE deamination. Conclusions: We developed a simple analysis workflow on the Ion Torrent sequencing platform with an AmpliSeq panel to estimate mutation load from FFPE and fresh frozen tumor research samples. This solution will advance research in immuno-oncology.

#581

Feasibility test of KRAS mutation in urine circulating tumor DNA in metastatic pancreatic ductal adenocarcinoma.

Min Jeong Kwon,1 Min Kyeong Kim,2 Sang Myung Woo,2 Kyong-Ah Yoon,3 Yun Hee Kim,2 Boram Park,2 Jung Nam Joo,2 Sang Jae Park,2 Sun-Young Kong2. 1 _National Cancer Center Graduate School of Cancer Science and Policy, Goyang, Republic of Korea;_ 2 _National Cancer Center, Goyang, Republic of Korea;_ 3 _Konkuk University, Goyang, Republic of Korea_.

Circulating tumor DNA (ctDNA) has been known to be released from tumor cells and evaluated as potential biomarkers for therapeutic responses. In our previous study, we selected KRAS mutation and evaluated applicability as a prognostic marker through the quantitative analysis of plasma ctDNA. Then we proved that KRAS mutation of ctDNA from plasma is significant biomarker for prediction of clinical outcome in pancreatic ductal adenocarcinoma (PDA). Here we investigated whether KRAS mutation in urine ctDNA had the prognostic impact as shown of plasma ctDNA. Of the 106 patients enrolled, 67 were males and median age was 66 years. Total of 51 PDA has been enrolled in this study and the median age was 63 years old and 30 males (58.8%). Urine was separated by established centrifugation method and ctDNA were extracted using QIAamp Circulating Nucleic Acid Kit (Qiagen, Germany) from 4 mL of urine. Extracted ctDNA was quantified using the Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific, USA). QX200 Droplet Digital PCR System (Biorad, USA) was applied to measure frequency of KRAS mutation by KRAS screening multiplex droplet digital PCR kit, which covers seven common mutation sites (Biorad, USA). Mutant concentration and fractional abundance were analyzed by QuantaSoft software (Biorad, USA). There was no significant difference between resectable (N=21), locally advanced (N=11) and metastatic (N=19) groups. When compared high versus low KRAS mutation concentration and KRAS fractional abundance group in each stage, high KRAS mutation groups showed short overall survival (p = 0.0321) in metastatic group. However, the correlation between plasma and urine was low (r=0.193) in metastatic group. This study represented the possibility of KRAS mutation concentration and fractional concentration of urine ctDNA as prognostic factors in metastatic PDA. Urine cfDNA role has not been well investigated in PDA. Urine is easy to obtain and is not well known for its mechanism. Therefore, we have conducted a pilot study and we will explore KRAS mutation status in urine ctDNA in further studies. (This study was supported by National Cancer Center, Korea, Grant no. 1510203.)

#582

Accelerating drug development with a CLIA approved functional test using patient derived organoids.

Franz X. Schaub,1 Michael J. Churchill,1 Hallie A. Swan,1 Rachele Rosati,2 Roland M. Watt,1 Reid C. Shaw,1 Stephanie A. Murphy,1 Robert L. Diaz,1 Shalini C. Pereira,1 Carla Grandori1. 1 _SEngine Precision Medicine, Seattle, WA;_ 2 _Cure First, Seattle, WA_.

The pool of targeted therapies is expanding steadily providing novel treatment opportunities. However, the process of expanding indications and including additional cancer types after initial approval is slow. Here we present an in vitro clinical trial platform using the data from the first CLIA-approved Precision Medicine Platform using high-throughput screening (P.A.R.I.S. Assay, SEngine Precision Medicine, Seattle, WA).

Briefly, patient-derived tumor cells from biopsies are expanded as living organoids, and functionally evaluated using a high-throughput drug screen. The drug library currently includes FDA approved and experimental drugs. Results are integrated with known genomic information and reported to the clinician to highlight treatment options of an individual patient. Combining this data from multiple patients enables the discovery of novel indications and biomarkers. To date we have acquired data from a wide range of tumor types including hard-to-treat cancers, rare tumor types and tumors from heavily pretreated patients. In addition, we also have collected data from more than 100 patient derived cell lines.

Using the data analysis platform developed in R, the drug responses across all patient screens of a given drug can be analyzed to find common indicators of sensitivities. For instance, a BRD4 inhibitor (CPI-203), which was tested on 8 different primary tumor samples, was selectively active in a subset of the patients. Interestingly, most of the responding tumor types are ovarian and breast cancer cases with mutations in BRCA1 or EZH2. However, there are currently no clinical trials investigating the more developed BRD4 inhibitor CPI-0610 for breast or ovarian cancer. Non-responders, on the other hand, are spread between multiple cancer types.

The number of patients receiving the P.A.R.I.S. test is steadily increasing and these patient cases contribute to our knowledge base. As this test is used to inform clinicians about potential treatment opportunities, we are also accumulating information on how these patients respond to treatment. Overall, this platform will provide real world data for optimal drug combinations and novel biomarkers to quickly expand indications of existing drugs.

#583

Rapid release of ctDNA as a biomarker of treatment response in preclinical models of head and neck squamous cell carcinoma.

Ariana Rostami, Caberry Yu, Marco A. Di Grappa, Scott V. Bratman. _Princess Margaret Cancer Center, Toronto, Ontario, Canada_.

Purpose: High-dose radiotherapy (RT) is a standard treatment for locally advanced head and neck squamous cell carcinoma (HNSCC). Despite known molecular prognostic biomarkers in HNSCC, such as the presence of human papillomavirus (HPV) within tumor tissues, RT regimens remain one-size-fits-all without any patient-specific individualization. We hypothesize that rapid release of circulating tumor DNA (ctDNA) can act as a biomarker of treatment response in HNSCC by reflecting tumor cell death in response to RT.

Methods: Four HNSCC cell lines, two HPV- (Cal33, FaDu) and two HPV+ (HMS-001, 93-Vu147T), were evaluated for ctDNA release following single dose RT in vitro. Quantification of ctDNA was performed by quantitative polymerase chain reaction (qPCR) with primers amplifying the human long interspersed nuclear element 1 (hLINE1). RT-induced apoptosis was measured using a luminometric caspase 3/7 assay, and RT-induced senescence was evaluated using a fluorometric senescence associated β-galactocidase (SA-β-Gal) assay. The pan-caspase inhibitor, z-vad-fmk, was used to block RT-induced apoptosis. Subcutaneous cell-line xenografts were established in Nod-Scid-Gamma (NSG) mice, where plasma from serial blood draws was purified and quantified for ctDNA release using hLINE1 qPCR. Endpoint tumors were evaluated for mechanisms of cell death by histological staining.

Results: HNSCC cell lines exhibited variable magnitude and timing of ctDNA release and apoptosis. Maximal ctDNA release occurred between 72 and 144 hours post-RT. The release of ctDNA was not correlated with HPV status nor with apoptosis occurring following RT. To further interrogate the contribution of apoptosis to ctDNA release, we treated cells with a pan-caspase inhibitor and evaluated ctDNA release and caspase activity following RT. Although caspase inhibition resulted in a near complete reduction in RT-induced caspase activity (84.0% ±8.1%), a comparatively minor reduction in ctDNA release (28.9%±8.9%) was observed. To evaluate the impact of senescence on RT-induced ctDNA release, we measured SA-β-Gal activity post-RT. The degree of senescence following RT was inversely associated with ctDNA release. In cell line xenografts, maximal ctDNA release into mouse plasma occurred 96 hours following RT. Staining patterns of endpoint tumors for caspase-3, TUNEL, p21, and Ki67 are currently under analysis.

Conclusions: Our results demonstrate a robust and sensitive method for longitudinal evaluation of ctDNA release and cell death post-RT both in vitro and in vivo. Timing of ctDNA release demonstrate peaks around 72-96 hours post-RT, highlighting the potential for ctDNA to depict early response to therapy. Ongoing studies aimed at further investigating ctDNA release in association with mechanisms of cell death in vivo will help further elucidate the potential for ctDNA as a biomarker of treatment response.

#584

Conquering undruggable oncogenic K-RAS-driven incurable metastatic cancer, and delivering precision medicine at neoadjuvant settings.

Amy H. Tang,1 Robert E. Van Sciver,1 Elizaveta Svyatova,1 Kevin Kanda,1 Michael P. Lee,1 Caroline Dasom Lee,1 Lauren L. Siewertsz Van Reesema,1 Alex C, Lafever,1 Amber L. Collier,1 Apoorva S. Iyer,1 L.D. Britt,1 Janet S. Winston,2 Cynthia A. Allen,3 David Z. Chang,4 Gloria M. Petersen,5 Richard A. Hoefer6. 1 _Eastern Virginia Medical School, Norfolk, VA;_ 2 _Sentara Norfolk General Hospital, Norfolk, VA;_ 3 _Sentara Healthcare, Virginia Beach, VA;_ 4 _Virginia Oncology Associates, Newport News, VA;_ 5 _Mayo Clinic, Rochester, MN;_ 6 _Sentara Surgery Specialists, Newport News, VA_.

SIAH is a new and potent anti-K-RAS drug target in human cancer. The oncogenic EGFR/HER2/K-RAS pathway activation is pivotal in driving uncontrolled tumor growth and systemic metastasis. Thus, counteracting ERBB/K-RAS hyperactivation in attempt to reverse malignant transformation and inhibit latent tumor growth is an important area for new therapy development against late-stage and metastatic cancer. Guided by Drosophila studies, we found that SIAH (Seven-In-Absentia Homologue) is the most downstream "gatekeeper" required for proper K-RAS signaling. Based on its extraordinarily evolutionary conservation, SIAH E3 ligase is well positioned to serve as an ideal drug target for developing new anti-K-RAS and anticancer therapy. We have shown that anti-SIAH-based therapy is indeed effective in inhibiting tumorigenesis and metastasis of pancreatic, lung and breast cancer cells in xenograft models. Importantly, we have shown that anti-SIAH-based anti-K-RAS strategy is effective against well-established, super-large and late-stage pancreatic and triple-negative breast tumors in a xenograft model in vivo. Through these studies, we have successfully identified a new oncogenic K-RAS "vulnerability," SIAH, in high-grade metastatic cancer. We aim to design and develop potent SIAH inhibitor, and translate these findings to the clinic to benefit more cancer patients with therapy-refractory, relapsed and metastatic diseases in the future. SIAH is a therapy-responsive and prognostic biomarker in human cancer: SIAH expression can be used to monitor tumor responses, and identify resistant tumor clones post-NST. SIAH and EGFR outperform ER, PR, HER2 and Ki67 as two robust, sensitive and prognostic biomarkers to predict survival in breast cancer patients with lymph node metastases. The prognostic power of SIAH and EGFR, alone or in combination, is comparable to the clinical gold standards of clinical predictors (LN positivity, mammary tumor size, grade, stage and molecular subtypes in combination), and imaging-guided technology. A marked reduction in SIAH/EGFR expression post-NST would indicate effective therapy and increased survival, while persistent high SIAH/EGFR expression post-NST would indicate ineffective therapy and decreased survival. The therapy-induced changes in SIAH expression are prognostic in quantifying effective/ineffective therapies, differentiating partial responders, identifying resistant tumor clones, and predicting remission/relapse in breast cancer at neoadjuvant settings. The identification of therapy-responsive and prognostic biomarkers is of paramount importance to stratify patients and guide therapies in clinical oncology and personalized medicine. By validating the RAS/SIAH pathway-centered prognostic biomarkers, we hope to guide standard therapies and improve patient survival in the future.

#585

Exosomal EphA2 transmits chemoresistance and predicts pancreatic cancer patient responses to therapy.

Jia Fan,1 Qian Wei,2 Eugene J. Koay,3 Yang Liu,1 Zhen Zhao,4 Ye Hu1. 1 _Arizona State University, Tempe, TX;_ 2 _Tianjin Medical University Cancer Institute and Hospital, China;_ 3 _University of Texas M.D. Anderson Cancer Center, TX;_ 4 _Weill Cornell Medicine, NY_.

Exosomes are small extracellular vesicles secreted by most cells that are found in blood and other bodily fluids, and which contain cytoplasmic material and membrane factors corresponding to their cell type of origin. Exosome membrane factors and contents have been reported to alter adjacent and distant cell behavior in multiple studies, but the impact of cancer-derived exosomes on chemoresistance is less clear. We therefore analyzed exosomes from the human pancreatic cancer (PC) cell lines PANC-1, MIA PaCa-2 and BxPC-3 for their ability to transmit chemoresistance, finding that PANC-1 exosomes increased the gembicitabine (GEM) resistance of MIA PaCa-2 and BxPC-3 cell cultures. Comparative proteomics determined that PANC-1 exosomes overexpressed Ephrin type-A receptor 2 (EphA2) versus exosomes of less chemoresistant PC cell lines MIA PaCa-2 and BxPC-3. EphA2-knockdown in PANC-1 cells inhibited their ability to transmit exosome-mediated chemoresistance to MIA PaCa-2 and BxPC-3, while treatment of MIA PaCa-2 and BxPC-3 cells with soluble EphA2 did not promote chemoresistance, indicating that membrane carried EphA2 was important for the EphA2 chemoresistance effect. EphA2 levels on circulating exosomes were markedly increased in mice bearing PANC-1 tumors and PC patients who subsequently revealed poor responses to treatment. We thus concluded that exosomal EphA2 expression could transmit chemoresistance and may serve as a minimally-invasive predictive biomarker for PC treatment response. Further work should address whether additional exosomal factors regulate resistance to other cancer therapeutic agents for PC or other cancer types.

#586

Sera levels of the immunomodulatory protein the progesterone induced blocking factor (PIBF) are not useful in determining which patients with non-small cell lung cancer will respond to progesterone receptor modulators, e.g., mifepristone.

Jerome H. Check,1 Diane Check,2 Maya Srivastava,3 Ann DiAntonio2. 1 _Cooper Medical School of Rowan University, Camden, NJ;_ 2 _Cooper Institute for Reproductive Hormonal Disorders, Mt. Laurel, NJ;_ 3 _SUNY at Buffalo, Buffalo, NY_.

Human leukemia cancer cell line studies have demonstrated the presence of a 34 kDa intracytoplasmic splice variant of the parent 90 kDa nuclear protein associated with the centrosome that has significant immunosuppressive activity, especially, but not limited to natural killer (NK) cells. One mechanism of suppressing NK cell cytotoxicity is by stabilizing perforin granules. The 34 kDa intracytoplasmic isoform has been shown to be present only in rapidly growing cells, e.g., embryonic, mesenchymal, trophoblast, and cancer cells. During the luteal phase in ovulating women, and throughout pregnancy, there is a rise in the serum level of this immunomodulatory protein. Thus, it is called the progesterone induced blocking factor (PIBF). Interestingly, the abortafacient, mifepristone, has been shown to suppress the 34 kDa intracytoplasmic concentration of PIBF in cancer cell lines, but it does not lower serum PIBF. Based on the demonstration of significant increase in quality and length of life in controlled animal studies, and anecdotal experience in compassionate use treatment of human patients with a variety of advanced cancer types, the FDA has granted an investigator initiated salvage study of 40 patients with advanced non-small cell lung cancer (NSCLC) who have failed a minimum of two rounds of chemo or immunotherapy to be treated with single agent oral mifepristone (www.clinicaltrials.gov). The first 2 patients are doing extremely well (2 years and 1 year so far) with a high quality life without significant disease progression. Sera PIBF levels were obtained on these patients every 2 months using an experimental ELISA assay. For patient 1, a man with stage IV NSCLC with brain metastasis, his PIBF levels (ng/ml) were all in the range of normal males and women in the follicular phase (27, 38, 53, 59, 52, 46, 39, 45, 44, and 49). For patient 2, who is a female positive for the PD-L1 marker, and who progressed despite 3 rounds of chemotherapy and 1 round of nivolumab, her levels were 10, 30, 12, 21, 23, 50, 64, and 30. Thus, these results suggest that measuring serum PIBF levels in patients with very advanced NSCLC has little value in determining which patients will respond to the progesterone receptor modulator mifepristone, or be able to monitor their response to progesterone receptor modulator therapy.

#587

Small solute carrier and sirtuin genes are potential prognostic biomarkers for renal cell carcinoma.

Andre R. Jordan, Naureen Mullani, Asif Talukder, Vinata B. Lokeshwar. _Augusta University, Augusta, GA_.

Introduction: Five-year survival of metastatic renal cell carcinoma (mRCC) patients is < 10% and African American (AA) males have the highest incidence. In the kidney, Small Solute Carrier (SLC) and Sirtuin (SIRT) gene families regulate redox signaling, oxidative stress responses, transport of glycolysis and Krebs cycle metabolites, etc. We examined the prognostic potential of eleven SLC and SIRT genes to predict clinical outcome in RCC patients and evaluated the biological functions of SLC13A3 in RCC cells.

Methods: Expression of SLC (13A3, 22A6, 47A1, 5A10) and SIRT 1-7 transcript levels was evaluated in a TCGA RCC-dataset (n=604 patients). 13A3 expression was measured by quantitative RT-PCR (qPCR) in matched normal and RCC specimens from 53 patients. Biomarker levels were correlated with clinical outcome, using Mann-Whitney test. RCC cells stably-expressing SLC13A3, were examined for proliferation and cell cycle under normoxia and hypoxia.

Results: In the TCGA dataset, SIRT4, SIRT5, SLC22A6, SLC5A10, and SLC47A1 downregulation and SIRT2, SIRT6 and SIRT7 upregulation correlated with higher tumor-stage and metastasis (P< 0.001). Kaplan-Meier plots showed that these biomarkers stratified patients into low- or high-risk for survival (P<0.001). qPCR showed 40-fold downregulation of SLC13A3 expression in RCC tissues when compared to normal kidney (P< 0.0001). Downregulation was 40-fold in White/Hispanic patients, but 198-fold in AA patients (P=0.0049) and correlated with tumor stage and metastasis (P=0.009). SLC13A3 expression in RCC cells increased GSH transport by 2.5-fold and inhibited cell proliferation by 3-4-fold (P<0.001), due to cell-cycle arrest at G1-S or G2-M checkpoints. Treatment of RCC cells with DNA-demethylating agent 5-Azacytidine and Trichostatin A, induced SLC13A3 expression by 50-fold (P<0.001).

Conclusion: SLC/SIRT family members are potential prognostic biomarkers for RCC. This first study on SLC13A3 reveals that it is a potential tumor suppressor and its downregulation in RCC tissues correlates with metastasis and racial disparity.

### Clinical Risk Factors and Outcomes in Solid Tumors / Survivorship Research

#588

Neutrophil to lymphocyte ratio as strongest predictor of early death in metastatic triple negative breast cancer.

Gabriel A. De la Cruz Ku,1 Zaida Morante,2 Joseph Pinto,3 Jhajaira Araujo,3 Daniel Enriquez,4 Eduardo Eyzaguirre,1 Antonella Saavedra,1 Maria Lujan,1 Renato Luque,1 Cesar Rázuri,5 Hugo Fuentes,2 Silvia Neciosup,2 Henry Gómez Moreno2. 1 _Universidad Científica del Sur, Lima, Peru;_ 2 _Instituto Nacional de Enfermedades Neoplasicas, Lima, Peru;_ 3 _Oncosalud, Lima, Peru;_ 4 _Instituto Nacional de Enfermedades Neoplásicas, Lima, Peru;_ 5 _Hospital Nacional Dos de Mayo, Lima, Peru_.

Background: Metastatic triple-negative breast cancer (mTNBC) is a heterogeneous disease with poor prognosis where recent studies suggest that the immune system plays a critical role the mechanisms of disease progression and metastasis. In the other hand, a high Neutrophil to Lymphocyte Ratio (NLR) is associated with poor survival in patients diagnosed with several types of cancer. Our aim was to evaluate the prognostic value of NLR in mTNBC.

Methods: We retrospectively reviewed 130 patients diagnosed with mTNBC between 2000-2014 at "Instituto Nacional de Enfermedades Neoplasicas". NLR group was divided in <2.5 and ≥2.5. Sites of metastasis were identified by TAC or magnetic resonance imaging. Survival rates were estimated by the Kaplan-Meier method and differences were calculated by Log-rank test. The Cox Regression was used for prognostic factors.

Results: The median age was 50 years (range: 23-92 years). From our study population, 41.2% were premenopausal, and just 10 patients (9.5%) had a breast-ovary cancer familiar background, 62.5% were diagnosed at clinical stage T3-T4 and 86.1% had lymph node involvement. From 130 patients, 41 and 64 had NLR <2.5 and ≥2.5, respectively, while there was no association between characteristics and NLR, only patients with malnutrition had NLR ≥2.5 (9.1% vs 40.8%, p=0.002) and solid organ involvement (62.4% vs 81.3%, p=0.04). Furthermore, lung and bone were the most frequent sites of metastasis. Those patients with lower ratio (<2.5) had better response to chemotherapy (p <0.001). With a median follow-up of 6.25 years, at 1-year OS was lower among patients with NLR ≥2.5 (59% vs 20%, HR: 2.77, 95%CI:1.77-4.33, p<0.001). In addition, at univariate analysis malnutrition also showed an impact on OS (HR: 1.79, 95%CI: 1.20-2.67, p=0.005). At multivariate analysis, between age, T stage, N stage, NLR and solid organ involvement, just NLR was the best and only predictor of worse OS (HR: 3.12, 95%CI:1.94-5.03, p<0.001).

Conclusion: In mTNBC, an NLR ≥2.5 was a strong and accessible biomarker of poor OS. Lower values of NLR could help us to predict chemotherapy response.

#589

A systematic review of randomized clinical trials for therapeutic outcomes of anti-EGFR agents used in combination with other targeted therapies.

Sonali Kurup,1 Pavlina Liskova,1 Paiboon Jungsuwadee,2 Prashant Sakharkar,1 Soniya Tambe1. 1 _Roosevelt University, Schaumburg, IL;_ 2 _Fairleigh Dickinson University, Florham Park, NJ_.

Introduction: The epidermal growth factor receptor (EGFR) plays a prominent role in cancer cell proliferation and metastasis in non-small-cell lung cancer (NSCLC). Targeted cancer therapies include agents that block tumor-specific molecular targets and thereby interfere with tumor growth, progression and metastasis. This study focuses on a systematic review of outcomes from phase II and III clinical trials for standard anti-EGFR therapy plus a targeted therapy to EGFR monotherapy.

Methods: Studies were identified through a thorough literature search using, EMBASE, Pubmed, Scifinder Scholar, clinicaltrials.gov and the Cochrane library using the following terms: "epidermal growth factor receptor kinase", "combination therapy", "non small cell lung cancer". Eligible studies included patients of any stage and mutation status. Data abstraction included patient demographic characteristics; sample size, patient inclusion and exclusion criteria, overall response rate (ORR), median progression free survival (PFS), median overall survival (OS), and adverse effects (AE).

Results: Literature search resulted in the identification of 1543 studies of which 153 were found to be relevant for this review and 31 fit the inclusion criteria. Of the 31 studies, 28 included erlotinib and 3 studies included gefitinib as the control. Twenty-five of the studies focused on kinase inhibitors targeting VEGFR, EGFR, MET, IGF-1R, AKT, mTOR and HER3 as the combination agent. The remaining six studies focused on alternate pathways of targeted inhibition including hormonal agents and histone deacetylase inhibitors. The identified studies included patients (N=7552) of mean age (63 ± 2), female (22-80%), smokers in combination therapy (12-96%) versus monotherapy (8-95%). Median OS (months) for patients on the EGFR-based combination regimen (range 5.7-24.7) vs monotherapy (range 4.5-21.8). Median PFS (months) for patients on the EGFR-based combination regimen (range 1.3-16.0) vs monotherapy (1.5-9.7). A greater incidence of AE was observed for EGFR-based combination therapy compared to EGFR monotherapy.

Conclusions: Several different targeted therapies have been evaluated as part of clinical trials for NSCLC in combination with EGFR-based therapies, and compared to EGFR-based monotherapy. For patients with NSCLC, a higher range for median OS and median PFS was observed for EGFR-based combination regimens compared to EGFR monotherapy.

#590

Marital status and survival among breast cancer patients.

Yan Lu, Bradford E. Jackson, Bassam Ghabach, Rohit P. Ojha. _JPS Health Network, Fort Worth, TX_.

Background: Recent evidence suggests improved survival for married breast cancer patients compared with unmarried patients, but unclear is whether this survival benefit extends to socioeconomically disadvantaged women. We aimed to assess the association between marital status and breast cancer survival among socioeconomically disadvantaged breast cancer patients and breast cancer patients from the general population.

Methods: We used data from the John Peter Smith (JPS) Oncology Registry, which comprises cancer patients in Tarrant County, TX treated at a safety-net institution, and the Texas Cancer Registry (TCR), a statewide population-based registry. Our eligible population included females aged 18 – 64 years diagnosed with primary breast cancer between January 2008 and June 2013 with follow-up through 2016. We categorized marital status into three groups: single, married, or separated/divorced/widowed. We estimated the 36-month restricted mean survival time, which allows survival comparisons within a specified period.

Results: Our study populations comprised 614 JPS and 32,985 TCR patients. The age distribution at diagnosis was similar for both groups (JPS: median=51 years; TCR: median=52 years). JPS patients were more often non-White (JPS=62%; TCR=42%) and uninsured (JPS=56%; TCR=9.6%). A higher frequency of TCR patients were married (TCR=65%; JPS=39%). Survival was shorter for JPS patients (JPS=33.1 months; TCR =34.8 months) after 36-months (Table 1). Married women had longer survival than unmarried women in both populations.

Conclusion: Our results suggest similar magnitudes of improved survival for married women with breast cancer compared with unmarried women in both populations, but socioeconomically disadvantaged patients have consistently lower survival estimates across marital subgroups compared with patients from the general population. Our findings warrant further studies that explore mechanisms of how marital status influences breast cancer survival in different populations.

Table 1. Restricted mean survival time (RMST) after breast cancer diagnosis based on marital status

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|

Disadvantaged Cancer Patientsa | Cancer Patients from General Populationb

|

RMSTc

(months) | Difference in RMST (95% CLd)

(months) | RMST

(months) | Difference in RMST (95% CL)

(months)

Separated/Divorced/Widowed | 32.2 | -0.89 (-2.8, 1.0) | 34.1 | 0.01 (-0.23, 0.25)

Married | 33.8 | 0.56 (-0.96, 2.1) | 35.1 | 0.54 (0.36, 0.71)

Single | 33.0 | 0 (Reference) | 34.1 | 0 (Reference)

aCancer patients from JPS Oncology Registry

bCancer patients in the Texas Cancer Registry

cBased on 36-month restricted mean survival

dCL: Confidence limits

#591

Clinical profile and outcome associated with breast cancer recurrence after pathologic complete response to neoadjuvant chemotherapy: A multicenter study.

Mariko Asaoka,1 Takashi Ishikawa,2 Eiichi Sato,2 Kazutaka Narui,3 Takashi Chishima,4 Akimitsu Yamada,5 Nobuyasu Suganuma,6 Tsutomu Kawaguchi,1 Kazuaki Takabe1. 1 _Roswell Park Cancer Institute, Buffalo, NY;_ 2 _Tokyo Medical University Hospital, Tokyo, Japan;_ 3 _Yokohama City University Hospital, Yokohama, Japan;_ 4 _Yokohama Rosai Hospital, Yokohama, Japan;_ 5 _Chigasaki Municipal Hospital, Kanagawa, Japan;_ 6 _Kanagawa Cancer Center, Yokohama, Japan_.

Background: Neoadjuvant chemotherapy (NAC) is a widely accepted treatment not only for locally advanced breast cancer, but also for earlier-stage operable disease. The main purpose of it is to downstage the tumor and to avoid the extensive operation and increase breast conservation surgery. Recent studies have indicated that patients who achieve a pathologic complete response (pCR) after NAC show better outcomes than patients with residual disease in breast cancer (non-pCR). On the other hand, there are some cases in which cancer recurred despite achieving pCR (pCR-rec). This retrospective study investigates the clinical profiles and outcome associated with the patients whose tumor recurred after achieving pCR after NAC.

Patients and Methods: All the clinical and pathologic data were collected from 4 institutions in Japan: Yokohama City University Hospital, Kanagawa Cancer Center, Yokohama Rosai Hospital and Tokyo Medical University Hospital. Total of 1,599 operable breast cancer patients who underwent NAC were enrolled. The median follow-up period was 41 months. Vast majority of the patients were treated with anthracycline followed by taxane-based chemotherapy, with or without trastuzumab as neoadjuvant therapy. We defined pCR as no residual invasive cancer in the breast and axially nodes, and non-pCR was divided into 2 groups as Grade 1 (no to mild response) and Grade 2 (moderate response).

Results: Of 1,599 patients enrolled in the study, 363 patients achieved pCR (22.7%). Among the pCR patients, 41 were Luminal type, 93 were Luminal-HER2 type, 109 were HER2 type, and 120 were triple-negative (TN) type (pCR rate was 5.94%, 30.4%, 49.3% and 31.3%, respectively). Cancer recurrence after resection was observed in 18 patients (pCR-rec, 4.9%). Axial nodal metastasis diagnosed before NAC associated to cancer recurrence among the pCR patients (p= 0.0003). pCR patients showed significantly better outcome compared to non-pCR, consistent with the previous reports among all patients. However, pCR-rec showed significantly worse DFS compared to those with Grade 1, Grade 2, or pCR (P<0.005 ; 2-year disease-free rate was 33.3%, 79.1%, 88.9% and 98.6%, respectively), as well as HER2 and TN cohort showed consistent results (P<0.005; 2-year disease-free rate was 14.5%, 63.1%, 83.7%,and 95.0%, and P<0.005; 2-year disease-free rate was 17.9%, 55.7%, 81.7% and [no data]).

Conclusions: Although achieving pCR after NAC improved outcome, patients with cancer recurrence after pCR show significantly unfavorable outcome even compared to non-pCR patients. In further study, identifying these patients before treatment could improve their poor survival.

#592

The potential effect of smoking cessation on mortality among patients diagnosed with smoking-related cancers.

Bradford E. Jackson,1 Tracey E. Barnett,2 Yan Lu,1 Bassam Ghabach,1 Rohit P. Ojha1. 1 _JPS Health Network, Fort Worth, TX;_ 2 _UNT Health Science Center, Fort Worth, TX_.

Background: The benefit of smoking cessation on mortality after cancer diagnosis is unclear, particularly given ethical restrictions of randomizing patients who are smokers to a smoking cessation intervention or continued smoking. We thus aimed to assess the effect of a hypothetical smoking cessation intervention on mortality among patients diagnosed with smoking-related cancers using observational data.

Methods: We used data from the JPS Oncology Registry, for which the source population is cancer patients in Tarrant County, TX who received care at a public health care system regardless of their ability to pay. Our eligible population included patients diagnosed with primary lung, head and neck, or colorectal cancer between January 2008 and June 2013 with follow-up through 2016, and self-reported current or former use of cigarette, cigar, or pipe tobacco products. We used a potential outcomes framework to estimate cancer-specific effects of a hypothetical smoking cessation intervention on 1- and 3-year all-cause mortality. Under certain assumptions, the estimated marginal risk ratios and risk differences from this approach are analogous to intention to treat estimates from a randomized controlled trial.

Results: Our study population comprised 1,058 patients diagnosed with lung (n=582), head and neck (n=248), or colorectal cancer (n=228). The median age at diagnosis overall was 57 years (interquartile range=51-63). Patients were predominantly male (64%), non-Hispanic White (63%), and uninsured (50%). Modeled smoking cessation decreased mortality for head and neck cancers but not lung or colorectal cancers (Table 1).

Conclusion: Albeit with certain limitations, our findings suggest that a hypothetical smoking cessation intervention initiated at the time of cancer diagnosis may not have homogeneous effects on mortality across cancer types. These results provide insight about target populations for whom smoking cessation may benefit cancer survival.

Table 1. Risk ratios and differences for the effect of hypothetical smoking cessation intervention

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|

1-year mortality | 3-year mortality

Site | RRa (95% CL)b | RDc (95% CL) | RR (95% CL) | RD (95% CL)

Lung | 0.98 (0.86, 1.1) | -0.01 (-0.10, 0.08) | 1.0 (0.95, 1.1) | 0.02 (-0.05, 0.08)

Head and Neck | 0.71 (0.45, 1.1) | -0.11 (-0.25, 0.03) | 0.78 (0.58, 1.0) | -0.14 (-0.29, 0.01)

Colorectal | 0.87 (0.48, 1.6) | -0.03 (-0.16, 0.10) | 1.1 (0.73, 1.5) | 0.02 (-0.14, 0.19)

aRisk Ratio < 1 indicate lower risk of mortality if all patients adhered to a hypothetical smoking cessation intervention.

bConfidence Limits

cRisk difference < 0 indicate a lower risk of mortality if all patients adhered to a hypothetical smoking cessation intervention.

#594

Strategies to improve repeat CRC screening in rural community clinics using the fecal immunochemical test (FIT).

Connie L. Arnold,1 Terry C. Davis,1 James Morris,2 Alfred Rademaker,3 Glenn Mills1. 1 _LSU Feist-Weiller Cancer Ctr., Shreveport, LA;_ 2 _LSUHSC - Shreveport, Shreveport, LA;_ 3 _Feinberg School of Medicine, Northwestern University, Chicago, IL_.

Introduction: Colorectal cancer (CRC), the second leading cause of cancer death in the United States, can be significantly reduced if it is detected early. Although overall CRC screening rates have increased significantly, disparities persist among low income individuals, adults with low literacy and those living in rural areas.

Objectives: To assess the effectiveness of two health literacy informed telephone follow-up strategies to improve annual repeat screening with Fecal Immunochemical Test (FIT) in rural community clinics.

Methods: A two-arm, randomized controlled trial is being implemented in four community clinics. Clinics reported CRC baseline screening rates of 1% to 3%. Eligible patients, age 50-75, were recruited at the clinic prior to a scheduled appointment. A research assistant (RA) conducted a baseline structured interview. The RA then recommended screening and gave brief literacy and culturally appropriate education using a pamphlet (4th grade level), the FIT kit with pre-addressed envelope, simplified instructions (3rd grade level) and a demonstration of how to use it, At four weeks patients who had not returned their kit receive either 1) a personal follow-up call (PC) from a central prevention coordinator using motivational interviewing skills and reminding them to complete and mail FIT kits; or 2) an automated follow-up call (AC) using plain language and motivational messages encourages patients to complete and mail the FIT. During years 2 and 3, FIT kits were mailed to patients along with previous literacy and culturally appropriate education. Follow-up call procedures previously used at weeks 4 and 8 were followed.

Results: 620 patients not up-to-date with screening were enrolled; 66% were African American, 55% women; 40% had limited literacy.

Screening results:

1st Year Screening: 620 patients enrolled - (6 withdrew before completing test)

AC Arm (n=308)

213 (69%) completed kits [23 (7%) positive]

124 (40%) people needed at least one follow-up call [29 (24%) returned FIT after call]

PC Arm (n=306)

205 (67%) completed kits [21 (7%) positive]

127 (41%) people needed at least one follow-up call [26 (20%) returned FIT after call]

2nd Year Screening: 568 Second kits mailed (4 withdrew and no previous positives included)

AC Arm (n=285)

111 (40%) completed kits [9 (8%) positive]

212 (74%) people needed at least one follow-up call [40 (19%) returned FIT after call]PC Arm (n=283)

104 (37%) completed kits [8 (8%) positive]

209 (74%) people needed at least one follow-up call [31 (15%) returned FIT after call]

Discussion: Simplified instructions accompanied by a face-to-face demonstration of FIT, use of "teach back" to confirm understanding with a follow-up call if needed, facilitated completion rates of all patients, particularly those with limited literacy. The less costly and time consuming automated call was equally effective as a personal call.

#595

Perioperative risk factors for postoperative pneumonia after major oral cancer surgery: A retrospective analysis of 331 cases.

Jieyun Xu, Pei Yu, Weiwang Wang, Zehang Zhuang, Xiqiang Liu. _Sun Yat-sen Univ., Guangzhou, China_.

Objective: Postoperative pneumonia (POP) is common and results in prolonged hospital stays, higher costs, increased morbidity and mortality. However, data on the incidence and risk factors of POP after oral and maxillofacial surgery are rare. This study aims to identify perioperative risk factors for POP after major oral cancer (OC) surgery.

Methods: Perioperative data and patient records of 331 consecutive subjects were analyzed in the period of April 2014 to March 2016. We individually traced each OC patient for a period to discharge from the hospital or 45 days after surgery, whichever occur later.

Results: The incidence of POP after major OC surgery with free flap construction or major OC surgery was 11.6% or 4.5%, respectively. Patient-related risk factors for POP were male sex, T stage, N stage, clinical stage and preoperative serum albumin level. Among the investigated procedure-related variables, incision grade, mandibulectomy, free flap reconstruction, tracheotomy, intraoperative blood loss, and the length of the operation were shown to be associated with the development of POP. Postoperative hospital stay was also significantly related to increased incidence of POP. Using a multivariable logistic regression model, we identified male sex, preoperative serum albumin level, operation time and postoperative hospital stay as independent risk factors for POP.

Conclusion: Several perioperative risk factors can be identified that are associated with POP. At-risk oral cancer patients should be subjected to intensified postoperative pulmonary care.

#596

Impact of histologic type on survival in primary thyroid lymphomas.

Taha Al-Juhaishi, Victor Yazbeck. _VCU Health, Richmond, VA_.

Background:

Primary Thyroid Lymphoma (PTL) is a rare disease constituting less than 2% of all extranodal lymphomas; and approximately 2-8% of all thyroid malignancies. In this study, we sought to describe the impact of histologic types on survival.

Methods:

The Surveillance, Epidemiology and End Results (SEER) database was used to identify patients (≥18 years) diagnosed with PTL between 1973 and 2014. B-cell and T-cell lymphomas of all stages were included. Overall survival (OS) was estimated using the Kaplan-Meier method, and compared using Log-Rank test. Cox proportional hazards models were used for adjusted survival analyses.

Results:

Total of 2397 patients were included. Median age was 65 years (56-77); majority were female (n=1734, 72%) and caucasian (n=2178, 91%). Diffuse large B cell lymphoma was the most common histology (DLBCL, N=1351, 56%), followed by marginal zone (MZL, N=334, 14%), follicular (FL, N= 214, 8.9%), Burkitt (BL, N=43, 1.8%), chronic/small lymphocytic leukemia/lymphoma (CLL/SLL, N=41, 1.7%), Hodgkin lymphoma (HL, N=33, 1.4%), T cell lymphomas (TCL, N=14, 0.6%), Lymphoplasmocytic lymphoma (LPL, N=11, 0.4%), and Mantle cell (MCL, N=8, 0.3%). 349 patients (14.6 %) were reported with unclear histology. Total of 1502 (63%) had surgery, 1417 (59%) had chemotherapy and 1153 (48%) had radiation. At a median follow-up of 10.5 years, 1187 (50%) patients died. Median overall survival was 11.4 [10.6-12.3; 95% CI] years. Survival was different by histology type (table, P<0.001). Compared with DLBCL, follicular lymphoma (HR 0.72 [0.57-0.92], P=0.008) and MZL (HR 0.62 [0.48-0.78], P<0.001) were associated with lower mortality, independently of age, sex, race, surgery, chemotherapy, radiation and year of diagnosis. Adjusted mortality decreased by a relative 1% annually (HR 0.99 [0.98-0.99], P<0.001).

Conclusion:

PTL is a rare disease mostly affecting elderly caucasian women. Outcomes vary depending on histologic type. Follicular, MZL and Hodgkin's lymphoma seem to have a favorable prognosis.

Overall Survival by Histology

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Histology | Number (%) | Mean Survival in Years (95% CI) | 1-year survival | 5-year survival

DLBCL | 1351 (66%) | 12.6 [11.7-13.4] | 80% | 66%

Burkitt | 43 (2.1%) | 15.9 [13.5-18.3] | 79% | 79%

CLL/SLL | 41 (2%) | 13 [9.5-16.5] | 78% | 67%

Follicular | 214 (10.4%) | 17.8 [15.1-20.6] | 97% | 83%

Hodgkin | 33 (1.6%) | 25 [20-30] | 94% | 84%

LPL | 10 (0.5%) | 14.2 [7.4-21] | 100% | 76%

MZL | 334 (16.3%) | 13 [11.9-14.1] | 95% | 83%

Mantel-Cell | 8 (0.4%) | 8.7 [3.9-13.5] | 87% | 56%

T-cell | 14 (0.7%) | 10.7 [3.9-17.5] | 70% | 53%

#597

Lung tumor histology as prognostic factor for short-term postoperative outcomes.

Roger Gerard,1 Frank Velez-Cubian,1 Carla Moodie,2 Joseph Garrett,2 Jacques Fontaine,2 Eric Toloza2. 1 _University of South Florida Health Morsani College of Medicine, Tampa, FL;_ 2 _Moffitt Cancer Center, Tampa, FL_.

Introduction: Based on the 2015 World Health Organization (WHO) Classification of Lung Tumors, we sought to investigate the relationship of common lung tumor histologies and their utility as prognostic clinical correlates of short-term perioperative outcomes.

Methods: We retrospectively analyzed patients who underwent robotic-assisted video-thoracoscopic lobectomy by one surgeon over 81 months. Patients were grouped based on tumor histology on their final surgical pathology report. Patients' demographics, smoking history, lobar distribution, extent of resection, intraoperative outcomes, perioperative complications, and hospital length of stay were compared among the groups.

Results: Among 420 study patients, the most common tumor type was adenocarcinoma (AD, 59.8%), followed by squamous cell carcinoma (SQ, 19.3%) and neuroendocrine carcinoma (NEC, 11.7%). Final histology for the remainder of the cohort consisted of adenosquamous carcinoma (AdSq, 2.1%) and pulmonary metastasis (7.1%). All patients with AdSq tumor histology had a positive smoking history, which was a significantly higher proportion than for those with NEC tumor type (100% vs 71.4%; p<0.001). Those with AdSq tumor histology were both significantly older and developed significantly larger tumors by time of resection (p<0.001 and p=0.003, respectively). Patients with AdSq tumors required more extensive resections compared to the remaining groups (p<0.004). They also experienced significantly greater estimated blood loss (EBL) and significantly longer operative times (p=0.005 and p=0.018, respectively) than the remaining groups. Patients with SQ or pulmonary metastasis had slightly more postoperative complications, but this was not statistically significant (p=0.990).

Conclusion: Patients with AdSq histology type were older and had larger tumors, which put them at risk of intraoperative difficulties, with more extensive resections, longer operative times, and greater EBL. Despite this, AdSq histology did not affect the postoperative course.

#598

Utility of cell therapy to control relapsed acute myeloid leukemia following allogeneic stem cell transplantation: Does cell source matter.

Woochan Park,1 Inho Kim,1 Sung-soo Yoon,1 Seonyang Park,2 Youngil Koh1. 1 _Seoul National University Hospital, Seoul, Korea, Republic of Korea;_ 2 _Inje University Haeundae Paik Hospital, Seoul, Korea, Republic of Korea_.

Introduction: Although allogenic hematopoietic stem cell transplantation (Allo-SCT) is curative therapy for acute myeloid leukemia (AML), many patients experience relapse following allo-SCT. For these patients, it is well known that donor lymphocyte infusion (DLI) could rescue a certain proportion via graft versus leukemia effect. On the other hand, with the advance of abundant stem cell harvesting techniques, residual stem cell after allo-SCT is also frequently used as a rescue therapy for AML relapse. However, clinical utility of residual stem cell is not well established yet. Hence, in this study, we compare the outcome of DLI with residual stem cell infusion in relapsed AML after allo-SCT.

Materials and Methods: We retrospectively reviewed the AML patients who underwent DLI or residual stem cell infusion for relapsed disease after allo-SCT from 2001 to 2017 in Seoul National University Hospital. We analyzed factors including overall survival (OS), cell counts, disease status at cell therapy, and GVHD development. Our primary outcome was to compare OS after cell therapy between DLI group and residual stem cell group.

Results: A total of 81 patients were analyzed. There were 50 patients (25 males and 25 females) who received cell therapy using DLI, and 31 patients (11 males and 20 females) who received residual stem cell infusion. There was no difference in age and duration from allo-SCT to cell therapy between the two groups. Majority of patients received cell therapy for first relapse after allo-SCT (63% in DLI, 93% in residual stem cell group). Median CD3 cell count infused was not different between DLI and residual stem cell product (median value of 0.967 X 108/kg and 0.964 X 108/kg). Median CD34 cell dose was 2.34 X 106/kg in residual stem cell group.

There was no difference in OS between the two groups (median OS 8.5 months for DLI and 10.2 months for residual stem cell group, p-value 0.673). GVHD occurred in 44% in DLI group and 41.9% in residual stem cell group. When multivariate analysis was performed, disease status at stem cell therapy or duration from allo-SCT to relapse did not show any significant difference. Characteristics of long-term survivors after cell therapy following relapse after allo-SCT could be summarized as whether to acquire bone marrow CR after therapy.

Conclusion: This study demonstrates that clinical utility of DLI and residual stem cell infusion is similar in relapsed AML after allo-SCT. Hence, residual stem cell could be used as an alternative source for T-cell therapy following allo-SCT.

#599

Secondary malignancies in temozolomide-treated metastatic pancreatic neuroendocrine tumors.

Nicole Balmaceda,1 Sunil Abhyankar,2 Joaquina Baranda1. 1 _University of Kansas Cancer Center, Kansas City, KS;_ 2 _University of Kansas Medical Center, Kansas City, KS_.

Purpose: To determine the incidence of secondary malignancies in patients treated with temozolomide (TMZ) for metastatic pancreatic neuroendocrine tumors (PNET).

Background: TMZ is an oral alkylating agent used to treat glioblastoma multiforme (GBM), refractory anaplastic astrocytoma (AA), and metastatic PNET. This imidazotetrazine analog of dacarbazine lacks the ability to directly crosslink DNA and is thought to be less leukemogenic than other alkylators. Given either alone, or in combination with other therapies, TMZ is associated with improved clinical outcomes. However, serious hematologic adverse events (HAEs) like agranulocytosis, lymphopenia and aplastic anemia are not uncommon. Until recently, metastatic PNET was primarily managed with somatostatin-analogs, but with more reports demonstrating therapeutic activity of TMZ-based regimens, it is anticipated that more patients with metastatic PNET will be exposed to TMZ.

Methods: To determine the incidence of secondary malignancy in TMZ-treated PNET, a systematic review of all known clinical trials, case reports, and other relevant literature regarding PNET and TMZ published before September 2017 was conducted using PubMed, Embase, Cochrane Library, and the FDA database.

Results: Twenty-one publications, including clinical trials, meta-analyses, case reports, and cohort studies were analyzed. HAEs ranged from agranulocytosis to myelodysplastic syndrome. No publications reported any secondary malignancies. Incidentally, at the University of Kansas Medical Center, 3 patients with TMZ-treated PNET developed hematologic malignancies. A 29-year-old female with metastatic PNET was treated with TMZ and subsequently developed acute myeloid leukemia (AML) with cytogenetics consistent with therapy-related leukemia. The second patient with TMZ-treated metastatic PNET developed diffuse large B-cell lymphoma. These two patients both had aggressive disease that was not responsive to multiple rounds of treatment. They succumbed to their hematologic malignancy, and not from metastatic PNET. The third patient is a 29-year-old who was recently diagnosed with high-grade T-cell lymphoblastic lymphoma and is currently undergoing treatment for his lymphoma.

Conclusion: This review did not find any cases of secondary malignancy in TMZ-treated metastatic PNET. Yet, at our own institution we have identified 3 cases of secondary hematologic malignancies in patients treated with TMZ for PNET. We believe that the leukemogenic potential of TMZ is underreported and anticipate increased reports of secondary malignancy as the use of TMZ increases. It is important for treatment guidelines to address this risk in the decision to pursue TMZ treatment. Appropriate dosing, proper follow-up and surveillance, especially in patients who are able to live long enough to develop these hematologic cancers, is crucial.

#600

Telomere length-associated genetic variants and the risk of thyroid cancer after childhood cancer: A report from the Childhood Cancer Survivor Study (CCSS).

Philip J. Lupo,1 Lindsay M. Morton,2 Yutaka Yasui,3 Wendy M. Leisenring,4 Gregory T. Armstrong,3 Leslie L. Robison,3 Smita Bhatia,5 Maria M. Gramatges1. 1 _Baylor College of Medicine, Houston, TX;_ 2 _National Cancer Institute, Bethesda, MD;_ 3 _St. Jude Children's Research Hospital, Memphis, TX;_ 4 _Fred Hutchinson Cancer Research Center, Seattle, WA;_ 5 _UAB School of Medicine, Birmingham, AL_.

Purpose: We previously reported an association between reduced telomere content and increased risk for radiation-related thyroid subsequent neoplasms (SNs) in childhood cancer survivors. We now extend this work to investigate associations between single nucleotide polymorphisms (SNPs) in the National Human Genome Research Institute-European Bioinformatics Institute (NHGRI-EBI) GWAS Catalog identified to influence telomere length and thyroid SN in a cohort of childhood cancer survivors.

Methods: The population included 5,324 5-year survivors of childhood cancer survivors participating in the CCSS with genome-wide SNP data generated using the Illumina HumanOmni5Exome array, who (1) developed thyroid SN (n=117) or (2) did not develop thyroid SN (n=5,207), censored by date of any SN, death, or last follow-up. Ancestry was estimated using HapMap data as the fixed reference population. Imputation was performed based on the 1000 Genomes Project reference haplotypes. For the analysis, we focused on SNPs in the NHGRI-EBI GWAS Catalog previously identified to influence telomere length (statistical significance threshold P<1.0x10-5). Assuming an additive genetic model, we used multivariable Cox proportional hazards models to estimate the hazard ratio (HR), 95% confidence interval (CI), and P value for the association between each SNP and thyroid SN risk. Covariates evaluated included sex, primary cancer diagnosis, radiation exposure, and exposure to alkylating agents. Analyses were restricted to those of European ancestry to limit bias due to population stratification. Results were also stratified on radiation exposure and primary diagnosis, and the false discovery rate (FDR) was used to account for multiple comparisons.

Results: We identified 30 SNPs in the NHGRI-EBI GWAS Catalog associated with telomere length. Based on the sample size and the number of SNPs evaluated, we had 80% power to identify HRs between 1.7 and 2.0, based on minor allele frequencies (MAFs) of 10-40%. In multivariable analyses, when evaluating associations among those exposed to radiation to the neck, the top hit was rs621559 (HR=1.94, 95% CI: 1.16-3.24, P=0.01), an intronic SNP in WDR65. However, after applying the FDR at <0.05, no SNPs were associated with thyroid SN risk overall, nor after stratifying on radiation exposure or primary diagnosis.

Conclusions: There is an inverse relationship between telomere content and thyroid SN among childhood cancer survivors. Our study was unable to identify an association between common SNPs influencing telomere length and thyroid SN. The observed relationship between reduced telomere content and thyroid SN could possibly be due to alternative molecular mechanisms such as rare or novel variants, or defects in specific genes related to telomere maintenance.

#601

Cognitive sequelae of adjuvant endocrine therapy in older women treated for early-stage breast cancer.

Emily A. Underwood,1 Kathleen I. Pritchard,1 Katarzyna Jerzak,1 Gerald Lebovic,2 Christine Elser,3 Paula Rochon,4 Mary C. Tierney1. 1 _Sunnybrook Health Sciences Centre, Toronto, Ontario, Canada;_ 2 _University of Toronto, Toronto, Ontario, Canada;_ 3 _Princess Margaret Cancer Centre, Toronto, Ontario, Canada;_ 4 _Women's College Research Institute, Toronto, Ontario, Canada_.

Background: Approximately 75% of women with BC have hormone receptor-positive (HR+) disease and are treated with anti-estrogen endocrine therapy (ET) in the adjuvant setting. Consistent with estrogens' well-established roles in neuroprotection and cognitive functioning, accumulating evidence suggests ET has adverse effects on cognition. However, there is a paucity of data regarding the cognitive effects of ET in older women, despite the fact that they are at greater risk of BC, HR+ disease, and cognitive impairment than their younger counterparts. Purpose: We conducted a prospective study to examine the cognitive effects of ET in older BC patients with a specific focus on verbal memory, the cognitive domain known to be most sensitive to the effects of estrogen deprivation. Methods: Forty-two chemotherapy-naïve non-demented HR+ BC patients aged 60+ underwent neuropsychological assessment before starting ET, and again after one year of treatment. This within-subjects design allowed us to control for pre-treatment effects of BC on cognition. The neuropsychological battery consisted of standardized measures assessing cognitive functioning across six domains: verbal memory, visual memory, processing speed, executive function, language function, and perceptual reasoning. Raw neuropsychological test scores were converted to age-adjusted z-scores to control for the effects of age on cognition. Change in mean-level performance from pre-treatment to post-treatment within each domain was examined using paired t-tests. Results: Participants exhibited significant decline from baseline pre-treatment performance in verbal memory over the year (t = 2.60, p = 0.01). This corresponds to a small to moderate effect size (d = 0.40). Performance on other domains did not change significantly over the year (all p > 0.05). Conclusions & Implications: Our findings suggest specific adverse effects of ET on verbal memory, which is consistent with previous evidence that performance on verbal memory measures is highly sensitive to the effects of estrogen deprivation. This finding is of considerable clinical relevance given that multiple large-scale studies have found performance within this domain to be an early predictor of cognitive impairment and dementia. Moreover, that significant verbal memory decline occurred after just one year of treatment is particularly concerning given that patients are currently treated with these agents for at least five years, with extended treatment durations of up to 10 years becoming increasingly common. In light of the frequency with which ET is prescribed to older women, it is critical that the potential longer-term effects of ET on cognitive functioning are examined in future larger-scale studies. Our findings should be interpreted with caution because we did not include untreated HR+ BC controls, which are not feasible to recruit given current prescribing guidelines.

#602

The α7 nicotinic acetylcholine receptor silent agonist R-47 prevents and reverses paclitaxel-induced peripheral neuropathy without enhancing the proliferation of lung cancer cells or interfering with paclitaxel-induced antitumor activity.

S Lauren Kyte,1 Wisam Toma,1 Ganeshsingh Thakur,2 M Imad Damaj,1 David A. Gewirtz1. 1 _Virginia Commonwealth University, Richmond, VA;_ 2 _Northeastern University, Boston, MA_.

While cancer chemotherapy continues to significantly contribute to the number of cancer survivors, exposure to these drugs can often result in chemotherapy-induced peripheral neuropathy (CIPN), a consequence of peripheral nerve fiber dysfunction or degeneration. CIPN is characterized by sensory symptoms such as numbness, burning, and allodynia, resulting in an overall decrease in quality of life. Paclitaxel (Taxol), a microtubule poison that is commonly used to treat breast, lung, and ovarian cancers, has been found to cause CIPN in 59-78% of cancer patients. There is currently no effective preventative or therapeutic treatment for this side effect, which can be a dose-limiting factor for chemotherapy or delay treatment. Our laboratories have previously shown that nicotine, the prototypical nicotinic acetylcholine receptor (nAChR) agonist, can prevent and reverse paclitaxel-induced peripheral neuropathy without increasing lung tumor growth in mice. Our current studies have demonstrated that the α7 nAChR silent agonist R-47 may be a promising preventative and therapeutic treatment for CIPN as well. R-47 (10 mg/kg, p.o.) was administered to male C57BL/6J mice treated with paclitaxel (8 mg/kg, i.p., every other day for a total of four injections). Von Frey filament testing revealed that R-47 can both prevent and reverse paclitaxel-induced mechanical allodynia; the latter effect can be inhibited by the α7 nAChR antagonist methyllycaconitine. In support of these findings, R-47 also prevents the reduction of intra-epidermal nerve fibers in the hind paws of paclitaxel-treated mice. In addition, α7 nAChR knockout mice exhibit a greater and sustained increase in mechanical allodynia after paclitaxel administration when compared to wild-type mice, suggesting the involvement of this receptor subtype in hindering CIPN development and/or maintenance. With regard to cancer, R-47 fails to increase A549 and H460 non-small cell lung cancer cell viability, colony formation, or proliferation alone, and does not interfere with paclitaxel-induced growth arrest, apoptosis, or DNA fragmentation. Most importantly, R-47 does not increase the growth of A549 tumors or interfere with the antitumor activity of paclitaxel in tumor-bearing mice. These data suggest that the use of an α7 nAChR silent agonist may be a safe and efficacious approach for the prevention and treatment of CIPN.

#603

Immune response to the influenza vaccination and heart rate variability in breast cancer survivors.

Annina Seiler, Angie S. LeRoy, Christopher P. Fagundes. _Rice Univ., Houston, TX_.

Background: Cancer treatment dysregulates immune function. These effects can persist long after the cessation of therapy and may diminish the immune system's ability to effectively mount an adequate immune response. Latent herpesvirus reactivation may mark a poor immune response to the influenza vaccination. Cancer treatment can also cause adverse effects on cardiac autonomic function as assessed by heart-rate variability (HRV), which is associated with higher morbidity and decreased survival in patients with cancer. Objective: To determine immune response to seasonal influenza vaccination as measured by human cytomegalovirus (CMV) IgG antibody titers and HRV in breast cancer survivors treated with radiation relative to healthy aged-matched controls. Methods: Serum samples for 13 breast cancer survivors within the first year of post-treatment and 13 healthy age-matched controls (mean age 44.7 years) were assayed for CMV IgG antibody titers and heart-rate variability was assessed using MindWare technologies. Group differences between cancer survivors versus healthy individuals were estimated using analysis of covariance (ANCOVA), with adjustment for age, body mass index (BMI) and race. Results: One-way ANCOVA modeling showed that breast cancer survivors treated with radiation had significantly higher antibody titers to CMV relative to age-matched healthy controls (F = 9.15 ; p <.01). While heart rate did not differ among the two groups (F = .36 ; p = .56), high frequency values were significantly lower in breast cancer survivors relative to the healthy controls (HF: F = 10.40 ; p <. 01). Conclusion: Breast cancer survivors manifested a dampened immune response to influenza vaccination and cardiovascular imbalance compared to healthy age-matched controls. These findings have important implications for long-term treatment outcomes.

#604

Metabolomics in predicting breast cancer treatment responses.

Carolina Puyana Barcha,1 Eunkyung Lee,2 Cristiane Takita,2 Jean L. Wright,3 Jennifer Hu2. 1 _University of Illinois at Chicago, Chicago, IL;_ 2 _University of Miami Miller School of Medicine, Miami, FL;_ 3 _Johns Hopkins University, Baltimore, MD_.

Breast cancer is the most frequently diagnosed cancer in women and the second leading cause of cancer death in American women. Postoperative adjuvant radiotherapy (RT) significantly improve local-regional recurrence and survival. Therefore, there has been increasing usage of adjuvant RT in early-stage breast cancer patients. Although well tolerated by some patients, 48% patients in our study experience RT-induced early adverse skin reactions (EASRs) that negatively impact quality of life. Therefore, we evaluated urine metabolomics in predicting RT-induced EASRs and precision intervention. Based on the pilot data of 478 metabolites in 60 patients, we used the MetaboAnalyst 3.0 (www.metaboanalyst.ca) to conduct metabolomic data analysis, visualization and interpretation. Using KEGG pathway analysis, we have identified 7 pathways that are significantly associated with RT-induced EASRs (i.e., False Discovery Rate (FDR) p less than 0.05). The alanine, aspartate and glutamate metabolism pathway has the most significant FDR p-value and the highest impact value of 0.60 in predicting RT-induced EASRs. In summary, to the best of our knowledge, this is the first study in breast cancer patients associating glutamate metabolism pathway to RT-induced EASRs. Our research will have significant clinical impact because: (i) many tumor cells depend on glutamine-glutamate as energy source for growth and survival, (ii) breast cancer cells secrete high levels of glutamate often metastasize to bone with severe pain issue, (iii) glutaminase inhibitors enhance chemotherapy in triple negative breast cancer, (iv) two thirds of cancer patients receive RT, and (v) glutamate metabolism is associated with RT-induced EASRs. With increasing recent interest in targeting tumor metabolism, we anticipate that glutaminase inhibitors will also have application in preventing RT-induced EASRs in addition to their anticancer activities in triggering metabolic crisis in tumor cells and enhancing chemotherapy.

#605

Investigating the potential impact of dose-banding for systemic anticancer therapy (SACT) in the pediatric setting based on pharmacokinetic evidence.

Melanie White-Koning,1 Caroline Osborne,2 Angelo Paci,3 Alan V. Boddy,4 Etienne Chatelut,1 Gareth J. Veal5. 1 _University of Toulouse, Toulouse, France;_ 2 _Alder Hey Children's NHS Foundation Trust, Liverpool, United Kingdom;_ 3 _Institut Gustave Roussy, France;_ 4 _University of Sydney, Sydney, Australia;_ 5 _Northern Inst. for Cancer Research, Newcastle-upon-Tyne, United Kingdom_.

Background: In order to make systemic anticancer therapy (SACT) preparation more practicable, dose-banding approaches are currently being introduced in many clinical centers. This involves the individual dose for a particular patient being calculated according to a single body surface area (BSA) value per band, usually the mid-point of the band in which the actual BSA of the patient lies. Advantages of such an approach include a rationalization of chemotherapy provision, with the preparation of drugs with sufficient long-term stability feasible well in advance of treatment, reduced potential for medication errors and improved capacity planning of pharmacy production. The current study aimed to determine the potential impact of utilizing recently developed National Health Service in England (NHSE) dose-banding tables in a pediatric setting, according to pharmacokinetic criteria determined from previously-published clinical trials for a range of commonly used anticancer drugs. Methods: Using pharmacokinetic parameters obtained from 385 drug administrations in 352 children aged from 1 month to 18 years, treated with 5 drugs (dactinomycin, busulfan, carboplatin, cyclophosphamide, etoposide), individual exposures (area under the curve; AUC) obtained using doses rounded according to the published NHSE tables (or banded using the same method) were calculated and compared to those obtained by standard dose calculation methods. The patients included in the study were treated for a wide range of tumors including neuroblastoma, B cell non-Hodgkin's lymphoma, Wilms tumor, rhabdomyosarcoma and Ewing's sarcoma. Results: For all five drugs, the relative variation between the NHSE dose and the recommended dose (standard individually calculated dose) was between -6% and +5% as expected. Indeed, more marked differences were observed between the actual dose administered and the recommended dose for these retrospective datasets, particularly for carboplatin and dactinomycin. In terms of AUC, there was no statistically significant difference in precision between exposures obtained by the recommended dose and those obtained with dose-banding (absolute value of relative difference 15-34%). Conclusion: Based on pharmacokinetic data from 352 children, the results generated from the current study support the implementation of NHSE dose-banding tables, at least for the five drugs investigated here. Indeed, inter-patient variability in drug clearance and exposure would appear to far outweigh the impact of relatively small drug dose changes associated with dose-banding. Further evaluation of the effect of using dose-banding for other drugs would be needed to confirm these results and extend them to additional anticancer drugs.

#606A

Biological predictors and the impact of cancer among African American women.

Claudia M. Davis. _California State University-San Bernadino, San Bernardino, CA_.

African American women have a lower incidence of breast cancer yet, they have an overall higher mortality rate compared with women of other racial groups. Furthermore, some African American women are more likely to be diagnosed with breast cancer at an advanced stage when compared to other women and are also more likely to have biomarkers that are estrogen and progesterone receptor negative and have a greater propensity to be diagnosed with variant forms of breast cancer when compared with other women. These aforementioned factors may affect a women's quality of life. While the literature is robust with psychosocial and behavioral factors that contribute to the quality of life that African American women experience; to date, there has been a paucity of empirical data that have investigated the role of biological predictors and its effect on the lives of these women. To that end, the specific aim of this study was to test a model and investigate the role of biological risk factors estrogen receptors (ER), progesterone receptors (PR), triple negative breast cancer, Breast CAncer gene (BRCA1/2), Tumor Protein 53 gene (TP53, p53), age <45, Basal Metabolic Index (BMI), and age of first pregnancy that may affect the quality of life, thus having an impact on the lives of African American breast cancer survivors. These factors are hypothesized as impacting the quality of lives of these women and their families. The Impact of Cancer is defined as negative or positive occurrences/after-effects along the breast cancer continuum. The Impact of Cancer (IOCv2) captures the multidimensional aspect of the cancer experience that is specific for cancer survivors and their quality of life.

Methods: As part of a larger cross-sectional study, breast cancer survivors completed a demographic data questionnaire inclusive of biological risk factors and the impact of cancer scale. A multiple linear regression was performed to determine the relationship between the two variables. Utilizing recruitment flyers, support groups and word-of-mouth, we recruited and screened 169 women from breast cancer support groups and community dwellers in Southern and Northern California. One hundred and sixty women were eligible and n=155 participated. Inclusion criteria were women who self-identified as African/Black American, age 25 year or older, diagnosis of breast cancer and at least 1-year post-active treatment.

Results: Among the biological predictors, none were statistically significant as predictors of the impact of cancer in the lives of these survivors. The results revealed a trend in survivors who were diagnosed before age 45 (p=.077) that their breast cancer experience had a positive impact on their lives.Conclusion: Understanding biological predictors and the unique interplay of these roles may be necessary along the survivorship trajectory. Additional exploration of psycho social predictors and inclusion of specific moderators may be warranted.

### Immune Response to Therapies 1

#607

Inhibition of CDK4/6 and Pi3Kɣ modulates mammary tumor immune microenvironment to enhance response to immunotherapy.

Stacey Mont,1 Kevin Black,2 Sheau-Chiann Chen,1 Gregory D. Ayers,1 Ann Richmond,1 Anna E. Vilgelm1. 1 _Vanderbilt University, Nashville, TN;_ 2 _Meharry Medical College School of Medicine, Nashville, TN_.

An antitumor immune response is critical in achieving immunotherapy-driven tumor regression. Despite remarkable clinical advances in immunotherapies, patients with breast cancer have been only moderately responsive. In this study, we tested whether CDK4/6 inhibition in combination with Pi3Kɣ inhibition can augment the immunotherapy response. Tumor growth was inhibited in the MMTV-PyMT breast cancer model when treated with palbociclib (CDK4/6i) in combination with anti-CD137, an antibody agonist of T cell co-stimulatory receptor 4-1BB. We also assessed the effect of palbociclib alone in a postsurgical model, where treatment began 5 days after injecting 37,000 MMTV-PyMT cells into the mammary fat pad, and show a significant inhibition of both average tumor weight and volume. In addition, we demonstrate that palbociclib can be effective in reducing total tumor burden in the PyMT-FVB spontaneous breast cancer model. Furthermore, mice treated with palbociclib in this model had an increase in CD3+ cell infiltrate in tumor lung metastasis. We used the Pi3Kɣ-null mice treated with palbociclib or vehicle control to assess the effect of dual inhibition of CDK4/6 and Pi3Kɣ on MMTV-PyMT tumors. We found an increase in CD45+ cells within the tumor microenvironment and an increase in MHC class II+, antigen-presenting macrophages upon palbociclib treatment. Additionally, gene expression analysis of chemokines in palbociclib-treated MCF-7 cells showed an increased expression of CCL4, CCL5, CXCL9, CXCL10, and CXCL11, each of which is implicated in T-cell tumor-homing. Overall, our results suggest that dual inhibition of CDK4/6 and Pi3Kɣ may enhance tumor immunogenicity. Drugs targeting CDK4/6 and Pi3Kɣ are of high clinical relevance and our findings indicate that they may be used in combination with immunotherapies for the treatment for breast cancer.

#608

IFN-γ elicits stimulatory MHC class I isotypes in human colorectal carcinoma cell lines with genetic features of microsatellite stable.

Yi-Hsin Liang, Kuan-Yu Chan, Chang-Cheng Lee, Te-Jung Chen, Ann-Lii Cheng, Kun-Huei Yeh. _National Taiwan Univ. College of Medicine, Taipei, Taiwan_.

Background:

Immunotherapy, especially anti-programmed cell death protein 1 (PD-1) antibodies, has yielded significant and durable tumor response in melanoma, renal cell carcinoma, and other cancer types. In contrast, immunotherapy applied major breakthrough achievement only on patients with microsatellite instability high (MSI-H) metastatic colorectal cancer (mCRC). However, MSI-H mCRC comprised only 1.8-4% of total mCRC patients. It is crucial to investigate immune pathways to develop a new strategy for immunotherapy in treatment of microsatellite stable (MSS) mCRC. Recently, some evidences indicate that interferon (IFN)-γ pathway is critical for anti-PD-1 therapy. In this study, we emphasized on evaluating the response of MSS CRC cell lines to IFN-γ.

Methods:

We characterized two MSS CRC cell lines, namely SW480 (KRAS G12V mutation, BRAF wild type) and COLO320 (KRAS wild type, BRAF wild type) for our studies. The impacts of interferon-γ (IFN-γ) on cell surface expressions of different isotypes of major histocompatibility complex (MHC) class I and MHC class 1 related molecule A/B (MICA/B, the NK cell ligand) were explored. Different concentration of IFN-γ with different incubation time were also examined.

Results:

Both cell lines demonstrated low baseline human leukocyte antigen (HLA)-ABC and HLA-BC expression and the expression significantly increased in response to IFN-γ stimulation. We further focused on the SW480 cell line. The SW480 demonstrated low expression of all MHC class I isotypes, including HLA-ABC, HLA-BC, HLA-A, HLA-C, HLA-E, HLA-F, HLA-G and NK cell ligand MICA/B. The IFN-γ specifically stimulated the expression of HLA-A, but all other isotypes were not responsive to IFN-γ stimulation. In summary, IFN-γ significantly stimulates the expression of HLA-ABC, HLA-BC and HLA-A without stimulating HLA-C. The NK cell ligand MIC A/B was also not responsive to IFN-γ stimulation. The stimulatory effect of HLA-ABC and HLA-A in response to IFN-γ stimulation was positively correlated with IFN-γ dosage, which demonstrated highest expression level in response to 800U/ml IFN-γ stimulation. The stimulatory effect in response to IFN-γ stimulation was also positively correlated with increased incubation time with IFN-γ.

Conclusion:

These findings reveal that regardless of their KRAS mutation status, both MSS CRC cell lines possess a phenotype that may be not responsive to anti-PD-1 therapy due to

low expression of MHC class I molecules. The IFN-γ specifically elicits the expression of stimulatory isotype, HLA-A and HLA-B, without eliciting all other inhibitory MHC class I isotypes. These results indicated that IFN-γ demonstrated pure adaptive immune stimulatory effect on CRC cell lines. Analysis of T- and NK cell-responsive immune markers along with IFN-γ signaling pathway may help us to survey possible combination therapy with anti-PD-1 treatment in CRC.

#609

Neutrophils play a critical role in mediating antitumor activity of a combination therapy of radiation and immunotherapy.

Mi-Hua Tao, Chia-Jen Wu, Yi-Ting Tsai, Ping-Yi Wu. _Academia Sinica - Inst. of Biomedical Sci., Taipei, Taiwan_.

The goal of cancer immunotherapy is to elicit robust tumor-specific immune responses that can generate durable tumor regression and/or eradication. Recent clinical trials using immune checkpoint blockades specific to programmed cell death-1/ligand-1 (PD-1/PD-L1) have shown durable clinical responses in various cancer types but benefit only a small portion of patients. We previously demonstrated that combination of radiation and immunotherapy of interleukin 12 (IL-12) and granulocyte macrophage colony-stimulator factor (GM-CSF) greatly improved the therapeutic efficacy than either single therapy alone. In analysis of the tumor-infiltrating leukocytes post combination therapy, we found that the number of CD11b+F4/80-/Ly6G+ neutrophils were greatly increased. In this study, we characterized these tumor-associated neutrophils (TANs) before and after treatment and investigated their role in mediating the antitumor activity of the combination therapy. We isolated TANs from the regressing tumors and demonstrated that these TANs were impaired in their immunosuppressive activities. Flowcytometric analysis revealed that several co-stimulatory molecules, including class II major histocompatibility complex, CD40 and CD86, were increased on these TANs. Accordingly, these TANs gained the ability to stimulate T cell proliferation. Antibody-depletion experiments showed that TANs were required for tumor regression in RT/12GM combination therapy. Together, our data demonstrate that combination of radiotherapy and IL-12/GM-CSF improved antitumor immunity through enhancing antigen presentation activity and reduced immunosuppressive functions of TANs.

#610

Dynamic immune landscape of the peripheral blood mononuclear cells from recurrent hepatocellular carcinoma patients undergoing radiofrequency ablation treatment.

Dexi Chen,1 Yang Zhao,1 Yabo Ouyang,1 Kai Liu,1 Wei Rao,2 Feili Wei,1 Xiaoni Liu,1 Ying Shi,1 Shanshan Wang,1 Lijun Pang,1 Luxin Qiao,1 Yunjin Zang,2 Xiaoming Yin3. 1 _Beijing You An Hospital, Capital Medical University, Beijing Institute of Hepatology, Beijing, China;_ 2 _The Affiliated Hospital of Qingdao University, Qingdao, China;_ 3 _Department of Pathology and Laboratory Medicine, Indiana University School of Medicine, Indianapolis, IN_.

Hepatocellular carcinoma (HCC) is one of the most common malignancies and the third most lethal malignancy worldwide. Radiofrequency ablation (RFA) is the most commonly used curative treatment for the patients not suitable for surgical resection or liver transplantation. It can cause regional and global anti-tumor immune response, but is insufficient to prevent HCC recurrence in many patients. Peripheral blood mononuclear cells (PBMC) are considered to be the best target for the study of the global immune status after RFA. Previous studies on PBMC were mainly based on traditional flow cytometry, which could not differentiate the complicated immune cell subtypes. In this study, we used mass cytometry to comprehensively characterize the phenotypic and functional alterations of the PBMC in recurrent HCC patients who had received RFA treatment. Fifty-four (54) blood samples from 13 patients were analyzed. Manual gating, SPADE analysis and viSNE analysis were used to interpret the mass cytometry data. The percentage of monocytes showed a fast increase after RFA, and reduced to the normal level before the HCC recurrence (P=0.026). A long-lasting reduction of B cells count were also found after the first RFA (P=0.021), and the B cell count remained at a low level before and after the secondary RFA. The percentage of CD4+ T cells in CD45+ cells was significantly reduced after the first RFA, then restored to the pretreatment level at the time of recurrence, and reduced again after the second RFA(P=0.024). CD8+ T cells showed the same alteration trend as CD4+ cells (P=0.03). We also examined the expression of functional markers in T cells and Tregs. Alterations of CD28 expression were most frequently found in T cells. The Treg population showed a large heterogeneity among the patients with TIM3, LAG3 or CTLA4 positive Tregs being the largest populations. These results suggest a more specific treating modality should be used in combination with the RFA treatment.

#611

**PD-1-based combination immunotherapy reinvigorates CD8** + **T cells in metastatic pancreatic cancer patients with improved survival.**

Annie A. Wu,1 Takahiro Tsujikawa,2 Gina Choe,2 Teresa Beechwood,2 Lisa M. Coussens,2 Jennifer N. Durham,1 Elizabeth M. Jaffee,1 Dung T. Le1. 1 _Johns Hopkins Medical Institute, Baltimore, MD;_ 2 _Oregon Health & Science University, Portland, OR_.

Background: Metastatic pancreatic ductal adenocarcinoma (PDAC) is lethal with a 5-year survival rate of 2%. Single-agent immunotherapies fail to show clinical activity due to a complex tumor microenvironment (TME) and lack of effector T cells. We previously showed that an irradiated, granulocyte-macrophage colony-stimulating factor (GM-CSF)-secreting, allogeneic PDAC vaccine (GVAX) given as neoadjuvant recruited T cells into the tumor and upregulated the PD-1/PD-L1 pathway. Here we described the first testing of GVAX prime given with attenuated listeria monocytogenes expressing mesothelin (CRS-207) boost given with or without nivolumab to block PD-1 signaling and evaluated changes in the TME.

Experimental Design: Metastatic pancreatic tumor biopsies were obtained at baseline and after 2 GVAX prime and 1 CRS-207 boost from vaccinated patients. Nivolumab was administered with each vaccine in patients randomized to the nivolumab arm. Biopsies containing high density of tumor cells (>30%) were chosen for multiplex immunohistochemistry (IHC) to examine changes in the immune cell subtypes and their signals in tumors. We did a comparative analysis looking at lymphoid, myeloid complexity, and immune functional status in the tumor microenvironment of patients who have overall survival <6 months [short OS] and >6 months [long OS].

Results: Post-immunotherapy changes in immune cell profiles correlated with overall survival. After prime-boost, expansion of CD8+ T cells was observed in the long OS group. Evaluation of the functional status of CD8\+ T cells after prime-boost of short OS group showed an EOMES+PD1+ exhausted phenotype. In the myeloid compartment, low CSF1R+ tumor associated macrophages and CD68+ CD163+ and CD163- myeloid frequency in post-immunotherapy tumors were associated with less exhaustion of CD8+ T cells and long OS. The clinical trial is ongoing and blinded to study arm; thus, we have not yet analyzed whether nivolumab influences results. These data will be available at the time of abstract presentation.

Conclusion: This study provides evidence that longitudinal changes in immune cell complexity profiles can be correlated with overall survival. Increases in CD8+ early effectors and decreases in monocytes in baseline versus post-immunotherapy tumors are associated with improved survival. The exhausted CD8+ T cell profile in short OS patients may predict early responders versus nonresponders to vaccine containing immunotherapy.

#612

Nivolumab-induced changes associated with patient outcomes are disparate in metastatic melanoma patient Tregs versus conventional T cells.

David M. Woods. _NYU Langone Health, New York, NY_.

The PD1 blocking antibody nivolumab induces durable responses in metastatic melanoma patients. However, many patients do not respond and biomarkers differentiating patient response remain unidentified. To address this need, we investigated the effects of nivolumab on peripheral blood conventional CD4+ (Tcons) and regulatory T-cells (Tregs) of metastatic melanoma patients receiving nivolumab as adjuvant therapy or for metastatic disease. An increased ratio of PD1 expression on Tregs compared with Tcons was found at baseline patients with no evidence of disease (NED), while relapsing patients had a lower ratio (p<0.05). Intriguingly, NED patients had an increase in circulating Treg percentages post-nivolumab (p<0.05), which was not observed in relapsing patients. In vitro treatment of isolated T-cells with αPD1 resulted in increased proliferation (p<0.001). RNA-seq analysis of Tcons and Tregs showed distinct gene expression changes based on cell type and patient outcome. Pathway analysis demonstrated: upregulation of proliferation associated genes in NED Tregs, but not in relapse Tregs; downregulation of oxidative phosphorylation associated genes in NED patient Tregs and upregulation in NED Tcons; upregulation of heme metabolism associated genes in NED Tcons and downregulated in relapsing patient Tcons; and similar differences in MTORC1, heme metabolism and hypoxia pathways (all pathways q<0.01). Both Tcons and Tregs from non-relapsing patients had induction of phosphoSTAT3 (pSTAT3) expression post-nivolumab (p=0.012), which was absent in relapsing patients. This was also observed in a cohort of active disease patients (p=0.022), and the magnitude of change in pSTAT3 was positively correlated with patient survival (R2=0.49, p<0.05). Enhanced pSTAT3 expression was recapitulated in vitro in isolated T-cells (p<0.05). Post-nivolumab Tregs from patients with positive outcomes also had reduced suppressive function in mixed lymphocyte reactions (p<0.05), while patients with negative outcomes had no changes. In vitro treatment of Tregs with PD1 blocking antibodies resulted in a reduction in suppressive function (p<0.0001), while addition of a STAT3 inhibitor enhanced suppressive function (p<0.0001). These results suggest that nivolumab alters Treg signaling and metabolism pathways, hindering immune suppressive function. Collectively, our data demonstrate distinct effects of nivolumab on Tcons vs. Tregs, and highlight several potential biomarkers of patient benefit, warranting further exploration.

#613

X4P-001, an orally bioavailable CXCR4 antagonist, enhances immune cell infiltration and activation in the tumor microenvironment of melanoma.

Robert H. Andtbacka,1 Robert H. Pierce,2 Jean S. Campbell,2 Melinda Yushak,3 Mohammed Milhem,4 Merrick Ross,5 Kenneth Grossmann,1 Kam Sprott,6 Eleni Tsiroyanni,6 Katie Niland,6 Lu Gan,6 Sudha Parasuraman,6 Yan Wang6. 1 _Huntsman Cancer Institute, Salt Lake City, UT;_ 2 _Fred Hutchison Cancer Research Center, Seattle, WA;_ 3 _Emory University School of Medicine, Atlanta, GA;_ 4 _University of Iowa, Iowa City, IA;_ 5 _MD Anderson Cancer Center, Houston, TX;_ 6 _X4 Pharmaceuticals, Cambridge, MA_.

Background: The CXCR4/CXCL12 pathway plays a central role in the trafficking of key immune cells in the tumor microenvironment (TME). X4P-001 is an oral, selective, allosteric CXCR4 inhibitor. We hypothesize that the disruption of CXCR4/CXCL12 signaling by X4P-001 will favor an improved response to checkpoint inhibitors by modulating the immune cell profile within the TME and increasing CD8+ T cell infiltration. A biomarker-driven phase 1b clinical study is being conducted in melanoma patients to test this hypothesis (NCT02823405).

Materials and Methods: The primary objectives for the study are to evaluate the safety and tolerability of X4P-001 as a single agent and in combination with pembrolizumab in patients (pts) with metastatic melanoma, and to characterize the effects of X4P-001 alone and in combination with pembrolizumab on tumor immune cell infiltrates. Serial biopsies of cutaneous or subcutaneous melanoma lesions, peripheral blood mononuclear cells (PBMCs), and serum samples were collected pre-dose, after 3 weeks of X4P-001 treatment, and after 6 weeks of combination treatment. Biopsies were assessed by immunohistochemistry (IHC) and multiplexed immunofluorescence (mIF) for multiple markers, including CD3, CD8, FoxP3, PD-L1 and Granzyme B, and by NanoString® analysis for changes in gene expression. PBMCs were analyzed by flow cytometry for both lymphoid and myeloid cells. In addition, multiple serum markers will be assessed using the multi-analyte profile (MAP) platform.

Results: As of September 15, 2017, 13 pts have been enrolled, and 11 have completed the study. The median age was 73 years (range 53-90). Of the evaluable pairs of biopsies, X4P-001 treatment alone increased CD8+ T cells, increased granzyme B signal, increased antigen-presenting machinery such as HLA-DR, and increased IFN-gamma gene expression signature scores in the TME. These biomarker responses were further enhanced when X4P-001 was combined with pembrolizumab. X4P-001 was well tolerated. AEs assessed as related to either X4P-001 or pembrolizumab at any time were diarrhea, maculopapular rash, fatigue, chills, and acute kidney injury. These data, along with additional biomarker measurements, will be presented.

Conclusions: Evidence of enhanced immune cell infiltration and activation is observed in the TME with X4P-001 treatment alone. Increased IFN-gamma gene expression signature scores after single-agent X4P-001 treatment support the use of X4P-001 to increase the likelihood of a response when combined with anti-PD-1 therapy. X4P-001 as a single agent and in combination with pembrolizumab is generally safe and well tolerated. Further in-depth biomarker analysis is ongoing as enrollment nears completion.

#614

Resiquimod, a Toll-like receptor agonist promotes melanoma regression by enhancing plasmacytoid dendritic cells and T cytotoxic activity as a vaccination adjuvant and by direct tumor application.

Jorge A. Borin Scutti, Luis M. Vence, Richard E. Royal, Tara C. Wray, Janice N. Cormier, Jeffrey E. Lee, Anthony Lucci, Jeffrey E. Gershenwald, Merrick I. Ross, Jennifer Wargo, Karen A. Millerchip, Rodabe N. Amaria, Michael A. Davis, Adi Diab, Isabella C. Glitza, Wen Hwu, Sapna Patel, Scott E. Woodman, Willem W. Overwijk, Patrick Hwu. _MD Anderson Cancer Center, Houston, TX_.

Introduction: Cancer immunotherapy is a modern strategy aiming at restoring the capacity of the immune system to target tumors in cancer patients. Toll-like receptor (TLR) agonists may enhance vaccination or direct immune activation at the tumor microenvironment. This clinical trial evaluated the biologic effects of Resiquimod, a TLR agonist that can activate both myeloid (TLR 8) and plasmacytoid (TLR 7) dendritic cells, on advanced stage melanoma. Methods: Subjects with in-transit melanoma metastases or high risk for recurrence and appropriate HLA were treated with peptide vaccination (class 1 restricted peptide GP100(g209-2m) and, if HLA-DP4+, class 2 restricted peptide MAGE-3243-258). Half of the patients were randomized to receive Resiquimod as an adjuvant applied to the GP100 vaccination site. Subjects with in-transit disease were then treated with resiquimod topically on half of the target lesions. To evaluate the T cell function, fresh PBMC and single cell tumor suspension were analyzed by flow cytometry using gp100-specific dextramer staining. RNA from the vaccination site was also analyzed using real-time PCR. Results: All patients (n=47) underwent GP100(g209-2m) vaccination, a majority (39) also received the MAGE-3243-258 peptide. Type 1 interferon pathway protein profiles of vaccination sites showed activation of plasmacytoid dendritic cells in patients with Resiquimod, but not in its absence. Nineteen subjects had in-transit disease at entry into the trial. In response to peptide vaccination alone, tumor regression was more likely in patients who received Resiquimod (group A) compared to those who did not (group B). (4/9 vs 0/10). In group A, 5 patients continued treatment with Resiquimod topically on the tumors, and all had tumor response (4PR, 1CR). In group B, 5 continued to tumoral resiquimod and 3 had regression (3 PR). Type I interferon (as measured by MxA and IRF7) IFN-gamma and TNF-alpha increased at the vaccination site 24 hrs after vaccination only at the sites where Resiquimod was applied. In blood, Resiquimod increased gp100-specific CD8 T cells frequency at week 8 (p=0.03) only in patients who received Resiquimod at the vaccination site. Conclusions: Resiquimod activates plasmacytoid dendritic cells at a peptide vaccination site and augments peptide vaccination sufficiently to mediate regression of in-transit melanoma metastasis. Resiquimod on in-transit melanoma, in vaccinated hosts, drives regression of metastases, regardless of previous exposure at vaccination site. An increased amount of cytokines such type I interferon, IFN-gamma, TNF-alpha, and T specific cytotoxic frequency were increased at the vaccination site after patients received Resiquimod.

#615

Increased somatic hypermutation in the immunoglobulin sequences of melanoma patients who have durable response to checkpoint inhibitor therapy.

Ngan Nguyen,1 Alusha Mamchak,1 Mariano Severgnini,2 Kevin S. Williamson,1 Xinqi Wu,2 Elliott F. Drabek,1 Michael Manos,2 Xiaomu Chen,1 Xiaobin Tang,1 Zoe Amiri,1 Chantia Carroll,1 Yvonne Leung,1 Dongkyoon Kim,1 Wayne Volkmuth,1 Norman Greenberg,1 Daniel Emerling,1 William H. Robinson,3 Guy Cavet,1 F. Stephen Hodi2. 1 _Atreca Inc., Redwood City, CA;_ 2 _Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA;_ 3 _Stanford University School of Medicine, Stanford, CA_.

Patient immune response to tumor can drive profound and durable clinical benefit, but the contribution of antibodies to this benefit is poorly understood. To further our understanding of the role of the humoral response, we compared antibody sequence repertoires of durable responder and non-responder melanoma patients before and during checkpoint inhibitor treatment.

We collected pre- and on-treatment peripheral blood from 26 melanoma patients treated with pembrolizumab (9), ipilimumab (8) or nivolumab+ipilimumab (9). Of the 26 patients, 14 were durable responders (RECIST stable disease, partial response, or complete response for at least 6 months) and 12 were non-responders. We generated natively paired heavy and light chain antibody sequences from individual IgG plasmablasts (CD19+CD20-CD38+CD3-CD14-IgA-IgM-IgD-). Sequences were obtained from a total of 26,725 plasmablasts. These sequences were used to reconstruct lineages (sets of plasmablasts likely derived from a common progenitor B cell) where each lineage shares heavy and light V genes, CDR3 length and 80% sequence similarity in CDR3s.

Durable responders exhibited an increase in somatic hypermutation (SHM) after initiation of treatment. In contrast, no significant change in SHM was observed in non-responders. Higher SHM was also observed in on-treatment repertoires of responders compared with those of non-responders suggesting that T cell checkpoint inhibitors promote activation of humoral immunity in clinical responders. Furthermore, persistent antibody lineages (observed both before and during treatment) showed higher SHM than lineages observed at only a single time point.

Comparison of lineages between patients identified antibodies with high sequence similarity suggesting these antibodies may have arisen from convergent selection, i.e., different patients raising antibodies against shared or similar epitopes. These putative convergent antibodies were enriched in IgG2. IgG2 usage was also higher overall in durable responders than non-responders, even in samples taken prior to treatment. The higher levels of IgG2 observed in responders and in the potentially convergent sets of antibodies suggest that IgG2 antibodies may play a role in effective anti-tumor responses.

In conclusion, our analysis of plasmablast repertoires from melanoma patients suggests that treatment with checkpoint inhibitors promotes SHM in durable responders, and that responder plasmablasts are enriched in IgG2. Moreover, patients make potentially convergent antibodies that are also enriched for the IgG2 subclass. Our observations demonstrate that the humoral immune response is remodeled in patients with anti-tumor responses and support a role for the humoral arm of the immune response in driving clinical benefit in patients treated with checkpoint inhibitors.

#616

Interferon gamma induced transformation of the cold tumor microenvironment in patients with NY-ESO-1 expressing sarcomas.

Shihong Zhang,1 Sara Cooper,1 Bailey Donahue,1 Venu G. Pillarisetty,2 Robin L. Jones,2 Stanley R. Riddell,2 Brian A. Van Tine,3 Seth M. Pollack2. 1 _Fred Hutchinson Cancer Research Center, Seattle, WA;_ 2 _Fred Hutchinson Cancer Research Center, University of Washington Medical Center, Seattle, WA;_ 3 _Washington University in St. Louis, St. Louis, MO_.

Synovial sarcoma (SS) and myxoid/round cell liposarcoma (MRCL) cells homogeneously express high levels of NY-ESO-1 and other cancer/testis antigens, which makes these sarcoma types good candidates for T cell immunotherapy. However, these tumors demonstrate a "cold" microenvironment, with low cell surface expression of human leukocyte antigen (HLA) molecules and few infiltrating T cells. Interferon gamma (IFNγ) has been used both as a single agent and in combination with other immunotherapies in cancers with "hot", highly inflamed tumor microenvironments such as melanoma. To date, little work has explored its impact on immunologically quiet tumors. In order to increase the expression of HLA on the tumor cell surface and improve the T cell recognition and cytotoxicity, we initiated a pilot "window of opportunity" clinical trial, 6 SS/MRCL patients were treated with subcutaneous systemic interferon gamma (IFNγ) weekly for 2 or 4 weeks at a dose of 100 mcg/m2. The primary objective of the study was to determine if HLA expression increased on post-treatment biopsies in comparison to pre-treatment biopsies. The treatment was relatively well-tolerated, patients experienced flu-like symptoms with no severe adverse effects. Patients' sera, PBMCs and tumor biopsies were collected pre- and post-IFNγ treatment for further analysis. Several cytokines including interleukin-16, microphage migration inhibitory factor and CXCL-10 etc. changed considerably in patients' periphery. Transcriptome profiling on the PBMCs showed significant changes in some immune regulatory genes in T cells and monocytes. Flow cytometry analysis of the tumor biopsies showed significant increase of both class I and class II HLA on the tumor surface in all treated patients. Tumor infiltrating immune cell frequencies increased by 3-10 times, of which, the majority were T cells. Functional assay of expanded tumor infiltrating lymphocytes (TIL) showed an increased and broadened tumor-associated peptide recognition capability. However, in all evaluable tumors there was an increase in PD-L1 either on tumor or on infiltrating macrophages. In conclusion, weekly IFNγ treatment is well tolerated in SS/MRCL patients and it can significantly enhance the expression of HLA on tumor surface, and increase T cell infiltration; however, increased PD-L1 expression on tumor cells and infiltrating immune cells may help tumors evade T cell elimination. Strategies combining PD-1 blockade and IFNγ should be explored.

#617

Nivolumab efficacy and safety in veterans with and without HIV infection.

Elaine Chang, Aaron P. Thrift, Donna L. White, Jennifer Kramer, Anita L. Sabichi, Christine Hartman, Kathryn E. Royse, Peter A. Richardson, Elizabeth Y. Chiao. _Baylor College of Medicine, Houston, TX_.

The objective of this study was to compare the efficacy and safety of nivolumab in the HIV-infected and HIV-uninfected veteran populations.

WHAT IS NEW: Most clinical trials highlighting checkpoint inhibitors excluded people living with HIV (PLWH). This is the first study to report equivalent nivolumab tumor response rates in PLWH, but higher rates of pneumonitis.METHODS: We searched the Corporate Data Warehouse (CDW) to identify all HIV-positive patients who used the Veterans Health Administration (VA) between 2000 and 2016. For this cohort of 46,916 PLWH, we obtained a 4:1 age-matched HIV-negative control cohort. We used pharmacy database to identify all nivolumab recipients through July 26, 2017. We reviewed patients' electronic medical records using Compensation and Pension Records Interchange (CAPRI) to obtain age, sex, geographic location, cancer type, number of nivolumab doses received, previous cancer therapy, adverse events, and response to therapy. We calculated summary descriptive measures for both groups and compared the proportion of patients experiencing adverse event or pneumonitis in groups by HIV status, using chi-square or Fisher's exact test.

RESULTS: Sixteen PLWH and 68 HIV-negative controls received nivolumab during the study period. Only 51 controls had accessible data. Median age at nivolumab initiation was 65 years in both groups (range, 47-85 [IQR, 59-68] in PLWH; range, 42-79 [IQR 59-68] in controls); all patients were male. Overall, 63% received nivolumab for non-small cell lung cancer (NSCLC): 8/16 (50%) in PLWH, 34/51 (67%) in controls. Other indications in PLWH included renal cell carcinoma (RCC) (2/16 [13%]), Hodgkin lymphoma (HL) (2/16 [13%]), hepatocellular carcinoma (HCC) (2/16 [13%]). Other indications in controls were RCC (9/51 [18%]), melanoma (5/51, [10%]), and head and neck squamous cell carcinoma (3/51 [6%]).Regardless of HIV status, the median number of prior lines of therapy was 1, median number of nivolumab doses received was 6, and median progression-free survival (PFS) in NSCLC was 2.75 months. Half of each NSCLC group received radiation; fewer PLWH had COPD (50%, vs. 82% of controls). PLWH had a trend towards more adverse events (AEs) (6/15 [40%], vs. 13/51 [26%] of controls, p=0.28). A significantly higher proportion of PLWH experienced pneumonitis (4/15 [27%], vs. 2/51 [4%] of controls, p=0.007).Other AEs among PLWH were rash, hypothyroidism, and autoimmune diabetes mellitus. Other AEs among controls were fatigue, colitis, rash, hypothyroidism, arthritis, and hepatitis (1-3 patients each).

CONCLUSION: Among veteran PLWH, rates of nivolumab administration, and PFS in NSCLC, were comparable to those of HIV-uninfected counterparts. Pneumonitis was significantly more frequent in PLWH. Further studies should investigate the mechanism of pneumonitis in this population, confirm the high frequency in larger cohorts of PLWH, and identify clinical risk factors for pneumonitis.

#618

Application of clinical whole-exome sequencing as a predictor of clinical benefit of PD1/PDL1 blockade in a prospective study of advanced cancer patients.

Guan Wang, Cheng Chen, Jinwang Wei, Angela Wu, Chun Dai, Xiaoman Xu, Xin Cai, Qiang Xu. _GenomiCare Biotechnology, shanghai, China_.

Background: The effectiveness of PD1/PDL1 checkpoint blockade immunotherapy is influenced by several genetic factors, including microsatellite status, tumor mutation burden (TMB) and chromosomal status. Systemic understanding of why immunotherapy is effective may predict long lasting responses and resistance to immunotherapy.

Methods: Over 1,600 cancer patients from 72 hospitals across 20 provinces in China were recruited and the whole exome of tumor/blood samples of each patient were sequenced. We applied genomic checkpoint immunotherapy predict (GCIP) model that developed based on CWES data associated with histopathologic features to predict checkpoint immunotherapy effectiveness score (CIE score) for each patients. Clinical decision roadmap recommendations including targeted therapy and tumor immunotherapy were provided to each patient and their treating physician. Based on the latest follow-up results, 22 advanced cancer patients including 6 lung, 5 biliary system, 3 soft tissue sarcomas and 8 others tumor types received anti-PD1/PDL1 treatment following their physician's recommendations.

Results: Of these 22 prospective real-world enrolled patients, the disease control rate (DCR) was 72.7% and Objective response rate (ORR) was 40.9%, with four (18.1%) patients having a complete response (CR) by investigator assessment. Nine patients with TMB-high (3 CR, 4 PR and 2 PD) had better ORR and DCR than those with TMB-medium (77.7% vs. 14.2% and 77.7% vs. 70.0%). Interestingly, the highest TMB patient with primary resistance to anti-PD-1 therapy presented CDKN2A and IFN-α family somatic co-deletion at chromosome 9p21 segment which also be found by tumor biopsy from another patient with acquired resistant after an extraordinary 18-month response. Of 6 progressive disease (PD) patients, chromosomal arm-level aberrations have been identified in 4 patients and 5 somatic mutated genes (total 148) in histocompatibility complex class I (MHC-I) molecules have been detected in one patient with MSI-high. Notably, four patients with both the highest chromosomal stability and either the TMB-high, or Indel proportion high, but neither alone, exhibited the longest clinical benefit (CR lasts longer than 12 months). These findings would be the mechanisms of tumor escape from the host's immunity.

Conclusions: GCIP model can effectively predict checkpoint immunotherapy response, durable clinical benefit and genetic resistance features for most of the advanced cancer patients. Our findings demonstrate the importance of tumor genomic data, especially chromosomal status; those important clues may potentially overcome primary and acquired resistance to immunotherapy.

#619

ALT-803 enhances antibody-dependent cell-mediated cytotoxicity (ADCC) mediated by NEO-201 against human carcinoma cells.

Massimo Fantini,1 Justin M. David,1 Hing C. Wong,2 Christina M. Annunziata,3 Philip M. Arlen,1 Kwong Yok Tsang1. 1 _Precision Biologics, Inc., Rockville, MD;_ 2 _Altor BioScience Corporation, Miramar, FL;_ 3 _Women's Malignancies Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD_.

Background: ALT-803 is a novel IL-15 superagonist complex consisting of an IL-15 mutant (IL-15N72D) bound to an IL-15 receptor α/IgG1 Fc fusion protein. It has been reported that ALT-803 exerted a significant anti-tumor activity in murine myeloma, breast, colon carcinomas, in rat bladder cancer, and in human bladder, ovarian carcinomas in vivo. ALT-803 was also found to enhance ADCC against wide range of human carcinoma cells in vitro. NEO-201 is a novel humanized IgG1 monoclonal antibody (mAb) which was found to react against a variety of cultured human carcinoma cell lines and tumor tissues. NEO-201 can mediate ADCC against human carcinoma cells.

Methodology: We investigate the ability of ALT-803 to modulate the ADCC mediated by NE0-201, employing NK cells isolated from normal donors as effectors. Human NK cells were treated with ALT-803 at different concentrations for 48h prior to be used as effector cells, and human carcinoma cell lines expressing the NE0-201 antigen were utilized as targets in an in vitro non-radioactive ADCC assay. The ability of ALT-803 to affect the phenotype of NK cells and to modulate NK cells gene expression was evaluated by flow cytometry and by using the Nanostring analysis respectively.

Results: The treatment with ALT-803 significantly enhanced the ADCC activity mediated by NEO-201 against NEO-201 positive carcinoma cells. The effect ALT-803 was dose-dependent and achieved statistical significance at all doses tested compared to vehicle control treatment. Treatment of NK cells with ALT-803 enhanced ADCC activity also from donors with minimal ADCC activity and lowered the effective dose of NEO-201 required to initiate the ADCC response compared to untreated NK cells. ADCC activity can be blocked by using anti-CD16 and anti-TIM3 blocking antibody. Phenotypic analysis of NK cells treated with 25 ng/ml of ALT-803 for 48h demonstrated that ALT-803 can enhance the expression of TIM3 and NKG2D and the mean fluorescence intensity (MFI) of granzyme B and CD107a in CD16/CD56 positive NK cells. Nanostring analysis of human NK cells treated with ALT-803 at different concentrations for 48h, showed that ALT-803 was able to modulate mRNA expression of 62 genes (1.6 log2 fold change compared to vehicle control was considered significant). ALT-803 up-regulated the mRNA expression of 43 genes, including NK activating receptors, factors involved in the NK cytotoxicity, cytokines and their receptors, and down-regulated the mRNA expression of 19 genes, including NK inhibiting receptors and factors involved in the activation of apoptosis.

Conclusions: ALT-803 can enhance ADCC activity mediated by NEO-201 against human carcinoma cells. The enhancement of the ADCC activity may be in part due to the increase in the expression of TIM3, NKG2D, granzyme B, and CD107a positive NK cells, as well as to the modulation of transcripts that are involved in the NK activation and cytotoxicity.

#620

Associations of genomic alteration, tumor mutational burden with PD-L1 expression and response to immune checkpoint inhibitors in Chinese lung patients.

Jing Hu,1 Bixun Li,2 Bing Zou,3 Senming Wang,4 Ye Qiu,2 Maolin Yan,5 Zhiming Zeng,6 Han Yang,7 Yan Guan,8 Lei Zhang,9 Wei Chen,10 Yuan Zhang,11 Lei Mei,11 Xiaowei Dong,11 Ming Yao,11 Kai Wang11. 1 _First People's Hospital of Yunnan Province, Yunnan, China;_ 2 _Affiliated Tumor Hospital of Guangxi Medical University, Nanning, China;_ 3 _Shandong Cancer Hospital Affiliated to Shandong University, Shandong Academy of Medical Sciences, Jinan, China;_ 4 _Zhujiang Hospital of Southern Medical University, Guangzhou, China;_ 5 _Fujian Provincial Hospital, Fuzhou, China;_ 6 _The First Affiliated Hospital of Guangxi Medical University, Nanning, China;_ 7 _First Affiliated Hospital of Guangxi Medical University, Nanning, China;_ 8 _Shandong Cancer Hospital Affiliated to Shandong University, Shandong Academy of Medical Sciences, Jinan, China;_ 9 _The Second Hospital of Shanxi Medical University, Taiyuan, China;_ 10 _Qingdao municipal hospital, Qiandao, China;_ 11 _OrigiMed,Inc., Shanghai, China_.

Background: For advanced or metastatic lung cancer patients (pts) in the absence of actionable genetic alterations, whose disease progressed after chemotherapy, the effective treatment options are limited. Recently, PD-1/PD-L1 inhibitors have demonstrated improved survival in advanced lung cancer. However, sufficient evidence is still lacking in Chinese lung cancer pts.

Methods: Twenty lung cancer pts including 12 adenocarcinomas and 5 squamous cell carcinomas who received anti-PD-1/PD-L1 treatments as first- to fourth-line therapy were retrospectively reviewed. The majority of the pts were stage IV (17 pts). PD-L1 expression was analyzed by immunohistochemical staining. Genomic alterations were detected by using a next-generation sequencing (NGS) assay targeting 450 cancer genes. Tumor mutational burden (TMB) and microsatellite instability (MSI) status were acquired by NGS algorithm.

Results: The median age of the 20 pts was 57 years (range: 36-69) and male vs. female was 3. The median time from date of diagnosis to the date of the first anti-PD-1/PD-L1 treatment was 9.5 months (range: 1-69 months). The overall response rate was 89%, including 8 partial response (PR), 9 stable disease (SD) and 2 progressive disease (PD), and the median PFS (mPFS) was not reached (NR) (range: 0.5-NR). Seven pts had one or more targetable oncogenic mutations including EGFR L858R (2) and amplification, PIK3CA (1), BRAF, ERBB2, PTEN, and ROS1 fusion. The median TMB value of the 20 pts was 11 muts/Mb (range: 2.4-38 muts/Mb). 45% of the pts had TMB greater than 20 muts/Mb, and 60% had TMB greater than 10 muts/Mb. Among the 9 pts who had the TMB greater than 20 muts/Mb, 5 pts achieved PR, 4 achieved SD, mPFS was not reached. Also, 5 pts including 2 PR and 3 SD were identified as MSI-high. In the PD-L1 positive subgroup (N=11), 5 pts achieved PR, 5 achieved SD and 1 had a PD. Furthermore, we found 5 pts with NFE2L2 mutations including 2 adenocarcinomas and 3 squamous cell carcinomas, associated with high TMB values (>20 muts/Mb) and PD-L1 positivity; all 5 pts had response to anti-PD-1/PD-L1 treatment, which include 3 PR and 2 SD with mPFS not reached (range: 6-NR).

Conclusion: Our preliminary results based on a small number of pts showed a significant association between high TMB values and response of immune checkpoint inhibitors in Chinese lung cancer pts, and a strong correlation between high TMB values and high PD-L1 expression. We also reported that NFE2L2 mutation was tightly associated with high TMB value (P=0.011) and PD-L1 positivity (P=0.038), which may serve as a new predictive biomarker for advanced or metastatic lung cancer pts receiving anti-PD-1/PD-L1 treatments. Further larger cohort study is warranted.

#621

Evaluating the compatibility of tumor treating electric fields with key anti-tumoral T cell functions.

Gil Diamant, Hadar Simchony, Tamar Shiloach, Anat Globerson-levin, Zelig Eshhar, Rachel Grossman, Zvi Ram, Ilan Volovitz. _Tel-Aviv Medical Center, Tel Aviv, Israel_.

Background: Tumor Treating Fields (TTFields) are low-intensity electric fields that target proliferating cells by hindering mitotic spindle formation and charged organelle translocation. TTFields are FDA-approved for treatment of newly diagnosed and recurrent glioblastoma. Combining TTFields with immunotherapy is a rational approach due to their different mechanisms of action (MOA) and to TTFields' ability to induce immunogenic cell death (ICD). Conversely, TTFields may interfere with immune functions critical for effective T cell responses. Here, we evaluated the effects of TTFields on select pivotal anti-tumoral T cell functions.

Methods: T cells from healthy donors' peripheral blood or from viably dissociated fresh glioblastoma samples were cultured under normal or TTFields conditions, with or without superantigen stimulation. Multiparametric flow cytometry (8 colors) was used to assess T cell responses by monitoring select pivotal anti-tumoral functions: proliferation (CFSE dilution), IFNγ secretion (intracellular staining), cytotoxic degranulation (CD107a surface expression) and activation/exhaustion (PD1 expression). T cell viability under TTfields was assessed in a dedicated assay. Direct cytotoxicity under TTFields was evaluated using a chimeric antigen receptor (CAR) T cell-based assay.

Results: The viability of superantigen-stimulated T cells that attempted to proliferate decreased under TTFields, in line with TTFields' MOA. Small or no reductions in viability were found in activated T cells that did not attempt to proliferate and in unstimulated T cells.

The functionality of peripheral-blood T cells and tumor-infiltrating T cells (TILs) stimulated by superantigen under TTFields was unhindered: T cells exhibited comparable PD1 upregulation, IFNγ secretion and CD107a surface-expression as controls. T cell polyfunctionality was previously reported to be associated with effective anti-tumoral responses. A polyfunctionality analysis conducted on our data demonstrated that T cells that had lost the capacity to proliferate under TTFields conditions, retained all other polyfunctional combinations. PD1+ TILs, a subset containing most of the tumor antigen-specific T cells, exhibited unaltered viability and functionality when cultured under TTFields. Lastly, the cytotoxic capability of CAR T-cells (which utilize the same killing machinery as normal T cells) was unaffected by TTFields.

Conclusions: All anti-tumoral T cell functions examined, with the exception of proliferation, were unhindered by TTFields. Our findings, together with the plausible synergism of TTFields

with systemic anti-tumoral immune responses, warrant the further preclinical and clinical investigation into the combination of these therapeutic approaches.

#622

**Differential impact of pembrolizumab and ipilimumab on IFNgamma and IL-10 production from** in-vitro **monocyte-derived DC and T cell co-cultures.**

Tina Tang, Fay Stewart, Alexander Roberts, John Gordon, John Curnow, Nicholas Barnes. _Celentyx Ltd, Birmingham, United Kingdom_.

Despite the success with checkpoint inhibitors in-vivo, demonstrating efficacy in-vitro using human primary immune cells remains a challenge. Our aim was to determine the impact of pembrolizumab and ipilimumab on proliferation, as well as IFNγ and IL-10 production, from monocyte-derived dendritic cell and T cell co-cultures.

CD14+ monocytes from the peripheral blood of healthy donors were isolated before differentiating into immature dendritic cells (iDC) with IL-4 and GM-CSF. Immature dendritic cells were subsequently matured (mDC) by exposure to IL-1β, IL-6, TNFα and PGE2. Allogeneic purified CD4+ T cells (from healthy donors) were co-cultured with either iDC or mDC in the absence (media alone or isotype control) or presence of either pembrolizumab or ipilimumab. T cell proliferation and IFNγ and IL-10 secretion were subsequently assessed.

Whilst there was no consistent impact of either pembrolizumab or ipilimumab on the proliferation of CD4+ T cells in co-culture with iDC or mDC, IFNγ secretion by CD4+ T cells was increased in iDC- and mDC-co-cultures by pembrolizumab (percentage increase at 1μg/ml compare to isotype control: 524.5±196.1, P=0.037; 200.7±80.6, P=0.047, respectively, n=7) and reduced in iDC cultures with ipilimumab (percentage increase at 1μg/ml compared to isotype control: -63.4±11.7, P=0.012, n=4). IL-10 secretion was also impacted upon by pembrolizumab, whereby it increased in both iDC and mDC cultures with CD4+ T cells (percentage increase at 1μg/ml compare to isotype control: 175.3±27.0, P=0.003; 183.2±16.7, P<0.001, respectively, n=4). Ipilimumab however, whilst having no evident effect in iDC-T cell co-cultures, reduced IL-10 production of mDC co-cultures (percentage decrease at 1μg/ml compare to isotype control: -27.7±6.3, P=0.022, n=4).

These data support the positive in-vivo results with pembrolizumab and ipilimumab, and demonstrates a reliable method for investigating the impact of checkpoint inhibitors in immune cells.

### Modifiers of the Tumor Microenvironment 1

#625

Cytotoxic T lymphocyte antigen 4 (CTLA-4) expression in ductal carcinoma in situ (DCIS) of the breast.

Jorge E. Novo, Lauren Rosen, Julianne M. Ubago, Jennifer Pincus, Luis Blanco, Kalliopi P. Siziopikou. _Northwestern University Feinberg School of Medicine, Chicago, IL_.

Background: Ductal carcinoma in situ (DCIS) consists of a heterogeneous group that can be subclassified by molecular signatures. High grade DCIS is commonly associated with periductal inflammation, a feature associated with increased risk for microinvasion. We previously assessed the immune microenvironment of DCIS and reported that HER2+ DCIS has increased numbers of tumor infiltrating lymphocytes (TILs), with significant expression of PD-L1. CTLA-4 is a supplementary immunomodulatory marker, which serves as a negative regulator of T-cell response. Targeted therapy is currently available against both PD-L1 and CTLA-4, and studies suggest an enhanced response with combination therapy as these therapies act though different mechanisms. In this study we evaluated the expression of CTLA-4 in both TILs and tumor cells in DCIS.

Design: The study population consisted of 74 cases of DCIS treated with surgical excision from 2008-2012. The molecular subtypes of DCIS were determined based on ER and HER2 expression. Tissue microarrays were constructed (3 cores/case) to account for tumor heterogeneity. The TMAs were immunostained for CTLA-4 and the expression CTLA-4 was assessed.

Results: Of the 74 cases, 41 were classified as Luminal A (ER+/HER2-), 17 as Luminal B (4 as Luminal B HER2- [ER+/HER2-/high Ki-67] and 13 as Luminal B HER2+ [ER+/HER2+]), 11 as HER2+ (ER-/HER2+) and 5 as basal (ER-/HER2-). Overall, the DCIS tumor cells expressed CTLA-4 in 58% of Luminal B (HER2+ 53% [7/13]; HER2- 75% [3/4]) and 36% of HER2+ (4/11), while only 39% of Luminal A (16/41), and one basal DCIS (1/5) did. In addition, the presence of CTLA-4 positive TILs was strongly associated with HER2+ expression (Luminal B HER2+=21.8%, HER2+=13.4%), compared to HER2- DCIS (Luminal B HER2-=8.5%, Luminal A=7.3%, Basal=6.4%) (p=0.026).

Conclusion: 1. CTLA-4 expression in TILs is increased in HER2+ DCIS. 2. A significant percent of tumor cells express CTLA-4 in all DCIS subtypes. Our results further advance our understanding of the role of the immune microenvironment in DCIS. Additional studies are needed to further elucidate the complex interplay between the host immune system and DCIS, which may offer insights into breast carcinoma progression and possible use of novel immune-based therapies.

#626

Bromodomain inhibition in ovarian cancer and the tumor microenvironment to improve PARP inhibitor response.

Andrew J. Wilson,1 Alyssa Hoover,2 Whitney Harris,2 Esther Liu,2 Dineo Khabele,3 Fiona Yull2. 1 _Vanderbilt University Medical Center, Nashville, TN;_ 2 _Vanderbilt University, Nashville, TN;_ 3 _Kansas University Medical Center, Kansas City, KS_.

Background: Ovarian cancer is the most lethal gynecologic malignancy. While women with BRCA-deficient tumors show sensitivity to PARP inhibitors (PARPi), new treatment options are needed for PARPi-resistant tumors. An emerging strategy to improve PARPi response is combination therapy with epigenetic drugs. A newly recognized epigenetic drug target in ovarian cancer is the bromodomain and extraterminal (BET) protein family. BET proteins such as BRD4 promote oncogenic transcription of progrowth and survival genes, including the established link between inflammation and cancer, nuclear factor-kappaB (NF-κB). A complementary strategy to targeting cancer cells with cytotoxic drugs is to activate normal immune processes in the tumor microenvironment (TME). In syngeneic mouse ovarian cancer models, we have shown that M2-like protumor macrophages are a prominent component of the TME, and that NF-κB inhibition reduces the M2 population. Thus, BET inhibitors (BETi) have the potential to induce transcriptional reprogramming in both tumors and macrophages for therapeutic benefit.

Objective: To determine the cellular and molecular effects of combining BETi and PARPi in mouse ovarian cancer and peritoneal macrophage cell lines.

Methods: Cultured wild-type and CRISPR-modified (TP53 and TP53/BRCA2 knockout) ID8 mouse ovarian cancer cells, and PMJ2-PC mouse peritoneal macrophages, were treated with vehicle, the PARPi olaparib, the first-in-class BETi JQ1 or the JQ1/olaparib combination for 24-72h. Sulforhodamine B (SRB) assays assessed cell growth. Immunofluorescence assays assessed adherent cell number, DNA damage (pH2AX) and cell cycle indices. Protein levels of pH2AX and the apoptosis marker cleaved PARP were assessed by Western blot. NF-κB activity was measured by luciferase assays of a transiently transfected reporter plasmid.

Results: Combined JQ1 and olaparib treatment synergistically reduced cell growth in SRB assays in wild-type and TP53 knockout ID8 cells. TP53/BRCA2 knockout cells showed greater responses to PARPi alone and no synergism was observed. Consistent with these results, the JQ1/olaparib combination cooperatively reduced the number of adherent cells and cells in S phase, and increased the G0/G1 population, DNA damage and apoptosis. In contrast, the drug combination had minimal effects on DNA damage or apoptosis in PMJ2-PC macrophages, while NF-κB activity was reduced in both cancer cells and macrophages.

Conclusions: BETi sensitize mouse ovarian cancer cells to the cytotoxic effects of PARPi. Combined drug treatment also has potential to inhibit NF-κB in both cancer cells and macrophages. Our novel immunomodulatory strategy will be tested in ID8 syngeneic ovarian cancer models. We believe BETi combination treatment could expand the use of PARPi in ovarian cancer patients, with the potential to benefit a substantial number of women with this devastating disease.

#627

Antitumor effect of an oligosaccharide API in a genetically engineered mouse-derived allograft (GEDA).

Shalini V. Rao,1 Tonje S. Steigedal,2 Aarthi Gopinathan,1 Synnøve N. Magnussen,3 Sabina S. Strand,2 Duncan Jodrell,1 Fran Richards,1 Kurt I. Draget,2 Catherine T. Nordgård2. 1 _Cancer Research UK University of Cambridge, Cambridge, United Kingdom;_ 2 _Norwegian University of Science and Technology, Trondheim, Norway;_ 3 _UiT-The Arctic University of Norway, Tromsø, Norway_.

The use of oligosaccharides as active pharmaceutical ingredients lags behind that of other biological molecules such as proteins and nucleic acids. However, there is now a growing understanding of the complex biological functions of saccharide structures. This has resulted in an increased interest in exploring the pharmaceutical applications of both oligosaccharides and glycoconjugates.

Despite recent advances in the treatment of pancreatic adenocarcinoma (PDAC), the median survival remains <12 months. Patients typically present with late stage disease and are often unable to tolerate drug combination regimens due to the associated toxicity.

Here we report a novel oligosaccharide drug candidate, RiXOVA (G-blocks). The term "G-blocks" refers to highly defined and specialized short oligomers comprising α-L-guluronate residues derived from alginates extracted from brown seaweed. The G-block oligomers are well tolerated at i.v./i.p. doses >100mg/kg in mice. These oligomers have previously demonstrated the ability to alter matrices of biological macromolecules including mucins, giving rise to the hypothesis that they may have similar effects on the dense desmoplastic tissue within tumours. We here describe the anti-tumour properties of RiXOVA in different PDAC mouse models.

Initial efficacy studies were performed in a xenograft model using the CAPAN-2 cells. RiXOVA was found to be non-toxic and non-immunogenic (I.P, 25mg/kg Q3D10 ) in mice. Relative to the start of dosing, the tumour growth in RiXOVA treated mice was 160% +/-30 (n=10) compared to 240% +/- 61 (n=10, p value=0.001) in the vehicle treated mice. Further testing was carried out in a Genetically Engineered Mouse-Derived Allograft (GEDA). The GEDA model recapitulates the dense desmoplastic stroma of the original donor (KPC mouse), unlike cell line-based allografts. KPC (LSL-KrasG12D; LSL-Trp53R172H; Pdx1-cre) mouse tumour fragments (2 donors) were implanted subcutaneously in the flank of recipient LSL-Trp53R172H; Pdx1-cre (PC), immunocompetent mice. Relative to the start of dosing, the tumour growth in the RiXOVA treated group (25 mg/kg TIW) was 260% +/- 160 (n=10), compared to 540% +/- 210 (n=8) for the Vehicle (p = 0.001) and 130 % +/- 57 (n=9) for the combination of RiXOVA + gemcitabine (100 mg/kg BIW p <0.001) compared to the vehicle. Ex vivo analysis of tumour tissue revealed changes in the expression and distribution of ECM proteins. Collectively, these

results indicate RiXOVA to be a non-toxic oligosaccharide, that acts at the level of the tumour microenviroment to inhibit tumour growth.

Whilst these results are in a preliminary stage they indicate G-block oligomers may represent a novel treatment modality in PDAC.

#628

Immunometabolic signature of papillary thyroid carcinoma.

Vivian L. Weiss, Naira Baregamian. _Vanderbilt University Medical Center, Nashville, TN_.

Introduction: Papillary thyroid carcinoma (PTC) is the most common subtype of thyroid cancer with rapidly rising incidence. The interface between PTC tumor metabolic reprogramming and metabolic immunomodulation remains poorly understood.

Experimental Procedures: We sought to investigate the immunometabolic interaction patterns of PTC tumors and to determine whether a correlative relationship exists between tumor-infiltrating immune cell phenotypes and the expression of tumor metabolic enzymes. We performed a computational immunogenomic analysis to calculate leukocyte fractions within 496 PTC tumors from The Cancer Genome Atlas (TCGA). Immunogenomic analysis was performed using CIBERSORT (Newman AM et al., Nature Methods2015), a deconvolution method to calculate leukocyte fractions using RNA gene expression signatures. The PTC immune cell fractions were then compared to the RNA expression of tumor metabolic enzymes within the TCGA data using regression analysis. Matching targeted metabolomic analysis (Metabolon, Inc.) of 20 human PTC tumors was performed to determine tumor metabolite profiles associated with perturbed tumor cellular metabolic pathways based on the TCGA data analysis.

Results: The immunogenomic and PTC metabolic pathway enzyme expression analysis demonstrated significant correlation between immune cell types and major metabolic pathway enzymes. Specifically, tumor glycolytic enzymes upstream of the TCA cycle correlated with CD8 (hexokinase-1, p<0.0001) and T regulatory (enolase, p<0.0001) cell infiltrates. Enhanced pyruvate cycling into the TCA cycle correlated with the presence of several immune cell types (malic enzyme 2 vs. follicular helper T cells, p<0.0001; pyruvate carboxylase vs CD8 T cells, p<0.0001). The expression of TCA cycle enzymes correlated with the fraction of macrophages (aconitase, p<0.0001; IDH1, p<0.05). Significant differences in PTC tumor and normal thyroid tissue metabolite profiles were observed in glycolysis and TCA cycle, further supporting our findings of immunometabolic plasticity it PTC. Together, these findings highlight the central role TCA cycle activity plays in PTC tumor immunometabolism.

Conclusion: We demonstrate, for the first time, a distinct immunometabolic tumor signature supported by tumor metabolic reprogramming in PTC tumors. This novel characterization approach provides an important foundation for future functional metabolic studies within the tumor and immunologic microenvironment. A mechanistic understanding of the complex integration of immune networks and cellular metabolism can be harnessed for future therapeutic immunometabolic modulation in PTC.

#629

Delivering type I interferon to dendritic cells empowers tumor eradication and immune combination treatments.

Jan H. Tavernier,1 Anje Cauwels,1 Sandra Van Lint,1 Franciane Paul,2 Geneviève Garcin,2 Alexander Van Parys,1 Nikolai Kley,3 Gilles Uzé2. 1 _University Ghent / VIB, Ghent, Belgium;_ 2 _CNRS UMR 5235, Université Montpellier, France;_ 3 _Orionis Biosciences, Ghent, Belgium_.

An ideal generic cancer immunotherapy should mobilize the immune system to destroy tumor cells without harming healthy cells and remain active in case of recurrence. Furthermore, it should preferably not rely on tumor-specific surface markers, as these are only available in a limited set of malignancies. Despite approval for treatment of various cancers, clinical application of cytokines is still impeded by their multiple toxic side effects. Type I interferon (IFN), for instance, has a long history in the treatment of cancer, but its multifaceted activity pattern and complex side effects prevent its optimal clinical use. Here we develop AcTakines (Activity-on-Target cytokines), optimized (mutated) immunocytokines that are up to 1000-fold more potent on target cells, allowing specific signaling in selected cell types only. As conventional Dendritic Cells (cDC) are essential for IFN antitumor efficacy, we targeted type I IFN-derived "AcTaferon (AFN)" to Clec9A+ cDC. Clec9A-AFN therapy displayed strong antitumor activity in murine melanoma (B16), breast carcinoma (4T1) and lymphoma models (A20), as well as against human RL lymphoma in immunodeficient NSG mice reconstituted with a human immune system. In sharp contrast to wild-type IFN therapy, the antitumor efficacy of Clec9A-AFN was not accompanied by any detectable toxicity, assessed by body weight and several hematological parameters. Clec9A-AFN effects were lost in CD8-depleted or Batf3-/- mice, and depended on IFN signaling in cDCs but not in T lymphocytes. Combined with α-PDL1 immune checkpoint blockade, Treg-depleting anti-CTLA4 + anti-OX40 therapy, immunogenic chemotherapy, or low-dose TNF, complete tumor regressions and long-lasting tumor immunity (memory) were obtained, still without any adverse effects. Our findings thus indicate that DC-targeted AFN provides a highly efficient, off-the-shelf and safe cancer immunotherapy, with possible application in a broad range of malignancies.

#630

Novel hexavalent HVEM agonist HERA-LIGHT promotes T cell activation and expansion.

Julian P. Sefrin. _Apogenix AG, Heidelberg, Germany_.

The tumor necrosis factor superfamily (TNFSF) member LIGHT (TNFSF14) plays an important role in regulating the activity of immune cells, especially T cells. Several studies have shown that engagement of HVEM (herpesvirus entry mediator), one of the known receptors of LIGHT, can deliver a co-stimulatory signal to support T cell activation and expansion and promote tumor clearance. The HERA technology platform developed by Apogenix generates fully human hexavalent TNFSF fusion proteins that mimic the natural receptor binding mode in order to co-stimulate T cells. HERA ligands are pure agonists whose signaling capacity does not rely on secondary Fcγ-receptor crosslinking. Here we report the in vitro and in vivo properties of a novel HERA-LIGHT construct. Similar to all HERA fusion proteins, HERA-LIGHT has been engineered as a perfect molecular mimic of the natural ligand with high clustering capacity for the cognate receptor. The core unit consists of a single chain polypeptide comprising the three minimal LIGHT-subsequences necessary for folding into a functional trivalent receptor binding domain (RBD). By fusing a silenced IgG1 Fc-domain as a dimerization scaffold to the C-terminus of the RBD we generated HERA-LIGHT, a hexavalent fusion protein. HERA-LIGHT was expressed in CHO suspension cells followed by a lab-scale purification process including AFC- and SEC-based polishing, resulting in homogenous, aggregate-free protein lots. HERA-LIGHT was proven to bind both the human and murine HVEM receptor, as determined by ELISA. Qualitative analytics revealed excellent stability following heat- and pH-stress as well as freeze-thaw cycles. Analyzing serum samples from a PK study in CD1-mice, the terminal half-life of the compound was 36.5 hours. This short half-life, relative to antibodies, allows for fast-in/fast-out dynamics essential for improving combination therapy and reducing serious side effects associated with immune system overstimulation. In order to test biological activity, T cells were isolated by magnetic sorting from human PBMCs and treated with HERA-LIGHT in vitro. Flow cytometric analysis revealed that HERA-LIGHT enhanced activation and proliferation of naïve effector T cells (Teff) following stimulation with anti-CD3 antibody, as determined by CFSE dilution. Importantly, co-stimulation with HERA-LIGHT prevented regulatory T cell (Treg)-mediated suppression of Teff proliferation. In vivo, treatment with a murine surrogate of HERA-LIGHT resulted in significant tumor growth inhibition in a pilot study using the syngeneic CT-26 colorectal cancer model.In summary, the unique hexavalent design of HERA-LIGHT mediates efficient co-stimulation of Teff even in the presence of Treg cells and independent of secondary crosslinking events. Being true agonists, all HERA molecules are unique from current antibody-based concepts rendering them attractive candidates for cancer immunotherapy.

#631

Intratumoral activation of STING with a synthetic cyclic dinucleotide elicits antitumor CD8 T-cell immunity that effectively combines with checkpoint inhibitors.

Anthony L. Desbien,1 Kelsey Sivick Gauthier,1 Leticia Corrales,1 Gabrielle Reiner,1 Laura Hix Glickman,1 George Katibah,1 Thomas E. Hudson,1 Uyen Vu,1 Natalie H. Surh,1 Brian Francica,1 Weiwen Deng,1 David B. Kanne,1 Justin J. Leong,1 Chudi Ndubaku,1 Ken Metchette,1 Jeffery M. McKenna,2 Steven L. Bender,3 Meredith L. Leong,1 Thomas W. Dubensky Jr.,1 Andrea van Elsas,1 Sarah M. McWhirter1. 1 _Aduro Biotech, Berkeley, CA;_ 2 _Novartis Institutes for BioMedical Research, Cambridge, MA;_ 3 _Genomics Institute of the Novartis Research Foundation, San Diego, CA_.

Activation of the STING pathway by intratumoral (IT) injection of synthetic cyclic dinucleotides (CDNs) induces stable tumor regression in preclinical models, yet the underlying immune correlates are not fully understood. ADU-S100, a CDN under clinical evaluation, was administered IT with an optimized dosing regimen to explore the immune requirements for antitumor efficacy in mouse syngeneic tumor models. We show that CD8+ T cells are necessary and sufficient for durable antitumor immunity elicited by ADU-S100 and that activation of STING in hematopoietic cells mediates CD8+ T cell induction. Both type I IFN and TNFα, which are induced by STING pathway activation, influence the antitumor immune response. The combination of ADU-S100 and anti-PD1 treatment enhances CD8+ T cell-dependent, noninjected tumor control that correlates with an enhanced effector profile of CD8+ T cells in the tumor. Combination of ADU-S100 with checkpoint inhibition also enhances durable immunity in a poorly immunogenic tumor model. Together, these results elucidate the immune correlates to STING-mediated antitumor efficacy and highlight the potential of combining STING agonists with checkpoint inhibition in the clinic.

#632

The metabolic requirements for effective immunotherapy.

Nicole E. Scharping, Ashley V. Menk, Greg M. Delgoffe. _University of Pittsburgh, Pittsburgh, PA_.

Introduction: CD8+ tumor-infiltrating T lymphocytes (TIL) in the tumor microenvironment (TME) are unable to kill their tumor targets due to tumor cell & regulatory immune cell-mediated suppression. Poor metabolite availably has also been found to negatively impact TIL, as T cells have high energetic demands that are not met in the TME due to competition with surrounding tumor cells. Consequently, tumor growth is allowed to progress as TIL becomes exhausted & dysfunctional. Immunotherapy, such as PD-1 checkpoint blockade, has been highly successful at treating many types of human cancer, but the majority of patients do not respond for reasons which are still unclear. Cancer cells have been shown to have heterogeneous, dysregulated metabolism, which could be a barrier to immunotherapy. Therefore, we hypothesize that TIL dysfunction & thus resistance to immunotherapy is driven in part by metabolic insufficiency, & either improving TIL metabolism or normalizing tumor metabolism can lower the barrier to immunotherapy to improve cancer outcomes.

Methods: Metabolic capacity was measured using flow cytometry & flux analysis in murine and human tumor samples. T cell reprogramming was performed by retroviral transduction on antigen-specific T cells before adoptive transfer into tumor-bearing mice. TME reprogramming was performed on tumor-bearing mice that were treated with metformin every other day & anti-PD1 3x weekly.

Results: We found that exhausted TIL exhibit a profound loss of functional mitochondria. This is due in part to repression of the transcriptional co-activator PGC1α resulting in suppressed mitochondrial biogenesis. Enforcing mitochondrial biogenesis in T cells not only led to increased mitochondrial mass, but improved TIL functionality, decreased tumor burden, & increased survival in mouse melanoma. To understand the effects of T cell-extrinsic metabolic insufficiency, we explored the TME factor hypoxia on TIL function. CD8 T cells function poorly in hypoxia, thus causing a possible barrier to function. We first compared the energetics of different murine tumor lines & found that tumor cells with a more deregulated oxygen metabolism (i.e. those that created more hypoxia), tended to be resistant to immunotherapy. When tumor cells were treated with metformin in vivo, a compound that decreases tumor mitochondrial oxygen consumption, overall tumor hypoxia was decreased. Metformin alone did not improve tumor clearance, but when used in combination with PD-1 checkpoint blockade, TIL effector function was dramatically increased, 80% of mice experienced tumor regression, & 70% of mice became tumor-free.

Conclusions: TIL metabolic insufficiency may provide rationale as to why some patients do not benefit from PD-1 monotherapy. Decreasing TIL intrinsic or extrinsic metabolic insufficiency may improve upon existing cancer immunotherapies, as well as allow for development of targeted therapies to greatly improve cancer outcomes.

#632A

Microbiome diversity in sputum and circulating exosomes of women with breast cancer and nontuberculous mycobacterial lung disease.

Julie V. Philley, David E. Griffith, Santanu Dasgupta. _UT Health Science Ctr. at Tyler, Tyler, TX_.

The nontuberculous Mycobacterial lung disease, primarily caused by Mycobacterium avium complex is an alarming health problem in the United States and worldwide. Being prevalent among Caucasian women with a history or current diagnosis of breast cancer, Nontuberculous Mycobacterial lung disease poses a significant challenge towards treatment. A large community of resident microbes, including bacteria, virus, Archeas and Fungi living in the human body are being increasingly recognized as the key components of controlling human health and disease. In this study, we examined microbiome diversity in sputa and circulating exosomes derived from women with both breast cancer and nontuberculous Mycobacterial lung disease, women with nontuberculous Mycobacterial lung infection only and healthy women by 16S rDNA sequencing. Our study identified a diverse microbiome community in the sputa and the exosomes dominated by Streptococcus, Haemophillus, Veillonella, Neisseria, Prevotella, Fusobacterium, Bacteroides, Allistipes, Faecalibacterium and Staphylococcus in women with both breast cancer and nontuberculous Mycobacterial lung disease and nontuberculous Mycobacterial lung disease only. Some of these genera, including Fusobacterium, Bacteroides, Allistipes have estrobolome activity and are associated with the development of breast and other neoplasm. This work confirms the presence of a distinct microbiome other than the Mycobacteriome in the sputa and the circulating exosomes of these patients. Further studies investigating the role of the sputa and exosome microbiome linking nontuberculous Mycobacterial pathogenesis and breast cancer are warranted. 

### Molecular Diagnostics 1: Cytogenetics, Clinical Molecular Genetics, and Clinical Imaging

#633

Application of a novel analytical metric that quantifies copy number alterations in low-coverage, genome-wide sequencing of cell-free DNA to monitor and differentiate response to immunotherapy in cancer patients.

Taylor J. Jensen,1 Aaron M. Goodman,2 Christopher K. Ellison,1 Shumei Kato,2 Gregory A. Daniels,2 Lisa Tran,2 Prachi Nakashe,1 Erin McCarthy,1 Amin R. Mazloom,1 Graham McLennan,1 Daniel S. Grosu,1 Mathias Ehrich,1 Razelle Kurzrock2. 1 _Sequenom, Inc., San Diego, CA;_ 2 _Center for Personalized Cancer Therapy, University of California, San Diego, Moores Cancer Center, San Diego, CA_.

Inhibitors of the PD-1/PD-L1/CTLA4 immune checkpoint pathway have transformed the cancer treatment paradigm. It has been observed that even some patients with advanced, refractory malignancies achieve durable responses; however, only a minority of patients benefit, demonstrating the importance of developing new biomarkers to predict patient outcome. The most commonly used biomarkers for predicting response to checkpoint inhibitors are the expression of PD-1/PD-L1, microsatellite instability, and tumor mutational burden. While these markers have been shown to have varying degrees of predictive power, they have not been used to monitor and differentiate response during treatment and require invasive procedures. Interrogating cell-free DNA (cfDNA) isolated from plasma (liquid biopsy) provides a promising noninvasive method for monitoring response. We describe the use of low-coverage (~0.3X) genome-wide sequencing of cfDNA, validated extensively for detecting chromosomal abnormalities in non-invasive prenatal testing (NIPT) and previously shown to enable incidental detection of occult maternal malignancies, to detect tumor-specific copy number alterations; and the development of a new metric--the genome instability number (GIN)--to monitor response to these drugs. Using a series of more than 450 plasma aliquots taken from more than 75 patients throughout their treatment, we demonstrate how the GIN can be used to discriminate clinical response from progression, differentiate progression from pseudoprogression, and identify hyperprogressive disease, as early as 4-6 weeks after treatment initiation. Finally, we provide evidence for a delayed therapeutic response to checkpoint inhibitors relative to targeted therapies. While this study is still ongoing, these initial data provide proof of concept for using this method for monitoring treatment outcome in cancer patients receiving immunotherapy.

#634

SOX11 is a potential clinical marker for hormone receptor negative ductal carcinoma in situ.

Kazuharu Kai, Wei Lu, Fei Yang, Ximing Tang, Ignacio I. Wistuba, Subrata Sen, Alastair Thompson. _UT MD Anderson Cancer Ctr., Houston, TX_.

Background: Ductal carcinoma in situ (DCIS) comprises 20-25% of screen-detected breast cancer and, like invasive ductal carcinoma (IDC), is heterogeneous in terms of underlying biology, presentation and outcome. There are limited potential biomarkers of outcome for DCIS, although estrogen receptor (ER)-positive (pos), progesterone receptor (PgR)-pos, and HER2-negative (neg) DCIS appears to have a better outlook than DCIS neg for ER and PgR but pos for HER2. The aim of this study was to identify markers for DCIS to develop a panel to stratify DCIS into DCIS at low or high risk of further DCIS.

Methods and results: To identify genes driving DCIS evolution followed by the progression to IDC, we first used transcriptional data sets (GSE788, GSE16873) which had data from both normal mammary glands (NMG) and DCIS and performed class comparison (NMG vs DCIS). For both GSE7882 and GSE16883, the number of over- and under-expressed genes was 297 and 187. Over-expressed genes in DCIS represented a mitotic/proliferative feature annotated as mitotic spindle and condensed chromosome. Under-expressed genes represented loss of epithelial features annotated as epithelial cell differentiation and development. A total of 484 differentially expressed genes in DCIS were further correlated with recurrence events of IDC using Kessler's breast cancer data set to identify genes contributing aggressive feature across DCIS and IDC. Genes correlating to recurrence events were selected. From 484 genes, 99 were significantly associated with recurrence events of IDC (with P<0.003). Among these 99 genes, proliferation-related genes were depleted but component genes of the Oncotype DCIS score and genes reported as relevant to DCIS biology, such as SOX11, were included for Nanostring transcriptional analysis. The final number of genes-of-interest was 58 including 5 housekeeping genes. 40 DCIS and 8 NMG lesions were macro-dissected from formalin-fix paraffin-embedded blocks (FFPE) and extracted transcripts were subjected for Nanostring analysis. Gene expression data was clustered in an unsupervised manner using R software. Two sample clusters were identified: an ER/PgR-neg cluster and an ER/PgR-pos cluster. Over-expression of SOX11 and HER2 were exclusively seen in the ER/PgR-neg cluster within which samples were further subcategorized into HER2low/SOX11+ and HER2high/SOX11+. These RNA expression findings are undergoing confirmation by immunohistochemistry of FFPE sections. A further independent series of DCIS which has recurred as DCIS or IDC is currently under investigation for validation of an ER/PgR/HER2/SOX11 signature of poor prognosis DCIS.

Conclusion: SOX11 is exclusively overexpressed in ER/PgR-neg DCIS and is a candidate clinical marker for recurrence of DCIS or progression of ER/PgR-neg DCIS to IDC.

#635

Characterizing PD-L1 expression in tumor-associated cells and CTC using IncellDx and Celsee molecular diagnostic technologies.

Fariba Fazeli,1 Vishal Premdev Sharma,2 William Chiro Chow,2 Amanda Chargin,1 Priya Gogoi,2 Kalyan Handique,2 Bruce Patterson1. 1 _IncellDx,Inc., El Camino, CA;_ 2 _Celsee, Inc., Plymouth, MI_.

Detection of PD-L1 expression in NSCLC cancer patients is increasingly becoming a widely used molecular diagnostic tool. Evidence also suggests that PD-L1 can be induced with radiotherapy and may be an immune escape mechanism in cancer. Current methods analyze PD-L1 expression in either solid tumor biopsies or circulating tumor cells (CTC), independent of each other. In order to design a comprehensive PD-L1 treatment regime, it is imperative to look at the PD-L1 expression in cancer cells of the tumor as well as in CTCs. In this study we present a comprehensive profiling of PD-L1 expression in solid tumor biopsy and CTC of 10 lung cancer patients. We further extend the study by evaluating the co-relation of the PD-L1 expression with other immune cell infiltration markers in the lung biopsy samples. There is no single technology that can provide a comprehensive profiling of PD-L1 expression in lung biopsies as well as the CTCs isolated from the peripheral blood. Using two very different and unique molecular diagnostic technologies, IncellDx OncoTect iO Single Cell Quantitative PD-L1 assay and automated CTC analysis Celsee C-PREP GENESIS, we present the first report discussing the PD-L1 expression in tumor cells of the solid and liquid biopsies and its correlation with DNA content and infiltrating immune cell markers. Nine out of the ten patient samples evaluated showed strong correlation between DNA content and PD-L1 expression in tumor-associated cells and CTC. One aneuploid patient sample showed very low expression of PD-L1 in tumor-associated cancer cells; however, it still showed a positive staining for PD-L1 in CTC. Obtaining a comprehensive PD-L1 expression and associated immune cell marker profile in solid tumor biopsy and circulating tumor cells may be needed to design a more effective and lasting immunotherapy to treat NSCLC lung cancer patients. The results discussed in this study will strongly impact the current understanding and implementation of the molecular diagnostics tools to evaluate cancer patients in order to define a robust therapeutic regimen.

#636

Accounting for processed pseudogene-related artifacts improves specificity of clinical cancer diagnostic sequencing.

Carlo G. Artieri, Marcin Sikora, Alex Artyomenko, Elena Helman, Darya Chudova, Richard Lanman, AmirAli Talasaz. _Guardant Health, Redwood City, CA_.

Background: Duplicated genomic regions pose a challenge to accurate variant calling in clinical sequencing applications as duplicate-specific variants may incorrectly be assigned to the target. One source of duplicated coding sequences are processed pseudogenes (PPGs) that can originate from LINE-mediated reverse transcription and genomic integration of processed mRNA, resulting in partial or complete copies of the original gene, lacking intronic sequences. False-positive variants resulting from pseudogenes found in the reference genome, such as those of PIK3CA and PTEN, have been well studied; however, the recent discovery of rare and even individual-specific cancer-related PPGs demonstrates a need for more systematic interrogation and mediation of PPG-related clinical artifacts on a sample-by-sample basis. Methods: We analyzed germline and somatic alterations in over 15,000 clinical circulating cell-free DNA samples sequenced using a 73-gene panel (Guardant Health, CA) focused primarily on clinically-relevant somatic alterations. All targeted genes were interrogated for two key hallmarks of PPGs: 1) split sequencing reads mapping to adjacent exons without intervening intronic sequence and 2) atypically increased sequencing coverage of exons relative to adjacent introns.Results: Applying a strict cutoff of ≥5 unique molecules supporting any putative intron-skipping event, we observed evidence of 94 PPGs in 91 of 15,315 analyzed samples (0.59%), affecting SMAD4 (57 samples), CDK4 (20), GNAS (6), HRAS (2), KRAS (1), TP53 (1), ESR1 (1), STK11 (1), RB1 (1), MAPK3 (1), MAPK1 (1), NF1 (1), and GATA3 (1). To determine the impact of PPGs on specificity, we applied a resampling test to assess whether somatic variant calling without PPG aware detection of SNVs results in over-representation of calls near splice junctions in samples harboring PPGs. Indeed, SMAD4 somatic SNVs were identified in 30% of samples containing SMAD4 PPGs as compared to an expected 0.7% of non-PPG samples (two-tailed p < 10E-4). Similarly, 10% of samples containing CDK4 PPGs also showed somatic CDK4 SNVs, whereas only 0.9% would be expected by chance (two-tailed p = 0.03). These results strongly suggest that PPG-related artifacts may be misclassified as true somatic events in these contexts. Algorithmically restricting variant-calling only to those reads containing intronic sequence can maintain high specificity with only minimal cost to sensitivity in most exons. Conclusions: Our analysis reveals the existence of rare PPGs derived from over a dozen common clinical cancer sequencing targets and provides strong evidence that these may produce false positive SNV calls in an appreciable number of cases. Importantly, we have also shown that PPGs can be detected in a sample-specific manner based on the presence of intron-skipping split reads, enabling algorithmic detection and elimination of artifacts.

#637

Quantitative screening of serum protein biomarkers with reverse phase protein arrays.

Zhizhou Kuang,1 Ruochun Huang,1 Zhiqiang Lv,2 Jingqiao Lv,1 Jian Wu,3 Ruo-Pan Huang1. 1 _RayBiotech, Inc., Norcross, GA;_ 2 _RayBiotech, Inc., Guangzhou, China;_ 3 _The Third Affiliated Hospital, Sun Yat-Sen University, Guangzhou, China_.

Screening biomarkers in serum samples for different diseases has always been of great interest because it presents an early, reliable, and, most importantly, noninvasive means of diagnosis and prognosis. Reverse phase protein arrays (RPPAs) are a high-throughput platform that can measure single or limited sets of proteins from thousands of patients' samples in parallel. They have been widely used for detection of signaling molecules involved in diseases, especially cancers, and related regulation pathways in cell lysates. However, this approach has been difficult to adapt to serum samples. Previously, we developed a sensitive method called the enhanced protein array to quantitatively measure serum protein levels from large numbers of patient samples. Here, we further refine the technology on several fronts: 1. simplifying the experimental procedure; 2. optimizing multiple parameters to make the assay more robust, including the support matrix, signal reporting method, background control, and antibody validation; and 3. establishing a method for more accurate quantification. Using this technology, we quantitatively measured the expression levels of 6 proteins: AFP, GP73, GDF15, IGFBP2, B2M and OPN from serum samples of 132 hepatocellular carcinoma (HCC) patients and 78 healthy volunteers. We found that all 6 protein expression levels are significantly increased in HCC patients. Statistical and bioinformatical analysis has revealed decent accuracy rates of individual proteins, ranging from 0.617 (B2M) to 0.908 (AFP) as diagnostic biomarkers to distinguish HCC from healthy controls. The combination of these 6 proteins as a specific HCC signature yielded a higher accuracy of 0.923 using LR, LDA, RF and SVM predictive model analyses. Our work reveals promise for using reverse phase protein arrays for biomarker discovery and validation in serum samples.

#638

Microbubble enrichment of CTCs.

Thomas Jones,1 Leo J. Ostruszka,1 Shamileh Fouladdel,2 Ebrahim Azizi,2 Jaroslav Slamecka,3 Brandon H. McNaughton,1 John Younger,1 Max Wicha,2 Steven McClellan3. 1 _Akadeum Life Sciences, Ann Arbor, MI;_ 2 _University of Michigan, Ann Arbor, MI;_ 3 _Mitchell Cancer Institute, Mobile, AL_.

Despite numerous new cell analysis technologies, contaminating red- and white blood cells remain a central barrier to the efficient characterization of circulating tumor cells. Microbubbles, which capture and separate cells by flotation, recently have been described as an alternative or adjunct to magnetic beads or microfluidic methods for CTC sample preparation. This technology may find particular value in applications where mechanical force needs to be minimized or magnetization of cells needs to be avoided. Here, we evaluate novel RBC-targeting microbubbles for buoyancy-based enrichment of CTCs. Cultured human pancreatic cancer cells (line CFPAC-1, ATCC CRL 1918) were spiked into standardized samples representative of common CTC workflows (106 RBC : 5x104 WBC : 1.5x102 tumor cells). Microbubbles bearing antibodies to human erythrocytes were added at various Bubble:RBC ratios and mixed via trituration for 1 minute. Samples were then centrifuged, the microbubble-captured RBCs removed, and the remaining pelleted cells analyzed for the fraction of RBCs removed and the extent of off-target depletion of tumor cells. Cells were quantified with an automated cell counter, and all experimental conditions were performed in at least triplicate. RBC removal rose with increasing Bubble:RBC ratio ( p < 0.001 ). At a ratio of 1:1, 92 +/- 1% of RBCs were removed with one minute of mixing; at a ratio of 1.5:1, 97 +/- 1% of RBCs were removed. Off-target binding of CFPAC-1 cells was minimal at a Bubble:RBC ratio of 1:1, with 0 +/- 6% of tumor cells captured. Off-target capture increased at a Bubble:RBC ratio of 2:1, with 12 +/- 9% tumor cell loss from the experimental samples. Using a standardized blood sample with a clinically relevant low concentration of cultured tumor cells, microbubbles targeting RBCs and dosed at a ratio of one bubble per target erythrocyte were able to achieve > 90% removal without statistically detectable loss of a cultured cancer line. Higher bubble concentrations resulted in more exhaustive RBC removal, but with some loss of cultured cells. Microbubble-based cell separation may have a useful role in CTC purification protocols.

#639

Salt-inducible kinase 3 expression identifies long-term survivors of serous ovarian cancer.

Keng-Fu Hsu,1 Yu-Ling Liang,1 Chin-Han Wu,1 Neng-Yao Shih2. 1 _National Cheng Kung Univ. Hospital, Tainan, Taiwan;_ 2 _National Health Research Institutes, Tainan, Taiwan_.

Background: Epithelial ovarian cancer (EOC) has a high tumor-associated mortality rate among the gynecological cancers because EOC is usually diagnosed during the advanced stage. Cancer antigen (CA) 125 is the most well-studied biomarker for ovarian cancer screening. However, CA125 is also elevated in numerous conditions, resulting in decreased specificity. Materials and Method: We collected ovarian cancer specimens from 204 patients and examined the SIK3 and CA125 expression levels. The expression percentage of SIK3 or CA125 in the tumor cells, pre-surgery serum CA125 was divided into two groups and compared with patient overall survival, progression-free survival, cancer stage, cancer type and chemotherapeutic resistance. Results: A total of 30.9% of the patients had stage I disease, 7.8% had stage II disease, 52.4% had stage III disease, and 8.9% had stage IV disease. The median OS and PFS were 49.5 months (range 0.25-205 months) and 28.4 months (0.25-182.4), respectively. The mean age of the patients was 52.8 years (range 25-82 years). The histological classification was based on the four main ovarian cancer types: serous (51.5%), endometrioid (14.2%), clear cell (20.6%), and mucinous (9.3%). In all stages of the disease, high expression of SIK3 (SIK3-H) was associated with a markedly better OS, compared with low expression of SIK3 (SIK3-L) (127.0 months and 46.0 months, respectively; hazard ratio [HR] for death, 0.59; 95% confidence interval [CI], 0.41 to 0.86; P=0.005). SIK3-H had a markedly better PFS rate than SIK3-L (66.0 months and 26.0 months, respectively; HR for disease progression or recurrence, 0.68; 95% CI, 0.47 to 0.99; P=0.04). The same results were evident for serous ovarian cancer, where SIK3-H had a better OS rate than SIK3-L (75.0 months and 34.0 months, respectively; HR for death, 0.57; 95% CI, 0.36 to 0.91; P=0.02) and a better PFS rate than SIK3-L (44 and 13 months, respectively; HR, 0.52; 95% CI, 0.32 to 0.83; P=0.006). Notably, for advanced stage serous ovarian cancer, Only SIK3-H had a better OS rate than SIK3-L (48.0 months and 28.0 months, respectively; HR for death, 0.56; 95% CI, 0.33 to 0.96; P=0.03) and PFS rate (32 and 13 months, respectively; HR, 0.57; 95% CI, 0.34 to 0.95; P=0.03). Tissue CA125 expression and the pre-surgery serum CA125 level did not show significant differences in OS and PFS. Our results demonstrated that high SIK3 expression correlated with better OS and PFS rates in ovarian cancer patients, especially in patients with advanced serous ovarian cancer. Conclusion: High expression of SIK3 indicates a better prognosis in primary ovarian cancer and serous disease, especially in advanced serous disease.

#640

Histopathologic and molecular features in a subset of pilocytic astrocytomas in a tertiary African hospital.

Michael C. Nweke,1 Gabriel O. Ogun,1 Clement A. Okolo,1 Amos D. Adeleye,1 Adekunle M. Adesina2. 1 _University College Hospital, Ibadan, Ibadan, Nigeria;_ 2 _Texas Children's Hospital, Houston, TX_.

Background: Pilocytic astrocytomas are WHO grade I astrocytomas. Worldwide, they have been documented to be the second most common gliomas after WHO grade IV glioblastomas. Definitive diagnosis may be made with traditional hematoxylin and eosin. Increasingly, a number of cases are atypical deceptive in clinical and histopathologic presentation, requiring ancillary immunohistochemical and molecular studies. This study evaluated a subset of pilocytic astrocytomas at the University College Hospital Ibadan. Nigeria.

Methods: All cases of Pilocytic Astrocytomas were identified retrospectively within our institutional database over a 12-year period. Inclusion criteria was by availability of the FFPE block. Astrocytic origin was confirmed by GFAP immunohistochemistry; previously stained hematoxylin and eosin slides were reviewed with newly re-sectioned hematoxylin and eosin stained slides along with ancillary Ki-67/MIB immunohistochemistry labelling index. ATRX immunohistochemistry was carried out on all re-sectioned slides. Tissue scrolls were taken for DNA extraction and pyro-sequencing for BRAFV600E BRAF mutation using a Qiagen pyrosequencer. This work was carried out in the molecular neuropathology laboratory at the Texas Children's Hospital, Houston Texas. Results were analyzed using SPSS version 24.

Results: 40 cases (30.7%) of all astrocytic neoplasms were documented over this 12-year period. 23(57.5%)of these cases were suitable for analysis in this study. Males accounted for 65.2% of cases. Male: female ratio was 1:0.5. The mean age of all the cases was 14.22+/- 12.90 years, median age of 8 years with an age range of 42 years. 65.2% of all cases were within the 0-15-year age group, 17.4% were within the 16 - 30-year age group while the remaining 17.4% were within the 31 - 45-year age group. Most cases occurred infratentoriallysupratentorially (69.6%). The cerebellum was the commonest location accounting for 47.8%, followed by the supra-sellar region (17.4%). Most cases were centrally sited (47.8%). All cases (100%) showed positive ATRX expression. 3 cases (20%) of BRAF V600E expression were recorded. This included 1 case with malignant/anaplastic features which was previously misdiagnosed as a glioblastoma with suggestive radiologic imaging. Mean age of tumors with BRAF positive expression was 13.95 +/- 12.95years, while the mean age for BRAF negative cases was 17.00 +/-16.97 years. The difference between these ages was not statistically significant.

Limitations: The quality of available blocks limited DNA amplification and further analysis in 8 of the cases.

Conclusion: This study aims to underscores the importance of BRAF V600E mutations among pilocytic astrocytomas in our environment with regards to its diagnostic and prognostic significance.

#641

Cell free DNA in the supernatant of pleural effusion can detect driver and resistance mutations and can guide TKI treatment decisions.

Karlijn Hummelink,1 Mirte Muller,1 Dorothe Linders,1 Vincent van der Noort,1 Petra Nederlof,1 Sjaak Burgers,1 Gerrit Meijer,1 Michel van den Heuvel,2 Daan van den Broek,1 Kim Monkhorst1. 1 _Netherlands Cancer Institute, Amsterdam, Netherlands;_ 2 _Radboud University Medical Center, Nijmegen, Netherlands_.

Introduction:

Molecular profiling of tumors has become the mainstay of diagnostics for metastasized solid malignancies and guides personalized treatment, especially in non-small cell lung cancer (NSCLC). In current practice it is often challenging to obtain sufficient tumor material for reliable molecular analysis. Cell free (cf) DNA in blood or other bio-sources could present an alternative approach to obtain genetic information from the tumor. In a retrospective cohort we analyzed the added value of cfDNA analysis in pleural effusions for molecular profiling.

Methods:

We retrospectively analyzed both the supernatant and the cell pellet of 44 pleural effusions sampled from 39 patients with KRAS (23) or EGFR (16) positive tumors for the original driver gene mutation as well as for EGFR T790M resistance mutations. Patients were diagnosed with either NSCLC (32), colon carcinoma (4), appendiceal carcinoma (2) or adenocarcinoma of unknown primary (1). Samples collected in the context of routine clinical care were stored in the NKI-AVL biobank. We used Bio-Rad QX200 droplet digital PCR for analysis.

Results:

The original driver gene mutation could be detected in 37 of the 44 pleural effusions by analysis of both supernatant (35/44 positive) and cell pellet (29/44 positive). In 7 out of 20 pleural effusions from patients with EGFR mutation positive tumors, a T790M mutation was detected. All 7 supernatants were positive as were 5 of the 7 cell pellets. The EGFR T790M mutation was confirmed in all supernatants (4/4) and in 3 of the 4 cell pellets sampled from patients with T790M positive tumors (4).

Conclusions:

Cell free DNA in pleural effusion proved to be a valuable bio-source and can be used to detect driver gene mutations as well as resistance mechanisms like EGFR T790M in pleural effusion.

#642

**Comparison between in situ hybridization and digital polymerase chain reaction methods for detecting** c-MYC **gene copy number gain in tissue and cell-free plasma samples of colorectal cancer patients.**

Soo Hyun Seo, Kyu Sang Lee, Soo Kyung Nam, Kyoung Un Park, Heung-Kwon Oh, Duck-Woo Kim, Sung-Bum Kang, Hye Seung Lee. _Seoul National University Bundang Hospital, Seongnam, Republic of Korea_.

Introduction: We focused on the utility of the droplet digital polymerase chain reaction (ddPCR) for detecting c-MYC gene copy number (GCN) gain in cell-free plasma and tumor tissue of CRC patients.

Methods: c-MYC GCN was determined by using dual-color silver in situ hybridization (SISH) and ddPCR in retrospective cohort of 193 CRC patients (cohort 1) and prospective cohort of 64 CRC patients (cohort 2). Additionally, c-MYC GCN was analyzed by ddPCR method in cell-free plasma samples of cohort 2.

Results: In cohort 1, c-MYC GCN gain, defined as mean c-MYC copies/nucleus ≥ 4.0 in SISH analysis, was observed in 34 (17.5%), and c-MYC GCN gain by ddPCR method in 7 (3.6%). c-MYC GCN by SISH was significantly correlated with ddPCR results (ρ= 0.532, P < 0.001). Forty cases (20.7%) showed intratumoral genetic heterogeneity, but it did not affect the concordance between SISH and ddPCR results (P > 0.05). c-MYC GCN gain by both SISH and ddPCR methods had the worst prognosis (P = 0.001). In cohort 2, c-MYC GCN by SISH was significantly associated with tissue ddPCR (ρ=0.349, P = 0.005), but not with plasma ddPCR (P = 0.620). Intratumoral genetic and regional heterogeneity of c-MYC status have been found in 29 (45.3%) and 8 (12.7%). c-MYC status in plasma was associated with tissue ddPCR, but borderline statistical significance (ρ= 0.246, P = 0.050). Conclusions: Our findings suggested that results by SISH and ddPCR methods were discordant. Therefore, detecting method should be carefully selected to determine c-MYC GCN status.

#643

**An ultra-sensitive multiplex allele-specific real-time PCR (Udx-PCR) assay for detection of** KRAS **/** BRAF **/** NRAS **mutations in colorectal cancer.**

Jianxin Zhai,1 Youmin Wu,2 Xuan Luo,1 Xinsheng Li,1 De-Hua Yu1. 1 _USK Bioscience, Shenzhen, China;_ 2 _Xiaolan Renmin Hospital, Zhongshan, China_.

RAS and BRAF gene mutation assessment is essential for therapy decision in metastatic colorectal cancer patients with anti-EGFR monoclonal antibody treatment. However the currently approved assays in clinic is mainly focusing on tissue samples with detecting sensitivity lower than 1% in 10ng genomic DNA background. The emerging analysis of ctDNA offers advantages in clinical diagnosis as a feasible method, which requires much more sensitive methodology due to low abundance in plasma samples. Herein, we developed a multiplex allele-specific real-time (ARMS) PCR assay for detection fourteen mutations of KRAS/BRAF/NRAS in an 8-tube PCR strip. The reagent and format of mutation detection were particularly optimized to reach high specificity and sensitivity. Performance evaluation showed that the assay is remarkably sensitive with lower detection limit of 0.1% in a background of 10ng wild-type genomic DNA. A total of 136 CRC FFPE samples were screened by using the assay. Sixty-two samples were positive for KRAS/BRAF/NRAS mutations, and fifty-eight were confirmed by the Sanger sequencing. The four discrepant samples were tested further by ddPCR and the results revealed that the allelic frequencies in these samples were as low as 0.1% to 5%. The evaluation of ctDNA detection is being carried in metastatic colorectal cancer patients. The multiplex allele-specific real-time PCR assay provides a rapid, reliable and low-cost diagnostic tool for accurate detection of KRAS/BRAF/NRAS mutations for clinical application.

#644

Validation of PD-L1 staining by IHC in solid tumors: A journey from xenograft model to ex vivo culture of clinical samples.

Xiaoqian lin,1 Jennifer Carlson,2 Megan Quast,2 Yuliang Sun,2 Tyler Jepperson,2 Casey Williams,2 Luis RojasEspaillat,2 Benjamin Solomon,2 David Starks,2 Amy Krie,2 Pradip De,2 Raed Sulaiman,2 Nandini Dey,2 Brian Leyland-Jones2. 1 _Avera Cancer Institute, Sioux falls, SD;_ 2 _Avera Cancer Institute, Sioux Falls, SD_.

Background: Blockade of tumor immune evasion by immune checkpoints inhibitors (ICI) which target PD-L1/2 and PD-1 signaling axis ensured durable clinical benefit in solid tumors including melanoma, Non-Small Cell Lung Cancer, and colorectal cancers which led to the approval of Atezolizumab, Nivolumab, and Pembrolizumab. Studies revealed that response to PD-1/PD-L1 blockade might correlate with PD-L1 expression in tumor cells (Herbst et al., Nature 2014; Robert et al., NEJM 2015a, 2015b; KEYNOTE-001; -012; -040 Trials). Currently, assays for the prognostic/predictive role of tumor PD-L1 expression are "not standardized with respect to either quantity or distribution of expression" (McLaughlin et al., JAMA Oncol. 2016).

Objective: We aimed to standardize, validate and set up criteria for the evaluation of PD-L1 protein expression using conventional immunohistochemistry (IHC) staining to demonstrate PD-L1 protein distribution in cultured ex vivo tumors obtained from surgically resected tumor samples.

Methods: We used cell lines, their xenografts tumors, and Formalin-Fixed Paraffin-Embedded tumor samples from patients with different solid tumors to compare results from two different PD-L1 antibodies (Rb Anti-Human PD-L1/CD274 Mono-Ab Clone SP142 from SPRING BIOSCIENCE CATALOG# M4420; Mono-Mouse Anti-Human PD-L1 [Concentrate] Clone 22C3 from DAKO Code: M3653).TNBC (MDA-MB231, MDA-MB468, SUM149) and NSCLC (H1975 and H2228) cell lines, human term placenta and tonsil whole tissue sections were used for antibody validation. PD-L1 protein expression in tumor and stroma was assessed using diaminobenzidine chromogenic IHC in FFPE sections/ fixed cells. Tumor-infiltrating lymphocytes (TILs) in tissue and ex vivo samples were scored in hematoxylin-eosin slides using current consensus guidelines. Finally, we validated PD-L1 expression in ex vivo cultured live tumor samples from patients with lung cancers.

Results: With both PD-L1 antibodies, we find that PD-L1 IHC expression was heterogeneous. When validated using human tonsil sections, a focal and sporadic predominantly membranous positivity was observed in xenograft tissues and tumor cells. Both antibodies showed limited concordance, significant intra-assay heterogeneity, and significant interassay discordance. We also observed a markedly high non-specific staining in both necrotic tumor areas and scars.

Conclusion: We had standardized and validated IHC method for the PD-L1 protein expression in (1) xenografts of several breast cancer cell lines, (2) FFPE from different solid tumors and (3) finally in ex vivo cultured tumors from lung cancer patients. Expression of PD-L1 is currently being correlated with TILs / inflammatory response in tumors the result of which will be presented at the meeting. This evaluation of PD-L1 expression based on ex vivo model may help to stratify patients for the treatment with ICI.

#645

Targeted next generation sequencing in molecular diagnostics of solid tumors.

Greta Grassini, Valeria De Pascali, Ilaria Francaviglia, Gilda Magliacane, Elena Dal Cin, Anna Talarico, Chiara Iacona, Claudio Doglioni, Lorenza Pecciarini, Maria Giulia Cangi. _San Raffaele Hospital, Milano, Italy_.

Cancer genomics role in patients management underlines the importance of rigorous validation of molecular diagnostics approaches to guide treatment decisions. Nowadays only next generation sequencing (NGS) assays can satisfy the need to match a comprehensive tumor molecular profile to the right therapies. Nevertheless, given the complexity, cost, and outcome implications for patients, a clinically valid NGS assay requires careful consideration of gene panel size, inclusion of appropriate markers, ability to detect multiple genomic aberration types, performance with low quality/quantity of nucleic acids, and workflow feasibility. In this study we describe the introduction of the Oncomine Comprehensive Assay (OCA) into a Molecular Pathology diagnostic framework. The OCA analyzes 143 genes of which 73 oncogenes are interrogated for mutational hotspots and 26 tumor suppressor genes for all exons. In addition, the OCA detects copy number variations in 49 genes and fusion drivers in 22 genes. Multiplexed PCR-based DNA and RNA libraries were sequenced on Ion S5 and data analysis was performed by the Torrent Suite using the Oncomine pipeline. The OCA was clinically validated analyzing a cohort of 200 tumor samples of 4 solid tumor types (100 Non Small Cell Lung Cancers, 40 Colon Adenocarcinomas, 40 Pancreatic Adenocarcinomas, 20 Esophageal Adenocarcinomas), for which routine molecular analysis was available. We successfully identified relevant somatic point mutations, indels, and high-level CNAs: TP53, KRAS and EGFR were the genes most frequently mutated though alteration frequencies varied among different tumor types, as expected. OCA DNA profiling demonstrated 95% concordance with standard methods for detecting commonly targeted somatic variants with highly concordant observed variant allele frequencies for detection of KRAS, EGFR and BRAF mutations. Also high-level CNAs strongly correlated with available routine standard tests. The concordance of OCA analysis and standard tests at the RNA level was definitely lower (80%): a subset of 45 lung samples were tested for ALK, RET, ROS1 rearrangements by OCA RNA assay and we successfully detected 15 of the 24 expected fusions, indicating the feasibility of identifying them by this NGS panel, but also stressing the limits of a targeted approach to detect widely variable breakpoints in the analyzed gene fusions. In order to detect all the ALK, ROS1, and RET fusions variants, we perform the parallel validation of the Archer FusionPlex RNA-based ALK, RET, and ROS1 gene fusion assay in 8 lung adenocarcinomas. Results are under evaluation. We conclude that the OCA DNA analysis can be a valuable tool in solid tumor molecular diagnostics being able to identify both common and additional relevant variants beyond current routine practice, and represents a broadly applicable targeted NGS assay which allows advanced precision oncology for different solid tumors types.

#646

Immune checkpoints and inflammation in colon tumors from African Americans.

Jenny E. Paredes,1 Ping Ji,2 Maria Munoz-Sagastibelza,1 Sayed Imtiaz,1 Kaylene Barrera,1 Raavi Gupta,1 Maksim Agaronov,3 Henry Talus,1 Jovanny Zabaleta,4 Jennie Williams,2 Laura Martello-Rooney1. 1 _SUNY Downstate Medical Ctr., Brooklyn, NY;_ 2 _Stony Brook University, Stony Brook, NY;_ 3 _Kings County Hospital, Brooklyn, NY;_ 4 _Louisiana State University Health Sciences Center, New Orleans,, LA_.

Colorectal cancer (CRC) is the third most common cancer among African Americans (AA) and when compared to Caucasian Americans (CA), they present more advanced CRC disease and lower survival rates. Recent findings suggest that this may be related to the differential expression in genes linked to inflammation and immune response. Therefore, we aimed to investigate if tumors from AA colon cancer patients diverge in their immunologic profile from CA and if the immune response of a CRC cell line derived from an AA tumor will differ from a CA CRC cell line. Additionally, we are recording the genetic profiles of colon tumors and outcomes from AA patients at our institution.

Methods: Using DESeq2 we evaluated the differential gene expression pattern by whole transcriptome sequencing (Illumina) of 10 CRC tissues (and matching adjacent non-tumor tissue) from both AA and CA individuals. We focused on genes involved in immune checkpoints and inflammation. We also examined the secretion of Interleukin 8 (IL-8) in plasma from our AA CRC patients. For the in vitro experiments, we used the AA tumor-derived colon cancer cell line SB-521, generated in Dr. Williams' laboratory, and the CA colon cancer cell line HT-29 to determine if the cell lines expressed the Programmed death-ligand 1 (PD-L1). Lastly, we analyzed the microsatellite (MSI) status and MMR mutations in tumors from AA colon cancer patients at our institution and correlated their genetic analysis to response to chemotherapies and survival.

Results: The genomic data revealed that AA and CA tumors had a significant difference of expression in a total of 221 genes. Remarkably, some of these genes included PD1, IL1B, IL17A, IL10, IL5, CD80 and FOXP3. The cytokine IL-8 concentration detected by ELISA in plasma of these patients revealed a differential expression between early stages (I, II) and late stages (III, IV). As hypothesized, the MSI and AA tumor-derived cell line SB-521 expressed PD-L1 and showed an increase in protein levels in response to TNF-α treatment (the CA cell line HT-29 did not express PD-L1). Lastly, our retrospective data (N=200 patients) demonstrated that up to 20% of our AA colon cancer patients have MSI and/or MMR mutations.

Conclusions: Altogether, our results suggest that the immune profiles of the tumors from AA patients differ from CA and these differences could be used as biomarkers and to guide therapeutic strategy for these populations. Also, since the AA cell line presented distinct inflammatory patterns and when compared to the HT-29 CA cell line, it is a potential model to study MSI and PD-L1 in AA. Hence, we aim to supplement our preliminary data on AA patients with MSI and MMR mutations at Downstate and to elucidate what other genomic differences exist and cytokines' secretion patterns observed. In conclusion, we will address the immune and molecular biology of CRC tumors in AA through genomic and in vitro studies, and generate patient's data on AA diagnosed with colon cancer.

#647

Identification of novel and known hotspot variants in African Americans with colorectal cancer.

Hassan Brim, Payaam Tavakoli, Mohammad Reza Daremipouran, Babak Shokrani, Edward Lee, Mehdi Nouraie, Adeinko O. Laiyemo, Hassan Ashktorab. _Howard Univ., Washington, DC_.

Background: The recent introduction of the cancer hotspot panel can help enhance targeted cancer therapy, but requires further refinement to identify which genes have significant clinical value for the population of interest. We aim to determine such genes by identifying somatic alterations in the 48 cancer gene panel in African Americans.

Methods: Tumor matched normal tissues (n=16) from African American colorectal cancer patients were analyzed using a 48 hotspot cancer panel by Ion Torrent sequencing. Variants of the genes found in TruSeq Amplicon - Cancer Panel (TSACP) were identified and validated. The type of variant was determined using bioinformatic pipelines including Ensembl's Variant Effect Predictor. The genes with relevant clinical value were determined based on frequency of somatic variants and results from previous studies.

Results: Somatic variants were detected in 39/48 (82%) genes. Of the 39 genes, the 7 with the most clinical value were selected for further analysis. Overall, 46 novel and known hotspot somatic variants were determined in the following 7 genes: KRAS (7/46 [15%]), NRAS (2/46 [4%]), MLH1 (1/46 [2%]), PIK3CA (8/46 [18%]), SMAD4 (6/46 [13%]), RET (9/46 [20%]), and TP53 (13/46 [28%]). Of the KRAS variants, 57% (4/7) were found in codons 12 and 13 while the other 43% (3/7) were found in codons 117 and 146. Out of the 46 somatic variants, 30% (14/46) were in hotspot regions. Of the KRAS hotspot variants, 100% (4/4) were in codons 12 and 13. The 2 NRAS variants were in codons 59 and 61. Lastly, the 9 genes without variants in our population are CDKN2A, SMARCB1, AKT1, ALK, FGFR2, IDH2, CSF1R, HNF1A and MPL.

Conclusion: We have identified novel and known hotspot somatic variants in African Americans with colorectal lesions. Patients with significant hotspot variants, primarily missense alterations, had a worse prognosis. As a result, cancer gene panel hotspot profile should be taken into consideration when treating African American CRC patients.

#648

Digital PCR in genetic diagnosis of NUT midline carcinoma.

Shunsuke Okumura, Shin-ichi Chiba, Masatoshi Sado, Nana Takahashi, Takaaki Sasaki, Masahiro Kitada, Yoshinobu Ohsaki. _Asahikawa Medical Univ., Asahikawa, Japan_.

Background; NUT midline carcinoma (NMC) is a rare, highly aggressive tumor with t(15;19) translocation involving NUT midline carcinoma family member 1 (NUTM1) gene. NMC is genetically defined and diagnosed by RNA sequencing or by immunohistochemistry for NUT protein expression instead. However, it can be challenging to perform these assays on small biopsy specimens. We, here, report the utility of digital PCR (dPCR) assay for diagnosis of NMC. Methods; The QuantStudio 3D dPCR system (Thermo Fisher Scientific) was used. We designed optimal dPCR probes for the BRD4-NUT fusion gene and evaluated the dPCR method using NMC cell lines (HCC2429 and Ty82) and clinical samples. Total RNA was extracted from the cell lines and FFPE tissue samples from patients with thoracic tumors (RecoverAll total nucleic acid isolation kit, Thermo Fisher Scientific). cDNA was synthesized with SuperScript VILO cDNA kit (Thermo Fisher Scientific). PCR was performed using ProFlex PCR system (Thermo Fisher Scientific). Data was analyzed with QuantStudio 3D AnalysisSuite Cloud Software (Thermo Fisher Scientific). Results; We identified the BRD4-NUT fusion gene in HCC2429 and Ty82 cells using the dPCR system, which allowed us to detect the fusion gene at a prevalence < 0.1%. The fusion genes were detected in the clinical samples, and we successfully found NMC patients using the dPCR assay. Conclusion; We conclude the dPCR assay is an appropriate method for diagnosis of NMC.

#649

Development of a novel RNA sequencing approach that identifies aberrant splicing in cancer predisposing genes.

Anne M. Jansen,1 Heleen M. van der Klift,2 Jaap D. van Eendenburg,2 Carli M. Tops,2 Juul T. Wijnen,2 Frederik J. Hes,2 Ajay Goel,1 Hans Morreau,2 Tom van Wezel2. 1 _Baylor Scott & White Research Institute, Dallas, TX; _2 _Leiden University Medical Center, Leiden, Netherlands_.

Background: High-throughput sequencing efforts in molecular tumor diagnostics detect increased numbers of novel genetic variants, including the ones that are predicted to affect splicing. Current approach for predicting whether a specific variant may result in aberrant RNA transcripts often consists of in-silico prediction using bioinformatics prediction tools, which is sometimes followed by RT-PCR analysis of the RNA extracted from blood, or functional splicing reporter assays. Although high-quality patient RNA analysis is usually preferred, such RNA is not always available, or the analysis is hampered due to degradation of aberrant transcripts through nonsense-mediated decay. To overcome this issue, we investigated the possibility of analyzing RNA isolated from formalin-fixed paraffin-embedded (FFPE) tissues, with the underlying hypothesis that formalin fixation may inhibit RNA degradation, enabling the detection of aberrant RNA products. Methods: RNA analysis was performed for 13 canonical splice site variants predicted or known to affect splicing in the cancer predisposition genes MLH1, MSH2, MSH6, APC and BRCA1. Of 11 variants, total nucleic acid was obtained from FFPE tumor tissues. For two variants, no FFPE material was available, but EBV-transformed B-cells carrying a splice variant were cultured. Primers were designed to amplify the exon-exon boundary predicted to be affected. Housekeeping gene expression was assessed to determine presence of RNA. Results: Total nucleic acid was successfully isolated for 10 variants from eight FFPE tumor tissues and two EBV-transformed B-cell lines. Aberrant splicing was confirmed in all six variants known to result in splicing. Of one known splice variant, present in the B-cell line, the known aberrant splice product could only be detected after formalin fixation of the cells, suggesting that formalin fixation possibly inhibited RNA degradation. Conclusion: Aberrant splicing was successfully identified in all known splice variants and three of four variants predicted to affect splicing, of which the splice effect was previously unknown. Using a RNA sequencing approach, somatic splice variants could be easily and rapidly analyzed, enabling retrospective analysis of variants predicted to result in splicing when only FFPE material is available in clinical settings.

#650

Effect of antigen retrieval on immuno-based microdissection methods.

Michael Tangrea,1 Meeiyueh Liu,2 Adam Roberge,1 Kristen Noyes,1 Kennedy Sanders,1 Michael Emmert-Buck,3 Donald J. Johann2. 1 _Sinai Hospital of Baltimore, LifeBridge Health, Baltimore, MD;_ 2 _UAMS, Little Rock, AR;_ 3 _Avoneaux Medical Institute, Baltimore, MD_.

Introduction - Immunohistochemical staining (IHC) of histological sections is a well-established and useful technique for a large number of research studies and clinical applications, and is increasingly used as a targeting strategy for procurement of labeled cells via tissue microdissection, including immuno-LCM, immuno/computer-LCM, expression microdissection, and other techniques. The antigen retrieval (AR) process employed at the beginning of most IHC protocols increases epitope availability and improves staining characteristics; however, the procedure can damage DNA to an unknown extent.

Experimental Procedures - To better understand the effects of AR on DNA quality and quantity, model system samples (lung cancer PDX tissue) and clinical specimens (KRAS gene mutation positive cases) were subjected to commonly employed AR methods [heat induced epitope retrieval (HIER), protease digestion] and the effects on DNA were assessed by Qubit, Fragment Analyzer, agarose gels, kappa qPCR, digital droplet PCR, NGS library preparation, and targeted sequencing.

Data Summary - The results showed that HIER resulted in optimal staining characteristics, but induced significant damage to DNA, producing extensive fragmentation and decreased yields. Variation in the HIER protocol mitigated but did not eliminate the negative effects. In contrast, the consequences of protease treatment on DNA were relatively minor and immunostaining quality was acceptable, although diminished in intensity compared to HIER treated sections. Apart from the AR procedure, the IHC process itself also resulted in a decreased yield of DNA. However, in spite of the observed deleterious effects on DNA, none of the AR methods combined with IHC negatively affected PCR amplification of small amplicons, and KRAS gene mutations were successfully identified in the clinical cases under all conditions.

Conclusions - The data indicate that DNA recovered from histology slides after standard AR and IHC processing can be successfully employed for the majority genomic applications, but studies that require larger DNA fragments such as long-read sequencing should avoid the use of HIER.

#651

**Development of an RNA** in situ **hybridization assay for the detection of prostate cancer cells in urine.**

Jillian N. Eskra, Daniel Rabizadeh, Christian P. Pavlovich, Leslie Mangold, Elizabeth Fabian, William B. Isaacs, Jun Luo. _Johns Hopkins Medicine, Baltimore, MD_.

Urine-based detection of prostate cancer represents a promising resource for non-invasive diagnostics. The value of urine as a biospecimen in the management of prostate cancer has already been established, but the utility of urine has yet to be fully explored. The objective of our study was to develop a RNA in situ hybridization (RISH) method for detecting prostate cells in urine and to evaluate the feasibility of using this assay for cancer detection in clinical specimens. We hypothesized that robust and specific labeling of prostate cancer cells could be achieved using this approach, providing benefit over existing urine detection methods, namely the FDA-approved PCA3 test, by allowing for the direct visualization and molecular characterization of individual cells. We optimized the following collection, processing, and preparation procedure for detection of prostate cells in fresh urine specimens collected from patients following digital rectal exam (DRE): the entire urine volume was centrifuged to collect the sediment, which was then formalin fixed, cytocentrifuged onto two slides, and slides were stored at -20°C until proceeding to staining and cytological analysis. Cellularity and sample adequacy was assessed by Papanicolaou staining. For identification of prostate cells in post-DRE urine via RISH, single-probe chromogenic labeling for NKX3.1 or HOXB13 was performed. Pretreatment conditions and staining parameters for RISH were optimized using urine samples spiked with 22Rv1 cells prior to staining patient specimens. We were able to readily identify cells of prostate origin in both spiked urines and patient samples. To further discriminate normal prostate cells from cancer cells, fluorescent multiplex RISH was pursued. We screened a candidate panel of prostate-specific RNA targets that included NKX3.1, HOXB13, KLK3, PRAC1, PRAC2, MALAT1, HOXC6, AMACR, and PCA3. Based on detection specificity, NKX3.1 and PRAC1 were selected as markers to identify cells of prostate origin and PCA3 as a marker of malignancy. Multiplex RISH with these three markers was carried out on 19 patient specimens. Prostate cells, classified as those positive for PRAC1 and/or NKX3.1, were identified in 11 samples, 6 of which were also positive for PCA3. These observations demonstrate that multiplex RISH can be used for the specific detection and visualization of prostate cancer cells in post-DRE urine samples. Although further refinement and validation are necessary, this study provides the first evidence supporting the application of a urinary RISH assay as a potential non-invasive method for prostate cancer detection. Ongoing and future studies will include characterizing performance of additional biomarker panels and validating clinical utility of this technique in a diagnostic setting.

#652

Mutational profile of papillary thyroid microcarcinoma with extensive lymph node metastasis: Comparison of primary thyroid cancer and metastatic lymph nodes.

Min Ji Jeon, Won Gu Kim, Sung Min Chun, Dong Eun Song, Tae Yong Kim, Young Kee Shong, Won Bae Kim. _Asan Medical Center, Seoul, Republic of Korea_.

Introduction: Papillary thyroid microcarcinoma (PTMC) has excellent clinical outcomes, but distant metastasis of PTMC can be fatal. We previously reported that change to an aggressive pathologic subtype of metastatic lymph nodes (LNs) were associated with distant metastasis of PTMC. This study aimed to evaluate and compare the mutational profile of primary tumor and metastatic LNs of PTMC. Method: We included 16 PTMC patients with lateral cervical LN metastases. One of them has distant metastases. DNAs from formalin-fixed, paraffin-embedded archival samples of 16 sets of the normal thyroid tissue, primary PTMC, and the largest metastatic LN were used for targeted next-generation sequencing by Oncopanel AMC version 2.0. Results: A total of 7 somatic variants were confirmed in primary PTMC compared to normal thyroid tissue. BRAF V600E mutation was the most common and seen in 12 primary PTMCs (75%) and 11 metastatic LNs (69%). Missense mutation of KMT2A, RAF1, and ROS1 was detected only in one primary PTMC (6.3%), respectively. Frame-shift mutation of JAK2, inframe deletion of ACVR2A and nonsense mutation of AR was detected one primary PTMC (6.3%) with its metastatic LN, respectively. When we compare the mutational profile of primary tumor and metastatic LNs, there were no newly developed somatic mutations in metastatic LNs. We also found ALK/STRN rearrangement in one PTMC with its metastatic LN (6.3%). CCDC6/RET rearrangement and RET intergenic rearrangement was detected only in 2 metastatic LNs (12.5%) and one metastatic LN (6.3%), respectively. BARD1 deletion was detected in one primary PTMC with its metastatic LNS (6.3%) and FLT4 deletion was detected only in one primary PTMC (6.3%). We also found JAK1, AURKC, and TP53 deletions, which were detected only in one metastatic LN, respectively (6.3%). Conclusion: The mutation frequency of PTMC was really low even in patients with extensive LN metastasis. The mutational status of primary tumor and their regional metastatic LNs were not significantly different and this suggests the minor role of genetic mutations in the process of LN metastases.

#653

A comparative analysis of pathological features and molecular genetics between salivary duct carcinoma and adenocarcinoma, not otherwise specified.

Yoshinori Imamura,1 Naomi Kiyota,1 Ken-ichi Nibu,2 Chihoko Ikeda,3 Tomoo Itoh,3 Ryohei Sasaki,4 Kazuko Sakai,5 Kazuto Nishio,5 Masanori Toyoda,1 Hironobu Minami,1 Naoki Otsuki2. 1 _Kobe University Hospital, Medical Oncology/Hematology, Kobe, Japan;_ 2 _Kobe University Hospital, Otolaryngology-Head and Neck Surgery, Kobe, Japan;_ 3 _Kobe University Hospital, Diagnostic Pathology, Kobe, Japan;_ 4 _Kobe University Hospital, Radiation Oncology, Kobe, Japan;_ 5 _Kindai University Faculty of Medicine, Department of Genome Biology, Sayama, Japan_.

Salivary duct carcinoma (SDC), an aggressive subtype of malignant salivary gland tumors with distinct histopathological features, is often characterized by over-expression of androgen receptor (AR) and HER2. Adenocarcinoma, not otherwise specified (ANOS), is another subtype with ductal differentiation without diagnostic histopathological features. The aim of this study was to compare the pathological features and genetic alterations (GAs) between SDC and ANOS.

We reviewed the 128 pathologically confirmed major salivary gland carcinoma cases and found 15 SDC and 20 ANOS cases, and each of 15 specimens were available for this comparative analysis. The clinicopathological characteristics of the 30 cases were retrospectively reviewed. Immunohisitochemical (IHC) analysis including AR, GCDFP-15, HER2, etc. was performed. Next generation sequencing (NGS) was performed, using multiplex PCR for enrichment of cancer related gene loci covering hotspots of 50 cancer genes.

There were 22 men and 8 women with a median age of 64 years (range, 18-87). The main results summarized in the table below. On IHC analysis, SDC and AR positive (+ve) ANOS showed distinct over-expression of GCDFP-15 and HER2. HER2 amplification (FISH) was detected only in SDC and AR +ve ANOS. The most frequent GAs was observed in TP53, followed by genes involved in PI3K-AKT-mTOR pathway and ERBB2. These frequencies were similar between SDC and AR +ve ANOS. In survival analysis among 25 advanced cases with definitive surgery, AR +ve ANOS appeared to have poorer prognosis than AR -ve ANOS. Univariate analysis revealed that HER2-positivity (IHC 3+, FISH positive, or both) tended to have a poorer prognosis (hazard ratio 3.2, 95% confidence interval, 1.0-10.6).

In conclusion, we found similarity in clinicopathological features and GAs between SDC and AR +ve ANOS. Screening AR and HER2 for ANOS is critical due to the therapeutic and prognostic considerations, as is SDC.

Immunohisitochemical patterns, genomic alterations, and clinical outcomes

---

|  | SDC | AR +ve ANOS | AR -ve ANOS

IHC (N=30) | Estrogen receptor (%) | 2/15 (13) | 1/9 (11) | 0/6 (0)

|

Progesterone receptor (%) | 3/15 (20) | 3/9 (33) | 0/6 (0)

|

Androgen receptor (%) | 15/15 (100) | 9/9 (100) | 0/6 (0)

|

Prolactin (%) | 0/15 (0) | 0/9 (0) | 0/6 (0)

|

GCDFP-15 (%) | 14/15 (93) | 8/9 (88) | 0/6 (0)

|

PSA (%) | 4/15 (27) | 0/9 (0) | 0/6 (0)

|

HER2 ≥ 2+ (%) | 8/15 (53) | 8/9 (88) | 0/6 (0)

|

EGFR ≥ 2+ (%) | 9/15 (60) | 9/9 (100) | 5/6 (83)

|

Ki-67 > 30% (%) | 7/8 (87) | 3/6 (50) | 2/5 (40)

FISH (N=26) | HER2 (%) | 5/14 (36) | 4/7 (57) | 0/5 (0)

NGS (N=29) | TP53 (%) | 3/15 (20) | 2/8 (25) | 2/6 (33)

|

PI3K-AKT-mTOR pathway (%) | 3/15 (20) | 2/8 (25) | 0/3 (0)

|

ERBB2 (%) | 2/15 (14) | 2/8 (25) | 0/6 (0)

Survival analysis (N=25) | 5-year overall survival | 61% (N=14) | 29% (N=8) | 75% (N=3)

|

5-year recurrence-free survival | 64% (N=14) | 14% (N=8) | 50% (N=3)

#654

Application of comprehensive genomic profiling (CGP) to predict therapeutic response to immune checkpoint inhibitors (ICI).

Ravi K Narra, Arun K Singavi, Jonathan Thompson, Smitha Menon, James Thomas Thomas, Carolyn Oxencis, Mathew Riese, Paul Ritch, Deepak Kilari, Aniko Szabo, Ben George. _Medical College of Wisconsin, Milwaukee, WI_.

Introduction : Immune check point inhibitors(ICIs) are widely used to treat various solid tumors, but there is a paucity of biomarkers that reliably predict response to such therapy. We sought to identify somatic alterations (SAs) that can predict response to ICI. Further, we investigated the effect of these SAs on clinical outcome. Methods: We retrospectively reviewed medical records of all patients (pts) that received ICI at our institution (2012-2017) and had CGP performed on pre-treatment biopsies. DNA was extracted from formalin fixed paraffin embedded clinical specimens and CGP was performed on hybrid-capture, adaptor ligation based libraries to a mean coverage depth of >600 unique reads utilizing the Foundation Medicine platform (315 gene panel). Twenty-four SAs, occurring in at least 5% of the pts were correlated with response using the Wilcoxon-Mann-Whitney test and p-values were adjusted for multiple testing using Benjamini-Hochberg's false-discovery rate (FDR) method. Cox proportional hazards regression was used to investigate the effect of baseline covariates on progression-free survival (PFS); The effect of SAs was analyzed using the exact log rank test. The effect of response on progression and death was investigated using a 4-state model with "ICI therapy", "response", "progression", and "death" as possible states. Results: Among the 76 pts that met criteria, 71 (lung-25, urothelial-6, esophageal-6, gynecologic-6, renal-5, sarcoma-5, melanoma-4, colorectal-4, prostate-3, head and neck-3, other-4) had an evaluable response and were included in the analyses. Median age was 62 years, 6 (8.5%) pts received ICI as first line therapy, while 65 (91.5%) pts received a median of 1 line of therapy prior to ICI; median of 5 doses of ICI therapy were administered. Complete Response (CR), Partial Response (PR), Stable Disease (SD), and Progressive Disease (PD) were noted in 3 (4.2%), 11 (15.5%), 10 (14.1%) and 47 (66.2%) pts respectively. SAs in RBM10 (p = 0.0024), PIK3CA (p = 0.0027), ARID1A (p = 0.0039), and SMARCA4 (p = 0.0047) correlated with response in a statistically significant fashion (adjusted p value = 0.028). PFS and overall survival (OS) of the entire cohort was 3.7 and 11.4 months respectively. Pts with SAs in RBM10 (p = 0.0011), ARID1A (p = 0.0102), and PIK3CA (p = 0.0013) demonstrated a statistically significant improvement in PFS (median not met, not met, and 9.3 months, respectively). Response to ICI significantly reduced the hazard of progression (HR=0.11, p=0.0006), but not the hazard of non-relapse mortality (NRM) (HR=0.41, p=0.53). Conclusion: CGP was able to identify SAs predictive of response to ICI and improved PFS. The significant reduction in the hazard of progression with response to ICI emphasizes the predictive value of the identified biomarkers. The validity and putative mechanistic relevance of these predictive SAs need further elucidation.

#655

Hyperdiploidy in plasma cell disorders using multi-parametric flow cytometry (MFC) vs. FISH.

Surbhi Sidana, Nidhi Tandon, Dragan Jevremovic, Rhett P. Ketterling, Angela Dispenzieri, Morie A. Gertz, Francis K. Buadi, Martha Q. Lacy, William Morice, Curtis A. Hanson, Michael Timm, Patricia Greipp, Linda B. Baughn, David Dingli, Suzanne R. Hayman, Wilson I. Gonsalves, Prashant Kapoor, Robert A. Kyle, Nelson Leung, Ronald S. Go, John A. Lust, S.Vincent Rajkumar, Shaji K. Kumar. _Mayo Clinic, Rochester, MN_.

Introduction: Hyperdiploidy on FISH portends a good prognosis in myeloma. We compared hyperdiploidy determined by MFC to that by conventional FISH.

Methods: We studied 1711 patients with plasma cell disorders who had simultaneous FISH and DNA index testing. Monotypic plasma cells in bone marrow are identified by MFC with antibodies to CD19, CD38, CD45, CD138, kappa and lambda; followed by staining by DAPI. DNA index is determined by dividing the measured DNA content of the G0/G1 abnormal plasma cells by the DNA content of the normal G0/G1 plasma cells present. Plasma cells with DNA content index of <0.95 are hypodiploid and G0/G1 DNA content index of >1.05 are considered hyperdiploid.

Results: Distribution DNA index and FISH results are described in Table 1. Sensitivity and specificity of DNA index by hyperdiploidy was 86% and 83%. Cohen's Kappa coefficient for concordance was 0.69. Sensitivity increased to 94% for those with 2 or more trisomies and 97% for 3 or more trisomies. Of the 104 patients with trisomy and non-hyperdiploid DNA index, 62% patients had monosomy (mono) or deletion (del) 13q. Overall, DNA index was lower in patients with mono 13/del 13q (1.01; 0.97-1.11) compared to those without (1.07; 1.0-1.19), p<0.001. After excluding patients with mono 13/del 13q, sensitivity and specificity of hyperdiploidy by DNA index were 92% and 82%, kappa = 0.73.

Hyperdiploidy was seen in 55% of 272 patients with newly diagnosed myeloma. Sensitivity and specificity were 88% and 93%, kappa = 0.78. After median follow-up of 2.3 yrs, those with hyperdiploid DNA index had better progression free survival at 2,3 and 5 years, 62%, 45% and 21% compared to 50%, 33% and 13% in non-hyperdiploid group, p=0.05. Median overall survival (OS) was not reached; estimated 2, 3 and 5 year OS was 83%, 76% and 63% vs. 76%, 64% and 44%, respectively; p=0.08.

Conclusions: DNA index by MFC is a rapid method to determine hyperdiploidy, with potential for replacing trisomy testing by FISH, especially when coupled with monosomy FISH probes. | |  | |

|

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

|

Hyperdiploid

N=768

n/N (%) | Diploid

N=762

n/N (%) | Hypodiploid

N=78

n/N (%) | P1 | P2

Any trisomy  | 619/765 (81) | 96/758(13) | 8/76 (11) | <0.001 | <0.001

One chromosome | 42 | 58 | 7 | |

7 | 3 | 3 | 1 | |

9 | 14 | 29 | - | |

11 | 13 | 6 | 2 | |

Others | 12 | 20 | 4 | |

Two chromosomes | 85 | 25 | - | |

Three or more chromosomes | 492 | 13 | 1 | |

Any translocation | 124/744 (17) | 566/748 (76) | 53/74 (72) | <0.001 | <0.001

t(4;14) | 10 | 63 | 13 | |

t(11;14) | 22 | 401 | 27 | |

t(14;16) | 5 | 32 | 5 | |

t(14;20) | 3 | 8 | - | |

t(6;14) | 8 | 10 | 4 | |

Unknown partner | 76 | 52 | 4 | |

Trisomy + Translocation | 95/743 (13) | 60/744 (8) | 6/74 (8) | 0.009 | 0.002

Monosomy 13/17 or deletion 13q/17p | 252/759 (33) | 295/744 (40) | 54/74 (73) | <0.001 | 0.0001

Monosomy 13/Deletion 13q | 208/717 (29) | 282/718 (39) | 20/70 (29) | |

Monosomy 17/Deletion 17p | 70/749 (9) | 50/735 (7) | 20/72 (28) | |

P1= three groups; P2= hyperdiploid vs. non-hyperdiploid

#656

Distinct pattern of alterations in TP53 mutated/deleted and wild-type high risk acute myeloid leukemia (AML) patients: Identification of new "targetable" genes/pathways.

Anna Ferrari, Eugenio Fonzi, Andrea Ghelli Luserna Di Rorà, Maria Chiara Fontana, Marco Manfrini, Carmen Baldazzi, Cristina Papayannidis, Vincenza Solli, Antonella Padella, Giovanni Marconi, Stefania Paolini, Valentina Robustelli, Enrica Imbrogno, Eugenia Franchini, Perricone Margherita, Maria Chiara Abbenante, Giorgia Simonetti, Nicoletta Testoni, Emanuela Ottaviani, Michele Cavo, Giovanni Martinelli. _Institute of Hematology, Bologna, Italy_.

Background: The reported TP53 mutation rate in AML is relatively low (7.5-9%, TCGA) and predict a poor prognosis. In 2017 European LeukemiaNet recommended for AML to add TP53 mutations (muts) in the risk stratification. Specific chromosomal aneuploidies are closely correlated with each other and with presence of TP53 muts. Aims: Considering that TP53 mut AML pts have HRisk and no target therapy, we would identify genes/pathways that are mainly CNA-affected (Copy Number Alteration) in the mut TP53 group compared to the wt one. Patients and Methods: 358 adult AML pts were screened for TP53 muts. 219/358 samples were genotyped with SNP arrays. CNA analyses were performed using two software to confirm or integrate karyotype data. Fisher's exact test and pathway enrichment analyses were performed. Results: We detected TP53 muts in 52/358 (14.5%) pts. Mostly (34/52) of the TP53 mut pts (65.4%) had complex karyotype. TP53 alterations were significantly associated with poor outcome (OS and EFS p<0.0001). On TP53 locus, we matched CNA and cytogenetic analyses results. We identify 23 mutated pts that were also deleted (alt) and 7 pts that presented only a TP53 deletion. Therefore 44.2% of mut pts present a concomitant deletion. OS of TP53 alt pts is not statistically inferior respect to mut pts (p=0.77). Comparing 52 TP53 alt and 167 TP53 wt pts CNAs results that: a) chrs significantly altered are 5q and 17p but there are also highly significant »Spot» losses (7q, 20p, 21q, 22q, 19q); b) TP53 CNAs are present in the 44% of TP53 alt vs 0.63% of wt pts; c) over 9013 genes are differentially involved (mainly in Loss, 93.1%); d) that pathway categories mainly enriched are Immune System, Metabolism, Signal Transduction; e) TP53 deletion seems less deleterious (in terms of OS) than TP53 mutation or TP53 alt; f) some TP53 protein-protein interacting genes like SKP1, CDK5, PPP2CA, CSNK1G3 and STAT5B are highly altered and drug target. Conclusions: TP53 muts with or without deletion were predicted to be deleterious and significantly correlated with worse prognosis. For these reasons, TP53 mutation/deletion screening should be recommended. Different alterations groups have been identified in terms of genes, pathway enrichment and protein-protein interaction between ALT and Wt; needed a deeper investigation to better focus on few targetable nodes of this complex network. Three groups comparison (Wt,TP53 Mut,TP53 MutDel pts) analyses would give us the opportunity to select a more appropriate target therapy in these pts. Different pattern of alterations in alt and wt groups have to be deeper investigated to discover targetable nodes of this complex network. ELN, AIL, AIRC, PRIN, Regione-Università 2010-12 (L. Bolondi), FP7 NGS-PTL; HARMONY.

#657

ESR1 **mutations abundance in local recurrence of endocrine-treated breast cancer.**

Adi Zundelevich, Maya Dadiani, Smadar Kahana-Edwin, Moran Gadot, Tal Sela, Irina Marin, Daniela Necula, Anya Pavlovsky, Iris Barshack, Bella Kaufman, Einav N. Gal-Yam. _Chaim Sheba Medical Center, Tel Hashomer, Ramat Gan, Israel_.

Emerging mutations in the ESR1 gene that encodes for estrogen receptor (ER) have been recently associated with resistance to endocrine therapy in ER positive metastatic breast cancer patients. These mutations promote the active conformation of the receptor, conferring resistance to endocrine therapy, and were associated with shorter progression free survival. ESR1 mutations were found to rarely exist in primary tumors (~1%) but are relatively common in metastatic, endocrine therapy-resistant lesions, with an estimated prevalence of ~10-50%. Nevertheless, not much is known about the incidence of these mutations in local recurrence (breast and adjacent lymph nodes) and its clinical significance. The objective of this study is to determine the prevalence of ESR1 mutations in local recurrence of endocrine-treated breast cancer patients. To this end, we collected a cohort of breast cancer patients that had at least one local or loco-regional recurrence during or after adjuvant endocrine treatment for primary breast cancer. We analyzed the 5 most common ESR1 hotspot mutations (D538G, L536R, Y537S/N/C) using droplet digital PCR technology in 44 samples from 34 patients (some of the patients had more than one recurrence). ESR1 mutations were identified in 10/34 (29%) patients. Specifically, mutations were distributed between D538G (5/34), Y537S+ D538G (4/34) and L536R (1/34). ESR1 Mutations developed after or on Tamoxifen only treatment in 50% of the positive patients (5/10 patients) and after or on Tamoxifen + Aromatase Inhibitor (AI) treatment in the other 50% (5/10 patients). Notably, one patient developed ESR1 mutation while on neoadjuvant endocrine therapy. ESR1 mutations persisted in local recurrence samples of mutation positive patients that had more than one local recurrence and the chance of detecting a mutation in patients that had more than one recurrence was higher than in those with a single recurrence. This study demonstrates that ESR1 mutations are common in local recurrence of hormone receptor positive breast cancer. Further studies to establish the clinical significance of these mutations and their impact on survival are underway. Since local recurrences are amenable to curative therapy, the identified unexpected high prevalence of ESR1 mutations at this stage of the disease may have important consequences for choosing the optimal adjuvant treatment option for these patients.

#658

Translational theranostic imaging of lymphoma using radiolabeled αCD19-antibodies.

Dominik Sonanini, Johannes Schwenck, Julia Schmitt, Andreas Maurer, Sergios Gatidis, Christian Seitz, Gerald Reischl, Manfred Kneilling, Gundram Jung, Peter Lang, Lothar Kanz, Konstantin Nikolaou, Rupert Handgretinger, Christian la Fougère, Bernd J. Pichler. _University of Tuebingen, Tuebingen, Germany_.

CD19 proved to be an excellent target in B cell leukemia and lymphoma, especially in patients, refractory or not eligible to αCD20 monoclonal antibody (mAb) therapy. With the CD3-CD19 bispecific mAb Blinatumumab and αCD19 chimeric antigen receptor (CAR) T cells, two promising therapeutics have recently found their way into clinical application with impressive results. However, therapy decisions are mainly based on histological CD19 staining at initial diagnosis disregarding tumor heterogeneity and temporal expression alterations. To address these limitations, Theranostic Imaging approaches using radiolabeled antibodies are emerging tools to advance targeted cancer therapies. Positron-Emission-Tomography (PET) enables non-invasive whole-body visualization of specific target expression perfectly suitable for therapy stratification and to monitor response to targeted drugs. Furthermore, by following antibody biodistribution and tumor targeting in vivo over time modes of action and resistance mechanisms can be uncovered. We here report on the generation, preclinical characterization, and clinical evaluation of a radiolabeled αCD19 antibody for Theranostic Imaging of Non-Hodgkin lymphoma (NHL). Fc-optimized αCD19-mAb (4G7SDIE) with enhanced antibody-dependent cell cytotoxicity was manufactured at our university and successfully tested to treat minimal residual disease of childhood acute lymphoblastic leukemia. For Theranostic Imaging, this antibody was chelator conjugated with DOTAGA at an antibody-to-chelator ratio of 1:15. Radiolabeling with Copper-64 (64Cu) yielded radiochemical purity of >90 %. For clinical application, the radioimmunoconjugate was produced in accordance to GMP. In vitro cell labeling studies revealed specific binding to the target cells and immunoreactive fraction was 30 % after radiolabeling. Three different xenograft NHL mouse models were established in CD1 nude mice for in vivo imaging studies. PET/MRI was performed 6 h, 24 h, and 48 h after i.v. injection confirming specific targeting of 64Cu-DOTAGA-αCD19-mAb compared to CD19-negative tumors. Administration of the radioimmunoconjugate was then carried out in four NHL patients based on compassionate use program to evaluate eligibility of CD19-targeted therapy. Cervical, mediastinal, and abdominal lymphoma sites as well as bone marrow infiltration were detected by PET. Limited uptake was observed in some lesions including bulk tumors indicating partly insufficient amount of injected antibody. To our knowledge, we demonstrate for the first time the feasibility of a radiolabeled αCD19-mAb for lymphoma targeting in human. This theranostic approach might serve as a novel tool to predict response to upcoming CD19-targeting therapies including antibody-based or CAR T cell therapies. Further clinical studies will follow to test optimal dose regime and predosing strategies for enhanced tumor targeting.

#659

A novel manganese MRI contrast agent (PyC3A) for the evaluation of hepatic neoplasms.

Derek J. Erstad, Ian Ramsay, Mozhdeh Sojoodi, Christian Farrar, NIcholas J. Rotile, Shen Li, Sarani Ghoshal, Lanuti Michael, Peter Caravan, Kenneth K. Tanabe, Bryan C. Fuchs, Eric Gale. _Massachusetts General Hospital, Boston, MA_.

Introduction:

Magnetic resonance imaging (MRI) is routinely used to diagnosis hepatic neoplasms. However, Gadolinium (Gd) is contraindicated in patients with severe renal impairment, and recent evidence also suggests Gd accumulates in brain tissue after repeated contrast injections. Manganese (Mn2+) is a paramagnetic ion that is cleared via biliary excretion, and provides an ideal alternative to Gd-based MR contrast agents. We present a novel Mn-based contrast agent, [Mn(PyC3A)(H2O)]- (referred to as PyC3A), and compare its efficacy to gadoxetic acid (Eovist) by imaging orthotopically implanted colorectal cancer hepatic metastases in mice.

Methods:

Male BALB/c mice (n = 8) were injected in the left lobe of the liver with 5x104 MC26/MCA-26 murine colon carcinoma cells, and were imaged on postoperative day 10 using a 4.7T MR scanner with PyC3A and gadoxetic acid (Eovist) 24 hrs apart. Signal contrast-to-noise ratio (CNR) was calculated for normal liver relative to tumor for each probe and compared.

Results:

Relaxivity of PyC3A in blood plasma was shown to be comparable to commercial Gd contrast agents. Biodistribution analysis confirmed that PyC3A clears via a mixed renal/hepatobiliary pathway with greater than 99% clearance within 24 hours. For imaging hepatic metastases, all mice survived to the study endpoint and developed radiographically discernible tumors (5 ± 2 mm diameter) without evidence of carcinomatosis. Calculated CNRs for liver relative to tumor for all recorded times are shown in Table 1. There was no difference in CNR between gadoxetic acid and PyC3A for all time points, suggesting near-equivalent enhancement of normal liver and tumor by both probes.

Conclusions:

PyC3A provides resolution of metastatic liver metastases that is comparable to gadoxetic acid. PyC3A may provide a safe and effective alternative to Gd constrast agents for imaging hepatic neoplasms in patients with renal impairment.

Table 1. Contrast-to-Noise Ratio (CNR) of Normal Liver Relative to Tumor  | |

|

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

Time

(minutes post injection) | Gadoxetic Acid | PyC3A | p-value

0 | 2.61 ± 1.08 | 2.60 ± 1.64 | 0.92

3 | 7.72 ± 4.67 | 7.53 ± 3.02 | 0.93

8 | 7.85 ± 3.89 | 8.85 ± 4.43 | 0.66

13 | 8.02 ± 3.98 | 7.62 ± 5.49 | 0.88

18 | 9.79 ± 4.17 | 5.60 ± 3.08 | 0.06

23 | 8.72 ± 4.16 | 7.03 ± 2.06 | 0.36

#660

Integration of radiomics with "omic" analyses to predict survival in newly diagnosed IDH-1 wild-type glioblastoma.

Paul M. Daniel. _McGill University, Montreal, Quebec, Canada_.

Glioblastoma (GBM) is the most commonly diagnosed glioma and has the poorest median survival of all brain tumours at only 14 months. Despite intensive characterisation of this disease through 'omic' investigations which has led to the identification of critical molecular drivers of malignancy, only a limited selection of biomarkers have proven to be clinically relevant in predicting patient survival. One reason for this lack of success is the existence of intratumoural heterogeneity, where individual tumours can harbour spatially separated molecular and phenotypic diversity, thereby limiting the insight which 'omic' analysis of small biopsy specimens can impart. Radiomics involves high-throughput mining of quantitative image features via image acquisition, region of interest segmentation and feature extraction from standard MRI scans and relating extracted feature values to molecular, phenotypic and clinical properties. Notably, radiomics derives features from the whole tumour mass and therefore overcomes limitations associated with intratumoural heterogeneity. As such, we sought to investigate the ability for radiomics to describe tumour characteristics and integrate radiomics alongside genomics and transcriptomics to derive a prognostic model for IDH1 wild-type GBM. We observed 2 radiomic clusters in IDH1-wild-type GBM which each had an equivalent distribution of each of the four molecular subtypes. Whilst radiomic clusters themselves were not predictive of survival, three radiomic features were able to separate patients into long and short survival groups. Integration of genomic and transcriptomic features alongside radiomic analysis increased the predictive strength of our model. Finally, we found an association between longer survival in patients with IDH1 wild-type GBM and increased expression of the complement system. Our observations suggest that integration of radiomics alongside 'omic' investigations offer a greater ability to predict survival in IDH1 wild-type GBM and suggests a role for the innate immune system in driving patient outcome.

#661

CT scan reveals that marked loss of muscle, visceral fat or subcutaneous fat after gastrectomy was a poor prognostic marker in advanced gastric cancer.

Si Won Lee, Hyung Soon Park, Seung Hoon Beom, Sun Young Rha, Woo Jin Hyung, Song-Ee Baek, Minkyu Jung. _Yonsei Cancer Center, Seoul, Republic of Korea_.

Background: There is increasing interest in the influence of body composition on oncologic outcomes. Using computed tomography (CT) scan, we evaluated the role of skeletal muscle and fat among patients with gastric cancer (GC) who underwent gastrectomy with or without adjuvant chemotherapy, as well as those changes' associations with survival outcomes.

Methods: The present study evaluated 136 patients with GC who were enrolled in the CLASSIC trial at Yonsei Cancer Center. Baseline body compositions including skeletal muscle area, Hounsfield units (HU), visceral fat area, and subcutaneous fat area were measured by preoperative CT scan. The L3 region contains the psoas, paraspinal muscles, and abdominal muscles, which were quantified using the pre-established HU thresholds for skeletal muscle tissue (HU: -30~150). Visceral fat and subcutaneous fat areas (HU:-150~-30) were measured using axial slices at the level of the lumbar vertebra (L3). CT before and after the gastrectomy was used to determine the 6-month relative changes in body composition parameters. Continuous variables were dichotomized according to the best cut-off values by Contal and O'Quigley method.

Results: Seventy-three patients (53.7%) underwent surgery alone, and 63 patients (46.3%) underwent surgery followed by adjuvant chemotherapy. The median baseline values were 46.8 cm2/m2 for SMI, 47.5 HU, 33.9 cm2/m2 for VFI, and 34.6 cm2/m2 for SFI. The baseline body composition parameters were not associated with disease-free survival (DFS) or overall survival (OS). However, patients with marked loss of skeletal muscle mass (<-11%), visceral fat area (<-48%), or subcutaneous fat (<-47%) after gastrectomy (the value was determined by best cut-off point) had poorer DFS and OS compared to patients with lesser changes. Patients with a marked loss in at least one significant body composition parameter had significantly shorter DFS (hazard ratio: 2.9, 95% confidence interval: 1.7-4.8, P<0.001) and OS (hazard ratio: 2.9, 95% confidence interval: 1.7-5.0, P<0.001).

Conclusion: Marked loss in body composition parameters significantly predicted shorter DFS and OS among patients with GC who underwent gastrectomy. Postoperative nutrition and active health care interventions could improve the prognosis of these GC patients. CT data are readily obtainable during routine clinical practice, which makes this approach useful for identifying prognostic biomarkers.

#662

IMA_Detect: Mass spectrometry guided development and clinical application of a companion diagnostic for adoptive cellular therapy against tumor associated HLA peptides.

Jens Fritsche,1 Arun Satelli,2 Helen Hörzer,1 Barbara Rakitsch,1 Franziska Hoffgaard,1 Norbert Hilf,1 Oliver Schoor,1 Harpreet Singh-Jasuja,2 Weinschenk Toni1. 1 _Immatics Biotechnologies GmbH, Tübingen, Germany;_ 2 _Immatics US, Houston, TX_.

Adoptive cellular therapy (ACT) has dramatically changed the landscape of cancer immunotherapy. ACTolog® and ACTengine® are actively personalized ACT approaches employing T-cell receptor products based on a warehouse of human leucocyte antigen (HLA)-bound peptide targets. Selecting the relevant target candidates requires the establishment of biomarkers predictive for HLA peptide presentation and their development into companion diagnostic devices.

Here we describe the development of IMA_Detect, a diagnostic test based on gene expression analysis of primary patient tumor material by qPCR which is predictive for presentation of a target peptide by HLA. To establish mRNA expression levels which indicate actual peptide presentation, data of Immatics' XPRESIDENT® target discovery platform was used by integrating quantitative immunopeptidomics data (label-free LC-MS) with paired transcriptomics data (RNA-Seq). The peptide-specific correlation between peptide presentation and expression of the coding exons was verified for each target individually. The resulting RNA-Seq thresholds were mapped to qPCR thresholds using calibration curves followed by validation of the qPCR assay.

The IMA_Detect test is performed in a CLIA/CAP approved setting and is currently being applied in the phase I ACT trials IMA101-101 and IMA201-101 to determine whether a patient's tumor expresses any of the tested targets at levels considered sufficient for potential benefit from the administered T-cell therapy. IMA101-101 uses autologous T-cell products (ACTolog®) for patients with solid cancers while IMA201-101 is based on TCR-engineered T Cells (ACTengine®) in NSCLC and HNSCC patients. We will present first results of the patient screening and the personalized target selection.

### Radiation Oncology

#663

HDAC6's functional regulation of Chk1, and its implications on the radiosensitivity of non-small cell lung cancer.

Niko Moses, Mu Zhang, Xiaohong Zhang. _Wayne State University, Detroit, MI_.

Lung cancer is a devastating disease; it is the second most diagnosed cancer type in both men and women, but accounts for more deaths than breast, prostate, and colon cancer combined. The 5-year survival for lung cancer is ~17% due to the late stage of progression many patients present with and the aggressive nature of this disease. Novel targeted approaches are necessary to improve the prognosis of patients that present with stage III and IV disease, and our group proposes to accomplish this by assessing the efficacy of radiosensitization of lung tumors with targeted HDAC6 inhibition. HDAC6 is a class IIb histone deacetylase, a largely cytoplasmic HDAC whose substrates include α-tubulin, cortactin and HSP90, and has been implicated in regulating cell migration and motility. This HDAC is unique in that it contains two deacetylation domains, termed DAC1 and DAC2, and DAC1 has been demonstrated by our lab to possess E3 ubiquitin ligase activity. Unpublished tissue microarray data reveals that HDAC6 is upregulated across all three subtypes of non-small cell lung cancer, suggesting its role as an oncogene that can be specifically targeted by inhibitors currently in clinical trials. Here, we extend our HDAC6 findings into the realm of ionizing radiation (IR) and demonstrate that HDAC6 knockdown enhances radiation-induced damage. Specifically, we find that the induction and persistence of γ-H2AX foci is exacerbated in HDAC6 KD lung cancer cells when compared to control knockdown cells, and PARP-1 cleavage is subsequently enhanced. Our previous work has revealed that nuclear HDAC6 can IP a ~2,000kDa complex including the MRN complex, one of the first responders to DSBs. Further work has confirmed that HDAC6 interacts with the MRN complex and imparts posttranslational modifications on MRN component NBS1, indicating that this interaction could impact the ability of the MRN complex to promote resolution of double-stranded breaks via homologous recombination. In addition, protein half-life assays have indicated that HDAC6 knockdown dramatically increases protein stability of Chk1, the gatekeeper of the G2/M checkpoint whose timely degradation is essential for the progression of the cell cycle. Specifically, accumulating Chk1 post-IR in our lung cancer cell lines is specifically phosphorylated on Ser317, indicating that this population was activated by ATM phosphorylation in an NBS1-dependent manner. Previous reports have indicated that cells arrested in G2/M are sensitive to a variety of genotoxic insults, thus Chk1 persistence could also be leading to enhanced sensitivity to radiation-induced damage in our lung cancer cells. Here, we propose that HDAC6 inhibition will lead to increased tumor radiosensitivity, and the mechanism behind this sensitivity involves a regulatory role for HDAC6 in the function of DNA damage response proteins with particular emphasis on the MRN complex and Chk1.

#664

Surgery plus adjuvant radiation as a valid treatment option for primary central nervous system lymphoma (PCSNL).

Connor J. Kinslow,1 Ali I. Rae,1 Alfred I. Neugut,1 Christopher M. Adams,1 Sameer A. Sheth,1 Guy M. McKhann,1 Michael B. Sisti,1 Jeffrey N. Bruce,1 Fabio M. Iwamoto,1 Adam M. Sonabend,2 Tony J. Wang1. 1 _Columbia University College of Physicians & Surgeons, New York, NY; _2 _Northwestern University Feinberg School of Medicine, Chicago, IL_.

Background: Recent studies of primary central nervous system lymphoma (PCNSL) have found an association between cytoreductive surgery and survival, challenging the traditional notion that surgery is not beneficial and potentially harmful. However, no studies have examined outcomes after surgery plus adjuvant treatment. We investigated treatment-related outcomes for surgery plus radiation using data from the Surveillance, Epidemiology, and End Results (SEER) Program.

Methods: The SEER database was queried to collect cases of histologically confirmed non-Hodgkin's lymphoma within the CNS diagnosed between 1995-2014. Median survival times were determined by the Kaplan-Meier method and compared using the log-rank test. Predictors of overall survival and cause-specific survival were determined using the Cox proportional hazards regression model. Treatment modalities were categorized as biopsy alone, biopsy plus radiation therapy (RT), surgery alone, and surgery + RT. Biopsy alone was used as the reference category. Subgroup analysis stratified patients by extent of surgical resection and recursive partition analysis (RPA) risk group.

Results: We identified 5,417 cases that met search criteria, 39% of which received surgical resection. Median survival times for biopsy alone (n=1,824, 34%), biopsy + RT (n=1,460, 27%), surgery alone (n=1,222, 23%), and surgery + RT (n=911, 17%) were 7, 8, 20, and 27 months, respectively (p<0.0001). On multivariable analysis, RT after surgery was associated with a 13% incremental increase in overall survival (Hazard Ratio [HR]=0.71, p<0.0001 vs. HR=0.58, p<0.0001). When analyzing by extent of resection, median survival times for subtotal resection alone (n=111, 2%), gross-total resection alone (n=583, 11%), subtotal resection + RT (n=98, 2%), and gross-total resection + RT (n=459, 8.5%) were 10, 20, 20, and 27 months, respectively (p<0.0001). RT after surgery was independently associated with increased survival in the gross-total resection subgroup (HR=0.85, p=0.046) and in all patients who received surgery (HR=0.87, p=0.017). RT after surgery was also associated with an incremental increase in survival in the RPA class I (HR=0.47, p<0.0001 vs. 0.40, p<0.0001) and class II-III (HR=0.77, p<0.0001 vs. HR=0.66, p<0.0001) subgroups. All findings were confirmed by multivariable analysis of cause-specific survival.

Conclusion: Surgical resection of PCNSL in the general population is more common than previously thought. Radiation therapy after surgery is associated with increased survival, regardless of the extent of surgical resection or the patient's RPA risk category. Neurotoxicity, dosing, and effects of concurrent chemotherapy should be addressed in future studies.

#666

Gut microbiota SCFA modulates DCs antigen presentation and impacts tumor response to radiotherapy.

Andrea Facciabene, Stavros Rafail, Luis Gil de Gomez, Stefano Pierini, Mireia Uribe-Herranz, Kyle Bittinger. _University of Pennsylvania, Philadelphia, PA_.

Alterations in gut microbiota modulate host physiologic functions, including immune responses, and they play a role in the pathophysiology of several diseases, including cancer. Radiotherapy (RT), an established curative and palliative cancer treatment, exerts potent immune modulatory effects, inducing tumor-associated antigen (TAA) cross-priming with antitumor CD8+ T cell elicitation and abscopal effects. Herein, we tested whether the gut microbiota modulates antitumor immune response following RT. Vancomycin, an antibiotic that acts mainly on gram-positive bacteria and is restricted to the gut, potentiated the RT-induced antitumor immune response and tumor growth inhibition. This synergy was dependent on tumor-associated antigen cross-presentation enanchement, cytolytic CD8+ T cells and on IFN-g. Notably, butyrate, a metabolite produced by the vancomycin-depleted gut bacteria, abrogated the vancomycin effect. In conclusion, gram-positive bacteria depletion by vancomycin enhances the antitumor activity of RT, which has important clinical ramifications.

#667

The radioprotector GC4419 ameliorates radiation induced lung fibrosis while enhancing the response of non-small cell lung cancer tumors to high dose per fraction radiation exposures.

Brock J. Sishc,1 Elizabeth Polsdofer,1 David A. Bloom,1 Collin Heer,2 Douglas R. Spitz,2 Debabrata Saha,1 Michael D. Story1. 1 _UT Southwestern Medical Center, Dallas, TX;_ 2 _University of Iowa, Iowa City, IA_.

Stereotactic Ablative Radiotherapy (SAbR) has revolutionized the treatment of non-small cell lung cancer (NSCLC). Despite these advances, treatment-limiting normal tissue toxicities preclude the use of fully potent radiation prescriptions in large or centrally located tumors. Radiation-induced lung fibrosis (RILF), thought to be generated by the production of superoxide in irradiated tissues, is a natural target for agents to limit RILF and thus allow for fully potent SAbR. Superoxide dismutase (SOD) catalyzes the conversion of superoxide into hydrogen peroxide, which then may be converted to water and oxygen via catalase, protecting normal cells from oxidative damage. GC4419 (Galera Therapeutics, St. Louis, MO), a selective small molecule SOD-mimetic acts as both a protector and mitigator of radiation-induced superoxide damage. A single pretreatment with GC4419 significantly reduced the fibrotic density of focally irradiated murine lung tissue (54 Gy, single dose). Furthermore, daily post-irradiation (54 Gy, single dose) use of GC4419 also increasingly reduced lung fibrosis based upon the total duration of daily delivery. Since tumor protection could be a concern, animals with H1299, A549, and HCC827 lung tumor xenografts were treated with GC4419 30 minutes prior to the tumors being irradiated with a single 18 Gy dose, followed by 4 additional daily doses of GC4419. Tumor growth was significantly delayed (p = 0.0022) with the majority of mice apparently tumor-free at X days. Similar enhancements in tumor radiation response were seen with syngeneic lung (LLC) and breast (4T1) tumor models. Subsequent Tumor Cure Dose (TCD50) assays demonstrated that GC4419 enhanced the efficacy of radiation by a factor of 1.67. The dose enhancement seen with GC4419 was associated with the size of the dose per fraction, and if the single 18 Gy dose fractionation scheme was altered to include the biologically equivalent dose schedules of daily irradiations of 2 Gy for 16 days, 4.8 Gy for 5 days, 7.3 Gy for 3 days, or 9.9 Gy for 2 days, the radiation enhancing properties of GC4419 were more pronounced as the dose per fraction increased. Our hypothesis that the tumor response was driven by the overwhelming and persistent production of H2O2 is supported by the fact that the GC4419-enhanced radiation response of tumors derived from H1299CAT cells (doxycycline-driven catalase overexpressing), and treated accordingly with doxycycline, is completely abrogated. These findings suggest that GC4419 not only displays clinical potential as a normal tissue radiation protector and mitigator, but has the additional advantage of enhancing SAbR.

#668

**Electron paramagnetic resonance (EPR) pO** 2 **image-guided tumor biopsies to analyze hypoxia-induced proteins.**

Richard Miller,1 Boris Epel,1 Martyna Elas,2 Martyna Krzykawska-Serda,2 Matthew Maggio,1 Eugene Barth,1 Mihai Giurcanu,1 Howard Halpern1. 1 _University of Chicago, Chicago, IL;_ 2 _Jagiellonian University, Krakaw, Poland_.

Stereotactic MCa4 and FSa tumor tissue biopsies registered with electron paramagnetic resonance (EPR) O2 imaging were used to identify sampling sites suitable to measure protein levels of three hypoxic cell biomarkers, hypoxia-induced factor 1-alpha (HIF-1α), carbonic anhydrase IX (CA9), and vascular endothelial growth factor (VEGF). EPR imaging provided quantitative localization of pO2 levels in tumors grown in the legs of C3H mice. Since hypoxic cells (pO2 <10 torr) are up to three times more resistant than normoxic cells to radiation lethality, identification of hypoxic populations to predict radiotherapeutic outcome is important. The oxygen broadening of narrow EPR spectral lines, or, equivalently, the increase in relaxation rates of electron magnetization, displays pO2 with 1-2 torr resolution in image voxels less than 1 mm3. The pO2 reporter molecule OX063 was used to image hypoxic areas in tumors. The molecule is selectively retained in tumors with a half-life of ~30 minutes. We used inversion recovery electron spin echo (IRESE) to measure the T1 rate of the trityl inside the tumor-bearing leg of mice. Voxel maps with O2 levels less than 10 torr (HF10) were used to guide biopsy cannulas to obtain tumor cell samples. Each of these biopsy cores were further subdivided into samples from which protein amounts could be determined via ELISA. Results from MCa4 and FSa tumor cells indicate a strong correlation between EPR pO2-identified hypoxic areas (<10 torr) and HIF-1α, CA9, and VEGF protein. The activation of various genes, including stem-cell activation, angiogenesis, vasodilation, glucose metabolism, reduced apoptotic activity, and cell cycle changes, contributes to poor radiotherapeutic outcome. EPR pO2 imaging may be used to precisely identify hypoxic areas harboring cells that protect tumor tissue from the toxicity of conventional radiation leading to radiotherapeutic failure.

Grant Support: This work was supported by grants from the NIH, including R01 CA98575 and P41 EB002034.

#669

The regulation of ALOX15 expression in colorectal cancer to overcome the resistance to radiation therapy.

Yoo Jin Na, Dae-Hee Lee, Bo Ram Kim, Jung Lim Kim, So Yeon Jeong, Seong Hye Park, Min Jee Jo, Yoon A Jeong, Hye Kyeong Yun, Sang Cheul Oh. _Korea Univ., Seoul, Republic of Korea_.

Colorectal cancer (CRC) is the third most common cancer and the third leading cause of 'cancer related death' world-wide. In case of CRC patients who have large sized tumor, radiation therapy is essential to decrease tumor size without having to eliminate the anus. However, radiation effects vary according to patients. Also, the association between genes and radiation sensitivity is still rarely researched. In this study, we elucidated that the expression level of ALOX15 regulates radiation sensitivity by TNFα signaling pathway in CRC cells. ALOX15 is known as the main metabolic enzyme for linoleic acid and arachidonic acid. Interestingly, stable cells which express low ALOX15 inhibit radiation-induced apoptosis. Mechanistically, inhibition of ALOX15 increases NF-kB under radiation therapy condition. NF-kB that plays a key role in the cellular response to DNA damage is a reason for ALOX15 to act as a radiation sensitizer. Taken together, our results indicate that ALOX15 is suggested as a prediction marker for radiation therapy. In addition, screening natural compound for elevating ALOX15 would be used for the purpose of combination therapy in radiation-resistant CRC cells.

#670

Specific ROS generation and monitoring in cancer using radioactive gold nanoparticle.

Jihye Choi,1 Yoon Woo Koh,1 Hyung Kwon Byeon,1 Guillem Pratx,2 Jaemoon Yang1. 1 _Yonsei University College of Medicine, Seoul, Republic of Korea;_ 2 _Stanford University School of Medicine, Palo Alto, CA_.

Radiotherapy is a key treatment and is beneficial in the treatment of about 50% of all cancer patients. Such treatment relies on the deposition of energy (the dose) in tumour cells, typically by irradiation with either high-energy gamma rays or X-rays (photons), or energetic beams of ions, sufficient to damage the cancer cells or their vasculature and thus induce tumour death or nutrient starvation. However, like chemotherapy, photon radiotherapy is non-specific, since a significant dose can be delivered to healthy tissue along the track of the photons, in front and behind the tumour. Therefore, recently several research groups have proposed the use of specifically targeted noble-metal nanoparticles irradiated by radiation as an effective therapeutic tool for treating various malignancies with minimum collateral damage to healthy tissue. In general, the photoelectric absorption cross sections of radiosensitive materials directly depend on atom radius, and thus their radiotherapy effects also depend on atom radius. This physical principle determines that gold (atomic-number Z=79) has a stronger radiation enhancement effect by photoelectric effect. Loading with gold nanoparticles (AuNPs) is one of the promising candidates in this area. Gold NPs have a larger atomic radius than gold atom (~0.17 nm), and thus should have a stronger radiation enhancement effect than gold atom. During irradiation, this results in an enhancement of the energy deposition in the vicinity of the gold particles generate the ROS on the surface due to the generation of photoelectrons, Auger electrons, and characteristic X-rays. ROS play important roles in various cellular process. In particularly, at sufficiently high concentrations, ROS could become an effect that is utilized for various therapeutic application such as apoptosis. Herein, we synthesized the radiosensitizer PEGylated gold nanoparticles (RPAuNPs) for generation and measuring of ROS on surface by radiation exposure. First, gold nanoparticles (AuNPs) was prepared by one-pot synthesis and the sharpness and size were efficiently controlled by adjusting the amount of reducing agent. Furthermore, they were replaced with heterobifunctional PEG (COOH-PEG-SH) not only to serve as a biocompatible stabilizer (PAuNPs) and but also to conjugate Dihydrorhodamine 123 (DHR 123) (RPAuNPs) as ROS sensor. Well-engineered formation of RPAuNPs was verified by transmission electron microscopy (TEM) showing their overall size of 50 nm. In addition, UV-Vis data strongly demonstrated successful synthesis. We evaluated their functional use in inducing ROS and the results showed that our radiosensitizing agents exhibited the feasibility of effective ROS generation under x-ray radiation. And we investigated there in vitro/in vivo imaging and therapeutic efficacies, optical properties, biocompatibility for tumor cell.

#671

Anemia in cancer patients undergoing radiotherapy and chemotherapy in National Hospital, Abuja, Nigeria.

Simeon C. Aruah, Z. M. Jawa, O. Ogbe, S. I. Ezikeanyi, B. Odume. _National Hospital Abuja, FCT-Abuja, Nigeria_.

Introduction: Many cancer patients present with anemia prior to radiotherapy and chemotherapy or may experience anemia /worsening of anemia at some point during treatment.

Aims and Objectives: The aim of the study was impact of anaemia in cancer patients undergoing Radiotherapy and Chemotherapy.

Methodology: 201 cancer patients of both sexes with histopathologically confirmed malignancies (solid cancers). Patient's pre-treatment Hb was taken. Patients were distributed into Radiotherapy, Chemotherapy and Chemoradiation. Their Hb were measured once every 2 weeks. The blood film pictures of the patients were examined. The whole process was terminated after 3 consecutive Hb reading. Anemia was classified into: Less than 10g/dl - Severe anemia 10 - 10.9g/dl - moderate anemia 11 - 12 g/dl - mild anemia 12 g/dl and above - no anemia.

Results and Analysis: Out of 201 cancer patients, 86.1% were female and 13.9% were male. Age range, 25 - 75 years, 100 patients were on Chemotherapy, 63 patients on Radiotherapy and 38 patients on Chemoradiation. Most of the patients presented with stage III disease. The prevalence in anaemia in cancer patients was found to be 63% as shown by blood film picture. At the end of therapy, 62% (100) patients on Chemotherapy and 55.6% (63) patients on Radiotherapy had their Hb level between 11-12g/dl, 39.5% (38) cancer patients on Chemoradiation arm had Hb value of 10-10.9 g/dl. At P- value > 0.05, there was no statistical significance on distribution of mean Hb, standard deviation based on sex and treatment type.

Conclusion: Prevalence of anemia in the study was 63% while 37% had adequate haemoglobin (Hb) after the therapy. At 95% confidence interval, Chemotherapy had greatest impact on Hb level during therapy. Thus Chemotherapy; 9.60-10.62g/dl, Radiotherapy; 11.52-12.1 3g/dl, Chemoradiation therapy; 10.98-11.3 6g/dl.

#672

Targeting metabolic pathways to radiosensitize pancreatic cancer.

Parthasarathy Seshacharyulu, Ramakrishna Nimmakayala, Satyanarayana Rachagani, Shuo Wang, Sicong Li, Geoffrey Talmon, Sukhwinder Kaur, Moorthy P. Ponnusamy, Maneesh Jain, Chi Lin, Surinder K. Batra. _University of Nebraska Medical Center, Omaha, NE_.

Background: Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer related mortality killing 117 PC patients every day in the United States. The efficacy of systemic chemotherapy and radiotherapy (RT) is tempered by the acquired and innate therapeutic resistance resulting in limited survival benefits to PDAC patients. Our bioinformatics and high throughput database analysis revealed that cholesterol biosynthesis (CBS) pathway is involved in radioresistance (RR) of PC patients. In addition, our previous studies suggested the involvement of FDPS, a CBS pathway enzyme, in imparting radioresistance to PC cells. Here, we tested the utility of Zoledronic acid (ZOL), a specific inhibitor of FDPS, in radiosensitization (RS) of PC cells in vitro and in vivo and investigated the underlying RS mechanisms. Material and Methods: RR PC cell lines (CD18/HPAF and Capan-1) were developed using fractionated irradiation (a total of 20 Gray) and validated using functional assays. RT-PCR and western blotting analysis were performed to determine the expression of CBS pathway associated transcripts and proteins. Stable CRISPR-Cas9 mediated knockout of FDPS in PC cells was developed to examine its role in PC radio sensitivity. Feasibility of pharmacologically inhibiting FDPS in in vitro and in vivo was tested using zoledronic acid (ZOL). Effect of ZOL on FDPS activity was studied by measuring intracellular cholesterol and by observing its inhibition on protein prenylation. Radiosenstization effects of ZOL on PC cells was analyzed using in vitro (FACS, cell cycle and clonogenic survival assay) and in vivo (orthotopic model) assays. Histological and immunohistochemistry analysis were performed to determine the levels of DNA damage markers on PC cells and pancreatic xenograft tissues. Results: Persistent exposure of PC cells to RT (20 Gy) resulted in altered cellular morphology, G2/M arrest and enriched side population accompanied by increased levels of FDPS and cell cycle regulatory proteins. Knockout of FDPS in PC cells enhanced radio-sensitivity. Pharmacological inhibition of FDPS in conjunction with radiation resulted in reduced number of colonies, increased apoptosis and abrogated radiation induced G2/M arrest. ZOL pre-treatment resulted in inhibition of prenylation of CDC42 and RAC1 GTPases and decreased accumulation of intracellular cholesterol. The combined therapy of ZOL and radiation resulted in significant reduction in tumor growth and metastasis as compared to ZOL and radiation alone treatment. Finally, we observed marked reduction in DNA repair proteins such as RAD50 and phosphorylation of P95 in cell lines knockout for FDPS/treated with ZOL and/or RT and xenograft tissues excised from single and combination treatment regimens. Conclusion: FDPS inhibition radiosensitizes PC cells by inducing G2/M cell cycle arrest and by abrogating radiation induced DNA repair mechanism thereby resulting in enhanced apoptosis.

#673

Preoperative radiation therapy in advanced carcinoma colon with post-colostomy intestinal obstruction: The first report.

Amit Jain, Elumalai L, Punam Goswami. _Valentis Cancer Hospital, Meerut, India_.

Introduction We report the use of preoperative radiotherapy in a moribund patient with advanced carcinoma colon with complete intestinal obstruction who had undergone colostomy for a previous episode of intestinal obstruction. Methods Fifty-five-year-old man presented in Dec 2016 with acute intestinal obstruction with pain in abdomen and vomiting for 3 months. On exploratory laparotomy, a 12x15 cm hard mass in splenic flexure of colon was found involving root of mesentery, superior mesenteric vein and third part of duodenum and abutting tail of pancreas. Finding it unresectable, loop transverse colostomy was performed. He was started on Capecitabine. In Mar 2017, he presented to us in emaciated condition weighing 28 kilograms with acute intestinal obstruction with a large abdominal mass. He was started on nasogastric aspiration with total parenteral nutrition. Imaging revealed a 14x19 cm large abdominal mass involving pancreas, duodenum, spleen and transverse colon with dilated stomach. In view of poor general condition and advanced disease, the prognosis was discussed with the family and the idea of radiotherapy to relieve intestinal obstruction was discussed with family. After taking the consent, he was started on radiation therapy with 1.8 Gy per fraction after three-dimensional conformal radiation planning to spare maximum bowel. After 11 fractions, the patient started passing stools through the stoma. He was gradually started on enteral nutrition and was discharged to continue radiation therapy on outpatient basis. After 15 fractions, planning PET-CT scan was taken for adaptive therapy to make changes as per the reducing tumor size. He was also started on capecitabine as he was taking orally well. He completed 45 Gy in 25 fractions over 5 weeks. One month after completion of radiation therapy, he was found to be doing well and was planned for definitive surgery. Six weeks after the radiation therapy completion, extended left hemicolectomy with distal pancreatico-splenectomy and colostomy closure was performed. Histopathology showed residual viable poorly differentiated adenocarcinoma invading full thickness of colonic wall into pericolic soft tissues with a discontinuous microscopic nodular tumor deposit in pericolic soft tissue with all margins and nodes negative. Postoperatively, he developed biliary leak which healed with conservative management. Six weeks after the surgery, he recovered completely and was started on adjuvant Capecitabine. Five months after the surgery, he is doing well and is on oral Capecitabine. Conclusion To our knowledge, this is the first reported case of preoperative radiation therapy in advanced carcinoma colon and also in the setting of intestinal obstruction. Radiotherapy should be further tested in setting of malignant intestinal obstruction due to unresectable carcinoma colon and may spare patients with ileo/colostomy.

#674

Clinical testing of the novel predictive biomarker for monitoring adverse reactions to radiation therapy.

Paul Okunieff,1 Elena Peletskaya,2 Steven Swarts,1 Jinwei Du,2 Larry Pastor,2 Michael Powell,2 Aiguo Zhang2. 1 _University of Florida, Gainesville, FL;_ 2 _DiaCarta, Inc., Richmond, CA_.

Introduction: RadToxTM is a unique, first generation patented technology that measures acute tissue damage following first radiation exposure, using only a few microliters of blood. The assay can be used both for research and clinical testing of plasma samples for patients undergoing radiation therapy.

Methodology: To evaluate the clinical utility of RadToxTM test, cfDNA measurements for 54 cancer patients were obtained from prospectively collected plasma samples at different times before and during radiation therapy for prostate cancer. RadToxTM data comparison between patients receiving x-ray (greater exposure of non-target tissue) versus proton (more focused intergral dose). Peak levels of RadTox, (minus pre-irradiation levels) during the first 7 days after starting radiation were significantly higher in the x-ray group (49.4 vs 25.8 ng/mL, p<0.05). Similarly, average levels (5 daily post-irradiation RadToxTM levels corrected for pretreatment levels during that first week were also significantly higher in the x-ray group (26.0 vs 12.8 ng/mL, p<0.05). Based on the clearance kinetics of RadToxTM measured in non-human primates with total body irradiation from previous work, the peak in cfDNA level was predicted to occur approximately 48 hr after the first dose of radiation. We analyzed RadToxTM data looking only at the 2nd day RadToxTM measurements from the 54 patient plasma sample sets (minus the pre-irradiation values from each patient). The average increase in RadToxTM was 14.8 ± 4.0 ng/mL in the proton group and 32.2 ± 11.8 ng/mL in the x-ray group 48 hr after the first dose of radiation (P=0.018). Three patterns of changes were seen among the patients. Many patients had little change in their RadToxTM during the first week of radiation. These have preliminarily been termed "low risk" and defined to have a peak/pre RadToxTM ratio of <1.5. Most patients had a slow rise of their RadToxTM, and thus are termed "average risk" with a peak/pre ratio of ≤2. A few patients (≈3 of 54) had larger increases in their RadToxTM ratio, and are being considered "high risk". Correlation of these RadToxTM data with objective short and medium term toxicity data are in progress.

Conclusion: RadToxTM was successfully tested against the "gold standard" predictor of toxicity - dose volume histography. A few patients, ≈6%, had very high RadToxTM levels, and may prove to be the subset of patients with greater treatment induced toxicity. Extended long-term follow-up of patient bowel and urinary function is underway. Follow-up data and future studies will allow us to determine if RadToxTM can stratify patients into toxicity-risk groupings and allow oncologists to develop safer cancer treatments.

#675

Analysis of post mastectomy radiation outcomes in breast cancer patients with 1-3 positive axillary lymph nodes : A review of the National Cancer Database.

Anu Paul, Runhua Shi, Prakash Peddi, Gary Burton. _LSU Health, Shreveport, LA_.

Background: The benefit of Post mastectomy radiation (PMRT) has been confirmed in breast cancer patients with ≥4 positive axillary lymph nodes (PALN). The role of PMRT in patients with 1-3 PALN is not well defined.

Methods: We identified 102,103 breast cancer patients with mastectomy and 1-3 PALN registered to the National Cancer Database (NCDB) between 2004- 2012. Primary outcome was overall survival. Adjusted variables include age, race, comorbidity, insurance, income, education, distance travelled, diagnosing/treating facility, treatment delays, hormone receptor status, regional lymph nodes, stage and treatment modalities. 8 treatment modalities were defined: +/- radiation, +/- adjuvant chemotherapy, +/- adjuvant hormone therapy. Univariate and multivariate analyses were performed to identify factors that predict overall survival.

Results: Patients with 1, 2, and 3 PALN comprised 55.7%, 27.6% and 16.6% of the population, respectively. 58.4% of patients received radiation. Younger age (<50years), Asian race, private insurance, annual income >$46,000, lower AJCC tumor stage, hormone receptor positive status and lower number of PALN were independently associated with a lower risk of death in multivariate analysis. Compared to patients who did not receive additional therapy, radiation alone was associated with a non-significant increased risk of death HR 1.10 (95% CI [0.92-1.31]). The addition of hormone and chemotherapy significantly reduced the risk of death HR 0.36 (95% CI [0.33- 0.3]). Stratified by regional lymph nodes, patients with 1 or 2 PALN had a trend towards increased risk of death with radiation alone compared to patients who did not receive radiation, hormone or chemotherapy. In patients with 3 PALN, there was a trend towards reduced mortality with radiation alone HR 0.89 (95% CI [0.63- 1.23]).

Conclusions: To our knowledge this retrospective study is the largest such review of the outcomes of breast cancer patients with 1-3 PALN receiving adjuvant radiation following mastectomy. Our study found no measurable reduction in risk of death for those patients who received radiation therapy compared to those who did not. We also note that, with an increasing number of PALN, patients receiving PMRT either alone, or with chemotherapy or hormonal therapy, appeared to have a decrease in the risk of death compared to the populations that had not received PMRT. Based on this analysis, the routine addition of radiation following mastectomy is not indicated in patients with 1-3 PALN. The addition of adjuvant hormone and/or chemotherapy was associated with a significant survival advantage.

#676

Preliminary investigation into the dosimetric impact of tumor treating field arrays on concurrent radiotherapy for newly-diagnosed glioblastoma.

Gregory C. Stachelek, Jimm Grimm, Joseph Moore, Juan F. Jackson, Kristin Redmond, Chetan Bettegowda, Lawrence Kleinberg. _Johns Hopkins Hospital, Baltimore, MD_.

Rationale:

Glioblastoma (GBM) is the most common and deadly primary CNS malignancy in adults. The addition of tumor treating fields (TTFields), a non-invasive anti-mitotic therapy approved for use in newly-diagnosed or recurrent GBM, following standard resection and chemoradiation has been shown to significantly improve both overall and progression-free survival in a randomized phase III clinical trial.

Preclinical evidence suggests that TTFields may disrupt mitotic spindles and exhibit a similar synergism with radiotherapy (RT) to other microtubule inhibitors. The ability to treat patients concurrently with RT and TTFields is thus of considerable clinical interest. The TTFields device array applicator worn on the scalp may affect radiation dose distribution, an effect that may be mitigated but also be made more difficult to account for by the routine repositioning of the applicator during therapy to avoid skin toxicity. In preparation for a clinical trial, we have therefore undertaken a phantom investigation to evaluate the effect of varying TTFields array position on RT dosimetry and planning target volume (PTV) coverage.

Methods:

Radiation beams and contours were copied to an anthropomorphic phantom from the treatment plan of a newly-diagnosed GBM patient receiving standard adjuvant RT. Four CT scans of the phantom were employed: one lacking the TTFields array and three with the array placed in varying positions over the cranium of the phantom to simulate routine replacement and repositioning of the array as would occur in clinical use.

Dosimetric recalculations were then performed for each scan using the Philips Pinnacle treatment planning system and the dosimetric average of the three array positions was calculated using Varian Velocity software.

Results:

Recalculation of the treatment plan of our test patient onto the phantom wearing the TTFields array resulted in reduction of the isodose covering 95% of the PTV by 5.5 cGy/day over the course of treatment as compared to the phantom with no array (to 51.6 Gy from 53.4 Gy). This small difference in PTV coverage is unlikely to be of clinical significance, obviating the need for time-intensive replanning each time the skin position of the array has changed.

Conclusion:

We plan to confirm this novel finding by further investigating the impact of repositioning TTFields arrays on the radiation treatment plans of a larger, sequential cohort of newly-diagnosed GBM patients. These preliminary studies will ultimately be the foundation of a clinical trial evaluating the concurrent use of TTFields during standard radiotherapy.

#677

TTK: A novel target for radiosensitization in triple-negative breast cancers.

Benjamin C. Chandler,1 Leah Moubadder,1 Cassie Ritter,1 Yashar Niknafs,1 Eric Olsen,1 Meleah Cameron,1 Meilan Liu,1 Kari Wilder-Romans,1 Shyam Nyati,1 Powel Brown,2 Arul Chinnaiyan,1 Corey Speers1. 1 _University of Michigan, Ann Arbor, MI;_ 2 _University of Texas MD Anderson Cancer Center, Houston, TX_.

Background: Increased rates of locoregional recurrence leading to poorer clinical outcomes have been observed in triple-negative breast cancer (TNBC) despite the use of radiation therapy (RT), therefore approaches that result in radiosensitizaton in TNBC are critically needed. Our previous work identified a group of cell cycle kinases differentially expressed in estrogen receptor negative (ER-) versus estrogen receptor positive (ER+) breast cancer (BC). Here we described the impact of elevated expression of one of these identified kinases, TTK, on radiation response and patient outcomes in TNBC.

Methods: TCGA breast cancer datasets were used to determine TTK expression in the intrinsic subtypes of BC. TTK RNA and protein levels were measured using qPCR and western blot at baseline and after radiation treatment. Clonogenic survival assays were used to determine the radiosensitization in several cells after TTK inhibition. DNA damage was quantified using γH2AX staining in TNBC cell lines. Kaplan-Meier analysis was used to determine the impact of TTK expression on locoregional recurrence (LRR) and overall survival (OS). A Cox proportional hazards model was constructed to identify potential factors of LRR-free survival in univariate (UVA) and multivariable analyses (MVA).

Results: TTK expression is elevated in breast cancer tissue compared to normal breast tissue (Q-VAL: 8.70 E-291) and is most highly expressed in basal-like tumors. TTK is overexpressed in ER- versus ER+ tumors in both breast cancers patient samples (p-value: <0.0001), and cells lines (p-value: <0.0001). TTK expression is significantly correlated with intrinsic radioresistance in a panel of 23 BC cell lines (R=0.58, p-value=0.0035). TTK RNA and protein levels are significantly increased at 12 and 24 hours after RT in TNBC cell lines. Genomic (siRNA knockdown) or pharmacologic (NMS-P715) inhibition of TTK increased radiosensitivity in vitro in 3 different TNBC cell lines. This sensitization is mediated, at least in part, through impaired DNA damage repair. Increased γH2AX foci was found after combination treatment of RT and TTK inhibition compared to only TTK inhibition, or RT. Clinically, breast cancer patients treated with breast conserving surgery and RT whose tumors have higher than median expression of TTK had worse local-recurrence free (LRF) survival and overall survival (HR for local recurrence 1.7 as continuous variable, p-value 0.004) compared to patients with higher than median expression, and this pattern held when considering quartile expression. TTK expression was associated with poorer LRF survival in UVA and in MVA only TTK expression and grade were significantly associated with worse LRF survival in 3 independent datasets.

Conclusion: Our results support the rationale for developing translational clinical trials to investigate TTK inhibition as a novel radiosensitizing target in TNBC.

## CANCER CHEMISTRY:

### Chemical and Structural Biology

#678

Raf-1 cysteine-rich domain (CRD) supports active orientations of KRas4B/Raf-1 at the membrane.

Hyunbum Jang, Ruth Nussinov. _National Cancer Institute at Frederick, Frederick, MD_.

Ras is a small GTPase, controlling signal transduction pathways and promoting cell proliferation and survival. In cancer, there are two independent pathways in tumor proliferation: one includes Raf/MEK/ERK (MAPK) and PI3K/Akt/mTOR. KRas4B is the most abundant oncogenic isoform. In the MAPK pathway, KRas4B preferentially recruits Raf-1 and activates it. The high-affinity interaction of Ras binding domain (RBD) of Raf with Ras was solved, but the relative position of Raf's cysteine-rich domain (CRD) in the Ras/Raf complex at the membrane and the key question of exactly how it affects Raf signaling are daunting. Here, we employ all-atom MD simulations to investigate membrane-anchored Raf-1 CRD and suggest a model of KRas4B/Raf-1 complex at the anionic lipid bilayer composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dioleoyl-sn-glycero-3-phosphoserine (DOPS) in mole ratio 4:1. Active Ras modulates Raf activation, with KRas4B preferentially activating Raf-1. We show that the Raf-1 CRD stably binds anionic lipid bilayers inserting a positively-charged loop into the amphipathic interface. The loop contains key basic residues, responsible for the membrane association. Importantly, when in complex with Ras/RBD, covalently-connected CRD presents the same membrane interaction mechanism, with CRD locating at the space between the RBD and membrane, suggesting that CRD is an intrinsic membrane binding domain of Raf kinase. To date, CRD's role was viewed in terms of stabilizing Raf-membrane interaction. Our observations argue for a key role of CRD that Raf-1 CRD not only offers an additional anchor for the KRas4B/Raf-1 complex, but by reducing the fluctuations of Ras/RBD it also increases the population of Ras/RBD at the membrane and enhances the already high affinity between Ras and RBD. In the KRas4B/RBD-CRD complex, Raf-1 CRD supports the active-state Ras orientation at the membrane, releasing the catalytic domain from the transiently confined orientation. This promotes MAPK signaling, the key Ras proliferative pathway, above other pathways. Even though in the absence of CRD the Ras/Raf-1 RBD interaction can elicit MAPK signaling, the enhanced stability of the complex at the membrane rendered by CRD can considerably intensify it, by helping in Raf's dimerization. Our data reveal these significant roles of CRD at the membrane in the Raf activation. Funded by Frederick National Laboratory for Cancer Research, National Institutes of Health, under contract HHSN261200800001E.

#679

ABI-231: A novel small molecule suppresses tumor growth and metastatic phenotypes of cervical cancer cells via targeting HPV E6 and E7.

Vivek K. Kashyap, Bilal B. Hafeez, Qinghai Wang, Neeraj Chauhan, Prashanth K. Nagesh, Nirnoy Dan, sonam kumari, Shabnam Malik, Saini Setua, Aditya Ganju, Murali M. Yallapu, Duane D. Miller, Wei Li, Meena Jaggi, Subhash C. Chauhan C. Chauhan. _University of Tennessee Health Science Center, Memphis, TN_.

Objective: Cervical cancer is one of the most common and deadly cancers among women worldwide and is associated with persistent Human Papillomavirus (HPV) infection. Human papilloma virus (HPV) expressing E6 and E7 oncoproteins involved in carcinogenesis through their interactions with the p53 and pRB pathways, respectively. Therefore, non-toxic agents that have potential to inhibit the expression of E6 and E7 oncoproteins and their regulated oncogenic signaling pathways could be used in the management of cervical cancer. Microtubule targeting agents including paclitaxel, colchicine and vinca alkaloids are widely used in the treatment of various cancers but most of these agents have toxic side effects and develop chemoresistance. Herein, we investigated the potential anti-cancer effects of a novel tubulin targeting agent (ABI-231) in in vitro and in vivo model systems.

Method: ABI-231 ((2-(1H-indol-3-yl)-1H-imidazol-4-yl)(3,4,5-trimethoxyphenyl))- methanone was synthesized in our home institution. Human cervical cancer cells (CaSki and SiHa) were used as in vitro model system. To determine the effect of ABI-231 on cell proliferation, migration and invasion, we performed MTS and wound healing assays. Effect of ABI-231 on the expression of HPV E6/E7 was determined by Western blot, qRT-PCR, and confocal microscopy. Xenograft study was performed to determine the effect of ABI-231 on cervical tumor growth.

Results: ABI-231 treatment significantly (P<0.01) inhibited growth, clonogenic, invasive and migratory potential of cervical cancer cells. ABI-231 treatment was resulted in inhibition of HPV E6 and E7 expression at both mRNA and protein levels in both Caski and SiHa cells. ABI-231 inhibited phosphorylation of STAT3 at both Tyr705 and Ser727 residues. ABI-231 arrested cell cycle in G2/M phase as determined by flow cytometry and inhibited protein levels of cyclin B1, p21 and p27. Moreover, ABI-231 treatment SiHa and Caski was resulted in induction of apoptosis which was analyzed by enhance Annexin V staining. Western blot results demonstrated cleavage in PARP protein, Bid, and Bim. ABI-231 treatment also showed significant (P<0.01) inhibition of xenograft tumors in athymic nude mice. Excised xenograft tumors tissues were also analyzed for oncogenic signaling components which showed potent inhibition of aforementioned oncogenic signaling components compared to control.

Conclusions: Taken together, our results demonstrate the potential anti-cancer efficacy of ABI-231 in in vivo and in vivo. ABI-231 can be explored as a potent therapeutic agent for the treatment of cervical cancer.

#680

FLLL12 is a small molecule JAK2 inhibitor that inhibits JAK-STAT3 pathway in head and neck cancer.

ARM Ruhul Amin,1 ASM Anisuzzaman,2 Abu B. Siddique,3 James R. Fuchs4. 1 _Marshall University, Huntington, WV;_ 2 _Emory University, Atlanta, GA;_ 3 _University of Louisiana at Monroe, Monroe, LA;_ 4 _Ohio State University, Columbus, OH_.

Purpose: We have recently reported that FLLL12, a synthetic curcumin analog, induces apoptosis of lung cancer cells through a death receptor (DR) 5-dependent pathway and in squamous cell carcinoma of head and neck (SCCHN) by modifying multiple Bcl-2 proteins. However, the direct molecular target (receptor) of FLLL12 is unknown. The purpose of the current study is to identify the receptor of FLLL12.

Methods: Computer-aided molecular docking was used to predict molecular interaction. Isothermal titration calorimetry and thermal shift assays were used to confirm molecular interactions in a cell free system. SCCHN cell lines were used to study the effect of this interaction on JAK-STAT3 pathway. Protein expression levels were measured by Western blotting.

Results: Computer-aided molecular docking modeling of FLLL12 and JAK2 (PDB code: 5CF5) predicted that FLLL12 fit well into the JAK2 catalytic pocket. Although curcumin also fit into the JAK2 catalytic pocket, the binding sites were different for FLLL12 and curcumin. Docking score for FLLL12, natural ligand and curcumin were -7.2, -12.1 and -6.9, respectively. FLLL12 also docked into the STAT3 catalytic pocket (PDB code: 5AX3). However, the docking score was worse than those for natural ligand and curcumin (-5.1, -7.3 and -7.4 respectively). We confirmed that FLLL12 interacted with JAK2 in a cell free system using recombinant JAK2. Isothermal titration calorimetry assay revealed one binding site for both FLLL12 and the FDA-approved JAK inhibitor ruxolitinib. The Ka values were 3.84E6±1.03E5 and 2.31E7±6.78E6 M-1, respectively. FLLL12 and JAK2 interactions were further confirmed by thermal shift assay. Kd values were 1.12±0.14 μM and 2.24 ±0.18 μM for ruxolitinib and FLLL12, respectively. Two hour pretreatment with FLLL12 also inhibited IL-6 induced phosphorylation of JAK2 and STAT3. Finally, FLLL12 strongly inhibited constitutively active STAT3 phosphorylation in SCCHN cell lines, a downstream effector of JAK.

Conclusions: Our results strongly suggest that FLLL12 is a novel small molecule inhibitor for JAK2 and FLLL12-JAK2 interaction inhibits IL-6-induced as well as constitutively active STAT3 phosphorylation, thus has great promise for cancer prevention and therapy. Grant Support: Start-up fund from Department of Hematology and Medical Oncology, Winship Cancer Institute of Emory University and from Department of Pharmaceutical Sciences and Research, Marshall University.

#681

Targeting diacylglycerol kinases for immuno-oncology.

Ku-Lung Hsu, Hsu Lab at the University of Virginia. _University of Virginia, Charlottesville, VA_.

Diacylglycerol kinases (DGKs) are integral components of signal transduction cascades that regulate cell biology through phosphorylation of the ubiquitous secondary messenger diacylglycerol. Emerging clinical evidence supports DGK-alpha (DGKA) as a promising target for cancer immunotherapy because of its critical role in regulating lipid signaling necessary for proper T cell activation. Methods for direct evaluation of DGK activity in native biologic systems are lacking and needed to develop chemical probes for studying isoform-specific functions. Here, I will discuss efforts from my group to utilize ATP acyl phosphate activity-based probes and quantitative mass spectrometry to map previously undefined ATP- and small molecule-binding sites of representative members from all subtypes of the DGK family. We use our chemical proteomics strategy to discover an unusual binding mode for DGKA inhibitors, including interactions at a novel binding site remote from the ATP binding pocket. I will also describe our efforts towards discovery of new lead inhibitors for future development of highly potent and selective DGKA inhibitors. Collectively, our studies illustrate the power of chemical proteomics to site-specifically profile protein-small molecule interactions and reveal key ligand binding sites for selective inactivation of the DGK family of lipid kinases.

#682

The mechanism of PI3Kα activation by SH2 release.

Mingzhen Zhang, Hyunbum Jang, Ruth Nussinov. _National Cancer Institute at Frederick, Frederick, MD_.

Phosphoinositide 3-kinase α (PI3Kα) is a ubiquitous kinase mediating signaling cascades important for cell survival, proliferation and protein synthesis. It phosphorylates PIP2 to PIP3, to which downstream Akt kinase binds to execute singling. PI3Kα dysfunction has been extensively implicated in cancers. The kinase activity of PI3Kα is controlled by the interactions between its catalytic p110 and regulatory p85 subunits. The interactions of the SH2 domains in the p85 subunit, particularly nSH2, with p110 are key factors in PI3Kα inhibition. Release of the SH2 domains from the p110 subunit, is promoted by factors including phosphotyrosine peptides (RTK), calmodulin (CaM), and oncogenic mutations, thereby activating PI3Kα. Our recent data show that both phosphorylated and unphosphorylated CaMs are capable of binding to the SH2 domains, nSH2 and cSH2, thus promoting the PI3K activation. Despite numerous seminal publications over the past two decades, the mechanism of PI3Kα activation is still a mystery. The large distance between the PIP2 binding sites and the ATP pocket in the crystal structure suggests that the solved PI3Kα structure represents the inactive conformation. What is the activated conformation of PI3Kα, and how PI3Kα switches from the inactive to the active conformation in response to stimulus are the significant mechanistic questions. We employ the large-scale molecular dynamics (MD) simulations to visualize the PI3Kα activation stimulated by the release of SH2 domains in the p85 subunit at the atomic resolution. Our trajectories show that release of the nSH2 domain initiates a "switch-like" conformational change of the iSH2 domain, extracting the ATP from its pocket to the PIP2 binding sites, eventually leading to an activated conformation. This dynamic process is initiated by the break of salt-bridges between iSH2 and helical domains induced by the nSH2 release, and further facilitated by the various residues in activation loop of p110 subunit including His-917, Lys-802 and Lys-776 and in iSH2 of p85 subunit including Arg-461 and Arg-465. The release of the cSH2 domain in PI3Kα has a minor effect. This work, which for the first time uncovers the mechanism of PI3Kα activation at atomic resolution, is expected to promote cancer drug design from a mechanistic dynamic standpoint view. Funded by Frederick National Laboratory for Cancer Research, National Institutes of Health, under contract HHSN261200800001E.

#683

SOS1 allosterically linked conformational changes modulate KRas4B activation.

Tsung-Jen Liao,1 Hyunbum Jang,2 David Fushman,1 Ruth Nussinov2. 1 _University of Maryland, College Park, MD;_ 2 _National Cancer Institute, Frederick, MD_.

Son of sevenless homology 1 (SOS1) is a robust guanine nucleotide exchange factor (GEF) that exchanges GDP by GTP and activates Ras. SOS1 is a large multidomain protein with molecular weight of ~153 kDa. The C-terminal catalytic region, composed of RAS exchanger motif (REM) and CDC25 domains, is responsible for Ras activation. SOS1 offers two Ras binding sites; one at the allosteric site in REM and the other at the catalytic site in CDC25. Experimental studies provided a Ras-SOS1-Ras ternary complex and suggested that Ras binding to REM allosteric site is essential to activate GDP-bound Ras at the CDC25 catalytic site. However, details of SOS1 conformational changes due to different binding modes of Ras at the allosteric and catalytic sites in Ras activation are not well understood. Here, using molecular dynamics (MD) simulations, we examine Ras activation mechanism for SOS1 systems containing GDP- and GTP-bound, and nucleotide-free KRas4B. Among the three Ras isoforms, including HRas, NRas, and KRas (with two splice variants of KRas4A and KRas4B), KRas4B is highly oncogenic, the most frequently mutated in RAS-driven cancers. We observed that the REM domain inherently and sterically obstructs the Ras binding site of CDC25 domain. Upon binding to the REM allosteric site, KRas4B-GTP relieves the steric occlusion, making the CDC25 catalytic site capable of associating with KRas4B-GDP, leading to the nucleotide exchange. Allosteric effects lead to the dislocation of the helix-hairpin motif at the CDC25 catalytic site. This large movement of the helix-hairpin motif causes the Ras catalytic site to open and liberates GDP. Allosteric signals propagating through the REM-CDC25 tandem domains of SOS1 can explain the communication between two Ras proteins modulating the large conformational changes. Our simulations provide insight into the structural correlation of KRas4B-SOS1 as well as the allosteric effect. The conformational changes of SOS1 correspond to the allosteric signaling between two KRas4B at REM and CDC25 domains, promoting Ras activation.

#684

Inhibition of translation by aglaiastatins: Mechanism of action.

Rayelle Itoua Maïga,1 Regina Cencic,1 Jennifer Chu,1 Lauren E. Brown,2 Daniel Dirck Waller,1 Mònica Gómez Palou,1 Michael Sebag,1 John A. Porco,2 Jerry Pelletier3. 1 _McGill University, Montreal, Quebec, Canada;_ 2 _Boston University, Boston, MA;_ 3 _McGill University, Goodman Cancer Research Centre, Montreal, Quebec, Canada_.

Background: Secondary metabolites from plants of the Aglaia genus consist of several classes of compounds, including the cyclopenta[b]benzopyrans, benzo[b]oxepines and cyclopenta[b]benzofurans (rocaglates). The best characterized of these is silvestrol; a rocaglate, which has been shown to target eukaryotic initiation factor 4A (eIF4A), the RNA helicase subunit of the eukaryotic initiation factor 4F (eIF4F) complex. The formation of this complex is regulated by the PI3K/mTOR and Ras-MAPK pathways. Hence, being at the nexus of important oncogenic pathways, eIF4F represents an attractive target for cancer therapy. Silvestrol and its analogs have demonstrated potent activity in human tumor cell lines and xenograft models. Some of the most responsive mRNAs are those encoding oncogenic proteins such as Myc and Mcl-1. This places this group of compounds as promising therapeutic agents against Myc-driven cancers.

Purpose of the study: The purpose of this study is to characterize a sub-group of Aglaia secondary metabolites known as aglaiastatins, which are characterized by the presence of a pyrimidone subunit fused to the cyclopenta[b]benzofuran structure, resulting in a pentacyclic skeleton.

Method: Using in vitro and in vivo assays, we assessed the potency of representative aglaiastatins towards inhibition of protein synthesis and cytotoxicity of tumor cells.

Results: We showed that aglaiastatins induce a specific inhibition of cap-dependent translation by interfering with eIF4A's RNA binding activity, similar to rocaglates. This strong correlation in the mechanism of action was further demonstrated in an eIF4AF163L rocaglate-resistant cell line. Aglaiastatins also demonstrated single agent potency in vitro against a diverse panel of human lymphoma cell lines as well as primary patient samples. In vivo, the compound of interest was found to have a chemosensitization capability, by reversing chemoresistance to doxorubicin in a pre-clinical murine lymphoma model.

Conclusion: Our results indicate that the aglaiastatins also target eIF4A, similarly to rocaglates. Their activity against rocaglate-resistant cells indicates a high similarity in drug target binding pattern. Moreover, the targeting of cap-dependent translation through the RNA helicase allows for the potent activity of the drug against several lymphoma lines, regardless of their mutational landscape, thus providing a potential therapeutic opportunity against difficult-to-treat hematological malignancies.

#685

Structural study of the 3'-end G-quadruplex formed in the human PDGFR-β promoter: Insight into a transcriptional inhibitor element.

Buket Onel, Clement Lin, Danzhou Yang. _Purdue University, West Lafayette, IN_.

Aberrant expression of PDGFR-β promotes multiple hallmarks of cancer, and its transcriptional regulation provides an attractive means to inhibit the PDGFR-β signaling pathway. The human PDGFR-β promoter nuclease hypersensitive element (NHE) can form multiple G-quadruplexes. While the most stable G-quadruplex formed in the human PDGFR-β promoter NHE region is the 5'-mid G-quadruplex, the 3'-end sequence that contains a 3'-GGA run forms a less stable G-quadruplex. Interestingly, the G-quadruplex formed in the 3'-end region of the NHE is recently found to be a transcriptional repressor of PDGFR-β, and a small molecule ellipticine, GSA1129, is shown to selectively stabilize the 3'-end G-quadruplex and repress PDGFR-β activity in cancer cell lines and in a preclinical animal model. Therefore, understanding the molecular structure of the 3'-end G-quadruplex is important for small molecule targeting in PDGFR-β gene regulation. Using Nuclear Magnetic Resonance (NMR) spectroscopy, we found that the 3'-end G-quadruplex formed in the extended PDGFR-β NHE 3'-end sequence consists of two novel intramolecular end-insertion G-quadruplexes, one with a 3'-non-adjacent flanking guanine inserted into the 3'-external tetrad (3'-insertion-G4), and another with a 5'-non-adjacent flanking guanine inserted into the 5'-external tetrad (5'-insertion-G4), respectively. The two guanines in the GGA-run move up or down within the G-quadruplex to accommodate the inserted guanine. The two end-insertion G-quadruplexes co-exist in equilibrium under physiological salt conditions. In the 3'-insertion-G4, the 3' non-adjacent guanine G20 is involved in the formation of the 3'-external G-tetrad, with the two guanines from the GGA-run involved in the formation of the 5'-external and middle G-tetrads, thereby inducing the formation of a bulge loop structure from the 3'-flanking strand. In the 5'-insertion-G4, the 5'-non-adjacent flanking guanine G0 is involved in the formation of the 5'-external G-tetrad, with the two guanines from the GGA-run involved in the formation of the middle and 3'-external G-tetrads, thereby inducing the formation of a lateral loop from the 5'-flanking residues on top of the 5'-external tetrad. The loop interactions and capping structures of the two end-insertion G-quadruplexes appear to be different. For the 3'-insertion-G4, only the 5'-flanking bases stabilize the structure. For the 5'-insertion-G4, the flanking residues at both the 5'- and 3'-ends stabilize the structure, and a favorable capping structure appears to be adopted by the 5'-lateral loop. Significantly, the formation of two equilibrating end-insertion G-quadruplexes appears to depend on the GGA-run and the guanine-containing flanking sequences. The unique end-insertion structures and capping conformations may provide a specific platform for small molecule targeting.

#686

**Drugging an undruggable pocket: The biochemical mechanism of covalent KRAS** G12C **inhibitors.**

Rasmus Hansen,1 Ulf Peters,1 Anjali Babbar,1 Yuching Chen,1 Jun Feng,1 Matthew R. Janes,1 Liansheng Li,1 Pingda Ren,2 Yi Liu,2 Patrick P. Zarrinkar1. 1 _Wellspring Biosciences, Inc., San Diego, CA;_ 2 _Kura Oncology, Inc., San Diego, CA_.

We describe the biochemical mechanism of the covalent KRASG12C inhibitors ARS-853 and ARS-1620. Activating mutations in KRAS are among the most common mutations found in cancer. The KRASG12C mutation in particular is observed in approximately 15 % of non-small cell lung adenocarcinoma, 3 % of colorectal adenocarcinoma and 1 % of pancreatic adenocarcinoma. Until recently, KRAS had been considered undruggable due to the lack of clearly defined pockets that might support binding of small molecules, and the difficulty of targeting the nucleotide binding site due to the high affinity of GDP and GTP. However several years ago small molecules were discovered that bind an inducible pocket near the switch II region and covalently target the mutated cysteine in KRASG12C, trapping KRASG12C in a nonproductive GDP-bound state. Subsequent optimization of these compounds yielded the recently described inhibitors ARS-853 and ARS-1620, the first compounds that directly inhibit KRAS with high potency in cells and animals. While the biological activity of the inhibitors has been described, the biochemical mechanism of how the compounds achieve potent inhibition remained incompletely understood. We now show through biochemical kinetics studies that the activity of ARS-853 and ARS-1620 is primarily driven by KRAS-mediated catalysis of the chemical step of covalent bond formation with cysteine 12 in KRASG12C, rather than by high reversible binding affinity. The reversible inhibition constant (Ki) for both ARS-853 and ARS-1620 is well above the highest compound concentration tested (64 µM, to avoid solubility limitations), likely in the hundreds of micromolar range, while the rate of the chemical step (kinact) is fast. We confirm by several independent means that there is no detectable reversible binding affinity of the inhibitors for KRAS up to at least 32 µM, and show that the rapid chemical reaction is not due to high inherent reactivity of cysteine 12 in KRAS, nor to high intrinsic reactivity of the inhibitors. The results imply that the inhibitors do bind reversibly to KRAS to enable bond formation, but that binding is weak and primarily serves to orient the electrophile. The KRAS-dependent activation of covalent bond formation of ARS-853 and ARS-1620 with the mutated cysteine 12 is reminiscent of mechanism-based or suicide covalent enzyme inhibition, and explains both the high selectivity of the inhibitors for this cysteine relative to other cellular cysteines, and their potent overall activity despite exhibiting poor reversible affinity. The mechanism described here therefore resolves how an induced, shallow and dynamic pocket that is not expected to support high affinity binding of small molecules can nevertheless be targeted with potent inhibitors, and may be applicable to other targets conventionally considered undruggable.

#687

A dGMP fill-in G-quadruplex forms in the PDGFR-β promoter that serves as a unique target for drug design.

Kaibo Wang, Clement Lin, Guanhui Wu, Danzhou Yang. _Purdue University, West Lafayette, IN_.

Platelet-derived growth factor receptor beta (PDGFR-β) is a cell-surface-receptor tyrosine kinase implicated in platelet-derived growth factor (PDGF) signaling pathways, and overexpression of PDGFR-β is associated with tumor growth, angiogenesis, and migration, making PDGFR-β an attractive target for cancer therapy. The formation of DNA G-quadruplexes in the GC-rich nuclease hypersensitivity element of the human PDGFR-β gene promoter has been found to inhibit PDGFR-β transcriptional activity. We previously determined the major G-quadruplex structure formed in the PDGFR-β promoter using a 22-mer sequence of pu22m1, which adopts a folding pattern containing a unique broken G-strand with the 3' tetrad-guanine coming from a distant site connected by a five nucleotide lateral loop. However, extension of the 3' end flanking segment of pu22m1 results in destabilization of the G-quadruplex, suggesting that this structure may be unstable in vivo. Recently, it has been reported that a unique type of intramolecular G-vacancy-bearing G-quadruplex (GVBQ) can be formed with three G3 tracts and one G2 tract and has an incomplete G-tetrad with a G-vacancy. A guanine derivative, such as GMP or GTP, can fill the G-vacancy with Hoogsteen hydrogen bonding and complete the G-tetrad, resulting in G-quadruplex stabilization. The unique broken-strand structure of PDGFR-β pu22m1 suggests a possibility of the formation of a GVBQ structure in this sequence, which can be completed and stabilized by guanine and guanine derivatives. To test this hypothesis, we examined the formation and stability of G-quadruplex formed in pu22m1 variants and the potential of guanine derivatives to fill in the incomplete G-tetrad and stabilize the overall G-quadruplex, using nuclear magnetic resonance spectroscopy, circular dichroism spectroscopy and DMS footprinting experiments. Our results showed that a GVBQ could form in in the PDGFR-β promoter sequence and that dGMP could insert and complete the incomplete G-tetrad to induce the formation of a stable and well-defined dGMP-fill-in-GVBQ in potassium solution. In addition, we discovered two natural plant alkaloids, berberine and chelerythrine, which could stabilize this dGMP-fill-in-GVBQ, with an increase in melting temperature by more than 10 °C. Therefore, it appears that dGMP could interact and stabilize the broken-stranded PDGFR-β promoter G-quadruplex, which may play a role in PDGFR-β transcriptional regulation. Elevated levels of guanine and guanine derivatives/metabolites are found in tumor cells, suggesting that the GVBQs may play important regulatory roles in tumor cell pathological processes, such as PDGFR-β transcriptional regulation. Furthermore, our results suggest that this unique dGMP-fill-in G-quadruplex in the PDGFR-β promoter may serve as a novel anticancer drug target for modulating PDGFR-β gene regulation.

#688

Triphenylstannyl 2-(benzylcarbamoyl)benzoate (Ch-319) modulates AKT/FOXO3a signaling and inhibits progression of prostate cancer.

Durdana Waseem,1 Deeba N. Syed,1 Gul Majid Khan,2 Ihsan-ul Haq,2 Hasan Mukhtar1. 1 _Univ. of Wisconsin-Madison, Madison, WI;_ 2 _Quaid-i-Azam University, Islamabad, Pakistan_.

Prostate cancer (PCa) is the most commonly diagnosed non-cutaneous malignancy in men in the United States. Moreover, its incidence is predicted to surge in many parts of the world due to increase in aging population and change in dietary habits. The PTEN/AKT axis plays a critical role in regulating PCa growth and survival. Synthetic and natural compounds have been explored in preclinical models as potential inhibitors of AKT pathway that can suppress proliferation or induce apoptosis in PCa cells. Triphenyltin compounds are complexes of tin with three phenyl groups attached to hydrocarbon ligands. Structural modifications have been attempted to yield organotins with enhanced cytotoxicity against cancer cells. Here, we synthesized a series of triphenyltin (IV) carboxylate derivatives and evaluated their efficacy against PCa. From this library, triphenylstannyl 2-(benzylcarbamoyl)benzoate (Ch-319) was selected for further studies on the basis of its selective cytotoxicity towards cancer cells. Treatment of PTEN-null PC3M and DU145 PCa cells with Ch-319 (250-500 nM) resulted in reduced viability and induction of cell cycle arrest associated with downregulation of PI3K and dephosphorylation of AKT and PRAS40. In parallel, elevation of FOXO3a expression, activation of pro-apoptotic Bax and downregulation of anti-apoptotic Bcl2 protein was observed. Flowcytometry revealed increased population of apoptotic cells in Ch-319 treated cells. Presensitization of PCa cells with Ch-319 augmented the effect of cabazitaxel, a commonly used taxane in patients with castration-resistant PCa. We next evaluated the effect of Ch-319 in a prostate-specific Pten-/- model where AKT activation is the major driving force for development of prostate adenocarcinoma. Mice treated with Ch-319 (5 µg/animal:intraperitoneally) for 6 weeks showed decreased tumor weights as compared to DMSO treated controls. Histological studies indicated a decrease in the number and size of foci in Ch-319 treated tumors. Consistently, reduced expression of androgen receptor, decreased phosphorylation of AKT and PRAS40 and increase in FOXO3a was observed in Ch-319 treated animals with no signs of organ toxicity. Our studies identify AKT/FOXO3a signaling as a target of triphenyltin (IV) carboxylate derivative Ch-319 and provide a molecular basis of its growth inhibitory effect in PCa cells. We propose that Ch-319 has potential to be developed as an anticancer agent against PCa.

#689

Identification and optimization of novel chemical matter via a structure-based approach resulting in a probe for MTH1.

Marcus Bauser,1 Anja Giese,1 Manuel Ellermann,1 Judith Guenther,1 Ashley Eheim,1 Stefanie Bunse,1 Roland Neuhaus,1 Joerg Weiske,1 Maria Quanz,1 Andrea Glasauer,1 Katrin Nowak-Reppel,1 Benjamin Bader,1 Horst Irlbacher,1 Hanna Meyer,1 Nina Queisser,1 Andrea Haegebarth,1 Matyas Gorjanacz,1 Lionel Tresaugues,2 Tobias Ginman,2 Fredrik Rahm,2 Martin Andersson,2 Ulrica Ericsson,2 Rickard Forsblom,2 Johan Lindstroem,2 Camilla Silvander,2 Jenny Vicklund2. 1 _Bayer AG, Berlin, Germany;_ 2 _Sprint AB, Stockholm, Sweden_.

Cancer cells can form reactive oxygen species (ROS) due to altered redox regulation that affect desoxynucleosides triphosphates (dNTP) in particular. 8-oxo-2'-deoxyguanosine-5'-triphosphate (8-oxo-dGTP) and 2-hydroxydeoxyadenosine-5'-triphosphate (2-OH-dATP) are the two most abundant oxidative nucleotide lesions in this respect. These undesired nucleoside triphosphates are sanitized by the hydrolase MTH1 (also known as NUDT1) in order to prevent their incorporation into replicating DNA. Sprint Bioscience created a series of drug-like, potent and selective MTH1 inhibitors using fragment-based drug discovery methods. In collaboration with Bayer, these inhibitors were extensively profiled, both in vitro and in vivo, to allow for the selection of a probe molecule with attractive properties for in vivo target validation studies. Herein, we would like to share novel chemical matter and it's binding to MTH1 in protein co-crystal structures. Furthermore, we describe the consecutive, stepwise structure-based optimization process. Extensive SAR elaboration clearly revealed the essential moieties for high potency and favorable ADME properties. We are able to report for the first time how we identified BAY-707 as a very potent and highly selective MTH1 inhibitor representing a potential probe to further evaluate the scope and limitations of MTH1 inhibition for therapeutic applications.

#690

Improvement of prostate cancer diagnosis using a multiplex test of PSA, GDF-15 (NAG-1) and glycan-binding auto-IgG in plasma.

Hyesook Kim,1 Julia Matzenbacher Santos,1 Aby Joiakim,1 David Kaplan,1 Ben Rybicki,2 Alan Dombkowski,3 David A. Putt1. 1 _Detroit R &D, Inc., Detroit, MI; _2 _Henry Ford Hospital, Detroit, MI;_ 3 _Wayne State University, Detroit, MI_.

The blood prostate-specific antigen (PSA) assay is used for prostate cancer diagnosis but specificity of the assay is not satisfactory. Previously a combined PSA and GDF-15 (NAG-1) score was shown to improve specificity of prostate cancer detection. Our hypothesis was that, in prostate cancer, glycoproteins were secreted from tumor tissues into blood and induce autoimmune immunoglobulin G (auto-IgG) production and, thus, measurement of the glycan-auto-IgG in blood would improve prostate cancer diagnosis. The 24 glycan-containing microarray analyses have been carried out with plasma samples obtained from 35 prostate cancer patients and 46 healthy subjects to identify glycan-binding auto-IgG biomarker candidates by incubating the auto-IgG captured by glycans spotted on the slide with biotinylated anti-human secondary IgG/streptavidin-Alexa647. Among the 24 glycans, GlcNAc-polyacrylamide (PAA) (G09) and Fucα1-3GlcNAcβ-PAA (G24) glycans showed lower signals of the auto-IgG in the prostate cancer after quantile normalization. β-D-Galactose-PAA (G04) and L-rhamnose-PAA (G08) glycans showed higher signals of the auto-IgG in prostate cancer. No auto-IgM signal was detected. Subsequently, a 5-glycan subarray analysis was developed and lower signals of G09 and G24 glycan-binding auto-IgG were verified by the subarray analysis using 35 prostate cancer plasma samples compared with 54 controls. A higher signal of Neu5Acα2-8Neu5Acα2-8Neu5Acα-sp-PAA (G81)-binding auto-IgG in prostate cancer was detected by the subarray analysis. When the result obtained with the G81-binding auto-IgG was combined with levels of PSA and NAG-1, the prediction rate of prostate cancer increased to 86.2% from 78.2% with PSA levels alone, improving diagnostic accuracy of prostate cancer. The G81 glycan-binding auto-IgG was isolated from prostate cancer and control plasma samples using G81 glycan-affinity chromatography. Western blot analysis of the auto-IgG eluate with a secondary IgG antibody revealed that the level of the 50 kDa heavy chain of the auto-IgG obtained from the prostate cancer patient was ~3-fold higher than the control sample. The 50 kDa fragment was identified as an IgG heavy chain variable region by N-terminal sequencing (Edman's degradation). Our result demonstrated that a multiplex biomarker diagnostic consisting of glycan-binding auto-IgG, PSA and NAG-1 increased specificity and sensitivity of prostate cancer diagnosis. Supported by NCI SBIR Phase I, CA159721. 

## IMMUNOLOGY:

### Inflammation, Immunity, and Cancer

#691

Associations of genetically-predicted circulating immunoglobulin traits with breast cancer risk.

Shuyang Wang,1 Shuyang Wang,2 Yaohua Yang,1 Jirong Long,1 Xiao-ou Shu,1 Wei Zheng,1 Qiuyin Cai1. 1 _Vanderbilt University Medical Cneter, Nashville, TN;_ 2 _Fudan University, Shanghai, China_.

The adaptive immune system plays an important role in cancer development. B cell infiltration has been observed in breast cancer tissues. However, it is unclear whether levels of circulating immunoglobulin (Ig) secreted by B cells may be related to breast cancer risk. A recent study has discovered 35 genetic variants that contribute to variability in eight circulating immunoglobulin traits, including three immunoglobulin levels (IgA, IgG, and IgM) and five composite immunoglobulin traits (total immunoglobulin levels (AGM) and ratios of IgAIgG/IgM, IgA/IgM, IgG/IgM and IgA/IgG) in serum. We utilized the genetic variants for these eight circulating immunoglobulin traits as instruments to evaluate their relationship with breast cancer risk. Beta coefficients and standard errors for the genetic variants for these eight immunoglobulin traits were extracted (Jonsson et al. Nat Genet, 2017). Summary statistics data for association of these genetic variants with breast cancer risk were obtained from 122,977 breast cancer cases and 105,974 controls of European descent in the Breast Cancer Association Consortium (BCAC). Associations of genetically-predicted levels of these eight immunoglobulin traits with breast cancer risk were evaluated using an inverse-variance weighted method. Significant associations with breast cancer risk were observed at Bonferroni-corrected threshold (P < 6.2×10-3) for IgA/IgM ratio and IgG/IgM ratio, two isotype-specific class switching proxy immunoglobulin traits that have distinct effector functions during an immune response. The genetically-predicted value of the IgA/IgM ratio was associated with an increased risk of breast cancer with odds ratio (OR) of 1.13 per unit increase in genetic risk score (P = 2.67x10-5). The genetically-predicted IgG/IgM ratio was associated with a decreased risk of breast cancer with OR of 0.55 per unit increase in genetic risk score (P = 2.83x10-12). We also conducted stratified analyses to evaluate the associations of predicted immunoglobulin traits with breast cancer risk by estrogen receptor (ER) status. The associations for both IgA/IgM ratio and IgG/IgM ratio were observed for ER-positive disease, with ORs of 1.15 and 0.55 and P values of 1.62x10-4 and 2.65x10-9, respectively. For ER-negative disease, a significant association was observed for the IgG/IgM ratio with OR of 0.63 and P value of 2.37x10-3, but the association for the IgA/IgM ratio did not reach significance (P =0.11). Our study provides first evidence that isotype-specific class switching proxy immunoglobulin traits may be related to the risk of breast cancer.

#692

Systemic inflammation and survival in colon and rectal cancer patients: Results from the ColoCare Study.

Jennifer Ose. _Univ. of Utah Huntsman Cancer Inst., Salt Lake City, UT_.

BACKGROUND

Systemic inflammation is an established risk marker for colorectal cancer that may also predict survival. We investigated associations between systemic inflammation and overall survival in a prospectively followed cohort of colorectal cancer patients.

METHODS

Pre-operatively collected serum samples from n=228 newly diagnosed colorectal cancer patients (stage I-IV) from the ColoCare Study were utilized to measure CRP, SAA, IL-6, IL-8, sICAM-1 and sVCAM-1 (Meso Scale Discovery platform). We used Cox proportional hazard models to investigate associations of biomarkers with overall survival after 24 months follow-up overall, and in analyses stratified by colon (n=92) and rectal tumors (n=136). Analyses were adjusted for age, sex, tumor stage and neo-adjuvant therapy (y/n).

RESULTS

After 24 months follow-up n=45 patients (20%) were deceased. CRP was significantly associated with increased risk of death in colon (HR:1.27; 95% CI: 1.03-1.57; pValue=0.02) but not rectal cancer (HR:1.06; 95% CI: 0.87-1.29; pValue=0.55). A doubling of circulating sICAM and sVCAM concentrations was significantly associated with increased risk of death in colorectal cancer (sICAM: HR:2.82; 95% CI: 1.57-5.05; pValue=0.0005; sVCAM: HR:3.70; 95% CI: 1.93-7.10; pValue<0.0001). For both biomarkers we observed stronger associations in rectal compared to colon cancer (e.g., sVCAM: rectal cancer (HR:8.13; 95% CI: 3.13-21.11; pValue<0.0001); colon cancer (HR:1.42; 95% CI: 0.68-2.98; pValue=0.35). Circulating IL-6 and IL-8 were not associated with increased risk of death in colon and/or rectal cancer.

CONCLUSIONS

Inflammatory biomarkers are significantly associated with increased risk of death in colorectal cancer patients. Our data suggest that the role of inflammation in disease progression may be particularly strong relevant in rectal cancer.

Keywords: colorectal cancer; inflammation, biomarkers, overall survival,

#693

Calcium intake and risk of colorectal cancer according to tumor-infiltrating T cells: Results from two large U.S. prospective cohorts.

Wanshui Yang,1 Li Liu,2 NaNa Keum,2 Zhi Rong Qian,2 Jonathan A. Nowak,2 Tsuyoshi Hamada,2 Mingyang Song,2 Yin Cao,2 Katsuhiko Nosho,2 Stephanie A. Smith-Warner,2 Shui Zhang,2 Yohei Masugi,2 Kimmie Ng,2 Keisuke Kosumi,2 Yanan Ma,2 Wendy S. Garrett,3 Molin Wang,3 Hongmei Nan,3 Marios Giannakis,4 Jeffrey A. Meyerhardt,4 Andrew T. Chan,4 Charles S. Fuchs,5 Reiko Nishihara,4 Kana Wu,4 Edward L. Giovannucci,3 Shuji Ogino,4 Xuehong Zhang4. 1 _JIangnan University, Wuxi, China;_ 2 _Harvard Medical School, Chestnut Hill, MA;_ 3 _Harvard T.H. Chan School of Public Health, Boston, MA;_ 4 _Harvard Medical School, Boston, MA;_ 5 _Yale School of Medicine, New Haven, CT_.

Background: The relationship between calcium intake and colorectal cancer risk remains inconclusive. Calcium may enhance T-cell proliferation and differentiation, and contribute to T cell-mediated antitumor immunity. Investigating the calcium and colorectal cancer association according to tumor immunity status may provide additional insights into the role of calcium in colorectal carcinogenesis.

Methods: We thus investigated whether the association between calcium intake and colorectal cancer risk differs by tumor subtypes according to the density of tumor-infiltrating CD3+ cells, CD8+ cells, CD45RO (PTPRC)+ cells, or FOXP3+ cells. A total of 88,509 U.S. female registered nurses from the Nurses' Health Study and 47,740 U.S. male professionals from the Health Professionals Follow-up Study were included in the analysis. Total calcium intake from food and supplemental sources was collected at baseline and every 4 years using validated food frequency questionnaires. The densities of tumor-infiltrating T-cell subsets (CD3+, CD8+, CD45RO+, or FOXP3+ cell) was assessed using immunohistochemical and computer-assisted image analysis.

Results: We identified 736 incident colorectal adenocarcinoma cases during follow-up with available data on T-cell infiltration in tumor tissue. Compared to calcium intake of <600 mg/day, total calcium intake of ≥1200 mg/day was associated with multivariable hazard ratio of 0.55 (95% confidence interval, 0.36-0.84) for CD8+ Tcell-low tumors and of 1.02 (95% confidence interval, 0.67-1.55) for CD8+ T cell-high tumors. Similarly, the corresponding HRs for low vs. high T-cell tumors were 0.63 (0.42 to 0.94; ptrend=0.01) and 0.89 (0.58 to 1.35; ptrend=0.20) for CD3+; 0.58 (0.39 to 0.87; ptrend=0.006) and 1.04 (0.69 to1.58; ptrend=0.54) for CD45RO+; and 0.56 (0.36 to 0.85; ptrend=0.006) and 1.10 (0.72 to 1.67; ptrend=0.47) for FOXP3+. We note that the difference by these subtypes was not statistically significant (all p values for heterogeneity>0.01, with the adjusted α of 0.01 by Bonferroni correction). Additionally, these differential associations appeared to persist regardless of sex, source of calcium intake, tumor location, and tumor microsatellite instability status (MSI).

Conclusions: Higher calcium intake appears to be primarily associated with lower risk of colorectal cancer containing low densities of T cells, but not with cancers containing high densities of T cells. This finding supports a possible role of calcium in cancer immunoprevention via modulation of T-cell function.

#694

The Chilean Gastric Cancer Task Force: An update of results.

Erica C. Koch,1 Mauricio P. Pinto,1 Ignacio N. Retamal,1 Maria L. Bravo,1 Maria J. Maturana,1 Miguel Cordova-Delgado,1 Diego Romero,1 Nathaly De La Jara,1 Javiera Torres,1 Manuel Espinoza,1 Carlos Balmaceda,1 Matias Freire,2 Valentina Garate-Calderon,2 Ricardo Avila,2 Fernando Crovari,1 Alejandro H. Corvalan,1 Ricardo Armisen,2 Gareth I. Owen,1 Marcelo Garrido1. 1 _Pontificia Universidad Católica de Chile, Santiago, Chile;_ 2 _Center of Excellence of Precision Medicine (CEMP), Santiago, Chile_.

Background: Gastric cancer (GC) is the world's second leading cause of cancer death. Gene alterations, treatment responses and mortality rates are geographically heterogeneous. In Chile, GC is the leading cause of cancer death. It affects 17.6/100,000 people and causes >3,000 deaths per year. Therapy response and clinical outcomes are highly heterogeneous and thus a better patient stratification may improve patient outcomes. The Gastric Cancer Task Force (GCTF) is a Chilean collaborative, non-interventional, non-concurrent, prospective study that seeks to stratify gastric adenocarcinomas (GACs) using clinical data, genomic and protein alterations in a cohort of 200 patients.

Methods: A total of ~100 patients were included in this preliminary analysis. Five different Single Nucleotide Polymorphisms (SNP) for the MTHFR and DPYD genes were assessed using genomic DNA. Tissue Microarray (TMA) analysis included: p53, p16, HER2, PDL1, MSI, and EBV. The analysis also included 120 clinical parameters for these patients.

Results: Preliminary results show that 63.6% (n=100) of patients were male. A 38% of patients were stage I-II, 39.55% were stage III, and 22.3% were stage IV. Tumor location was predominantly at the stomach body (48.5%), a 30% of patients had comorbidities and 50% had a cancer family history. Median overall survival (OS) was 29.0 months. In stage IV patients, median OS was 14.0 months. Regarding TMA data a 26% were PDL1+, 13% were EBV+, MSI was present in 13% of patients, 38% were p16+, a 48% had a mutated p53 and 9.0% were HER2+++. Finally, one out of the 4 DPYD SNPs was present in all analyzed patients (100%). Using a multivariate Cox-regression model we found that PDL1+ patients had a 15-fold increase in the risk of death. In contrast, EBV+ patients had a significant reduction in their risk of death. Both were adjusted by sex, age & TNM.

Conclusion. Our preliminary data suggest an elevated proportion of EBV+ among GC patients in the Chilean population compared to similar studies in other geographical regions. On the other hand, majority of patients are males and classified as stage III/IV, which is in-line with previous reports in South America. Finally, PDL1+ and EBV+ correlate with an increase and a reduction in the risk of death, respectively. ClinicalTrials.gov identifier: NCT03158571, Registered May 18th, 2017

#695

Exposure to World Trade Center (WTC) dust initiates a pro-cancer inflammatory signature in mice.

Lin Wang,1 Andriy Kobryn,1 Kyeongah Kang,2 Emanuela Taioli,1 William Oh,1 Paolo Boffetta,1 Shu-Hsia Chen,3 Stuart Aaronson,1 David J. Mulholland1. 1 _Icahn School of Medicine at Mount Sinai, New York, NY;_ 2 _Immunotherapy Research Institute, Houston, TX;_ 3 _Houston Methodist Research Institute, Houston, TX_.

The World Trade Center destruction exposed many individuals to chemicals associated with inflammation and cancer including insulation fibers, asbestos, glass fibers and dioxins. Though the exposure to WTC dust was short, responders experienced an acute intake of harmful dust particles may be at risk for developing long term health problems. To date, responders have shown higher rates of prostate and thyroid cancer compared to non-exposed individuals. Curiously, rates of lung cancer have not shown to increase. To model the effects of WTC dust exposure on inflammation and cancer, we have taken a genetic approach including treatment of Wt C57/Bl6j mice and mice with genetic predisposition to prostate or lung cancer. Using a dosing strategy consisting of 4 consecutive exposures to WTC dust (3 mg/30 ul in PBS dosed via nasal instillation), we assayed for changes in immune and inflammatory gene expression at short term (7 days post exposure) and longer (3 wks. post exposure) time points. RNAs from normal prostate, spleen, lung and blood were assessed using RT2 Profiler PCR Arrays (Inflammation Panel). Organs harvested from WTC dust treated mice showed increased expression of markers including prostate (Ctla4, Cxcl5, IL17a, Il6), lung (Cxcl5, Cxcr1, Bcl2l1), spleen (Ccl20, Cxcl11, Ccl28, Il17a) and blood (Il6) when normalized to PBS controls. As an extreme test of WTC dust effects in a target organ, WTC dust was introduced directly to the mouse prostate (4 mg, orthotoptic injection), resulting in significantly increased prostate size associated with marked elevation of inflammatory/immune gene expression (Cxcl5, Il17a, Ccl20, Il6, Ifng). Histological analysis showed massive immune cell infiltration as well as increased proliferation of prostate epithelia (5-7% Ki67+) compared to PBS controls (0.1%). Collectively, these data indicate that WTC dust can induce a strong inflammatory response systemically or by contact with the cells of the prostate. To determine whether WTC dust exposure promotes cancer progression, we applied two genetically mouse (GEM) models of cancer susceptibility. These include use of prostate specific Cre recombinase (Probasin Cre), to induce susceptibility to prostate cancer by loss of function of key tumor suppressors (Ptenloxp/Wt, p53loxp/Wt) or activation of oncogenes (Krasloxp-G12D) but also to induce cancer susceptibility in lung in a GEM model with Adeno cre virus mediated excision of p53loxp/Wt and KrasG12D. These studies indicate that WTC dust exposure is capable of inducing a strong inflammatory response in mice including elevated expression of multiple cytokines that are associated with human cancer progression. Current studies will determine whether WTC dust exposure can induce cancer or promote progression in responders with or without genetic susceptibility to tumorigenesis.

#696

Patterns of TIGIT expression in normal lymphatic tissue, inflammation and cancer.

Niclas C. Blessin,1 Ronald Simon,1 Martina Kluth,1 Kristine Fischer,2 Claudia Hube-Magg,1 Wenchao Li,1 Georgia Makrypidi-Fraune,1 Björn Wellge,1 Tim Mandelkow,1 Nicolaus F. Debatin,1 Guido Sauter,1 Waldemar Wilczak,1 Andrea Hinsch1. 1 _Univ. Medical Ctr. Hamburg-Eppendorf, Hamburg, Germany;_ 2 _dianova GmbH, Hamburg, Germany_.

T cell immunoreceptor with Ig and ITIM domains (TIGIT) is an inhibitory immune checkpoint receptor and a putative target for novel immune therapies. To study patterns of TIGIT expression, "microenvironment tissue microarrays" containing 4mm tissue spots were constructed from lymph nodes of healthy (n=3) and HIV infected individuals (n=2), normal tonsils (n=3), Hashimoto thyroiditis (n=10), sarcoidosis (n=10), lichen sclerosus (n=2), IgG4-pancreatitis (n=2), rheumatoid arthritis (n=2) colorectal (n=5) and lung cancers (n=2) and analyzed for expression of TIGIT, PD-1 and standard lymphocyte markers by brightfield and multiplex fluorescence immunohistochemistry. TIGIT expression was seen in CD8+ cytotoxic T cells, CD4+ T-helper cells, FOXP3+ regulatory T cells and in NK cells, but not in CD11c+ dendritic cells, CD68+ macrophages and CD20+ B lymphocytes. TIGIT expression paralleled that of PD-1. More than 70% of TIGIT positive cells were PD-1 positive and more than 90% of the PD-1 positive cells were TIGIT positive. Expression of both proteins varied between different tissue compartments. TIGIT expression in tonsil gradually increased from the interfollicular area over the marginal/mantle zone to the germinal center in all T cell subtypes. Stronger expression of TIGIT and PD-1 than in tonsil germinal centers (2-3 fold) was only found in Hashimoto thyroiditis germinal centers. Marked location specific variations were also seen in other inflammatory diseases and in cancers. In general, TIGIT and PD-1 expression was higher in lymphocyte-dense compartments, such as areas of lymphocytic infiltration in sarcoidosis, IgG4 pancreatitis or rheumatoid arthritis, than in areas containing fewer and scattered lymphocytes. In lung and colorectal cancers, the density of TIGIT and PD-1 expressing T cells was highest at the invasion front. Also, the TIGIT and PD-1 expression levels were typically stronger in tumor adjacent stromal CD8+ cells than in tumor infiltrating CD8+ cells. In conclusion, TIGIT is regularly expressed in a large subset of T cells. The variable expression levels of TIGIT and PD-1 between cell types and tissue compartments illustrates the high complexity of immune response. The frequent co-expression of TIGIT and PD-1 may offer an opportunity for co-targeting these proteins with checkpoint inhibitor drugs.

#697

Comparison of tumor-infiltrating lymphocytes between primary and metastatic tumors in breast cancer.

Miseon Lee,1 Hee Jin Lee,1 In Hye Song,1 In Ah Park,1 Sun-Hee Heo,2 Young-Ae Kim,3 Won Seon Bang,3 AReum Lee,3 Hyeonjin Lee,3 Heejae Lee,3 Gyungyub Gong1. 1 _University of Ulsan College of Medicine, Asan medical center, Seoul, Republic of Korea;_ 2 _Asan Center for Cancer Genome Discovery, Asan Institute for Life Sciences, Seoul, Republic of Korea;_ 3 _Asan Center for Cancer Genome Discovery, Asan Institute for Life Sciences, Seoul, Republic of Korea_.

Background: The level of tumor-infiltrating lymphocytes (TILs) and the presence of tertiary lymphoid structures (TLSs) are significant prognostic and predictive factors in primary breast cancer. However, understanding about the differences of TILs and TLSs among different metastatic sites or between primary breast tumors and metastatic sites is limited.

Methods: A total of 337 cases of metastatic breast cancer with available hematoxylin and eosin slides of metastatic sites (biopsy, n=185; operation, n=152) were included. We analyzed the percentage of TILs (defined as the percentage of invasive carcinoma stroma infiltrated by lymphocytes in 10% increments; if less than 10% of stroma was infiltrated by TILs, 1% or 5% criteria were used; all available full sections were evaluated) and the presence of TLSs (defined as the lymphoid aggregation with high endothelial venules) in primary and metastatic sites.

Results: The mean percentage of TILs in the lung (19.5%) was significantly higher than those in the liver (4.2%), brain (8.3%), and ovary (3.4%, p<0.001). The level of TILs in metastatic tumors of the lung was higher than those in primary tumors (Wilcoxon signed-rank test, p<0.001), while metastatic tumors of the liver (p=0.024) and brain (p=0.095) showed lower level of TILs than in primary tumors. TLSs were found only in the lung (27%) and liver (13%). Patients with lung metastasis (p<0.001), higher level of TILs (p=0.001), and presence of TLS (p=0.002) showed better overall survival. In patients with lung metastasis, who showed best clinical outcome, those with 10% or more of TIL had better overall survival than those with less than 10% (p=0.008).

Conclusion:. Metastatic breast tumors in the lung had more TILs than other sites and matched primary tumors. Underlying mechanism of differences of TILs among metastatic sites should be investigated.

#698

The loss of SATB2 promotes ulcerative colitis-related colon cancer progression through altered localized inflammatory response.

Sudipto Das,1 Kirsha Naicker,2 Rut Klinger,2 Yue Fan,2 Girish Mallya,2 Fredrick Ponten,3 Karin Jirström,4 Jacintha O'Sullivan,5 William M. Gallagher,2 Donal J. Brenan,2 Darran P. O'Connor1. 1 _Royal College of Surgeons in Ireland, Dublin, Ireland;_ 2 _University College Dublin, Dublin, Ireland;_ 3 _Uppsala University, Uppsala, Sweden;_ 4 _Lund University, Malmo, Sweden;_ 5 _Trinity College Dublin, Dublin, Ireland_.

SATB2 is a member of a family of special AT-rich binding proteins and a novel transcription factor that orchestrates gene expression in a tissue-specific manner by regulating higher-order chromatin structure. Here, we assess the precise functional role of SATB2 in the development and progression of colorectal cancer, initiating from the proinflammatory phenotype. Differential expression of SATB2 was observed in colorectal cancer, with loss of expression occurring sequentially along the adenoma-carcinoma sequence, with the early preneoplastic tissue demonstrating highest SATB2 levels, which diminishes within adenomas followed by complete loss of SATB2 expression in metastatic disease. Functionally, we demonstrate that siRNA-mediated knockdown of SATB2 in SW480 cells was associated with the acquisition of an aggressive phenotype. Consistent with these findings, loss of SATB2 protein and mRNA expression was determined as an independent predictor of poor prognosis in 3 independent CRC cohorts (n=776). Furthermore, gene set enrichment analysis in two of these independent colorectal cancer cohorts revealed that loss of SATB2 mRNA expression was associated with a TH2 cytokine response and checkpoint genes like CTLA4 and PD1 and modulation of SATB2 expression in CRC cell lines led to altered expression of proinflammatory cytokines. Within this context, following classification of the patient samples within these cohorts into the recently published consensus molecular subtypes (CMS) revealed that SATB2 levels were significantly lower in the CMS1 subtype that effectively represents the immune subtype of colorectal cancer. In addition, tissue microarray (TMA) analysis of a sigmoidal colorectal cancer TMA representing 350 colorectal cancer patients demonstrated that lower SATB2 levels with poor outcome and inversely correlated with CD68+ infiltrates. Intriguingly, SATB2 expression was associated with chromosomal stability as knockdown of SATB2 resulted in an increase of anaphase bridging, which was independent of telomere length and directly related to the spindle assembly complex. Given the relationship between SATB2, chromosomal instability and local inflammatory response, SATB2 protein expression was assessed in a large cohort of patients with ulcerative colitis (UC), some of whom developed UC-related carcinoma. SATB2 protein expression decreased across the disease spectrum from normal to UC to dysplasia to carcinoma, suggesting that SATB2 expression can be used to monitor UC patients at risk of developing CRC. The findings from this study for the first time demonstrate the role of SATB2 as potential master immune-regulator in colon cancer and its pivotal role in regulating key inflammatory factors facilitating the progression of colorectal cancer initiating from a proinflammatory phenotype.

#699

Pd-l1 gene alterations identify a subset of diffuse large B cell lymphoma harboring a T cell inflamed phenotype.

Justin Paul Kline, James Godfrey, Sravya Tumuluru, Girish Venkataraman, Riyue Bao, Sonali M. Smith. _Univ. of Chicago, Chicago, IL_.

Programmed death – ligand 1 (PD-L1) expression on malignant cells is a dominant immune escape mechanism across a variety of human cancers, and classically occurs as an adaptive response to the presence of activated T cells and interferon gamma (IFN-γ) in the tumor environment. Interestingly, a unique genetic mechanism underlying PD-L1 up-regulation has been described in classical Hodgkin lymphoma (cHL) and in uncommon variants of diffuse large B cell lymphoma (DLBCL), in which the chromosomal region (9p24.1) encoding the PD-1 ligands is highly amplified. While chromosome 9p24.1 copy number alterations (CNA) are ubiquitous in cHL, which is exquisitely sensitive to PD-1 blockade therapy, they also occur in DLBCL, albeit with a lower incidence. Here, FISH was utilized to identify DLBCLs harboring pd-l1 CNA, thereby enabling a characterization the immunogenomic landscape of these lymphomas compared to those lacking pd-l1 CNA. Among 76 DLBCLs analyzed, 25% harbored pd-l1 CNA (12 copy gain, 6 amplified, 1 translocation). Pd-l1 CNA were highly enriched among DLBCLs with an activated B cell (ABC) phenotype (75%). PD-L1 cell surface expression was strikingly enhanced in DLBCLs with pd-l1 CNA versus those without, as assessed by PD-L1 H score. Furthermore, pd-l1 CNA were associated with a pronounced infiltration of DLBCLs by T cells with a restricted T cell receptor (TCR) – beta (β) repertoire, suggesting that lymphoma antigen-specific T cells had expanded in these samples. RNA-sequencing of pd-l1 gene-altered versus non-altered DLBCLs revealed differential expression of pd-l1, as well as genes involved in T cell and cytokine signaling in the former, indicating that pd-l1 gene-amplified DLBCLs have been subjected to host immune surveillance. Lastly, whole exome sequencing (WES) of pd-l1 gene altered versus non-altered DLBCLs to identify potential differences in mutational loads and profiles is ongoing. Collectively, our results indicate that pd-l1 CNA identify a unique biological subset of DLBCL that has activated an endogenous anti-lymphoma immune response, and that may be more likely to respond to checkpoint blockade therapy with anti-PD-1 antibodies. We propose that a straightforward FISH analysis for pd-l1 gene amplification may be useful as a predictive biomarker for immunotherapy responsiveness in DLBCL.

#700

Characterization of new AML PDX models: Engraftment kinetics and mutational profile.

Pali Kaur,1 Victoria Sachs,1 Amanda L. Christie,2 David M. Weinstock,2 James G. Keck1. 1 _The Jackson Laboratory, Sacramento, CA;_ 2 _Dana-Farber Cancer Institute, Boston, MA_.

Acute myeloid leukemia (AML) is a genetically heterogeneous cancer of progenitor cells in myeloid hematopoiesis. Due to heterogeneity, most of AMLs are not responsive to targeted therapy. In addition, treatment outcome varies greatly depending upon patient age, disease etiology and mutational status. Previously, a large repository of well-characterized patient-derived- xenografts (PDXs) of hematologic malignancies was established (Townsend et al., 2016). This repository was generated by injecting primary bone marrow and blood samples from patients with leukemia into NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) mice. An average success rate of 23.2% engraftment was observed in first passage (P0). In this study, we have characterized ten AML PDX models in NSG-SGM3 mice by serial transplantation. NSG-SGM3 (NOD.Cg-Prkdcscid Il2rgtm1Wjl Tg(CMV-IL3,CSF2,KITLG)1Eav/MloySzJ) mice have been found to have increased proliferation and survival of engrafted AML cells along with decreased disease latency (Wunderlich et al., 2010). Leukemic engraftment in these models was confirmed by flow analysis for hCD45+ hCD33+ in the peripheral blood, bone marrow and spleen of engrafted mice. Kinetics of engraftment varied from one model to another; and ranged from 10% - 80% of hCD33+ in peripheral blood 8 week post AML cell injection (1X106 cells/mouse). Four models displayed positive response to Cytarabine (Ara-C) treatment with reduction in levels of human CD33+ cells following a 5-day treatment cycle. Data will be presented on model characterization to show distinct engraftment kinetics, correlation with disease mutations and response to AML standard of care drug Cytarabine. These models have varying mutational profiles and treatment histories. Mutations include FLT3, FLT3-ITD, NPM1, TP53, DNMT3A and CUX1 alone or in combination. Treatment phases at the time of collection varied from untreated to relapse post-chemotherapy. These AML PDX models are useful tools for testing new experimental therapeutics. Diversity of these AML PDX models will facilitate focus on specific AML targets, thus providing an invaluable tool set to support drug discovery.

#701

An inflammation paradox: Obesity vs. neutrophil-lymphocyte ratio in immunotherapy.

Wungki Park, Vaia Florou, Alfredo Torres, Diana Saravia, Sandra Algaze, Gilberto Lopes, University of Miami. _University of Miami, Miami, FL_.

Background: The dual roles of inflammation in malignancy has been well-described. Neutrophils play a key role in inflammation and carcinogenesis. We previously showed that high NLR may confer resistance to PD1/PDL1 inhibitors in lung cancer patients. In addition, obesity has been shown to produce chronic inflammation and neutrophil dysfunction have been described. The interplay between obesity and neutrophil in solid tumor patients treated with immunotherapy is unknown. Methods: We retrospectively evaluated 408 patients with solid tumors who treated with immunotherapies at the University of Miami. Cox regression was performed to evaluate correlation of obesity (BMI >30) and NLR <5 with Progression Free Survival (PFS) and Overall Survival (OS). Pearson Correlation was used for correlation between BMI and NLR. Results: Median follow-up was 8.6 months. There were head, neck and thoracic (n=257), gastrointestinal (n=32), genitourinary (n=58), skin and musculoskeletal (n=61). There were 230 males, mean age was 65.8. Treatments include nivolumab (n=204), pembrolizumab (n=128), atezolizumab (n=35), and combination with ipilimumab (n=41). There were underweight (BMI<18.5, n=27), normal weight (18.5≤BMI<25, n=186), overweight (25≤BMI<30, n=117), and obese patients (BMI≥30, n=78). Obesity (BMI≥30) and NLR <5 were strongly correlated with better OS with HR: 3.031 and HR: 2.424, respectively. (Table 1) There was trend of better PFS in obese patients, however it was not statistically significant. BMI and NLR showed inverse correlation. (r= -0.172, p=0.001) Conclusions: Surrogates of inflammation, such as obesity and NLR in patients with solid cancer may predict immunotherapy responses. High NLR was associated with adverse clinical outcomes and failure to checkpoint inhibitors, perhaps due to unfavorable tumor-associated neutrophil phenotypes. There was also a strong correlation between obesity and overall survival in these solid tumor patients.

Table 1. Multivariate analysis | |  | |  | |

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

|

PFS | OS

Factor | HR | 95% C.I. | p-value | HR | 95% C.I. | p-value

ECOG>1 | 2.188 | (1.561-3.068) | 0.0001 | 3.031 | (1.937-4.742) | 0.0001

BMI<30 | 1.136 | (0.806-1.601) | 0.468 | 2.934 | (1.484-5.801) | 0.002

NLR≥5 | 1.704 | (1.301-2.230) | 0.0001 | 2.424 | (1.661-3.538) | 0.0001

#702

IMLEC: A population of innate myelolymphoblastoid effector cell expanded by inactivation of mTOR complex 1.

Fei Tang,1 Peng Zhang,1 Peiying Ye,1 Christopher A. Lazarski,1 Qi Wu,2 Ingrid L. Bergin,2 Timothy P. Bender,3 Michael N. Hall,4 Ya Cui,5 Liguo Zhang,5 Taijiao Jiang,5 Yang Liu,1 Pan Zheng1. 1 _Children's National Medical Center, Washington, DC;_ 2 _University of Michigan Medical School, Ann Arbor, MI;_ 3 _University of Virginia, Charlottesville, VA;_ 4 _University of Basel, Basel, Switzerland;_ 5 _Institute of Biophysics, Chinese Academy of Sciences, Beijing, China_.

Adaptive autoimmunity is restrained by controlling population sizes and pathogenicity of harmful clones, while innate destruction is controlled at effector phase. We report here that deletion of Rptor in hematopoietic stem/progenitor cells causes self-destructive innate immunity by massively increasing the population of previously uncharacterized innate myelolymphoblastoid effector cells (IMLECs). IMLECs are CD3-B220-NK1.1-Ter119\- CD11clow/-CD115-F4/80low/-Gr-1\- CD11b+, but surprisingly express high levels of PD-L1. Although they morphologically resemble lymphocytes and actively produce transcripts from Immunoglobulin loci, IMLECs have non-rearranged Ig loci, are phenotypically distinguishable from all known lymphocytes, and have a gene signature that bridges lymphoid and myeloid leukocytes. Rptor deletion unleashes differentiation of IMLECs from common myeloid progenitor cells by reducing expression of Myb. Importantly, IMLECs broadly overexpress pattern-recognition receptors and their expansion causes systemic inflammation in response to Toll-like receptor ligands. Our data unveil a novel leukocyte population and an unrecognized role of Raptor/mTORC1 in innate immune tolerance. Moreover, since IMLECs constitutively express high levels of PD-L1, it is interesting to investigate their functions in cancer immunotherapy.

#703

The anti-cancer drug BPM 31510 elicits immune-modulatory effects through regulation of IL-10.

Louisa Dowal, Maria D. Nastke, Nidhi Gaur, Richa Singh, Samantha Fowler, Shyamali Jayashankar, Anne R. Diers, Stephane Gesta, Vivek Vishnudas, Niven R. Narain, Rangaprasad Sarangarajan. _BERG, LLC, Framingham, MA_.

Mitochondria have emerged as important immune-regulatory organelles, supporting biosynthetic and bioenergetic needs of immune cells and controlling cytokine production. BPM31510 is a clinical stage, nanodispersion of the mitochondrial electron transfer molecule ubidecarenone (CoQ10). Given that cytokine secretion orchestrates immune cell effector function in the tumor microenvironment and importance of mitochondria in this process, the ability of BPM31510 to modulate cytokine secretion was investigated. An ex vivo model using phytohemagglutinin-activated healthy donor peripheral blood mononuclear cells (PBMCs) treated with increasing concentrations of BPM31510 was used to assess cytokine secretion profiles. Analysis of supernatants by multi-analyte ELISA platform revealed effector cytokines IL-2 and IFN-γ to be dose-dependently increased with BPM31510 treatment while IL-10, a key immune-regulatory cytokine, was decreased. Analysis of monocyte depleted PBMCs compared to monocytes alone demonstrated that the observed changes in IL-2 and IFN-γ secretion occur only when both lymphocytes and monocytes are present; however, monocytes alone were identified as the significant source for IL-10. To define the mechanism of BPM31510-induced IL-10 attenuation, two monocytic lineage models were employed in vitro, LPS-stimulated THP-1 monocytes and PMA-differentiated U937 macrophages. Similar to monocytes isolated from PBMCs, treatment of LPS-stimulated THP-1 monocytes with increasing doses of BPM 31510, resulted in a decrease in IL-10 secretion as did treatment of U937 macrophages. The contribution of canonical IL-10 regulatory pathways, ERK, PI3K and STAT3, was assessed by co-treating U937 macrophages with BPM31510 and inhibitors of these pathways (U0126, Wortmannin, and Stattic, respectively). Co-treatment resulted in a further decrease in IL-10 secretion than any inhibitor treatment alone, suggesting that regulation of IL-10 secretion by BPM31510 is not exclusively through canonical signaling pathways. Nevertheless, further investigation into the activation status of these pathways revealed a decrease in STAT3 phosphorylation with BPM31510 treatment, but not of ERK or Akt. Consistent with decreased STAT3 activation, levels of IL-10 transcript, a STAT3 target gene, were also decreased in these samples, indicating that BPM31510 partially regulates IL-10 secretion via inhibition of the STAT3 signaling cascade. Taken together, these data demonstrate BPM31510 attenuates IL-10 secretion from the monocytic lineage through both canonical and non-canonical IL-10 regulatory pathways; this may have important implications for macrophage-mediated immunosuppression and immune cell crosstalk in the tumor microenvironment more broadly upon treatment with BPM31510.

### Response and Resistance to Immune Checkpoint Blockade

#704

Tumor-experienced NK cells inhibit T cell proliferation and activation through PD-L1.

Jessica M. Sierra, Ximena L. Raffo Iraolagoitia, Florencia Secchiari, Andrea Ziblat, Sol Y. Nuñez, Nicolas I. Torres, Maria V. Regge, Carolina I. Domaica, Norberto W. Zwirner, Mercedes B. Fuertes. _IBYME-CONICET, Buenos Aires, Argentina_.

Natural killer (NK) cells play a key role in cancer immunosurveillance and in the elimination of infected cells, however, NK cells from cancer patients show an altered phenotype and impaired effector functions. Moreover, evidence of a regulatory role for NK cells is emerging in diverse models of viral infection, transplantation and autoimmunity. In the tumor context, we have recently described an immunoregulatory population of PD-L1-expressing NK cells arising in tumor-bearing mice that inhibits CD8+ T cell priming. Those results led us to examine PD-L1 expression on human NK cells after tumor recognition and their regulatory potential. First, we analyzed PD-L1 expression on tumor-infiltrating or peripheral blood NK cells (CD56+CD3- cells) from patients with kidney cancer by flow cytometry. We found an increased frequency of PD-L1+ tumor-infiltrating NK cells compared to autologous peripheral blood NK cells. Moreover, PD-L1 was up-regulated on peripheral blood NK cells from those cancer patients compared to healthy donors. Further experiments were performed in vitro to investigate the mechanisms involved in tumor-induced PD-L1 up-regulation on NK cells. After culture of human PBMCs from healthy donors with the susceptible K562 cell line, PD-L1 expression was induced on NK cells. Transwell and receptor blockade experiments showed a dependence on cell-to-cell contact that involved NKG2D engagement. PD-L1 expression on isolated NK cells cultured with K562 tumor cells could be further increased by soluble factors derived from tumor-experienced PBMCs; neutralization of different cytokines identified IL-18, produced by monocytes, as the main factor responsible for PD-L1 up-regulation. Analysis of NK cells after incubation with K562 cells showed that relative to PD-L1- NK cells, PD-L1+ NK cells expressed increased levels of the activation markers CD25 and CD69 and the effector molecules TRAIL, FasL, IFN-γ and CD107a, suggesting that PD-L1 is preferentially up-regulated on activated NK cells. Finally, to evaluate the immunosuppressive potential of these NK cells, CFSE-labeled autologous T cells were stimulated with anti-CD3/anti-CD28 antibodies (Ab) and cultured in the presence of sorted tumor-experienced NK cells or control NK cells, and in the absence or in the presence of anti-PD-L1 blocking Ab. After 5 days, T cell proliferation and activation (evaluated as CFSE dilution and CD25 expression by flow cytometry) were diminished in the presence of tumor-experienced NK cells compared to control NK cells, and this inhibition was reverted by PD-L1 blockade. Our results indicate that tumor recognition results in upregulated expression of PD-L1 on NK cells leading to a direct inhibition of T cell priming trough PD-L1. Thus, rational manipulation of these regulatory cells could lead to improved anti-tumor immunity in vivo.

#705

**Nonuniform T-cell infiltration induced by PD-1 checkpoint blockade,** ex vivo, **predicts distinct clinical response.**

Munisha Smalley,1 Basavaraja Shanthappa,1 Hans Gertje,2 Mark Lawson,2 Baraneedharan Ulaganathan,2 Allen Thayakumar,2 D.C. Doval,3 Anurag Mehta,3 S. P. Somashekhar,4 Padhma Radhakrishnan,2 Pradip Majumder,2 Aaron J. Goldman5. 1 _Mitra Biotech, cambridge, MA;_ 2 _Mitra Biotech, MA;_ 3 _Rajiv Gandhi Cancer Institute & Research Centre, New Delhi, New Delhi, India; _4 _Manipal Hospitals, Bangalore, India;_ 5 _Harvard Medical School, cambridge, MA_.

Background: The presence and activity of lymphocytes within the tumor is critical for clinical response to cancer immunotherapy, such as immune checkpoint blockade. Tumors with poor T-cell inflamed phenotypes, often referred to as a 'cold' tumor, is associated with modest clinical response. High baseline infiltration of effector T-cell lymphocytes is considered 'hot', and patients are predicted to respond more favorably to treatment. However, patient-to-patient response and durability remains highly variable. There is an urgent gap in available methods to study lymphocyte infiltration, trafficking and spatial heterogeneity induced by different cancer immunotherapies in individual patients. Moreover, there is a poor correlation between therapy-induced lymphocyte infiltration with clinical response, which could be shaped using personalized approaches to therapy. Methods: Here, we used CANscriptTM, an ex-vivo human tumor model that recapitulates and preserves the native, patient-autologous tumor microenvironment, including autologous patient-derived peripheral blood mononucleated cells (PBMC). Utilizing tissue from breast cancer patients classified as either 'cold' or 'hot', we studied lymphocyte infiltration under pressure of a-PD-1 immune checkpoint blockade (pembrolizumab) over a 72h time course. Using fluorescent labelling and flow cytometric analysis we characterized infiltrating lymphocytes, studying the role of T-cell repertoires under different environmental and immunotherapy pressures. We coupled these analyses with multiplex immunohistochemistry (CD3+, CD4+, CD8+, pan-cytokeratin, DAPI) to map spatial heterogeneity of tumor cells and lymphocytes before and after treatment, ex-vivo. Results: We determined that immune checkpoint blockade induced unique patterns of migration and infiltration of effector T-cells (Teff) and T-regulatory (Treg) cells in 'hot' vs 'cold' tumors. Furthermore, we determined that, in some instances, 'cold' tumors can be driven towards a 'hot' phenotype characterized by trafficking of active immune lymphocytes following treatment, which corresponded to differential ratio of Teff to Treg compared to baseline. Concluding remarks: Taken together, these data demonstrate the utility of CANscriptTM as a platform to characterize response to immunotherapy in a spatial context, providing insight into the migratory patterns of immune cell subsets at the individual patient level. Such an advance in our preclinical methods to study immuno-modulators may help guide treatment decisions for clinicians while simultaneously functioning as a platform to study and discover mechanisms of clinical efficacy for emerging drug combinations.

#706

**In vivo** **tumor-associated mutation screen identifies PI3K activation as a mechanism of resistance to PD-1 blockade.**

Natalie B. Collins,1 Brian C. Miller,1 Kevin Bi,1 Rose Al Abosy,1 Kathleen Yates,1 Yashaswi Shrestha,2 John Doench,2 Jesse Boehm,2 W Nicholas Haining1. 1 _Dana-Farber Cancer Institute, Boston, MA;_ 2 _Broad Institute, Boston, MA_.

Known genomic correlates of response to immunotherapy do not perfectly predict clinical outcome, supporting the existence of unknown mechanisms of resistance to tumor immunity. We hypothesize that somatic, cancer-associated mutations account for heterogeneity in spontaneous response to tumors and response to immunotherapy. We have undertaken a systematic in vivo screen to identify mechanisms of resistance to tumor immunity in order to define a comprehensive set of therapeutic targets and provide biomarkers of sensitivity to immunotherapy. Mouse tumor cell lines (MC38 colon carcinoma or B16 melanoma) were engineered to express a library of barcoded open reading frames (ORFs) mutagenized to encode known cancer-associated somatic mutations. Tumor-bearing animals were treated with anti-PD-1. A mutation in Phospho-Inositol 3 Kinase (PI3K), PIK3CA c.3140A>G, increased in representation in anti-PD-1 treated tumors but not in immunodeficient animals, suggesting that activity of the mutant allele conferred selective growth advantage in the setting of tumor immunity. This mutation encodes a constitutively active mutant catalytic domain, PIK3CA H1047R. MC38 tumors homogeneously expressing H1047R and implanted into wild-type mice failed to respond to anti-PD-1 therapy, while tumors expressing a control gene regressed. Pharmacologic PI3K inhibition resensitized tumors to treatment with anti-PD-1. PD-1-treated PIK3CA H1047R tumors had fewer infiltrating CD8+ T cells as measured by immunohistochemistry and flow cytometry. Single-cell RNA-seq of tumor-infiltrating immune cells revealed a population of myeloid cells expressing known immune inhibitory proteins that differentially enriched in PIK3CA H1047R-expressing tumors. Our data suggest that PI3K has, in addition to its well-described oncogenic role, a role in tumor immune evasion mediated by establishment of an inhibitory myeloid microenvironment. As such, activating mutations in PI3K may be useful as a biomarker of poor response to immunotherapy, and these studies provide a rationale for therapeutic combination trials of PI3K inhibition with checkpoint blockade and other myeloid-targeting immunotherapies.

#707

Circulating immune profile in childhood neuroblastoma displays an activated response with simultaneous expression of checkpoint proteins by T cells and monocytes.

Flavia B. Oliveira,1 Luísa M. Magalhães,2 Livia S. Passos,2 Joaquim C. Aguirre Neto,1 Álvaro P. Dutra,1 Patricia R. Martins,3 Paulo G. Oliveira Salles,3 Walderez O. Dutra,2 Kenneth J. Gollob4. 1 _Hospital Santa Casa-Belo Horizonte, Belo Horizonte, Brazil;_ 2 _UFMG, Belo Horizonte, Brazil;_ 3 _Instituto Mario Penna, Belo Horizonte, Brazil;_ 4 _AC Camargo Cancer Center, Sao Paulo, Brazil_.

Neuroblastoma (NB) is a rare childhood cancer typically affecting children under five and arising from neural crest sympathoadrenal linage cells. Despite its rarity, it is the most frequent solid tumor in children under five. Determination of the circulating immune response in childhood NB before treatment has begun can help in the identification of potential cells and pathways involved in disease development and suitability for immunotherapy. These studies were designed to investigate the cellular and soluble immune response (cytokines) using flow cytometry with peripheral blood samples in a group of seven NB patients and four non-NB solid tumor patients between the age of 20-42 months. These data of the systemic immune response were compared to the intensity of tumor infiltrating lymphocytes and disease outcome after 18 months of treatment. Analysis of peripheral blood cellular immune status revealed that the NB group displayed an increase in the frequency of effector memory T cells (p <0.05), as well as an increase in the expression of inhibitory molecules CTLA-4 and PD-1 by CD4 + T lymphocytes (p < 0.05), and CTLA-4 by CD8+ T cells (p <0.06). Moreover, an increase in PD-L2 was seen on transitional (CD14+CD16+) and non-classical (CD14-CD16+) monocytes from NB (p <0.05). Further studies analyzing circulating cytokines (IL-2, 4, 5, 6, 9, 10, 13, 17A, 17F, 21, 22, IFN-gamma and TNF-alpha) in the plasma of the same patients showed an overall pattern of increased cytokines from the NB group as compared to the control group with increased IL-5, IL-6, IL-9 and IL-10 (p < 0.10). Lastly, the NB group showed an intense tumor infiltrating lymphocyte response in both the septa region and tumor nests, which was correlated with circulating TNF-alpha levels (R2 = 0.82, p = 0.06). Overall these findings of the systemic immune response in this rare childhood cancer indicate an active immune response with profiles consistent with both a down modulatory response and an overall activated profile based on soluble cytokines and tumor infiltrating lymphocyte intensity. These findings point to a systemic immune-modulation profile of the cellular response associated with NB disease.

#708

Increase of senescent mesenchymal stromal/stem cells is predictive of unresponsiveness to the treatment with immune checkpoint inhibitors.

Chie Kudo-Saito, Takahiro Miyamoto, Mami Kawamura, Yamato Ogiwara, Kazunori Aoki. _National Cancer Center, Tokyo, Japan_.

Purposes: The therapeutic efficacy induced by immune inhibitory checkpoint inhibitors is still limited to a part pf the treated patients, and the methods effective in overcoming the treatment failures have not been established yet. A main issue under discussion is immune exhaustion as well as immune senescence with age, which is caused by persistent and chronic inflammation due to both internal and external stimuli ultimately leading to immune dysfunction in cancer patients. In literature, aging has been reported as a strong risk factor not only for causing cancers, but also for inducing hyper progression after treatment with immune checkpoint inhibitors in clinical settings. This suggests that a distinct strategy from releasing the brake on immune suppression might be needed for properly organizing anti-tumor immunity in aged hosts. In this study, we attempted to elucidate the molecular mechanisms underlying the immune exhaustion with age.

Results: We used young (1 to 4-month-old) and aged (18 to 24-month-old) C57BL/6 mice, and comprehensively analyzed cell population and immune responses of spleen cells and bone marrow cells obtained from the mice. With age, CD45-CD166+ mesenchymal stromal/stem cells (MSCs) increased correlatively with increase of immunosuppressive CD4+CTLA4+Foxp3+ Tregs and CD11b+Gr1+ MDSCs, and dysfunctional CD3+PD1+Tim3+ T cells in mice. These cells were further expanded by implanting with tumor cells such as melanoma B16-F10 and lung cancer 3LL, and immune checkpoint inhibitors were ineffective in suppressing tumor progression in aged mice. As compared to the young MSCs, the aged MSCs generated much more dysfunctional T cells in vitro and in vivo, and differentiated into hypertrophic adipocytes with a large lipid droplet containing a huge amount of inflammatory adipokines. These suggest that the increasing MSCs play a key role in disturbing anti-tumor immunity both before and after differentiation in aged hosts. Utilizing cDNA microarray analysis, we identified the specific molecules those are significantly upregulated in the aged MSCs, but not young MSCs. Knockdown of the gene expressions in the aged MSCs using the specific siRNAs deprived of the morbid properties, and generated potent tumor-specific CTLs through balancing immunity in the aged mice.

Conclusions: These results demonstrate that increase of the senescent and inflammatory MSCs are, at least in part, responsible for the unresponsiveness to the treatment with immune checkpoint inhibitors. Elimination of these MSCs may be a promising strategy for successfully controlling immunity against cancer.

#709

Anti-PD-1 therapy reduces bone lesion growth in a novel syngeneic bladder cancer bone metastasis model.

Tiina E. Kähkönen,1 Mari I. Suominen,1 Jussi M. Halleen,1 Jenni Bernoulli,1 Arne Scholz2. 1 _Pharmatest Services, Turku, Finland;_ 2 _Bayer AG, Berlin, Germany_.

Bladder cancer is a common and aggressive type of cancer occurring both in women and in men. At diagnosis, lymph node involvement is frequently observed and in some cases metastatic spread can already be seen at early stage. With advanced stages, the metastatic incidence increases and the most common metastatic sites are bones, lung, liver and peritoneum. Bone metastases count about 50% of all metastases and the 5-year survival rate in metastatic disease is only 15%. Bone metastases are incurable but can be treated to reduce tumor burden in bone, and new therapies such as immunotherapies hold the potential to treat patients with bone metastatic bladder cancer. The aim of the study was to establish a novel syngeneic model for bladder cancer bone metastasis that could be used to study efficacy of new immunotherapies, and to test efficacy of anti-PD-1 therapy in the model.

In the model establishment study, an intratibial injection of varying number of murine MBT-2 bladder cancer cells was given to 5-6 weeks old female C3H/HeN mice. Tumor-induced bone changes were followed by X-ray imaging once a week. At sacrifice, tumor-bearing tibias were collected and evaluated by histology. In the efficacy study, 5x105 MBT-2 cells were used. The mice were stratified to treatment groups based on similar bone lesion areas at 10 days after cancer cell inoculations and treated with anti-PD-1 therapy (RMP1-14) or isotype control (rat IgG2a, 200 µg per dose) at Q3D schedule. Bone lesion growth was followed by X-ray imaging one week after start of treatment and at sacrifice after three weeks on treatment.

In the model establishment study, tumor take rate of 50% was observed. MBT-2 cells induced an osteolytic bone reaction resulting in substantial bone loss in the mice. The osteolytic bone lesions started to be visible between 7-14 days and the maximum duration of the study was 4 weeks from the cancer cell inoculations. Histology confirmed a large tumor and decreased bone mass in the inoculated tibia. In the efficacy study, treatment with anti-PD-1 reduced bone lesion growth. A trend towards decreased bone lesion area was seen already at one week on treatment and a significant reduction of bone lesion area was seen at sacrifice.

A novel syngeneic bladder cancer bone metastasis model was established. According to the observed significant effects on bone lesion growth by anti-PD-1 treatment this model can be used in preclinical efficacy assessment of new immunotherapies as monotherapy or in combination with other therapies.

#710

CD73 inhibitors (CD73i) reverse the AMP/adenosine-mediated impairment of immune effector cell activation by immune checkpoint inhibitors (ICI).

Annette Becker, Nell Narasappa, Fangfang Yin, Kristen Zhang, Daniel DiRenzo, Timothy Park, Jaroslaw Kalisiak, Ken Lawson, Jenna Jeffrey, Jay P. Powers, Ulrike Schindler, Matthew J. Walters, Joanne B. Tan. _Arcus Bioscienes, Hayward, CA_.

INTRODUCTION: CD73 catalyzes the extracellular generation of adenosine (ADO) from adenosine monophosphate (AMP). ADO suppresses immune responses, including those of T cells, NK cells and dendritic cells through activation of A2aR and A2bR receptors. Exhausted T cells and NK cells express high levels of several immune checkpoint proteins, including PD-1 and TIGIT. We present here preclinical data on the ability of CD73i to reverse effector cell suppression from exposure to ADO even in the presence of ICI.

METHODS: CD73i effects in a monotherapeutic setting were assessed by CD3/CD28/CD2 T cell stimulation and cytolytic assays. Combinatorial settings were assessed using mixed lymphocyte reactions (MLRs). In vivo effects of CD73i + ICI were determined using syngeneic tumor models.

RESULTS: CD73 is expressed across a wide range of tumor types, including those with limited response to anti-PD-1 therapy. CD73i completely rescued AMP-mediated inhibition of T cell proliferation and effector function as well as NK cell cytolytic function. AMP abrogated the enhanced allogeneic CD4+ T cell activation and IFN-γ production mediated by blocking PD-1/PD-L1 and TIGIT, an effect that was reversed by CD73i. Mechanistically, addition of AMP in MLRs repressed expression of activation markers and immune checkpoint proteins. Thus, activation of the adenosinergic pathway may limit the efficacy of ICI. TCGA data from anti-PD-1-treated melanoma patients identified CD73 expression as a negative prognostic factor. Finally, co-administration of a CD73i with an anti-PD-1 mAb resulted in significant reduction of tumor volume associated with increases in immune cell infiltration.

CONCLUSIONS: CD73 inhibition, alone or in combination with anti-PD-1 and anti-TIGIT antibodies, translates into potent enhancement of immune cell activation in a variety of studies. These data provide a rationale for CD73i + ICI combinations.

#711

Intratumoral immunotherapy with TLR7/8 agonist MEDI9197 modulates the tumor microenvironment leading to enhanced activity when combined with other immunotherapies.

Stefanie R. Mullins,1 John Vasilakos,2 Katharina Deschler,1 Iwen Grigsby,2 Song Ren,3 Matthew J. Elder,1 Simon J. Dovedi,1 Andrew J. Leishman,1 Patricia Ryan,3 Zachary Cooper,3 James Elvecrog,2 Ronald Herbst,3 Rakesh Kumar,3 Mark Tomai,2 Robert W. Wilkinson1. 1 _Medimmune, Cambridge, United Kingdom;_ 2 _3M, MN;_ 3 _Medimmune, MD_.

Despite the recent breakthrough of checkpoint blockade antibodies targeting T cells to combat cancer, it has become apparent that the majority of patients are not fully responsive. Engaging innate immune cells is a promising way to fully exploit the anti-cancer potential of the immune system. Our strategy is to stimulate TOLL-like receptors (TLRs) with MEDI9197, a potent TLR7 and TLR8 agonist, which stimulates the release of interferon-alpha (IFN-α) from plasmacytoid dendritic cells (pDCs) and interleukin-12 (IL-12) and other cytokines from myeloid DCs. While several compounds including MEDI9197, CpG and STING agonists can induce the production of IFN-α, we show that only MEDI9197 is able to induce high levels of interferon-gamma (IFN-γ) and IL-12p70 in human PBMCs, a hallmark of TLR 7/8 agonists. We also demonstrate in vitro that MEDI9197 upregulates activation markers on both innate and adaptive human immune cells, repolarizes macrophages and skews immunity from a Th2 towards a Th1 phenotype in a PHA assay, thereby increasing the functional activity of NK and cytotoxic CD8 cells. MEDI9197 is an imidazoquinoline with a unique lipid tail and formulation designed for intratumoral (IT) dosing and retention at the site of injection to minimize unwanted systemic exposure. Mass spectrometry imaging of MEDI9197 in mouse tumors and whole-body autoradiography in rats confirm local retention of MEDI9197, with < 5% of the initial IT dose detectable in the rat serum. We show in the B16-OVA mouse syngeneic tumor model that a single IT dose increases the percentage of tumor infiltrating lymphocytes (TILs), enhances activation of T cells, and leads to a qualitative shift from single to dual cytokine production (IFN-γ and Tumor necrosis factor alpha, TNF-α). Gene expression analysis further reveals increased expression of activatory innate and adaptive immune gene signatures consistent with increased inflammation. As our mouse flow cytometry studies show elevated levels of PD-1 and PD-L1 on TILs, the combination of MEDI9197 with anti-PD-L1 mAb was also evaluated and resulted in enhanced efficacy compared to MEDI9197 alone. We further demonstrate in vitro that combining MEDI9197 with durvalumab (anti-PD-L1) augments cytokine production (e.g. IL-2, IFN-γ) in a co-culture of human DCs and allogenic T cells. These data showcase MEDI9197 as a potent modulator of the innate and adaptive immune system in the fight against cancer and support the rationale for evaluating MEDI9197 in combination with checkpoint blockade and costimulatory agonists in the clinic. MEDI9197 is currently being evaluated in human clinical trials for safety and efficacy (NCT02556463).

#712

Immune checkpoint inhibitor induced tumor gene expression changes in murine syngeneic colon cancer models.

Patrick Fadden, Douglas Weitzel, Kelli Davis, Thi Bui, David Hurtado, Hannah Haines, Rhiannon Roark, Ashleigh Derrick, Beverly Godfrey, Ian Belle, Katherine Krontz, Cristina Sokolowski, Aidan Synnott, Edgar Wood. _Charles River Laboratories, Morrisville, NC_.

The success of immune checkpoint therapy with anti-PD-1 and anti-CTLA-4 antibodies has generated broad interest in immuno-based interventions for oncology. Preclinical efficacy assessment of these novel immune-oncology therapies requires a functional immune system and therefore is frequently performed in murine syngeneic tumor models. In addition to tumor growth inhibition, syngeneic tumor models provide a platform to investigate the mechanism of action for a given novel therapeutic target. Characterization and mechanism of action for immune-based cancer therapies in murine syngeneic tumor models is often assessed by flow cytometry; measuring changes in tumor infiltrating leukocyte (TIL) populations following treatment induction. We assessed whether transcriptomic gene expression analysis could identify additional relevant biomarkers for immunotherapy response. Mice bearing subcutaneous syngeneic colon cancer tumors were dosed intraperitoneally with either vehicle alone, anti-CTLA-4, anti-PD-1, or a combination of the two immune checkpoint inhibitors on days 1, 4, and 8. Tumors were harvested on day 9 and half from each group were assessed for TIL population composition by flow cytometry and half by Clariom D mouse transcriptome array analysis. We identified a number of immune cell-related genes with differential expression that correlated to the therapeutic response and were consistent with changes identified in TIL populations by flow cytometry. These results provide an additional metric for assessing immunotherapy response in vivo. In addition, these data suggest that a smaller, defined panel of gene targets may be developed as biomarkers for specific immune system responses to immune checkpoint therapies in murine syngeneic models.

#713

Upregulation of immune checkpoint molecules on Treg cells in tumor microenvironment reinforces immune exhaustion in cancer patients.

Hye Ryun Kim,1 Hyo Jin Park,2 Jimin Son,2 Hyo Sup Shim,1 Byoung Chul Cho,1 Sun Young Rha,1 Sang-Jun Ha2. 1 _Yonsei Univ. Cancer Ctr., Seoul, Republic of Korea;_ 2 _Yonsei Univ., Seoul, Republic of Korea_.

Purpose: Regulatory T (Treg) cells perform the immune suppressive function in cancer, but their suppressive mechanism in tumor microenvironment (TME) has not been clearly elucidated. We aim to identify the phenotype and functional mechanism of Treg cells in TME from cancer patients.

Experimental Design: We collected 72 malignant effusion (ME) and peripheral blood (PB) specimens from stage IV cancer patients and 10 tumor tissue (TM) and PB from lung cancer patients who underwent surgery. Lymphocytes from ME, TM, and PB were analyzed for subtype of Tconv and Treg cells, and their expressions of immune checkpoint (IC) molecules including programmed death (PD-1), cytotoxic T lymphocyte antigen-4 (CTLA-4), and T cell immunoglobulin containing molecule-3 (TIM-3) using by flow cytometry. To examine the functional role of PD-1 expressed Treg cells, TC-1 lung cancer mouse model was used.

Result: Upregulation of PD-1 on Treg cells in TM and ME compared to PB was even more distinguishable than that on CD4+ and CD8+ Tconv cells. To clarify the characteristics of tumor infiltrating Treg cells, we comprehensively examined the characteristics of Treg cells from tumor, peri-tumor, and PB in lung cancer patients by analyzing the expression of IC molecules including PD-1, TIM-3, CTLA-4, and TIGIT, of which PD-1 is the highest expressed on tumor infiltrating Treg cells. To investigate the mechanism by which Treg cells mediate immune suppression, we compared the suppressive activity of Treg cells expressing high and low levels of PD-1 by co-culturing each population with naïve CD8+ T cells with or without αCD3/CD28 stimulation. This was more potently inhibited in co-cultures with PD-1high tumor infiltrating Treg cells than in those with PD-1low Treg cells. PD-1 blocked tumor infiltrating Treg cells demonstrated a significantly decreased suppressive function in the proliferation of CD8+ cells and their IFN-γ production. These results implicate that PD-1 expressed tumor infiltrating Treg confers the suppressive function of proliferation in CD8+ T cells through PD-1: PD-L1 interaction.

Conclusion: Our study evidently demonstrated that upregulated PD-1 on tumor infiltrating Treg cells and their PD-1: PD-L1 interaction could be potential cause of T cell suppression, which might be helpful to completely understand their suppressive mechanism in cancer patients.

#714

A case for combining immunotherapy and targeted small molecule inhibitors: Immunoregulation by primary melanoma cells.

Rachana R. Maniyar, Sanjukta Chakraborty, Neha Y. Tuli, Ghada Ben Rahoma, Sarnath Singh, Marc Wallack, Jan Geliebter, Raj K. Tiwari. _New York Medical College, Valhalla, NY_.

Metastatic melanoma, leads to the highest number of skin cancer related deaths. Checkpoint inhibitor therapy has witnessed a high success rate in melanoma patients with anti-CTLA-4 and anti-PD-1. However, checkpoint inhibitor molecules and their compensatory stimulatory counterparts are widely expressed on T cells and antigen presenting cells suggesting a robust redundancy in these molecules as clinical targets. Some of these molecules that include CTLA-4, PD-1, HVEM, VISTA, 41-BB, OX-40 and CD226 are also expressed on tumor cells. Their role in regulating an immune response whether it is cell killing or immune evasion remains to be elucidated. We isolated and characterized five primary patient derived melanoma cell lines: MEL-2, MEL-V, 3MM, KFM and GLM2. We screened these cells for the expression of a comprehensive panel of twenty-five co-stimulatory and co-inhibitory molecules by RT-PCR which revealed significant heterogeneity in expression of these molecules compared to normal adult melanocytes under normal conditions; underscoring the importance of understanding tumor tissue pleiotropy prior to designing a therapeutic regimen. Surprisingly, inhibitory molecules including PD-1, VISTA and LAIR1 and stimulatory molecules including 4-1BB, HVEM and ICOS were upregulated differentially in these cell lines by metabolic stress brought on by starvation conditions. Some of these molecules were restored to basal levels of expression on treatment with 10μM vemurafenib (PLX4032), a BRAFV600E inhibitor, for 24 hours. However the treatment led to concurrent upregulation of molecules such as LAG3, BTLA, CD226 and TIM1, suggesting a compensatory mechanism that could aid melanoma adaptation and escape from immune recognition. Exposing melanoma cells to classical activated dendritic cell cytokines, IL-6 and IL-12, led to a differential expression of these molecules. Additionally, experiments using tumor lysate loaded dendritic cells to study activation revealed an ability to modulate immune activation correlating with unique stimulatory and inhibitory molecule expression profile of each primary cell line. Our results underscore the importance of understanding the profile of co-stimulatory and co-inhibitory molecules expressed in tumor cells. With eighty percent of melanoma patients being positive for the BRAFV600E lesion, we make a case for designing a combinatorial therapeutic regimen, with targeted immunotherapies as well as targeting specific genetic lesions with small molecule inhibitors.

#715

Expression of tumor cell-associated immune checkpoint molecules in multiple human solid tumors.

Annelies Laeremans, Na Li, Jeff Kim, Xiao-Jun Ma, Emily Park. _Advanced Cell Diagnostics, Newark, CA_.

Cancer immunotherapy using monoclonal antibodies targeting inhibitory immune checkpoint molecules is being established as a new paradigm in cancer treatment. This crucial strategy involves inhibition of the interaction between immune checkpoint receptors, expressed on the immune cells, and their respective ligands, expressed on antigen-presenting cells. While this receptor-ligand concept is well accepted in infection immunology, there have been numerous reports on the expression of immune checkpoint molecules in tumor cells. In addition to well-characterized tumor-intrinsic expression of PD11 and PD-L12, many other checkpoint molecules are associated with tumor cells3, conferring important impact on tumor biology and clinical consequences. In this study, we evaluated in situ single-cell expression profiles of immune checkpoint molecules in the tumor microenvironment (TME) of archived FFPE tissues from multiple solid cancers, including breast, kidney, ovarian, stomach, colorectal, and non-small cell lung cancer. Applying the RNAscope® assay, a highly specific and sensitive in situ hybridization (ISH) technology, tissue microarrays consisting of 20-35 FFPE tumors per each cancer type were stained for key therapeutic targets including PD1, PD-L1, TIM3, and LAG3. We observed that, in addition to PD-L1, all evaluated immune checkpoint molecules were expressed in the tumor cells of multiple tumor types though at a lower level than in immune cells. While there was notable heterogeneity of expression in different tumor regions in some samples, the single-cell expression pattern of each checkpoint molecule across all tumor cells in each region suggested a potential tumor-intrinsic mechanism for the expression of these molecules. In selected lung and ovarian cancers, fluorescence multiplex ISH-IHC staining confirmed that multiple different checkpoint molecules were co-expressed together in individual tumor cells. These findings add to the growing evidence that immune checkpoint genes can be expressed in tumor cells in addition to immune cells, which will be missed by gene expression analysis techniques using bulk tissue. The RNAscope® ISH platform is especially well suited for this type of analysis since it allows for assessing cell type-specific expression of any gene within the native tissue architecture even when it is expressed at very low levels. The approach illustrated in this study may help to better understand the anti-tumor activities of immune checkpoint inhibitors and provide new insights into better predictive biomarkers and therapeutic strategies. References

1. Kleffel et al. Cell. 2015 2. Clark et al. Cancer Res. 2016 3. Marcucci et al. BBA - Review. 2017

### Vaccines 1

#716

A novel virus-like particle vaccine presenting HER-2 extracellular domain elicits strong immune responses against mammary carcinoma.

Arianna Palladini,1 Susan Thrane,2 Christoph M. Janitzek,2 Jessica Pihl,2 Stine B. Clemmensen,2 Wilhelm A. de Jongh,3 Thomas M. Clausen,2 Giordano Nicoletti,4 Lorena Landuzzi,4 Manuel L. Penichet,5 Tania Balboni,1 Marianna L. Ianzano,1 Veronica Giusti,1 Thor H. Theander,2 Morten A. Nielsen,2 Ali Salanti,2 Pier-Luigi Lollini,1 Patrizia Nanni,1 Adam F. Sander2. 1 _University of Bologna, Bologna, Italy;_ 2 _University of Copenhagen, Copenhagen, Denmark;_ 3 _Expres2ion Biotechnologies, Horsholm, Denmark;_ 4 _Rizzoli Orthopaedic Institute, Bologna, Italy;_ 5 _University of California, Los Angeles, Los Angeles, CA_.

Overexpression of human epidermal growth factor receptor-2 (HER-2) occurs in about 20% of invasive breast cancers. Anti-HER-2 monoclonal antibody therapy is effective, but its utility is limited by high costs, side effects and development of resistance, thus underlining the need of new therapeutic approaches. A novel anti-HER-2 vaccine made of virus-like particles (VLPs) displaying the extracellular domain (ECD) of the human oncogene/antigen HER-2 induced protective immune responses against transgenic mouse mammary carcinomas expressing human HER-2. We have developed a versatile antigen display platform that, unlike existing technologies, effectively facilitates directional covalent attachment of large antigens at high density on the surface of VLPs (J. Nanobiotechnology 14: 30, 2016). The system uses a tag/catcher conjugation system that was developed by splitting the CnaB2 domain from the fibronectin-binding protein FbaB of Streptococcus pyogenes into a highly reactive peptide (SpyTag) and a protein (SpyCatcher) binding partner. Interaction between SpyTag and SpyCatcher results in the spontaneous formation of an isopeptide bond, occurring at high efficiency in a wide variety of protein contexts and buffer conditions. Here, we genetically fused with SpyCatcher the full extracellular domain (subdomains I-IV) of human HER-2, and bound the fusion antigen (SpyCatcher-HER2) to the surface of VLPs (derived from the AP205 phage), each presenting 360 SpyTag peptides. The vaccine, referred to as HER2-VLP, effectively overcame immune tolerance and induced Th1 cytokines and high-titer, high affinity, therapeutically potent anti-HER-2 antibodies which inhibited tumor growth in wild-type FVB mice implanted with transgenic mammary carcinomas expressing human HER-2. Furthermore, vaccination with HER2-VLP prevented spontaneous development of human HER2-positive mammary carcinomas in tolerant transgenic mice. Vaccination with a control preparation of untagged VLP and HER-2 ECD did not induce protective immune responses. Polyclonal IgG antibodies elicited by HER2-VLP vaccination had an affinity for human HER-2 comparable to trastuzumab and inhibited the 3D growth in vitro of both trastuzumab-sensitive and trastuzumab-resistant BT-474 human breast cancer cells. In conclusion, the HER2-VLP vaccine has the potential to become a tool in the fight against HER-2-positive human cancer. The results also provide strong proof-of-concept for the use of the versatile VLP platform to develop a variety of vaccines against other tumor antigens. Supported by grants from the Italian Association for Cancer Research (AIRC), the University of Bologna, the Danish Research Council, the Eurostars program and the European Research Council (ERC).

#717

Mouse model for the development of preventive and therapeutic vaccines against microsatellite-unstable cancers.

Matthias Kloor,1 Mine Oezcan,1 Aysel Ahadova,1 Yan Yuan,1 Peer Bork,2 Shizuko Sei,3 Robert Shoemaker,3 Oezkan Gelincik,4 Steven Lipkin,4 Johannes Gebert,1 Magnus von Knebel Doeberitz1. 1 _University Hospital Heidelberg, Heidelberg, Germany;_ 2 _EMBL Heidelberg, Heidelberg, Germany;_ 3 _National Cancer Institute, MD;_ 4 _Weill Cornell University, NY_.

Microsatellite-unstable (MSI) cancers occurring in the context of Lynch syndrome elicit pronounced tumor-specific immune responses. These immune responses are specifically directed against frameshift peptide (FSP) neoantigens, which result from mismatch repair (MMR) deficiency-induced insertion/deletion mutations in coding microsatellites (cMS). We have recently completed a clinical phase I/IIa trial that successfully demonstrated safety and immunogenicity of an FSP neoantigen-based vaccine in MSI colorectal cancer patients (Clinical trial number: NCT01461148). To further develop a vaccine against MSI cancers in Lynch syndrome, we aimed to establish a preclinical mouse model. A systematic database search was performed to identify cMS sequences in the murine genome. Subsequently, intestinal cancers obtained from Lynch syndrome mice (Msh2flox/flox VpC+/+) were evaluated for mutations affecting these candidate microsatellites. Thirteen candidate cMS were detected that presented with a mutation frequency of 15% or higher. The cMS most frequently affected by frameshift mutations was located in the Nacad gene (75% of tested tumors). Epitope prediction using the netMHC4.0 algorithm was performed, and ten most promising FSP neoantigens were synthesized. Immunogenicity was evaluated after vaccination of C57BL/6 mice using IFN-gamma ELISpot. Four FSP neoantigens derived from cMS mutations in the genes Nacad, Maz, Xirp1, and Senp6 elicited strong antigen-specific cellular immune responses. CD4-specific T cell responses were detected for Maz(-1), Nacad(-1), and Senp(-1), all of which also induced humoral immune responses. CD8-positive T cells were detected for Xirp(-1) and Nacad(-1). Preliminary epitope mapping indicated that there was no cross-reactivity with the respective wild type proteins. Based on mutation data, a vaccine with the four FSP neoantigens has a predicted coverage of up to 75% of intestinal tumors in the Msh2flox/flox VpC+/+ Lynch mouse model. In summary, we have identified 4 immunogenic FSP neoantigens derived from commonly mutated cMS in murine Lynch syndrome colorectal cancers. These results provide the basis for evaluating the concept of cancer-preventive FSP vaccines in a mouse model of Lynch syndrome. This model allows longitudinal monitoring of immune responses and tumor development using different vaccination schemes, adjuvants and combination with chemoprevention.

#718

Nanoparticle-based tumor cell lysate vaccine for cancer immunotherapy.

Hyunjoon Kim, Peter Larson, Tamara A. Kucaba, Katherine A. Murphy, David M. Ferguson, Thomas S. Griffith, Jayanth Panyam. _University of Minnesota, Minneapolis, MN_.

Introduction Combination of tumor-associated antigen (TAA) and toll-like receptor (TLR) agonists can facilitate a potent anticancer immune response. Synthetic peptides, which are derived from antigens that are highly elevated or only expressed in tumor tissue, have been investigated as a source of TAA. However, these peptides are not available for all tumor types and often poorly immunogenic. Whole tumor cell lysate is a promising alternate antigen source that seeks to overcome some of the limitations associated with peptide-based vaccines. In this study, we utilized whole tumor cell lysate as antigen source and a novel TLR 7/8 agonist encapsulating nanoparticles as adjuvant to formulate the cancer vaccine and examined its efficacy against subcutaneous (S.C.) tumor models.

Methods A new imidazaquinoline-based TLR 7/8 agonist (termed '522') was encapsulated in poly(lactide-co-glycolide) (PLGA) nanoparticles using emulsion solvent evaporation method to formulate the vaccine adjuvant. The adjuvant (522NP) was used in combination with whole tumor cell lysate (CL) prepared from murine bladder carcinoma (MB49) cells. Immunocompetent C57BL/6 mice were immunized subcutaneously with 5 doses of cell-lysate vaccine or various control treatments. Two days after the final vaccination dose, mice were sacrificed and organs were harvested. Splenocytes were analyzed using flow cytometry to measure antigen (Ag)-specific CD8 T cell response. We investigated whether prophylactic and therapeutic CL+522NP immunization can delay tumor growth and improve survival in MB49 S.C. tumor models.

Results CD11ahighCD8low CD8 T cells were examined as they represent Ag-experienced CD8 T cells that can respond to Ag. Frequency of CD11ahigh CD8low CD8 T cells increased in mice vaccinated with CL+522NP. In the prophylactic model, CL+522NP significantly delayed tumor growth compared to that in other control groups. On d 30, average tumor volumes reached ~1000 mm3 in untreated mice compared to ~500 mm3 in CL alone and CL+Soluble522 treated groups. However, CL+522NP vaccinated mice showed remarkably very little tumor growth (~122 mm3) over the same time period. In the therapeutic model, only CL+522NP treatment was effective in delaying tumor growth. Unlike in the prophylactic study, where CL alone had a moderate effect, therapeutic effects were not observed from CL alone or CL+Soluble522 groups.

Conclusion CL+522NPs vaccination triggered a robust anti-cancer immune response that resulted in the expansion of CD11ahigh CD8low CD8 T cells, which suggests that 522NPs enhance the immunogenicity of cell lysate and elicit Ag-specific CD8 T cell response. Furthermore, CD8 T cells generated by CL+522NPs vaccination appeared to be effective in killing tumors in both prophylactic and therapeutic tumor models. These results suggest that 522NPs are effective vaccine adjuvants capable of improving the outcome of cancer immunotherapy.

#719

A tumor mitochondria vaccine protects against experimental renal cell carcinoma.

Stefano Pierini, Andrea Facciabene, Mireia Uribe-Herranz, Stavros Rafail, Chongyun Fang, Janos Tanyi. _University of Pennsylvania, Philadelphia, PA_.

Mitochondria provide energy for cells via oxidative phosphorylation. Reactive oxygen species, a byproduct of this mitochondrial respiration, can damage mitochondrial DNA (mtDNA), and somatic mtDNA mutations have been found in all colorectal, ovarian, breast, urinary bladder, kidney, lung, and pancreatic tumors studied. The resulting altered mitochondrial proteins or tumor-associated mitochondrial Ags (TAMAs) are potentially immunogenic, suggesting that they may be targetable Ags for cancer immunotherapy. In this article, we show that the RENCA tumor cell line harbors TAMAs that can drive an antitumor immune response. We generated a cellular tumor vaccine by pulsing dendritic cells with enriched mitochondrial proteins from RENCA cells. Our dendritic cell-based RENCA mitochondrial lysate vaccine elicited a cytotoxic T cell response in vivo and conferred durable protection against challenge with RENCA cells when used in a prophylactic or therapeutic setting. By sequencing mtDNA from RENCA cells, we identified two mutated molecules: COX1 and ND5. Peptide vaccines generated from mitochondrial-encoded COX1 but not from ND5 had therapeutic properties similar to RENCA mitochondrial protein preparation. Thus, TAMAs can elicit effective antitumor immune responses, potentially providing a new immunotherapeutic strategy to treat cancer.

#720

Anti-OX-40 agonist antibody significantly increases therapeutic efficacy of TVGV-1 vaccine for HPV-positive cancers.

Yin-Ching Lin,1 Yi-Tsui Chiu,1 Yi-Chia Lin,1 Jiun-Ming Wu,1 Chien-Hung Chen,2 Wen-Fang Cheng,3 Chia-Mao Wu1. 1 _TheVax Genetics Vaccine Co. Ltd., Taipei city, Taiwan;_ 2 _National Taiwan University Hospital, Taipei city, Taiwan;_ 3 _National Taiwan University College of Medicine Graduate Institute of Oncology, Taipei city, Taiwan_.

Background: Human papillomavirus 16 (HPV16) E7 protein is a potential target antigen for HPV-positive cancers due to its interaction with pRb tumor suppressor protein. The clinical-stage therapeutic vaccine, TVGV-1, comprises of the PE-E7-K3 fusion protein and the adjuvant GPI-0100, has shown therapeutic effect preclinically by stimulating T cell response targeting HPV16 E7 protein. TVGV-1 was well tolerated clinically in more than 30 HPV+ cervical intraepithelial neoplasia (CIN) patients. Agonistic antibodies targeting OX40 (CD134) have shown remarkable single agent anti-tumor effect by modulating T cell response surrounding tumor environment, as well as the ability to combine with other immunotherapies in several mouse tumor models. Here we evaluated the synergistic therapeutic potential of TVGV-1 vaccine combined with anti-OX-40 agonist antibody for HPV-positive cancer therapy.

Methods: Therapeutic and immune efficacy of TVGV-1 in combination with anti-OX-40 agonist antibody was tested in E7 antigen expressing TC-1 tumor mouse model. Mice with various treatments were assessed for tumor growth and survival. Peripheral and tumor-infiltrating immune cell profiles with various treatments were also assessed by flow cytometry.

Results: Our results revealed that both the HPV16 E7-specific humoral and cell-mediated immunities solely elicited by TVGV-1 vaccination was synergistically enhanced more than two-fold by concomitant anti-OX-40 agonist antibody treatment. In a TC-1 tumor model, tumor growth and overall survival was not protected by OX-40 antibody treatment alone, and mice was protected with prolonged survival benefit when treated in combination of TVGV-1 and OX-40 antibody. In TC-1 tumor bearing mice, about 10% decrease of peripheral CD3+ T cell was observed upon TC-1 tumor development, and restoration was only observed when mice were treated with combined TVGV-1 and OX-40 antibody. When tumor infiltrating lymphocytes recognizing HPV E7 antigen were prepared from these mice, the amount of tumor-infiltrating mononuclear cells and CD3+ T cells were dramatically increased with combination treatment when compared to control group or to treatment alone groups.

Conclusions: We demonstrated efficacy with combination approach between TVGV-1 vaccine and anti-OX-40 agonistic antibody in a pre-clinically mouse model. This enhanced activity is likely due to modulation of T-cell population in tumor and in periphery. This result will guide to a better design to perform clinical studies in order to obtain better outcome with TVGV-1 cancer vaccine intended for treating HPV E7 bearing tumors.

#721

Development of a therapeutic HPV vaccine based on mass spectrometry-verified target epitopes.

Sebastian Kruse,1 Maria Bonsack,2 Sara Becker,1 Nitya Mohan,1 Alina Steinbach,2 Stephanie Hoppe,2 Renata Blatnik,2 Angelika B. Riemer2. 1 _German Cancer Research Center (DKFZ), Heidelberg, Germany;_ 2 _German Cancer Research Center (DKFZ) and German Center for Infection Research (DZIF), Heidelberg, Germany_.

For rational therapeutic vaccine design, detailed knowledge about target epitopes that are truly presented on cancer cells is essential. Many potential tumor-specific epitopes, such as viral epitopes or mutation-derived neoepitopes, are presented at low abundance. Thus, detection of these epitopes remains a challenge. We have developed a high-sensitivity targeted mass spectrometry (MS) approach for direct detection of low-abundant epitopes. We used human papillomavirus (HPV) as a model system, as high-risk HPVs cause over 600,000 cervical, anogenital and oropharyngeal cancer cases per year. Moreover, the HPV oncoproteins E6 and E7 are essential for the induction and maintenance of the malignant phenotype, and thus are ideal targets for immunotherapy.

Potential HPV16 E6 and E7 target epitopes were predicted in silico and their HLA-binding verified in cellular binding assays. HLA-peptide complexes were immunoprecipitated from HPV16-positive cancer cells and the purified peptides were analyzed by MS. Identified HPV epitopes were assessed for immunogenicity in vitro with PBMC from healthy donors and in vivo in the MHC-humanized mouse model A2.DR1.

The binding assays resulted in the identification of known and novel HPV16-derived HLA-binding peptides. Furthermore, we used this data to formulate rules on how to optimally use epitope prediction tools to increase the chances of predicting true HLA ligands. 14 HLA-A2-restricted HPV16 epitopes were detected on the surface of CaSki and 866 cervical cancer cells, 9 of which were novel. 13 out of the 14 MS-detected peptides were immunogenic in ELISpot assays. Vaccination of A2.DR1 mice with one selected epitope induced high numbers of specific cytotoxic T-cells, and caused anti-tumor effects in a newly developed HPV16 E6/E7-expressing A2.DR1-compatible tumor model.

We conclude that our epitope detection and validation approach is suitable for validating even low-abundant candidate epitopes to be true immunotherapy targets. MS-detection of epitopes appears to be a good predictor of immunogenicity, which makes this approach an attractive platform for determining target epitopes for therapeutic cancer vaccines.

#722

Apoptosis inhibitor 5 (API5) induces cytotoxicity CD8+ T cells mediated anticancer effect though TLR4 signaling of dendritic cells.

Young Seob Kim. _Konkuk university, Seoul, Republic of Korea_.

Dendritic cells(DCs) among innate immune system are important to induce adoptive immune response such as CD8+ cytotoxic T cells activation. Immunotherapy using dendritic cells is a promising one of cancer therapy. Because most of adjuvants that activate immune cells are derived from bacteria, restricted to the use of DC-based immunotherapy. Here we report adjuvant effect of novel human derived API5 protein. API5 regulating survival is generally localized at nuclear, upregulated in various cancer cells and released during damage or death as one of them for damage associated molecular pattern (DAMP). Recombinant API5 induce maturation and activation of mouse DCs and human macrophage. DC vaccine using API5 enhance Th1 type immune response increase number of tumor antigen specific CD8+ T cells and have a strong prevention and treatment against cancer. The activity of extracellular API5 is mediated through Toll-like receptor 4 of APC. In conclusion, our results suggest novel human derived adjuvant protein that have immune activity through TLR4 and can be used for cancer immunotherapy.

#723

Targeting phosphorylated p53 to elicit tumor-reactive T helper responses against head and neck squamous cell carcinoma.

Kenzo Ohara,1 Takumi Kumai,1 Tatsuya Hayashi,1 Takayuki Ohkuri,1 Nobuyuki Bandoh,2 Hiroya Kobayashi,1 Yasuaki Harabuchi1. 1 _Asahikawa Medical University, Asahikawa, Japan;_ 2 _Hokuto hospital, Obihiro, Japan_.

The p53 is a DNA-binding and transcription activation domain containing tumor suppressor that upregulates growth arrest and apoptosis-related genes. In response to DNA damage, p53 is phosphorylated at multiple sites including serine 33 or 37. Additionally, abnormal phosphorylation of cellular proteins is a hallmark of malignant transformation. The human immune system is capable of distinguishing between the normal components and these posttranslational modifications. Therefore, the existence of posttranslational modifications increases the diversity of potential immune reactions to a determinant antigen. We identified phosphorylated peptide epitopes from tumor protein p53 that could elicit effective T helper-cell responses. Two epitope peptides, p5322-41/Phospho-S33 and p5322-41/Phospho-S37, induced T helper responses against tumor cells expressing phosphorylated p53 protein. Moreover, cisplatin and doxorubicin augmented the responses of such CD4+ T cells via upregulation of phosphorylated p53. Importantly, we elucidated the presence of peripheral blood lymphocytes specific for these peptide epitopes in the PBMCs of head and neck squamous cell cancer patients. These results predict that epitopes p5322-41/Phospho-S33 and p5322-41/Phospho-S37 are immunogenic for anti-tumor responses and could be beneficial for cancer immunotherapy.

#724

A novel heterologous prime boost vaccine system drives tumor specific and potent CD8 T cell responses for cancer immunotherapy.

Wade Blair,1 Gijsbert Grotenbreg,1 Ciaran Scallan,1 Amy Rappaport,1 Renee Greer,1 Leonid Gitlin,1 Kieu Lam,2 James Heyes,2 Anne Van Abbema,1 Hadley Hanson,1 Petra Schreiner,2 karin Jooss1. 1 _Gritstone Oncology, Emeryville, CA;_ 2 _Arbutus Biopharma, Burnby, British Columbia, Canada_.

Tumor-specific neoantigens (TSNAs) are present in a majority of tumor types and are key targets for T cells released by immune checkpoint blockade therapy. Given that TSNAs are non-self antigens, they are particularly attractive cancer vaccine targets. We have developed a potent heterologous prime/boost immunization approach to deliver predicted TSNAs to patients, which is comprised of a replication incompetent chimpanzee adenoviral vector (ChAdV) for the prime vaccination and a self-replicating, synthetic viral vector (srRNA) for repeated boost vaccinations. The ChAdV vector is similar in design to other adenoviral vectors that have demonstrated induction of high titer, polyfunctional and durable CD4 and CD8 T-cell responses against non-self antigens that protected humans against infections. The srRNA vector is based on Venezuelan equine encephalitis virus (VEE), where sequences encoding the structural proteins of VEE were deleted and replaced by a TSNA expression cassette. For delivery in vivo, the srRNA is formulated with a lipid nanoparticle (LNP), which facilitates efficient cellular uptake of the RNA and enhances antigen expression as well as the resulting immune response. We demonstrate that the srRNA vector effectively replicates in vitro and in vivo resulting in durable and high levels of antigen expression. To characterize the vectors in pre-clinical animal models, a prototypical expression cassette that encodes multiple mouse MHC class I tumor antigens as well as Mamu-A01 restricted class I antigens for monitoring immune responses in mice and non-human primates (NHPs) was introduced into both vector systems. We demonstrate that immunization of mice with either vector results in strong antigen-specific CD8 T-cell responses against the encoded murine epitopes. The heterologous prime/boost approach provided a statistically significant survival advantage to tumor bearing mice when compared to untreated mice. The potency of the immunization platform was also assessed in Indian rhesus macaques and the platform demonstrated a quick onset of T-cell responses 1 week post ChAdV prime vaccination with peak T-cell responses against all delivered Mamu-A01 restricted class I antigens at 2-3 weeks. Such ChAdV primed T-cell responses were effectively boosted by the LNP formulated srRNA vector. Co-administration of anti-CTLA4 with the vaccine demonstrated enhanced vaccine induced immune response. These data demonstrate that the heterologous prime/boost platform effectively programs robust T-cell immunity toward encoded non-self antigens in NHPs, which is a highly predictive model of vaccine responses in humans. This vaccine platform is targeted for entry into clinical trials in mid 2018.

#725

Targeting the skin microenvironment for effective cancer vaccines.

Jack R. You, Rong Hao, Louis D. Falo, Guo Chen, Xingxing Hao, Cara Carey, Zhaoyang You, Louis D. Falo. _Univ. of Pittsburgh School of Medicine, Pittsburgh, PA_.

Skin, the most accessible organ, harbors keratinocytes and immunocytes that contribute to both innate and adaptive immunity, rendering it an ideal anatomic target for immunization. Keratinocytes can drive skin inflammation by producing pro-inflammatory mediators that promote cutaneous immune responses. Thus, targeting accessible keratinocytes to express transgenic antigens and increase production of pro-inflammatory mediators is a salient and attractive approach to vaccination, but remains a critical challenge to achieve. Toward this goal, we evaluated the effects of transient overexpression of the stress response factor x-box binding protein 1 (XBP1) in keratinocytes on the skin microenvironment. In vitro, keratinocytes genetically engineered to overexpress XBP1 increased production of both co-transduced exogenous antigen and pro-inflammatory mediators. In vivo, overexpression of XBP1 and transgenic antigen in keratinocytes induced both protective and therapeutic antitumor immunity that was associated with enhanced cutaneous expression of pro-inflammatory mediators and co-delivered antigen, increased skin-infiltrating immunocytes including dendritic cells (DCs), and the induction of durable antigen-specific CD8\+ T cells and skin-resident memory CD8\+ T cells. Importantly, in human skin, correlative effects were observed: transient overexpression of XBP1 in skin triggered the expression of genes linked to a pro-immunogenic microenvironment, increased the expression of the co-delivered antigen, and promoted the activation and IFN-γ-producing T cell-stimulating function of skin DCs. These findings provide novel insights into the rational design of cancer vaccines with improved immunogenicity through strategic modulation of the skin microenvironment.

#726

Type-I interferon and epigenetic modulators enhance the anti-tumor efficacy of a dendritic-cell targeting MIP3α-antigen vaccine in the B16F10 mouse model.

James Gordy, Richard Markham. _Johns Hopkins School of Public Health, Baltimore, MD_.

Background: The chemokine MIP-3α (CCL20) binds to CCR6 found on immature dendritic cells. DNA vaccines fusing MIP-3α to melanoma-associated antigens have been shown to be effective in therapeutically reducing melanoma tumor burden in mouse models. However, the anti-tumor efficacy was not complete. To optimize the therapy, our laboratory has added agents designed to overcome immunoregulatory mechanisms of the tumor microenvironment. Here, we report that the combination of type-I interferon therapy (IFN) with 5-Aza-2'-deoxycitidine (Aza), which has been shown to prevent silencing of interferon responsive genes, profoundly enhanced the therapeutic anti-melanoma efficacy of a MIP-3α-antigen DNA vaccine. Methods: The current studies utilize the B16F10 syngeneic, transplantable, mouse melanoma model system. The MIP-3α-antigen DNA vaccine is administered intramuscularly (i.m.) into the tibialis muscle, followed immediately by i.m. electroporation. The primary construct utilized includes MIP-3α fused to immunogenic regions of gp100 and tyrosinase related protein 2 (TRP-2). Vaccinations are given thrice at one week intervals, beginning at day 5 post challenge. Two days post vaccination, CpG adjuvant is administered into the vaccinated muscle. Aza is given i.p.at 1mg/kg on the first two vaccination days. IFN therapy is given in a series of one high dose followed by three days of low doses, beginning on the first two vaccination days. Tumor sizes, growth, and survival were all assessed. Vaccine antigens and interferon stimulated gene expression levels were explored by RT-PCR. Results: With this therapeutic protocol, we demonstrate for the first time that the addition of IFN therapy with Aza enhances the anti-tumor efficacy of a MIP-3α-gp100-TRP2 vaccine. Combination therapy has led to significantly reduced tumor burden and overall increases in mouse survival, increasing median survival of vaccine alone by 39% and negative control by 86%. Importantly, this increase in efficacy was dependent on the presence of all three components, including vaccine, IFN, and Aza. All permutations of one or two treatments provided inferior efficacy to the triple therapy. The immunological details of this model are under current investigation. Conclusions: Efficient targeting of antigen to immature dendritic cells with a chemokine fusion vaccine offers a potential alternative approach to the ex vivo dendritic cell antigen loading protocols currently undergoing clinical investigation. Combining this approach with IFN therapy enhanced by Aza treatment significantly improved vaccine efficacy. Further potential therapy optimization currently undergoing investigation offers promise for this line of investigation to become a novel melanoma therapy.

#727

A novel rMVA combination immunotherapy triggers potent innate and adaptive immune responses against established tumors.

Jose Medina-Echeverz, Maria Hinterberger, Marco Testori, Marlene Geiger, Raphael Giessel, Paul Chaplin, Hubertus Hochrein, Henning Lauterbach. _Bavarian Nordic, Planegg, Germany_.

Virus-based vaccines and appropriate costimulation enhance potent antigen-specific T cell immunity against cancer. However, the tumor microenvironment exerts intrinsic and extrinsic mechanisms to evade tumor destruction. In the present study we exploit both innate and adaptive immune responses triggered by a novel recombinant modified vaccinia virus Ankara (rMVA) encoding costimulatory CD40L against solid tumors in combination regimes to overcome tumor-induced resistance to immunotherapy. Therapeutic treatment with rMVA-CD40L resulted in strong antitumor effects in unrelated established tumor models. Tumor infiltration was composed of non-exhausted, antigen-specific CD8+ T cells with proliferative capacity after rMVA-CD40L immunization. Strikingly, this antitumor effect was not entirely dependent on cross-presenting CD8α+ DC -induced CD8+ T cell expansion, as experiments with MVA encoding for T. gondii profilin (TLR11 ligand) and using Batf3-/- tumor bearers depicted. Indeed, rMVA-CD40L-induced tumor control did not depend on cytosolic DNA sensor STING. Interestingly, rMVA-CD40L induced strong NK cell activation and thereby potent Antibody Dependent Cell Cytotoxicity (ADCC) against Tumor-Associated Antigen (TAA) targeting antibodies. Hence, the combination of TAA targeting antibodies and rMVA-CD40L resulted in increased therapeutic antitumor efficacy. We describe a novel and translationally relevant therapeutic synergy between viral vaccination and CD40L costimulation. We connect CD40 ligation to cross-presenting CD8α+ DC -mediated expansion of non-exhausted CD8\+ T cells in the tumor microenvironment. Taking advantage from intrinsic MVA-induced NK cell activation and further improved NK cell function by CD40 ligation, we show strengthened antitumor immune responses when both rMVA-CD40L-induced innate and adaptive immune mechanisms are exploited by combining immunotherapeutic regimes. This finding has a direct potential impact in clinical trials where TAA targeting antibodies are currently under evaluation.

#728

Optimization of peptide vaccines to induce robust antitumor CD4 T-cell responses.

Takumi Kumai,1 Hussein Sultan,2 Hiroya Kobayashi,1 Yasuaki Harabuchi,1 Esteban Celis3. 1 _Asahikawa Medical Univ., Asahikawa, Japan;_ 2 _Augusta University, Augusta, GA;_ 3 _Augusta University, GA_.

Substantial evidence indicates that immunotherapy is a feasible and effective approach for the treatment of numerous types of cancer. Among various immunotherapy options, peptide vaccines to generate antitumor T cells appear as promising candidates, because of their cost effectiveness and ease of implementation. Nevertheless, most peptide vaccines are notorious for being weekly immunogenic and, thus, optimization of the vaccination strategy is essential to achieve therapeutic effectiveness. In addition, effective peptide vaccines must stimulate both CD8 cytotoxic and CD4 helper T lymphocytes. Our group has been successful in designing effective peptide vaccination strategies for inducing CD8 T-cell responses in mouse tumor models. Here, we describe a somewhat similar, but distinct, peptide vaccination strategy capable of generating vast CD4 T-cell responses by combining synthetic peptides with toll-like receptor (TLR) agonists and OX40/CD40 costimulation. This vaccination strategy was efficient in overcoming immune tolerance to a self-tumor- associated antigen and generated significant antitumor effects in a mouse model of malignant melanoma. The optimized peptide vaccine also allowed the expansion of adoptively transferred CD4 T cells without the need for lymphodepletion and IL-2 administration, generating effective antimelanoma responses through the enhancement of proliferative and antiapoptotic activities of CD4 T cells. These results have practical implications in the design of more effective T-cell-based immunotherapies.

#729

CD40 and 4-1BB pathway activation using oncolytic viruses kills tumor cells by oncolysis and CD40-mediated apoptosis while activating immune cells.

Emma Eriksson, Jessica Wenthe, Sedigheh Naseri, Ann-Charlotte Hellström, Angelica Loskog. _Uppsala University, Uppsala, Sweden_.

LOAd703 is a tumor-selective oncolytic adenovirus (serotype 5/35) encoding two immunostimulatory transgenes: trimerized membrane-bound CD40L and 4-1BBL. We have previously shown the immunostimulatory effect of LOAd703 in pancreatic cancer models and a clinical trial enrolling patients with pancreatic cancer is ongoing. LOAd703 can potentially be used in a variety of cancers and its applicability across various cancer types was investigated herein. The immune function of the transgenes was further elucidated, including the tumor-specific apoptotic effect of TMZ-CD40L. A panel of human tumor cell lines including B-cell lymphoma (Karpas-422, BC-3, Daudi, DG-75, U-698), multiple myeloma (RPMI-8226), colorectal (HT29), ovarian (SKOV3), lung (A549), neuroendocrine (H727), bladder (T24) and pancreas (Panc01) was selected for evaluation of the LOAd703 oncolytic capacity. The killing capability of LOAd viruses was analyzed using CellTiter96AQueous One Solution Cell Proliferation Assay and Incucyte. Further, LOAd703 was investigated in xenograft models (one per indication). Replication was confirmed by virus-specific quantitative PCR in selected lines. Immunomodulatory capacity on myeloid cells including dendritic cells and macrophages was evaluated in in vitro cultures by flow cytometry and cytokine assays. Apoptosis was determined using flow cytometry. LOAd703 infection resulted in robust virus expansion and killing of tumor cell lines across indications while primary cultures obtained from the pancreas, lung, heart and kidney were not affected. Cell lines such as T24 expressing CD40 showed enhanced sensitivity to LOAd703 that contains TMZ-CD40L compared to LOAd(-) lacking transgenes. The enhanced cell death was due to increased apoptosis frequency. In contrast, the CD40 negative cell lines such as SKOV-3 was equally sensitive to LOAd703 and LOAd(-). Likewise, LOAd703 could effectively control established tumors in xenograft models and the effect was more pronounced in CD40+ cell lines. Interestingly, infected B cell lines showed an enhanced immunogenic phenotype (MHC molecules, Fas, ICAM-I, CD80 and CD86) post infection likely due to their origin as antigen-presenting cells. Stimulation of immature DCs with LOAd703 forced differentiation into a mature phenotype with high capacity of antigen-presentation and expansion of both T cells and NK cells while stimulation of macrophages favored a high M1:M2 ratio. In conclusion, the LOAd703 oncolytic virus can kill a broad range of tumor cells both by CD40-mediated apoptosis and replication-induced oncolysis and the effect is most potent in CD40+ tumor cells. CD40+ myeloid cells such as DCs and macrophages are instead activated by LOAd703. Hence, LOAd703 is a potent multi-functional oncolytic virus.

#730

Neoantigen identification using the ATLAS T cell profiling platform highlights the need to empirically define neoantigens.

Jason Dobson, Huilei Xu, Johanna Kaufmann, James Foti, Jin Yuan, Michael O''Keeffe, Crystal Cabral, James Loizeaux, Christopher Warren, Ning Wu, Erick Donis, Kyle Ferber, Pamela Carroll, Jessica B. Flechtner, Wendy Broom. _Genocea Biosciences, Cambridge, MA_.

Neoantigens arise from tumor-specific, somatic mutations and have the potential to be recognized by T cells that are associated with anti-tumor immune responses. Since they are non-self, they are hypothesized to provide an attractive therapeutic modality because T cells that can respond to those sequences have not undergone thymic selection. The ATLASTM platform enables identification of biologically relevant CD4+ and CD8+ T cell neoantigens in any subject in an unbiased manner, overcoming the limitations of conventional in silico predictive approaches. The ATLAS platform utilizes matched patient tumor biopsy and blood samples to identify recall T cell responses to tumor specific mutations. From patient peripheral blood, CD14+ monocytes were isolated and differentiated into dendritic cells (MDDCs), and T cells were sorted into CD4+ and CD8+ populations and non-specifically expanded. Tumor-specific changes (single nucleotide variants and insertion/deletions) were identified through whole exome sequencing and cloned into E. coli expression vectors with and without co-expressed listeriolysin O to enable presentation via MHC class I or class II, respectively. For each patient, their unique clones were co-cultured with autologous MDDCs in an ordered array, then their CD4+ or CD8+ T cells were added and incubated overnight. T cell activation was determined by measurement of TNF-α and IFN-γ levels in the supernatants by a Meso-Scale Discovery assay. Neoantigens were defined as clones that elicited cytokine responses >2 median absolute deviations from the median of negative control clones. Historically, ATLAS has identified CD4+ and CD8+ T cells responses to up to 15% of mutant polypeptide sequences. Here we will present ATLAS profiling of T cell responses to >2,500 potential neoantigens, across a broad cohort of patients with different tumor types, including tumors with a wide range of mutational burden. T cell responses detected by ATLAS challenge assumptions in the field, with the majority of empirically identified neoantigens not predicted by algorithms, and many predicted neoantigens demonstrating "inhibitory" activity. When exploring neoantigens selected by ATLAS by tumor type, no patterns in overall mutational burden, RNA expression level, or DNA mutant allele frequency have yet been identified. We will also present broader functional analysis, including pathway analysis of proteins containing neoantigens, review of the immunogenicity of known oncogenes and features of immunogenic peptide sequences. The ATLAS platform empirically defines which potential neoantigens created by somatic mutations elicit immune responses in individual patients independently of a patient's HLA type and T cell receptor repertoire. This approach provides the opportunity to identify better targets to include in a personalized vaccine formulation.

#731

A minigene platform to validate novel immunogenic peptides arising from somatic mutations as therapeutic cancer vaccines.

Xiaoshan Shi, Vasumathi Kode, Kayla Lee, Amit Chaudhuri, Papia Chakraborty. _MedGenome Inc, Foster City, CA_.

Cancer vaccines have shown promise in treating late-stage cancers that have failed traditional therapies. Somatic mutations provide a rich source of potential cancer vaccines with minimal T-cell tolerance. We have built a robust screening assay to test somatic mutations for CD8+ T cell activation in a T cell-dendritic cell co-culture assay with synthetic peptides added from outside. Our screening assay revealed that non-mutated wild-type peptides in many instances induced T-cell activation, although their HLA binding affinity was weak. We suspect that addition of high concentration of the peptides from outside may overcome their weak HLA-binding property, allowing them to bind HLA and activate T cells. To reduce the bias of artificial HLA presentation, we have built a minigene platform to screen pairs of wild-type and mutant peptides to assess their immunogenicity. We applied our proprietary cancer vaccine prediction platform to a set of somatic single-nucleotide variants (SNVs) and indels and selected immunogenic peptides by a combination of features that include TCR binding, HLA binding, gene expression and proteasomal processing. Predicted mutant immunogenic peptides and their wild-type counterpart were cloned as minigenes and expressed in HLA restricted antigen-presenting cells (APCs). The minigene-expressing APCs were co-cultured with allogenic CD8+ T cells and activation was monitored by the combined expression of IFN-gamma, TNF-alpha and granzyme-A (CTL phenotype of T cells) by flow cytometry. Our screening assay reveals that a significant number of indels are not presented when they are expressed as a longer peptide because of the stability of the transcripts. However, their presentation and immunogenicity are preserved when they are expressed as short 9-mer peptides with engineered proteasomal cleavage sites. Our study also reveals a range of HLA-binding affinities for peptide presentation. In conclusion, our analysis demonstrates that the two approaches for investigating immunogenicity of peptides—minigene approach and external addition of peptide approach—have differential utilities for testing and validating the immunogenicity of indels and SNVs.

#732

A practical strategy to pancreatic cancer immunotherapy using resected human tumor lysate vaccines remodeled to express α-gal epitopes.

Masahiro Tanemura,1 Kenta Furukawa,1 Eiji Miyoshi,2 Hidetoshi Eguchi,2 Hiroaki Nagano,3 Katsuyoshi Matsunami,4 Satoshi Nagaoka,2 Manabu Mikamori,1 Kentaro Kishi,1 Hiroki Akamatsu,1 Masaki Mori,2 Yuichiro Doki2. 1 _Osaka Police Hospital, Osaka, Japan;_ 2 _Osaka University Graduate School of Medicine, Osaka, Japan;_ 3 _Yamaguchi University Graduate School of Medicine, Ube, Japan;_ 4 _Hiroshima University Graduate School of Biomedical and Health sciences, Hiroshima, Japan_.

Cancer immunotherapy is a potential treatment for pancreatic ductal adenocarcinoma (PDAC) patients. As the predominant natural Ab found in humans, anti-Gal accounts for approximately 1% of immunoglobulins. The ligand of anti-Gal, "α-gal epitope (Galα1-3Galβ1-4GlcNAc-R)," is a major carbohydrate antigen, expressed by non-primate mammals, New World monkeys. One useful application of anti-Gal abundance is the enhancement of the immunogenicity of tumor-associated antigens (TAAs) that promote effective uptake by antigen-presenting cells (APCs). To develop an effective vaccine-based immunotherapy for PDAC, we hypothesized that resected tumor tissue lysates from patients might be an attractive source of PDAC-associated TAAs vaccination. This study presents a novel immunotherapy expressing α-gal epitopes using freshly obtained human PDAC tumor tissue homogenates from patients. Tumor and normal pancreatic tissue specimens were obtained from 10 patients at the time of surgical exploration for primary PDAC. To synthesize α-gal epitopes on either tumor membranes or normal pancreatic tissue membranes, we employed recombinant α1,3 galactosyltransferase (α1,3GT). PDAC membranes or normal pancreatic tissues were homogenized, and were incubated with UDP-Gal and α1,3GT. α1,3GT KO mice were immunized with pig tissues to produce anti-Gal Ab. The high anti-Gal KO mice were vaccinated by i.p. injection with unprocessed or processed PDAC tumor lysate (α-gal(-) PDAC-ly or α-gal(+) PDAC-ly). The high anti-Gal KO mice were also vaccinated with unprocessed normal or processed normal pancreatic tissue lysate (α-gal(-) N-ly or α-gal(+) N-ly). Effective synthesis of α-gal epitopes was demonstrated after processing of PDAC tumor lysates in Western blot analysis. α-gal(+) PDAC-ly vaccines elicited significant antibody production against multiple TAAs, assessed by ELISPOT (anti-MUC1; α-gal(-) PDAC-ly vs. α-gal(+) PDAC-ly=28.7 vs. 151.8 spots [P=0.0008], anti-mesothelin; α-gal(-) PDAC-ly vs. α-gal(+) PDAC-ly=36.5 vs. 97.2 spots [P=0.029]) and activated multiple tumor-specific T cells, assessed by ELISPOT (MUC1; α-gal(-) PDAC-ly vs. α-gal(+) PDAC-ly=146.0 vs. 828.0 spots [P=0.0009], mesothelin; α-gal(-) PDAC-ly vs. α-gal(+) PDAC-ly= 250.7 vs. 988.0 spots [P=0.021], α-gal(-) N-ly and α-gal(+) N-ly groups did not display significant spots.). To demonstrate in vivo effectiveness, splenocytes from vaccinated KO mice were prepared, and these isolated cells were transferred by i.p. injection into NOD/SCID mice. Then, live PANC1 cells were challenged with s.c. injection. The survival time of NOD/SCID mice received from α-gal(+) PDAC-ly groups was significantly extended (95.0 days) compared with α-gal(-) PDAC-ly groups (45 days, p<0.01). We conclude that α-gal(+) PDAC-ly vaccination may be a practical and effective new immunotherapeutic approach for treating PDAC.

#733

**Modulating T cell immunity in tumors by targeting PD-L1 and neoantigens using a live attenuated oral** Salmonella **platform.**

Sébastien Wieckowski,1 Lilli Podola,2 Heiko Smetak,3 Anne-Lucie Nugues,4 Philippe Slos,4 Amine Adda Berkane,5 Ming Wei,5 Klaus Breiner,1 Albrecht Meichle,2 Philipp Beckhove,3 Marc Mansour,1 Heinz Lubenau2. 1 _VAXIMM AG, Basel, Switzerland;_ 2 _VAXIMM GmbH, Mannheim, Germany;_ 3 _Regensburg Center for Interventional Immunology, Regensburg, Germany;_ 4 _Oncodesign, Dijon, France;_ 5 _CellVax, Romainville, France_.

Significant progresses have been achieved recently in cancer vaccines that aim at engaging or reengaging tumor directed T cells, in particular in the rapidly growing field of personalized immunotherapy. Yet novel immunization solutions to produce effective tumor-associated (neo)antigens T cell responses while simultaneously overcoming the immunosuppressive tumor microenvironment are urgently needed. We are developing a unique and versatile oral T-cell vaccination platform based on the FDA-approved live-attenuated Salmonella Typhi strain Ty21a vaccine Vivotif®, capable of delivering tumor-associated antigens encoded in DNA expression construct to the gut-associated lymphoid tissue, breaking immune tolerance and inducing anti-tumor immunity. This study summarizes the immunogenicity and antileukemia efficacy of VXM10 vaccines based on the live-attenuated Salmonella Typhimurium strain SL7207, transformed with a eukaryotic expression plasmid encoding the murine programmed death-ligand 1 (PD-L1) protein. It also describes for the first time the systemic immunogenicity of Salmonella based polyepitope oral vaccines, supporting the design of Salmonella based neoantigen vaccines. The antileukemia activity of VXM10 was evaluated in the FBL-3 disseminated model of leukemia, in which the tumor cells express high levels of PD-L1. Oral administration of VXM10 produced a strong anti-tumor effect in the FBL-3 leukemia model, with 100% of surviving animals 80 days after leukemia challenge in the highest dose groups. In contrast, administration of the empty vector control did not show any anti-cancer effect. Moreover, 100% of long-term surviving mice resisted re-challenge with FBL-3 cells, demonstrating that vaccination with VXM10 generated a potent memory T cell response against the leukemia. Importantly, full leukemia control was achieved in both prophylactic and therapeutic settings. The anti-tumor effect observed in these experiments was as strong as in previous experiments using a Salmonella Typhimurium transformed with a plasmid encoding Wilm's tumor 1 (WT1). The anti-tumor efficacy was accompanied by an increased systemic antibody response, and the activation of T cells directed against PD-L1 epitopes. Finally, different polyepitope vaccines encoding model epitopes from VEGFR2, Mesothelin, WT1, CEA, and Ovalbumin, induced a significant systemic immunogenicity for up to 6 out of 9 epitopes, 10 days after vaccination of healthy C57BL/6 mice via the oral route, as measured in the spleen by flow cytometry using peptide-MHC class I pentamers. These studies demonstrate that the Salmonella Ty21a platform can be used to generate anti PD-L1 antibody and T cell responses, as well as CD8-positive T cell responses against a majority of MHC class I epitopes of a polyepitope construct. We are now designing a clinical neo-antigen based vaccine that employs the concepts presented.

## EXPERIMENTAL AND MOLECULAR THERAPEUTICS:

### Antibody-Drug Conjugates: Agents and Technology

#734

Thienoindoles: New highly promising agents for antibody-drug conjugates generation.

Michele Caruso,1 Fabio Gasparri,1 Barbara Valsasina,1 Clara Albanese,1 Italo Beria,1 Ilaria Candiani,2 Marina Ciomei,1 Nicoletta Colombo,1 Sabrina Cribioli,1 Ulisse Cucchi,1 Eduard Felder,1 Ivan Fraietta,1 Arturo Galvani,1 Antonella Isacchi,1 Aurelio Marsiglio,1 Paolo Orsini,1 Rita Perego,1 Simona Rizzi,1 Attilio Tomasi,2 Sonia Troiani,1 Carlo Visco,1 Daniele Donati1. 1 _Nerviano Medical Sciences, NERVIANO, Italy;_ 2 _NerPharMa, NERVIANO, Italy_.

Thienoindoles are a new proprietary class of highly potent DNA minor groove alkylating agents.

This class is characterized by a fused thiophene ring whose intrinsic electron-withdrawing character provides a nearly optimal increase in stability and potency of alkylating subunits. Furthermore, the presence of a solubilizing moiety on the minor groove portion provides compounds with physicochemical properties highly compatible with deployment as antibody payloads.

Extensive optimization of this class has led to the identification of the highly potent toxin NMS-P528 with sub-nanomolar IC50 in proliferation assay across a large panel of tumor cell lines.

NMS-P945, a drug-linker generated from NMS-P528, is highly suited for conjugation with targeted antibodies, consistently yielding ADCs with favorable drug-antibody ratios in the absence of significant antibody aggregation and with full maintenance of antigen binding capability.

Proof of concept efficacy and mechanism of action studies were performed using NMS-P945 conjugated with trastuzumab, observing for the resulting conjugate excellent in vitro target-directed cytotoxicity in HER2-positive vs. negative cancer cell lines. In animal efficacy studies against HER2-positive human breast cancer tumors, trastuzumab-NMS-P945 ADC administration yielded complete tumor regression with no effects on body weight gain, while unarmed trastuzumab and armed control antibody showed little and no effect, respectively. PK data indicated long plasma half-life of our novel ADCs in the monkey. Promising results were also obtained with a range of different antibody/antigen systems, e.g. with an antibody directed to the ALK receptor tyrosine kinase.

Extensive process research has been completed for the production of NMS-P528 and NMS-P945, resulting in an efficient stereoselective method for the preparation of GMP materials on the 100g scale and both products are proposed as a licensing opportunity for conjugation with tumor-targeting antibodies.

#735

SAR of amanitin and optimization of linker-amanitin derivatives for solid tumors.

Michael Kulke, Anikó Pálfi, Christoph Müller, Werner Simon, Susanne Werner-Simon, Christian Lutz, Torsten Hechler, Andreas Pahl. _Heidelberg Pharma Research GmbH, Ladenburg, Germany_.

Background:

ATACs (Antibody Targeted Amanitin Conjugates) comprise a new class of antibody-drug conjugates using amanitin as toxic payload. Amanitin binds to the eukaryotic RNA pol II and thereby inhibits the cellular transcription process at very low concentrations. We accomplished the chemical synthesis of amanitin and were able to synthesize amanitin variants in order to optimize the toxin structure for different tumors and antibodies. We will present in vitro and in vivo data of eight different linker-amanitin constructs attached to three different antibodies targeting solid tumors.

Material and methods:

Cell lines: JIMT-1, SKBR-3, BT474 and NCI-N87 (used for anti-Her-2-ATACs); LnCap, 22RV1, MDA-PCa2b and C4.2 (used for anti-PSMA-ATACs); Raji, Raji Luc, Nalm-6 and MEC-2 (used for anti-CD19-ATACs)

Antibodies: Anti-Her-2 (cysteine engineered monoclonal antibody, Heidelberg Pharma); humanized anti-PSMA (Albert Ludwig University Freiburg, medical center; humanization at Lonza Group AG; cysteine engineered monoclonal antibody, Heidelberg Pharma); chimeric anti-CD19 (DKFZ Heidelberg, Germany; cysteine engineered monoclonal antibody, Heidelberg Pharma).

Toxic warhead: Cysteine reactive linker-amanitin constructs were synthesized at Heidelberg Pharma and conjugated site-specifically to the antibodies.

Cell proliferation assay: Quantitative determination of cell viability was performed by CellTiter Glo 2.0 assay (Promega).

Animal models: Subcutaneous Mouse xenograft tumor models (Her-2-, PSMA- and CD19-positive cell lines) were performed in single-dose experiments. Tolerability was assessed in mice and will be assessed in non-human primates (NHP).

Results:

Eight different amanitin-linker constructs were synthesized. They differed in the attachment site of the linker at the amanitin as well as in the toxin core structure. All ATACs showed in vitro cytotoxicity on target positive cell lines in the picomolar range.

In mouse xenograft models, ATACs with four of the eight linker-amanitin derivatives caused dose-dependent tumor regression and complete remission after a single i.v. dose of 2.0 mg/kg in s.c. xenografts irrespective of the antibody and target used. In contrast the other four linker-amanitin derivatives were only poorly effective in vivo while showing comparable in vitro activities. When comparing subcutaneous and intravenous xenograft models using the same cancer cell line, the different efficacy of the eight linker-amanitin variants was only detected in subcutaneous but not in intravenous xenografts. Mouse tolerability studies of ATACs showed a MTD of at least 10mg/kg for all linker-amanitin variants.

Conclusions:

Different efficacy of linker-amanitin derivatives with regard to mouse xenograft models was detected. An SAR profile of amanitin could be established which enabled the selection of optimized linker-amanitin variants for the use of ATACs in solid tumors.

#736

Pyridinobenzodiazepines (PDDs): A new class of sequence-selective DNA mono-alkylating ADC payloads with low hydrophobicity.

Nicolas Veillard,1 Paolo Andriollo,1 Julia Mantaj,1 Keith R. Fox,2 K Miraz Rahman,1 George Procopiou,1 Francesco Cascio,1 David B. Corcoran,1 Ilona Pysz,1 Patricia A. Cooper,3 Steven D. Shnyder,3 Yawen Ju,4 Edwin Tan,4 William M. Schopperle,4 Paul J. Jackson,1 David E. Thurston1. 1 _Femtogenix, Welwyn Garden City, United Kingdom;_ 2 _University of Southampton, Southampton, United Kingdom;_ 3 _University of Bradford, Bradford, United Kingdom;_ 4 _CureMeta, Boston, MA_.

Although four ADCs have been approved and over sixty others are in development, the majority contain payloads belonging to two classes; tubulin inhibitors and DNA cross-linkers. Challenges in the development of ADCs include managing off-target toxicity and hydrophobicity. Some payload classes (e.g., PBD dimers) are notably hydrophobic leading to problems (e.g., aggregation) during conjugation. Thus, there is interest in developing novel payloads which retain the potency of DNA cross-linkers but have lower hydrophobicity and a wider therapeutic window when part of an ADC. The pyridinobenzodiazepines (PDDs) are a new class of sequence-selective, DNA mono-alkylating ADC payload which contain a polyheterocyclic chain with sufficient span to guide them to specific DNA sequences (e.g., transcription factor binding sites). The lead PDD payload, FGX-2-62, has a different sequence-selectivity profile to other DNA-interactive agents, spanning 8-9 base-pairs compared to 6-7 for a PBD dimer, and DNA footprinting experiments indicate a preference for 5'-XGXWWWWXX-3' sequences (X is any base; W is A/T). Transcription factor array studies have shown that the molecule inhibits DNA-binding of oncogenic transcription factors (e.g., NF-κB and GATA). In in vitro cell line studies, FGX-2-62 has low pM cytotoxicity in a diverse cell line panel, including stem cells, cells from both solid and blood cancers (e.g., 9 pM in HL-60) and MDR-resistant tumours, and arrests the cell cycle at the G0/G1 phase compared to G2-M arrest for PBD dimers. It is compatible with attachment to most linker technologies, and is significantly less hydrophobic than other payload classes. Initial MTD studies were carried out by separately conjugating (with negligible aggregation) FGX-2-62 and the PBD dimer Talirine to a THIOMAB® version of trastuzumab (DAR = 2). In female athymic nude mice, a greater tolerance was observed for the THIOMAB®-(FGX-2-62) ADC compared to the THIOMAB®-PBD dimer (i.e, MTD >8 mg.kg-1 versus 4 mg.kg-1). In an efficacy study, FGX-2-62 was conjugated to a cancer stem cell-targeting IgG1 antibody (Bstrongximab) with DAR 1.9. Initial evaluation afforded IC50 values of 0.67 nM and 0.47 nM in two antigen positive cell-lines, and an MTD of 6 mg.kg-1 in mice. In an antigen-positive embryonal carcinoma stem cell CDX mouse model, complete regression was observed at a dose of 2 mg.kg-1 (Q7Dx3). In a cholangiocarcinoma PDX model, complete tumour regression was observed out to 80 days (when experiment was terminated) at a dose of 5 mg.kg-1 (Q7Dx3), with no observed toxicity. The favourable hydrophobicity profile of the PDDs and ease of conjugation, along with their novel mechanism of action, significant in vitro cytotoxicity, in vivo efficacy and tolerability in MTD studies suggest that they represent a promising new class of ADC payloads.

#737

Antibody drug conjugates with anthracycline payload induce tumor-selective antitumor immunity and exhibit a favorable safety profile in cynomolgus monkey toxicology studies.

Ulf Grawunder. _NBE-Therapeutics AG, Basel, Switzerland_.

Background: Antibody Drug Conjugates (ADCs) target highly potent small molecule cytotoxic agents to tumors via tumor-specific antibodies. The therapeutic index of these drugs is strongly affected by a multitude of parameters, including -most importantly- (i) the quality of the targeting antibody, (ii) the stability of the covalent linker between mAb and payload, as well as (iii) the potency and MOA of the cytotoxin. In contrast to tubulin-binding cytotoxic payloads, which are comprised in the majority of clinical-stage ADCs, recently DNA-damaging cytotoxic payloads, like PBDs (pyrrolo-benzo-diazepines), IBDs (indilino-diazepines), or enediynes (e.g. calicheamicin) have taken center-stage, because these toxins show significantly higher anti-tumor efficacy. However, with the advent of these ultrapotent DNA damaging agents, linker-stability and homogeneity of the ADCs ensuring high tolerability and predictable PK/PD properties, respectively, is crucial for achieving a favorable therapeutic index. Methods: Homogeneous ADCs targeting either HER2 or ROR1 have been generated by site-specific conjugation using sortase-enzyme mediated antibody conjugation (SMAC-TechnologyTM) with an ultra-potent, DNA damaging anthracycline toxin, based on the nemorubicin derivative PNU-159682. These ADCs have been evaluated for in vivo efficacy in various HER2 and ROR1 positive PDX-models of solid tumors, but also in syngeneic EMT-6 orthotopic breast cancer models. Furthermore, tolerability of these ADCs was assessed in rodents (HER2 and ROR1 ADCs), as well as in a non-GLP-toxicology study in cynomolgus monkeys (ROR1). Results: Homogeneous PNU-containing HER-2 and ROR1 ADCs show very high anti-tumor efficacy in vivo, both in PDX as well as in syngeneic solid tumor models - in case of HER-2 targeting, HER-2-PNU-ADCs exceeded efficacies of T-DM1 used as a benchmark ADC. In syngeneic breast cancer models, both HER-2, as well as ROR1 ADCs resulted in the induction of a long-lasting tumor selective anti-tumor immunity involving activated CD8 T cells. While strong anti-tumor efficacies are achieved at dose-levels ranging from 0.5-2 mg/kg, these ADCs doses are well tolerated in rodents. An exploratory non-GLP toxicology study in cynomolgus monkey confirmed the safety of PNU-ADCs also in a non-rodent species. Conclusion: Homogeneous PNU-containing HER-2 and ROR1 ADCs exhibit potent anti-tumor immunity in various in vivo tumor models and induce tumor-specific immunity in immune-competent syngeneic tumor models. In addition the ADCs display a very favorable therapeutic index and represent promising and safe drug candidates for treatment of various solid tumors.

#738

Development of an HPLC method for the assessment of hydrophobicity of ADC payloads.

Ilona Pysz,1 Paul J. Jackson,2 Khondaker M. Rahman,1 David E. Thurston1. 1 _King's College London, London, United Kingdom;_ 2 _Femtogenix Ltd, Welwyn Garden City, United Kingdom_.

Antibody-Drug Conjugates (ADCs) are antibodies attached to cytotoxic agents (payloads) through chemically labile linkers. The development of ADCs can fail at various stages, a common cause being a lack of reproducibility in the methods used to attach the payload to the antibody (i.e., the conjugation process) to provide a good yield and uniform drug-antibody ratio (i.e., the DAR). One of the major problems associated with conjugation is the hydrophobicity of a payload which can cause aggregation. The aim of this study was to create a hydrophobicity model to study the physicochemical properties of payloads at an early stage. This should allow their hydrophobicity to be evaluated and optimised at the design stage. A predictive model has been developed based on High Performance Liquid Chromatography (HPLC). Initial work involved development of a gradient reversed-phase HPLC method, and its use in the analysis of multiple reference compounds to establish their relative retention times. This involved the evaluation of a range of reversed-phase columns and solvent systems in order to obtain reproducibility. These initial studies, which utilized a C18 monolithic column for optimum results, demonstrated a clear relationship between the retention time (RT) of the reference compounds and their experimental LogP values obtained from the literature (r = 0.983). Next, a number of commercially available ADC payloads were analysed using the same HPLC method to determine their RT values, and their LogP values were calculated using the ChemDraw software. Payloads studied included Monomethyl Auristatin E (MMAE), Talirine and Tesirine (PBD dimers), Mertansine (DM1) and Calicheamicin. For this group of compounds, the results confirmed that their RT values correlated well with their calculated LogP values (r = 0.94). For example, the payload Talirine, which has a calculated LogP of 3.77, had an RT value of 17 min on the HPLC system equating to a LogP of 3.13 based on the reference compounds. Finally, some novel DNA-interactive payloads (e.g., the pyridinobenzodiazepine FGX-2-62) were evaluated using the same methodology. For FGX-2-62, the calculated LogP varied significantly between different software packages (i.e., 1.47, 3.38 or 4.46 for ChemSketch, MarvinSketch or ChemDraw, respectively). However, its HPLC retention time using the same method was 1.88 min, equating to a LogP of 1.63. This suggests that FGX-2-62 is significantly less hydrophobic than Talirine (e.g., RTs of 1.88 min versus 17 min, respectively), an observation consistent with the ease of conjugation of FGX-2-62 to antibodies without significant aggregation. Overall, these results suggest that HPLC retention time derived from a suitable HPLC method is more reliable than the currently available software-based LogP prediction methods in providing a realistic assessment of the hydrophobicity of novel payloads and their propensity to cause aggregation during conjugation.

#740

Preclinical evaluation of an anti-PSMA antibody-targeted amanitin conjugate (ATAC).

Anikó Pálfi, Christian Breunig, Torsten Hechler, Christoph Müller, Christian Lutz, Andreas Pahl, Michael Kulke. _Heidelberg Pharma Research GmbH, Ladenburg, Germany_.

Background:

ATACs (antibody-targeted Amanitin conjugates) comprise a new class of antibody-drug conjugates using Amanitin as toxic payload. Amanitin binds to the eukaryotic RNA pol II and thereby inhibits the cellular transcription process at very low concentrations. In the current study, in vitro and in vivo data of an ATAC targeting PSMA (prostate specific membrane antigen) are presented. PSMA is predominantly expressed on malignant prostate cells in prostate carcinoma and correlates with tumor progression. Hence it is considered an interesting target for Amanitin based ADCs.

Material and methods:

PSMA cell lines: MDA-PCA-2B, LnCap. PC3 cell line served as PSMA negative control.

Antibody: humanized anti-PSMA and cysteine engineered monoclonal antibody (Albert Ludwig University Freiburg, medical center; humanization at Lonza Group AG, derivatization and production at Heidelberg Pharma).

Toxic warhead: A cysteine reactive amanitin-linker was conjugated site-specifically to engineered cysteine residues of the anti-PSMA antibody yielding an ATAC with a DAR of 2.0.

Cell proliferation assay: Quantitative determination of cell viability was performed by CellTiter Glo 2.0 assay (Promega).

Animal models: Subcutaneous Mouse xenograft tumor models with PSMA-positive cell lines MDA-PCA-2B and LnCap were performed in single-dose and multiple-dosing experiments. Tolerability was assessed in mice and non-human primates (NHP).

Results:

The anti-PSMA ATAC showed in vitro cytotoxicity on PSMA+ cell lines in picomolar range, whereas no cytotoxic activity on PSMA- cells was observed.

In mouse xenograft models, the anti-PSMA ATAC caused dose-dependent tumor regression. Complete remission was achieved after a single i.v. dose of 4.0 mg/kg and after repeated i.v. doses of 2.0 mg/kg in s.c. xenografts.

Safety profiling in Cynomolgus monkeys revealed a good tolerability and therapeutic index after sequentially applied doses of 0.3, 1.0 and 3.0 mg/kg. Hematology and clinical chemistry parameters were unaffected except liver enzymes and LDH: A moderate and transient increase was observed. The half-life of the ATAC in serum was 7-10 days; the free toxin was detectable at levels close to the lower limit of quantification only (LLOQ = 1.2 nM).

Conclusions:

Targeted cytotoxic drug delivery to PSMA positive prostate cancer cell lines was achieved by using an anti-PSMA ATAC. The mode of action of the payload Amanitin led to an efficient anti-tumor potential in vitro and in vivo with good tolerability in NHP studies. The use of ATACs in the therapy of PSMA positive prostate cancer is a promising approach, especially by using a cytotoxic agent whose mode of action differs from other commonly used toxins.

#741

Targeting trastuzumab and T-DM-1 resistant breast cancer cells with EV20/MMAF, an antibody drug-conjugate against HER3.

Gianluca Sala,1 Lucia Gandullo,2 Emily Capone,1 Maria Elena Diaz-Rodriguez,2 Daniela D'Agostino,1 Vincenzo De Laurenzi,1 Jean-Frederic Sauniere,3 Stefano Iacobelli,3 Atanasio Pandiella2. 1 _University of Chieti, Chieti, Italy;_ 2 _University of Salamanca, Salamanca, Spain;_ 3 _Mediapharma srl, Chieti, Italy_.

Trastuzumab and its derivative Trastuzumab-emtansine (T-DM1) are drugs approved to treat HER-2+ tumors. Despite the impressive clinical efficacy observed in many patients, intrinsic and acquired resistance to both drugs has emerged as a challenge. The HER-3 receptor is emerging as an attractive molecule in therapeutics because of its overexpression across many human cancers and because of its role in resistance to certain anticancer drugs. We recently showed that the EV20 humanized anti-HER-3 antibody has the ability of selectively and efficiently deliver the plant toxin Saporin to tumor cells. Here, we describe the antitumor activity of a novel ADC derivative of EV20 coupled to the cytotoxic drug monomethyl auristatin F (MMAF). This HER-3/ADC (EV20/MMAF) demonstrated a powerful and target-dependent killing activity in a panel of HER2+ breast cancer cells, including those resistant to trastuzumab and T-DM1 with an IC50 in the pico/nanomolar range.

EV20/MMAF mediated potent antitumor activity in mice bearing trastuzumab resistant xenograft models. Notably, we found that the novel conjugate was well tolerated and caused tumor shrinkage in 100% of tumor-bearing mice. In summary, these findings open the possibility to use EV20/MMAF as a novel ADC targeting HER-3 and support its clinical development for malignancies, such as HER-2+ breast cancer, in which HER-3 is frequently overexpressed.

#742

Nonclinical safety evaluation of STRO-001, a site-specific anti-CD74 antibody-drug conjugate for the potential treatment of B-cell malignancies.

Willy Solis, Venita De Almeida, Abigail Yu, Maureen Bruhns, James Zawada, Adam Galan, Shannon Matheny, Arturo Molina, Trevor Hallam, Mark Lupher. _Sutro Biopharma, South San Francisco, CA_.

Antibody-drug conjugates (ADCs) constitute an expanding class of therapeutic molecules in preclinical and clinical development for multiple oncology indications. These antibody-based therapeutics are designed to selectively deliver cytotoxic payloads to tumor targets to improve efficacy and minimize toxicity. Cluster of differentiation 74 (CD74), a membrane-bound glycoprotein, is an antigen amenable ADC targeting because of its low expression in normal tissues and over expression in B-cell malignancies (multiple myeloma and non-Hodgkin's Lymphoma). STRO-001, a well-defined, homogenous, and site-specific ADC, is comprised of maytansinoid cytotoxic drugs conjugated to an anti-CD74 aglycosylated antibody (SP7219) through a noncleavable linker at two engineered non-natural amino acids. In an in vitro immunological activation assay, STRO-001 did not elicit cytokine production when incubated with human PBMCs for 48 hours, suggesting a low risk for immunological response in cancer patients. In a tissue cross-reactivity study with human tissues, STRO-001-specific membranous staining was detected primarily in lymphoid organs and lymphocytic infiltrates in multiple non-lymphoid tissues. In an exploratory nonclinical safety study in cynomolgus monkeys, a pharmacologically relevant model for toxicity testing, STRO-001 was tolerated at 1, 3, and 10 mg/kg, but not at 30 mg/kg, when intravenously administered on days 1 and 15 followed by a 4-week observation period. Dose-dependent and transient decreases in B-cells and monocytes were observed at tolerated doses (on-target pharmacology), with no effects on NK/T-cells. At tolerated doses, the main findings included reversible toxicities to hematopoietic and lymphoid tissues that correlated with cytopenias. Hematopoietic toxicity is considered antigen-independent, clinically manageable, and consistent with that of other ADCs with similar payloads. Toxicokinetic analysis confirmed drug exposures (total antibody, ADC, and a prominent linker-maytansine catabolite) with evidence of non-linearity suggesting target-dependent clearance. In summary, STRO-001 ADC has a favorable nonclinical safety profile and Phase I study of STRO-001 in patients with B-cell malignancies is planned.

#743

**ABT-806-derived antibody-drug conjugates (ADCs) inhibit growth of malignant mesothelioma** in vivo **.**

Puey Ling Chia,1 Diana Cao,1 Angela Rigopoulos,1 Hui Gan,1 Ed Reilly,2 Andrew Phillips,2 Thomas John,1 Andrew M. Scott1. 1 _Olivia Newton-John Cancer Research Institute, Heidelberg, Australia;_ 2 _Abbvie Cancer Discovery, North Chicago, IL_.

Introduction: Malignant mesothelioma (MM) is an aggressive malignancy of the pleura with limited therapeutic options, and is associated with a poor prognosis. EGFR is known to be highly over-expressed in mesothelioma with reported EGFR overexpression between 44 to 97%. We have developed an anti-EGFR antibody (ABT-806), which is tumour specific and robustly inhibits EGFR-expressing tumours. We aimed to establish the validity and feasibility of targeting tumour expressed EGFR in MM using ABT-806 novel ADCs (ABT-414 and ABBV-221).

Methods: We evaluated EGFR and mAb 806 immunohistochemistry in 4 MM cell lines (MSTO-211H, NCI-H2052, NCI-H28, NCI-H2452) and performed in-vitro cell proliferation assays (CPA) to evaluate the antineoplastic potential of mAb806-related ADCs. In-vivo therapeutic studies using the biphasic mesothelioma cell line (MSTO-211H) were conducted with treatment groups involving ABT-414, ABBV-221, ADC control, cisplatin chemotherapy. We also performed quantitative biodistribution and imaging of mAb806 ADC uptake (89Zr mAb806 ADC) to allow correlation of mAb806 ADC concentration in tumours.

Results: mAb806 elicited strong binding to three of four MM cell lines (MSTO-211H, NCI-H2052 and NCI-H28). CPAs also demonstrated ABT-414 and ABBV- 221 had significant cell growth inhibition demonstrated in the range between 1 to 10ug/ml for MM cell lines. In MSTO-211H xenograft model significant anti-tumour response to both ABT-414 and ABBV-221 (p<0.01), was demonstrated. High, specific targeting of 89Zr-ch806 to MM tumour in-vivo was also shown.

Conclusion: ABT-806 ADCs show potent anti-tumour activity in MM model, and warrant further exploration as a potential therapy for MM.

#744

ADCT-701, a novel pyrrolobenzodiazepine (PBD) dimer-based antibody-drug conjugate (ADC) targeting DLK1-expressing tumors.

Francesca Zammarchi,1 Karin Havenith,1 Simon Chivers,1 Paul W. Hogg,1 Charlie Britten,1 Sandamali Dissanayake,1 Peter Tyrer,2 Francois Bertelli,2 Ian Hutchinson,2 Luke Masterson,2 Phil Howard,2 John A. Hartley,3 Patrick H. van Berkel1. 1 _ADC Therapeutics, London, United Kingdom;_ 2 _Spirogen, Medimmune, London, United Kingdom;_ 3 _University College of London, London, United Kingdom_.

Delta-like 1 homolog protein (DLK-1) is an EGF-like membrane bound protein consisting of six tandem EGF-like repeats, a juxtamembrane region with a TACE (ADAM17)-mediated cleavage site, a transmembrane domain, and a short intracellular tail. DLK-1 is strongly expressed during fetal development, while its expression is highly restricted in adults. Conversely, DLK-1 gets re-expressed in several tumors, such as neuroblastoma, hepatocellular carcinoma (HCC), rhabdomyosarcoma, small cell lung cancer, myelodysplastic syndrome and acute myeloid leukemia. Interestingly, in HCC DLK-1 has been shown to be a marker of cancer stem cells, a subpopulation of cells responsible for tumor initiation, growth, metastasis, and recurrence. Altogether, DLK-1 represents an attractive target for an antibody-drug conjugate (ADC) approach based on its selective expression in a wide range of malignancies and restricted expression in healthy organs, as well as its expression on HCC cancer stem cells. ADCT-701 is an ADC composed of a humanized IgG1 antibody against human DLK-1, site-specifically conjugated using GlycoconnectTM technology to PL1601, which contains a valine-alanine cleavable linker and the PBD dimer cytotoxin SG3199. The purpose of this study was to characterize the in vitro and in vivo anti-tumor activity of ADCT-701 in human cancer cell lines and patient-derived xenograft (PDX) models and to determine its safety and tolerability in the rat. In vitro, ADCT-701 demonstrated potent cytotoxicity in a panel of human cancer cell lines of different origin and levels of DLK-1, while its potency was strongly reduced in DLK-1-negative cell lines. In vivo, ADCT-701 showed potent anti-tumor activity in the DLK1-expressing neuroblastoma-derived SK-N-FI xenograft in which a single dose of ADCT-701 at 0.5 or 1 mg/kg showed dose-dependent anti-tumor activity compared to the vehicle- and isotype control ADC-treated mice. At 1 mg/kg, ADCT-701 resulted in 1/9 partial responders (PR) and 4/9 complete responder (CR), one of which was tumor-frees survivors (TFS) at the end of the study on day 60. Moreover, ADCT-701 showed dose-dependent anti-tumor activity in a HCC PDX model when tested as a single dose at 0.1, 0.3 or 1 mg/kg. At the highest dose tested, ADCT-701 resulted in 3/8 PR and 2/8 CR, while none of the mice treated with the vehicle or the isotype-control ADC (1 mg/kg, single dose) showed any activity. ADCT-701 was stable, well tolerated and showed a favorable pharmacokinetic profile in the rat with a half-life of 9 days and a maximum tolerated dose of at least 5 mg/kg. In conclusion, ADCT-701 demonstrated potent and specific in vitro and in vivo anti-tumor activity in DLK-1-expressing cancer-derived models and it was stable and well tolerated in the rat, warranting further development of this ADC into the clinic.

#745

Development of a novel ron receptor targeted antibody-drug conjugates using cysteine bridging technology for potential treatment of pancreatic cancer.

Sreedhar Reddy Suthe,1 Hang-Ping Yao,2 Paul C. Trippier,1 Ming-Hai Wang1. 1 _Texas Tech University Health Sciences Center, Amarillo, TX;_ 2 _Zhejiang University School of Medicine, Hangzhou, Zhejiang, China_.

Therapeutics targeting known oncoproteins have been applied for pancreatic cancer treatment but clinical outcomes are not promising. Hence, there is an urgent need to identify novel targets and develop effective drugs to improve pancreatic cancer therapeutic index. Antibody-drug conjugates (ADC) represent a promising class of drugs for targeted cancer therapy. Here we developed a novel ADC targeting RON receptor tyrosine kinase for potential pancreatic cancer treatment. To this end, we have synthesized a bis-alkylating linker (BL), attached to a lysosomal protease-cleavable dipeptide with payload Monomethyl auristatin E (MMAE). The BL-MMAE was then conjugated to Zt/g4 (anti-RON mAb) through cysteine bridging technology to produce Zt/g4-BL-MMAE with a homogeneous conjugation profile and an antibody to drug ratio of 1:4. Zt/g4-BL-MMAE showed significant improvement in drug conjugation homogeneity and serum stability over conventional ADCs prepared through maleimide based linkers. In pancreatic cancer cell lines overexpressing RON, Zt/g4-BL-MMAE specifically targeted RON-expressing tumor cells and was effective in rapid induction of cell surface RON endocytosis. Functional analysis revealed that Zt/g4-BL-MMAE caused cell cycle arrest at G2/M phase, reduction of cell viability and subsequently resulted in massive cell death. The calculated IC50 is in the range of 1 to 2 µg/ml. We conclude that Zt/g4-BL-MMAE is a novel anti-RON ADC with excellent conjugation profile, serum stability, and selective cytotoxicity for pancreatic cancer cells. This work provides a pharmaceutical opportunity for evaluating potentials of RON-targeted ADCs in pancreatic cancer treatment in the future.

#746

Development of novel formats of anti-AXL ADCs.

Maria L. Jaramillo,1 Myriam Banville,1 Alma Robert,1 Luc Meury,1 Maurizio Acchione,1 Anne Marcil,1 Christine Gadoury,1 Cunle Wu,1 Yves Fortin,1 Kevin Henry,2 Joseph Schrag,1 The-Minh Tu,1 Binbing (Erica) Ling,2 Jacqueline Slinn,2 Maria Moreno2. 1 _National Research Council, Montréal, Quebec, Canada;_ 2 _National Research Council, Ottawa, Ontario, Canada_.

AXL is a member of the Tyro3- AXL -Mer (TAM) receptor tyrosine kinase subfamily which has been associated with several cellular functions including growth, migration, aggregation, and anti-inflammation (Li Y et al., 2009) via activation through its ligand, Gas6. Originally identified as an oncogene in chronic myelogenous leukemia, expression of Axl has been reported to be upregulated in a variety of cancers including breast, gastric, prostate, ovarian, and lung. AXL expression has been shown to be negatively associated with patient survival and implicated in epithelial-to-mesenchymal transition, a process closely linked with invasive motility and metastasis of malignant cells. Several recent studies have reported that AXL is overexpressed during resistance to chemotherapy and other molecularly targeted therapies, as well as during hypoxia (Schoumacher M et al 2017). Consequently, Axl is promising therapeutic target for development into ADCs for the first or second line treatment of cancer.

At the National Research Council (NRC) of Canada, we have selected mouse monoclonal (mAbs) or llama derived single domain antibodies (sdAbs) that are selective for the extracellular domain of human Axl and which have been shown to be internalized into Axl expressing cells for antibody drug conjugate (ADC) development using a surrogate ADC screen. These antibodies were also characterized based on their binding domains and ability to compete with Gas6, the extracellular ligand for Axl. A panel of Axl-specific ADCs was subsequently directly conjugated to maytansine, the microtubule disrupting agent through a noncleavable linker. Axl-DM1 ADCs were shown to efficiently induce cytotoxicity in vitro, in Axl expressing cells including breast (MDA-MB 231), lung (NCI-H292) and ovarian (SKOV3) cell lines with sub nM potencies achieved with both sdAb and conventional mAb-based ADCs. When tested in vivo using ovarian (SKOV3) and breast cancer (MDA-MB 231) xenograft models, Axl-DM1 ADCs were shown to have potent anti-tumor activity.

Biparatopic antibodies (i.e. those which target two non-overlapping epitopes) directed against HER2 have been previously shown to induce target clustering and promote robust internalization, lysosomal trafficking, and degradation (LI JY et al 2016). Since these properties are important in the mechanism of action of ADCs, we have further tested anti-Axl antibodies in various configurations and have identified potent biparatopic antibodies for further development into ADCs.

#747

A new class of DNA alkylating indolino-benzodiazepine agents (BIAs) linked with a DNA binding moiety for use with antibody-drug conjugates (ADCs).

Michael L. Miller, Emily E. Reid, Katie E. Archer, Manami Shizuka, Molly A. McShea, Erin K. Maloney, Olga Ab, Leanne Lanieri, Alan J. Wilhelm, Jose F. Ponte, Nicholas C. Yoder, Ravi V. Chari. _ImmunoGen, Inc., Waltham, MA_.

We have previously disclosed antibody-drug conjugates (ADCs) that incorporate our highly potent novel DNA alkylating indolino-benzodiazepine (termed IGN) pseudo dimer. ADCs of these DNA alkylating IGNs were found preclinically to demonstrate better tolerability and an improved overall therapeutic index (TI) compared with those of DNA crosslinking IGNs (Miller, et al., AACR 2017 #53). In our ongoing effort to further explore DNA alkylating effector molecules for ADCs, we disclose here a new class of IGNs (termed BIAs) in which one IGN monomer subunit is connected to a bis-aryl moiety with affinity for the DNA binding pocket. Our initial structure activity relationship (SAR) studies around a series of BIAs identified a set of scaffolds that met our requirement for high in vitro potency. These scaffolds were subsequently modified to incorporate functionalities that allow linkage to an antibody using various linker chemistries. These linkable BIAs displayed high in vitro potency across a panel of cell lines, indicating that the incorporation of a linker was not detrimental to their overall potency. Furthermore, we found that the potency of these linkable BIAs could be modulated by careful selection of substituents on the bis-aryl moiety providing enhanced binding affinity with DNA. Upon identification of lead BIA molecules, conjugates with a folate receptor-α (FRα)-binding antibody were prepared. These ADCs displayed potent, antigen-specific in vitro activity across a panel of FRα-expressing cell lines. In vivo, these ADCs demonstrated potent efficacy in xenograft models at doses well below the maximum tolerated dose. In light of these findings, BIA ADCs represent a promising new class of DNA alkylating effector molecules for use in the development of ADCs.

#748

Non-internalizing site-specific antibody-drug conjugates based on maytansinoids display curative properties.

Gianluca Sala,1 Emily Capone,1 Enza Piccolo,2 Sara Ponziani,2 Roberta Gentile,2 Rodolfo Ippoliti,3 Francesco Giansanti,3 Stefano Iacobelli2. 1 _University of Chieti, Chieti, Italy;_ 2 _Mediapharma srl, Chieti, Italy;_ 3 _University of l'Aquila, l'Aquila, Italy_.

Antibody-Drug Conjugates (ADCs) are highly potent biopharmaceuticals that use the targeting ability of monoclonal antibodies to selectively bind to tumor cells where the conjugated cancer drug is released. Traditionally, the anticancer efficacy of ADCs relies on their selective internalization by cancer cells. However, it has recently been shown that non-internalizing, linkerless ADCs targeting the tumor extracellular environment can display a potent therapeutic activity. LGALS3BP (aka Mac-2 BP or 90K) represents an appealing target for non-internalizing ADC development, as the protein is largely secreted by the majority of human tumors, while being virtually undetectable in normal adult tissues except for the prostate and the colorectum. Additionally, once secreted the protein stops from spreading and clusters on the plasma membrane where it binds to some endogenous ligands. Here, we show that an engineered variant of the humanized 1959 antibody to human LGALS3BP selectively localizes around the tumor cells and once site-specifically coupled to the maytansinoid drugs DM3 or DM4 by means of disulfide linker can display a potent therapeutic activity. Both the 1959sss-DM3 and -DM4 conjugates mediated potent antitumor activity in mice bearing human tumor xenografts. Quantitative biodistribution studies confirmed a preferential accumulation of 1959sss-DM3 and -DM4 conjugates around the tumor cells. Notably, we found that both conjugates were well tolerated and cured 100% of tumor-bearing mice. Our findings offer a preclinical proof-of-concept for curative non-internalizing ADC endowed with an innovative mechanism of action.

#749

Antibody internalization assays for cancer drug discovery.

Nicola J. Bevan, Tim J. Dale, Derek J. Trezise. _Essen BioScience Ltd, Welwyn Garden City, United Kingdom_.

Monoclonal antibodies (mAb) and antibody-drug conjugates (ADCs) are widely used as anti-cancer therapeutics. A key property of these biologics is the extent and rate of internalization into different cells, which governs their efficacy, safety and pharmaco-dynamic profile. Quantifying and comparing the internalization rates of different Ab drug candidates and production batches is therefore a critical step in the biopharmaceutical selection and optimization process. Here, we describe novel and enabling cell-based Ab internalization assays that are turnkey, medium throughput and geared toward industrial biologics discovery. Internalization measurements are made over time on 96-well microplates using live-cell analysis (IncuCyte S3) and a fluorescent pH-sensitive dye coupled antibody-binding fragment (FabFluor) that binds the test mAb Fc region using a single step, no wash labeling protocol. An increase in fluorescence signal is observed as the mAb complex is internalized into the acidic lysosome. To validate this approach trastuzumab (Herceptin, Her-2) or rituximab (Rituxan, CD20) were mixed with the hFabFluor reagent (1: 3 molar ratio, 15min), serially diluted in complete media (1:2) and added to pre-plated BT-474 or Raji cells (no wash). Cell images (10-20x) were taken and automatically analysed for fluorescence area every 30min for up to 48h. Both trastuzumab (BT-474) and rituximab (Raji) caused clear time- and concentration-dependent internalization (EC50 values 2.1 and 2.6nM, respectively). The fluorescence signal was punctate, outside of the nucleus and strongly co-localized with a lysosomal marker (LysoSensor). In line with known marker expression profiles, specific internalization of mAbs to CD45, CD71 and CD3, but not CD20, was observed in Jurkat T-lymphocytes and CD45, CD71 and CD20, but not CD3 in Raji B cells. As a proof of concept for screening and direct comparison of test mAbs, 6 commercially available CD71 (transferrin receptor) Abs were labeled with mFabFluor reagent, serially diluted (1:2, 4.6-10000ng mL-1) and added to HT1080 osteosarcoma cells. 3 of the Abs produced a large internalization signal with detection less than 50ng mL-1, whilst the other 3 were internalized weakly with signal only visible at higher concentrations. A mean Z' value of 0.82 was calculated from control wells indicating a microplate assay with high precision and robustness. Taken together these data support the validation of a simple, integrated and quantitative solution for directly studying internalization of mAbs into cells which can easily be scaled to compare multiple Abs in parallel. This method enables mAb and ADC internalization measurements to be implemented at early stages of the biologics discovery process and will prove valuable in efficacy, safety and pharmacokinetic optimization.

#750

Modular coiled-coil masking domains for tumor-specific antibody activation.

Vivian H. Trang, Xinqun Zhang, Melissa M. Dominguez, Weiping Zeng, Julia H. Cochran, Ivan J. Stone, Peter D. Senter, Matthew R. Levengood. _Seattle Genetics, Bothell, WA_.

The power of monoclonal antibody therapeutics arises from their remarkable selectivity for a particular antigen. However, antibody therapies can still be limited by serious side effects that result from target-mediated toxicity when the antigen is also expressed on non-diseased tissue. An emerging concept in the field of therapeutic antibodies is to restrict antibody binding in healthy tissues while empowering the antibody at sites of disease. This has previously been accomplished by fusing masking groups to the antibody through cleavable sequences that can be activated upon hydrolysis by disease-associated proteases. Here, we present a novel approach to mask antibodies utilizing a variety of leucine zipper coiled-coil peptides fused to the N-termini of antibody heavy and light chains. An optimized heterodimeric coiled-coil masking domain was identified that significantly impaired the binding of multiple antibodies to their cognate receptors with no antibody-specific optimization required. Protease-cleavable linkers were introduced between the coiled-coil peptides and antibody termini, and protease-catalyzed removal of the coiled-coil domains restored antibody binding affinity to that of the unmasked parent antibody. The coiled-coil mask did not impair production of antibody-drug conjugates (ADCs) of varying drug-to-antibody ratios, and masked antibodies and ADCs had similar stability and pharmacokinetic profiles to unmasked counterparts. However, coiled-coil masked ADCs and antibodies displayed decreased functional activity in biological assays, commensurate with the decrease in affinity afforded by masking, and this activity could be restored upon cleavage of the mask. Lastly, coiled-coil masked antibodies and ADCs had been tested in vivo and had demonstrated improved safety benefits as well as equivalent antitumor activity compared to the unmasked counterparts. Coiled-coil masked antibodies represent an advance in the field of antibody pro-drugging, as this modular domain can be rapidly applied to an array of therapeutic antibodies and ADCs.

#751

A controlled affinity-guided ADC-linker (MAGNET) technology.

Nimish Gupta,1 Goutam Biswas,1 Aniruddha Sengupta,1 Monideepa Roy,1 Sudip Roy,1 Shiladitya Sengupta2. 1 _Akamara Therapeutics, Inc., India;_ 2 _Brigham and Women's Hospital, Harvard Medical School, MA_.

Antibody-drug conjugates (ADCs) have emerged as a powerful technology in cancer therapy. However, challenges with current linker technologies have limited the repertoire of ADCs. Currently-used linkers result in a heterogeneous mixture, unstable conjugates and/or involve complex processes such as genetic modification of the antibodies. Here, for the first time, we have explored high affinity non-covalent interactions between antibody and affinity ligands to design novel linkers that overcome the above challenges. The affinity ligands-based linkers coupled with payload, enable strong non-covalent association with the antibodies owing to their high specificity and affinity for antibodies without the need for any chemical reaction with the antibody. With a prototype affinity ligand, we demonstrated that the affinity ligand is able to guide the payload to six precise locations on the antibody conferring site-specificity to the resulting ADCs. Significantly, despite non-covalent interactions, the conjugates were found to be stable in presence of albumin and in human plasma. The ADC exhibited excellent antigen-specific internalization and nanomolar potency in vitro and were effective in suppressing tumor growth in a lung adenocarcinoma xenograft model with minimal systemic toxicity. This novel technology, which we term MAGNET (multivalent, affinity-guided antibody-empowerment technology) overcomes the existing limitations of ADC engineering by imparting trinity of (1) site-specificity, (2) stability and (3) homogeneity, while simplifying the ADC synthesis process. The self-assembled MAGNET ADCs pave the way for exploration of non-covalent conjugation of payloads to any biomolecule, and heralds a new paradigm for ADC generation.

#752

CovisoLinK: New bacterial transglutaminase Q-Tag substrate for the development of site specific antibody-drug conjugates.

Eva Sivado,1 Vincent Thomas,2 Meddy El Alaoui,2 Anne-Catherine Jallas,1 Mike R. Dyson,3 John McCafferty,3 Said El Alaoui,2 Sandrine Valsesia-Wittmann1. 1 _Centre Leon Berard, Lyon, France;_ 2 _Covalab, Villeurbanne, France;_ 3 _Iontas, Cambridge, United Kingdom_.

CovIsoLink™ (Covalently Isopeptide crosslinking) relates to methods for enzymatic covalently coupling drugs and other compounds through transglutaminase site specific generated in the targeted proteins including, polypeptides, proteins and immunoglobulins (patent pending1). Transglutaminases (TGases) catalyze covalent cross- linking of specific glutamine residues to the primary amine of peptide-bound lysine residues or primary amines of other compounds such as polyamines2. Using an in house peptide library and the transglutaminase colorometric assay3, we identified several amino acid sequences that were recognized as glutamine donor substrates. The optimum peptide sequences were selected and we further confirmed that these sequences have improved affinity compared with the conventional small peptides Z-QG. In different experiments we engineered Fc-containing polypeptide at the C-terminal domain and showed that mTG incorporates with high efficiency several types of amine donors to the engineered antibodies. CovIsoLink™ is now used to develop new antibody drug conjugates (ADCs) since the major advantage of this method is to obtain a homogenous immunoconjugate with uniform stoichiometry. We developed and characterized different recombinant anti Her2 IgG1 Mab carrying optimized enzymatic substrates (tag) by genetic insertion in the coding sequence of MAb. We then evaluated the best linkers and conformation to incorporate different compounds through bacterial Transglutaminase (mTG) enzymatic reaction. We set up experimental conditions, production, purification, HPLC/MS analysis and control of the immunoreactivity of CovIsoLink™ Her2-ADC. Using mTG, we obtained site specific conjugation of different modified drugs with optimized linker on the anti Her2 IgG1 antibody. By HIC analysis, we validated a specific and reproducible DAR reaching DAR2 depending on drugs and experimental conditions. In vitro and In vivo characterization and dose response studies of CovIsolink-ADC specificity and reactivity are currently performed in Her2 positive models demonstrating an improved efficacy by comparison with Kadcyla (T-DM1).1) PCT/EP2014/0792278, 2) El Alaoui S et al, Int J Cancer, 1991; 3) Perez AM et al, Anal Biochem, 2009.

#753

Development of an in vivo model system to assess the interplay between the various drivers of antibody-drug conjugate (ADC) activity.

Leanne Lanieri, Steven Boule, Ling Dong, Tara Drake, Rassol Laleau, Jenny Lee, Fenghua Liu, Qifeng Qiu, Neeraj Kohli, Jose Ponte, Yulius Setiady, Richard Gregory. _ImmunoGen, Inc., Waltham, MA_.

Among the many factors determining the clinical efficacy of antibody-drug conjugates (ADCs) are dose and schedule, target antigen expression (both on tumor and normal tissues), antibody affinity, drug-to-antibody ratio (DAR), bystander killing activity, the ability of the ADC to penetrate and distribute into the tumor, and interaction with the host immune system. Traditional ADC efficacy studies use cell line-derived (CDX) or patient-derived xenografts (PDX) implanted into immuno-compromised mice. These traditional methods are limited in predicting clinical efficacy because they are typically performed using ADCs that lack cross-reactivity to normal mouse tissue antigens, thus increasing drug exposure to the tumor and over-estimating in vivo activity. In addition, they are limited in their ability to assess interplay between the ADC mechanism of action and the immune system. To better understand the variables that impact ADC efficacy, we have built a cross-reactive murine efficacy model, beginning with the generation of an anti-murine folate receptor alpha (FRα) antibody. Rabbits were immunized with a mouse FRα expressing stable cell line. The resulting antibodies were screened for cross-reactivity to human FRα to enable use in efficacy studies with mouse and human tumor models. A cross-reactive antibody with similar binding affinity to mouse and human FRα was identified and chimerized to a muIgG2a backbone creating rmFR1-12. Additionally, we have engineered the completmentarity-determining regions (CDRs) of rmFR1-12 to generate variants that bind to FRα with varying affinities to assess the impact of affinity on activity. The rmFR1-12 antibody has been successfully conjugated to maytansinoid (DM) and indolino-benzodiazepine (IGN) payloads and demonstrates specific in vitro cytotoxicity to multiple murine and human cell lines with varying levels of mouse or human FRα expression. Biodistribution studies revealed substantial FRα expression on normal tissue that significantly impact the pharmacokinetic profile of rmFR1-12. In vivo efficacy studies comparing equivalent doses of rmFR1-12 ADCs to ADCs generated with an antibody that is not cross reactive to mouse FRα demonstrated a significant decrease in anti-tumor activity in mice treated with rmFR1-12 ADCs. In summary, we have developed a novel mouse tumor model to determine the interplay of various factors on ADC activity.

#754

Unique pharmacologic properties of Dolaflexin-based ADCs—a controlled bystander effect.

Susan M. Clardy, Alex Yurkovetskiy, Mao Yin, Dmitry Gumerov, Ling Xu, Elena Ter-Ovanesyan, Charlie Bu, Alex Johnson, Marina Protopopova, Qingxiu Zhang, Natalya Bodyak, Marc Damelin, David H. Lee, Donald Bergstrom, Timothy B. Lowinger. _Mersana Therapeutic, Cambridge, MA_.

Antibody-drug conjugates (ADCs) are designed to bind tumor-associated antigens and deliver conjugated cytotoxic payloads to antigen-positive cells. Some ADCs also kill neighboring cells (including antigen-negative cells) by a mechanism referred to as the bystander effect. This effect can be beneficial when the antigen has heterogeneous expression among cells in a solid tumor, but it can also increase off-target toxicity of ADCs. Herein, we report on a unique pharmacologic property of the Dolaflexin platform, which provides a controlled bystander effect that retains the benefits of the bystander effect with respect to antitumor cytotoxicity but reduces the off-target toxicity. The controlled bystander effect, termed "Dolalock," was achieved through design of a payload, auristatin F-hydroxypropylamide (AF-HPA), that is membrane-permeable and capable of bystander killing but is further catabolized to membrane-impermeable auristatin F (AF). This catabolism of the payload "locks" the highly potent AF in the cell. Using Dolaflexin-based ADCs, we investigated the extent of intracellular AF-HPA and AF release, tumor cell retention and bystander activities in vitro and in vivo. We observed both auristatin species within cells. Co-culture assays with HER2-positive and HER2-negative cells confirmed the cell permeability and bystander-killing capabilities of AF-HPA released from a Dolaflexin-based ADC. Biodistribution studies of Dolaflexin-based ADCs revealed time-dependent concentrations of AF-HPA and AF as well as significant accumulation of AF in xenografted tumor cells, consistent with the Dolalock mechanism. An additional benefit of AF formation was seen in multidrug-resistant transporter studies, which demonstrate that AF, in contrast to AF-HPA, is not a P-glycoprotein 1 (Pgp) substrate. This property may offer additional benefit in Pgp-expressing tumors. In summary, we have shown that the novel AF-HPA payload used in the Dolaflexin platform allows for a controlled bystander effect that likely contributes to the enhanced efficacy and lack of neutropenia we have observed with Dolaflexin-based ADCs.

#755

Synthesis and evaluation of sugar alcohol based molecules for drug conjugation and delivery.

Yumei Huang,1 Shuyan Chen,1 Binyuan Sun,1 Amanda Griffin,1 Nathanial Sweet,1 Mingqian Feng,2 Mitchell Ho2. 1 _CellMosaic Inc., Woburn, MA;_ 2 _National Cancer Institute, NIH, Bethesda, MD_.

Hydrophilic linkers, which are available in different motifs, have been used as agents of drug delivery, as aqueous solubility enhancers for hydrophobic molecules, and as tethers or spacers in conjugation. Polyethylene glycol (PEG) is one of the most commonly used hydrophilic linkers and commercially available. Lower molecular weight (MW) heterobifunctional PEG linkers are the most frequently used linkers for linking hydrophobic compounds, whereas high MW polymers have been used to conjugate proteins, peptides, oligonucleotides, siRNA, and other therapeutic biopolymers. Here, we describe a new type of biomaterial based on sugar alcohols: AqueaTether™ (AqT™) molecules. Various AqT™ linkers with different lengths and end groups were synthesized and their hydrophilic nature and ability to enhance the water solubility of hydrophobic compounds compared to different commercial and PEG linkers. Various antibody drug conjugates (ADCs) with water soluble AqT™-drugs were prepared. An ADC made with AqT™-monomethyl auristatin E (MMAE) with only two sugar alcohol units attached and an average drug-to-antibody ratio (DAR) of 4 demonstrated significant improvements in minimizing the aggregation of antibodies and long-term stability during storage. Due to increased water solubility, AqT™ linkers also allow higher loading of drugs per antibody, enabling the use of less toxic drugs, potentially increasing the overall efficacy and safety profile of an ADC. For example, ADCs with average DARs of 8 and 15 have been made using AqT™-doxorubicin with only two sugar alcohol units attached. In peptide-based drug discovery, discrete high MW AqT™ linker is used to label peptide, resulting in an increased half-life in human serum. In summary, AqT™ molecules are by far the most hydrophilic and versatile linkers currently in development. AqT™ molecules represent a great tool for drug conjugation and delivery.

#756

Development of next-generation antibody-drug conjugates for resistant HER2-positive tumors.

Vincent Lacasse,1 Jeffrey V. Leyton,1 Simon Beaudoin,1 Mark Barok,2 Heikki Joensuu2. 1 _Université de Sherbrooke, Sherbrooke, Quebec, Canada;_ 2 _University of Helsinki, Helsinki, Finland_.

Antibody-Drug Conjugates (ADC) represent a promising therapeutic modality for improving the clinical management of cancer. However, there now exists resistant mechanisms by cancer cells that are effective at ultimately reducing the cellular accumulation of the delivered drug and hence, reducing cytotoxic effectiveness. Kadcyla® (trastuzumab-emtansine [T-DM1]) is a benchmark as it is the first solid-tumor approved ADC. Despite this new therapeutic option, approximately 43% of patients achieve complete or partial response and most who initially respond develop resistance soon after. In order to transform current ADCs to more effective products improved intracellular delivery strategies could provide solutions. Our group developed is a natural composite compound (termed Accum) that conjugates to surface lysines and enables antibodies to escape endosomal entrapment followed by active routing to the nucleus. Modified antibodies retain high tumor cell selectivity, enhanced cellular accumulation, and improved tumor delivery of molecular payloads in vivo. We asked whether the modification of Accum-modified T-DM1 maintained its intracellular delivery mechanism and cytotoxic effectiveness in T-DM1-resistant cells. The cancer cell lines N87, OE19 (gastric), JIMT1, SK-BR-3 (breast), SKOV3 and SKOV3.IP1 (ovarian) were treated with increasing concentrations of T-DM1 in a 3 day on, 10 days off fashion for a period of 1 to 2 months. Resistant clones were identified. Parental and resistant cells were placed and passaged in SILAC (Stably Labeled Amino acid for Cell culture) media for proteomic analysis by liquid chromatography tandem mass spectrometry (LC-MS/MS). Cells were also treated with Accum-T-DM1 and mechanistic underpinnings determined. The IC50 of Accum-T-DM1 was increased 50- and 10-fold relative to T-DM1 in parental OE19, and JIMT1 cells, respectively. T-DM1 resistant cell lines were successfully generated for each tumor type. Typically, the T-DM1 IC50 values were increased ≥100-fold in resistant cells compared to parental cells. Preliminary proteomic studies revealed HER2 expression is marginally reduced in resistant cells. IP of Accum-T-DM1 co-precipitated showed the interaction with α-importin. This study is a work in progress of modifying T-DM1 with Accum and demonstrating enhanced cytotoxicity in T-DM1 resistant HER2-positive tumor cells. Ongoing studies will determine DM1 accumulation levels, potential resistance mechanisms, and ultimately cytotoxic effectiveness.

#757

Novel warheads for targeted therapies of cancer: The concept and design of oxime-ether-based pro-PBDs.

Iontcho Vlahov, Albert Felten, Ning Zou, Kevin Wang, Hari K. Santhapuram, Paul Kleindl, Spencer Hahn, Jeremy Vaughn, Christopher Leamon. _Endocyte, W. Lafayette, IN_.

Pyrrolo[2,1-c][1,4]benzodiazepine (PBD) antibiotics are a class of natural products produced by various actinomycetes bacteria. PBD-dimers function as sequence-selective DNA-crosslinking, alkylating compounds which are considerably more potent than systemic chemotherapeutic agents. PBDs recognize and bond to specific sequences in the DNA minor groove. After DNA insertion, a covalent aminal bond is formed through nucleophilic attack of the N-2 of a guanine (G) base on the electrophilic C-11 imine of PBD. The end result is obstruction of tumor cell division without any significant distortion of the DNA's helical structure, thus potentially avoiding the common phenomenon of drug resistance.

In recent years, synthetic PBD-dimers have emerged as a promising class of payloads for conjugates in the field of targeted cancer therapies. Unfortunately, many PBD conjugates preserve the strongly alkylating (electrophilic C-11) imine moiety within their structural framework, which can react in a deleterious manner while in circulation on their way to their intended targets. As an elegant solution to this inherent problem, we designed diazepine-ring-opened conjugated prodrugs lacking the imine functionality. In our design concept, we replaced this moiety with its synthetic precursors: an aldehyde and an amine. The later provided an attachment point to targeting ligands through self-immolative linker systems, whereas the former was masked as an oxime ether. Such oxime ethers are known to exhibit enhanced stability under physiological conditions. Once the prodrug (pro-PBD) conjugate enters a targeted cell, cleavage of the linker system triggers the generation of an aromatic amine. As we discovered, an intramolecular ring-closure subsequently takes place as the aromatic amine adds directly into the sp2 center of the oxime ether to form the imine condensate and ultimately the 1,4-diazepine ring in a PBD framework.

In our pro-PBDs, we envision exploiting their aromatic amines as a part of an arsenal of self-immolative linker systems for small molecule conjugation. To prove the range of applications for this new class of latent DNA-alkylators, we modified their linkers to tailor the kinetics of prodrug release and drug formation. The utility of these novel warheads and linker systems towards the design of Small Molecule Drug Conjugates (SMDC) for targeted cancer therapies will also be discussed in the presented poster. One can predict that any of the presented pro-PBDs could be easily incorporated as novel payloads in Antibody-Drug Conjugates (ADCs).

#758

Head-to-head comparison of ADC and SMDC products, alone and in combination with antibody-IL2 fusion proteins.

Samuele Cazzamalli,1 Alberto Dal Corso,2 Fontaine Widmayer,2 Dario Neri2. 1 _Philochem AG, Otelfingen, Switzerland;_ 2 _Swiss Federal Institute of Technology, Zurich, Switzerland_.

In an attempt to improve the therapeutic index of cancer chemotherapy, monoclonal antibodies and small organic ligands have been proposed as delivery vehicles, allowing the construction of antibody-drug conjugates (ADCs) and of small molecule-drug conjugates (SMDCs). Four ADC products have gained marketing authorization for cancer therapy, while SMDCs are still being investigated in clinical trials. SMDCs could in principle offer certain advantages compared to ADCs, such as a more rapid and uniform diffusion into the tumor mass, lower cost-of-goods and lack of immunogenicity, but a direct head-to-head comparison has not yet been reported.

Here we describe a novel small molecule-drug conjugate, based on a high-affinity ligand specific to carbonic anhydrase IX. The product features a peptide linker, suitable for cleavage in the tumor extracellular environment, and monomethyl auristatin E as cytotoxic payload. We compared the therapeutic efficacy of the novel SMDC and of an anti-CAIX ADC, which were injected at equimolar doses in nude mice bearing SKRC-52 renal cell carcinoma xenografts. Both products showed a potent anti-cancer activity, although no complete tumor eradication was observed. Instead, when the SMDC was administered together with L19-IL2 (a clinical-stage fusion protein capable of delivering interleukin-2 to the tumor neo-vasculature), all treated mice in the combination group could be rendered tumor-free, in a process which favored the influx of natural killer cells into the tumor mass. The combination of L19-IL2 and the new small molecule-drug conjugate also eradicated cancer in 100% of immunocompetent mice, bearing subcutaneously-grafted CT26 colorectal cancer cells, which stably expressed carbonic anhydrase IX. These findings may be of clinical significance, since carbonic anhydrase IX is over-expressed in the majority of clear-cell renal cell carcinomas and in approximately 30% of colorectal cancers. The targeted delivery of interleukin-2 helps potentiate the action of targeted cytotoxics leading to cancer eradication in models that cannot be cured by conventional chemotherapy.

#759

Preclinical evaluation of a GFRA1 targeted antibody-drug conjugate in breast cancer.

Emily E. Bosco, Ronald J. Christie, Rosa Carrasco, Darrin Sabol, Jiping Zha, Karma DaCosta, Lee Brown, Sandrina Phipps, Qun Du, Binyam Bezabeh, Shannon Breen, Cui Chen, Molly Reed, MaryJane Hinrichs, Haihong Zhong, Zhan Xiao, Rakesh Dixit, Ronald Herbst, David A. Tice. _MedImmune, LLC, Gaithersburg, MD_.

Despite recent advances in treatment, breast cancer remains the second-most common cause of cancer death among American women. A greater understanding of the molecular characteristics of individual tumors could ultimately lead to improved treatment options, particularly for subsets of breast cancer patients with unmet needs. Using an unbiased genomics approach to identify membrane-localized tumor associated antigens (TAAs), we have identified a novel breast cancer TAA that can potentially be exploited through antibody-drug conjugate (ADC) therapy. Here, we describe the development of a GFRA1-targeted ADC that demonstrates activity in GFRA1-positive cell lines and PDX tumor models. Work in non-Good Laboratory Practice (GLP) rat toxicology models revealed acceptable toxicity profiles and supports further evaluation of GFRA1-PBD in GFRA1-positive tumors.

#760

**Conjugation of daratumumab with** 225 **actinium greatly increases its antitumor activity against multiple myeloma tumors.**

Wojciech Dawicki,1 Kevin Allen,1 Rubin Jiao,2 Mackenzie Malo,1 Keisha Thomas,3 Mark S. Berger,3 Ekaterina Dadachova1. 1 _University of Saskatchewan, Saskatchewan, Canada;_ 2 _Univeristy of Saskatchewan, Saskatchewan, Canada;_ 3 _Actinium Pharmaceuticals, Inc., New York, NY_.

Daratumumab is a human cytolytic antibody specific for CD38 that is used clinically for treatment of patients with multiple myeloma (MM). Current therapeutic regimens require multiple injections over months of treatment. Increasing the potency of daratumumab to shorten the length of treatment would be beneficial. 225Ac is an alpha-particle emitting radionuclide that has potent cytotoxic activities over short distances, allowing for precise targeting of a lethal dose of radiation. Previously we have established that labeling daratumumab with 225Ac increased more than 10-fold its ability to kill MM cell lines in vitro (ASH 2017, Poster Number 4427). In this study we evaluated the ability of 225Ac-daratumumab to kill established tumors in mice. Mice deficient in T- and B-cells were injected subcutaneously with human MM tumor cells and once tumors reached an average volume of ~200 mm3, mice were treated with the 225Ac-daratumumab. To determine the localization of daratumumab within the tumor-bearing mice it was labeled with 111In, an γ-emitting radioisotope. 111In-daratumumab can be easily imaged and is used as a surrogate to estimate the localization of 225Ac-daratumumab. The distribution of the 111In-daratumumab was then followed for 10 days using a microSPECT/CT scanner. To evaluate the antitumor ability of the 225Ac-daratumumab, tumor-bearing mice were injected with 225Ac-daratumumab at a dose of of 400 nCi/0.3 μg of antibody, and 200 nCi/0.3 μg of antibody. As a control, mice were injected with either saline or an equivalent amount of unlabeled daratumumab. In addition, a group of mice was also treated with 30 times greater dose of unlabeled daratumumab (10 μg) - a dose which was previously shown to be effective against established tumors. 111In-daratumumab begun to accumulate in the tumor 24 hours after intraperitoneal injection and by 7 days was exclusively present in the tumor. The growth of the tumors in mice treated with 400 nCi/0.3 μg was significantly retarded compared to mice treated with equal concentration of unlabeled daratumumab or saline. Tumor growth was similar between mice treated with 400 nCi/0.3 μg of 225Ac-daratumumab and mice treated with 10 μg of unlabeled daratumumab. In conclusion, this study shows that labeling daratumumab with 225Ac increases its antitumor activity ~30-fold. This study suggests that approach to increase the potency of daratumumab via 225Ac labeling could greatly reduce the amount of daratumumab treatment needed in the clinic. This study also highlights the potential of targeting α-emitters to tumors as a viable therapeutic approach.

### Biomarker Identification and Novel Methods

#761

The role of PI3 kinase pathway in the the skin of Sezary syndrome.

Cristina Cristofoletti,1 Antonella Bresin,1 Mario Picozza,2 Maria Cristina Picchio,1 Francesca Passarelli,1 Francesca Monzo,1 Mauro Helmer Citterich,1 Alessandro Monopoli,1 Roberto Benucci,1 Maria Cantonetti,3 Alessandra Frezzolini,1 Enrico Scala,1 Elisabetta Caprini,1 Giandomenico Russo,1 Maria Grazia Narducci1. 1 _Ist. Dermopatico dell'Immacolata-IRCCS, Rome, Italy;_ 2 _Fondazione Santa Lucia-IRCCS, Rome, Italy;_ 3 _'Università degli Studi di Roma "TOR VERGATA", Rome, Italy_.

Sezary Syndrome (SS) is an aggressive variant of cutaneous T cell lymphoma (CTCL) characterized by an accumulation of malignant T lymphocytes - the Sezary (SS) cells - in the skin, lymph nodes and blood. Until now, most studies on the characterization of this lymphoma have been performed on circulating SS cells because of the easy availability of tumor material from this compartment. For this reason, the role of skin microenvironment has not been characterized although it might play a fundamental role in the pathogenesis and in the disease progression of this neoplasia. According to this view, we recently demonstrated that PTEN downregulation, due to genetic loss and microRNA deregulation, commonly observed in SS patients, mainly activates AKT in skin-resident respect to blood-derived SS cells. This data underline how microenvironment signals amplify genetic abnormalities. Based on these observations, we investigated the effect of the skin microenvironment on SS cells in vivo. Comparing skin and blood-derived SS cells concurrently obtained from SS patients, we demonstrate that skin milieu promotes proliferation and PI3K/AKT/mTORC1 activation, a pathway that we prove to have multiple copy number (CN) alterations in our cohort of forty-three patients, thus indicating these genetic lesions as valid therapeutic targets to treat SS. At the end, we also demonstrate that the progressive increase of these CN alterations is associated to the shortening of overall survival of SS patients.

#762

Disrupting the glucose-influx induced β-catenin activation for the treatment of hepatocellular carcinoma.

Man-Hsin Hung,1 Li-Ju Chen,2 Pei-Yi Chu,3 Yao-Li Chen,4 Ming-Hsien Tsai,2 Feng-Shu Hsieh,2 Kuen-Feng Chen2. 1 _Taipei Veterans General Hospital, Taipei, Taiwan;_ 2 _National Taiwan University Hospital, Taipei, Taiwan;_ 3 _Show Chwan Memorial Hospital, Taiwan;_ 4 _Changhua Christian Hospital, Taiwan_.

Background: Reprogramming of glucose metabolism is a critical hallmark in hepatocellular carcinoma (HCC), and alteration of WNT/β-catenin signaling is one of the most common molecular alterations observed in patients with HCC. Interestingly, some studies reported that high glucose exposure leaded to activation of WNT/β-catenin signaling. But, for HCC, whether the accelerated glucose metabolism affects β-catenin, and whether this singling pathway reserves potential for the development of novel anti-cancer treatment have not yet been explored.

Method:

HCC cells were exposed to different glucose conditions and various glucose transporter inhibitors, including Phloretin, WZB117, Canagliflozin, Dapagliflozin and Empagliflozin. Cells were examined for cell viability and molecular signaling after treatments. Huh7 xenografted tumor model was used for in vivo testing. Clinical HCC tumor tissues were examined by immunohistochemical stain.

Result:

HCC cells exposed to high glucose environment showed higher proliferation rate and upregulated β-catenin as comparing to those cultured in lower glucose-median. Notably, a similar association was observed in the clinical samples; HCC patients with higher glycemic level had stronger expressions of β-catenin in their tumors (p=0.034). Above data suggested that high glucose condition induced activation of β-catenin. Next, we asked whether blocking glucose influx attenuates this glucose-influx-mediated β-catenin activation and inhibits HCC cell growth. By treating HCC cells with various glucose transporter inhibitors, we found that canagliflozin, Phloretin and WZB117 attenuated

glucose influx of HCC cells, but only canagliflozin showed potent growth inhibition against HCC. Furthermore, treatment of canagliflozin leaded to a dose-dependent downregulation of β-catenin in HCC cells. Using cycloheximide and MG-132, we proved that canagliflozin treatment promoted the proteasome-mediated degradation of β-catenin protein. Since the phosphorylation of β-catenin is the initial step for its degradation, we examined the expression of p-β-catenin and found that canagliflozin treatment increased the expressions of p-Ser33/Ser37/Thr41-β-catenin and p-Ser45-β-catenin. Furthermore, the activity of protein phosphatase 2A (PP2A) was decreased in canagliflozin-treated HCC cells. The roles of glucose-influx and PP2A/p-β-catenin mediating the anti-HCC effects of canagliflozin were validated. Moreover, in vivo tumor growth inhibition of canagliflozin treatment was shown.

Conclusion:

Our results showed that high glucose upregulated beta-catenin signaling in HCC. Canagliflozin, by direct promoting the degradation of β-catenin protein and attenuating glucose-influx, inhibits the glucose-influx-mediated β-catenin activation and produces anti-HCC effects in vitro and in vivo.

#763

PLAC1 interacts with FGF7 and promotes phosphorylation of FGFR2 and AKT.

Diana Barea Roldan,1 Christoph Hartmann,2 Stefanie Hubich-Rau,1 Tim Beissert,1 Claudia Paret,3 Giuseppe Cagna,4 Christoph Rohde,4 Stefan Wöll,4 Ugur Sahin,1 Özlem Türeci5. 1 _TRON-Translational Oncology at the University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany, Mainz, Germany;_ 2 _Formerly of TRON – Translational Oncology at the University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany;_ 3 _University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany;_ 4 _Formerly of Ganymed Pharmaceuticals GmbH, Mainz, Germany;_ 5 _CI3 – Cluster of Individualized Immune Intervention, Mainz, Germany_.

Background PLAC1 is a protein predominantly expressed in the placenta with no substantial presence in other human tissues; however, PLAC1 is aberrantly expressed in various human cancers (eg, breast cancer), and is functionally involved in the motility, migration, and invasion of cancer cells. While these functions have been linked to the PI3K/AKT pathway, the role of PLAC1 in this signaling pathway is unclear. AKT mediates cell proliferation induced by fibroblast growth factor 7 (FGF7), a specific mitogen for epithelial cells which has a significant expression in estradiol receptor-positive breast cancer. The objectives of these nonclinical studies were to investigate if/how PLAC1 is linked to the FGF7/fibroblast growth factor receptor 2IIIb (FGF2IIIb) axis, evaluate the possible role of PLAC1 in PLAC1-expressing tumor cells, and to assess the potential of PLAC1 as a therapeutic target.

Methods PLAC1 protein expression and localization, as well as its interactions with FGF7 and FGFR2, were studied in human cancer cell lines by immunohistochemistry, Western blotting, and co-immunoprecipitation. To compare cell proliferation and protein phosphorylation in PLAC1-expressing versus non-expressing cells, PLAC1 expression was knocked down in the choriocarcinoma cell line, BeWo, as well as the breast cancer cell lines, SkBr3 and T47D.

Results Consistent with a previous report by Massabbal et al (2005), PLAC1 was found to be coexpressed with FGF7 and FGFR2 in placenta. Increased PLAC1 expression was noted in cell lines of trophoblastic, breast, and pancreatic lineage compared with adenocarcinoma cell lines. PLAC1 localized on the cell surface and was released into the extracellular matrix where it exhibited a strong affinity for heparin, which binds to FGFs and FGF receptors facilitating FGF-receptor binding and activation of the FGFR tyrosine kinase. In cultured cells, PLAC1, FGF7, and the FGFR2 isoform FGFR2IIIb formed a trimeric complex; only FGF7, but not other FGFs, was able to interact with PLAC1, suggesting a highly specific interaction. PLAC1 knockdown models showed substantially reduced cell proliferation and phosphorylation of AKT and FGFR2 after stimulation with FGF7 compared with control cells, indicating the involvement of PLAC1 in the FGF7-induced AKT signal transduction pathway that leads to cell proliferation.

Conclusions PLAC1 may play a role in tumorigenesis through the FGF7-induced pathway of cell proliferation by promoting phosphorylation of FGFR2 and AKT through interactions with FGF7, FGFR2, and heparin. Because of this function and its cancer-selective, cell-surface expression in adult human tissue, PLAC1 may be an attractive target candidate for therapeutic anticancer antibodies.

#764

GSTP1 knockdown and inhibition impairs pancreatic ductal adenocarcinoma (PDAC) growth.

Rahul Raj Singh, Katie M. Reindl. _North Dakota State University, Fargo, ND_.

Pancreatic ductal adenocarcinoma (PDAC) is the third-leading cause of cancer-related mortalities in the Western world and continues to be a major unresolvable health problem at the start of the 21st century. Resistance to the currently available treatment options has led to development of new approaches, such as personalized medicine and immunotherapy. However, new therapeutic strategies based on the unique molecular biology and physiology of pancreatic cancer hold the greatest promise. Glutathione S-transferase pi 1 (GSTP1) is a key detoxification enzyme which metabolizes xenobiotic compounds and byproducts of cellular metabolism. GSTP1 is overexpressed in many tumors, particularly ovarian, non-small cell lung, breast, colon, and pancreas. Moreover, GSTP1 is overexpressed in drug-resistant cancer cell lines. The reasons for increased expression ratios compared to normal tissues or wild-type cell lines are not well understood.

To investigate the role of GSTP1 in PDAC pathogenicity, we generated two knockdown lines of GSTP1 in metabolically diverse PDAC cells. We showed that GSTP1 knockdown impairs the growth and proliferation of PDAC cells. Additionally, GSTP1 knockdown cells exhibit elevated reactive oxygen species (ROS) levels and a prolonged G0/G1 phase of the cell cycle. Intrigued by these results, we next examined whether pharmacological inhibition with a selective GSTP1 inhibitor, Ezatiostat (TLK199), also impaired PDAC pathogenicity. Ezatiostat is a small molecule drug and is novel glutathione analog, which selectively binds and inhibits GSTP1. Ezatiostat treatment in PDAC cells recapitulated the proliferation impairments observed with genetic inactivation of GSTP1 and elevated ROS levels. Orthotopic implantation of GSTP1 knockdown cells in athymic nude mice resulted in reduced tumor weight and volume compared to the control. The growth trajectory of the tumors was monitored via Vevo-3100 ultrasound imaging system. Our preliminary data indicate enhanced sensitivity of glycolytic cancer cells towards an ROS-inducing agent and a GSTP1 inhibitor, piperlongumine (PL). Interestingly, we have found PL is less cytotoxic to cells with reduced GSTP1 levels, indicating that PL primarily works by inhibiting GSTP1 activity. Together, these data suggest that GSTP1 knockdown and inhibition impairs the growth and survival of phenotypically diverse PDAC cells in vitro and in vivo. Moreover, GSTP1 knockdown results in elevated ROS levels and an extended G0/G1 phase of the cell cycle. With these data, we propose that GSTP1 is a novel therapeutic target for PDAC.

#765

Multiple signalling pathways are active in BRAF/NRAS wild type melanomas.

Zizhen Ming, Su Yin Lim, Richard F. Kefford, Helen Rizos. _Macquarie University, Sydney, Australia_.

In the last five years, the treatment of advanced melanoma has been revolutionised by the introduction of selective kinase inhibitors and immunotherapy. Selective inhibition of the mitogen activated protein kinase (MAPK) pathway has significantly improved the survival of patients with BRAFV600-mutant melanoma, and combination MEK and CDK4 inhibition is currently being trialed in patients with NRAS-mutant melanoma.

Almost a quarter of all melanomas, however, are wild type for BRAF and NRAS and have no known actionable mutations. Currently, patients with wild type melanoma are treated with immunotherapies, which produce prolonged responses, but only in a small subset of patients. Overall, the prognosis for patients with wild type melanomas remains poor, and there is an urgent need to establish better therapeutic strategies for these patients.

In order to define new therapeutic targets in BRAF/NRAS wild type melanoma, we sought to characterize the key proliferative and survival pathways in wild type melanomas. Wild type (n=14) and BRAFV600 (n=7) melanomas were treated with the MEK inhibitor trametinib at increasing doses from 0.5nM to 5000nM for 72h and sensitivity to MEK inhibition was assessed using viability assays. Signaling pathway activity in response to trametinib (10nM, 24h) was also examined in our panel of melanoma cells using the Human Phospho-Kinase Antibody Array (R&D Systems), which detects 43 different phosphorylated kinases.

As expected, BRAFV600 melanomas were exquisitely sensitive to trametinib treatment (IC50=0.8-2.4nM). We also found that although a subset of BRAF/NRAS wild type melanomas (5/14; 36%) were sensitive to trametinib (IC50 0.5-4.9nM), the majority of these melanoma cells (9/14; 64%) were resistant to trametinib (IC50 10.5-264.8nM).

Analysis of kinase activation status revealed little difference in signaling pathway activity between the sensitive and resistant wild type melanomas at baseline. Resistant wild type melanomas had higher activity of STAT3 compared to the sensitive cell lines. However, all these wild type melanomas showed variable activation of multiple signaling pathways that were not active in BRAFV600 melanomas. Specifically, at baseline, wild type melanomas showed pronounced activation of the JNK and p38 MAPK pathways, the FAK/Src pathway (i.e. FAK, Lck, Lyn, Fgr and Yes), the epidermal growth factor receptor tyrosine kinase and

β-catenin compared to BRAFV600 melanomas.

Activation of multiple signaling pathways in wild type melanomas may contribute to innate resistance or limit the efficacy of MEK inhibition, and we are currently exploring the impact of these pathways on the survival and MEK dependence of wild type melanomas.

#766

Refractory and relapsed predictive biomarkers in Hodgkin lymphoma and potential directed-therapy targets.

Angélica M. Gamboa-Cedeño,1 Victoria Otero,2 Natalia Schutz,2 Dorotea Fantl,2 Hernán Rivello,2 Federico Jauk-Vitali,2 Myriam Nuñez,3 Stella Maris Ranuncolo1. 1 _Instituto Universitario Hospital Italiano (IUHI), Buenos Aires, Argentina;_ 2 _Hospital Italiano de Buenos Aires (HIBA), Buenos Aires, Argentina;_ 3 _Facultad de Farmacia y Bioquímica (FFyB), Buenos Aires, Argentina_.

Despite 90% stage I Hodgkin Lymphoma (HL) patients can respond to current therapy, this drops to 60% when diagnosed in late stages. Nevertheless and independently of the lymphoma stage, the real challenge when treating these patients, is the refractory and relapsed disease. There is no biomarker to identify patients that would be non-responsive to conventional treatment or that would relapse. Furthermore, rescue chemotherapy schemes for refractory and relapsed patients, associate with acute and late toxicity high risk. This highlights the need to deeper understand the HL molecular biology and the screening for predictive biomarkers as well as potential therapeutic directed-targets. We have previously reported that HL relies on the alternative NFkB pathway, mediated by relB and NIK, to survive. Depletion of either RelB or NIK by shRNAs or pharmacological NIK inhibitors induce HL cell death. ChIP-Seq analysis uncovered relB target genes showing relB bound to BCL2 promoter. A significant downregulation of BCL2 mRNA and bcl2 protein levels, following relB or NIK knockdown was observed, indicating relB regulation. Our molecular studies suggested that NFkB alternative pathway constitutive signaling could at least partially explain the non-responding HL cases. We aimed to analyze whether mediators of this pathway could be useful as predictive biomarkers and would represent potential targetable factors in both refractory and relapsed patients. We analyzed NIK and bcl2 citoplasm expression in Hodgkin Reed-Sternberg cells (HRS) in lymphatic node biopsies of 96 patients by inmunohistochemistry [50 female Md age and (range) 59 (6-82), 46 male 42 (9-78)]. The univariate analysis showed no correlation between NIK or bcl2 expression and the prognosis clinical and pathological parameters, neither the molecular markers routinely assayed. A positive correlation was found between NIK and bcl2 expression (p=0.01). NIK and bcl2 correlated with lack of response to conventional therapy and both early and late disease progression. The analysis of survival, applying the Kaplan-Meier Curves, showed > 60% NIK positive HRS cells associated with shorter Disease Free Survival (DFS) [Log Rank Test (p=0.000)] and predicted overall survival (OS) as well [Log Rank Test (p=0.01)]. Furthermore, > 60% bcl2 positive HRS cells correlated with poor prognosis in terms of OS [Log Rank Test (p=0.002)]. The statistical significance was maintained in the multivariate analysis [Cox Regression and Logistic Regression (p=0.001)]. NIK and bcl2 performed successfully as useful predictive markers to identify refractory or risk of relapse HL patients at diagnosis. They represent attractive molecules to further analyse their potential as directed-therapy targets, since we have already reported that HL is sensitive to NIK inhibitors and bcl2 blockers have already been approved for clinical use in other hematological pathologies.

#767

Identification of cell surface proteins expressed exclusively on mutant KRas-dependent cancer cells.

Vijayendra Agrawal, Bin Fang, Eric A. Welsh, John Koomen, Said M. Sebti. _Moffitt Cancer Center, Tampa, FL_.

Mutant KRas is a major contributor to human oncogenesis and confers resistance to therapy. Identification of cell surface proteins that are expressed exclusively in mutant KRas-driven tumors could lead to novel approaches for diagnosing and treating mutant KRas-dependent tumors. To this end, we used a biotinylation/mass spectrometry approach to capture cell surface proteins from mutant KRas-dependent and -independent human cancer cells. KRas was depleted from both mutant KRas-dependent and -independent cells, and the cells were subjected to cell surface protein biotinylation using EZ-Link Sulfo-NHS-SS-Biotin reagent and captured via neutravidin column. The efficiency of cell surface protein capture was confirmed by the presence of membrane proteins EGFR, IGF1R and absence of cytosolic proteins RhoGDIa in biotin-pull down fractions (western blot analysis). Mass spectrometric and proteomics analyses of biotinylated proteins yielded 20 highly probable hits with very high expression in mutant KRas-dependent cells and very low expression in mutant KRas-independent cells. Upon KRas depletion, the level of these proteins was significantly reduced in mutant KRas-dependent cells (up to 25 fold) but not in mutant KRas-independent cells. Currently, we are analyzing these 20 hits to determine if any of these are required for the survival of mutant KRas-dependent cells. Results from these studies have the potential to lead to novel approaches for diagnosing and treating mutant KRas-driven cancers.

#768

Identification of signal transduction pathways specific for mutant KRas-dependent tumors.

Oorvashi Roy Puli,1 Hua Yang,1 Bin Fang,1 Hong Yuan (Rays) Jiang,2 John Koomen,1 Said M. Sebti1. 1 _Moffitt Cancer Center, Tampa, FL;_ 2 _University of South Florida, Tampa, FL_.

Targeting KRas directly has proven difficult. Therefore, identifying vulnerabilities specific for mutant KRas tumors is an important alternative approach to target mutant KRas-driven tumors. Towards this goal, we used phosphoproteomics to dissect key differences in signaling pathways between mutant KRas-dependent and mutant KRas-independent human cancer cells. Mass spectrometry analysis led to the identification of 25 proteins with significantly higher (up to 410 fold) phosphorylation levels in mutant KRas-dependent cells. Among these, 25 proteins- 8, 8 and 3 were tyrosine (Y), Serine (S) and Threonine (T) phosphorylated, and 6 were phosphorylated on both S and T. Presently, CRISPR-Cas9 knockout and siRNA depletion studies are ongoing to identify which of these 25 hits are required for the survival of mutant KRas-dependent human cancer cells. These studies can lead to identifying signaling circuits that can be targeted to treat mutant KRas-driven cancers.

#769

Zeb-1 contributes to the development of chemotherapeutic resistance in mantle cell lymphoma.

Sudjit Luanpitpong,1 Jirarat Poohadsuan,1 Montira Janan,1 Kanjana Thumanu,2 Yon Rojanasakul,3 Surapol Issaragrisil1. 1 _Mahidol University, Bangkok, Thailand;_ 2 _Synchrotron Light Research Institute, Nakhon Ratchasima, Thailand;_ 3 _West Virginia University, Morgantown, WV_.

Despite progress overall in improving cancer treatments, the complete response of mantle cell lymphoma (MCL) is still limited due to the inevitable development of drug resistance. Understanding how MCL cells acquire resistance at the molecular level may be a key innovation to targeted therapy. We established a series of bortezomib (BTZ)-resistant MCL cells and performed gene/protein profiling along with metabolic fingerprinting using synchrotron-based Fourier-transform infrared spectroscopy. Zeb-1, an epithelial-mesenchymal transition-inducing transcription factor better known for promoting metastasis in solid carcinomas, was found to be aberrantly expressed in the resistant MCL cells in accordance with an excessive production of lipids. Ectopic expression of Zeb-1 in parental MCL cells rendered the cells to acquire apoptosis resistance to BTZ. Here we demonstrated, for the first time, two possible encompassing mechanisms for Zeb-1-mediated BTZ resistance: (i) Zeb-1 induces cancer stem cells, as evaluated by tumor spheres and aldehyde dehydrogenase activity; and (ii) Zeb-1 induces lipid reprograming that may interfere with cell growth and drug response. Together, our results unveil a novel role of Zeb-1 in MCL aggressiveness. These findings have a clinical significance insight to the development of chemotherapeutic resistance of MCL.

#770

Identification of MASTL as novel mitotic vulnerability of thyroid tumor cells.

Elena Cetti,1 Maria Chiara Anania,1 Katia Todoerti,2 Giuseppe Mauro,1 Antonino Neri,3 Angela Greco1. 1 _Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy;_ 2 _IRCCS-CROB, Referral Cancer Center of Basilicata, Rionero in Vulture, Italy;_ 3 _Fondazione IRCCS Ca' Granda, Ospedale Maggiore Policlinico and Department of Oncology and Hemato-oncology, University of Milan, Milan, Italy_.

Background: Despite thyroid tumors are generally curable, a fraction of patients develops resistance to therapy and tumors progress towards undifferentiated forms, which have a poor prognosis and whose treatment is still a demanding challenge. To identify potential novel targets for thyroid cancer treatment, especially for those aggressive forms, we faced the non-oncogene addiction paradigm, which states that tumor cells become addicted to stress support pathway genes, not required to the same degree by normal ones. To this aim, in a previous study we screened a siRNA library on normal and tumor thyroid cell lines and found several genes essential for the growth of tumor but not normal cells; among the top-ranking hits we found MASTL, a serine/threonine kinase involved in mitosis regulation. Here, we investigated the mechanisms of growth inhibition mediated by MASTL silencing in thyroid tumor cells, in order to identify whether it could represent a potential target for tumor treatment.

Methods: MASTL gene expression profile was assessed in 58 normal thyroid tissue samples and 72 papillary (PTC), 17 poorly differentiated (PDTC) and 31 anaplastic (ATC) thyroid carcinoma samples, derived from a meta-analysis of different publicly available datasets. HTC/C3 and 8505C cell lines (ATC-derived) were transfected with MASTL and control siRNAs and tested for cell growth, nuclear aberrations, DNA damage and cell death.

Results: By gene expression data analysis, we found that MASTL expression levels in PTC and PDTC samples were similar to those observed in control group, while the highest levels were evidenced in ATC. We next tested by different growth assays the effects of MASTL depletion on HTC/C3 and 8505C cells and found that MASTL silencing impaired their proliferation capacity. Notably, MASTL depletion increased the percentage of cells presenting nuclear anomalies, such as micronuclei, multiple nuclei and lobular nuclei, which are indicative of mitotic catastrophe. We observed that MASTL depletion was associated with increased phosphorylation of CDK1 (Y15), supportive of impairment of cell growth, and of H2AX (Ser139), indicative of increase of DNA damage. Furthermore, MASTL depletion was associated with increased expression of the apoptotic markers cleaved caspase 3 and cleaved PARP, thus suggesting that MASTL inhibition may cause thyroid tumor cell death through apoptosis. These finding was further corroborated by Annexin V assay, which showed an increase of apoptotic and necrotic cells upon MASTL silencing, compared to control.

Conclusions: Our results demonstrated that MASTL inhibition is able to impair the growth of thyroid tumor cell lines and is associated with mitotic catastrophe and tumor cell death, thus suggesting that it may represent a valuable candidate for validation as therapeutic target in thyroid cancer.

#771

HPRT: A biomarker and potential target for detection and treatment of colorectal cancer.

Michelle H. Townsend, Eric C. Olsen, Evita G. Weagel, Edwin J. Velasquez, Abigail M. Felsted, Weston Burrup, K Scott Weber, Richard Robison, Kim L. O'Neill. _Brigham Young University, Provo, UT_.

This study aims to evaluate the expression of the salvage pathway enzymes DCK, APRT, and HPRT in colorectal cancer cells to investigate their potential as biomarkers for diagnosis and treatment. As the third most common cancer diagnosed in the United States, there remains a need to identify biomarkers capable of detecting and characterizing colorectal cancers. Because of their involvement in nucleotide synthesis and their role in cell cycle regulation and proliferation, we hypothesized an increase in these enzymes within malignancy. In order to quantify expression, we utilized two colorectal cancer cell lines (HT29 and SW620) as well as healthy and malignant patient tissue samples. Expression in tissues was evaluated using immunohistochemistry (IHC), and surface presence of the enzymes was assessed via confocal microscopy, flow cytometry, and scanning electron microscopy. Staining of malignant tissue samples using IHC showed upregulation of HPRT in 59% of patients when compared to control tissues derived from healthy patients (p=0.001). Surface analysis was performed utilizing flow cytometry, confocal microscopy, and scanning electron microscopy. Upon flow cytometry evaluation no significant presence of either DCK or APRT was found on the membranes of SW620 and HT29 cells (p=0.234 and 0.93, respectively), while HPRT expression was significantly increased on the surface of both SW620 and HT29 cells (p=0.013). Cell population fluorescence increased 58% and 28% in SW620 and HT29 cells, respectively, compared to controls. Confocal microscopy images showed direct overlap between SW620 cells stained with membrane-specific dye and anti-HPRT antibody, suggesting localization on the membrane. To further confirm the surface presence of HPRT, cells were treated with gold-labelled HPRT antibody and visualized under a scanning electron microscope to determine if surface expression was randomly dispersed across the cell surface. When treated with anti-HPRT antibodies, there was an increase in gold weight percentage on both SW620 (p=8.14x10 -6) and HT29 (p=1.74x10 -4) cells compared to controls, confirming HPRT presence and showing random surface localization patterns. Surface analysis was also conducted on malignant samples of patients with colorectal cancer. Of three samples, one of them exhibited significant HPRT surface localization. This supports the variation found within the tissues, as HPRT overexpression is not found in every patient. One patient did exhibit significant levels of HPRT on the surface of their malignant cells; this indicates HPRT as a potential target in patients that exhibit upregulation of the enzyme. These results suggest a relationship between colorectal cancers and HPRT, and indicate HPRT as a potential target for the detection and treatment of colorectal cancers.

#772

High MDC1 expression in cervical cancer cells can affect the chemo- and radiotherapeutic response as its depletion leads to increased cell death.

Neeru Singh, Rashmi Bhakuni, Sivapriya Kirubakaran. _Indian Inst. of Technology-Gandhinagar, Gandhinagar, India_.

Cervical cancer is the third most frequent cancer and common cause of death in women worldwide. The work presented identifies mediator of DNA damage checkpoint 1 (MDC1) as an important molecular target to increase sensitivity of cervical cancer cells to chemo or radiotherapy. MDC1 functions as an adaptor protein for recruitment and retention of many other DNA damage repair proteins in ataxia telangiectasia mutated (ATM) pathway for double-stranded DNA damage repair. It is reported to be highly expressed in cervical cancer cells. Also, its expression tends to increase with increase in malignancy. We have studied in detail MDC1 mRNA expression in three cervical cancer cell lines, HeLa, SiHa and CasKi, in response to various genotoxic stresses including some known inhibitors, UV exposure or gamma irradiation through quantitative PCR. The cellular response to the DNA damage resulted in increase in MDC1 expression, which declined with increase in treatment time period. Protein expression and activation by Western blotting with anti-MDC1 and anti-phosphoMDC1 antibody indicated a higher level of phosphorylated as compared to unphosphorylated MDC1. The significance of this increase in MDC1 expression was studied by generating stable cell lines knocked down for MDC1 expression. The modified cell lines were assessed for apoptosis through various assays, including flow cytometry, and showed greater cell death in response to DNA damage. In summary, high MDC1 expression can significantly affect chemo or radiotherapeutic response and its inhibition can improve sensitivity to these treatments.

#773

A heterochromatin gene signature unveils HP1α mediating neuroendocrine prostate cancer development and aggressiveness.

Xinpei Ci,1 Jun Hao,1 Xin Dong,1 Stephen Y. Choi,1 Hui Xue,1 Sifeng Qu,1 Ladan Fazli,1 Francesco Crea,2 Christopher Ong,1 Amina Zoubeidi,1 Housheng H. He,3 Martin E. Gleave,1 Colin C. Collins,1 Dong Lin,1 Yuzhuo Wang1. 1 _University of British Columbia, Vancouver, British Columbia, Canada;_ 2 _The Open University, Milton Keynes, United Kingdom;_ 3 _University of Toronto, Toronto, Ontario, Canada_.

Neuroendocrine prostate cancer (NEPC) is a lethal subtype of prostate cancer (PCa) arising mostly from adenocarcinoma via NE transdifferentiation following androgen deprivation therapy. However, mechanisms contributing to both NEPC development and its aggressiveness remain elusive. In light of the fact that hyperchromatic nuclei is a distinguishing histopathological feature of NEPC, we identified 36 heterochromatin-related genes that are significantly enriched in NEPC by transcriptomic analyses of our patient-derived xenograft (PDX) models, multiple clinical cohorts, and genetically engineered mouse models. Longitudinal analysis using our unique, first-in-field PDX model of adenocarcinoma-to-NEPC transdifferentiation revealed that, among those 36 heterochromatin-related genes, heterochromatin protein 1α (HP1α) expression is increased early and steadily during NEPC development and remain elevated in the developed NEPC tumor. Its elevated expression is further confirmed in multiple PDX and clinical NEPC samples. Functional studies showed that HP1α knockdown in the NCI-H660 NEPC cell line dramatically inhibits proliferation, completely ablates colony formation, and induces apoptotic cell death, ultimately leading to tumor growth arrest. Its ectopic expression significantly promotes NE transdifferentiation in adenocarcinoma cells subjected to androgen deprivation treatment. Mechanistically, HP1α reduces expression of androgen receptor (AR) and RE1 silencing transcription factor (REST), two crucial transcription factors silenced in NEPC, and enriches the repressive histone mark trimethylation of histone H3 at Lys9 (H3K9me3) on their respective gene promoters. These observations indicate a novel mechanism underlying NEPC development mediated by abnormally expressed heterochromatin genes, with HP1α as an early functional mediator and a potential therapeutic target for NEPC prevention and/or management.

#774

Anti-Müllerian hormone type II receptor (AMHRII) found expressed in human non-gynecological solid tumors, suggesting potential broader applications for anti-AMHRII-based therapy.

Jean-Marc M. Barret,1 Didier Meseure,2 Guillaume Bataillon,2 Noora Andersson,3 Aurélie Auguste,4 Olivier Dubreuil,1 Anniina Färkkilä,3 Emeline Perrial,5 Celine Bossard,6 Emily Loison,7 Anne Jarry,8 André Nicolas,2 Fanny Lemée,1 Solenne Gaillard,1 Mehdi Lahmar,1 Mikko Anttonen,3 Gabriel Champenois,2 Charles Dumontet,5 Isabelle Ray-Coquard,9 Alexandra Leary,4 Isabelle Tabah-Fisch,1 Anne Vincent-Salomon,2 Jean-François F. Prost1. 1 _GamaMabs Pharma, Toulouse, France;_ 2 _Curie Institute, Paris, France;_ 3 _Helsinki University Hospital, Helsinki, Finland;_ 4 _Gustave Roussy Cancer Centre, Villejuif, France;_ 5 _Cancer Research Center Lyon, Lyon, France;_ 6 _Nantes University Hospital, Hôtel-Dieu, Nantes, France;_ 7 _Hospices Civils Lyon, Lyon, France;_ 8 _INSERM U1232, Nantes, France;_ 9 _Léon Bérard Cancer Centre, Lyon, France_.

The anti-Müllerian hormone (AMH) belongs to the TGF-β family and plays a key role during fetal sexual development. It has been shown that AMH inhibits proliferation and induces apoptosis of AMH type II receptor (AMHRII) -positive tumor cells, especially progenitor cancer cells. Moreover, various reports have described the expression of AMHRII in human gynecologic cancers including ovarian tumors. Based on qRT-PCR test results, which were confirmed by a specific In-Situ Hybridization (ISH) assay, AMHRII mRNA could be detected only in normal ovary, testis and adrenal gland tissue, and at lower levels in pancreas and stomach. However, when a tissue microarray of solid tumor samples was tested with ISH assay, AMHRII mRNA was surprisingly detected in several primary cancer tissues including melanoma, lung, head and neck, colorectal and breast cancers. For example, a large ISH study involving 58 colorectal cancer samples demonstrated AMHRII expression in 55% of cases. AMHRII protein expression was confirmed by ImmunoHistoChemistry (IHC) in gynecological cancer samples including 179 out of 210 (85%) Granulosa Cell Tumor samples and 55 out of 80 (69 %) Epithelial Ovarian Cancers. In patients with gynecological cancers pre-screened by IHC for inclusion in a Phase Ia/Ib clinical trial of GM102, an anti-AMHRII monoclonal antibody, AMHRII positive membrane expression was detected in 116 out of 166 (70%) archived tumor tissues. Using the same IHC protocol, over 1000 frozen samples covering 16 different cancer types were tested, which demonstrated AMHRII expression in over 50% of hepatocarcinoma, colorectal, lung and renal cancer samples. No AMHRII expression was observed in 75 neuroendocrine lung tumor samples nor in 18 non-Hodgkin lymphoma samples tested. On a small panel of frozen ovarian (11) and colorectal (9) cancer samples using an immunofluorescence assay with an AlexaFluor488-conjugated GM102 antibody, AMHRII expression was confirmed on the matching fresh tissue samples, previously detected by IHC in their fixed counterpart samples. Preliminary FACS analysis of fresh ovarian and colorectal tumor samples have demonstrated comparable expression levels with mean values of 50,000 and 40,000 AMHRII receptors per cell, respectively. In conclusion, the results presented above, using a variety of detection methods and systems, have consistently shown that AMHRII is expressed in many different histological types of solid tumors. These results suggest that this embryonic receptor could be a suitable target for selective antitumor agents such as GM102.

#775

**HIPStA, a high-throughput alternative to CETSA** ® **.**

Kelvin F. Cho,1 Christopher Rose,2 Taylur Ma,2 Donald Kirkpatrick,2 Robert A. Blake2. 1 _Stanford University, CA;_ 2 _Genentech, Inc., CA_.

The measurement of drug - target interaction in the cellular context is critical to many drug development programs. The Cellular Thermal Stability Assay (CETSA®) represents an established broadly applicable method for measuring drug target interaction. However, in its classic format, it has some limitations that make it difficult to scale to the throughput typically required for a drug development project. It requires heating samples to different temperatures and centrifugation and / or filtration steps which limit throughput. The HSP90 Inhibitor Protein Stability Assay (HIPStA) is a novel method for measuring drug target interaction. Like CETSA®, HIPStA is based on the premise that the binding of a ligand to a target protein can influence that protein's stability. Instead of using heat to destabilize a protein, HIPStA uses a Heat Shock Protein 90 inhibitor (HSP90i) to cause protein instability. Instead of scanning a range of different temperatures to establish a thermal denaturation curve, HIPStA applies a range of concentrations of an HSP90i to determine an HSP90i induced denaturation curve, and ultimately measures the ability of a compound to stabilize a protein. We present data demonstrating the proof of concept for the HIPStA method, using 3 different classes of drug discovery targets: Receptor tyrosine kinases, Nuclear Hormone Receptors and Cytoplasmic Protein Kinases. HIPStA represents a scale-able method for detecting drug-target interaction in cells.

#776

Utilising genetic susceptibility and big data to inform novel cancer therapies.

Elizabeth A. Coker, Ben Kinnersley, Amit Sud, Patrizio Di Micco, Bissan Al-Lazikani, Richard Houlston. _Inst. of Cancer Research, London, United Kingdom_.

Despite a move towards personalised medicine, attrition rates for new cancer drugs remain unacceptably high. The pharmaceutical industry has also shown a preference for well-studied targets and pathways, as evidenced by 'me too' drugs. Together with the challenge of inadequate pre-clinical models, this indicates a need for novel, evidence-based therapeutic targets. Genome-wide association studies (GWAS) have identified over 450 robust genetic variants associated with increased cancer risk. Genes implicated through GWAS are often mutated somatically and therefore represent attractive therapeutic targets. Examples include the target of venetoclax in chronic lymphocytic leukaemia, BCL2.

We exploit this principle more generally by integrating genetic associations for common cancers with drug target data and druggability using the canSAR drug discovery knowledgebase (https://cansar.icr.ac.uk). By harnessing the power of Big Data we aim to both identify opportunities for repurposing of existing drugs, and prioritise novel targets for cancer drug discovery.

We mined the NHGRI-EBI Catalog of published GWAS for all cancer risk SNPs. We annotated candidate target genes through overlapping topologically associating domains (TADs), a more sensitive technique than previously published methods using linkage disequilibrium. We used canSAR to identify target genes for which there is no FDA-approved small molecule drug, and the resource Probe Miner to identify targets for which high-quality chemical probes exist. We also utilised canSAR's machine learning algorithms to assess the druggability of target genes by structure-, ligand-, precedence- and network-based approaches.

We additionally analysed results from cancer drug databases to ascertain whether there is an enrichment of 'drug target-indication pairs' at successive stages of the drug development pathway for which supporting evidence from GWAS exists: this indicates potential 'stumbling blocks' that may present a risk for future drug development projects.

7 257 protein-coding genes mapped within TADs overlapping cancer risk SNPs. Of these, 98 were pre-existing targets for which there is an FDA-approved small molecule drug. For the remaining 7 159 genes we performed multi-faceted druggability analyses incorporating assessments of the 3D structure of the target and any protein complexes it exists in, chemical properties of known ligands of the target, and the target's position and role within the human interactome. We comprehensively rank our target-indication pairings by criteria including novelty relative to existing targets and predicted attrition risk.

Mapping approved drug targets back to cancer GWAS signals enables identification of both novel drug targets and patient populations. Collectively our findings show the value of investigating germline cancer genetics as part of interdisciplinary, data-driven approaches to inform drug discovery.

#777

Malic enzyme 3 as a collateral lethality target in pancreatic cancer.

Prasenjit Dey,1 Joelle Baddour,2 Youngsoo Kim,3 Robert Macloed,4 Florian Muller,1 Chia Chin Wu,1 Huamin Wang,1 Andrea Viale,1 Haoqiang Ying,1 Giulio Draetta,1 Anirban Maitra,1 Alan Wang,1 Deepak Nagrath,5 Ronald DePinho1. 1 _UT MD Anderson Cancer Ctr., Houston, TX;_ 2 _Rice University, Houston, TX;_ 3 _Ionis Pharmaceuticals Inc., CA;_ 4 _Ionis Pharmaceuticals Inc, CA;_ 5 _University of Michigan, MI_.

Cancer genomes possess many deletion events targeting tumor suppressor genes (TSG) and neighboring genes in these loci. These deletion patterns prompted us to consider a systematic approach, termed "collateral lethality", designed to identify cancer-specific vulnerabilities resulting from the deletion of neighboring genes. These bystander genes do not appear to be involved in cancer pathogenesis, yet encode cell-essential functions and are members of multi-gene families that are functionally redundant and co-expressed. Homozygous deletion of SMAD4 is a frequent event in pancreas cancer and other cancer types, totaling >30,000 cases in US annually. SMAD4 deletion often results in co-deletion of the neighboring mitochondrial malic enzyme 2 (ME2) gene. In mammalian cells, two genes (ME2 and ME3) encode redundant cell-essential mitochondrial ME activity. Together, ME2 and ME3 function to generate pyruvate to fuel the TCA cycle, and NADPH to maintain ROS homeostasis. These observations prompted us to hypothesize that the genetic or pharmacological extinction of ME3 activity in a ME2 null cell would specifically compromise cancer cells yet be tolerated in normal host cells possessing ME2 activity. Inducible shRNA strategies were employed to genetically deplete ME3 in ME2-null versus ME2-intact cells followed by apoptosis measurements, integrated metabolomics, and molecular investigations. In collaboration with IONIS Pharmaceuticals, we tested and validated anti-sense oligonucleotide (ASO) to target ME3. Genetic depletion of ME3 in ME2 null, but not ME2 intact, cells resulted in apoptosis and blocked tumorigenic potential. Mechanistically, integrated metabolomic and molecular investigation of mitochondrial ME-deficient cells revealed diminished NADPH production and consequent high ROS, which activates AMP activated protein kinase (AMPK) and which in turn directly suppresses sterol regulatory element-binding protein 1 (SREBP1)-directed transcription of its direct targets including the BCAT2 (Branched chain amino acid transaminase 2) gene. We also determined that mitochondrial MEs regulate the utilization of branched chain amino acid (BCAA) via BCAT2, a transaminase required for BCAA catabolism. Notably, enforced expression of BCAT2 can restore tumorigenic potential of ME2/3 deficiency, and free nucleotides can restore proliferation in cell culture. Specific targeting of ME3 using ASOs could dramatically reduce tumor burden in a subQ tumor model of pancreatic cancer. Thus, a key mechanism driving cancer cell lethality involves BCAAs as crucial metabolites under the critical regulation of the mitochondrial MEs. These studies reveal a collateral lethal vulnerability in pancreas and other cancers that can be targeted pharmacologically in genotype-defined patient populations. We propose that highly specific ME3 inhibitors could provide an effective therapy across a substantial number of cancer patients.

#778

Potential drug targets for adenoid cystic carcinoma elucidated by proteogenomic analysis.

Sheeno P. Thyparambil,1 Yeoun Jin Kim,1 Andrew G. Chambers,1 Dongyao Yan,1 Shankar Sellappan,1 Andrew J. Sedgewick,2 Yulia Newton,2 J Zachary Sanborn,2 Charles J. Vaske,2 Stephen C. Benz,2 Fabiola Cecchi,1 Hyunseok Kang,3 Todd A. Hembrough1. 1 _NantOmics, Rockville, MD;_ 2 _NantOmics, Santa Cruz, CA;_ 3 _Johns Hopkins Medical Institutions, Baltimore, MD_.

Background: Adenoid cystic carcinoma (ACC) is a rare cancer of secretory glands accounting for 10% of salivary gland malignancies and 1% of head and neck cancers. About 1,200 new cases are diagnosed annually in the United States. ACC is typically chemoresistant and clinical trials of multiple targeted agents found few responders. Studies of ACC oncogenesis have described frequent fusion of MYB-NFIB genes and other infrequent genomic mutations. ACC has not been proteomically characterized. We hypothesized that proteogenomic analysis of ACC tumor tissues would identify clinically relevant molecular differences between ACC and other tumor types. The objective of this study was to identify altered disease pathways and potential drug targets in ACC.

Methods: Clinical tumor samples of ACC and squamous cell carcinoma (SCC) of the head and neck were analyzed using the GPS Cancer diagnostic test, which includes whole genome and transcriptome sequencing, and proteomic expression analysis using mass spectrometry. To quantify proteins, tumor areas of formalin-fixed paraffin-embedded tissue sections were marked by a pathologist, microdissected and solubilized. The resulting lysate was analyzed with mass spectrometry to quantitate 30 clinically relevant proteins. Samples were further analyzed with a global proteomics platform intended to discover targetable protein biomarkers. Proteins that were overexpressed or underexpressed (defined as a 1.5-fold difference between ACC and SCC) were subjected to pathway analysis to identify perturbed pathways and potential drug targets. Finally, the mRNA expression in ACC tumors was compared with RNA-seq data from various solid tumor types using the k-nearest neighbors algorithm.

Results: In 14 tumor samples, unsupervised hierarchical clustering analysis of 4,002 proteins revealed a clear separation between ACC (n=8) and SCC (n=6) tissues. Four of 8 ACC samples harbored an MYB-NFIB fusion and single samples had fusions of MYBL-NFIB or AHI1-NFIB. Mutational burden in ACC and SCC samples was 1.35 and 3.53 mutations per megabase, respectively. Pathway analysis found enrichment of ACC genes in essential cell cycle processes. CDK6 protein expression was 4-fold higher in ACC samples than SCC samples by mass spectrometry (p=0.0036) and 3-fold higher at the mRNA level. Expression of p16 protein was 3-fold lower in ACC than in SCC (p=0.0289) and corresponding p16 mRNA levels were 17-fold lower in ACC than SCC. All ACC samples harbored intact retinoblastoma (RB1) gene. Expression of mRNA in 15 ACC samples was compared with that of 25 different tumor types from TCGA and the authors' clinical laboratory; breast cancer was found to be the nearest neighbor to ACC.

Conclusions: Proteogenomic analysis revealed CDK6 overexpression in ACC. The combination of CDK6 overexpression, p16 underexpression and RB1 proficiency suggests that ACC tumors may respond to treatment with CDK6 inhibitors.

#779

Integrative analysis reveals therapeutic targets to the DNA methyltransferase inhibitor SGI-110 in hepatocellular carcinoma.

Minmin Liu,1 Toshinori Hinoue,1 Wanding Zhou,1 Hitoshi Ohtani,1 Anthony El-Khoueiry,2 John Daniels,2 Casey O'Connell,2 Tanya B. Dorff,2 Daniel J. Weisenberger,2 Gangning Liang2. 1 _Van Andel Research Inst., Grand Rapids, MI;_ 2 _University of Southern California, Los Angeles, CA_.

Objective There is an urgent need for developing more effective therapies for Hepatocellular carcinoma (HCC) because of its aggressiveness. Guadecitabine (SGI-110) is a second-generation DNA methyltransferase inhibitor (DNMTi) currently in clinical trials for HCC and shows greater stability and performance over first generation DNMTi. The aim of this study is to identify potential therapeutic targets of SGI-110 for clinical trials.

Design HCC cell lines (SNU398, HepG2 and SNU475) were used to evaluate effects of SGI-110 by an integrative analysis of DNA methylation, nucleosome accessibility and gene expression profiles following the transient SGI-110 treatment and its clinic relevant by comparing HCC clinic data from TCGA.

Results These HCC cell lines represent the three DNA methylation subtypes of primary HCC tumors based on TCGA data. After SGI-110 treatment, all cell lines were sensitive to SGI-110 with prolonged anti-proliferation effects. Up-regulated genes including tumor suppressors were positively correlated with nucleosome accessibility and negatively correlated with gene promoter DNA methylation, while the down-regulated genes, such as oncogenes, were negatively correlated with nucleosome accessibility and positively correlated with gene body DNA methylation. Furthermore, SGI-110 down-regulated PRC2 complex genes by demethylating their gene bodies, resulting in re-activation of PRC2 repressed gene promoters which are independent on DNA methylation. In addition, SGI-110 up-regulated endogenous retroviruses (ERVs) to reactivate immune pathways. Finally, about 45% of frequently altered genes in primary HCC tumors can be re-shaped into a "normal like" expression status through the treatment.

Conclusion Our integrative analysis has successfully linked the anti-tumor effects of SGI-110 to detailed epigenetic alterations in HCC cells and identified potential therapeutic targets. We also provide rationale of combination treatment with immune checkpoint therapies.

#780

Molecular chaperone is a novel biomarker for disease outcome in multiple myeloma.

Yingqi Li,1 Ping L. Zhang,2 Zihai Li,1 Bei Liu1. 1 _Medical University of South Carolina, Charleston, SC;_ 2 _William Beaumont Hospital, Royal Oak, MI_.

Multiple myeloma (MM) is an incurable plasma cell neoplasm whose pathogenesis is closely linked to dysregulated unfolded protein response (UPR) in the endoplasmic reticulum (ER). MM typically evolves from an asymptomatic premalignant stage called monoclonal gammopathy of undetermined significance (MGUS), to an intermediate asymptomatic smoldering MM (SMM), to eventually symptomatic MM. However, no single pathologic or molecular biomarker can be used to distinguish MM patients from SMM, who have clonal premalignant plasma cells from those with clonal myeloma cells. Therefore, there is a compelling need for further development of novel diagnosis and prognosis biomarkers for MM. Heat shock protein grp94 is a key downstream chaperone in the ER to mediate UPR. Using genetic approaches, we have demonstrated that grp94 is an obligate chaperone for Wnt co-receptor low-density lipoprotein receptor-related protein 6 and that it is required for multiple myeloma cell survival, which is mediated in part by the Wnt target survivin. Moreover, we found that grp94 is highly expressed in malignant plasma cells in MM, and the higher level of grp94 is significantly associated with more advanced stage of this disease. Additionally, the grp94 expression is significantly associated with the levels of serum M protein in MM patients, but not in patients with MGUS and SMM. Thus, our studies demonstrated that molecular chaperone grp94 is a novel diagnostic and prognostic biomarker for MM as well as a potential target for treatment of this disease.

#781

Comparative analysis of androgen receptor phosphorylation in castrate resistant prostate cancer using conventional immunohistochemistry and immunofluorescence.

Milly McAllister,1 Pamela McCall,1 Mark Underwood,2 Hing Leung,3 Joanne Edwards1. 1 _University of Glasgow, Glasgow, United Kingdom;_ 2 _Queen Elizabeth University Hospital, Glasgow, United Kingdom;_ 3 _Cancer Research UK Beatson Institute, Glasgow, United Kingdom_.

Despite the increase in new hormonal therapies available to treat prostate cancer, over 10,000 men die per year in the UK from treatment resistant disease, therefore highlighting the need to develop effect predictive and prognostic markers for these patients. Therefore, we aim to identify the most accurate method of determining the association of androgen dependent (pARser81) and androgen independent (pARser213) phosphorylation with patient survival.

109 patients with castrate resistant prostate cancer were immunohistochemically characterised for pARser81 and pARser213 expression to determine the association of androgen receptor phosphorylation with time to death from biochemical relapse. Immunofluorescence was employed on formalin fixed paraffin embedded tissue to determine the localisation of pARser81 and pARser213 and whether phosphorylation of the AR at these sites were occurring within the same cell or neighbouring cells.

High pARser213 immunoreactivity was associated with a reduced time to death from biochemical relapse when compared to those with low pARser213 expression (p=0.001). However pARser81 expression was not associated with survival (p=0.068). Patients expressing both high pARser81 and high pARser213 had a reduced time to death from biochemical relapse when compared to those with low expression of one or both sites (p=0.000297). A subgroup of these patients were then selected and dual immunofluorescence employed to assess pARser81 and pARser213 expression. The presence of pARser81 expression within the nucleus reduced time to death from biochemical relapse when compared to those with no pARser81 nuclear expression (p=0.029). Punctate nuclear pARser81 expression (which was not observed using standard immunohistochemistry) was also associated with reduced time to death from biochemical relapse when compared to those with no pARser81 expression (p=0.008). pARser213 nuclear expression was associated with reduced time to death to death from biochemical relapse when compared to those with no pARser213 expression (p=0.000082). When dual immunofluorescence was employed, expression of both pARser81 and pARser213 within the nucleus of the same cell was associated with reduced time to death from biochemical relapse when compared to those with no expression of either sites (p=0.00003).

In conclusion, dual expression of pARser81 and pARser213 are associated with reduced time to death from relapse using either immunohistochemistry or immunofluorescence. However immunofluorescence appears to be more precise, identifying the importance of pARser81 expression in castrate resistant patients that would otherwise of been missed via traditional immunohistochemistry along with highlighted the importance of personalised medicine in prostate cancer with the potential of dual targeting patients.

#782

Development of a high throughput in vitro screening platform to identify novel inducers of immunological cell death.

Didier Grillot, Akanksha Gangar, Raphaelle Guillard-Huet, Eric Boursier, Florent Potvain, Guillaume Serin, Jean-François Mirjolet. _Oncodesign S.A., Dijon Cedex, France_.

Immunological cell death (ICD) is a form of cancer cell death induced by radiotherapy, photodynamic therapy and a few chemotherapeutic agents such as Doxorubicin, Mitoxantrone, and Oxaliplatin. Unlike apoptosis or necrosis, ICD can induce an effective immune response directed against the tumor whereby both dendritic cells and T lymphocytes are mediators of this response. Dying cancer cells recruit and activate immune cells by releasing damage-associated molecular patterns (DAMPS) that help and promote the immune response to antigenic tumor neo-epitopes. Three key DAMPS are associated with the ICD process: calreticulin exposition on the cell surface, ATP secretion and high-mobility group box 1 (HMGB1) release. In order to identify new therapeutic agents that promote ICD in malignant cells, we developed a screening strategy facilitated by an automated in vitro platform with four assays on three different tumor cell lines (human osteosarcoma U-2 OS, human breast MDA-MB-231 and murine liver Hepa 1-6). ICD inducers Doxorubicin and Mitoxantrone used as positive controls increased ATP secretion by 2 to 10-fold at a non-cytotoxic dose after 72 hours incubation on the three cell lines. Both compounds also increased calreticulin exposition by 2 to 4-fold (determined by immunofluorescence using the Operetta High-Content Imaging System) and HMGB1 release by two-fold on the three cell lines. Here we will present recent data from the screening of Oncodesign's Nanocyclix® library using this platform to identify novel ICD inducers.

#783

**Impact of KRAS and TP53 co-mutations on outcomes following 1** st **-line therapy among patients with LKB1/STK11 mutated stage IV NSCLC.**

Erin M. Bange, Melina E. Marmarelis, Wei-Ting Hwang, Yu-Xiao Yang, Jeffrey C. Thompson, Joshua Bauml, Christine A. Ciunci, Evan W. Alley, Tracey L. Evans, Jennifer J. Morrissette, Roger B. Cohen, Corey J. Langer, Erica L. Carpenter, Charu Aggarwal. _University of Pennsylvania, Philadelphia, PA_.

* Authors E. M. B. and M. E. M. contributed equally

Introduction: LKB1/STK11 is a tumor suppressor and a negative regulator of mTOR signaling. NSCLC patients with STK11 mutations (MT) commonly also have other co-mutations. We evaluated the impact of STK11 MT on outcomes following chemotherapy for metastatic NSCLC, and the role of common co-existing MTs in KRAS and TP53.

Methods: We conducted a retrospective review of patients (pts) with NSCLC and STK11 MT treated at the University of Pennsylvania. STK11 MT was identified through next generation sequencing (NGS) in tissue or using the Guardant 360TM platform for plasma. Four treatment groups were analyzed: STK11 alone (A), and 3 co-MT groups: STK11/KRAS (B), STK11/TP53 (C), and STK11/KRAS/TP53 (D). Chi-square analysis was used to assess differences in baseline characteristics between the MT groups. Cox proportional hazard models (HR) were used to determine the relationship of STK11 co-MT to survival. Kaplan-Meier analysis was used to estimate overall survival (OS) and progression-free survival (PFS).

Results: Between 2/14/13 and 12/1/16, 77 pts with STK11 MT who received systemic therapy were identified (56 tissue, 21 plasma): median age at diagnosis 66 yrs, 51.5% male, 85% ECOG PS of 0 or 1, 94% of pts received 1st-line chemotherapy. There were no significant differences in baseline characteristics among mutation groups. Pts with STK11/KRAS had a worse median PFS and OS vs. pts with STK11 alone (Table 1). Pts with STK11/TP53 had a significantly better OS compared to STK11/KRAS patients.

Conclusion: Among STK11 mutant NSCLC pts treated with chemotherapy, co-MT with KRAS was associated with a significantly worse PFS and OS compared to patients with STK11 alone. By contrast, co-MT with TP53 conferred a better prognosis. These results warrant further validation in a larger study.

Table 1: Outcomes by mutation status

---

|

A:STK11alone n=16 (24.2%) | B:STK11/KRAS n=21 (31.8%) | C:STK11/TP53 n=18 (27.3%) | D:STK11/KRAS/TP53 n=11 (16.7%)

Median PFS | 5.3 mo | 2.4 mo* | 9.9mo | 4.9 mo

Median OS | 13.1 mo | 6.9 mo** | 22.2 mo*** | 13.9 mo

|  | *compared to A, HR 2.8, 95% CI 1.1 to 6.9, p = 0.026 | **compared to A, HR 6.4, 95% CI 2.4 to 17.3, p<0.001 | ***compared to B, HR 0.12, 95% CI 0.04 to 0.31, p<0.001

#784

NCI CPTAC phase III, proteogenomic analysis of 10 cancers.

Mathangi Thiagarajan. _Leidos Biomedical Research Inc., Rockville, MD_.

The National Cancer Institute's Clinical Proteomic Tumor Analysis Consortium (CPTAC) is an international effort to accelerate the understanding of the molecular basis of cancer through the application of large-scale proteomic and genomic analysis, or proteogenomics. Leidos Biomedical Research, a prime contractor to NCI, provides a robust infrastructure for supporting the collection and sequencing of high-quality biospecimens and data, in addition to project and subcontract management for this program. CPTAC applies the understanding of the molecular basis of cancer to identify biomarker candidates. CPTAC phase III began late 2016 to collect and analyze 200 cases of each of ten additional cancers. The cancers include sarcoma, pancreatic ductal adenocarcinoma, head and neck squamous cell carcinoma, lung adenocarcinoma, lung squamous cell carcinoma, uterine corpus endometrial carcinoma, clear cell renal cell carcinoma, glioblastoma multiforme, and acute myeloid leukemia. The program completed the discovery phase collection of 100 cases for 3 tumor types in the first year. The study entails collection and pathology evaluation of biospecimens, high-quality clinical data and images from clinical sites around the world. A biorepository evaluates and processes the biospecimens, sending nucleic acids to sequencing centers and tissues to proteomics groups. Data are combined and analyzed by proteogenomic data analysis and translational centers. Genomic data are made available to the research community through the NCI Genomic Data Commons. Proteomic and proteogenomic data are made available through the Data Coordinating Center. Imaging data are made available through NCI imaging archive. We report here on progress in collection of tissues and clinical data, the preliminary analysis trends and the integration of biorepository, proteomics and genomics and analysis centers.

#785

Application of CRISPR/Cas9 gene editing to primary T cells.

Xi Li, Wanbing Tang, Chenjie Zhou, Yulin Yang, Zhengang Peng, Wenrong Zhou, Qunsheng Ji, Yong Cang. _Oncology and Immunology BU, Research Service Division, WuXi AppTec (Shanghai) Co., Ltd, Shanghai, China_.

T cells mediate a broad range of immune responses, especially generation of adaptive immunity for cancer cell clearance. Identification of genetic components that modulate T cell proliferation, differentiation, migration and cytotoxicity would facilitate development of more effective cancer immunotherapy. CRISPR/Cas9, as a tool, could generate gene knockout with high specificity and efficiency and has been widely used in genome-wide screening for therapeutic targets in many tumor cell models. However, its application to primary cells especially T cell has not been extensively investigated. Here, we described how to employ the CRISPR/Cas9 tools for immune-oncology target identification in primary mouse T cells. Cas9/sgRNA could be transduced into T cells with high efficiency and low toxicity using a lentivirus based delivery system. The transduced T cells acquired Cas9 expression but no DNA editing was observed. However, we achieved gene editing in primary T cells isolated from a Cas9 transgenic mouse strain after lentiviral delivery of guide RNA's. In summary, we provide a new method to generate DNA editing in primary T cell using CRISPR/Cas9 technology. It could be used for gene knockout, knockin and even genome-wide screening in primary T cell for immune checkpoint regulators.

#786

To examine the effect of limiting heme bioavailability on lung tumor growth & progression.

Poorva Ghosh,1 Sarada Preeta Kalainayakan,1 Sancharika Dey,2 Sagar Sohoni,2 Li Zhang2. 1 _University of Texas at Dallas, Dallas, TX;_ 2 _University of Texas at Dallas, Richardson, TX_.

Numerous epidemiological studies in the past have shown that increased heme intake is associated with higher risk of several cancers, including lung cancer. Previous studies from our lab have demonstrated a significant increase in heme uptake, synthesis, and expression of oxygen utilizing hemoproteins in non -small cell lung cancer cells (NSCLCs) as compared to normal cells. Also, inhibition of heme synthesis was shown to negatively impact proliferation of NSCLCs specifically. This led us to hypothesize that heme fuels proliferation of NSCLC cells and promotes lung tumor growth. Heme is a central molecule for mitochondrial function and for all processes involved in oxygen utilization. Also, it serves as a prosthetic group in several oxidative phosphorylation enzymes and other oxygen-utilizing hemoproteins. Most human cells synthesize heme de novo and uptake heme from the circulation. The purpose of the study is to employ various strategies to limit bioavailability of heme and evaluate whether altering heme availability impacts NSCLC tumor growth and development in vivo.

To limit heme availability, we designed an agent (Heme targeting agent 1or HTA1) to sequester heme from circulation and thereby limit heme available for uptake. A well-known inhibitor of heme synthesis was used to limit heme synthesis. NSCLC cells that express luciferase was implanted in NOD/SCID mice to generate subcutaneous and lung orthotopic xenografts. The mice were treated with the HTA1 alone and HTA1 in combination with an inhibitor. We designed another agent (Heme targeting agent 2 or HTA2) that is thought to have higher heme binding affinity compared to HTA1. Treatment with HTA2 caused significantly greater reduction in growth and proliferation of NSCLCs in vitro as compared to HTA1. To assess its anti-tumor efficacy in comparison to HTA1 in vivo above-mentioned mice were treated with HTA2. Tumor growth was monitored by non-invasive bioluminescence imaging (BLI) using Perkin Elmer's IVIS Lumina III Imager.

In lung orthotopically implanted xenografts, BLI data show that there is a significant reduction in radiance (total flux in photons per second) in the treatment groups that received the HTA1 in combination with the inhibitor compared to HTA1 alone. Also, HTA2 caused a more effective reduction in radiance compared to HTA1. Immunohistochemistry was performed on lung tissues from the mice treated with the heme limiting agents mentioned above.

Our results indicate that combination of heme limiting agents works effectively in inhibiting lung tumor growth and progression by limiting both heme uptake and synthesis. Further studies to evaluate the potential of these heme limiting agents in inhibiting lung cancer growth and progression are underway.

#787

Cytosolic TRIM24 characterizes an aggressive subset of ER- PR- and TP53 mutant breast cancer.

Lalit R. Patel,1 Jurgen Mitsch,2 Grazziela P. Figueredo,2 Philip Quinlan,2 Lee B. Jordan,3 Colin A. Purdie,3 Savitri Krishnamurthy,1 Michelle C. Barton,1 Alastair M. Thompson1. 1 _University of Texas MD Anderson Cancer Center, Houston, TX;_ 2 _University of Nottingham, Nottingham, United Kingdom;_ 3 _Ninewells Hospital and Medical School, Dundee, United Kingdom_.

Introduction: Tripartite Motif Containing 24 (TRIM24) is a PhD/Bromodomain containing steroid receptor co-activator that targets p53 for proteosomal degradation, transforms human mammary epithelial cells, and promotes treatment resistance in preclinical models. While bromodomain inhibitors and proteolysis targeting chimeras have been developed against TRIM24, the disease subtype it is most relevant to remains under explored. In this study, we characterize a uniquely annotated cohort of invasive ductal carcinomas for tumor expression of TRIM24 protein by immunohistochemistry (IHC) to assess its relationship with molecular and clinicopathological features.

Methods: Tissue microarrays (TMAs) representing 198 tumors with 6 cores/tumor were obtained from the Tayside Biospecimen Repository and stained by IHC for TRIM24. TMAs were scored for nuclear and cytosolic intensity and the proportion of tumor cells with staining. These values were combined into Histo-scores (H-Scores) and averaged as a semi-quantitative metric for tumor TRIM24 expression. Statistical associations between TRIM24 H-Scores, clinicopathological annotations, and existing molecular profiles generated from the same TMA were determined using SciPy and SPSS.

Results: TRIM24 has four distinct expression patterns in the 170 tumors with scorable cores on the TMA: nuclear (55), cytosolic (38), nuclear and cytosolic (35), and negative (42). Non-parametric analysis revealed associations between TRIM24 expression pattern and ER (χ2=21.3, p=0.000), PR (χ2=14.8, p=0.002), invasive grade (χ2=14.9, p=0.021), and TP53 mutation (χ2=9.55, p=0.023). No significant association was found with HER2 (χ2=1.51, p=0.68). Higher nuclear H-scores are observed in ER+ (p=0.0008) and PR+ (p=0.001) tumors. Higher cytosolic but not nuclear H-scores are observed in Grade 3 (p=0.003), triple negative (TNBC, p=0.004), and TP53 mutant (p=0.0289) cases. Kaplan-Meier analysis revealed high cytosolic (p=0.037) but not nuclear (p=0.601) H-scores associated with poor survival in ER- patients. No significant survival difference was found in ER+ patients stratified by nuclear (p=0.781) or cytosolic (p=0.683) H-scores. Similar analysis of TNBC was underpowered. TP53 mutant (p=0.142) but not TP53 wild type (p=0.378) disease with high cytosolic H-scores trend toward diminished survival. No such trend is observed in cases stratified by nuclear H-scores and TP53 status.

Conclusions: TRIM24 is overexpressed in most human breast cancers. Nuclear expression is more common in ER+/PR+ tumors but does not stratify outcome. Cytosolic expression occurs in Grade 3, ER-, and TP53 mutant tumors and associates with poor clinical outcome in ER- disease. This suggests TRIM24 may be a more relevant drug target in ER-/PR- than luminal breast cancer and emphasizes the need for studies investigating the cytosolic functions of TRIM24 in ER-/PR- and TP53-mutant breast cancer.

#788

Modeling tumor microenvironmental heterogeneity identifies CREBBP as a novel tumor suppressor in breast cancer.

Barrie Peck, Philip J. Bland, Patty T. Wai, Hannah Cottom, Sarah L. Maguire, Eamonn Morrison, Holly E. Barker, Divya Kriplani, Rebecca Marlow, Kalnisha Naidoo, Gareth Muirhead, Syed Haider, Frances Daley, Frederik Wallberg, Andrew N. Tutt, Rachael C. Natrajan. _Inst. of Cancer Research, London, United Kingdom_.

Solid tumors display significant histological, genetic and micro-environmental intra-tumor heterogeneity that can change substantially over the course of their evolutionary trajectory. In particular, changes in the micro-environmental complexity within breast cancer such as hypoxic and nutrient deplete environments are associated with aggressive disease and a poor patient outcome.

We sought to identify novel driver alterations in aggressive disease by employing a functional genomics screen in a 3-dimensional model of breast cancer progression that more accurately recapitulates in vivo micro-environmental heterogeneity. Screening of the top 200 recurrently mutated genes in breast cancer in cancer cell line spheroids identified several genes whose silencing impacted growth. A second targeted validation screen in a larger panel of triple negative cell line models showed that silencing of the histone acetyltransferase CREBBP, promoted growth in 3D but had limited effect under traditional 2D culture conditions.

Investigation of TCGA and METABRIC datasets showed that CREBBP was more frequently mutated in triple negative breast cancers (TNBCs) and at least a third of TNBCs also displayed gene haploinsufficiency or complete loss of CREBBP. Interrogation of expression and proteomic datasets showed that loss of CREBBP resulted in the upregulation of the pro-proliferative transcription factor FOXM1. Significantly, this conserved FOXM1-driven transcriptional programme was also seen in multiple solid tumors with CREBBP alterations including lung, oesophageal, bladder and endometrial cancers. This was recapitulated in several CREBBP deficient cells where we identified that FOXM1 is driving altered metabolism, allowing cancer cells to grow under nutrient stress conditions.

In summary, CREBBP is a bona fide tumor suppressor in up to a third of TNBCs, as well as a wide range of other solid tumors. CREBBP-altered tumors display up-regulation of FOXM1, which alters cancer cell metabolism under nutrient stress conditions. Moreover, CREBBP-altered tumors are selectively sensitive to small molecule inhibitors that target FOXM1 activity, suggesting that this maybe a viable targeted therapeutic approach for CREBBP altered cancers.

#789

Nuclear IGFBP-3 is a potential biomarker for response to EGFR-sphingosine kinase targeted therapy in basal-like triple-negative breast cancer (TNBC).

Sohel M. Julovi, Janet L. Martin, Robert C. Baxter. _Kolling Institute, Sydney, Australia_.

Background: TNBC comprises about 15% of breast cancer cases and, with no approved targeted therapy, is commonly treated with adjuvant chemotherapy. We have described a novel targeted therapy for basal-like TNBC combining EGFR and sphingosine kinase (SphK) inhibitors, which induce a strongly synergistic cytostatic effect in six TNBC cell lines (Martin et al, Breast Cancer Res 19:90, 2017). IGFBP-3, which is highly expressed in basal-like TNBC and prognostic for poor recurrence-free survival, initiates an oncogenic pathway involving SphK1 and EGFR activation. Since IGFBP-3 downregulation in vitro prevents the synergistic cytostasis caused by EGFR and SphK inhibitors, we aimed in this study to evaluate nuclear IGFBP-3 as a response biomarker in TNBC tumors after treatment with the inhibitor combination, with or without doxorubicin (dox), a common component of TNBC chemotherapy.

Methods: Human basal-like TNBC cell lines HCC1806 and MDA-MB-468 were grown as orthotopic xenograft tumors in female BALB/c nude mice, and treated when tumors reached 100 mm3 with the SphK inhibitor and S1P receptor modulator, fingolimod, plus the EGFR kinase inhibitor, gefitinib (F+G) ± dox at the maximum tolerated dose of 2 mg/Kg weekly. Tumors were analyzed by immunohistochemistry (IHC). Cell proliferation was studied in vitro by IncuCyte live-cell imaging.

Results: In both cell lines in vitro, F+G at doses that caused minimal cytostasis, acted synergistically with a sub-cytostatic dox dose (10 nM), to cause almost complete inhibition of proliferation. In contrast, in xenograft mouse models, in which F+G administered 3x weekly significantly inhibited tumor growth and enhanced mouse survival, dox administration for 6 weeks showed minimal growth inhibition alone, and no significant incremental effect with F+G. Examined by IHC, TNBC tumor IGFBP-3 staining showed predominantly nuclear localization, was positively correlated with Ki67, and was significantly downregulated by F+G treatment, with no added dox effect. By Kaplan-Meier analysis, high (> median) nuclear IGFBP-3 IHC scores were strongly associated with worse mouse survival, similar to high Ki67 scores, while high apoptosis (cleaved caspase-3) scores were associated with better survival.

Conclusion: The synergism between dox and combination F+G, observed in vitro, was not seen in vivo, possibly because of dose-limiting dox toxicity. This might be improved by alternative dox delivery methods. However, the F+G combination alone is highly inhibitory to basal-like TNBC tumor growth in both xenograft models. IGFBP-3 is known to interact with nuclear hormone receptors and DNA damage response intermediates in the nucleus. Nuclear IGFBP-3 staining may have utility as a biomarker of treatment response in TNBC, alone or together with Ki67 and cleaved caspase-3. Supported by Cancer Council NSW.

#790

Vitamin K pathway functions in triple-negative breast cancer.

Sarah Beaudin, Leila Kokabee, JoEllen Welsh. _University at Albany, Rensselaer, NY_.

The vitamin K pathway comprises three major enzymes: gamma-glutamyl carboxylase (GGCX) and the vitamin K oxidoreductases VKORC1 and VKORC1L1. GGCX requires the reduced form of vitamin K as a co-factor to post-translationally generate γ-carboxylated (GLA) residues in proteins. Either VKORC1 or VKORC1L1 can reduce the oxidized form of vitamin K to maintain pathway activity. Interrogation of TCGA data indicates a subset of invasive breast cancer tumors harbor mutations in GGCX (8%), VKORC1 (9%), and/or VKORC1L1 (9%), with nearly all alterations being amplifications. As these data suggest upregulation of these genes is associated with aggressive breast cancer, we measured their expression in a variety of established breast cancer cell lines. qPCR results correlated with the TCGA data indicating the highest expression of GGCX, VKORC1 and VKORC1L1 expression in the two triple-negative breast cancer (TNBC) cell lines, Hs578T and SUM159PT. Since the function of these genes is to generate GLA residues in proteins, we used Western blotting to detect GLA-modified proteins in TNBC cells cultured in the presence or absence of vitamin K. In lysates from cells maintained in vitamin K, multiple bands representing GLA-modified proteins were observed between 40-60 kDa and 80 kDa. These bands were not present in lysates of cells grown in standard media that are typically devoid of vitamin K. Treatment with the VKOR antagonist, warfarin, decreased expression of these GLA-modified proteins in cells cultured in vitamin K. Thus, our data suggest that vitamin K acts in TNBC cells to produce GLA-modified proteins, and that these effects are abrogated by warfarin. To confirm the presence of a functional vitamin K pathway, we used a vitamin K-dependent reporter protein (FIXgla-PC) to measure γ-carboxylation activity. TNBC cells stably transfected with the FIXgla-PC reporter were cultured in the presence and absence of vitamin K, with or without warfarin, and secretion of γ-carboxylated FIX-gla was measured by ELISA. FIX-gla γ-carboxylation was enhanced in the presence of vitamin K and blocked by warfarin in both Hs578T and SUM159PT cell lines. These data confirm that the classical vitamin K pathway is activated in these representative TNBC cell lines when cultured in vitamin K-supplemented media. To assess the biologic relevance of this pathway in TNBC, mammosphere assays were performed with Hs578T and SUM159PT cells. Both cell lines formed significantly more mammospheres when cultured in the presence of vitamin K, suggesting that γ-carboxylation may enhance stemness. Further studies to identify the GLA-modified proteins generated in TNBC cells treated with vitamin K and their mechanisms of action may lead to new therapeutic targets for aggressive TNBC. 

### Experimental Agents and Combinations for Hematologic Malignancies 1

#791

CG'806, a first-in-class pan-FLT3/pan-BTK inhibitor, demonstrates superiority to other FLT3 and BTK inhibitors against primary patient samples.

Stephen E. Kurtz,1 Kevin Watanabe-Smith,1 Dan Bottomly,2 Beth Wilmot,2 Shannon Mcweeney,2 Andrea Local,3 Hongying Zhang,3 Stephen Howell,4 William Rice,3 Brian J. Druker,5 Jeffrey W. Tyner6. 1 _Knight Cancer Institute, Division of Hematology and Medical Oncology, Oregon Health & Science University, Portland, OR; _2 _Knight Cancer Institute, Division of Bioinformatics and Computational Biology, Oregon Health and Science University, Portland, OR;_ 3 _Aptose Biosciences, Inc, San Diego, CA;_ 4 _UC San Diego Moores Cancer Center, San Diego, CA;_ 5 _Knight Cancer Institute, Oregon Health & Science University, Portland, OR; _6 _Oregon Health & Science University, Knight Cancer Institute, Portland, OR_.

Introduction: While acute myeloid leukemia (AML) is a complex and heterogeneous malignancy, the most common mutation is the internal tandem duplication (ITD) of FLT3 occurring in ~30% of AML patients. Several FLT3 inhibitors have transient clinical benefit, lasting only 3-4 months, often due to emergence of drug resistant clones with additional mutations in FLT3. Thus, there is need for a pan-FLT3 inhibitor to control all mutant forms of FLT3, and to suppress diverse oncogenic clones. Likewise, overexpression of Bruton's tyrosine kinase (BTK) is a driver of chronic lymphocytic leukemia (CLL). Ibrutinib, a covalent BTK inhibitor approved for CLL and certain other B-cell malignancies, is limited by resistance resulting from mutation at cysteine residue 481 to serine (BTK-C481S), prompting a need for new agents to inhibit all forms of BTK.

CG'806 is a small molecule that inhibits wild type (WT) FLT3, FLT3-ITD and tyrosine kinase domain (TKD) point mutations, including D835G, D835Y, D835H, F691L; it also inhibits BTK WT and BTK-C481S. We profiled CG'806 against primary samples from patients with AML, CLL and other hematologic malignancies to determine its activity and potency relative to other FLT3 inhibitors. CG'806 and ibrutinib were compared directly for sensitivities on primary CLL samples and various B-cell and other hematologic malignancies.

Methods: Inhibitor activity was assessed by an ex vivo assay to determine sensitivities of CG'806 and other FLT3 and BTK inhibitors on freshly isolated primary patient samples. Cell viability was assessed after 72-hour culture using a tetrazolium-based MTS assay and IC50 values calculated as a measure of drug sensitivity. For AML samples, mutational status of ITD and TKD in FLT3 were assessed by a PCR assay for ITD and by whole exome sequencing for TKD.

Results: CG'806 has more potent activity against a broader subset of AML samples relative to other FLT3 inhibitors, including midostaurin, gilteritinib, quizartinib, sorafenib, crenolanib, and dovitinib. This was especially true in FLT3-ITD and FLT3-TKD positive cases, although enhanced activity was also observed in FLT3 WT samples. CG'806 potency correlated with FLT3-ITD allele frequency as cases with higher allelic frequency generally had greater sensitivity in comparison to lower allelic frequency cases. CG'806 had greater activity on primary CLL samples than ibrutinib, which may be due to the dual activity of CG'806 against CSF1R (FMS) (IC50 = 0.6nM), a recently described target in CLL that provides a pro-tumor signal from nurse-like monocyte/macrophage lineage cells.

Conclusions: CG'806 is a potent and broadly active FLT3 inhibitor in AML and BTK inhibitor in CLL. These data reveal CG'806's enhanced activity relative to kinase inhibitors currently approved for each indication and support further clinical development of CG'806 for both AML and CLL.

#792

A novel covalent reversible BTK inhibitor for B cell malignancies with enhanced selectivity and in vivo efficacy.

Xi Chen,1 Shaoqiang Huang,1 Fred Hu,2 Jun Xian,3 Jian Yang,1 Jing Qiang,2 Meijie Le,2 Yuchuan Wu1. 1 _Sinovent Pharma, Beijing, China;_ 2 _Sinovent Pharma, Suzhou, China;_ 3 _Yukon Pharma, Southborough, MA_.

Bruton's tyrosine kinase (BTK), a member of Tec family kinase, plays a pivotal role in the regulation of BCR signaling, which is important for B cell development and function. Ibrutinib, which has been approved by FDA, is an effective treatment option for multiple types of B cell neoplasms. However, the treatment associated adverse events (AEs) are one of the major concerns due to its poor kinase selectivity . Here, we present a novel covalent, but reversible BTK inhibitor, XNW-1011, which demonstrated enhanced potency, better selectivity, improved drug-like properties and superior in vivo efficacy with a favorable safety profile. XNW-1011 was developed structurally and biochemically differently from current BTK inhibitors, being more potent and highly selective. In the in vitro assay, unlike Ibrutinib, it did not inhibit EGFR, ITK, JAK3, LYN and SRC. It completely suppressed the cell proliferation of a panel of lymphoma cell lines such as TMD-8 and Rec-1. Besides, XNW-1011 inhibited cellular phosphorylation of BTK and calcium efflux, confirming the inhibition of activation of BTK in vitro. We have shown that XNW-1011 formed covalent bond with cysteine in a cysteine reactivity assay. In addition, a washout assay by Western Blot confirmed the reversibility of inhibition of XNW-1011 to BTK. XNW-1011 has a good prolife of ADME, which translated into improved in vivo PK properties over Ibrutinib (low clearance and prolonged half-life). XNW-1011 was further evaluated in several animal models such as REC-1 and TMD-8 tumor xenografts. XNW-1011 inhibited tumor growth with as low as 3.125 mpk by BID and resulted in tumor regression with 12.5 mpk by BID. Interestingly, XNW-1011 completely inhibited the tumor growth of a patient derived xenograft (PDX) with Myd88 mutation. The dose proportionality was confirmed through determination of the plasma concentration and in vivo BTK occupancy in the last day of treatment. In summary, XNW-1011 is a potent, selective, covalent reversible BTK inhibitor with improved ADME prolife and in vivo efficacy, which makes it suitable for further evaluation in clinical setting.

#793

The novel Bruton's tyrosine kinase inhibitor ARQ531 disrupts survival signaling and triggers apoptosis in AML cells.

soncini debora,1 stefania orecchioni,2 antonia cagnetta,1 veronica retali,1 samantha ruberti,1 paola minetto,1 paola contini,1 alessio nencioni,1 fiammetta monacelli,1 Terence Hall,3 marco gobbi,1 sudharshan eathiraj,3 briam schwartz,3 francesco bertolini,2 roberto lemoli,1 michele cea1. 1 _University of Genoa, genova, Italy;_ 2 _European Institute of Oncology, Milan, Italy;_ 3 _ARQULE, Burlington,, MA_.

Introduction: Currently available therapeutics against Acute Myeloid Leukemia (AML) have improved patient outcome. However, resistance develops even to novel therapies and patient overall survival remains low, especially for patients who are not eligible for allogeneic bone marrow transplantation. Therefore, there is an urgent need to overcome the biologic mechanisms underlying drug resistance in AML, to both enhance the efficacy of existing treatments and to facilitate the design of novel approaches. The Bruton's tyrosine kinase (BTK) is emerging as new therapeutic target in a wide range of hematologic malignancies including AML, especially those carrying FLT3-ITD mutation. ARQ 531 is an ATP competitive, orally bioavailable, potent inhibitor of BTK and other relevant kinases. Herein we present preclinical data with ARQ 531 in AML and its efficacy compared with the standard BTK inhibitor Ibrutinib.

Methods: Inhibitory effects of ARQ531 on cell viability were investigated in a panel of AML cell lines as well as primary tumor cells. The effect of ARQ531 on BCR signaling was investigated by western blot. Specific transcriptomic profiling of ARQ531 treated AML cells was performed by RNA-Seq. NSG mice engrafted with primary AML cells were used to determine anti-AML activity of ARQ531 in vivo.

Results: ARQ531 blocked phosphorylation of BTK and downstream protein PLCγ in a panel of AML cell line regardless of their genetic background. ARQ531 also inhibited cell viability of AML cell lines (n=11) and primary AML cells (n=12) with IC50 values lower than reference compound (0.9±1 µM compared with 19±0.6 µM of Ibrutinib). Moreover, ARQ531 effects were not reduced in presence of normal or leukemic mesenchymal stromal cells (MSCs) and, more importantly such treatment showed greater therapeutic window than standard: IC50 value on CD34+ cells from healthy donors was >10µM and ±3 µM after ARQ531or ibrutinib, respectively. A transcriptome profiling analysis revealed a reversion of the oncogenic MYC-driven transcriptional program as specific event triggered by ARQ531. As result, Myc-targets inhibition was observed in AML cells treated with ARQ531 compared with ibrutinib. Finally, the anti-tumor activity of ARQ 531 was determined in AML-PDX model. At 31st day after cell transfer, flow cytometry evaluation of the circulating human CD45+ cells in the murine PB revealed a significant lower leukemia burden after ARQ531 treatment compared with Ibrutinib (10 ± 0.1% and 40 ± 0.01%, respectively; p=0.006)

Conclusion: The novel BTK inhibitor ARQ 531 is a highly potent kinase inhibitor with promising activity against AML in preclinical models.

#794

CG'806, a first-in-class pan-FLT3/pan-BTK inhibitor, targets multiple pathways to kill diverse subtypes of acute myeloid leukemia and B-cell malignancy in vitro.

Hongying Zhang,1 Andrea Local,1 Khalid Benbatoul,1 Peter Folger,1 Susan Sheng,1 Cheng-Yu Tsai,2 Stephen B. Howell,2 William G. Rice1. 1 _Aptose Biosciences, Inc, San Diego, CA;_ 2 _University of California San Diego, San Diego, CA_.

Small molecules targeting the FLT3 kinase with an internal tandem duplication mutation (FLT3-ITD) and the Bruton's Tyrosine Kinase (BTK) have shown great potential to treat hematologic malignancies. However, acquired mutations have emerged in resistant/relapsed patients treated with the FLT3 inhibitor quizartinib (Smith et al., 2012) or the BTK inhibitor ibrutinib (Woyach et al., 2017), respectively. This study explored the potency and molecular mechanisms of CG'806, a pan-FLT3/pan-BTK inhibitor, in hematologic malignancies relative to other FLT3 or BTK inhibitors commercialized or in development.

In FLT3-ITD AML cells, CG'806 induced apoptosis through inhibition of FLT3 signaling (decreased phospho-FLT3, -STAT5 and -ERK) and promotion of G0/G1 cell cycle arrest determined by immunoblotting and flow cytometry, and CG'806 was approximately 10-fold more potent than quizartinib. Although FLT3-ITD is found in 25-30% of AML patients, most AML patients express wild type (WT) FLT3. CG'806 had an IC50 = 11 nM against FLT3 WT-transfected Ba/F3 cells and was superior to quizartinib, gilteritinib and crenolanib FLT3 inhibitors (1,956, 500 and 2,617 nM, respectively). In FLT3-WT AML cell lines, or Ba/F3 cells transfected with FLT3-WT, D835Y, ITD+D835Y, or ITD+F691L, CG'806 markedly decreased phosphorylation of BTK, aurora kinases (AURK) and H3S10, resulting in G2/M arrest or polyploidy, and apoptosis with less or no effect on FLT3-WT activity. In contrast, quizartinib did not affect BTK or AURK signaling, and CG'806 was >2,000-fold more effective than quizartinib on FLT3-WT AML cells in MTS based cell proliferation assays.

In B cell malignancies, BTK signaling plays a pivotal pathogenic role. CG'806 decreased BTK phosphorylation in all malignant B cell lines tested (n =10) and inhibited cell proliferation and colony formation 50-6,000 times more potently than ibrutinib, an effect which could not be explained by the exclusive inhibition of BTK signaling. Further analysis revealed CG'806 effectively inhibited AURK signaling and caused polyploidy and apoptosis in B cell lines, sequelae which were not induced by ibrutinib. BTK-C481S is the most common mutation induced by treatment with ibrutinib and is associated with ibrutinib resistance in the clinic. CG'806 equivalently inhibited BTK-WT and BTK-C481S in HEK293 transfected cells, whereas ibrutinib was much less potent against the BTK-C481S mutant.

The ability of CG'806 to target all WT and mutant forms of FLT3 and BTK, and to inhibit multiple signaling pathways, produces killing of diverse subtypes of hematologic malignancies driven by different genomic aberrations. Considering the efficacy of CG'806 in the absence of observed toxicity in murine AML models, CG'806 appears superior to other FLT3 or BTK inhibitors and is suitable for development in patients with either AML or B-cell malignancy.

#795

Isatin analog for the treatment of acute myeloid leukemia.

Krishne Gowda,1 Charyguly Annageldiyev,1 Dhimant Desai,1 Shantu Amin,1 David Claxton,1 Arati Sharma2. 1 _Pennsylvania State University College of Medicine, Hershey, PA;_ 2 _Hematology/Oncology, Hershey, PA_.

Acute myeloid leukemia (AML) is a heterogeneous disease with therapeutic modalities relying on traditional cytotoxic therapies and hematopoietic stem cell transplantation. Current first-line cytotoxic chemotherapy exhibits limited success, with 50% of younger patients and 80% of older patients succumbing to the disease. There is an urgent unmet need for new therapeutics in acute myeloid leukemia (AML) as standard therapy has not changed in the past three decades and outcome remains poor for most patients. Our group has reported cytotoxic potential of dibromo-derivative of isatin, 5,7-dibromo-N-(p-thiocyanomethylbenzyl)isatin (KS99) against variety of solid tumors and multiple myeloma (MM) cells by inhibiting BTK, AKT and tubulin polymerization. In this study, we examined the ability of KS99, to inhibit the growth of panel of human AML cell lines and primary samples collected from AML patients. After 48h exposure, KS99 inhibited the growth of AML cell lines (IC50, 150-225nM) and primary blast samples (IC50, 600-1300nM) in a dose dependent manner. KS99 treatment also selectively reduced the clonogenicity of human acute myeloid leukemia patient cells compared to the hematopoietic stem cells. We also found that KS99 induced apoptosis by modulating expression of Mcl-1 and Bcl-2. Further studies will aim to determine the mechanism of action of KS99 and as a single agent and in combination with anti-leukemia agents and demonstrate the preclinical efficacy in AML animal models including AML patient derived xenografts. Together, these data demonstrate the therapeutic efficacy of Isatin analog and suggest that KS99 should be further developed as a novel therapeutic agent for AML.

#796

The BTK inhibitor BGB-3111 is synergistic with other anti-lymphoma targeted agents.

Chiara Tarantelli,1 Lu Zhang,2 Elisabetta Curti,1 Filippo Spriano,1 Eugenio Gaudio,1 Luciano Cascione,1 Alberto Arribas,1 Emanuele Zucca,2 Anastasios Stathis,2 Davide Rossi,1 Francesco Bertoni1. 1 _Università della Svizzera italiana, Institute of Oncology Research, Bellinzona, Switzerland;_ 2 _Oncology Institute of Southern Switzerland, Bellinzona, Switzerland_.

Introduction. BGB-3111 is a novel generation irreversible BTK inhibitor (Hu et al AACR2017) under active clinical investigation for lymphoid tumors. Here, we evaluated BGB-3111 in combination with other anti-cancer agents on a panel of human lymphoma cell lines.

Methods. Cell lines derived from activated B cell (ABC) diffuse large B cell lymphoma (DLBCL) (n=3), mantle cell lymphoma (MCL) (n=2) and chronic lymphocytic leukemia (CLL) (n=2) were exposed to increasing doses of BGB-3111 alone and in combination with increasing doses of other compounds (72h). Synergy was assessed with Chou-Talalay combination index (CI): synergism (<0.9), additive (0.9-1.1), antagonism/no benefit (> 1.1). Cell cycle analysis was performed after 24h of treatment.

Results. As single agent BGB-3111 showed anti-tumor activity in the nanomolar range in two ABC-DLBCL (TMD8, IC50 0.4 nM; OCI-LY-10, 1.5 nM) and in one MCL (REC1, IC50 0.9 nM) cell lines, while the remaining four cell lines resulted resistant (IC50s > 5µM). The pattern of activity was similar to what seen with ibrutinib and other 2nd generation BTK inhibitors (Gaudio et al, ENA 2016).

BGB-3111 was then valuated in combination with targeted agents. In ABC-DLBCL, synergism was achieved in 3/3 cell lines when BGB-3111 was combined with the MEK inhibitor pimasertib or with BCL2 inhibitor venetoclax. The combination with BET inhibitor OTX-015 was synergistic in 2/3 cell lines, while the combination with the XPO1 antagonist selinexor was beneficial in 2/3 (1 synergism, 1 additive).

In CLL cell lines, the best combinations were BGB-3111 with OTX015 or with selinexor with 2/2 synergisms. The results of the combinations with pimasertib or venetoclax were discordant (pimasertib, 1 synergism, 1 no benefit; venetoclax, 1 synergism, 1 no benefit).

Both MCL cell lines achieved synergism combining BGB-3111 with pimasertib, or selinexor, or venetoclax. The combination with OTX-015 was also beneficial, but synergism was observed in only one of the two cell lines, and additive in the remaining.

The improved anti-tumor activity of the combination versus the single agents were confirmed performing cell cycle analysis in an ABC-DLBCL (OCI-LY-10) with an increased subG0 phase when BGB3111 was combined with venetoclax, pimasertib and OTX-015.

Conclusion. BGB-3111 was active as single and the combination with inhibitors of key regulatory pathways in cell lines derived from ABC-DLBCL, CLL and MCL.

#797

Targeting HDAC3 reactivates immunosurveillance and enhances immunocheckpoint activity in B-cell lymphoma.

Patrizia Mondello,1 Matt Teater,1 Lorena Fontan,1 Matthew Durant,1 Elisa de Stanchina,2 Giorgio Inghirami,1 Michael Green,3 Ari Melnick1. 1 _Weill Cornell, New York, NY;_ 2 _Memorial Sloan Kettering Cancer Center, New York, NY;_ 3 _MD Anderson Cancer Center, Houston, TX_.

Somatic mutation of the histone acetyltransferase CREBBP occurs in ~40% of diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL) patients. CREBBP mutation leads to transcriptional repression of genes involved in B-cell differentiation, antigen presentation and cell growth, and this repressive effect is mediated specifically through HDAC3. Hence CREBBP mutant DLBCL cells are biologically dependent on HDAC3 for both cell autonomous effects on cell proliferation and evasion of immune surveillance. Because of this we hypothesize that CREBBP mutant DLBCL would be highly responsive to HDAC3 specific inhibition.Therefore we treated a panel of DLBCL cell lines with increasing dose of the clinically relevant HDAC3 selective inhibitor (HDAC3i) KDAC0001 (from 0.1 to 20 μM), which resulted in a dose- and time-dependent growth inhibition and cell death. HDAC3i treatment demonstrated a stronger efficacy in cells carrying CREBBP mutation compared to the wild type with an IC50 20-30 fold lower at 72 Hrs. Knockdown by shRNA or loss of function mutations introduced via CRISPR in CREBBP wt cell lines (MD901, OCI-LY18 and RL) led to increased sensitivity to KDAC0001 compared to the parental lines. Along these lines we placed six primary DLBCL specimens (3 GCB and 3 ABC) in our 3D organoid culture system and observed potent growth suppression by KDAC001 vs vehicle after 6 days. To identify the genes through which HDAC3 drives the lymphoma phenotype we performed RNA Seq analysis using 3 DLBCL cell lines (OCI-LY1 CREBBP wt, OCI-LY19 and OZ CREBBP m) treated with either DMSO or KDAC0001 at 10 μM for 24 Hrs (a timepoint prior to cells manifesting any response to drug). We observed induction of over 1100 genes whereas 74 genes were downregulated. Gene pathway analysis showed that HDAC3 induced genes corresponded to those that are repressed in CREBBP mutant lymphomas, and regulated by BCL6-HDAC3 complexes linked to gene enhancers, including their known downstream p53 and NFKB target genes. On the other hand KDAC0001 treatment also broadly induced MHC class II and antigen presentation genes. We confirmed the increase in MHC class II genes, CIITA, PD-1 and PD-L1 not only at the transcriptional level, as determined by qPCR, but also at the protein level, as determined by flow cytometry. Co-culturing OCI-LY18 DLBCL cells and human lymphocytes with increasing amounts of HDAC3i was associated with increased cytotoxicity in a time-dependent manner. We engrafted three CREBBP wild type primary human PDX in NSG mice and treated with either vehicle or KDAC0001 at 25 or 50 mh/kg daily for 21 days. Even thought these PDX were not CREBBP mutant, there was still cell autonomous activity against all these tumors. In conclusion, HDAC3 inhibitor KDAC0001 has both cell autonomous and immune related activities.Therefore, it can be used as a novel form of "epigenetic immunotherapy" to restore immune clearance of resistant DLBCL and FL.

#798

Pathway-directed high-throughput drug screen identifies PI3K inhibitors that synergistically potentiate antitumor activity of HDAC inhibitors in cutaneous T-cell lymphoma.

Chi-Heng Wu,1 Chen-Yen Yang,2 Taha Rakhshandhroo,3 Laura Pincus,1 Sourav Bandyopadhyay,1 Frank McCormick,1 Weiyun Ai1. 1 _University of California, San Francisco, San Francisco, CA;_ 2 _Pharmacyclics, Inc., Sunnyvale, CA;_ 3 _University of California, San Diego, La Jolla, CA_.

Background: Cutaneous T-cell lymphoma (CTCL) represents a type of malignancy in the skin that is incurable. Recent studies demonstrated dysregulation of several signaling pathways in CTCL, including PI3K/AKT, JAK/STAT, and NFκB pathways. We performed a high-throughput drug screen to determine the potential of novel agents targeting these pathways for the treatment of CTCL.

Methods: We compiled a library of 94 compounds targeting pathways known to be relevant in cancer biology, such as kinases involved in growth factor receptor signaling, HDACs, proteasome, DNA repair and regulators of apoptosis. The compounds were screened for antiproliferative activity against four CTCL cell lines in high-throughput proliferation assays. PI3K inhibitors were selected from the "hits" of the screen and were further studied in xenograft models of CTCL and in primary T-cell lymphomas cells from patients. To examine which PI3K isoform mediates antitumor effect in CTCL, isoform-specific siRNAs were used to knock down each isoforms as well as the combination of doublet isoforms. Based on this experiment, we tested antitumor activity of isoform-specific inhibitors. Promising candidates were also tested in combination assays using a matrix block method designed to detect synergistic activities.

Results: From the high-throughput screen, we identified 14 compounds with antiproliferative activity in CTCL, including multiple inhibitors of the PI3K pathway. Validation assays confirmed the class effect of PI3K inhibitors in growth inhibition. From this class, a pan-PI3K inhibitor BKM120 was selected for in vivo studies. In a xenograft model of CTCL, BKM120 exhibited striking antitumor activity measured by a marked suppression of tumor growth and prolonged survival of tumor-bearing mice compared with vehicle control. To investigate which isoform of the PI3K mediates the antitumor effect in CTCL, we performed isoform-specific siRNA assays and demonstrated that knocking down isoform ð exhibited the most potent growth inhibition. Copanlisib targets α and δ isoforms of PI3K and is newly approved by the FDA for low-grade B-cell lymphoma. We demonstrated that copanlisib exhibits potent antiproliferation activity in CTCL cell lines. Furthermore, combination of copanlisib and LBH, a HDAC inhibitor, display synergistic antiproliferative effects across a broad dose gradient for each agent.

Conclusion: A pan-PI3k inhibitor, BKM120, is highly active in CTCL cell lines and exnograft models. Inhibition of Isoform δ exhibits the most potent growth inhibition. Furthermore, an isoform-specific inhibitor, copanlisib, synergistically potentiates the effect of HDAC inhibitors against CTCL. These are highly promising approaches for the treatment of CTCL and warrant clinical investigation.

#799

The novel histone deacetylase inhibitor pracinostat is an effective anti-lymphoma agent.

Afua A. Mensah,1 Filippo Spriano,1 Eugenio Gaudio,1 Chiara Tarantelli,1 Luciano Cascione,1 Luca Aresu,2 Emanuela Lovati,3 Emanuele Zucca,4 Anastasios Stathis,4 Claudio Pietra,3 Francesco Bertoni1. 1 _Università della Svizzera italiana, Institute of Oncology Research, Bellinzona, Switzerland;_ 2 _Department of Comparative Biomedicine and Food Science, University of Padua, Padua, Italy;_ 3 _Helsinn Healthcare SA, Lugano, Switzerland;_ 4 _IOSI Oncology Institute of Southern Switzerland, Bellinzona, Switzerland_.

Background. Currently, 3 histone deacetylase inhibitors (HDACi) are clinically approved for the treatment of T-cell lymphomas: vorinostat, romidepsin and belinostat. Pracinostat (SB939) is a class I, II and IV HDACi (Novotny-Diermayr et al, Mol Cancer Ther 2010), in phase 3 in combination with azacitidine for acute myeloid leukemia patients who are unfit to receive intensive remission induction chemotherapy (NCT03151408). Here, we tested it on a large panel of lymphoma cell lines alone or in combination with a BCL2 inhibitor, venetoclax and with 5-azacitidine.

Methods. 60 cell lines derived from T-cell lymphoma (10), diffuse large B cell lymphoma (DLBCL, 25), mantle cell lymphoma (10), marginal zone lymphoma (5), Hodgkin lymphoma (4), chronic lymphocytic leukemia (2), primary mediastinal B-cell lymphoma (1), murine (2) and canine (1) lymphomas were exposed to increasing doses of pracinostat and vorinostat alone or to pracinostat in combination with venetoclax or 5-azacitidine for 72h. Cell proliferation was assessed using the MTT assay. Chou-Talalay index was used to determine synergism. For apoptosis and cell cycle analyses, cells were treated with 250 nM pracinostat for 72h then stained with Annexin V and 7-AAD (apoptosis) or fixed then stained with 7-AAD (cell cycle) before flow cytometry.

Results. All lymphoma subtypes responded well to pracinostat (median IC50 250 nM; 95%C.I., 171-324 nM). Pracinostat compared favorably with vorinostat (306 nM; 95%C.I., 241-363 nM), and the anti-proliferative activity of the two compounds was correlated (R=0.8, P<0.0001).

Comparison of consensus cluster classification DLBCL subgroups showed that BCR DLBCL were more sensitive to HDACi than OxPhos DLBCL (pracinostat median IC50 BCR: 171 nM, OxPhos: 865 nM, P 0.001; vorinostat median IC50 BCR: 246 nM, OxPhos: 829 nM, P 0.01. No differences were observed between the DLBCL subtypes based on the cell of origin.

Pracinostat mostly provoked cell cycle arrest in the S and G2/M phases with S-phase arrest associated with OxPhos and G2/M arrest with BCR DLBCL. Pracinostat caused pronounced apoptosis in 2/8 DLBCL cell lines.

Functional annotation analysis to compare DLBCL with IC50 values < 200 nM (n=12) and DLBCL with IC50 > 400 nM (n=7) revealed an enrichment of mitochondrial metabolic pathways, DNA repair and cell cycle regulation in DLBCL with IC50 >400 nM. Combination with venetoclax was beneficial in 3/3 cell lines, while the addition of 5-azacitidine benefited 2 out of 3 cell lines.

Conclusions. Pracinostat robustly inhibits the proliferation of lymphoma cells with a similar IC50 range to vorinostat. Its anti-proliferative activity can be cytotoxic or cytostatic. Combination of pracinostat with other compounds further inhibits lymphoma cell proliferation. In DLBCL, the OxPhos phenotype and an enrichment of mitochondrial metabolism, DNA repair and cell cycle pathways are associated with a poorer response to pracinostat.

#800

ABBV-744, a first-in-class and highly selective inhibitor of the second bromodomain of BET family proteins, displays robust activities in preclinical models of acute myelogenous leukemia.

Xiaoyu Lin,1 Xiaoli Huang,1 Richard Bellin,1 Emily Faivre,1 Paul Hessler,1 Lloyd Lam,1 Mai Ha Bui,1 Denise Wilcox,1 Tamar Uziel,1 Debra C. Ferguson,1 Terrance J. Magoc,2 Daniel H. Albert,1 Keith F. McDaniel,1 Warren Kati,1 Yu Shen1. 1 _AbbVie Inc., North Chicago, IL;_ 2 _Abbvie retiree, IL_.

Many small-molecule inhibitors that target both bromodomains of the BET family proteins (pan BET inhibitors) are undergoing studies in clinical trials. Emerging data are beginning to suggest that clinical responses to these pan BET inhibitors in subsets of hematologic malignancies may be modest and short lived, perhaps due, at least in part, to tolerability issues that limit dosing levels. We hypothesized that selective inhibition of four of the eight bromodomains in BET family proteins might retain the anticancer activities in certain tumor subsets while alleviating some of the tolerability liabilities of pan BET inhibitors, thus possibly providing better therapeutic benefits. ABBV-744 is a highly selective inhibitor for the second bromodomain (BDII) of the four BET family proteins, exhibiting greater than 300-fold more potent binding affinity to the BDII bromodomain of BRD4 relative to the first bromodomain (BDI) of BRD4. In contrast to the broad antiproliferative activities observed with pan BET inhibitors, ABBV-744 only displayed significant antiproliferative activities in a limited number of cancer cell lines, including AML and androgen receptor (AR)-positive prostate cancer. Studies in AML xenograft models demonstrated antitumor efficacy for ABBV-744 that was comparable to the pan-BET inhibitor ABBV-075 but with improved tolerability. Taken together, these results suggest that ABBV-744 could be a promising second-generation BET inhibitor for AML therapy.

Affiliation: Oncology Discovery, AbbVie Inc., 1 North Waukegan Rd., North Chicago, IL 60064. Disclosures: All authors are employees of AbbVie. The design, study conduct, and financial support for this research were provided by AbbVie. AbbVie participated in the interpretation of data, review, and approval of the publication.

#801

Cellular pharmacokinetics and molecular pharmacodynamics studies of a novel BET inhibitor NHWD870 in sensitive and resistant leukemic cell lines.

Maria E. Riveiro,1 Samuel Huguet,2 Marie-francoise Gauzan,2 Mohamed Bekradda,1 Nenghui Wang,3 François Lokiec,2 Olivier Madar,2 Keyvan Rezai2. 1 _Early Drug Development Group, Boulogne-Billancourt, France;_ 2 _Institut Curie-René Huguenin Hospital, Saint Cloud, France;_ 3 _Ningbo Wenda Pharma Technology LTD, Zhejiang, China_.

Background: NHWD870 (Ningbo Wenda Pharma, China) is a novel oral bromodomain and extraterminal (BET) protein family inhibitor. BET proteins are transcriptional co-activators that interact with multiple co-regulatory molecules at gene promoters and enhancers. BET inhibitors disrupt transcriptional regulatory complexes and have broad anticancer activity. The relation between the cellular pharmacokinetics (PK) and targeted-gene regulation governing the biological effects of BET inhibitors is still largely not understood. We analyzed cellular PK properties and CMYC and related genes modulation in NHWD870-sensitive and resistant leukemic cell lines.

Materials and Methods. Established human cell lines from acute and chronic myeloid leukemia (HL-60, U937, K562), acute lymphoblastic leukemia (Jurkat, MOLT-3) and SET-2 a JAK2 V617F mutated-megakaryoblastic cell line were studied. Anti-proliferative effects of NHWD870 were assessed by MTT after 72h-exposure. For cellular PK studies, cells were seeded at 2.106cells/ml and exposed to 245.5 ng/ml (500nM) NHWD870 for 0, 3, 10, 30, 60, 120 and 180 min. At each time point, NHWD870 extracellular and intracellular concentrations were analyzed in cell supernatants and pellets respectively, using Ultra Performance Liquid Chromatography with tandem Mass Spectrometry (concentration range 0.1-100 ng/mL). In cell pellets, protein modulation was analyzed by Western blot using commercial antibodies.

Results: IC50 values were between 20-600 nM in HL60, U937, SET-2 and Jurkat cells and were ≥ 1,000 nM for other cell lines. Cellular uptake of NHWD870 was rapid (3 min) in both sensitive and resistant leukemic cell lines, with a mean concentration of ~5.0 ng/106 cells (3-8.5 ng/106cells) and ~3.1 ng/106 cells (2.8-3.5 ng /106cells) for HL60 and K562 cell line respectively, whereas extracellular levels of NHWD870 were stable with up to 3h exposure. In the resistant cell line, K562, we observed CMYC mRNA downregulation after 2h exposure, even though NHWD870 intracellular levels were detected after 5 min exposure. A rapid downregulation of CMYC and P21 proteins were observed in the sensitive cell lines, HL60, U937, SET-2 and Jurkat, whereas other BET-targeted genes, such as HEXIM and BCL2 levels were modulated after longer exposure time points in sensitive cell lines.

Conclusions: NHWD870 intracellular accumulation occurred to a similar extent and timing in NHWD870 sensitive and resistant leukemic cell lines. Rapid modulation of NHWD870-target genes was observed at the protein levels only in sensitive cell lines.

#802

Targeted inhibition of KDM1A and BET proteins or DNMT1 exerts synergistic lethal activity against human AML blast progenitor cells.

Warren C. Fiskus,1 Christopher P. Mill,1 Dyana T. Saenz,1 Agnieszka J. Nowak,1 Baohua Sun,1 David N. Saenz,1 Steven M. Kornblau,1 Sunil Sharma,2 Kapil N. Bhalla1. 1 _The University of Texas MD Anderson Cancer Center, Houston, TX;_ 2 _Huntsman Cancer Institute, Salt Lake City, UT_.

KDM1A is an FAD-dependent amine oxidase that demethylates H3K4Me1/2 chromatin, which regulates enhancer maintenance and transcription in stem/progenitor cells. GFI1 is a zinc-finger transcriptional repressor that binds and recruits KDM1A-CoREST-HDAC complex to repress loci regulating lineage differentiation in hematopoiesis. In the present studies, we evaluated the anti-AML efficacy of KDM1A inhibition alone and in combination with BET protein inhibitor (BETi) or DNMT1 inhibitor. Tet-inducible shRNA to KDM1A depleted mRNA and protein levels of KDM1A, repressed c-Myc, de-repressed p21, CD11b, CD86 and CEBPα, as well as inhibited colony growth and modestly induced lethality in genetically diverse cultured AML cell lines, including MOLM13 and OCI-AML5 cells. Following knockdown of KDM1A in the AML cells, RNA-seq followed by gene-set enrichment analysis linked mRNA perturbations to pathways involving signal transduction, metabolism, RNA POL II transcription and its regulation. Treatment with the reversible KDM1A inhibitor SP2577 or the irreversible inhibitor ORY-1001 also inhibited cell proliferation and clonogenic survival, associated with induction of genes involved in myeloid differentiation, of AML OCI-AML5, MOLM13, THP1 and MV4-11 cells. KDM1A inhibitors also induced loss of cell viability in patient-derived CD34+ AML BPCs as well as in CD34+CD38- Lin- AML stem cells. SP2577 or ORY-1001 also disrupted binding of KDM1A with Co-REST in OCI-AML5, MOLM13 and THP1 cells. Utilizing CRIPSR/Cas9 technology, we gene-edited KDM1A in OCI-AML3 cells. Surviving clones exhibited ~50% KDM1A as well as decreased c-Myc and DNMT1 expression compared to control OCI-AML3 cells. By utilizing RNA-seq mRNA signatures from KDM1A-depleted AML cells, we also queried for expression mimickers (EMs) through LINCS1000-CMap (Connectivity Mapping) analyses. Among the top hits were pan-histone deacetylase inhibitors (HDIs), which we have previously reported to exert in vitro synergistic lethality in combination with KDM1A inhibitor (Leukemia 2014;28:2155-64). Utilizing publicly available chIP-seq data with anti-BRD4, anti-H3K27Ac and RNA Pol-II antibodies, we identified that the LSD1 promoter is occupied by BRD4 in the AML MOLM14 cells. Treatment with the BETi OTX015 depleted KDM1A expression in AML cells. Co-treatment with SP2577 and OTX015 synergistically induced apoptosis of cultured and PD CD34+AML BPCs and CD34+CD38- Lin- AML stem cells (combination indices < 1.0). Notably, co-treatment with KDM1A inhibitor (SP2577 or ORY-1001) and the DNMT1 inhibitor decitabine also resulted in synergistic growth inhibition and lethality of cultured and PD CD34+ AML BPCs (combination indices < 1.0). These findings strongly support further in vivo testing and preclinical development of KDM1A inhibitor-based combinations with BETi and DNMT1 inhibitor against AML BPCs.

#803

Bromodomain and extra-terminal BET inhibitors induce TP53 independent apoptosis, maturation and oncoprotein degradation in NPM1 mutated acute myeloid leukemia.

Thorsten Braun,1 Marie-Magdelaine Coudé,2 Jeannig Berrou,2 Hanene Djamai,2 Mélanie Dupont,2 Anna Kaci,2 Marc Delord,3 Raphael Itzykson,3 Emmanuel Raffoux,2 Caroline Berthier,4 Hugues de Thé,4 André Baruchel,5 Claude Gardin,1 Hervé Dombret2. 1 _Avicenne Hospital/Paris XIII University/Laboratoire de Transfert des Leucémies, EA3518, Bobigny, France;_ 2 _Saint Louis Hospital/Paris VII University/Laboratoire de Transfert des Leucémies, EA3518, Paris, France;_ 3 _Saint Louis Hospital/Paris VII University, Paris, France;_ 4 _INSERM/CNRS/University Paris Diderot, UMR 944/7212, Paris, France;_ 5 _Robert Debré/Paris VII University/Laboratoire de Transfert des Leucémies, EA3518, Paris, France_.

Background: Differentiation based therapy by all trans retinoic acid (ATRA) and arsenic trioxide (ATO) results in cure of >90% of patients with acute promyelocytic leukemia (APL). ATRA+ATO is highly biologically active in NPM1c AML, accounting for 30-40% of AML patients. ATO/ATRA induces proteasomal degradation of NPM1c, differentiation, growth arrest and TP53 dependent apoptosis in NPM1c cells. Furthermore, ATRA/ATO exposure restores nuclear localization of NPM1wt and significantly reduces blasts in NPMc AML patients. It was shown that the BET inhibitors OTX015/MK-8628 and JQ1 yield antileukemic activity and here we demonstrate their effects in NPM1c leukemia cells compared to ATRA/ATO. Methods : NPMc OCI-AML3 cell line or patient bone marrow (BM) blast cells obtained after informed consent were exposed to ATRA/ATO or OTX015/MK-8628 and JQ1. Apoptosis was assessed by annexin V/PI and caspase 3/PARP cleavage by WB. TP53 expression was detected by WB. Knock down of TP53 was performed with siRNA. Differentiation of OCI-AML3 cells was studied by CD11b surface expression and morphologic studies after MGG stain. Gene expression profiling was performed with GeneChip Array (Affymetrix®). NPMc expression was assessed by WB (+/- bortezomib) and cellular localization of NPMc/NPMwt was studied by immunofluorescence. Results : Exposure of OCI-AML3 cells to OTX015/MK-8628 and JQ1 was more potent to induce apoptosis as compared to ATRA/ATO. All treatments lead to caspase 3 and PARP cleavage. In OCI-AML3 cells, ATO-ATRA induced strong upregulation of genes of the TP53 dependent pathway (BAX/GADD45) while the anti apoptotic gene BCL2 was downregulated. In contrast, treatment with BET inhibitors lead to strong down regulation of the TP53 dependent pathway. In line, ATRA/ATO induced TP53 protein expression and TP53 knock down by siRNA decreased significantly ATRA/ATO induced apoptosis suggesting that apoptosis induced by BET inhibitors is TP53 independent. As compared to ATRA/ATO, OTX015/MK-8628 and JQ1 were more potent to induce differentiation as detected by CD11b surface expression and by morphologic analysis of OCI-AML3 cells. Interestingly, gene expression profiling of human leukocyte differentiation pathways in OCI-AML3 cells revealed different expression profiles for exposure to BET inhibitors compared to ATRA/ATO. Treatment of OCI-AML3 cells either by OTX015/MK8628, JQ1 or ATRA/ATO lead to proteosomal degradation of the NPMc protein. Exposure of OCI-AML3 cells and primary BM blasts of patients either by OTX015/MK8628, JQ1 or ATRA/ATO led to nuclear relocalization of NPMwt protein to the nucleus. Conclusion : BET inhibitors induce TP53 independent apoptosis, differentiation, proteasomal degradation and NPMwt relocalization in NPMc cells. Thus, clinical testing of bromodomain inhibitors in NPMc AML is indicated.

#804

Antitumor activity of the potent and novel FLT3 inhibitor HM43239 in acute myeloid leukemia.

Miyoung Lee, Young Eun Ha, Mi Jin Moon, Joo-Yun Byun, HyunKyung Yu, SeokJong Kang, JaeHo Lee, Kyuhang Lee, Eunkyung Kim, Eunyoung Kim, Ho Jeong Lee, YoungHoon Kim, YoungGil Ahn, KweeHyun Suh, Sun-Jin Kim. _Hanmi Pharm. Co., Ltd., Hwaseong-si, Republic of Korea_.

Introduction: Acute myeloid leukemia (AML) with FMS-like tyrosine kinase 3 (FLT3) mutations is associated with poor prognosis with a high risk of relapse after therapy and reduced overall survival. Activating mutations of internal tandem duplication (ITD) and tyrosine kinase domain point mutations (TKD) of FLT3 have been reported in approximately 30% of AML as oncogenic driver mutations. Currently, FLT3 inhibitors showed clinical benefits in the corresponding AML patients. In this study, we have characterized a novel FLT3 inhibitor, HM43239, and assessed the potential as a novel therapy for AML patients. Materials and Methods: HM43239 is a novel, highly potent FLT3 kinase inhibitor. This compound showed tight binding to FLT3 kinase in in silico docking model as a reversible Type I inhibitor. In vitro kinase assay was performed to identify kinase selectivity of HM43239. Standard proliferation assay, immunoblotting, and apoptosis analysis were carried out to validate the potency of HM43239 in AML cell lines. HM43239 was evaluated in MV-4-11 and MOLM-13 xenograft mice models. Tumor sizes were measured and tumor samples were analyzed of the mechanisms of action. Results: Among 191 kinases biochemically assayed, HM43239 showed the high selectivity toward FLT3 and AML associated other kinases (e.g. SYK, JAK and TAK1). IC50s' of HM43239 against FLT3 WT, FLT3 ITD and FLT3 D835Y kinases were 1.1 nM, 1.8 nM and 1.0 nM, respectively. HM43239 potently inhibited the growth of AML cell lines harboring FLT3 ITD mutation, such as MV4-11 (IC50: 1.3 nM), MOLM-13 (5.1 nM) and MOLM-14 (2.9 nM). Furthermore, HM43239 effectively inhibited the phosphorylation levels of FLT3 and of downstream kinases related with cell proliferation. In addition, caspase 3/7-dependent apoptosis was induced by HM43239 in AML cell lines expressing FLT3 ITD mutant. And HM43239 inhibited proliferation and induced apoptosis of leukemic stem cell (LSC) marker-expressing KG1a cells (CD34+/CD38- cells) suggesting that the possibility for targeting LSC. HM43239 showed the excellent dose proportional antitumor activity in mouse models xenografted with both MV4-11 and MOLM-13 cell line without any significant toxicity. Moreover, we identified in vivo modulation of related targets (p-FLT3 & p-STAT5) in AML cell with FLT3 mutant. Conclusion: Taken together, HM43239 has demonstrated the potential therapeutic efficacy for the treatment of AML patients and implicated the mechanism of overcoming resistance and preventing relapse.

#805

NMS-P088, a FLT3-KIT-CSF-1R inhibitor with activity on FLT3 F691L as a novel agent in AML.

Marina Ciomei, Elena Ardini, Gemma Texido, Rachele Alzani, Wilma Pastori, Dario Ballinari, Sabrina Cribioli, Fabio Gasparri, Nilla Avanzi, Daniele Casero, Daniele Donati, Arturo Galvani, Andrea Lombardi Borgia, Antonella Isacchi. _Nerviano Medical Sciences, Nerviano, Italy_.

FLT3, KIT and CSF1R are members of the class III receptor tyrosine kinase family, characterized by an autoinhibitory juxtamembrane (JM) domain that docks within the kinase domain to stabilize a catalytically inactive conformation. Activating rearrangements of the JM domain of FLT3 (FLT3-ITD) occur in 20-25% of AML and represent a driver of disease and a negative prognostic factor. Another 5-7% of AML harbours an activating D835 mutation in the activation loop of the kinase domain. Several FLT3 inhibitors, including quizartinib, crenolanib and gilteritinib, are currently in advanced clinical testing. Moreover midostaurin, a multikinase inhibitor with activity on FLT3, has been recently approved in FLT3 mut AML in combination with standard chemotherapy. Unfortunately, the clinical response to FLT3 inhibitors is shortened by the emergence of resistance mutations at critical residues such as D835 and F691, the so called "gatekeeper" residue. F961L mutations have been described to induce resistance to quizartinib, crenolanib and gilteritinib, making F691L an unmet medical need in AML. NMS-P088 is a potent and selective inhibitor of FLT3 and KIT kinases, including variants with both primary and secondary resistance mutations, as well as of CSF1R, with exquisite cellular selectivity for cell lines dependent on these targets. NMS-P088 has remarkable (subnanomolar) activity against the MOLM-13 and MV4-11 AML cell lines harbouring the FLT3-ITD rearrangement. On a panel of BA/F3 cells harbouring FLT3-ITD and its mutant forms NMS-P088 showed high potency and very significant activity on F691L, superior to that of key comparators tested in parallel. In the disseminated MOLM-13 AML model, repeated oral administration of NMS-P088 as single agent was able to significantly increase survival time, and showed synergy with cytarabine. The outstanding in vitro activity of NMS-P088 on BA/F3_FLT3-ITD harbouring the F691L mutation was also confirmed in vivo, with high TGI for NMS-P088, while quizartinib tested in parallel resulted not active. NMS-P088 has activity on CSF1-dependent macrophages both in vitro and in vivo and showed single agent efficacy in a syngeneic tumor model done in immunocompetent mice, with robust reduction of CSF-1R positive intratumoral macrophages, providing a rationale for testing in clinical setting as a modulator of host vs tumor response. GLP toxicity studies revealed good tolerability at efficacious exposures, with no cardiac effects and excellent BBB penetration. Thus NMS-P088, a potent FLT3 inhibitor with activity on the gatekeeper mutation, is a preclinical candidate with potential to address an unmet medical need in AML, both as single agent and in combination, as well as for testing in different solid tumors sensitive to immunomodulation.

#806

KI2011 is a novel potent FLT3 tyrosine kinase inhibitor in acute myeloid leukemia cells with FLT3-mutation.

Hwani Ryu, Jie-Young Song, Jiyeon Ahn. _Korea Inst. of Radiological & Medical Sci., Seoul, Republic of Korea_.

More than 30% of patients with acute myeloid leukemia (AML) harbor a mutation with internal tandem duplication (ITD) in the juxtamembrane domain and point mutations in the tyrosine kinase domain of FMS-like tyrosine kinase-3 (FLT3), which results in aberrant activation FLT3 by auto-phosphorylation, and confers poor prognosis. To develop novel FLT3 inhibitors, ten novel oxazole amine derivatives were designed and synthesized based on FLT3 protein structure. Among the synthesized small molecule compounds, KI2011 exhibited selectively inhibitory activities of the class III receptor tyrosine kinases, especially FLT3, wile-type, FLT3-D835Y mutant and FLT3-internal tandem duplication (ITD) mutant isoforms with IC50 values of 25, 9, and 1.2 nM, respectively, determined by an in vitro kinase activity assay. The compound decreased FLT3 phosphorylation and its downstream signaling in MV4-11 cells harboring FLT3-ITD mutation. Treatment of KI2011 selectively inhibited cell proliferation and induced apoptosis of MV411 cells in a concentration-dependent manner, but not in K562 (FLT3 negative) cells. In addition, we found that KI2011 enhanced radiosensitivity of MV4-11 cells. The results suggest that KI2011 is a novel potent FLT3 inhibitor that may be further developed in the preclinical studies as a therapeutic agent in AML treatments.

#807

Mechanism of action of synergistic activity of EZH2 inhibition and IMiDs in preclinical multiple myeloma models.

Allison E. Drew, Vinny Motwani, Lindsey Eichinger, Jesse Smith, Alejandra Raimondi. _Epizyme, Inc., Cambridge, MA_.

The EZH2 inhibitor tazemetostat (EPZ-6438) is currently being evaluated in phase 2 clinical trials for the treatment of non-Hodgkin lymphoma (NHL). EZH2 inhibitors have shown anti-proliferative effects in multiple preclinical models of NHL, mesothelioma, and molecularly defined solid tumors. Objective clinical responses have been reported in patients with B-cell lymphomas in phase 1 and in preliminary analysis of phase 2 studies of tazemetostat. Mounting evidence suggests that EZH2 regulates the maturation of both normal B-cells and B-cell lymphomas and may be an important mediator of cell fate in other B-cell malignancies. Consistent with this hypothesis, recent preclinical studies suggest that EZH2 may be a therapeutic target for treatment of multiple myeloma (MM), a disease arising from late stage B-cell lymphocyte plasmablasts. Inhibition of EZH2 has shown potent anti-proliferative effects in both in vitro and in vivo preclinical models of MM. Here, we show that synergistic anti-proliferative activity was observed when EZH2 inhibitors were combined with small molecule immune system modulators in both in vitro and in vivo multiple myeloma models and describe the mechanism of action of this enhanced effect. In particular, combination with IMiDs led to dramatic synergy across multiple MM cell lines. Like EZH2, Ikaros/Aiolos, the two important targets of IMiD response, are repressors of transcription. This led us to investigate the effects of the combination of tazemetostat and IMiDs on gene expression. Previous reports have indicated that both EZH2 and Ikaros/Aiolos play important roles in the regulation of interferon signaling via the IRF4 axis. We found that both agents singly elicited changes in interferon target genes, a phenomenon that could be enhanced when the two drugs were added in combination. Combination treatment also led to an increase in apoptosis as would be expected following downregulation of the IRF4 axis which is critical for MM cell survival. In this study, we provide evidence that EZH2 and Ikaros/Aiolos share a set of target genes, including IRF4, suggesting that these overlapping pathways may, in part, mediate the synergy between EZH2 inhibitors and IMiDs on cell viability. Based upon these in vitro findings, we conducted a xenograft study using the RPMI-8226 myeloma model and observed a notable enhancement in tumor growth inhibition with the combination EZH2 inhibitor and pomalidomide compared to the effect of either single agent. These data provide evidence of synergy between pomalidomide and EZH2 inhibitors in both in vitro and in vivo preclinical models, and provide a rationale for clinical development of this combination strategy for patients with multiple myeloma.

#808

Opportunity for therapeutic expansion in mantle cell lymphoma: Tazemetostat combination synergy status in preclinical MCL models.

Tami L. Hood, Kat Cosmopoulos, Allison Drew, Kelli Armstrong, Jennifer Johnson, Smith Jesse, Alejandra Raimondi. _Epizyme, Cambridge, MA_.

Tazemetostat (EPZ-6438) is a potent, selective, orally bioavailable small molecule inhibitor of EZH2, the enzymatic subunit of the polycomb repressive complex 2, which is currently being evaluated in multiple phase II clinical trials for the treatment of non-Hodgkin lymphoma, mesothelioma and molecularly defined solid tumors. Objective clinical responses have been observed in phase II studies of tazemetostat, including patients with B-cell lymphomas.

EZH2 has been shown to play a key role in the maturation of B-cells and, consistent with this phenomenon, multiple B-cell malignancies are dependent on EZH2 for survival. In preclinical studies in mantle cell lymphoma (MCL), a disease arising from mantle zone cells surrounding germinal centers, treatment with EZH2 inhibitors, in in vitro and in vivo models demonstrated antiproliferative activity, suggesting EZH2 may be a promising therapeutic target for MCL.

MCL is a distinct B-cell non-Hodgkin lymphoma characterized by the chromosomal translocation t(11;14) that leads to overexpression of cyclin D1. The disease most often presents at an advanced stage, and while initial responses occur, nearly all cases relapse or become resistant to frontline therapy and progress with poor prognosis. Novel targeted therapies are currently entering the clinic in combination with standard of care treatments, but heterogeneous response rates and durations of response show that there remains an unmet medical need in this population.

The most widely used MCL treatment regimens include combinations of two or more Standard of Care (SOC) agents. In order to support potential future clinical investigations with tazemetostat we established in vitro assays to assess its combinatorial activity with 24 traditional and emerging therapies across seven MCL cell lines. All compounds were evaluated for their single agent activity in order to determine the appropriate starting doses to investigate synergy. We then looked at both a seven-day pretreatment with tazemetostat alone followed by a four-day cotreatment with combination partner as well as a seven-day cotreatment including both drugs simultaneously. Synergy status was determined via Loewe, BLISS and additional mathematical models. Robust synergy was observed with glucocorticoid receptor agonists, immunomodulatory drugs, venetoclax and a variety of B-cell receptor pathway modulators in both dosing regimens. In addition, we show that EPZ011989 (an EZH2 tool compound) induces tumor growth inhibition in a MCL xenograft model, and has combination benefit with ibrutinib.

Preclinical data suggest that the sensitivity to tazemetostat as a single agent in MCL cell lines can be greatly enhanced in combination with new and emerging therapies. These combination approaches may be worthy of exploration in the clinic for the treatment of MCL.

### Growth Factors and Protein Kinases as Targets

#809

Afatinib restrains K-RAS driven lung tumorigenesis.

Herwig P. Moll,1 Klemens Pranz,2 Monica Musteanu,3 Luka Brcic,4 Helmut Popper,4 Judit Moldvay,5 Balázs Dome,1 Emilio Casanova2. 1 _Medical University of Vienna, Vienna, Austria;_ 2 _Ludwig Boltzmann Institute for Cancer Research, Vienna, Austria;_ 3 _Spanish National Cancer Research Centre (CNIO), Madrid, Spain;_ 4 _Medical University of Graz, Graz, Austria;_ 5 _Semmelweis University, Budapest, Hungary_.

Lung cancer is still the leading cause of cancer deaths worldwide. While it is well documented that patients suffering from epidermal growth factor receptor (EGFR) mutated non-small cell lung cancer (NSCLC) benefit from (EGFR) inhibition, there are still no successful targeted therapies for oncogenic mutations in the V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) gene available. Indeed, based on clinical trials using first generation EGFR tyrosine kinase inhibitors (TKI) it became a doctrine that K-RAS mutations drive resistance to EGFR inhibition in non-small cell lung cancer (NSCLC).

Conversely, we demonstrate here that EGFR signaling is engaged in K-RAS driven lung tumorigenesis both in human and in mice. Genetic mouse models revealed that deletion of Egfr quenches mutant K-ras activity and transiently reduces tumor growth. However, EGFR inhibition initiates a resistance mechanism which involves non-EGFR ERBB family members. This escape mechanism not only clarifies the disappointing outcome of first generation TKI in clinical trials, but also suggests therapeutic potential of pan-ERBB inhibitors. Indeed, by using several experimental models, including genetically engineered mouse models, (patient derived-) xenografts and in vitro experiments, we demonstrate that the FDA approved pan-ERBB inhibitor afatinib formidably reduces K-RAS driven lung tumorigenesis, whereas first generation TKI erlotinib and gefitinib remained ineffective in our models.

Altogether, our data strongly suggests revisiting the potential of EGFR TKI in clinical trials for the treatment of K-RAS mutated NSCLC, with a particular focus on pan-ERBB inhibitors, which are readily available.

#810

Anti-tumor effects of Sym015 in HGF overexpressing gastric cancer cell lines.

Hyun Jeong Kim,1 Sun Kyoung Kang,2 Michael Kragh,3 Ivan D. Horak,3 Woo Sun Kwon,2 Tae Soo Kim,2 Inhye Jeong,1 Joong Bae Ahn,4 Hyun Cheol Chung,4 Sun Young Rha4. 1 _Songdang Institute for Cancer Research, Brain Korea 21 PLUS Project for Medical Science, Yonsei University College of Medicine, Seoul, Republic of Korea;_ 2 _Songdang Institute for Cancer Research, Yonsei University College of, Seoul, Republic of Korea;_ 3 _Symphogen A/S, Ballerup, Denmark;_ 4 _Songdang Institute for Cancer Research, Brain Korea 21 PLUS Project for Medical Science, Division of Medical Oncology, Department of Internal Medicine, Yonsei Cancer Center, Yonsei University Health System, Seoul, Republic of Korea_.

MET, hepatocyte growth factor receptor (HGFR), has received considerable attention as a potential target for cancer therapy. MET and its ligand, HGF are associated with poor outcomes and significantly shorter overall survival in various cancer types including gastric cancer (GC). Although MET inhibitors have been shown to be sensitive to tumors with MET amplification, it is relatively rare that incidence is 4-5 % in GC and no MET inhibitor has succeeded in clinical trials so far. Therefore, development of better MET inhibitors and new biomarkers to identify proper patients to benefit from MET inhibitors is needed. We previously analyzed the sensitivity of a novel MET inhibitor, Sym015, a mixture of two human monoclonal antibodies directed at MET, according to MET/HGF status in 49 GC cell lines. Interestingly, two HGF overexpressing cell lines, IM95m and SNU484 were sensitive to 100 nM Sym015 with inhibition rate 52.3 and 23.4 %, respectively. HGF expression in lysate was 7,970 and 2,908 pg/ml and both cell lines had low levels of c-MET and p-MET. To evaluate the influence of HGF overexpression on the antitumor effect of Sym015, sensitivity was measured by CCK-8 assay and cell proliferation was determined by counting cells. Invasiveness was assessed through Transwell assay. Cell viability, invasiveness and related downstream molecules were assessed after HGF silencing by siRNA. In vivo, both cell lines were injected into BALB/c nude mice and Sym015 was applied (50mg/kg, 3qw) and tumor volume and weight were measured every other day. As a result, median inhibition rate of Sym015 in the two HGF overexpressing cell lines was 37.9 %, which was similar to the 44.9 % seen with MET amplified cell lines. The cell proliferation was delayed about 1.6 times and 1.2 times and also invasiveness was decreased by 12.9 and 29.5 % in IM95m and SNU484, respectively. After HGF silencing, inhibition rate at 100 nM Sym015 was significantly decreased (p=0.007) from 50.9 to 15.1 % due to AKT pathway activation in IM95m. There was no change in inhibition rate (from 12.2 to 14.8 %) and downstream pathway molecules in SNU484. Also, after silencing HGF expression, the inhibition effect of Sym015 on invasiveness was reduced from 29.8 % to no inhibition in SNU484 and there was no HGF silencing effect of Sym015 in invasiveness in IM95m. In vivo, xenograft models of IM95m and SNU484 both showed a significant reduction of 53.4 and 30.7 % in tumor volume at time of sacrifice, respectively (p<0.0001). Especially Sym015 effectively inhibited the growth of tumor in IM95m than SNU484. Downstream molecular changes are under evaluation. Consequently, Sym015 was effective in HGF overexpressing cell lines and HGF overexpression induced Sym015 sensitivity was shown in IM95m and the inhibitory effect of invasiveness was shown in SNU484. We suggest that HGF overexpression influences the sensitivity of MET inhibitor and could be a potential predictive marker in GC.

#812

New c-KIT mutations in GISTs: Bad for good.

Erik Laurini,1 Suzana Aulic,1 Domenico Marson,1 Maurizio Fermeglia,1 Roberta Riboni,2 Marco Lucioni,2 Elena Dellera,2 Mario Alessiani,3 Vittorio Perfetti,3 Sabrina Pricl1. 1 _Univ. of Trieste, Trieste, Italy;_ 2 _Fondazione IRCSS Policlinico San Matteo, Pavia, Italy;_ 3 _Ospedale SS Annunziata, Pavia, Italy_.

Gastrointestinal stromal tumors are mesenchymal tumors arising in the stomach and small bowel and more rarely in the rectum, esophagus, peritoneum and retroperitoneum. These tumors are characterized by KIT or PDGFRA mutations. KIT mutations are all in frame and lead to constitutive tyrosine kinase domain activation without ligand binding. Mutations concern four exons (9, 11, 13 and 17) but mainly exon 11.

In this work we report a case of a GIST patient with wild-type PDGFRA but bearing two mutations in KIT exon 9, i.e., the insertion mutation Y503_F504insAY and the missense mutation K509N.

The patient was treated with Imatinib at standard regimen dose (400 mg/day), and responded well.

Accordingly, in silico experiments were performed to disclose the effects exerted by these new mutations on the protein structure, its thermodynamic stability, and the ability to promote/interfere with Imatinib binding.

Direct drug binding thermodynamics and kinetics of the mutant KIT were measured using isothermal titration calorimetry. FInally, in vitro kinase assays were performed to monitor the phosphorylation status of this new mutant KIT.

The prototypical KIT mutations Δ559 and T670I were also analyzed in parallel for comparison.

To our knowledge, only AY 502-503 duplication/insertion and K509I substitution have been previously reported.

#813

Development of anti-Globo H ADC against cancer.

Wei-Ting Sun, Shih-Hsien Chuang, Chao-Pin Lee, Yi-Jen Chen, Win-Yin Wei, Ying-Shuan Lee, Chuan-Lung Hsu, Yu-Chin Nieh, Chia-Cheng Wu. _Development Center for Biotechnology, New Taipei City, Taiwan_.

Globo H antigen is a hexasaccharide originally isolated as a ceramide-linked glycolipid from the human breast cancer cell line MCF-7. Globo H is also highly expressed in many other cancers such as colon cancer, ovarian cancer, gastric cancer, pancreatic cancer, lung cancer, and prostate cancer. DCBD16001 is an ADC from humanized anti-Globo H antibody DCBPR1101. DCBPR1101 and DCBD16001 both show good binding affinities against Globo H antigen. DCBD16001 also shows high cytotoxicity in Globo H overexpressing cell line MCF-7 and HCC-1428 and shows no cytotoxicity in Globo H negative cell line BT-474. DCBD16001 can internalize to target cell more than 50% within 4.0 hours. In vivo evaluation data indicates that DCBD16001 shows acceptable PK profiles and good efficacy. It shows nearly 80% tumor growth inhibition in HCC-1428 xenograft model. DCBD16001 is a candidate under pre-clinical development and expected to apply IND submission within two years.

#814

Aurora kinase: A target modulating invasiveness of lung adenocarcinoma.

Abhilasha Sinha, Seungyeul Yoo, Hideo Watanabe, Jun Zhu, Charles A. Powell. _Icahn School of Medicine at Mount Sinai, New York, NY_.

Introduction: Lung cancer is the major cause of cancer-related deaths in United States and lung adenocarcinoma (LUAD) is its most common subtype exhibiting highly variable molecular signature. Identification of patients at high risk of developing invasive tumor is important for their better management.

Experimental procedure: We obtained 53 early-stage LUAD patient's tumor samples, histopathologically classified into 32 indolent (adenocarcinoma in situ, minimally invasive and lepidic predominant adenocarcinoma) and 21 invasive (acinar predominant, papillary predominant, micropapillary predominant and solid predominant adenocarcinoma) subtypes and profiled their transcriptomes by RNA sequencing. We clustered these samples in an unsupervised manner followed by additional supervised clustering into 2 groups.

Using our signature genes, we categorized 51 lung adenocarcinoma cell lines from Cancer Cell Line Encyclopedia database based on their relative invasiveness scores derived from elastic network trained in our dataset. We chose NCI-H1792, NCI-H1373, SK-LU-1 and NCI-H2009 representing high while HCC1833, Calu3 and NCI-H3255 representing low invasive signature for our in vitro functional assays.

Summary: Our clustering significantly overlapped with the groups classified by histology and we defined 21 tumors as "invasive" and 32 as "non-invasive" early stage LUAD. We identified 1,322 differentially expressed genes between two groups with 526 upregulated and 796 downregulated genes in invasive tumors. Among the enriched signature genes, AURKA and AURKB were significantly upregulated in the "invasive" group. We used two potent aurora kinase inhibitors AMG 900 and PF-03814735 to determine the effect of aurora kinase inhibition on invasiveness of LUAD cells. AMG 900 and PF-03814735 significantly inhibited Aurora kinase (Aur-) A and B activity, but did not affect cell viability (IC50 ≥2μM) of all cell lines regardless of their invasiveness score. Of note, both drugs significantly suppressed wound healing, migration and invasion of all four "invasive" cell lines at much lower 100nM. Moreover, the cells with low invasive signature used in the study showed little migration and invasion thus confirming the validity of our elastic network analysis for their relative invasiveness.

Conclusion: Here, we have identified a robust gene signature that distinguishes invasive to indolent non-invasive early-stage LUADs, which may lead to development of assays on clinical specimens such as biopsy to identify patients at high risk of developing invasive LUAD at an early stage. This gene signature also suggests Aur-A and B promote invasiveness in early stage LUAD. Our in vitro functional data suggest that targeting Aur-A and B could be a promising approach for management of early stage LUAD patients who are more likely to develop an invasive tumor.

#815

New approach for old target: W0101 antibody drug conjugate effectively inhibits tumor growth in preclinical models of IGF-1R overexpressing solid tumors.

Barbara Akla,1 Noureddine Loukili,1 Alain Robert,1 Charlotte Beau-Larvor,2 Martine Malissard,1 Jean-Francois Haeuw,1 Alain Beck,1 Michel Perez,2 Cyrille Dreyfus,1 Mariya Pavlyuk,2 Eric Chetaille,2 Nathalie Corvaia1. 1 _Institut de Recherche Pierre Fabre, Saint-Julien-en-Genevois, France;_ 2 _Institut de Recherche Pierre Fabre, Toulouse, France_.

Insulin-like growth factor type 1 receptor (IGF-1R) has been recognized for decades for its role in tumorigenesis and growth1. In addition, overexpression of this receptor has been largely documented in numerous tumor tissues. However, so far, previous therapeutic approaches based on monoclonal antibodies and tyrosine kinase inhibitors failed to show clinical benefit in the overall patient population2. Here we disclose for the first time a novel IGF-1R targeted Antibody Drug Conjugate (ADC), W0101, designed to deliver highly potent cytotoxic drugs selectively to IGF-1R over-expressing tumor cells.

The naked antibody for our ADC was selected on the basis of its specific binding properties to IGF-1R compared to insulin receptor (IR), and for its internalization properties.

After coupling of a proprietary auristatin derivative to the naked antibody, neither the binding nor the internalization properties of W0101 evaluated by FACS analysis, were modified.

Interestingly, the capacity of W0101 to induce cytotoxicity was evaluated in vitro in various cell lines and was demonstrated to be independent of effector functions.

Mouse efficacy studies with various doses and schedules of administration were conducted in several models expressing different level of IGF-1Rin order to determine their sensitivity to W0101. The sensitivity of W0101 was correlated to the expression level of IGF-1R evaluated by IHC. And in a 3+ MCF-7 breast cancer model, a single injection of W0101 at 3mg/kg led to more than 90% tumor growth inhibition (TGI), associated with a modulation of the IGF-1R expression on the tumor cells (IHC study).

The first in-human trial of W0101 is currently on going to address clinical safety. We believe that W0101 will bring a new therapeutic option for patients that overexpress IGF-IR.

1Pollak M. Insulin and insulin-like growth factor signaling in neoplasia. Nat Rev Cancer, 2008,8:915-928.

2Corvaia N, Beck A, Caussanel V, Goetsch L. Insulin-like growth factor receptor type I as a target for cancer therapy. Frontiers in Bioscience, Scholar, 5, 439-450, January 1, 2013

#816

Antibody-drug conjugate targeting glypican-1 shows potent antitumor effect in pancreatic cancer.

Satoshi Serada,1 Takahiko Nishigaki,2 Takahito Sugase,1 Yurina Saito,2 Tsuyoshi Takahashi,2 Kosuke Hiramatsu,1 Minoru Fujimoto,1 Masaki Mori,2 Yuichiro Doki,2 Tetsuji Naka1. 1 _Kochi Medical School, Kochi University, Nankoku-city, Kochi, Japan;_ 2 _Osaka University Graduate School of Medicine, Suita-city, Osaka, Japan_.

Pancreatic cancer is the most lethal malignancy; thus developing new treatment options is urgently required. We confirmed that enhanced expression of glypican-1 (GPC1) was observed in pancreatic cancer. In this study, we aimed to develop novel antibody-drug conjugate (ADC) targeting GPC1 as a new therapy for pancreatic cancer. By the immunohistochemical analysis, enhanced expression of GPC1 was observed in pancreatic cancer. Among pancreatic cancer cell lines, BxPC3 and T3M4 cells expressed high expression of GPC1 compared to SUIT2 cells. We developed new anti-GPC1 monoclonal antibody (mAb) and selected a clone having highly internalizing activity. The anti-GPC1 mAb was conjugated with cytotoxic agent monomethyl auristatin F (MMAF). Compared with the control ADC, this GPC1-ADC showed potent antitumor effect toward BxPC3 and T3M4, while little activity in SUIT2 cells. In BxPC3 xenograft model, GPC1-ADC had significant and potent tumor growth inhibition in a dose dependent manner. In addition, using patient derived tumor xenograft (PDX) model with GPC1-positive pancreatic cancer, GPC1-ADC also showed significant tumor growth inhibition. In the tumor tissue, GPC1-ADC-mediated G2/M phase cell cycle arrest was detected in GPC1-ADC treated mice compared to control-ADC treated mice. In summary, our newly developed GPC1-ADC showed significant tumor growth inhibition against not only GPC1-positive pancreatic cell line but also patient derived GPC1-positive pancreatic cancer. Our preclinical data demonstrated that targeting GPC1 by ADC is a promising therapy for patients with GPC1-positive pancreatic cancer.

#817

Overcoming FGFRs-mediated drug resistance by inhibiting myristoylation of FRS2α.

Houjian Cai, Qianjin Li. _University of Georgia, Athens, Athens, GA_.

Fibroblast growth factor (FGF)/fibroblast growth factor receptor (FGFR) signaling facilitates tumor initiation and progression. Although there are currently approved inhibitors and agents in clinical trials targeting FGFR kinase activity that show therapeutic benefit, over-expression and/or mutations of FGFRs eventually confer drug-resistance and bypass the mechanisms of kinase inhibitors in a variety of cancer types. We hypothesize that a compound blocks the myristoylation of FRS2α, thus suppressing its interaction with FGFRs or FGFRs drug resistant mutants (FGFRsDRM) and downstream proteins. As a result, it inhibits oncogenic signaling and tumor progression mediated by FGF/FGFRs or FGFRsDRM. The goal of this study is to investigate if the novel lead compound inhibits FGF/FGFRs oncogenic signaling and overcomes FGFRsDRM mediated drug resistance. We used CRISPR/Cas9 mediated gene knockout to demonstrate that FRS2α is essential for FGF/FGFR signaling. The myristoylation of FRS2α was detected by Click chemistry. Additionally, inhibition of cell proliferation, migration, invasion assays and cell cycle analysis were analyzed in numerous cancer cells. Prostate regeneration assay and xenograft tumor models were applied for in vivo studies. We demonstrate that loss of myristoylation of FRS2α, a scaffold protein key in FGFRs signaling, inhibits FGF/FGFRs-mediated oncogenic signaling and FGF10-induced tumorigenesis. Additionally, a previously synthesized myristoyl-CoA analog, B13, which targets the activity of N-myristoyltransferases, suppresses FRS2α myristoylation, and modulates the phosphorylation and localization of FRS2α at the cytoplasmic membrane. B13 exhibited an inhibitory effect on oncogenic signaling induced by wild type FGFRs and their drug resistant mutants (FGFRsDRM). The single agent or in collaboration with an FGFR inhibitor suppressed FGF induced wild type FGFR or FGFRDRM initiated PI3K and/or MAPK signaling, subsequently inhibiting cell proliferation and migration in a variety of cancer cell types by inducing cell cycle arrest. Finally, B13 significantly inhibited growth of xenograft tumors without pathological toxicity to the liver, kidney, or lung in vivo. Our study demonstrates that B13, a myristoyl-CoA analog, inhibits FGF/FGFR-mediated oncogenic signaling and tumor progression. This study provides a novel therapeutic approach for inhibiting cancer progression mediated by FGF/FGFRs and their drug resistant mutants.

#818

NG-HER2 ADC (PF-06804103) is superior to trastuzumab emtansine in a mouse 'avatar' head-to-head clinical trial.

Matthew S. Sung,1 Christine Hopf,1 Erik Upeslacis,1 Jonathon Golas,1 Mark Kaplan,1 Kiran Khandke,1 Manoj Charati,1 Frank Kotch,1 Frank Loganzo,1 Ken Geles,1 Judy Lucas,1 Hans-Peter Gerber,2 Puja Sapra,1 Edward Rosfjord1. 1 _Pfizer Inc., Pearl River, NY;_ 2 _Maverick Therapeutics, Brisbane, CA_.

Patient-derived xenografts (PDXs) are resected human tumors engrafted into mice and represent personalized murine 'avatars' of those tumors. PDXs are valuable tumor models for drug development since they recapitulate the complexity of the human tumor microenvironment more extensively than cell line xenografts (CLXs). Unlike CLXs, PDXs are never passaged in vitro, and therefore they more faithfully recapitulate native tumor biology and response to therapeutics. Thus, PDXs may more accurately predict clinical activity of therapeutic compounds than traditional CLXs. We are developing a next generation (NG)-HER2 antibody-drug conjugate (ADC), PF-06804103, that employs a proprietary site-specific conjugation technology that chemically links the clinically-validated linker-payload "ValCit-Aur0101" to an anti-HER2 antibody. The site-specific conjugation enables enhanced exposures and reduced off-target toxicities as previously described. We utilized our PDX collection to evaluate the breadth-of-efficacy of PF-06804103 versus trastuzumab emtansine (T-DM1), an FDA-approved ADC for metastatic breast cancer patients with high HER2 expression. Efficient cleavage of the 'ValCit' linker and payload release in the early endosome is not impacted by the rapid recycling properties of the HER2 receptor, unlike T-DM1 which requires catabolism in the within the lysosomal milieu to efficiently release its payload. To date, we have enrolled >20 HER2-expressing breast, gastric and non-small cell lung cancer PDXs, with varying low to high HER2 expression levels, in a head-to-head 'mouse avatar clinical trial' comparing activity of a single cycle of T-DM1 (6 mg/kg) to PF-06804103 (3 mg/kg). Impressively, PF-06804103 had more durable complete responses and a higher objective response rate (ORR) than T-DM1 (84% vs. 4%), including in low-moderate HER2 expressers. PF-06804103 prolonged median overall survival (OS) of mice with HER2-expressing tumors compared to T-DM1 (100 vs 45 days). Biomarker analysis showed that tumors at all levels of HER2 expression were more likely to receive more benefit with PF-06804103 than T-DM1 (HR < 0.45). By leveraging our novel PDX 'avatar' clinic, we were able to demonstrate that PF-06804103 displays superior in vivo breadth-of-efficacy compared to T-DM1.

#819

**High response rates with entospletinib in patients with t(v;11q23.3);** KMT2A **rearranged acute myeloid leukemia and acute lymphoblastic leukemia.**

Alison R. Walker,1 John C. Byrd,2 William Blum,2 Tara Lin,3 Howland E. Crosswell,4 Danjie Zhang,5 Jie Gao,5 Arati V. Rao,5 Mark D. Minden,6 Wendy Stock7. 1 _The Ohio State University, Columbus, OH;_ 2 _Winship Cancer Institute of Emory University, Atlanta, GA;_ 3 _University of Kansas Medical Center, Kansas City, KS;_ 4 _Bon Secours St. Francis Health System, Greenville, SD;_ 5 _Gilead Sciences, Inc., Foster City, CA;_ 6 _Princess Margaret Cancer Centre, Toronto, Ontario, Canada;_ 7 _The University of Chicago Medicine, Chicago, IL_.

Introduction: Targeted therapy for KMT2A (mixed lineage leukemia [MLL]) rearranged acute leukemia (AL) is lacking. KMT2A regulates leukemic stem cell transcription factors HOXA9 and MEIS1. Spleen tyrosine kinase (SYK) signaling induces MEIS1 in conjunction with HOXA9. We hypothesize that this regulatory loop may be sensitive to SYK inhibition with Entospletinib (ENTO) a highly selective, oral SYK inhibitor. In this analysis, we evaluate the efficacy of ENTO in patients with acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) with t(v;11q23.3) KMT2A/MLL gene rearrangements.

Methods: Patients with KMT2A rearranged AL in two different clinical trials were included. AML patients (NCT02343939) received ENTO 400mg BID monotherapy for up to 14 days prior to and with induction chemotherapy (cytarabine 100 mg/m2 for 7 days plus daunorubicin 60 mg/m2 for 3 days) if previously untreated, or ENTO monotherapy alone if relapsed/refractory (R/R). R/R B-ALL patients received ENTO monotherapy for 7 days prior to and with vincristine and dexamethasone in a phase 1 study (NCT02404220).

Results: 18 patients with KMT2A rearranged AL were treated. Untreated AML (n=10) patients had a median age of 49 years. One patient was in a morphologic leukemia-free state after ENTO monotherapy (before chemotherapy). After induction chemotherapy plus ENTO, seven patients had morphologic (CR) and cytogenetic (CRc) remission, but two of them had incomplete count recovery (CRi). The composite CR rate (CR/CRi/CRc) was 90%: 5 CRc, 3 CRi (CR/CRc with incomplete count recovery), and 1 CR. Six patients underwent allogeneic stem cell transplantation (SCT) in CR1. After median follow-up of 9.9 months one patient who achieved CR relapsed, with persistence of t (6;11) and an NRAS mutation. Median overall survival and event-free survival have not been reached. In R/R AML (n=6), patients had a median age of 48 years, with 2 median prior therapies. One had CR and one had CRi (but normal cytogenetics) and both received SCT. In all, 3 AML patients (1 newly diagnosed, 2 R/R) responded to ENTO monotherapy alone. In R/R ALL (n=2), median age was 59 years. Both patients had t (4;11) along with other cytogenetic abnormalities with 2 prior therapies. Both patients achieved CR with loss of KMT2A on cytogenetic testing, and one patient received SCT. Overall, ENTO was safe and well tolerated, even in combination with chemotherapy.

Conclusions: KMT2A rearranged AL is sensitive to ENTO with CR observed on monotherapy in AML and high response rates in AL patients treated with combination therapy. This represents the first documentation of CR with small molecule monotherapy (n=3) in this genetic subgroup, which typically portends a poor prognosis. Correlative biomarker studies evaluating HOXA9/MEIS1 in patients with t(v;11q23.3) KMT2A rearranged leukemia treated with ENTO are pending.

#820

Preclinical development of MGC018, a duocarmycin-based antibody-drug conjugate targeting B7-H3 for solid cancer.

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

Introduction: B7-H3, a member of the B7 family of immunomodulatory molecules, is overexpressed in a wide range of solid tumors; tumor overexpression has been correlated with disease severity and poor outcome in several cancer types. MGC018 is an antibody-drug conjugate (ADC) targeted against B7-H3 and comprised of the cleavable linker-duocarmycin payload, valine-citrulline-seco DUocarmycin hydroxyBenzamide Azaindole (vc-seco-DUBA), conjugated to an anti-B7-H3 humanized IgG1/kappa monoclonal antibody through reduced interchain disulfides, with an average drug-to-antibody ratio of ~2.7. Previous studies indicated MGC018 exhibited a favorable preclinical profile, with strong reactivity toward tumor cells and tumor-associated vasculature, limited normal tissue reactivity, potent cytotoxicity in vitro and antitumor activity in vivo toward a range of B7-H3-expressing tumor cell lines representing several cancer types. Based on these preliminary results, expanded preclinical development of MGC018 was undertaken to support clinical development.

Methods: vc-seco-DUBA conjugation to obtain MGC018 ADC was performed by Synthon Biopharmaceuticals B.V. Single- and repeat-dose in vivo efficacy studies were conducted in CD-1 nude mice with human tumor xenografts that express B7-H3 to explore the relationship between Cmax, exposure and anti-tumor activity, and to define the minimal efficacious dose in these models. A GLP toxicology study was conducted in cynomolgus monkeys in which MGC018 was administered at dose levels of 1, 3, 6 and 10 mg/kg every three weeks for a total of three doses.

Results: MGC018 demonstrated specific, dose-dependent in vivo antitumor activity toward B7-H3-positive tumor xenografts representing breast, lung and ovarian cancers, and melanoma. Fractionated MGC018 dose studies were consistent with antitumor activity driven by the total exposure (AUC) rather than peak drug exposure (Cmax). MGC018 was tolerated in cynomolgus monkeys at all dose levels tested, with 10 mg/kg, the highest dose administered, defined as the highest non-severely toxic dose (HNSTD).

Conclusion: MGC018, a preclinical candidate comprised of a humanized mAb targeting B7-H3, conjugated to the potent DNA alkylating payload DUBA via a cleavable peptide linker, exhibited a favorable preclinical profile. MGC018 demonstrated potent antitumor activity in vivo toward B7-H3-expressing tumor xenografts at clinically relevant dose levels. MGC018 was tolerated in cynomolgus monkeys, a relevant toxicology model, at exposure levels in excess of those required for antitumor activity. Our findings support the clinical development of MGC018 to evaluate its potential as an ADC therapeutic for B7-H3-expressing solid cancers.

#821

B7-H3 is a potential antibody drug conjugate target for the treatment of prostate cancer.

Gareth C. Davies,1 Kelli Ryan,1 Alison J. Smith,1 Chris Lloyd,1 Ravinder Tammali,2 Noel Monks,2 Arthur Lewis,1 Robert W. Wilkinson1. 1 _MedImmune, Cambridge, United Kingdom;_ 2 _MedImmune, Gaithersburg, MD_.

B7-H3 is a member of the B7 family of immune-regulatory ligands and has been reported to be overexpressed in a broad range of human cancers. In order to understand more comprehensively the expression of B7-H3 on both normal tissues and on tumors, we developed an immunohistochemistry (IHC) assay for the detection of human B7-H3. Using this, we confirmed a restricted normal tissue expression pattern and demonstrated expression of B7-H3 in a range of solid tumors. In particular, a high proportion of prostate cancer samples showed strong homogenous B7-H3 expression. This low normal tissue expression and upregulation in cancer makes B7-H3 an attractive target for an antibody drug conjugate (ADC). To evaluate this approach, antibodies to B7-H3 were isolated by screening a phage display library for binding to B7-H3. These antibodies specifically bind to and are internalised by B7-H3 expressing cancer cells. ADCs were generated by site-specific conjugation of either a DNA-damaging pyrrolobenzodiazepine dimer or a microtubule-disrupting tubulysin payload. These ADCs selectively inhibited the growth of B7-H3 expressing cell lines including androgen-insensitive prostate cancer cell lines such as LNCAP-AI in vitro. Furthermore, these ADCs caused durable tumour regression in human tumor xenograft models in vivo. Together, these data suggest that an ADC targeting B7-H3 may have potential to be used for the treatment of a range of solid tumors including prostate cancer.

#822

Predicting response to kinase inhibitors based on tumor genomics in NF1-related MPNSTs.

Jamie Grit,1 Matt Pridgeon,1 Elizabeth Tovar,1 Zachary Madaj,1 Jackie Peacock,1 Julia Wulfkuhle,2 Emanuel Petricoin,2 Carrie Graveel,1 Matt Steensma1. 1 _Van Andel Research Institute, Grand Rapids, MI;_ 2 _George Mason University, Fairfax, VA_.

Malignant Peripheral Nerve Sheath Tumors (MPNSTs) are aggressive, chemoresistant sarcomas that develop in patients with the genetic disorder Neurofibromatosis Type 1 (NF1). Loss of TRP53 and amplification of the receptor tyrosine kinase (RTK) MET is observed 25-50% of cases. To investigate the role of MET and p53 in MPNST progression, we developed three MPNST mouse models: an NF1 null and MET overexpression MPNST model (NF1-MET), an NF1 and TRP53 heterozygous knockout model (NF1-p53), and a NF1 null model (NF1). Using orthotopic tumorgrafts from these models, we tested the efficacy of MET (capmatinib) and MEK (trametinib) inhibition on MPNST growth. Trametinib moderately inhibited tumor growth in all of the models; however, combined inhibition of MEK and MET was highly effective in both the NF1-MET and NF1 models. NF1-p53 tumors were the least responsive to combined inhibition and upregulated AKT signaling in response to single agent therapy. These data suggest that alternate RTK signaling pathways downstream of MET may compensate for MEK inhibition, and that p53 loss promotes kinome reprogramming in response to targeted inhibition in MPNSTs. To determine the effects of MEK, MET, PI3K, and mTOR inhibition on kinome signaling, we generated MPNST cell lines from each of our MPNST models. Western blot analysis revealed that NF1-MET MPNST cells are highly sensitive to MET inhibition, NF1-p53 MPNST cells maintain ERK and AKT activity in the presence of MET inhibition, and NF1-p53 MPNST cells uniquely upregulate MEK signaling in response to PI3K/mTOR inhibition. These results suggest p53 plays a unique role in kinome reprograming and resistance to kinase inhibitors. Currently, we are determining the effects of combined kinase inhibition on proliferation of MPNST cells and utilizing reverse phase protein arrays to evaluate the effect of MET and MEK inhibition on the phospho-proteome in our MPNST models. By understanding these complex signaling networks we will identify combinations of targeted therapies that can be used to effectively treat MPNSTs.

#823

Anti-melanoma effects of novel Cripto-1 CFC small peptide mimetics.

Emanuela Iaccarino,1 Gustavo Untiveros,2 Annamaria Sandomenico,3 Andrea Caporale,3 Luisa Calvanese,3 Lucia Falcigno,3 Gabriella D'Auria,3 Menotti Ruvo,3 Luigi Strizzi2. 1 _Universita' Degli Studi Della Campania, L. Vanvitelli, Italy;_ 2 _Midwestern University, Downers Grove, IL;_ 3 _Universita' Federico II di Napoli, Italy_.

The diagnosis of melanoma is increasing and current therapies for advanced disease remain unsuccessful. Thus, research aimed at developing novel targeting approaches is needed. Cripto-1 (CR-1), an epidermal growth factor (EGF)-like developmental morphogen, functions as a co-receptor for the tumor growth factor (TGF)-beta related molecule, Nodal. CR-1/Nodal signaling pathways can induce cell survival, proliferation and migration of cancer cells. Studies have shown that Nodal is expressed at high levels in advanced melanoma. CR-1 has also been shown to be expressed in melanoma but limited to a small subpopulation of melanoma cells with more stem cell-like characteristics. Recent work has demonstrated that targeting Nodal in melanoma is associated with reduction of a significant population of melanoma cells. However, a residual population continues to persist. We hypothesize that interference with CR-1/Nodal receptor binding dynamics will negatively affect downstream Nodal-dependent signaling events important for cancer growth and further reduce the number of surviving melanoma cells. To test this hypothesis, we have developed small peptides that mimic CR-1 CFC binding domain and prevent CR-1 from binding to the ALK4 receptor necessary for proper CR-1 co-receptor function for Nodal. We show that in vitro treatment of human melanoma cells with our prototype CFC mimetic B3 reduced melanoma cell viability and negatively affected melanoma cell cycle progression. We also show that treatment with B3 resulted in reduction of active Smad2/3 and ERK1/2, known to be involved in downstream Nodal signaling, as a result of B3 interference with CR-1 co-receptor function. These preliminary data provide the scientific rationale for the development of anti-CR-1 and Nodal therapeutics that can synergize with or complement current treatment approaches in melanoma.

#824

Targeting epidermal growth factor receptors and urokinase-type plasminogen activator receptors for sarcoma therapy.

Ashley J. Graef, Jong-Hyuk Kim, Daniel A. Vallera, Jaime F. Modiano. _University of Minnesota, Minneapolis, MN_.

eBAT is a bispecific angiotoxin consisting of truncated, deimmunized Pseudomonas exotoxin fused to epidermal growth factor (EGF) and the amino terminal fragment of urokinase. This drug was designed to use epidermal growth factor receptors (EGFR) and urokinase-type plasminogen receptors (uPAR) as baits to deliver the lethal toxin into the cells, rather than as a means to interrupt signaling by the receptors. We previously showed that eBAT is remarkably safe, and that it reduced tumor burden in mice with established human rhabdomyosarcoma xenografts. It also was safe and showed a strong efficacy signal when used in the adjuvant setting in a clinical trial for dogs with splenic hemangiosarcoma. Nevertheless, the therapeutic mechanism of action of eBAT remains to be elucidated. Our goal is to identify patterns of uPAR expression in sarcomas that promote sarcoma growth and that mediate sensitivity for therapeutic activity by eBAT. We generated uPAR-knockout (KO) cells from MC1A-C1 mouse fibrosarcoma cell line using the CRISPR-Cas system to target exon 2 of the uPAR gene. The resulting cell lines D10 and H6 are clonal uPAR knockouts, whereas K02 and K06 represent mixtures where approximately 80% and 30% of the cells retain uPAR expression. uPAR expression in K02 and K06 has remained stable over numerous passages, indicating that loss of uPAR does not create selective pressure to enhance or inhibit cell growth in vitro. Furthermore, the growth kinetics of D10 and H6 are comparable to parental MC1A-C1 cells. eBAT treatment significantly inhibited proliferation of parental cells, but not of uPAR KO cells. In contrast, all of the cells were resistant to treatment with EGF- and uPA-monospecific toxins. The engraftment efficiency and tumor growth of uPAR KO cells in syngeneic hosts was substantially diminished when compared to K02 and K06 uPAR+ cells, suggesting that, even while uPAR-KO cells might be more resistant to eBAT therapy, uPAR expression appears to be necessary to create the tumor niche and/or to promote tumor growth in vivo. In our ongoing work, we are investigating how uPAR expression in stromal cells and in cells of the innate immune system influence sarcoma growth and the response to eBAT.

#825

Identification of molecular therapeutic targets that enhance the antitumor activity of neratinib in breast cancer cells.

Bora Lim,1 Jangsoon Lee,1 Troy Pearson,1 Liu Huey,1 Minha Hwang,1 Kuicheon Choi,1 Francesca Avogadri-Connors,2 Richard E. Cutler,2 Debu Tripathy,1 Naoto T. Ueno1. 1 _UT MD Anderson Cancer Ctr., Houston, TX;_ 2 _Puma Biotechnology, South San Francisco, CA_.

Background: Aberrant expression of members of the ErbB family of receptor tyrosine kinases and its downstream pathways is known to play pivotal roles in breast cancer malignancy. HER2-positive breast cancer has an aggressive nature that is driven by HER2 gene amplification. Inflammatory breast cancer is a rare but the most aggressive clinical subtype of breast cancer. Triple-negative breast cancer is an aggressive receptor subtype of breast cancer. More than 30% of cases of each of these groups express EGFR. Emerging therapeutic resistance continues to be a challenge in recurrent and/or metastatic disease. Neratinib is a potent irreversible EGFR, HER2, and HER4 tyrosine kinase inhibitor that blocks signal transduction generated via these receptors. In this study, we sought to identify the optimal targets in different subtypes of breast cancer and synergistic partners to maximize the antitumor effect of neratinib against breast cancer cells in vitro and in vivo.

Methods: Expression of EGFR and HER2 was assessed via Western blot in 31 breast cancer cell lines, including HER2-targeted drug-resistant cell lines (14 triple-negative breast cancer and 10 HER2-positive). The antiproliferative effects of neratinib were measured using cell proliferation and anchorage-independent colony-formation (soft agar) assays. A reverse-phase protein array was used to profile and validate the signaling networks induced by neratinib-based treatment. To identify the synergistic targets, RNA interference screening of a 779-kinome library was performed under multicellular tumor spheroid culture conditions.

Results: In vitro proliferation data demonstrated that neratinib had a nanomolar range of half-maximal inhibitory concentrations in most of the tested cell lines, including T-DM1-resistant HER-2 breast cancer cell lines. Neratinib inhibited colony formation in a dose-dependent manner (P = 0.0001). Reverse-phase protein array data revealed that the efficacy of neratinib correlated with phosphorylated EGFR (R2 = 0.6055) or phosphorylated HER2 (R2 = 0.6734) expression level. Statistical analysis identified the 25 most relevant targets in the RNA interference screening, which we will validate for combinational antitumor effects with neratinib in in vitro and in vivo studies.

Conclusion: We determined the efficacy of neratinib and identified target molecules to enhance neratinib efficacy in different breast cancer subtype cell lines with ErbB alterations. Our data inform new combined treatment strategies with neratinib for breast cancer that will lead to innovative designs using optimized neratinib combinations in clinical trials.

#826

Th1 cytokines and EGFR inhibition: A combinatorial therapeutic strategy in TNBC.

Amrita Basu, Krithika Kodumudi, Doris Wiener, Brian Czerniecki. _Moffitt Cancer Center, Tampa, FL_.

Oncodrivers are a promising target in novel breast cancer therapy development, owing to oncogene addiction of breast cancer cells to sustain their malignancy. We have previously shown progressive loss of CD4+ T-helper type 1 response to oncodrivers in triple negative breast cancer (TNBC) patients. TNBC is marked by lack of cell surface ER, PR and Her2 receptors and is the most aggressive subtype of breast cancer. Lack of therapeutic targets, resistance to existing hormone therapy, metastasis and poor survival in TNBC patients underline the need for novel TNBC therapy development. In this study, we investigated the effect of EGFR inhibitors (cetuximab, erlotinib and lapatinib) alone or combination with Th1 cytokines (IFN-γ and TNF-α) on growth and proliferation of MDA-MB-231 and MDA-MB-468 TNBC cells. We observed decrease in EGFR expression in TNBC cells when treated with IFN-γ alone and in combination with EGFR inhibitors. In addition, IFN-γ alone, in combination with TNF-α and all three EGFR inhibitors, markedly increased STAT1 phosphorylation, indicating suppression of growth and proliferation in TNBC cells. Decreased STAT3 phosphorylation by combination treatment in TNBC cells may induce apoptosis and inhibit proliferation further. Combination of cetuximab and IFN-γ increased cleaved caspase-3 expression in MDA-MB-231, but not in MDA-MB-468 TNBC cells. EGFR inhibition and Th1 cytokine treatment showed combination treatment resulted in severe cell loss and morphological alteration, while Th1 cytokines alone did not induce significant senescence in MDA-MB-231 cells in senescence associated β-galactosidase assay. We are currently investigating activation status of other signaling pathways in TNBC cells, following Th1 cytokines treatment with EGFR inhibition. Our data suggests a combinatorial treatment approach, including DC1 vaccination to elicit Th1 immune response and inhibition of EGFR oncodriver, may lead to an effective and novel therapeutic strategy for triple negative breast cancer.

#827

Anti-tumor efficacy of BA3011, a novel Conditionally Active Biologic (CAB) anti-AXL-ADC.

Leslie L. Sharp, Cathy Chang, Gerhard Frey, Jing Wang, Haizhen Liu, Charles Xing, Safak Yalcin, Marlena Walls, Yong Ben, William J. Boyle, Jay M. Short. _BioAtla LLC, San Diego, CA_.

AXL is a TAM family receptor tyrosine kinase that has been implicated in the pathogenesis of many cancer types. The high level of expression on the cancer cell surface and increased expression in various settings such as PD-1 resistant tumors makes AXL a particularly attractive target for antibody therapeutics. However, AXL is expressed on many normal tissues and has been implicated in wide ranging requisite biological processes including response of endothelial cells to vascular injury, hematopoiesis, and regulation of immune responses. This normal tissue expression combined with the presence of soluble AXL may limit AXL as a target for antibody-drug-conjugates (ADC). The Conditionally Active Biologics (CAB) technology is a patented, proprietary platform that generates antibodies that reversibly bind to target antigen in the context of diseased tissues, but not normal tissues, by taking advantage of the unique cancer microenvironment that is produced largely as a result of the Warburg effect. Using our CAB technology, we have identified anti-AXL CAB Abs that reversibly bind to recombinant AXL and AXL expressing cells under conditions that are present in the tumor microenvironment, but not in normal tissues.

BA3011 is a CAB-AXL-ADC. The pharmacological properties of BA3011 were characterized in a number of in vitro and in vivo pharmacology studies. BA3011 binds selectively to human and cyno AXL in conditions reflective of the tumor microenvironment, but has reduced binding under normal tissue conditions. BA3011 demonstrated the ability to induce cytotoxicity of human tumor cell lines expressing AXL in vitro and inhibit tumor growth in LCLC-103H (lung), DU145 (prostate), and MIAPaCa-2 (pancreatic) human tumor xenografts and in selected gemcitabine resistant pancreatic cancer patient derived xenograft models in vivo.

In conclusion, our data is consistent with our work on CAB-EGFR-ADC, CAB-ROR2-ADC, CAB-PD-1, and other CAB programs, and suggests that ADCs generated using the CAB technology provide biologics with increased therapeutic index. Specifically, the CAB-AXL-ADC is an excellent candidate for evaluation as a treatment for human cancers that are AXL positive.

#828

**Targeting the** MET **oncogene by concomitant inhibition of receptor and ligand by an antibody-'decoy' strategy.**

Cristina Basilico, Chiara Modica, Federica Maione, Elisa Vigna, Paolo M. Comoglio. _Candiolo Cancer Institute, Candiolo, Italy_.

Background: MET, a master gene sustaining 'invasive growth', is a relevant target for cancer precision therapy. In a limited number of cases, a MET genetic lesion drives the malignant phenotype in ligand-independent manner (oncogene 'addiction'). In the vast majority of tumors, however, wild-type MET behaves as a 'stress-response' gene and relies on the ligand (HGF) to sustain cell 'scattering', invasive growth and apoptosis protection (oncogene 'expedience'). In this context -i.e. ligand-dependent MET activation- concomitant targeting of MET and HGF could be crucial to reach effective inhibition. Methods: To test this hypothesis we combined an anti-MET antibody (MvDN30), characterized by the property of inducing 'shedding' (i.e. removal of MET from the cell surface), with a 'decoy' (i.e. the soluble extracellular domain of the MET receptor) endowed with HGF-sequestering ability. To avoid antibody/decoy interaction -and subsequent neutralization- we identified by site directed mutagenesis a single aminoacid in the extracellular domain of MET -lysine 842- that is critical for MvDN30 binding, and engineered the corresponding recombinant decoyMET (K842E). The effect of MvDN30 and decoyMETK842E in combination on the inhibition of MET phosphorylation and on the impairment of different MET driven biological responses (motility, invasion, cell proliferation, apoptosis and anchorage independent cell growth) was evaluated on a panel of cancer cells (lines and patient derived-primary cultures) expressing wild-type MET sensitive to HGF stimulation. Therapeutic efficacy was assessed in an orthotopic model of pancreatic cancer generated in SCID mice engineered to express human HGF. Results: The decoyMETK842E retains the ability to bind HGF with high affinity and inhibits HGF-induced MET phosphorylation. In HGF-dependent cellular models, MvDN30 antibody and decoyMETK842E used in combination cooperate in restraining invasive growth, and synergize in blocking cancer cell 'scattering'. The antibody and the decoy unbridle apoptosis of colon cancer stem cells grown in vitro as spheroids. In the preclinical model of MET 'expedience' concomitant treatment with antibody and decoy significantly reduces metastatic spread. Conclusions: The data reported indicate that vertical targeting of the MET/HGF axis results in powerful inhibition of ligand-dependent MET activation, providing proof of concept in favor of combined targeted therapy of MET 'expedience'.

#829

SAS1B protein: Determining whether ASTL or SAS1B has a role in tumor progression.

Zunair S. Khokhar, Eusebio S. Pires, Alexander J. Kim, Ryan D'Souza. _University of Virginia School of Medicine, Charlottesville, VA_.

The matrix metalloproteinase Sperm Acrosomal SLLP1 Binding (SAS1B) is an ideal molecular and cellular candidate for both female contraception and for cancer therapies. SAS1B translation is restricted among adult tissues to the ovary and oocytes, and SAS1B first appears in oocytes of follicles at the primary‐secondary transition and also in tumors of varied types and stages. Research conducted using SAS1B antibodies suggested that the protein is localized at the cell membrane of cancer cells and can get internalized in a classical endocytosis assay and can be killed using an immunotoxin linked onto antibodies. The current study aims at deciphering whether the ASTL gene or the SAS1B protein is important for tumor progression, invasion and metastasis through cell biology and molecular biology tools and ensuring tumor cell-killing properties via an ADC-mediated killing using biochemistry and molecular biology tools. PCR, immunohistochemistry, Immunofluorescence, and Western blots revealed that a noncancerous breast cell line, MCF10A5E, is SAS1B null and can be used to study cancer-like properties once stably transfected with the SAS1B construct. Previous work in the lab indicated that an SAS1B-ADC strategy is a viable immunotherapy that could benefit patients who have failed to respond to chemotherapy, if indeed SAS1B plays a role in cancer development and progression. The outcomes of an SAS1B-ADC in a breast cancer model will be presented.

#830

ISU104, a fully human antibody targeting a specific epitope on the ErbB3, displays potent inhibition of tumor growth in multiple xenograft tumor models.

Miyoung Kim, Youngmi Hur, Sohyeon Seo, Heynjeong Lim, Kyungyong Kim, Youngsoo Sohn, Seung-Beom Hong, Donggoo Bae. _ISU ABXIS, Bundang-gu Seongnam-si, Republic of Korea_.

Members of the epidermal growth factor receptor family (ErbB family) are known as potent mediators in the development and progression of cancer. Activated ErbBs recruit various adaptors and signaling molecules through the phosphorylated cytoplasmic domain, which further leads to activation of downstream oncogenic signaling pathways. There are approved therapeutics for ErbB1 (EGFR) and ErbB2 (HER2) in the treatment of human cancers, while monoclonal antibodies targeting ErbB3 are just undergoing clinical trials. ISU104 is a fully human anti-ErbB3 antibody isolated from phage display antibody library. We performed the hydrogen/deuterium exchange mass spectrometry (HDX-MS) analysis with ErbB3 extracellular domain and ISU104, and that indicated that ISU104 mainly binds with domain 3 and weakly interacts with domain 1 of ErbB3. The binding property of ISU104 induced dose-dependent inhibition of ligand (heregulin, HRG) binding, blocking of dimerization of ErbB3 with other ErbBs and subsequently inactivated the downstream signaling of ErbB3. Also, ISU104 occasioned internalization of ErbB3 from plasma membrane, and downregulated the expression level of ErbB3. We demonstrated the biologic effect of ISU104 in several ErbB3-expressing cancer cell lines, including head and neck squamous cell carcinoma (HNSCC) and breast cancers. ISU104 completely suppressed the HRG-induced ErbB3/AKT phosphorylation, reduced cell proliferation and survival. Next, we evaluated efficacy of ISU104 in multiple xenograft models. Mice were treated with 10 mg/kg of ISU104 twice weekly. ISU104 regressed tumor growth in FaDu HNSCC model, and showed more than 70% tumor growth inhibition (TGI) in CAL27 (HNSCC), BxPC3 (pancreatic cancer), MDA-MB-468 (breast cancer), A549 (skin cancer), and BT474 (breast cancer) models. Our results suggest that ISU104 effectively blocks activation of ErbB3 and the downstream pathway by ErbB3, and may provide clinical benefit to ErbB3-activated patients.

#831

Reproducible, mechanism of action-reflecting reporter based bioassays to enable drug development of biosimilars and biobetters.

Richard L. Somberg, Rich Moravec, Dun Li, Aileen Paguio, Mei Cong. _Promega, Madison, WI_.

The demand for biosimilars and bioassays for biosimilars are increasing as the patent cliff approaches for many blockbuster biologics. Recently, the expanded approval of Actemra (Tocilizumab) to treat CAR T-cell-induced cytokine release syndrome (CRS) demonstrated the role of anti-IL-6 blocking drugs as critical for the treatment of serious diseases such as cancer and autoimmune diseases. Here we describe a portfolio of luciferase reporter-based bioassays to support the development and potency determination of biosimilar drugs targeting cytokines such as IL-2, IL-6, IL-12, IL-15, IL-12/23, IL-17, VEGF, RANK, Epo, IFNs, etc. The availability of quantitative, functional bioassays in Thaw-and-Use format provides the benefit of convenience, reproducibility, and transferability. We demonstrate these assays are able to measure relative potency for antibody biologics and detect potency changes for stressed antibody samples. In summary, the reporter-based bioassay portfolio for biosimilars provides a valuable tool for the development, stability testing, and potency determination in manufacture of biosimilars and biobetters.

#832

An AXL-specific antibody-drug conjugate shows preclinical anti-tumor activity in non-small cell lung cancer, including EGFR-inhibitor resistant NSCLC.

Louise A. Koopman,1 Maarten L. Janmaat,1 Kirstine Jacobsen,2 Mikkel Green Terp,2 Elke Gresnigt-van den Heuvel,1 Ulf Forssman,3 Andreas Lingnau,1 Paul W. Parren,4 Henrik Ditzel,2 Esther C. Breij1. 1 _Genmab, Utrecht, Netherlands;_ 2 _University of Southern Denmark, Odense, Denmark;_ 3 _Genmab, Copenhagen, Denmark;_ 4 _Leiden University Medical Center, Leiden, Netherlands_.

Enhanced AXL expression has been observed in tumor tissues obtained from non-small cell lung (NSCLC) patients, both in drug-treatment naïve patients and in patients with acquired resistance to EGFR tyrosine kinase inhibitors (EGFRi). In this study, we have evaluated the antitumor activity of the novel AXL-targeting antibody-drug conjugate (ADC) AXL-107-MMAE (HuMax-AXL-ADC) in non-small cell lung cancer (NSCLC) in vitro and in vivo. The antitumor activity of AXL-107-MMAE (4 mg/kg) was evaluated in vivo in a mouse patient-derived xenograft (PDX) clinical trial (1 mouse per group), using a collection of 57 NSCLC-derived PDX models, encompassing the typical NSCLC histological subtypes and mutational genotypes. AXL-107-MMAE induced responses, defined by a decrease in relative tumor growth compared to an untreated control tumor, in 35/57 (61%) of PDX models. Potent anti-tumor activity, i.e. tumor stasis or tumor regression, was observed in 16 out of 57 (28%) models. Potent anti-tumor activity was associated with higher AXL RNA expression compared to models showing intermediate response or non-responders (p<0.001). The therapeutic activity of AXL-107-MMAE (2 and 4 mg/kg) was confirmed in a panel of NSCLC PDX or cell line-derived xenograft models using 6-8 mice per group. AXL-107-MMAE induced dose-dependent, single agent anti-tumor activity in 8 out of 9 models. These included 2 EGFR-mutant PDX models that were resistant to the EGFR inhibitor erlotinib, including one model containing L858R/T790M EGFR mutations and one model with a L858R EGFR mutation. To specifically evaluate opportunities for AXL-107-MMAE in EGFR-mutant, treatment-resistant NSCLC, we established a panel of EGFR-mutant NSCLC cell lines with acquired resistance to the EGFRi erlotinib, gefitinib, or osimertinib. In general, cell lines showed enhanced AXL protein expression upon acquiring resistance to EGFRi. Moreover, the AXL-expressing, EGFRi-resistant cell lines were efficiently killed by AXL-107-MMAE in vitro, whereas the parental, EGFR-TKI sensitive NSCLC cell lines, which expressed little or no AXL, did not respond to treatment with AXL-107-MMAE. In summary, we show that AXL-107-MMAE has therapeutic activity as a single agent in the majority of NSCLC-derived models evaluated in vivo, representing different NSCLC histological and mutational subtypes. Moreover, AXL-107-MMAE induced efficient cytotoxicity in NSCLC cell lines that showed enhanced AXL expression upon acquired resistance to EGFRi. Finally, AXL-107-MMAE induces anti-tumor activity in EGFR-mutant, EGFRi resistant NSCLC PDX models.

#833

Anti-tumor efficacy of BA3021, a novel Conditionally Active Biologic (CAB) anti-ROR2 ADC.

Leslie L. Sharp, Cathy Chang, Gerhard Frey, Jing Wang, Haizhen Liu, Charles Xing, Safak Yalcin, Marlena Walls, Yong Ben, William J. Boyle, Jay M. Short. _BioAtla, San Diego, CA_.

ROR2 is a developmentally regulated member of the receptor tyrosine kinase orphan receptor (ROR) family and is a non-canonical receptor for selected Wnt family members. Many of the activities associated with ROR2 in development have also been implicated in cancer including cell migration and invasion. In many cancer types, expression of ROR2 correlates with advanced disease or poor prognosis. The high level of expression on the cancer cell surface has made it an attractive target for antibody-drug-conjugates (ADC). The Conditionally Active Biologics (CAB) technology is a patented, proprietary platform that selects antibodies that reversibly bind to target antigen in the context of diseased tissues, but not normal tissues, by taking advantage of the unique cancer microenvironment that is produced largely as a result of the Warburg effect. Using our CAB technology, we have identified anti-ROR2 selective Abs that reversibly bind to recombinant ROR2 and ROR2 expressing cells under conditions that are present in the tumor microenvironment, but not in normal tissues.

BA3021 is a CAB-ROR2-ADC. The pharmacological properties of BA3021 were characterized in a number of in vitro and in vivo pharmacology studies. BA3021 binds selectively to human and cyno ROR2 in conditions reflective of the tumor microenvironment, but has reduced binding under normal tissue conditions. BA3021 demonstrated ability to induce cytotoxicity of cell lines expressing ROR2 in vitro and inhibit tumor growth in LCLC-103H (lung), MDA-MB-436 (breast), HT1080 (sarcoma) and SK-MEL-5 (melanoma) human tumor xenografts and in selected sarcoma cancer patient derived xenograft models in vivo.

In conclusion, our data is consistent with our work on CAB-EGFR-ADC, CAB-AXL-ADC, CAB-PD-1 and other CAB programs and suggests that ADCs generated using the CAB technology provide biologics with increased therapeutic index. Specifically, the CAB-ROR2-ADC is an excellent candidate for evaluation as a treatment for human cancers that are ROR2 positive.

#834

FLT3 inhibitors and PP2A-activating drugs synergistically induce cytotoxicity in AML cells with FLT3-ITD through enhanced Pim-1 and Myc proteasomal degradation.

Mario Scarpa, Shivani Kapoor, Sikemi Ibikunle, Andre Rush, Patrick R. Baldwin, Danilo Perrotti, Maria R. Baer. _Univ. of Maryland Greenebaum Comprehensive Cancer Ctr., Baltimore, MD_.

Background: In 30% of acute myeloid leukemia (AML) patients, the presence of fms-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) causes constitutive FLT3 signaling, and these patients relapse rapidly following response to chemotherapy. FLT3 inhibitors have limited and transient efficacy, but their efficacy may be enhanced by combination with other drugs targeting FLT3 signaling. The oncogenic serine/threonine kinase Pim-1 is transcriptionally upregulated downstream of FLT3-ITD, contributes directly to its proliferative and antiapoptotic effects and also phosphorylates and stabilizes it in a positive feedback loop. FLT3 activation also inhibits the tumor-suppressor protein phosphatase 2A (PP2A), causing stabilization of Pim-1 protein. FLT3 inhibitors and PP2A-activating drugs (PADs) are known to synergistically induce cytotoxicity in cells with FLT3-ITD, and here we studied mechanisms underlying this effect.

Methods: Ba/F3-ITD, 32D-ITD, MV4-11 and MOLM-14 cells, with FLT3-ITD, were cultured with a FLT3 inhibitor, gilteritinib (ASP2215) or quizartinib (AC220), and the PAD fingolimod (FTY720) at pharmacologically relevant concentrations, or DMSO control. Apoptosis was measured by Annexin V/propidium iodide staining. Drug combination indexes were determined by the Chou-Talalay method with CompuSyn software. Pim-1 kinase, Myc, MycSer62, MycThr58, STAT5, phospho-STAT5, PP2A and phospho-PP2A levels were measured by immunoblotting. Cycloheximide was used to assess protein stability.

Results: Concurrent treatment with ASP2215 or AC220 and FTY720 decreased growth and enhanced apoptosis of cells with FLT3-ITD, relative to single drugs, and produced synergistic cytotoxicity. Concurrent treatment sequentially decreased expression of the 44 kDa Pim-1 isoform (Pim-1L) and then c-Myc, via decreased Pim-1L and c-Myc protein stability. Co-incubation with the proteasome inhibitor MG-132 prevented drug-induced degradation and restored Pim-1L and Myc expression in cells co-treated with FLT3 inhibitor and PAD. Drug-induced Myc destabilization/proteasomal degradation was associated with decreased MycSer62 and increased MycThr58 levels. Treatment with the pan-Pim kinase inhibitor AZD1208 prior to ASP2215 or AC220 and FTY720 treatment markedly increased MycThr58 levels but did not affect MycSer62, consistent with both Pim-dependent and -independent effects of FLT3 inhibition and PP2A activation on c-Myc expression.

Conclusions: Combined FLT3 inhibitor and PAD treatment synergistically induces cytotoxicity in cells with FLT3-ITD by enhancing Pim-1 and Myc proteasomal degradation, and effect on Myc is both Pim-1-dependent and -independent. The data support in vivo testing of FLT3 inhibitor and PAD combinations.

#835

TRPH-222, a novel anti-CD22 antibody drug conjugate (ADC), has significant anti-tumor activity in NHL xenografts and reduces B cells in monkeys.

Ann P. Maclaren, Nancy Levin, Henry Lowman. _Triphase Accelerator, La Jolla, CA_.

CD22 is a cell surface protein with expression restricted to mature B lineage cells. It is highly expressed on a wide range of B cell malignancies including NHL (non-Hodgkin's lymphoma), DLBCL (diffuse large B cell lymphoma), follicular lymphoma and MCL (mantle cell lymphoma). CD22 is a clinically validated target for an antibody-drug conjugate (ADC) with approval in B cell acute lymphoblastic leukemia (B-ALL). In contrast, in NHL CD22 targeted ADCs have been limited clinically due to a poor therapeutic index. Subsequently the ADC field has focused on stable and site-specific conjugation of payloads for increased therapeutic index. TRPH-222 comprises an anti-CD22 antibody site-specifically modified at one site per heavy chain to express formylglycine (FG), allowing site-specific conjugation of a maytansinoid payload, a protease-insensitive spacer, and a functional group for coupling to an aldehyde on antibody FG residues. This SMARTAG™ site specific non-cleavable conjugation enforces a maximum drug to antibody ratio (DAR) of 2 with high stability enabling an improved therapeutic index. Studies described here characterize the anti-tumor activity of TRPH-222 in human NHL xenograft models and evaluate the pharmacodynamic effects in peripheral B cells. In WSU-DLCL2 xenografts once weekly intravenous (IV) dosing with either 3 or 10 mg/kg TRPH-222 reduced tumor volume by 100% after 28 days, as did a once every three-week 10mg/kg dose. In SU-DHL-2 and SU-DHL-4 xenograft models, TRPH-222 resulted in dose- and schedule-dependent anti-tumor activity with 91% and 100% tumor growth inhibition respectively at 10 mg/kg once weekly dosing. In the Granta-519 xenograft model, weekly 10 mg/kg dosing resulted in 87% tumor growth inhibition and also significantly inhibited tumor growth following escape from R-CHOP. Cross-species assessment of TRPH-222 binding revealed that TRPH-222 bound to immobilized human but not rat CD22. Similarly, TRPH-222 bound strongly to human and monkey peripheral B cells whereas no binding was observed to rat B cells. The pharmacodynamic effects of TRPH-222 on circulating rat and monkey B cell levels were evaluated following either a single (rat) or repeat (monkey) IV doses of TRPH-222 up to either 60 mg/kg or 50mg/kg respectively. In rats, TRPH-222 did not result in a reduction in absolute B cell numbers in any of the treatment groups. In contrast, B cells in cynomolgus monkey were reduced up to 70% from baseline in peripheral blood at all doses evaluated. In conclusion, the studies reported here demonstrate that TRPH-222 has significant anti-tumor activity in all NHL lymphoma models tested and has activity following R-CHOP escape. TRPH-222 demonstrated a CD22-mediated pharmacodynamic effect in monkeys that was consistent with target binding. IND-enabling safety studies are currently ongoing with TRPH-222 to support clinical development in 2018.

#836

ISU104, a fully human anti-ErbB3 antibody, overcomes acquired cetuximab resistance.

Miyoung Kim, Youngmi Hur, Mirim Hong, Youngsoo Sohn, Kum-Joo Shin, Kyungyong Kim, Seung-Beom Hong, Kum-Joo Shin, Donggoo Bae. _ISU ABXIS, Bundang-gu Seongnam-si, Republic of Korea_.

ErbB3 is noted as one of major causes of acquired cetuximab resistance in colorectal and head and neck cancers. ErbB3 causes activation of alternative signaling pathways that bypass the original target and sustained PI3K/AKT activation, and these are associated with cetuximab resistance. We confirmed the induced ErbB3 activation by the cetuximab treatment in FaDu head and neck squamous-cell carcinoma (HNSCC) xenograft model. Immunoblot analysis was shown that twice weekly 10 mg/kg of cetuximab treatment upregulated ErbB3 expression and phosphorylation even though tumor growth was well controlled. To investigate whether ErbB3 activation by ligand, heregulin (HRG,) might induce cetuximab resistance, two cetuximab-sensitive colorectal cancer cell lines, DiFi and LIM1215, were treated 0-25 ng/mL of HRG. HRG induced dose-dependent cetuximab resistance in cell proliferation assay, and this was reversed via ISU104 treatment. To evaluate if ISU104 could overcome resistance to cetuximab in vivo, acquired cetuximab-resistant FaDu xenograft model was established. Tumors that had acquired resistance to 5 mg/kg of cetuximab treatment were significantly regressed by replaced treatment of ISU104 alone (10 mg/kg) or combination treatment of ISU104 and cetuximab, while mice continued on cetuximab only showed uncontrolled tumor growth like vehicle-treated group. Our results suggest that ISU104 effectively overcomes cetuximab resistance and may provide clinical benefit to cetuximab-resistant patients.

#837

Antitumor effects of anti-EGF antibodies generated by vaccination in NSCLC tumor cells.

Silvia Garcia-Roman,1 Jordi Codony-Servat,1 Miguel Angel Molina-Vila,1 Jordi Bertran-Alamillo,1 Ana Gimenez-Capitán,1 Niki Karachaliou,2 Erik d'Hondt,3 Rafael Rosell2. 1 _Laboratory of Oncology / Panagaea Oncology, Barcelona, Spain;_ 2 _Quirón-Dexeus University Institute, Barcelona, Spain;_ 3 _Bioven, Aberdeen, United Kingdom_.

Background: Immunization against Epidermal Growth Factor (EGF) has demonstrated clinical efficacy in a phase III trial including unselected NSCLC patients. We are currently analyzing if anti-EGF antibodies generated by vaccination (anti-EGF VacAbs) showed antitumor activity in EGFR-mutant, Kras-mutant (mut) and Anaplastic Lymphoma Kinase (ALK) translocated non-small cell lung cancer cells (NSCLC), alone or in combination with tyrosine kinase inhibitors (TKI). In an EGFR-mut and in an ALK translocated cell lines we are also studying if the anti-EGF VacABs can delay the emergence of resistance to TKIs.

Methods: Anti-EGF VacAbs were obtained by immunizing rabbits with recombinant EGF. Cell lines were treated with anti-EGF VacABs alone and in combination with TKIs in EGFR (PC9, H1975) and ALK translocated (H3122, H2228) cell lines. In KRAS-mut cells (A549, H23) the combination was with chemotherapy agents. Cell viability was analyzed by MTT and apoptosis and cell cycle by fluorescence-activates cell sorting analysis (FACS). Changes of total and phosphorylated proteins were determined by Western blot. Sera from advanced NSCLC patients immunized with anti-EGF vaccine were also tested

Results: Anti-EGF VacAbs suppressed EGF-induced cell proliferation and inhibited downstream EGFR signaling in all cell lines tested. In combination, the anti-EGF VacAbs significantly enhanced the antitumor activity of gefitinib, erlotinib, osimertinib and afatinib in EGFR-mut cells, potentiated Erk ½ phosphorylation, arrested cell cycle progression and increased apoptosis. In addition, anti-EGF VacAbs blocked the activation of STAT3, downregulated the expression of proteins related to EGFR resistance, such as AXL, and significantly delayed the emergence in vitro of clones resistant to EGFR TKI. Sera from patients vaccinated against EGF also suppressed Erk ½ phosphorylation. Results for the combination of ALK TKIs and chemotherapy agents in ALK translocated and KRAS-mut cell lines will be presented at the meeting.

Conclusions: Anti-EGF VacAbs decreased cell proliferation and inhibited the activation of EGFR-pathway downstream proteins in EGFR-mut, KRAS-mut and ALK translocated cell lines. Also, they potentiate the effects of TKIs and prevent the emergence of resistance in EGFR-mut NSCLC cells. Two clinical trials are currently testing anti-EGF vaccination in EGFR-wt and EGFR-mut advanced NSCLC patients.

#837A

Combining CRISPR/Cas9 mutagenesis and a small-molecule inhibitor to probe the function of MELK in cancer.

Christopher J. Giuliano,1 Ann Lin,1 Joan C. Smith,2 Ann C. Palladino,3 Jason M. Sheltzer3. 1 _Stony Brook Univ., Stony Brook, NY;_ 2 _Google, New York, NY;_ 3 _Cold Spring Harbor Laboratory, Cold Spring Harbor, NY_.

The Maternal Embryonic Leucine Zipper Kinase (MELK) has been identified as a promising therapeutic target in multiple cancer types. MELK over-expression is associated with aggressive disease, and MELK has been implicated in numerous cancer-related processes, including chemotherapy resistance, stem cell renewal, and tumor growth. Based on these preclinical results, the MELK inhibitor OTS167 is currently being tested as a novel chemotherapy agent in several clinical trials. Here, we report that cancer cell lines harboring CRISPR/Cas9-induced null mutations in MELK exhibit wild-type growth in vitro, under environmental stress, in the presence of cytotoxic chemotherapies, and in vivo. Furthermore, MELK-knockout lines remain sensitive to OTS167, suggesting that this drug blocks cell division through an off-target mechanism. By combining our MELK-knockout clones with a new highly-specific MELK inhibitor, we further demonstrate that the acute inhibition of MELK results in no specific anti-proliferative phenotype. Analysis of gene expression data from cancer patients identifies MELK expression as a correlate of mitotic activity, explaining its association with poor prognosis. In total, our results demonstrate the power of CRISPR/Cas9-based genetic approaches to investigate cancer drug targets, and call into question the rationale for treating patients with anti-MELK monotherapies. 

### Modulators of Ionizing Radiation and Other Radiotherapeutics

#838

**Synergistic** in vivo **activity of the ATR inhibitor BAY 1895344 in combination with the targeted alpha therapy radium-223 dichloride in a preclinical tumor model mimicking bone metastatic castration-resistant prostate cancer (mCRPC).**

Antje Margret Wengner,1 Gerhard Siemeister,1 Ulrich Luecking,1 Julien Lefranc,1 Arne Scholz,1 Mari Suominen,2 Kirstin Meyer,1 Eleni Lagkadinou,1 Dominik Mumberg1. 1 _Bayer AG, Berlin, Germany;_ 2 _Pharmatest Services Ltd., Turku, Finland_.

The integrity of the genome in eukaryotic cells is secured by complex signalling pathways, known collectively as DNA damage response (DDR). Recognition of DNA damage activates DDR pathways resulting in cell cycle arrest, suppression of general translation, induction of DNA repair, cell survival, or even cell death. Proteins that directly recognize aberrant DNA structures recruit and activate kinases of the DDR pathway, such as ATR (ataxia telangiectasia and Rad3-related). The ATR kinase is activated by a broad spectrum of DNA damages, including double-strand breaks (DSBs) and lesions derived from interference with DNA replication as well as increased replication stress. Therefore, inhibition of ATR kinase activity could be the basis for a novel anti-cancer therapy in cancers characterized by increased DNA damage, deficiency in DNA damage repair or replication stress. Radium-223 dichloride (Xofigo®) is the first approved targeted alpha therapy. It is indicated for the treatment of patients with castration-resistant prostate cancer (CRPC), symptomatic bone metastases and no known visceral metastatic disease, based on improvement of overall survival. It is a calcium-mimetics that selectively binds to hydroxyapatite targeting areas of high bone turnover such as bone metastases, thereby exhibiting strong cytotoxic effects on adjacent cells via induction of DNA DSBs. We assessed the anti-tumor efficacy of combination treatment with ATR inhibitor (ATRi) BAY 1895344 and radium-223 dichloride in an intratibially injected prostate cancer model mimicking CRPC with bone metastases. In vivo analyses addressed the optimization of the dosing schedule as well as dose-response of BAY 1895344 in radium-223 combination setting. Levels of intra-tumor DNA damage (P-H2AX) were assessed to demonstrate the proposed mode-of-action. Here, we show that combination treatment with ATRi BAY 1895344 and radium-223 exhibits synergistic anti-tumor activity in the intratibial LNCaP xenograft model of mCRPC, achieving best efficacy when BAY 1895344 is applied 24 hours after treatment with radium-223, as indicated by direct reduction of tumor burden in the bone, lower serum tumor marker (PSA), and smaller areas of tumor-induced changes in bone. With this optimized schedule, a very low dose of BAY 1895344 (3% of single-agent MTD) was effective in combination with radium-223. Our findings strongly suggest that the combination of DNA damage induction by radium-223 with DDR inhibition by ATRi BAY 1895344 creates a potential new treatment option for CRPC patients with bone metastases. BAY 1895344 is currently under clinical investigation in patients with advanced solid tumors and lymphomas (NCT03188965).

#839

Treatment and signaling contexts for the application of the imipridone ONC201 to prostate cancer cells.

Francesca Amoroso,1 Adam Pickard,2 Kimberly Glass,1 Rohinton Tarapore,3 J. E. Allen,3 Ian G. Mills1. 1 _Queen's Univ. Belfast, Belfast, United Kingdom;_ 2 _University of Manchester, Manchester, United Kingdom;_ 3 _Oncoceutics Inc., Philadelphia, PA_.

Prostate cancer is the most common non-cutaneous cancer in men and a significant cause of cancer mortality. In the majority of the cases localised disease is treated with surgery or radiotherapy and whilst androgen deprivation therapy can extend survival in the subset of cases that progress, metastatic disease is incurable. We have previously reported that oncogenic drivers of prostate cancer (e.g. PTEN loss or mutation), as well as therapy, lead to changes in the activation of prostate cancer cell stress signalling pathways and in particular the unfolded protein response (UPR)1. Here we assess whether the activation of the UPR in response to radiation mirrors the immunogenic/inflammatory response, which is known to characterize the initially acute apoptotic phase before resolving to a chronic level. Further we have evaluated the impact of combining radiation with ONC201, a novel anti-cancer small molecule in clinical trials that is known to activate the pro-apoptotic stress response signalling2. We find that ONC201 has a delayed cytotoxic effect as a single agent in prostate cancer cells, with significant dose-dependent reductions in cell viability occurring 72 hours post-treatment. However activation of multiple arms of the UPR occurs earlier and is detectable at 24 hours for ATF4, ATF6 and IRE1-XBP1 at the protein/mRNA levels in the absence of impacts on cell viability. This delayed impact on viability offers a time window within which to evaluate short-term administration of ONC201 as a 'primer' to enhance the cytotoxic response to radiation. We show that 'priming' does indeed enhance radiotherapy response as assessed by cell viability and clonogenic assays. Since PTEN-loss is a high-incidence event in the development and progression of prostate cancer and has been linked to prognosis, we have also evaluated the impact of altering PTEN expression in our models as well as the effect of knocking down core regulators of the UPR. Cumulatively this work represents a further progression to positioning ONC201 in an appropriate molecular context to have durable effects when combined with other therapies to treat prostate cancer.

1.Sheng, X. et al., EMBO Molecular Medicine 7, 788-801 (2015).

2.Allen, J. et al. Discovery and clinical introduction of first-in-class imipridone ONC201. Oncotarget (2016). doi:10.18632/oncotarget.11814

#840

ATR inhibition is a promising radiosensitizing strategy for chemotherapy-resistant triple negative breast cancer (TNBC).

Robert W. Mutter, Mohamed M. Kahila, Xinyi Tu, Qin Zhou, Jia Yu, Krishna R. Kalari, Liewei Wang, Judy C. Boughey, Matthew P. Goetz, Jann N. Sarkaria, Zhenkun Lou. _Mayo Clinic, Rochester, MN_.

Purpose: TNBC represents an aggressive subtype characterized by earlier relapse and worse survival compared to non-TNBC. Patients with residual TNBC after neoadjuvant chemotherapy (NAC) have a high risk of locoregional recurrence despite aggressive subsequent local therapies, including RT. Thus, new radiosensitizers that address chemoresistant TNBC (i.e residual disease after NAC) are needed. ATR plays a key role in the cellular response to replication stress and DNA damage. TNBC is characterized by G1 checkpoint loss, homologous recombination deficiency (HRD), and other features which increase dependence on ATR pathway signaling. We hypothesized that VX-970, a selective inhibitor of ATR, would effectively radiosensitize TNBC.

Methods: Cell lines representing varying subtypes of TNBC (MDA-MB-231, HCC1806, and BT-549) and the normal breast epithelial cell line MCF10a were investigated in clonogenic survival, cell cycle, and DNA damage signaling and repair assays. In addition, Mayo Clinic (MC)TNBC1 and MCTNBC2, patient derived xenograft models (PDXs) generated prospectively from baseline and chemoresistant residual disease surgical specimens after NAC, respectively, of unique patients in the Breast Cancer Genome Guided Therapy Study were analyzed. Tumors were injected into hind-legs of athymic nude mice and animals were randomized to: 1) Vehicle 2) RT 3) VX-970 4) RT + VX-970. RT was delivered in 5 daily fractions of 2 Gy. VX-970 was administered daily 1 hour prior to RT at a dose of 60 mg/kg. Exome sequencing was assessed for germ-line and/or somatic alterations in HR genes and an ex-vivo RAD51 foci formation assay was applied to confirm the functional status of HR in each PDX model.

Results: In vitro, VX-970 preferentially inhibited ATR-Chk1-CDC25a signaling, abrogated the RT-induced G2/M checkpoint, delayed resolution of ƔH2AX and 53BP1 foci and reduced colony formation after RT in MDA-MB-231, HCC1806, and BT-549 relative to MCF10a. In vivo, VX-970 did not exhibit single agent activity at the dose administered but markedly sensitized both MCTNBC1 and MCTNBC2 to RT, suggesting that VX-970 could overcome chemoresistant TNBC biology. For MCTNBC1 and MCTNBC2, the median time to tumor tripling was 26 vs 49 days (p=0.002) and 25 vs 43 days (p=0.006) for the RT and RT + VX-970 groups, respectively. Ex-vivo RAD51 foci formation was robust in MCTNBC1, a PDX without HR gene alterations. In contrast, there was no RT-induced RAD51 foci formation in MCTNBC2, suggesting a BRCA1 mutation in the corresponding human tumor resulted in loss of HR function in that model.

Conclusion: VX-970 + RT effectively radiosensitized TNBC PDXs established from both pre-treatment biopsy specimens and chemoresistant residual tumor samples and in both HR deficient and HR proficient settings. ATR inhibition should be investigated in the clinic in combination with RT for patients with residual TNBC after NAC.

#841

Protection of normal tissues by DIM (3,3'-Diindolylmethane) during radiation therapy.

Renxiang Chen, Lijun Li, Loretta Y. Lin, Albert J. Fornace, Heng-Hong Li. _Georgetown Lombardi Comp. Cancer Ctr., Washington, DC_.

96 Normal 0 false false false EN-US ZH-CN X-NONE /* Style Definitions */ table.MsoNormalTable {mso-style-name:"Table Normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-parent:""; mso-padding-alt:0in 5.4pt 0in 5.4pt; mso-para-margin:0in; mso-para-margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:12.0pt; font-family:"Calibri",sans-serif; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-fareast-language:EN-US;} Despite advances in precision targeting of tumors by radiation therapy, local recurrence is still a serious problem due to the inability to sometimes deliver a sufficiently high dose of radiation to achieve tumor control without unacceptable late normal tissue and organ toxicity. DIM (3,3'-diindolylmethane), a commercially available dietary supplement, has long been studied as a cancer prevention and anti-tumor agent. We are investigating DIM's differential effects after radiation in tumor and normal tissues using a mouse model approach. We monitored tumor growth after localized radiation in a tumor engraftment model of MDA-MB-231 human breast carcinoma cells using immunocompromised NSG mice. The fractionated regimen consists of a total dose of 24 Gy in four fractions (6 Gy per fraction given every other day) with administration of DIM or vehicle at one hour prior to each fraction. DIM showed no significant effect on the growth of irradiated tumors, i.e. DIM did not affect the treatment efficacy of radiation on tumors. On the other hand, we are investigating the molecular mechanism of DIM's radio-protective effects in normal tissues. Our transcriptomic data indicate that DIM treatment increases expression of some stress-responsive genes without causing DNA damage. Results to date indicate that DIM might delay radiation-induced cell cycle arrest and apoptosis. The ultimate goal is to develop DIM as a clinical radioprotector in order to improve the therapeutic index by allowing higher doses of radiation to improve local tumor control by reducing late dose-limiting normal tissue toxicity by radiation.<!--EndFragment-->

#842

Increased irradiation-induced senescence in Fanconi anemia (FA) mice.

Aranee P. Sivananthan, Xichen Zhang, Wen Hou, Donna Shields, Renee Fisher, Michael Epperly, Joel S. Greenberger. _University of Pittsburgh, Pittsburgh, PA_.

Senescence is an irreversible cell cycle arrest induced by various triggers, including aging and irradiation. Fanconi Anemia (FA) is a genetic disorder associated with defects in 22 proteins in the scaffold DNA double strand break repair. FA mice develop anemia and marrow failure and have been considered as a potential model for accelerated aging. Fancd2-/- murine bone marrow (BM) stromal cell lines are radiosensitive in clonogenic survival curves compared to wild type (Berhane et al, Radiat Res, 2014). We tested the Fanca-/- mouse model for irradiation induced senescence. 129Sv Fanca+/+ and Fanca-/- BM stromal cell lines were irradiated to 5Gy, then assayed for senescence at various timepoints. Cells were stained with a chromogenic beta-galactosidase assay. Cells were also stained by immunofluorescence (IF) to determine percent of cells positive for p16 and p21. For total body irradiation (TBI) studies, 129Sv Fanca+/+ and Fanca-/- mice were given 7Gy. Organs were harvested at Day 7, then assayed for senescence. Statistical analysis was carried out using student's t-test. Baseline levels of beta-gal positive Fanca-/- BM stromal cells were not significantly different from Fanca+/+ (respectively, 0.88% ± 0.22% vs 0.74% ± 0.23%). Fanca-/- and Fanca+/+ IF also showed no significant differences in baseline p16 levels (4.98%±2.5 vs 14.37%± 4.06%) and p21 levels (14.57% ± 7.61% vs. 11.7% ± 3.48%). In contrast, Fanca-/- cells showed significantly higher beta-gal staining compared to Fanca+/+ at days 1, 3, and 5 after irradiation (p=0.0042, p<0.0001, p=0.0011 respectively). By day 3 after 5Gy irradiation, Fanca-/- cells showed significant senescence increase compared to control non-irradiated cells for p16 (29.54%± 4.11%) and p21 (84.27±10.40). At day 3 after 5Gy irradiation, In Fanca+/+ cells there was less profound but also significant induction of p16 (17.78%± 3.99%) and p21 (62.08±84.27). At Day 7 after irradiation, beta-gal positive cells were significantly increased compared to control non-irradiated in WT (5.943% ± 0.96%, p<0.001) and Fanca-/- (9.07% ± 1.45%, p<0.0001) cells. At Day 7 after 7Gy TBI, kidney, liver, lung, skin, and brain did not show induction of beta-gal staining. The spleen, a radiosensitive organ, displayed increased beta-gal staining, with a larger increase in Fanca-/- spleen compared to Fanca+/+. Expression of p21 mRNA was significantly induced in Fanca-/- (5.8x increase, p=0.0305) and Fanca+/+ (7.3x increase, p=0.0357) compared to non-irradiated control spleens. Expression of p16 mRNA was increased in both genotypes. Therefore, Fanca-/- mouse organs in vivo and cell lines in vitro display increased irradiation induced senescence compared to Fanca+/+ mice. Sorting live senescent cells will confirm the increase in irradiation-induced senescent cells, and functional outcomes in FA mice.

#843

Assessment of high-dose radiation efficacy administered to patient-derived xenografts based on the genomic reclassification of endometrial carcinomas.

Chieh-Hsiang Yang,1 David K. Gaffney,2 Elke A. Jarboe,3 Margit M. Janát-Amsbury1. 1 _Department of OB/GYN, University of Utah, Salt Lake City, UT;_ 2 _Department of Radiation Oncology, University of Utah, Salt Lake City, UT;_ 3 _Department of Pathology, University of Utah, Salt Lake City, UT_.

Adjuvant treatment of patients with endometrial cancer (EC) is tailored to clinical-pathologic prognostic factors. Pelvic radiation therapy for stage I EC provides a highly significant improvement of local control, but without survival advantage, and the use of adjuvant radiotherapy (RT) is limited to patients with high-intermediate or high-risk factors. Due to a rising incidence rate of EC there is an immediate need to improve how adjuvant treatments are being selected. The exploration of four genomic clusters by The Cancer Genome Atlas (TCGA) Network provides molecular insights and opportunity to refine current management strategies. However, no prospective validation of the relationship between cluster-association and treatment response has been reported. In this study, we evaluated the therapeutic efficacy of high-dose rate (HDR) radiation on the basis of genomic clusters.

Objectives: To evaluate treatment response of HDR radiation according to TCGA classification using a panel of endometrial cancer patient-derived xenografts (EC-PDXs).

Methods: Thirty-two EC-PDXs upon histologic and genomic characterization were orthotopically transplanted and propagated. Subcutaneously grafted PDX were subjected to a PDX clinical radiation trial. Animals were placed under a lead shield for irradiation once tumors reached 150-200 mm3 and 7Gy of radiation was delivered weekly for three weeks. Two additional weeks were given to investigate potential tumor regrowth. Histologic evaluation was conducted upon necropsy.

Results: The overall response rate was 78% (25/32), in which high-grade ECs exhibited increased sensitivity to radiation compared to low-grade ECs (overall response rate 89% for high-grade versus 62% for low-grade). Copy number high (CNH) EC-PDXs showed the best response rate (100%), microsatellite instable (MSI) lines responded moderately (73%), and copy number low (CNL) lines exhibited the lowest response rate (42.9%). Nonresponders were noted among CNL and MSI clusters featuring low-grade endometrioid histology. Further evaluation of the proliferation index confirmed that all responders had a >90% decrease on H-score compared to the nonresponders, which either showed a slight decrease or no changes at all.

Conclusions: This is the first radiation treatment study selecting candidates within a PDX clinical trial based on TCGA cluster affiliation. Our findings suggest that EC-PDXs categorized as CNH or with higher histologic grade seem more sensitive to radiotherapy. Therefore, special attention and reconsideration of treatment recommendation should be given to patients harboring tumors with MSI and CNL genomic features. Further studies towards the identification of biomarkers or additional subclusters within responders and nonresponders will benefit physicians and patients to direct therapy.

#844

Preclinical activity of PSMA-TTC, a targeted alpha therapeutic in patient-derived prostate cancer models.

Stefanie Hammer,1 Urs B. Hagemann,1 Sabine Zitzmann-Kolbe,1 Aasmund Larsen,2 Christine Ellingsen,2 Oliver von Ahsen,1 Jenny Karlsson,2 Roger M. Bjerke,2 Olav B. Ryan,2 Pascale Lejeune,1 Hartwig Hennekes,1 Alan Cuthbertson,2 Karl Ziegelbauer,1 Dominik Mumberg1. 1 _Bayer AG, Berlin, Germany;_ 2 _Bayer AS, Oslo, Norway_.

Targeted alpha therapy (TAT) agents deliver high linear energy transfer (LET) alpha-radiation selectively to tumors. The first TAT to be approved is radium-223 which prolongs overall survival in metastatic castration resistant prostate cancer (mCRPC) patients with symptomatic bone metastasis. Radium-223 shows a selective uptake in newly formed bone matrix such as bone metastasis and binds to hydroxyapatite. The PSMA targeted thorium-227 conjugate PSMA-TTC represents another TAT approach in mCRPC. It consists of a fully human PSMA IgG antibody covalently linked to the chelator moiety (3,2 HOPO). This antibody-chelator conjugate is radiolabeled with thorium-227, which decays with a half-life of 18.7 days to radium-223 via alpha-particle emission.

Herein we describe tumor targeting and anti-tumor activity of PSMA-TTC in two PSMA positive patient derived xenograft (PDx) models of prostate cancer with different characteristics. In vivo biodistribution and anti-tumor efficacy were analyzed after i.v. injection of PSMA-TTC at radioactive doses from 75-500 kBq/kg and protein doses of either 0.14 or 0.43 mg/kg to tumor bearing mice.

Initially, the PDx model KuCap1 (provided by Prof. O. Ogava, University of Kyoto, Japan), a prostate cancer model resistant to the second generation antiandrogen enzalutamide, was analyzed. In this model PSMA-TTC showed strong dose dependent tumor growth inhibition starting at a single dose of 75 kBq/kg. Moreover, after a single i.v. administration of PSMA-TTC at 300 kBq/kg 9 out of 10 mice (90%) showed either stable disease or tumor regression for at least 33 days after treatment. The observed activity was highly selective, as injection of a radiolabeled control conjugate at 300 kBq/kg showed only limited tumor growth inhibition.

Next, PSMA-TTC was tested in the hormone- and enzalutamide-sensitive prostate cancer PDx model ST1273 (South Texas Accelerated Research Therapeutics, San Antonio, Texas). A single i.v. injection of PSMA-TTC resulted in significant tumor accumulation of thorium-227 for more than 3 weeks, whereas a radiolabeled isotype control conjugate did not show tumor uptake. In addition, single i.v. administration of PSMA-TTC showed strong dose dependent anti-tumor activity while limited tumor growth inhibition was observed for a radiolabeled isotype control conjugate. Single doses of either 250 or 500 kBq/kg resulted in a 100 % response rate 4 weeks after treatment, with all animals showing partial or even complete regression of tumor growth. No significant effects on body weight were detected compared to vehicle treated animals.

In summary, PSMA-TTC shows strong anti-tumor activity in patient derived prostate cancer models which were either sensitive or resistant to standard of care drugs. These data warrant further clinical investigation of this targeted alpha pharmaceutical investigational agent.

#845

Acute irradiation exposure induces long-term cardiac adverse effects in the spontaneously hypertensive rat.

Elliot T. Rosen,1 Dmitry Kryndushkin,1 Baikuntha Aryal,1 Yanira Gonzalez,1 Leena Chehab,1 Jennifer Dickey,1 Steven Mog,2 V Ashutosh Rao1. 1 _FDA-CDER, Silver Spring, MD;_ 2 _FDA-CFSAN, College Park, MD_.

Radiation-induced heart disease (RIHD) presents a significant challenge in the oncology setting. For instance, patients receiving acute doses of irradiation to the thoracic cavity for Hodgkin's lymphoma or breast cancer are subject to an elevated risk of RIHD. Excessive oxidative stress, at least in part, is responsible for some of the tissue damage. The ability to monitor and mitigate the long-term cardiac effects of radiation exposure in these populations by modulating oxidative stress has the potential to lead to improved health outcomes. We utilized the spontaneously hypertensive rat (SHR), which has proven a good model for drug-induced cardiotoxicity, to evaluate irradiation-induced heart damage. SHRs were exposed to low, medium, or high doses of full-body irradiation. Necropsy was performed to collect serum and tissue samples at two and four weeks post-irradiation, as well as followed for one year. Four weeks following gamma irradiation, both males and females showed decreased heart mass and decreased whole body mass after exposure to medium and high doses of radiation and these decreases were sustained in the high dose groups after one year. Serum cardiac troponin I and T analysis revealed signs of cardiomyopathy in both the medium and high dose groups at early time points and this signal was sustained in some animals up to one year later. Echocardiography revealed functional changes in cardiac performance including an increase in left ventricular volume at diastole in males and a decrease in left ventricular mass. Low red blood cell count indicated sustained anemic conditions in both males and females receiving the highest dose of irradiation, yet males appeared to be more sensitive. Levels of pro-inflammatory IL-6 were elevated at four weeks in males receiving the high dose. Finally, multiple animals receiving radiation developed spontaneous tumors after one year. We are currently investigating the oxidative stress-mediated molecular mechanisms behind the radiation-induced cardiac damage.

#846

Co-targeting of Axl and MerTK improves radiation response in HNSCC.

Mahyar Abbariki,1 Mahmoud Toulany,2 Mari Iida,1 Alecia Morgan,1 Kwang Nickel,1 Nellie McDaniel,1 Grace Kang,1 Chunrong Li,1 Paul Harari,1 Deric Wheeler1. 1 _University of Wisconsin-Madison, Madison, WI;_ 2 _University of Tuebingen, Tuebingen, Germany_.

Axl and MerTK are members of the TAM family (Tyro, Axl and MerTK) of receptor tyrosine kinases and have been reported to be altered in several types of cancer including Head and Neck Squamous Carcinoma (HNSCC). It is well known that aberrant expression or activity of Axl is associated with resistance to various therapies. Therefore, the translational potential of targeting AXL in HNSCC is worthy, however poorly defined. We recently identified an adaptive feedback mechanism of resistance to AXL-targeting agents mediated by upregulation of MerTK that reveals a direct role for MerTK in resistance to Axl inhibition. In this study, we investigated potential radiosensitization by dual targeting of Axl and MerTK in combination with radiation in human HNSCC cell lines and xenografts (SCC47 and TU138). Immunoblot analysis demonstrated that endogenous levels of Axl or MerTK expression were distinct between these two cell lines. SCC47 cells expressed higher endogenous levels of Axl compared to Tu138. Tu138 expressed higher levels of MerTK compared to SCC47 cells. Radiation induced Axl phosphorylation in SCC47 cells but not in Tu138 cells in vitro. Next, inhibitors of Axl (R428) and/or MerTK (UNC2025) were utilized in combination with single dose radiation in vitro. Clonogenic assays showed that the Axl inhibitor alone acted as a potent radiosensitizer in SCC47 cells. To better define Axl's role in radiation response in HNSCC, we developed HN30-Vector and HN30-Axl stable cells and exposed them to radiation. Phosphorylation of Axl was increased 1 hour post-radiation in Axl overexpressed HN30 compared to parental HN30-Vector which expresses low endogenous levels of Axl. We also found that the HN30-Axl cell line was associated with radioresistance by clonogenic assays. Tumor xenograft studies demonstrated that the combination of R428 and UNC2025 inhibitors improved the efficacy of radiation in Tu138 but not in SCC47. Interestingly, radiation therapy alone was more effective in SCC47 tumors than TU138 tumors. These results reveal that the co-targeting of Axl and MerTK enhanced tumor growth delay in TU138 xenograft mouse models in vivo. Mechanisms of radiosensitization are currently under investigation.

#847

Optimization of radiation regimen in a carcinogen-induced mouse model for lung cancer.

Sagar Bhayana, Joseph Liu, Arnab Chakravarti, Naduparambil K. Jacob. _The Ohio State University, Columbus, OH_.

Purpose: Lung cancer remains the most common cause of cancer-related mortality in United States and worldwide. The major cause of lung cancer is attributed to carcinogens present in tobacco smoke, air pollution, diet and food supplements, occupational and other environmental exposures. Lung Cancer Mutation Consortium has identified many recognized "driver mutations" in nearly two-thirds of the patients who developed advanced form of lung cancer. Our primary goal is to understand the mechanism underlying the variations in radiation sensitivity with driver mutations and optimize radiation dose and treatment regimen. A better understanding of the most frequent driving mutations is required for progression towards personalized therapy. Methods: Urethane, a carcinogen injected via intraperitoneal route into A/J mice induced multiple lung tumors by week 25 that resembled non-small cell lung carcinoma. These lung tumors were assessed via MRI and each tumor was longitudinally tracked via surface rendered 3D imaging following X-ray exposure using small animal radiation research platform (SARRP). We employed acute high single dose (12 Gy) and clinically relevant fractionated dose (3 X 5 Gy). Individual lung tumor was also dissected from non-irradiated and irradiated mice followed by collagenase digestion and then cultured in a specific media for in-vitro mutational analysis. Results: Urethane induced multiple lung tumors (20-25 tumors with 1mg/kg urethane) of varying size (0.5 - 2mm) were detectable by MRI at 25 week following injection. Longitudinal MRI performed on irradiated tumor bearing mice showed significant regression after radiation with commitment survival benefit. Mice performed better when exposed to fractionated regimen than high single dose with relatively low level radiation toxicity. Primary lung cancer cells were prepared from the explanted tumors for prospectively examining relevant driver mutations and studying the genetic determinants of resistance and to identify novel targets. We also have collected body fluids for investigating changes in molecular profile of exosomes at time points after irradiation. Conclusions: Our study thus far set up a platform for longitudinal 3D tracking of individual lung tumors following treatment and recurrence. Tumor regression was evident after a week of radiation exposure. Primary lung tumor cell cultures were successfully established for molecular analysis and mechanistic studies

#848

**In vitro and in vivo evaluation of the beta-emitting lutetium-177 labeled anti-CD37 antibody radionuclide conjugate** 177 **Lu-NNV003 in DLBCL, CLL and MCL models.**

Astri F. Maaland,1 Helen Heyerdahl,1 Adam O'Shea,1 Bergthora Eiriksdottir,2 Jostein Dahle1. 1 _Nordic Nanovector, Oslo, Norway;_ 2 _ArcticLAS ehf, Reykjavík, Iceland_.

The aim of this study was to evaluate the cytotoxic and anti-tumor efficacy of the beta-emitting lutetium labelled NNV003 antibody (177Lu-NNV003) in non-Hodgkin lymphoma (NHL) and Chronic Lymphocytic Leukemia (CLL) models.

Antibody-Dependent Cellular Cytotoxicity (ADCC) and Complement-Dependent Cytotoxicity (CDC) were measured in REC-1 (MCL), MEC-2 (CLL) and DOHH-2 (DLBCL) cells using FcRγIIIa immortalized NK92 cells as effector cells. Antibody-Dependent Cellular Phagocytosis (ADCP) was measured using a commercially available FcRγIIa-H reporter kit. Binding and internalization of 177Lu-NNV003 after up to 18 hours incubation were measured by a stripping buffer method. Cytotoxicity effects were evaluated by incubating cells with increasing concentrations of 177Lu-NNV003 and measuring cell proliferation. Biodistribution of 177Lu-NNV003 was measured in CB17 SCID and NSG mice with DLBCL and CLL xenografts, respectively. Therapeutic and toxic effects of 177Lu-NNV003 were measured in different strains of immunocompromised mice bearing systemic DLBCL (in RAG-2), CLL (in RAG-1) or MCL (in NOD SCID) xenografts, and compared with NaCl and unspecific 177Lu-labelled isotype control groups.

NNV003 elicited ADCC lysis in all cell lines and induced ADCP in MEC-2 and DOHH-2. Minimal CDC activation was observed. 177Lu-NNV003 showed activity dependent antiproliferative effect in all cell lines. Maximum tumor uptake in vivo was 45% IA/g in MEC-2 tumors and 15% IA/g in DOHH-2 tumors. Survival was significantly extended in DOHH-2 xenograft-bearing mice treated with NNV003 (2-30 mg/kg) and 177Lu-NNV003 (200-400 MBq/kg) compared to control groups (p<0.0001). In the MEC-2 model, 200 MBq/kg 177Lu-NNV003 extended survival compared to the NNV003 and the 177Lu-isotype treatment (p<0.025). In mice bearing REC-1 xenografts, both 50 and 100 MBq/kg 177Lu-NNV003 improved survival compared to control groups (p<0.02). Transient hematological toxicity was observed in all groups treated with radioactivity, and one mouse treated with 200 MBq/kg in the MEC-2 study died of radiation toxicity. 177Lu-NNV003 showed best anti-tumor effect in the two NHL models, and less effect in the CLL model despite the high tumor uptake, which might be explained by the very aggressive growth of the MEC-2 cells in vivo.

In conclusion, in vitro studies have shown that the unlabelled NNV003 antibody have an immunological cytotoxicity effect on tumor cells, mainly through ADCC, and that 177Lu-NNV003 inhibits cell growth. 177Lu-NNV003 has shown significant tumor uptake and therapeutic effect in vivo in all three models tested. The results warrant clinical testing in patients suffering from CD37-expressing B-cell malignancies.

#849

Synergistic effect of MSLN-TTC in combination with DNA damage response inhibitors.

Katrine Wickstroem,1 Urs B. Hagemann,2 Antje M. Wengner,2 Alexander Kristian,1 Christine Ellingsen,1 Gerhard Siemeister,2 Roger M. Bjerke,1 Jenny Karlsson,1 Olav B. Ryan,1 Lars Linden,3 Bertolt Kreft,2 Dominik Mumberg,2 Hanno Wild,2 Karl Ziegelbauer,2 Alan Cuthbertson1. 1 _Bayer AS, Oslo, Norway;_ 2 _Bayer AG, Berlin, Germany;_ 3 _Bayer AG, Wuppertal, Germany_.

Targeted Thorium-227 Conjugates (TTCs) consist of the alpha particle emitter thorium-227 bound with high affinity by a 3,2-HOPO chelator covalently attached to a tumor-specific antibody. Thorium-227 has a half-life of 18.7 days and decays to the alpha particle emitter radium-223 (half-life of 11.4 days), a calcium-mimetic used in the treatment of CRPC [Henriksen et al. J Nucl Med, 2003]. The mechanism of action for alpha emitters is based on the induction of clustered DNA double strand breaks and G2 cell cycle arrest. In principle, inhibitors of DNA damage response (DDR) should sensitize cancer cells to TTCs. Mesothelin (MSLN) is a 40 kDa membrane-anchored glycoprotein known to be overexpressed in mesothelioma, ovarian, lung, triple-negative breast (TNBC) and pancreatic cancers. This study describes the evaluation of combination treatment with anti-MSLN targeted thorium-227 conjugate (MSLN-TTC) in combination with DDR inhibitors. In vitro cytotoxicity experiments were performed on a set of cancer cell lines from different tissue origins, including Ovcar-3 (ovarian), NCI-H226 (mesothelioma), Capan-2 (pancreas) and mesothelin transfected HT29 (colon) cells. DDR inhibitors targeting Poly [ADP-ribose] polymerase 1 (PARP1), ataxia telangiectasia and Rad3-related (ATR), Ataxia-telangiectasia mutated (ATM) and DNA-dependent protein kinase (DNA-PK) included olaparib, BAY 1895344, AZD0156, and VX-984, respectively. Synergistic activity of MSLN-TTC was observed in vitro for all four DDR inhibitors with ATRi BAY 1895344 exhibiting the strongest increase in potency. We further evaluated the in vivo anti-tumor efficacy of MSLN-TTC monotherapy and combination treatment with DDR inhibitors ATRi BAY 1895344 or PARPi olaparib in Ovcar-3 xenografts in nude mice. Monotherapy of the MSLN-TTC yielded a dose dependent and specific tumor growth inhibition as compared with a radiolabeled isotype control. In the combination treatment an increased anti-tumor effect of MSLN-TTC at the lowest dose was observed when combined with a non-efficacious dose of either ATRi BAY 1895344 or olaparib, with a more pronounced effect for the combination of MSLN-TTC with ATRi BAY 1895344, resulting in tumor stasis. The present data support the rationale for combining the MSLN-TTC with DDRi's based on their individual mode of actions and bears strong potential for future cancer therapies in tumor indications characterized by overexpression of mesothelin, such as mesothelioma, ovarian, pancreatic, lung or triple-negative breast (TNBC) cancers.

#850

Mesothelin targeted thorium-227 conjugate (MSLN-TTC): Preclinical evaluation of a new targeted alpha therapeutic in mesothelin-positive cancers.

Urs B. Hagemann,1 Alexander Kristian,2 Christine Ellingsen,2 Katrine Wickstroem,2 Anne Mobergslien,2 Jenny Karlsson,2 Roger M. Bjerke,2 Christoph Schatz,1 Christoph Kneip,1 Joachim Schuhmacher,3 Liv-Ingrid Oedegaardstuen,2 Hartwig Hennekes,1 Anna Tafuri,1 Dominik Mumberg,1 Hanno Wild,3 Karl Ziegelbauer,1 Alan S. Cuthbertson2. 1 _Bayer Ag, Berlin, Germany;_ 2 _Bayer AS, Oslo, Norway;_ 3 _Bayer Ag, Wuppertal, Germany_.

Targeted Thorium-227 Conjugates (TTCs) represent a new class of targeted alpha therapy. In this compound family a 3,2-HOPO chelator, which binds thorium-227 with high affinity, is covalently attached to an antibody. This enables the specific delivery of the alpha particle emitter thorium-227 to tumor cells. Thorium-227 has a half-life of 18.7 days and decays via emission of an alpha particle to radium-223 (half-life of 11.4 days), a calcium-mimetic used in the treatment of CRPC [Henriksen et al. J Nucl Med, 2003]. The high linear energy transfer from the alpha-emitter thorium-227 induces clustered DNA double-strand breaks. Its short penetration range of 2-10 cell diameters limits the damage to the normal tissue surrounding the tumor. We present the preclinical evaluation of a mesothelin targeted thorium-227 conjugate (MSLN-TTC), the first TTC that will enter clinical development in MSLN-positive solid tumor indications, based on the fully human anti-MSLN monoclonal antibody anetumab. MSLN is a 40 kDa membrane-anchored glycoprotein with prominent overexpression in mesothelioma, ovarian, pancreatic, lung and breast cancer. In normal tissue, MSLN is confined mainly to the mesothelial cells of pleura, peritoneum and pericardium. In vitro, the mode of action of MSLN-TTC in cellular assays was demonstrated to induce DNA double strand breaks, leading to cell cycle arrest and subsequent reduced cell viability. In vivo, MSLN-TTC demonstrated potent tumor growth inhibition administered as a single-dose in cell- and patient-derived xenograft tumor models. Similar anti-tumor activity to single dose application was observed when the MSLN-TTC was applied at fractionated doses. A trend for dependence of anti-tumor activity on MSLN expression levels in preclinical tumor models was observed. Biodistribution studies evaluated the tumor accumulation of MSLN-TTC in xenograft models. These studies served to develop a mechanistic PK/PD model, which was used to predict the efficacious dose in humans. The initiation of clinical investigation of the MSLN-TTC in mesothelin positive cancers' is planned for 2018.

#851

Therapeutic combination of radiolabeled CLR1404 with external beam radiation in head and neck cancer murine xenograft models.

Chunrong Li, Jenna M. Mylin, Jamey P. Weichert, Justin J. Jeffery, Ashley M. Weichmann, Kwang P. Nickel, Lindsey J. Abel, Reinier Hernandez, Joseph J. Grudzinski, Ian R. Marsh, Bryan P. Bednarz, Shari M. Piaskowski, Paul M. Harari. _Univ. of Wisconsin School of Medicine, Madison, WI_.

Radiation therapy is a central treatment modality for head and neck cancer (HNC). Although significant technical advances have been made in delivering highly conformal radiation, normal tissue toxicity remains dose limiting. CLR1404 is a radiolabeled alkylphophocholine analog with theranostic potential as a PET imaging agent (CLR 124, labeled with 124I) and as a radiotherapy agent (CLR 131, labeled with 131I). CLR1404 exhibits preferential uptake in human cancers and provides tumor-selective internal delivery of radiation to complement external beam radiation (XRT) in the treatment of cancer. In this study, we investigated the antitumor effect of CLR 131 in combination with external beam radiation (XRT) in HNC. Using the fluorescently labeled CLR1404 analog, CLR 1501, in conjuntion with fluorescence microscopy and flow cytometry, we first confirmed CLR1404 uptake in 20 HNC cell lines in vitro. Thereafter, we examined CLR 124 uptake and retention in 12 HNC xenograft and patient-derived xenograft (PDX) mouse models via in vivo PET/CT imaging. Our results showed significant tumor uptake and retention of CLR 124 and limited retention in normal tissues. These results were corroborated by ex vivo gamma counting of excised tissues from tumor-bearing mice injected with CLR 131. We further assessed tumor response following single-dose administration of CLR 131 combined with fractionated XRT in 6 HNC xenograft models. Mice treated with non-radiolabeled CLR1404 displayed unchecked tumor progression. Mice treated with either CLR 131 or XRT showed partial inhibiton on tumor growth. Mice treated with CLR 131 combined with XRT exhibited enhanced inhibitory effect on tumor growth compared with either single treatment, confirming augmentation of XRT response by CLR 131 treatment. Targeted radionuclide therapy (TRT) is an attractive approach that employs radiolabeled molecules to specifically deliver radiation to primary and metastatic tumors. We hypothesized that TRT combined with reduced-dose XRT could reduce the normal tissue toxicity profile compared with high-dose XRT alone. In this study, we demonstrated uptake of CLR 131 across multiple HNC cell lines and xenograft models with enhanced antitumor effects when CLR 131 is combined with XRT. These results suggest the potential value of TRT via CLR 131 combined with reduced dose XRT in HNC patients, which will be further tested in a phase I clinical trial.

#852

Specificity of PSMA-617 radiotherapy for prostate cancer.

Joseph A. Reddy, Melissa Nelson, LeCun Xu, Elaine Westrick, Marilynn Vetzel, Leroy Wheeler, Albert Felten, Hari Santhapuram, Iontcho Vlahov, Christopher P. Leamon. _Endocyte, Inc., West Lafayette, IN_.

Prostate-specific membrane antigen (PSMA) is a biomarker that is overexpressed on prostate cancer as well as on the neovasculature of many non-prostate solid tumors. Lately, several small molecular PSMA binding ligands chelated to various radionuclides have been studied for imaging and treatment of prostate cancer lesions. One such clinically tested agent is PSMA-617, a DOTA derivative of a PSMA-specific targeting ligand. In our hands, radiochemical yields of PSMA-617 labeled with either 177Lu (β emitter) or 225Ac (α emitter) were greater than 97%. Binding experiments with 177Lu and 225Ac chelates of PSMA-617 and D-Glu PSMA-617 (a predicted non-PSMA binding, D-glutamic acid isomer of PSMA-617) were performed using the PSMA-positive, 22RV1 cell line. PSMA-617, was found to bind in a concentration dependent manner with very high affinity, whereas the cellular association of D-Glu PSMA-617 was found to be > 60-fold lower. The in vivo biodistribution of these two agents were investigated in BALB/c nu/nu mice bearing subcutaneous 22RV1 xenografts. Organ distribution revealed specific PSMA-617 uptake in the 22RV1 tumors and the PSMA+ kidneys, while the uptake of D-Glu PSMA-617 remained near background in both of these tissues. Different from tumor, PSMA-617 exhibited a rapid clearance from the kidneys to yield a high tumor-to-background contrast (tumor:blood = 300; tumor:muscle = 290; and tumor:kidney = 15) at 24 h post dose. Complementary antitumor activity studies were performed with 225Ac-labeled PSMA-617. Here, significant tumor growth inhibition was observed without detectable hematologic or renal toxicity to the mice. These data provide additional support for a phase 3 prospective trial currently being planned to evaluate 177Lu-PSMA-617 in patients with metastatic PSMA+ prostate cancers, along with the continued clinical investigation of 225Ac-PSMA-617 in a similar patient population.

#853

Investigation of the cumulative number of chromosome aberrations induced by three consecutive CT scans.

Yu Abe,1 Hideyoshi Noji,1 Misaki Sugai,1 Yumiko Kurosu,1 Takashi Ohba,1 Aki Yanagi,1 Yukari Yanai,1 Naohiro Tsuyama,1 Tetsuo Ishikawa,1 Tomisato Miura,2 Kenji Kamiya,3 Mitsuaki A. Yoshida,4 Akira Sakai1. 1 _Fukushima Medical University School of Medicine, Fukushima, Japan;_ 2 _Hirosaki University Graduate School of Health Sciences, Hirosaki, Japan;_ 3 _Hiroshima University, Hiroshima, Japan;_ 4 _Hirosaki University, Hirosaki, Japan_.

Purpose The relationship between radiation exposure by CT and the risk of leukemia and brain tumor has been reported in children, in which the risk of leukemia was tripled at a radiation dose of 60 mGy to bone marrow (Pearce MS, Lancet 2012). Therefore, there is a possibility that CT scans induce chromosome aberrations (CAs) causing malignant tumors. When chromosomes are cleaved by radiation, dicentric chromosomes (Dics) and chromosome translocations (Trs) are formed in the same ratio. We have reported that breakage of chromosomes was induced after a single CT scan in adults by Dic assay (DCA) (Scientific Rep 2015). On the other hand, the number of Trs formed also increased after a single CT scan, but, no significant change of the numbers was observed before and after the CT scan (J Rad Res 2016). We then investigated whether cumulative CAs would be observed when an annual CT scan was performed for 3 years.

Methods Peripheral blood samples were collected from 5 patients (4 males and 1 female; age range 51-73 years; mean age, 64 years) 3 times before and after a CT scan at annual examinations. They included 4 patients with malignant lymphoma who had not been treated for more than 3 years after the end of treatment and 1 patient followed due to an abnormal lung shadow without treatment. We performed analysis of Dic and Tr formation by centromere-FISH and painting-FISH using isolated lymphocytes, respectively, and counted the number of CAs equivalent to 2,000 metaphases per sample. The effective radiation dose of a CT scan was calculated by computational dosimetry system (WAZA-ARI).

Results Regardless of CT scans, the number of Trs formed was higher than that of Dics in all patients. The dose of radiation exposure in one CT scan was 22.0 to 73.5 mSv (mean, 42.3 mSv). Both DCA and Tr analyses showed an increase in Dic and Tr formation after all CT scans, except that their number decreased in one patient after the second CT scan. However, the increase in these CAs decreased after about one year, and no cumulative increase in the number of Dics and Trs formed due to three consecutive CT scans was observed.

Conclusion A single CT scan tended to produce both Dic and Tr formation, but there was no cumulative increase in CAs after three consecutive CT scans. The reason that the number of Trs formed was basically higher than that of Dics is considered to be related to such factors as age, smoking status, and past medical radiation exposure especially in adults because Tr is a stable chromosome.

#854

Validation of a viability assay for assessing radiation response and investigating drug/radiation combinations.

Meghan Lambie, Venkata S. Manem, Benjamin Haibe-Kains, Scott V. Bratman. _Princess Margaret Cancer Centre, Toronto, Ontario, Canada_.

Purpose: Radiotherapy (RT) is frequently used either alone or in combination with chemotherapy for curative treatment of squamous cell carcinomas (SCC) originating from the head and neck, lung, esophagus, and cervix. Treatment outcomes for SCCs are heterogeneous, in part due to variable degrees of resistance to ionizing radiation and modest benefit of existing radiosensitizing drugs. Preclinical research into intrinsic cancer cell radiosensitivity biomarkers and novel radiosensitizing drugs has been hampered by a lack of robust high throughput assays of radiation response. We sought to validate a recently published high throughput viability assay as a surrogate of clonogenic survival and extend its use to drug-radiation combinations.

Experimental Methods: Clonogenic assays—the gold standard for assessing radiation survival in vitro—and viability assays were performed on 19 SCC cell lines (16 head and neck, 3 esophageal). The clonogenic assay was performed according to published procedures (Franken et al. Nat. Protoc, 2006) with colonies counted using ImageJ. The viability assay was adapted from Abazeed et al. (Cancer Res., 2013). Cells were seeded with a range of densities in 96 well plates then treated with a range of radiation doses (0, 2, 4, and 8 Gy); viability was measured using CellTitre Glo reagent after 9 days. Agreement between clonogenic and viability assays was measured using modified concordance index (C-index) and Pearson correlation. For the addition of drug, cells were allowed to adhere to plates for 2 hours before drug administration. Cells were irradiated 16 hours later again at a range of doses (0, 2, 4, and 6 Gy for drug analysis), and then quantified as above. All assays were performed with technical and biological triplicates.

Results: There was a high degree of agreement between the area under the curve with the clonogenic assay and the viability assay (C-index = 0.80, p = 3.03x10-7; Pearson r = 0.70, p = 1.10x10–3). Significant concordance was also seen at 2 Gy and 4 Gy dose points (Pearson r = 0.62 and 0.73 respectively), however not at 8 Gy (Pearson r<0.5). The viability assay decreases the overall time for the experiment, as it has a 9-day endpoint rather than the 14 days typical for the clonogenic assay. The viability assay also eliminates cell counting time, as results can be analyzed in minutes. Survival curves with and without radiation with clinically utilized chemotherapeutic drugs cisplatin and paclitaxel were developed which showed an additive effect for drug combinations, demonstrating the ability of the assay to be combined with chemotherapeutic agents and allows for the investigation into dose-response effects.

Conclusion: The proliferative assay recapitulates the clonogenic assay within our cohort of SCC cell lines. Novel higher throughput methods to analyze radiation response will allow for a more efficient measure of drug and radiation combinations.

#855

Increased in vitro potency and in vivo efficacy of FGFR2-targeted thorium-227 conjugate (FGFR2-TTC) in combination with the ATR inhibitor BAY 1895344.

Katrine Wickstroem,1 Urs B. Hagemann,2 Antje M. Wengner,2 Anette Sommer,2 Alexander Kristian,1 Christine Ellingsen,1 Roger M. Bjerke,1 Jenny Karlsson,1 Olav B. Ryan,1 Lars Linden,3 Bertolt Kreft,2 Dominik Mumberg,2 Hanno Wild,2 Karl Ziegelbauer,2 Alan Cuthbertson1. 1 _Bayer AS, Oslo, Norway;_ 2 _Bayer AG, Berlin, Germany;_ 3 _Bayer AG, Wuppertal, Germany_.

Targeted Thorium-227 Conjugates (TTCs) consist of the alpha emitter Thorium-227 (227Th) coupled, by a 3, 2-HOPO chelator, to a tumor specific antibody. The alpha particles release high energy over a short range (2- 10 cell diameters), resulting in a potent local irradiation of the tumor with limited damage to surrounding tissue. Here, we describe the in vitro and in vivo evaluation of an FGFR2 targeted thorium-227 conjugate (FGFR2-TTC) in combination with the ATR inhibitor BAY 1895344. FGFR2 (fibroblast growth factor receptor 2) is a receptor tyrosine kinase and overexpression of FGFR2 has been described in different cancers, while its expression in healthy human tissues is moderate to low. This renders FGFR2 an attractive antigen to explore the concept of targeted alpha therapy (TAT). The mode-of-action of TTCs is based on the induction of clustered DNA double strand breaks and G2 cell cycle arrest. We hypothesized that combination of FGFR2-TTC with inhibitors of DNA damage response (DDRi's) may enhance potency and efficacy. The ataxia telangiectasia and Rad3-related protein (ATR) kinase is a central mediator of DDR. ATR kinase responds to a broad spectrum of DNA damage, including double-strand breaks (DSB) and lesions derived from interference with DNA replication as well as increased replication stress. Inhibition of ATR kinase activity induces cell death especially in tumors with increased DNA damage, deficiency in DNA damage repair or replication stress. Therefore, we investigated whether the combination of the FGFR2-TTC with the ATRi BAY 1895344 results in enhanced tumor sensitivity in vitro and in vivo. In in vitro cytotoxicity assays, the combination of FGFR2-TTC and BAY 1895344 resulted in increased potency of the FGFR2-TTC on three different cancer cell lines (KATO III (gastric), MFM-223 (triple negative breast cancer), SUM52-PE (triple negative breast cancer)). Mechanistic analysis demonstrated that the combination treatment resulted in reduced levels of G2 arrest and increased levels of DNA damage in comparison to single agent treatments. The combination was further evaluated in vivo using the MFM-223 breast cancer xenograft model. An increased anti-tumor efficacy of a low dose of FGFR2-TTC (100 kBq/kg) was observed in combination with BAY 1895344 compared to animals treated with vehicle. The presented data support the mechanism-based rationale for combining DNA damage induction by FGFR2-TTC with DNA damage repair inhibition using ATRi BAY 1895344. Our findings warrant further exploration of TTCs in combination with BAY 1895344 for cancer therapy.

#856

Selection of radiosensitizers based on HRAS mutation in bladder cancer.

Michael M. Fisher, Adam D. Swick, Kwangok P. Nickel, Randall J. Kimple. _University of Wisconsin School of Medicine and Public Health, Madison, WI_.

Purpose/Objective(s): The ability of activated HRAS to promote radiation resistance and predict for sensitivity to potential radiosensitizers was tested in a bladder cancer model system.

Materials/Methods: Mutations in HRAS, KRAS, and NRAS were analyzed using cBioPortal in a bladder cancer cohort (TCGA). We employed a panel of urothelial carcinoma cell lines-T24 (HRASG12V), J82 and 5637 (RASWT). Cell growth and survival was tested with colony formation and proliferation assays. Radiation survival was assessed by clonogenic survival assay. Immunoblots were used to confirm mutant HRAS expression, and target activation/knockdown in overexpression and knockdown studies. Radiosensitization of mutant-HRAS bladder cancer was tested using a flank cell line xenograft model in nude mice.

Results: Approximately 10% of bladder cancers feature alterations in one of three RAS genes; mutations in HRAS comprise greater than 50% of these. An HRAS activating (G12V) mutation was detected in T24. Consistent with known roles for activated RAS, T24 is relatively insensitive to both cetuximab and radiation. Using both in vivo and in vitro studies, cetuximab exhibited no radiosensitization of T24. Treatment with cetuximab lowered AKT, but not ERK activation. Direct inhibition of the MEK/ERK or PI3K/MTOR pathway by selumetinib or BEZ235, respectively, decreased target protein activation and resulted in significant growth inhibition compared to control (p<0.05). Treatment with either selumetinib or BEZ235 radiosensitized HRASG12V expressing cells (SER 1.3-1.8), but had more modest effects on cells with wild-type HRAS. siRNA knockdown of HRAS radiosensitized T24, but not J82 or 5637 cells relative to non-targeting control. The combination of selumetinib and radiation therapy delayed the growth of T24 tumors in vivo.

Conclusions: Over 10% of bladder cancers feature alterations in the RAS family of genes. These mutations can lead to resistance to cetuximab, either as a single treatment or as a radiosensitizer. Inhibition of downstream MEK/ERK and PI3K/MTOR pathways can radiosensitize tumors with activating mutations in HRAS. Identification of additional mutation/drug combinations that result in radiosensitization may be valuable to advance the design of personalized radiation therapy.

#857

Metal oxide nanoparticles as adjuvant for radiation therapy.

Nadine Wiesmann,1 Martin Kluenker,2 Wolfgang Tremel,2 Juergen Brieger1. 1 _University Medical Center Mainz, Mainz, Germany;_ 2 _Johannes Gutenberg University Mainz, Mainz, Germany_.

Background: Radiation therapy comprises a fundamental component of modern tumor treatment. Unfortunately, its success is limited by the development of radiation resistances. The emerging field of nanotechnology offers great opportunities for diagnosing, imaging, as well as treating cancer. Metal oxide nanoparticles in particular zinc oxide nanoparticles (ZnO-NP) have been shown to display a selective cytotoxic effect on tumor cells via a yet unknown mechanism. Most likely the generation of reactive oxygen species (ROS), breakdown of mitochondria and DNA damage are involved. The success of radiation therapy equally relies on the generation of ROS, which develop their cytotoxic potential by damaging DNA, proteins and membranes in a fashion that exceeds the repair capacity of tumor cells. Thus, our aim was to evaluate the applicability of ZnO-NP as radiosensitizer to restore the response of tumor cells to radiation therapy.

Methods: An AlamarBlueTM assay was used to evaluate the cellular metabolic activity (CMA) of A549 adenocarcinoma cells after treatment with ZnO-NP. To assess cellular viability and cell death a flow cytometric apoptosis assay was applied and genotoxicity of ZnO-NP was analysed by γH2AX foci analysis. The performance of ZnO-NP as radiosensitizer was assessed by a colony formation assay (CFA).

Results: We were able to show that ZnO-NP-mediated cytotoxicity is conveyed by dissolved Zn2+ ions as well as by the particles themselves. Treatment with ZnO-NP resulted in double-strand breaks (DSB) of the DNA measured by γH2AX foci analysis. The apoptosis assay could show induction of apoptosis in A549 cells after treatment with 100 µg/ml ZnO-NP for four hours. The colony formation assay showed that irradiation with 2 or 4 Gray, according to dosages that are typically applied in clinical settings, in combination with ZnO-NP treatment could enhance tumor cell death and reduce clonogenic survival. This revealed that ZnO-NP could improve the success of radiation therapy.

Conclusions: Our study proves that ZnO-NP exert a cytotoxic and genotoxic effect on human tumor cells. Additionally, we could show, that ZnO-NP are able to serve as radiosensitizer. Combined treatment of human tumor cells with ZnO-NP and irradiation resulted in reduction of tumor cell survival. Since nanoparticles can in principle be targeted selectively to tumor tissue, ZnO-NP could be used to specifically sensitize tumor cells for irradiation while sparing healthy tissue. All in all, the study shows that ZnO-NPs could be a promising antitumor agent.

#858

Enzalutamide versus abiraterone as a radiosensitizer in hormone-sensitive prostate cancer cells.

Maryam Ghashghaei, Thierry Muanza, Moulay Alaoui-Jamali, Miltiadis Paliouras, Mohammad Tamim Niazi. _Jewish general hospital-McGill university, Montreal, Quebec, Canada_.

Prostate cancer treatment is based on the estimated risk of recurrence in the U.S. Combined androgen deprivation therapy (ADT) with radiation therapy (XRT) is the standard of care for high-risk localized PCa. However, a large percentage of tumors are resistance to ADT due to continued AR signaling. Abiraterone (ABI), an androgen synthesis inhibitor, and Enzalutamide (ENZA), a potent AR antagonist, are new treatment options for metastatic castration resistance prostate cancer (mCRPC) patients. The aim of this study is to compare the efficacy of ENZA or ABI as a radiosensitizer in XRT therapy on PCa cells.

Methods: The effect of ENZA or ABI alone or in combination with XRT was assessed on hormone-sensitive (LNCaP, PC3-AR-T877A) and insensitive (PC3, PC3-AR V7) PCa cells using cell viability (MTT) and also clonogenic assays in different scheduling regimens: A- drug 24 h before XRT, B-drug 2h before XRT, or C- XRT followed by 24h later drug.

Results: We first determined the effect of ENZA or ABI on MTT assays in androgen-dependent (AD) and androgen-independent (AI) PCa cell lines. The results of MTT assay showed that ENZA inhibited the growth of the four different cell lines, LNCaP, PC3-T877A, PC3 and PC3 AR-V7 with IC50 values of 20, 22, 50, and 45μmol/L, respectively, after 24 hours of treatment. The same effect was observed on ABI treated cell lines. Radiosensitivity was not significantly increased in AD and AI PCa cell lines by ABI (DEF=1.00, in all cases) while there was a supra-additive dose enhancement factor (DEF= 1.75±0.08) for hormone-sensitive cells treated with ENZA (Table 1).

Conclusion: Our data indicates that ENZA acts as a much stronger radiosensitizer compared to ABI through different probable mechanisms of radiosensitivity.

Table 1. Dose Enhancement Factors calculations by scheduling Protocol | |  | |  | |

|

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

|  | A | |

B | |

C

|

|  | ENZA | ABI | ENZA | ABI | ENZA | ABI

AD | LNCaP | 1.35±0.02 | 1.05±0.01 | 1.75±0.08 | 1.00 | 1.30±0.05 | 1.00

|

PC3-AR T877A | 1.30±0.03 | 1.00 | 1.65±0.01 | 1.00 | 1.35±0.06 | 1.05±0.02

AI | PC3 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00

|

PC3-AR V7 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00

#860

Experimental alpha-radioimmunotherapy for liver metastasis of gastric cancer.

Huizi Keiko Li,1 Sumitaka Hasegawa2. 1 _National Institutes for Quantum and Radiological Science and Technology, Japan Society for Promotion Science, Japan;_ 2 _National Institutes for Quantum and Radiological Science and Technology, Japan_.

Gastric cancer (GC) is one of the leading causes of cancer-related death worldwide. Early stage of GC is curable by surgical therapy or endoscopic surgery. However, previous studies reported that 35% of patients is present with evidence of distant metastasis at the time of diagnosis of GC. Metastatic GC is fetal and the patients with metastatic GC has extremely poor prognosis because current therapeutic approaches to metastatic GC are still limited. Therefore, the development of new therapeutic options has been eagerly awaited. Liver metastasis of GC (LMGC) is often observed during GC progression. It has been shown that 4-14% of the patients has LMGC. It is believed that LMGC is occurred by blood-borne metastasis of GC cells via blood stream. A previous study reported that approximately 70% of LMGC was positive for human epidermal growth factor receptor 2 (HER2). Therefore, HER2 may be a reliable therapeutic target for LMGC. Alpha-particle radioimmunotherapy (alpha-RIT) is an emerging new therapy, in which radioisotopes emitting cytotoxic alpha-particle radiation are delivered to target cancer cells to kill the cells by target-specific antibody. Among alpha-particle emitters, astatine-211 (At-211) is a promising emitter because it emits highly cytotoxic alpha-particles during its decay process with a half-life of 7.2 hours. The aim of this study is to evaluate the therapeutic efficacy and toxicity of At-211-labled anti-HER2 antibody trastuzumab ([At-211]trastuzumab) in a preclinical mouse model of HER2-positive LMGC. We established a mouse model of LMGC by injection of luciferase-labelled HER2-positive human metastatic GC cells (NCI-N87) into splenic vein of severe combined immunodeficiency mouse. Biodistribution studies revealed that the maximum tumor uptake of [At-211]trastuzumab was about 12% of injected dose per tissue gram 24 hours after injection. In experimental therapies, a systemic injection of [At-211]trastuzumab (1 MBq) significantly reduced a tumor burden in the liver and prolonged the survival. Transient but recoverable leukocytopenia was observed in mice received 1 MBq of [At-211]trastuzumab at 5-7 days after injection. No body weight loss was found during the observation period. In this study, we provides a preclinical evidence that alpha-RIT using [At-211]trastuzumab may be a new therapeutic option against LMGC.

#861

Angiopoietin-2 blocking antibodies improve tumor growth inhibition and survival in mice treated with low doses of radiation.

Pauliina Kallio, Elina Jokinen, Suvendu Das, Jenny Högström, Sarika Heino, Marianne Lähde, Kari Alitalo. _Univ. of Helsinki, Helsinki, Finland_.

Almost 50 % of all cancer patients receive radiation therapy during their treatment, yet several tumor types show radiation therapy resistance that compromises treatment efficacy. Many radiation sensitizers have been tested for improvement of the outcome of radiation therapy. For example, inhibitors of VEGF and VEGF receptors were tested in attempt to normalize the tumor vasculature and to improve the outcome of radiation therapy. Here, we have analysed if antibodies blocking the angiogenic factor angiopoietin-2 could improve radiation therapy of mouse tumor isografts and human tumor xenografts in mice. To test if the anti-tumor effect of small doses of radiation can be increased by Ang2 blocking antibodies, we analysed the radiation sensitivity of subcutaneous tumor isografts in C57bl/6JRj mice, and human xenografts in NOD scid gamma mice. In several models, the combination treatment with anti-Ang2 plus radiation was superior to the monotherapies in decreasing tumor growth and increasing the lifespan of the tumor-bearing mice both in immunocompetent and immunocompromised mice. Based on our results, the anti-Ang2 blocking antibody treatment could be tested in the clinics to improve the effect of radiation therapy, especially in those cases in which an optimal radiation dose cannot be given because of the radiation sensitivity of the surrounding tissues.

#862

Effects of fixation and antigen retrieval on the RNA integrity number.

Dennis Otali,1 Maitlyn Mullen,2 He Qinghua,3 Denise Oelschlager,1 William E. Grizzle1. 1 _Univ. of Alabama at Birmingham, Birmingham, AL;_ 2 _Samford University, Birmingham, AL;_ 3 _Auburn University, Auburn, AL_.

The effect of fixation and antigen retrieval (AR) on RNA as measured by the RNA integrity number (RIN) has not been adequately explored. RIN is an index (pseudo-measure) used to measure stability of mRNA; however, RIN is an actual measure of the stability of ribosomal RNA and not an actual measure of mRNA. RIN values range from 1 for highly degraded to 10 for intact RNA.

Most surgical specimens are fixed in 10% neutral buffered formalin (3.7% formaldehyde) because of its ability to preserve morphological detail and prevent degradation. However, formaldehyde fixation causes changes in proteins through formation of crosslinks, which masks the immunorecognition of some antigens. In immunohistochemical evaluations, antigen retrieval (AR) approaches e.g., boiling of tissue sections under pressure are used to reverse these effects of fixation.

It is believed that fresh or snap frozen specimens serve as most the suitable source of intact mRNA, DNA and other macromolecules for molecular analysis. However, more recent studies using rt-Q PCR indicate that RNA and DNA can be measured in formalin fixed paraffin embedded (FFPE) tissue samples which usually have RIN values of 3 or less. The question arises as to what is the usefulness of RIN in FFPE tissues.

This study used three cancer cell lines DU145 (prostate), MDA231 (breast) and S2VP10 (pancreas) to examine the relationship between 10% NBF fixation, RIN and AR. The first objective was to analyze whether the duration of fixation of cells in 10% NBF correlated with decreases in RIN. Three fixation times i.e., 5 minutes, 20 minutes and 120 minutes were studied. The second objective was to investigate the effects of two AR approaches on RIN. Two AR conditions at pH 6 and pH 9 were used on the same cell lines and fixation times.

For each cell line and treatment i.e., no fixation, or fixation at different times (with and without AR), RNA was extracted and RIN analyzed using the Agilent 2100 Bioanalyzer. RIN values obtained from the Agilent 2100 Bioanalyzer were statistically analyzed. T-tests show that the large decreases to about 3 in RIN values after fixation (5, 20 and 120 minutes) are statistically significant for all cell lines. All p-values are less than 0.0001. This study confirms that fixation of cells in 10% NBF lowers RIN to about 3 the value observed for fixed tissues. RIN did not vary significantly with time of fixation in 10% NBF and RIN did not change from the fixed values after either condition of AR. Thus RIN is not a useful measure of RNA stability in 10% NBF fixed tissue.

#863

Targeted α-therapy for treatment of metastatic melanoma.

Jessie Nedrow, Greg McCarty, Angel Cortez, Sanchita Roy, George Sgouros. _Johns Hopkins Univ., Baltimore, MD_.

Background Very late antigen 4 (VLA-4; also called integrin α4β1) is overexpressed in melanoma tumors with an active role in tumor growth, angiogenesis, and metastasis. This makes VLA-4 an ideal antigenic target for targeted alpha therapy. The expression of VLA-4 on primary melanomas in human correlates with the development of metastasis and has been associated with high risk of metastasis and tumor progression. Furthermore, up-regulation of VLA-4 in melanoma is associated with a more aggressive, metastatic phenotype. Here in we evaluated an 225Ac-anti-VLA-4 conjugate as well as its 111In-labeled companion imaging agent for targeted radiotherapy in an aggressive mouse melanoma model. Methods An anti-VLA-4 antibody was conjugated to DOTA for 225Ac-labeling and DTPA for 111In-labeling. The resulting agents, 225Ac- or 111In-labeled anti-VLA-4 were evaluated in vitro, including binding affinity, internalization, and clonogenic assays. Furthermore, the therapeutic efficacy of 225Ac-anti-VLA-4 will be evaluated in multiple metastatic animal models of melanoma. Results 111In-DTPA-anti-VLA-4 demonstrated high affinity for VLA-4 in B16F10 cells, having a Kd of 0.18 ± 0.05 nM and approximately 46% was internalized at 6 hours. For delivery of an α-emitting radionuclide to VLA-4 positive tumor cells, the DOTA conjugate was labeled with 225Ac with specific activity of 3.5 MBq/nmol and >95% radiochemical purity, and demonstrated selective uptake in VLA-4 positive B16F10 cells. Clonogenic assays demonstrated a decrease in the surviving fraction of B16F10 cells treated with 225Ac-DOTA-anti-VLA-4 compared to controls. Therapeutic efficacy studies are currently underway. Conclusion 111In- and 225Ac-labeled anti-VLA-4 antibody conjugates are capable of selectively delivering radionuclides for SPECT imaging and targeted alpha therapy to melanoma tumor cells that overexpresses VLA-4. The agents presented here have the potential to identify and treat patients who have metastatic melanoma.

### New Therapy with New Mechanism of Action

#864

Repurposing FDA-approved drug bazedoxifene as a novel inhibitor of IL-6 signaling for triple-negative breast cancer.

Xiang Chen,1 Shengling Fu,1 Jilai Tian,1 Yang Cao,2 Chenglong Li,3 Jiayuh Lin1. 1 _University of Maryland, Baltimore, MD;_ 2 _Huazhong University of Science and Technology, Wuhan, China;_ 3 _University of Florida, Gainesville, FL_.

Triple-negative breast cancer (TNBC) is a major cause of death among breast cancer patients and is the only subtype of breast cancer that is still lacking effective therapeutic options. IL-6 is one of the principal oncogenic mediators in breast cancer and systemic IL-6 levels correlates with poor prognosis, advanced disease, and metastases. Importantly, growth of TNBC cells are replied on autocrine cytokines including IL-6 and TNBC cells secret the highest levels of IL-6 comparing to other subtypes of breast cancer. Therefore, IL-6 signaling represents a novel approach with a potential to improve the therapeutic efficacy. To date, however, no small molecules that target IL-6 signaling are available for clinical cancer therapy. IL-6 binds to IL-6 Rα, then recruits Glycoprotein 130 (GP130) to form hexamer consisting two IL-6/IL-6 Rα/GP130 heterotrimers to trigger a signaling cascade downstream including JAK/STAT3, PI3-K/AKT/mTOR, and MEK/ERK. Therefore, it is possible to target IL-6 signaling by blocking IL-6 binds to GP130 or IL-6 Rα and thus inhibiting its signaling cascade downstream.

We have utilized a novel drug discovery approach combining Multiple Ligand Simultaneous Docking and drug repurposing to target GP130 and have identified a FDA-approved drug Bazedoxifene (for the prevention of the osteoporosis in postmenopausal women) with a novel function to inhibit IL-6 and GP130 proteinprotein interactions and thus blocking IL-6 signaling cascade downstream. In this study, we examined the therapeutic efficacy of Bazedoxifene in combination with paclitaxel on human TNBC cells and TNBC tumor model, aiming to provide experimental results of Bazedoxifene and paclitaxel combination for TNBC therapy.

Our data showed Bazedoxifene inhibited increased STAT3 phosphorylation induced by IL-6. Bazedoxifene also inhibited phosphorylation of STAT3, AKT, and ERK in TNBC cells that produced IL-6. In addition, combination of Bazedoxifene with paclitaxel exhibited more significant inhibition on cell viability and colony formation and induction of apoptosis than single agent alone in TNBC cells in vitro. Moreover, Bazedoxifene inhibits cell migration and combination of Bazedoxifene with paclitaxel more significantly inhibit cell migration than single agent alone. We also showed bazedoxifene could inhibit increased proliferation of TNBC cells induced by IL-6. The combination of Bazedoxifene with paclitaxel also showed significantly inhibition of tumor growth of TNBC in vivo furtherly supported the therapeutic effects of Bazedoxifene. The results support the significant therapeutic efficacy of Bazedoxifene and paclitaxel combination for further TNBC therapy.

#865

Small molecule-mediated modulation of Ras elicits inhibition of phospho ERK signaling through negative feedback on SOS1.

Jennifer E. Howes, Denis T. Akan, Michael C. Burns, Qi Sun, Andrew J. Little, DeMarco V. Camper, Jason R. Abbott, Jason Phan, Taekyu Lee, Olivia W. Rossanese, Alex G. Waterson, Stephen W. Fesik. _Vanderbilt University, Nashville, TN_.

Oncogenic mutation of Ras is responsible for more than 30% of all human tumors. Therefore, pharmacological modulation of Ras has attracted great interest as a therapeutic strategy. Our laboratory has recently discovered small molecules that activate Son of Sevenless (SOS)-catalyzed nucleotide exchange on Ras and paradoxically inhibit downstream signaling. Here we describe how pharmacologically targeting SOS1 induces biphasic modulation of Ras-GTP and downstream ERK levels. We consistently observed this phenotype in a variety of cell lines expressing different Ras mutant isoforms, and using multiple chemotypes of compound. In elucidating the molecular mechanism, we observed that compound treatment caused an increase in phosphorylation at ERK consensus motifs on SOS1 that was not observed with the expression of a non-phosphorylatable S1178A SOS1 mutant, or after pre-treatment with an ERK inhibitor. Phosphorylation at S1178 on SOS1 is known to inhibit the association between SOS1 and GRB2 and disrupt SOS1 membrane localization. Consistent with this, we show that wild-type SOS1 and GRB2 dissociated in a time dependent fashion in response to compound treatment, and conversely, this interaction was enhanced with the expression of a S1178A SOS1 mutant. Pre-treatment with an ERK inhibitor prevented compound-induced inhibition of the association between SOS1 and GRB2. Furthermore, in cells expressing either S1178A SOS1 or a constitutively membrane-bound CAAX box tagged SOS1 mutant, we observed elevated Ras-GTP levels over time in response to compound, as compared to the biphasic changes in Ras-GTP exhibited in cells expressing wild-type SOS1. These results show that small molecule agonists of SOS1 cause paradoxical inhibition of Ras-GTP and MAPK signaling in Ras mutant cancer cells, through negative feedback on SOS1. Overall, these compounds provide us with the unique opportunity to better understand the biological functions of SOS and Ras in cancer cells, and may aid in the discovery of small molecules to treat Ras-driven tumors.

#867

Blocking breast cancer metastasis by targeting HuR-FOXQ1 signaling axis.

Xiaoqing Wu,1 Gulhumay Gardashova,1 Lan Lan,1 Yu Zhan,1 Jiajun Liu,1 Dan A. Dixon,2 Jeffrey Aubé,3 Danny R. Welch,2 Liang Xu1. 1 _Univ. of Kansas, Lawrence, KS;_ 2 _Univ. of Kansas Medical Center, Kansas City, KS;_ 3 _Univ. of North Carolina, Chapel Hill, NC_.

As most of treatment options do not work very well for metastatic cancer. Patients with metastatic cancer have a greatly lower survival rate compared with patients with local cancer. Metastasis remains a life-threat to cancer patients and an unmet medical need. The RBP Hu antigen R (HuR) is overexpressed in virtually all malignancies tested, including breast cancer. Cytoplasmic HuR accumulation correlates with high-grade malignancy, poor distant disease-free survival and serves as a prognostic factor for poor clinical outcome in breast cancer. HuR promotes tumorigenesis by promoting mRNA stability and translation of proteins implicated in proliferation, survival, angiogenesis, invasion, and metastasis. We found that silencing of HuR inhibited cell invasion in vitro in breast cancer. Using RIP-seq (ribonucleoprotein immunoprecipitation-sequencing), a transcription factor FOXQ1, which is recently revealed to implicate in breast cancer invasion and metastasis processes, is found to be a direct HuR target. Furthermore, exogenous introduction of FOXQ1 can rescue cell invasive ability inhibited by HuR knockout. Taken together, HuR-FOXQ1 signaling axis is a potential target for blocking breast cancer metastasis. RNA-binding proteins had previously been considered "undruggable" due to lack of a well-defined binding pocket for target RNAs. Using high throughput screening followed by structure-based rational design and lead optimization, we have identified small molecules that inhibit HuR-mRNA interaction at nM to sub-µM potency. Our lead compound, KH-3, potently inhibits breast cancer cell growth and decreases cell invasion in vitro similar to HuR knockout, as well as increasing the expression of epithelial marker E-cadherin. FOXQ1 overexpression abolishes the effect of KH-3 on blocking metastasis in breast cancer cells, demonstrating that the HuR inhibitor KH-3 inhibits cell metastasis by blocking FOXQ1 function. Moreover, KH-3 treatment disrupts HuR-FOXQ1 interaction in RNP-IP and FOXQ1 3′-UTR luciferase reporter assays. In vivo efficacy studies show that KH-3 not only exhibits potent antitumor efficacy in orthotopic xenograft models of breast cancer, but also efficiently blocks lung metastasis in experimental metastatic cancer model. In conclusion, we identified a potent and specific small molecule disrupter of HuR-FOXQ1 interaction for potential novel anti-metastatic therapy of breast cancer with HuR overexpression.

#868

The pan-ErbB inhibitor dacomitinib but not the dual EGFR/ErbB2 inhibitor labatinib disrupts membrane localization of the EGFR family of receptor tyrosine kinases.

Maitreyee K. Jathak,1 Thomas M. Steele,1 Salma Siddiqui,2 Benjamin A. Mooso,1 Leandro S. D'Abronzo,1 Christiana M. Drake,1 Paramita M. Ghosh1. 1 _University of California Davis, Sacramento, CA;_ 2 _VA Northern California Health Care System, Mather, CA_.

Introduction: Despite ample evidence for the overexpression/overactivation of the epidermal growth factor receptor (EGFR) family, the dual EGFR/ErbB2 inhibitor lapatinib failed in patients with castration-resistant prostate cancer (CRPC). Our preclinical data show that the pan-ErbB inhibitor dacomitinib succeeds where lapatinib fails by decreasing membrane-localized EGFR family protein expression. Experimental Procedures: Nude mice were implanted with CWR22 tumors (human-patient-derived, androgen-dependent) and its CRPC subline 22Rv1 (relapsed CWR22). Mice were castrated or left intact and gavaged daily with 100 mg/kg lapatinib or vehicle. Tumors were analyzed for EGFR/ErbB2/ErbB3/androgen receptor (AR) proteins by both immunohistochemistry in paraffin-embedded tumor sections and Western blots in tumor lysates. Observations were validated in human-derived androgen-dependent and CRPC cell lines treated with lapatinib or dacomitinib by immunoblots and by immunofluorescence. Results: Intratumoral EGFR and ErbB2 (but not ErbB3) increased in intact, lapatinib-treated 22Rv1 tumor-bearing mice compared to placebo-treated ones. Elevated EGFR/ErbB2 was also observed in vitro in lapatinib-treated LNCaP, C4-2, PC-346C cells compared to vehicle-treated cells. EGFR phosphorylation increased from 1/5 intact, lapatinib-treated Rv1 tumors (20%) to 3/5 castrated, lapatinib-treated 22Rv1 tumors (60%). Lapatinib caused an increase in cytoplasmic EGFR and ErbB2 in LNCaP (androgen-dependent) and 22Rv1 (CRPC) cells. Low, physiological doses of dacomitinib overcame lapatinib resistance by suppressing cell viability and this effect was heightened by EGFR family knockdown or when AR activity was simultaneously inhibited. High-magnification microscopy revealed that dacomitinib possibly exerted its effects by decreasing the presence of membrane-localized EGFR, ErbB2 and ErbB3 proteins in comparison to lapatinib. Conclusions: We hypothesize that increased EGFR/ErbB2 heterodimerization is one cause of lapatinib resistance. Activated EGFR family proteins are typically localized in the plasma membrane and dacomitinib, by disrupting this membrane-localization, decreases cell viability. Future studies will attempt to refine the mechanism by which lapatinib and dacomitinib induce changes in RTK localization and how this may impact therapeutic response and prostate tumor progression.

#869

Depletion of blood arginine with pegzilarginase (AEB1102) in combination with anti-PD-L1 increases tumor infiltration by immune cells and enhances antitumor activity.

Giulia Agnello, Mark D. Badeaux, Danlee Enzler, Leslie Priddy, Jason F. Wiggins, Christopher L. Daige, Scott W. Rowlinson. _Aeglea BioTherapeutics, Inc, Austin, TX_.

Tumors unable to synthesize L-Arginine (arginine) due to decreased expression of enzymes of the arginine biosynthetic pathway show increased sensitivity to arginine depletion. Pegzilarginase (AEB1102) is a bioengineered human PEGylated arginase 1 with enhanced pharmacological properties that enables marked depletion of arginine in plasma and slows tumor growth in pre-clinical in vivo models. Extracellular depletion of arginine directly affects tumor cells, increasing protein turnover, inhibiting proliferation, inducing apoptosis, and increasing autophagy (PMID:27109103); however, the impact of arginine availability on tumor immunogenicity has not been clearly established. Depletion of extracellular arginine induces autophagy, and given the known relationship between autophagy and antigen processing for MHC presentation (PMID:29058602) we hypothesized that pegzilarginase could trigger an enhanced recruitment of immune cells to the tumor microenvironment.

Pegzilarginase, both as monotherapy or in combination with anti-PD-L1 mAb (10F.9G2), was administered to Balb/c mice bearing palpable, syngeneic, subcutaneous CT26 tumors. At pre-determined time points, tumor measurements were taken and tumor cell viability and immunophenotyping were assessed via flow cytometry.

Treatment with both pegzilarginase or anti-PD-L1 mAb alone slowed tumor growth compared to control. Combination treatment of pegzilarginase and anti-PD-L1 mAb resulted in an enhancement of anti-tumor activity with 12.5% complete response (CR) observed in the anti-PD-L1 mAb monotherapy group and 25% CR observed in the combination therapy group. All mice with CR were re-challenged with fresh CT26 cells and failed to develop new tumors, consistent with induction of an immune memory response. We observed a decrease in tumor cell viability in all treatment groups on days 7 and 17, with the combination treatment group showing the greatest reduction in viable cells and tumor volume at day 17. The observed anti-tumor activity in monotherapy and combination therapy groups was accompanied by an increase in CD45+ tumor-infiltrating cells, with the combination therapy group showing the highest proportion of CD45+ tumor-infiltrating cells, including total T cells, macrophages and dendritic cells, and an increase in serum IFN-γ.

Combination of pegzilarginase and anti-PD-L1 mAb results in synergistically greater anti-tumor activity than either monotherapy, and is accompanied by an increase in tumor-infiltrating immune cells. The enhanced infiltration of immune cells into tumor following depletion of arginine with pegzilarginase monotherapy and combination therapy challenges some of the prevailing theories on the role of arginine in immune cell signaling and cancer biology.

#870

Effects of epigenetic modifier inhibitors on AML cell sensitivity to differentiation therapy.

Kalsi K. Heimdal, Edjay Ralph A. Hernandez, Heidi J. Gill Super. _Minot State University, Minot, ND_.

Human acute myeloid leukemia (AML), the most common type of acute leukemia, has approximately eight subtypes, many of which have poor prognosis. Most of these subtypes are associated with specific, recurrent chromosome translocations. These translocations result in fusion genes, which encode oncoproteins that block differentiation and promote proliferation of immature cells. The Myeloid Lymphoid Leukemia gene (MLL) is frequently involved in these translocations, and is considered a driver of the AML. Differentiation promoting drugs, such as all-trans-retinoic acid (ATRA) are an attractive alternative to cytotoxic chemotherapy, but few types of AML, other than acute promyelocytic leukemia (APL), respond to ATRA. We hypothesize that specific genes must be activated or inhibited in AML for drugs like ATRA to induce differentiation, and that gene activation or inhibition may be the result of specific epigenetic modification. We also hypothesize that AML with different genetic alterations may respond differently to specific epigenetic inhibitors. Our initial studies have focused on one AML cell line, MV4;11, with alterations in the MLL gene, and one non-MLL related AML cell line, U937. MV4;11 and U937 were treated with specific epigenetic modifiers, including tranylcypromine (TCP), an inhibitor of histone demethylase KMD1A/LSD1, and CI-994, a general histone deacetylase inhibitor. Evidence for differentiation was noted in a variety of assays, including reduced cell proliferation as measured directly and by MTT assay, upregulation of myeloid-specific cell surface markers such as CD11b as measured by fluorescence-activated cell sorting (FACS), and myeloid-related nuclear morphological changes noted with cytospin analysis of cells. Both CI-994 and TCP seemed to sensitize U937 cells to differentiate when treated with ATRA as measured by all indicators of differentiation. MV4;11 cells were sensitized to differentiation by CI-994 with or without ATRA, although there was no indication of CD11b upregulation. However, MV4;11 was not sensitized by TCP to differentiate with ATRA. These experiments suggest epigenetic inhibitors may increase sensitivity to differentiation therapy, but that the response may still be dependent on the specific genetic alteration driving the AML. Our studies suggest that in MLL-driven leukemia, the MLL fusion oncoprotein may override genetic manipulation resulting from treatment with epigenetic inhibitors, preventing differentiation by ATRA.

#871

BR101801: A first-in-class dual inhibitor of PI3Kδ and DNA-PK in non-Hodgkin's lymphoma.

Mi Kwon Son, Byeongwook Jeon, Jin Sang Wang, Baek kyung Kim, Bo Ram Lee, Yeon seo Choi, Nam-Hoon Kim, Jayhyuk Myung, Dal-Hyun Kim. _Boryung Pharmaceutical, Republic of Korea_.

Introduction: Inhibition of PI3Kδ (idelialisib, TGR-1202) has been reported as having limited clinical benefits for the treatment of indolent non-Hodgkin's lymphoma such as chronic lymphocytic leukemia, small lymphocytic lymphoma or follicular lymphoma but not aggressive NHL such as relapsed/refractory Diffuse Large B-Cell Lymphoma (RR DLBCL). Recently, up-regulation of γ-H2AX, a sensor of DNA damage, was reported to be caused by hyper-proliferation burden in aggressive NHL cells under protective action of the DNA repair system. This means that DNA repair system targeting therapy in aggressive NHL could lead to high genomic instability toward cell death. In this study, BR101801, a first-in-class dual inhibitor of DNA-PK and PI3Kδ, will be introduced as a novel candidate of RR DLBCL.

Methods: Cell-based selectivity assay for PI3K isotypes was performed using a specific ligand-induced phospho-AKT in various cells. To assess the effect of BR101801 on DNA-PK in LoVo cells, cells were pre-incubated with etoposide for 2 hours and then exchanged into new media containing BR101801 or other PI3Kδ inhibitors without etoposide. Activity of BR101801 was evaluated based on prolongation of γ-H2AX using Western blotting. GI50 was tested in vitro by WST-based viability assay in various NHL cell lines.

Results: BR101801 showed higher selectivity for p110δ and DNA-PK compared to the other PI3K class I enzymes by 7- to 100-fold (IC50 p110δ = 4 nM; IC50 DNA-PK = 7 nM; p110α, p110β, and p110γ IC50 values were 368, 398, and 52 nM, respectively), whereas no activity was observed for the other 394 human kinases at 1 μM. The inhibitory level of phospho-AKT by BR101801 was analyzed by stimulation with PDGF (p110α), LPA (p110β), IgM (p110δ), and C5a (p110γ), resulting in IC50 values of 146, 50, 1.2, and 110 nM, respectively. BR101801 prolonged etoposide-induced γ-H2AX but not idelalisib and showed potent growth inhibition in DLBCL cell lines compared to PI3Kδ inhibitors such as idelalisib and TGR-1202. SU-DHL-10 cells represented low sensitivity (IC50≒4.46 μM) when idelalisib was treated alone. However, a combination of idelalisib and NU7441 was used to improve the combination index (CI) to below 0.5 in a dose-dependent manner. This result indicates that the inhibition of both DNA-PK and PI3Kδ lead to synergistic effect through targeting each independent pathway like as BR101801. Next, several DNA-damaging inducers known to boost γ-H2AX, including bendamustine, doxorubicin, and etoposide, were combined with BR101801. In vitro synergy activity was confirmed based on a CI value less than 0.5, and in vivo synergy was assessed in a SU-DHL-10 xenograft model.

Conclusions: BR101801 is a first-in-class dual inhibitor of PI3Kδ and DNA-PK. DNA-PK targeting has shown to be efficient with PI3Kδ inhibitor or DNA-damaging inducers. Therefore, the strategy of dual targeting with DNA-PK and PI3Kδ suggests further investigation as a novel option of RR DLBCL.

#872

IDH1-mutated cancer cells are sensitive to cisplatin and an IDH1-mutant inhibitor counteracts this sensitivity.

Mohammed Khurshed, Remco J. Molenaar, Johanna W. Wilmink, Hanneke W. van Laarhoven, Cornelis J. van Noorden. _Cancer Center Amsterdam at Academic Medical Center, Amsterdam, Netherlands_.

Isocitrate dehydrogenase 1 (IDH1) is mutated in various types of human cancer and predicts improved response to treatment with irradiation or chemotherapy. Mutated IDH1 enzymes catalyze neomorphic conversion of α-ketoglutarate (α-KG) to the oncometabolite D-2-hydroxyglutarate (D-2HG) with concomitant consumption of NADPH, resulting in decreased reducing power needed for detoxification of e.g. reactive oxygen species (ROS). We report that a small-molecule inhibitor of IDH1-mutation (IDH1MUT) that is being investigated for cancer therapy may limit efficacy of treatment when co-administered with cisplatin.

In the present study, we investigated whether the efficacy of treatment with cisplatin, which is a widely-used chemotherapeutic agent, induces DNA strand breaks and oxidative damage in IDH1MUT cancer cells. We found that exposure to cisplatin induced higher levels of ROS, DNA double-strand breaks, and cell death in IDH1MUT cancer cells as compared to IDH1 wild-type (IDH1WT) cancer cells. Besides these cytotoxic effects, mechanistic investigations revealed that cisplatin treatment causes dose-dependent reduction of oxygen consumption and thus the oxidative respiration in lDH1MUT cells and not in lDH1WT cells, which was accompanied by disturbed mitochondrial proteostasis and impaired mitochondrial activity. These effects were abolished by the IDH1MUT inhibitor AGI-5198 and were recapitulated by treatment with D-2HG. Thus, our study shows that altered oxidative stress responses due to a vulnerable oxidative metabolism underlie the sensitivity of IDH1MUT cancer cells to cisplatin. Furthermore, our data offer an explanation for the relatively longer survival of patients with IDH1-mutated tumors, and imply that administration of IDH1MUT inhibitors in these patients limit efficacy of cisplatin treatment.

#873

BPM 31510, a clinical stage metabolic modulator, demonstrates therapeutic efficacy in glioblastoma models of temozolomide chemo-sensitive and resistance by targeting mitochondrial function.

Tulin Dadali,1 Shreya Kulkarni,2 Ryan Ng,1 Pallavi Awate,1 Saie Mogre,1 Anne R. Diers,1 Taichang Jang,3 Milton Merchant,3 Jiaxin Sun,3 Stephane Gesta,1 Khampaseuth Thapa,1 Seema Nagpal,3 Lawrence Recht,3 Niven R. Narain,1 Rangaprasad Sarangarajan1. 1 _BERG, Framingham, MA;_ 2 _University of Massachusetts Medical School, Worcester, MA;_ 3 _Stanford University, Stanford, CA_.

BPM31510 is a metabolic modulating agent composed of a parenteral nanodispersion of ubidecarenone which is currently in clinical studies for glioblastoma. Glioblastoma is a highly metabolic and aggressive malignancy with limited treatment options and dismal median survival. Temozolomide (TMZ) as a first line treatment option, however, 90% of recurrent gliomas acquire TMZ chemoresistance. Recently, acquisition to TMZ resistance has been correlated to alterations in mitochondrial metabolism. Thus, in the present study we sought to investigate whether BPM31510 could elicit anti-cancer activity in TMZ naïve and TMZ-chemoresistant glioma models. In vitro, in a 2D model BPM31510 treatment demonstrated anti-cancer activity in a panel of glioma cell lines (rat C6 and human U251-MG and U87-MG), and this effect was translatable in spheroidal 3D cultures. Importantly, in an aggressive rat C6 orthotopic glioma model, treatment with BPM31510 (50mg/kg/day, b.i.d) starting between 4 and 8 days post-implantation resulted in a 32% cure rate compared to 0% in controls (P < 0.001, Fisher's exact test), demonstrating an improved survival (P < 0.01, log rank survival), despite producing a minimal change in median survival (13 vs. 12 days). A marked increase in caspase3 staining was observed in tumors from BPM31510 treated animals compared to controls assessed at a similar time point post-tumor implantation, suggesting a strong apoptotic effect of this agent in vivo. Next, BPM31510 was examined in a cellular model of acquired TMZ resistance (TMZ-R) generated by exposing parental (chemosensitive naïve) U251-MG and U87-MG cells to increasing concentrations of TMZ for 9-12 months. Similar to parental cells, BPM31510 displayed anti-cancer activity in both TMZ-R cell models, as decreased cell viability and an increase in the percentage of apoptotic cells was observed upon BPM31510 treatment. Consistent with prior studies, compared to parental cells, TMZ-R cells demonstrated metabolic rewiring characterized by increases in mitochondrial function parameters and decreased extracellular acidification rate, indicative of glycolytic flux. Regardless of chemosensitivity, BPM31510 decreased mitochondrial substrate oxidation (e.g., succinate, glycerol-3-phosphate) at doses which induce cell death. Concomitantly, increases in the reactive oxygen species production were observed with BPM 31510 treatment in both parental and TMZ-R cell lines. Together, these data define a link between regulation of mitochondrial function and the anti-cancer activity of BPM31510 in both TMZ chemo-sensitive and resistant glioblastoma models, demonstrating a distinct approach in targeting mitochondrial metabolism for the treatment of this clinically intractable disease.

#874

Pharmacologically induced RAS-GTP levels and CRAF-BRAF hetero-dimerization drive sensitization to Type II pan-RAF inhibitors in KRAS mutant cancer.

Christiaan N. Klijn, Ivana Yen, Frances Shanahan, Mark Merchant, Christine Orr, Thomas Hunsaker, Matthew Durk, Hank La, Xiaoling Zhang, Scott Martin, Eva Lin, John Chan, Yihong Yu, Dhara Amin, Amy Gustafson, Scott Foster, Joachim Rudolph, Shiva Malek. _Genentech, South San Francisco, CA_.

Although effective in BRAF V600 mutant melanoma, Type 1.5 RAF inhibitors such as vemurafinib and dabrafenib have not proven to be successful in KRAS mutant cancers, neither as single agent nor in combination with MEK inhibitors. Through large-scale cellular compound combination screening we found that Type II RAF inhibitors such as AZ-628 do show synergistic activity with MEK inhibitors in multiple KRAS mutant indications, including NSCLC, colorectal cancer and ovarian cancer. The combination of Type II RAF inhibitors and MEK inhibitors demonstrates robust and durable abrogation of MAPK signaling both on canonical markers of MAPK activity such as pERK and pRSC as well as transcriptional output. We also observe synergistic in vivo tumor growth inhibition in two independent models of KRAS mutant cancer by this combination treatment. We found that treatment with MEK inhibitors alone drives the increase of active RAS-GTP levels and induces CRAF:BRAF hetero-dimerization. These induced dimers are active and able to phosphorylate MEK in vitro. This increased dimerization renders cells sensitive to Type 2 RAF inhibitors. We find that this effect is not limited to KRAS mutant cells, as a subset of KRAS wild-type cells show increased RAS-GTP levels upon MEK inhibitor treatment. These cells also show synergistic sensitivity to Type 2 RAF inhibition. Additionally, we observed significantly higher synergy and higher RAS-GTP levels in KRAS G13D mutant cells, which have intrinsically high GDP exchange and low intrinsic GTP hydrolysis. Finally, we show that GDC-0941 and GDC-0032, two broad PI3K inhibitors, also induce RAS-GTP levels in cells independent of PIK3CA or KRAS mutation status. We subsequently observed a synergistic sensitivity to Type 2 RAF inhibitors in these PI3K inhibitor-treated cells. Overall, we demonstrate that pharmacologic inhibition of MEK or PI3K increases RAS-GTP levels and drives increased CRAF:BRAF hetero-dimerization. This in turn sensitizes cells to Type 2 RAF inhibition, leading to a synergistic drug effect. Combination of these inhibitors may be a viable therapeutic approach in KRAS mutant cancer, and may be especially effective in KRAS G13D-carrying tumors.

#875

Chemical biology approach to phenotypic intra-tumor heterogeneity in high-grade serous ovarian cancer.

Daria R. Bulanova,1 Katja Kaipio,2 Tarja Lamminen,2 Aleksandr Ianevski,1 Tero Aittokallio,1 Olli Carpen,2 Krister Wennerberg1. 1 _University of Helsinki, Helsinki, Finland;_ 2 _University of Turku, Turku, Finland_.

Objective: High-grade serous ovarian cancer (HGSOC) remains the most lethal gynecologic malignancy. More than 50% of high-grade serous ovarian cancer (HGSOC) patients die due to recurrent disease within 5 years. Recurrent tumor clones are selected by chemotherapy treatment from heterogeneous subpopulations of cancer cells, and differ phenotypically. The objective of current study is to develop a high-throughput chemical biology approach to analyze phenotypic heterogeneity in HGSOC following progression of the disease.

Methods: We utilize the Drug Sensitivity and Resistance Testing (DSRT) platform to assess the effects of 528 approved and investigational anti-cancer agents on primary HGSOC isolates. Samples are collected from ascites and/or solid high-grade ovarian tumors from the patients at different disease stages. DSRT profiling of the samples integrates viability and toxicity readouts with high-content imaging in order to assess the drug responses of phenotypically different cell subpopulations of epithelial cancer cells and stromal component.

Results: Drug sensitivity profiling of ten primary and six relapse patient samples revealed heterogeneous drug sensitivity profiles characterized by common responsiveness to HDAC inhibitors, CDK inhibitors, and PI3K/mTOR inhibitors (mean drug sensitivity score (DSS) > 10). Relapse HGSOC samples demonstrated general trend towards resistance to a broad spectrum of chemotherapeutics and targeted drugs. Imaging-based DSRT profiling revealed that in both primary and relapse ascites-derived cancer cells the response to platinum drugs and DNA damaging agents associated with upregulation of transcriptional regulators of stemness, including BMI1, SOX2 and NANOG. The ascites-derived CK18-expressing epithelial cells collected from the same patient a) before chemotherapy, b) after 6 cycles of Carboplatin treatment and c) at the relapse demonstrated an increasing sensitivity to aldehyde dehydrogenase inhibitor disulfiram. These preliminary findings suggest that increase in a subpopulation of cancer cells with stem-like characteristics may accompany recurrence in HGSOC.

Conclusions: Compound sensitivity testing combined with high-content imaging appears an informative tool to explore therapeutic vulnerabilities in chemoresistant subpopulations of ovarian cancer cells, and improves the accuracy of drug sensitivity profiling. Chemical biology screening of prospectively collected primary HGSOC samples can also help identifying phenotypic changes associated with chemoresistance development.

#876

Effective reduction of PD-L1 expression by simultaneous blockade of EGFR and HER3 (ErbB3) in head and neck cancer.

Zhuo Georgia Chen,1 Dongsheng Wang,1 Christopher C. Griffith,1 Sreenivas Nannapaneni,1 Conor Steuer,1 Mihir R. Patel,1 Mark W. El-Deiry,1 Xu Wang,1 Yuzi Zhang,1 Zhengjia Chen,1 Diego Alvarado,2 Dong M. Shin,1 Nabil F. Saba1. 1 _Emory Univ. Winship Cancer Inst., Atlanta, GA;_ 2 _Celldex Therapeutics, Inc, Hampton, NJ_.

Background: We previously reported that simultaneous blockade of EGFR and HER3 by antibodies cetuximab and MM-121, respectively, could more potently reduce tumor growth in both squamous cell carcinoma of head and neck (SCCHN) cell lines and patient derived xenograft (PDX) models (Jiang et al, MCT, 13:1826-36, 2014; Wang et al., CCR, 23:677-86, 2016). Recently, we tested a new anti-HER3 antibody CDX-3379 (provided by Celldex Therapeutics, Inc) in combination with cetuximab for treatment of SCCHN. The effects of this combination on expression of the immune check point regulator PD-L1 and regulatory pathways underlying these effects were examined. In fact, a Phase II clinical trial combining CDX-3379 with cetuximab in checkpoint and cetuximab refractory patients has already been initiated. Method: Antitumor effects of cetuximab and CDX-3379 combination were investigated in SCCHN PDX animal models. Both HPV positive and negative SCCHN cell lines, UM-SCC47 (HPV+), 93Vu147T (HPV+), FaDu (HPV-), and JHU-022 (HPV-), were used for western blot analysis to examine EGFR and HER3 downstream pathways and PD-L1 expression in the presence and absence of interferon γ (IFNγ) after treatment with cetuximab, CDX-3379, or both agents in combination. Results: The PDX study indicated that, similar to MM-121, combining CDX-3379 with cetuximab inhibited tumor growth more potently than cetuximab alone or the untreated control (control vs. combination: p < 0.0001; cetuximab vs. combination: p < 0.0001). Mechanistic in vitro studies demonstrated that in addition to co-blocking ERK and AKT pathways, and regardless of HPV status, the cetuximab and CDX-3379 combination reduced PD-L1 expression more effectively than any of the single agents in the presence or absence of IFNγ, which has been previously reported to be secreted by natural killer (NK) cells through cetuximab-mediated antibody-dependent cell mediated cytotoxicity, leading to induction of PD-L1 expression in vivo. Using an AKT inhibitor, we confirmed that the AKT pathway was one of the major regulatory pathways involved in regulation of PD-L1. Considering that HER3 exerts its inhibitory effect mainly through regulation of the AKT pathway, reducing AKT activity by addition of CDX-3379 to cetuximab contributed significantly to the reduction of PD-L1 expression. Conclusion: In addition to its anti-tumor effect, the combination of cetuximab and CDX-3379 significantly reduced PD-L1 expression. The role of this combination in the regulation of tumor immunology and the tumor microenvironment in SCCHN deserves further investigation.

#877

Sigma 2 ligands kill pancreatic cancer cells via disruption of lysosomal cholesterol transport.

Linda X. Jin, Suwanna Vangveravong, Narendra V. Sankpal, Darren R. Cullinan, Peter Goedegebuure, Dirk Spitzer, William G. Hawkins. _Washington University in St. Louis, Saint Louis, MO_.

Introduction: The sigma 2 receptor is overexpressed in pancreatic cancer and has been an attractive target for development of cancer selective therapies. However, the mechanism of sigma 2 mediated cancer cell death remains elusive. The sigma 2 receptor has been recently identified as TMEM97, a lysosomal/endoplasmic reticulum protein involved in regulation of cholesterol export in conjunction with NPC1.

Methods: Uptake of SW43 in pancreatic cancer cells was visualized using a NBD fluorophore conjugated ligand. Intracellular cholesterol staining was visualized using filipin staining. Protein expression was quantified by both Western blot and RT qPCR. Cell death was assayed in triplicate using Titer-Glo viability assay.

Results: We hypothesized that sigma 2 ligands may kill pancreatic cancer cells through disruption of lysosomal cholesterol export. Using ASPC1 pancreatic cancer cells, we first demonstrate that sigma 2 ligand SW43 is rapidly taken up and targets to endo-lysosomal compartments, and that treatment with SW43 causes a rapid and dose dependent sequestration of cholesterol in lysosomal compartments as visualized by filipin cholesterol staining. This correlated to an increase in TMEM97 and corresponding decrease in NPC1 expression following SW43 treatment. We next demonstrate that this cholesterol sequestration phenotype is required for cell death, and might be related to the free amine side chain of SW43, as its N-phthalimide derivative fails to induce both lysosomal cholesterol sequestration and cell death despite high doses of ligand used. Lysosomal cholesterol sequestration from sigma 2 ligand treatment sensitizes cells to further targeting of cholesterol metabolism. Using low doses of simvastatin and SW43, we demonstrate that targeting both cholesterol export and cholesterol synthesis displays significant synergism in killing ASPC1 pancreatic cancer cells.

Conclusions: These findings provide further insight into how the sigma 2 receptor/ligand interaction mediates cancer cell death and provides the basis for rationally combined targeted therapy regimens to be tested in vivo.

This work was supported by NIH R01CA163764 and NIH T32CA009621-26A1 and the SUS Foundation Resident Research Award.

#878

Mitochondria-targeted drugs stimulate mitophagy and abrogate colon cancer cell proliferation.

Kathleen A. Boyle, Jonathan VanWickle, Jacek Zielonka, Gang Cheng, Balaraman Kalyanaraman, Michael B. Dwinell. _Medical College of Wisconsin, Milwaukee, WI_.

In the US, colorectal cancer is predicted to afflict 1 out of every 20 people and is the third major cause of cancer. The first line of treatment for localized colorectal tumors is surgical resection, while perioperative chemo- and/or radiation-therapy is administered upon disease metastasis. These cytotoxic treatments may have pronounced side-effects and off-target consequences. A major caveat to these treatment regimens is that their efficacy is restricted to tumors that express wild-type KRAS protein. KRAS is a common genetic mutation, accounting for ~50 % of colorectal cancer oncogenic mutations. Thus, there remains a significant need for therapies with increased efficacy, reduced toxicity and directed tumor specificity, particularly in the context of KRAS oncogene expression. With the continuing demonstration that mitochondrial metabolism is a key player in cancer development, progression and metastasis, it is rational to exploit this observation and explore mitochondrial targeted compounds as novel, untapped therapeutics. Indeed, several reports have shown that compounds targeted to the mitochondria induce anti-proliferative and cytotoxic effects in tumor cells without a significant impact on normal cells. We sought to exploit mitochondria-targeted cationic drugs that selectively inhibit energy metabolism in cancer cells as treatment of KRAS mutant colorectal cancer. The anti-cancer mechanisms of Mito-CP and Mito-Met, two mitochondria-targeted drugs we have shown to block bioenergetic metabolism, were examined in wild-type and oncogenic KRAS-expressing colon cancer cells. Mitochondrial function, intracellular ATP levels, cellular uptake, energy sensor signaling and functional effects on cancer cell growth were assayed. Both Mito-CP and Mito-Met depleted intracellular ATP and caused prolonged inhibition of ATP-linked oxygen consumption in colon cancer cells. The anti-proliferative effects reflected the activation of AMPK and the phosphorylation-mediated inhibition of the mTORC1-target p70S6K. Moreover, Mito-CP and Mito-Met released the autophagy regulator ULK1 from mTORC1-mediated inhibition, leading to mitophagy. Inhibition of AMPK and mTORC1 signaling mitigated the anti-proliferative effects of these mitochondria-targeted compounds. These data are the first demonstration that drugs selectively targeting the cancer cell mitochondria induce mitophagy. Given the resistance of KRAS colon cancer tumors to immunotherapeutic agents, targeting bioenergetic metabolism using mitochondria-targeted agents to stimulate mitophagy provides an attractive novel approach for therapeutic intervention.

#879

Investigating the activity of neratinib in human gastric cancer and gastric cancer cells, implications on clinical outcome and chemotherapy resistance.

W. G. Jiang,1 Tracey A. Martin,1 Sioned Owen,1 Lin Ye,1 Andrew J. Sanders,1 Yuxin Cui,1 Meng Xie,1 Shiqin Jia,2 Yongning Jia,2 Fiona Ruge,1 Francesca Avogadri-Connors,3 Alshad S. Lalani,3 Richard P. Bryce,3 Jiafu Ji2. 1 _Cardiff University, Cardiff, United Kingdom;_ 2 _Peking University Cancer Hospital, Beijing, China;_ 3 _Puma Biotechnology, Los Angeles, CA_.

Background. The EGF receptor (EGFR) kinase family plays important role in tumour growth and progression and has been a validated therapeutic target for lung cancers. Here, we examined the activity of an irreversible pan-HER tyrosine kinase inhibitor, neratinib, on kinase phosphorylation and cellular response in human gastric cancer. The study also examined the expression of the neratinib responsive kinases in relation to chemoresistance in human gastric cancer. Method. Tumour and normal gastric tissues from a cohort of 85 patients were obtained along with clinical outcome and therapeutic response to chemotherapy. The level of expression of a panel of protein kinases of interest, including EGFR family members, was quantitatively analysed using a protein based kinase array. Human gastric cancer cells (AGS and HGC27) were assessed for their sensitivity to neratinib, inhibitors of other candidate kinases and combinations thereof. Cellular growth, migration and matrix adhesiveness were evaluated using multiple in vitro platforms. Result. We identified a panel of kinases that were highly responsive to the treatment by neratinib in cell lines. In the gastric cancer cohort, expression of these kinases significantly correlated with the overall survival of the patients (p<0.001) and, together with node involvement, invasion, staging and type of surgery, was an independent prognostic indicator for the clinical outcome. Expression of the EGFR kinase family members EGFR, HER2, and HER4 was linked to poor clinical outcome. Expression of both neratinib response kinases and EGFR family members was also associated with therapeutic resistance to chemotherapies in the cohort of patients analyzed. In vitro, neratinib caused a concentration dependent inhibition of migration, matrix adhesiveness and growth in human gastric cancer cells. The migratory pace of the gastric cancer cells was particularly sensitive to neratinib. The role of two neratinib response kinases, Protein Kinase C iota (PKCi) and Mouse Double Minute 2 homologue (MDM2), was then further investigated. Interestingly, both PKCi and MDM2 were aberrantly expressed in gastric cancer tissues. In addition, the PKCi inhibitor Oncrasin-1 and the MDM2 antagonist Nutlin-3 both demonstrated synergy with neratinib in inhibiting adhesiveness and migration of the gastric cancer cells. Conclusion. Our data indicates that EGFR family members and other neratinib responsive kinases are aberrantly expressed in human gastric cancer and may be of prognostic value for the patients. In addition, gastric cancer cells are sensitive to neratinib, and displayed reduced migration when combined with inhibitors of PKCi or MDM2, two of the neratinib responsive kinases.

#880

Anti-tubulin antibody drug conjugates potentiate venetoclax activity in non-Hodgkin lymphoma by targeting MCL-1.

Dhara N. Amin, Jason Oeh, Anuradha Zindal, Lisa Musick, Jamie Hirata, Mehrdad Mobasher, Andy Polson, Deepak Sampath, Ingrid E. Wertz. _Genentech, South San Francisco, CA_.

Venetoclax, a potent, orally bioavailable inhibitor that selectively targets BCL-2 and induces intrinsic apoptosis, is currently approved for the treatment of relapsed/refractory chronic lymphocytic leukemia with 17p deletion (R/R 17p del CLL) and also has profound efficacy in broad R/R CLL as a single agent or in combination with obinutuzumab (anti-CD20 antibody). However, evidence of robust efficacy by venetoclax as a single agent in certain subtypes of non-Hodgkin lymphoma (NHL) has been limited. For example, diffuse large B-Cell lymphoma (DLBCL) is the most common sub-type of NHL, yet the overall response rate of DLBCL patients to venetoclax was 18% in contrast to 61% in other NHL sub-types. It has been proposed that expression of other pro-survival BCL-2 family members may limit single-agent venetoclax efficacy and is thus considered a potential major resistance factor. To this end, we have observed that low sensitivity to venetoclax monotherapy is correlated with MCL-1 expression in NHL cell lines. We have previously reported that anti-tubulin agents promote MCL-1 degradation. Given that polatuzumab vedotin is a novel antibody drug conjugate targeted to CD79b and delivers an auristatin anti-tubulin agent, we hypothesized that combining polatuzumab with venetoclax presents a mechanism-driven combination strategy for maximizing anti-tumor responses in NHL. In NHL patients, both polatuzumab vedotin and venetoclax have acceptable and non-overlapping toxicities and the combination of both drugs may provide an improved therapeutic index relative to conventional chemotherapy. Here, we demonstrate that polatuzumab vedotin synergizes with venetoclax both in vitro and in vivo. Mechanistically, the combination of both drugs promotes apoptosis in NHL cell lines as indicated by caspase 3/7 activation and dependence on BAX/BAK expression, and decreases MCL-1 protein levels. Furthermore, the combination of venetoclax with polatuzumab vedotin is more efficacious in vitro than the combination of venetoclax with a selective MCL-1 small molecule inhibitor (AMG 176). The combination of polatuzumab vedotin with venetoclax results in durable tumor regressions in diffuse large B-cell and mantle cell lymphoma xenograft models at tolerated doses and is more efficacious than the combination of venetoclax plus bendamustine, a standard chemotherapeutic agent. Based on our pre-clinical data and the strong mechanistic rationale, a drug combination regimen of venetoclax with polatuzumab vedotin and obinutuzumab is currently being evaluated in Phase1b clinical trials in R/R FL and DLBCL (ClinicalTrials.gov identifier NCT02611323).

#881

Identifying the context of vulnerability to MLN4924 in glioblastoma (GBM).

Leena Chaudhuri,1 Rita L. Bybee,1 Lauren K. Hartman,1 Sen Peng,1 Darren Finlay,2 Kristiina Vuori,2 Michael E. Berens,1 Harshil D. Dhruv1. 1 _Translational Genomics Research Institute, Phoenix, AZ;_ 2 _Sanford Burnham Prebys, CA_.

Neddylation is a post-translational mechanism that marks proteins for degradation through activity of NEDD8 Activating Enzyme (NAE). NAE activates cullin-RING ligases (CRL), which ubiquitylate selected substrates and mark them for proteosomal degradation. MLN4924, or Pevonedistat, targets NAE and inhibits Neddylation and induces apoptosis in sensitive cells. To assess the preferential sensitivity of cell lines to MLN4924, we performed a 10-point drug dose response (DDR) assay on long-term established GBM cell lines. Efficacy of MLN4924 in glioma cell lines was evaluated by measuring cell viability (CellTiterGlo®) and cell cycle progression (flow cytometry with propidium iodide staining). To identify mechanism of differential response to MLN4924 treatment, cell cycle regulatory pathway and DNA damage were also examined by Western blotting. GB1 (IC50 = 0.28 μM) & LN18 (IC50 = 0.19 μM) were established as sensitive and M059K (IC50 = 5.5 μM) & SNU1105 (IC50 = 20.9 μM) as non-sensitive cell lines based on the IC50 values. Flow cytometry analysis of DNA content revealed significant arrest of cells in G2/M even at low doses of 100 nM of MLN4924 preferentially in GB1 and LN18. This was consistent with an increase in CRL substrates p21, p27 and WEE1 in GB1 and LN18 possibly contributing to the G2/M arrest. While CDT1 accumulation was observed starting at 2h post MLN4924 treatment in sensitive cell lines, it took upto 8h for CDT1 accumulation in the non-sensitive cell lines. Increases in CDT1 induced re-replication causing massive arrest in G2/M phase lead to increased DNA damage, validated by higher expression of γH2AX in the sensitive cell lines. Additionally, we also investigated the efficacy of MLN4924 against orthotopic glioma PDX models in vitro and in vivo to validate our findings. In a cohort of glioblastoma PDX models we discovered that GBM PDX models with lower Neddylation gene set enrichment score (GBM116, GBM59, SF7300) were markedly more vulnerable to MLN4924 than GBM PDX models with higher Neddylation gene set enrichment score (GBM91 and GBM102) in vitro and in vivo. Orthotopic PDX models of selected GBM revealed survival prolongation of GBM116, but minimal survival benefit to GBM102 tumors. Validation of the predictive markers of vulnerability to MLN4924 in additional PDX models will set the stage for prospective clinical trials of MLN4924 in glioblastoma patients.

#882

The anti-claudin 6 antibody, IMAB027, induces antibody-dependent cellular and complement-dependent cytotoxicity in claudin 6-expressing cancer cells.

Özlem Türeci,1 Maria Kreuzberg,2 Korden Walter,2 Stefan Wöll,2 Ramona Schmitt,2 Rita Mitnacht-Kraus,2 Ikumi Nakajo,3 Tomohiro Yamada,3 Ugur Sahin4. 1 _CI3 – Cluster of Individualized Immune Intervention, Mainz, Germany;_ 2 _Formerly of Ganymed Pharmaceuticals GmbH, Mainz, Germany;_ 3 _Astellas Pharma, Inc, Tokyo, Japan;_ 4 _TRON – Translational Oncology at the University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany_.

Background Claudin 6 (CLDN6) is a tight junction membrane protein whose expression in normal tissue is confined to embryonic cells, but aberrantly expressed in various human cancer types, including some with a high medical need (eg, ovarian and uterine cancers). This tumor-specific expression in adult organs makes CLDN6 an attractive drug target; as such, IMAB027, an anti-CLDN6 monoclonal antibody (mAb), was developed. This report describes the preclinical characteristics of IMAB027.

Methods IMAB027 was generated by hybridoma technology; the discovery process was set up so that mAbs that were good binders as well as inducers of the immune effector mechanisms of antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) activity would be detected. ADCC and CDC were assessed in vitro. Apoptosis of CLDN6\+ cells was assessed by caspase 3/7, annexin V, and TUNEL assays. Xenografted mouse tumors with human CLDN6+ cells were generated to investigate the in vivo antitumor effects of IMAB027 both as a single agent and in combination with chemotherapeutic agents such as paclitaxel.

Results: IMAB027 binds specifically to CLDN6 without cross-reactivity with other closely related family members, such as Claudins 3, 4, and 9. IMAB027 induced target-selective ADCC and CDC in a number of CLDN6+ ovarian and testicular cancer cell lines; median EC50 values for ADCC and CDC were of the order of ng/mL in representative cell lines. Direct induction of apoptosis did not appear to be a contributor to the antitumor effect of IMAB027. In cancer cell lines that heterogeneously expressed CLDN6, but not in those with high or homogeneous CLDN6 levels, pretreatment with chemotherapeutic agents upregulated CLDN6 expression. Increased CLDN6 expression sensitized the cells to IMAB027-induced ADCC, resulting in increased cell lysis. In vivo, treatment with IMAB027 was associated with reduced tumor growth and increased overall survival in different mouse tumor models. These potent antitumor effects were observed in both early and advanced ovarian cancer xenografts. IMAB027, in combination with paclitaxel administered to mice with CLDN6+ xenografts, also prolonged survival compared with paclitaxel alone.

Conclusions In these preclinical studies, binding of IMAB027 was CLDN6 specific. IMAB027 as a single agent induced cell death in CLDN6+ cancer cells via ADCC and CDC, thereby exerting in vitro and in vivo antitumor activity. Further, in tumors that have heterogeneous CLDN6 expression, chemotherapy sensitizes cells to IMAB027-induced ADCC, and the combination of IMAB027 and chemotherapeutic agents may enhance the antitumor effect of chemotherapy.

#883

Alvocidib-mediated inhibition of CDK9 upregulates BIM via suppression of miR17-92.

Hillary Haws,1 Hubert F. Arokium,1 James L. Bogenberger,2 Adam Siddiqui-Jain,1 David J. Bearss,1 Raoul Tibes,2 Steven L. Warner,1 Clifford J. Whatcott1. 1 _Tolero Pharmaceuticals, Inc., Lehi, UT;_ 2 _Mayo Clinic, Scottsdale, AZ_.

The positive-transcription elongation factor (P-TEFb) complex is critical in stimulating the transcription of non-abortive transcripts by RNA polymerase II. The CDK9/cyclin-T heterodimer comprises the P-TEFb complex and promotes transcription elongation through phosphorylation of serine 2 of the heptapeptide repeats of the c-terminal domain of RNA Polymerase II (RNAPII-CTD). Alvocidib, a potent CDK9 inhibitor, has been shown to suppress expression of MCL-1 and Myc mRNAs, and clinical pharmacodynamic data suggest that alvocidib's activity is primarily mediated through the suppression of MCL-1 mRNA and protein expression. In addition to MCL-1 suppression, we hypothesized that CDK9 inhibition affects other RNA Polymerase II target RNAs, such as microRNAs. Recently, it was reported that inhibition of bromodomain and extraterminal domain (BET) protein by JQ1 suppresses miR17-92 expression. miR17-92 negatively regulates expression of the pro-apoptotic BH3-only protein, BIM, leading to suppression of BIM expression, thereby decreasing the cells' ability to induce apoptosis. It was hypothesized that CDK9 kinase activity is key to miR17-92 expression and suppression of BIM expression and that targeting CDK9 would lead to a decrease in miR17-92, and increases in BIM. MV4-11, OCI-AML3, MOLM13, and THP1 AML cell lines were used to determine the effects of alvocidib treatment on microRNAs. RT-qPCR was utilized to determine microRNA expression levels of miR17-92 and mRNA levels of BIM and other markers. Protein changes were determined using standard gel electrophoresis and immunoblotting technique. CellTiter-Glo and Caspase-Glo were used for all cell viability and apoptosis assays interrogating alvocidib. With CDK9 inhibition mediated via alvocidib, a dose- and time-dependent decrease in miR17-92 expression in MV4-11, OCI-AML3, MOLM13, and THP1 cells was observed. A decrease was observed even 3 hours post-treatment, persisting for up to 24 hours. MicroRNA suppression following treatment ranged in magnitude, with a maximal effect of between 3.3 to 4.2-fold suppression, depending on the cell line and timepoint. This suppression coincided with an increase in BIM mRNA and protein expression in the MV4-11, OCI-AML3, and MOLM13 cells. However, BIM protein increase was not observed in THP1 cells. Maximal increase in BIM mRNA levels reached 5.4-fold in the MV4-11 cell line. Our data suggest that CDK9 inhibition suppresses RNA Polymerase II-mediated expression of miR17-92, which in turn leads to increased expression of BIM. Combined with MCL-1 reduction, increased BIM protein expression mediated by alvocidib leads to enhanced apoptosis. Taken together, the data provide additional understanding of CDK9 as a potential therapeutic target, and are consistent with the hypothesis that CDK9 activity is necessary for miR17-92 expression.

#884

Mechanistically-driven investigation of pegcrisantaspase combination therapy against acute myeloid leukemia.

Hannah Kaizer,1 Binny Bhandary,1 Brandon A. Carter-Cooper,1 Eun Yong Choi,1 Bandish Kapadia,1 Elizabeth T. Chang,1 Rena G. Lapidus,1 Ronald B. Gartenhaus,2 Ashkan Emadi2. 1 _Univ. of Maryland School of Medicine, Baltimore, MD;_ 2 _Univ. of Maryland Greenebaum Comprehensive Cancer Center, Baltimore, MD_.

Introduction: Novel, more efficacious, and better tolerated therapy for acute myeloid leukemia (AML) is needed, as current treatment options result in only 35-40% 5 year survival in patients younger than 60 years of age. Targeting cellular metabolism, specifically by disrupting glutamine metabolism, is a promising approach to treat AML since altering metabolism appears to affect AML cell growth more compared to normal cells. L-asparaginases, which are approved for use in acute lymphoblastic leukemia, has been shown to alter glutamine metabolism in AML cells. In a recently published clinical trial, we found that Erwinaze, Erwina chrysanthemi asparaginase, is capable of depletion of plasma glutamine levels and is well tolerated in adult patients with AML with no occurrence of dose-limiting toxicity (DLT). Here, we report the anti-AML activity of pegcrisantaspase, a novel long-acting Erwinia chrysanthemi L-asparaginase, in combination with clinically relevant chemotherapeutic agents in vitro and in vivo.

Materials and Methods: In this study we investigated the cytotoxic effect of pegcrisantaspase along with 12 other clinically relevant chemotherapeutic agents. In vitro anti-proliferative activity of each agent was determined in 7 different AML cell lines (MV4-11, MOLM-14, MonoMac-6, U937, THP-1, HL60, K562) using the WST-1 cell proliferation reagent. In combination studies, cell survival was measured by Trypan blue staining. For determination of synergism, results of the WST-1 cell proliferation assay were analyzed by median effect analysis and used to determine a combination index (CI), with synergism defined as CI values <0.9. In vivo anti-AML activity of pegcrisantaspase was determined in NSG mice harboring luciferase, expressing the MV4-11 cell line.

Results: Pegcrisantaspase inhibited AML cell growth in six of seven cell lines tested, with IC50s ranging from 0.0001 IU/mL to 0.049 IU/mL. At higher concentrations, pegcrisantaspase was synergistic with the proteasome inhibitors ixazomib and carfilzomib. When pegcrisantaspase was combined with the Bcl-2 inhibitor, venetoclax, pegcrisantaspase potentiated the effect of venetoclax in AML cells by more than 10 fold. Additionally, pegcrisantaspase showed significant anti-AML effects compared to control in our mouse xenograft model.

Conclusions and Future Directions: Our data demonstrates the meaningful anti-AML activity of pegcrisantaspase in vitro and in vivo. The results also suggest that combination of pegcrisantaspase with proteasome and Bcl-2 inhibitors is potentially efficacious at treating AML. Mechanistic experiments to examine the phosphorylation state of key downstream targets of mTOR are ongoing. Furthermore, future in vivo studies will involve combining the above chemotherapies with pegcrisantaspase in our mouse xenograft model.

#885

Anthracycline-induced apoptosis through caspase3/7 activity is p53-dependent in bladder cancer cells.

Sony Pandey,1 Leonard Lothstein,2 Maria Cekanova1. 1 _The University of Tennessee Knoxville, Knoxville, TN;_ 2 _The University of Tennessee Health Science Center Memphis, Memphis, TN_.

Doxorubicin (Dox) is a chemotherapy drug widely used for treatment of several cancers including bladder cancer. However, cardiovascular side effects caused by doxorubicin-induced cardiotoxicity has necessitated the synthesis and validation of next generation chemotherapy drugs that are as effective but not cardiotoxic. AD 312 (N-Nitrosureidodaunorubicin; Daunomustine®, Paradox Pharmaceuticals, Inc.) and AD 198 (N- benzyladriamycin-14-valerate; Benzarubicin®, Paradox Pharmaceuticals, Inc.) are novel anthracyclines with confirmed non-cardiotoxicity in mice. Compared to Dox, AD 312 and AD 198 induce apoptosis in cells through distinct mechanisms. The tumor suppressor gene p53 is a key regulator of cell-cycle arrest and apoptosis. Dysregulation of p53 expression and function was identified in over 70% of the bladder cancers and reported as a potential target for therapy. As a result, small molecule compounds that can restore mutated p53 function to wild-type, including PRIMA-1, are under intensive investigation. In this study, we have compared the efficacy of AD 312 and AD 198 to Dox to induce apoptosis in human bladder transitional cell carcinoma (TCC) cells by MTT assay and assessed the possible mechanisms of anthracyclines-induced apoptosis in TCC cells expressing wild-type (wt) p53 (RT4, SW780) and mutated (mt) p53 (UM-UC-3, 5637, J82, and TCCSUP) protein. We have also investigated the effects of co-treatment of anthracyclines with PRIMA-1 in J82 cells, as a representative mt-p53 TCC cell line, and compared the co-treatment effects in RT4, as a representative wt-p53 cell line. Our results demonstrated that anthracyclines inhibited cell viability of all tested TCC cells in vitro, with AD 198 demonstrating highest efficacy. Furthermore, anthracyclines upregulated p53 protein expression in wt-p53 RT4 and SW780 cells, but not in tested mt-p53 UM-UC-3, 5637, J82, and TCCSUP cells. A strong correlation was observed between p53 mutation status and activation of caspase 3/7 and cleavage of PARP. The upregulated caspase 3/7 activity and PARP cleavage was observed in wt-p53, but not in mt-p53 TCC cells. AD198 blocked c-myc expression in mt-p53 TCC cells and no expression of c-myc was detected in wt-p53 TCC cells. Furthermore, sensitivity to AD 198 treatment in mt-p53 J82 cells was significantly improved by a co-treatment with PRIMA-1. However, PRIMA-1 had no effect on cell viability in RT4, the wt-p53 TCC cells. Overall, our results demonstrated that AD 312 and AD 198 are potent inhibitors of bladder cancer cells in vitro and are promising candidates for new therapeutic strategies to replace Dox for bladder cancer treatment. PRIMA-1 even further sensitized mt-p53 cells to AD 198 treatment and thereby this co-treatment improved the inhibition of bladder cancer in vitro.

#886

Metformin effect on multidrug resistant leukemia cells: metalloproteinases 2 and 9.

Ligia P. Oliveira, Camila Perea, Caroline Baldi, Rodrigo F. Prata, Débora L. Renó, Luana P. Lima, Rodrigo Curvello, Ana Carolina S. Souza. _UFABC, Santo André, Brazil_.

Besides the progress in studies of cancer, the rates of morbidity and mortality due to this disease still considerably elevated. Chronic myeloid leukemia (CML) is a hematological neoplasm, therefore chemotherapy is the main therapeutic approach used, however most patients develop resistance to chemotherapy. Several factors contribute to this scenario, among them the lack of selective treatment, the emergence of tumor cells resistant to the wide variety of cytotoxic agents during treatment and the formation of metastases, which accounts for the majority of cancer deaths. Therefore, application of new drugs in antitumor therapies has been constantly studied. Studies have shown that metformin, an oral euglycemic used in the treatment of type II diabetes, has potential use in the treatment of cancer, potentializing the effects of standard chemotherapy used in treatment. In this work the effects of metformin on the metastatic capacity of K562 (non-resistant leukemic cell), Lucena and FEPS (leukemic cells resistant to multiple drugs) were evaluated through the study of MMP2 and MMP9 metalloproteinases, enzymes essential for the metastatic process. Thus, the present project aimed to evaluate the gene expression and enzymatic activity of MMP2 and MMP9 metalloproteinases in CML cells, before and after treatment with metformin, seeking to verify the effects of metformin on the expression and activity of these enzymes. Gene expression was evaluated using quantitative PCR and the quantification of enzymatic activity was performed by the substrate degradation using the zymography method. The results showed that metformin has the potential to modulate the expression and activity of the studied metalloproteinases.

#887

**The effect of anticancer chemotherapeutics on immune microenvironment of lung adenocarcinoma with EGFR mutation in vivomicroenvironment of lung adenocarcinoma with EGFR mutation** in vivo **.**

Young-Ah Suh,1 Hyemin Mun,1 Sun-Hye Lee,1 Se-Young Jo,1 Ju-Hee Oh,1 Chu-Hee Lee2. 1 _Univ. of Ulsan Asan Medical Ctr., Seoul, Republic of Korea;_ 2 _School of Medicine, Yeungnam University, Daegu, Republic of Korea_.

Lung cancer is the leading cause of cancer related deaths worldwide. Molecular targeting therapy is an attractive treatment for lung cancer, although it eventually develops resistance in most cases. In this report, we tested taxan antitumor effect on lung adenocarcinomas in vitro and in vivo. It could inhibit cell growth of many NSCLC cells tested, exhibiting reduced expression of c-MET and STAT3. The nascent effect of taxan on lung tumor cells were also proved on patient-derived primary lung tumor cells and patient-derived xenograft model that highly expresses c-MET. The antitumor effect of this chemotherapeutics was also observed on T790M mutant cells. Interestingly, the antitumor effect of taxan was synergistic in combination of c-Met inhibitor on lung tumor cells as well as patient-derived primary lung tumor cells. However, single treatment of taxan was sufficient for halting tumor growth in vivo, as no combinational effect of c-Met inhibitor was obvious. Not only c-Met inhibitor but also taxan reduced phosphorylated c-MET and STAT3, although cytokines on downstream of c-MET and STAT3 were explosively increased in vitro setting resulting in high expression of chemokines/cytokines. Importantly, the effect was not true in in vivo setting, in which the expressions of chemokines/cytokines as well as phosphorylated c-MET and STAT3 were influenced with this agent. These results suggest that taxane is one of the comparative adjuvant for lung tumors exhibiting targeted drug resistance, especially for enhanced signaling of MET-STAT-immuno-microenvironment.

#888

A novel assay for the evaluation of ADCC in 3D-culture.

Macarena Irigoyen,1 Andrea Alms,2 Yoko Ejiri,2 Satoru Ayano,2 Gonzalo Castillo1. 1 _BIOENSIS, Bothell, WA;_ 2 _Kuraray Co. Ltd., Japan_.

In the last few years, one of the most innovative areas of cancer research has been immuno-oncology where significant advancements have been seen, particularly, in the use of monoclonal antibodies to treat cancers. The regulation of immune-mediated killing by monoclonal antibodies is an important mechanism of action, like in the case of trastuzumab, a humanized monoclonal antibody used in the treatment of HER-2 positive breast cancers. Binding of the humanized antibody to the receptor induces antibody-dependent cellular cytotoxicity (ADCC). Assays that monitor this activity are historically difficult to execute suffering from low sensitivity and reproducibility. Furthermore, experiments are typically done in suspension of cells grown as monolayers; often, showing limited clinical translatability when studying solid tumor models. Unencumbered by the limitations of 2D assays, the tumor microenvironment of 3D-cultures includes cell-cell interactions and formations of metabolic gradients, which are vital to understanding drug efficacy and resistance. To study the ADCC triggered by trastuzumab in 3D-cellular models, we developed a novel method to evaluate cellular toxicity in spheroids. In this study, we demonstrated the quantification, with specificity and precision, of the ADCC activity elicited by trastuzumab in 3D-culture. The high HER2-expressing BT474 cell line (target cells) was pre-labeled and formed into spheroids utilizing Kuraray's 384-well Elplasia's microplates. PBMCs (effector cells) isolated from healthy donors were added in the presence or absence of the antibody, trastuzumab, and incubated overnight. Cytotoxicity of the tumor-derived cells was evaluated by counting stained dead cells using a cell imager. In parallel, a 2D-platform for ADCC assay was run and the pharmacology of trastuzumab was characterized. BT474 cells grown as spheroids show an IC50 of 0.09 µg/mL, when the study was done in 2D-culture an IC50 of 0.028 µg/mL was obtained. The ability to evaluate ADCC in both 2D- and 3D- may help the development of more translatable models in the clinic.

#889

Metformin treatment inhibits proliferation and induces apoptosis in medulloblastoma cells.

Kristen C. Payne,1 Abigail Hunter,1 W Paul Bowman,1 Jeffrey C. Murray,2 Riyaz Basha,1 Umesh T. Sankpal1. 1 _Univ. of North Texas Health Science Ctr., Fort Worth, TX;_ 2 _Cook Childen's Health Care System, Fort Worth, TX_.

Medulloblastoma is the most common malignant brain tumor in children that typically requires a patient specific combination of surgical resection, chemotherapy, and radiation. Up to 80% of medulloblastoma patients respond to treatment and survive long-term. However, survivors often suffer from severe long term sequelae secondary to treatment that affects their neurocognitive and growth potential. Therefore, there is an urgency to develop less toxic, more focused treatment strategies that target specific pathways or molecules involved in medulloblastoma development and progression. Metformin is a widely used non-toxic anti-diabetic drug that has mild, generally well-tolerated side effects. Studies have shown that it can reduce cancer risk and improve the prognosis of certain malignancies. However, the mechanism underlying its anti-cancer effect is still unclear and varies depending on the type of cancer. There are reports that metformin can target the specificity protein 1 (Sp1) transcription factor. Sp1 regulates the expression of a variety of proteins involved in regulation of cancer cell proliferation including survivin, an Inhibitor of Apoptosis Protein family member. Expression of survivin is increased in medulloblastoma cells and often correlates with poor prognosis. We studied the anti-cancer activity of metformin on medulloblastoma using DAOY and D283 cells. Metformin treatment resulted in a dose and time dependent reduction in cell proliferation in both cell lines. This growth inhibition was accompanied by the induction of apoptosis as determined by annexin-V staining, PARP cleavage, and activation of caspase3/7. Metformin also induced cell cycle arrest in G0/G1 phase. Western blot analysis using extracts from cells treated with metformin revealed that the anti-proliferative and apoptotic effects of metformin in medulloblastoma were accompanied by a reduction in the expression of survivin protein. Overexpression of survivin is reported to be associated with chemo- and radio-resistance. This study suggests the use of metformin as a novel anticancer agent in combination with standard therapy for the treatment of medulloblastoma.

#890

Calmodulin governs nuclear entry of fusion PAX3/FOXO1 oncoprotein, a target in alveolar rhabdomyosarcoma.

Emad Darvishi,1 Cheryl Lynn Thomas,1 Berkley Eric Gryder,2 John Frederick Shern,2 Javed Khan,2 Girma M Woldemichael3. 1 _Molecular Targets Program, National Cancer Institute, NIH, Frederick, MD;_ 2 _Genetics Branch, National Cancer Institute, NIH, Bethesda, MD;_ 3 _Basic Science Program, Leidos Biomedical Research, Inc., Molecular Targets Program, Frederick, MD_.

Reciprocal chromosomal translocations resulting in the fusion of the DNA binding domain of the transcription factor PAX3 and the transactivational domain of FOXO1 underlie most cases of alveolar rhabdomyosarcoma (aRMS). Testing of compound MTPF63 against a panel of sarcoma cell lines showed that it had an inhibitory effect on proliferation of PAX3-FOXO1 translocation-positive cells. Further evaluation of its activity in Rh4 cells transduced with a PAX3-FOXO1 luciferase reporter showed that it attenuated reporter activity. Additional confirmation for the compound's inhibitory impact on PAX3-FOXO1 activity was obtained by detecting significant attenuation in the levels of Alk1 and MyoD1 proteins whose expression is driven by PAX3-FOXO1 in aRMS cells. Cell viability assays performed under 2D and 3D culturing conditions demonstrated that MTPF63 inhibits cell growth more effectively in translocation-positive alveolar RMS than in translocation-negative embryonal RMS. A hypothesis for MTPF63's mode of action was generated using the complete pool of barcoded essential heterozygous (~1150 strains) and homozygous (~4800 strains) diploid gene deletion mutants of Saccharomyces cerevisiae to identify gene deletions that confer sensitivity to the compound. Gene ontology enrichment analysis of 255 sensitive mutants whose growth rates were significantly inhibited in the presence of the compound compared to control, not only revealed calmodulin as main target of the compound but also illustrated the compound's interference with calmodulin-dependent nuclear protein import pathway, suggesting that it might impact PAX3-FOXO1 nuclear localization. Immunostaining of nuclear-localized PAX3-FOXO1 oncoprotein in two aRMS cell lines revealed cytoplasmic localization. Immunoblot analysis of fractionated cytosolic and nuclear protein lysates for PAX3-FOXO1 in MTPF63 treated cells also showed increased amounts in the cytosolic fractions. Transient overexpression of calmodulin was shown to partially rescue the inhibitory effect of the compound on PAX3-FOXO1-driven luciferase expression in RH4 reporter cells. Taken together, the data demonstrate the involvement of calmodulin in the nuclear import of PAX3-FOXO1 and introduce calmodulin antagonists as potential therapeutics against translocation-positive childhood alveolar rhabdomyosarcoma.

#891

A novel quinazolin-4(3H)-one/Schiff base hybrid phosphodiesterase 4 inhibitor as a potential therapeutic agent for leukemia.

Alexander Richard,1 Hamdy M. Abdel-Rahman,2 Jung Hyun Kim,1 Lana Vukadin,1 Gary A. Piazza,1 Eun-Young E. Ahn1. 1 _University of South Alabama, Mitchell Cancer Institute, Mobile, AL;_ 2 _Assiut University, Egypt_.

Cyclic adenosine monophosphate (cAMP) is a secondary messenger that regulates cell proliferation, differentiation, and apoptosis. Phosphodiesterases (PDEs) are enzymes that catabolize cyclic nucleotides to relay cellular signals. Among them, PDE4 is a cAMP-specific PDE isozyme family and several PDE4 inhibitors have been developed to treat a diverse group of diseases, such as asthma and chronic obstructive pulmonary disease. Although PDE4 inhibitors have been demonstrated to induce cell death in solid tumors, their therapeutic potential in treatment of hematopoietic malignancies has not been well studied. Our analysis on the expression pattern of PDE family demonstrated that PDE4B is abundantly expressed in blood cancers compared to other solid tumors, implicating PDE4 as a potential therapeutic target for leukemia. N-acylhydrazone and nitraquazone are potent PDE inhibitors that are structurally unrelated to rolipram, a prototypical PDE4 inhibitor. To develop novel PDE4 inhibitors with increased scaffold rigidity, we hybridized N-acylhydrazone with Nitraquazone, and designed a series of quinazolin-4(3H)-one/schiff base hybrid compounds. Among those, compound 23 (CPD23) showed the best activity in PDE4B inhibition, and was selected for further studies. Our results showed that CPD23 treatment leads to growth inhibition and cell cycle arrest of several human leukemia cell lines. Since cAMP is a well-studied secondary messenger for megakaryopoesis and apoptosis, we continued to elucidate CPD23's mechanism of action in several megakaryoblastic leukemia cell lines. While other well-known PDE4 inhibitors, such as rolipram, did not show any effect in cell viability, CPD23 significantly decreases the viability of megakaryoblastic cell lines. Further studies on molecular actions revealed that CPD23 caused double-stranded DNA breaks, which was detected by increased ϒH2AX levels. Quantitative RT-PCR experiments also demonstrated that CPD23 induces transcriptional activation of megakaryocytic differentiation markers, such as CD61, in selective cell lines. Taken together, our study shows that CPD23, synthesized as a quinazolin-4(3H)-one/schiff base hybrid PDE4 inhibitor, decreases proliferation, induces double-stranded DNA breaks, and differentiation in megakaryoblastic leukemia cell lines. Further studies will enhance our knowledge on detailed molecular actions of CPD23 and its therapeutic value for leukemia.

#892

Doxorubicin-induced cardiotoxicity in iPSC-cardiomyocytes: Altered mitochondrial gene expression and function.

Monica E. Reyes,1 Rashida Callender,1 Jianzhong Ma,2 Megan L. Grove,2 Alanna C. Morrison,2 Michelle A. Hildebrandt1. 1 _The University of Texas MD Anderson Cancer Center, Houston, TX;_ 2 _School of Public Health, The University of Texas Health Science Center at Houston, Houston, TX_.

Introduction: Cardiomyocytes are highly vulnerable to anthracycline-induced toxicity, which may lead to heart failure. This includes doxorubicin, which is a commonly used chemotherapeutic agent. Although mitochondrial function has been implicated as a mechanism of anthracycline-induced toxicity in rodent heart cells, the precise genes that regulate this response in humans remain to be elucidated. We hypothesized that doxorubicin significantly alters expression of mitochondrial genes in human cardiomyocytes, which impairs mitochondrial function.

Methods: Human inducible pluripotent stem cell (iPSC)-derived cardiomyocytes were treated with doxorubicin or left untreated for control to assess changes in gene expression using RNAseq. A total of 169 genes involved in mitochondrial function, as defined by Ingenuity Pathway Analysis and KEGG, were analyzed for significant differences between untreated and treated conditions using DESeq2 and GenePattern 2.0. Mitochondrial respiration was measured in control and doxorubicin-treated cells using the Seahorse Bioscience XFe96 Cell Mito Stress Test kit. We used a Spearman's partial correlation coefficient analysis to correlate gene expression levels with mitochondrial basal respiration, ATP production, maximal respiration, and spare respiratory capacity for untreated and doxorubicin-treated iPSC-cardiomyocytes.

Results: Of the 169 mitochondrial genes analyzed, we identified 25 genes that were significant in our global differential expression analysis across all conditions (P<0.05). Seven of these genes (ATP5D, COX5A, CYC1, HSD17B10, NDUFB10, NDUFS8, UQCRC1) remained significant in pairwise analyses between control and doxorubicin treated cells. We observed a decrease in mitochondrial respiration following treatment with doxorubicin. Maximal respiration (r=-0.929; P=0.022) and spare respiratory capacity (r=-0.98;P=0.0025) negatively correlated with NDUFS8 expression in doxorubicin-treated iPSC-cardiomyocytes.

Conclusion: Our findings underscore a role for mitochondrial function in the development of doxorubicin-induced cardiotoxicity and implicate specific genes in this process. Doxorubicin-altered gene expression in cardiomyocytes may provide insight into how impaired mitochondrial function leads to heart failure in cancer survivors.

#893

Hyperthermia regulates both SLC22A16 expression and ABCG2 expression via ROS production to enhance the cytotoxicity of doxorubicin.

Hirofumi Matsui. _University of Tsukuba, Tsukuba, Japan_.

Introduction: Hyperthermia (HT) is a non-invasive cancer therapy. Treatment temperature between 41°C to 44°C has no cytotoxic damage in normal cells, however shows cytotoxicity in cancer cells because of the underdeveloped vascular system. HT often used with other cancer therapy such as radiation therapy and chemotherapy. However mechanism of synergistic effect using these therapies remains unclear. Compared to 37°C, 42°C is mild heat stress for cells, thus superoxide anion is released from tissue. Superoxide anion is produced by mitochondrial electron transport chain. Reactive oxygen species (ROS), produced by mild heat stress, can be released from mitochondria. We have previously reported that ATP-binding cassette sub-family G member 2 (ABCG2) expression was suppressed by increasing mitochondrial ROS, andinduction of the cancer specific porphyrin accumulation. ABCG2 is a transporter of doxorubicin (DOX), therefore we hypothesized that synergistic effect of HT and chemotherapy would be induced by down-regulation of ABCG2 expression via intracellular ROS increase. In this study, we investigated if cytotoxic effect of breast cancer cell using DOX can be enhance by HT via intracellular ROS increase. Materials and methods: The murine breast cancer cell line, 4T1E was incubated at 37°C or 42°C for 1h. Intracellular ROS generation after HT treatment was detected by electron spin resonance (ESR). Twenty four hours after HT treatment, cells were incubated in medium containing 0, 0.1 and 1 μM DOX for 24 h. Cell viability was measured using the Cell Counting Kit 8, a water-soluble tetrazolium-8 based colorimetric assay. ABCG2 expression in whole cells was analyzed by Western blotting. Results and discussion: ESR signal peak with HT treatment became high as compared to without HT treatment, indicating intracellular ROS level was increased by HT treatment. Cell viability and ABCG2 expression were decreased by DOX exposure and by HT treatment. The enhancement of HT treatment effect by DOX is considered to be result of down-regulation of ABCG2 expression by ROS. Conclusion: HT treatment involved intracellular ROS production and down-regulated the expression of ABCG2 protein. HT treatment also enhanced the cell damage by DOX.

### Strategies to Reversing Drug Resistance

#894

Overexpression and extended degradation rate of Axl in acquired gefitinib-resistant non-small cell lung cancer cells.

Donghwa Kim, Duc-Hiep Bach, Ji-Young Hong, Hyen Joo Park, Sang Kook Lee. _Seoul National University, Seoul, Republic of Korea_.

Non-small cell lung cancer (NSCLC) is a major type of lung cancer which accounts for approximately 80-85% of all lung cancers. Epidermal growth factor receptor (EGFR)- targeted tyrosine kinase inhibitors (TKIs), such as gefitinib have improved the survival of NSCLC patients with activating mutations in EGFR. However, the drug responses are only transient because tumors acquire resistance via secondary mutations in EGFR (T790M) or amplification of other receptor tyrosine kinases (RTKs) including MET or AXL. Therefore, the precise mechanisms of chemoresistance and finding the novel ways of targeting cancer cells are needed to be identified. AXL, a TAM (Tyro3, AXL, Mer) family member, has been studied as a tumorigenesis, chemoresistance, and EMT marker. Also, we found that AXL is overexpressed in EGFR mutated gefitinib-resistant non-small lung cancer cells (PC9-gef) compared to its parent cells (PC9). Moreover, the AXL degradation rate was found to be suppressed. These findings suggest that the modulation of AXL turnover can be a therapeutic target to overcome gefitinib-resistance. In this study, we confirmed the possibilities of overcoming gefitinib-resistance through the down-regulation and turnover acceleration of AXL by employing yuanhadine, an anti-tumor agent derived from Daphne Genkwa. Yuanhuadine was also effectively suppressed the tumor growth in nude mouse xenograft models implanted with PC9 or PC9-gef cells. Therefore, regulation of AXL expression by modulating its degradation rate can be a therapeutic strategy to overcome acquired gefitinib-resistance in NSCLC.

#895

Noncanonical c-Met activation mediates de novo and acquired resistance to polo-like kinase 1 inhibitor-induced apoptosis in non-small cell lung cancer.

Ratnakar Singh, Pavitra Viswanath, Shaohua Peng, Vaishnavi Sambandam, Li Shen, Lerong Li, Jing Wang, Bingliang Fang, Faye M. Johnson. _MD Anderson Cancer Center, Houston, TX_.

Introduction: Plk1 is a serine-threonine protein kinase that is overexpressed in cancer cells, and plays a major role in regulating tumor growth. Plk1 inhibitors are well tolerated, but only a few unselected patients with non-small cell lung cancer (NSCLC) respond to single-agent therapy. Our lab discovered that mesenchymal NSCLC cell lines are more sensitive to Plk1 inhibitors than epithelial cell lines in vitro and in vivo. However, mechanisms of resistance to Plk1 inhibitors have not been elucidated and this unknown is a major gap in knowledge.

Experimental procedure: To study the mechanisms of Plk1 inhibitor-induced apoptosis we used 3 pairs of isogenic epithelial NSCLC cell lines induced to a mesenchymal phenotype with TGF-β. These isogenic pairs were treated with the Plk1 inhibitor volasertib for 24 h and levels of 301 proteins and phosphoproteins were simultaneously measured using reverse phase protein array (RPPA). Volasertib acquired resistance (VAR) cell lines were generated by exposing cells to increasing doses of volasertib.

Results: The induction of a mesenchymal phenotype using TGF-β increased Plk1 inhibition-induced apoptosis in all 3 cell lines. To further elucidate mechanisms of resistance, we compared protein expression in these isogenic cell lines, 24 h after Plk1 inhibition. There were 33 proteins differentially regulated following Plk1 inhibition in parental vs TGF-β induced isogenic cells (p-value < 0.05). Notably, phosphorylated c-Met (Y1234/1235), FAK (Y397) and Src (Y416) were consistently inhibited following Plk1 inhibition in the mesenchymal lines. These changes were confirmed by Western blotting. Total c-Met, FAK and Src protein levels were not affected, implicating a post-translational changes. Likewise, VAR cell lines exhibited an epithelial phenotype and c-Met phosphorylation was persistent even after Plk1 inhibition. Simultaneous c-Met and Plk1 inhibition or silencing increased apoptosis in NSCLC cell lines tested compared to single agent inhibition or silencing. Combination of Plk1 and c-Met inhibitors decreased tumor volume and increased mouse survival in vivo in patient derived and cell line xenograft models. Similarly VAR cells also showed more apoptosis when treated with combination of Plk1 and c-Met inhibitors. Levels of the c-Met ligand HGF were unchanged after Plk1 inhibition and further mechanistic studies are on-going.

Conclusion: NSCLC cell lines have diverse sensitivities to Plk1 inhibition, which is consistent with the results of clinical trials of Plk1 inhibitors in solid tumors. This study reveals a novel mechanism of non-canonical c-Met activation in resistant epithelial NSCLC after Plk1 inhibition. We demonstrate a profound effect of combination Plk1 and c-Met inhibition in vivo in multiple mouse models that could be a novel therapy for NSCLC patients.

#896

Adjutant therapeutic strategy for sorafenib-resistant hepatocellular carcinoma.

Syue-Wei Peng,1 Yi-Heng Lin,1 Ming-Hao Teng,1 Hung-Cheng Lai,2 Te-Sheng Chang,3 Yen-Hua Huang1. 1 _Department of Biochemistry and Molecular Cell Biology, School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan;_ 2 _Department of Obstetrics and Gynecology, School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan;_ 3 _Division of Gastroenterology and Hepatology, Department of Internal Medicine, Chang Gung Memorial Hospital, Chiayi, Taiwan, Taiwan_.

Purpose: Hepatocellular carcinoma (HCC) is the most common primary liver cancer and is the leading cause of cancer mortality worldwide. To date, Sorafenib remains the standard of care for advanced HCCs not amenable to curative therapies such as surgery or local ablation. Unfortunately, clinical efficacy of Sorafenib remains marginal as most HCC patients receiving Sorafenib treatment develop Sorafenib resistance. The recently approved regorafenib is the only rescue therapy for patients who fail Sorafenib treatment. However, the effect of regorafenib is also far from satisfactory. Therefore, new strategies to overcome sorefenib resistance are critically warranted. Recently, one FDA-approved drug, here we tentatively named as "FA", was shown to suppress different cancer malignancy by various mechanisms including inhibiting the development of drug resistance in cancer. These findings provide a rationale for combination treatment of this FA drug with Sorafenib in Sorafenib-resistant HCC. Results: Two Sorafenib-resistant HCC cell lines of Hep3B and HepG2215 were established. The Sorafenib resistance characteristics of the HCC cells were further verified by western blotting assay detecting protein tyrosine phosphorylation. We also demonstarted the resistant HCC cells expressed stemness-related genes and showed high ability of cell migration and metastasis. Combination use of FA drug and Sorafenib significantly suppressed the cell viability as well as the stemness-related properties of Sorafenib-resistant HCC cells, including stemness-related gene/protein expression, secondary sphere formation, and the cell migration. Conclusion: Combination use of FA drug and Sorafenib would be a potential therapeutic strategy for Sorafenib-resistant HCC treatment.

#897

A novel fusion gene responsible for colon cancer drug resistance.

Jingwu Xie, Dongsheng Gu, Ruolan Song. _Indiana University School of Medicine, Indianapolis, IN_.

Colorectal cancer is a leading cause of cancer-related mortality worldwide. Patients with advanced colorectal cancer eventually succumb to the disease, despite the fact that some patients respond initially to therapies (chemotherapy and targeted therapy). Thus, identifying molecular mechanisms responsible for drug resistance will help design novel strategies to treat colorectal cancer. In this study, we identified a novel mechanism by which colorectal cancer cells acquired 5-FU resistance. Through exome sequencing, we discovered a novel fusion gene common in 5-FU-resistant colorectal cancer cells. This fusion gene, once ectopically expressed in the sensitive cancer cells, promotes drug resistance to 5-FU treatment. Further analyses have revealed a signaling network linking this fusion gene to drug resistance. Taken together, we have revealed a novel mechanism for drug resistance in colorectal cancer, which may be a novel target to overcome drug resistance in colorectal cancer.

#898

Clear cell renal cell carcinoma resistance to RTKs inhibitors is mediated by c-Met receptor and MCPIP1.

Paulina Marona, Judyta Górka, Jolanta Jura, Katarzyna Miękus. _Jagiellonian University, KRAKÓW, Poland_.

Introduction

Clear cell renal cell carcinoma (ccRCC) treatment with small molecules that inhibit multiple receptor tyrosine kinases (RTKs),showed in approximately 38% of patients significant tumor control. However, despite the efficacy of treatment, ccRCC often develops resistance to targeted drugs and the majority of patients who receive such treatment exhibit progressive disease after 1 year. Several hypotheses have been proposed regarding the mechanisms underlying resistance to RTKs inhibitors, but the precise pathways have not yet been fully elucidated.

Aim

The main objective of our study was to determine the role of c-Met and anti-inflammatory protein MCPIP1 in the acquisition of resistance to RTKs inhibitors in ccRCC.

Materials and methods

ccRCC cell lines (Caki-1 and Caki-2) as well as normal epithelial cell lines (Hek293, RPTEC/TERT1) were treated with sunitinib, sorafenib or SU11274 constantly for 24h, 1 and 3 weeks. The level and localization of key proteins were examined using western blots and IF staining. ccRCC patient samples were divided into four histological grades, and levels of genes or proteins were investigated using microarray analysis, western blots, protein arrays, mass spectrometry and real time PCR.

Results

Our study shows that short term (24h) and long term (3 weeks) stimulation with sunitinib or sorafenib induced phosphorylation of c-Met receptor, STAT3 and Src kinase. Furthermore, sunitinib treatment resulted in the acquisition of cancer stem cells features and the formation of clones. In addition, after sunitinib and sorafenib treatment we observed a decrease in the protein level of MCPIP1, postulated tumor suppressor. Interestingly, after MCPIP1 overexpression, we observed c-Met downregulation, regulated by RNase activity of MCPIP1. Moreover, our results show that in patient samples, together with MCPIP1 downregulation, level of phosphorylated and total c-Met receptor increase.

Conclusions

We showed that acquisition of therapy resistance in ccRCC may be affected by MCPIP1 decrease, together with phopsphorylation of c-Met receptor and it can be partially reversed by overexpressing the MCPIP1 protein, which may act as a potent tumor suppressor. Proposed research may help in understanding the mechanisms responsible for tumor resistance to targeted therapy. Obtained results may contribute to increased understanding of the biology of clear cell renal cell carcinoma, which in the future may help in identifying new, more effective therapeutic purposes or improving existing ones.

Acknowledgment

This study was supported by research grant from the National Science Centre 2013/09/D/NZ/00249, 2017/25/N/NZ5/03014 and grant from Jagiellonian University BMN 16/2017.

#899

Phosphorylated mTOR by signaling through EphB3 confers resistance to a selective FGFR inhibitor in SNU-16 gastric cancer cells.

Suk-young Lee,1 YooJin Na,1 YoonA Jung,1 JungLim Kim,1 Sang Cheul Oh,1 Dae-Hee Lee2. 1 _Korea University Guro Hospital, Seoul, Republic of Korea;_ 2 _College of Medicine, Korea University, Seoul, Republic of Korea_.

Because about 10% of gastric cancers show amplification of FGFR2, inhibition of FGFR2 activation has been regarded as one of therapeutic targets. AZD4547, a selective inhibitor of the FGFR1-3 tyrosine kinases, was developed to inhibit FGFR signaling; however, its efficacy is limited by emergency of the acquired resistance. We tried to clarify a resistance mechanism against the FGFR inhibitor. The cell line resistant against AZD4547 was established using SNU-16 (SNU16R), an FGFR2-amplified gastric cancer cell line, by culturing with increasing concentration of AZD4547. The expression levels of FGFR or phosphorylated FGFR (pFGFR), EphB3 and downstream signaling molecules, proteins involved in epithelial-mesenchymal transition (EMT) and apoptosis, were determined by Western blot. Cell viability was measured with EZ-Cytox kit reagent (DoGen, Seoul, Korea). Relative level of tyrosine phosphorylation of receptor tyrosine kinases (RTKs) was evaluated with Proteome ProfilerTM array by human phosphor-RTK kit (R&D Systems). Apoptosis was determined by Annexin V-FITC apoptosis detection kit (BioBud, Seoul, Korea). Cell cycle assay was performed using Beckman Coulter flow cytometer and WinMDI for determination of cell numbers in G1, S, G2/M phases. Here we show the following: 1) establishment of a gastric cancer cell line (SNU16) resistant to AZD4547 (SNU16R); 2) expression of EphB3 receptor in SNU16R and induction of apoptosis and cell cycle arrest as well as inhibition of cell proliferation and suppressed expression of EMT markers under the treatment EphB3 inhibitor in SNU16R; 3) demonstration of mTOR as a downstream signaling cascade molecule of EphB3 by showing increased expression of phosphorylated (p-) mTOR and its downstream molecules in SNU16R and suppressed expression of p-mTOR by the EphB3 inhibitor. Cell cycle arrest, inhibition of cell proliferation and suppressed expression of p-mTOR under treatment with everolimus were also shown. We concluded that phosphorylation of mTOR through activation of EphB3 receptor is one of the mechanisms that confer resistance to AZD 4547, a selective FGFR inhibitor, in gastric cancer cells.

#900

Dianhydrogalactitol (VAL-083) has the potential to overcome major challenges in the treatment of DIPG.

Anne Steino,1 Beibei Zhai,2 Jeffrey A. Bacha,1 Dennis M. Brown,3 Mads Daugaard,2 Sabine Mueller4. 1 _Delmar Pharmaceuticals Inc., Vancouver, British Columbia, Canada;_ 2 _Vancouver Prostate Center, University of British Columbia, Vancouver, British Columbia, Canada;_ 3 _Delmar Pharmaceuticals Inc., Menlo Park, CA;_ 4 _University of California, San Francisco, San Francisco, CA_.

Despite decades of clinical trials, children with diffuse intrinsic pontine gliomas (DIPG) continue to have a very poor prognosis and dismal survival. DIPG is inoperable and standard treatment is radiation alone. Major obstacles to the successful treatment of DIPG include 1) an intact blood brain barrier impeding drug penetration, 2) inherent tumor cell resistance mechanisms to conventional chemotherapeutics, and 3) a lack of drug-induced potentiation of radiotherapy. VAL-083 is a structurally unique bi-functional DNA targeting agent that has the potential to overcome these barriers. VAL-083 readily crosses the blood-brain barrier and accumulates in brain tumor tissue where it rapidly forms interstrand DNA crosslinks at guanine-N7, leading to persistent DNA double strand breaks, cell cycle arrest, and cancer cell death. VAL-083 has cytotoxic activity in several pediatric brain tumors as assessed in NCI-sponsored clinical trials, both as a single agent and in combination with other chemotherapeutics. We recently completed a Phase I/II clinical trial in refractory GBM and established a well-tolerated dosing regimen of VAL-083 in adult brain tumor patients. We have previously demonstrated that VAL-083 cytotoxicity is independent of both the primary, DNA repair protein O6-methylguanine DNA methyltransferase (MGMT), and the secondary, DNA mismatch repair (MMR) system, mechanisms of resistance to temozolomide (TMZ) in vitro. TMZ is commonly used in combination with radiation for the treatment of adult brain tumors but has failed to improve the effect of radiation in DIPG. Importantly, VAL-083 potentiates the effect of radiation and overcomes TMZ resistance in TMZ-resistant adult glioblastoma cancer stem cells (CSCs) and non-CSCs at concentrations that are physiologically achievable in the CNS. Additionally, VAL-083 demonstrated synergistic efficacy in prostate and lung cancer cells with topoisomerase (Topo) inhibitors camptothecin and etoposide, commonly used in the treatment of pediatric brain tumors. In conclusion, VAL-083 is able to 1) cross the blood brain barrier, 2) overcome resistance mechanisms to chemotherapeutics commonly used in the treatment of adult gliomas, and 3) potentiate the effects of radiation in adult glioma cells at physiologically achievable concentrations. Thus, VAL-083 may have the potential to overcome major challenges in DIPG treatment as a single agent, in combination with radiotherapy, or as part of a combination regimen with Topo inhibitors. In the present study, we investigated the effects of VAL-083 in combination with radiation, irinotecan (Topo 1 inhibitor), or Wee1 inhibitor AZD1775 in a panel of DIPG cell lines with varying genetic profiles. The results will provide guidance for the design of animal models studying VAL-083 treatment of DIPG, either as part of a chemo-radiation regimen or in combination with Topo or Wee1 inhibitors.

#901

Antibody-mediated blockade of annexin A3 (ANXA3) synergistically enhances the efficacy of sorafenib and regorafenib for hepatocellular carcinoma.

Man Tong,1 Noelia Che,1 Lei Zhou,1 Phillis W. Kau,1 Steve T. Luk,1 Xin Chen,2 Jin Ding,3 Terence K. Lee,4 Stephanie Ma1. 1 _The University of Hong Kong, Hong Kong, Hong Kong;_ 2 _University of California, San Francisco, CA;_ 3 _Eastern Hepatobiliary Surgery Hospital/Institute, Second Military Medical University, Shanghai, China;_ 4 _Hong Kong Polytechnic University, Hong Kong, Hong Kong_.

Advanced hepatocellular carcinoma (HCC) is a lethal malignancy with limited treatment options. Sorafenib is the only FDA approved first-line targeted drug for advanced HCC, but its effect on patients' survival gain is limited and patients ultimately present disease progression. Molecular mechanism underlying acquired resistance are still far from being well understood. A better understanding of causes of sorafenib resistance, enhancing the efficacy of sorafenib and finding a reliable predictive biomarker are crucial to achieve efficient control of HCC. In this study, we uncovered a role of annexin A3 (ANXA3) in conferring ability of HCC cells to resist sorafenib. ANXA3 was found highly enriched in sorafenib-resistant HCC cells and patient-derived xenografts. Mechanistically, we found overexpression of ANXA3 in sorafenib-resistant HCC cells to suppress PKCδ/p38 associated apoptosis and activate autophagy for cell survival. ANXA3 expression correlated positively with LC3B, an indicator of autophagy induction, in HCC specimens and was associated with a worst overall survival in HCC patients who went on to receive sorafenib treatment. Anti-ANXA3 therapy, alone or combined with sorafenib and regorafenib, the standard of care for advanced HCC, impaired tumor growth in vivo and significantly increased survival. In sum, we found anti-ANXA3 therapy to exhibit abilities to reduce tumor growth and sensitize HCC cells to sorafenib and regorafenib treatment via suppressing autophagy and activating apoptosis. Inhibition of ANXA3 would benefit a subset of patients after sorafenib progression. ANXA3 can also serve as a useful predictive biomarker for patient response to sorafenib treatment.

#902

**In vitro and** in vivo **characterization of resistance to lorlatinib treatment in ALK mutated cancers.**

Geeta G. Sharma, Sara Redaelli, Monica Ceccon, Marina Zappa, Carlo Gambacorti-Passerini, Luca Mologni. _University of Milano-Bicocca, Monza, Italy_.

The development of targeted therapy has significantly improved the treatment of Anaplastic Lymphoma Kinase (ALK) dependent neoplasias such as Anaplastic Large Cell Lymphoma (ALCL) and Non-Small Cell Lung Cancer (NSCLC). Despite the efficacy of ALK inhibitor crizotinib, acquired resistance is a major challenge. Lorlatinib is a second generation inhibitor with improved activity and selectivity against ALK. To investigate resistance mechanisms that may arise on lorlatinib therapy, we selected in vitro and in vivo lorlatinib resistant tumor cells.

In vivo, 10 mice carrying subcutaneous Karpas299 xenografts received vehicle or lorlatinib at increasing doses, starting at 0.1 mg/kg BID. As expected, tumor size was significantly reduced in lorlatinib group compared to controls. After initial partial responses, tumors relapsed and mice were shifted to an increased dose (0.25 mg/kg). Tumors relapsed again and mice were randomized into three groups, to achieve a maximum dose of 0.5, 1 or 2 mg/kg. In all cases tumors regrew after an initial response, indicating acquired resistance to lorlatinib. Upon mice sacrifice, tumors were collected and characterized. All ex vivo cell lines were resistant to lorlatinib as assessed by proliferation assay and 2/10 showed moderate drug addiction. However, in several cases, ALK phosphorylation did not fully correlate with the observed high drug resistance, suggesting the involvement of ALK-independent mechanisms. ALK kinase domain sequencing revealed N1178H mutation in 5/10 mice, alone or in combination with other mutations and L1196M substitution in 2/10 cases. Since both mutants have been described as sensitive or moderately resistant to lorlatinib in murine BaF3 cells, further characterization of these cell lines was performed. ALK N1178H mutant was found to be predominantly localized in the cytoplasm, possibly mimicking overexpression, in human cells. Whole-exome and mRNA sequencing of cells carrying the L1196M substitution showed significant alterations in the PI3K/AKT and RAS/MAPK pathways, as well as perturbations of RAC1-related small GTPases. Functional validation by small molecule inhibitors confirmed the involvement of these pathways in resistance to lorlatinib.

In vitro selected lorlatinib-resistant cell lines, representing ALCL, NSCLC and neuroblastoma, showed both ALK mutations, including G1202R and C1156F/L1198F, and ALK-independent mechanisms of resistance. In one case, NSCLC cells acquired hyper-activation of EGFR; its blockage by erlotinib restored sensitivity to lorlatinib. Whole-exome sequencing and proteomic profiling of sensitive and resistant neuroblastoma cell lines revealed NF1 and EP300 mutations, as well as hyper-activation of ErbB4, in lorlatinib-resistant cells.

Altogether, our data provide an extensive characterization of resistance mechanisms that may arise in different ALK positive cancers upon lorlatinib exposure.

#903

Upregulation of DARPP32/PPP1R1B is a critical mediator of acquired lapatinib resistance in ErbB2-overexpressing breast cancer cells.

Zhikun Ma, Erin W. Howard, Amanda B. Parris, Xiaohe Yang. _North Carolina Central University, Kannapolis, NC_.

ErbB2/Her2 overexpression is detected in approximately 30% of human breast cancers and is associated with poor prognosis. Lapatinib, a small molecule dual inhibitor targeting ErbB2 and EGFR, is FDA-approved for the treatment of ErbB2-positive (ErbB2+) advanced or metastatic breast cancer. However, lapatinib resistance (LR) is emerging as a critical issue in clinical oncology. To understand the molecular mechanisms of LR, we first developed a lapatinib-resistant cell line from ErbB2-overexpressing BT474 breast cancer cells by prolonged exposure of parental BT474 cells to gradually increasing concentrations of lapatinib (up to 8 µM). We performed microarray analyses on parental and LR cells to identify novel factors/pathways that contribute to LR. Our microarray data indicated that the gene expression profile of the BT474/LR cells is significantly different from the parental BT474 cells. We found that DARPP32/PPP1R1B is one of the most significantly upregulated genes in the BT474/LR cells. To investigate the role of DARPP32 in LR development, we examined the effect of DARPP32 knockdown via lentiviral shRNA on BT474/LR cell proliferation and apoptosis. Results from MTT, clonogenic, cell cycle, and cleaved caspase-3 and PARP analyses indicated that DARPP32 knockdown significantly sensitized BT474/LR cells to lapatinib. We also found that DARPP32 knockdown renders MDA-MB-361 breast cancer cells, which express high endogenous levels of DARPP32, more sensitive to lapatinib. These data demonstrate that DARPP32 overexpression contributes to LR. To understand the mechanism of DARPP32 overexpression-mediated LR, we focused on the regulation of transcription factor cAMP response element-binding protein (CREB) in this process. As such, DARPP32 overexpression was generally associated with CREB upregulation. We also found that CREB-mediated transcription was increased in BT474/LR cells, and DARPP32 knockdown in BT474/LR and MDA-MB-361 cells significantly downregulated CREB protein levels and CREB-mediated transcription, as indicated by luciferase reporter assays. CREB overexpression rendered BT474 cells resistant to lapatinib and reversed DARPP32 knockdown-induced sensitization in BT474/LR cells. In contrast, CREB knockdown sensitized the cells to lapatinib. Further analysis with MG132 blockage suggests that DARPP32 may regulate CREB protein levels through the proteasomal degradation pathway. Taken together, we have identified DARPP32 overexpression as a critical mediator of acquired lapatinib resistance. The underlying mechanisms involve the deregulation of CREB protein and activity levels. These novel findings advance our understanding of the molecular mechanisms of LR and provide fundamental support for testing the DARPP32-CREB axis in clinical settings for the management of LR breast cancers, which is of significant translational value.

#904

The novel C-terminal truncated 90-kDa isoform of topoisomerase IIα, TOP2α/90, is a determinant of etoposide resistance in K562 leukemia cells via heterodimerization with the TOP2α/170 isoform.

Ragu Kanagasabai, Soumendra Karmahapatra, Yang Yu, Victor A. Hernandez, Corey A. Kientz, Evan E. Kania, Terry S. Elton, Jack C. Yalowich. _Ohio State Univ. College of Pharmacy, Columbus, OH_.

DNA topoisomerase IIα (170 kDa, TOP2α/170) is essential in proliferating cells since it resolves DNA topologic entanglements during chromosome condensation, replication, and segregation. We previously characterized a C-terminally truncated TOP2α/90 isoform, detectable in human leukemia K562 cells but more abundantly expressed in a clonal subline, K/VP.5, with acquired resistance to the anticancer agent etoposide (J Pharmacol Exp Ther 2017;360:152-63). TOP2α/90 (786 amino acids) is the translation product of a TOP2α mRNA that retains a processed intron 19. TOP2α/90 lacks the active-site tyrosine-805 (Tyr805) required to generate double-strand DNA breaks as well as the nuclear localization signals present in the TOP2α/170 isoform (1531 amino acids). The function of TOP2α/90 is unknown. Here, we found that TOP2α/90, like TOP2α/170, was detectable in the nucleus and cytoplasm of K562 and K/VP.5 cells. Importantly, co-immunoprecipitation of endogenous TOP2α/90 and TOP2α/170 demonstrated heterodimerization of these isoforms. Forced expression of TOP2α/90 in K562 cells suppressed, while siRNA-mediated knockdown of TOP2α/90 in K/VP.5 cells enhanced, etoposide-mediated DNA strand breaks compared with similarly treated K562 or K/VP.5 cells transfected with empty vector or control siRNAs, respectively. In addition, forced expression of TOP2α/90 in K562 cells inhibited etoposide cytotoxicity assessed by soft agar colony formation assays. qPCR and immunoassays demonstrated expression of TOP2α/90 mRNA and protein in normal human tissues/cells and in leukemia cells from patients. Together, results strongly suggest that TOP2α/90 expression decreases drug-induced TOP2α-DNA covalent complexes and is a determinant of chemoresistance through a dominant-negative effect related to heterodimerization with TOP2α/170. Alternative processing of TOP2α pre-mRNA, and subsequent synthesis of TOP2α/90, may be an important mechanism regulating the formation and/or stability of TOP2α/170-DNA covalent complexes in response to TOP2α-targeting agents.

#905

RICTOR modulation regulates cisplatin sensitivity in lung cancer.

Haiying Cheng,1 Ni Fan,2 Alain Borczu,3 Huijie Liu,2 Aditi Singh,1 Feng Wang,2 Balazs Halmos,1 Roman Perez-Soler1. 1 _Montefiore Medical Center/Albert Einstein College of Medicine, Bronx, NY;_ 2 _Albert Einstein College of Medicine, Bronx, NY;_ 3 _Weill Cornell Medical College, New York, NY_.

Background: Despite treatment advances, most lung cancer patient will still receive platinum-based chemotherapy and platinum resistance remains a serious clinical problem. RICTOR is a key component of the mTOR complex 2. Activation of RICTOR signaling has been suggested to play critical roles in regulating cancer cell migration, invasion and metastasis. A recent study indicates that RICTOR contributes to cisplatin resistance in human ovarian cancer cells. We hypothesize that activation of RICTOR signaling is associated with platinum resistance in lung cancer. Thus, we tested platinum sensitivities in lung cancer cells and xenograft mouse models with different RICTOR levels. Results: RICTOR expression was found in 79% (99/125) of primary lung tumors and 67% (24/36) lung cancer cells by immunohistochemistry. The expression of RICTOR was significantly higher in squamous cell lung cancer in comparison to lung adenocarcinoma (P<0.05). Overexpression of RICTOR in the Ba/F3 cells resulted in significant cisplatin resistance, whereas down-regulation of RICTOR in H23, HCC827 and H1703 lung cancer cells led to significantly improved cisplatin sensitivity. In addition, the combination of cisplatin and RICTOR knockdown significantly promoted more apoptosis than single treatment. To test the in vivo effects of RICTOR on platinum sensitivity, we performed HCC827 xenograft mouse experiments. Cisplatin alone and RICTOR knockdown alone significantly inhibited tumor growth. Moreover, RICTOR knockdown significantly increased the sensitivities to cisplatin in the xenografts and the combination led to more pronounced tumor reduction than either alone (P<0.05). Conclusion: Our study provides a novel resistance mechanism to platinum treatment in lung cancer and may serve as basis to target RICTOR to overcome platinum resistance.

#906

Development of novel preclinical models of secondary resistance downstream B cell receptor in marginal zone lymphoma.

Alberto J. Arribas,1 Eugenio Gaudio,1 Luciano Cascione,1 Chiara Tarantelli,1 Antonella Zucchetto,2 Francesca Rossi,2 Andrea Rinaldi,1 Ivo Kwee,1 Anastasios Stathis,3 Valter Gattei,2 Emanuele Zucca,3 Davide Rossi,3 Francesco Bertoni1. 1 _Università della Svizzera Italiana, Institute of Oncology Research, Bellinzona, Switzerland;_ 2 _Centro di Riferimento Oncologico di Aviano – CRO, Aviano, Italy;_ 3 _IOSI Oncology Institute of Southern Switzerland, Bellinzona, Switzerland_.

Background. Targeting downstream signaling to B-cell receptor (BCR) is one of the promising therapeutic approaches in lymphoma. PI3Kδ-inhibitor (i) idelalisib (IDEL) and BTK-i ibrutinib (IBRU) have been approved by FDA for different indications and are currently used to treat indolent lymphomas including marginal zone lymphoma (MZL). However, a subset of patients relapses due to acquired resistance. A better understanding of resistance mechanisms could help to design improved therapies. With this aim, we generated MZL cell lines resistant to either IDEL or IBRU.

Materials and Methods. VL51 and Karpas1718 cell lines were exposed to IC90 concentration of IDEL or IBRU for several months until they acquired specific drug resistance (RES). In parallel, lines were cultured upon similar conditions in the absence of drug (parental, PAR). Proliferation of acquired stable resistance was tested by MTT assay (72 hrs) in RES and PAR after 2-weeks of drug-free culture. Multi-drug resistance phenotype was ruled out. RES and PAR cells underwent transcriptome profiling by RNA-Seq, whole exome sequencing and were exposed to IDE or IBRU alone or in combination with a library of 348 compounds. Protein expression of p-AKT, p-BTK, p-PLCG2, p-mTOR and p-ERK was determined by FACS.

Results. Six lines with IC50s 5-10 fold times higher than PAR counterparts were generated: two VL51 and two Karpas1718 IDEL-RES and two VL51 IBRU-RES. IDEL-RES-VL51 showed mutations in AKT-mTOR regulators, DNA damage genes and Ca2+/Calmodulin partners while IDEL-RES-K1718 exhibited variants affecting kinases and chromatin remodeling genes. IBRU-RES-VL51 harbored mutations involving kinases and cell cycle genes. IDEL-RES-VL51 showed enrichment in RNA-Seq signatures related to BCR and RAS-RAF signaling while IDEL-RES-K1718 was enriched in cell cycle and HDAC targets suggesting different mechanisms of resistance. IBRU-RES-VL51 exhibited overexpression of PI3K signaling and proliferation signatures. Consistent with RNA-Seq, upon 1µM IDEL, IDEL-RES showed increased p-AKT and p-BTK, while upon 1µM IBRU, IBRU-RES showed enhanced p-PLCG2. Single treatment with DOTL1-i, JAK-STAT-i and TGFB-i decreased cell viability in IDEL-RES. IDEL-combo with tyrosine kinase-i (ABL/SRC, cKit), EZH2-i and RAF-i overcame resistance in IDEL-RES. IBRU-combo with cell cycle modulators and VEGFR-i exhibited synergism in IBRU-RES-VL51, which showed sensitivity to PI3K/mTOR-i, JAK/STAT-i and NFKB-i.

Conclusions. We created three novel models, derived from MZL, of secondary resistance: two to the PI3Kδ-i idelalisib, and one to BTK-i ibrutinib. We also identified potential active treatments for IDEL-RES or IBRU-RES that are worth of further investigations. These models, apparently driven by different biologic processes, will help in clarifying mechanisms of resistance to the drug and to evaluate alternative therapeutic approaches.

#907

Targeting glycolysis enzyme, PFKFB3, in endocrine therapy resistant breast cancers.

Surojeet Sengupta, Catherine M. Sevigny, Xiaoyi Liu, Lu Jin, Paula R. Pohlmann, Robert Clarke. _Georgetown Lombardi Comp. Cancer Ctr., DC_.

Reprogrammed glucose metabolism is recognized as one of the hallmarks of cancer. High energy demand in cancer cells leads to increased glycolysis to maintain anabolic processes that may be driven by altered enzyme levels. The second committed step in the glycolysis pathway is catalyzed by phosphofructokinase1 (PFK1), which converts fructose-6 phosphate (F6P) to fructose 1,6-bisphosphate (F1,6BP). Another enzyme, 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3), converts F6P to fructose 2,6-bisphosphate (F2,6BP) that then functions as an allosteric activator of PFK1 and drives glycolysis. PFKFB3 enzyme is upregulated in many cancers including breast cancers. PFKFB3 is regulated by estrogen in ER-positive breast cancer cells but its role in endocrine-therapy resistant breast cancer cells is largely unknown. We investigated the expression of PFKFB3 mRNA and protein in estrogen-responsive MCF7 cells and compared it with LCC9 cells that are estrogen-independent and resistant to 4-hydroxytamoxifen (4OHT) and fulvestrant (Fulv). Twofold higher PFKFB3 mRNA and protein levels were expressed in LCC9 cells compared to MCF7 cells. In addition, the estrogen-mediated stimulation of PFKFB3 that was evident in MCF7 cells was not observed in LCC9 cells.

We further studied the effect of pharmacological inhibition of PFKFB3, on the growth of therapy-resistant breast cancer cells using a compound PFK158, a potent inhibitor of the PFKFB3 enzyme. Treatment with PFK158 inhibited the growth of LCC9 cells as well as another cell line, MCF7:5C that is also resistant to 4OHT and partially resistant to Fulv. PFK158 alone was effective in suppressing the growth of both the cell lines at a concentration of 5 µM or higher. Notably, combining PFK158 with either 4OHT or Fulv significantly potentiated the effect of PFK158 treatment in blocking the growth of the cells. In MCF7:5C cells the combination treatment of 2.5 µM PFK158 and Fulv (500nM) or 4OHT(500nM) inhibited cell growth by ~70% as compared to ~30% inhibition in the presence of PFK 158 alone. In LCC9 cells >70% of growth was inhibited in the combination treatment as compared to no inhibition when PFK158, 2.5 µM or 4OHT or Fulv alone was used. In publicly available datasets of ER+, node-negative breast cancer patients, high expression of PFKFB3 was associated with adverse recurrence-free survival (hazard ratio = 4.12 and p= 5.5x10-5).

Our study shows an increased basal expression of PFKFB3 mRNA and protein in estrogen-independent, endocrine-therapy resistant breast cancer cells as compared to estrogen-responsive breast cancer cells. Targeting PFKFB3 in combination with anti-estrogens should be explored for potential therapeutic intervention in endocrine therapy-resistant breast cancers.

#908

Overexpression of nicotinamide N-methyltransferase confers gemcitabine resistance in bladder cancer.

Kelly A. Martin,1 Aimy Sebastian,1 Nicholas R. Hum,1 Deepa K. Murugesh,1 Chong-xian Pan,2 Ai-Hong Ma,3 Ralph W. de Vere White,2 Gabriela G. Loots1. 1 _Lawrence Livermore National Laboratory, Livermore, CA;_ 2 _UC Davis Comprehensive Cancer Center, Sacramento, CA;_ 3 _UC Davis Comprehensive Cancer Center, Livermore, CA_.

Bladder cancer is among the top ten most common cancers diagnosed, with about ~380,000 new cases and ~150,000 deaths per year reported worldwide. Platinum-based chemotherapy in combination with gemcitabine, a nucleoside analog, is a widely used treatment option for advanced bladder cancer. It has been shown that only ~50% of the patients with advanced bladder cancer respond to platinum-based therapy. Cancer cells often become non-responsive to therapy that once proved efficacious and are now drug resistant. Drug resistance represents a significant, ongoing challenge in eradicating cancer.

We have employed a patient-derived bladder cancer xenograft (PDX) platform to further investigate the molecular mechanisms that contribute to gemcitabine-induced drug resistance in advanced bladder cancer. Transcriptome profiling of passage 4 bladder cancer xenograft tumors from 2 gemcitabine sensitive PDX lines (BL0440 & BL0293) was performed using RNA sequencing (RNAseq) analysis, before and after a 21-day cisplatin/gemcitabine drug treatment regimen. Key regulatory pathways and genes contributing to drug resistant bladder cancer have been identified and validated by overexpression in a 5637 bladder cancer cell line.

RNA sequencing data has indicated significant differences between the transcriptional profiles of drug-sensitive and drug-resistant tumors. PDXs retained morphology and shared 92-97% of genetic alterations of parental cancer cells. We identified 106 genes >1.5 fold up-regulated and 45 genes >1.5 fold down-regulated in the drug resistant tumors compared to their drug sensitive counterparts. Of the genes that were significantly upregulated, two methyltransferase enzymes were further validated: Nicotinamide N-methyltransferase (NNMT) and methionine methyltransferase 1A (MAT1A). These genes were found to be contributors to gemcitabine-mediated drug resistance. 5637 cells overexpressing NNMT yielded a 200-fold increase in gemcitabine resistance relative to parental strain. In conclusion, chemoresistance to gemcitabine is associated with differential expression of genes and alteration of downstream signaling pathways. Upregulation of NNMT & MAT1A can potentially be used as novel biomarkers for subpopulations of drug resistant bladder cancer for which improved therapeutics can be developed. Future direction will likely include studies to elucidate exact molecular mechanisms by which cancer cells utilize methyltransferases to no longer respond to gemcitabine therapy.

This work was conducted under the auspices of the USDOE by LLNL (DE-AC52-07NA27344).

#909

Development and characterization of androgen receptor knockout prostate cancer cell lines by CRISPR-Cas9.

Yezi Zhu, Pei Zhao, Changxue Lu, Shihua Chen, Jun Luo. _Johns Hopkins University, Baltimore, MD_.

Background: Androgen receptor (AR) is the key drug target in prostate cancer. AR splice variant-7 (AR-V7) is the most relevant AR variants implicated in castration-resistant prostate cancer (CRPC). Detection of AR-V7 in both liquid biopsies and tissue biopsies predicts poorer response to novel AR-targeting drugs. However, because AR-V7 always coexists with the full-length AR (AR-FL), the functional importance of AR-V7 needs to be dissected in the context of AR-FL. Lack of relevant cell line models presented a hurdle for detailed dissection of the role for AR-V7. It is essential to develop cell lines that express endogenous AR-V7 at a level comparable to clinical specimens, with or without AR-FL, to facilitate functional studies. The goal of this study is to develop and characterize LNCaP95 lines following specific and complete knock-out of AR molecules by CRISPR-Cas9 system.

Methods: Different AR isoforms were knocked-out in parental LNCaP95 cells by CRISPR-Cas9. AR-FL was knocked-out through sgRNAs targeting AR exon5 or exon6. AR isoforms with functional N-terminal domain (total AR) were knocked-out through sgRNA targeting AR exon1. Complete knockout of AR-FL and total AR was achieved in LNCaP95 cells. The on-target editing of AR genomic regions were verified by Sanger Sequencing.

Results: Complete knockout of AR-FL protein and total AR protein was confirmed in two clones carrying homologous indels, ARfl-KO#1/2 and AR-KO#1/2. Antibodies against different AR domains were used to differentiate expression of different AR isoforms, by Western blot and immunohistochemistry (IHC). Cell proliferation was not affected by androgen in AR-FL or total AR knockout cells, as shown in cell growth and clonogenic assays. The expression of AR-target genes such as prostate-specific antigen (PSA) were analyzed by realtime-PCR. PSA was expressed at similar level compared to parental cells in AR-FL knockout cells, which was totally lost in total AR knockout cells. Interestingly, total AR knockout cells are more resistant to ionizing radiation and PARP inhibitor olaparib compared to parental cell line.

Conclusion: We have successfully developed the first prostate cancer cell line expressing endogenous AR-V7 at a level comparable to clinical specimens without AR-FL, allowing dissection of the functions mediated by AR-FL and AR-V7. Initial functional characterization suggested AR-V7 maintained the baseline level of AR signaling activity, which was not dependent on androgen. The chromatin-binding pattern of AR-V7 in the absence of AR-FL is under investigation. We have also developed a novel total AR knockout cell model from the AR-positive parental cell line. This model suggested prostate cancer cells were able to survive in an AR-null manner, although loss of AR significantly inhibited cell proliferation. Preliminary studies have found loss of total AR confers resistance to ionizing radiation and PARP inhibition.

#910

Mechanisms of Ewing sarcoma resistance to LSD1 inhibition.

Kathleen I. Pishas,1 Sunil Sharma,2 Stephen L. Lessnick1. 1 _Nationwide Children's Hospital, Columbus, OH;_ 2 _Huntsman Cancer Institute, Salt Lake City, UT_.

Given the modest efficacy of current sarcoma therapeutic modalities, there is a major unmet clinical need for the translation of novel agents to tackle the burden of Ewing sarcoma (pediatric and young adolescent bone cancer). We have recently shown that therapeutic targeting of LSD1, a highly expressed lysine specific demethylase, with the reversible small molecule inhibitor SP-2509, comprehensively disrupts the global transcriptional function of EWS-FLI, and significantly impairs the growth of Ewing sarcoma cell lines both in vitro and in vivo. Prior to clinical trial evaluation, the goal of this study was to elucidate mechanisms of Ewing sarcoma SP-2509 drug resistance.

In order to investigate SP-2509 resistance, A673 Ewing sarcoma cells were cultured in escalating concentrations of SP-2509 for 8 months. Cell Titer Glo analysis revealed that these SP-2509 drug resistant (DR) cells were 54.9 fold more resistant to SP-2509 compared to parental A673 cells (72hr IC50 0.138μM versus 7.586μM). SP-2509 DR cells also exhibited a significant decrease in their ability to form anchorage-independent colonies in soft agar. Whole exome (250X coverage) and RNA seq analysis of parental and SP-2509 DR A673 cells revealed 25 drug induced mutations, with 88% of the whole cell population harboring a Glu53* stop-gain mutation in MRPL45 (mitochondrial ribosomal protein L45). In addition, SP-2509 DR cells displayed a distinct transcriptomic profile, with 3250/2376 genes significantly up/down regulated (≥1.5 fold) in SP-2509 DR cells compared to parental controls. KEGG Pathway analysis revealed that the genes were highly enriched for hepatic stellate cell activation/interferon signaling and cholesterol biosynthesis respectively. Lastly, SP-2509 DR cells were shown to be equally sensitive to etoposide, resistant to vincristine/doxorubicin and surprisingly more sensitive to the HDAC inhibitors vorinostat and entinostat compared to parental A673 cells.

Together, our preliminary findings provide key insights into the mechanisms of SP-2509 drug resistance, information of which can be used to find combinatorial agents that can potentially be used to circumvent resistant clones to achieve maximal therapeutic effect.

#911

Targeting the mutant KRAS/BRAF-induced enhancer as an approach to overcome resistance to 5-fluorouracil-based chemotherapy in colorectal cancer.

Rentian Wu, Qian Nie, Phillip A. Kubica, Zhiquan Wang, Shikshya Shrestha, Colbren S. Trogstad-Isaacson, Calvin R. Jerde, Erin E. Tapper, Kelly J. Bouchonville, Steven M. Offer, Robert B. Diasio. _Mayo Clinic, Rochester, MN_.

Colorectal cancer (CRC) is the third most common cancer and is expected to cause approximately 50,000 deaths in the US in 2017. The antimetabolite 5-fluorouracil (5-FU) is the cornerstone of first-line adjuvant chemotherapy regimens for CRC. Resistance to 5-FU-based chemotherapies develops over time and is the greatest obstacle to effective management in this disease. However, the resistance mechanisms of 5-FU are largely unknown. Approximately 50% of the CRC cases have activating mutations in KRAS or BRAF. Based on recent clinical data, it is now appreciated that KRAS/BRAF mutations are associated with worse survival in 5-FU-based chemotherapies for CRC. Therefore, there is an urgent unmet need for improved therapeutic options to treat mutant KRAS/BRAF tumors. We analyzed the differential activation status of enhancers in KRAS/BRAF mutant and wildtype CRC cell lines. Enhancers that associated with genes regulating pyrimidine metabolism were significantly enriched in KRAS/BRAF mutant CRC cells. Among them, the differential activation of an enhancer located ~400kb upstream of the DPYD promoter was most significant. By using CRISPR-dCas9 system, specifically targeting of p300 to the enhancer significantly increased DPYD expression, and Ezh2 significantly decreased expression. Knockout (KO) of this enhancer resulted in reductions in DPD expression and increased resistance to 5-FU. In mouse xenograft studies, enhancer-KO cells responded to doses of 5-FU that were otherwise ineffective against control cells. Treating KRAS mutant CRC cells with CBP30, H3K27 acetyl-transferase inhibitor, significantly reduced DPYD expression. Synergistic interactions was identified between CBP30 and 5-FU. Finally, mouse xenograft studies strongly suggest that inhibition of H3K27ac may significantly improve 5-FU effectiveness against KRAS mutant tumors. In summary, the present studies advance our understanding of how mutant KRAS/BRAF epigenetically regulates DPYD expression and results in 5-FU resistance in CRC. We showed that clinical treatment of CRC might be significantly improved by the development of future pharmacologic approaches targeting enhancers that are activated by mutant KRAS/BRAF.

#912

uPA as a topoisomerase-1 protective agent in lung fibroblast cells.

Liang Ge,1 Manu Gnanamony,1 Victoria Stepanova,2 Dzung H. Dinh,1 Christopher S. Gondi1. 1 _Univ. of Illinois College of Med. at Peoria, Peoria, IL;_ 2 _University of Pennsylvania Perelman School of Medicine, Philadelphia, PA_.

Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-related death in the United States, with approximately 53,000 people in the U.S. diagnosed with the disease every year. The Gondi lab has previously shown that poor survival rates in pancreatic cancer are correlated with increased expression of urokinase plasminogen activator (uPA), a serine protease. Our previous data has shown for the first time that uPA binds to topoisomerase-1 (TOPO-1), in addition to the interaction of uPA with various transcription factors. Our current research is aimed at elucidating the mechanism of the interaction between uPA and TOPO-1, with the goal of demonstrating a protective role for uPA in the regulation of TOPO-1 function in the presence of TOPO-1 inhibitors. Methods: Two knock-out lung fibroblast cell lines were used. One cell line was a uPA knock-out line, while the other was a uPA knock-out line with a uPA expression vector transfected into the cells. Transfection was performed using Chariot transfection vector. The cell lines were treated with topoisomerase inhibitors TX1111 and SN38. DNA replication activity was measured using flow cytometry as a surrogate for TOPO-1 activity. Controls included untreated cells, cells treated with empty Chariot, and cells treated with Chariot containing TXC peptide. Results and Discussion: In the presence of topoisomerase inhibitors TX1111 and SN38, uPA knock-out lines demonstrated incomplete cell division relative to controls. In uPA-expressing lines, cell division activity was retained in the presence of topoisomerase inhibitors. Our data demonstrates for the first time that uPA serves a protective role in preventing the inhibition of TOPO-1 by topoisomerase inhibitors. This indicates uPA as a potential new drug target in the treatment of chemoresistant pancreatic cancer. Further studies to be performed include visualizing the degree of DNA-replication using DNA combing, and subsequently expanding this study to pancreatic cancer cells.

#913

**MENA** INV **expression identifies a de novo** **chemotherapy-induced prometastatic subpopulation in breast cancer.**

George S. Karagiannis, Luis Rivera Sanchez, Yarong Wang, Ved P. Sharma, Joseph Burt, Sara Brizio, David Entenberg, Maja H. Oktay, John S. Condeelis. _Albert Einstein College of Medicine, Bronx, NY_.

Neoadjuvant chemotherapy (NAC) is used for treatment of localized breast cancer to decrease tumor size and improve surgical outcome. However, Karagiannis et al. (2017) has uncovered a previously unrecognized side-effect, which involves the induction of pro-metastatic changes in the primary breast cancer microenvironment, in response to NAC treatment. In particular, NAC promotes the assembly of structures that serve as doorways for intravasation of tumor cells called tumor microenvironment of metastasis (TMEM), and increases the proportion of the highly invasive and migratory MenaINV-hi/Mena11alo (MenaCalc+) tumor cells, which utilize the TMEM sites for hematogenous dissemination (Karagiannis et al., 2017). MenaINV expression in particular is associated with decreased sensitivity of cancer cells to receptor tyrosine-kinase (RTK) and tyrosine-kinase (TK) inhibitors and with dramatically increased TMEM-dependent intravasation. The mechanism behind these pro-metastatic changes was not known until recently. Here, we report that NAC induces an influx of macrophages in the tumors, resulting in increased contact between macrophages and tumor cells leading to MenaINV expression in the cancer cells, and MenaINV\- and TMEM-dependent tumor cell intravasation. Multichannel IF imaging and distance analysis algorithms demonstrate that MENAINV-hi cells have a higher probability of being in direct cell contact with infiltrating macrophages in chemotherapy-treated tumors. Specific depletion of the macrophage lineage in transgenic MMTV-PyMT mice developing spontaneous breast carcinoma and patient-derived xenografts results in a significant suppression of the prometastatic MenaINV-hi cancer cell subpopulation. Moreover, chemotherapy-induced MenaINV expression in tumor cells is not seen in such macrophage-depleted mice, indicating that macrophage influx is both necessary and sufficient for the generation of the de novo prometastatic tumor cell subpopulation. These data provide an explanation as to why mice and patients with breast cancer do not always respond with a decrease in circulating tumor cells (CTCs), or even respond with a slight increase, following cytotoxic NAC treatment. Our work has uncovered a previously unrecognized mechanism behind pro-metastatic changes in response to cytotoxic chemotherapy and the markers that predict these changes (TMEM, MenaCalc and MenaINV).

Reference: Karagiannis GS, et al. Neoadjuvant chemotherapy induces breast cancer metastasis through aTMEM-mediated mechanism. Sci Transl Med, 2017; 9 aeen0026.

#914

COX-2 expression is associated with chemoresistance through cancer stemness property in head and neck squamous cell carcinoma.

Shin Saito,1 Hiroyuki Ozawa,1 Yorihisa Imanishi,2 Mariko Sekimizu,1 Yoshihiro Watanabe,3 Fumihiro Ito,4 Yuichi Ikari,1 Nana Nakahara,1 Kaori Kameyama,1 Kaoru Ogawa1. 1 _Keio University, School of Medicine, Tokyo, Japan;_ 2 _Kawasaki Municipal Kawasaki Hospital, Tokyo, Japan;_ 3 _Saiseikai Central Hospital, Tokyo, Japan;_ 4 _National Hospital Organization Tokyo Medical Center, Tokyo, Japan_.

Introduction: COX-2 is one of the two isoforms of cyclooxygenase, an enzyme that catalyzes the conversion of arachidonic acid to prostaglandins. COX-2 is considered to be associated with progression in various cancers, and its expression has been reported to have an impact on poor prognosis in head and neck squamous cell carcinomas (HNSCCs). Furthermore, COX-2 expression has been shown to be associated with resistance to anticancer drugs. However, the precise mechanism of COX-2 for chemoresistance in HNSCC is not fully elucidated. The aim of the present study was to investigate the impact of COX-2 on cancer stem cell property, and to reveal its effect on chemoresistance by in vitro and clinicopathological assays. Methods: We examined the chemoresistance to docetaxel with or without COX-2 inhibitor (celecoxib) in HNSCC cell lines (FaDu, Detroit512) by MTS assays. To evaluate the association of COX-2 expression with stemness property, we assessed alteration of expression levels of the genes related to cancer stem cell (CSC) property after exposure to celecoxib by quantitative real-time PCR. We also performed a sphere formation assay using ultra low attachment dishes and microscopic imaging. Moreover, we analyzed immunohistochemical expressions of COX-2 and clinicopathological factors by using matched samples of pre-treatment biopsy and surgical specimens from hypopharyngeal carcinoma patients who had undergone trans-oral tumor resection with preoperative induction chemotherapy including docetaxel. Results: Cell survival rate under exposure to docetaxel was decreased by addition of celecoxib. COX-2 inhibitor downregulated the expression of Oct3/4 and Nanog. Sphere formation was inhibited by co-culture with COX-2 inhibitor, especially in FaDu cells. Immunohistochemical study in biopsy specimens revealed a negative correlation between COX-2 expression in biopsy specimens and pathological effect of induction chemotherapy in surgical specimens. Conclusion: The present study suggests that COX-2 expression is associated with cancer stemness property, and inhibition of COX-2 has a possibility to enhance chemosensitivity in HNSCCs. Furthermore, initial COX-2 expression in biopsy specimens may predict the effect of chemotherapy in HNSCCs. These results indicate that COX-2 can be an attractive target for the treatment of HNSCC.

#915

AKT inhibition sensitizes TNBC cells with acquired drug resistance to UM-164.

Nathan M. Merrill, Eric J. Lachacz, Rabia A. Gilani, LiWei Bao, Zhi Fen Wu, Sofia D. Merajver, Matthew B. Soellner. _University of Michigan, Ypsilanti, MI_.

One of the major clinical challenges in breast cancer treatment is the rapid development of resistance to targeted therapies. In light of this, there is significant interest in identifying markers of drug resistance. In particular, markers of drug resistance are sought that can be targeted to overcome acquired drug resistance. To this end, we have used reverse phase protein array (RPPA) analysis to identify differences in protein expression levels between cell lines that are sensitive or have acquired resistance to UM-164, a mechanistically novel, dual c-Src/p38 kinase inhibitor with potent anti-TNBC activity. UM-164 resistant cells acquire increased resistance also to many diverse chemotherapeutics and targeted agents relative to their drug-sensitive counterparts. We analyzed RPPA data for both UM0164-sensitive and UM-164-resistant cells and identified an increase in phospho-AKT, phospho-ERK, and phospho-FAK levels in the resistant cells. Using inhibitors of each upregulated pathway, we found that combination of UM-164 with MK2206, an inhibitor of AKT, resulted in an increased sensitization of cells to treatment with UM-164. Furthermore, formal Chou-Talalay synergy analyses demonstrate that AKT inhibitors + UM-164 are effectively synergistic in reducing the proliferation of drug-resistant cells. We further evaluated this combination treatment in mice, combining UM-164 and MK2206 in a xenograft of UM-164-resistant cells, showing significant anti-tumor efficacy. Finally, we show that the combination treatment resulted in increased cell death in ex vivo cultures of patient derived TNBC materials. Together, we have demonstrated that hyperactivation of AKT leads to UM-164 resistance and AKT inhibitors can be used to re-sensitize TNBC cells to UM-164. These results demonstrate that RPPA analyses are a valuable tool for the systematic and unbiased identification of truly actionable markers of therapeutic resistance.

#916

A screen to target EGFR (C797S) in osimertinib-resistant lung cancer identifies brigatinib-based combinations that synergistically inhibit cell growth.

Matthew J. Martin, Nicolas Floc'h, Chrysiis Michaloglou, M Raymond Finlay, Richard A. Ward, Paul D. Smith, Darren A. Cross. _AstraZeneca, Cambridge, United Kingdom_.

Up to 20% of non small-cell lung cancers are driven by activating mutations in EGFR (EGFRm). While EGFR tyrosine kinase inhibitors (TKIs) have proven highly effective in the clinic (e.g. gefitinib in first line EGFRm and osimertinib in T790M post-TKI resistance), acquired resistance mechanisms present an obstacle to curing EGFR-driven disease. To-date, approximately 20% of osimertinib-resistant cases in the 2nd line setting are EGFRm/T790M/C797S, and there is currently no approved therapy for these "triple-mutant" (TM) patients. We have obsrerved that the ALK inhibitor brigatinib shows moderate, equipotent activity against both EGFR-TM and the single activating mutant. We used this information to screen a panel of compounds to identify brigatinib-based combinations that could effectively target EGFR-TM-expressing cells. Among the most potent combination partners were selumetinib and other inhibitors of the MAP kinase pathway. These agents were able to synergistically impair growth of TM cells when combined with brigatinib in vitro, and accordingly show prolonged suppression of downstream pathway biomarkers in combination- vs. brigatinib monotherapy-treated cells. Further, we have shown effective combinations can dramatically enhance apoptotic cell death compared to brigatinib treatment alone. Critically, neither monotherapy treatment with effective combination partners, nor combinations based on osimertinib showed growth inhibition in the EGFR triple-mutant setting. Taken together, these data suggest novel therapeutic strategies for patients in this critical area of unmet need that warrant further preclinical testing.

#917

Defects in multiple DNA damage repair pathways render endocrine treatment resistance in ER+ breast cancer patients.

Meenakshi Anurag,1 Nindo Punturi,1 Jeremy Hoog,2 Matthew N. Bainbridge,3 Matthew J. Ellis,1 Svasti Haricharan1. 1 _Baylor College of Medicine, Houston, TX;_ 2 _Washington University School of Medicine, Houston, TX;_ 3 _Rady Children's Institute for Genomic Medicine, San Diego, CA_.

Endocrine therapy resistance results in the death of majority of women diagnosed with breast cancer in the USA every year. Although a few markers can predict endocrine treatment failure, comprehensive understanding of the underlying cause and early detection of resistant population remains biggest challenge in the field. Recently, deficient expression or mutation in MutL mismatch repair components was found to cause 20% of intrinsic resistance in primary ER+ HER2- breast cancers. The role of other DNA damage repair (DDR) defects in inducing endocrine resistance therefore requires exploration. To address this question, a systematic study of associations between DDR dysregulation at DNA and RNA level and response to endocrine therapy was conducted using aggregated data from patient tumors from several neoadjuvant aromatase inhibitor (NeoAI) clinical trials. METABRIC, TCGA and Loi et al. datasets were used as validation datasets. Statistically significant correlations between endocrine therapy resistance and mutations in two single strand break repair (SSBR) pathways, i.e. nucleotide excision repair (NER; p=0.04) and base excision repair (BER; p=0.03), and one double strand break repair (DSBR) pathway, i.e. non-homologous end joining (NHEJ; p=0.004) were identified. A second set of analyses identified loss of expression of three genes: CETN2 and ERCC1 from the NER pathway and NEIL2 from BER as specifically associating with poor outcome despite endocrine treatment, emphasizing potential connections between NER and BER defects and endocrine treatment response. The functional role of CETN2, NEIL2 and ERCC1 loss in inducing intrinsic endocrine therapy resistance was experimentally validated in two breast cancer cell lines, and in ER+ PDX models. Finally, a DDR signature score was devised from baseline expression levels of candidate genes and was able to predict endocrine treatment failure in >30% of patients.In conclusion, this study describes a screening strategy for identifying novel predictive markers of endocrine therapy resistance in primary patient tumors. Three DDR pathways, NER, BER and NHEJ, were identified in clinical data as associating with endocrine resistance and validated using experimental model systems. Results reveal an understudied but impactful role for DDR pathways that may explain up to half of all endocrine therapy failure and open new avenues for early screening, predictive diagnostics and personalized medicine.

#918

**A secondary** RET **mutation allosterically conferring resistance to vandetanib.**

Takashi Nakaoku,1 Takashi Kohno,1 Mitsugu Araki,2 Seiji Nino,3 Rakhee Chauhan,4 Phillip P. Knowles,4 Katsuya Tsuchihara,5 Shingo Matsumoto,3 Yoko Shimada,1 Sachiyo Mimaki,5 Genichiro Ishii,5 Hitoshi Ichikawa,6 Yasushi Okuno,2 Kiyotaka Yoh,3 Neil Q. McDonald,4 Koichi Goto3. 1 _National cancer center research Institute, Tokyo, Japan;_ 2 _Graduate School of Medicine, Kyoto University, Kyoto, Japan;_ 3 _National Cancer Center Hospital East, Kashiwa, Japan;_ 4 _The Francis Crick Institute, London, United Kingdom;_ 5 _Exploratory Oncology Research and Clinical Trial Center, National Cancer Center, Kashiwa, Japan;_ 6 _Exploratory Oncology Research and Clinical Trial Center, National Cancer Center, Tokyo, Japan_.

Targeted therapy against oncogenic ALK and ROS1 fusion using type I tyrosine kinase inhibitors (TKIs), which bind to the ATP-binding cleft of kinases, is highly effective in lung adenocarcinoma (LADC); however, such cancers inevitably acquire resistance, which severely limits the efficacy of cancer treatments. The identification of these resistance mutations led to the development of novel TKIs to overcome acquired resistance. Here we report resistance to vandetanib, a type I RET kinase inhibitor, developed in a patient with metastatic LADC harboring a CCDC6-RET fusion that initially exhibited a response to treatment. The resistant tumor acquired a secondary mutation resulting in substitution of an amino acid located outside the nucleotide binding cleft. The mutation conferred resistance to vandetanib by increasing the ATP affinity and auto-phosphorylation activity of RET kinase. A crystal structure of the mutant revealed a small hydrophobic core around the mutated amino acid likely to enhance basal kinase activity by stabilizing an active conformer. In addition, a thermal shift assay suggested that the mutation was less able to bind vandetanib. Molecular dynamics simulation supported the mutant underwent conformational changes decreasing the binding affinity of vandetanib. Our findings indicate that the missense mutation is able to increase kinase activity and confers drug resistance through allosteric effects.

#919

Endogenous STING inhibition induces breast cancer cell death.

Laura Cheradame,1 Hery Ratsima,1 Olivier Déas,1 Jean-Gabriel Judde,1 Vincent Goffin,2 stefano cairo1. 1 _XenTech, Evry, France;_ 2 _Inserm U1151 / Institut Necker Enfants Malades (INEM), Paris, France_.

Several studies have recently indicated the (re)activation of the immune system against tumor cells as an effective strategy to inhibit tumor growth. A current cancer immunotherapy strategy proposes the use of STING agonists to boost the patient's immune system through the cytokine-mediated enhancement of tumor infiltration and killing by immune cells. We recently showed that genotoxic treatment in vitro of the ER-positive breast cancer cell line MCF7 triggered the STING pathway. Of interest, genetic inhibition of this pathway increased genotoxic treatment efficacy by promoting cell death and delaying cell colony regrowth, indicating that STING pathway intrinsically promotes MCF7 cell resistance to treatment. While STING is commonly described as an endoplasmic reticulum resident protein that localizes to Golgi and/or perinuclear vesicles upon activation, we showed that STING is constitutively present in the nucleus of MCF7 cells and, upon activation, forms nuclear foci that co-localize with gH2AX at DNA breaks. The aim of this study was to investigate whether these observations could be extended to cell lines derived from human breast cancer xenografts that are more representative of the disease.

To this aim, STING pathway was monitored in HBCx-3 (derived from the homonymous parental ER-positive breast cancer PDX) and HBCx-39 (derived from the homonymous triple negative breast cancer PDX) cells using cell fractionation, western blot, immunofluorescence and viability assays. Similarly to what we observed in MCF7 cells, STING was invariably present in the cytoplasm as well as the nucleus of HBCx cells. In these models, STING formed clusters together with gH2AX at DNA breaks even in the absence of genotoxic treatment. Finally, we showed that STING silencing decreased cell viability of HBCx-3 and HBCx-39 cells irrespective of genotoxic treatment. Interestingly, another ER-positive PDX-derived cell line (HBCx-19) and two well-known triple negative breast cancer cell lines (BT20 and HCC1937) showed similar sensitivity to STING downregulation in a siRNA screening assay.

These studies show that STING clustering at DNA breaks in the nucleus is a general feature of breast cancer cells, and that the STING pathway is a cell-intrinsic mechanism of cell survival of many breast cancer cell lines including those derived from patient-derived xenografts. Our preliminary results indicate that STING-gH2AX foci can also be observed in the nucleus of TC122A cells derived from a human colon tumor xenograft, suggesting this mechanism may be common to other types of cancer.

To gain insight into the potential antagonistic roles of STING pathway modulation in tumors in vivo, we have transduced MCF7 and PDX-derived cell lines with an inducible shRNA directed against STING, and are evaluating the impact of STING inhibition or activation on tumor growth in immune cells-humanized PDX models.

#920

COP1 E3 ligase regulates response to oncogenic MAPK pathway inhibition.

Manasi K. Mayekar, Luping Lin, Trever G. Bivona. _University of California San Francisco, San Francisco, CA_.

Oncogenically activated RAS-MAPK pathway is the driver of several cancers including the majority of non-small cell lung adenocarcinomas (NSCLC). RAS-MAPK pathway is activated in NSCLC tumors via diverse genetic alterations in upstream receptor tyrosine kinases such as EGFR and ALK as well as in RAS, BRAF, MEK and RAS GTPase activating protein (GAP) and tumor suppressor, NF1. Molecular targeting of players of RAS-MAPK pathway can elicit an initial tumor response in many patients. However, most patients show an incomplete response and some fail to respond despite the presence of RAS-MAPK pathway activating genetic lesion in the tumor. Hence, it is important to understand the molecular basis of response and resistance to targeted therapies inhibiting the RAS-MAPK pathway in lung adenocarcinomas in order to improve patient survival. We conducted a genetic screen to identify regulators of response to MAPK pathway inhibition in lung adenocarcinomas. Our genetic screen uncovered the E3 ubiquitin ligase COP1/RFWD2 as a novel modulator of response to inhibition of RAS-MAPK pathway. We found that depletion of COP1 and members of its complex, as well as proteasomal subunits, confers resistance to RAS-MAPK pathway inhibition in human lung adenocarcinoma cells with oncogenically activated RAS-MAPK pathway. Interestingly, oncogenic targets of COP1 include MAPK pathway effectors, presence of which has been shown to support the survival of cancer cells with oncogenically activated MAPK upon inhibition of the RAS-MAPK pathway. Hence, we tested if depletion of COP1 alters the levels of those oncogenic substrates. Excitingly, we observed a substantial impact of COP1 depletion on the levels of certain effectors in the presence of RAS-MAPK inhibitors in genetically diverse NSCLC cells and also in oncogenic-BRAF driven melanoma cells. Our studies suggests that depletion of COP1 confers resistance to MAPK pathway inhibition in RAS-MAPK pathway driven cancers through accumulation of specific MAPK pathway effectors. This work has improved our understanding of the molecular basis of tumor cell resilience during initial treatment as well as of primary treatment resistance. Additionally, we are examining if levels of COP1 could be a biomarker for predicting response to RAS-MAPK pathway inhibitor therapy. We will also determine if the resistance-conferring COP1 substrate could be cotargeted along with RAS-MAPK pathway to improve the patient response in RAS-MAPK pathway driven lung adenocarcinomas and other cancers with low levels of COP1.

#921

**High-throughput functional evaluation of variants of unknown significance in** ERBB2 **.**

Masaaki Nagano, Shinji Kohsaka, Toshihide Ueno, Shinya Kojima, Masachika Ikegami, Ikuko Takeda, Hiroyuki Mano. _The University of Tokyo, Tokyo, Japan_.

Erb-b2 receptor tyrosine kinase 2 (ERBB2) gene amplification is present in a wide variety of human cancers. ERBB2-targeted therapies have been developed and improved the outcome in patients with ERBB2-positive cancers. Recently, the advent of next-generation sequencing technology has enabled to identify numerous ERBB2 somatic mutations across a variety of cancers, but many of the mutations are still variants of unknown significance (VUS) that await further investigation of clinical relevance. In addition, the sensitivities of those ERBB2 mutations to ERBB2-targeted drugs have not been evaluated systematically. Here we assessed comprehensively the transforming activities as well as drug sensitivities of ERBB2 mutations in a high-throughput manner using the mixed-all-nominated-in-one (MANO) method. We evaluated 55 non-synonymous ERBB2 mutations which are reported recurrently in COSMIC database, and discovered several novel activating mutations which probably drive tumorigenesis. Furthermore, ERBB2 mutations showed varying drug sensitivities to ERBB2-targeted inhibitors. Thus, the MANO method may be a novel approach for assessing VUS, and our findings in this study will be beneficial to deliver personalized medicine to cancer patients harboring ERBB2 mutations. 

## CLINICAL RESEARCH:

### Pediatric Cancer: Poster Discussion

#1629

Targeting resistance mechanisms to CDK4/6 inhibitors in Ewing sarcoma with an IGF1R inhibitor drug combination strategy.

Lillian M. Guenther,1 Neekesh V. Dharia,1 Linda Ross,1 Amy S. Conway,1 Amanda L. Robichaud,1 Alanna J. Church,2 Rajarshi Guha,3 Mindy I. Davis,3 Gabriela Alexe,1 Jaume Mora,4 Federica Piccioni,5 Kimberly Stegmaier1. 1 _Dana-Farber Cancer Institute, Boston, MA;_ 2 _Boston Children's Hospital, Boston, MA;_ 3 _National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, MD;_ 4 _Hospital Sant Joan de Déu, Barcelona, Spain;_ 5 _Broad Institute, Cambridge, MA_.

Novel targeted drug combinations have transformed the care of subsets of patients with cancer. In pediatric malignancies, however, where tumor genomes are typically simple with infrequent targetable mutations, there have been few new FDA-approved targeted drugs. The CDK4/6 pathway, which has been extensively validated as a target in estrogen receptor-positive breast cancer, recently emerged as a dependency in Ewing sarcoma, an aggressive pediatric bone tumor. Given the heightened efficacy of CDK4/6 inhibitors (CDK4/6i) in combination with other targeted drugs in breast cancer, as well as the known propensity of resistance to emerge with single agent targeted cancer therapy, we aimed to identify CDK4/6i resistance mechanisms and biologically relevant drug combinations in Ewing.

To identify candidate resistance mechanisms, we performed a genome-wide lentiviral open-reading frame (ORF) rescue screen in two Ewing cell lines sensitive to the CDK4/6 inhibitors palbociclib and ribociclib and looked for genes that confer resistance to these drugs. This revealed IGF1R as a gene whose overexpression promoted drug escape. In parallel to the ORF screen, we established resistance to CDK4/6i in Ewing sarcoma cell lines by chronic ribociclib exposure. We found elevated levels of phospho-IGF1R in resistant cells by immunoblotting, also supporting that this pathway's activation is relevant to acquired resistance. We performed CRISPR knockout of IGF1R in these drug resistant cells and demonstrated rescue of sensitivity to CDK4/6i. Furthermore, resistant lines maintained high sensitivity to an IGF1R inhibitor.

Concurrently, in a small molecule screen, an IGF1R inhibitor scored as synergistic with CDK4/6i in Ewing cells. We thus tested ribociclib and the IGF1R tool compound AEW541 in a panel of six Ewing cell lines in vitro. We observed strong synergy utilizing the Chou-Talalay method. This finding was next validated in an in vivo cell line xenograft mouse model, where we demonstrated prolonged survival and decreased tumor volumes with combination drug treatment. In order to investigate mechanism of CDK4/6-IGF1R inhibitor synergy, we used proteomic analysis by reverse phase protein array (RPPA) in single and combined drug treated Ewing cells. RPPA revealed enhanced suppression of the PI3K/mTOR pathway in the combination, a central cancer cell signaling axis. Taken together, these results suggest that IGF1R activation is an escape mechanism to CDK4/6i in Ewing and that dual targeting of CDK4/6 and IGF1R provides a candidate synergistic drug combination for this disease.

#1630

FLT3 chimeric antigen receptor T cell therapy induces B to T cell lineage switch in infant acute lymphoblastic leukemia.

Christopher D. Chien,1 Lila Yang,1 Sang M. Nguyen,2 Christopher T. Sauter,3 Kazusa Ishii,1 Feng Shen,3 Sarah K. Tasian,3 Terry J. Fry1. 1 _NIH, Bethesda, MD;_ 2 _University of California Riverside School of Medicine, Riverside, CA;_ 3 _University of Pennsylvania, Philadelphia, PA_.

Though childhood acute lymphoblastic leukemia (ALL) is highly treatable, there remain subsets of pediatric ALL with very poor prognoses. Infant ALL, found in children under the age of 1, is difficult to treat due to the scarcity of cases impeding the ability of even the largest pediatric oncology centers from gaining experience in treating the disease, the more aggressive initial clinical presentation, as well as the inability for these young patients to tolerate toxicities associated with chemotherapeutic regimens and procedures. Despite being only 5% of total ALL cases, 80% of infant ALL cases are marked by mixed lineage leukemia (MLL/KMT2A) rearrangements. In KMT2A rearranged (KMT2Ar) ALL, FLT3 is the most differentially expressed gene that distinguishes KMT2Ar ALL from non-KMT2Ar ALL making FLT3 an attractive target for infant ALL. CD19 and CD22 targeting chimeric antigen receptor (CAR) T cell therapy has demonstrated outstanding responses in phase 1 clinical trials for relapsed/refractory B ALL, leading to tremendous interest in testing other CAR targets. Here we explore FLT3 CAR as a potential treatment for B ALL and the unexpected finding that FLT3 CAR T cells induce lineage switch of an infant ALL from a B to T cell phenotype. We generated a FLT3-targeting CAR consisting of a FLT3 binding domain derived from a well-characterized anti-human FLT3 antibody coupled to 4-1BB costimulatory and CD3-zeta activation domains. In vitro studies confirmed that human T cells expressing the FLT3 CAR produced interferon-gamma and interleukin-2 after co-culture with KMT2Ar B ALL SEM and infant B ALL KOPN8. FLT3 CAR T cells eliminated ALL in vivo in NOD-SCID-IL2Rγc-/- (NSG) mice engrafted with high FLT3 expressing SEM. KOPN8, which expresses lower levels of FLT3 when treated with FLT3 CAR T cells showed an initial clearance of leukemia in NSG mice, however relapsed with ALL that no longer expressed FLT3 or B cell marker CD19. Interestingly, this loss of FLT3 and CD19 happened concurrently with gain of T cell markers (CD3+ and either CD4+ or CD8+). The durability of this T cell phenotype was transient because when T lineage switched KOPN8 was cultured ex vivo without immune pressure, the KOPN8 cells reverted to the parental B ALL phenotype (FLT3+, CD19+, CD3 neg, CD4 neg, CD8 neg) suggesting that the ability to lineage switch is not a selection of a clone that genetically does not express B cell markers while expressing T cell markers, but rather a potential epigenetic mechanism driving the cell lineage change. Contrary to reports from CD19 CAR treated KMT2Ar B ALL that switched to a myeloid phenotype, these cells did not upregulate myeloid markers (CD33, CD11b). Taken together these data imply that lineage switch driven by CAR T cell immune pressure may cause different types of lineage switch based on the target of the CAR. Furthermore, using CAR T cell immunotherapy we may be able to interrogate the biology of leukemia.

#1631

T cell dysfunction in pediatric cancer patients at diagnosis and after chemotherapy can limit chimeric antigen receptor potential.

Rajat K. Das, Julie Storm, David M. Barrett. _Children's Hospital of Philadelphia, Philadelphia, PA_.

Cellular therapy using engineered T cells has demonstrated clinical efficacy in the treatment of hematologic malignancies. Many patients, however, have T cells that result in a poor clinical product that either fails manufacture or does not proliferate in the patient. We have prospectively characterized peripheral blood T cells from 157 pediatric cancer patients and identified key metabolic changes associated with CAR T cell potential. We collected peripheral blood samples from 157 pediatric patients with acute lymphoblastic leukemia (ALL), non-Hodgkin Lymphomas (NHL) , neuroblastoma, osteosarcoma, rhabdomyosarcoma, Wilms, Hodgkins, chronic myelogenous leukemia and Ewings sarcoma at diagnosis and after each cycle of chemotherapy. We depleted the adherent cells from this collection, quantified the CD3+ population using flow cytometry, and expanded these T cells using CD3/CD28 stimulatory beads as in CAR T cell manufacturing. We noted very poor CAR T cell potential in all tumor types (<30% pass) except ALL (80% pass) and Wilms tumor (50% pass) in the pre-chemotherapy samples. We noted a decline in CAR T cell potential with cumulative chemotherapy in all cases, though this was particularly significant in children less than 3yo. Nanostring RNA profiling of metabolic pathways revealed a bias towards glycolysis and away from fatty acids in the poor performing samples. In vitro assays of chemotherapy exposure reveal that certain classes are especially toxic to Naïve T cells versus memory T cells, and that this correlates with poor potential as CAR T cells. In particular, cyclophosphamide and doxorubicin containing regimens severely depleted CAR T cell potential in patients. These chemotherapy agents were associated with mitochondrial dysfunction both in vitro and in patient T cells after in vivo chemotherapy. Oxygen consumption rate (OCR) analysis also reveals a poor spare respiratory capacity (SRC) in the T cell samples that go on to perform poorly as CAR T cells. Enforced use of fatty acids such as palmitate helped restore the SRC in chemotherapy exposed T cells.

These data represent the most detailed analysis of T cell number, phenotype and metabolic function from pediatric cancer patients. Low abundance of Naïve T cells (whether pre-chemotherapy or post), reliance on glycolytic pathways and poor SRC define patients with poor potential to have an active CAR T cell product made. This data raises important questions for the development of CAR T cell therapies for cancers other than ALL in pediatrics, as inherent T cell dysfunction and more intensive chemotherapy regimens in solid tumors may represent new barriers to efficacy.

#1632

Suppression of EWS-FLI1 transcription using a combination therapy of mithramycin and cyclin-dependent kinase 9 inhibition.

Guillermo Flores,1 Joel Everett,2 Brandon Oswald,1 Natasha Caplen,3 Lee Helman,3 Zachary Madaj,1 Patrick Grohar1. 1 _Van Andel Institute, Grand Rapids, MI;_ 2 _Vanderbilt University School of Medicine, Nashville, TN;_ 3 _National Cancer Institute, Bethesda, MD_.

Background: Ewing sarcoma (ES) is a pediatric soft tissue tumor with a poor prognosis. ES is dependent on the presence and activity of the oncogenic transcription factor, EWS-FLI1, that dysregulates gene expression at hundreds of targets. We have previously identified mithramycin (MMA) as a potent inhibitor of EWS-FLI1 driven transcription. However, only sub-therapeutic concentrations have been achieved in pediatric patients. To maximize inhibition, we conducted an siRNA screen to identify gene targets that potentiated the effects of MMA in ES. Knock-down of several transcriptionally related genes significantly sensitized ES cells to the effects of MMA. We next developed a drug matrix screening platform to identify commercially available transcriptional inhibitors that also potentiated the effects of MMA on EWS-FLI1 driven transcription and ES cell viability. PHA-767491, a cyclin-dependent kinase 9 inhibitor (CDK9i), reverses the effect on EWS-FLI1 on several targets at both the mRNA and protein level across multiple ES cell lines when combined with MMA.

Methods: We utilize matrix drug screening to identify that PHA-767491 synergizes with MMA in terms of a reduction in cell viability as measured by MTS and cell growth as measured by time-lapse microscopy. We use RT-qPCR to measure changes in gene expression across multiple well characterized targets of EWS-FLI1 driven transcription. We use western blot analysis to measure changes at the protein level for these targets as well. We then use an orthotopic xenograft mouse model to measure changes in tumor size after administration of our MMA-CDK9i combination therapy.

Results: Our combination of MMA and PHA-767491 displays strong synergy as measured by Bliss-Independence. Multiple ES cell lines become unviable, with minimal effect on non-ES cells, and this effect is stable after removal of the compounds. This synergy is recapitulated as a reversal of EWS-FLI1 driven transcription across multiple targets at both the mRNA and protein levels. We are currently evaluating the combination therapy in our animal model comparing MMA-CDK9i to control or either agent alone. Importantly our MMA-CDK9i combination uses drug concentrations that are clinically achievable.

Conclusions: We describe an MMA-CDK9i combination that displays excellent activity against EWS-FLI1 driven transcription. We confirmed this using multiple independent assays in both in vitro and in vivo models. We complete this work with the hope that it can eventually be translated to patients.

#1633

Chemical proteomics identifies druggable proteins in ALK-driven neuroblastomas.

Smita Matkar,1 Renata Sano,1 Colleen Larmour,1 Kateryna Krytska,1 Gabriela M. Witek,1 Mark Gerelus,1 Tim J. Stuhlmiller,2 Gary L. Johnson,3 Yael P. Mosse1. 1 _Children's Hospital of Philadelphia, Philadelphia, PA;_ 2 _SPYRYX Biosciences, Apex, NC;_ 3 _University of North Carolina, Chapel Hill, NC_.

The discovery of mutations in the ALK oncogene as the genetic etiology of familial neuroblastoma (NB) and as somatically acquired in 14% of patients with the most aggressive form of the disease has positioned ALK as the major tractable oncogene product for targeted therapy in NB. ALK tyrosine kinase domain mutations are found mostly at three hotspots (F1174, F1245 and R1275), with the F1174* and F1245* mutations conferring broad resistance to crizotinib. We have recently demonstrated that lorlatinib, a novel ATP-competitive macrocyclic ALK inhibitor, overcomes de novo resistance and exerts unprecedented activity as a single agent in patient-derived xenografts (PDXs) harboring F1174L or F1245C mutations, while also inducing durable responses in R1275Q xenografts. The objective of our work was to identify differences in reprogramming of the kinome upon ALK inhibition with crizotinib and lorlatinib. We hypothesize that identification of the targets through which lorlatinib mediates its superior anti-tumor effects will provide insights into the mechanisms by which full-length ALK inhibition abrogates tumorigenesis, activates compensatory pathways, and uncover novel targets to overcome resistance to ALK inhibition. We used Multiplexed Inhibitor Beads coupled with Mass Spectrometry (MIB/MS) to quantitatively measure kinase activity dynamics on a proteomic scale. Three PDX models harboring F1174L (COG-N-453x) or the other two most-common mutations, R1275Q (NB1643) and R1245C (Felix) were treated with either crizotinib (100 mg/kg/day) or lorlatinib (10 mg/kg/day) for 2.5 days or 6.5 days prior to MIB/MS analysis. Lorlatinib more potently inhibited ALK and preferentially inhibited a series of other tyrosine kinases, correlating with the superior efficacy of lorlatinib in pre-clinical studies. Moreover, a number of kinases involved in G2/M cell cycle transition including PLK1, CHEK1 and aurora kinases were significantly inhibited by lorlatinib, proposing that this multi-kinase inhibition is responsible for the robust anti-tumor activity observed in vivo with lorlatinib. We interrogated the role of SHP2, a ubiquitously expressed SH2 domain-containing tyrosine phosphatase recently implicated in the proliferation of receptor-tyrosine kinase driven cancers and shown to have a direct role in the G2/M checkpoint. Inhibition of ALK by treatment with lorlatinib as well as shRNA knockdown of ALK in SY5Y (ALK F1174L, PTPN11 T507K) and NB1643 NB cell lines showed decreased levels of pSHP2. Depletion of SHP2 using CRISPR constructs significantly inhibited cell proliferation in SY5Y, suggesting that SHP2 plays a crucial role in ALK driven NB. Additionally, knockdown of ALK in SY5Y downregulated PLK1 expression and arrested cells in G2/M phase. Whether SHP2 is regulated primarily downstream of ALK or jointly through alternative or compensatory signaling networks warrants further investigation.

#1634

BRCA-like phenotype constitutes hallmark of osteosarcoma.

Michal Kovac,1 Sebastian Ribi,1 Claudia Blattmann,2 Michaela Nathrath,3 Daniel Baumhoer1. 1 _The Institute of Pathology, Basel, Switzerland;_ 2 _Klinikum Stuttgart Olgahospital, Stuttgart, Germany;_ 3 _Klinikum Kassel, Kassel, Germany_.

Background: Osteosarcomas are aggressive bone tumours with a high degree of genetic heterogeneity, which limits treatment options in patients who do not respond to standard chemotherapy.

Methods: We sequenced 104 exomes and low-coverage genomes from 14 time-series consisting of chemotherapy-naïve biopsies, resection specimens, locally recurring tumours and/or distant metastases. We then identified cancer driver mutations, signatures of mutation processes and reconciled clonal structures of tumours with phylogenetic trees, thus building and evolutionary history of each tumour. Where possible, we searched for the evidence of selection acting on the tumours and molecular hallmarks that could be therapeutically exploited.

Results: Interrogating genomic data from time series, we identified pathogenic mutations in at least 10 cancer genes, but there was no universal cancer driver event present in the majority of tumours at any given time point. However, >80% of osteosarcomas exhibited specific combination of single-base substitutions, LOH and large-scale genome instability characteristic of BRCA1/2-deficient tumours. In osteosarcoma, "BRCAness" developed recurrently through independent BRCA1/2 mutations and mutations in their binding partners such as PALB2 or CHEK2. We therefore investigated the sensitivity of a panel of osteosarcoma cell lines to the poly(ADP)-ribose polymerase inhibitor talazoparib. In combination with temozolomide, talazoparib led to the to good response of osteosarcoma cell lines, activation of BAX and BAK, loss of mitochondrial membrane potential, DNA fragmentation and caspase-dependent cell death.

Conclusion: The acquisition of a BRCA-like phenotype in osteosarcoma has implications for the development of novel treatment strategies using PARP inhibitors alone, or together with standard chemotherapy. 

## BIOINFORMATICS AND SYSTEMS BIOLOGY:

### Computational Methods and Resources for Cancer Research

#922

Access, visualize and analyze 5,000 whole-genomes from pediatric cancer patients on St. Jude Cloud.

Scott Newman,1 Xin Zhou,1 Clay McLeod,1 Michael Rusch,1 Gang Wu,1 Edgar Sioson,1 Shuoguo Wang,1 J. Robert Michael,1 Aman Patel,1 Michael N. Edmonson,1 Andrew Frantz,1 Ti-Cheng Chang,1 Yongjin Li,1 Robert I. Davidson,2 Singer Ma,3 Irina McGuire,1 Nedra Robison,1 Xing Tang,1 Lance Palmer,1 Ed Suh,1 Leigh Tanner,1 James McMurry,1 Keith Perry,1 Zhaoming Wang,1 Carmen Wilson,1 Yong Cheng,1 Mitch Weiss,1 Leslie L. Robison,1 Yutaka Yasui,1 Kim E. Nichols,1 David W. Ellison,1 James R. Downing,1 Jinghui Zhang1. 1 _St Jude Children's Research Hospital, Memphis, TN;_ 2 _Microsoft Research, Redmond, WA;_ 3 _DNAnexus, Mountain View, CA_.

While whole-genome (WGS), whole-exome (WES), and RNA-Seq data of patient samples are key resources for the development of precision medicine, major computing infrastructure is typically required to use them effectively. The St Jude Cloud (SJCloud, https://stjude.cloud), built in collaboration with DNAnexus and Microsoft, aims to remove this barrier by sharing genomic sequencing data generated at St Jude Children's Research Hospital, making complex bioinformatics pipelines easily accessible, and providing intuitive visualizations for data mining in the cloud. Over 5000 WGS, 6000 WES and 1500 RNA-Seq from >5,000 pediatric cancer patients mapped to the latest reference genome are securely available in SJCloud. These data were generated from three St Jude-funded genomic initiatives: the Pediatric Cancer Genome Project (PCGP), the St Jude Life Genome Project, and the Genomes for Kids Clinical Trial. SJCloud hosts BAM files, coding and non-coding somatic and germline SNVs and indels, copy number (CNV) and structural alterations (SV). Non-identifiable data (e.g. somatic alterations, genotype frequency, cancer diagnosis and demographics) can be viewed immediately using our interactive genome browser, while raw data and individual genotype access requires a simple online approval. Data synchronization and visualization enables novel discoveries by non-bioinformaticians. For example, a genomic view of the TERT locus shows enrichment of CNVs and SVs in neuroblastoma (NBL), consistent with reports of activation via rearrangement. The same view also shows a somatic promoter mutation, C228T, in one NBL; such mutations have not been reported in primary samples to our knowledge. This integrated view across somatic mutation types enables evaluation of the diverse genetic mechanisms deregulating cancer genes. SJCloud also facilitates data re-analysis. We ported the "MutationalPatterns" R package (Blokzijl et al. 2017) to the cloud to elucidate major mutational signatures in >500,000 PCGP WGS somatic variants. Inclusion of non-coding mutations was critical as the low number of exonic mutations in some pediatric cancers is insufficient for robust analysis. A surprising finding was a signature consistent with ultraviolet-induced DNA damage in a subset of B-acute lymphoblastic leukemia. End-to-end workflows to detect gene fusions, predict neoepitopes, classify mutations, process ChIP-seq, and identify differentially expressed genes are also freely accessible. By integrating analytic tools with the world's largest set of pediatric genomics data, SJCloud enables data sharing and mining, innovative genomic analysis, and development of new analytic methods. We anticipate that in 2019 we will host data from over 10,000 pediatric cancer patients, and we are actively exploring approaches to make this a federated data repository capable of interchange with the global pediatric cancer research community.

#923

The cBioPortal for Cancer Genomics: An intuitive open-source platform for exploration, analysis and visualization of cancer genomics data.

Jianjiong Gao,1 Tali Mazor,2 Ersin Ciftci,2 Pichai Raman,3 Pieter Lukasse,4 Istemi Bahceci,5 Alexandros Sigaras,6 Adam Abeshouse,1 Ino de Bruijn,1 Benjamin Gross,1 Ritika Kundra,1 Aaron Lisman,1 Angelica Ochoa,1 Robert Sheridan,1 Jing Su,1 Selcuk O. Sumer,1 Yichao Sun,1 Avery Wang,1 Jiaojiao Wang,1 Manda Wilson,1 Hongxin Zhang,1 Priti Kumari,2 James Lindsay,2 Karthik Kalletla,3 Kelsey Zhu,7 Oleguer Plantalech,4 Fedde Schaeffer,4 Sander Tan,4 Dionne Zaal,4 Sjoerd van Hagen,4 Kees van Bochove,4 Ugur Dogrusoz,5 Trevor J. Pugh,7 Adam Resnick,3 Chris Sander,2 Nikolaus Schultz,1 Ethan Cerami2. 1 _Memorial Sloan Kettering Cancer Center, New York City, NY;_ 2 _Dana-Farber Cancer Institute, Boston, MA;_ 3 _Children's Hospital of Philadelphia, Philadelphia, PA;_ 4 _The Hyve, Utrecht, Netherlands;_ 5 _Bilkent University, Ankara, Turkey;_ 6 _Weill Cornell Medical College, New York City, NY;_ 7 _Princess Margaret Cancer Centre, Toronto, Ontario, Canada_.

The cBioPortal for Cancer Genomics is an open source software platform that enables interactive, exploratory analysis of large-scale cancer genomics data sets. It integrates genomic and clinical data, and provides a suite of visualization and analysis options, including cohort and patient-level visualization, mutation visualization, survival analysis, alteration enrichment analysis, and network analysis. The user interface is user-friendly, responsive, and makes genomic data easily accessible to scientists and clinicians. The public site (http://www.cbioportal.org) hosts data from more than 165 studies, including data from large consortia (TCGA and ICGC) and individual labs. With newly released functionality, users can now explore and query these studies individually or can combine multiple studies into new "virtual studies". The main features of the portal include OncoPrints, a compact graphical representation of alterations in multiple genes across a cohort, mutational diagrams that show locations and frequencies of mutations in a single gene, Kaplan-Meier survival curves, plots that allow the visualization of correlation between different data types for a single or multiple genes (e.g. the correlation between DNA copy number and mRNA expression), among others. To facilitate interpretation of genomic data, the cBioPortal also now integrates annotations from several leading knowledgebases (OncoKB, CIViC, MyCancerGenome and COSMIC), as well as other resources that can guide variant interpretation (CancerHotspots, MutationAssessor, SIFT and PolyPhen).

The cBioPortal has been widely adopted by the cancer community, with dozens of private instances at academic institutions and pharmaceutical/biotechnology companies. The public portal is currently accessed by approximately 25K unique visitors per month. Another notable instance is the cBioPortal for AACR GENIE (http://www.cbioportal.org/genie/), which hosts 31,706 samples from AACR Project GENIE. The cBioPortal is fully open source and all code is available on GitHub (https://github.com/cBioPortal/) under a GNU Affero GPL license. Development is a collaborative effort among groups at Memorial Sloan Kettering Cancer Center, Dana-Farber Cancer Institute, Children's Hospital of Philadelphia, Princess Margaret Cancer Centre, and The Hyve, an open source bioinformatics company based in the Netherlands. Ongoing development efforts are focused on (1) building the open source community; (2) implementing architectural and performance improvements; (3) expanding user support, documentation and training resources; (4) developing novel features to support immunogenomics and immunotherapy; (5) enhancing visualization of patient timelines, multiple tumor profiles, and cohort response; and (6) releasing a new public Application Programming Interface (API).

#924

A mathematical-experimental approach for predicting host responses in a preclinical model for trastuzumab-treated HER2+ breast cancer.

Angela M. Jarrett, Meghan Bloom, Wesley Godfrey, Anum Syed, David A. Ekrut, Lauren I. Ehrlich, Thomas E. Yankeelov, Anna G. Sorace. _The University of Texas at Austin, Austin, TX_.

Introduction: Trastuzumab, a targeted therapy for human epidermal growth factor receptor 2 (HER2) positive breast cancer, induces cell cycle arrest and inhibits HER2 expression. Trastuzumab has been shown to improve vascular delivery of subsequent cytotoxic therapies, but the mechanism by which it regulates tumor-associated angiogenesis is not well characterized. Therefore, we developed an integrated, mathematical-experimental approach to systematically investigate the interactions of tumor-growth characteristics—vasculature, immune response, hypoxia, and necrosis—and to evaluate their effects on tumor response to treatment in a murine model of HER2+ breast cancer.

Experimental: Mice (N=102) were injected subcutaneously with HER2-overexpressing BT474 breast cancer cells and treated with trastuzumab (10 mg/kg) or saline. Tumor volumes for two separate cohorts of treated and control mice were recorded longitudinally, and either mice were quantitatively imaged or tumors extracted for histology over seven days. Necrosis, vasculature, and hypoxia were characterized by: immunohistochemistry for percent necrosis, dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) for the percentage of well-vascularized tissue, and 18F-fluoromisonidazole positron emission tomography (FMISO-PET) for percent hypoxia. Preliminary immune (myeloid) cell infiltration was quantified using immunofluorescence of F4/80 and CD11c.

Mathematical Model: A system of five coupled, ordinary differential equations describing the temporal variation in tumor growth, vasculature, hypoxia, necrosis, and immune response was calibrated using data for tumor volume and percentages of well-vascularized tissue, hypoxia, and necrosis. After validation using a second tumor volume data set, uncertainty analysis was used to verify plausible overlap between the model's simulations and the experimental data when considering calibration error, and sensitivity analysis identified critical parameters for experimental estimation and potential model reductions.

Results and Discussion: The calibrated model was used to predict immune response behavior over time and yielded different results between the two groups—stagnant versus increasing immune component values for control versus treated mice, respectively. Preliminary immunofluorescence data support the immunological predictions of the model; in particular, F4/80 and CD11c co-staining in the total tissue is greater in trastuzumab treated than control tumors (p < 0.03). Furthermore, differences in the calibrated parameter sets indicate several experimentally testable hypotheses, including increased cross-talk between immune cells and vasculature as a mechanism for vascular stabilization due to trastuzumab therapy.

We acknowledge the support of CPRIT RR160005 and NCI R01CA186193.

#925

In silico models accurately predict in vivo response for IL-6 blockade in head and neck cancer.

Fereshteh Nazari,1 Alexandra E. Oklejas,1 Jacques E. Nör,1 Alexander T. Pearson,2 Trachette L. Jackson1. 1 _University of Michigan, Ann Arbor, MI;_ 2 _The University of Chicago Medicine, Chicago, IL_.

The purpose of this study is to create and validate a mathematical model describing the effect of interleukin-6 (IL-6) on head and neck cancer growth and cancer stem cell fraction. Head and Neck Squamous Cell Carcinoma (HNSCC) is the seventh most-common solid tumor with about 55,000 new cases diagnosed every year. Malignant features of HNSCC cells are derived from a shift towards more stem-like features, as postulated by the cancer stem cell (CSC) hypothesis. CSCs encompass a unique cellular subpopulation characterized by multipotency, uniquely high tumorigenic potential, and self-renewal. These cells function as putative drivers of tumor initiation, therapeutic evasion, metastasis, and recurrence. Though they are an appealing conceptual target, CSC directed cancer therapies remain scarce.

High levels of both serum IL-6 and tumor IL-6 receptor (IL-6R) expression are strongly correlated with poor patient survival. Endothelial cell-secreted IL-6 enhances the tumorigenic potential and self-renewal of head and neck CSCs. Furthermore, interruption of the IL-6 pathway results in a decreased fraction of HNSCC CSCs. Efficacy and optimal administration of the FDA approved rheumatoid arthritis IL-6R antibody Tocilizumab in HNSCC is not known.

Here we used a multi-scale mathematical model that operates at the intracellular, molecular, and tissue level to investigate the impacts of endothelial cell-secreted IL-6 signaling on the crosstalk between tumor cells and ECs during tumor growth. This endothelial cell - tumor cell (EC-TC) model was used to study the effect of tocilizumab treatment on HNSCC, and particularly the CSC fraction. Our ordinary differential equation model is the first of its kind to include full occupancy dynamics between endothelial-cell produced IL-6, IL-6R, and the competitive IL-6R inhibitor Tocilizumab.

In order to biologically validate the utility of our mathematical model, we performed a series of in-vivo experiments using HNSCC cell lines and human endothelial cells co-embedded in a porous scaffold and implanted in SCID mice flank. Tumor volumes were measured serially. CSC fraction was determined in HNSCC cell lines using flow cytometry for aldehyde dehydrogenase (ALDH) high/CD44 high proportion. Without artificial tuning to the laboratory data, our model provided superb predictive agreement to the decrease in tumor volumes observed in TCZ treated mice (r = 0.947, p < 0.0001), as well as a decrease in CSC fraction. This predictive in-silico framework can serve to rapidly evaluate dosing strategies for IL-6 pathway modulation, as well as providing the basis for proposing combination treatments with IL-6 blockade and cytotoxic or other targeted therapies.

#926

Multi-Center Mutation Calling in Multiple Cancers: The MC3 Project.

Kyle Ellrott,1 Mathew Bailey,2 Gordon Saksena,3 Kyle Covington,4 Cyriac Kandoth,5 Chip Stewart,3 Michael McLellan,2 Heidi Sofia,6 Carolyn Hutter,6 Gad Getz,3 David Wheeler,4 Li Ding2. 1 _Oregon Health and Science University, Portland, OR;_ 2 _Washington University in St. Louis, St. Louis, MO;_ 3 _The Eli and Edythe L. Broad Institute of Massachusetts Institute of Technology and Harvard University Cambridge, Cambridge, MA;_ 4 _Baylor College of Medicine, Houston, TX;_ 5 _Memorial Sloan Kettering Cancer Center, New York, NY;_ 6 _National Human Genome Research Institute, Bethesda, MD_.

The Multi-Center Mutation Calling in Multiple Cancers (MC3) data set provides consistent variant calling and filtering across the 10K patients in The Cancer Genome Atlas (TCGA). The MC3 was a collaborative science effort, driven by a consortium of researchers across multiple institutions, to form a TCGA capstone project focused on cross-tumor type automated analysis. This dataset covers 33 different cancer types using an ensemble of 7 advanced mutation-calling algorithms with scoring and artifact filtering, implemented for sharing in a reproducible, portable, standardized workflow. The resulting dataset represents several million core-hours of computational time on over 400 TB of short read data using the current state-of-the-art variant calling and filtering methods. In the past decade, the precipitous drop in sequencing cost from $10M to $1000 has allowed for larger cohorts of data to be analyzed. In 2016 there were an estimated 1.6M new cancer diagnoses in the United States. Scaling computational systems and genomic analysis to work at this scale requires the coordination of many institutions, many experiments and many computational techniques. Aside from problems of scale, there are several issues that prevent large analyses: 1) deployment of reproducible computing methods in new computing environments, 2) the ability to deploy methods without manual intervention, 3) the biases of single methods and the need for consensus, and 4) the large amount of noise and false positives that come from data including both germline sequencing, heterogeneous tumor sequencing, technical artifacts, and low variant allele fraction of observed reads. The MC3 project dealt with these issues by 1) modeling the pipeline using Common Workflow Language (CWL) format with the required software packages deployed using Docker software containerization technology, ensuring the ability to deploy analysis on new computer systems; 2) crafting the entire pipeline using best practice parameters and applying them consistently, across the entire cohort; we have also analyzed validation data to determine particular cancer type where these parameters do not fit; 3) deploying an ensemble of 7 variant calling methods including MuTect, MuSE, Radia, Somatic Sniper, Pindel, Indelocator and Varscan2 (both indel and SNP calling); and 4) applying a robust set of additional filters, as well as applying meta-calling based on results of multiple callers, to reduce false-positive rates. Over 20 million variants were produced and the data generated by this work have formed the basis of the somatic exome variant analysis presented in the other papers from the TCGA PanCanAtlas project. A set of over 3 million high-quality variants from a public release of this data has been made available so that researchers may easily use this open resource.

#927

TCPA: An open access resource for cancer functional proteomics data.

Jun Li, Han Liang. _The University of Texas MD Anderson Cancer Center, Houston, TX_.

In recent years, tremendous efforts have been made to systematically characterize the molecular profiles of tumor tissues from individuals with cancer, laying a critical foundation for elucidating the molecular basis of tumorigenesis and developing biomarker-based diagnostic, prognostic and therapeutic approaches. In particular, cancer genomic data at the DNA or RNA level are being accumulated at an unprecedented speed. However, comprehensive cancer proteomic data have been relatively limited. Using reverse phase protein arrays (RPPA), we have generated a large collection of cancer protein expression data (>8,000 samples of 32 cancer types from The Cancer Genome Atlas and >650 independent cell lines across 19 lineages). To facilitate access of the broader research community to these RPPA datasets, we have developed a user-friendly data portal, TCPA (The Cancer Proteome Atlas), which can be accessed at http://tcpaportal.org.

TCPA currently has two web applications, one is for patient cohort data and the other is for cell line data. Both of them provide three types of modules: (i) "Summary": the summary module help users check the detailed information about all the datasets curated in our database and provide a user-friendly interface for downloading the data. (ii) "Analysis": this module provides common used analyses such as correlation, differential, survival and drug sensitivity analysis; and (iii) "Visualization" module provides approaches to examine the global patterns in a specific dataset. share some major analytic and visualization modules.

In summary, TCPA represents a highly valuable resource dedicated to cancer functional proteomic data. Compared with other proteomic databases, the primary advantage of TCPA is the availability of quantitative protein expression data over many large cohorts of well-characterized TCGA patient tumors. Moreover, TCPA provides an unprecedented opportunity to validate the findings from TCGA RPPA data through independent sample cohorts and to help select model tumor cell lines for further functional investigation. These unique features of TCPA greatly increase the utility of TCGA proteomic data for both basic research and clinical applications.

#928

Regulatory heterogeneity in glioblastoma multiforme informs novel drug target discovery.

Yunpeng Liu,1 Shan He,2 Michael T. Hemann,1 Aviv Regev3. 1 _Massachusetts Institute of Technology, Cambridge, MA;_ 2 _University of Birmingham, Birmingham, United Kingdom;_ 3 _Broad Institute of MIT and Harvard, Cambridge, MA_.

Glioblastoma multiforme (GBM) is one of the most malignant forms of cancer. Its heterogeneity has been assessed at multiple levels, including bulk and single-cell transcriptome profiling. The disease has been stratified into four molecular subtypes - Classical, Neural, Proneural and Mesenchymal according to their gene expression profiles, and each of these subtypes display distinct mutational signatures and histological features reflective of their nomenclature. However, the underlying regulatory circuitry that gave rise to such heterogeneity and its implications for specialized therapeutic options are unclear. To dissect subtype-specific regulatory programs, we have developed a nonlinear regression model to unveil key transcriptional rewiring across the four GBM subtypes. Using a backbone network of potential transcription factor (TF) - target gene pairs, we applied nonlinear regression using expression profiles of each subtype to derive subtype-specific regulatory parameters for each TF-gene pair. Coupled with a novel analysis pipeline, the model revealed a set of transcription regulators whose regulatory capacities for target genes are unique to each subtype. The target genes of these signature TFs are enriched for expression signature genes for the corresponding subtype, implying that subtype-specific rewiring of TFs play a significant role in shaping differential transcription landscapes. The model was also able to capture known physical and functional interactions between TFs, as well as identify potential new co-regulatory relationships. Detailed examination of both expression and co-regulatory profiles of signature TFs showed that altered regulatory behavior could be a result of differential expression of the TF itself, differential functional partnering of the TF with other TFs, or a combination of both mechanisms. Importantly, our model also demonstrates that differential regulatory programs could provide new options for targeted therapy in GBM. We simulated the effects of knockdown/inhibition of these co-regulatory partners by propagating changes in expression to the corresponding target genes and then to the protein signaling layer using a novel random walk-based algorithm. Using the apoptosis pathway as readout, we show that targeting the retinoic acid receptor RXRA may be specifically beneficial for killing tumor populations of the Neural subtype, whereas the HNF4A gene is a potentially beneficial drug target for cells of the Mesenchymal subtype.

## CANCER CHEMISTRY:

### Molecules Addressing Challenges in Drug Discovery

#929

**Discovery of novel covalent KRAS** G12C **inhibitors that display high potency and selectivity in vitro and in vivo.**

Liansheng Li,1 Matthew R. Janes,1 Jingchuan Zhang,1 Rasmus Hansen,1 Ulf Peters,1 Xin Guo,1 Yuching Chen,1 Anjali Babbar,1 Sarah J. Firdaus,1 Jun Feng,1 Jeffrey H. Chen,1 Shuangwei Li,1 Shisheng Li,1 Carol Thach,1 Yuan Liu,1 Ata Zarieh,1 Jeff M. Kucharski,1 Tao Wu,1 Ke Yu,1 Yi Wang,1 Yvonne Yao,1 Xiaohu Deng,2 Patrick P. Zarrinkar,1 Dashyant Dhanak,3 Matthew V. Lorenzi,3 Dana Hu-Lowe,1 Pingda Ren,2 Yi Liu2. 1 _Wellspring Biosciences, Inc., San Diego, CA;_ 2 _Kura Oncology, Inc., San Diego, CA;_ 3 _Janssen Research & Development, Spring House, PA_.

Activating mutations in KRAS have a high prevalence in human cancer. The codon 12 glycine to cysteine missense mutation (KRASG12C) is among the most common KRAS mutations, present in non-small cell lung adenocarcinoma (~15 %), colorectal adenocarcinoma (~3 %), and pancreatic adenocarcinoma (~1 %). KRASG12C was previously identified as potentially druggable by allele-specific covalent targeting of cysteine 12 near the allosteric switch II pocket (S-IIP). Building on this early work, we recently described the ARS-853 series of S-IIP KRASG12C inhibitors that covalently react with the GDP-bound state of KRASG12C, trapping KRASG12C in this inactive state. In cells, ARS-853 series compounds profoundly deplete the signaling competent GTP-bound state of KRASG12C, thereby inhibiting downstream RAS signaling. However, this series of covalent KRASG12C inhibitors exhibited modest cellular potency and/or poor pharmacokinetic properties, making them unsuitable for further evaluation of covalent KRASG12C inhibition in animal models.

We now describe in further detail the discovery and characterization of a new series of structurally distinct quinazoline based S-IIP KRASG12C inhibitors with substantially improved potency and pharmacologic properties that overcome limitations of the ARS-853 series. Through structure-guided medicinal chemistry optimization we identified compound ARS-1620, a potent, orally bioavailable covalent inhibitor of KRASG12C. The co-crystal structure of ARS-1620 covalently bound to KRASG12C reveals a distinct binding mode and additional interactions, compared to ARS-853. ARS-1620 rapidly engages KRASG12C, depletes KRASG12C-GTP in tumor cell lines, and inhibits downstream RAS signaling in a dose-dependent manner. The compound potently inhibits the growth of cell lines harboring the KRASG12C mutation with little or no effect on control cell lines. ARS-1620 demonstrates robust dose-dependent efficacy with once daily oral administration across a panel of KRASG12C-positive mouse cell line (CDX) and patient-derived (PDX) tumor xenograft models, with no response observed at all doses tested in KRASG12C-negative tumor models. The anti-tumor activity of ARS-1620 correlates with target engagement in the tumors as well as with inhibition of downstream RAS signaling. The in vivo efficacy and mutant selectivity observed with ARS-1620 across a wide range of KRASG12C mouse tumor models provides the first in vivo evidence that the S-IIP targeted approach may be a promising therapeutic strategy for patients with KRASG12C mutant cancers.

#930

Targeted delivery of cytotoxic NAMPT inhibitors using antibody-drug conjugates.

Christopher S. Neumann, Kathleen C. Olivas, Andrew B. Waight, David Meyer, Luke V. Loftus, Margo Zaval, Martha E. Anderson, Steven Jin, Julia H. Cochran, Jessica K. Simmons, Paul G. Pittman, Robert P. Lyon, Peter D. Senter. _Seattle Genetics, Bothell, WA_.

Nicotinamide phosphoribosyltransferase (NAMPT) is the rate-limiting enzyme involved in the salvage biosynthetic pathway of NAD from nicotinamide. Small-molecule inhibitors of NAMPT lead to rapid depletion of NAD(H) and subsequent depletion of ATP as cellular energy metabolism is interrupted. While these inhibitors can be potently cytotoxic in vitro, their application as cancer chemotherapeutics is limited by toxicity upon systemic administration. We have developed NAMPT inhibitors that are suitable for targeted delivery to cancer cells using our antibody-drug conjugate (ADC) platform. The inhibitors retain impressive biochemical potency against purified NAMPT, as well as potent in vitro cytotoxicity. Cleavable drug linkers were constructed using an enzyme-labile glucuronic acid moiety as the trigger for drug release. The resulting linkers are relatively hydrophilic, allowing ADCs with eight drugs per antibody to be prepared with no aggregation and favorable pharmacokinetic profiles. In vitro, treatment of cells with ADCs leads to depletion of cellular NAD. This pharmacodynamic effect is also observed in xenografted L540cy tumors, where NAD(H) is not detected in excised tumors 4 days after dosing. Across multiple tumor xenograft models, treatment with NAMPT-based ADCs leads to tumor regression. Exploratory toxicology studies in rats show excellent single-dose tolerability, with recoverable anemia and lymphopenia noted. Importantly, retinal and cardiac toxicities associated with systemic administration of NAMPT inhibitors in rodents were not observed with NAMPT-based ADCs. These data demonstrate the ability of NAMPT inhibitors to serve as a novel payload class for ADCs, and the potential to reintroduce this unique mechanism of action to the clinic with improved therapeutic windows.

#931

Discovery of ABBV-744, a first-in-class highly BDII-selective BET bromodomain inhibitor.

George S. Sheppard,1 Le Wang,1 Steven D. Fidanze,1 Lisa A. Hasvold,1 Dachun Liu,1 John K. Pratt,1 Chang H. Park,2 Mai-Ha Bui,1 Emily J. Faivre,1 Xiaoli Huang,1 Xiaoyu Lin,1 Denise M. Wilcox,1 Yu Shen,1 Daniel H. Albert,1 Terrance J. Magoc,2 Ganesh Rajaraman,2 Warren M. Kati,1 Keith F. McDaniel1. 1 _AbbVie Inc., North Chicago, IL;_ 2 _Former AbbVie employee, North Chicago, IL_.

The BET family of proteins consists of BRD2, BRD3, BRD4 and BRDT, with each of these proteins containing two distinct bromodomains (BDI and BDII). ABBV-075, like other first generation BET family bromodomain inhibitors currently under clinical development, binds to each of the 8 bromodomains with similar affinity and inhibits the proliferation of cancer cells that represent a wide range of tumor types. We hypothesized that selectively targeting specific subsets of the BET bromodomain might abolish this broad spectrum profile such that only the tumor types that are highly addicted to the subtype specific BET bromodomain-mediated transcription would remain sensitive to these selective agents. Both BDI and BDII are highly conserved across BET family members (&gt; 70% identity), suggesting that the generation of compounds that are selective for the BDI bromodomains or the BDII bromodomains might be achievable. Structure-based design targeting the Asp144/His 437 and Ile146/Val439 sequence differences (BRD4 BDI/BDII numbering) led to the identification of structural analogs of ABBV-075 demonstrating greater than 100X selectivity for BRD4 BDII over BRD4 BDI. Further elaboration led to compounds with improved BDII-selectivity and oral bioavailability and the identification of the clinical asset ABBV-744. Disclosures: All authors are employees or former employees of AbbVie. The design, study conduct, and financial support for this research were provided by AbbVie. AbbVie participated in the interpretation of data, review, and approval of the publication.

#932

**Structure-based design of selective inhibitors for the** β **-catenin/T-cell factor protein-protein interaction.**

Min Zhang, Zhen Wang, Cheng Mo, Haitao Mark Ji. _H. Lee Moffitt Cancer Center & Research Institute, Tampa, FL_.

INTRODUCTION: Significant compound screening efforts have been made to discover the inhibitors for the Wnt/β-catenin signaling pathway, but little success was obtained. Even so, most of the identified inhibitors act on Wnt upstream effectors, which can cause the cross-regulatory effects on the noncanonical Wnt signaling pathways, cannot confer the inhibitory activities to cancer cells that harbor downstream APC, Axin, or β-catenin activation mutations, and perturb the function of β-catenin in cell-cell adhesions. The formation of the β-catenin/T-cell factor (Tcf) complex in the cell nucleus is the penultimate step of canonical Wnt signaling. The aberrant formation of this protein-protein interaction (PPI) complex has been recognized as the key driving force for many cancers including triple negative breast cancers (TNBCs). The crystallographic and biochemical analyses reveal that the binding mode of Tcf, cadherin and adenomatous polyposis coli (APC) with β-catenin is identical. In the previous studies, we identified that the Tcf4 G13ANDE17 binding site of β-catenin can be targeted to design potent small-molecule inhibitors selective for the β-catenin/Tcf over β-catenin/cadherin and β-catenin/APC PPIs. EXPERIMENTAL PROCEDURE: In combination of peptidomimetic strategy, structure-based drug design, and chemical synthesis, and biochemical and cell-based characterizations, we successfully designed and synthesized potent and selective small-molecule inhibitors for the β-catenin/Tcf over β-catenin/cadherin and β-catenin/APC interactions. RESULTS AND DISCUSSION: The most potent inhibitor exhibited submicromolar inhibitory potency for disruption of the β-catenin/Tcf PPI. The potent inhibitors also exhibited dozens to hundreds folds selectivities for the β-catenin/Tcf PPI over the β-catenin/E-cadherin and β-catenin/APC PPIs. The binding mode of new inhibitors was characterized by the site-directed mutagenesis and structure-activity relationship studies. The cell-based studies demonstrated that new inhibitors passed the cell membrane, significantly attenuated canonical Wnt signaling in cancer cells, and suppressed growth of Wnt/β-catenin-dependent cancer cells. These inhibitors also exhibited cell-based selectivity for the β-catenin/Tcf over β-catenin/cadherin and β-catenin/APC PPIs. CONCLUSION: In TNBCs, the autocrine activation of Wnt ligands, the epigenetic silencing of Wnt suppressor genes, and the cross talks between signaling pathways stabilize beta-catenin in the dephosphorylated state, increase the level of nuclear β-catenin, and aberrantly activate the Wnt/β-catenin signaling pathway. The potent and selective β-catenin/Tcf inhibitors will provide the starting points for development of a class of targeted therapy for the treatment of triple negative breast cancers.

#933

Discovery of a potent dual ALK and EGFR T790M inhibitor.

Seock Yong Kang,1 Jaebong Jang,2 Jung Beom Son,1 So Young Kim,1 Hwan Geun Choi,1 Nathanael S. Gray2. 1 _New Drug Development Center, Daegu-Gyeongbuk Medical Innovation Foundation, Daegu, Republic of Korea;_ 2 _Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA_.

Anaplastic lymphoma kinase (ALK) and epidermal growth factor receptor (EGFR) are part of a family of proteins called receptor tyrosine kinases (RTKs). Oncogenic mutations and translocations of RTKs cause the development and progression of many cancers. Particularly, mutations that lead to ALK and EGFR overexpression/amplification have been associated with a number of cancers, non-small cell lung cancer (NSCLC). Several wild-type and mutant-directed inhibitors of both ALK and EGFR have been developed such as the first-generation ALK inhibitor, crizotinib and the first and second generation EGFR inhibitors, gefitinib, erlotinib and afatinib to treat NSCLC. The second generation ALK inhibitors, ceritinib, alectinib and brigatinib and the third generation EGFR inhibitor, osimertinib potently overcome the secondary resistant mutations. Despite extensive exploration of diverse chemotypes as inhibitors of ALK and EGFR, there have been no reports of small molecules that can act as dual inhibitors of both kinases. We report highly potent dual small molecule inhibitors of both ALK and EGFR, particularly the T790M mutant which confers resistance to first generation EGFR inhibitors. Based on our previous effort developing covalent T790M EGFR inhibitor, WZ4002, we have rationally designed and synthesized the potent dual inhibitor of ALK and EGFR, particularly the T790M resistant mutant, by combining the crucial functional groups of known ALK inhibitors ceritinib and brigatinib, and EGFR inhibitors WZ4002 and osimertinib, in the 2,4-diarylaminopyrimidine scaffold. Dual ALK/ EGFR inhibitors may provide an efficient approach to prevent resistance that arises as a consequence of clinically reported reciprocal activation mechanisms. Our lead compound 7c displayed remarkable inhibitory activities against both ALK and EGFR in enzymatic and cellular assays. We demonstrate that 7c is capable of recapitulating the signaling effects and antiproliferative activity of combined treatment with the approved ALK inhibitor ceritinib and T790M EGFR inhibitor osimertinib against patient-derived nonsmall cell lung cancer cell line, DFCI032 which harbors both EML4-ALK and activated EGFR.

#935

Generation and preclinical characterization of CD123-CPI antibody-drug conjugate (ADC).

Yoon-Chi Han,1 Fan Jiang,1 Nicole Piche-Nicholas,1 Madan Katragadda,1 Nadira Prashad,1 Manoj Charati,1 Wendy Hu,1 Mauricio Leal,1 Nathan Tumey,2 Andreas Maderna,1 Russell Dushin,1 Kenny Kim,3 LuAnna Lemon,1 Marc Damelin,4 HP Gerber,5 Lioudmila Tchistiakova,6 Frank Loganza,1 Chris O'Donnell,7 Puja Sapra1. 1 _Pfizer, Pearl River, NY;_ 2 _University of Binghamton, Pearl River, NY;_ 3 _N/A, Pearl River, NY;_ 4 _Maderna, MA;_ 5 _NA, Pearl River, NY;_ 6 _Pfizer, MA;_ 7 _Pfizer, Pearl River, CT_.

CD123 is expressed on cancer cells in a variety of hematologic malignancies including acute myeloid leukemia (AML). CD123 is frequently expressed on leukemic blasts and leukemic stem cells (LSCs), a cell population associated with relapse in patients, but there is minimal or no expression on most normal hematopoietic cells and solid tissues. Thus, CD123 is a promising target for AML. We have generated an anti-CD123 ADC that carries cyclopropylpyrroloindoline (CPI) payload that crosslinks DNA. A drug loading of 2 molecules of CPI per molecule of antibody was achieved by site-specific conjugation using our transglutaminase methodology. Upon binding to the CD123 antigen, CD123-ADC is internalized and delivered to the endosomal-lysosomal pathway whereupon the CPI payload is released from the antibody by proteolysis of the linker. The released CPI alkylates DNA, which activates ATR/ATM, CHK1, CHK2 and FANCD2, ultimately resulting in cell death. In vitro, CD123--CPI elicited cytotoxicity in a dose-dependent manner against several CD123-positive, but not against CD123-negative cell lines. Cell lines with higher CD123 expression level were more sensitive to the ADC. Long-Term Culture-Initiating Cells in vitro assay showed that AML patient bone marrow samples that naturally have high percentage of LSCs yielded substantially fewer colonies in CD123-ADC treated cells compared to control ADC. Importantly, CD123-ADC had no adverse effects in healthy donor bone marrow cells experimentally enriched in CD34+ primitive stem cells. This result suggests that CD123-ADC specifically inhibits the growth potential of leukemic blasts and progenitor cells. In vivo, robust antileukemic activity was observed in CD123-positive AML cell line-derived xenograft models. Low doses of CD123-ADC effectively regressed tumors whereas the tumor progressed in mice that received control ADC. Efficacy was also evaluated in disseminated AML PDX models (n≥7) established with patient samples of various cytogenetics and molecular abnormalities, and relapse/refractory. Flow cytometry analyses of leukemic load in peripheral blood and in bone marrow samples of mice showed that CD123-ADC was efficacious in reducing tumor burden even at the suboptimal doses. In summary, our data demonstrate that our CD123-ADC is highly active in a broad panel of primary AML samples. Currently, CD123-ADC in combination with other AML therapies is being tested in vivo. All these attributes of CD123-ADC make it an attractive agent to evaluate in clinical trials. 

## CLINICAL RESEARCH:

### Liquid Biopsy 1

#936

**Analysis of cell-free DNA from 32,991 advanced cancers reveals novel co-occurring activating** RET **alterations and oncogenic signaling pathway aberrations.**

Karen L. Reckamp,1 Thereasa A. Rich,2 Young Kwang Chae,3 Robert C. Doebele,4 Wade T. Iams,3 Michael Oh,3 Victoria M. Raymond,2 Richard B. Lanman,2 Thomas E. Stinchcombe,5 Vivek Subbiah,6 David R. Trevarthen,7 Oliver P. Gautschi8. 1 _City of Hope Cancer Center, Duarte, CA;_ 2 _Guardant Health, Redwood City, CA;_ 3 _Northwestern University Feinberg School of Medicine, Chicago, IL;_ 4 _University of Colorado Cancer Center, Aurora, CO;_ 5 _Duke Cancer Institute, Durham, NC;_ 6 _MD Anderson Cancer Center, Houston, TX;_ 7 _Comprehensive Cancer Care and Research Institute of Colorado, Englewood, CO;_ 8 _Cantonal Hospital Lucerne, Lucerne, Switzerland_.

Background

The receptor tyrosine kinase (RTK), RET, is an emerging oncogenic target. Inhibitors targeting activating RET alterations (alts), including on-target resistance alts, are being studied in solid tumors. We present a large cohort of patients (pts) with metastatic solid tumors and somatic RET alts detected by next generation sequencing (NGS) of cell-free DNA (cfDNA). cfDNA provides a global description of RET and other genomic alts in advanced disease, typically exposed to one or more lines of systemic therapy, complementing previous descriptions of RET alts in tissue which is often obtained from early stage/untreated tumors (e.g. TCGA).

Methods

Somatic activating RET alts (fusions/missense alts predicted to be oncogenic) were identified from 32,991 consecutive pts tested with a 68-73 gene cfDNA assay (Guardant Health, CA) between 02/2015-07/2017. This comprehensive NGS assay evaluates single nucleotide variants, and select indels, fusions, and copy number gains. Variants of uncertain significance and synonymous alts were excluded.

Results

175 somatic activating RET alts were detected in 170 pts with non-small cell lung (NSCLC, n=123), colorectal (n=15), breast (n=8), thyroid (n=8), or other cancers (n=13). 141 pts had 142 RET fusions involving 7 different partners, most commonly KIF5B (n=77). Nineteen unique activating RET missense alts were detected in 29 pts, the most common being M918T (n=8). Two pts had multiple activating RET missense alts.

Advanced cancers with activating RET alts commonly harbored additional alts in oncogenic signaling pathway genes: other RTKs (n=57 pts), cell cycle (n=48), MAPK (n=46), PI3K (n=27). The frequency and distribution of alts in these co-occurring pathway genes varied by specific RET alt. Missense RET alts had a lower proportion of co-occurring TP53 alts and a higher proportion of PIK3CA alts compared to RET fusions. Non-KIF5B-RET fusions were enriched for co-occurring alts in MAPK and other RTK genes relative to tumors with KIF5B-RET fusions. In pts with NSCLC, KIF5B-RET fusions were rarely observed with other bona fide oncogenic drivers whereas known EGFR driver alts were identified in pts with CCDC6-RET (10/31), NCOA4-RET (4/14), and TRIM24-RET (1/2) fusions. 12/15 had EGFR T790M and 3/12 had C797S mutations. Prior treatment with anti-EGFR therapy was confirmed in 5 pts. The RET fusion arose without (n=1), concurrent with (n=2), and subsequent to (n=2) emergence of T790M.

Conclusions

In the largest cohort of pts with advanced cancers harboring RET activating alts, we describe novel co-occurrences of oncogenic signaling pathway aberrations. Additionally, our findings suggest that CCDC6- and NCOA4-RET fusions may contribute to anti-EGFR therapy resistance in NSCLC. Understanding the clinical significance of co-occurring alts is essential to evaluating the efficacy of RET-directed therapies.

#937

**Longitudinal circulating-tumor DNA profiling of** EGFR **-mutated non-small cell lung cancer patients treated with EGFR-tyrosine kinase inhibitors.**

Sandra Ortiz-Cuaran,1 Aurélie Swalduz,2 Camille Léonce,1 Solène Marteau,1 Séverine Martinez,2 Gilles Clapisson,2 Virginie Avrillon,2 Luc Odier,3 Lionel Falchero,3 Pierre Fournel,4 Emma Green,5 Clive Morris,5 Maurice Pérol,2 Pierre Saintingy1. 1 _Centre Léon Bérard / Cancer Research Center of Lyon, Lyon, France;_ 2 _Centre Léon Bérard, Lyon, France;_ 3 _Hôpital Nord-Ouest, Villefranche-sur-Saône, France;_ 4 _Institut de Cancérologie Lucien Neuwirth, Saint-Étienne, France;_ 5 _Inivata Ltd., Cambridge, United Kingdom_.

Background: In EGFR-mutant non-small cell lung cancer (NSCLC), progression disease (PD) under 1st-generation EGFR tyrosine kinase inhibitors (EGFR-TKIs) is driven by the EGFR T790M mutation in about 50% of cases. This mutation is targeted with osimertinib, a specific and potent inhibitor that also showed superior efficacy to that of 1st-generation EGFR-TKIs in the 1st-line treatment EGFR+ NSCLC.

Strategy: We performed a longitudinal circulating-tumor DNA (ctDNA) analysis to evaluate 1) the dynamics of sensitizing or resistant mutations over time and 2) the genomic alterations associated with EGFR-TKI resistance, in NSCLC patients treated with either 1st-generation EGFR-TKIs (n=24) or with osimertinib (n=20). ctDNA sequencing was performed using InVisionSeq™, an amplicon-based NGS (36-gene panel) assay.

Results: Sensitivity was 80% and 100% for the detection of EGFR-activating mutations and the EGFR T790M mutation, respectively, at PD in ctDNA vs tissue biopsies. The type of progression appears to influence the detection of EGFR mutations in plasma. Patients (pts) with exclusively brain or thoracic PD had significantly lower allelic fractions (AFs) of EGFR-activating mutations than pts with systemic PD (P=0.006). AFs of both EGFR-activating and EGFR T790M mutations were highest in patients with progressive metastases in liver or bone. High cell-free DNA load was associated with the presence of TP53 mutations regardless of the time under treatment (n=248 samples). The total number of mutations detected in ctDNA was higher in pts treated with osimertinib vs. 1st-generation EGFR-TKIs (P=0.004), suggesting a potential increase of tumor heterogeneity over time. We observed that the AFs of mutant ctDNA were correlated with response to treatment. Complete clearance of EGFR T790M (AF detection limit: 0.01%) was observed in pts who presented a partial response to osimertinib (n=17), of these, EGFR-activating mutations were found in 4/17 pts (AF:0.05-1.03%). PD was evidenced in 36/44 pts. Emergence of mechanisms of resistance, before the confirmation of clinical PD, was evidenced in 5 and 3 pts under erlotinib and osimertinib, respectively. PD to 1st-generation EGFR-TKIs was mainly driven by EGFR T790M (86%), followed by mutations in PIK3CA or PTEN. We detected the emergence of heterogeneous potential mechanisms of resistance to osimertinib in 3/9 cases, including mutations in EGFR, KRAS, BRAF as well as HER2 and MET amplification. We did not identify genomic drivers of resistance in 16/36 pts, despite evidence of clinical PD. These pts had either exclusive brain or thoracic progression. ctDNA WES will be performed in these samples.

Conclusion: Our results suggest that ctDNA sequencing might be a complementary, noninvasive tool to monitor response to treatment and heterogeneous mechanisms of resistance in NSCLC pts treated with EGFR inhibitors.

#938

Detection of actionable mutations in plasma cfDNA samples from patients with non-small cell lung carcinoma using a novel amplicon-based Firefly NGS assay.

Lin Wang,1 Li Weng,2 Xiao Chen,3 Min Li,4 Qiaomei Guo,1 Wenjun Yu,1 Tobias Wittkop,2 Hongyan Wang,5 Malek Fahem,2 Shengrong Lin,2 Grace Q. Zhao,2 Jiatao Lou1. 1 _Shanghai Thoracic Hospital, Shanghai, China;_ 2 _AccuraGen, Menlo Park, CA;_ 3 _Jilin University Hospital, Jilin, China;_ 4 _Zhongshan Hospital, Guangzhou, China;_ 5 _AccuraGen, Shanghai, China_.

Background: Detection of EGFR, KRAS and BRAF mutations can help guide cancer treatment for non-small cell lung cancer (NSCLC) patients. To identify an easy to use, accurate, multiplex molecular diagnostic assay, we evaluated the performance of a novel next-generation sequencing (NGS)-based cell-free DNA (cfDNA) assay, Firefly assay, which employs a concatemer-based noise suppression mechanism with an amplicon workflow.

Methods: Performance of amplicon based Firefly assay, with a panel covering EGFR, BRAF, and KRAS mutations designed for targeted therapy selection of NSCLC was first evaluated using a cfDNA reference standard and blank control samples. This panel was then used to analyze plasma cfDNA samples from 134 NSCLC cancer patients and 50 non-cancerous controls, and results were compared with tumor tissue ARMS and cfDNA ddPCR results.

Results: Firefly assay demonstrated superior sensitivity and specificity with median detection of 100% at allele frequency of 0.1% for 20ng of cfDNA and zero false positive in all blank control samples. In cfDNA from plasma collected before treatment, EGFR mutation detection by Firefly assay was 94% concordant with tumor tissue ARMS. Firefly assay demonstrated strong per-variant detection-rate concordance (98%) and allele frequency concordance (R2 = 0.95) when compared with cfDNA ddPCR result.

Conclusions: The amplicon based Firefly assay offers multiplex capacity, de novo variant detection, high sensitivity and specificity. Thus, Firefly assay is a kitable NGS solution for cfDNA analysis, which can help guide targeted therapy selection, drug resistance detection, and disease monitoring in NSCLC and other cancer patients.

#939

Analytical validation of InVisionFirst™, a liquid biopsy assay for high-sensitivity broad molecular profiling of circulating tumor DNA using plasma samples of cancer patients.

Samuel Woodhouse,1 Vincent Plagnol,1 Karen Howarth,1 Stefanie Lensing,1 Matt Smith,1 Michael Epstein,1 Mikidache Madi,1 Sarah Smalley,2 Catherine Leroy,1 Jonathan Hinton,1 Frank de Kievit,1 Esther Musgrave-Brown,1 Colin Herd,1 Katherine Baker-Neblett,2 Will Brennan,2 Peter Dimitrov,1 Nathan Campbell,2 Nitzan Rosenfeld,1 James Clark,1 Davina Gale,1 Jamie Platt,2 John Calaway,2 Greg Jones,2 Tim Forshew1. 1 _Inivata Ltd, Cambridge, United Kingdom;_ 2 _Inivata Inc, Research Triangle Park, NC_.

Circulating tumor DNA (ctDNA) analysis enables minimally invasive assessment of somatic genetic alterations for cancer patients. ctDNA analysis is quickly being incorporated into cancer care; notably in profiling patients' tumors to guide treatment decisions. Patients have demonstrated a response to targeted therapies even when the actionable mutations detected in their plasma DNA was at low variant allele fractions (VAFs) (< 0.5%). Here we describe a detailed analytical validation study for InVisionFirst™, an NGS-based assay offering broad molecular profiling with exceptional sensitivity for analysis of ctDNA.

The InVisionFirst assay is based on enhanced tagged amplicon sequencing (eTAm-SeqTM) technology and profiles 36 genes commonly mutated in non-small cell lung cancer (NSCLC) and other cancer types for actionable genomic alterations. Analytical validation demonstrated the performance of this assay for detection of point mutations, indels, amplifications and gene fusions that commonly occur in NSCLC. Over 100 different contrived samples and 200 plasma samples were analyzed, representing a wide spectrum of genetic aberrations and variant allele frequency (VAF). Analysis was performed by multiple operators, at different times and using different reagent lots.

The InVisionFirst assay demonstrated an excellent sensitivity, with 99.48% sensitivity for SNVs present at VAF range 0.25%-0.33% and 92.46% sensitivity for indels at 0.25% VAF. DNA amplifications for ERBB2, FGFR1, MET and EGFR were also detected with high sensitivity and specificity. Greater than 50% of SNVs were detected down to a few molecules (0.06%-0.08% VAF), with the lower reportable range of 0.0125% for SNVs and indels. This high sensitivity was achieved while still retaining exceptional specificity (99.9997% per base). The assay also demonstrates, for the first time, detection of ALK and ROS1 gene fusions with an amplicon-based ctDNA technology. The novel methodology detected EML4-ALK and SLC34A2-ROS1 breakpoints at a VAF of 0.0625%. Comparison of VAFs between the InVisionFirst assay and ddPCR showed excellent concordance (R2 = 0.965).

This analytical validation study has evaluated the performance characteristics of the InVisionFirst assay across a range of genomic alterations, establishing it as a highly sensitive and specific assay that meets the analytical requirements for clinical applications. The InVisionFirst assay can be deployed as a liquid biopsy NGS assay for broad molecular profiling of plasma to aid in the management of cancer patients.

#940

Analytical and clinical validation of the Idylla™ ctKRAS and ctNRAS-BRAF liquid biopsy tests identifies mCRC patient groups with high and low ctDNA shedding.

Bart Jacobs,1 Bart Claes,1 Valentina Pomella,2 Sabine Tejpar,3 Jean-Baptiste Bachet,4 Pierre Laurent-Puig,5 Geert Maertens,1 Erwin Sablon1. 1 _Biocartis, Mechelen, Belgium;_ 2 _KU Leuven, Leuven, Belgium;_ 3 _UZ Leuven, Leuven, Belgium;_ 4 _Groupe Hospitalier Pitié Salpêtrière, Paris, France;_ 5 _Université Paris Descartes, Assistance Publique-Hôpitaux de Paris, Paris, France_.

Background: Ras mutation analysis is required for the administration of anti-EGFR therapies to mCRC patients. Incorporating ctDNA testing in routine diagnostics allows rapid detection of baseline RAS mutation status from a single blood draw. We validated ctDNA analysis of 21 KRAS, 18 NRAS and 4 BRAF ctDNA mutations using 2 ml of plasma on the Idylla™ platform. The tests were performed using the Idylla™ ctKRAS and ctNRAS-BRAF Mutation Tests, comprising all required reagents for fully automated and integrated ctDNA analysis from plasma with a turnaround time of approximately 2hrs.

Methods: Analytical validation was performed using EDTA plasma samples containing fragmented DNA containing mutant sequences of interest. For both assays the LOD was calculated using regression models. Reproducibility studies were conducted using the Acrometrix™ Idylla™ ctRAS Verification Panel consisting of 4 KRAS mutations, 4 NRAS mutations and 1 BRAF mutation. During between-laboratory reproducibility studies, the panel of reference samples was tested at three different sites using two different Idylla™ instruments. Between-lot reproducibility was assessed on three different lots. For clinical validation, pre-treatment plasma samples from 203 mCRC patients enrolled in the prospective multicenter RASANC study (NCT02502656) were retrospectively assessed. Proper informed consent and statistical analysis were included. As a comparator test, NGS analysis was performed on ctDNA according to Pécuchet et al. (2016). In addition, tissue RAS/BRAF results were available from standard of care testing.

Results: The between-lab reproducibility for the 4 mutations evaluated with the Idylla™ ctKRAS Mutation Test is with 95% confidence at least 98.7% Idylla™ and is with 95% confidence above 98.7% for the 5 mutations evaluated with the Idylla™ ctNRAS-BRAF Mutation Test. The between-lot reproducibility for the evaluated mutations is with 95% confidence at least 98.9% for the ctKRAS Test and 98.5% for the ctNRAS-BRAF Test. In the clinical study, overall RAS agreement between Idylla™ and NGS on plasma was 90.0% with a sensitivity of 89.5% and specificity of 90.4%. RAS concordance between FFPE tissue and plasma was 78.9% in the whole study population. Low amounts of cfDNA in the sample, absence of liver metastases and primary tumor resection were associated with discordant results. In the population with liver metastases showing sufficient cfDNA, overall RAS agreement between plasma and tissue RAS status was 92.5% with a sensitivity of 93.2% and specificity of 90.9%.

Conclusions: The Idylla™ ctKRAS and ctNRAS-BRAF Mutation Tests provide a sensitive, reliable and fast solution for RAS-BRAF ctDNA testing. Results confirm that RAS-BRAF mutation status can be adequately determined using plasma from metastatic colorectal cancer patients with liver metastases.

#941

Method for sensitive detection of tumor fingerprints in plasma.

Fangyan Yu,1 Mariana Fitarelli-Kiehl,1 Ka Wai Leong,1 Viktor Adalsteinsson,2 Heather Parsons,1 Mike G. Makrigiorgos1. 1 _Dana-Farber Cancer Inst., Boston, MA;_ 2 _Broad Institute, Boston, MA_.

Presence of excess unaltered, wild-type DNA providing no information of biologic or clinical value may often mask rare alterations containing diagnostic or therapeutic clues for cancer. There is a growing demand for removing unaltered DNA over large pools-of-sequences, especially for applications in liquid biopsies with circulating DNA (cfDNA). We recently developed nuclease-assisted minor-allele enrichment with probe-overlap (NaME-PrO), a single-step approach that removes WT-DNA prior to DNA amplification, following which current genomic analysis processes remain unchanged. NaME-PrO employs a double-strand DNA-specific nuclease (DSN) and overlapping oligonucleotide-probes interrogating multiple DNA targets. Following genomic-DNA denaturation, the temperature is lowered and the probes form transient double-stranded regions with their targets, and DSN is added to provide nuclease digestion to the selected sites. Mutations create mismatches that inhibit DSN digestion; thus, subsequent amplification yields DNA with alterations enhanced at multiple targets. In this manner, WT DNA at hundreds or thousands of DNA regions can be digested simultaneously. We also show that addition of organic solvents like DMSO in the NaME-PrO reaction enables DNA denaturation at lower temperatures that do not inactivate the enzyme (DSN). This enables DSN addition from the beginning of the reaction, enabling homogeneous, closed-tube application of the approach and avoids contamination-prone sample manipulations due to open-tube addition of DSN enzyme after denaturation. Using NaME-PrO ± DMSO we demonstrate several-hundred-fold multiplexed mutation enrichment in diverse human samples on multiple clinically relevant targets in 50-plex reactions. Application with targeted resequencing of tumor-circulating DNA demonstrated detection of mutations at the 0.01-0.1% levels with few sequence reads. In ongoing studies aiming to detect minimal residual disease using cfDNA in late-stage breast cancer, we used tumor exome sequencing to identify multiple clonal mutations for individual patients. We then performed multiplexed NaME-PrO to trace the "tumor fingerprint" in plasma. In ~50-plex reactions we showed that the majority (>60%) of the tumor-specific mutations were also detected in plasma. To evaluate the ultimate sensitivity of the method to identify such "tumor fingerprints," we applied NaME-PrO in serial dilutions of cfDNA from breast cancer patients into cfDNA from cancer-free, normal patients. We demonstrated that dilutions down to 1,000-10,000-fold, corresponding to mutations at <0.01% allelic frequency, lead to identification of at least one tumor-specific mutation in plasma. NaME-PrO can be combined with platforms that utilize molecular barcodes to provide "maximum efficiency sequencing," enabling sensitive detection of low-level mutations without requiring "deep sequencing" and excessive sequence reads.

#942

Sensitive detection of microsatellite instability (MSI) in tumors and liquid biopsies using nuclease-based enrichment.

Ioannis Ladas, Chen Song, Fangyan Yu, Ka Wai Leong, Katerina Troullinou, Matthew Kulke, Harvey Mamon, Mike G. Makrigiorgos. _Dana-Farber Cancer Institute, Boston, MA_.

The role of MSI in colorectal cancer (CRC) is well characterized, and colon tumors are classified as MSI-High or MSI-Stable by screening specific microsatellites. MSI-H compared to MSI-S is predictive for therapy outcome in chemotherapy and immunotherapy and has been associated with distinct characteristics and favorable results including better prognosis, a higher 5-year survival, and lesser metastasis. Several other tumors also present MSI changes reflecting mismatch repair deficiency. While tumor testing is the gold standard, a convenient approach to screen for MSI before and during cancer treatment is screening circulating DNA (liquid biopsy) using a blood draw, thereby interrogating 'systemic' MSI reflecting primary or secondary (occult) tumor status at the time of blood collection. However, presence of excess unaltered, wild-type DNA often masks alterations such as MSI. Using capillary electrophoresis or next generation sequencing for MSI detection presents challenges at low levels of MSI due to polymerase slippage ('stutter') that generate high false positive rates at positions of homo-polymers. We present a new approach for enrichment of altered micro-satellites prior to DNA-amplification thereby facilitating their detection. We recently developed nuclease-assisted minor-allele enrichment with probe-overlap, a single-step approach that removes WT-DNA and enriches mutation-containing alleles. Here we adapted NaME-PrO for detection of homopolymer indels, for MSI detection. The method employs a double-strand-DNA-specific nuclease and overlapping oligonucleotide-probes interrogating multiple micro-satellite targets. Following DNA denaturation, the probes form double-stranded regions with their targets, thereby guiding nuclease digestion to selected sites. Microsatellite indels create 'bulges' that inhibit digestion, thus subsequent amplification yields DNA with microsatellite alterations enhanced at multiple targets. The assay is applied at the genomic or circulating-DNA level prior to amplification, thereby avoiding polymerase-introduced 'stutter peaks' arising from WT DNA. Inclusion of organic solvents allows homogeneous application of the method in closed tube reactions. We validated the method by evaluating 5 MSI targets simultaneously, NR27, NR21, NR24, BAT25 and BAT26 using DNA from tumor biopsies and circulating-DNA from colorectal cancer patients. The technique enriched all altered targets and detected microsatellite alterations down to 0.01% altered allele frequency, thus improving detection sensitivity by >100-fold relative to current approaches. We anticipate application of this highly- multiplex-able method either with standard 5-plex reactions in conjunction with capillary electrophoresis or with NGS-based detection of MSI on thousands of targets to enable sensitive detection in tumors and liquid biopsies.

## ENDOCRINOLOGY:

### Novel Roles of Steroid Hormone Receptors

#943

SAR439859, an orally bioavailable selective estrogen receptor degrader (SERD) that demonstrates robust antitumor efficacy and limited cross-resistance in ER+ breast cancer.

Monsif Bouaboula, Maysoun Shomali, Jane Cheng, Natalia Malkova, Fangxian Sun, Malvika Koundinya, Zhuyan Guo, Stephane Poirier, Mikhail Levit, Dietmar Hoffman, Hui Cao, Laurent Bestret, Francisco Adrian, Christoph Winter, Youssef El-Ahmad, Sylvie Vincent, Frank Halley, Gary McCort, Laurent Schio, Vicky Richon, Hong Cheng, Karl Hsu, Chris Soria, Patrick Cohen, Joanne Lager, Carlos Garcia-Echeverria, Laurent Debussche. _Sanofi, Cambridge, MA_.

Despite resistance to endocrine therapies, estrogen receptor-positive (ER+) breast cancers (BC) still rely on ER. Eliminating ER by inducing its degradation with selective ER downregulators (SERD) should induce complete ablation of ER pathways. The clinical SERD fulvestrant, although it has demonstrated clinical benefits, is hampered by its poor drug properties and undesirable pharmacokinetics, undermining its optimal clinical activity. Therefore there is an unmet need for an improved therapy targeting ER. Here we describe SAR439859, a novel, nonsteroidal, orally bioavailable SERD currently in clinical trials. SAR439859 has a potent ER antagonist and ER degrading activity that translates in a robust inhibition of ER signaling in multiple ER+ breast cancer cell lines, including tamoxifen-resistant lines as well as cell lines harboring ER mutations. SAR439859 displays a broad and superior ER degradation activity across a large panel of ER+ cells. Importantly, based on its mechanism of action SAR439859 shows limited cross-resistance with other clinical SERD molecules. SAR439859 induces strong in vivo antitumor activity against a variety of BC cell lines and patient-derived xenografts, including models that harbor ERα mutations. The transcriptional profile analysis highlighted a crosstalk of ER mutant signaling and other oncogenic pathways. Interestingly, CDK4/6 inhibition by palbociclib induces partial activation of ER pathways as potential mechanism of tumor escape, which is completely abolished by the combination of SAR439859 with palbociclib. Finally, we demonstrate that SAR439859 in combination with palbociclib can lead to higher in vivo efficacy. This study highlights novel mechanism of ER degradation by SAR439859 that leads to profound inhibition of ER signaling as well as modulation of other oncogenic pathways and provides rationale for the ongoing clinical investigation of SAR439859 in ER+ breast cancer patients, both as a single agent and in combination with approved agents, such as CDK4 inhibitor.

#944

Glucocorticoid receptor (GR)-mediated activation of cyclic-adenosine monophosphate (cAMP) pathway gene expression following androgen receptor (AR) antagonism of prostate cancer.

Tiha M. Long, Eva Y. Tonsing-Carter, Wen-Ching Chan, Donald Vander Griend, Suzanne D. Conzen, Russell Z. Szmulewitz. _University of Chicago, Chicago, IL_.

Background: Despite the recent discovery of several potent androgen receptor (AR)-targeted therapies, progressive castration-resistant prostate cancer (CRPC) remains the second leading cause of male cancer deaths in the United States. While androgen-deprivation therapy (ADT) and AR antagonism continue to be the standard of care for progressive metastatic CRPC, identifying alternative molecular mechanisms is crucial. Our lab and others have shown that increased glucocorticoid receptor (GR) expression and activity can contribute to CRPC progression; this discovery has led to the development of selective GR modulators (SGRMs) as potential therapies for CRPC. Although GR clearly contributes to cell growth in AR-blocked PC cells, it is unclear whether GR serves solely to recapitulate AR-driven cancer-related mechanisms or if GR also regulates critical gene expression pathways unique to GR (and different from AR) allowing a novel growth advantage for tumor cells.

Methods: Primary PC and metastatic CRPC samples were normalized and utilized for gene expression analysis. LAPC4 and CWR-22Rv1 PC cell lines were treated with androgen +/- enzalutamide +/- dexamethasone and +/- SGRMs in order to activate or inhibit GR function in both AR active and AR-blocked conditions. For transcriptome analysis we performed next generation sequencing (NGS) of RNA followed by gene expression pathway analysis. To determine GR and AR chromatin binding sites, we performed GR and AR ChIP followed by DNA NGS.

Results: Transcriptome analysis of GR-mediated gene expression in LAPC4 PC cells following AR antagonism revealed that the majority of GR-regulated genes were unique to GR activity rather than overlapping with AR target genes.. Furthermore, GR and AR ChIP-sequencing indicated that while important overlap existed between GR and AR ChIP-seq, the majority of GR binding sites were distinct from AR binding sites. Analysis of the GR-unique transcriptomes of both enzalutamide-treated LAPC4 and CWR-22Rv1 cells predicted that cAMP-mediated signaling was activated by GR. Antagonism of GR with SGRMs blocked GR-unique cAMP pathway gene expression. Gene expression analysis of patient samples indicated that GR signaling is also activated in mCRPC and correlated with increased cAMP pathway gene expression.

Conclusions: These data suggest that GR activity in progressive CRPC results in novel GR-specific mechanisms of CRPC progression involving signaling pathways distinct from those driven by AR activation.

#945

Novel role for SIRT1 in non-canonical activation of AR signaling.

Shih-Bo Huang, Dinesh Thapa, Amanda Muñoz, Roble G. Bedolla, Ratnesh Srivastav, Rita Ghosh, Addanki P. Kumar. _University of Texas Health San Antonio, San Antonio, TX_.

Metastatic castrate resistance prostate cancer (CRPC) is most challenging to treat with a 5-year survival of less than 32%. Despite improved treatment landscape including use of second-generation androgen receptor (AR)-pathway inhibitors, emergence of acquired resistance is a significant problem facing both physicians and researchers. This is in part due to incomplete understanding of the compensatory molecular networks associated with therapeutic resistance. NAD+-dependent deacetylase, Sirtuin 1 (SIRT1), was traditionally known to modulate various cellular and pathological processes by regulating both histone and non-histone proteins. Furthermore, analysis of data from multiple prostate cancer cohorts indicated association of SIRT1 genetic alterations with poor clinical outcome. These findings suggest previously uncharacterized role for SIRT1 in prostate pathogenesis including therapeutic resistance. Based on these observations, we examined the role of SIRT1 in prostate pathogenesis using in vitro cell culture and in vivo preclinical models. Levels of SIRT1 are relatively higher in hormone-sensitive (LNCaP) cells compared to castrate resistant AR-positive (22Rv1) and androgen independent AR-negative (PC-3 & DU145) cells. Notably, stably knockdown (KD) of SIRT1 in LNCaP but not 22Rv1 cells showed downregulation of AR signaling pathway as evidenced by decreased levels and expression of subset of AR target genes (PSA, FKBP5), and secreted levels of PSA. Interestingly, SIRT1 KD reduced PSA reporter activity and secreted levels even under androgen depleted conditions with no significant effect on AR protein levels and nuclear translocation. Further, the observed SIRT1-mediated repression of AR signaling changes was independent of AR activation by androgen stimulation or suppression by enzalutamide. Biologically, these changes reflected in significantly decreased cell growth and migratory ability. Importantly, SIRT1 silenced LNCaP cells were more sensitive to growth suppression under conditions of androgen deprivation. Additionally, silencing of SIRT1 markedly inhibited tumor development in nude mice. Taken together, these findings led us to believe that SIRT1 could play a regulatory role in prostate pathogenesis in part by altering AR transcriptional network, independent of canonical AR signaling. Thus, our study identified previously uncharacterized role for SIRT1 in activation of AR signaling and promoting therapeutic resistance, offering avenues to develop SIRT1 or SIRT1 regulated pathways as novel therapeutic targets for PCA management. Supported by CPRIT RP150166 (APK) & CPRIT RP170345 (SH).

#946

Suppression of breast cancer metastasis and extension of host animal survival by a new adamantyl antiestrogen, K-07, in a preclinical breast cancer metastasis model driven by constitutively active mutant estrogen receptors.

Mary J. Laws,1 Sung Hoon Kim,1 Jian Min,1 Yuechao Zhao,1 Yvonne Ziegler,1 David Chu,2 Ben H. Park,2 John A. Katzenellenbogen,1 Benita S. Katzenellenbogen1. 1 _Univ. of Illinois at Urbana-Champaign, Urbana, IL;_ 2 _Johns Hopkins University School of Medicine, Baltimore, MD_.

Over one-third of human breast tumors that become resistant to endocrine therapies have acquired mutations in the estrogen receptor (ER alpha) that result in constitutive receptor activity in the absence of estrogen, and these tumors are much less sensitive to suppression by current standard-of-care antiestrogens such as tamoxifen and fulvestrant. We reported recently that the novel adamantyl antiestrogen, K-07, was effective in inhibiting the proliferation of breast tumor xenografts containing the two most frequent constitutively active mutant forms of the ER, Y537S ER and D538G ER, and that this antiestrogen reduced ER levels and the expression of ER-regulated genes (Zhao et al., Cancer Research 77(20):5602-5613, 2017). Because mutant ERs are usually enriched in recurrent, metastatic breast cancers compared to the primary breast cancer, we have now examined the effectiveness of compound K-07 in preventing the growth of breast cancer metastases and in extending survival in a metastatic tumor model. MCF-7 breast cancer cells expressing luciferase and Y537S ER or D538G ER (ca. 50% mutant ER and 50% wild type ER) were injected i.v. by the tail vein into NOD-SCID-gamma (NSG) female mice. The constitutively active ER-containing breast cancer cells established metastases in liver, bone and brain that increased in number and size over time (day zero – 80 days) as monitored by IVIS imaging and immunohistochemical (IHC) analysis. Daily oral treatment with K-07 (80 mg/kg orally for 30 days and then 40 mg/kg) versus oral vehicle greatly reduced the metastasis of mutant ER-containing breast cancer cells. Notably, mice with mutant ER-containing metastases treated with K-07 survived much longer than mice given daily control vehicle. In fact, by day 60, only 25% of vehicle treated mice with mutant ER breast cancers were alive whereas all K-07 treated D538G mice and 80% of Y537S mice were still alive. Hence, the findings indicate that this antiestrogen can reduce the in vivo metastasis of breast cancers driven by constitutively active mutant ERs and extend host survival. The findings suggest that the antiestrogen K-07 may be suitable for further translational and clinical examination of its efficacy in suppression of metastasis in patients with breast cancers containing constitutively active mutant ERs.

#947

Influence of abiraterone therapy on anti-tumor immunity in genetically engineered mouse prostate cancer models.

Eri Banno,1 Marco A. De Velasco,1 Yurie Kura,1 Naomi Ando,1 Noriko Sato,1 Masahiro Nozawa,1 Kazuhiro Yoshimura,1 Kazuko Sakai,1 Kazuhiro Yoshikawa,2 Kazuto Nishio,1 Hirotsugu Uemura1. 1 _Kindai University Faculty of Medicine, Osaka-Sayama, Japan;_ 2 _Aichi Medical University, Japan_.

Recent evidence has suggested that androgen deprivation therapies (ADTs) can influence tumor immune responses via androgen receptor (AR) regulation. On one hand, reports have indicated that certain ADTs can compromise T cell immune responses and enhance PD-L1 immune suppression, while others indicate that ADT can enhance anti-tumor responses via modulation of the apoptotic pathway. Abiraterone is a steroidal CYP17 inhibitor approved for the treatment of late-stage metastatic castration-resistant prostate cancer. In this study we use genetically engineered mouse models of prostate cancer (GEMMs-PCa) to investigate the antitumor activity of abiraterone and its influence on tumor immunity. In a mouse Pten-deficient prostate cancer model, chronic treatment with abiraterone acetate (40 mg/kg/d, 5 days on, 2 days off) reduced prostate tumor burden by 13.1% ± 9.0 (P=0.499) after four weeks of dosing and 30.5% ± 8.1 (P=0.0275) after eight weeks. Downregulation of classical mouse Ar-responsive genes (Fkbp5, Nkx3.1, Msmb and Timp4) confirmed the inhibition AR transcriptional activity after abiraterone therapy. In a model of advanced prostate cancer, driven by the conditional inactivation of Pten and Trp53, treatment with abiraterone after surgical castration modestly improved median overall survival from 7 days to 16 days vs. castration alone (P=0.240, n=8 mice/group). qRT-PCR-based analysis of a panel of 54 immune-responsive genes, revealed distinct expression signatures in abiraterone-treated tumors compared to tumors from orchidectomized mice. Relative to orchidectomized mice, tumors from abiraterone treated mice consistently demonstrated reduced mRNA levels of the T regulatory cell gene markers Cd4, Foxp3, Cd4, Tgfb1 and Il10. Furthermore, mRNA expression levels of representative immune checkpoint genes Cd274, Pdcd1lg2, Pdcd1 and Ctla4 were also lower in abiraterone treated mice. Follow-up immunohistochemical analysis showed a 1.8-fold increase of tumor infiltrating granzyme B-positive cells in tumors of mice treated with abiraterone compared to surgical castration. Our results show that abiraterone suppressed AR transcriptional activity and reduced tumor growth and progression in GEMMs-PCa. Our data also suggests that abiraterone induces lesser immunosuppressive responses than surgical castration and supports further investigation into developing rational combinations of ADT and immunotherapy in order to enhance therapeutic responses for patients suffering with prostate cancer.

#948

Preventing estrogen receptor alpha-positive breast cancer outgrowth with the use of hormone replacement therapy.

Anna G. Dembo, Emily S. Aledort, Geoffrey L. Greene. _University of Chicago, Chicago, IL_.

Menopause occurs in all women, usually between the ages of 45 and 55. As a result of menopause, women often experience undesirable vasomotor symptoms, which can be alleviated by hormone replacement therapy (HRT). The Women's Health Initiative (WHI) trial concluded that PremPro, a HRT formulation that combines conjugated equine estrogens (CE) with medroxyprogesterone acetate, increases the risk of breast cancer. Over the years, the number of women taking HRT has dramatically decreased due to the perceived risk based largely on the results of the WHI trial. Follow-up sudies suggest that breast cancer cases from PremPro treatment were primarily due to the outgrowth of occult tumors, not the formation of new disease. Duavee, a more recent form of HRT that combines CE and bazedoxifene (BZA), a selective estrogen receptor modulator (SERM) and degrader (SERD), has been approved by the FDA for treatment of moderate to severe hot flashes and to reduce the risk of osteoporosis. More importantly, this CE+BZA mixture not only relieves symptoms associated with menopause, but it also does not stimulate the breast or uterus. Several preclinical studies suggest that CE+BZA might be protective in the breast, however the mechanism of action of this new combination therapy is not known. Our goal, therefore, is to elucidate the underlying molecular mechanisms by which CE+BZA differentially affects estrogen receptor alpha (ERα) action in the mammary gland, using transcriptome and whole genome occupancy analysis in breast cancer cell lines. RNA-Seq and ChIP-Seq studies suggest that CE+BZA decreases pathways related to S-phase entry, cell cycle regulation and BRCA1. We are also studying the effects of CE+BZA on early mammary cancer progression in the estrogen-sensitive polyoma middle T antigen (PyMT) transgenic mouse model and have observed that treatment with CE+BZA delayed the onset of tumors and decreased their rate of growth. Mice treated with CE+BZA also survived longer. In addition, CE+BZA was able to decrease the rate of tumor growth in an ERα-positive patient-derived xenograft (PDX) mouse model. An improved understanding of the molecular mechanisms of CE+BZA action in hormone sensitive breast cancer cell and animal models should have important implications for women considering HRT.

#949

Progesterone receptor/IRS-1 cooperation promotes stem cell outgrowth and endocrine resistance in estrogen receptor-positive luminal breast cancer.

Amy R. Dwyer, Deepali Sachdev, Carol A. Lange. _University of Minnesota, Minneapolis, MN_.

Luminal breast cancers account for ~75% of all cases and while adjuvant hormone therapy targeting ERα has significantly improved overall survival for patients with ER+ tumors, acquired resistance remains a major clinical problem. Tamoxifen-resistant (TamR) breast cancer models show loss of IGF1R concomitant with increased insulin-induced growth, underpinned by insulin receptor (InsR) compensation for IGF1R loss. PR (gene name PGR) is an estrogen-regulated target gene whose expression is used as a clinical marker of ER activity. However, its de novo relevance to breast cancer is unclear. We have shown previously that post-translational modifications create unique PR species whose altered behavior drives an endocrine-resistant gene signature, in part by crosstalk with the IGFR pathway. We propose that phospho-PR target gene selectivity is mediated by cooperation between PR-B and InsR/IGF1R pathway components. Herein we show that phospho-PR-expressing T47D cells lose expression of IGF1R compared to control cells expressing WT PR-B. Furthermore, expression of the adapter protein IRS-1 requires PRB expression and these IRS-1+ cells were more sensitive to insulin in anchorage-dependent growth assays. Phospho-PR T47D cells exhibit increased ALDH+ and CD24-/CD44+ tumorsphere formation compared to wt PRB-expressing cells. Inhibitors targeting IRS-1 and InsR were used to test their requirement for tumorsphere growth and, surprisingly, IRS-1 perturbation reduced phospho-PRB but not wt PRB tumorsphere growth. Interestingly, IRS-1 replaces IGF1R in phospho-PRB-containing transcriptional complexes that are recruited to the CTSD promoter, which we previously identified as a phospho-PR target gene. Finally, breast cancer cells expressing phospho-PR species exhibited tamoxifen-resistant growth. Collectively our data suggest that phospho-PRB cooperates with IRS-1 downstream of the Ins/IGF1R system to promote outgrowth of endocrine-resistant cells that include ALDH+ stem-like cells capable of forming secondary tumorspheres in vitro. Targeting phospho-PR species in addition to the signaling components of PR-complexes may provide a means to block emergence of endocrine-resistant cancer cells during breast cancer progression.

## EXPERIMENTAL AND MOLECULAR THERAPEUTICS:

### Novel Therapeutic Agents and Combinations

#950

New strategy to sensitize pancreatic and bladder cancer to gemcitabine.

Simon Bayle, Francesca Vena, Andrii Monastyrskyi, William Roush, Derek Duckett. _The Scripps Research Institute, Jupiter, FL_.

Pancreatic Ductal Adenocarcinoma (PDAC), the major histological subtype of pancreatic cancer displays few symptoms for early detection and the majority of PDAC patients are diagnosed with advanced or metastatic disease. PDAC patients with advanced disease lack durable responses to chemotherapy and have a dismal prognosis. Gemcitabine is the cornerstone of care for advanced and metastatic PDAC. While the underlying mechanisms leading to gemcitabine resistance are likely to be multifactorial, there is a strong association between activating gemcitabine metabolism pathways and clinical outcome. Accordingly, strategies that lead to upregulation of signaling pathways and enzymes involved in generating the active metabolite may lead to improved outcome in PDAC.

We have developed a potent, selective, small molecule inhibitor of casein kinase-1 delta (CK1δ) and demonstrated that inhibition of CK1δ activity specifically compromises the growth, survival and invasion of breast cancer cells that overexpress CK1δ. Notably, our lead compound, SR-3029 provokes pre-clinical tumor regression of triple negative breast cancer (TNBC), including lung metastatic TNBC and basal-like PDX breast cancer models. We observed that CK1δ is upregulated in other cancers including bladder cancer and demonstrated that SR-3029 acts in synergy with DNA damaging cytotoxic agents. Notably, the combination of gemcitabine with SR-3029 resulted in synergistic antiproliferative activity and impaired colony formation in both pancreatic and bladder cancer cells. Mechanistic studies demonstrate that silencing CK1δ or inhibition of CK1δ activity by SR-3029 induces a significant upregulation of deoxycytidine kinase (DCK; rate limiting enzyme for gemcitabine activation) both at the RNA and protein levels, which results in increased cellular DCK and its active gemcitabine metabolite. We evaluated the effect of SR-3029 alone and in combination with gemcitabine in an orthotopic pancreatic tumor mouse model and observed much improved tumor growth inhibition with combination treatment, concomitant with increased expression of DCK.

In conclusion, our study suggests that CK1δ plays signaling roles in DNA metabolism and the combination of CK1δ inhibition with gemcitabine holds promise as a future therapeutic option for metastatic pancreatic cancer as well as other cancers.

#951

Direct small-molecule BAX activation in acute myeloid leukemia.

Denis Reyna,1 Thomas Garner,1 Andrea Lopez,1 Felix Kopp,1 Gaurav Choudhary,1 Ashwin Sridharan,1 Swathi-Rao Narayanagari,1 Kelly Mitchell,1 Baoxia Dong,1 Boris Bartholdy,1 Loren Walensky,2 Amit Verma,1 Ulrich Steidl,1 Evripidis Gavathiotis1. 1 _Albert Einstein College of Medicine, Bronx, NY;_ 2 _Dana-Farber Cancer Institute, Boston, MA_.

Resistance to apoptosis is a hallmark of human cancer. The BCL-2 protein family includes both pro- and anti-apoptotic proteins and has a central role in regulating mitochondrial apoptosis. Cancer cells most frequently ensure their survival and resistance to treatments by overexpression of anti-apoptotic BCL-2 proteins to maintain BAX and other pro-apoptotic members suppressed. Pro-apoptotic BAX is the cardinal executioner BCL-2-family member that, upon conformational activation and oligomerization at the mitochondrial outer membrane (MOM), causes permeabilization of the MOM and release of mitochondrial factors, e.g., cytochrome c, that activate the caspase cascade of apoptosis. Interestingly, the vast majority of cancer cells contain functional BAX in an inactive conformation or suppressed by anti-apoptotic proteins; mutations or alterations in BAX that may cause its inactivation to occur at a low frequency. Therefore, we hypothesized that induction of apoptosis by direct activation of BAX offers the possibility of a new anticancer strategy. Using a structure-based drug design approach we developed BTSA1, a pharmacologically optimized BAX activator that binds with high affinity and specificity to the N-terminal activation site (trigger site) and induces conformational changes to BAX, resulting in BAX mitochondrial translocation, oligomerization and mitochondrial dysfunction (Reyna et al., Cancer Cell 2017). BTSA1 induces activation of cytosolic BAX, leading to prompt and robust mitochondrial apoptosis in acute myeloid leukemia (AML) cell lines. The efficacy of BTSA1 is regulated by the availability of anti-apoptotic BCL-2 proteins to inhibit activated BAX and therefore, higher levels of cytosolic BAX monomer correlated with higher efficacy of BAX-mediated mitochondrial dysfunction. BTSA1-induced BAX activation promotes effectively apoptosis in primary AML samples and has significant antitumor activity in human AML xenograft models, increasing host survival. BTSA1 is orally bioavailable with excellent pharmacokinetics at therapeutically effective doses; it does not show any detectable toxicity in the hematopoietic system or other tissues. Importantly, BTSA1 is also effective in inducing apoptosis in leukemic stem cell-enriched fractions (CD34+CD38−) while sparing healthy counterparts. Consistent with their sensitivity to direct BAX activation, AML blasts and highly purified stem and progenitor cell populations from AML patients when compared to healthy counterparts displayed higher expression of BAX. Overall, our data provide proof of concept for BAX as a druggable target and demonstrate the therapeutic potential of direct BAX activation by BTSA1 as an effective treatment strategy in AML.

#952

MORAb-202, a folate receptor-alpha (FRA)-targeting antibody-eribulin drug conjugate (ADC), exhibits durable antitumor activity and payload-mediated bystander effects on the tumor microenvironment in triple-negative breast cancer.

Keiji Furuuchi,1 Xin Cheng,1 Katherine Rybinski,1 Tomoyuki Moriyama,2 James Fulmer,1 Chris Maddage,1 Andrew Milinichik,1 Keigo Tanaka,3 George Lai,2 Mary Lou Dula,1 Danielle Fernando,1 Luigi Grasso,1 Earl Albone,1 Toshimitsu Uenaka3. 1 _Morphotek, Inc., Exton, PA;_ 2 _Eisai, Inc., Andover, MA;_ 3 _Eisai, Co. Ltd, Tokyo, Japan_.

MORAb-202 is an antibody drug conjugate (ADC) consisting of farletuzumab (humanized anti-FRA mAb) and eribulin (as a payload, which are conjugated via a cathepsin B-cleavable linker. FRA is highly over-expressed in a large number of cancers including triple negative breast cancer (TNBC). Eribulin has been approved by Phase III study 305 for metastatic breast cancer patients who have previously received at least two chemotherapies. Moreover, exploratory subpopulation analysis from combined Phase III studies of 301 and 305 found that survival benefit by eribulin treatment was observed in HER2-negative and TNBC patients. Here we show the robust anti-tumor effects of MORAb-202 against patient-derived (PDX) TNBC xenograft models.

MORAb-202 demonstrated target selectivity and favorable cytotoxic drug delivery to tumors across a series of in vitro and in vivo studies. First, we found that MORAb-202 showed highly specific cytotoxicity on FRA positive cell lines at various IC50 values (0.001~23nM) according to their FRA expression levels, with little off-target killing (IC50 >100nM) on FRA negative cells. Moreover, it demonstrated significant in vitro bystander cytotoxicity (>140-fold) in FRA negative cells co-cultured with FRA positive cells when compared to mono-cultured FRA negative cells. In vivo, a single administration of MORAb-202 (1, 2.5, 5mg/kg) in FRA positive xenograft models showed dose-dependent antitumor activity with no observable toxicity. Single administration of MORAb-202 (5mg/kg) on high FRA-expressing TNBC PDX models showed long-lasting complete tumor regression (4/8), while near-MTD dose of eribulin (3.2mg/kg) showed minimum relative tumor volume of 96%. One dose of MORAb-202 (5mg/kg) elicits partial responses even on stroma-rich, low FRA-expressing (3.7%) TNBC PDXs. Immunofluorescent staining of tumor specimens both from the pre-treatment and five days post-treatment mice revealed that MORAb-202 achieved target specific engagement to FRA-positive regions in the tumor. Notably, MORAb-202 treatment diminished the network structure of cancer associated fibroblasts. This phenomenon was confirmed in an independent FRA-positive xenograft model, collected days 3, 5, 7 and 9 post-treatment of MORAb-202.

Finally, GLP toxicology studies in cynomolgus monkeys found no adverse effects on safety pharmacology endpoints (i.e., cardiovascular, CNS, respiratory function) in the intermittent repeated-dose (Q3W×2) of MORAb-202 nor changes in electrocardiography at doses up to 6 mg/kg. This dose is believed to be about five times higher than the projected effective dose based on PDX results and eribulin dose modelling.

Cumulatively, these data strongly support MORAb-202 as a promising ADC drug candidate for FRA-positive cancers.

#953

LRRC15 is a novel antigen in sarcoma and the therapeutic target of the antibody-drug conjugate (ADC) ABBV-085.

Eytan Ben-Ami,1 Ying Huang,1 Prafulla C. Gokhale,1 Benjamin Eschle,1 Lisa Durkin,2 Jonathan Hickson,3 Mien Sho,4 Susan Morgan-Lappe,3 Kurt Gish,4 Dominic W. Lai,4 Randy R. Robinson,4 Diane Hollenbaugh,4 Eric D. Hsi,2 Debra T. Chao,4 George D. Demetri,5 James W. Purcell4. 1 _Dana Farber Cancer Institute, Boston, MA;_ 2 _Cleveland Clinic, Cleveland, OH;_ 3 _AbbVie Inc, North Chicago, IL;_ 4 _AbbVie Biotherapeutics, Redwood City, CA;_ 5 _Dana Farber Cancer Institute/Ludwig Center at Harvard, Boston, MA_.

Background: Leucine rich repeat containing 15 (LRRC15) is a TGFβ-regulated structural protein that is highly expressed on cancer-associated fibroblasts (CAFs) in the stromal microenvironment of many solid tumors, as well as directly on cancer cells of mesenchymal origin. Soft tissue sarcomas (STS) and bone sarcomas (BS) represent a diverse family of mesenchymal malignancies that can develop at any anatomic site and that comprise more than 70 histopathologic subtypes. After screening LRRC15 expression across a variety of sarcoma histologies, we evaluated the antitumor activity of ABBV-085, an MMAE (monomethyl auristatin E) containing antibody-drug conjugate directed against LRRC15 (mouse, cyno, human), in patient-derived xenograft (PDX) models of selected sarcomas with varying levels of LRRC15 expression.

Methods: LRRC15 expression/intensity in sarcoma histologies was performed by immunohistochemical (IHC) staining (Leica Bond RX, Bond Polymer Refine Kit) with a human LRRC15 specific mouse IgG2b antibody. Based on LRRC15 expression levels, STS and BS tumor fragments (PDXs) were implanted into NSG mice. Mice with growing tumors were then selected to evaluate the in vivo efficacy of ABBV-085 monotherapy (6 mg/kg).

Results: LRRC15 expression was evaluated in 340 human sarcoma tumors representing a variety of STS and BS histologic subtypes. LRRC15 IHC expression was determined by scoring the percentage of positive cells, together with the intensity of staining (score of 0 for negative, 1 for weak, 2 for moderate, and 3 for strong staining), for the cancer cells and stroma, respectively. A stringent cut-off for strong LRRC15 positivity was defined as ≥2+ intensity in ≥50% of the cancer or stromal area. Strong LRRC15 expression was observed in several sarcoma subsets: 67% (14/21) of osteosarcoma (OS) tumor samples, 64% (23/36) of undifferentiated pleomorphic sarcoma (UPS), 18% (8/44) of leiomyosarcomas (LMS) and 17% (6/35) of liposarcomas (LPS). Significant antitumor activity including regressions and cures was induced by ABBV-085 in LRRC15-positive STS and BM PDX models, when compared with isotype-control treated mice. The ABBV-085-induced efficacy in osteosarcoma PDX models was superior to current standard-of-care therapies when dosed maximally in mice. In addition, ABBV-085 demonstrated efficacy in different LRRC15 positive STS subtypes including UPS, LMS and LPS. ABBV-085 was well tolerated with minimal to no body weight loss observed.

Conclusions: LRRC15 is highly expressed in the majority of human osteosarcomas and undifferentiated pleomorphic sarcomas, as well as in a range of other STS and BS subtypes. ABBV-085 demonstrates promising preclinical antitumor efficacy in LRRC15-positive PDX models of STS and BS. ABBV-085 is currently being investigated in an ongoing phase 1 study in soft tissue sarcomas (including undifferentiated pleomorphic sarcoma) and osteosarcoma.

#954

Predicting synergistic drug combinations and resistance mechanisms from genomic features and single-agent response profiles.

Matthew G. Rees, Lisa Brenan, Patrick Duggan, Cory M. Johannessen. _Broad Institute of MIT & Harvard, Cambridge, MA_.

Drug combinations promise to improve clinical responses and/or forestall drug resistance. To capitalize on this promise, we need to know which drugs to combine, and which patients to give them to based on the genetic or pathological features of their disease. However, progress towards this goal has been hindered by the infeasibility of performing comprehensive drug-combination studies across thousands of cellular contexts.

We hypothesized that the basal gene-transcription state of cancer cell lines, in concert with the cell-viability profiles of single-agent small molecules, might be leveraged to nominate specific synergistic drug combinations and identify mechanisms of drug resistance, eliminating the need to test all possible drug/drug combinations across cellular models. Specifically, we predicted that inhibiting the protein product of transcripts associated with drug resistance to a given small molecule might induce drug synergy.

To test this notion, we analyzed nearly 400,000 drug-sensitivity profiles in >800 cancer cell lines to identify candidate compound-gene pairs. We identified over 100 examples where outlier expression of a single transcript was correlated with resistance to a small molecule. Of these gene/drug pairs, 9 genes represented imminently druggable targets, including established clinically-relevant relationships between the alkylating agent temozolomide and MGMT expression, and between a subset of chemotherapeutics including paclitaxel and the efflux pump ABCB1.

Inhibition of candidate "co-targets", which included 3 previously characterized relationships and 6 novel relationships, resulted in cell-line-specific synergistic cell killing across multiple cell-line models. For validated compound-gene pairs, exogenous expression of the "co-target" was sufficient to confer resistance. For example, we found that high expression of MGLL, encoding monoglyceride lipase, was uniquely associated with lack of response to the histone lysine demethylase inhibitor GSK-J4. Endogenous or exogenous MGLL expression conferred resistance to GSK-J4, while MGLL-proficient cell lines could be sensitized to GSK-J4 up to 50-fold by co-treatment with an irreversible MGLL inhibitor.

These initial studies highlight the potential of integrating basal gene expression features with small-molecule response to nominate rational candidates for drug combinations. As public repositories of single agent response data from diverse cellular contexts continue to expand, so too will our repertoire of therapeutic combinations. Moreover, this approach permits the parallel identification of genomic features that indicate which patient populations are most likely to benefit from such combinations.

#955

**Combined inhibition of MEK and mTOR pathways is effective in** NRAS Q61K **mutant small cell lung cancer.**

Atsuko Ogino,1 Mika Lin,1 Prafulla C. Gokhale,2 Margaret K. Wilkens,2 Jihyun Choi,1 Antonio Calles,1 Man Xu,2 Marzia Capelletti,1 Geoffrey Oxnard,1 Nathanael S. Gray,1 Paul Kirschmeier,2 Pasi A. Jänne1. 1 _Harvard Medical School, Dana-Farber Cancer Institute, Boston, MA;_ 2 _Experimental Therapeutics Core and Belfer Center for Applied Cancer Sciences, Boston, MA_.

Background: Small cell lung cancer (SCLC) is a heterogeneous disease, consisting of a phenotypically diverse population of cells, often described as neuroendocrine (NE) and non-NE. Though previous studies suggest that aberrant RAS signaling is involved in the transition from NE to non-NE profile, the characteristics of SCLC with RAS mutations have not been fully examined due to the rarity of the occurrence and the lack of resources available to study its biology.

Methods: We created a cell line (DFCI168) from the pleural effusion of the SCLC patient with a rare NRASQ61K mutation. A patient-derived xenograft (PDX) model from this patient was successfully established by subcutaneous implantation of cancer cells in NSG mice. The efficacy of MEK and mTOR inhibitors was evaluated in DFCI168 and the commercially available lung cancer cell lines with NRAS Q61 mutations (SW1271, H1299, H2087, H2347). The Chou-Talalay combination index was used to evaluate drug synergy. Retroviral NRASQ61K transduction was performed on a classical SCLC cell line Glc16 to examine whether NRAS activation leads to acquisition of non-NE features.

Results: DFCI168 showed non-NE/mesenchymal features both morphologically and biologically, which is exemplified by an elongated spindle shape, mesenchymal marker (VIM) expression and silenced NE markers (SYP, ASCL1) expression. NRASQ61K transduction on Glc16 achieved the transition from NE to non-NE/mesenchymal phenotype. Although DFCI168 showed marked sensitivity to MEK inhibitors both in vitro (trametinib IC50=2nmol/L, selumetinib IC50=50nmol/L) and in vivo, the tumors regrew after withdrawal of treatment with trametinib in xenograft mice, and caused sustained activation of the mTOR pathway during MEK inhibitor treatment in vitro. The combination of MEK and mTOR inhibitors (trametinib and TORIN2) prevented mTOR activation at 100nmol/L and yielded synergistic effects (CI=0.61). The commercially available lung cancer cell lines (SW1271, H1299, H2087, H2347) also responded synergistically to the trametinib/TORIN2 combination except H1299, a cell line with a known PTEN promoter methylation.

Conclusion: We demonstrate that the oncogenic NRAS mutation defines a distinct molecular subset of SCLC, and is sufficient to cause the phenotypic switch from NE to non-NE/mesenchymal profile. Such tumors are likely to benefit from the dual targeted therapy with MEK and mTOR inhibitors. Our findings highlight the future potential of targeted therapies for SCLC patients with NRAS activating mutations.

#956

Systematic identification of the actionable kinase dependencies of chemotherapy-resistant triple-negative breast cancer.

Jean-Philippe F. Coppe, Pan Bo, Carolien van der Borden, Nina Koemans, Changjun Wang, Denise Wolf, Christina Yau, Sietske Bakker, Marij Hartog, Miki Mori, Ana Ruiz-Saenz, Zhongzhong Chen, Aleksandra Olow, Pei Rong Evelyn Lee, Laura van 't Veer. _University of California at San Francisco, San Francisco, CA_.

Triple-negative breast cancer (TNBC) accounts for approximately 15% of all breast cancer cases, with over 35,000 newly diagnosed women per year in the USA. TNBC patients are at high risk of recurrence, and neoadjuvant standard chemotherapy leads to pathologic complete response in only about 30% of patients. No targeted therapy has yet been conclusively established to improve outcome. The management of TNBC will significantly improve once mechanisms responsible for TNBC resistance to chemotherapies are identified. Here, we applied a new functional proteomic strategy to reveal which (dys)regulated phospho-signaling circuits are the effective dependencies of chemotherapy-resistant TNBC cells. The high-throughput kinase activity-mapping (HT-KAM) assay is our new screening technology to assess the catalytic activity of many kinases in parallel. HT-KAM relies on collections of biologic peptide probes that are computationally derived from PhosphoAtlas (Olow and Chen et al., Cancer Research 2016) and are physically used as combinatorial sensors to measure the activity of kinase enzymes in biologic extracts. The HT-KAM system provides access to a vast, untapped resource of meaningful measurements, whether readouts are interpreted irrespective of which enzymes phosphorylate which probes, or analyzed to convert global phospho-signatures into functional profiles of kinase activities. Kinome maps reveal how signaling networks are rewired by drug interventions in the context of different cellular backgrounds or exogenously mutated proteins/pathways, and provide insight into potentially targetable pathways. We previously successfully showed that the HT-KAM platform identifies new, actionable kinases responsible for intrinsic or acquired targeted-therapy resistance in BRAFV600E colorectal cancer cells and melanoma tumors from patients. To study TNBC, we used a 615-peptide sensor library that supports the mapping of >100 kinases and represents >900 functional kinase-substrate interactions relevant to tumor biology, and matches hundreds of druggable components of signaling circuits. We characterized the phospho-catalytic signatures of 10 TNBC cell lines, either untreated or treated with chemotherapeutic drugs (5-FU, carboplatin, doxorubicin) at IC50 concentrations. We also included a panel of 8 luminal BC cells. Based on our advanced preliminary data, we anticipate showing a comprehensive map of the oncogenic kinome of TNBC, and how it rewires its signaling network in response to chemotherapies. The most hyperactive and conserved pathways establish a priority map of strategic kinase hot spots to explore as therapeutic candidates. We find that chemotherapy-induced kinases such as SRC, AKT, CHEK, or regulating NFkB or PARP pathways can be inhibited to improve therapeutic sensitivity in TNBC.

## IMMUNOLOGY:

### Engineering Immunotherapies for Anticancer Activity

#957

Design of CD19-4-1BBL, a novel CD19-targeted 4-1BB ligand for combination therapy with CD20 T-cell bispecific antibodies and CD20 antibodies.

Wei Xu,1 Johannes Sam,1 Mario Perro,1 John Challier,1 Christina Claus,1 Stanford Chen,1 Claudia Ferrara Koller,1 Michael Mølhøj,2 Stella Tournaviti,2 Marina Bacac,1 Tom Moore,1 Christian Klein,1 Pablo Umana1. 1 _Roche Innovation Center Zurich, Schlieren, Switzerland;_ 2 _Roche Innovation Center Munich, Penzberg, Germany_.

Co-stimulation through 4-1BB has shown promising anti-tumor activity in preclinical models, but the development of 4-1BB agonistic antibodies in the clinic has been hampered by Fc-mediated liver toxicity. Here, we describe a novel CD19-targeted 4-1BB ligand designed to deliver a safe but potent 4-1BB agonist to effector T and NK cells with the goal to improve treatment of B cell malignancies. The antibody fusion protein is composed of split trimeric 4-1BB ligands and a tumor antigen targeting moiety recognizing CD19 fused to a silent Fc part (CD19-4-1BBL). The construct is devoid of FcgR-mediated crosslinking responsible for Fc-mediated toxicity and reintroduces 4-1BB hyperclustering upon binding to CD19 on B cells exclusively. In mice and cynomolgus monkeys, CD19-4-1BBL shows IgG-like pharmacokinetic properties. When cross-linked via CD19 on Non-Hodgkin Lymphoma cell lines or normal B cells, CD19-4-1BBL is biologically active in co-stimulating T cells. As 4-1BB is an inducible protein on activated T cells and NK cells, we combined CD19-4-1BBL with a T cell bispecific Ab targeting CD20 (CD20-TCB) to provide initial T cell activation while engaging with CD19+CD20+ tumor cells. In vitro, pre-treatment with CD20-TCB mediates the clustering of CD19-4-1BBL molecules to the interaction synapse of T cells and tumor cells. Live imaging revealed that the addition of CD19-4-1BBL induces significantly prolonged T cell-tumor contact (>30 min), leading to fast and efficient killing of tumor cells. In vivo in WSU-DLCL2-bearing human stem cell engrafted NSG mice (HSC-NSG) mice, CD20-TCB treatment quickly up-regulates 4-1BB on activated T cells, resulting in tumor growth inhibition. The combination of CD19-4-1BBL and CD20-TCB (used at a suboptimal dose) synergizes to eradicate the tumor completely. The combination induces strong T cell infiltration into the tumor, accompanied by an elevated CD8/Treg ratio, as compared to the monotherapies. Similarly, the combination also induces complete remission in a "difficult-to-treat" Nalm6 tumor model associated with low and patchy CD20 expression, mimicking patterns seen in the ABC subtype of DLBCL. Finally, the combination of CD19-4-1BBL and the ADCC-enhanced Type II CD20 antibody obinutuzumab induces complete tumor remissions in WSU-DLCL2 tumor-bearing HSC-NSG mice, confirming effective 4-1BB co-stimulation on NK cells. Taken together, tumor-targeted cross-linking of 4-1BB mediated by CD19-4-1BBL provides safe and efficient co-stimulation of T cells that are pre-activated by a TCB, or of NK cells pre-activated by an ADCC mediating antibody. This novel and effective combination immunotherapy warrants clinical investigation and offers possible "chemo-free" treatment of B cell malignancies.

#958

Anti-GD2 chimeric antigen receptor T cells as a potent immunotherapy regimen in xenograft models of histone 3 K27M mutant diffuse midline glioma.

Christopher W. Mount, Robbie Majzner, Shree Sundaresh, Evan P. Arnold, Meena Kadapakkam, Samuel Haile, Louai Labanieh, Pamelyn Woo, Skyler P. Rietberg, Hannes Vogel, Michelle Monje, Crystal L. Mackall. _Stanford University, Stanford, CA_.

Histone 3 K27M mutated diffuse midline glioma (H3K27M DMG) is a universally fatal pediatric brain tumor. Despite improved understanding of the molecular origins of this disease, translations to improvement in clinical outcomes have yet to materialize. To date, there has been little target exploration for immunotherapy applications in H3K27M DMG. Here we report substantial preclinical efficacy of chimeric antigen receptor (CAR)-bearing T cells targeting the disialoganglioside GD2 in patient-derived orthotopic xenograft models of H3K27M DMG. GD2 is highly and uniformly expressed in patient-derived H3K27M DMG cultures, and in vitro assays demonstrate substantial target-dependent cytokine generation and target cell killing. Two H3 WT pediatric high-grade gliomas (HGG) cultures do not express significant levels of GD2, and the H3K27M mutation is associated with increased synthesis of ganglioside pathway synthesis enzymes, suggesting expression of the GD2 antigen is driven by H3K27M-induced transcriptional dysregulation. Single-dose systemic administration of GD2-4-1BBz-CAR T cells in multiple orthotopic xenograft models of H3K27M DMG achieves potent and lasting antitumor efficacy within a 4-week period of administration compared to a CD19-4-1BBz-CAR T cell control cohort. In brainstem xenografts, GD2-CAR T cell therapy cleared established tumor as measured by in vivo bioluminescence imaging and follow-up histology, and significantly prolonged survival. Using a fluorescently-labeled GD2-CAR, we demonstrate tumoricidal GD2-CAR T cells infiltrating the brain parenchyma and sparing of local neurons during tumor clearing. Treatment-associated neuroinflammation in brainstem xenografts is accompanied by transient ventriculomegaly due to swelling of the targeted tumors, which is accompanied by transient weight loss followed by full recovery. Similar swelling in thalamic xenograft models is lethal in a significant fraction of animals, presumably due to uncal herniation. These results demonstrate potent antitumor effects of GD2-directed CAR T cells in H3K27M DMG, and suggest an important role for neuroanatomic site in neurotoxicity associated with CAR T cell therapy. If these results are predictive of human response, GD2-directed CAR T cell therapy in the setting of careful clinical management could have a transformative impact upon H3K27M DMG outcomes.

#959

Extracellular matrix-binding immunotherapies show enhanced antitumor efficacy and reduced adverse events.

Jun Ishihara, Ako Ishihara, Kazuto Fukunaga, Lambert Potin, Peyman Hosseinchi, Melody A. Swartz, Jeffrey A. Hubbell. _University of Chicago, Chicago, IL_.

Immune checkpoint inhibitors (CPI) and co-stimulatory agonist antibody therapies have exhibited considerable antitumor effects, but instances of severe side effects have been previously reported. We hypothesized that conjugation of an extracellular matrix (ECM)-binding peptide derived from placenta growth factor-2 (PlGF-2123-144) to CPI (combination of anti-CTLA4 antibody (αCTLA4) and αPD-L1) or agonistic αCD40 would accomplish local cancer immunotherapy, minimizing the antibodies' systemic exposure. Here, we show that enhanced tissue retention and lower antibody concentrations in blood plasma resulted from PlGF-2123-144 conjugation when the antibodies were administered peri-tumorally compared to unmodified antibodies administered by the same route at the same dose. This resulted in decreases in systemic cytokine release in the blood serum, and in liver tissue damage, as was assessed both biochemically and histologically, indicating lower systemic side effects after CPI and αCD40 therapies. Particularly, we show that PlGF-2123-144 conjugation reduces the risks of αPD-L1-induced autoimmune diabetes in the male nonobese diabetic mouse. Because PD-1/PD-L1-inhibition induced diabetes has been reported in the clinic, this supports the idea that PlGF-2123-144 conjugation to antibodies may reduce adverse events. This observation with localized PlGF-2123-144-antibodies could be an important breakthrough in treating patients who have discontinued immunotherapy due to the associated side effects. Regarding efficacy, we show that either PlGF-2123-144-CPI treatment or PlGF-2123-144-αCD40 treatment showed significantly higher antitumor activity compared to unmodified forms in multiple cancer models: B16F10 and Tyr:Cre-ER+/LSL-BrafV600E/Ptenfl/fl melanoma, CT26 colon carcinoma, and MMTV-PyMT breast cancer. Local injections of PlGF-2123-144-CPI increased the number of activated CD8+ and CD4+ T cells within the tumor, compared to normal CPI. PlGF-2123-144-αCD40 treatment increased the frequency of activated dendritic cells in the tumor-draining lymph node, activated CD8+ T cells within the tumor, and secretion of endogenous antibodies against tumor cells. This immune cell activation resulted in growth suppression of a distant tumor. These data suggest the feasibility of treating patients with oligometastatic tumors by administration of PlGF-2123-144\- PlGF-2123-144-CPI or PlGF-2123-144-αCD40 into one accessible metastasis. Our data suggest that local injection of either PlGF-2123-144-CPI or PlGF-2123-144-αCD40 efficiently activates tumor antigen-specific T cells while maintaining systemic immune homeostasis by avoiding influencing nontumor antigen-specific T cells. This simple approach of engineered ECM-binding immunotherapy antibodies may be clinically useful.

#960

Defined cell composition and precise control over JCAR017 dose enables identification of relationships between chimeric antigen receptor T cell product attributes, pharmacokinetics, and clinical endpoints in NHL.

Ryan P. Larson, Rachel Lower, Todd DeVries, Yue Jiang, Ronald J. Hause, Rich Getto, Brian Christin, Nathan K. Yee, Michael A. Bowen, Clinton Weber, Daniel Li, Tina Albertson, Claire Sutherland, Christopher G. Ramsborg. _Juno Therapeutics, Seattle, WA_.

JCAR017 is a CD19-directed 41BB chimeric antigen receptor (CAR) T cell product administered in a defined composition at a precise dose of CD8 and CD4 CAR T cells. JCAR017 manufacturing employs process controls that enable precise independent control of the infused dose of CD8 and CD4 cells. Preliminary safety data from JCAR017 administered in relapsed/refractory B cell non-Hodgkin lymphoma (NHL) demonstrated lower rates of cytokine release syndrome (CRS) and neurotoxicity (NT) compared to those reported for other CD19-directed CAR T cell therapies with heterogeneity in total infused dose and CD8 and CD4 composition. Multiple patient factors and blood biomarkers have been identified that correlate with JCAR017 CAR T cell in vivo expansion, antitumor activity, and toxicity (Heipel M et al. and Siddiqi T et al. ASH 2017). However, little is known regarding the contribution of CAR T cell product differentiation state to pharmacokinetics (PK), clinical outcome, or toxicity. Product characterization is executed on JCAR017 to define the CAR T cell memory phenotype composition and antigen-specific function. Product attributes were assessed for relationships with clinical response, safety, and PK through univariate, multivariate, and machine learning-based analyses. JCAR017 memory T cell composition demonstrated strong relationships with cytokine production profile observed following in vitro CD19 stimulation of the CAR T cell drug product. Specifically, products with elevated frequencies of CCR7+ central memory T cells exhibited increased production of IL-2 (Spearman ρ=0.55, P<0.0001), whereas patient drug product with increased frequencies of effector differentiated T cells demonstrated increased production of IFNγ (ρ=0.51, P<0.0001) and IL-13 (ρ=0.45, P<0.0001). The correlations between CAR T cell drug product memory phenotype and function translated to positive correlations between central memory subset composition and peak in vivo CAR T cell expansion (ρ=0.42, P=0.002) and progression-free survival (Kaplan-Meier survival estimate, P=0.0164). In addition, an increased frequency of a central memory subpopulation in drug product demonstrated relationships with CRS (P=0.0069) and severe NT (P=0.0014) events. The phenotype to functional links described above offer insights as to how specific memory subpopulations contribute to the complex CAR T cell mechanism of action. CAR T cell products with increased CCR7+ central memory composition demonstrated increased peak CAR T cell expansion and persistence, suggesting less differentiated CAR T cells contribute to PK and progression-free survival. These findings can be used to further define the next generation of gene-engineered T cell products (e.g., a fixed dose of CCR7+CAR+) that will decrease the dose-to-dose variability.

#961

Intralesional injection of anti-PD-L1 (pembrolizumab) results in increased T cell infiltrate in high risk DCIS.

Michael J. Campbell, Emma McCune, Jennifer Bolen, Scott VandenBerg, Jo Chien, Jasmine Wong, Laura Esserman. _UCSF, San Francisco, CA_.

Background. Accumulating evidence shows a correlation between tumor-infiltrating lymphocytes (TILs), in particular the presence of CD8+ T cells, in cancer tissue and favorable prognosis in various malignancies. Less is know about the role of the immune microenvironment in the context of pre-malignant lesions such as ductal carcinoma in situ (DCIS). In a previous study of women with DCIS, we observed that high numbers of activated CD8+ T cells within the DCIS lesions were associated with good outcomes while low numbers associated with high risk features and poor outcomes. These results suggest that manipulating the immune microenviroment in DCIS, for example via checkpoint blockade, could potentially alter disease progression. Methods. We recently initiated a phase 1 pilot study to investigate changes in the immune microenvironment of high risk DCIS after intralesional injection of anti-PD-L1 (pembrolizumab). Study participants were given 2 intralesional injections of pembrolizumab 3 weeks apart with surgery approximately 3 weeks after the 2nd dose. The study started with a dose of 2 mg/ml (1/100 of the standard 200 mg/ml iv dose), then escalated to 4mg/ml and 8 mg/ml, with 3 patients at each dose. FFPE tissue samples from pre-treatment biopsies and post-treatment surgical resections were immunostained using Opal reagent kits (Perkin Elmer) on a fully automated Ventana Discovery platform, imaged with a Vectra automated imaging system, and analyzed with inForm software (Perkin Elmer). A 7-plex panel, consisting of mAbs to CD3, CD8, CD68, PD-1, PD-L1, Ki67, and cytokeratins, was used. An algorithm for tumor/stroma segmentation developed in inForm was used to randomly select 7-10 high power fields (hpfs) for imaging. Cell phenotype maps were generated for each of these hpfs for each sample. Cell densities were determined per area of stroma, DCIS, or total tissue and averaged across all hpfs for a given case. Results. We are completing our 2nd cohort of patients, and there have been no adverse clinical events. The injections are easily administered and well tolerated. By MRI imaging, there was no change in the size of lesions after the treatment. All patients have proceeded to surgical resection. Following intralesional pembrolizumab injection, multiplex fIHC analyses indicated an increase in CD8+ T cells, which in some areas was striking. This increase was mainly observed within the epithelial DCIS regions, as opposed to a lesser increase in stromal CD8+ T cells. We also observed an increase in CD68+ macrophages. Conclusions. We have demonstrated the safety and feasibility of intralesional injection of an immune checkpoint inhibitor (pembrolizumab) in high risk DCIS. Further, we have shown that this immunotherapeutic intervention results in a change in the immune microenvironment, with an increase in both T cells and macrophages, even at a dose that is 1/100 of the standard intravenous clinical dose.

#962

Testing T cell co-potentiation as an antitumor therapeutic strategy in humanized mouse models.

Alfreda D. Nelson,1 Laura Elsbernd,2 Adam G. Schrum,1 Diana Gil Pages1. 1 _University of Missouri, Columbia, MO;_ 2 _Mayo Clinic College of Medicine, Rochester, MN_.

We recently described the concept of T cell co-potentiation as means to target antigen recognition by T cells to achieve antitumor immunotherapy in mouse models. Normally, T cell antigen receptor (TCR) engagement by a strong immunogenic antigen induces a CD3 conformational change that exposes a proline-rich sequence (PRS) in the cytosolic domain of CD3 epsilon (CD3e) to dock cytosolic proteins and contribute to the assembly of the signaling cascade that activates T cell immune function. While induction of CD3 conformational change is required by T cells to achieve productive TCR/CD3 signaling, this molecular event fails to occur when the TCR interacts with poorly immunogenic antigens, like most tumor-associated antigens (TAAs). We found that CD3 conformational change can be exogenously induced on T cells using monovalent Fab fragments (Mono-Fabs) specific for the CD3 complex. Anti-CD3 Mono-Fabs expose the CD3e PRS, but fail to crosslink the TCR/CD3, turning them incompetent to trigger T cell activation in the absence of additional stimulation. However, if TCR has bound a weak antigen, one that fails to induce CD3 conformational change intrinsically, then anti-CD3 Mono-Fabs enable TCR/CD3 signaling, and as a result co-potentiate T cell responses to weak antigenic stimulation. Using a mouse model for melanoma (B6 mice and B16-F10 melanoma cell line), we found that treatment of mice with anti-CD3e Mono-Fabs improved antitumor T cell responses against melanoma in an antigen-dependent manner, and dramatically improved the outcome of complementary T cell immunotherapies. Based on these observations, we hypothesize that T cell co-potentiation using anti-human CD3 Mono-Fabs can be exploited as a novel immunotherapy for cancer patients. We have recently identified one anti-human CD3 Mono-Fab that is capable to induce CD3 conformational change in human T cells and allows the co-potentiation of T cell response to weak antigenic stimulation. Here we present data from human T cells and humanized mouse models using this Mono-Fab that support the potential therapeutic value of exploiting T cell co-potentiation to treat cancer patients.

#963

Screening of neoantigen-specific T cells in head and neck cancer and establishment of T-cell receptor-engineered T cells with cytotoxic reactivity.

Lili Ren,1 Tatsuo Matsuda,1 Boya Deng,1 Kazuma Kiyotani,2 Taigo Kato,1 Jae-Hyun Park,1 Tanguy Seiwert,1 Everett Vokes,1 Nishant Agrawal,1 Yusuke Nakamura1. 1 _The University of Chicago, Chicago, IL;_ 2 _Japanese Foundation for Cancer Research, Tokyo, Japan_.

Neoantigen-reactive T cells are considered to be crucial mediators for anti-tumor activity in adoptive T cell transfer (ACT) immunotherapy although the efficacy of ACT using in vitro expanded tumor-infiltrating T lymphocytes (TILs) has been limited. Since characterization of T cell receptor (TCR) repertoires of TILs before and after in vitro expansion has not well investigated, we in present study performed whole exome sequencing (WES) and transcriptome analysis, and selected candidate neoantigen epitopes to induce cytotoxic T lymphocytes (CTLs) in 20 patients with head and neck cancer (10 frozen tissues and 10 freshly resected tumors). 36 potential neoantigen peptides including missense somatic mutations were examined for induction of neoantigen-reactive cytotoxic T cells in vitro using patients' derived dendritic cells. We have so far confirmed three neoantigen-reactive T cells and obtained the sequence information of a pair of TCR alpha and beta chains, which were identical to those identified in resident dominant TILs in the original tumors. We cloned the TCR genes into T lymphocytes and constructed the neoantigen-reactive T-cell receptor-engineered (TCR-engineered) T cells, which showed HLA-restricted neoantigen-reactive cytotoxic activity. In addition, we compared the TCR composition of TILs before/after in vitro expansion in 10 fresh head and neck cancers, and found that TCR clonotypes in original TILs and in vitro expanded TILs are drastically different except for those between expanded CD8 cells and resident TILs in three tumors, which had unusually high mutational burden due to mutations in the mismatch repair genes (MSH2, MSH6) or a DNA polymerase (POLE) gene. Our data also suggest that some expanded TILs are likely to be cancer-specific T cells and may be good cell sources for ACT immunotherapy and that the mutational load might be a good biomarker to screen effective TIL treatment. We demonstrate establishment of an effective and rapid protocol to generate neoantigen-specific T cells and to identify neoantigen-specific TCRs for individual patients, which makes TCR-engineered T cells applicable for the clinical use.

## MOLECULAR AND CELLULAR BIOLOGY / GENETICS:

### Cell Signaling Pathways

#964

Genome-wide CRISPR screen identifies potential therapeutic combination of EGFR and FGFR inhibitors in oral cancer.

Megan Ludwig, Andrew Birkeland, Sai Nimmagadda, Sue Foltin, Aditi Kulkarni, Hui Jiang, Thomas Carey, John Chad Brenner. _University of Michigan, Ann Arbor, MI_.

Oral squamous cell carcinoma (OSCC) has remained a disease with poor survival for decades. Current treatment options such as targeting EGFR has had limited success. We hypothesize that inhibiting compensatory pathways in combination with EGFR signaling may result in greater efficacy. To identify synergistic combinations with EGFR-targeted therapy, we introduced Genome-scale CRISPR-Cas9 KnockOut (GeCKO) libraries into OSCC cell lines. CRISPR-Cas9 generates individual genetic knockouts through targeted gene editing. We used a pool of CRISPRs targeting over 18,000 genes to perform genome-scale screening for drivers of sensitivity to EGFR-targeted therapy. Upon selection of the OSCC GeCKO pool, we identified gene knockouts in the FGFR pathway that increased sensitivity to the EGFR inhibitor gefitinib. Using resazurin viability assays we tested combinations of EGFR and FGFR inhibitors in 14 OSCC cell lines. Six/14 (43%) of the cell lines were responsive to the combination, indicating that the FGFR pathway is an alternate mechanism of resistance to EGFR-targeted therapy in some tumors. We advanced an OSCC cell line to test in vivo by establishing subcutaneous cell line xenografts in mice. After treatment for 21 days, the mice receiving the combination of EGFR and FGFR inhibitors, gefitinib and BGJ398, had significantly smaller tumors than mice receiving vehicle or either monotherapy. These results suggest targeting EGFR in combination with its compensatory pathway such as FGFR signaling could be effective in vivo. While OSCC remains a common and frequently lethal cancer, there is great potential for the development of novel personalized targeted therapies. Here, we describe the use of genome-wide CRISPR-Cas9 library to discover the synergistic combination of EGFR and FGFR inhibition. Further investigation suggests the FGFR pathway is a common compensatory mechanism to EGFR inhibition, and that targeting EGFR and FGFR in combination has efficacy in an in vivo model. Overall, targeting compensatory mechanisms in combination with EGFR-targeted therapy could generate novel, beneficial treatment plans for patients with OSCC.

#965

Deleterious effects of MAPK pathway hyperactivation in BRAF-mutant melanoma.

Grace P. Leung, Tianshu Feng, Matthew D. Shirley, Frederic D. Sigoillot, David A. Ruddy, Alyson K. Freeman, Mariela Jaskelioff, Darrin D. Stuart. _Novartis Institutes for BioMedical Research, Cambridge, MA_.

The most frequent genetic alterations in melanoma are gain-of-function mutations in BRAF, which result in addiction to RAF-MEK-ERK signaling. Given the centrality of this pathway in many cancers, inhibitors have been developed against various nodes in this signaling axis. Despite the success of these inhibitors in treating BRAFV600E mutant tumors, a major problem is the inevitable emergence of drug resistance. The mechanisms of acquired resistance often include reactivation of the MAPK pathway, thus efforts to combat resistance have used vertical combination strategies to inhibit multiple nodes of the pathway. Previous work from our group showed that vemurafenib-resistant melanoma tumors displayed increased levels of MAPK signaling as indicated by elevated phospho-ERK. Strikingly, cell lines derived from these resistant tumors were addicted to vemurafenib, and removal of drug caused loss in cell viability. Consistently, in vivo, the tumors regressed upon cessation of drug treatment, which was accompanied by elevated MAPK signaling. Recent studies have reported similar results for in vitro models of melanoma resistant to MAPK inhibitors, which are addicted to drug in an ERK2-dependent manner. Together these data suggest that extreme levels of MAPK signaling are not tolerated by tumor cells. To further characterize this phenomenon, we generated models of inducible MAPK hyperactivation in BRAFV600E melanoma cells. Using these model systems, we confirmed that increasing the levels of MAPK signaling can hinder tumor cell proliferation, and demonstrated that MAPK inhibitors can rescue this phenotype. Intriguingly, the effect of MAPK hyperactivation varied depending on the RAS/RAF mutation status of the tumor cells. Upon induction of MAPK hyperactivation in sensitive cells, there was no cell cycle arrest, but instead cell death occurred, which was accompanied by activation of caspases. On a global level, MAPK hyperactivation resulted in transcriptome changes that were opposite to those produced by treatment with an ERK inhibitor. This phenomenon of MAPK hyperactivation-induced cell death was also observed in vivo, where complete tumor regression was observed in a xenograft model. Ongoing studies seek to further define the mechanism of cell death, which could suggest potential avenues to leverage hyperactivation of MAPK signaling as a therapeutic approach.

#966

Oncogenic signaling pathways differentially regulate clathrin-mediated endocytosis in cancer cells.

Guan-Yu Xiao, Sandra Schmid. _UT Southwestern Medical Center, Dallas, TX_.

Clathrin-mediated endocytosis (CME), the major endocytic pathway, regulates the rates of internalization of receptors as well as their downstream signaling activities. Recently, our lab has discovered that signaling can reciprocally regulate CME in cancer cells. The crosstalk between signaling and CME contributes to abnormal trafficking of signaling receptors and altered downstream signaling, leading to enhanced cancer metastasis. Accordingly, it is important to understand the nature and extent of interactions between CME and intracellular signaling in cancer cell biology. To address this issue, we targeted a set of prioritized kinases that are often implicated in cancer-relevant signaling pathways using validated pharmacologic inhibitors and compared their effects on CME function using different models for human noncancerous and cancer cells. We found that inhibition of several kinases selectively affected CME function in cancer cells. Notably, ERK inhibition had the most significant and consistent effects on CME activity across different types of cancer cells, while not affecting CME in several noncancerous cell lines tested. We established that a second ERK inhibitor, and importantly inhibition of the essential upstream kinase MEK, had the same cancer cell-specific effects on CME activity. Furthermore, we found that inhibition of ERK dramatically reduced the rate of clathrin-coated pit (CCP) initiation selectively in cancer cells. Characterization of one potential ERK substrate, FCHSD2, showed that knockdown of FCHSD2 had the similar cancer cell-specific effects on CME activity and on CCP initiation rate and that the cells depleted for FCHSD2 were insensitive to ERK inhibition. Surprisingly, the expression level of FCHSD2 is positively correlated with higher cancer patient survival rate, suggesting that FCHSD2 might be a tumor suppressor regulated by ERK. Our study provides new insight into the mechanisms and consequences of the reciprocal regulation of signaling and CME in cancer cells.

#967

Targeting notch one notch above.

Deniz A. Ucar-Bilyeu,1 Margarite D. MATOSSIAN,2 VAN Hoang Barnes,3 Fokhrul M. Hossain,1 Mohit Gupta,4 HOPE E. BURKS,2 THOMAS D. WRIGHT,4 Jane Cavanaugh,4 Patrick Flaherty,4 Matthew E. Burow,2 Lucio Miele1. 1 _LSUHSC Stanley S. Scott Cancer Center, New Orleans, LA;_ 2 _Tulane University School of Medicine, New Orleans, LA;_ 3 _National Cancer Institute, Frederick, MD;_ 4 _Duquesne University, Mylan School of Pharmacy, Pittsburgh, PA_.

Triple negative breast cancer (TNBC) is a molecularly heterogeneous, clinically aggressive disease group that is highly prevalent among African-Americans and younger patients. Standard chemo/radio therapy often produces clinical responses, but recurrence and metastasis are unfortunately common. Metastatic disease is generally incurable. Chemo/radiotherapy has been shown to induce EMT and enrich a chemo-resistant cancer stem cell-like (CSC) population in TNBC. CSCs are thought to drive disease recurrence. Notch signaling, particularly Notch1, is critical for maintenance of TNBC CSC. Expression of Notch1 and its ligand Jagged1 are correlated with poor prognosis. Efforts to pharmacologically target Notch directly have been impaired by the systemic toxicity of the Gamma Secretase Inhibitors (GSI) used, and by the fact that Notch1 also plays a key role in anti-tumor adaptive immunity. Therapeutic agents that target Notch signaling in breast cancer cells indirectly and selectively are a potentially attractive strategy. However, no such target has been identified to date. We have found that the MAPK5-ERK5 kinase pathway, which contains at least two druggable targets, functions as a master regulator of Notch signaling in TNBC cells. ERK5 knockout TNBC cells have dramatically decreased expression of Notch receptors, ligands and targets. In vivo, these cells form barely detectable tumors that do not metastasize and express lower levels of Notch1 and its ligand Jagged1. Using in silico screening method, we have identified a small molecule compound that targets MAP2K5 (MEK5) and decreases phosphorylation of MAPK7 (ERK5). Expression of ERK5 is associated with poor prognosis in TNBC. Consistent with ERK5KO cells, suppression of ERK5 phosphorylation decreased the amount of Notch1 and Jagged1 protein and mRNAs. More importantly, a selective MEK5 inhibitor, SC-181, reversed EMT and reduced the CD44hi/CD24lo CSC population in TNBC cells without suppressing T-cell proliferation. Treatment with nanomolar concentration of this compound decreased the number and size of mammospheres in a dose- dependent manner. Our preliminary results suggest that targeting the MEK5-ERK5 pathway is a promising strategy to selectively target Notch signaling in TNBC CSC without systemic Notch inhibition.

#968

Targeting FAK inhibits YAP-dependent tumor growth in uveal melanoma.

Xiaodong Feng,1 Damiano Rigiracciolo,1 Joo-Sang Lee,2 Huwate Yeerna,1 Nadia Arang,1 Simone Lubrano,1 David D. Schlaepfer,1 Pablo Tamayo,1 Eytan Ruppin,2 J. Silvio Gutkind1. 1 _Moores Cancer Center, UCSD, La Jolla, CA;_ 2 _University of Maryland Institute for Advanced Computer Studies, MD_.

Activating mutations in GNAQ and GNA11 (known as GNAQ oncogenes), which encode constitutively active Gαq proteins, occur in ~90% of uveal melanoma (UM) and 4% of skin cutaneous melanoma (SKCM) respectively. UM is the most common primary cancer of the eye in adults, diagnosed in about 2,500 adults in the US every year. Approximately 50% of UM patients develop liver metastasis within 5-10 years after diagnosis, independently of the successful treatment of the primary lesions. There are effective therapeutic strategies for primary UM lesions, but UM metastatic disease is refractory to current chemotherapies and immune checkpoint inhibitors, and most patients with UM metastasis die within a year. The MEK inhibitor selumetinib has been recently approved for UM treatment, but MEK inhibition has nearly no impact on UM patient overall survival. To date, there are no effective treatment options for metastatic UM. We used bioinformatics approaches to identify systems vulnerabilities that can be exploited for UM treatment. Using the TCGA UM dataset (N=80) and SKCM samples that do not harbor Gαq genomic alterations (N=209) as control, (i) we first identified genes that are highly overexpressed in UM. We validated this approach by confirming that the genes over- and under-expressed in UM and Gαq-altered SKCM are highly overlapping (hyper-geometric p<1E-198 and p<1E-232 respectively). ii) We then filtered the genes for those whose inactivation leads to better patient survival in UM based on TCGA survival data. (iii) We finally used large datasets of gene essentiality and drug response screens in cancer cells to identify genes that are predicted to reduce cell viability when targeted in Gαq-expressing tumors. By filtering these genes for those that can be targeted by approved and experimental drugs (N=756), we identified 7 candidate druggable genes. Among them, the top was PTK2, which encodes a non- receptor tyrosine kinase known as FAK (focal adhesion kinase). By dissecting the Gαq signaling pathway, we found that Gαq actives FAK (p379-FAK) through a Trio-RhoA signaling pathway, independent of PLC-β regulated second messenger systems. RNA-seq of UM cells treated with a FAK inhibitor (FAKi, VS-4718) revealed that YAP gene-expression signatures were highly sensitive to FAKi. Of interest, YAP is a key regulator of cancer growth, and we have shown that YAP is highly activated by Gαq in UM, contributing to tumor growth (Cancer Cell. 2014). We found FAK regulates YAP activation by Gαq through a mechanism that involves YAP tyrosine phosphorylation on Y357. Interestingly, CRISPR-Cas9 KO of FAK and clinically relevant FAKi showed strong inhibition of UM growth in vitro and in vivo, concomitant with repression of YAP-regulated gene programs. Overall, our computational biology approach revealed that FAK may represent a novel precision therapeutic target human diseases initiated by aberrant Gαq signaling, including UM, the first identified Gαq-driven human malignancy.

#969

Tunneling nanotubes, a novel mode of tumor cell-macrophage communication in tumor cell invasion.

Samer Hanna, Kessler McCoy-Simandle, Edison Leung, John Condeelis, Dianne Cox. _Albert Einstein College of Medicine, Bronx, NY_.

The interaction between tumor cells and macrophages is crucial in promoting tumor invasion and metastasis. These two cell types are engaged in a mutual interaction in which tumor-associated macrophages produce epidermal growth factor (EGF) to activate tumor cells. In turn, tumor cells produce colony-stimulating factor-1 (CSF-1) that stimulates macrophages. The tumor cell - macrophage pairs formed in response to this paracrine signaling are then attracted toward blood vessels under an endothelial cell produced HGF gradient. This mutual signaling interaction leads to the co-migration and invasion of both cell types as imaged both in vitro and in vivo. . Recent studies have revealed an additional novel mechanism of intercellular communication between macrophages that can transmit signals over long distances through membranous actin-based tunneling nanotubes (TNTs). Our data demonstrates that heterotypic TNTs form between macrophages and tumor cells in co-culture. This novel interaction induced changes in tumor cell morphology consistent with a more invasive phenotype which was dependent on EGF-EGFR signaling. Moreover, the presence of these heterotypic TNTs was important for tumor cell invasion in an in vitro 3D invasion assay. Furthermore, reduction of M-Sec (TNFAIP2) in macrophages, a protein involved in TNT formation, inhibited tumor cell elongation and blocked the ability of tumor cells to invade. Using a modified 1D assay that mimics macrophage-dependent tumor cell streaming observed in vivo, we show a significant increase in long distance directional migration of tumor cells towards an endothelial-coated bead in a TNT dependent manner. We also employed an in vivo zebrafish model that recreates macrophage mediated tumor cell invasion in a more physiological fashion. The presence of macrophages increased tumor spread from the injection site, number of metastatic foci, and the distance of metastatic spread in a macrophage TNT-dependent manner. Overall, our studies support a role for TNTs as a novel means of interaction between tumor cells and macrophages that may lead to tumor progression and metastasis.

#970

Transforming NTRK2 and NTRK3 mutations as potential drivers of leukemia.

Sunil K. Joshi,1 Kristin Qian,1 Jacob P. Wagner,1 Monika A. Davare,2 Cristina E. Tognon,3 Brian J. Druker3. 1 _Knight Cancer Institute, Oregon Health & Science University (OHSU), Portland, OR; _2 _Papé Pediatric Research Institute, Oregon Health & Science University (OHSU), Portland, OR; _3 _Knight Cancer Institute/Howard Hughes Medical Institute, Oregon Health & Science University (OHSU), Portland, OR_.

The neurotrophic tyrosine receptor kinases (NTRKs) are a family of neuronal transmembrane receptors that signal through several pathways including JAK/STAT, PI3K/AKT, and MEK/ERK to promote proliferation, differentiation, and survival. Although much of the literature has focused on the importance of these receptors in neuronal development, over the past decade, gene fusions containing NTRK family members have been implicated in driving tumor growth in salivary gland, breast, lung, colon, and neuronal cancers. Moreover, expression of NTRK2 and NTRK3 has been reported in lymphoid and myeloid malignancies (Li, Z et al. Blood, 2009). Interestingly, NTRK3 expression has been associated with poorer prognosis. Despite these initial studies, the role of NTRK receptors largely remains under-investigated in the setting of hematologic malignancies. Herein, we describe somatic mutations in NTRK2 and NTRK3 that we identified using next-generation sequencing of primary leukemia patient samples. We evaluated changes in downstream signaling driven by these mutations in our previously validated Ba/F3 transformation model system. Lastly, we demonstrate that selective inhibition of NTRK2 and NTRK3 with entrectinib, a well-validated NTRK inhibitor currently in clinical trials for NTRK-fusion positive cancers, can inhibit proliferation and induce apoptosis of NTRK transformed cells. We found two point mutations in NTRK2 that were transforming — one in the extracellular domain (A203T) and one in the juxtamembrane domain (R458G). Similarly, we found two point mutations that were transforming in NTRK3. One was in the extracellular domain (E176D) and the other within the transmembrane domain (L449F). Immunoblot results suggest that expression of total and phosphorylated NTRK2 and NTRK3 is increased in mutant-transformed Ba/F3 cells relative to wild-type cells. Furthermore, NTRK2 and NTRK3 mutant-driven cells exhibited enhanced phosphorylation of AKT, SRC, and ERK compared to wild-type cells. While phosphorylation of STAT3 was significantly increased in Ba/F3 cells transfected with both NTRK3 mutants, it was increased only in one of the NTRK2 mutants (A203T). Upon treating NTRK mutant-transformed cells with entrectinib (0, 5, 25, and 100 nM), we observed a dose-dependent decrease in total and phosphorylated NTRK2 and NTRK3 in all mutants except for NTRK2 mutant R458G. With R458G, total and phosphorylated NTRK2 expression decreased up to 25 nM of entrectinib but that effect was diminished at 100 nM. Taken together, our data demonstrates that mutations in NTRK2 and NTRK3 have transformative potential to promote downstream survival signaling and leukemogenesis. More importantly, these activated pathways can be pharmacologically attenuated using entrectinib. Our findings contribute to ongoing efforts to understand molecular changes that drive hematological malignancies and could potentially alter current treatment modalities.

### Epigenetic Mechanisms of Tumor Progression

#971

Three-dimensional gene regulatory landscapes in normal and cancer cells.

Brian J. Abraham, Yang Eric Guo, Denes Hnisz, Charles H. Li, Abraham S. Weintraub, Daniel S. Day, Jurian Schuijers, Tong Ihn Lee, Richard A. Young. _MIT Whitehead Inst. for Biomedical Research, Cambridge, MA_.

Key oncogenes can be misregulated in tumor cells by the acquisition of tumor-specific enhancers that direct gene expression through physical contacts (1,2). Identifying the full complement of enhancers regulating each oncogene could identify additional vulnerabilities in tumor expression programs, but remains challenging, because a given enhancer can regulate multiple genes and each of its targets may be genomically distant. Here we use DNA interaction data to construct gene regulatory landscapes for all expressed genes in a tumor cell and demonstrate that sets of enhancers can be assigned to these oncogenes using direct looping and insulated neighborhood data. A subset of these landscapes contains an exceptional amount of transcriptional apparatus, reminiscent of SEs (3), so we term this subset 3D-SEs. Well-characterized oncogenes, including c-Myc, acquire 3D-SEs in tumor cells where a c-Myc¬-associated linear SE was not identified, suggesting SE acquisition by c-Myc and other oncogenes has been underestimated. Each gene's regulatory network can also surprisingly extend beyond the confines of insulated neighborhoods and incorporate additional distal enhancers. Visualizing instances of 3D-SEs with microscopy demonstrates that they are components of liquid-liquid phase-separated bodies in cells, suggesting gene regulatory landscapes underpin these transcriptional condensates. The interactions comprising gene regulatory networks and their target genes thus extend previous interpretations of the targets of enhancers, signaling pathways, and disease-associated enhancer variants.

References: 1. Hnisz, Abraham, Lee, et al. and Young, Cell 2013. 2. Mansour, Abraham, et al. and Young and Look, Science 2014. 3. Whyte, Orlando, Hnisz, Abraham, et al. and Young, Cell 2013.

#972

Defining the molecular context of MYC and WDR5 at chromatin.

Alissa D. Guarnaccia, William P. Tansey. _Vanderbilt Univ., Nashville, TN_.

MYC is an oncoprotein transcription factor that is overexpressed in the majority of malignancies and contributes to the deaths of ~100,000 Americans each year. The tumorigenic functions of MYC stem from its influence on the transcriptional program of a cell, promoting the expression of thousands of genes involved in cell cycle progression, growth, and metabolism. In order to understand how MYC promotes cancer, we must understand the mechanisms by which MYC selects its target genes. Epigenetic signatures, DNA sequence motifs, and protein-protein interactions have all been implicated to play a role in MYC target gene selection, but a deep molecular understanding of the determinants of target gene recognition by MYC is missing. Our laboratory discovered that the chromatin-associated scaffolding protein WDR5 directly interacts with MYC and co-localizes on chromatin at a majority of loci (Thomas et al., Mol. Cell, 58: 440-452, 2015). The MYC-WDR5 interaction is disrupted by point mutation of three charged residues in MYC. While this MYC mutant retains the ability to bind to naked DNA, the mutant does not bind target loci in the context of chromatin and is unable to form tumors in mice. From these findings we propose that WDR5 is a crucial factor for enabling MYC activity in cancer. Both MYC and WDR5 are known to be highly networked proteins with multiple simultaneous interactions. Our aim is to tease apart this complicated network and define the molecular context specifically for the MYC-WDR5 interaction. To do this, we are employing our WDR5-binding deficient MYC mutant as a tool to genetically distinguish MYC interactions that are dependent upon WDR5. Our approach is to use Stable Isotope Labeling of Amino acids in Cell Culture (SILAC) because it enables us to quantitatively compare wild-type and mutant MYC and reduces background noise common to non-quantitative MYC-centered proteomics. Once identified by SILAC, interacting proteins are validated with biochemical methods and by proximity ligation assay in cells. This dual validation confirms the interaction in two ways, and provides information on the sub-cellular localization of the interaction. Thus far we have identified a preliminary set of ~25 interacting proteins that are currently undergoing validation. Characterization of these new factors by genetic, genomic, and biochemical approaches will expand our understanding of MYC biology, and potentially identify new factors that can be therapeutically targeted in cancer.

#973

MEKK3 sustains EMT and stemness in pancreatic cancer by regulating YAP and TAZ transcriptional activity.

Geny Piro, Raffaela Santoro, Marco Zanotto, Carmine Carbone, Giampaolo Tortora, Davide Melisi. _University of Verona, verona, Italy_.

Background: Pancreatic cancer (PC) is one of the most threatening and poorly understood human malignancies. MEKK3 (MAP3K3) is an intracellular serine/threonine kinase activated by different signaling pathways, including IL1, TNFα and TLR8, which regulates in turn NF-κB, JNK and p38 activation.The transcriptional regulators Yes Associated Protein (YAP) and transcriptional coactivator with a PDZ binding domain (TAZ) are central determinants of malignancy and have been identified as a critical oncogenic effectors of KRAS in pancreatic cancer. We hypothesized that MEKK3 could sustain pancreatic cancer by inducing YAP/TAZ oncogenic activities. Methods: MEKK3 was knocked-out (KO) in AsPC1, Panc1, and MDA-Panc28 PC cell lines by CRISPR/Cas9. The expression of MEKK3, E-cadherin, YAP/TAZ and their target genes was measured by both Western blot and qPCR. Cell migration was assessed by transwell and wound healing assays. Stemness was measured by spheroid formation and percentage of CD44+/CD24+/EpCAM+ cancer stem cells (CSC). Effects of MEKK3 KO was evaluated in vivo in an orthotopic nude mouse model with GFP-expressing Panc1 PC cells. Expression of YAP/TAZ and their target genes in tumor samples was measured by IHC. Results: MEKK3 KO resulted in the reversion of both Epithelial-to-Mesenchymal Transition (EMT) and cell migration, as well as in the impairment of cell proliferation. More interestingly, the size of 3D colonies arising from MEKK3 KO cell lines was smaller than the ones formed by their respective controls, due to a reduced percentage of CD44+/CD24+/EpCAM+ CSC. In MEKK3 KO cells, the recruitment of YAP/TAZ onto the promoters of their target genes was significantly impaired and this paired with a reduction of the expression of the same genes. In nude mice, MEKK3 KO significantly reduced tumor growth and prolonged overall survival. Conclusions: Our study is the first to demonstrate that genetic silencing of MEKK3 is a valid approach to revert in vivo the aggressiveness of PC by modulating YAP and TAZ transcriptional activities. Inhibition of MEKK3 could represent an exciting therapeutic strategy for the treatment of PC, which should be warranted further development.

#974

Alternative polyadenylation of androgen receptor variants in castration-resistant prostate cancer.

Jamie L. Van Etten, Michael Nyquist, Yingming Li, Rebecca LaRue, Yeung Ho, Rendong Yang, Daniel F. Voytas, Christine Henzler, Scott M. Dehm. _University of Minnesota, Minneapolis, MN_.

The androgen receptor (AR), which is activated by androgens, functions in the normal prostate as a master transcriptional regulator. Accordingly, prostate cancer (PCa) presents as an androgen- and AR-dependent disease. Inhibition of AR transcriptional activity with androgen-deprivation therapy (ADT) is effective for patients with locally advanced and metastatic disease. All ADT suppresses AR via interference with its ligand binding domain (LBD)-dependent transcriptional activity. However, development of therapy resistance and transition to castration-resistant prostate cancer (CRPC) represents a major clinical challenge. CRPC circumvents ADT by expressing constitutively active AR variants (AR-Vs) that lack the LBD. Therapies targeting constitutively active AR variants are not currently available. To address this problem, we investigated the mechanisms underlying AR-V expression in CRPC. One particular AR-V, called AR-V7, consists of contiguously spliced exons 1, 2, 3, and a cryptic exon, CE3, and has been shown to be associated with clinical resistance to second-generation AR inhibitors abiraterone and enzalutamide. We noted that CE3 contains a consensus hexameric AAUAAA polyadenylation signal (PAS). We hypothesized that intronic polyadenylation promotes AR-V7 expression in CRPC. To test this, we developed a morpholino that sterically blocked the CE3 PAS to prevent splicing of AR-V7. We have demonstrated that the PAS in CE3 controls expression of AR-V7, as well as a broad array of AR variants arising by splicing of adjacent cryptic exons that use the shared CE3 PAS. Furthermore, our data demonstrate that the cleavage and polyadenylation complex (CPSF complex) is enriched in primary PCa tumors relative to normal tissues. We have also demonstrated that CPSF1, a member of the CPSF complex, controls expression of AR-Vs relative to full length AR and plays a global role in the determination of mRNA isoform length in prostate cancer cells. Collectively, these findings reveal alterations in the cellular machinery governing mRNA cleavage and polyadenylation in prostate cancer, and identify specific opportunities for therapeutic intervention.

#975

Functional CRISPR screen towards identifying novel epigenetic co-factors of oncogenic AR-activity.

Abhijit Parolia, Lanbo Xiao, Josh N. Vo, Marcin Cieslik, Xuhong Cao, Arul M. Chinnaiyan. _University of Michigan, Ann Arbor, MI_.

Prostate cancer (PCa) is the second leading cause of cancer-related mortality in North American men. In recent years, there has been mounting evidence establishing the centrality of epigenetic mechanisms in PCa initiation and progression. Accordingly, various epigenetic genes have been described to collaborate with the androgen receptor (AR) in enabling its oncogenic transcriptional program and aberrantly restoring its activity in metastatic castration resistant PCa. Concordantly, our laboratory has recently described two epigenetic genes, BRD4 and MLL2, as key co-activators of AR-signaling. Furthermore, we have demonstrated that inhibition of these genes synergistically work with AR antagonists to attenuate PCa progression in preclinical models. Thus, in a setting where all metastatic patients eventually progress to evolve resistance to anti-AR therapies, there exists a dire clinical need to identify novel therapeutic targets. To this end, using CRISPR-Cas9 technology, we have engineered a unique AR-reporter LNCaP model that harbors the mCherry gene directly downstream of the endogenous KLK3/PSA promoter (a canonical AR-target). Notably, validation experiments confirm that the mCherry reporter gene, akin to KLK3, is dynamically regulated by AR activity in response to treatment with dihydrotestosterone (DHT; a physiological AR agonist) or enzalutamide (an AR inhibitor). Furthermore, we have designed a focused, high-depth library of small-guide RNAs (sgRNAs) to target over 200 distinct epigenetic genes, both at transcriptional start sites and functionally-essential enzymatic domains. Overall, this library comprises of roughly 1400 distinct guide-RNAs that include 50 non-targeting negative controls. Using these molecular tools, we plan to perform a marker-based, functional screen that involves the sorting of sgRNA-infected AR-reporter cells into mCherryHIGH and mCherryLOW populations. Notably, we have successfully validated the stated screening design: Starting from a mixed pool of AR-targeting siRNAs or R1881 (a synthetic AR-agonist) treated reporter cells, we sorted them into LOW15% or HIGH15% fractions based on the intensity of mCherry fluorescence. As expected, transcriptomic analyses of these fractions confirmed that AR-signaling was indeed significantly inhibited and enhanced in the LOW15% and HIGH15% fractions, respectively, relative to the unsorted cells. Thus, for our CRISPR screen, we theorize that the genomic interrogation of sgRNAs enriched in the mCherryLOW population will reveal novel epigenetic genes that function as essential co-factors of oncogenic AR-activity. These epigenetic factors can be co-targeted to extort durable therapeutic responses in patients treated with the current line of anti-AR therapies, thus directly addressing an urgent clinical need.

#976

FOXA1 promotes a luminal growth program in prostate cancer.

Elizabeth J. Adams, Elizabeth Hoover, Wouter R. Karthaus, Charles L. Sawyers. _Memorial Sloan Kettering Cancer Ctr., New York, NY_.

FOXA1 is an important pioneering transcription factor for androgen receptor (AR), a key driver of prostate cancer. FOXA1 has emerged as one of the most recurrently mutated genes in primary and metastatic castrate resistant prostate cancer, with alterations frequencies ranging from 4-15% in various cohorts. Given the well-established role of FOXA1 in modulating AR function, we hypothesize that FOXA1 mutations contribute to prostate oncogenesis by altering the chromatin landscape, resulting in changes in the AR cistrome and AR transcriptional output. Here we use prostate organoids to show that acute FOXA1WT overexpression promotes lumen formation and drives luminal differentiation. Consistent with this, CRISPR/Cas9-mediated deletion of FOXA1 slows proliferation and organoids lacking FOXA1 fail to form lumens. Assays to evaluate the functionality of recurrent FOXA1 mutations (measured by cell viability, lumen formation, and lumen size) along with RNA-sequencing suggest there are distinct classes of FOXA1 mutations. The first are the gain-of-function mutations (such as FOXA1FE254-255del), which promote growth and lumen formation to a larger extent than wild-type FOXA1. Interestingly, a FOXA1 luciferase reporter assay we developed in HEK293 cells indicates that the majority of mutants tested behave as gain of function. The only mutant to score as loss of function, FOXA1R219S, is found nearly exclusively in prostate cancer, and is enriched in neuroendocrine prostate cancer cohorts. Expression of FOXA1R219S suppresses lumen formation and promotes both basal and epithelial-mesenchymal transition transcriptional programs. However, this mutant also provides a growth advantage beyond what is conferred by FOXA1WT. Taken together, these data suggest that FOXA1R219S is not simply a loss of function allele and instead may drive disease progression through a different set of signaling pathways than the FOXA1 alleles that promote luminal differentiation. Further study of these classes of mutations and how they relate to patient outcome, along with analysis of the FOXA1mut/AR cistromes, is ongoing to determine the genome-wide implications of FOXA1 alterations and how they promote prostate cancer progression.

#977

Glycosylation of estrogen receptor alpha by N-acetylgalactosaminyltransferase 6 in breast cancer.

Boya Deng,1 Yunus Emre Tarhan,1 Koji Ueda,2 Yo Matsuo,3 Jae-Hyun Park,1 Yusuke Nakamura1. 1 _the University of Chicago, Chicago, IL;_ 2 _Cancer Precision Medicine Research Center, Tokyo, Japan;_ 3 _Oncotherapy science Inc., Kawasaki, Japan_.

Alteration of protein O-glycosylation in various human cancers including breast cancer is well known, but molecular mechanisms of such aberrant modifications and their effects on cancer development have not been fully understood. We previously reported the critical roles of polypeptide N-acetylgalactosaminyltransferase 6 (GALNT6), which is upregulated in a great majority of breast cancers and is responsible for initiating mucin-type O-glycosylation. Suppression of GALNT6 expression by small interfering RNA to GALNT6 significantly enhanced cell-cell adhesion, induced mesenchymal-epithelial transition, and suppressed the growth of breast cancer cells. Here we further analyzed molecular functions of GALNT6 and found that GALNT6 could glycosylate an estrogen receptor alpha (ER-α) protein, a molecule playing a central role in proliferation of hormone-dependent breast cancer cells. We have used two breast cancer cell lines, T47D and MCF7, in which both the estrogen receptor and GALNT6 were highly expressed. Knockdown of GALNT6 expression by siRNA could significantly attenuate expression of ER-α at transcriptional and protein levels in these breast cancer cell lines in a condition with or without estradiol (P<0.05). In addition, immunocytochemical analysis clearly showed the dramatic decrease of the ER-α protein in the nucleus of cancer cells. Accordingly, the downstream genes of the ER-α pathway, such as MYC, CCND1, and CTSD were significantly downregulated after the GALNT6 knockdown (P=0.002, P=0.018, and P=0.026, respectively). Moreover, to evaluate the GALNT6 function in the breast cancer cell lines, we used a small molecular compound targeting the GALNT6 activity. Interestingly, as similar to siRNA, the ER-α protein expression level was decreased in a dose-dependent manner with this GALNT6 inhibitor. To further investigate the GALNT6-ER-α pathway, we transfected Hela cell lines with mock vector, wild-type GALNT6 or enzyme-dead GALNT6 (mock, GALNT6-WT, GALNT6-H271D, respectively) as well as ER-α-expressing plasmid, and examined the O-glycosylation of ER-α by an IP pull-down assay in combination of a Vicia villosa agglutinin (VVA)-lectin assay. The ER-α glycosylation band was detectable in GALNT6-WT cells, but not in mock cells or GALNT6-H271D cells, indicating GALNT6-dependent ER-α glycosylation. Subsequent LC-MS analysis confirmed that S573 in an F domain of ER-α was glycosylated by GALNT6. Our results clearly indicate that the glycosylation of ER-alpha at S573 by GALNT6 is essential for protein stability and nuclear localization of ER-alpha and that GALNT6 may play important oncogenic roles through glycosylation ER-alphain breast cancer cells. We suggest that targeting GALNT6 may be a promising therapeutic approach to ER/GALNT6-positive breast cancer patients.

## MULTIDISCIPLINARY:

### Radiation Science

#978

In vivo shRNA screening identifies synthetic cytotoxicity in CREBBP/EP300 mutant head and neck cancer.

Manish Kumar,1 Kathleen Bridges,1 David Molkentine,1 Liangpeng Yang,1 Aakash Sheth,1 Raymond Meyn,1 Mitchell Frederick,2 Jeffrey Myers,1 Curtis Pickering,1 Heath D. Skinner1. 1 _UT MD Anderson Cancer Ctr., Houston, TX;_ 2 _Baylor College of Medicine, Houston, TX_.

The p300-CBP1 coactivator family members (CREB-binding protein (CREBBP) and E1A binding protein p300 (EP300)) share significant homology and act as transcriptional co-activators for a large number of target genes. These genes are collectively mutated in approximately 14% of all head and neck squamous cell cancers carcinomas (HNSCC) and are targeted by multiple agents in clinical development. Because of this the p300-CBP1 coactivator family presents a promising target in HNSCC. Recently, our group performed in-vivo screening utilizing a barcoded shRNA library of targets of known anti-neoplastic agents. A total of 5 HNSCC cell lines of varying HPV status (HPV (-): Cal27, HN31, UMSCC22A; HPV (+): UMSCC47, UPCISCC152) were transduced with the shRNA library and allowed to form subcutaneous, flank tumors, which were then treated with IR (2 Gy/day) to achieve an approximate 20% reduction in tumor volume. The tumors were then harvested and barcodes were sequenced. Interestingly, this screen revealed that CREBBP or EP300 depletion led to dramatic sensitization to IR, but only in tumors harboring a mutation in either CREBBP or EP300, independent of HPV status. To investigate this phenomenon further, we utilized chemical inhibition of CREBBP as well as shRNA to CREBBP in multiple HNSCC cell lines of varying HPV and CREBBP mutational statuses. In those cell lines harboring a CREBBP mutation, we found significant in vitro sensitization to IR on clonogenic assay following CREBBP inhibition, however this phenomenon was not observed in CREBBP wild type cells. All cell lines tested were generally resistant to IR-induced apoptosis, as measured by TUNEL assay and PARP cleavage. However, IR combined with inhibition of CREBBP led to dramatically increased IR-associated apoptosis as well as increased γ-H2AX and decreased BRCA1 foci following radiation. We next examined CREBBP inhibition using two shRNA clones specific for CREBBP in an in vivo UMSCC47 xenograft model. In this model, inhibition of CREBBP alone or IR alone (2 Gy x 8 d) led to minimal tumor volume reduction (tumor growth delay (TGD) 6 and 2 days respectively). However, the combination of each shCREBBP clone and IR led to a TGD of 24 and 23 days (p<0.0001 for both). Analysis of these tumors following radiation identified an increase in PARP cleavage and decrease in BRCA1 in the combined treatment group. Finally, we examined clinical outcomes in a cohort of 75 HPV (-) HNSCC patients treated with surgery and radiation. Mutations in CREBBP or EP300 were significantly associated with poorer survival (median survival 18 mos vs. 62.8 mos in wild type; p=0.015). Our results suggest the synthetic cytotoxicity of combining CREBBP inhibition and IR in tumors harboring mutations in the p300-CBP1 coactivator family. As mutations in this family are relatively common in HNSCC, and are associated with worse survival, this strategy could dramatically improve patient outcome.

#979

EGLN inhibition reduces gastrointestinal radiation toxicity and improves survival in a murine model of locally advanced pancreatic cancer.

Cullen M. Taniguchi, Tara N. Fujimoto, Lauren E. Colbert, Jessica M. Moilkentine, Amit Deorukhkar, Laura J. Baseler, Ramesh Tailor, Gabriel O. Sawakuchi. _UT MD Anderson Cancer Ctr., Houston, TX_.

Rationale: Pancreatic cancer is almost always fatal since chemotherapy is only modestly effective and surgery is often not possible. Radiation therapy (RT) can be a definitive treatment in localized pancreatic cancer, but gastrointestinal toxicity to the nearby duodenum limits its efficacy. There are no known medications that can prevent or treat this gastrointestinal radiotoxicity, but we find that FG-4592, a small molecule inhibitor of EGLN proteins, reduces radiation damage to the intestines by selectively enhancing HIF expression within normal intestines but not within tumors.

Methods: We developed a murine model of spontaneous pancreatic cancer (KrasLSL/+; Trp53FL/+; Ptf1aCre/+) that behaves similarly to locally advanced disease. KPC mice were screened for tumors by weekly ultrasounds and upon diagnosis were sequentially assigned to receive one of four possible treatments: Vehicle only (VEH), FG-4592 (FG), RT concurrent with vehicle (RT+VEH), or RT concurrent with FG-4592 (RT+FG). Mice were enrolled on a rolling basis and the total time from diagnosis to treatment end was approximately three weeks. Radiation treatments were planned to administer a total of 75Gy in 15 daily fractions.

Results: A total of 70 KPC mice were evaluated. The administration of high-dose radiation therapy of >65Gy (high-dose) with or without radioprotection improved survival compared to all animals that received ≤65Gy (low-dose) of treatment or no RT (37 days [95% CI 33-41] vs. 15 days [10-21] vs. 15 days [10-20], respectively; p=0.005). KPC animals that received dose-escalated radiation lived significantly longer compared to unirradiated controls that received vehicle or FG-4592 alone. The median overall survival was compared amongst the treatment groups and was highest (p<.001) for mice that received RT with FG-4592 for radioprotection (43 days, [95% CI 39-47]) versus RT with vehicle (36 days, [95% CI 10-62]), FG alone (29 days, [95% CI 21-37]), or vehicle alone (9 days, [95% CI 5-13]). High-dose RT reduced death from local progression as determined by reduced jaundice and obstruction on necropsy which was present in 64% (25/39) of mice who did not receive RT versus 0% (0/29) who received RT with or without radioprotection (p=0.02). The addition of FG-4592 to RT further improved survival compared to RT + vehicle controls by eliminating radiation-induced gastrointestinal toxicity. Approximately 56% (5/9) of animals that received high-dose RT without radioprotection died from fatal GI bleeding, whereas no bleeding was seen in animals who received high dose RT+ FG-4592 (P<.0001). Thus, these experiments demonstrate a proof of concept that selective radioprotection of the intestinal tract by EGLN inhibition enables higher, and potentially curative, doses of cytotoxic therapy without any tumor protection.

#980

Molecular basis of adipose-derived stem cell (ASCs) therapy for management of radiation-induced fibrosis (RIF).

Asim Ejaz, Micheal Epperly, Renne Fisher, Xichen Zhang, Moriah Johngrass, Asher Schusterman, Lauren Kokai, Joel Greenberger, Peter Rubin. _University of Pittsburgh, Pittsburgh, PA_.

Radiation therapy doses above 50Gy to head and neck, breast cancer and soft tissue sarcomas can result in the rare late complication of RIF, which can significantly impair function. Several published case studies suggest injection of fibrotic sites with autologous adipose tissue stem cells can ameliorate RIF. In this study, we elucidated the molecular mechanism in an in vitro co-cultivation system and in vivo using a mouse model of hind limb fibrosis. In vitro Transwell co-cultures contained a bottom layer of irradiated human foreskin fibroblasts (HFFs), mouse or human cell lines derived from clinical fibrosis biopsies, and an upper layer containing freshly prepared ASCs. We quantitated levels of fibrosis -related genes in the lower layer cells and regulatory cytokines in the upper layer cells using quantitative real-time (qRT) PCR. In vivo studies: Female C57BL/6 mice were irradiated to the right flank to 35Gy in single fraction with 6Mv electron beam. The irradiated and contralateral unirradiated flank tissue was monitored for expression of fibrosis related genes at days 1 and 14 post irradiation (PI). Fibrosis was confirmed by histologic staining for collagen and range of limb motion measurements. Irradiated fibrotic sites were injected at day 28 with ASCs from luciferase\+ GFP\+ mice. RIF was uniformly detected at day 14 and amplified by day 28, and confirmed by histologic staining for collagen by Masson's Trichrome. At day 14 PI there was clear upregulation in biopsied tissue of fibrosis -related genes: TGF β (500 fold), CTGF (60 fold), collagen 1 (400 fold), collagen3 (500 fold) and collagen4 (500 fold) compared to nonirradiated contralateral hind limb tissue. Irradiation induced by day 28 a reduction in limb excursion with a range of limb extension of 11.4 ± 2.7 degrees compared to 57.0 ± 2.5 (p < 0.0001) degrees in the contralateral nonirradiated limb. A single ASCs injection at day 28 significantly restored the limb excursion to 42.5 ± 2.5 degrees (p = 0.0013). To measure possible systemic radiation mitigatory effects of ASCs PI, a single intraperitoneal injection of 1 million ASCs at 24 hrs after 9.25 Gy total body irradiation significantly increased mouse survival at 30 days (P = 0.047). In transwell co-cultures ASCs demonstrated significant downregulation of profibrotic genes (including collagen 1-4, and TGF β) in both acutely irradiated and irradiation-fibrosis tissue derived cell lines. Among the genes expressed in upper layer ASCs, hepatocyte growth factor (HGF) was a key secreted mediator in co-culture models. Addition of human recombinant HGF to irradiated HFFs significantly downregulated profibrotic gene transcripts. Thus, HGF secreted by ASCs is involved in the reduction of RIF in vivo and with in vitro models of radiation fibrosis. Further studies should elucidate the mechanism of HGF and other secreted and cell contact regulators of adipose tissue-based cell therapy of radiation fibrosis.

#981

Wee-1 kinase inhibitor AZD-1775 radiosensitizes esophageal cancer through targeting G2 checkpoint activation.

Linlin Yang, Cory Pettit, Andrew Hu, Tianyun Li, Terence M. Williams. _Ohio State Univ. Comp. Cancer Ctr., Columbus, OH_.

Background: Esophageal cancer is a deadly cancer that has a 5-year survival rate of 18%, which has remained unchanged for decades. Due to a high incidence of p53 mutation, esophageal cancer cells often lack a functional G1-S checkpoint, which increases reliance upon the G2-M checkpoint for adaptation to stress and DNA repair, making G2-M checkpoint inhibition a target for novel therapeutic development. AZD-1775, a potent inhibitor against the critical G2-M checkpoint protein Wee-1, is currently in clinical trials across a number of tumor types. The purpose of this study was to determine the capacity of AZD-1775 to abrogate the radiation-induced G2 checkpoint arrest and modulate radiosensitivity in esophageal cancer cell (ECC) line models. Materials and Methods: AlamarBlue assay and clonogenic assays were carried out in 4 ECC cell lines (squamous cells: Flo-1, KYSE-30; adenocarcinoma cells: OE-33, SK-4) to calculate IC50 and potential of AZD-1775 as a radiosensitizer in ECC cell lines, respectively. The effects of AZD-1775 on radiation induced checkpoint response of ECC cells were determined in vitro by analyzing cell cycle via flow cytometry, detecting alterations on cell cycle markers through immunoblotting. We also determined AZD-1775 function on radiation caused DNA damage response through mitotic catastrophe analysis by immunofluorescence staining, and p-H2A.x expression analysis by immunoblotting. The role of AZD-1775 in fractionated radiation in comparison to single fraction radiation was also investigated through colony formation assay. Eventually, human esophageal cancer cell xenografts were generated to explore the radiosensitization effect of AZD-1775 on ECC tumors in vivo. Results: AlamarBlue assay showed that the IC50 concentrations of AZD-1775 on ECC cells are between 300 - 600 nM, and exposure to 100 nM of AZD-1775 didn't significantly increase cytotoxicity. However, 100 nM of AZD-1775 was sufficient to effectively sensitize ECC cells to radiation therapy in clonogenic assays in 3 of 4 cell lines. Study of mechanisms underlying AZD-1775-mediated radiosensitization revealed that AZD-1775 significantly abrogates radiation induced G2 checkpoint arrest, and attenuates radiation induced phosphorylation of CDC2. Additionally, AZD-1775 altered expression of various mediators of the cell cycle, consistent with its functions of abrogating G2 checkpoint.AZD-1775 prevented effective repair of DNA damage after radiation, and eventually led to cell death, as supported by increased mitotic catastrophe and increased and persistent p-H2A.x expression after radiation.Conclusion: These results show that inhibition of WEE-1by AZD-1775 has the potential to be an effective strategy for radiation sensitization in esophageal cancer cells.

#982

Pharmacological DNA-PK inhibition induces ATM/p53 dependent premature senescence with immunomodulatory phenotype in irradiated cancer cells.

Yige Guo,1 Qing Sun,1 Xiaohong Liu,1 Janusz Puc,1 Frank Czauderna,1 Frank Zenke,2 Andree Blaukat,2 Lyubomir T. Vassilev1. 1 _EMD Serono, Billerica, MA;_ 2 _Merck Biopharma, Merck KGaA, Darmstadt, Germany_.

A new generation, clinical-stage DNA-PK inhibitor, M3814, potently and selectively blocks one of the two major pathways for repair of DNA double strand breaks (DSB) and synergizes with ionizing radiation (IR) and DSB-inducing chemotherapy. We show that by inhibiting DNA-PK catalytic activity in the presence of DNA DSBs, M3814 simultaneously suppresses DNA repair and a negative regulatory signal to ATM, leading to enhanced activation of the ATM dependent signaling, including CHK2 and p53-dependent cell cycle arrest. Combination treatment of proliferating p53 wild-type cancer cells (A549, A375, H460) with a single dose of ionizing radiation (2-5Gy) and sustained exposure to M3814 induced a complete G1/S and G2/M cell cycle block. Within 4-7 days of treatment cells acquired a typical senescence phenotype with large/flat morphology and β-Gal staining. Live cell imaging and BrdU labeling in A549 cells demonstrated that this phenotype is not reversible following M3814 removal, in contrast to a fully reversible senescence-like phenotype caused by selective p53 activation by MDM2 inhibitor Nutlin-3a. Isogenic p53-null A549 cells lost the ability to fully arrest their cell cycle, confirming the role of p53 in senescence induction. Analysis of mRNA from IR/M3814 induced senescent A549 and A375 cells by the Nanostring PanCancer Immune profiling panel revealed activation of a large group of genes from several immune response pathways, including interferon, cytokine/chemokine, and complement. Eighteen genes were commonly upregulated >3-150 fold compared to controls. These substantial changes in gene expression were built gradually and correlated with the development of senescence phenotype. Several proteins from the induced subset were measured in the cell media (MesoScale Discovery) and confirmed that they are secreted by senescent cells in the absence of M3814. Culture media from M3814-induced senescent cells showed increased immunomodulatory effect on human PBMC-derived immune cells, suggesting increased probability for clearance of IR+M3814 induced senescent cancer cells by the immune system.

#983

Metabolic radiosensitization: Overcoming the radioresistance of hypoxic tumors by targeting OXPHOS.

Martin Benej,1 Xiangqian Hong,2 Bing Yu,2 Ioanna Papandreou,1 Nicholas Denko1. 1 _The Ohio State University Wexner Medical Center, Columbus, OH;_ 2 _Marquette University and Medical College of Wisconsin, Milwaukee, WI_.

It has been recognized decades ago that lower oxygen tension decreases the efficiency of radiation therapy (RT). Strategies to clinically overcome hypoxia have been disappointing. We have addressed this issue by targeting the tumor metabolism. "Metabolic radiosensitization" is a concept of achieving a temporary decrease in the oxygen consumption (OCR) of tumor cells and therefore reduce hypoxia prior to RT. Inhibiting mitochondrial respiration will cause a decrease in the demand for oxygen that will in turn lead to a decrease of tumor hypoxia providing a therapeutic window to deliver RT. A number of FDA-approved drugs have been shown to inhibit mitochondrial OCR as an off-target effect. Repurposing one of these drugs with well-established safety profiles for radiosensitization would thus provide an exciting opportunity for clinical translation. We found that papaverine (PPV) showed the most promising results by rapidly achieving up to 40% inhibition of OCR within minutes in clinically achievable doses in the low micromolar range. Using near infra-red optical spectroscopy (NIRS) with a mouse flank xenotransplantation model we confirmed that a single dose of the drug caused a 1.3-1.4x increase of tumor oxygenation in vivo in the first 30 minutes, while the normal tissue oxygenation remained unaffected. We then determined that combination of papaverine followed by RT after 30 minutes enhanced the tumor radiation growth delay significantly by 2.1-3.9x compared to RT alone. Papaverine has been traditionally used as a vasodilator and anti-spasmodic and is thought to act through its ability to inhibit phosphodiesterase 10A (PDE10A). However, we found that increased cAMP did not inhibit OCR in vitro, suggesting that PPV has dual functions as PDE10A and OCR inhibitor. To mechanistically establish that PPV is radiosensitizing through inhibition of mitochondrial complex I, we generated tumor cells with PPV-resistant mitochondria. We used CRISPR/Cas9 to knock out a gene essential for complex I core activity. We then rescued partial mitochondrial function in these cells by introducing papaverine-resistant yeast complex I encoded by the NDI1 gene. Analysis of these cells by Seahorse confirmed that they were resistant to the effects of PPV on OCR. These PPV-resistant tumor cells were used to grow tumors, and in radiation growth delay experiments, NDI1-expressing tumors were resistant to PPV radiosensitization. These findings confirm that PPV radiosensitizes hypoxic tumors though inhibition of complex I. In conclusion, our data indicate that repurposing FDA approved papaverine for metabolic radiosensitization has a strong potential for translation into the clinics.

#984

Systematic annotation of genetic variants that determine sensitivity to radiation: A pan-cancer encyclopedia.

Brian D. Yard, Aaron P. Petty, Mohamed E. Abazeed. _Cleveland Clinic, Cleveland, OH_.

Cancer genome sequencing efforts have revealed tumor-associated gene variants that can direct therapy, functionally significant variants that do not currently direct therapy or variants that are of unknown functional significance. The impact of all of these categories of variants on the efficacy of radiotherapy remains largely unknown. To gain insight into the genetic landscape of radioresistance, we previously established a high-throughput platform that measures cell line survival to radiation. We then used this technology to analyze the radiosensitivity of more than 500 cancer cell lines annotated by the Cancer Cell Line Encyclopedia and discovered gene sets and pathways that regulate radiation survival. Here, we used the results from this study to identify gene variants (missense mutations, short inserts/deletions, and premature stop codons) that are associated with either radiosensitivity or radioresistance and functionally annotate a multitude of these variants.

Candidate variants from more than 100 ORFs were prioritized on the basis of their location within conserved protein domains (UniProt), predicted functional impact (MutationAssessor), and variant allele frequency. We used site-directed mutagenesis to generate mutant clones and transferred the ORFs into lentiviral vectors for stable expression under a PGK promoter in both SV40 and hTERT immortalized cell lines. For a subset of variants, expression of the endogenous gene was repressed by CRISPRi to model loss of heterozygosity. The effect of individual variants on radiation response was assessed using our high-throughput platform and benchmarked against the canonical, cyto- and radio-protective NFE2L2 E79K gain-of-function mutation and validated by colony forming assay.

We successfully adapted our high-throughput platform to measure the radiosensitivity of individual variants expressed in immortalized cell lines. Integral survival was significantly correlated between our high-throughput platform and clonogenic survival measurements. We discovered radioresistant conferring variants in genes involved in cell cycle transition, apoptosis (intrinsic), and MEK/ERK signaling. In contrast, variants associated with radiation sensitivity were identified in genes with established roles in DNA damage response pathways as well as in putative DNA repair factors.

Determining the impact of gene variants remains a major obstacle in the implementation of personalized radiotherapy. Here, we report on a large-scale profiling effort to identify and classify mutant alleles that govern radiation survival across multiple tumor lineages. Our results reveal new insights into the mechanisms of cellular survival to radiation and genome maintenance.

## TUMOR BIOLOGY:

### Patient-Derived Models of Cancer: Present and Future

#985

The EurOPDX EDIReX project: Towards a European research infrastructure on patient-derived cancer models.

Enzo Medico,1 Mohamed Bentires-Alj,2 Andrew V. Biankin,3 Alejandra Bruna,4 Annette T. Byrne,5 Carlos Caldas,4 Robert B. Clarke,6 Georges Coukos,7 Olivier Elemento,8 Manuel Hidalgo,9 Giorgio Inghirami,8 Steven de Jong,10 Jos Jonkers,11 Ales Krenek,12 Eleonora Leucci,13 Gunhild Mari Maelandsmo,14 Michaela Th. Mayrhofer,15 Terrence F. Meehan,16 Simone P. Niclou,17 Pier Giuseppe Pelicci,18 Alejandro Piris-Gimenez,19 Leo Price,20 Sergio Roman-Roman,21 Livio Trusolino,1 Luca Vezzadini,22 Alberto Villanueva,23 Emilie Vinolo,24 on behalf of the EurOPDX Consortium1. 1 _University of Torino, Candiolo, Italy;_ 2 _University Hospital Basel, Basel, Switzerland;_ 3 _Wolfson Wohl Cancer Research Centre, Institute of Cancer Sciences, University of Glasgow, Glasgow, United Kingdom;_ 4 _Cancer Research UK Cambridge Institute, Cambridge Cancer Centre, University of Cambridge, Cambridge, United Kingdom;_ 5 _Royal College of Surgeons in Ireland, Dublin, Ireland;_ 6 _Manchester Cancer Research Centre, University of Manchester, Manchester, United Kingdom;_ 7 _Lausanne Branch, Ludwig Institute for Cancer Research at the University of Lausanne, Lausanne, Switzerland;_ 8 _Cornell University, Weill Cornell Medical College, New York, NY;_ 9 _Hospital de Fuenlabrada, Fuenlabrada, Spain;_ 10 _University Medical Centre Groningen, University of Groningen, Groningen, Netherlands;_ 11 _The Netherlands Cancer Institute, Amsterdam, Netherlands;_ 12 _Masarykova Univerzita, Brno, Czech Republic;_ 13 _Katholieke Universiteit Leuven, Leuven, Belgium;_ 14 _Oslo University Hospital, Oslo, Norway;_ 15 _Biobanking and BioMolecular resources Research Infrastructure - European Research Infrastructure Consortium, Graz, Austria;_ 16 _European Bioinformatics Institute, Wellcome Genome Campus, Hinxton, Cambridge, United Kingdom;_ 17 _Luxembourg Institute of Health, Luxembourg, Luxembourg;_ 18 _European Institute of Oncology, Milan, Italy;_ 19 _Vall d'Hebron Institute of Oncology and CIBERONC, Barcelona, Spain;_ 20 _Ocello BV, Leiden, Netherlands;_ 21 _Institut Curie, PSL Research University, Paris, France;_ 22 _Kairos3D SRL, Torino, Italy;_ 23 _Catalan Institute of Oncology ICO, Bellvitge Biomedical Research Institute IDIBELL, L'Hospitalet de Llobregat, Spain;_ 24 _seeding science SPRL, Limelette, Belgium_.

Counteracting high attrition rates in anticancer drug development and providing optimal therapeutic management of cancer patients require preclinical models that properly recapitulate the complexity and diversity of human tumours. Patient-derived cancer xenografts (PDXs), developed by transplanting human tumor fragments into immunodeficient mice, retain the idiosyncratic characteristics of different tumors from different patients. The possibility of population-scale correlations between therapeutic response in PDXs and extensive, multidimensional molecular annotation has enabled the identification of several sensitivity and resistance biomarkers in a number of different tumor types, with immediate clinical relevance. To effectively recapitulate and therapeutically interrogate through patient-derived models the heterogeneity that typifies human cancer, 13 European cancer centres and university hospitals joined forces in 2013 to start EurOPDX, an academic research consortium that now gathers 19 institutions throughout Europe and in the US. EurOPDX includes world-renowned experts at the forefront of research in basic, preclinical, translational and clinical oncology across multiple pathologies, and displays a wide range of expertise in technological platforms. The primary goal of the Consortium is to maximize exploitation of PDXs and other patient-derived cancer models for cancer research by: (i) integrating institutional collections into an organic, multicentre collection of patient-derived models, now reaching 2,000 subcutaneous and orthotopic models for 30+ different pathologies (www.europdx.eu); (ii) defining common operating procedures to improve and standardize molecular and pharmacologic characterization of the models; (iii) sharing models within and outside the consortium to perform collaborative precision oncology "xenopatient" trials, to discover predictive biomarkers, new targets, and new strategies to overcome drug resistance. Towards these objectives, the EC granted 5 million euros under the H2020 programme for a "EurOPDX Distributed Infrastructure for Research on patient-derived Xenografts" - EDIReX EU project n. 731105, starting on February 2018. By teaming up with other key academic, technological and SME partners, our goal with EDIReX is to establish a cutting-edge European infrastructure offering transnational access to PDX resources to academic and industrial cancer researchers, including the distribution of cryopreserved samples to third parties, the structured biobanking of user-developed models, and the performance of drug efficacy studies. We will provide an overview of the current achievements of the Consortium and the objectives of the EDIReX project.

#986

The National Cancer Institute's patient-derived models repository (PDMR).

Yvonne A. Evrard,1 Michelle M. Gottholm Ahalt,2 Sergio . Y. Alcoser,2 Kaitlyn Arthur,1 Mariah Baldwin,1 Linda L. Blumenauer,2 Carrie Bonomi,1 Suzanne Borgel,1 Elizabeth Bradtke,1 Corinne Camalier,1 John Carter,1 Tiffanie Chase,1 Alice Chen,2 Lily Chen,1 Donna W. Coakley,1 Nicole E. Craig,1 Biswajit Das,1 Vivekananda Datta,1 Jordyn Davidson,1 Margaret R. DeFreytas,1 Emily Delaney,1 Michelle A. Eugeni,2 Raymond Divelbiss,1 Palmer Fliss,1 Thomas Forbes,1 Marion Gibson,1 Tara Grinnage-Pulley,2 Sierra Hoffman,1 Lilia Ileva,1 Paula Jacobs,2 Franklyn Jimenez,1 Joseph Kalen,1 Catherine Karangwa,1 Chris Karlovich,1 Candace Mallow,1 Chelsea McGlynn,1 Jenna E. Moyer,1 Michael Mullendore,1 Dianne L. Newton,1 Nimit Patel,1 Rajesh Patidar,1 Kevin Plater,1 Marianne Radzyminski,1 Lisa Riffle,1 Larry Rubinstein,2 Luke H. Stockwin,1 Mickey Williams,1 Melinda G. Hollingshead,1 James H. Doroshow2. 1 _Leidos Biomedical Research, Inc, Frederick, MD;_ 2 _National Cancer Institute, Bethesda, MD_.

The National Cancer Institute (NCI) has developed a Patient-Derived Models Repository (PDMR) comprised of quality-controlled, early-passage, clinically-annotated patient-derived xenografts (PDXs) to serve as a resource for public-private partnerships and academic drug discovery efforts. These models are offered to the extramural community for research use (https://pdmr.cancer.gov/), along with clinical annotation and molecular information (whole exome sequence, RNASeq), which is available in a publicly accessible database. The PDMR was established by NCI at the Frederick National Laboratory for Cancer Research (FNLCR) in direct response to discussions with academia and industry; the oncology community's highest priority need was preclinical models that more faithfully reflect the patient's tumor and are associated with the patient's treatment history. NCI has focused on generating models to complement existing PDX collections and address unmet needs in the preclinical model space. The PDMR generates the majority of its PDXs by subcutaneous implantation except for those histologies having better success rates in either orthotopic or alternate implant sites. All SOPs and quality-control standards developed by the PDMR as well as those shared by collaborators are posted to a public web site that houses the PDMR database. In May 2017, the public website (https://pdmr.cancer.gov/) went live with its first 100 models from histologies including pancreatic, colorectal, renal, head and neck, and lung squamous cell cancers as well as melanoma and adult soft tissue sarcomas. In early 2018, the PDMR will begin releasing models from gynecological cancers, small cell lung cancer, chondro/osteo sarcomas, lung adenocarcinoma, and squamous cell skin and Merkel cell carcinomas. In addition, wherever available germline sequence and somatic variant calls will be added to the existing molecular characterization data for each model. NCI has also increased its focus on creating PDXs from racial and ethnic minorities through several funding opportunities. The overall goal of NCI is to create a long-term home for at least 1000 models such that sufficient biological and clinical diversity is represented to allow researchers to ask questions regarding the impact of tumor heterogeneity on target qualification or clinical response, whether PDXs more faithfully represent the human tumor for pharmacodynamic assay and predictive marker development, or if adequately powered preclinical PDX clinical trials can lead to better evaluation of therapies for future clinical use. Moving forward the PDMR plans to distribute in vitro, early-passage tumor cell cultures and cancer-associated fibroblasts as well as releasing PDX drug response data for a panel of FNA-approved therapeutic agents. Funded by NCI Contract No. HHSN261200800001E

#987

Organoid-based characterization of patient tumors and microenvironments at single cell resolution.

Ameen A. Salahudeen,1 Junjie Zhu,2 Jihang Ju,1 Arpit Batish,1 Ken Sutha,1 James T. Neal,1 Valeria Giangarra,3 Luz Montesclaros,3 Jerald Sapida,3 Osman Sharifi,3 Josephine Lee,3 Grace X. Zheng,3 Dhananjay A. Wagh,1 John A. Coller,1 Joel W. Neal,1 Sukhmani K. Padda,1 Chiara Sabatti,4 Calvin J. Kuo1. 1 _Stanford School of Medicine, Stanford, CA;_ 2 _Stanford School of Engineering, Stanford, CA;_ 3 _10X Genomics, Pleasanton, CA;_ 4 _Stanford University, Stanford, CA_.

The advent of microenvironment directed cancer treatments such as antiangiogenic agents and recent immunotherapies have produced a pressing need for robust and systematic characterization of patient tumors and corresponding stromal populations. However, in vitro cultures of tumor biopsies do not typically preserve both the tumor epithelium and tumor microenvironment as an intact syngeneic unit. To address these limitations, we developed organoid cultures of surgically resected patient cancer biopsies that intrinsically retain diverse tumor microenvironmental cellular components without requiring reconstitution. We compared transcriptome profiles of fresh tumor cell populations to organoid cultures by droplet based single cell 5' RNA sequencing (scRNA-seq). Analysis of >50k single cell transcriptome profiles revealed tumor and stromal cell populations including tumor epithelia, fibroblasts and immune cells in fresh tumors. scRNA-seq of organoid cultures revealed a similar composition of native cancer cells and stromal components in both immune and non immune populations. In particular, 5' scRNA-seq enabled simultaneous characterization of paired T cell receptor alpha and beta chains as well as generalized gene expression in individual cells. We observed clonotype expansion of cytotoxic T cells in both fresh tumors and organoid cultures. The faithful recapitulation of tumor microenvironmental diversity within human organoid cultures should facilitate the in vitro exploration of immunotherapeutic agents and modeling of associated patient-specific responses.

#988

Short-term culture of organotypic tumor spheroids derived from patient xenografts in a novel 3D microfluidic chip predicts in vivo response of targeted therapies.

Amir Aref, Elena Ivanova, Emily Chambers, Andrew Portell, Russell Jenkins, Mika Lin, Man Xu, Jacob Haworth, Magda Bahcall, Cloud Paweletz, Pasi Janne. _Dana-Farber Cancer Institute, Boston, MA_.

Introduction: Patient-derived xenografts (PDX) are increasingly adopted to evaluate the efficacy of new treatment approaches, including the development of combination therapies and testing of predictive biomarkers hypotheses. However, PDXs can take months to generate and are impractical for screening large sets of drug combinations. Here we report on a novel 3D microfluidic platform that allows for evaluating ex vivo responses to targeted therapies from genotyped defined patient-derived tumor spheroids (XDOTS) (1).

Methods: Non-small cell lung cancer (NSCLC) PDXs with known genotype, including EGFR ex19 del, HER-2 ins, and BRAF V600E, were generated from either core needle biopsies or pleural effusions under an IRB-approved protocol. XDOTS (<100 μm and >40μm) were grown from PDX tumors using DAX-1 3D cell culture chips (AIM, Singapore) and treated with genotype matched inhibitors that are either approved (e.g., erlotinib for EGFR mut) or are being tested in the clinic (neratinib for HER-2 ins) at known peak plasma concentrations for 3 days. Live/death quantification was performed by dual labeling fluorescence microscopy using acridine orange for live and propidium iodide for dead cells. Cell type characterization was performed by immunofluorescence in the devices.

Results: We first characterized our XDOT system on models for which we had orthogonal validation. We show by dual labeling fluorescence microscopy that PC9GR4 (EGFR Del19/T790M) cells are sensitive to osimertinib (IC50 = 12nM), but not gefitinib (IC50 >10uM). We further show that HCC827GR6 (Del 19/MET Amp) and DFCI32 (EML4-ALK/EGFR/HER2) cells are sensitive to crizotinib/gefitinib combo, but not to each of the single agents. Results were validated using the IncuCyte Live Cell analysis. We then went on to show that genotype-specific responses can be recapitulated in tumor spheroids generated from PDXs. In DFCI 359, a PDX derived from a HER2 mutant (755_757 LRdelinsPR), we show the antitumor efficacy of afatinib and confirm that effect is specific to tumor cells and not stromal. We observe significant drug efficacy only on cells growing in our microfluidic devices and not on spheroids growing 3D in 384 well plates. Similar results were obtained for additional PDXs with known NSCLC driver mutations. Finally, we evaluate our system on a small cohort of direct patient specimens.

Conclusions: Taken together, our data show that short culture experiments using XDOTS are feasible and recapitulate key futures of in vivo response and resistance ex vivo.

Reference: 1. Jenkins RW, et al. Cancer Discov DOI:10.1158/2159-8290.

#989

Organoid cultures from normal and cancer-prone human breast tissues preserve complex epithelial lineages and can form chimeric mammary glands in vivo.

Jennifer M. Rosenbluth,1 Ronald Schackmann,1 Carman Li,1 Norman Sachs,2 Deborah Dillon,3 Andrea Richardson,3 Jane Brock,3 Judy Garber,4 Gary Kenneth Gray,1 Jason Zoeller,1 Mackenzie Boedicker,1 Hendrick Johannes Kuiken,1 Hans Clevers,2 Joan Brugge1. 1 _Harvard Medical School, Boston, MA;_ 2 _Hubrecht Institute, Utrecht, Netherlands;_ 3 _Brigham and Women's Hospital, Boston, MA;_ 4 _Dana-Farber Cancer Institute, MA_.

Use of cell culture models to investigate the development of cancer associated with familial breast cancer syndromes is challenging because many culture methodologies do not maintain all of the lineages that are present in vivo, particularly the progenitor cells that may be the specific cancer cell-of-origin. We evaluated the ability of organoid culture technology to preserve stem/progenitor and differentiated cell types via long-term propagation of normal human mammary tissues. Tissue samples were obtained from elective reduction mammoplasties or from prophylactic mastectomies for cancer prevention, and were histologically normal. These were grown using conditions similar to organoid cultures for other epithelial cell types, as has been recently described (Sachs et al., Cell 2018). We found that basal stem and luminal progenitor cells could be cultured long-term, and differentiated in culture to generate mature basal and luminal cell types. Analysis of matched organoid cultures and native tissues by mass cytometry (CyTOF) for 38 markers at single-cell resolution confirmed the presence of multiple mammary epithelial cell types in the organoids, and demonstrated that protein expression patterns of the tissue of origin were largely preserved in culture. In addition, we generated a panel of over 40 mammary organoids derived from patients harboring inherited mutations in the cancer predisposition gene BRCA1 and from unaffected controls. Despite interindividual variability, an expansion in luminal progenitor populations associated with BRCA1 mutation could be detected in cultured organoids. Human mammary organoids heterozygous for BRCA1 could be engrafted into the murine mammary gland, resulting in mixed ductal and acinar structures. These studies indicate that this new model system is well suited for studies of aberrant phenotypes associated with BRCA1 mutation and approaches to prevent cancer development in these genetically predisposed tissues.

#990

Single-cell profiling of small cell lung cancer circulating tumor cell-derived xenograft models reveals intratumoral heterogeneity among mediators of chemoresistance.

C. Allison Stewart,1 Carl M. Gay,1 Yuanxin Xi,1 Siva V.,2 Junya Fujimoto,1 Pan Tong,1 Lixia Diao,1 Lerong Li,1 Mohan Bolisetty,2 Neda Kalhor,1 Patrice Lawson,1 Mayra Vasquez,1 Hai Tran,1 Ignacio I. Wistuba,1 Bonnie Glisson,1 Jianjun Zhang,1 Stephen G. Swisher,1 Jack A. Roth,1 John V. Heymach,1 Paul Robson,2 Jing Wang,1 Lauren A. Byers1. 1 _UT MD Anderson Cancer Ctr., Houston, TX;_ 2 _The Jackson Laboratory for Genomic Medicine, Farmington, CT_.

Small cell lung cancer (SCLC) accounts for 14% of lung cancer diagnoses in the United States and is characterized by rapid onset of chemoresistance and poor clinical outcomes. Once considered a homogeneous disease, recent analyses of SCLC have identified intratumoral heterogeneity (ITH) with respect to NOTCH signaling, ASCL1/NEUROD1 balance and MYC amplification - all of which are potential mechanisms underlying SCLC's aggressive and refractory biology. Unfortunately, patient-derived models of SCLC with which to better characterize the molecular profiles of refractory SCLC are scarce. To address this, we generated circulating tumor cell-derived xenograft (CDX) models from liquid biopsies of patients with treatment-naïve or relapsed SCLC. Each CDX model underwent pathological review to confirm tumors were consistent with SCLC based on histology and standard immunohistochemical markers (e.g., TTF1, chromogranin A, synaptophysin, NCAM). Sequencing of these models revealed mutations typical of SCLC (e.g. TP53, RB1), which were maintained in vivo over multiple passages. Importantly, each model's in vivo response to cisplatin matched the patient's platinum response at the time of CDX generation. At the proteomic level, platinum-resistant models exhibited mTOR activation, increased SOX2 and ATM, and reduced E-cadherin, suggesting a shift toward EMT and cancer stem cell expansion may contribute to resistance. To investigate ITH, we analyzed single-cell gene expression profiles by RNAseq using a droplet-based Chromium Single Cell system that analyzed a filtered subsample of 2000 cells per tumor. Consistent with SCLC, all CDX models contained large numbers of cells expressing neuroendocrine-specific genes (SYP, CHGA). However, Principle Component Analysis revealed that cells from chemosensitive CDX models had distinct expression profiles from resistant models. Using our published EMT gene signature, we found that resistant models had higher proportions of mesenchymal (vs. epithelial) cells. Several other distinctions between sensitive and resistant models were detected at the single-cell level but not in bulk RNA and protein analyses, suggesting that single-cell resolution can identify occult platinum-resistant subpopulations. For example, higher proportions of ASCL1- and DLL3-expressing cells were associated with platinum sensitivity, whereas a shift toward predominant NEUROD1-expression was observed with resistance. Cells expressing each of these three genes were identified across all tumors, suggesting platinum-sensitive and resistant subpopulations are ubiquitous but that even subtle shifts in the fractional distribution of these subsets can exert significant impact on response. These data support further use of single-cell analysis to explore the role of ITH as a driver of drug resistance in SCLC.

#991

Development and characterization of patient derived xenografts from central nervous system metastasis reveal minor clone expansion linked with aggressive tumor behavior.

Ben Yi Tew,1 Christophe Legendre,2 Tim Triche,3 Gerald C. Gooden,1 Kyle N. Johnson,2 Rae Anne Martinez,2 Emanuel F. Petricoin,4 Mariaelena Pierobon,4 Joyce O'Shaughnessy,5 Cindy Osborne,5 Mark A. Schroeder,6 Daniel J. Ma,6 Mark Bernstein,7 Jann N. Sarkaria,6 Steven A. Toms,8 Bodour Salhia1. 1 _University of Southern California, Los Angeles, CA;_ 2 _The Translational Genomics Research Institute, Phoenix, AZ;_ 3 _Van Andel Research Institute, Grand Rapids, MI;_ 4 _George Mason University, Manassas, VA;_ 5 _Baylor University Medical Center, Texas Oncology, Dallas, TX;_ 6 _Mayo Clinic, Rochester, MN;_ 7 _University Health Network, Toronto, ON;_ 8 _Lifespan Physician Group, Providence, RI_.

Despite recent advances in cancer therapy, options for central nervous system metastasis (CM) remain limited and patient outcomes are dismal. The lack of tumor models that reflect the heterogeneity and complexity of human CM tumors is a barrier to understanding the underlying biology and to developing novel therapies. Here, we report the development and characterization of 39 patient derived xenograft (PDX) models of CM tumors with histological subtypes representing the disease spectrum. The PDX models successfully grew both in the flank and brains of mice, and retained their histology regardless of the site of implantation. Brain metastasis also formed in 5/12 models after intra-cardiac injection. Multi-omic analysis of the PDX tumor and the patient-matched CM tumor showed that PDXs largely retained the molecular profiles of the tumor from which they were derived, including the retention of aberrations in key driver genes and signaling pathways. PDXs differed primarily from the ungrafted patient tumors by a downregulation in immunity-related pathways, which was expected due to the use of an immuno-compromised host, arguing for the validity of using the flank as a site of implantation. Integrated analysis of mutations and gene expression changes in CM revealed an upregulation of neuronal signaling and DNA damage response, and downregulation of calcium signaling and inflammation. We also assessed the clonal evolution of the PDX from the original patient CM by tracing variant allele frequencies in copy number neutral regions. We found that the PDX tumors were heterogeneous and comprised of multiple tumor clones that were either unique or in common with the original tumor, and displayed evidence of clonal expansion. Two PDXs that underwent phylogeny assessment showed evidence of minor clone expansion. One such PDX was a tumor from a Her2+ breast cancer (CM13), which largely did not molecularly resemble its matched patient tumor but was confirmed to have some shared expressed variants. The PDX was nearly copy number neutral, which included loss of the Her2 amplicon, and had few mutations compared with its parent tumor. Most noteworthy, is that CM13 displayed unique molecular patterns, aggressive growth and was highly metastatic in vivo. From the flank, it led to multiple metastases, including micrometastasis in the brain. This suggests that minor clone takeover may confer tumor aggressiveness and potentiate brain metastasis. The large and diverse repertoire of PDXs developed and characterized in this study provides a new set of tools that will help deepen our understanding of CM and improve preclinical testing for CM therapies. Our data also show that expansion of minor clones could confer aggressive tumor behavior and may be important in tumor evolution in PDX models and human cancers.

### Building the Microenvironment through Crosstalk

#992

Pancreatic tuft cells resolve injury and restrain tumorigenesis.

Kathleen E. DelGiorno,1 Chi-Yeh Chung,1 Maya Ridinger,1 Wahida Ali,1 Crystal Tsui,1 Cynthia Ramos,1 Makoto Ohmoto,2 LinJing Fang,1 Uri Manor,1 Ichiro Matsumoto,2 Geoffrey M. Wahl1. 1 _The Salk Institute, La Jolla, CA;_ 2 _Monell Chemical Senses Center, Philadelphia, PA_.

Pancreatic ductal adenocarcinoma (PDA) is the 3rd leading cause of cancer-related deaths in the United States and is predicted to be 2nd by the year 2020. The overall 5-year survival rate is < 9% and median survival is only 4-6 months. Symptoms appear late in disease progression and metastasis has typically occurred by the time of diagnosis, making the study of early events in tumorigenesis essential. Intra-tumoral heterogeneity contributes to metastasis and engenders chemotherapeutic resistance in cancer. We have identified acinar-to-ductal metaplasia (ADM) as a source of heterogeneity in early pancreatic disease progression. ADM is the formation of highly reactive, dedifferentiated ducts from pancreatic acinar cells and is hypothesized to progress to pancreatic intraepithelial neoplasia (PanIN), a proposed precursor to PDA. Interestingly, we have identified phenotypically distinct cell populations in ADM, associated with both pancreatitis and PanIN, including a significant number of tuft cells. Tuft cells are solitary chemosensory cells found throughout the hollow organs of the respiratory and digestive tracts. Simultaneous expression of taste, inflammatory, and neuronal signaling pathways is thought to allow for monitoring of intraluminal content and local control of absorptive and secretory processes, though how they contribute to tissue injury and tumorigenesis remains a critical knowledge gap. Using imaging and sequencing techniques, as well as novel mouse models, we have found that tuft cells accumulate during chronic, caerulein-induced pancreatitis and form during the recovery phase of acute injury. Tuft cell ablation using Pou2f3 knockout mice significantly worsens chronic injury, including greater tissue loss and extracelluar matrix deposition, and impairs recovery. Similarly, tuft cell knockout mice experienced greater transformation and advanced tumorigenesis in a LSL-KrasG12D;Ptf1aCre/+ model of pancreatic tumorigenesis, suggesting that tuft cells abate disease progression. Tuft cell isolation and RNA sequencing revealed expression of several immune modulators including prostaglandin synthase Hpgds. We have found that treatment of primary pancreatic stellate cells and macrophages with Pgd2-family prostaglandins inhibits activation providing one mechanism by which tuft cells contribute to pancreatic disease and a possible therapeutic route to inhibit disease progression. In conclusion, chemosensory tuft cells form following tissue injury and in early pancreatic tumorigenesis and use paracrine signaling mechanisms to quell inflammation and ebb disease progression.

#993

Wnt-er is coming: WNT5A promotes a slow cycling phenotype via p53 in conditions of stress.

Marie R. Webster,1 Mitchell Fane,1 Amanpreet Kaur,1 Gretchen Alicea,1 Brett L. Ecker,2 Abibatou Ndoye,1 Curtis Kugel,1 Subhasree Basu,1 Alexander Valiga,1 Jessica L. Appleton,1 Maureen E. Murphy,1 Ashani Weeraratna1. 1 _Wistar Institute, Philadelphia, PA;_ 2 _University of Pennsylvania, Philadelphia, PA_.

Resistance to targeted therapy and chemotherapy increases with age. Previously, we found that aged skin fibroblasts secrete sFRP2, which decreases ß-catenin, MITF, and APE1 in melanoma cells, and promotes invasion. Loss of APE1, a key redox effector, increased DNA damage in melanoma cells and caused the cells to become resistant to targeted therapy. Here we show that the aging tumor microenvironment drives a slow cycling phenotype in melanoma cells via WNT5A and p53. We found WNT5A expression in melanoma cells correlates with p53 expression and a higher percent of slow cycling cells. Knocking down WNT5A decreases both p53 expression and the percent of slow cycling cells. When we isolated slow cycling cells by flow cytometry, we found that they have increased WNT5A, p53, and p21 expression. Analysis of patient tumors in the TCGA data base revealed that patients with higher than average p53 expression, also express WNT5A. To determine if WNT5A and p53 may be promoting the survival of metastatic melanoma in an aged microenvironment, we examined tumors from young and aged C57/BL6 mice generated using Yumm1.7 cells. We found that tumors in aged mice have a higher percentage of cells expressing p53 and increased WNT5A expression compared to tumors in young mice. Treatment of human melanoma cells with conditioned media from aged dermal fibroblasts decreases proliferation and increases the percent of slow cycling. Inhibition of p53 re-sensitizes human melanoma cells in conditioned media from aged fibroblasts to combination BRAFi/MEKi. These data suggest, it may be these slow cycling cells driven by WNT5A and p53, which are enriched in an aging microenvironment, that are driving resistance to targeted therapy in aged patients.

#994

Cancer cells induce a protumorigenic senescent phenotype in fibroblasts through MMP1 but not autophagy in large cell carcinoma of the lung.

Jordi Alcaraz,1 Marta Gabasa,1 Rafael Ikemori,1 Noemí Reguart,2 Evette Radisky,3 Derek Radisky3. 1 _Univ. of Barcelona, Barcelona, Spain;_ 2 _Hospital Clínic de Barcelona, Barcelona, Spain;_ 3 _Mayo Clinic, Jacksonville, FL_.

Large cell carcinoma (LCC) is among the most aggressive histologic subtypes of non-small cell lung cancer, but the mechanisms underlying such aggressive nature remain unknown. We recently showed that fibroblasts from LCC patients exhibit premature senescence in vitro, and that co-culturing LCC cells (but not cancer cells from other lung cancer subtypes) with normal fibroblasts in transwells is sufficient to induce senescence in the latter in an oxidative stress-dependent manner, supporting that fibroblast senescence is induced by a secreted factor(s) from LCC cells. Remarkably, we also found that senescent fibroblasts secrete factors that stimulate the growth and invasion of LCC cells beyond the stimulation elicited by nonsenescent fibroblasts, revealing that fibroblast senescence may contribute to the aggressive nature of LCC. Whole-genome transcriptional profiling of a panel of lung cancer cell lines identified MMP1 among the genes with larger expression in LCC compared to other lung cancer subtypes. Since MMPs can induce oxidative stress, we examined whether the excessive MMP1 expression in LCC cells was involved in the induction of fibroblast senescence in co-cultures. Knocking down MMP1 in LCC cells was sufficient to abrogate fibroblast senescence in co-cultures as well as the growth and invasion enhancement elicited by the conditioned medium of fibroblasts in LCC cells. In contrast, autophagy, which has been previously associated with fibroblast senescence in breast cancer, was not upregulated in fibroblasts upon co-culture with LCC cells. These results support that the selective aberrant expression of MMP1 in LCC cells plays a major role in their ability to induce a protumorigenic senescent phenotype in adjacent fibroblasts through a mechanism that is independent of autophagy. Moreover, our observations identify MMP1 as a potential therapeutic target against the aberrant cancer cell-fibroblast crosstalk in LCC.

#995

Tumor-stroma IL-1β-IRAK4 feedforward circuitry drives tumor fibrosis, chemo-resistance and is associated with poor prognosis in pancreatic cancer.

Daoxiang Zhang, Lin Li, Hongmei Jiang, Andrea Wang-Gillam, Jinsheng Yu, Richard Head, Jingxia Liu, Marianna B Ruzinova, Kian-Huat Lim. _Washington Univ., Saint Louis, MO_.

Targeting the desmoplastic stroma of pancreatic ductal adenocarcinoma (PDAC) holds promise to augment the effect of chemotherapy, but so far success remains limited in the clinic. Furthermore, preclinical mouse models suggest that near-depletion of cancer-associated fibroblasts (CAFs) carries a risk of accelerating PDAC progression. These concerns underscore the need to concurrently target the key signaling mechanisms that drive the malignant attributes of both CAFs and PDAC cells. We previously reported that inhibition of Interleukin-1 Receptor Associated Kinase 4 (IRAK4) suppresses NF-kB activity and promotes chemotherapy response in PDAC cells. In this study, we show that CAFs in PDAC tumors robustly express activated IRAK4 and NF-kb. The role of IRAK4 and NF-kB in PDAC CAFs has not been reported, and should be clarified before advancing IRAK4 inhibitors to the clinic. Using shRNAs and small molecular inhibitors, we found that IRAK4 is a key driver of NF-kB activity in CAFs. We showed that CAFs utilizes IRAK4 to drive tumor fibrosis, support PDAC cells proliferation, survival and chemo-resistance in vitro and in vivo. From cytokine array analysis of CAFs and microarray analysis of PDAC cells, we identified IL-1b as a key cytokine that activates IRAK4 in CAFs. Targeting IRAK4 or IL-1b renders PDAC tumors less fibrotic and more sensitive to gemcitabine in vivo.Moreover, high IL-1b expression by immunohistochemistry in PDAC samples is strongly associated with poor overall survival. Together, our studies established a tumor-stroma IL-1b-IRAK4 feedforward circuitry that can be therapeutically disrupted to render chemotherapy more effective in PDAC.

#996

Host-derived MCP-1 dictates prostate cancer skeletal metastasis in vivo.

Jie Meng,1 Weiping Liang,1 Haibo Tong,1 Evan T. Keller,2 Jian Zhang,3 Yi Lu3. 1 _Guangxi Medical University, China;_ 2 _University of Michigan, Ann Arbor, MI;_ 3 _Southern University of Science and Technology, Shenzhen, China_.

Overexpression of the tumor-derived monocyte chemotactic protein-1 (MCP-1) has been suggested to partially promote prostate cancer bone metastasis, but the function for host-derived MCP-1 in these processes is not well established. To understand the function of host-derived MCP-1 on bone metastasis, we developed a mouse model using intracardiac injection and in vivo selection to obtain tumor cell subpopulations with a higher propensity for bone metastasis. Here, we found that a loss of host MCP-1 (MCP-1 knockout) retarded tumor growth in bone and prolonged survival of mice by intracardiac injection of tumor cells in vivo. Moreover, a decrease in osteolytic bone lesion was observed in MCP-1 knockout mice by bone density analysis, as compared to wild-type control. Systemically, MCP-1 deficiency inhibited the proportion of PMN-and M-MDSC populations with immunosuppressive function. Together, this study indicates that the dysregulated immunity resulting from a loss of host MCP-1 signaling is sufficient to drive PCa bone metastasis, and provides a new perspective for targeting the endogenous MCP-1 pathway as an antimetastatic strategy.

Supported by NSFC Key Project 81130046; NSFC projects 81773146, 81272415; JCYJ20170412152943794, JCYJ20170412154619484, JCYJ20170307105128101, JCYJ2017030711041760; Guangxi Key Projects 2013GXNSFEA053004.

#997

The cholesterol metabolite 27-hydroxycholesterol promotes breast cancer progression by affecting immune responses.

Amy E. Baek, Sisi He, Hannah B. McDowell, Erik R. Nelson. _Univ. of Illinois at Urbana-Champaign, Urbana, IL_.

With upwards of 90% of mortalities associated with breast cancer being attributed to metastasis, there is an urgent need to better understand what drives this stage of disease. In this regard, it has been reported that an elevated concentration of circulating cholesterol is an independent risk factor for breast cancer recurrence. Supporting the relationship between cholesterol and breast cancer progression, the use of cholesterol lowering drugs (statins) has been demonstrated to improve recurrence-free survival. These clinical observations suggest that cholesterol influences the metastatic progression of breast cancer. Therefore, in this study we directly tested the impact of cholesterol on breast cancer metastasis, and interrogated the downstream mechanisms by which it may be doing so.

In strong support of our hypothesis, increased metastasis was observed when mice were placed on a high cholesterol diet. These findings highlighted recent work by us and others demonstrating that the primary metabolite, 27-hydroxycholesterol (27HC), was able to modulate the activities of two hormone receptors: the estrogen and the liver X receptors. Thus, we pursued a series of experiments to determine whether 27HC was responsible for the metastatic actions of cholesterol. 27HC is synthesized from cholesterol by the enzyme CYP27A1. The genetic ablation of CYP27A1 completely attenuated the effects of a high cholesterol diet, implicating 27HC as a primary mediator of cholesterol. Treatment with exogenous 27HC robustly increased metastasis. Importantly, genetic or pharmacologic inhibition of CYP27A1 also reduced the basal metastatic burden, indicating that this enzyme may be a suitable target for the prevention and/or treatment of metastatic breast cancer.

Intriguingly, we found that the pro-metastatic effects of 27HC also required the presence of myeloid cells, which was demonstrated by their ablation using clodronate-loaded liposomes. In the absence of myeloid cells, the ability of 27HC to promote metastasis was dramatically reduced. Whilst interrogating the distal metastatic site, we found that this oxysterol enriched Ly6G+/CD11b+ polymorphonuclear-neutrophils (PMNs) and γδ T cells. The induction of metastasis by 27HC was lost in models where either PMNs or γδ T cells were ablated. We further demonstrate that 27HC treatment results in a decrease in cytotoxic CD8+ T cells, suggesting that the net result of the 27HC enriched γδ T cells and PMNs is local immune suppression.

Collectively, our results demonstrate that cholesterol increases metastasis via the actions of its metabolite 27HC, which exerts its effects through γδ T cells and PMNs to suppress acquired immunity. Since 27HC acts on the host environment to promote breast cancer progression, these results strongly support the immediate translational potential of targeting the 27HC pathway for the prevention and treatment of metastasis.

#998

Radiation-induced immunosuppressive macrophages limit CD8 T-cell mediated tumor killing.

Keaton I. Jones, Jiske Tiersma, Jon Buzzelli, Alex Gordon-Weeks, Arseniy Yuzhalin, Jaehong Im, Ruth J. Muschel. _University of Oxford, Oxford, United Kingdom_.

Emerging pre-clinical data suggests that radiation both stimulates and suppresses tumor immunity. The role of tumor associated macrophages (TAMs) has been extensively investigated in the non-irradiated tumor microenvironment. We aimed to investigate the role of macrophages in the immune response to radiation. Single dose 10Gy irradiation to tumors generated from colorectal (MC38) and pancreatic (KPC) cell lines induced Colony Stimulating Factor (CSF-1). Coincident with the elevation in CSF1, significant increases of CD11b+ F480+ macrophages within the tumor microenvironment peaked five days following irradiation returning to baseline during tumor regrowth. These TAMs were skewed toward an immunosuppressive phenotpye (CD206hi iNOSlo) compared to those from controls. Furthermore, the TAMs from irradiated tumors were more effective in suppression of CD8 T cell expansion in ex vivo assays. As a result of these data, we investigated the effect of anti-Colony Stimulating Factor (αCSF-1), which reduced macrophages in both control and irradiated tumors. αCSF had no effect on the growth of control tumors, yet led to a significant enhancement of tumor growth delay after radiation. TAMs sorted from irradiated tumors after aCSF treatment had reduced inflammatory gene expression and increased expression of some genes associated with immune suppression (Arg-1, IL-10, CCL2). Following aCSF, there was a significant increase in CD8 T lymphocytes with greater expression of cytotoxic markers. Radiation is also immune-stimulatory to tumors consistent with enhanced recognition of tumor cell antigens by T cells isolated from irradiated tumors. CD8 depletion however abrogated the growth delay from aCSF further documenting the immunosuppressive effect of the macrophages within irradiated tumors. aCSF1 was given to mice bearing two tumors, one of which was irradiated. An increased growth delay was only observed in the irradiated tumours. These data document the immunosuppressive nature of macrophages generated in irradiated tumors. TAMs expressed high levels of PD-L1 and aCSF treatment resulted in a reduction in tumor PD-L1. As a result we determined the effect of checkpoint inhibition using anti-PD-L1 in combination with aCSF following 10Gy irradiation. Whilst combination treatment did not augment outcomes in mice bearing MC38 tumors, there was complete tumor regression in a minority of mice bearing KPC tumors. These results suggest that infiltrating macrophages limit the adaptive immune response initiated following of radiation. Whilst it has been demonstrated that tumor cell PD-L1 expression plays an important role in checkpoint blockade, our data suggests macrophages are an important additional source. This is of particular importance in the context of radiation which can elicit a potent immune response but in which the TAMs from irradiated tumors also suppress this in part via altered PD-L1 expression.

## MOLECULAR AND CELLULAR BIOLOGY / GENETICS:

### Targeting the Cell Cycle: Mechanism and Therapy-- Poster Discussion

#1522

The CDK4/6 inhibitor G1T38 enhances response to targeted therapies in preclinical models of non-small cell lung cancer.

Jessica A. Sorrentino, Daniel M. Freed, John E. Bisi, Jay C. Strum, Patrick J. Roberts. _G1 Therapeutics, Research Triangle Park, NC_.

The recent FDA approvals of palbociclib, ribociclib, and abemaciclib in breast cancer validate cyclin-dependent kinases 4 and 6 (CDK4/6) as key therapeutic targets, and warrant investigations in other tumor types with frequently observed alterations in the CDK4/6 pathway. Towards this end, we assessed the preclinical antitumor efficacy of G1T38, an oral, potent, and selective small-molecule CDK4/6 inhibitor in clinical development, in models of non-small cell lung cancer (NSCLC). Importantly, preclinical studies have shown that G1T38 has a differentiated and potential best-in-class profile since it can be dosed continuously without causing severe neutropenia, which could result in better tumor efficacy. Initial efficacy screening of G1T38 in 60 patient-derived NSCLC xenografts revealed significant tumor growth inhibition (TGI), primarily in lung adenocarcinomas harboring frequently observed oncogenic alterations in KRAS, EGFR, BRAF, and ALK. In vitro analyses show that CDK4/6 inhibition enhances the antiproliferative effect of inhibitors targeting these oncogenes (or their signaling pathways), suggesting a possible role for G1T38 in augmenting response and/or delaying acquired resistance to agents currently utilized in the clinic. These results were validated in vivo in mouse NSCLC xenograft models with defined oncogenic alterations, including EGFR mutations. For example, in H1975 xenografts harboring EGFR L858R/T790M (the T790M mutation is responsible for over 50% of cases of acquired resistance to first-generation EGFR inhibitors), combination of G1T38 with erlotinib resulted in 77% TGI after 18 days--a significant improvement over either therapy alone. Moreover, in H1975 xenografts, G1T38 delayed resistance to the second-generation EGFR inhibitor afatinib, and upon development of resistance to afatinib monotherapy, addition of G1T38 to afatanib treatment resulted in stabilization of tumor growth. G1T38 also augmented the response of EGFR-mutant NSCLC xenografts to the third-generation EGFR inhibitor osimertinib, significantly enhancing TGI compared to either therapy alone. As a result, a safety, pharmacokinetics, and efficacy study of G1T38 + osimertinib (G1T38-03) is scheduled for initiation in 1Q18 in patients with EGFR-mutant, T790M-positive NSCLC. Additional efforts are ongoing to investigate the combination of G1T38 with targeted therapies in NSCLC murine models harboring alterations in KRAS, ALK, and BRAF. Together, our results suggest a compelling rationale for utilizing G1T38 as a backbone for multiple targeted therapy combination regimens in NSCLC.

#1523

Repression of IRF4 and CYTIP unleashes anti-tumor interferon response in CDK4 inhibitor therapy in mantle cell lymphoma.

Maurizio Di Liberto, Peter Martin, Xiangao Huang, Priyanka Vijay, David Chiron, Scott Ely, Christopher Mason, Olivier Elemento, John Leonard, Selina Chen-Kiang. _Weill Cornell Medical College, New York, NY_.

Inhibition of CDK4/6 has emerged as an effective cancer therapy, but the underlying mechanism remains obscure. We have addressed this question in mantle cell lymphoma (MCL), a Non-Hodgkin's B cell lymphoma that remains incurable due to the development of drug resistance. As dysregulation of CDK4 (CDK6 is silenced) and cyclin D1 underlies unrestrained proliferation of MCL cells, targeting CDK4 also represents a rational approach to MCL therapy.

Previously we have demonstrated in preclinical studies that sustained inhibition of CDK4 with palbociclib led to prolonged early G1 arrest (pG1) that both prevented proliferation of MCL cells and reprogrammed them for killing by diverse agents. On this basis, we hypothesized that CDK4 inhibition may prolong and deepen the clinical response to its partner drug in combination therapy. Indeed, in a phase I clinical trial of palbociclib plus ibrutinib (BTK inhibitor) in recurrent MCL (n=28), complete response was observed in 43% of patients as compared with 23% in response to ibrutinib alone. Moreover, the responses were rapid and durable; only 4 of the 18 responding-patients have progressed in the 3.5 years since the trial opened.

To address how CDK4 inhibition enhances therapy vulnerability, in a separate phase I clinical trial palbociclib was administered to MCL patients for 12 days to induce pG1; bortezomib was given in pG1 and again after synchronous S phase reentry (pG1-S). At the optimal dose combination only one of 6 patients progressed and complete remission in one patient continues into its 6th year. Longitudinal integrative RNA and exome-sequencing (WTS/WES) of MCL cells isolated from lymph node biopsies at baseline, in pG1 (day 8) and in pG1-S (day 21) of individual patients showed that inhibition of CDK4 induced pG1 in all patients initially, regardless of the subsequent clinical response.

However, induction of pG1 led to a striking difference in cellular gene expression in pG1 (day 8/day 1) in MCL cells of responders (R) and non-responders (NR). There were 2041 genes upregulated and 1483 genes downregulated 1.5-fold or greater in R (N=4, EdgeR, FDR 0.05), but only 39 genes upregulated and 401 genes dowregulated in NR (N=3). Moreover, 5 genes were down regulated in R and upregulated in NR. Gene set enrichment analysis together with loss and gain of function studies revealed that among them, loss of IRF4 in R led to de-repression of IRF7 and induction of the interferon response. This in turn resulted in TRAIL mediated apoptosis due to repression of CYTIP downstream of IRF4 repression.

In summary, by longitudinal functional genomics of purified MCL cells and functional studies, we have discovered that CDK4 inhibition reprograms MCL for therapy vulnerability through induction of anti-tumor IFN response, and identified CYTIP and IRF4 as biomarkers that discriminate sensitivity from resistance to therapeutic targeting of CDK4.

#1524

A first-in-class CDK4 inhibitor shows excellent in vitro and in vivo efficacy against ovarian cancer.

Laychiluh B. Mekonnen,1 Solomon Zeleke,1 Jimma Lenjisa,1 Mingfeng Yu,1 Benjamin Noll,1 Gary Heinemann,1 Robert Milne,1 Hugo Albrecht,1 Martin K. Oehler,2 Shudong Wang1. 1 _University of South Australia, Adelaide, Australia;_ 2 _Royal Adelaide Hospital, Adelaide, Australia_.

Ovarian cancer is one of the most common malignancies, accounting for more deaths than any other cancer of the female reproductive system. Cyclin-dependent kinases 4 and 6 (CDK4/6) are fundamental drivers of the cell cycle and are required for the initiation and progression of various cancers. Over-expression of CDK4/6 &/or cyclin D1 is commonly observed in ovarian cancer and is associated with an aggressive phenotype and poor prognosis. Patients with advanced ovarian cancer whose tumor demonstrated Rb-positive & a low expression of p16INK4a have the worst clinical outcome, but these patients are likely to gain benefit from CDK4/6 inhibition. Here, we describe the preclinical development of 2-94; a potent, highly selective and orally bioavailable CDK4 inhibitor for the treatment of ovarian cancer. Specifically, 2-94 exhibits high selectivity for CDK4/D1 (Ki = 2 nM) over CDK6/D3 (Ki = 279 nM). Consistent with a CDK4-targeted mechanism, 2-94 potently inhibited proliferation of Rb proficient ovarian cancer cells. It induced G1 cell cycle arrest by reducing Rb phosphorylation at S780, S795 and S807/11 residues in a dose dependent manner. Apoptosis was induced with evident increase of caspase 3/7 activity and reduction of Bcl-2 protein levels in A2780 cells. In addition, 2-94 has shown synergistic activity with mTOR, MEK, PI3K and PARP inhibitors. In contrast, no apparent cell cycle effect, senescence or apoptosis was observed in human bone marrow mononuclear (hBMNCs) cells by 2-94 treatment unlike palbociclib and ribociclib which increased the G1 population of hBMNCs, suggesting the low toxicity of 2-94 compared to clinical compounds. Consistent with its favourable pharmacokinetic properties and high oral bioavailability, 2-94 delayed tumor growth and significantly increased the survival of A2780 tumor bearing nude mice by oral administration. 2-94 was more efficacious when compared to palbociclib, resulting in a T/C = 22% for 2-94 vs T/C = 31% for palbociclib. Moreover, 2-94 showed little or no effect on lymphocyte and neutrophil counts whilst palbociclib caused their reduction. This lower effect of 2-94 on the neutrophils and lymphocytes might be due to its lesser effect on CDK6-cyclin D3 compared to palbociclib. Both 2-94 and palbociclib didn't cause histopathological changes in the bone marrow, intestine, liver, heart and kidney of the mice. Pharmacodynamical studies confirmed that 2-94 reduced the Rb phosphorylation at S780, S795 and S807/811 in the tumour models. In conclusion, 2-94 is a first-in-class CDK4 inhibitor with high selectivity against a panel of 360 human kinases. This selectivity can be taken as a reason for the observed better safety of 2-94 compared to palbociclib. The favourable oral pharmacokinetics, robust antitumor efficacy and excellent safety profiles make 2-94 a highly attractive candidate for development towards the clinic.

#1525

SY-1365, a selective CDK7 inhibitor, exhibits potent antitumor activity against ovarian cancer models in vitro and in vivo.

Panagiotis A. Konstantinopoulos,1 Graeme Hodgson,2 Nisha Rajagopal,2 Liv Johannessen,2 Joyce F. Liu,1 Paul T. Kirschmeier,1 Shan Zhou,1 Cam Anh Tran,1 David Orlando,2 Christian Fritz,2 Emmanuelle di Tomaso,2 Ursula A. Matulonis1. 1 _Dana Farber Cancer Institute, Boston, MA;_ 2 _Syros Pharmaceuticals, Inc., Cambridge, MA_.

Introduction: CDK7, a key regulator of transcription and cell cycle progression, has been implicated in the pathogenesis of ovarian carcinoma. SY-1365, a potent and selective inhibitor of CDK7, was developed to exploit tumor dependencies driven by CDK7 and is in phase 1 clinical development. Here we report, for the first time, on the activity of SY-1365 in preclinical models of ovarian carcinoma and on potential biomarkers of SY-1365 response.

Methods: SY-1365 growth rate inhibition (GR) curves were determined in 314 solid tumor cell lines, including 22 ovarian carcinoma lines. Cell lines were clustered into cytotoxic and cytostatic response groups and assessed for RNA expression to identify markers associated with SY-1365 sensitivity. In vivo, SY-1365 activity was evaluated at the maximum tolerated dose in 17 independent ovarian PDX models derived from heavily pretreated patients; a signal-finding design was used in which tumor growth in SY-1365 treated mice (n=2-3 per PDX model) was compared to vehicle-treated mice or historical growth in untreated mice.

Results: SY-1365 induced cytotoxicity (GR <0) in 61% (190/314) of all tumor lines at <50nM. In ovarian carcinoma lines, 36% (8/22) were cytotoxic at <50nM, with deep responses (GRmax < -0.5) seen in 23% (5/22). Lower expression of BCL2L1, which encodes the mitochondrial apoptosis antagonist BCLXL, was the most predictive marker of sensitivity across all lines (accuracy = 70%; FDR=0.006). Reasoning that low BCL2L1 may be a marker of dependence on other mitochondrial apoptosis antagonists (MCL1 and/or BCL2) for survival, we evaluated expression of BCL2, BCLXL, and MCL1 protein at baseline and post-SY-1365 treatment in ovarian carcinoma lines. As with BCL2L1 RNA, BCLXL expression was lower at baseline in cytotoxic- versus cytostatic-responsive lines. In addition, SY-1365 induced MCL1 downregulation in cytotoxic, but not in cytostatic lines. In vivo, SY-1365 induced responses in 52% (9/17) of ovarian carcinoma PDX models. Of the 9 responders, 2 exhibited 40-60% tumor growth inhibition (TGI), 7 exhibited ≥ 100% TGI. As in cell lines, low BCLXL, in combination with low BCL2, was associated with SY-1365 response in PDXs. In addition, 7/9 responsive models had alterations in the RB signaling pathway including loss of RB1 expression (n=3), high CCNE1 expression (n=3), or CDKN2A loss (n=1).

Conclusions: SY-1365 induces cytotoxicity in ovarian carcinoma cell lines and induces robust responses in heavily pretreated (including PARP-inhibitor and platinum-resistant) ovarian PDX models. Alterations in expression of mitochondrial apoptosis antagonists and RB pathway regulators are associated with SY-1365 response and support exploration of these as predictive biomarkers of SY-1365 clinical activity in ovarian carcinoma. SY-1365 is currently being assessed in a phase 1 trial in adult patients with advanced solid tumors (NCT03134638).

#1526

WEE1 inhibition suppresses esophageal adenocarcinoma tumor growth both in vitro and in vivo.

Mylan Blomquist, Vashti M. Carson, Ross M. Bremner, Timothy G. Whitsett, Landon J. Inge. _St. Joseph's Hospital and Medical Center, Phoenix, AZ_.

Esophageal adenocarcinoma (EAC) has seen a 400% increase in incidence over the past 30 years. The 5-year survival rate is under 20% due to ineffective therapeutics and a lack of actionable oncogenic drivers, necessitating novel therapeutic avenues in this disease. Genomic analysis indicates that a subset of EAC tumors have alterations in the G2/M cell cycle check point and DNA damage response governed by mutations in TP53 or other hits in this pathway, suggesting targeting the G2/M pathway might be a viable option in EAC. We and others have demonstrated that the WEE1 inhibitor AZD1775 is effective both in vitro and in vivo against tumors with DNA damage response alterations, and this drug is currently in clinical trials across a number of tumor types. We hypothesized that inhibition of WEE1 would induce DNA damage and cell death in EAC tumors, and provide a rational therapeutic avenue against this deadly disease. Across multiple EAC cell lines, AZD1775 suppressed tumor cell growth either as a monotherapy or in combination with DNA-damaging therapies. The growth suppression was accompanied by persistent DNA damage as determined by γH2AX and the induction of apoptosis. FLO-1 cells, responsive to monotherapy AZD1775, showed a dramatic reduction in G2 cells 24 hours post AZD1775 exposure with concomitant increases in cells in G0 and S phases of the cycle; while SK-GT-4, resistant to monotherapy AZD1775, showed an initial reduction in cells in G2 but recovered to normal levels 24 hours post-exposure. To assess AZD1775 in vivo, we generated a patient-derived xenograft (PDX) model of an EAC tumor excised at our institution. Combinations of AZD1775 with radiation or cisplatin significantly reduced in vivo tumor growth compared to vehicle, and the combination of AZD1775 with cisplatin was as effective as standard-of-care treatment (cisplatin + docetaxel + radiation), a therapeutic avenue plagued by toxicity in humans. Exposure to AZD1775 in combination with cisplatin showed suppression of phospho-CDC2 (target-hit) and induction of γH2AX in vivo compared to vehicle. Towards understanding a mechanism for lack of response of certain EAC cell lines to AZD1775 monotherapy, we explored the PI3K pathway that has been previously implicated in AZD1775 resistance. SK-GT-4 cells, which are unresponsive to AZD1775 monotherapy, demonstrated elevated phospho-AKT and phospho-p70S6K protein levels compared to other cell lines and were found more sensitive to PI3K or MTOR inhibition, suggesting a dependence on this pathway for tumor cell survival. Collectively, our data suggest that AZD1775, both alone and in combination with standard-of-care DNA damage, may be an effective therapeutic strategy for those EAC tumors dependent on the G2/M checkpoint.

#1527

Splicing factors determine breast cancer cell mitosis through control of sister chromatid cohesion.

Esmee Koedoot,1 John Martens,2 Sylvia E. Le Dévédec,1 Bob van de Water1. 1 _Leiden University, Leiden, Netherlands;_ 2 _Erasmus MC, Rotterdam, Netherlands_.

Breast cancer is the mostly diagnosed cancer in women worldwide and is also the leading cause of death from cancer in women, mainly due to metastasis formation. Accumulating evidence suggests that RNA splicing is critical in breast cancer progression. To investigate the role of every single component of the splicing machinery (the spliceosome) in this process, we systematically evaluated the effect of siRNA-mediated knockdown of 244 splicing factors on proliferation in two highly proliferative breast cancer cell lines MDA-MB-231 and Hs578T. Hits of the primary screen were validated with single siRNAs and separated based on their effects on migration and proliferation respectively. Knockdown of nine validated proliferation hits, amongst which was SF3B1, a known breast cancer driver gene, resulted for all nine hits in a distinct poly-lobed nuclear phenotype in both cell types, accompanied by increased DNA content and higher number of cells in G1-S transition. Increased levels of phospho-Histone H3 and lack of metaphase alignment suggested a defect in mitosis, which was confirmed by decreased RNA expression of sister chromatid cohesion factors MAU2, ESPL1 and SMC1A and interestingly, increased CDCA5 RNA levels. Upon splicing factor knockdown, CDCA5 intron 1 was retained resulting in decreased CDCA5 protein levels, inaccurate metaphase alignment and finally cell death. To identify the direct partners of the splicing factors that control mitosis, we coupled pull-down of GFP fused splicing factors with mass spectrometry. Seven out of the nine splicing factors that were identified in our screen causing poly-lobed nuclear phenotypes, resided in the same protein complex together with proteins involved in snRNP assembly and some mitosis-related factors. In conclusion, we have identified several splicing factors that are critically determining breast cancer cell proliferation through modulating the expression of chromatid cohesion factors and thereby mediating the successful metaphase alignment and ultimate mitosis. 

# Monday, April 16, 2018

## TUMOR BIOLOGY:

### Adaptation and Checkpoints in Tumorigenesis

#999

Unravelling myeloid and T cell compartment interactions through a novel approach to tumor multimodal analysis combining whole slide multiplexed immunofluorescence and gene expression profiling.

Natalie Zwing. _Roche Innovation Center Munich, Penzberg, Germany_.

Understanding the role of myeloid cells in tumor biology requires a systems approach combining phenotypic and functional biomarkers. As a model system, colorectal cancer (CRC) tumors represent well-described clinically relevant immune subtypes. Distinct immune patterns have been reported involving the myeloid cells acting as immune suppressors for CD8 cytotoxic T cells. We hypothesized that myeloid derived suppressor cells (MDSCs) and CD8 T cells co-localize due to their direct interactions.

A cohort of 85 CRC primary tumors were analyzed by multiplexed immunohistochemistry/ immunofluorescence and subsequent quantitative and qualitative whole slide image analysis. To define the functional status of the identified immune cell populations, their corresponding signatures were analyzed by gene expression profiling.

Combining density and distribution of both myeloid cells and T cells as well as their distance to epithelial cancer cells, specific patterns were observed. In both microsatellite stable (MSS) and instable (MSI) tumors, MDSCs showed a comparable stromal distribution. By contrast, in MSI cases only, effector T cells tended to accumulate in the tumor epithelium suggesting they overcome the immunosuppressive environment. Additionally, the assessment of CD8 T cell morphology revealed changes potentially reflecting their cancer cell eliminating properties.

This multimodal analysis represents a novel approach to characterize and understand the role of the myeloid compartment through a combination of morphological and spatial characteristics of the T cell compartment.

#1000

PD-L1 binding peptide identified by phage peptide display inhibits PD-1/PD-L1 interaction and activates T cells.

Smriti Gurung, Fatima Khan, Soo-Woong Lee, Jaewon Yoon, Byungheon Lee. _Kyungpook National Univ., Daegu, Republic of Korea_.

Immune suppressive mechanism within the tumor microenvironment inhibits antitumor T cell function, leading to evasion of tumor from immune attack. One of the mechanisms is the up-regulation of programmed death-ligand 1 (PD-L1) expression on tumor cells which binds to programmed cell death protein 1 (PD-1) on activated T cells and inhibits T cell functions. Inhibiting the PD-L1/PD-1 interaction could reverse the tumor microenvironment and enhance the endogenous antitumor immune response. To identify a novel peptide that selectively binds to PD-L1, we screened a phage-displayed peptide library against cells transiently expressing PD-L1 at high levels. After multiple rounds of screening, selected phage clones were sequenced. Of these, two phage clones showed more selective binding to cells expressing PD-L1 compared to a control clone. The peptides displayed on the clones were synthesized for further study and named as PD-L1Pep-1 (9mer) and PD-L1Pep-2 (7mer). Fluorescein-conjugated PD-L1Pep-1 and PD-L1Pep-2 selectively bound to cells highly expressing PD-L1, whereas only negligible binding to cells expressing PD-L1 at low levels. The cell binding of the PD-L1-binding peptides was enhanced after treatment of cells with interferon-γ which is a potent inducer of PD-L1 in many types of cancer cells. FACS analysis showed that the pre-treatment of cells with an anti-PD-L1 antibody competed and reduced the cell binding of the PD-L1-binding peptides, suggesting the antibody and peptides bind to the same epitope on PD-L1. Co-culture experiments of CT26 mouse colon tumor cells with primary T cells isolated from a mouse spleen showed that, similar to the anti-PD-L1 antibody, the PD-L1-binding peptides increased the secretion of Interleukin-2 and interferon-γ by the T cells, indicating that the PD-L1-binding peptides can reactivate the T cells that were suppressed by the co-cultured tumor cells. In-vivo fluorescence imaging of BALB/c mice bearing subcutaneously transplanted CT26 mouse colon tumor cells showed tumor targeting ability of the PD-L1-binding peptides to PD-L1-expressing tumor. These findings suggest that PD-L1Pep-1 and PD-L1Pep-2 can inhibit PD-L1/PD-1 interaction and activate T cells in vivo and hold a potential as a cancer immunotherapeutic. Keywords: Colon tumor, immunotherapy, PD-L1, peptide, phage display

#1001

Gut microbiota modulate T cell trafficking into human colorectal cancer.

Eleonora Cremonesi,1 Valeria Governa,1 Jesus Garzon,2 Valentina Mele,1 Francesca Amicarella,1 Manuele G. Muraro,1 Raoul Droeser,1 Martin Bolli,3 Julia Slotta-Huspenina,4 Luigi Terracciano,1 Paul Zajac,1 Giulio C. Spagnoli,1 Serenella Eppenberger-Castori,1 Klaus-Peter Janssen,4 Lubor Borsig,5 Giandomenica Iezzi1. 1 _University Hospital of Basel, Basel, Switzerland;_ 2 _University of Zürich, Switzerland;_ 3 _Claraspital, Basel, Switzerland;_ 4 _Technical University of Munich, Munich, Germany;_ 5 _University of Zürich, Zurich, Switzerland_.

Introduction: Colorectal cancer (CRC) is a leading cause of cancer-related death. CRC infiltration by immune cells, including cytotoxic CD8+ T cells (CTLs), IFN-gamma-producing T-helper 1 cells (Th1), Foxp3+ regulatory T cells (Tregs) and CD16+ MPO+ neutrophils, is associated with favorable prognosis. However, chemokines driving these cell populations into the tumor site, their cellular sources and their microenvironmental triggers remain to be elucidated. Aim: We investigated the chemokine/chemokine receptor network promoting CRC infiltration by immune cells associated to favorable prognosis. Results: CRC infiltration by immune cells was associated with defined chemokine gene signatures, including CCL5, CXCL9 and CXCL10 for cytotoxic T lymphocytes and T-helper 1 cells, and CCL17, CCL22 and CXCL12 for T-helper 1 and regulatory T cells. Most of these chemokine genes were expressed by tumor cells upon exposure to gut bacteria in vitro and in vivo. Indeed, chemokine expression levels were significantly higher in orthotopic xenografts than in intraperitoneal tumors, and were drastically reduced by antibiotic treatment of tumor-bearing mice. Importantly, in human CRC samples, extents of chemokine production and immune cell infiltration was significantly associated with bacterial loads. Conclusion: Gut microbiota stimulates chemokine production by CRC cells, thus favoring T cell recruitment into tumor tissues.

#1002

Dysfunction of HPV16-specific CD8+ T cells derived from oropharyngeal tumors is related to the expression of Tim-3 but not PD-1.

Simona Partlova,1 Kamila Hladikova,1 Vladimir Koucky,1 Anna Fialova,1 Radek Spisek,1 Jan Boucek,2 Michal Zabrodsky,2 Michael Halaska,3 Ruth Tachezy,4 Marek Grega,5 Jean-François Fonteneau6. 1 _Sotio A.S, Prague, Czech Republic;_ 2 _1st Faculty of Medicine, Charles University and University Hospital Motol, Prague, Czech Republic, Prague, Czech Republic;_ 3 _3rd Faculty of Medicine and University Hospital Královské Vinohrady, Prague, Czech Republic, Prague, Czech Republic;_ 4 _Faculty of Science, Charles University in Prague, Prague, Czech Republic, Prague, Czech Republic;_ 5 _2nd Faculty of Medicine, Charles University and Motol University Hospital, Prague, Czech Republic, Prague, Czech Republic;_ 6 _CRCINA, INSERM, Université de Nantes, Nantes, France_.

Human papillomavirus (HPV) type 16 infection is one of the most important etiological agents of oropharyngeal squamous cell carcinoma. Patients with HPV-positive squamous cell carcinomas of the head and neck were reported to have a better clinical outcome than patients with HPV-negative tumors. Because HPV16 E6 and E7 oncoproteins are highly immunogenic and constitutively expressed within the tumor cells, HPV-specific T cell immunity may contribute to the better prognosis of HPV-positive tumors. We analyzed the frequency, phenotype and function of HPV16 E6/E7-specific tumor-infiltrating T cells (TILs) in oropharyngeal tumors and tested the effect of anti-PD1 mAb (nivolumab), soluble Tim-3 (sTim-3) and homeostatic in vitro expansion on these characteristics. We show that 73.1% of HPV-associated oropharyngeal tumors are positive for HPV16-specific T cells capable of producing IFNγ upon stimulation with E6/E7 peptide pools. These IFNγ-producing HPV-specific TILs were mainly PD-1+Tim-3-CD8+ T cells, identifying Tim-3 rather than PD-1 as a marker of T cell dysfunction. Consequently, specific IFNγ production was further enhanced by combined nivolumab plus sTim-3 treatment, but not with nivolumab alone. Our data provide the rationale for exploring additional setups of anti-PD-1 mAb treatment, including combinations with other check-point blockers, such as Tim-3 and/or with HPV16-directed therapeutic vaccines.

#1003

Improving the functional taxonomy of breast cancer-associated fibroblasts by single-cell RNA sequencing reveals clinically relevant cellular subsets.

Michael Bartoschek, Kristian Pietras. _Lund University, Lund, Sweden_.

Aim: The traditional tumor cell-centric view of cancer has been replaced in the past years with the appreciation of the importance of the tumor microenvironment for the malignant phenotype. Cancer-associated fibroblasts (CAFs) are the main constituent of the breast tumor microenvironment, although the exact origin and role of CAF subpopulations in disease initiation, progression and response to treatment remain unclear. Therefore, cancer therapies targeting CAFs are not available.

Methods: Here, we delineate the heterogeneity of CAFs through single-cell RNA sequencing of 768 EPCAM-CD31-CD45-NG2- cells from the MMTV-PyMT mouse model of breast cancer.

Results and discussion: Unbiased transcriptional clustering and differential gene expression analysis revealed four populations, with functionally distinct transcriptional profiles. The existence of these cell populations was further validated by immunostaining in experimental and human tumors revealing a preferential localization of each CAF subtype within the tumor microenvironment. Gene profiles specific to each CAF subpopulation held prognostic capability in clinical cohorts and correlated to known functional gene profiles. Our high-resolution comparison of single-cell transcriptomes in CAFs reveals previously unappreciated functional diversity within the tumor microenvironment.

Conclusions: Ultimately, our delineation of an improved taxonomy of breast CAFs reveals a previously unappreciated functional diversity within the tumor microenvironment and opens the possibility for biomarker-driven development of new drugs for precision medicine.

#1004

Tuft cell chemosensory signaling during pancreatic ductal adenocarcinoma.

Megan T. Hoffman, Howard Crawford. _University of Michigan, Ann Arbor, MI_.

Pancreatic ductal adenocarcinoma (PDA) is the second most common cause of cancer death in the US with a 10% 5-year survival rate and, as such, is one of the most common deadly cancers. PDA is characterized by a robust stromal reaction, a large portion of which is composed of infiltrating immune cells. This inflammatory response has been previously shown to promote both the initiation and progression of PDA. Current research has been to understand the interaction between tumor cells and infiltrating immune cells and the contribution to cancer progression. In this work we focus on a specific cell population present in the epithelium during pancreatic tumorigenesis called a tuft cell. Tuft cells have been found in luminal surfaces throughout the body, most notably characterized in the intestine where these cells have been shown to directly regulate the immune response to parasitic infection, but have also been found in the nasal, respiratory and bladder epithelium. These solitary chemosensory cells, as they are also known, use a common chemosensory pathway, previously characterized in type-II taste cells, in order to interact with the luminal environment. Interestingly, tuft cells in the metaplastic pancreas uniquely express TRPM5 and alpha-gustducin, both components of the chemosensory cascade, suggesting these cells have the potential to mediate a response to extracellular signals. Pancreatic tuft cells also make an array of cytokines and immunomodulatory factors that can directly alter the immune response in the tumor microenvironment. These data leads us to hypothesize that tuft cells utilize the chemosensory response pathway to remodel the tumor microenvironment in PDA. In order to answer this question, we ablated a key protein in the chemosensory cascade, alpha-gustducin, and found that this loss increases aggressiveness in a model of pancreatitis-associated tumorigenesis. We are currently exploring differences in the tumor microenvironment that may explain these results.

#1005

Combination T cell receptor immunosequencing and multiplex immunohistochemistry reveal novel insights into the immune response to human pancreatic cancer.

Yongwoo David Seo,1 Florencia Jalikis,1 Xiuyun Jiang,1 Kevin M. Sullivan,1 Marissa Vignali,2 Harlan Robins,3 Venu G. Pillarisetty1. 1 _University of Washington, Seattle, WA;_ 2 _Adaptive Biotechnologies, Seattle, WA;_ 3 _Fred Hutchinson Cancer Research Center, Seattle, WA_.

Background: Despite advancements in therapy, pancreatic ductal adenocarcinoma (PDA) remains an aggressive cancer with high mortality. It is characterized by dense inflammation, including many T cells; however, it is unclear whether these T cells signify true anti-tumor response. In the setting of disappointing results of immunotherapy in PDA, we sought to gain a deeper understanding of the intratumoral T cell response.

Methods: With IRB approval, we obtained archival resected PDA tumors in paraffin embedded blocks from 54 patients and performed T cell receptor (TCR) immunosequencing on extracted DNA. Productive clonality was defined as 1-Pielou's evenness; TCR fraction was calculated from number of observed templates versus housekeeping genes. Multiplex immunohistochemistry (IHC) was performed on slides immediately adjacent to blocks sent for TCR sequencing using the PerkinElmer Vectra system. False H&E images generated from autofluorescence signatures were analyzed by a pathologist to categorize peritumoral stroma versus lymphoid tissue. Image analysis was performed using FlowJo10, turning object fluorescence data into cytometric outputs. Correlation analysis between multiplex and TCR data was performed using Pearson's correlation.

Results: Mean TCR fraction was 0.27, and mean clonality was 0.15 (typical peripheral blood clonality is 0.08). TCR fraction was positively correlated with clonality (R2=0.23, p=0.007), but there was no survival difference between high or low TCR fraction or clonality. 3.1 million individual cells within the peritumoral stroma were phenotyped in total using multiplex IHC; within the stroma, CD4 T cells, CD8 T cells, and macrophages made up on average 4.6%, 6.8%, and 2.0% of all cells analyzed. 35.6% of CD8 T cells were positive for PD-1, while 20.8% of CD4 cells were FOXP3+ regulatory T cells (Treg). TCR fraction correlated positively with infiltration of CD8+ (R2=0.28, p=0.03) and CD68+ (R2=0.27, p=0.03) cells, but not CD4 cells. Only CD8 cells which were PD-1- correlated positively with clonality (R2=0.26, p=0.04). The presence of Treg correlated very strongly with CD8+PD-1+ cells (R2=0.44, p=0.0004).

Conclusion: Here we demonstrate a novel approach to describe the varying immunophenotypic landscape of human PDA depending on the clonal expansion of intratumoral T cells. T cell infiltration appear to be correlated mostly with CD8 T cells as well as the presence of macrophages. Patients with more clonal expansion also had more CD8 cells that lacked the exhaustion marker PD-1, and the presence of regulatory T cells accompanied environments where CD8 cells were positive for PD-1. These data suggest that cytotoxic T cell exhaustion is a phenomenon which may be regulated by Treg and have a negative impact on clonal expansion of intratumoral T cells in human PDA.

#1006

Adipsin enhanced clonogenicity and proliferation of human breast cancer patient-derived xenograft cells.

Hideaki Goto, Yohei Shimono, Toru Mukohara, Yohei Funakoshi, Yoshinori Imamura, Seishi Kono, Shintaro Takao, Akira Suzuki, Hironobu Minami. _Kobe University Graduate School of Medicine, Kobe, Japan_.

Introduction: Tumor microenvironment plays a key role in the development and progression of tumors. Although adipose tissue is a predominant component of tumor microenvironment in mammary tissue, and secretes various cytokines, chemokines and growth factors, roles of adipocytes in the breast cancers remain to be elucidated.

Methods: In this study, we identified adipsin, an adipokine predominantly expressed in the adipose-derived stem cells (ADSCs) of breast cancer patients, enhanced the clonogenicity, proliferation, and cancer stem cell-like properties of human breast cancer patient-derived xenograft (PDX) cells. Human breast cancer PDXs were established by the engraftment of surgical specimens of the breast cancer patients into immunodeficient mice. Human mammary adipose-derived stem cells (ADSCs) were isolated from the enzymatically dissociated adipose tissues of the surgical specimens of breast cancer patients.

Results: When ADSCs with diverse patient backgrounds were co-cultured with human breast cancer PDX cells, ADSCs significantly enhanced the clonogenicity of breast cancer PDX cells (n = 9; p < 0.05). We found that adipsin, an adipokine that is involved in the generation of C3a and C3b in the complement alternative pathway, was highly expressed in the cultured supernatants of ADSCs when compared to those of breast cancer PDX cells (n = 9; p < 0.05). Accordingly, the amount of C3a was significantly higher in the cultured supernatants of ADSCs when compared to those of breast cancer PDX cells (n = 9; p < 0.05). Suppression of adipsin by a specific inhibitor or knockdown in mammary ADSCs significantly suppressed the sphere-forming abilities of breast cancer PDX cells; reduced the mRNA expression levels of the cancer stem cell marker CD44 and the chemokine receptor CXCR4 (p < 0.01); and reduced the cell surface expression of CD44 in breast cancer PDX cells, suggesting that cancer stem cell-like properties were regulated by adipsin. Although the suppression of CXCR4 by a specific inhibitor showed a tendency to suppress the sphere-forming abilities of breast cancer PDX cells, the dual suppression of adipsin/C3a and CXCR4 by their specific inhibitors showed no additive effects on the suppression of the clonogenicity of breast cancer PDX cells.

Conclusion: These observations suggest that ADSCs are important components of tumor microenvironment in human breast cancers, and secrete adipsin that functions as regulators of the clonogenicity, proliferation, and cancer stem cell-like properties of human breast cancer cells.

#1007

Durvalumab + tremelimumab increase T cells and decrease expression of genes involved in angiogenesis, EMT, and the Th2 pathway in the NSCLC tumor microenvironment.

Li Cheng, Lydia M. Greenlees, Nicholas M. Durham, Fernanda Pilataxi, Todd Creasy, Brandon W. Higgs, Koustubh Ranade, Katie Streicher. _Medimmune Inc, Gaithersburg, MD_.

Background

Cancer cells utilize multiple mechanisms to suppress the development of an effective anti-tumor immune response. Overcoming these suppressive mechanisms to restore effector function to exhausted tumor infiltrating lymphocytes (TILs) is critical for response to checkpoint blockade; however, the specific changes associated with different IO treatments has yet to be fully characterized. We treated NSCLC tumor fragments or digests ex vivo with the anti-PD-L1 antibody durvalumab (D) alone or in combination with the anti-CTLA-4 antibody tremelimumab (T) to explore changes in gene and protein expression associated with checkpoint blockade.

Methods

10 NSCLC tumors (5 fresh and 5 frozen) were analyzed. Minced fresh tumor fragments or frozen tumor digests were seeded in media containing low dose interleukin-2 (IL-2) plus D and/or T or the appropriate isotype controls. After 1-2 weeks of treatment, TILs were evaluated for changes in cytokine production, as well as the expression of inhibitory receptors and other immune-related markers. Microarray gene expression analysis was performed on a subset (n = 2) of the NSCLC tumors evaluated.

Results

Of 10 NSCLC tumors tested, 4 showed a robust increase in IFNg production (> 2-fold) relative to isotype controls in both D and D + T treated conditions, with an additional 3 tumors showing smaller, but consistent 30-40% increases in IFNg production. 4 NSCLC tumors showed at least a 30% increase in CD4+ and CD8+ T cell proliferation following D and D+T, with a maximum increase of 120% in CD4+ T cells due to D+T. D and D+T reduced expression of the Th2 pathway genes IL-4, IL-5, and IL-9 (2-8 fold compared to isotype control) and > 2-fold increased expression of THEMIS and CXCL13, genes important for TCR signaling and T follicular helper cell mediated B cell recruitment, respectively. D + T treatment resulted in greater reduction compared to D in expression of genes involved in regulating angiogenesis (VHL, FGD5; > 2-fold), as well as metastasis (PFTK1; 2.2-fold), epithelial to mesenchymal transition (EMT) (CD133; 2-fold), and T reg function (TGFB2 and IL17RB; 1.8-2-fold).

Conclusions

Our results demonstrate that D + T augmented the effects of D in the microenvironment of this set of NSCLC tumors. The specific impact of D + T on the regulation of angiogenesis and TGFb-mediated immunosuppression warrants further evaluation in a larger set of tumors.

#1008

Peroxynitrite mediates the resistance of tumor cells to cytotoxic T cells by altering the MHC1-peptide repertoire on tumor cells.

Evgenii N. Tcyganov, Dmitry I. Gabrilovich. _The Wistar Institute, Philadelphia, PA_.

Therapeutic advances were able to significantly enhance antigen-specific T cell responses in cancer therapy. However, despite the obvious success, our ability to translate the generation of specific T-cell response into a clinical benefit remains limited. Recently, factors of tumor microenvironment such as hypoxia and oxidative agents were shown to dampen the efficiency of T-cell responses. Previously, we demonstrated that macrophages and myeloid-derived suppressor cells (MDSCs) produce a significant amount of highly oxidative agent, peroxynitrite (PNT), inside the tumor and revealed that PNT inhibited binding of processed peptides to tumor cell MHC class I (MHC I). As a result, tumor cells become resistant to antigen-specific cytotoxic T cells (CTLs). In other studies, it was shown that high levels of PNT (measured by nitrotyrosine level) in tumor tissues are associated with worse clinical outcome. We hypothesized that PNT affects peptide repertoire presented by tumor cell MHC I molecules and by this impairs the immune recognition of tumor cells by CTLs. To test this hypothesis we compared MHC I peptides isolated from PNT-treated and nontreated tumor cells. Quantitative LC-MS/MS analysis revealed that PNT treatment affected the peptide repertoire of MHC I bound peptides. A number of peptides were substantially underrepresented in PNT-treated cells ("nonstable" peptide subset), whereas others remained at the same level after PNT treatment ("stable" peptide subset). We generated CTLs specific to some peptides from "stable" or "nonstable" groups. PNT treatment significantly decreased the cytotoxic activity of T cells specific to "non-stable" but not to "stable" peptides, confirming our initial hypothesis. To further characterize "stable" and "nonstable" peptides we investigated their affinity to MHC I as well as the stability of the formed peptide-MHC I (pMHC) complexes. We found that peptides from both groups demonstrated equally high affinity to MHC I. However, we observed significantly higher dissociation rate of pMHC complexes formed by peptides from "nonstable" group than by "stable" peptides. PNT treatment disrupted proteasome function in tumor cells, suggesting that PNT may impair antigen-processing machinery and "nonstable" peptides quickly dissociating from MHC I were not recovered by malfunctioning proteasome. Consecutively, this could lead to the poorer peptide presentation on the tumor cell surface hindering tumor cell recognition and killing by CTLs. Our findings demonstrate a novel PNT-mediated mechanism of tumor cell resistance to specific CTLs and suggest potential therapeutic avenues for its neutralization.

#1009

Reciprocal roles of cancer associated fibroblasts and CD4+ T cells in ovarian cancer progression.

Ye Hu, Sandra Orsulic. _Cedars Sinai Medical Center, Los Angeles, CA_.

Recent evidence suggests that the efficacy of tumor immunotherapy is reliant on the signals received from the tumor microenvironment. The two prevalent cell types in the tumor microenvironment are immune cells and cancer-associated fibroblasts (CAFs). The presence of CAFs has been associated with poor survival in multiple cancer types and CAF-targeting in several experimental mouse cancer models has been shown to improve tumoral immune response and therapeutic efficacy.

By conducting immunohistochemistry in 42 patient-matched primary, metastatic, and recurrent ovarian tumors, We have shown that both CAF activation and CD4+ T cell infiltration increase during cancer progression. Upon co-culturing human CAFs with CD4+ T cells, We found that CAFs physically attract CD4+ T cells. In turn, CD4+ T cells activate CAFs, as indicated by the increased ability of CAFs to contract extracellular matrix. Further experiments showed more immune suppressive T cell subtypes (Th2 and Treg cells), when co-culture activated human CAFs with CD4+ Naive T cells.

A recent analysis of chemokines has shown that their expression is highly variable in primary ovarian tumors. Differential expression of chemokines in different areas of the tumor may result in a skewed distribution of immune cell types. We searched for genes that belong to the KEEG pathway 'cytokine-cytokine receptor interaction' that are differentially expressed in the mesenchymal and mesenchymal subtype-equivalent C1 subtypes compared to all other subtypes in the TGCA and GEO datasets. Our comparison of the top 50 differentially expressed cytokine-cytokine receptor interaction genes in the mesenchymal and C1 subtypes revealed high gene signature conservation, with 32 of 50 genes being shared, including CXCL12, CXCL14, CCL11, CCL26 and KDR. Flow cytometry results showed over-expression of some cytokines in CAFs could foster CAFs into more active state, which created an immune suppressive environment by turning more T cells to Th2 and Treg subtypes.

In summary, ovarian CAFs may have great impact on immune cells proliferation and differentiation, thus influence tumor progression.

#1010

Establishing lobular-like and interlobular-like fibroblast cell lines from human breast reduction mammoplasties.

Mikkel Morsing,1 Abbas Jafari,1 Moustapha Kassem,2 Ole William Petersen,1 Lone Rønnov-Jessen1. 1 _University of Copenhagen, Copenhagen, Denmark;_ 2 _University of Southern Denmark, Odense, Denmark_.

Introduction: The importance of epithelial-stromal interactions for tissue homeostasis and maintenance is well documented. We have recently provided evidence that two distinct fibroblast lineages in the human breast stroma, lobular and interlobular, respectively, can be prospectively isolated and stably propagated in serial-passage subculture. The aim of the present study was to generate immortalized and stable cell lines derived from lobular and interlobular fibroblasts with preserved lineage identity and functionality. The availability of such cell lines will allow for an as of yet unprecedented detailed characterization of mammary fibroblast lineages including their specific interactions with normal and malignant breast epithelium.

Results: Lobular- and interlobular-derived fibroblast cell strains in passage 8 were immortalized by retroviral introduction of human telomerase reverse transcriptase (hTERT) followed by neomycin selection. At abstract submission deadline, lobular and interlobular hTERT expressing fibroblasts had reached passage 56 corresponding to 60 and 100 population doublings, respectively, while mock-transfected controls had seized proliferating in passage 21. Lineage restriction of immortalized cells was maintained as lobular fibroblasts express higher levels of CD105 than interlobular fibroblasts. Also, lobular fibroblasts are able to undergo adipogenic and osteogenic differentiation upon relevant induction in culture, but fail to generate bone in an in vivo ectopic bone formation model, thus functionally distinguishing lobular fibroblasts from bone marrow-derived mesenchymal/skeletal stem cells. Upon co-culture with primary breast luminal progenitors, lobular fibroblasts support epithelial growth and branching morphogenesis, including correct apical-basal polarization.

Conclusion: Lobular and interlobular fibroblasts have been successfully immortalized while preserving both lineage restriction and functional characteristics of their primary counterparts. With 80% of breast cancers arising in lobular epithelium, these established fibroblast models may prove instrumental to unravel such focal susceptibility to breast cancer development.

#1011

Tumor cell intrinsic factors dictate T cell infiltration and therapeutic response in pancreatic ductal adenocarcinoma.

Katelyn T. Byrne,1 Jinyang Li,2 Robert H. Vonderheide,3 Ben Z. Stanger2. 1 _Parker Institute of Cancer Immunotherapy, University of Pennsylvania, Philadelphia, PA;_ 2 _Abramson Family Cancer Research Institute, University of Pennsylvania, Philadelphia, PA;_ 3 _Abramson Cancer Center, Parker Institute of Cancer Immunotherapy, University of Pennsylvania, Philadelphia, PA_.

The establishment of immune heterogeneity in the tumor microenvironment (TME) is poorly understood, despite recent data that the success of immunotherapies is dictated by the immune environment of the tumor site. Pancreatic ductal adenocarcinoma (PDA) is characteristically devoid of CD8 T cells and resistant to therapeutic intervention. However, a small subset of patients (15%) have tumors highly infiltrated by CD8 T cells, correlating with improved overall survival. To better elucidate the determinants of immune heterogeneity in the PDA TME, we generated clones from spontaneous tumors harvested from KrasG12D+/-;Trp53R172H+/-;Pdx-1 Cre (KPC) mice, a genetically engineered mouse model of PDA. Using a panel of 17 tumor clones, we found the clones segregated in to two groups with differential immune cell infiltration upon implantation in congenic C57BL/6 mice. 7/17 tumor clones were categorized as 'T cell high,' with an immune infiltrate comprising CD8 T cells, CD103+ dendritic cells (DCs), and a dearth of myeloid derived suppressor cells (MDSCs). In contrast, the remaining 10/17 tumor clones were categorized as 'T cell low' lines, with TME dominated by myeloid cells and macrophages, especially granulocytic MDSCs. Hypothesizing that increased T cell infiltrate would render PDA sensitive to therapy, we treated two T cell high and two T cell low tumor clones with combination immunotherapy and chemotherapy. Mice bearing T cell high clones responded to therapy (7/7 and 4/7 mice cured) and formed protective memory responses against secondary tumor challenge, while none of the mice bearing T cell low tumors responded to treatment (0/7 and 0/7 mice cured). At baseline, T cell high tumors had similar proportions of functional CD8 T cells as in T cell low tumors (62.9% vs. 50.2% IFN-g+ in T cell high vs low, p=0.06). However, the proportion of activated CD44hiPD-1+ CD8 T cells was significantly increased in T cell high tumors (62.2% vs. 35.1% in T cell low clones, p<0.0001), and predicted response to therapy across the panel of tumor clones. CD44hiPD-1+ CD8 T cells trafficked in to the tumor site independently of CXCR3 (53.8% in control vs. 50.0% in anti-CXCR3 treated tumors, p=0.8), in contrast to the bulk CD8 T cell population which required CXCR3 to enter the TME (7.5% in control vs 0.53% in anti-CXCR3 treated tumors, p<0.01). Furthermore, in Batf3 KO mice, T cell high tumors lacked CD8 T cells and resembled T cell low tumors, indicating the requirement for CD103+ DCs in the TME for T cell infiltration. These data reveal tumor cell intrinsic factors as major determinants of immune heterogeneity in the TME, and highlight the use of this panel of clonal tumor lines as a tool to probe the immune heterogeneity of the TME. Future studies will identify the specific factors regulating T cell infiltration and mechanisms to convert T cell low tumors to T cell high tumors, thereby improving responses to immunotherapy.

#1012

CD20 supports BCR signaling in an intra-clonal aggressive chronic lymphocytic leukemia subpopulation of cells and rituximab primarily targets these BCR-proficient B cells in vivo.

Gabriela Pavlasova,1 Marek Borsky,2 Veronika Svobodova,1 Jan Oppelt,3 Katerina Cerna,1 Jitka Novotna,2 Katerina Musilova,1 Vaclav Seda,1 Eva Vojackova,1 Yvona Brychtova,2 Michael Doubek,2 Sarka Pospisilova,1 Jiri Mayer,4 Marek Mraz1. 1 _Ceitec MU and University Hospital Brno and Faculty of Medicine MU, Brno, Czech Republic;_ 2 _University Hospital Brno, Brno, Czech Republic;_ 3 _Ceitec MU and National Centre for Biomolecular Research, Faculty of Science MU, Brno, Czech Republic;_ 4 _University Hospital Brno and Faculty of Medicine MU, Brno, Czech Republic_.

The hallmark of chronic lymphocytic leukemia (CLL) cells is their re-circulation between peripheral blood and immune niches to obtain pro-proliferative and pro-survival signals. CLL cells that have recently exited the immune niches to the peripheral blood are characterized by low cell-surface levels of chemokine receptor CXCR4 and high levels of activation molecule CD5. These CXCR4dimCD5bright CLL cells have a ~2-fold higher CD20 expression due to the activation of the CXCR4/SDF-1 axis (Pavlasova et al., Blood, 2016). We hypothesized that CD20 up-regulation in the context of a microenvironment is required for some functional regulation. We hypothesized that CD20 expression is of importance for B-cell receptor (BCR) signaling as we observed that CXCR4dimCD5brightCD20bright CLL cells have also ~2-fold higher surface IgM levels (P<0.0001). This was coupled with higher responsiveness to BCR-crosslinking with anti-IgM (P=0.0015). CD20 levels directly affect BCR-induced calcium flux and the phosphorylation of BCR/PI3K-associated molecules (LYN, SYK, ERK, GAB1) after BCR-crosslinking. The CXCR4dimCD5brightCD20bright subpopulation contains more proliferative (Ki67+) cells, higher levels of pAKT/pERK/pCD79a (P<0.001), and their gene expression signature (NGS Illumina) is significantly enriched for genes involved in BCR and MAPK signaling, migration, and actin cytoskeleton organization (P<0.0001). Finally, we have shown that rituximab primarily and potently eliminates the CXCR4dimCD5brightCD20bright CLL cells (P<0.0001). Overall, rituximab was ~9-fold more efficient in eliminating CXCR4dimCD5brightCD20bright CLL cells than CXCR4brightCD5dimCD20dim cells (P=0.03) during FCR therapy in vivo. Altogether, we described that higher CD20 expression supports BCR signaling and contributes to the activated phenotype and aggressiveness of an intra-clonal subpopulation of CXCR4dimCD5brightCD20bright cells. This is a first mechanistic explanation of CD20 function in CLL cells. Additionally, it is tempting to speculate that rituximab's clinical success is at least partially attributed to the preferential elimination of the intra-clonal proliferative subpopulation of BCR-proficient CLL cells. Supported by: the Ministry of Education, Youth and Sports of the Czech Rep. under the project CEITEC 2020 (LQ1601); Czech Science Foundation (project No. 16-13334Y); the Ministry of Health of the Czech Rep., grant No. 16-29622A. All rights reserved. This project has received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement No 692298. This study reflects only the author's view and the Research Executive Agency is not responsible for any use that may be made of the information it contains. MH CZ-DRO (FNBr, 65269705); MUNI/H/0865/2016; MUNI/A/0968/2017.

#1013

Analyzing infiltrating B cell populations in the tumor microenvironment using single cell transcriptomics.

Sarah E. Taylor, Stephane Boutet, Valeria Giangarrá, Grace X. Zheng, Alvaro M. Barrio, Luz Montesclaros, Josephine Lee, Samuel Marrs, Kevin J. Wu, Paul Ryvkin, Tarjei Mikkelsen, Deanna M. Church. _10x genomics, Inc., Pleasanton, CA_.

Understanding the complex interactions between malignant, stromal and infiltrating lymphocyte cell types within the tumor microenvironment is a critical component of personalized cancer treatments. We describe an approach that couples single-cell transcriptional profiling of tumors with high-resolution receptor profiling of tumor infiltrating lymphocytes (TILs). Using a fully integrated, droplet-based system for 5' single cell RNA sequencing (scRNA-seq), we simultaneously profiled the transcriptome and immune repertoire of the same cells from a primary colorectal cancer (CRC) tumor and a primary non-small cell lung cancer (NSCLC) tumor. Each tumor varied in type and proportion of its cellular components, noticeably in the proportion of TILs. The tumor cells in the CRC were mostly epithelial in nature, with the TILs comprising 21% of the total cells sequenced. This lymphocyte population consisted of both T (5% CD4+, 3% CD8+) and, interestingly, B cells (5% CD19+), with a number of plasma B cells (IGH high, CD138+) also identified. The NSCLC tumor had a high immune cell infiltrate, with lymphocytes comprising 43% of cells (19% T, 23% B, and 5% plasma B cells) indicating a robust adaptive immune response. To examine these cells further, CD45+ cells were subject to scRNA-seq. When compared to the unenriched sample, the enriched cell population had a similar B to T cell ratio with a reduction in the proportion of plasma B cells, likely due to exclusion of mature plasma B cells by the sort (47% T (44% CD4+, 29% CD8+), 42% B, and 2% plasma B cells). To fully characterize the B cell infiltrate we applied targeted 5' scRNA-seq to obtain full length, paired, B cell receptor heavy and light chain sequences. Despite the presence of plasma B cells in the CRC tumor, limited clonal expansion was observed. In the NSCLC tumor, clonal expansion of B cells was observed in the unenriched tumor sample but these clonotypes were not present in the CD45+ population, suggesting the clonally expanded cells are in the mature plasma B cell population. We also applied a targeted scRNA-seq approach to obtain full length paired T cell receptor alpha and beta sequences, however, no clear T cell expansion was observed in either sample. These findings emphasize the importance of examining receptor sequences rather than relying on the presence of B or T cells alone to determine if a robust immune response is being mounted. The presence of tumor-infiltrating B lymphocytes has been linked to a favorable clinical outcome in some types of cancers. Identification of these cells, their subsets and high resolution profiling of their receptor sequences using scRNA-seq allows a novel insight into the adaptive immune response in tumors. This technology will allow better characterization of tumor heterogeneity and the adaptive immune response to the tumor microenvironment and will serve as a foundation for future research into tumor immunology and immunotherapy.

#1014

Pharmacological modulation of PDAC stroma to enhance checkpoint blockade immunotherapy.

Jun Zhao, Zhilan Xiao, Willem Overwijk, Chun Li. _UT MD Anderson Cancer Ctr., Houston, TX_.

Sonic hedgehog signaling pathway is an important regulator during the initiation and progression of pancreatic cancer, and sustained activation of this pathway contributes to excessive deposition of tumor stroma and maintenance of tumor-initiating cells. Cyclopamine (CPA) is a potent inhibitor of the SHH pathway. In this work, we encapsulated both paclitaxel (PTX) and CPA in a long-circulating polymeric micelles to obtain M-CPA/PTX. In a genetically engineered mouse model of pancreatic ductal adenocarcinoma (PDAC) (KPC model), systemic administration of M-CPA/PTX significantly prolonged the median survival of mice with well-established tumors from 23 days with nab-paclitaxel or gemcitabine to 58 days post-enrollment (p<0.0001). Further studies indicated that M-CPA/PTX modulated PDAC stroma by softening extracellular matrix, increasing blood perfusion, and alleviating hypoxia without depletion of α-smooth muscle actin-positive tumor-associated fibroblasts. Significantly, combination of M-CPA/PTX and anti-PD-1 immunotherapy significantly prolonged the survival of PDAC-bearing KPC mice compare with monotherapies (p<0.01). In an immune-competent, orthotopic PDAC model bearing established murine PDAC, treatments with combined M-CPA/PTX and anti-PD-1 enhanced tumor infiltration of CD8+ T cells and IFNγ expression. Neutralization of either CD8 T cells or INFγ nullified the therapeutic gain achieved with combination therapy. Thus, M-CPA/PTX enhanced checkpoint blockade immunotherapy through modulation of PDAC stroma and enhanced tumor infiltration of effector immune cells.

#1015

Dual BRAF and VEGFA targeting in melanoma elicits antitumor immune response that is enhanced by PD-1 blockade.

Valentina Comunanza,1 Valentina Martin,2 Gabriella Doronzo,1 Federica Di Nicolantonio,1 Dario Sangiolo,1 Federico Bussolino1. 1 _University of Torino and Candiolo Cancer Institute-FPO, IRCCS, Candiolo, Italy;_ 2 _Candiolo Cancer Institute-FPO, IRCCS, Candiolo, Italy_.

The introduction of BRAF inhibitors (BRAFi) has improved response rate and overall survival of metastatic melanoma patients compared to standard chemotherapy. However, acquired drug resistance occurs in nearly all patients. The comprehension of cellular and molecular mechanisms underlying BRAF inhibitor resistance could help to identify novel actionable pathways in the treatment of BRAF dependent tumors.VEGFA is an attractive target for combinatorial cancer therapy and we have recently demonstrated that targeting VEGFA in melanoma and CRC xenografts could enhance the antitumor effect of BRAFi by normalizing the tumor vasculature, recruiting M1 macrophages and inducing a remodeling of the extracellular matrix characterized by a reduction in collagen I and in cancer-associated fibroblasts. While the previous proof of concept was obtained within an immunodeficient model, here we investigated the therapeutic effect of VEGFA targeting in association with PLX4720 (BRAFi) in a dedicated immunocompetent model. D4M cells, a BRAFV600E-mutant melanoma murine cell line, were subcutaneously injected in syngeneic C57BL/6J mice. We demonstrated that the association of BRAFi with DC101 (antibody anti VEGFR2) had a weak activity while we observed a synergistic antitumor effect when combined with B20 (murine anti-VEGFA neutralizing antibody). Although targeted inhibition of either BRAF or VEGFA delayed tumor growth, only combined inhibition of both pathways could induce regression of initial tumor size, with an evident apoptotic effect, and delayed the onset of acquired resistance to BRAF target therapy. Since VEGFA has a well characterized tumor immune-suppressive role, we further investigated whether contrasting VEGFA along with simultaneous BRAF inhibition could modulate both innate and adaptive immunity. Both flow cytometry and immunofluorescence analysis of tumors demonstrated that the combinatorial regimen activates the host immune system, inducing the tumor infiltration by cytotoxic CD8+ lymphocytes, macrophages with tumor suppressive features and NK cells. Moreover, the association between BRAF targeting and VEGFA removal reduced the number of circulating CD11b+Ly6ClowLy6G+ polymorphonuclear MDSCs (PMN-MDSCs). Based on this observation, we hypothesized that the therapeutic effect obtained by the simultaneous VEGFA blockade and BRAFi could be further exploited as a favorable platform for the association with immune-checkpoint inhibitor targeting PD-1. Although the addition of anti-PD-1 antibody improved the antitumor activity of either PLX4720 or B20, we observed striking tumor volume regression only when combining PD-1 blockade with dual BRAF and VEGFA inhibition. Our results offer the rationale for novel combinatorial approaches including co-targeting of signaling molecules, tumor angiogenesis and immune system.

#1016

Transcriptomic analysis of bulk tissues of large PDX collection as a novel platform discovering new TME target/drug.

Jia Xue, Wubin Qian, Sheng Guo, Xiaoyu An, Xuesong Ouyang, Henry Q. Li. _Crown Bioscience Inc., Taicang, China_.

Cancers are collections of diverse diseases of genetic and immunological abnormalities. The heterogeneous tumor microenvironment (TME), including immune components, and their interactions with tumor cells play critical roles in tumor progression and response to pharmaceutics, particularly immuno-oncology (I/O) therapy. However, investigating TME-specific components is rather challenging for the difficulty to separate stroma from tumor cells, either physically via microdissection or in silico via bioinformatics. Patient derived xenograft (PDX) may be a new system to investigate TME1, where human and mouse content can readily be separated in silico2. We have transcriptome-sequenced ~1600 bulk tumor tissues from subcutaneous PDXs grown in athymic mice3. By aligning reads to human and mouse genomes, we found that the average mouse-to-human sequencing read ratio is around 11% (5~20%), consistent with the previous report2. After removal of the low-expressed and less-variable genes and by deconvolution analysis of gene expression data, we identified all types of TME components, including adaptive and innate immune cells. The corresponding fractions vary across cancer types and individual models. Co-regulation analysis identified a huge number of intra-species interactions and also, a smaller number of inter-species interactions that vary greatly among different cancer types. The cross-species interactions observed are likely implicated in the growth of these tumors, and their numbers may also reveal the degree of the dependence of tumor growth on TME, which should be reversely correlated to the transplantation take-rate of corresponding type of PDX. Indeed, we have demonstrated this reverse-correlations (# interactions: take-rate %) with statistical-significance (p-value = 0.034) across our PDX collections, including melanoma (406:27%), lung (146:50%), colorectal (CRC) (157:68%) and pancreatic cancer (32:80%). The cancer type with the highest take-rate and lowest # interactions is pancreatic cancer that also has highest KRAS mutation rate (>90%), hinting the role of KRAS mutation in tumor growth independency on TME. This is further confirmed in KRAS mutant CRC (1:96%) vs. wild-type (98:53%). Moreover, some putative cross-species co-regulations in specific cancers were also observed in human tumors (e.g. in TCGA dataset), indicating potential importance in TME-tumor interaction and tumor development in human. Further investigation of each of these interactions may reveal novel TME-related disease pathways and thus novel targeting strategy for cancer therapy. In conclusion, transcriptomic analysis of large number of bulk PDXs provides a novel and unique platform to study TME, likely to facilitate new discovery of disease pathways and strategy to treat cancers involving the TME mechanism, particularly I/O strategy.

#1017

Vinorelbine, cyclophosphamide, and 5-FU effects on the circulating and intratumoral landscape of immune cells improve anti-PD-L1 efficacy in preclinical models of breast cancer and lymphoma.

Stefania Orecchioni, Giovanna Talarico, Valentina Labanca, Patrizia Mancuso, Francesco Bertolini. _European Inst. of Oncology, Milan, Italy_.

Checkpoint inhibitors (CIs) are active in many types of cancer. However, only a minority of patients achieve complete and/or long-lasting responses. We studied the effects of 4 different doses of 3 widely-used, orally-active chemotherapeutics (vinorelbine, V, cyclophosphamide, C, and 5FU) over local and metastatic tumor growth, and over the landscape of circulating and tumor-infiltrating immune cells. V, C and 5-FU were given alone or with anti-PD-1 or anti-PD-L1 CIs. Immunocompetent Balb/c mice were used to generate orthotopic models of breast cancer (BC, 4T1 cells) and B-cell lymphoma (NHL, A20 cells). V, C and 5-FU were administered at low-dose metronomic, medium, or maximum tolerable dosages. V and and C increased circulating monocytes, 5-FU reduced circulating monocytes. C increased MDSC count. C and 5-FU reduced circulating APCs, V increased circulating APCs. V and C reduced circulating Tregs. C at low doses and 5-FU increased circulating Tregs. C reduced circulating CD3+CD4+ and CD3+CD8+ T cells. V slightly reduced CD3+CD4+ T cells. V, C, and 5-FU reduced circulating B cells, with C showing the most significant effect. V reduced NK cells, C and 5-FU increased circulating NKs. In both BC and NHL models, anti-PD-L1 was significantly more effective than anti-PD-1. In BC models V, C and 5-FU were effective in reducing local and metastatic tumor growth. The association of V, C and anti-PD-L1 was the most effective combinatorial regimen in terms of local and metastatic BC control. Anti-PD-L1 treatment alone increased BC-infiltrating immune cells, in particular B cells. Treatment with C alone was associated with the largest tumor infiltration by NK, CD3+CD4+ T cells and m-MDSC, and with the lowest B cell infiltration. Combinatorial treatment with C+V+anti-PD-L1 was associated with the largest intratumoral B cell count and the lowest number of CD3+CD8+PD-1+ T cells. In NHL models, the association of C and anti-PD-L1 was the most effective regimen. Anti-PD-L1 alone was associated with the largest increase in infiltrating immune cells, and in particular in B cells. C alone was associated with increased NK infiltration. V decreased the immune cell infiltrate. The addition of anti-PD-L1 to V and C increased B cell infiltrate.

Taken together, our data indicate that chemotherapeutics have very complex effects on the circulating landscape of immune cells, with subtle but significant differences related to the dosage and duration of the administration of the drugs. Notably, these effects on circulating immune cells differ qualitatively and quantitatively from those observed in the intratumoral immune cell infiltrate. The present data also suggest that chemotherapy might add to the effect of CIs, even though different types and sites of cancer generate significantly different atlases of intratumoral infiltrates.

#1019

EphA2 receptor tyrosine kinase regulates programmed death ligand 2 (PD-L2) expression in tumor cells and inhibits immune infiltration.

Eileen Shiuan, Wenqiang Song, Deanna Edwards, Shan Wang, Sung Hoon Cho, Mark Boothby, Jin Chen. _Vanderbilt University, Nashville, TN_.

Background: Given the success of both targeted and immunotherapies, there is increasing utility for identifying targeted agents that also promote anti-tumor immunity. EphA2 is a receptor tyrosine kinase that contributes to tumor growth and metastasis in various cancer types and plays a role in inflammatory processes. Previous work in our lab demonstrates EphA2 is a viable target for non-small cell lung cancer (NSCLC) and breast cancer. Here, we examine how EphA2 affects programmed death-ligand (PD-L) expression and immune response in the tumor microenvironment.

Methods: Our preliminary studies suggest EphA2 regulates the expression of PD-L2 but not PD-L1 in human lung and breast cell lines. To investigate this, we induced PD-L2 expression in vitro via cytokines, overexpressed or knocked down EphA2, and measured PD ligand expression by flow cytometry. To evaluate the mechanism by which EphA2 affected PD-L2 expression, we curated the ENCODE database to identify transcription factor (TF) binding sites near the PD-L2 promoter and validated individual TF candidates with knockdown experiments. To study in vivo effects, we generated an EphA2-overexpressing murine NSCLC cell line from a primary mouse lung tumor. Both subcutaneous and tail vein injected lung tumor models were used to assess the impact of EphA2 overexpression on tumor burden and survival, as well as immune infiltration by flow cytometry.

Results: In human bronchial and mammary epithelial cells, PD-L2 was induced by IFNγ and, to a lesser extent, TNFα and IL-4, and knockdown of EphA2 decreased surface expression of PD-L2. TF binding sites for Myc and p65, among others, were found in the PD-L2 promoter region. Because our lab has previously shown that Myc and YAP are downstream effectors of EphA2, we pursued these TFs for validation studies, which are ongoing. In contrast to human epithelial cells, murine counterparts do not express PD-L2, even after IFNγ stimulation. While the EphA2-overexpressing murine NSCLC cell line did not display proliferative advantage over control cells in vitro, they developed larger tumors and had worse survival in both tumor models. Analysis of lung tumor immune infiltrate revealed decreased NK and T cells in the EphA2-overexpressing tumors.

Conclusion: Our studies suggest EphA2 upregulates PD-L2 in cancer cells and inhibits tumor infiltration of key lymphocytic populations. Ongoing investigations are determining the mechanisms behind these findings. Despite PD-L2's known role in immune tolerance, its impact on the tumor microenvironment is understudied compared to PD-L1. Thus, elucidation of EphA2's role in regulating PD-1 and PD-L2 interactions and immune recruitment will further our understanding of PD-1 and PD-L biology and mechanisms of immune evasion, as well as provide additional rationale for targeting EphA2 in cancer.

#1020

**Characterization of the immune landscape and analysis of tumor response after anti-PD-1 blockade in a 3D** ex vivo **system of non-small lung cancer.**

Melanie Mediavilla-Varela,1 Melba Marie Page,2 Jenny Kreahling,2 Soner Altiok2. 1 _Moffitt Cancer Center, Tampa, FL;_ 2 _Nilogen Oncosystems, Tampa, FL_.

Background: Immuno-oncology drugs face the challenge that cell lines and animal models cannot recapitulate the full intimacies of the tumor microenvironment. Although these models have aided in the discovery of new medical modalities, only 8% of these models have had a successful translation. Furthermore, the phenotype of the microenvironment of various tumor types has not been assessed in depth. Nilogen Oncosystems' proprietary 3D-EX vivo drug screening platform analyzes fresh patient tumor tissue that remains embedded in its natural environment. With our model, the characteristics of the tumor microenvironment can be accurately revealed in response to checkpoint inhibitors in non-small cell lung cancer (NSCLC) and correlates can be found among different platforms.

Methods: For the 3D-EX vivo platform, microspheres were produced from procured fresh tumor tissue from NSCLC cancer patients. They were then treated with Keytruda ex vivo and treatment-mediated changes in TIL subpopulations were analyzed using flow cytometric analysis, cytokine release by Bio-Rad's 17-plex cytokine assay as well as gene expression by NanoString's 770 gene Immune Panel.

Results: Ex vivo treatment of the 3D microspheres with Keytruda, showed significant changes in T-cell activation and immune cell populations in 26% of NSCLC tumors. This was observed by the simultaneous increase in IFN-γ and TNF-α upon cytokine analysis. Furthermore, we found a differential expression of signature genes such as CD8, CXCL10, CXCL9, EOMES, Granzyme A/B, IFN-γ, related to T-cell subpopulations via Nanostring analysis, which was accompanied by an increase in Granzyme B via flow cytometry.

Conclusion: The positive and negative associations between expression of immune function genes, TIL activation, and cytokine production by ex vivo treatment shows that Nilogen Oncosystems' 3D-Ex vivo platform can accurately recapitulate the tumor microenvironment and exhibits the importance of a comprehensive analysis of the tumor immune microenvironment for a better understanding of the mechanism of action of immuno-oncology drugs that may aid in developing biomarkers that can be used for patient selection.

#1021

Clinical significance of tertiary lymphoid structures (TLS) and tumor-infiltrating plasma cells in ovarian cancer.

Masayo Ukita, Junzo Hamanishi, Tsukasa Baba, Ryusuke Murakami, Kaoru Abiko, Masaki Mandai. _Kyoto University Graduate School of Medicine, Kyoto, Japan_.

Objective: Tertiary lymphoid structures (TLS) are transient ectopic lymphoid aggregates that reflect lymphoid neogenesis mainly through CXCL13 and develop under chronic inflammation. Recently TLS have been detected in several types of cancers; however, there are few reports about TLS in ovarian cancer. We investigated the clinical significance of TLS in ovarian cancer.

Methods: 28 patients who were diagnosed with high-grade serous ovarian cancer and treated surgically in our hospital were selected from microarray dataset (KOV-75; GSE39204/55512). We evaluated TLS, tumor-infiltrating lymphocyte (TIL) subsets (CD4+ T cell, CD8+ T cell, CD20+ B cell, CD38+ plasma cell), and the correlation between expression of CXCL13 and TIL subsets by immunohistochemical staining. We analyzed the correlation between gene expression of CXCL13 and the survival time of the patients (pts) by using KOV-75 and the cancer genome atlas (TCGA) datasets.

Results: Immunohistochemical staining revealed that TLS were detected in 64% (18 pts) of ovarian cancer patients, and the numbers of tumor-infiltrating CD8+ T cells, CD20+ B cells and CD38+ plasma cells were significantly higher in cases with TLS (p<0.05). The numbers of tumor-infiltrating CD4+ T cells, CD8+ T cells, CD20+ B cells and CD38+ plasma cells were significantly higher in the CXCL13-high tumor than in the CXCL13-low tumor (p<0.001). In the group with high intratumoral infiltration of CD8 T cells and plasma cells, progression-free survival and overall survival were both significantly extended (p<0.05, respectively). In microarray analyses of KOV-75 and TCGA, high CXCL13 gene expression correlated significantly with both favorable progression-free survival and overall survival (p<0.05, respectively).

Conclusion: CXCL13 contributes to better prognosis in ovarian cancer patients by inducing TLS and various subsets of TIL. Induction of CXCL13 might be a new therapeutic target for ovarian cancer patients.

#1022

Evaluation of potential factors contributing to the exhaustion of T lymphocytes in the tumor microenvironment.

Annelies Laeremans, Na Li, Jeff Kim, Xiao-Jun Ma, Emily Park. _Advanced Cell Diagnostics, Newark, CA_.

The presence of immunosuppressive molecules and cells in the tumor microenvironment (TME) can lead to T cell dysfunction. CD8-positive cytotoxic T cells (CTLs) are ineffective in killing tumor cells primarily due to upregulated expression of inhibitory checkpoint molecules and decreased production of effective cytokines. In addition, immune suppressive cell types such as regulatory T cells (Tregs) and tumor associated macrophages (TAMs) are recruited to the TME, further establishing a suppressive immune environment. In this study, we evaluated expression profiles of key immunosuppressive molecules and cell types by applying RNAscope® assay, a highly specific and sensitive in situ hybridization (ISH) technology, and dual ISH-IHC staining. First, we evaluated CD8-positive cell infiltration in TME of archived human tissues from non-small cell lung cancer and ovarian cancer. Selected tissues with either high or low CD8-positive cell number (CD8-high or CD8-low) were evaluated for (1) the presence of Tregs (FOXP3+CD4+) and TAMs (CD163+, including IL10 and CCL22), (2) the expression of immune checkpoint molecules including PD1, PD-L1, TIM3, and LAG3, (3) the expression of immune suppressive molecules IDO1 and TGFβ, and (4) IFNγ expression in CD8-positive subsets. Examination of immune inhibitory molecules expressed in single cells in the tumor and stromal microenvironment revealed that in general, CD8-high tissues expressed higher level of immune checkpoint molecules, often co-expressed in the same individual cells in the same TME, while the expression of IDO1 and TGFβ was independent of CD8-positive cell inflammation. Tregs and TAMs often co-existed with IFNγ-positive CTLs in the same TME. Unexpectedly, all investigated inhibitory molecules were expressed in both tumor cells and stromal/immune cells in some tumors. Beyond PD-L1, immune checkpoint molecules PD1, TIM3, and LAG3 were frequently expressed in tumor cells but at lower level than in immune cells. Expression of IDO1 and TGFβ was observed in many cell types, including tumor cells, with various expression levels in each tumor cell. The single-cell tumor expression profiles suggest a potential tumor-intrinsic mechanism of expression for these inhibitory molecules. This exploratory study highlights the potential of RNAscope® ISH to better understand the cellular and molecular suppressive mechanisms associated with T cell dysfunction and exhaustion in the TME. The robust RNAscope® ISH platform is well suited for evaluating critical secreted factors and other key molecules in a highly sensitive and cell type-specific manner. As multiple therapeutic approaches to augment the CTL function are being developed, the presented method may facilitate the identification and development of key biomarkers to stratify patients based on their specific tumor and immune cell states.

#1023

Release of cancer cells from the prostate tumor microenvironment to promote neoantigen exposure and enhance anti-tumor immune response.

Kenneth C. Valkenburg, Kenneth J. Pienta. _Johns Hopkins University School of Medicine, Baltimore, MD_.

Prostate tumors are widely considered to be immunologically silent and resistant to immunotherapy. PCa is widely considered to be an immune desert, with little infiltration of cytotoxic T lymphocytes (CTLs). However, anti-tumor immune responses have been seen in PCa in the right context. We believe we can boost immune response and T cell infiltration into PCa tumors by inhibiting the stromal derived factor 1-CXC chemokine receptor 4 (SDF1-CXCR4) signaling axis and releasing tumor cells from the immune-suppressive bone marrow niche. We have shown that CXCR4 antagonism causes PCa disseminated tumor cells (DTCs) to leave the bone marrow niche and enter the circulation as released circulating tumor cells (CTCs). Our hypothesis is that the release of PCa cells from the tumor microenvironment will enhance chemotherapy-induced cell death and the subsequent presentation of tumor-associated antigens, allowing increased anti-tumor response to immunotherapies. I generated a novel selection-free immunofluorescence-based protocol to confirm this in dissemination models of cancer in mice. After CXCR4 inhibition, I observed an approximate five-fold increase in the number of released CTCs in the blood of mice 24 hours after treatment. Very little research has been performed on the proliferation of CTCs or DTCs, particularly when released from the bone marrow niche. I have been able to mark proliferating cells in blood and bone marrow with EdU and quantify proliferating CTCs. We determined that while released cancer cells are proliferating more than DTCs, the ratio of released proliferating cells is not sufficient to cause a robust response to chemotherapy alone. This led to the hypothesis that released cells could provide an excellent substrate for the immune system. Evidence to support this idea is that we consistently observe the engulfment of CTCs by CD45-positive immune cells from the blood of mice, indicating that immune cells may respond to CTCs but not DTCs. Our approach is unique in that we are using CXCR4 inhibition to release cancer cells into the bloodstream to expose them to immune cells and initiate an immune response via activated CTL infiltration into PCa tumors. We believe this represents an innovative polyvalent autologous vaccine approach as it does not rely on a single antigen to stimulate CTLs, and also relies on each individual patient's cancer cells and immune cells. We have also generated some immunofluorescent protocols to determine the immune composition of circulating cells in relation to CTCs. While we believe that CTLs are responsible for most of the anti-tumor immune response, we are using this opportunity to discover novel immune cell types that may be present before and after therapy. This approach, when combined with classic cancer therapy and immunotherapy, may represent a novel way to boost response to cancer therapies in metastatic PCa.

#1024

Tumor intrinsic properties associate with differential effects on CD8+ tumor-infiltrating lymphocyte density and immune gene expression in non-small cell lung cancer (NSCLC) samples.

Cyrus Hedvat,1 Keyur Desai,1 Dimple Pandya,1 Peter Szabo,1 Johannes Zimmermann,2 Jan Lesniak,2 Scott Ely,1 Sujaya Srinivasan,1 Xi-Tao Wang,1 Michele French,1 Robin Edwards1. 1 _Bristol-Myers Squibb, Princeton, NJ;_ 2 _Definiens AG, Munich, Germany_.

Introduction: Anti-tumor immune response is controlled by a complex interaction between the immune system, tumor cells, and associated stroma. Expression of major histocompatibility complex class I (MHC I) and MHC class II (MHC II) antigens may be dysregulated in cancer, leading to alterations in the tumor antigen presentation profile. Stage I–III resected NSCLC tumor samples were profiled to study the relationship between MHC I, MHC II, and programmed death ligand 1 (PD-L1) tumor cell (TC) expression with tumor-associated inflammation. Here we report the potential impact of respective profiles on tumor immune response.

Methods: 53 adenocarcinoma (NSCLC-AD) and 51 squamous cell carcinoma (NSCLC-SQ) stage I–III resected formalin-fixed, paraffin-embedded tumor specimens from commercial sources were stained by immunohistochemistry for MHC I (HLA-A,B,C), MHC II (HLA-DP,DQ,DR), and PD-L1 and TC expression was assessed by manual pathologist review. CD8+ cell density was quantified using Definiens image analysis algorithms for the intraepithelial tumor region. RNA extracted from the samples was analyzed by RNAseq, with data available for 48 NSCLC-AD and 46 NSCLC-SQ specimens. A 25-gene IFN-gamma gene signature (IFNG score) was used to study the association of T-cell inflammation with other biomarkers.

Results: Of all NSCLC-AD and NSCLC-SQ specimens, 85% showed either complete (<20% TC) or partial (20–80% TC) loss of TC MHC I, while TC MHC II was increased (>1%; MHCIIhi) in 60% of NSCLC-AD and 18% of NSCLC-SQ. Quantitative analysis of CD8 in the tumor microenvironment (TME) revealed a significantly reduced intraepithelial density of CD8+ tumor-infiltrating lymphocytes (TILs) in NSCLC-AD (P=0.009) and NSCLC-SQ (P=0.01) with complete MHC I loss. In contrast, intraepithelial CD8+ TILs were significantly increased in MHCIIhi tumors for both NSCLC-AD (P=0.004) and NSCLC-SQ (P=0.006). Tumors displaying both MHCIIhi and retained MHC I demonstrated the highest TIL density. Gene expression associated with IFN-gamma response was increased in tumors with retained MHC I (P=0.0036) and MHCIIhi samples (P<0.001). In NSCLC-AD and NSCLC-SQ, 38% and 59% had PD-L1 expression ≥1%, respectively. PD-L1 expression correlated with CD8 density and IFNG score in NSCLC-AD, but not in NSCLC-SQ.

Conclusions: Understanding the role of the antigen presentation machinery in immune activation and evasion in the TME is important to predict responses to immunotherapy. These data suggest that the expression of MHC I, MHC II, and PD-L1 by tumor cells may play a role in guiding the localization of CD8+ T cells in the TME with differential effects dependent on

histologic subtype. The lack of correlation between inflammation and PD-L1 expression in stage I–III NSCLC-SQ suggests an alternate PD-L1 induction mechanism in a subset of those tumors.

#1025

Clinical outcomes and differential tumor immune microenvironment in patients with small cell lung cancer and paraneoplastic syndromes.

Wade T. Iams,1 Eileen Shiuan,2 Catherine B. Meador,3 Marc Roth,4 Jennifer Bordeaux,5 Christine Vaupel,5 Lucy L. Wang,6 Joseph T. Schneider,6 Jeremy L. Warner,7 Zhiguo Zhao,8 Christine M. Lovly4. 1 _McGaw Medical Center of Northwestern University, Chicago, IL;_ 2 _Vanderbilt University Cancer Biology Graduate Program, Nashville, TN;_ 3 _Vanderbilt University School of Medicine, Nashville, TN;_ 4 _Vanderbilt University Department of Medicine, Nashville, TN;_ 5 _Navigate Biopharma Services, Inc., a Novartis Subsidiary, Carlsbad, CA;_ 6 _Vanderbilt University Department of Biomedical Informatics, Nashville, TN;_ 7 _Vanderbilt University Departments of Medicine and Biomedical Informatics, Nashville, TN;_ 8 _Vanderbilt University Department of Biostatistics, Nashville, TN_.

Background

Among patients with small cell lung cancer (SCLC), approximately 20% develop a paraneoplastic syndrome (PNS). Neurologic PNS are immune-mediated phenomena predicated on host recognition of an onconeural antigen, while endocrinologic PNS are attributed to ectopic tumor secretion of normal hormones. The presence of neurologic PNS improves prognosis and endocrinologic PNS worsens prognosis in patients with SCLC. We hypothesized that tumors from patients with neurologic PNS may have increased TILs and PD-1/PD-L1 expression compared to tumors from patients with endocrinologic PNS and this improved immune recognition accounts for prognostic differences.

Methods

We searched electronic medical record text stored in the Vanderbilt University Medical Center (VUMC) clinical data warehouse to identify SCLC patients with and without a PNS. We obtained clinical information through manual, retrospective chart review using an IRB-approved protocol. Overall survival (OS) and progression free survival (PFS) were compared using a log rank test. Archived formalin fixed, paraffin embedded samples were obtained from the Vanderbilt University Pathology Tissue Repository. We performed multiplexed fluorescence immunohistochemistry combined with automated quantitative analysis (AQUA® Technoloy; Navigate BioPharma Services, Inc.) to assess PD-1, PD-L1, CD4, and CD8 expression. A PD-1/PD-L1 interaction score was calculated by measuring the total area of PD-1 positive cells within the proximity of PD-L1 positive cells. This area was then divided by the total area of all non-tumor nucleated cells in the image and multiplied by a factor of 10,000. CD4, CD8, and PD-1/PD-L1 interaction scores were compared using a two sample t-test or Wilcoxon Rank Sum test.

Results

A total of 145 SCLC patients were identified, 55 with a PNS (25 neurologic and 30 endocrinologic) and 90 control patients. Patients with neurologic PNS exhibited significantly improved OS and PFS compared to patients with endocrinologic PNS and control patients (median OS 24mo, 95% CI 16.4mo-not reached (NR), vs 12mo, 95% CI 8.3mo-15.5mo, vs 13mo, 95% CI 12.2mo-16mo; median PFS 14mo, 95% CI 9.3mo-NR vs 6mo, 95% CI 4.6mo-9.5mo, vs 7mo, 95% CI 6.6mo-8.1mo, respectively). Tumors from patients with neurologic PNS (n=9) had statistically significantly higher PD-1/PD-L1 interaction scores (p=0.02), and increased CD4 (p=0.01) and CD8 (p=0.003) T cell infiltrates compared to tumors from patients with endocrinologic PNS (n=11).

Conclusion

Our study of tumor tissue from patients with SCLC and PNS demonstrated a statistically significant increase in immune modulation markers' expression in patients with neurologic PNS. Tumor immunomodulation may be the driver of the improved prognosis that has been observed in ours and other retrospective cohorts of patients with SCLC and neurologic PNS.

#1026

A multiplex IHC evaluation of multiple immune checkpoint receptors and mismatch repair proteins in colorectal carcinoma.

George Yang, Sara Figueroa, David Tacha, Cristin Douglas. _Biocare Medical, Pacheco, CA_.

Introduction: The recent success of checkpoint inhibitor therapies in clinical oncology practice has generated overwhelming enthusiasm for immunotherapeutics. Recent studies have suggested that patients with mismatch repair deficient (dMMR)/microsatellite instability-high (MSI-H) colorectal carcinoma (CRC) may benefit from anti-PD-1/PD-L1 inhibitors. In this study, we have evaluated a large panel of immune checkpoint receptors (ICRs) on CRC by multiplex immunohistochemistry (IHC). Design: Tissue sections were initially reviewed by H&E to confirm the presence of adequate pathological features including histology, grade and abundance of tumor-infiltrating lymphocytes (TILs). An MMR protein panel (MLH1, MSH2, MSH6, PMS2) was tested by IHC to determine dMMR/MSI-H status through loss of MLH1/PMS2 or MSH2/MSH6. Multiple ICRs, including CD4, CD8, CD137, CTLA-4, FOXP3, GITR, IDO1, LAG-3, PD-1, and PD-L1, were then tested by multiplex IHC on 18 CRC cases consisting of MSI-H CRCs (n=8) and microsatellite stable (MSS) CRCs (n=10) using four chromogenic end-points. Expression of ICRs in TILs was determined at four sites: tumor, invasive margin, peri-tumor, and tertiary lymphoid structures (TLS). ICR expression levels were determined by counting and averaging positive TILs in three high-power fields (400X) and categorized in the following manner: 0 (negative, no expression), 1 (weak, 1-25 cells), 2 (moderate, 26-50 cells), 3 (high, >50 cells). Results: Five cases of MSS (50%) and two cases of MSI-H showed 10-30% PD-L1 positive tumor cell expression. The remaining cases were negative for PD-L1. All cases of MSI-H and MSS demonstrated high expression of CD8 in tumor, invasive margin, peri-tumor and TLS. Five MSI-H cases showed moderate to high PD-1, and three MSI-H cases were weak for PD-1. All MSS cases showed weak to moderate PD-1 expression. CD4, FOXP3, CTLA-4, IDO1, LAG-3, and CD137 were weak to moderate in all MSI-H and MSS cases. Within the TLS, the predominant cellular components were CD8+, CD4+ and PD-1+ T cells in all cases. Conclusion: In the present study, a greater number of MSS CRCs expressed PD-L1 than MSI-H CRCs. PD-1 was positive in all CRC cases with variable expression between MSI-H and MSS CRCs. There appears to be no significant difference in TIL expression of additional immune checkpoint receptors tested in MSI-H colorectal carcinomas and MSS colorectal carcinomas. Multiplex technology appears to allow for superior evaluation of the synergistic relationship between immune checkpoint receptors as well as the interconnection between inhibitory barriers to CD8+ effector T cells and regulatory T cells developed by immune checkpoint receptors within each of the tumoral regions. Further study of expression patterns of immune checkpoint receptors may provide useful information for clinical management of patients with colorectal carcinoma.

#1027

Regulatory T-cell genes drive altered immune microenvironment in adult solid cancers and allow for immune contextual patient subtyping.

Jurriaan Brouwer,1 Wei-Yi Cheng,1 Anna Bauer-Mehren,2 Daniela Maisel,2 Katharina Lechner,2 Emilia Andersson,2 Joel T. Dudley,3 Francesca Milletti1. 1 _Roche, New York, NY;_ 2 _Roche, Penzberg, Germany;_ 3 _Icahn School of Medicine at Mount Sinai, New York, NY_.

The tumor microenvironment is an important factor in cancer immunotherapy response. To further understand how a tumor affects the local immune system, we analyzed immune gene expression differences between matching normal and tumor tissues. We analyzed previously published and new gene expression data from solid cancers and isolated immune cell populations. We also determined the correlation between CD8, FoxP3 immunohistochemistry (IHC) and immune-related genes. Across solid TCGA cancers, we observed that regulatory T-cells (Tregs) were one of the main drivers of immune gene expression differences between normal and tumor tissues. A tumor-specific CD8 signature had slightly lower scores in tumor tissues compared to normal of most (12 of 16) cancers, while a Treg signature score was higher in tumor tissues of all cancers except liver. We clustered TCGA colorectal samples (626 patients) and a new separate testing data set (60 patients) into two groups according to Treg gene signature expression. The High Treg cluster had more colorectal tumors that were Consensus Molecular Subtype 1/4, right-sided and microsatellite-instable, compared to the Low Treg cluster. Finally, we determined the correlation between CD8, FoxP3 immunohistochemistry (IHC) and our gene signatures and found that in this small data set correlation between signature and IHC overall was low, but samples in the High Treg cluster had significantly more CD8+ and FoxP3+ cells compared to the Low Treg cluster. We conclude that high Treg signature expression scores correlate with high overall immune gene expression. Using this novel way of classifying patients, more colorectal tumors with high immune activation were identified compared to other colorectal subtyping methods. Further research will reveal if this Treg-based subtyping improves the identification of patients that may benefit from cancer immunotherapy. 

### Advances in the Generation and Analysis of Patient-Derived Xenografts

#1028

Patient-derived xenografts undergo mouse-specific tumor evolution.

Uri Ben-David, Gavin Ha, Yuen-Yi Tseng, Noah F. Greenwald, Coyin Oh, Juliann Shih, James M. McFarland, Bang Wong, Jesse S. Boehm, Rameen Beroukhim, Todd R. Golub. _Broad Inst. of MIT and Harvard, Cambridge, MA_.

Patient-derived xenografts (PDXs) have become a prominent cancer model system, as they are presumed to faithfully represent the genomic features of primary tumors. Here we monitored the dynamics of copy number alterations (CNAs) in 1,110 PDX samples across 24 cancer types. We observed rapid accumulation of CNAs during PDX passaging, often due to selection of pre-existing minor clones. CNA acquisition in PDXs was correlated with the tissue-specific levels of aneuploidy and genetic heterogeneity observed in primary tumors. However, the particular CNAs acquired during PDX passaging differed from those acquired during tumor evolution in patients. Several CNAs recurrently observed in primary tumors gradually disappeared in PDXs, indicating that events undergoing positive selection in humans can become dispensable during propagation in mice. Importantly, the genomic stability of PDXs was associated with their response to chemotherapy and targeted drugs. These findings have important implications for PDX-based modeling of human cancer.

#1029

The PDX Data Commons and Coordinating Center (PDCCC) for PDXNet.

Anurag Sethi,1 Anuj Srivastava,2 Xingyi Woo,3 Vishal Sarsani,3 Ziming Zhao,2 Javad Noorbakhsh,2 Christian French,1 Jack DiGiovanna,1 Ogan D. Abaan,1 Steve Neuhauser,3 Peter Robinson,2 Yvonne A. Evrard,4 Carol J. Bult,3 Jeffrey A. Moscow,5 Brandi Davis-Dusenbery,1 Jeffrey H. Chuang2. 1 _Seven Bridges Genomics, Cambridge, MA;_ 2 _The Jackson Laboratory for Genomic Medicine, Farmington, CT;_ 3 _The Jackson Laboratory, Bar Harbor, CT;_ 4 _Frederick National Laboratory for Cancer Research, Frederick, MD;_ 5 _National Cancer Institute, Bethesda, MD_.

Patient-Derived Xenografts (PDX) are powerful models to study tumors' drug-response in the context of personalized medicine. In the PDX model settings, by virtue of expanding the patient's tumor sample, testing multiple drug or drug-combinations can be executed rapidly and has no ethical limitations. However, there are major issues around standards that need to be addressed to make these models widely accessible and usable.

The overarching goal of the PDXNet is to coordinate the development of appropriate PDX models and methods for preclinical drug testing to advance CTEP clinical development of new cancer agents. In an effort to standardize protocols for PDX generation as well as data analysis and metadata harmonization, we are building a data storage, sharing, and analysis platform that harmonizes PDXNet data with other large datasets and analysis workflows. The PDX Data Commons is built on top of existing NCI resources, leveraging the Cancer Genomics Cloud maintained by Seven Bridges Genomics, where PDXNet data is co-located with TCGA and other large-scale datasets. The PDCCC is co-led by experts from The Jackson Laboratory, providing scientific leadership in xenograft methods and cancer biology to ensure the promulgation of standards that are well-suited for the PDX community. In addition, the PDCCC is responsible for establishing studies to identify best-practices for PDX data analysis and metadata schemas. The data collected as part of the PDXNet is currently stored on the PDXNet portal that has a query interface for identifying models for pre-clinical trials. Simultaneously, we administer training activities and research pilots to build synergies within the PDXNet, enhancing the ability of the PDXNet to develop clinical trials from PDX studies.

In PDXNet, besides the PDCCC, there are 4 PDX Development and Trial Centers (PDTCs) responsible for executing specific pre-clinical trials focused around cancer types including breast cancer, melanoma, and lung cancer. Data generated by the PDTCs will be hosted by the PDCCC, and metadata will be collected based on schemas developed by the network for systematic ontological analysis. These PDX models, in coordination with the NCI Patient-Derived Models Repository (PDMR) at the Frederick National Laboratory for Cancer Research (FNLCR) will be shared with the broader community. In addition, PDTC's will collaborate with non-PDXNet investigators for PDX studies through an administrative supplement program supported by the NCI.

The PDXNet is a strong step toward building a consensus around PDX models, so that the power for discovery can be expanded by making multi-institutional PDX cohorts a reality. The PDCCC is a central part of this process to systematically capture and analyze the variables most influential to PDX models and share protocols and tools to make PDXs an interchangeable research currency for pre-clinical discovery.

#1030

Circulating tumor cells from patients' blood induce tumors in a personalized xenograft mouse model: A functional approach to assess the patients' premetastatic disease state.

Netta R. Shraga,1 Amihai Lieberman,1 Neta Moskovits,2 Alejandro Livoff,3 Evgeny Solomonov,4 Inbar Ben Shachar,4 Aron Popovtzer,5 Salomon M. Stemmer*,5 Izhak Haviv*1. 1 _The Azrielyi Faculty of Medicine, Bar Ilan University, Safed, Israel;_ 2 _Institute of Oncology, Davidoff Centre, Rabin Medical Centre and Bar Ilan University, Petah Tikva, Israel;_ 3 _Barzilai Medical Center, Ashkelon, Israel;_ 4 _Ziv Medical Center and Azrieli Faculty of Medicine, Bar Ilan Univeristy, Safed, Israel;_ 5 _Institute of Oncology, Davidoff Centre, Rabin Medical Centre and Sackler Faculty of Medicine, Tel-Aviv University, Petah Tikva, Israel_.

Background: Circulating tumor cells (CTCs) originate from both primary and metastatic solid tumors and infiltrate the blood system. Although the metastatic potential of the single CTC cell is unknown, CTCs in circulation have been reported to have prognostic value as early predictors of relapse in cancers including ovarian, breast, colorectal, prostate, lung, bladder, head and neck and melanoma. As metastatic spread remains the major problem in cancer treatment, it is of great importance to characterize the subpopulations of CTCs with the highest metastatic potential.

Methods: Peripheral blood mononuclear cells (MNCs) or bone marrow MNCs, were collected from each of over 60 cancer patients and grafted separately to immunodeficient NOD.Cg-Prkdc.scid.Il2rgtm1Wjl/SzJ (NSG) mice. Additionally, CTCs were enriched by either CD8 T-cell depletion or by a size-based filtration system. Tumors that developed in the mice were confirmed to be human tumors by demonstrating that they have human HLA expression and by comparing their tumor type by an independent pathologist to that of the donor patient's. The CTC-derived tumors were passaged to naive NSG mice to establish the scale up potential for treatment-groups. To facilitate future research of CTC-derived tumors, we have established a Biobank for CTC-derived tumors with matched samples of the patient's MNCs, plasma, tumor biopsy(s) and patient-biopsy derived xenograft (PDX) tumors. Additional characterization of the CTC-derived tumors was performed, such as comparison to the driver-mutations of the original tumor-biopsies and expression of epithelial or mesenchymal markers. The clinical relevance of the CTC-derived tumors was assessed by long term follow-up of each patient's disease progression.

Results: CTC-derived tumors were detected in xenografts in 15 cases, within an average of eight months post grafting, representing a 25% success rate. The CTC-derived tumors were from a variety of cancers including: breast (6), colon (1), lung (2), pancreatic (1), ovarian (1), malignant-mesothelioma (1) head and neck (1) hepatocellular carcinoma (1) and sarcoma (1). The patients, whose blood or marrow contained tumorigenic CTCs, developed metastasis. Interestingly, there was a correlation between sites of CTC-derived tumors in the mice and sites of metastatic spread in the patients. CTC-derived tumors were successfully transferred to subsequent PDX mice, enabling the establishment of large treatment groups to study drug reactivity of these tumors.

Summary: This study describes a functional assay to characterize the CTCs subtypes with a high metastatic potential, which utilizes the capacity of CTCs to form tumors in a permissive immune-free environment. CTC-derived tumors may offer a new tool to predict the significance of these malignant cells and prevent metastatic spread.

#1031

XenoSarc: Patient-derived xenograft (PDX) models of soft tissue sarcoma (STS)—an update on a preclinical platform for early drug testing.

Agnieszka Wozniak,1 Jasmien Cornillie,1 Yemarshet K. Gebreyohannes,1 Yannick Wang,1 Jasmien Wellens,1 Ulla Vanleeuw,1 Daphne Hompes,2 Marguerite Stas,2 Friedl Sinnaeve,2 Hazem Wafa,2 Maria Debiec-Rychter,1 Raf Sciot,1 Patrick Schöffski1. 1 _KU Leuven and University Hospitals Leuven, Leuven, Belgium;_ 2 _University Hospitals Leuven, Leuven, Belgium_.

Background: STS constitutes a rare family of mesenchymal tumors with more than 70 subtypes described. The limited treatment options available for advanced STS underline the need for reliable preclinical models to test novel therapeutic strategies.

Methods: Panel of PDX models was established by subcutaneous implantation of fresh tumor specimens in immunodeficient, athymic nude NMRI mice. Once tumor growth was observed, pieces of tumor were re-transplanted to next generations of animals. At each passage tumor fragments were collected for detailed characterization. A model was considered established after observing stable histological and molecular features for at least two passages.

Results: Between 09/2011 and 11/2017, 228 STS samples from 203 consenting patients treated at University Hospitals Leuven, Belgium, have been transplanted. Thirty-three stable PDX models have been established, maintaining the histopathological and molecular features of the original tumor. Detailed clinical information about a donor patient, including sensitivity to given therapy, is linked to every model. Higher engraftment rate was observed in samples collected from patients who developed metastasis throughout the course of disease (38% vs. 23%, p<0.05). Moreover, patients whose tumor successfully engrafted had significantly poorer overall survival (OS) than those whose tumor did not grow in mice (median OS 83 vs. 259 months; p<0.05). XenoSarc platform includes models of gastrointestinal stromal tumor (6 models), myxofibrosarcoma (6), leiomyosarcoma (5), dedifferentiated liposarcoma (4), malignant peripheral nerve sheath tumor (2), synovial sarcoma (1), pulmonary artery intimal sarcoma (1), CIC-DUX4 fusion-positive round cell sarcoma (1), epithelioid haemangioendothelioma (1), mesenchymal chondrosarcoma (1), pleomorphic rhabdomyosarcoma (1), telangiectatic extraskeletal osteosarcoma (1), and high-grade undifferentiated pleomorphic sarcoma (3). These models are well-characterized, with detailed data on copy number changes and expression profile. In addition we have constructed tissue microarray (TMA), which can be used for target identification. Some of these models have already been successfully used for in vivo testing of novel agents, including both targeted and cytotoxic (pro-)drugs, and results served as a rationale for several prospective clinical trials. In addition, 17 other xenografts are still in early stages of engraftment, not yet fulfilling our criteria of an "established model".

Conclusion: Our XenoSarc platform contains a number of well-annotated models, characterized by stable histological and molecular features. This platform is a reliable tool for the evaluation of new anticancer treatments for STS and for studying the biology of these rare diseases. The platform is made available to collaborators from academia and industry.

#1032

Novel mouse models of high-risk HPV-related oral cancers.

Miranda B. Carper,1 Scott Troutman,2 Kevin M. Byrd,1 Bethany Wagner,1 Erin C. Henry,1 Stephanie A. Montgomery,1 Scott E. Williams,1 Joseph L. Kissil,2 Antonio L. Amelio1. 1 _UNC-Chapel Hill, Chapel Hill, NC;_ 2 _The Scripps Research Institute, Jupiter, FL_.

The rate of HPV-induced head and neck squamous cell carcinoma (HNSCC) is steadily increasing and implicated in approximately 60% of all oropharyngeal carcinomas. The advent of whole genome, transcriptome, and proteome analyses have aided in identifying altered signaling pathways in HPV-induced HNSCCs, however, additional tools such as mouse models are needed to study the role of HPV oncogenes in oral tumor initiation and progression. Current inducible models of HPV-driven oral cancer do not accurately recapitulate the levels, stoichiometric ratios, or anatomic location of oncoprotein expression. To address these limitations, we developed a tractable genetically engineered mouse model (GEMM) that enables directed expression of high-risk HPV16 E6 and E7 oncogenes (Rosa26-loxP-STOP-loxP-E7iresE6; "H") in the oral epithelium. Analysis of mouse embryonic fibroblasts treated with Cre recombinase confirmed E6 and E7 expression and changes in mRNA expression of known E6 and E7 targets. We crossed our H mice to several tissue-specific transgenic Cre-driver mouse strains including the EBV lytic promoter (ED-L2-Cre recombinase; "L") to drive robust expression in the oropharyngeal squamous epithelia. Analysis of epithelial tissues isolated from LH mice displayed increased oral volumes and cutaneous epithelial thickening associated with hyperplasia, dysplasia, accumulation of neutrophils and recruitment of cytotoxic and regulatory T cells. A second cross to the keratin 14 promoter (KRT14-Cre; "K") line enabled targeted expression to the basal epithelial layer (KH) and confirmed increased oral volumes and cutaneous epithelial thickening associated with increased suprabasal proliferation and expression of canonical E7 targets. Lastly, a third cross to a tamoxifen-inducible Cre (CreERT2) line driven by the keratin 14 promoter (KRT14-CreERT2; "iK") enabled conditional and inducible post-natal E6 and E7 expression in basal epithelia (iKH). To prevent systemic oncogene expression in K14+ cells, we validated a method for submucosal delivery of tamoxifen to the tongue. We confirmed that this approach prevents tamoxifen spread and anatomically restricts activation oncogene expression to intra-lingual regions using an optical reporter mouse recently generated by the Amelio lab (Rosa26-loxP-STOP-loxP-LumiFluor; "F") crossed to the iK line ("iKF"). Notably, direct intra-lingual injection yields higher recombination and reporter activation using 40% less overall tamoxifen than traditional i.p. administration routes. Moreover, immunofluorescent staining revealed mosaic transgene activation in the oropharynx, an important feature for modeling HPV-induced OSCC. In fact, intra-lingual injection of tamoxifen in iKH mice coupled with 4-nitroquniloline 1-oxide (4NQO) administration led to E6 and E7 expression and dysplasia in the tongue epithelia. Ongoing studies are evaluating cooperating mutations in driving HNSCC development.

#1033

Patient-derived xenografts for prognostication and personalized treatment for head and neck squamous cell carcinoma.

Christina Karamboulas, Jeffrey P. Bruce, Andrew J. Hope, Jalna Meens, Shao Hui Huang, Jie Su, Fei-Fei Liu, Trevor J. Pugh, Scott V. Bratman, Wei Xu, Laurie E. Ailles. _Princess Margaret Cancer Centre, Toronto, Ontario, Canada_.

Overall outcomes for HPV-negative head and neck squamous cell carcinoma (HNSCC) remain poor with 5-year overall survival rates of 50-60%. Oral squamous cell carcinoma (OSCC), the most common subtype of HPV-negative HNSCC, is typically treated with surgery, and clinico-pathologic features are used to identify patients in need of adjuvant therapies such as radiation therapy (RT), or radiation plus concurrent chemotherapy (CRT). It is clear from the rate of loco-regional or distant failures that more accurate methods of risk stratification would greatly improve outcomes for OSCC patients. This requires biomarkers to identify patients that will benefit from adjuvant RT or CRT but currently there are no validated molecular biomarkers that have been clinically implemented for the personalized treatment of OSCC. In addition to biomarkers for better risk stratification, there is also a need for novel therapeutic strategies leading to improved outcomes. Recently patient-derived xenografts (PDXs) have been shown to faithfully recapitulate human tumor biology and predict drug responses, supporting their relevance as preclinical models for new drug development.

Upon subcutaneous implantation of HNSCC specimens into NOD/SCID/IL2Rγ-/- mice, 161 of 243 samples (66%) successfully formed patient-derived xenografts (PDX). Using univariable and multivariable analyses, the ability to form a PDX correlated significantly with adverse clinical outcomes, and specifically, patients with palpable PDX-formation within 8 weeks experienced particularly poor outcomes (hazard ratio for overall survival = 3.0). A cohort of engrafting and non-engrafting samples were sequenced using a targeted sequencing panel designed for both mutational and copy number alteration detection. The overall frequency of somatic genomic alterations detected was similar to The Cancer Genome Atlas cohort and interestingly, successful engraftment correlated to amplification of the CCND1 gene. Twelve PDX models were treated with the CDK4/6 inhibitor, abemaciclib; 5 of 6 models with CCND1 amplifications and/or CDKN2A mutations responded to abemaciclib treatment, whereas only 1 of 6 models lacking these alterations responded. These results demonstrate the potential of using PDX models to identify novel targeted therapies for HNSCC patients who have the poorest outcomes. In the future, PDX avatars could also be exploited to individualize treatment for patients at high risk of relapse following definitive treatment.

#1034

Genotyping of patient-derived xenograft tumors using short tandem repeats: Challenges and observations in ensuring concordance across passages for effective preclinical studies.

Bridgette Sisson,1 Andrew Hesse,1 Melissa Soucy,1 Daniel Bergeron,1 Shelbi Burns,1 Kevin Kelly,1 Emily Jocoy,2 Margaret Bundy,2 Honey V. Reddi1. 1 _The Jackson Laboratory, Farmington, CT;_ 2 _The Jackson Laboratory, Sacramento, CA_.

Patient derived xenograft (PDX) tumor models represent the closest step to in-human trials for cancer drug research and are therefore an invaluable resource. Implantation of a cancerous tissue from a patient's primary tumor directly into an immune-deficient mouse, generating a xenograft tumor potentially mimics the tumor microenvironment in the patient and allows for the evaluation of therapies specific to a particular tumor. One of the significant challenges associated with PDX tumors is the potential loss of the human component or the take-over by mouse tissue with every passage into a new mouse model. Short tandem repeats (STRs) are short intronic sequences of DNA that are highly polymorphic and contain a pattern of nucleotides repeated in tandem. STRs can be found in distinct regions of both prokaryotes and eukaryotes organisms, with each organism containing a unique combination of the STR allelic frequencies that allow for individuals to be unambiguously identified. STR analysis is routinely used to genotype PDX tumors comparative to the original resected human cancerous tissue, ensuring concordance with original tissue and to identify mouse tissue contamination with progressive passages. The Jackson Laboratory for Genomic Medicine's CLIA laboratory has evaluated over 170 PDX tumor samples from multiple mouse model passages across 105 diverse PDX mouse models. Of those samples analyzed, genomic DNA for 33 had been extracted from fresh frozen tissue and for 143 extracted from Formalin Fixed Paraffin embedded tissue (FFPE). The current study presents the challenges and discrepancies observed in our evaluation relative to number of passages for each tumor, sample type (FFPE vs fresh frozen) - reflective of tumor heterogeneity and also potential encroachment by mouse tissue. We also present results of a cross institutional comparative STR analysis study in 123 samples wherein discordance observed in certain cases were a result of differing sample types, suggestive that tumor heterogeneity can affect the STR results and could be a leading cause for cross institutional discordant genotyping results.

#1035

A comprehensive patient-derived tumor xenograft (PDX) collection representing the heterogeneity of kidney, prostate and bladder cancers.

Hervé Lang,1 Claire Béraud,2 Myriam Lassalle,3 Isabelle Bernard-Pierrot,4 Véronique Lindner,5 Yves Allory,6 Michel Soulié,7 Xavier Gamé,7 Pascal Rischmann,7 Eric Potiron,8 François Radvanyi,4 Philippe Lluel,3 Thierry Massfelder9. 1 _Department of Urology, Strasbourg University Hospital, Strasbourg, France;_ 2 _Urolead, Toulouse, France;_ 3 _Urolead and Urosphere, Toulouse, France;_ 4 _UMR144 CNRS – Institut Curie, Paris, France;_ 5 _Department of Pathology, Strasbourg University Hospital, Strasbourg, France;_ 6 _Department of Pathology, Hôpital Henri Mondor, Créteil, France;_ 7 _Department of Urology, Hôpital Rangueil, Toulouse, France;_ 8 _Clinique Urologique Atlantis, Nantes, France;_ 9 _INSERM U1113 Team 3, Strasbourg, France_.

Kidney, prostate and bladder cancers (KCa, PCa and BCa, respectively) represent 1 700 000 cases and 450 000 deaths worldwide per year, with an incidence rising yearly by 1-10%. Surgery is usually curative at early and localized stages but there are no efficient therapies at advanced and metastatic stages for any of them. Although genetically-modified and/or chemically-induced avatar models do exist for these cancers and may help to identify new therapeutic targets, they suffer from a lack of an extended biological concordance with the natural history and heterogeneity of the diseases. Patient-derived tumor xenograft models are now well recognized as reliably reproducing tumor heterogeneity and have become over the past few years the preclinical tools of choice to test drugs and identify biomarkers. Since 10 years, we are continuously developing a unique panel of PDX models for these major urological cancers. Tumor tissues along with normal corresponding tissues were obtained from patients at surgery. Patient informed consent and clinical history are available for all patients. Tumor tissues pieces were xenografted subcutaneously in the interscapular space of nude mice, and serially passaged into mice after the first engraftment, up to passage 10. To ensure model stability between primary tumors and tumors growing in mice but also from passage to passage, we performed various analyses at histopathological, genetic (short tandem repeat fingerprinting) and molecular (cDNA profiling) levels. In addition, growth characteristics and responses to standards of care (SOCs) were examined. Finally, specific molecular characteristics were also explored including expression of the androgen receptor, PSA and pan-cytokeratin for PCa models and hotspot mutations of FGFR3, PIK3CA, K/N/H-RAS for BCa models. Up to now, we have xenografted 336 (on 569 samples), 247 and 152 KCa, PCa and BCa tumor tissues, and developed 30 (8.9% success rate), 6 (2.1%) and 30 (19.7%) PDX models, respectively. We recently published part of the KCa PDX models collection (Lang et al., Oncotarget, 2016). Characterization studies showed that PDX models are stable at all levels analyzed considering concordance to primary tumors and from passage to passage; and less than 5% of genes were differentially expressed between the primary tumors and PDX tumors at various passages. Responses to SOCs recapitulated the clinical state. Only for KCa PDX models, the take rate was correlated to tumor stage and grade, and sarcomatoid components. Importantly, several molecular subtypes were defined in our collection of BCa PDX models including PDXs with FGFR3 mutations and PDXs of basal subtype, the most aggressive one. Overall, this panel of PDX models for urological cancers should definitely help to find molecularly guided targeted therapies for these still incurable cancers at metastatic stages.

#1036

Establishment and molecular profiling of patient-derived xenograft models of Korean patients with primary lung cancer.

Tae Ho Kim,1 Hyunjin Heo,1 Soo Jung Lee,1 Seungje Lee,1 Eunjoo Hwang,1 Jinseon Lee,1 Heekyoung Lee,2 Kevin Koo,2 Hanna Lee,2 Seungjae Lee,2 Hwanseok Rhee,2 Jong Eun Lee,2 Jhingook Kim1. 1 _Samsung Medical Center, Seoul, Republic of Korea;_ 2 _DNA Link, Seoul, Republic of Korea_.

Non-small cell lung cancer is the most prevalent type of lung cancer, and one of its subtype, squamous cell carcinoma (SCC) has the genomic complexity and high overall mutation due to the chemical carcinogens so that limited therapeutic options are available in comparison with adenocarcinoma. We constructed the patient-derived xenograft models of Korean primary lung cancer and then focused on the SCC PDX models for this particular ethnic subgroup. PDX models were established using the tissues of patients who underwent surgery as primary lung cancer at Samsung Medical Center during the period between October, 2014 and September, 2017. Briefly, tumor tissues from patients were subcutaneously engrafted and passaged two more times in NOD-scid-IL2Rγnull mouse. Models were selected whose tumor tissues showed the histopathology and genomics similar to the patient tumor tissues. Statistics of established PDX models are summarized in the Table. | |

---|---|---

|

Successful engraftment | Success rate (%)

Primary lung cancer (n=532) | 199 | 37.41

Histology

Non-small cell lung cancer (n=524)

\- Adenocarcinoma (n=355)

\- Squamous cell carcinoma (n=135)

\- Other NSCLC (n=34)

Small cell carcinoma (n=8) | -

194

89

83

22

5 | -

37.02

25.07

61.48

64.71

62.5

Type of resection

Curative lung resection (n=458)

Biopsy (n=74) | -

174

25 | -

37.99

33.78

Pathologic staging of ADC (n=355)

I (n=162)

II (n=58)

III (n=68)

IV (n=67) | -

40

15

19

15 | -

24.69

25.86

27.94

22.39

In order to help facilitate the target drug development of SCC, we concentrated on the construction of comprehensive database of fourteen SCC PDX models integrating the information from whole exome sequencing, whole transcriptome sequencing and clinical profiles. We detected mutations commonly reported in SCC genes including EGFR, BRAF, CUL3, DDR2, TP53, MYD88, PIK3CA, PTEN, MED12, SMAD4, AKT1, CTNNB1, IDH1, HRAS, KRAS, NRAS, SMARCA4, GNAS, CDKN2A, APC, CDKN2A and RRAS2. Specifically potential therapeutic targets such as EFGR, BRAF, CUL3, DDR2, TP53 and KRAS which are currently under several clinical trials were found in our SCC PDX models, and intriguingly one model had five mutations in CUL3, KRAS, TP53, and EGFR simultaneously. We expect these SCC PDX models could be valuable resource for target drug development and interpretation of their therapeutic responses in the pre- or co-clinical trials. Detailed information on pathology and genomic information on all PDX models will be available at DNA Link PDX webpage.

#1037

Comprehensive molecular profiles of gastric cancer patient derived xenograft (PDX) models and its implication in precision cancer medicine.

Jae Eun Lee,1 Yoon Young Choi,2 Ju Yeon Lim,1 Su-Jin Shin,3 Gunho Lee,1 Eun Young Kim,1 Taeil Son,2 Hyoung-Il Kim,2 Woo Jin Hyung,2 Sung Hoon Noh,2 Hyunki Kim,4 Minkyu Jung,5 Sangwoo Kim,1 Soonmyung Paik,1 Jae-Ho Cheong2. 1 _Yonsei Biomedical Research Institute, Yonsei University college of medicine, Seoul, Republic of Korea;_ 2 _Department of Surgery, Yonsei University college of medicine, Seoul, Republic of Korea;_ 3 _Department of Pathology, College of Medicine, Hanyang University, Seoul, Republic of Korea;_ 4 _Department of Pathology, Yonsei University college of medicine, Seoul, Republic of Korea;_ 5 _Division of Medical Oncology, Department of Internal Medicine, Yonsei University college of medicine, Seoul, Republic of Korea_.

Background: Recent cancer research is focused on precision medicine with the advent of next generation sequencing (NGS) technology and patient tumor derived model systems. Here, we present molecular characteristics of gastric cancer patient derived xenograft (PDX) models and explore the potential of molecularly defined PDX model based drug development.

Materials and Methods: We generated PDX models from patient tumors with advanced gastric cancer. The genomic alterations of tumors were profiled by whole exome sequencing (WES), RNA sequencing (RNAseq), targeted sequencing, in-situ hybridization (ISH) and immunohistochemistry (IHC). Further, we developed overcoming strategy of chemotherapy resistance mechanism by combination of signaling pathway inhibitor and standard chemotherapy regimen in "N-of-1" PDX trial.

Results: Thirty-five PDX models were successfully established and categorized into four subgroups of The Cancer Genome Atlas (TCGA) and Asian Cancer Research Group (ACRG) classifications: EBV (2.9%), MSI (20.6%), GS (23.5%) and CIN (52.9%) subtypes by TCGA while MSI type (11.4%), MSS/EMT type (17.1%), MSS/TP53+ (31.4%) and MSS/TP53- type (40.0%) by ACRG. In the protein levels by IHC, there were 21 cases (60.0%) of any RTK proteins overexpression; nine of HER2 (25.7%), 14 of EGFR (40.0%), and 16 of c-MET (45.7%). Five tumors (14.3%) were related to PTEN loss and 22 tumors (62.9%) showed p53 overexpression or null. Targeted sequencing identified that ERBB2 (25.7%), KRAS (11.4%), and CCND1 (11.4%) were found frequently amplified gene while PIK3CA (11.4%) and CTNNB1 (8.6%) were found most mutated genes. N-of-1 PDX trial demonstrated that the response to FOLFOX in PDX tumor was concordant with that of corresponding patient. In FOLFOX resistant tumors, multiple signaling pathways were up-regulated and inhibition of these signaling pathways was regressed tumor growth.

Conclusions: The utilization of molecularly catalogued gastric cancer PDX models will guide precision medicine for cancer therapy and be a useful tool for drug development and repurposing.

#1038

Xenograft-associated B cell lymphoproliferative disease as a surrogate model to study Epstein-Barr virus (EBV) driven lymphoma of the elderly.

Tomas Vilimas,1 Gloryvee Rivera,1 Brandie Fullmer,1 Wiem Lassoued,1 Lindsay Dutko,1 William Walsh,1 Amanda Peach,1 Corinne Camalier,1 Li Chen,1 Rajesh Patidar,1 Suzanne Borgel,2 John Carter,1 Howard Stotler,1 Raymond Divelbiss,1 Jesse Stottlemyer,1 Margaret Defreytas,1 Michelle M. Gottholm-Ahalt,2 Michelle A. Crespo-Eugeni,2 Sean McDermott,1 Yvonne A. Evrard,2 Melinda G. Hollingshead,2 Biswajit Das,1 Chris Karlovich,1 Vivekananda Datta,1 James H. Doroshow,3 P. Mickey Williams1. 1 _Frederick National Lab for Cancer Research / Leidos Biomed. Research, Inc., Frederick, MD;_ 2 _National Cancer Institute at Frederick, Developmental Therapeutics Program, Frederick, MD;_ 3 _National Cancer Institute, Division of Cancer Treatment and Diagnosis, Bethesda, MD_.

Background: Patient-derived tumor xenografts (PDX) are powerful tools to study cancer biology, cancer genomics and developmental therapeutics. A common problem in the development of PDX models is proliferation of atypical lymphocytes at the implant site, which often overtake or limit the growth of the original tumor. This atypical proliferation has been described as Xenograft-Associated B cell Lymphoproliferative Disease (XABLD) in our PDX models. In this study, we characterized XABLD cases by morphology, immunophenotyping and genomic profiling. We hypothesize that XABLD tumors are morphologically and phenotypically similar to EBV-driven lymphoma of the elderly and may function as a surrogate model for that lymphoma. Materials and Methods: Models were generated from patient tissue collected under NCI Tissue Procurement Protocol (clincialtrials.gov: NCT00900198) and CIRB Tissue Procurement Protocol 9846 for development of models for NCI's Patient-Derived Models Repository (https://pdmr.cancer.gov). Specimens were implanted subcutaneously in NOD/SCID/IL2Rg null (NSG) mice and animal health was monitored throughout the study. Tumors in mice with suspected XABLD were harvested and reviewed by histology and immunohistochemical analysis for CD45, B and T cell markers and EBV status. All samples in this study were classified by the Lymph2Cx NanoString cell of origin assay and transcriptome profiling. Results: XABLD-associated mice had rapidly growing CD45-positive tumors at the implantation site. Histopathological features were consistent with EBV-driven diffuse large B-cell lymphoma (DLBCL) primarily of polymorphous subtype. All XABLD specimens were diffusely positive for CD20 and EBNA, and most cases contained tumor infiltrating CD8-positive T-cells. Out of 42 cases, 36 were PD-L1-positive and 26 were PD-1-positive by IHC. 39 cases exhibited an activated B cell (ABC) phenotype, which is predominant in EBV-positive DLBCL. Conclusion: XABLD development has been seen across multiple patient histologies from both solid tumor and circulating tumor cells tissues of origin. The clinical presentation, morphology and molecular characteristics of XABLD cases were similar to EBV-driven DLBCL. As DLBCL is an aggressive disease with limited treatment options, our early-passage XABLD models may be useful in the preclinical evaluation of new therapies for EBV-positive DLBCL. Grant Support: This project has been funded in part with federal funds from the National Cancer Institute, National Institutes of Health, under Contract No. HHSN261200800001E. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government.

#1039

PDX models generated from a patient with metastatic colon adenocarcinoma display both spatial and temporal tumor heterogeneity.

Biswajit Das,1 Chris Karlovich,1 Corrine E. Camalier,1 Rajesh Patidar,1 Li Chen,1 Vivekananda Datta,1 William D. Walsh,1 Sean P. McDermott,1 Tomas Vilimas,1 Palmer Fliss,1 Justine N. McCutcheon,1 Amanda Peach,1 Michelle Ahalt-Gottholm,2 Carrie Bonomi,1 Kelly Dougherty,1 John Carter,1 Shivaani Kummar,3 Yvonne A. Evrard,1 Melinda G. Hollingshead,2 Paul M. Williams,1 James H. Doroshow4. 1 _Leidos Biomedical Research Inc., Frederick National Laboratory for Cancer Research,, Frederick, MD;_ 2 _National Cancer Institute at Frederick, Developmental Therapeutics Program, Frederick, MD;_ 3 _Stanford University School of Medicine, Palo Alto, CA;_ 4 _National Cancer Institute, Division of Cancer Treatment and Diagnosis, Bethesda, MD_.

Background: Patient-derived Xenograft (PDX) models are being widely used in preclinical studies to identify biomarkers of drug response and to enhance our understanding of cancer biology. Since patients with metastatic cancer have both intra-tumor and inter-site heterogeneity, PDX models generated from different tumor sites may provide a way to study tumor heterogeneity. Characterization of the genomic landscape in these models may also provide better insights into treatment response or resistance. It is rare to have multiple PDX models generated from a single patient over multiple time points during a treatment trajectory. Here, we report the genomic profiles of PDX models generated from 4 distinct tissue specimens over a 7-month period from a patient with metastatic colon adenocarcinoma. The first 2 PDX models were generated from circulating tumor cells (CTCs) and a liver biopsy prior to treatment with a combination pan-AKT + MEK inhibitor regimen. A third PDX model was generated from a liver biopsy while on-treatment and a fourth from an adrenal gland resection at progression. Clinically, all reported metastatic sites, except the adrenal gland, responded to the combination therapy.

Results: Genomic characterization of the specimens obtained from these 4 PDX models led to the following observations: 1) PIK3CA E545K and KRAS G12D are present in all the specimens tested for all 4 models and are likely truncal driver mutations; 2) exclusive inter-model SNVs (single nucleotide variants) were identified, and may be model-specific variants representing inter-site heterogeneity in the patient; 3) variants involved in known resistance mechanisms to MEK inhibition were not present in any specimens; 4) overexpression of AKT3 has been reported as a resistance mechanism to a pan-AKT inhibitor and was observed in the adrenal tissue from the patient but not in any other PDX model derived from this patient; 5) intra-model and inter-model heterogeneity in whole genome CNV (copy number variant) profiles was observed between individual PDXs obtained from the pre-treatment CTC-derived model and the on-treatment liver biopsy model. Interestingly, one of the PDXs from the CTC-derived model presented a sub-clonal tumor fraction closely related to the on-treatment liver biopsy model. The multiple inter-model CNV profiles in the liver biopsy derived PDX models represent temporal heterogeneity within a tissue.

Conclusions: We observed genomic heterogeneity in PDXs generated from specimens from a patient with metastatic colon adenocarcinoma. Both truncal and sub-clonal variants were identified representing various tumor fractions in these models. This case study illustrates how genomic profiling of multiple tumor sites at different times during course of treatment can provide insight into the complexity of tumor heterogeneity and tumor evolution in patients with metastatic disease.

#1040

Patient derived xenografts of hematologic malignancies for translational research.

Antje Siegert,1 Bernadette Brzezicha,1 Stephan Fuhrmann,2 Leonid Karawajew,3 Martin Janz,4 Clemens Schmitt,4 Iduna Fichtner,1 Jens Hoffmann1. 1 _EPO Berlin-Buch GmbH, Berlin, Germany;_ 2 _Helios Klinikum Berlin-Buch GmbH, Berlin, Germany;_ 3 _Charité Campus Buch, Berlin, Germany;_ 4 _MDC for Molecular Medicine, Berlin, Germany_.

Acute leukemias and lymphomas represent a very heterogeneous group of hematologic malignancies and pose an important challenge in the clinical routine. They frequently develop resistance to the treatment with standard-of-care (SoC) drugs and have a high incidence of disease recurrence. Recent progress in molecular profiling has helped to identify new potential drivers for the different leukemia and lymphoma subtypes, with some of them being potential therapeutic targets. Further target validation and drug development projects are highly dependent on corresponding preclinical models representing the different clinical subtypes. Therefore, we started to establish and characterize new patient derived xenografts (PDX) of AML, ALL and lymphomas for drug development and translational research.

AML- and ALL-PDX were derived from bone marrow aspirates or peripheral blood samples, from primary or relapsed acute leukemia patients. Purified cells were transplanted either intravenously (i.v.) and/or subcutaneously (s.c.) into immunodeficient mice. Some mice developed a systemic AML, which was monitored by flow cytometric analysis of blood samples. Non-Hodgkin- or Hodgkin lymphoma-PDX were derived from peripheral blood, lymph node extirpations or core needle biopsies, and were usually transplanted subcutaneously into immunodeficient mice.

For further characterization, established PDX models were treated with SoC and investigational drugs. In parallel, gene expression profiles as well as mutations were analyzed within the first in vivo passages.

More than 20 new PDX models from AML, ALL, NHL and HL have been successfully established and characterized. Highly individual response to the treatments were observed, correlation analyses with mutations and gene expression are ongoing.

Our newly and extensively characterized PDX models from hematologic malignancies are suitable tools for preclinical drug development. They provide an exceptional platform for the identification and validation of new targets and allow the preclinical screening of new compounds and combinations for translational research projects.

#1041

XenofilteR: Computational dissection of mouse and human reads in PDX and xenograft sequence data.

Oscar Krijgsman,1 Roelof JC Kluin,1 Kristel Kemper,1 Thomas Kuilman,1 Julian R. de Ruiter,1 Vivek Iyer,2 Josep V. Forment,2 Paulien Cornelissen-Steijger,1 Iris de Rink,1 Petra ter Brugge,1 Ji-Ying Song,1 Sjoerd Klarenbeek,1 Ultan McDermott,2 Jos Jonkers,1 Arno Velds,1 David J. Adams,2 Daniel S. Peeper1. 1 _Netherlands Cancer Institute, Amsterdam, Netherlands;_ 2 _Wellcome Trust Sanger Institute, Hinxton, United Kingdom_.

Mouse xenografts from (patient-derived) tumors (PDX) or tumor cell lines are widely used as models to study various biological and preclinical aspects of cancer. However, analysis of their RNA and DNA profiles is challenging, because they comprise reads not only from the grafted human cancer but also from the murine host. The reads of murine origin can result both in the generation of false positives in mutation analysis of DNA samples and obscure gene expression levels when sequencing RNA. Therefore, we developed the open-source R-package XenofilteR, which separates mouse from human sequence reads based on the number of discordant base pairs between each read and the reference genomes. To assess the accuracy of XenofilteR, we generated sequence data by in silico mixing of mouse and human whole genome and whole exome DNA sequence data. This analysis revealed that XenofilteR removes >99.9% of sequence reads of mouse origin while retaining sequence reads of human origin. The filtering allowed for mutation analysis of PDX samples with accurate variant allele frequencies, and retrieved all non-synonymous somatic mutations present in the original tumor. These findings were further validated in breast cancer and melanoma PDX samples, confirming the retrieval of accurate variant allele frequencies and somatic mutations. In conclusion, XenofilteR accurately dissects sequence reads from mouse and human origin in PDX sequence data, thereby outperforming currently available tools.

#1043

**Patient-derived and cell line xenograft growth in the B6;129-** Rag2tm1FwaIL2rgtm1Rsky **/DwlHsd (R2G2) mouse model.**

Jamie L. Naden. _Envigo, Indianapolis, IN_.

Immunodeficient mouse models are helping to advance the field of oncology. A new model on the market, the B6;129-Rag2tm1FwaIL2rgtm1Rsky/DwlHsd (R2G2) knockout mouse, lacks responsiveness to common gamma chain cytokines, including IL-2, IL-4, IL-7, IL-9 and IL-15. In addition, this model exhibits defects in lymphoid development and so lacks mature lymphocytes of the B, T, and natural killer (NK) cell lineages. Herein we describe growth of multiple patient-derived (PDX) and tumor cell line xeno- (CDX) and allografts in the R2G2 immunodeficient mouse model. The PDXs examined include colorectal and head and neck cancers. The CDXs studied include esophageal (OE33 and FLO1) and stomach cancer (AGS). Tumor growth data were also collected from two allografts of murine colorectal cancer (CT26) and B-cell lymphoma (A20) cells. Colorectal PDX tissue was subcutaneously implanted bilaterally into five male R2G2 and five male NSG mice. Growth was comparable between the R2G2 and the NSG mouse models; however, the standard error was much lower in the R2G2 strain. Head and neck PDX 626 and 635 was transplanted in 2.2 mm2 tissues into 4 sections of each of 2 R2G2 mice each (n=2/PDX), and 100% of mice developed either one or two tumors. The human esophageal adenocarcinoma OE33 cells were implanted into the left and right flanks of three each of R2G2, athymic nude and SCID mice. There was a 100% take rate in R2G2 mice, 0% in SCID mice and 17% in athymic nude mice. The human esophageal adenocarcinoma FLO1 cells were examined in two studies. In both studies, cells were injected into both flanks of R2G2 and SCID mice. Study A also examined growth in athymic nude mice. In study A, no tumor growth was seen in athymic nude or SCID mice, whereas the take rate was 100% in R2G2 mice. In study B, the take rate was 100% in both the R2G2 and the SCID mice, although differences were seen in growth rate. Human gastric adenocarcinoma AGS cells were implanted in both flanks of four each of R2G2 and SCID mice. The take rate was 75% in R2G2 mice and 0% in SCID mice. Head and neck squamous cell carcinoma SQ20b cells were implanted in twenty R2G2 mice and take rate was 90%. Growth of two allogeneic tumor lines was also examined. The mouse colon carcinoma CT26 cells were implanted in ten R2G2 mice and took in 100% of the mice. Mouse B-cell lymphoma A20 cells were implanted in ten R2G2 mice and take rate was 100%. Both allografts grew to 1000mm3 by 13 days post implantation. These data provide evidence that the R2G2 mouse model is a valuable tool for oncology programs including cell line tumor models research, with high take rates and quick growth of allogeneic models.

#1044

Establishment and characterization of rare breast patient-derived xenograft models as a potential resource for personalized medicine.

Hellen Kuasne,1 Paul Savage,1 Constanza Martinez Ramirez,1 Leah Liu,1 Valentina Muñoz-Ramos,1 Virginie Pilon,1 Anie Monast,1 Radia Johnson,1 Nicholas Bertos,1 Jamil Asselah,1 Nathaniel Bouganim,1 Kevin Petrecca,1 Sarkis Meterissian,1 Atilla Omeroglu,1 Mark Basik,2 Morag Park1. 1 _McGill, Montreal, Quebec, Canada;_ 2 _Jewish General Hospital, Montreal, Quebec, Canada_.

Breast cancer (BC) exhibits a wide range of morphologic phenotypes and gene expression profiles. Most of the studies that led to the identification of intrinsic molecular subtypes in BC were limited to invasive ductal carcinomas of the breast and did not take rare histopathologic subtypes into account. Rare histopathologic BC subtypes (collectively less than 2% of all breast cancer) have particular prognostic and clinical characteristics. There is no current established treatment that takes into account the specificity of rare BC subtypes. This is mainly due to the absence of clinical trials to determine the optimal management of these rare pathologies. The establishment of relevant preclinical models and molecular characterization of rare BC subtypes is essential for identifying directed and suitable therapeutic regimens for BC patients diagnosed with these rare histopathologic variants. Patient-derived xenograft (PDX) has been recognized as a valuable method to evaluate the clinical diversity of breast cancer. These models were shown to be predictive of clinical outcomes and are being used for preclinical drug evaluation, biomarker identification, and personalized medicine strategies. We developed a cohort of eight BC rare histopathologic subtypes, including four metaplastic, one adenoid cystic, one IDC pleomorphic, one neuroendocrine, and one mucinous BC subtype. These PDXs and their primary tumors were submitted to whole-genome sequencing (WGS) and RNA sequencing. We also evaluated a total of 255 proteins by reverse phase protein array (RPPA) in these PDX samples. We are currently performing conditional reprogramming experiments to generate cell lines from these rare BC PDXs. Our preliminary results indicate that pathways related to PI3K/AKT, ERK/MAPK, mTOR, HGF, ERBB, AMPK and IL3 signaling are disrupted in rare BC subtypes. Several genes belonging to these pathways are dysregulated in rare BC tumors, and therefore represent potential therapeutic targets for personalized treatment.

#1045

Development of patient derived orthotopic xenografts from metastatic colorectal cancer.

Tiago Goss dos Santos, Bruno Roque, Caroline Roque, Maria Dirlei Begnani, Eduardo Lima, Felipe Coimbra, Rubens Chojniak. _A.C.Camargo Cancer Center, Sao Paulo, Brazil_.

The occurrence of liver metastasis is the major cause of death in colorectal cancer patients. The treatment consists of surgery and chemotherapy. However, the patient outcome is very unfavorable. Thus, the development of new therapeutic regimens to treat this condition is urgent and demand the establishment of good animal models. Among the models that have being used, the patient-derived xenografts (PDX) have emerged as one of the most promising. PDXs are preclinical models that faithfully represent the individuality of human cancer, by grafting fresh human tumor in severely immunodeficient mice which allows for higher tumor implantation rates and growth. Metastatic disease could be also modeled using orthotopic implantation into the affected organ. The patient-derived orthotopic xenografts (PDOX) can be interrogated for different treatment strategies based on the individualized gene signature of the human tumor, and the results can inform clinically relevant treatment strategies for the patient. With the objective to establish a PDOX from metastatic colorectal tumors in athymic nude mice, we implanted fresh tumor fragments into mouse liver parenchyma and propagated tumors from three patients which could be serially implanted in second and third mice generations. The morphological and immunohistochemical characterization indicate that xenografts recapitulate the tumor architecture and the expression of mismatch repair genes (MLH1, MSH2, MSH1, PMS2). After tumor implantation in first passage, the time of tumor growth decrease from 150-250 days to 30-100 days without loss of tumor identity. The growth of a post-transplantation lymphoproliferative disease (PTLD) was observed in one case. This pilot study was successful to establish the institutional PDX preclinical platform to study new therapeutic strategies, biomarkers of disease progression and treatment responsiveness.

#1046

Developing patient-derived xenograft tumor models that recapture clinical manifestation of inflammatory breast cancer patients.

Wenan Qiang,1 Youbin Zhang,2 Demirkan Gursel Gursel,2 Jian-Jun Wei,2 Charles David James,1 Thomas V. O'Halloran,1 Massimo Cristofanilli2. 1 _Northwestern Univ., Evanston, IL;_ 2 _Northwestern Univ., Chicago, IL_.

Inflammatory breast cancer (IBC) is the most aggressive clinical manifestation of breast cancer (BC), with striking erythema of the overlying skin, and exhibiting features of early metastatic and treatment-refractory disease. Malignant cells and infiltrating leukocytes, e.g. tumor-associated microphages (TAMs) commonly designated "M2" macrophages, play a major part in metastasis and treatment resistance. TAMs help promote tumor growth and progression. There is a significant need for preclinical models that retain the characteristics of the original patient tumor for investigating mechanisms of IBC metastasis and for testing candidate therapies.

Six samples of pleural effusion-derived tumor cells or circulating tumor cells (CTCs) from five IBC or metastatic breast cancer (MBC) patients were obtained from Northwestern Memorial Hospital. IBC cells were used for 3D tumor spheroid cultures, and for developing PDX tumor models in immunodeficient mice. Short Tandem Repeat profiling against with original patient tumor DNA was conducted to establish patient DNA identify, and to serve as a reference for authenticating derivative tumor models.

The tumor genomic mutation analysis indicated the most common alterations of five IBC or MBC patients were TP53 (75%), CCND1 (75%), BRCA2 (50%), NF1 (50%), FGFR1 (50%), CDK6 (50%), EGFR (50%), MYC (50%), BRAF (50%). Examination IBC pleural effusion cellular composition indicated ~60% were tumor cells and ~40% were M2 TAMs (CD163+). In early 3D spheroid IBC culture, a symbiotic phenomenon was observed between tumor cells and attached TAMs; Anti-cancer drugs GW2580 and Pexidartinib inhibited 32% and 68% of cell proliferation, respectively, when added to spheroid cultures at 100 nanomolar.

Histopathologic analysis of derivative IBC and MBC PDX revealed highly heterogeneous characteristics and metastatic features characteristic of corresponding patient tumors. Subcutaneous tumors also demonstrated metastasis to liver and lung tissue. NSG mice engrafted with original IBC pleural effusion cells or derived spheroid cultures manifested a variety of inflammatory clinical symptoms. Mice engrafted with original pleural effusion cells developed palpable tumor and manifested most IBC-characteristic including reddish and ulcerated skin lesions necessitating euthanasia within 2 months of cell engraftment. Mice engrafted with > 6 passage or <4 passages of spheroid cultures (lower % of TAMs present) manifested lesser skin inflammation and lesion, and developed tumors of ~ 1 cm within 2-3 months. This may indicate the role of TAMs in the development of IBC skin lesion.

Our results indicate IBC 3D spheroid cultures and PDX tumor models recapitulate clinical characteristics observed in IBC patients, and will prove useful for studying mechanisms of IBC metastasis as well as responsiveness to investigational therapies.

#1047

Advanced Lung Adenocarcinoma Cell Bank (ALACB) : A comprehensive preclinical platform.

Seok Young Kim,1 Dong Hwi Kim,1 Hyeong-Seok Joo,1 Ji Yeon Lee,2 Mi Ran Yun,1 Han Na Kang,1 Byoung Chul Cho,2 Hye Ryun Kim2. 1 _JE-UK Laboratory of Molecular Cancer Therapeutics, Seoul, Republic of Korea;_ 2 _Yonsei University College of Medicine, Seoul, Republic of Korea_.

Purpose: Patients with advanced lung adenocarcinoma often lack large clinical specimens required for molecular testing, and are limited in next therapeutic options. Patient-derived cells (PDC) derived from malignant effusion (ME) of the patient can predict patient drug responses and provide a valuable tool for studying mechanisms of drug resistance. In this study, we created an Advanced Lung Adenocarcinoma Cell Bank (ALACB) consisting of 27 unique PDCs established from patients who progressed on various TKIs including osimertinib.

Experimental Design: ME samples were tested for malignancy, processed, observed by light microscopy, and cultured. The establishment of a PDC was considered complete when it was free of normal cells, maintained known patient tumor mutation, and recapitulated patient's response. PDCs were cryopreserved to maintain a cell bank and analyzed by next-generation sequencing before passage number 25.

Results: A total of 110 malignant effusion samples were obtained from 89 patients with advanced lung adenocarcinoma. Sixty-seven patients had EGFR mutations, 9 with ALK rearrangements, 1 with both EGFR mutation and ALK rearrangement, 7 with ROS1 rearrangements, and 5 with unknown driver oncogenes. 62 (56%) samples were positive for tumor. A high correlation between malignancy and spheroid positivity was observed (47/62; 76%). Most PDCs (26/27) were established from malignancy-positive/spheroid-positive samples, 12 of which were serially established from 5 patients along the clinical course. Of 36 cases that were positive for malignancy but failed in the establishment, 27 (75%) were lost due to a paucity of tumor cells, and 8 (22%) lost due to rapid cell senescence before passage number 10. Establishment of ROS1-positive PDCs showed a higher success rate (53%) than PDCs with EGFR-mutations (21%) or ALK rearrangements (23%). Median passage number of all PDCs was 24, while 3 EGFR-mutant PDCs displayed slow growth rates. Of 15 EGFR-mutant PDCs, 7 were derived from gefitinib-resistant tumors, 1 from afatinib-resistant tumor, and 6 from osimertinib or olmutinib-resistant tumors. Three PDCs harbored rare EGFR mutations including exon 20 insertion, L861Q, or G719X/S768I, respectively. Three ALK-positive PDCs were derived from crizotinib-resistant, alectinib-resistant, or ceritinib-resistant tumor. Of 8 ROS1-positive PDCs, 3 were derived from TKI-naïve tumors, and 5 from crizotinib-resistant tumors. Whole-exome sequencing and RNA sequencing were performed on 13 and 6 cases, respectively.

Conclusions: Cytology positivity and spheroid formation were crucial factors in the successful establishment of ALACB, which reflects genomic profiles and drug response of advanced lung adenocarcinoma. ALACB is a renewable resource for biologic assays, generating in vitro drug-resistant models, and evaluation of novel drugs that will advance lung cancer translational research.

#1048

Preservation of tumor architecture and heterogeneity in long-term cultures of patient-derived explants.

Sofia Abreu,1 Fernanda Silva,2 Sara da Mata,3 Teresa F. Mendes,1 Marta Teixeira,1 Bruno Filipe,3 Sónia Morgado,3 Inês Francisco,3 Marta Mesquita,3 Cristina Albuquerque,3 Paula Chaves,3 Ricardo Fonseca,4 Jacinta Serpa,5 Isadora Rosa,3 Ana Felix,5 Erwin R. Boghaert,6 Vítor E. Santo,7 Catarina Brito1. 1 _iBET, Instituto de Biologia Experimental e Tecnológica & Instituto de Tecnologia Química e Biológica António Xavier - Universidade Nova de Lisboa, Oeiras, Portugal; _2 _CEDOC - Centro de Estudos de Doenças Crónicas, Faculdade de Ciências Médicas, Universidade Nova de Lisboa, Lisboa, Portugal;_ 3 _IPOLFG, Instituto Português de Oncologia Francisco Gentil de Lisboa, Lisboa, Portugal;_ 4 _IPOLFG, Instituto Português de Oncologia Francisco Gentil de Lisboa & FMUL, Faculdade de Medicina da Universidade de Lisboa, Lisboa, Portugal; _5 _CEDOC - Centro de Estudos de Doenças Crónicas, Faculdade de Ciências Médicas, Universidade Nova de Lisboa & IPOLFG, Instituto Português de Oncologia Francisco Gentil de Lisboa, Lisboa, Portugal; _6 _Abbvie, IL;_ 7 _iBET, Instituto de Biologia Experimental e Tecnológica & Instituto de Tecnologia Química e Biológica António Xavier - Universidade Nova de Lisboa,, Oeiras, Portugal_.

The tumor microenvironment plays an important role on tumor drug sensitivity; thus the incorporation of microenvironment features on cancer models is expected to improve their predictive power. Patient-Derived Explants (PDE) have been proposed as potential models; however there are typically short term cultures. Our goal was to improve culture longevity and vitality to evaluate efficacy of repeated drug treatments, taking advantage of dynamic culture systems.

Fresh ovarian and colorectal cancer (OC and CRC, respectively) samples were mechanically dissociated into PDE and cultured in dynamic conditions. The cell population dynamics, cell viability and proliferation were assessed by a panel of readouts, e.g. immunohistochemistry, rezasurin reduction capacity, PDE concentration and morphometric measurements.

To this date, 20 OC and 18 CRC were successfully cultured as PDE, retaining the original tumor architecture and main cellular components: epithelial cells, fibroblasts and immune cells for at least 28 days (OC) or 7 days (CRC). OC samples included all main malignant ovarian carcinoma types (endometrioid, clear cell, mucinous, undifferentiated, serous low and high grade) and CRC samples included adenocarcinomas' stage I to IIIB. Epithelial, stromal and immune cells were maintained for the duration of OC PDE cultures For CRC, epithelial neoplastic cells were preserved, in some cases up to 3 months, and stromal components were progressively lost along culture. The status of the original tumors was preserved for the majority of cases in terms of driver mutations of CRC and of microsatellite stability. To validate the model, OC-PDE cultures were exposed to cyclic chemotherapy treatment (paclitaxel, carboplatin and the combination of both). After two cycles (1 cycle of 24h per week), PDE showed low cellularity and cell viability, compared to untreated controls, reflecting the action of the compounds.

Altogether, we established PDE dynamic cultures in which tumor architecture and heterogeneity is preserved, replicating the original tumor features. Moreover, we demonstrated the feasibility of performing ex vivo drug efficacy studies employing cyclic drug exposure regimens.

This work was partially supported by FCT (iNOVA4Health – UID/Multi/04462/2013, PD/BD/105768/2014 and SFRH/BD/52208/2013).

#1049

Orthotopic implantation of aggressive breast cancer achieves better engraftment and larger tumor in patient-derived xenograft models.

Maiko Okano, Tsutomu Kawaguchi, Kazuaki Takabe. _Roswell Park Cancer Institute, Buffalo, NY_.

Background: Breast cancer patient-derived xenograft (PDX) has come into the limelight to address the issue of intratumoral heterogeneity in preclinical studies; however, there are still some unsolved problems regarding its practical usage in translational research. There are no systematic studies that clarify the advantage of orthotopic implantation of the tumor as opposed to ectopic, or of the type of source tumor. Thus, we investigated the difference of tumor growth caused by these variables among breast cancer PDX model mice.

Methods: We xenografted 10 patient breast cancer tumors into NSG mice. 2 tumors were derived from brain metastasis (B-met), whereas the others were from primary. 3 tumors were ER(+) HER2(-) and 7 tumors were triple negative (TN). Both of b-met tumors are ER(+) HER2(-). Using 2-3 mice per each patient sample, about 1mm3 tumor fragments were implanted surgically into four subcutaneous (SQ) sites ectopically and four sites (bilateral 2nd and 4th mammary fat pad) orthotopically (Orth). Tumors were passaged to another 2-4 mice as the next generation when the largest tumor grew to 1.5cm in diameter. Tumor "take" was defined as tumorigenesis of palpable tumor after implantation regardless of time it took.

Results: PDX tumors were established in 7 out of 10 patient tumors. Histologic grade 3 tumors have significantly higher take rate than grade 2 tumors in 1st generation (3.4% [1/29 site] vs 51.9% [56/108 site], p<0.001) (excluding B-met), which is same as previous reports. The average take time for primary tumor was significantly longer in 1st generation compared to 2nd or 3rd generation (116.7 vs 61.7 days, p<0.001) (excluding B-met), but there is no significant difference between 2nd and 3rd generation. The overall take rate of primary tumor was 55.1% (190/345 site), which was significantly better in Orth compared to SQ (64.9% [120/185] vs 43.8% [70/160], p<0.001). Tumor weights were significantly heavier in Orth compared to SQ (0.65 vs 0.11g, p<0.001). Orth tumors significantly grow faster than SQ tumors (p<0.001), and have significantly more mitotic figures than SQ in H&E staining (19.2 vs 7.9, p<0.01, mean of 10 high power field). Take rates from primary TN and ER-positive tumors were 59.9% (187/312 site) and 3.4% (1/29 site), respectively, but that of ER positive B-met was 82.4% (112/136 site). In B-met, take rate was significantly better in Orth than SQ (94.1%, 64/68 vs 70.6%, 48/68, p=0.001), and tumor weights were significantly heavier in Orth compared to SQ (0.57g vs 0.25g, p<0.001). B-met tumors significantly grow faster than primary tumors (p<0.001).

Conclusions: Orthotopic implantation showed better take rate and greater tumor size and weight than heterotopic implantation, regardless of the cancer subtypes and their sources.

#1050

Efficient derivation and expansion of tumor cell lines from primary and xenotransplanted pancreatic tumors.

David Agorku,1 Anne Langhammer,1 Elisa M. Noll,2 Christian Eisen,2 Silke Schult,1 Andreas Bosio,1 Martin R. Sprick,2 Andreas Trumpp,2 Olaf Hardt1. 1 _MILTENYI BIOTEC GMBH, BERGISCH GLADBACH, Germany;_ 2 _HI-STEM, Heidelberg Institute for Stem Cell Technology and Experimental Medicine, Heidelberg, Germany_.

Cancer cell lines are used to study tumor biology and as models for testing of novel anti-cancer therapeutics. The vast majority of this work has been done using established cell lines that have been cultured for decades. Upon extensive in vitro propagation, cancer cell lines acquire multiple genetic and epigenetic alterations, lose initial heterogeneity present in the parental tumor, and tend to lack tumor initiating as well as multi-lineage differentiation capacity. Consequently, it has been shown that 'classical' tumor cell lines only insufficiently resemble the characteristics of a tumor. In order to generate improved models for cancer research, novel cell lines are derived from primary tissues and used at low passages. However, this process is very inefficient for most tumor entities. In addition, most of the media used include largely undefined serum, such as FBS, which has been shown to drive primary tumor cell cultures to a more differentiated state when used over multiple passages.

We have developed an advanced, serum-free medium for derivation and expansion of tumor cell lines from pancreatic tumors (Noll et al., 2016). This medium has been further optimized concerning formulation, stability, and usability. It allows for efficient generation of primary cell lines from both, patient and xenotransplanted tumors. Primary cell lines derived with this medium closely resembled essential characteristics of the parental tumor, including expression of subtype-specific markers, cellular heterogeneity, as well as genetic and epigenetic signatures. The derived cell lines have been used for genetic engineering and the developed medium allowed for the subsequent expansion of single-cell clones, a pre-requisite for reliable downstream assays. Importantly, culturing with our medium retained the tumorigenic potential as shown by xenotransplantation in immunodeficient mice. The resulting tumors closely and reliably resembled the initial patient tumor. This has been shown on the histomorphological as well as functional level, for example by conservation of tumor specific resistance mechanisms.

Taken together, we have developed a serum-free medium for efficient derivation and expansion of tumor cell lines from primary and xenotransplanted pancreatic tumors, allowing for the establishment of easily accessible in vitro models. Moreover, the cell lines could also be used to generate corresponding xenograft models facilitating the translation of in vitro findings directly into a pre-clinical in vivo setting.

#1051

Mutation detection in patient-derived xenografts by WES and RNAseq.

Wubin Qian, Henry Q.X. Li, Sheng Guo. _Crown Bioscience, Jiangsu, China_.

Patient derived xenograft (PDX) is widely used in preclinical cancer research for drug efficacy evaluation, biomarker discovery and validation, drug mechanism and resistant mechanism study, tumor biology studies and so on. Many PDX libraries are now available to researchers1-4. A good library is composed of PDXs with high-quality characterization for genomics, pharmacology, histology, and clinical information associated with the originating patient tumors. Various technologies are used for genomic profiling of PDX models. For example, RNAseq is used for transcriptomic profiling to obtain data on gene expression, RNA-level mutations, gene fusion; whole exome sequencing (WES) is used to infer DNA-level mutations and gene copy numbers. In this study, we compared mutations inferred by RNAseq and WES for 600 PDX models with both datasets.

We found that about 50% of mutations detected by WES were uncovered by RNAseq. The other half are missing because these mutation alleles were covered by insufficient reads (<5) or the mutations are located in poly-N regions, which make it hard to call mutations. By contrast, 99% of the RNAseq-based mutations were detected by WES. It is not surprising to see such difference. WES amplifies exons at DNA level, and is only affected by experimental variations such as hybridization efficacy. RNAseq yields more reads for genes with higher expression, therefore, mutations in genes with no or low expression cannot be reliably detected. Our results also indicate that there is limited RNA editing. We also examined mutation ratios and found good agreement between WES and RNAseq. Pearson's correlation coefficient is 0.91 and 0.81 between the two mutation ratios when homologous mutations are included and excluded, respectively.

Overall, our study shows that RNAseq data is reliable to infer mutations in highly expressed genes and can be readily used for such when WES data is not available.

References

1. Budinská E, Popovici V. The EurOPDX consortium: Sharing patient tumor-derived xenografts for collaborative multicentric preclinical trials. Molecular cancer therapeutics 2014;12:A8-A

2. Gao H, Korn JM, Ferretti S, Monahan JE, Wang Y, Singh M, et al. High-throughput screening using patient-derived tumor xenografts to predict clinical trial drug response. Nature medicine 2015;21:1318-25

3. Ledford, H. (2016). Us cancer institute to overhaul tumour cell lines. Nature, 530.

4. Guo, S., Qian, W., Cai, J., Zhang, L., Wery, J. P., & Li, Q. (2016). Molecular pathology of patient tumors, patient derived xenografts and cancer cell lines. Cancer Research, 76(16), 4619.

#1052

Identification of signal transduction pathway activity in patient-derived xenograft models in comparison with their originating clinical samples of a variety of human cancer types.

Wim Verhaegh,1 Anja van de Stolpe,1 Nevisa Caushaj,2 Manuel Landesfeind,2 Angelika Zaremba2. 1 _Philips Research, Eindhoven, Netherlands;_ 2 _Charles River, Freiburg, Germany_.

Background. Targeted drugs are directed against cellular signal transduction pathways, such as the PI3K pathway, and efficacy depends on the active pathway in the tumor. Development and clinical application of targeted drugs for personalized cancer treatment require tests which identify pathway activity in cancer cells/tissue. Patient-derived xenograft (PDX) models are becoming the cornerstone of preclinical drug development. Characterization of PDX models and clinical samples for pathway activity is needed for successful preclinical drug testing and patient drug response prediction. Inference of signal transduction pathway activity from cancer genomics information is often unreliable. A novel analysis method has been developed which enables identification and quantification of activity of signal transduction pathways, based on Bayesian reasoning models which infer an activity score from mRNA levels of pathway target genes (Verhaegh et al, Cancer Res 2014:2936-45). For the AR, ER, PI3K, Hedgehog, TGFbeta, Wnt, and NFkB pathways, models have been biologically validated on a number of different cell/tissue types. Methods. For a number of different cancer types (among which colon, breast, lung, ovarian, bladder, osteosarcoma, melanoma, esophagus), AR, ER, PI3K, Hedgehog, TGFbeta, Wnt, and NFkB pathway activity was inferred from Affymetrix HG-U133Plus2.0 microarray data, available from clinical studies (GEO database) and from PDX models (Charles River). Clinical and xenograft pathway activity scores were compared and where possible correlated to genomic datato identify associated pathway driving alterations. Results. Different cancer types were analyzed and compared. Pathway activity patterns for different cancer types in PDX models resemble pathway activity in clinical patient cohorts. Furthermore, known associated cancer mutations are reproduced in PDX models, such as APC mutations in colon PDX and ovary PDX, and an activating beta-catenin mutation in breast PDX, that show an activated Wnt pathway. Conclusion. Signal transduction pathway activity in PDX models resembles pathway activity in patient cohorts with corresponding cancer types and corresponds with known driving mutations. Functional pathway activity testing in PDX models is expected to increase xenograft model utility for (1) further validation of pathway tests for clinical diagnostic use and for (2) preclinical testing of targeted drug efficacy.

#1053

Highly characterized patient-derived PDX breast cancer collection for preclinical efficacy studies.

Michele Melton. _Horizon Discovery, Saint Louis, MO_.

We have acquired a world-class collection of highly characterized patient-derived breast cancer models (PDX) from Washington University, St. Louis, MO (1). We have established growth curves for 17 of these lines, 6 ER+, 2 HER2+ and 4 triple-negative (TNBC) models beyond what was originally published. Continued characterization of these models is ongoing in our lab to ensure the integrity of the models is being maintained over multiple rounds of propagation. Short tandem repeat (STR) analysis, sequencing, quantitative RTPCR and Western blots were run to confirm genotypic and phenotypic fidelity of many of these model. To further characterize these models, RNA samples have been run using NanoString PanCancer gene expression analysis with 770 genes from 13 cancer-associated pathways including: MAPK, STAT, PI3K, RAS, Cell Cycle, Apoptosis, Hedgehog, Wnt, DNA Damage Control, transcription regulation, chromatin modification and TGF-β. Results: These data sets show that we have maintained the integrity of the WHIM PDX models over several passages in mice and provided confidence in the use of these models for preclinical efficacy studies.

Reference: 1. Li et al., Cell Reports 2013;4:1116-30.

#1054

Genomic representation of metastatic breast cancer patients by commercially available cell lines.

Rick Nicoletti,1 Pamela Shaw,2 Naghmeh Salimi,2 Agoston T. Agoston,3 Elin S. Agoston2. 1 _RN Informatics, Boston, MA;_ 2 _Cellaria Biosciences, Cambridge, MA;_ 3 _Brigham and Women's Hospital, Harvard Medical School, Boston, MA_.

We probed the clinicopathologic context of commercially available cell lines using sequencing data from the recently released Metastatic Breast Cancer (MBC) Project. Additionally, we identified cohorts in the MBC with similar profiles to the Wood cell model we recently derived and investigated alignment between clinicopathologic characteristics of these patients and the patient from which the model was derived.

Using a validated cancer panel, we analyzed 592 genes in the Wood cell model detecting mutations and CNAs. We then accessed mutation and CNA data from 1) the MBC Project for 103 primary and metastatic samples from 78 patients and 2) the Cancer Cell Line Encyclopedia (CCLE) database for 56 breast cancer cell lines. We performed unsupervised clustering on the 159 total specimens, incorporating both mutation and copy number data in the clustering analysis by labeling genes either altered or unaltered. MBC patient specimens were grouped based on the clustering results and assessed for trends across multiple clinicopathologic data categories. Specimens representing metastatic sites and the primary site from the same patient were both included in the analysis.

Clustering analysis revealed three major groups representing high, medium and low alteration levels (means of 237, 104 and 29 panel genes altered). The High mutation load group had 34 cell lines and 6 patient samples, the Medium group had 15 cell lines and 71 patent samples, and the Low group had 7 cell lines (including Wood) and 26 patient samples. The 6 patient specimens in the High group represented 5.8% of the MBC data, yet were represented by a majority (60.7%) of CCLE breast cancer lines. All patients in the High group self-reported extraordinary response to treatment; the other two groups had even distribution. None of the specimens in the High group had evidence of DCIS reported in the path report, whereas observed DCIS was distributed equally outside of the High group. Expectedly, markers of aggressiveness of disease were lower in the Low group compared to the rest of the MBC patients, including histologic grade (p=0.00072) and Ki67 percentage (p=0.026). The Low group MBC samples were more likely to be HER2(-) (p=0.02) and ER(+) (p=0.037), consistent the Wood patient. The eight patients clustering most closely with the Wood cell model had low N-stage disease when compared to the rest (p=0.036) which was consistent with the Wood patient N0 status.

CCLE breast cancer cell lines over-represent a small MBC patient subset with a very high load of genomic alterations that also trends toward exceptionally good response to treatment- consistent with the notion that cancer cell lines over-predict drug efficacy. Complete lack of DCIS in the High group is an interesting observation that may describe a disease progression resulting in absent or minimal DCIS precursor. As more MBC patient data are released, these analyses will be repeated to increase statistical power.

#1055

Lymphotoxin-alpha plays key roles in lymphoproliferative lesions arising in patient-derived cancer xenografts.

Tsuyoshi Chijiwa,1 Daisuke Komura,2 Mizuha Haraguchi,3 Haruo Hashimoto,4 Hiroshi Suemizu,4 Makoto Katayama,5 Yoshiyasu Nakamura,6 Daisuke Furukawa,7 Takayuki Isagawa,2 Hiroto Katoh,2 Takashi Moriya,1 Shumpei Ishikawa,2 Yohei Miyagi,6 Masato Nakamura7. 1 _Saitama Medical Center, Jichi Medical University, Saitama, Japan;_ 2 _Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan;_ 3 _Nihon Koukan Hospital, Tokyo, Japan;_ 4 _Central Institute for Experimental Animals, Kawasaki, Japan;_ 5 _Kawasaki Municipal Hospital, Kawasaki, Japan;_ 6 _Research Institute, Kanagawa Cancer Center, Yokohama, Japan;_ 7 _Tokai University School of Medicine, Tokyo, Japan_.

Patient-derived xenograft (PDX) mouse models from engrafting fresh surgical specimens directly onto immunodeficient mice have enabled the development of more relevant in vivo models for human cancers. PDX models retain similar morphology, heterogeneities, and molecular signatures with the original cancers. Moreover, PDXs with clinical information, termed "cancer xenopatients", are remarkable systems in personalized medicine for cancer. During the establishment process, a transplantable lymphoproliferative lesion (LPL) that replaces the original tumor cells sometimes occurs in severely immunodeficient mice. Although the occurrence of LPL is the most problematic aspect of the establishment of PDX, its mechanisms are unknown. We previously reported that the LPL phenomenon resembled the graft-versus-host disease (GVHD)-like mechanism and was suppressed by anti-GVHD agents. In the present study, we genomically analyzed the detailed mechanism of LPL in PDX mouse models. In our previous study, we established three LPL-PDX/NOG lines. These LPL-PDX lines were re-transplanted onto NOG mice and the xenograft tumors were then harvested for study. Genome sequencing and comprehensive analyses of tumor-stroma interactions (CAncer-STromal INteractome analysis, CASTIN) were performed on mRNA of these LPL-PDX/NOGs. Immunohistochemistry demonstrated that LPL tissues consisted of B-cells, T-cells, and differentiated plasma cells. CASTIN revealed tumor-stroma interactions in PDX/NOG comprehensively and quantitatively at the gene expression level by distinguishing gene arrangements in human tissue (cancer or LPL) from mouse tissue (stroma) by signal strength and dependency. Some strong interactions were found for these LPL-PDX/NOGs; VEGFB-FLT1, LTA-LTBR, LTA-TNFRSF1A, IL13-IL4R, IL13-IL13RA1, and SEMA4F-NRP2. Suppression of LPL-PDX/NOG by the anti-GVHD agent FK506 (1.0 mg/kg/day, 4 weeks) decreased interactions of LTA-LTBR and LTA-TNFRSF1A. Based on these results, LPLs during establishment of PDX occur due to a lymphotoxin-alpha-related mechanism. Controlling lymphotoxin-alpha related interactions may improve the establishment rate of PDX in severely immunodeficient mice.

#1056

Cancer avatars are sensitive diagnosticians of the pleural space.

Gwendolyn B. Kuzmishin, Priyanka Gopal, Mohamed E. Abazeed. _The Cleveland Clinic, Cleveland, OH_.

The identification of malignant cells in pleural fluid has critical prognostic and therapeutic implications but is frequently a diagnostic challenge. The first step in determining the cause of a suspected malignant pleural effusion, thoracentesis, has an unsatisfying low sensitivity (~60-70%). Patient-derived xenografts (PDX) retain the principal characteristics of the tumor of origin. However, there has been little progress in the application of these models to guide diagnostic and staging strategies. We assessed the feasibility, cellular yield and comparative diagnostic accuracy of pleural-derived xenografts in patients with effusions.

An institutional review board-approved single-institution prospective registry of patients was used to identify patients undergoing diagnostic and/or therapeutic thoracentesis. Cells isolated from the pleural fluid of these patients were injected into NSG mice. A diagnosis of the cancer in the xenograft was confirmed by a staff pathologist with expertise in the correspondent cancer type.

All of the cases that were clinically positive for tumor cells in the pleural space demonstrated tumor engraftment. Samples taken from patients with known benign etiologies did not result in tumor engraftment. The developed xenografts provided ample tissue that can be made available for extensive molecular testing, even in cases where the cytological samples consisted of very few cells. Critically, two of 10 cases resulted in engraftment of the tumor despite no evidence of malignant cells on cytological examination, indicating a 20% improvement in the statistical power of pleural-based xenografting for the detection of malignancy.

Our study indicates that pleural-derived xenografts can enhance the sensitivity of malignant cell detection, reflecting the inherent limitations of cytological analyses of a few malignant cells. Moreover, the amplification of cellular material provides ample source material that can be used to satisfy the increasing demands for tissue-based molecular testing. To our knowledge, this is the first effort to effectively use patient-derived xenografts for the purpose of cancer staging and diagnosis.

#1057

Characterization and functional consequences of neuronal invasion in oral cancer.

Nicole N. Scheff, Richard Klares, Brian L. Schmidt. _New York University, New York, NY_.

Most oral cancer patients suffer severe, chronic pain promoted by oral cancer-induced changes in sensory nerves innervating the oral cavity. However, the impact of sensory neurons on carcinogenesis beyond nociceptive signaling is an understudied problem in oral cancer. We hypothesize that oral cancer and sensory neurons interact, such that, oral cancer induces sensitization, sprouting and plasticity in neurons, and efferent neuronal activity promotes oral carcinogenesis. Using an oral cancer subcutaneous xenograft model, we tested the impact of peripheral nerve presence on squamous cell carcinoma tumor growth in the mouse hindpaw. Mice that received a sciatic nerve axotomy one week prior to HSC-3 oral cancer cell inoculation developed significantly smaller tumors (28.23 ± 6.6 % of total paw area) compared to mice that received sham surgery (50.71 ± 2.48 %). These data suggest that peripheral neuron innervation supports oral cancer growth. Additionally, neuropeptides are released from primary afferent neurons and suppression of the immune response has been linked to peripheral neurotransmission. Using flow cytometry, we tested whether inhibition of peripheral sensory neurons impacted inflammation in response to oral cancer. Temporary nerve block with 1% bupivacaine injection into the lingual nerve prior to injection of supernatant from HSC-3 oral cancer cells resulted in significantly more CD45+ immune cell infiltration (21.66 ± 3.4 %) into the tongue compared to HSC-3 supernatant with intact nerve function (9.10 ± 1.2 %). These results suggest neuron-mediated suppression of cancer-evoked inflammation and support the premise that neurons and neuronal activity modulates the oral cancer microenvironment. High-resolution imaging of neuronal innervation into tongue cancer and its remodeling in response to pathological changes is challenging due to dense neuronal innervation in the tongue. We utilized a recently developed optical clearing protocol (CLARITY) that preserves protein integrity and three-dimensional structure of tissue and high-resolution microscopy to visualize and characterize sensory neuron innervation in thick 1 mm tongue sections from an orthotopic xenograft mouse model of oral cancer. In a preliminary study of neuronal innervation in a tongue HSC-3 xenograft model, we observed that PGP9.5+ neuronal fiber density was greater in the cancer environment and diminished with distance from the cancer. These data suggest that oral cancer induces plasticity and sprouting of innervating peripheral neurons. A better understanding of the nerve-cancer interaction has potential for development of new therapeutic strategies to treat oral cancer and oral cancer pain by targeting sensory neurons.

### Biomarkers of Metastasis

#1058

CRLF1 promotes proliferation and metastasis in papillary thyroid carcinoma via activating MAPK/ERK and PI3K/AKT pathway.

QIAN ZHONG,1 Shitong Yu,2 Xiaoming Huang,2 Musheng Zeng1. 1 _Sun Yat-sen University Cancer Center, Guangzhou, China;_ 2 _Sun Yat-sen Memorial Hospital, Guangzhou, China_.

Papillary thyroid cancer (PTC) is the most common cancer in endocrine system which accounts for the majority component of thyroid cancer during the past decades. PTCs usually are indolent and curable with 5-year survival rate over 95%. However, some of PTCs will dedifferentiate and become aggressive with a poor prognosis. Current established biomarkers may not fully explain the mechanisms of the development of PTC. Therefore, it is still urgent to find novel biomarkers of PTC to better predict patients' prognosis and lead to the personalized therapies for PTC patients. By analyzing The Cancer Genome Altas (TCGA) database, we identified Cytokine Receptor Like Factor-1(CRLF1) as a potential oncogene. We identified CRLF1 is highly expressed in papillary thyroid carcinoma (PTC) compared with normal thyroid tissues both at mRNA and protein level. High CRLF1 level was associated with aggressive clinicopathological features and worse disease-free survival rate. By loss-of-function and gain-of-function assays, we found that CRLF1 not only boosts capacity of cell migration and invasion in vitro, but promotes tumor growth both in vitro and in vivo. Besides, CRLF1 can induce epithelial-mesenchymal transitions. Overexpressing CRLF1 could activate ERK1/2 and AKT pathways. The oncogenic effects induced by CRLF1 were suppressed by treating the cells with the MEK inhibitor U0126 or the AKT inhibitor MK-2206. These results suggest an oncogenic role CRLF1 plays in PTC tumorigenesis by regulating MAPK/ERK and PI3K/AKT signaling pathways. These results suggest that CRLF1 is a potential biomarker in PTC patients and it may be a valuable therapeutic target for PTC in the future.

#1059

EpCAM differentially expressed on circulating and disseminated tumor cells promotes tumor metastasis in breast cancer patients.

Peter P. Lin,1 Junjian Li,2 Daisy Wang,1 Olivier Gires,3 Hongxia Wang2. 1 _Cytelligen, Inc., San Diego, CA;_ 2 _Shanghai General Hospital, Shanghai, China;_ 3 _Ludwig Maximilians University, Munich, Germany_.

To investigate the role of EpCAM expressed on both circulating tumor cells (CTCs) in peripheral blood and disseminated tumor cells (DTCs) in bone marrow with respect to tumor metastases, the integrated subtraction enrichment and immunostaining-FISH (SE-iFISH) was performed to quantify, phenotypically and karyotypically characterize cytogenetically abnormal CTCs and DTCs expressing EpCAM in breast cancer patients. In a cohort of 34 recruited late stage patients, consisting of 32 metastatic and 2 non-metastatic subjects, the average number of detected CTCs in metastatic vs non-metastatic patients was 25 and 18 cells, respectively. Among those CTCs in metastatic subjects, 23.4% of them showed EpCAM+, whereas none of EpCAM+ CTCs was detected in non-metastatic patients. There were total of 49 CTC clusters detected in metastatic patients, and 33% of those clusters were EpCAM+, however, only 3 CTC clusters were found in non-metastatic patients, and none of them was EpCAM+. In the case of DTCs, the average number of 2224 and 364 DTCs per subject was detected in metastatic vs non-metastatic subjects, with EpCAM+ ratio of 66.53% in metastatic and 8% in non-metastatic patients. Analysis of DTC clusters indicated that the average of 226 and 21 DTC clusters/subject were identified in metastatic and non-metastatic patients, respectively, and metastatic subjects showed 62% of DTC clusters EpCAM+, whereas 37% of clusters were EpCAM+ in non-metastatic patients. Comparing to the breast cancer serum biomarker CA15-3 detected in 32 metastatic cancer patients, showing positivity (≥28 ng/ml) of only 41% (13 out of 32), both CTCs (≥5) and DTCs (≥5) had a positivity rate of 81 (26 out of 32) and 97% (31 out of 32), respectively. Obtained results indicated that EpCAM+ CTCs and DTCs are reliable markers in terms of evaluating therapeutic efficacy and predicting cancer patients' prognosis.

#1060

Longitudinal profiling of plasma derived extracellular vesicles (EVs) from women presenting with metastatic triple-negative breast cancer (mTNBC) informs on metastatic location and treatment outcome.

Sinéad Lindsay,1 Jennifer McIntyre,2 Luke Gubbins,1 Martina Smith,3 Laura Kerr,4 Emma Kavanagh,1 Michaela J. Higgins,3 Bryan Hennelly,4 Paul Dowling,5 Hugh Byrne,2 Amanda McCann6. 1 _Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Dublin, Ireland;_ 2 _FOCAS Research Institute, Dublin Institute of Technology, Dublin, Ireland;_ 3 _Mater Misericordiae University Hospital, Dublin, Ireland;_ 4 _Department of Electronic Engineering, National University of Ireland Maynooth, Maynooth, Ireland;_ 5 _Biology Department, National University of Ireland Maynooth, Maynooth, Ireland;_ 6 _School of Medicine, Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Dublin, Ireland_.

Background: Women presenting with Triple Negative Breast Cancer (TNBC) have an increased risk of recurring at distant visceral metastatic sites such as brain, lungs and liver rather than bone compared to oestrogen-receptor-positive breast cancer. (Rakha et al., 2011, Wood 2004). The preferential homing of TNBC cancer cells to these sites is not clearly understood, but is increasingly being attributed to the ability of extracellular vesicles (EVs) released by chemoresistant cancer cells to circulate to distant sites creating metastatic niches and paving the way for organ-specific metastases.

Hypothesis: We hypothesise that the physical EV properties, macromolecular and proteomic content of these EVs could potentially determine the risk of metastasis and align with these "preferred" metastatic locations.

Methodology: This project uses nanoparticle tracking analysis (NTA), infra-red and mass spectroscopy to generate unique patient-specific EV longitudinal signatures of women diagnosed with organ defined metastatic TNBC. These signatures hold potential as "unique identifiers" of clinically documented organ-specific metastases. All profiles generated are compared longitudinally to the patient's initial baseline sample and also to age matched female control plasma samples.

Results and Conclusions: Preliminary results have developed SOPs to (i) successfully enrich for EVs from patient plasma samples and (ii) employ nanoparticle tracking analysis, infra-red and mass spectroscopic profiling to develop unique longitudinal patient specific EV signatures. The longitudinal profiling of EV signatures from patients with metastatic TNBC will potentially (a) predict response to therapy, (b) uniquely identify risk of and potential location of metastatic site/sites and (c) inform on patient outcome. Following this multidisciplinary profiling of our mTNBC, our remit is to recapitulate this profiling into the primary TNBC setting to identify potential sites of organ specific metastases ahead of any detection on an x-ray or scan, thereby informing on clinical management.

#1061

Advanced quantitative mass-spectrometry-based SILAC proteome profiling of Y-box binding protein-1 (YB-1) overexpressing breast cancer cells unravels proteins involved in metastasis.

Jia Pei Lim,1 Sunitha Nair,2 Sukanya Shyamasundar,1 Jayantha Gunaratne,2 Boon Huat Bay1. 1 _National University of Singapore, Singapore;_ 2 _Institute of Molecular and Cell Biology, Agency for Science, Technology and Research, Singapore_.

Metastasis is responsible for the majority of cancer related deaths and remains a global medical challenge in cancer treatment. The Y-box binding protein-1 (YB-1), a DNA and RNA binding protein, is known to regulate a plethora of fundamental cellular processes, including cell proliferation, chemoresistance and DNA repair. There is also cumulative evidence that YB-1 enhances breast cancer spread although the exact mechanism of YB-1-mediated metastasis is still not clear. Hence, we aim to determine the role of YB-1 overexpression in breast cancer progression. We first stably overexpressed YB-1 in non-aggressive MCF7 breast cancer cells (MCF7-YB-1) using a turboGFP tagged plasmid and performed phenotypic assays. We observed a significant increase in cell migration in MCF7-YB-1 in the Boyden chamber migration assay, which was substantiated by performing live imaging of the cells in a wound healing assay. Furthermore, a significant decrease in cell proliferation with G1 phase arrest was observed by the alamarBlue assay and flow cytometry analysis of propidium iodide staining, respectively for MCF7-YB-1 cells. We then performed a whole proteome analysis by stable isotope labelling with amino acids in cell culture (SILAC), a powerful proteomics platform. The whole proteome analysis identified 122 upregulated proteins and 120 downregulated proteins in the MCF-7-YB-1 cells. Bioinformatics analysis of the differentially regulated proteins using the Metacore software revealed the involvement of proteins that promote cell migration and invasion, such as those belonging to the S100 family which are enriched in the extracellular vesicles and exosome compartment, providing some mechanistic insights into the metastasis promoting role of YB-1. Taken together, our results showed that YB-1 could enhance the migratory potential of breast cancer cells, an important step in the metastatic cascade and therefore warrants a more in-depth study.

Acknowledgement: Jia Pei Lim is a recipient of Ong Hin Tiang Scholarship in Cancer Research. This research was supported by Ministry of Education Grant (MOE2013-T2-1-129).

#1062

Downregulation of miR-200c stabilizes XIAP mRNA and contributes to invasion and lung metastasis of bladder cancer.

Zhongxian Tian, Honglei Jin, Jingxia Li, Chao Huang, Chuanshu Huang. _Nelson Institute of Environmental Medicine, Department of Environmental Medicine and Urology, Tuxedo Park, NY_.

Our previous studies have demonstrated that X-linked inhibitor of apoptosis protein (XIAP) plays a regulatory role in cancer invasion via mediation of RhoGDIα protein SUMOylation at Ser138 in colorectal cancer HCT116 cells. However, the alteration/association of XIAP in invasion, progression, and lung metastasis of bladder cancer (BC) as well as the molecular mechanisms leading to this XIAP alteration during BC development have not been explored yet. Herein, we found that XIAP was overexpressed in human high grade BCs, high metastatic human BC T24T cells, and in mouse invasive BCs. Mechanistic studies indicated that XIAP overexpression in the highly metastatic T24T cells was due to increased mRNA stability of XIAP in comparison to the parental non-metastatic T24 cells. Results from XIAP mRNA stability regulation revealed that miR-200c was profoundly downregulated in T24T cells and in highly invasive BCs, and that such miR-200c downregulation was mediated by CREB inactivation. Furthermore, miR-200c downregulation reduced its binding to the 3'-UTR region of XIAP mRNA, and resulted in its mRNA stabilization, protein overexpression, and invasive capabilities in T24T cells. Moreover, our results from XIAP-specific shRNA and miR-200c ectopic expression showed that suppression of XIAP profoundly attenuated lung metastasis of T24T cells in nude mice. In conclusion, our findings demonstrate the molecular basis leading to XIAP overexpression and the crucial role of XIAP in BC invasion in vitro and lung metastasis in nude mice.

#1063

A clear cell renal cancer metastasis model identifies novel mediators of tumor aggressiveness and predictors of patient survival.

Felix Geist,1 Teresa Dolt,1 Thomas Höfner,2 Corinna Klein,3 Vanessa Vogel,3 Albrecht Stenzinger,4 Tim Holland-Letz,1 Ornella Kossi,1 Wilko Weichert,5 Peter Schirmacher,4 Sascha Pahernik,4 Markus Hohenfellner,4 Andreas Trumpp,1 Martin Sprick3. 1 _German Cancer Research Center (DKFZ), Heidelberg, Germany;_ 2 _Mainz University Medical Center, Johannes Gutenberg University, Mainz, Germany;_ 3 _Heidelberg Institute for Stem Cell Technology and Experimental Medicine (HI-STEM), Heidelberg, Germany;_ 4 _University Heidelberg, Heidelberg, Germany;_ 5 _TUM School of Medicine, Technical University of Munich, München, Germany_.

Clear cell renal cell carcinoma (ccRCC) is the most common subtype of renal cell carcinoma with a 5-year survival rate of only 10.5%, and chemotherapy and radiotherapy regimen have shown limited efficacy. On the molecular level, significant inter- and intra-tumor heterogeneity hamper identification of drug targets, biomarkers and unraveling of disease mechanisms.

We developed a novel patient-derived model system of ccRCC that recapitulates the heterogeneity of the originating cancer enabling us to study ccRCC on a functional level. In five rounds and in three biological replicates of an in vivo selection, we transplanted the metastases of orthotopically transplanted tumor cells into the renal capsules of NOD scid gamma (NSG) mice. The tumor was enriched for cells with higher growth and metastatic potential compared to the initial heterogeneous population. Comparative gene-expression analysis revealed candidate genes associated with enhanced malignant growth and metastasis. Absolute shrinkage and selection operator (LASSO) regression identified a gene signature that can robustly predict patient survival. The prognostic power of our signature was additionally verified in independent patient cohorts suggesting that this approach leverages efficient stratification of patients into distinctive risk groups.

One of the hallmark genes in this signature is known to alter cellular signaling properties. Therefore, we hypothesized that this gene contributes to tumor growth and metastasis and thus to aggressiveness of ccRCC. In fact, in knockdown and overexpression xenografts experiments we could confirm an essential role for tumor aggressiveness in vivo suggesting that the gene and associated downstream signaling pathways are attractive targets for treatment of clear cell renal cancer.

#1064

Evaluation of ex vivo and in vivo biomarkers in different stages of prostatic cancer.

Gerit Theil, Carlotta Schietinger. _Martin Luther Univ. of Halle-Wittenberg, Halle/Saale, Germany_.

Clinical factors such as PSA and pathological factors such as Gleason grading and TNM staging are well known as prognostic markers in prostatic cancer. These tools are often insufficient for an accurate risk stratification of each patient. It is therefore essential for therapy monitoring to provide qualifed surrogate biomarkers for survival, the ability to characterize the changing biology of the tumor at the patients level, and qualified predictive biomarkers which can guide treatment selection based profiles derived from characterization. The detection of circulating tumor cells in the blood of patients with prostate cancer might therefore, in addition to their prognostic value, serve as liquid biopsy, complementing or replacing PSA determination in predicting and monitoring the response to different therapies. Here, we present data of a pilot study comparing different biomarkers. CTCs are the in vivo biomarker isolated by the CellCollector, which enables for the vivo isolation of CTCs from the peripheral blood. The multiplex assay was used for the determination of the ex vivo biomarkers the following analytes HER-2, IL-6, IL-8, Leptin, uPA and uPAR were measured in serum samples. We enrolled 34 PCa patients, 21 with localized (PCa-l) and 13 with metastasized prostate cancer (PCa-m). At multiple time points of treatment, CTCs and a Luminex assay were determined. The CellCollector was inserted in a cubital vein for 30 minutes. The interaction of target CTCs with the CellCollector was mediated by an antibody to the epithelial cell adhesion molecule The Luminex Assay is a bead-based principle which requires a small sample volume (≤ 50 µl) for the determination. The number of in vivo captured CTC in PCa-m patients varied from 0-820, a mean of 17.9 CTCs, median 5 and in PCa-l patients from 0-8 CTCs, a mean of 1.6 CTCs, median 0. The CTC count, the PSA level and the Gleason grading were significant different in our two groups. Apart from the leptin level showed levels of HER-2, IL-6, IL 8, uPA, uPAR no significant differences appeared in our groups. Interestingly, significant correlations between PSA and uPAR; HER-2 ; IL-8 ; uPA r; Leptin; IL-6 r= in metastasized prostate cancer patients were demponstrated. Unfortunately, the CTC numbers showed and included no correlation between the ex vivo Biomarkers. The overall survival for metastasized patients with < 5 CTCs was significant better than for patients with ≥ 5 CTCs. Some personalized marker profiles of the patients show a correlation between the different ex vivo Biomarker and in vivo biomarkers. The in vivo captured CTCs seem to be a useful prognostic marker because of the significant better survival of patients with <5 CTCs. Although there was no general validity, single patient analyses showed several, partly significant correlations between CTC count, PSA level and ex-vivo parameters. So these markers could be useful in individual risk stratification and therapy option decision-making.

#1065

IL-10 as an indicator of metastatic potential in colorectal carcinoma.

Michelle H. Townsend, Taylor P. Cox, Abigail M. Felsted, Steven R. Piccolo, Richard A. Robison, Kim L. O'Neill. _Brigham Young University, Provo, UT_.

The purpose of this study was to evaluate the expression of TGF-β and IL-10 in primary and metastatic tumors to determine the contribution that these cytokines may have to metastasis. Interleukin-10 (IL-10) is an anti-inflammatory cytokine that inhibits antigen-presenting cells and the secretion of pro-inflammatory cytokines. Because tumors typically thrive in a pro-inflammatory microenvironment, the anti-inflammatory properties of IL-10 have been shown to decrease the metastatic ability of tumors. IL-10 has also been shown to suppress the anti-tumor immune response, thereby increasing metastatic potential. These conflicting conclusions led us to investigate the possible correlation IL-10 expression may have on the metastatic capabilities of colorectal cancer. TGF-β was also chosen for this study because of the observed connection between cytokine expression and metastasis. In order to evaluate cytokine expression, we utilized standard immunohistochemistry procedures. Human tissues were stained with antibodies against TGF-β and IL-10, along with GAPDH as a positive control and a universal negative isotype control. We tested colon adenocarcinoma (n=30), metastatic colon adenocarcinoma (n=30), cancer adjacent normal tissue (n=20), normal colon tissue (n=10), and tubular adenoma (n=10). Using ImageJ software, samples were analyzed by converting tissue images to a grayscale and then assigning a threshold to ensure analysis was not affected by negative space within the tissue. Gray intensity values above 115 were determined to be 'negative' for expression, while samples with gray intensity values below 115 were 'positive' for expression. Our results show that IL-10 expression was significantly higher in patients with metastatic colon cancer when compared to patients with non-metastatic colon cancer, suggesting a connection between the over expression of IL-10 and metastatic capability. In colon adenocarcinoma samples, 20% of patients expressed high levels of IL-10 (average gray intensity = 111.46), while 80% of patients expressed low levels of IL-10 (average gray intensity = 125.51). In metastatic colon adenocarcinoma samples the proportion of high cytokine expression was higher, with 53% of patients expressing high levels of IL-10 and 47% of patients expressing low levels of IL-10. Additionally, IL-10 levels were significantly greater (p<.0001) in patients with metastatic colorectal carcinoma, with an average gray intensity of 106.42 in Metastatic IL-10 High patients and 119.95 in Metastatic IL-10 Low patients. TGF-β expression appeared to remain consistent between normal and cancerous tissues, showing no significant variation between primary tumor and metastatic tissue samples. We have concluded that while TGF-β expression showed little variability between primary and metastatic tumors, IL-10 may be an effective biomarker for characterizing tumors and determining their metastatic potential.

#1066

L1CAM as a pivotal mediator for disease progression and prognostic biomarker in gastric cancer patients.

Yoshinaga Okugawa, Yuji Toiyama, Yasuhiko Mohri, Akira Yamamoto, Tsunehiko Shigemori, Chengzeng Yin, Aya Narumi, Shozo Ide, Takahito Kitajima, Hiromi Yasuda, Hiroyuki Fujikawa, Junichiro Hiro, Shigeyuki Yoshiyama, Masaki Ohi, Koji Tanaka, Toshimitsu Araki, Masato Kusunoki. _Mie University, Tsu, Mie, Japan_.

Background/Aims: Although significant progress has been made in the diagnosis and treatment of Gastric cancer (GC) patients, GC is the second leading cause of cancer-related deaths worldwide. Identification of critical factors driving the progression of metastasis and new prognostic biomarkers are urgently required to improve both early detection of recurrence and prognosis of patients with advanced GC. L1 cell adhesion molecule (L1CAM) has been observed to be highly expressed in several types of tumors, which may play a vital role in tumor progression. The present study investigated the clinical significance of L1CAM, in the development and progression of gastric cancer.

Experimental Design: At first, the effects of L1CAM alteration were investigated using proliferation, migration, invasion, and anoikis and xenograft model. Next, we analyzed L1CAM expression in surgical tissue speciemens from 131 gastric cancer patients (Cohort 1) by quantitative real-time reverse transcription-PCR and 309 patients (Cohort 2) by immunohistochemistry.

Results: Knockdown of L1CAM using small interfering RNA in GC cell lines resulted in reduced cell proliferation, invasion, migration, and anoikis in gastric cancer cells. Furthermore, Knockdown of L1CAM inhibited metastasis in a xenograft model. Interestingly, L1CAM expression in cancer tissues was significantly higher in patients with lymph node metastasis and distant metastasis. The expression of L1CAM was significantly associated with poor prognosis. Furthermore, L1CAM emerged as an independent prognostic predictor and a predictor of lymph node metastasis in GC patients.

Conclusions: Our data highlight that L1CAM appear to play an important role in disease development and L1CAM expression may serve as a novel predictor for the identification of patients who are at high risk for tumor recurrence and strict surveillance in curative GC patients.

#1067

Tenascin C in colorectal cancer stroma is a predictive marker for liver metastasis and is a potent target of miR-198 as identified by microRNA analysis.

Tomohiro Murakami, Hirotoshi Kikuchi, Hisahito Ishimatsu, Sanshiro Kawata, Amane Hirotsu, Tomohiro Matsumoto, Yusuke Ozaki, Yoshihiro Hiramatsu, Kinji Kamiya, Megumi Baba, Satoshi Baba, Masatoshi Kitagawa, Takanori Sakaguchi, Hiroyuki Konno, Hiroya Takeuchi. _Hamamatsu Univ. School of Medicine, Hamamatsu, Japan_.

Background: Liver metastasis in colorectal cancer (CRC) is still one of the major determinants of survival. Tumor stroma has important roles in the development of colorectal cancer (CRC) metastasis. MicroRNAs (miRNAs) are known to regulate the expression of genes involved in many physiological conditions, including cancer. Previous studies reported important roles of miRNAs in cancer cells in CRC development and metastasis. However, only few studies have focused on miRNA expression in CRC stroma. In this study, we revealed the miRNA expression profiles in tumor stroma by miRNA array analysis using Laser captured microdissection (LCM) to identify miRNAs and their target genes as novel molecular markers involved in CRC liver metastasis.

Methods: Tumor stroma was isolated from formalin-fixed, paraffin-embedded tissues of primary CRCs with or without liver metastasis by LCM, and miRNA expression was analyzed using TaqMan miRNA arrays.

Results: Hierarchical clustering classified 16 CRCs into two groups according to the existence of synchronous liver metastasis. Combinatory target prediction identified tenascin C (TNC) as a predicted target of miR-198, one of the top 10 miRNAs downregulated in tumor stroma of CRCs with synchronous liver metastasis. Immunohistochemical analysis of TNC in 139 primary CRCs revealed that a high staining intensity was correlated with synchronous liver metastasis (P<0.001). TNC staining intensities in the tumor stroma of 83 CRC liver metastases revealed a higher staining in liver metastases than in primary CRCs independently of preoperative chemotherapy. Univariate and multivariate analyses revealed that the TNC staining intensity was an independent prognostic factor to predict postoperative overall survival (P=0.005; n=139) and liver metastasis-free survival (P=0.001; n=128). To evaluate the biological link between miR-198 and TNC, we first checked expression levels of miR-198 in the 16 samples in miRNA array data, and compared them with TNC staining intensities. The CRC samples expressing miR-198 tended to have lower levels of TNC expression in comparison with those without miR-198 expression, although the difference was not statistically significant (P=0.237). Next, we transfected a miR-198 mimic or a miR-198 inhibitor into SW620 human colon cancer cells and CCD-18Co human colon fibroblast cells and analyzed the expression change of TNC protein by western blotting. The relative protein expression of TNC was suppressed by treatment with the miR-198 mimic and increased by miR-198 inhibitor.

Conclusions: Alterations of miRNAs in CRC stroma appear to form a metastasis-permissive environment that can elevate TNC to promote liver metastasis. TNC in primary CRC stroma has the potential to be a novel biomarker to predict postoperative prognosis.

#1068

LAMB3 mediates metastatic tumor behavior in papillary thyroid cancer by regulating c-MET/Akt signals.

Jae Won Chang, Bon Seok Koo. _Chungnam National Univ. College of Medicine, Daejeon, Republic of Korea_.

Laminin subunit beta-3 (LAMB3) encodes one of the three subunits of LM-332, a protein of the extracellular matrix secreted by cultured human keratinocytes. While LAMB3 is involved in the invasive and metastatic abilities of several tumor types, including those found in the colon, pancreas, lung, cervix, stomach, and prostate, its mechanism of action in thyroid cancer has not been investigated previously. Our results show that LAMB3 is upregulated in papillary thyroid cancer, and that its suppression reduces cell migration/invasion via downregulation of epithelial‒mesenchymal transition-associated proteins (N-cadherin, vimentin, slug) and inhibition of matrix metalloproteinase 9. LAMB3 suppression also significantly decreases Akt phosphorylation and inhibits the transcription of c-MET, reducing its activation. These results suggest that LAMB3 leads to tumor invasion via Akt activation induced by the HGF/c-MET axis in papillary thyroid cancer cells. Our findings reveal a novel mechanism of action for LAMB3 in papillary thyroid cancer cells.

#1069

Cxcl12 is involved in liver metastasis of intrahepatic cholangiocarcinoma.

Tatsunori Miyata,1 Yo-ichi Yamashita,1 Tomoharu Yoshizumi,2 Masayuki Shiraishi,2 Masayuki Ota,2 Susumu Eguchi,2 Shinichi Aishima,3 Hideo Baba,1 Hikaru Fujioka2. 1 _Department of Gastroenterological Surgery, Kumamoto University, Kumamoto city, Japan;_ 2 _Kyushu Liver Surgery Study Group, Japan;_ 3 _Department of Diagnostic Pathology, Saga University, Japan_.

Background: Recurrences often happen in patients with intrahepatic cholangiocarcinoma (ICC) after radical hepatectomies. In addition, ICC has a poor sensitivity for chemotherapy. Most of the recurrence in ICC is liver metastasis, and how to control liver metastasis should be important to improve patients' prognosis.

Purpose and Methods: Samples of ICC (primary and metastatic lesions) were collected as a multicenter study of the Kyushu Liver Surgery Study Group. cDNA microarray was performed using five frozen samples of ICC primary and metastatic sites; primary A - liver metastasis A1, re-liver metastasis A2, and primary B - liver metastasis B. Protein expression of the five high or low expression genes was evaluated by immunohistochemical staining using 25 primary ICC and 33 metastatic lesions; 22 cases of liver, 7 cases of lymph node, 2 cases of lung, 1 case of bone, and 1 case of adrenal gland. In in vitro experiments, ICC cell lines of the epithelial type (HuCCT-1, RBE) and the spindle type (SSP-25, HuH-28) were used.

Results: Using cDNA microarray, genes of high expression (KRT83, CXCL12) and low expression (REG3G, OLFM4) were extracted in the metastatic site as compared with the primary tumor, evaluated by the sum of three pairs: primary A - liver metastasis A1, primary A - liver metastasis A2, and primary B - liver metastasis B. In addition, as a result of analyzing the results of the cDNA microarray with gene set enrichment analysis (GSEA), it was associated with a gene group related to the cell cycle, and CD168 was identified to be strongly correlated among them. In immunohistochemical staining, CXCL12 was significantly increased in liver metastasis compared to primary tumor (p = 0.0329), but there was no significant difference in the other 4 genes. Furthermore, in immunohistochemical staining, CXCR7, a receptor for CXCL12, was significantly increased in liver metastatic sites (p=0.0011). Next, in vitro experiments showed that gene expression of CXCL12 -CXCR4/CXCR7 differed depending on morphology: high in the spindle type cells (SSP25, HuH28) and low in the epithelial type cells (HuCCT-1, RBE). There was no significant difference in proliferative capacity between the two types of morphology, but the invasive ability was significantly higher in the spindle-shaped cell lines (p <0.05). By suppressing CXCL12, invasive ability was suppressed in both SSP-25 and HuH28 cells.

Conclusions: CXCL12 may be involved in hepatic metastasis of ICC with organ specificity. Currently, the possibility of therapeutic application against CXCL12 in vivo is under consideration.

#1070

Discoidin domain receptor 1 (DDR1): A potential suppressor of prostate cancer progression.

R. Daniel Bonfil,1 Anjum Sohail,2 Semir Vranić,3 Daniel S. Oliveira,2 Dongping Shi,2 Wei Chen,2 Hyejeong Jang,2 Allen D. Saliganan,2 Benjamin D. Wasinski,2 Hyeong-Reh C. Kim,2 Rafael A. Fridman2. 1 _Nova Southeastern University, Fort Lauderdale, FL;_ 2 _Wayne State University School of Medicine, Detroit, MI;_ 3 _Qatar University, Doha, Qatar_.

Discoidin domain receptors DDR1 and DDR2 are the only receptor tyrosine kinases that bind to and signal in response to collagen. In cancer, DDRs have been shown to play a key role in mediating the crosstalk between tumor cells and the stromal collagen matrix. Because prostate cancer (PCa) preferentially metastasizes to bone, a collagen-rich microenvironment, we set out to investigate the role of DDR1 in intraosseous growth of PC3 cells, a human PCa cell line that expresses DDR1 but not DDR2. PC3 cells were engineered to express short hairpin RNAs (shRNAs) against DDR1, or a scrambled shRNA as a control. These cells were inoculated into the tibiae of male SCID mice, and then bone response and intraosseous tumor growth evaluated by X-ray and histomorphometry. Whereas no differences were observed in bone response (osteolytic lesions), downregulation of DDR1 in PC3 cells was associated with a significant increase in intraosseous tumor growth when compared to control PC3 cells (P<0.05, Mann-Whitney test). To further evaluate the role of DDR1 in PC3 tumor growth, human DDR1a was overexpressed in PC3 cells. The engineered cells were inoculated subcutaneously into SCID mice and tumor growth was monitored over time. Tumors formed by DDR1a-overexpressing PC3 cells were significantly smaller than those obtained with control cells (P<0.005, at final time point, Mann-Whitney test). We also evaluated the expression of DDR1 in a 200-case grade/stage tissue microarray (TMA) with matched normal tissue obtained from The Prostate Cancer Biorepository Network (PCBN). Tissues were subjected to immunohistochemistry (IHC) with an antibody to human DDR1. A predominant membranous DDR1 immunostaining was observed in most epithelial prostate cells, with sporadic cytoplasmic or nuclear immunoreactivity. We found no differences in DDR1 expression between normal and benign prostate glands and low-grade PCa (Gleason score ≤7 [3+4]). However, a significantly lower DDR1 expression was observed in high-grade PCa (Gleason score ≥7 [4+3]) when compared to low-grade PCa (P=0.002, Fisher's exact test). These results suggest that reduced DDR1 expression in PCa is associated with a more aggressive disease. Collectively, these data highlight an unreported role of DDR1 in PCa progression whereby DDR1 may elicit a tumor-suppressive activity, as shown by its ability to reduce intraosseous and subcutaneous growth of PC3 cell xenografts. These data also suggest a differential role for DDRs in PCa bone metastasis whereby DDR1, as opposed to DDR2 (as reported previously), restrains intraosseous PCa growth. Because DDRs are being considered as potential novel therapeutic targets in cancer, defining their precise contributions in PCa progression is of critical importance for the development of effective therapies in this cancer type.

#1071

Overexpression of SHH and GLI1 contributes to poor prognosis and peritoneal metastases in gastric adenocarcinoma.

Yan Xu,1 Yuan Li,2 Bovey Liu,1 Melissa Pool Pizzi,1 Yongxi Song,2 Kazuto Harada,1 Ailing Scott,1 Lang Ma,1 Jiankang Jin,1 Xiaochuan Dong,1 Ying Wang,1 Brian D. Badgwell,1 Jeannelyn S. Estrella,1 Roy-Chowdhuri Sinchita,1 Fatemeh G. Amlashi,1 Zhenning Wang,2 Shumei Song,1 Jaffer A. Ajani1. 1 _MD Anderson Cancer Center, Houston, TX;_ 2 _First Hospital of China Medical University, Shenyang, China_.

Background: The prognosis of gastric adenocarcinoma (GAC) patients with metastases is very poor. Understanding of molecular biology is limited. Hedgehog (Hh) signaling plays an important role in many tumor types and expression of Shh/Gli-1, two major molecules in Hh pathway has been documented in GAC. However, their clinical impact on GAC patients particularly in peritoneal metastasis remains elusive. Methods: Expression of Gli1 and Shh were examined using IHC in tissue microarrays containing more than 500 cases of GAC tissues with clinical annotation. The prognostic variables were determined using univariate and multivariate Cox regression analyses. GAC cell lines, patient-derived peritoneal metastatic cells and novel PDX metastatic model were used to determine the functional role of Shh/Gli-1 in vitro and in vivo. Genetic knockout Gli-1 using LentiCRISPR/Cas9 and Hh inhibitor GDC0449 as well as BET inhibitor were used to test their antitumor activities in GAC cell line and patient-derived cells. Cell proliferation, colony formation, invasion, tumor sphere assays and immunofluorescence were performed to evaluate their functionality and effects of targeted therapy. Results: Both Gli1 and Shh expression are significantly overexpressed in GAC tissue. Among 519 GAC cases, 80.76% and 87.02% were positive for nuclear Gli-1 and cytoplasmic Shh expression respectively, while the strong nuclear expression rate for Gli-1 is 69.56% and 50.10% for Shh. In the univariate Cox analysis, the overall survival was shorter for patients with high Gli-1 (p=0.018) or high Shh expression (p=0.038). In the multivariate cox analysis for both markers, only Gli-1 remained as an independently prognostic for short survival. We also observed high Gli-1 nuclear expression correlated with the presence of lymph node metastasis (p=0.032). Gli1 was highly expressed in most human malignant ascites cells. Interestingly, Gli-1 was significantly upregulated in mouse PDX-ascites cells compared to primary mice tumors. Genetic knockdown Gli-1 or pharmacologically inhibition of Gli-1 by GDC0449 Hh inhibitor or BET inhibitor JQ1 decreased Gli-1 and restored E-cadherin expression and significantly suppressed malignant cell properties and reduced population of cancer stem cells (ALDH1+ or CD133+) in patients' derived metastatic cells. Conclusions: These findings indicate that overexpression of Gli1 and Shh plays an important role in progression of peritoneal metastases of GAC. Targeting Gli1/Hh signaling may provide novel therapeutic strategies for GAC patients with peritoneal metastases. Keywords:Hh pathway, Gli1, Shh, Gastric cancer, metastasis

#1072

Potential biomarkers correlated with poor survival rate of melanoma patients.

Alice S. Morais,1 Ana C. Monteiro,1 Fernando Andrade,2 Lucas Goedert,3 Enilza Espreáfico,3 Regine Schneider-Stock,4 André Fujiota,2 Miriam G. Jasiulionis1. 1 _Universidade Federal de São Paulo, São Paulo, Brazil;_ 2 _Universidade de São Paulo, São Paulo, Brazil;_ 3 _Universidade de São Paulo, Ribeirão Preto, Brazil;_ 4 _Friedrich-Alexander Universitat, Erlangen, Germany_.

Malignant melanoma is an extremely aggressive tumor, responsible for 90% of skin cancer mortality. Widespread metastasis is the main cause of death in melanoma patients. Therefore, the identification of molecules related to the degree of melanoma malignancy could be used as prognostic biomarkers. In the present study, using a murine model of melanoma progression, different genes, such as Snai1, Chd1, Gata4, Id4, Cbs, Vav1, and Itga4, were first identified as presenting aberrant DNA methylation in their promoters along melanoma progression by Methylight analysis. The gene expression pattern and epigenetic regulation were validated in these murine cell lines by RT-qPCR and 5AzaCdR treatment, respectively. The genes Angpt2 and Fgf1 were also identified as differentially expressed along tumor progression in this murine model. The expression profile of these genes was also evaluated in primary human melanocytes and RGP (radial growth phase), VGP (vertical growth phase) and metastatic melanoma cell lines. Clinical and gene expression data from 460 patients were obtained from TCGA (The Cancer Genome Atlas). Using an univariate test, patients presenting tumors with low levels of Chd1, Vav1, Id4 and Fgf1, and high levels of Snai1 and Angpt2 were shown to exhibit poor overall survival rate. Indeed, the multivariate analysis using Cox regression model identified high Snai1 (HR=1.217; 95% CI 1.075-1.377; p=0.002) and Angpt2 (HR=1.186; 95% CI 1.059-1.329; p=0.003) expression and low Chd1 (HR=0.757; 95% CI 0.575-0.996; p=0.047), Fgf1 (HR=0.858; 95% CI 0.780-0.944; p=0.002) and Vav1 (HR=0.859; 95% CI 0.764-0.967; p=0.011) expression as independent prognostic factors for the overall survival rate of patients with melanoma. Also using a multivariate analysis, the low levels of Id4 promoter methylation (HR=0.689; 95% CI 0.531-0.891; p=0.045) was shown to be an independent predictive factor for poor survival rate, although no correlation was found between Id4 expression and survival rate. In conclusion, the upregulation of Snai1 and Angpt2, and the downregulation of Chd1, Vav1 and Fgf1 are associated with unfavorable survival outcome, indicating they might be valuable prognostic biomarkers for melanoma. Supported by CNPq and FAPESP.

#1073

Breast cancer intravasation signature from patient fine-needle aspirates.

Gargi Bandyopadhyaya,1 Srinjoy Goswami,1 John S. Condeelis,2 Maja H. Oktay,2 Sumanta Goswami2. 1 _Yeshiva University, New York, NY;_ 2 _Albert Einstein College of Medicine, Bronx, NY_.

A key step in the progression of metastatic breast cancer is the process of blood vessel intravasation. Understanding the molecular mechanisms that underlie this crucial step is essential in designing strategies to block the same with an aim to prevent metastatic disease. Recently our group has developed and published an in vitro intravasation assay (1) that recapitulates the process of intravasation and allows the use of patient derived breast cancer cells. Previously our group had identified a breast cancer invasion signature that helps facilitate clinical decision making in breast cancer patients (2).

Here using fine needle aspirates from breast cancer resections we collected viable cancer cells which were labeled green using vital dyes and mixed with a macrophage cell line labeled red and layered on the top compartment of a transwell. The bottom of the transwell membrane contains a layer of extracellular matrix and a tight layer of HUVECs. Green cancer cells were separated one at a time using a florescent microscope and a micromanipulator. The cancer cells capable of intravasation are divided into two groups, one group that has crossed the entire layer and fallen to the bottom of the well, and a second group that crossed the endothelial cells but are still bound to the bottom of the endothelial layer. Cancer cells from top of the transwell which did not intravasate were also collected.

RNA was extracted from the few cells collected by micromanipulation from all the three groups, amplified and converted to Cy3 labeled cDNA. The labeled cDNA was hybridized to whole genome microarrays. Gene expression was compared between the three groups of cells. Both unsupervised and supervised data analysis was performed on the three groups of cells from five patients. Significance Analysis of Microarrays, Ingenuity Pathway Analysis and Gene Set Enrichment Analysis was performed on these datasets. Supervised analysis and validation of the altered genes was performed using qRT-PCR.

Preliminary data analysis indicates the upregulation of the genes associated with Breast Cancer Stem cells and DNA repair. This is the first report of whole genome gene expression analysis from cells collected by FNA from patients, separated in vitro into intravasation competent and intravasation incompetent and unsupervised whole genome analysis performed to identify intravasation specific genes and pathways. Once unique targets and pathways are identified in intrvasation competent cells, a number of additional function blocking steps will be designed and undertaken.

(1) Pignatelli J., Goswami S., et. al. Invasive breast carcinoma cells from patients exhibit MenaINV- and macrophage-dependent transendothelial migration. Sci Signal. 2014 Nov 25;7(353)

(2) Karagiannis GS., Goswami S., Jones JG., Oktay MH., and Condeelis JS. Signatures of breast cancer metastasis at a glance. J Cell Sci. 2016 May 1;129(9):1751-8.

#1074

Expression of p39 and Rb in lung cancer and its correlation with clinicopathologic parameters.

Jaileene Perez-Morales,1 Stephanie Rivera,1 Bryan Torres,2 Xavier Rodriguez,2 Angel Isidro,1 Pedro Santiago-Cardona1. 1 _Ponce Health Sciences University, Ponce, Puerto Rico;_ 2 _University of Puerto Rico-Ponce, Ponce, Puerto Rico_.

Lung cancer is characterized by its aggressiveness and proclivity for early metastasis. A predictor of metastasis of NSCLC rely on post-resection evaluation of tumor histology, which is a severe limitation since only 15% of the patients are diagnosed with resectable disease. Hence, there is a need to characterize biomarkers with metastasis-predicting value in pre-resection small biopsy specimens. Rb is a tumor suppressor inactivated due to hyper-phosphorylation in NSCLC. Our preliminary data relates the Rb S249 phosphorylation to an epithelial-to-mesenchymal transition (EMT), a trait strongly associated to metastasis and increase in CDK5 activator p39. Here, we examined the prognostic significance of Rb S249 and p39 as biomarkers of metastasis staging in non-small cell lung cancer. Our hypothesis is that Rb hyper-phosphorylation in S249 can have prognostic value by being associated with a metastatic phenotype and EMT. To determine p39 and Rb phosphorylation in lung cancer, immunohistochemistry analysis was performed on two lung cancer microarrays. Three pathologists observed the microarrays and scored them using the all-red scoring system. P39 and Rb phosphorylation in S249 were more present in lung tumors than adjacent normal tissue. P39 positive expression had a positive correlation with staging, lymph node involvement, and metastasis. Rb phosphorylation in residue S249 had a positive correlation with histological grading. Using general linear model, we determined that staging can be predicted when combining phosphorylation of Rb S249 and p39. Our analysis suggests that p39 and Rb S249 are likely to be diagnostic biomarkers for lung cancer metastasis.

#1075

CD99 expression in astrocytomas and functional analysis in glioblastoma cell line.

Lais Cavalca Cardoso, Suely Kazue Marie, Roseli Silva Soares, Sueli M. Oba-Shinjo. _School of Medicine of University of São Paulo, São Paulo, Brazil_.

Glioblastoma (GBM) is the most frequent high grade brain tumor in adults. Treatment consists of a combination of maximal surgical resection, radiotherapy and chemotherapy, but prognosis is still poor. Therefore, new therapeutic strategies with other biologically-based therapies is essential to improve the survival of patients with GBM, currently restricted to 15-17 months. This urgent need for better therapies has motivated many studies to pursuit new targets in GBM. Our laboratory demonstrated higher expression of CD99 mRNA in GBM samples when compared to lower grade astrocytomas and non-neoplastic brain tissue. CD99 is a membrane molecule and acts as a key regulator in several biological processes, including cell adhesion, migration, apoptosis, differentiation of T cells and thymocytes, diapedesis of lymphocytes to inflamed vascular endothelium, maintenance of cellular morphology and regulation of intracellular membrane protein trafficking. CD99 expression has been described in many types of cancers, with distinct function and signaling pathways, and its role is not completely elucidated yet, including in GBM.We confirmed the higher CD99 expression in TCGA dataset of GBM cases when compared to grade II and III astrocytomas. Furthermore, amongst GBM cases, Proneural molecular subtype presented lower CD99 expression compared to Mensechymal, Classic and Neural subtypes. We performed functional assays knocking down CD99 with two different shRNAs in the human GBM cell line U87MG. The CD99 silencing efficiency was 80 and 97%, for shRNA_1 and shRNA_2, respectively, confirmed at transcriptional level by quantitative real time PCR and at protein level by Western blot. Significant reduction of migration and invasion of CD99 knocked-down U87MG cells was observed. Additionally, cell shrinkage with relevant reduction of cell area was detected after the silencing of this target, with decreased CD99 localization at lamellipodia by immunocytochemistry. These findings pointed out the cytoskeleton rearrangement with reduction in adhesion sites. Further expression profile analysis after CD99 knockdown by a cancer panel RNAseq confirmed its involvement in cell adhesion, regulation of actin cytoskeleton and ECM-receptor interaction in the KEGG pathways analysis. Interestingly, CD99 knockdown impacted in the reduction of cell adhesion in a distinct quantitative pattern, suggesting that the result is dose dependent. Refinement of this aspect is ongoing in further in vivo assays which will better clarify the CD99 role in tumor cell migration/invasion. Altogether, our results suggest CD99 as a potential new therapeutic target to control GBM infiltrative process, allowing a more efficient treatment outcome.

#1076

Identifying biomarkers of metastasis through biosynthetic tagging.

Yiqiang Zhang,1 Xiaojie Yu,1 Shaimar Gonzalez,1 Sarah Almasri,1 Leonidas Bleris,2 Alexander Pertsemlidis1. 1 _UT Health Science Center at San Antonio, San Antonio, TX;_ 2 _UT Dallas, Dallas, TX_.

Introduction: Short, noncoding RNAs called microRNAs (miRNAs) have demonstrated huge potential as diagnostic and therapeutic agents. One hurdle to their application as biomarkers is that they are not labeled by cell of origin, making it impossible to distinguish host- from disease-specific species. The current proposal is a novel combination of chemical and genetic tools to identify the tumor-specific miRNA secretome. We leverage murine models of the extremes of neuroblastoma and lung adenocarcinoma metastatic behavior, a protozoan enzyme that can selectively incorporate a modified base into RNA, and expression profiling by next-generation sequencing to identify miRNA markers of metastasis.

Methods: To biosynthetically label RNA in vivo by cell of origin (tumor versus host), we exploit a nucleotide salvage enzyme used by the protozoan parasite Toxoplasma gondii to yield uridine monophosphate (UMP). When the uracil analog 4-thiouracil (4TU) is provided as a substrate, UPRT couples ribose-5-phosphate to the 4TU N1 nitrogen, resulting in 4-thiouridine-monophosphate (4TUMP). 4TUMP is then incorporated into RNA, providing a molecular handle by which RNA species from UPRT-expressing cells can be manipulated. The thiol-labeled ("tagged") RNA can be separated from untagged RNA by thiol-specific biotinylation followed by affinity purification on streptavidin-coated magnetic beads. Importantly: (1) thiol-containing nucleotides do not naturally occur in eukaryotic mRNAs, (2) multicellular eukaryotes lack UPRT activity, and (3) expression of UPRT has little effect on cellular physiology. Targeted expression of UPRT therefore allows cell-specific tagging of miRNAs with 4TU. This approach is highly efficient; nearly all human and murine miRNAs are taggable.

Results: Cell lines derived from metastatic and nonmetastatic models of adult and pediatric cancers were stably transfected to express the UPRT enzyme and injected into syngeneic mice to induce tumor formation. The mice were fed a diet supplemented with 4TU. Since the tumor cells expressed UPRT, they synthesized TU-tagged miRNAs, while host cells, which did not express UPRT, did not. miRNAs released by the tumor into the blood or adjacent tissue were TU-tagged. Separation of the miRNAs present in tumor, normal adjacent tissue and serum into labeled and unlabeled fractions followed by small RNA sequencing enabled the controlled comparison of the profile of miRNAs released into the blood specifically by the tumor relative to the profile of miRNAs from the host.

Conclusions: Preliminary experiments have allowed us to identify miRNAs that are biomarkers of cancer metastasis that we are investigating as therapeutic targets in the same mouse models, extending to models based on human cell lines, evaluating in patient populations, and developing as clinical tools.

#1077

Genomic alterations in plasma cell-free DNA predict treatment response to abiraterone acetate and prednisone and survival in metastatic castration-resistant prostate cancer.

Meijun Du,1 Manish Kohli,2 Chiang-Ching Huang,3 Liang Wang1. 1 _Medical College of Wisconsin, Milwaukee, WI;_ 2 _Mayo Clinic, Rochester, MN;_ 3 _University of Wisconsin, Milwaukee, WI_.

Background: Abiraterone acetate/prednisone (AA/P) is used for treatment of metastatic castration-resistant prostate cancer (mCRPC), but response is variable and there are no known molecular predictive factors.

Methods: We prospectively collected germline and plasma specimens from 72 mCRPC patients progressing on androgen-deprivation therapy (ADT) before initiating AA/P treatment (baseline) and serially after 12 weeks of treatment. After extracting cell-free DNA (cfDNA), we performed copy number analysis using low pass whole-genome sequencing approach. The primary endpoints were to determine association with response and survival. Cox regression and Kaplan-Meier survival analyses were used to associate the CNV with overall survival (OS) and progression-free survival (PFS) with statistical significance at p≤0.05. For determining primary resistance to AA/P, responders and nonresponders were defined as patients showing no progression or progression after 12 weeks treatment. T-test was used to evaluate the CNV with AA/P response at 12 weeks.

Results: Whole-genome sequencing generated approximately 8.0 million mappable sequence reads per subject (total plasma samples sequenced, n=144). By comparing cfDNA to germline control DNA, our copy number analysis showed significant genomic abnormalities in 37 of 144 plasma samples. We observed the most frequent amplification of 1q, 8q, Xq and deletion of 2p, 6q,10q, 13q. Among these CNVs, amplification of MYC at 8q24.21, AR at Xq12, deletion of FOXP1 at 3p14, PTEN at 10q23.31, RB1 at 13q14.2, TP53 at 17P13.1, and gene fusions (TMPRSS2:ERG) were consistent with previous reports from tumor tissue-based studies. Survival analysis showed significant association of a fragment deletion at 13q14-32 with both OS and PFS (FDR= 0.0207/0.0141). The most significant fragment showing association with OS was the deletion of fragment at 2p22.1-p14 (FDR=0.00219). Genomic gains of MYC at 8q24.21 and NCOA2 at 8q13.3 were associated with PFS with FDR= 0.0085 and 0.0072, respectively. By comparing baseline CNVs between responder and nonresponders, we identified significant association of the deletion of PTEN at 10q23.31 (p= 0.0003), RB1 at 13q14.2 (p= 0.0008) and amplification of NCOA2 at 8q13.3 (p= 0.0030) with AA/P response/resistance at 12 weeks. Although statistical analysis showed the significant CNV change at AR locus between baseline and 12-week post-AA/P treatment (p=0.0002), the CNV change was not significantly different between responders and nonresponders (p=0.1697).

Conclusions: cfDNA CNV may serve as a sensitive biomarker for prediction of treatment response to AA/P. Further detailed CNV and clinical association analyses are needed to fully validate the potential clinical utilities of cfDNA-based liquid biopsy for clinical management.

#1078

Identification of peptides binding to mesenchymal subtype breast cancer from phage display peptide library.

Kelvin M. Jones,1 Rajeev Samant,2 Shree Singh,3 Deepa Bedi1. 1 _Tuskegee University, Tuskegee, AL;_ 2 _University of Alabama at Birmingham, Birmingham, AL;_ 3 _Alabama State University, Montgomery, AL_.

Most available therapeutic drugs efficiently treat tumors; however, absence of specificity has deleterious effects on patients. Identifying a tumor ablation method that has specificity with minimal to no side effects is needed. Phage display allows for expression of peptide and protein libraries on the surface of phage with a diversity of 109 peptides of different permutations, which leads to the selection of peptides and proteins, with high affinity and specificity. Phage display can be used as a tool to identify specific targets using a pIII phage library for identification of cell surface markers. To do so, we performed an initial depletion assay to isolate specific phages that express an affinity to human cells. We isolated a phage diversity of 10-2 from 109. Subsets of this population were used to identify phage and breast cancer cell interaction in vitro using an ELISA assay. Briefly, phages were exposed to epithelial and mesenchymal breast cancer cells and fibroblast cells. We have identified a phage profile that selectively targets tumor cells based on their transition from epithelial to mesenchymal. Interestingly, PE-26, a phage subset, indicated a greater affinity for more mesenchymal-like tumor cells. Moreover, we identified a 5-fold increase in the binding affinity for MDA-MB-231 and MCF-7 (overexpressed with TGF-β) in comparison to the control fibroblast cells. Additionally, PE-21 demonstrates a 6-fold increased affinity for breast cancer cells when contrasted with our control fibroblast cells. Our data provide further support that the identified phage profiles exhibit a preferential affinity for more mesenchymal-like tumor cells, which are known to be aggressive. This suggests their potential chemotherapeutic capability. Our future endeavors include further validation of selected phages and development of peptides to be employed as imaging agents to study EMT in vivo and as diagnostics to detect circulating tumor cells.

#1079

PCDH7 is overexpressed in advanced prostate cancer and modulates aggressive phenotype in prostate cancer cells.

Gauri Shishodia, Prakash Srinivasan Shanmugam, Sweaty Koul, Hari Koul. _LSU Health Sciences Center - Shreveport, Shreveport, LA_.

Introduction: Most of prostate cancer (PCa) deaths are a result of distant metastasis and due to emergence of castrate resistant PCa (CRPC). Protocadherins (PCDHs) are members of the cadherin superfamily that regulate cell adhesion. Their extracellular domains contain cadherin-like repeats, but they differ significantly from classical cadherins with respect to their unique cytoplasmic domains that lack the conserved motifs for binding β-catenin and p120-catenin. PCDH7, a cadherin superfamily transmembrane protein that is known to function in cell-cell recognition and adhesion, is reported to be overexpressed in breast cancer and non-small cell lung cancer and promoted tumor metastasis. The role of PCDH7 in PCa is investigated for the first time in the present investigation.

Materials and Methods: PCa cells- LNCaP, C4-2b, DU145, PC3, 22Rv1 and RWPE1 were used in this study. PCDH7 protein and mRNA levels were checked by Western Blotting and quantitative real time PCR respectively. Immunohistochemistry was done to check expression in TRAMP mice FFPE prostate tissue sections and human prostate tissues. Publically available data set (Trento 2016) was used to analyze PCDH7 expression in NEPC patients. PCDH7 lentiviral knock down was performed using PCDH7 shRNA in PC3 cells. Cell migration and invasion were done using IncuCyte® Scratch Wound Cell Migration and Invasion System. Colony formation was assessed by staining with 0.4% crystal violet after 3 weeks of cell seeding.

Results: PCDH7 mRNA and protein is overexpressed in CRPC cells (C4-2b, 22Rv1, DU145 and PC3 cells as compared to castrations sensitive LNCaP cells as well as normal prostate RWPE1 cells. We observed highest levels for PCDH7 in enzalutamide refractory 22Rv1 cells. We also observed a significant increased expression of PCDH7 during tumor progression (30wk vs 12wk) in prostate tissues of TRAMP mice. Our results also show that PCDH7 is amplified in 9% of PCa patients and overexpressed in 43% patients. Immunohistochemical analysis of PCDH7 revealed high expression of PCDH7 in human prostate cancer tissues. EGF treatment enhanced ERK and AKT activities in time-dependent manner and the phosphorylation remain sustained even at late time points in PCDH7 positive PC3 cells as compared to decline in phosphorylation PCDH7 negative RWPE1 cells. shRNA-mediated knockdown of PCDH7 reduced ERK and AKT activities. Knockdown of PCDH7 also resulted in decreased cell migration, reduced cell invasion, and decreased colony formation. Collectively, these data suggest PCDH7 plays a crucial role in advanced PCa.

Conclusion: Our results are the first direct demonstration of PCDH7 expression in PCa. Our exciting observations reveal a critical role for PCDH7 in prostate cancer progression. These results suggest that PCDH7 may be an attractive target for therapeutic intervention in PCa, in general, and CRPC in particular.

#1080

**The effect of germline polymorphisms on somatic hotspot mutations in** TP53 **for the treatment of high-grade serous ovarian carcinoma.**

Cristabelle Madona N. De Souza. _University of New Mexico, Albuquerque, NM_.

Introduction:

Due to frequent recurrences and acquired resistance to platinum-based chemotherapy in high-grade serous ovarian cancer (HGS OvCa), it is important to identify novel targets and pathways to overcome drug resistance and improve the outcome of patients with ovarian cancer. TP53, which encodes the transcription factor p53, is mutated in 96% of HGS OvCa . Majority of the studies that establish the relationship between mutations in TP53 and cancer have focused on hotspot somatic mutations. In addition to somatic mutations, nonsynonymous germline single nucleotide polymorphisms (SNPs) at codon 72 play a role in altering the function of p53 and a cancer susceptibility. However, the effect of codon 72 (P72R) polymorphism on somatic mutations in TP53 is not well characterized with regard to the Epithelial to Mesenchymal phenotype and cancer progression.

Objective:

-Characterize the effect of somatic mutations in TP53 in the presence of the P72R SNP on cellular morphology and the Epithelial to Mesenchymal transition (EMT) transcriptional network.

-Determine the effect of somatic mutations in TP53 in the presence of the P72R SNP on proliferation and the cell cycle checkpoint transcriptional network

Experimental Procedures:

We generated 8 pairs of hotspot mutations in TP53 in the context of the P72 or R72 SNP into p53-null SKOV3 cells. Cell morphology was recorded by fluorescence microscopy. Using CHIP-seq, RNA-seq and immunoblotting, we studied the effect of the polymorphism + mutation on EMT markers like BMP1, CALD1, CAMK2N1, CDH2 that are up-regulated during EMT and genes like CAV2, CDH1, DSP, FGFBP1, IL1RN, KRT19, MITF that are down-regulated during EMT. We used techniques like chromatin immunoprecipitation ChIP-seq, gene expression profiling, and computational methods to construct blueprints of the transcription factors like Cyclin D1, p21 that play a role in cell cycle checkpoint regulation which help understand compromised pathways due to the presence of somatic mutations in combination with germline polymorphisms.

Summary:

\- Hotspot mutants with the R72 SNP cause changes in cell morphology resembling mesenchymal cells whereas mutants with the P72 SNP resemble epithelial cells. This effect can be accounted for by changes in the EMT network.

\- The hotspot missense mutants with R72 have a greater growth potential in comparison to their P72 counterpart which could explain the Gain of Function (GOF) effect of the former.

Conclusions: Germline polymorphisms at codon 72 in the p53 protein plays a significant role in regulating the Gain-of-Function (GOF) effect of TP53 somatic hotspot mutations. This is significant because it will be useful in designing a treatment plan for patients with specific somatic mutations and germline polymorphisms.

#1081

TCF12 **regulates metastasis-promoting genes in pancreatic cancer.**

Michelle M. Maurin,1 Justin Y. Newberg,1 Mik A. Black,2 Devin J. Jones,3 Nancy A. Jenkins,4 Neal G. Copeland,4 Karen M. Mann1. 1 _Moffitt Cancer Center, Tampa, FL;_ 2 _University of Otago, Otago, New Zealand;_ 3 _Houston Methodist Research Institute, Houston, TX;_ 4 _MD Anderson Cancer Center, Houston, TX_.

Pancreatic ductal adenocarcinoma (PDAC) is a highly metastatic disease with poor patient outcome. While acquired oncogenic activation of KRAS is an initiating event, disease progression requires functional perturbation of additional genes and cellular pathways. We have taken a comparative genomics approach to identify progression drivers using a mouse model of metastatic PDAC driven by Sleeping Beauty (SB) insertional mutagenesis in the context of oncogenic Kras (SB PDAC). Statistical enrichment analysis of high read depth insertions from SB PDAC tumors defined 48 drivers predicted to contribute to disease progression. Among these drivers, Tcf12 was associated with increased metastatic incidence in the SB PDAC mouse model. We demonstrated significant increases in primary tumor burden and metastatic spread by single gene knockdown of TCF12 in an orthotopic mouse model of pancreatic cancer. TCF12 encodes a basic helix-loop-helix (bHLH) transcription factor that partners with other bHLH transcription factors in different cellular contexts, including TWIST, a transcription factor involved in epithelial-to-mesenchymal transition (EMT), PTF1A, a pancreas-specific transcription factor with essential roles in pancreas development and cancer, and SMAD3, a TGFß-related transcription factor involved in cellular migration. TCF12 resides in a chromosomal region (15q21) frequently lost in pancreatic cancer, and decreased TCF12 expression is associated with poor patient prognosis. RNASeq analysis identified genes with TCF12 consensus binding sites within 10 kb of their promoter regions that are differentially expressed upon TCF12 knockdown. A subset of these genes function in Wnt or TGF-beta signaling pathways and show consistent decreases in expression upon TCF12 knockdown in orthotopic tumors. ChipSeq analysis and functional validation studies are ongoing to confirm these genes are direct TCF12 transcriptional targets involved in pancreatic tumor metastasis.

#1082

Loss of desmoglein 2 promotes tumor growth and progression through the activation of Src and facilitates the internalization of EGFR in biliary tract carcinoma cells.

Sang-Hyun Lee, Jeong-Ki Min. _Korea Research Institute of Bioscience and Biotechnology, Daejeon, Republic of Korea_.

Understanding the molecular carcinogenesis pathways is critical to establish novel individualized molecular targeted therapies and finally to improve overall clinical outcomes of patients with a highly heterogeneous group of malignancies such as biliary tract cancers (BTCs). In this context, we investigated the patho-physiological roles of a desmosomal component, desmoglein 2 (Dsg2) and found that Dsg2 functions as a tumor suppressor protein in BTC, as Dsg2 loss induced a number of cellular biological activities including proliferation, motility, invasion, transendothelial migration in vitro and tumor growth and metastasis in vivo. Clinico-pathological analyses of BTC patients indicated that lower expression of Dsg2 is associated with a poor prognosis of patients as perineural and lymphatic invasion were highly increased in those patients. In addition, loss of Dsg2 showed a significant increase of EGFR clearance from the cell membrane in BTC cells and patients' tumor tissues. Those cells were significantly decreased in the EGF-driven activation of EGFR and highly resistant to the anti-EGFR antibody treatment. Src appears to be involved in the Dsg2-mediated clearance of EGFR, as this significantly blocked by the suppression of cSrc expression in Dsg2-depleted cells, possibly through the direct binding of Dsg2 preferentially with an inactive form of Src through its SH2 domain. Suppression of cSrc by Src inhibitors or siRNA dramatically reduced BTC tumor growth in vitro and in vivo. In comparison with the anti-EGFR antibody therapy, Src-targeted therapies produced consistent anti-tumor activities independent with the expression status of EGFR in the BTC tumors. In conclusion, targeting Src would be a promising therapeutic approach to patients with advanced BTC and potentially useful in overcoming resistance to current anti-EGFR therapies, although further clinical studies are essentially required to translate the current preclinical knowledge into clinical practice.

#1083

GAP43, a novel potential metastases promoter in non-small cell lung cancer.

Fanrong Zhang,1 Jiaoyue Jin,1 Junzhou Wu,1 Kaiyan Chen,1 Minran Huang,1 Herbert Yu,2 Lisha Ying,1 Dan Su1. 1 _Zhejiang Cancer Hospital, Hangzhou, China;_ 2 _University of Hawaii Cancer Center, Honolulu, HI_.

Metastasis is a serious sequela with dismal prognosis in non-small cell lung cancer (NSCLC). To identify novel biomarkers for predicting NSCLC metastasis, we performed high-throughput Luminex assays to profile the transcriptional level of 36 genes in 37 operable NSCLC patients who had distant metastases within 3 years after surgery and 33 paired patients without relapse after operation. Among the 36 genes, growth associated protein 43 (GAP43), an axonal membrane protein, was identified related to distant metastases. Multivariate cox regression analysis showed that NSCLC patients with elevated GAP43 had a 3.71-fold increase in risk for metastasis than those with low levels (95% confidence interval 1.69-8.14, P = 0.001). Analysis of a patient cohort of 1,926 NSCLC revealed that patients with high levels of GAP43 had worse progression-free and overall survival. In vitro experiments showed that GAP43 knockdown suppressed NSCLC cell migration and invasion, while GAP43 overexpression facilitated their migration and invasion. Immunofluorescence assay indicated that the linear polymer microfilament F-actin became cracked after GAP43 knockdown. Furthermore, animal studies using left ventrical injection demonstrated that GAP43 silenced NSCLC cells were less likely to metastasize to brain and bone than the controls. Together, our findings suggest that GAP43 may be an independent predictor for NSLCL metastasis, and it may facilitate distant metastasis through targeting F-actin.

#1083A

An ex vivo 3D bone metastasis model for prostate cancer.

Jenny Zilberberg,1 Saba Choudhary,2 Eugenia Dziopa,1 Cirian Mannion,1 Yair Kissin,1 Erika Parasido,3 Christopher Albanese,3 Woo Lee2. 1 _Hackensack University Medical Center, Hackensack, NJ;_ 2 _Stevens Institute of Technology, Hoboken, NJ;_ 3 _Georgetown University, Washington, DC_.

Introduction: Bone is the preferred site for prostate cancer (PCa) metastases and there is virtually no cure once the tumor is established within this niche. The development of therapeutic strategies against metastatic PCa has been limited by the lack of an all-human preclinical model to: (1) study PCa-bone interactions under physiologically relevant three dimensional (3D) microenvironments, (2) understand how these interactions result in the development of drug resistance, and (3) explore paradigm-changing precision medicine concepts.

Methods: Conditionally reprogrammed primary PCa cells (CR-PCa, derived from a lymph node metastatic site) were integrated with our ex vivo human 3D bone tissue platform, as a transformative approach for modeling tumor behavior at the bone niche. The 3D bone tissue was engineered using primary osteoblastic (OSB) cells from human bone samples. These cells were biomimetically assembly with biphasic calcium phosphate microbeads (BCPm) in perfusion microfluidic culture devices. BCPm were used to replicate the 3D cellular network of OSTs in human bone. This 3D tissue model was successfully used to support the osteocytic differentiation of primary human OSB cells within the BCPm structure, while maintaining a single cell layer of osteoblasts on the surface of the 3D tissue resembling the bone endosteum.

Results: Integration of the 3D bone tissue with CR-PCa cells resulted in a compromised endosteal layer after only 4 days of coculture, with more apoptotic OSTs in the tissues with CR-PCa vs. no PCa cells controls (P<0.01). For functional analysis, we stained 5 μm sections of the 3D tissues for alkaline phosphatase (ALP, an OSB marker) and sclerostin, an osteocytic marker and major signaling molecule released by embedded OSTs that suppresses osteoblastogenesis. Quantification of immunofluorescence staining revealed that ALP was significantly increased (P<0.05), whereas sclerostin significantly decreased (P<0.01) with the introduction of CR-PCa cells. This OSB phenotype, induced by PCa-bone interactions ex vivo, is entirely consistent with the OSB prostate cancer-bone metastasis niche seen in patients with advanced disease.

Conclusions: Our model recapitulates: (1) an OSB monolayer closely resembling the endosteum at the bone/ bone marrow interface, (2) the complex microphysiological features and functions of 3D-networked OSTs, and (3) the interactions of primary PCa cells with osteoblasts and OSTs. 

### Mechanisms Underlying Metastasis 2

#1084

TRAF4-mediated NGF receptor TrkA ubiquitination regulates prostate cancer metastasis.

Ramesh Singh,1 Dileep Karri,2 Jiangyong Shao,1 Subhamoy Dasgupta,3 Shixia Huang,1 Dean P. Edwards,1 Bert W. O'Malley,1 Ping Yi1. 1 _Baylor College of Medicine, Houston, TX;_ 2 _University of Texas Southwestern Medical School, Dallas, TX;_ 3 _Roswell Park Cancer Institute, Buffalo, NY_.

Receptor tyrosine kinases (RTKs) are important drivers of cancers. In addition to genomic alterations, aberrant activation of wild type RTKs play an important role in driving cancer progression. However, the underlying mechanisms of how RTKs drive prostate cancer remains incompletely characterized. Here we show that non-proteolytic ubiquitination of RTK regulates its kinase activity and contributes to RTK-mediated prostate cancer metastasis. TRAF4, a RING domain E3 ubiquitin ligase, is highly expressed in metastatic prostate cancer. We demonstrated here that it is a key player in regulating RTK mediated prostate cancer metastasis. We further identified TrkA, a neurotrophin receptor tyrosine kinase, as a TRAF4-targeted ubiquitination substrate that promotes cancer cell invasion and genetic ablation of TrkA abolished TRAF4-dependent cell invasion. TRAF4 promoted atypical K27 and K29-linked ubiquitination at the TrkA kinase domain and increased its kinase activity. Mutation of TRAF4-targeted ubiquitination sites abolished TrkA tyrosine auto-phosphorylation and its interaction with downstream effector proteins. TRAF4 knockdown also suppressed nerve growth factor-stimulated TrkA downstream p38 MAPK activation and subsequent invasion-associated gene expression. Furthermore, elevated TRAF4 levels significantly correlated with increased NGF-stimulated invasion-associated gene expression in prostate cancer patients, indicating this signaling axis is significantly activated during oncogenesis. Our results revealed a post-translational modification mechanism contributing to aberrant non-mutated receptor tyrosine kinase activation in cancer cells.

#1085

Prostate cancer secreted microRNAs influence early steps in bone metastasis via osteoclast precursors.

L. Ayse Erozenci,1 Ning Wang,2 Albert A. Geldof,1 Jorn Mulder,1 Connie R. Jimenez,1 R. Jeroen van Moorselaar,1 Allison Gartland,2 Teun J. de Vries,3 Irene V. Bijnsdorp1. 1 _VU University Medical Center, Amsterdam, Netherlands;_ 2 _The Mellanby Centre for Bone Research, The University of Sheffield, Sheffield, United Kingdom;_ 3 _Academic Centre for Dentistry Amsterdam (ACTA), Amsterdam, Netherlands_.

BACKGROUND: Bone metastasis is a complex process involving reciprocal interplay between cancer and bone cells. Metastatic prostate cancer (PCA) cells secrete extracellular vesicles (EVs), that contain microRNAs (miRNAs), and these can be found in blood. We hypothesize that precursor osteoclasts scavenge PCA EVs, leading to a manipulation of their behavior thereby driving metastasis.

METHODS: To test how EV-miRNAs affect metastasis, we stably overexpressed two metastasis-related miRNAs in PCA cells. Bone metastases were induced by inoculating miRNA overexpressing PCA cells into nude mice via intracardiac route with or without the presence of subcutaneous tumors. Tumor progression was monitored by F-luciferase signal (IVIS) while bones were analyzed by microCT ex vivo. miRNAs expression was analyzed by qRT-PCR on precursor osteoclasts isolated from mice blood. Osteoclast differentiation assays were performed using human peripheral mononucleated blood cells on bone chips together with RANKL and M-CSF to drive differentiation.

RESULTS: In vivo, intracardiac injection of PC3 cells that overexpress selected miRNAs accelerated the formation of bone metastases (0-85%) compared to control PC3 cells (43%). To determine whether EVs secreted by PCA cells affect osteoclasts, human monocytes were allowed to differentiate to osteoclasts while exposing them to PCA EVs. EVs secreted by control PCA cell lines increased osteoclast differentiation by 22.5% (p<0.01), compared to EVs secreted by RWPE1 noncancerous prostate cells. Exposure to EVs secreted by the miRNA overexpressing PCA cells further enhanced osteoclast formation by an additional 10% (p<0.05). To test whether pro-metastatic miRNAs enhance metastases when mouse osteoclast precursors are pre-stimulated (via EVs or soluble factors), subcutaneous tumours of pro-metastatic cells were grown for 2 weeks. In circulating osteoclast precursors of mice that were pre-stimulated with PCA factors that were secreted by subcutaneous tumors (2 weeks), the miRNA levels were increased by 1.5 - 4 fold (p<0.05), demonstrating that these cells scavenge the secreted tumor miRNAs. Subsequently, mice received an intracardiac injection with matching cells to induce metastases. Trabecular bone volume of >15% was used as indicator for metastasis induced bone loss. Mice that grew both a subcutaneous tumor and also received an intracardiac injection to drive metastasis formation led to an ~30% increased number of mice with >15% bone loss (p<0.01). This indicates that osteoclasts may be manipulated by the cancer cell-secreted factors to form a pro-metastatic niche.

CONCLUSION: PCA miRNAs secreted via EVs stimulate osteoclast formation, potentially increasing the formation of overt metastases. Further investigation is needed to unravel novel pathways that can be targeted to prevent bone metastasis.

#1086

Phosphoproteomic analysis of signaling pathways driving bone metastasis in prostate cancer.

Benjamin Abbott, Andrew Pierce, Anthony D. Whetton, Paul A. Townsend. _University of Manchester, Manchester, United Kingdom_.

Osteoblasts play a fundamental role in prostate cancer bone metastasis, whereby dysregulated bone remodelling results in bone pain, fractures and increased mortality. Although it remains unclear how early on in the disease pathway prostate cancer cells populate the endosteal niche, osteoblasts almost certainly play a role in the homing of tumor cells to this microenvironment. In support of this, prostate cancer cells have been shown to home to osteoblast-rich areas in animal models of bone metastasis and human prostate cancer bone metastases are typically osteoblastic on radiography. We have shown that conditioned medium from mature, mineralising human hFOB1.19 osteoblasts (hFOB-CM) is rich in pro-inflammatory cytokines including IL-6, IL-8 and various chemokine ligands. hFOB-CM promotes a strong migratory and invasive phenotype in a panel of prostate cancer cell lines (including androgen-sensitive and -insensitive lines), with invading cells displaying increased actomyosin contractility and amoeboid-like traits, namely a rounded cell morphology, cytoskeletal reorganization and phosphorylation of myosin light chain. We now aim to comprehensively interrogate the intracellular signaling pathways driving endosteal homing in prostate cancer cells by systems-wide ITRAQ mass spectrometry-based phosphoproteomic analysis of prostate cancer cells treated with hFOB-CM. This approach will identify individual signaling pathways or combinations of pathways to target with existing kinase inhibitors to inhibit osteoblast-driven invasion and endosteal homing during prostate cancer metastasis. Ultimately, targeting tumor cell responses to stromal signals may prove critical in the search for better therapies to treat this frequently lethal disease.

#1087

Semaphorin 3C drives invasiveness in prostate cells through epithelial-to-mesenchymal transition and stemness.

Kevin J. Tam,1 Daniel H. Hui,1 Wilson C. Lee,2 Mingshu Dong,1 Tabitha Tombe,1 Ivy Z. Jiao,1 Shahram Khosravi,1 Ario Takeuchi,2 James W. Peacock,1 Larissa Ivanova,1 Igor Moskalev,1 Martin E. Gleave,1 Ralph Buttyan,1 Michael E. Cox,1 Christopher J. Ong1. 1 _University of British Columbia, Vancouver, British Columbia, Canada;_ 2 _Vancouver General Hospital, Vancouver, British Columbia, Canada_.

Our work sets out to show that semaphorin 3C is a driver of epithelial-to-mesenchymal transition and stemness in prostate cancer. Prostate cancer (PCa) is among the most commonly-occurring non-cutaneous cancers in men. Local non-invasive PCa is highly treatable but limited treatment options exist for those with locally-advanced and metastatic forms of the disease underscoring the need to identify mechanisms mediating PCa progression. The semaphorins are a large grouping of membrane-associated or secreted chemotactic proteins whose normal functions reside in embryogenesis and development where they are responsible for directing cell movement. Semaphorins act through autocrine, paracrine, and juxtacrine signaling and have been implicated in a broad range of biological functions ranging from tissue morphogenesis to immunity; altered semaphorin expression has also been observed in numerous cancers. One member of the class 3 semaphorins, semaphorin 3C (SEMA3C), has been implicated in several forms of cancer and its increased expression is correlated with prostate cancer severity. Additionally, SEMA3C has been shown to be upregulated in response to chemotherapy and radiation treatment, promote metastasis to the lung, and promote tumourigenicity of glioma cells. SEMA3C has also been documented to increase cell proliferation and migration, decrease apoptosis, and promote integrin signaling and VEGF secretion in endothelial cells. SEMA3C was shown to drive migration of breast cancer cells and recent studies have highlighted the importance and prognostic value of SEMA3C in prostate cancer. Given SEMA3C's roles in development and its augmented expression in PCa, we hypothesized that SEMA3C promotes cancer progression by driving mesenchymal and stem-like phenotypes. Other class 3 semaphorins have been shown to drive EMT and the link between SEMA3C and stemness has been established in glioma cells. In the present study, using gain of function studies coupled to gene expression (qPCR, Western blot, FACS, immunofluorescence microscopy) and functional studies (migration, invasion, and sphere-forming assays) we show that ectopic expression of SEMA3C in RWPE-1, a normal prostate epithelial cell line, promotes epithelial-to-mesenchymal transition and stemness. Specifically, we find that overexpression of SEMA3C leads to an upregulation of EMT markers and migratory and invasive phenotypes. SEMA3C overexpression was also associated with an upregulation of the cancer stem cell marker, CD44, and heightened sphere-forming capabilities. Additionally, using ultrasound-guided intracardiac injection of SEMA3C-overexpressing cells, we show that SEMA3C drives cell dissemination in vivo. We conclude from our studies that SEMA3C is a driver of prostate cancer by promoting epithelial-to-mesenchymal transition and stemness.

#1088

Defining bone metastatic potential of Abi1 CRISPR prostate cancer cell lines.

Alexander Nappi, Dawn Post, Disharee Das, Ishita Joshi, Megan Oest, Gennady Bratslavsky, Leszek Kotula. _SUNY Upstate Medical Univ., Syracuse, NY_.

Prostate cancer is the second most common cancer in American men, and it is the third leading cause of cancer-related deaths in American men. Distant stage and metastatic disease is often treated with androgen-deprivation therapy (ADT); however, this treatment ultimately fails, leading to castration-resistant prostate cancer (CRPC). This further emphasizes the need for better therapeutic targets. Our work focuses on understanding the role of the Abi1 gene in prostate cancer tumor progression. Our current in vitro data demonstrate an invasive phenotype with our CRISPR-mediated Abi1 knockout (KO) cell lines. In total, using CRISPR-mediated technology we have generated four Abi1 gene KO cell lines, PC-3, DU145, LnCaP, and RWPE-1. Importantly, the androgen-dependent cell line, LNCaP, demonstrated upregulation of Abi1 to the androgen receptor stimulation and the opposite response to the receptor inhibition upon treatment with enzalutamide. These data indicated that low Abi1 level could be induced by ADT therapies and are consistent with our previous observation of low Abi1/WAVE complex in prostate tumor samples. To learn more about this mechanism, we set out to investigate the role of Abi1 in castrated mice. The overall goal of the experiment is to compare the ability of cells lacking the Abi1 gene to engraft in bone under androgen deprivation condition and compare it to the control cells and examine any change in the engraftment phenotype. It is hoped that this research will lead to better understanding of the Abi1/WAVE complex role in CRPC.

Supported by NCI R01 CA16101.

#1089

Role of high mobility group A2 (HMGA2) in prostate cancer.

Ohuod A. Hawsawi, Liza Burton, Jodi Dougan, Ana Cecillia Millena, Peri Nagappan, Shafiq Khan, Valerie Odero-Marah. _Clark Atlanta Univ., Atlanta, GA_.

Prostate cancer is the most highly diagnosed cancer in men and the second leading cause of death in the United States. Epithelial-mesenchymal transition (EMT) plays a critical role in cancer progression and metastasis. High mobility group A (HMGA2), a non-histone protein, has been shown to promote EMT in separate studies. Interestingly, wild-type HMGA2 and truncated (lacking the 3'UTR) HMGA2 isoforms are overexpressed in many cancers, but have not been investigated in prostate cancer. Jun-D is a transcriptional factor that might be regulated by HMGA2. Moreover, the function for each individual isoform is still not well understood. We hypothesize that each of the two isoforms of HMGA2 in will have different functions in prostate cancer. We stably overexpressed wild-type HMGA2 and truncated HMGA2 in LNCaP cells and measured the expression and the localization of EMT markers. We also measured reactive oxygen species (ROS) levels using DCFDA dye, since ROS has been associated with EMT. We performed migration and cell viability assays as well as Jun-D knockdown, a putative downstream effector of HMGA2. Our results showed that the overexpression of HMGA2 in LNCaP cells led to an increase in cell viability and migration for both wild-type and truncated HMGA2. We observed that wild-type, but not truncated HMGA2 promoted EMT. Additionally, Jun-D and ROS levels increased for truncated, but not significantly for wild-type HMGA2. Jun-D knockdown with siRNA decreased ROS and cell migration in cells with truncated HMGA2, but not in cells overexpressing wild-type HMGA2. Therefore, wild-type HMGA2 is the isoform that induces EMT, while the truncated form appears to have an alternate role in upregulating ROS via Jun-D. In conclusion, we show a link between HMGA2 and JunD/ROS for the first time, and data that wild-type and truncated HMGA2 may have differing functions that converge to increase cell proliferation and migration, and may in future instruct on more precise therapeutic targeting in patients with these isoforms.

GRANT SUPPORT: NIH 1P20MD002285 and NIH/NCRR/RCMI G12RR003062-22

#1090

The role of connexin43 in prostate cancer motility.

Abdulaziz A. Aloliqi. _Kent State Univ., Kent, OH_.

Prostate cancer (PC) is one of the most common male cancers in United States. PC lethality is due to metastatic growth, when cancer cells move from the primary tumor site to a secondary site. A key step in metastsis is the acquisition of motility. Connexins (Cx) play a major role in cell-cell communication and growing evidence demonstrates their contribution to cell motility. Cx43 is up-regulated in cultured PC3 metastatic prostate cancer cells. To understand the role of Cx43 in migration we asked whether silencing Cx43 would decrease PC3 cell migration. Using cells in which short interfering RNA (sh-RNA) silenced Cx43 mRNA, we showed that silencing Cx43 decreased motility of sh-Cx43 PC3 cells in Boyden chamber assays. To confirm these results in another biological system, we performed cellular wound healing assays. Silencing Cx43 decreased motility of PC3 cells reducing movement to wounded area, leaving more area open in the Cx43 inhibited cell lines. The wound healing assay showed that knockdown Cx43 altered directional movement, velocity and distance. These results supported those of our Boyden assays showing that loss of Cx43 decreases the migratory ability of PC3. Conversely, the overexpression of Cx43 in LNCaP cells, non-migratory prostate cancer cells that lack Cx43 expression, increased their motility. A comparison of cell lines engineered to overexpress or to silence Cx43 confirms the importance of Cx43 in the motility of prostate cancer cells in vitro. Although the mechanism whereby Cx43 regulates motility is still unclear, our preliminary data revealed that Cx43 co-immunoprecipitated with N-cadherin, suggesting that they form a complex in PC3 cells. Cell fractionation studies of PC3 control cells showed that N-cadherin was located in the cell membrane and not detected free in the cytoplasm, but in sh-Cx43 PC3 cells that lack Cx43, the distribution of N-cadherin protein was altered and significant levels of N-cadherin were also detected in cytoplasmic fractions. Whether changes in N-cadherin distribution is a direct or indirect result of loss of Cx43 in sh-Cx43 PC3 cells, is unclear. However, modulation of Cx43 levels in prostate cancer cells does alter motility and N-cadherin distribution. These results indicate that Cx43 and N-cadherin are in close association in PC3 cells and their interaction may play an important role in prostate cancer cell motility. A better understanding of the requirements of migratory PC cells may lead to more effective targeting of key steps in PC progression to lethal metastatic disease.

#1091

Role of growth differentiation factor-15 in prostate cancer-bone environment: Understanding on bone metastasis.

Jawed A. Siddiqui, Sakthivel Muniyan, Parthasarathy Seshacharyulu, Satyanarayana Rachagani, Suprit Gupta, Mohd W. Nasser, Kaustubh Datta, Surinder K. Batra. _University of Nebraska Medical Center, Omaha, NE_.

Background: Prostate cancer (PCa) cells preferentially metastasize to bone and skeleton resulting in the poor 5-year survival of less than 1%. Growth differentiation factor-15 (GDF15) is overexpressed in several cancers, including PCa. However, the role of GDF15 in prostate cancer bone metastasis remains elusive. The interaction between cancer cells and the bone microenvironment forms a vicious cycle that increases bone deformities and drives tumor growth in the bone. Therefore, we question whether GDF15 secreted by the PCa cells alters the bone environment by uncoupling bone formation and resorption. Methods: Murine calvarial-derived osteoblast (MCO) cells were used for the functional effect of GDF15 on differentiation, proliferation, and mineralization. Mineralization of mouse bone marrow stromal cell (BMSCs) derived osteoblasts was determined by Alizarin Red S staining. Bone marrow macrophages (BMMs) were isolated from C57 mice and cultured along with M-CSF and RANKL to identify osteoclasts. Results: Conditioned media (CM) from PCa (LNCaP and C4-2B) cells and rhGDF15 increased osteoblast proliferation and differentiation in MCO cells. However, GDF15 deletion in PCa (LNCaP and C4-2B) cells prevented PCa-mediated osteoblast differentiation. Further, CM from PCa and rhGDF15 increased the alkaline phosphatase+ CFU-F (Colony Forming Unit- Fibroblast) in mouse bone marrow stromal cells culture. Supportively, stable knockout of GDF15 decreased mineralized nodule formation in MCO. qRT-PCR assay revealed that the PCa and rhGDF15 increased mRNA expression of osteoblast-related genes such as alkaline phosphatase, Runx-2, collagen type-1 and osteocalcin. PCa cells derived CM and rhGDF15 increase the osteoblast-derived osteoclastic signal (RANKL/OPG ratio), suggesting that GDF15 promotes RANKL-dependent osteoclastogenesis. Furthermore, rhGDF15 treatment increased TRAP-positive multinucleated cells, pit resorption and osteoclast-specific genes such as TRAP, Cathepsin K, Carbonic anhydrase and NFATc1 in BMMs. Conclusions: Altogether, our results suggest that GDF15 is crucial for PCa-mediated osteoblast differentiation and mineralization. GDF15 also increased bone marrow osteoclast formation and its function. Our finding suggests that GDF15 mediated increase in osteoblastic-RANKL further enhance osteoclast activity and favor PCa cell to metastasize to bone.

#1092

Defining the molecular phenotypes of metastatic castration-resistant prostate cancer sensitive to FGF pathway inhibition.

Mark P. Labrecque,1 Lisha G. Brown,1 Ilsa M. Coleman,2 Lawrence D. True,1 Lori Kollath,1 Bryce Lakely,1 Yu C. Yang,2 Holly M. Nguyen,1 Eva Corey,1 Peter S. Nelson,2 Colm Morrissey1. 1 _University of Washington, Seattle, WA;_ 2 _Fred Hutchinson Cancer Research Center, Seattle, WA_.

Background: Widespread and long-term use of first-and second-line androgen-deprivation therapy (ADT) is changing the molecular and phenotypic landscapes of prostate cancer. Observations made through our longstanding rapid autopsy and patient-derived xenograft (PDX) programs at the University of Washington support a shift in metastatic castration-resistant prostate cancer (mCRPC) towards androgen receptor (AR)-null phenotypes, such as neuroendocrine (NEPC) and double negative (DNPC). Currently, there are no effective therapies for AR-null mCRPC. We showed previously that DNPC (AR-null, NE-null) bypasses AR-dependence through fibroblast growth factor (FGF) signaling. However, the role of the FGF pathway in other molecular mCRPC subtypes remains to be determined.

Methods and Results: Here, we define four mCRPC subtypes that can be categorized by the presence or absence of functioning AR or RE1-silencing transcription factor (REST). Transcriptomic analysis of mCRPC specimens showed that AR and REST activity define four emerging mCRPC phenotypes: adenocarcinoma (AR+/REST+), amphicrine (tumor cells that co-express AR and NE markers, AR+/REST-), DNPC (AR-/REST+) and NEPC (AR-/REST-). Immunohistochemistry of mCRPC and PDX models for AR, prostate specific antigen, synaptophysin, chromogranin, and other clinically relevant markers accurately reflected the AR/REST transcriptomic signature classifications. Furthermore, loss of REST activity can, at least in part, be attributed to alternative splicing of REST mRNA by serine/arginine repetitive matrix protein 4 (SRRM4), leading to the translation of a truncated REST protein. PCR analysis of mCRPC identified the REST splice variant exclusively in amphicrine and NEPC specimens. RNA sequencing/GSEA, qPCR and immunoblot analyses determined that overexpression of SRRM4 or siRNA knockdown of REST in C4-2B (AR+) and PC-3 (AR-) prostate cancer cells promotes expression of neuroendocrine markers. Finally, we are conducting preclinical testing of the FGFR inhibitor CH5183284 in multiple PDX models representing the four mCRPC subtypes described above to delineate the impact of FGF pathway inhibition in all mCRPC subtypes.

Conclusions: Our data highlight the importance of AR and REST transcriptional programs in maintaining phenotypic stability in mCRPC and explain the phenotypic heterogeneity of mCRPC in the post-abiraterone/enzalutamide era. Understanding the mCRPC subtypes that depend on the FGF pathway for survival and proliferation will inform treatment and lead to the development of novel therapies for advanced disease.

#1093

Epithelial-to-mesenchymal transition conveys resistance to restrictive growth conditions through increased autophagic flux.

Lei Zhao,1 Jones Nauseef,1 Marisa Buchakjian,1 Matthew Barnes,2 Michael Henry1. 1 _University of Iowa, Iowa City, IA;_ 2 _University of San Francisco, San Francisco, CA_.

Tumor progression requires cancer cells to adapt to varying microenvironments both in the primary tumor and in metastatic end organs. Metastasis is enhanced by epithelial-to-mesenchymal transition (EMT), which facilitates invasiveness and confers resistance to local stresses, such as hypoxia and nutrient limitation, although EMT is not necessarily required for these processes. Among these stressors, growth-factor (GF) deprivation may induce or select for cells that bear the hallmarks of EMT (EMT-like cells). The mechanisms by which EMT-like cells survive GF deprivation (restrictive growth conditions), which may occur when metastatic cancer cells are removed from GFs resident in the primary tumor microenvironment, remain largely uncertain. Herein we show that EMT confers upon cells the ability to maintain proliferative potential, following extended serum deprivation. We found that EMT-like prostate cancer cells (TEM 4-18) demonstrate elevated survival and reduced senescence compared to their more epithelial counterparts (PC-3E) in both short- and long-term exposure to serum withdrawal. In addition, resistance to anchorage-dependent growth-induced apoptosis (Anoikis) due to elevated autophagy is also evident in EMT-like cells. Forced expression of snail1 conferred survival advantages to PC-3 cells. Conversely, CRISPR-mediated knockout of Zeb1 reversed EMT status in TEM4-18 cells and survival advantage in restrictive growth conditions. We found that EMT-like cells exhibit elevated autophagic flux. Genetic interference with autophagy in EMT-like cells reversed their survival advantage and increased senescence in restrictive growth conditions as well as anoikis resistance. Our results suggest that the metastatic capabilities of EMT-like cells depend not only on their established invasive characteristics, but also on increased autophagy tied to the EMT-like state, and its role in surviving restrictive growth conditions.

#1094

A role for olfactory-like chemosensory signaling in prostate cancer metastasis.

Hong Lam, Wanqu Zhu, Hailun Wang, Jun Luo, Qizhi Zheng, Ken Valkenburg, Paula Hurley, Jennifer L. Pluznick, Phuoc T. Tran, Angelo M. De Marzo, William B. Isaacs, Kenneth J. Pienta, Steven S. An. _Johns Hopkins University, Baltimore, MD_.

Odorant sensing G protein-coupled receptors (ORs) are the largest signaling family encoded by the mammalian genome. Historical understanding suggests these specialized receptors are only expressed on sensory neurons in the main olfactory epithelium and respond to external ligands involved in smell perception. More recently, olfactory-like chemosensory signaling has been found in a variety of non-sensory tissues and shown to be evoked by endogenous metabolic byproducts of anaerobic bacteria and hypoxia. Here we screened the full complement of ORs in the prostate and studied their expression and function in the context of metastatic-invasion of prostate cancer (PCa). In the germline of men with lethal PCa, we detected a large number of nonsynonymous coding changes in ~400 different OR genes including specific missense and loss-of-function mutations that are present in higher rates than would be predicted. RNA-Seq of clinical prostate samples showed higher OR51E2 expression in localized PCa than normal prostate. Most striking, we detected a low abundance of the OR51E2 transcript in PCa metastases to the bone relative to localized. Similarly, we found a marked reduction of OR51E2 expression in PCa cell lines that were derived from bone metastases, or those that had undergone an epithelial-mesenchymal transition (EMT) phenotype through co-culture with IL4-treated CD14+ monocytes. Using mouse models of PCa metastasis, we confirmed the overexpression of OR51E2 in localized tumors generated by subcutaneous injection while observing a marked reduction in PCa cells metastatic to the bone that also demonstrated a mesenchymal cellular phenotype. Together these findings establish, for the first time, a novel role for 'sensory GPCRs' in PCa progression to metastasis.

Grant supports: The Johns Hopkins Discovery Award; The Johns Hopkins Catalyst Award; Maryland Cigarette Restitution Fund

#1095

Insulin-like Growth Factor Binding Protein-3 (IGFBP-3) regulates mitochondrial dynamics, EMT and angiogenesis in progression of prostate cancer.

Arpit Dheeraj,1 Dhanir Tailor,2 Gagan Deep,3 Rana P. Singh1. 1 _Jawaharlal Nehru University, New Delhi, India;_ 2 _Stanford University, Palo Alto, CA;_ 3 _Wake Forest Baptist Medical Center, Winston-Salem, NC_.

Prostate cancer (PCa) is a major epithelial cancer among men and with the second highest incidence rate, worldwide. The high ratio of IGF-1/IGFBP-3 correlates with increased risk of many cancers including prostate cancer. In present study, we have evaluated the role of IGFBP-3 and effect of fisetin, a phytochemical, active constituent of strawberry, apple etc. in the progression of prostate cancer. The exogenous expression of IGFBP-3 moderately decreased the cell growth and it further decreased with addition of fisetin in DU145 and LNCaP cells. The restoration of IGFBP-3 in DU145 cells inhibited the clonogenic potential which is further decreased with fisetin treatment. Increased ROS content was observed in IGFBP-3 overexpression condition, however fisetin treatment reversed the ROS content in DU145, shows antioxidant behaviour. Morphological examination of mitochondria revealed that IGFBP-3 overexpression destabilizes the mitochondrial dynamics by reduction in active DRP1 level which has been reversed by fisetin treatment. We evaluated the effect on mitochondrial mass which was decreased in IGFBP-3 overexpressing DU145 cells which further decreased with addition of fisetin at 12 and 24 h. IGFBP-3 overexpression decreased the migratory potential of DU145 cells under normoxic conditions and under hypoxic condition it increased the migration of DU145 cells. Furthermore, IGFBP-3 overexpression decreased the VEGF expression as compared to vector control which can inhibit the expansion of tumor cells. Under the hypoxic conditions (1% oxygen), cells showed increased levels of IGFBP-3 when compared to normoxic conditions (21% Oxygen) in time dependent manner. The down-regulation of IGFBP-3 in PC3 cells, increased the expression of E-cadherin, a biomarker of epithelial to mesenchymal transition. The wound scratch assay showed the pro-migratory role of IGFBP-3 in PC3 cells and treatment with fisetin inhibited the migration of these cells under the hypoxic condition. The knockdown of IGFBP3 resulted in the decreased invasive potential of PC3 in comparison to cells in hypoxic state. Together with these observation, IGFBP-3 have shown biphasic character depending on normoxic and hypoxic condition in controlling the prostate cancer progression.

#1096

STAT3 pathway regulates the cancer-bone microenvironment interactions mediated by Snail.

Veronica M. Henderson,1 Ohuod Hawsawi,1 Liza J. Burton,2 Kennedi Trice,1 Jodi Dougan,1 Simone M. Howard,1 Valerie A. Odero-Marah1. 1 _Clark Atlanta University, Atlanta, GA;_ 2 _Emory University, Atlanta, GA_.

Prostate Cancer (PCa) is the second leading cause of cancer death in American men. PCa patients' mortality is mainly attributed to complications caused by metastasis of the disease to organs critical for survival, such as bone. As such, it is important to understand cancer-bone microenvironment interactions in order to develop therapeutics that will slow or halt the process of cancer metastasis. Snail1 is a zinc-finger transcription factor that induces epithelial-mesenchymal transition (EMT) which is associated with cell migration and metastasis in cancer. We hypothesized that cancer cell-bone interactions would promote higher calcium release from bone, more specifically by cancer cells overexpressing Snail, which would lead to increased paracrine cell signaling and migration. For this study, we utilized various prostate cancer cell lines: LNCaP (Snail-low), C4-2 (Snail-high), E006AA (Snail-high), E006AA HT (Snail-high) and C4-2 with stable Snail knockdown. Cancer cells were co-cultured with Hydroxyapatite (HA; inorganic component of bone) of different densities to represent the African American vs Caucasian bone ratio (since African Americans have higher bone density than any other race). The conditioned media was then used to assay calcium levels, perform paracrine migration assays using LNCaP cells, and examine paracrine signaling effects of the various conditioned media on LNCaP cells by western blotting. We observed that calcium levels were elevated in conditioned media from cancer cell-bone co-cultures, compared to media or cancer cells alone, and this could be antagonized by EGTA, a calcium chelator. C4-2 cancer-bone co-culture conditioned media increased paracrine cell migration which was decreased by Snail knockdown as well as lower bone density. We also observed increased STAT3 phosphorylation and paracrine cell migration in LNCaP cells incubated with conditioned media from C4-2, E006AA or E006AA HT cells co-cultured with HA; this phosphorylation and cell migration could be antagonized by Snail knockdown or STAT3 inhibitor (WP1066). An in vivo study was also done using nude mice that were surgically implanted with either 40mg HA or 100mg HA and subcutaneously injected with either C4-2 NS or C4-2 Snail shRNA cells into a position close to the shoulder from the surgical HA implant site in the mouse. At week 2 and week 4, Snail shRNA cells injected mice showed larger tumors than C4-2 NS injected mice, however, mice implanted with cancer cells plus higher bone density resulted in larger tumors than those with lower bone density. In conclusion, our study shows that Snail can mediate cancer-bone microenvironment interactions via STAT3 signaling, that can possibly promote increased paracrine cell migration towards bone of high mineral density. Therefore, targeting cancer-bone micronenvironmental interactions is an important avenue to consider for therapeutic targeting of prostate cancer.

#1097

The Phlpp2 phosphatase protects Myc and is a target for the prevention of prostate cancer metastasis.

Vincent D'Andrea,1 Dawid Nowak,2 Kaitlin Watrud,2 Alexandra Ambrico,2 Irene Casanova-Salas,2 Lloyd Trotman2. 1 _Zucker School of Medicine at Hofstra/Northwell, Hempstead, NY;_ 2 _Cold Spring Harbor Laboratory, Cold Spring Harbor, NY_.

Prostate cancer (PC) is one of the most prevalent cancers among men, causing almost 30,000 deaths per year in the US alone. Death caused by PC is mainly due to metastasis, and the 5-year survival rate of metastatic PC is only 28%. In contrast, the 5-year survival rate of PC confined to the prostate is nearly 99%. In contrast to primary PC tumors, metastatic tumors commonly present with targeted, bi-allelic mutations of Pten and Tp53 tumor suppressor genes. Whereas 49% of metastatic PC tumor samples have Pten/Tp53 co-deletions, these same co-deletions arise in only 10% of primary tumors. Because of the scarcity of human samples from metastatic disease, our lab has developed a genetically engineered mouse model (GEMM), in which Pten/Trp53 co-deletion using lentiviral plasmids gives rise to metastatic PC. As a result of the propensity of these mice to quickly develop metastatic PC, the model is termed RapidCaP. In the setting of our model for metastatic PC, we sought to investigate other genes which could be regulating the switch from a relatively benign primary PC to a highly lethal metastatic PC. Interestingly, most candidate tumor suppressor genes in PC are part of large recurrent hemizygous deletions, such as the common chromosome 16q-deletion, which involves the AKT-suppressing phosphatase, Phlpp2. Using our RapidCaP model, we find evidence that complete loss of Phlpp2 in the setting of Pten/Tp53 co-deletion paradoxically blocks prostate tumor growth and metastasis burden in RapidCaP mice. In fact, when Phlpp2 is deleted using a lentiviral vector, only 7% of the mice have PC tumors with metastasis compared to 60% in Pten/Tp53 co-deletion alone. Furthermore, we show that the Phlpp2 phosphatase activates Myc, a key driver of PC metastasis, by regulating Myc stability. Phlpp2 deletion in embryonic fibroblasts (MEFs) derived from RapidCaP mice does not affect Myc transcript levels by qPCR, but decreases Myc protein expression by over 75% as measured by Western blot. Using the same MEFs, we further show that the half-life of the Myc protein decreases from 19.3 minutes to 12.7 minutes upon deletion of Phlpp2 using lentiviral vectors. We propose that Phlpp2 dephosphorylates the threonine 58 (T58) site of Myc, thus directly increasing its stability. Importantly, we show that small molecule inhibitors of the Phlpp2 phosphatase can suppress Myc and cause cell death. Using the MEFs derived from the RapidCaP mice, Myc concentration decreases to an undetectable level in a dose-dependent manner following treatment with a Phlpp2 inhibitor between the concentrations of 0-200 υM, which coincides with a dose-dependent increase in cell death as measured by PI staining. A similar trend is seen in the PC3 cell line derived from human PC. In sum, our findings reveal that the frequent hemizygous deletions on chromosome 16q present a possible druggable vulnerability for targeting the Myc protein in metastatic PC with Phlpp2 phosphatase inhibitors.

#1098

Innate immune response to primary breast cancer prevents metastasis-initiating cell colonization.

Zafira Castano. _BWH/Harvard Medical School, Boston, MA_.

Current lack of insight into mechanisms governing breast cancer metastasis has precluded development of curative therapies. Previous reports, including our own, have demonstrated that metastatic success is due to tumor cell intrinsic properties as well as microenvironmental and systemic factors (Castano, et al., Cancer Discovery, 2013). The goal of our current study was to determine whether metastasis initiating cells (MIC), i.e., cells enriched for metastatic capacity, also rely on systemic cues. Using a preclinical model of the early phases of breast cancer metastasis, when cells are disseminated in the presence of the primary tumor (McAllister, et al., Cell 2008), we were surprised to find that certain primary tumors inhibit progression of disseminated MICs into overt tumors. In the presence of the primary tumor, MICs were maintained in a Zeb1hi/CDH1lo state and were less proliferative and poorly differentiated relative to those that formed in the absence of the primary tumor. We established that the primary tumor induces mobilization of IL1beta-expressing innate immune cells that are recruited to sites where MICs reside. At those sites, signaling via the IL1 receptor maintains MICs in the Zeb1hi/CDH1lo state, thus promoting disease indolence. Confirming our xenograft results, we established that expression of IL1beta in patient tumors is associated with metastasis free survival. Collectively, these data highlight the profound impact a primary tumor can exert on metastasizing cells - in this case, by altering the systemic environment to the detriment of secondary tumor growth-. Moreover, our data highlight a central role for IL1 pathway in modulating tumor cell plasticity and suggest it may provide a novel avenue for targeting recurrent disease.

#1099

Molecular drivers of neutrophil recruitment to primary non-small cell lung cancer.

Claire Wang,1 Roni Rayes,1 Jack Mouhanna,1 Betty Giannias,1 Arvind Chandrasekaran,2 Rachel Mot,2 Christopher Moraes,2 Sidong Huang,2 Jonathan Cools-Lartigue,1 Nicholas Bertos,1 Lorenzo Ferri,1 Jonathan Spicer1. 1 _Ri-MUHC, Montreal, Quebec, Canada;_ 2 _McGill University, Montreal, Quebec, Canada_.

Neutrophils are associated with developing cancer lesions and are the main immune component of primary non-small cell lung cancer (NSCLC). Multiple studies support the notion that tumor associated neutrophils (TANs) can promote tumor progression. We hypothesize that there is a hierarchy of molecular cues produced by developing lung cancers that guide circulating neutrophils to infiltrate the tumor microenvironment and become TANs. Identifying these cues may permit modulation of neutrophil infiltration within developing lung cancers and may thereby act as an immunotherapeutic tool to suppress cancer progression and improve response to existing therapeutics. To this end, we profiled 5 established NSCLC cell lines representing the common NSCLC subtypes using a qRT-PCR 84 gene panel (A549; KRAS mutant, PC9; EGFR mutant, H1993; MET amplification, H3122; EML4-ALK translocation, HCC78; ROS1 translocation). We focused on 4 of the most commonly upregulated genes in all cell lines, which were osteopontin (Spp1), vascular endothelial growth factor A (VEGF-A), macrophage inhibitory factor (MIF), and C-X-C motif ligand 1 (CXCL1). After confirming protein expression of these targets by western blot, we performed shRNA knock down (KD) of these genes and tested the migration of neutrophils towards treated and control cell lines in a novel microfluidic device that allows increased throughput studies of neutrophil attractants. Findings from KD experiments were confirmed via antibody-mediated inhibition. We observed a 3-fold increase of neutrophil migration to the A549 cancer cell line compared to the serum free control (p=0.0265). Furthermore, this increase was inhibited in Spp1 (64% decrease), MIF (84%), VEGF (82%) KDs and their corresponding neutralizing antibodies. We have therefore identified 4 proteins that play a key role in neutrophil recruitment to NSCLC cell lines in vitro and have demonstrated the application of a simple microfluidic device to test neutrophil migration patterns. This data provides the basis for in vivo investigations to elucidate the key molecular cues for neutrophil infiltration within developing lung cancers.

#1100

Screening the chemical library against granulocytic MDSCs: Critical player in breast cancer metastasis.

Hasan Korkaya,1 Eunmi Lee,1 Raziye Piranioglu,1 Thomas Albers,1 Maria Ouzounova,1 Charlie Weeks,1 Riley Rodier,1 Ahmet K. Korkaya,1 Khaled A. Hassan2. 1 _Augusta Univ., Augusta, GA;_ 2 _University of Michigan, Ann Arbor, MI_.

We recently demonstrated that infiltration of granulocytic MDSCs in distant organs is a critical step in metastatic process. The mouse transcriptome analysis of in vitro co-cultures and samples from syngeneic mouse model revealed that granulocytic subset of myeloid-derived suppressor cells (gMDSCs) from metastatic 4T1 tumor bearing mice are regulated a distinct set of genes. Two genes, S100A8 and S100A9 were highly upregulated in gMDSCs isolated from mice with metastatic 4T1 tumors compared to mice with non-invasive tumors. Using the recombinant single S100A8, S100A9 proteins or the S100A8/S100A9 heterotetramer (calprotectin), we demonstrated that calprotectin is a critical player in gMDSC induction and infiltration in distant organs particularly in lungs. Furthermore, injection of recombinant calprotectin but not single S100A8 or S100A9 proteins via tail vein enhanced the metastatic ability of nan-invasive EMT6 tumors in mice. We therefore performed computational screen of NCI compounds against the crystal structure of calprotectin and identified over 100 lead compounds. In vitro gMDSC differentiation assay identified 3 compounds with strong gMDSC inhibitory activity. We therefore designed a pilot experiment to test these compounds in preclinical mouse models. We also compared the activity of these compounds against tasquinimod, a small molecule targeting S100A9 only. However, tasquinimod treatment of 4T1 tumor-bearing mice had a moderate anti-tumor activity which may be due to a limited activity on granulocytic MDSC accumulation. In addition, we determined that there was a significant upregulation of S100A8 in MDSCs from tasquinimod treated mice. This data suggested that inhibiting only S100A9 leads to activation of S100A8 and thus may be ineffective targeting of MDSCs. In line with these data, one of the compounds (#10) showed a potent activity against gMDSCs in 4T1 tumor-bearing mice and significantly inhibited pulmonary metastasis. Moreover, this compound eliminated the cytotoxicity caused by cyclophosphamide and animals were free of cancer. In summary, we have identified a molecular target which regulates gMDSC induction and play critical role in metastatic process and our findings from in vitro screens and preclinical testing provide a strong rationale for targeting MDSCs.

#1101

Visualization of epithelial-mesenchymal transition in inflammatory microenvironment-colorectal cancer network in vitro and in vivo.

Hiroshi Tazawa,1 Takeshi Ieda,2 Shuya Yano,2 Satoru Kikuchi,2 Shinji Kuroda,1 Toshiaki Ohara,2 Kazuhiro Noma,2 Hiroyuki Kishimoto,2 Masahiko Nishizaki,2 Shunsuke Kagawa,2 Takashi Saitou,3 Takeshi Imamura,3 Toshiyoshi Fujiwara2. 1 _Okayama Univ. Hospital, Okayama, Japan;_ 2 _Okayama Univ. Grad. Sch., Okayama, Japan;_ 3 _Ehime Univ. Grad. Sch. Med., Toon, Japan_.

Background: Epithelial-mesenchymal transition (EMT) is a biologic process by which epithelial cancer cells acquire mesenchymal phenotype with malignant properties for invasion and metastasis, leading to poor prognosis. Inflammatory microenvironment has been shown to be responsible for the development and progression of colorectal cancer (CRC). However, the precise role of inflammatory microenvironment in the EMT-related tumor progression remains unclear because EMT network is regulated by complex crosstalk between tumor and normal cells. In this study, we developed a fluorescence-guided live-cell imaging system for the spatiotemporal visualization of EMT state, and assessed the in vitro and in vivo visualization of EMT state in human CRC cells under the inflammatory microenvironment.

Methods: We used two human CRC cell lines, HCT116 and RKO. The promoter of mesenchymal marker vimentin was used for inducing the EMT-dependent red fluorescence protein (RFP) expression in human CRC cells. The RFP expression was quantitatively analyzed to evaluate its association with EMT-related markers and malignant phenotype. Inflammatory cytokines, IL-1β (1 ng/ml) and TNF-α (20 ng/ml), or co-culture with inflammatory mouse macrophage cell line RAW264.7 stimulated by lipopolysaccharide (LPS) (200 ng/mL) were used as an inflammatory microenvironment. Biodistribution of EMT cells was analyzed using in vivo experiments with primary and metastatic CRC xenograft tumors.

Results: Inflammatory cytokines reversely induced the RFP expression in association with EMT-related malignant phenotype, such as morphologic change, EMT marker modulation, and invasion ability, in HCT116 and RKO cells. Co-culture with LPS-stimulated inflammatory macrophages also induced the RFP expression by secreting inflammatory cytokines. In vivo experiments revealed that EMT cells were detected within tumor tissues at colon, liver and peritoneum.

Conclusions: Our results suggest that inflammatory microenvironment has a great potential to induce EMT program in CRC cells. This fluorescence-guided EMT imaging system is a useful tool for monitoring the EMT state in the inflammation microenvironment-CRC network in vitro and in vivo.

#1102

IL-6/STAT3 activation in hepatocytes drives pro-metastatic niche formation in the liver.

Jae W. Lee,1 Stacy K. Thomas,1 Chad A. Komar,1 Whitney L. Gladney,1 Xia Hua,1 Dong Xin,1 Abraham Shaked,1 Mitesh J. Borad,2 Ramesh K. Ramanathan,2 Ailing Ji,3 Nancy R. Webb,3 Maria C. de Beer,3 Frederick C. de Beer,3 Paige M. Porrett,1 Gregory L. Beatty1. 1 _University of Pennsylvania, Philadelphia, PA;_ 2 _Mayo Clinic, Phoenix, AZ;_ 3 _University of Kentucky, Lexington, KY_.

The liver is the most common site of metastasis in pancreatic ductal adenocarcinoma (PDAC). This metastatic tropism is dependent, at least in part, on the formation of a "pro-metastatic" niche that supports tumor cell seeding and colonization in the liver. However, mechanisms that direct the formation of this niche remain poorly understood. We show using the LSL-KrasG12D/+;LSL-Trp53R172H/+;Pdx-1-Cre (KPC) model of PDAC that pancreatic tumor development enhances the susceptibility of the liver to metastatic seeding by inducing recruitment of F4/80+ and Ly6G+ myeloid cells and fibrosis within the liver. 3' mRNA sequencing (QuantSeq) on RNA isolated from the liver of KPC mice versus control PC mice revealed that the liver produces a specific set of myeloid chemoattractants, particularly serum amyloid A1 and A2 (SAA1/2), early during PDAC development. In addition, gene set enrichment analysis (GSEA) on genes upregulated in the liver of KPC mice demonstrated a significant enrichment of the interleukin 6 (IL-6)/Signal Transducer and Activator of Transcription 3 (STAT3) signaling pathway. Consistent with this finding, phosphorylation of STAT3 was detected in 20-30% of F4/80+ myeloid cells and 80-90% of hepatocytes. A requirement for IL-6/STAT3 signaling in the formation of a pro-metastatic niche was determined by comparing the metastatic potential of wild type mice, Il-6 knockout (Il-6-/-) mice, and mice treated with anti-IL-6 receptor (IL-6R) antibody after orthotopic implantation of KPC-derived PDAC cells. Compared to wild type mice, the liver of Il-6-/- mice and mice treated with anti-IL-6R antibody was less susceptible to metastatic seeding and showed significantly less accumulation of myeloid cells, fibrosis, and production of SAA1/2 in the liver. We obtained similar results with mice that lack Stat3 specifically in hepatocytes (Stat3flox/flox Alb-Cre), demonstrating that IL-6/STAT3 signaling in hepatocytes is necessary for the formation of a pro-metastatic niche in the liver. Further, using Saa1/2 double knockout (Saa-/-) mice, we found that SAA1/2 production by hepatocytes was required for formation of the pro-metastatic niche in the liver and increased susceptibility to metastatic seeding. Patients with a history of liver metastasis also showed higher levels of SAA1/2 in the plasma compared to normal donors, and SAA overexpression was detected in hepatocytes in liver biopsy samples collected from PDAC patients. Collectively, our study reveals a novel role for hepatocytes in directing the formation of a pro-metastatic niche in the liver during PDAC development and identifies IL-6/STAT3/SAA1/2 signaling as a promising therapeutic target for prevention of metastasis in PDAC.

#1103

Colorectal cancer-derived small extracellular vesicles establish an inflammatory premetastatic niche through macrophage polarization in liver metastasis.

Yingkuan Shao, Ting Chen, Xi Zheng, Shu Zheng. _Zhejiang University, Hangzhou, China_.

Liver metastases develop in more than half of patients with colorectal cancer (CRC) and indicate a poor prognosis. In this study, we aim to investigate CRC-derived small extracellular vesicles (CRC-sEVs) participating in liver-specific metastasis. Fluorescence-labeled CRC-sEVs were found to accumulate in macrophages in mouse liver and prime macrophage polarization toward an interleukin-6 (IL-6)-secreting pro-inflammatory phenotype (macrophage expresses different programs in response to different microenvironmental signals called macrophage polarization). By analyzing miRNA expression profiles of the CRC-sEVs and their parental cells (SW480 and SW620), we identified miR-21-5p (miR-21) as the top ranked miRNA that was highly upregulated both in the sEVs and their parental CRC cells. The majority of macrophage-secreted inflammatory signaling pathways are activated by Toll-like receptor (TLR). Macrophages treated with miR-21 analogs exhibited enhanced expression of pro-inflammatory cytokines such as IL-6 and TNF-α. Silencing either miR-21 in CRC-sEVs, or the Toll-like receptor 7 (TLR7) in macrophages which miR-21 binds to, abolished the effect of CRC-sEVs in inducing pro-inflammatory macrophage polarization. Upon intravenous injection, CRC-sEVs promoted miR-21 expression and macrophage infiltration in the mouse liver, followed by the up-regulation of inflammatory genes. Accordingly, the multiplicity of liver metastases was increased upon transplantation of CRC cells in mice treated with CRC-sEVs. Furthermore, miR-21 expression in plasma-derived sEVs positively correlated with tumor stage and liver metastasis in CRC patients. Finally, co-expression of the miR-21 and IL-6 macrophages markers was observed in liver metastatic tissues from CRC patients. Together, our data point to a pivotal role of CRC-sEVs in inducing an inflammatory pre-metastatic niche through the miR-21-TLR7-IL6 axis. Moreover, sEVs-miR-21 represents a potential prognostic marker and therapeutic target for CRC patients with liver metastasis.

#1104

Tumor-associated macrophages-secreted CXCL1 promotes breast cancer metastasis via activating NF-κB/SOX4 signaling.

Zhiyu Wang, Neng Wang, Weiping Liu, Yifeng Zheng, Shengqi Wang. _Guangzhou Univ. Of Chinese Medicine, Guangzhou, China_.

Purpose: Secreted cytokines play a significant role in modulating cancer metastatic microenvironment, especially for tumor associated macrophages (TAMs). The current study aimed to determine the significance of TAMs-secreted CXCL1 in regulating cancer metastasis and its underlying mechanisms. Experimental Design: Cytokine array was utilized to screen the highest chemokine secreted from TAMs. In vivo immunohistochemistry assay and in vitro wound healing or transwell experiments were carried out to demonstrate the metastatic-promoting effects of TAMs/CXCL1. QPCR profile screening was used to identify CXCL1 targeting metastasis-related gene. In vivo luciferase imaging experiments were designed to confirm the therapeutic significance of CXCL1 silencing on breast cancer metastasis. The clinical significance of CXCL1 was analyzed by human tissue microarray and bioinformatic analysis. Results: CXCL1 was validated as the highest secreted protein released from TAMs, and TAMs-derived CXCL1 could promote breast cancer migration and invasion ability, as well as epithelial-mesenchymal transition (EMT) process. QPCR screening validated SOX4 as the most responsive gene following CXCL1 administration. Mechanistic study revealed that CXCL1 activates SOX4 transcription via NF-κB pathway. CXCL1 silencing in TAMs resulted in a significant reduction in breast cancer growth and metastatic burden. Clinical investigation suggests that high CXCL1 expression was significantly correlated with breast cancer lymph node metastasis, poor overall survival and basal-like subtype. Conclusions: TAMs/CXCL1 promotes breast cancer metastasis via NF-κB/sox4 activation, and CAV1 based therapy might become a novel strategy for breast cancer metastasis prevention.

#1105

Tumor cells of the early metastatic process show up regulation of cytokine genes involved in migration of inflammatory cells.

Coya Tapia, Xiaoping Su, Ximing Tang, Lakshmi Kakaral, Aysegul Sahin, Wei Lu, Ignacio I. Wistuba. _UT MD Anderson Cancer Center, Houston, TX_.

Background: Patients with metastatic breast cancer have poorer prognosis and distant metastasis is the main cause of breast cancer (BC) deaths. Hence, better understanding of the early metastatic process is needed. Most BC metastasize primarily lymphogenic and tumor cells within the lympho-vascular space are the first clear visible step in the metastatic process. However, these cells are hardly investigated since the tumor area is very small and therefore difficult to analyze. In this study we wanted to show that tumor cells within the lympho-vascular space can be analyzed for gene expression and that differences compared between main tumor mass and lympho vascular invasion (LVI) occur. Methods: We included 20 BCs diagnosed at UT MD Anderson Cancer Center between 2010 and 2015. The cohort showed the following characteristics: Invasive ductal (n=18), lobular (n=1), ductulo-lobular (n=1), ER positive (n=18), ER negative (n=2), PgR positive (n=15), PgR negative (n=5), Her2 negative (n=16), and Her2 positive (n=4). Suitable FFPE tissue blocks were identified for all BCs. Ten µm sections were performed, air-dried and stained with methyl-green for visualization of the morphology. Macro-dissection and measurement of the tumor area (LVI) was done under a stereo microscope. Afterwards, HTG Seq quantitative assay of the oncology biomarker panel (2,560genes) was performed with some modifications to the standard protocol. Results: The macro-dissection was successful for both, LVI and main tumor mass, in 19 BCs. The dissected areas were between 11-60mm2. Eighteen samples could be included for RNA expression analysis. The main tumor mass and LVI showed clearly different expression patterns (clustering). LVI showed down regulation of genes coding for collagens (COL3A1, COL5A1), or genes involved in the interaction with the extracellular matrix (SPARC, VCAN). Up regulation was seen for genes coding for chemotactic factors involved in migration of inflammatory cells such as CCL18 for B-lymphocytes, CCL27 that seems to play a role in homing of lymphocytes to the skin, or CCL2 that is involved in recruiting monocytes and basophils. Conclusion: We showed that RNA expression analysis is feasible on small amount of tumor cells dissected from LVI that were stained with methyl green. Additionally, we could show that tumor cells from the main tumor mass and LVI have different gene expression levels and show clear clustering. Down regulation was seen in genes interacting with the extracellular matrix may indicating that these interactions are not needed when floating in lymphatic vessels. Interestingly, LVI showed upregulation of genes involved in the migration of white blood cells what could be an indication that tumor cells might relay on the same cytokines for migration in lymphatic vessels as inflammatory cells.

#1106

A synthetic pre-metastatic niche mimic alters the primary tumor and tumor microenvironment.

Brian A. Aguado,1 Rachel M. Hartfield,2 Grace G. Bushnell,2 Joseph T. Decker,2 Samira M. Azarin,3 Dhaval Nanavati,4 Matthew J. Schipma,4 Shreyas S. Rao,5 Robert S. Oakes,2 Yining Zhang,2 Jacqueline S. Jeruss,2 Lonnie D. Shea2. 1 _University of Colorado Boulder, Boulder, CO;_ 2 _University of Michigan, Ann Arbor, MI;_ 3 _University of Minnesota, Minneapolis, MN;_ 4 _Northwestern University, Chicago, IL;_ 5 _University of Alabama, Tuscaloosa, AL_.

Background: Immune cells at the primary tumor (PT) and pre-metastatic niche (PMN) sites are critical to metastasis progression. Recently, synthetic biomaterial scaffolds used as PMN mimics were shown to capture both immune and metastatic tumor cells in vivo (1-3). The redirection of tumor cells toward the implant also reduces tumor burden and provides a survival benefit in orthotopic breast cancer mouse models (4). Given the scaffold reduces tumor burden, we hypothesized the scaffold modulates PT and immune cell phenotypes to generate an invasion-suppressive tumor microenvironment (TME).

Methods: Female NOD scid gamma (NSG) mice inoculated with tdTomato+ MDA-MB-231BR cells in the mammary fat were used as orthotopic human breast cancer models. Poly(lactide-co-glycolide) scaffolds were implanted in the intraperitoneal fat pad 7 days post-tumor inoculation (3). Mock surgery mice did not receive an implant. After 28 days, mock vs. scaffold PTs were harvested, digested, and live-cell sorted to obtain tdTomato+ tumor cells and CD45+ immune cells. The PT cell transcriptome was analyzed using RNAseq and GO analysis was performed in Metascape. CD45+ immune cells were cultured to obtain conditioned media (CM) for secretomics analysis, in vitro tumor cell invasion assays, and transcription factor activity arrays (2). Immune cells, specifically tumor associated macrophages (TAMs) (5), were characterized using flow cytometry and RT-PCR.

Results: RNAseq analysis identified 892 differentially expressed genes in tumor cells in response to the scaffold implant, and Metascape GO analysis revealed signaling pathways relevant to invasion. The scaffold immune-CM decreased tumor cell invasion more than two-fold relative to mock CM. Secretomics analysis showed an increase in the pan-metastasis inhibitor decorin and a decrease in invasion-promoting CCL2 in the scaffold immune cell CM relative to mock CM. Reduced NFkB, SRF, and RAR transcription factor activity in tumor cells treated with scaffold CM relative to mock CM indicate immune secreted factors contribute to the invasion-suppressive scaffold-influenced TME. Finally, analysis of PT immune cells identified a recruited TAM population whose transcriptomic profile may contribute to the invasion-suppressive TME in scaffold bearing mice.

Discussion: We demonstrate that implanted scaffolds can distally modulate secretomic and transcriptional profiles of immune cells in the TME and may reduce PT cell invasion. Our work suggests scaffolds have an active role in modulating tumor burden and may provide a foundation for developing an effective implantable therapeutic tool.

References: (1) Aguado et al, Acta Biomater 33, 13-24, 2016. (2) Aguado et al, Sci Rep 5, 17566, 2015. (3) Azarin et al, Nat Commun 6, 8094, 2015. (4) Rao et al, Cancer Res 76, 5209-5218, 2016. (5) Franklin et al, Science 344, 921-925, 2014.

#1107

Cell-surface vimentin-positive macrophages like CTCs as novel biomarkers of metastatic gastrointestinal stromal tumors.

Heming Li,1 Qing H. Meng,2 Hyangsoon Noh,2 Neeta Somaiah,2 Keila E. Torres,2 Xueqing Xia,2 Izhar Singh Batth,2 Cissimol P. Joseph,2 Zachary A. Mulder,2 Ruoyu Wang,1 Shulin Li2. 1 _Affiliated Zhongshan Hospital of Dalian University, Houston, TX;_ 2 _UT MD Anderson Cancer Ctr., Houston, TX_.

Background: Gastrointestinal stromal tumors (GISTs) are the most common gastrointestinal tract sarcomas and are heavily infiltrated with tumor-associated macrophages (TAMs). Biomarkers specifically capturing circulating tumor cells (CTCs) that can be used to noninvasively determine the disease status in GIST patients have been lacking.

Patients and Methods: To fill this gap, we captured both regular CTCs and TAMs from GIST patients' blood and tumor samples using our proprietary cell-surface vimentin (CSV) antibody 84-1 and the TAM markers CD14 and CD68 based on our previous established CTC detection method from 104 localized or metastatic sarcoma patients and 10 healthy donors. Also, freshly procured metastatic GIST tissues were obtained from four patients.

Results: We defined a rare population of CSV+ macrophage-like CTCs (ML-CTCs) in metastatic GIST patients' blood samples with expression of CD14 and CD68 but not CD45. Also, we demonstrated CSV+ ML-CTCs to be tumor microenvironment-derived. Metastatic GIST patients had markedly higher numbers of CSV+ ML-CTCs than localized GIST patients and healthy blood donors. However, regular CTC counts could not predict metastasis of GISTs but could for other types of sarcoma. Notably, CSV+ ML-CTCs were represented as M1-like macrophages in their phenotype and function.

Conclusions: CSV+ ML-CTCs are novel biomarkers for prediction of metastatic risk and therapeutic response in GIST patients. Routinely monitoring these cells will be an important approach to liquid biopsy analysis of GISTs.

#1108

M2-polarized macrophages increase invasiveness of EBV-associated nasopharyngeal carcinoma by inducing invadopodia formation.

Wilson Wing Chung Tang,1 Chan Ping You,1 Gareth E. Jones,2 George Sai-Wah Tsao,1 Anna Chi Man Tsang1. 1 _The University of Hong Kong, Hong Kong, Hong Kong;_ 2 _King's College London, London, United Kingdom_.

Cancer metastasis is a major cause of mortality, accounting for approximately 90% of cancer related deaths. Emerging evidences suggest that tumour microenvironment plays an indispensable role in cancer metastasis. Recruitment of innate and adaptive immune cells to the tumour results in chronic inflammation which favours tumour growth and progression. Particularly, tumour associated macrophages (TAMs) are found to promote the initiation, growth, and metastasis in several types of cancers. Nasopharyngeal carcinoma (NPC) is one of the highly invasive and metastatic cancers and is closely associated with EBV infection. We postulate that TAMs may increase the invasiveness of NPC by enhancing the formation of invadopodia, which are membranous actin-rich protrusions with digestive ability of extra-cellular matrix. In this study, monocyte THP-1 cells were polarized to M2-macrophages, and co-cultured with nasopharyngeal cells in a contained chamber-insert. The stimulated nasopharyngeal cells then formed massive numbers of invadopodia and digested larger area of gelatin compared with the unstimulated counterparts. A cytokine antibody array was used to identify the upregulated cytokines in the co-culture medium of the M2-macrohphages and the NPC cells. Tumour necrosis factor alpha (TNFα) was revealed to be the responsible cytokine to induce the formation of invadopodia and digestion of gelatin. Besides, western blot analysis identified that several actin-associated proteins such as Src, Erk, and cortactin were involved in the TNFα-mediated invadopodia formation. We also found that the expression of latent membrane protein 1 (LMP1), an EBV protein that has similar structure to TNF receptor was elevated in NPC cells after co-culturing with the M2-polarized macrophages. Through transient transfection and stable expression of LMP1 in the NPC cells, LMP1 could also promote the formation of invadopodia through cdc42 activation. Taken together, these data indicate macrophages can promote the invasiveness of EBV-infected NPC cells by enhancing their ability in forming invadopodia through an activated TNFα signaling axis and LMP1 upregulation.

#1109

S100A10 contributes to cancer stemness, invasion and metastasis of head and neck cancer cells.

Nobuyukit Tanaka, Taketo Nishikawaji, Naoko Ogama. _Miyagi Cancer Ctr. Research Inst., Natori, Japan_.

Head and neck cancer is one of the most common cancers worldwide, and an innovating therapy is awaited. We attempted to identify proteins associated with cancer stem cell phenotypes, and found an EF-hand protein S100A10 is highly expressed in the stem cell population. Together with its binding partner, Annexin-A2, S100A10 regulates a spectrum of dynamic membrane-related events, such as actin cytoskeleton control, vesicle formation and intercellular interaction. Immunohistochemical analysis using clinical head and neck cancer specimens showed that elevated S100A10 expression is typically present in the invasion front of tumors, and S100A10-high cases were associated with poor prognosis. S100A10 knockdown and knockout resulted in significant reduction of cell migration and invasion in vitro. Cell movement was slowed down by S100A10 knockdown. Accordingly, in vivo tumor formation was impaired by S100A10 depletion. Cell cycle progression was delayed by the depletion, suggesting its potential role in proliferation controls. These results suggest that S100A10 has a significant role in head and neck cancer cell proliferation and invasion.

#1110

Ganglioside GM2 mediated tumor growth, progression, and metastasis involves YAP-dependent transcriptional program.

Barun Mahata, Abhisek Sarkar, Elora Khamrui, Sohini Chakraborty, Zhumur Ghosh, Kaushik Biswas. _Bose Institute, Kolkata, India_.

Purpose: Recent report from our laboratory showed a novel role of tumor derived ganglioside, GM2 in mediating tumor cell migration as well invasion. In the present study we report a novel role of GM2 in mediating anchorage independent growth (AIG) and epithelial-mesenchymal transition (EMT) leading to tumor growth, progression and metastasis, through a HIPPO-YAP dependent transcriptional program.

Experimental procedures : Permanent GM2-synthase knockout (KO) cells were generated using targeted genome editing tool, TALEN. TALEN was used to generate Renca-vGM2-syn KO cells (from a GM2-over-expressing variant of a mouse kidney cancer cell line, Renca-v). Renca-vWT as well as Renca-vGM2-syn KO cells were used to define the role of GM2 in AIG, EMT and tumorigenesis both in vitro and in vivo. DNA microarray analysis was used to elucidate the mechanism behind GM2-mediated AIG and EMT. To define involvement of YAP in GM2-mediated EMT, real time PCR was used to validate YAP-target gene expression in GM2-synthase KO or silenced (siRNA) cells as well as in cells treated with exogenous GM2, in the presence or absence of specific inhibitors of the Hippo-YAP signaling pathway. CRISPR-Cas9 mediated YAP/TAZ-double KO (HelaYAP/TAZ KO) cells were used to conclusively prove the involvement of YAP/TAZ in GM2-mediated EMT.

Results : Renca-vGM2-syn KO cells show reduced AIG, higher cell adhesion possibly through increased anoikis sensitivity, indicating a critical role of GM2-synthase and complex gangliosides in GM2-mediated EMT. Furthermore, Renca-vGM2-syn KO cell line shows reduced tumor growth potential, significant reduction in experimental lung metastasis with increased median survival in a syngeneic mice tumor model. Gene expression profiling using DNA microarray with GM2-synthase silenced cells reveal significant modulation of YAP target genes, suggesting a critical role of the Hippo-YAP signaling axis. Molecular insight reveals that GM2-synthase knockout or siRNA mediated knockdown results in significant downregulation of YAP activated genes namely Ctgf, Cyr 61, Pdgf-c, Lox, etc. and upregulation of YAP repressed gene Ddit4, while exogenous addition of GM2 in GM2 deficient cells (Renca, MCF-7 and Hela) show opposite results. Verteporfin mediated disruption of YAP transcriptional program abrogated GM2 mediated modulation of YAP target genes. Finally, significant decrease in YAP-target gene expression in YAP/TAZ-double KO cells, but not in either YAP or TAZ (paralogue of YAP) single KOs confirm the definitive involvement of YAP/TAZ in GM2-mediated EMT.

Conclusion : Our findings confirm a novel role of GM2 in triggering EMT by targeting YAP, ultimately leading to increased tumorigenic potential and metastatic activity in tumor cells.

#1111

Role of a prometastatic miRNA as a negative regulator of the key metastasis suppressor genes in renal cell carcinoma.

Nadeem S. Bhat,1 Altaf A. Dar,2 Sharanjot Saini,1 Varahram Shahryari,1 Soichiro Yamamura,1 Yuichiro Tanaka,1 Taku Kato,1 Yutaka Hashimoto,1 Marisa Shiina,1 Priyanka Kulkarni,1 Pritha Dasgupta,1 Z Laura Tabatabai,1 Guoren Deng,1 Rajvir Dahiya,1 Shahana Majid1. 1 _UCSF VA Medical Ctr., San Francisco, CA;_ 2 _CPMC, Research Institute, San Francisco, CA_.

Background: Renal cell carcinoma (RCC) is among the ten leading cancer types in United States. The 5-year relative survival rate of regional or localized RCC is 65-92%, while that of metastatic RCC is only 12%. Tumor recurrence and metastasis represent two major obstacles in the successful treatment of cancer. Emerging lines of evidence suggest that cancer aggressiveness is associated with epithelial to mesenchymal transition (EMT). Therefore, it is of critical importance to regulate EMT and to develop effective therapeutic strategies for the treatment of recurrent and metastatic cancer. Critical regulators of EMT include transcription repressors and microRNAs that target key proteins involved in EMT. MicroRNAs are implicated in regulating cancer progression and metastasis. Here we show that miR-720 is positively associated with RCC by negatively regulating key metastasis suppressing genes.

Methods: We performed a series of in vitro and in vivo experiments including qRT-PCR, FACS cell cycle and apoptosis, chemotactic transwell migration and invasion, immunoblotting and luciferase reporter assays along with intratumoral xenograft mouse model.

Results: Elevated levels of miR-720 were observed in a panel of RCC cell lines and clinical tissues compared to non-malignant cell line and normal samples. Loss of miR-720 function inhibited proliferation, migration and invasion and induced apoptosis in RCC cell lines in vitro and repressed tumor growth in xenograft mouse model. Conversely, gain of miR-720 function in non-malignant HK-2 cells induced pro-cancerous characteristics. Silencing of miR-720 caused a marked induction in the levels of endogenous αE-catenin and E-cadherin protein levels in anti720 transfected cells compared to control. Whereas, miR-720 overexpression in RCC cell lines reduced activity of a luciferase reporter gene fused to the wild-type αE-catenin or E-cadherin 3' UTR compared to non-specific 3' UTR control indicating that αE-catenin-E-cadherin complex is a direct and functional target of miR-720 in RCC. We also observed attenuation of β-Catenin, CD44 and Akt expression in RCC cells transfected with miR-720 inhibitor compared to control. Further, miR-720 exhibited clinical significance in RCC. Expression of miR-720 significantly distinguished malignant from normal samples. Elevated miR-720 levels positively correlated with higher Fuhrman grade, pathological stage and poor overall survival of RCC patients.

Conclusion: These findings uncover a new regulatory network in RCC involving metastasis-promoting miR-720 that directly targets expression of key metastasis-suppressing proteins E-cadherin and αE-catenin complex. These results suggest that therapeutic regulation of miR-720 may provide an opportunity to regulate EMT and metastasis in RCC.

#1112

AKT2 loss impairs BRAF mutant melanoma metastasis.

Siobhan K. McRee,1 Jodie R. Pietruska,1 Philip N. Tsichlis,2 Phil W. Hinds1. 1 _Tufts Univ., Boston, MA;_ 2 _Tufts Medical Center, Boston, MA_.

Despite recent advances in treatment, melanoma remains the deadliest form of skin cancer, due to its highly metastatic nature. Many melanomas with oncogenic BRAFV600E lose the tumor suppressor PTEN, leading to unconstrained PI3K/AKT signaling and a drastic increase in melanoma invasiveness, but the contribution of AKT to melanoma metastasis has not been fully explored. Differential roles for AKT1 and AKT3 have been suggested in many cancers, but a requirement for AKT2 has not been described in melanoma. We report here that shRNA-mediated, conditional knockdown of AKTs in a variety of human melanoma cell lines reveals that selective AKT2 depletion inhibits migratory and invasive phenotypes in vitro, and prevents metastasis in vivo. Our results support a critical role for AKT2 in stimulating melanoma cell migration and invasion and in supporting growth of metastatic lesions. As AKT inhibitors advance in clinical trials for melanoma, our findings could inform therapeutic strategies to improve treatment options and outcome for this devastating disease.

#1113

PODXL1 as a critical accelerator for pancreatic cancer metastasis.

Eisaku Kondo, Ken Saito. _Niigata Univ. Grad. School of Medical Dental Sci., Niigata, Japan_.

Podocalyxin-like 1 (PODXL1) is an anti-adhesive transmembrane protein belonging to the CD34 family and also stem cell marker that has been associated with an aggressive tumor phenotype and poor prognosis in several forms of cancer. In this study, we report the biological role of PODXL1 in progression of pancreatic invasive ductal adenocarcinoma (PDAC), especially focusing to invasion and metastasis of PDAC cells. Knockout of PODXL1 gene in three different human PDAC lines, MiaPaCa-2, AsPC1 and Panc-1, almost completely abrogated metastatic lesion in liver-metastatic mouse model in vivo. Molecular analysis revealed that PODXL1 functioned as a critical activator to multiple cytokine receptors expressed on cancer cells, and its interaction facilitated PDAC cell invasion. Endogenous expression of PODXL1 was strongly detected on cancer cells at the invasive front of the PDAC tissues, where coincidently expressed specific cytokine receptor that critically mediates cancer metastasis in vivo. Thus, we identify the novel functional aspect of PODXL1 and its biological role on PDAC as one of the most intractable malignancies.

### Regulation of Stemness in Cancer

#1114

IGFBP7 reduces acute myeloid leukemia stem cell survival without affecting normal hematopoiesis.

Han J. Verhagen, Noortje van Gils, Anna van Rhenen, Arjo Rutten, Marjon Smit, Mei-Ling Tsui, Louise L. de Vos Klootwijk, Renee X. Menezes, Meyram Cil, Margaretha G. Roemer, Fabio Brocco, Fedor Denkers, Eline Vermue, Stan Heukelom, Sonja Zweegman, Jeroen J. Janssen, Gert J. Ossenkoppele, Gerrit Jan Schuurhuis, Linda Smit. _VU University Medical Center, Amsterdam, Netherlands_.

Only 30-40% of acute myeloid leukemia (AML) patients survive five years after diagnosis. This poor prognosis is mainly caused by treatment failure due to chemotherapy resistance and relapse. Leukemic stem cells (LSCs) are thought to be the major determinants of AML relapse due their potential for self-renewal and chemotherapy resistance. It has been demonstrated that LSC frequency and expression of LSC gene expression signatures is highly predictive of therapy failure in AML patients, indicating the clinical importance of LSCs. LSCs co-reside with residual normal hematopoietic stem cells (HSCs) in the diseased bone marrow. Increasing the dose of chemotherapy might eliminate LSCs, however will inevitable result in non-specific elimination of HSCs, delaying or preventing recovery of normal hematopoiesis after treatment. To significantly improve AML patients outcome, there is an urgent need for identification of alternative therapies that specifically eliminate LSCs while sparing HSC. We generated gene expression profiles of HSCs, LSCs and leukemic progenitors all derived from the same AML bone marrow and identified insulin growth factor binding protein 7 (IGFBP7) as one of the top differentially expressed genes. We found that low IGFBP7 is a feature of LSCs, associated with reduced chemotherapy sensitivity and that IGFBP7 is frequently downregulated at relapse as compared to AML diagnosis. Together, these results suggest that there is a survival advantage of IGFBP7low AML cells during chemotherapy treatment. To test whether increasing IGFBP7 levels might be a strategy to deplete leukemic (stem) cells, we overexpressed IGFBP7 or added recombinant human IGFBP7 (rhIGFBP7) to primary AML cells. Strikingly, IGFBP7 overexpression or addition of rhIGFBP7 led to induction of differentiation and cell death in AML patient cells. Moreover, rhIGFBP7 reduces leukemic engraftment in an AML xenograft mouse model, reverses a stem-like gene signature, and inhibits AML blast and leukemic stem/progenitor cell survival in vitro and in vivo, while it had no influence on normal hematopoietic (stem) cell survival. Our findings define an association between chemotherapy sensitivity and IGFBP7 expression levels in primary AML cells, and indicate that treatment of AML patients with a combination of rhIGFBP7 and chemotherapy might reduce AML residual disease and LSC survival.

#1115

A CRISPR/Cas9 kinome screen identifies GRK5 as a novel regulator of rhabdomyosarcoma (RMS) self-renewal.

Thao Q. Pham, Terra Vleeshouwer-Neumann, Eleanor Chen. _University of Washington, Seattle, WA_.

Rhabdomyosarcoma (RMS) is a devastating pediatric soft tissue sarcoma. Clinical outcome for patients with relapsed or metastatic disease remains poor. However, current treatment options remain relatively unchanged over the last two decades, so there remains an urgent need for more effective treatment options for disease relapse. Cancer stem cells (CSCs), also known as tumor-propagating cells (TPCs), have the capacity for self-renewal and have been proposed to be responsible for tumor relapse and metastasis. RMS has been shown to follow the CSC model of tumor progression. We have previously performed an siRNA library screen against the human kinome (714) and a secondary CRISPR/Cas9 screen of top candidates to narrow down to 11 novel kinase candidates that are potentially essential for self-renewal of human RMS cells. GRK5, a G-protein coupled receptor kinase, was among the top candidate genes. Preliminary data from in vitro cell-based assays and in vivo limiting-dilution experiments in xenografts showed that CRISPR/Cas9-mediated targeted disruption of GRK5 resulted in significant reduction in the self-renewal capacity of RMS cells. GRK5-deficient RMS cells also displayed significant reduction in growth secondary to cell cycle arrest at the G2/M checkpoint. The functional effects on tumor growth and self-renewal appeared unique to GRK5 among the GRK kinase subfamily, as targeted disruption of closely-related GRK4 and GRK6 did not significantly affect RMS tumor cell growth and self-renewal. We propose that targeted disruption of GRK5 by CRISPR/Cas9 results in elevated p53 protein stability and activity, leading to inhibition of proteins required for cell cycle progression. We are also characterizing the loss-of-function effects of GRK5 in a RMS TPC reporter cell line created using a CRISPR/Cas9 knockin approach that will allow for direct assessment of GRK5 and other candidate gene function within the TPC population. By characterizing the biologic role of TPC as well as the function of GRK5 in RMS, we hope to provide new biologic insights into cancer stem cell biology and GRK5 as a novel therapeutic target for the treatment of advanced RMS.

#1117

Norrin signaling is protumorigenic in glioblastoma progression.

Ahmed A. Elsehemy,1 Hayden Selvadurai,2 Katherine Rowland,2 Peter Dirks,2 Valerie Wallace1. 1 _University of Toronto, University Health Network, Toronto, Ontario, Canada;_ 2 _The Hospital for Sick Children, Toronto, Ontario, Canada_.

Norrin signaling is protumorigenic in GBM progression

A-Introduction:

Norrin, a secreted factor encoded by the Norrie Disease Protein gene (NDP), is an atypical WNT ligand that specifically binds the FZD4 receptor and activates the canonical WNT signaling pathway in endothelial cells to regulate vascular development and barrier function in the brain. Recently, our group identified a novel tumor inhibitory function of NDP/FZD4 signaling in the endothelium in mouse models of medulloblastoma. Here we investigated the function of the NDP/FZD4 signaling axis in glioblastoma (GBM) progression.

B-Experimental approaches

GBM patients' survival data was obtained from TCGA and analyzed using the cBioportal server. Low passage mitogen expanded GBM cancer stem cell lines were established from primary tumors using standard conditions. NDP expression in experimental cell lines was modulated using lentiviral delivery of short hairpins or full length cDNA. Cell number was assessed by trypan blue cell counting, and sphere forming cell frequency was tested using extreme limited dilution assay (ELDA). For in vivo assessment of tumorigenesis, experimental and control cell lines were xenografted intracranially in NOD/SCID/Gamma (NSG) mice and symptomatic animals were euthanized and brains collected for histological analysis.

C-Results

Analysis of GBM data from TCGA showed that survival and NDP expression are negatively GBM. Gene expression analysis indicated variable expression levels of NDP/FZD4 signaling pathway components in a panel of 10 primary GBM stem cell lines. In vitro gain and loss of function studies showed that NDP promotes proliferation and sphere formation in GBM. Moreover, the effects of are recapitulated in xenografts. NDP appears to control GBM differentiation, as it affects the proportion of cycling (Ki67+) cells and the level of stemness (Sox2 levels). Finally, NDP function is independent of FZD4 and canonical Wnt signaling, indicating that it functions through an alternative pathway.

D-Conclusions Our in vitro and in vivo results indicate that Norrin might have an oncogeneic role in GBM. In addition, the effect of Norrin is FZD4- and WNT-independent. Our results indicate that targeting Norrin GBM might be a potential therapeutic approach.

#1118

DOT1L epigenetically regulates cancer stem cell properties and tumor progression in glioblastoma brain tumor stem cells.

Danielle Bozek, Xiaoguang Hao, H. Artee Luchman, Samuel Weiss. _University of Calgary, Calgary, Alberta, Canada_.

The median survival for patients diagnosed with Glioblastoma (GBM) is only 14 months, due to recurrence, despite current treatment options of surgery, radio- and chemo-therapies. Brain tumor stem cells (BTSCs) are thought to underlie disease recurrence and lethality as they exhibit self-renewal, multipotency and tumorigenesis. The epigenetic regulation of BTSCs are relatively un-characterized and unlike genomic mutations are potentially reversible. Disrupter of telomeric silencing-1-like (DOT1L) is the only known histone methyltransferase responsible for histone-3-lysine-79 methylation (H3K79me), an epigenetic mark associated with active gene transcription. Previous studies investigating the therapeutic implications of targeting DOT1L in cancer have shown that its inhibition in leukemia results in cancer cell death and, in solid cancers, decreases metastasis. We investigated the role of DOT1L in GBM BTSCs. We find that short-term DOT1L inhibition in BTSCs in vitro has limited effects on viability but alters growth morphology, as neurospheres become flattened and adherent. Furthermore, inhibition decreases BTSC invasion and promotes differentiation. Long-term inhibition of DOT1L had a more pronounced effect on sphere morphology and decreased BTSC proliferation and survival. Pre-treatment with the DOT1L inhibitor EPZ-5676 followed by orthotopic xenografts of BTSCs led to slowed tumor growth and improved overall survival. Initial H3K79me2 ChIP-sequencing and RNA-sequencing results show that H3K79me2 levels and gene expression are decreased for stem and progenitor cell markers SOX2 and OLIG2 respectively, in BTSCs treated with EPZ-5676. These results suggest that DOT1L may be an important regulator of GBM cancer stem cell properties and tumor progression. Current studies aim to further investigate how DOT1L regulates BTSCs by overlaying H3K79me2 ChIP-sequencing, RNA-sequencing and ATAC-sequencing in a panel of BTSCs following DOT1L inhibition and overexpression. These findings highlight the potential clinical implications of epigenetic targeted therapies for GBM.

#1119

Quaking (Qki) loss enhances stemness of glioma stem cells through regulating intracellular vesicle trafficking system.

Jian Hu, Takashi Shingu. _UT MD Anderson Cancer Ctr., Houston, TX_.

Glioma stem cells (GSCs) have higher self-renewal capacity than neural stem cells (NSCs), yet the underlying molecular mechanism is less clear. Here we report the generation of a novel conditional KO allele of Quaking (Qki), a tumor suppressor in glioblastoma with RNA binding activity. When Qki was deleted in Nestin-CreERT2;QkiL/L mice, the NSC population, which is characterized by long-term BrdU retention and GFAP+/Nestin+ double positive staining, was greatly increased, indicating that Qki deletion enhances NSC self-renewal. To determine whether Qki deletion promotes gliomagenesis, we generated a Nestin-CreERT2;QkiL/L;PtenL/L;p53L/L cohort, in which 92% of the mice developed glioblastoma with a median survival of 105 days. However, the Nestin-CreERT2;PtenL/L;p53L/L cohort did not develop any glioma up to a year; therefore Qki deletion greatly promotes gliomagenesis. Transcriptomic and proteomic profiling coupled with PAR-CLIP (Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation) analyses revealed that genes involved in intracellular vesicle trafficking were greatly enriched by Qki deletion. Specifically, 34% of the genes up-regulated by Qki deletion are involved in receptor delivery, and 39% of the genes down-regulated by Qki deletion are subunits of receptor degradation machineries such as endosomes and lysosomes. High level of receptor delivery and low level of receptor degradation concomitantly enrich receptors on the membrane and enhance the activity of the receptors that are involved in maintaining stemness, including RTK, Notch1 and Frizzled. Lower lysosomal activity induced by Qki deletion also leads to defective mitophagy, which consequently leads to accumulation of damaged mitochondria, high level of ROS, and genomic instability in Qki-deficient NSCs. We identified that genomic instability induced by Qki deletion led to accumulation of classical glioblastoma-associated mutations such as copy number gains of PDGFRa, Cyclin D1 and Cyclin D3. Taken together, our data suggest that Qki loss-induced endolysosomal defects promotes gliomagenesis through both reducing receptor degradation and inducing genomic instability.

#1120

Wnt/β-catenin mediated enrichment of EpCAM positive cancer stem cells promote drug resistance in hepatocellular carcinoma.

Harshul Pandit, Yan Li, Gouzhen Cui, Qianqian Zheng, Suping Li, Robert Martin. _University of Louisville, Louisville, KY_.

Introduction: Hepatocellular carcinoma (HCC) attains resistance to anti-cancer drugs which accounts for most of the therapeutic failures, and is one of the major factors for poor prognosis and poor overall survival of HCC patients. Cancer stem cells (CSCs) are sub-population of cells that bear stem-like properties, and are believed to contribute in tumor initiation, drug resistance and recurrence in tumor microenvironment. We hypothesize that canonical Wnt/β-catenin mediated CSC enrichment is responsible for acquired drug resistance in HCC. We aimed to study HCC CSCs for aberrant Wnt/β-catenin signaling, possible dedifferentiation, and drug resistance property. Methods: Total n=26 human HCC specimens with adjacent controls were analyzed for EpCAM and β-catenin expression by histology and western blot. In vitro CSC enrichment was achieved by treating murine (Hepa1-6) and human (HepG2, Hep3B) HCC cells in serum-free condition. CSC enrichment was confirmed by analyzing for established surface markers (EpCAM, CD44, CD133, CD90) and functional markers (Aldeflour assay and Hoechst-33342 efflux). For possible dedifferentiation, we analyzed gene expression profile during enrichment process using qRT-PCR for dedifferentiation and stem cell specific genes. Drug resistance property of CSCs was studied for Doxorubicin and Sorafenib by XTT assay. To investigate CSC activation, Wnt/β-catenin signaling was studied by analyzing expression of β-catenin, GSK3β, p-GSK3β, EpCAM, ABCG2; and downstream targets (C-Myc,Cyclin-D1,TCF1) and findings were confirmed by β-catenin knockdown, inhibitors of Wnt pathways (LiCl, XAV939, FH535) and TOP flash reporter assay. To study in vivo tumorigenesis of HCC CSCs, immunocompetent mouse model was established using C57L/J mouse and copGFP expressing Hepa1-6 cells for possible lineage tracking. Tumor growth was monitored using high-frequency ultrasound and animals were euthanized after 18 days. Histology and ICC analysis was performed to confirm the tumors and findings. Results: Human specimens showed concomitant EpCAM and beta-catenin expression. Spheroid forming HCC CSCs showed significant higher β-catenin and EpCAM expression, and acquired drug resistance compared with control HCC cells. Silencing β-catenin by knockdown abolish drug resistance property of CSCs for Doxorubicin and reverse spheroids to adherent phenotype. Our in vivo findings confirmed EpCAM+/+ CSCs have shown significant tumorigenesis compared with no tumors in EpCAM-/- (n=9, p<0.005). Conclusions: Our findings suggest that possible dedifferentiation by canonical β-catenin confer drug resistance and CSC properties to HCC cells. Sorted EpCAM+ CSCs showed very aggressive in vivo tumor initiation and growth in immunocompetent orthotopic mouse model. This EpCAM+ CSCs population could potentially responsible for HCC recurrence and therapeutic failure.

#1121

**Profiling patient-specific glioblastoma drug response** in vitro **using complex 3D microtumors.**

Ashley M. Smith,1 Melissa Millard,1 Lillia Holmes,1 Michael T. Lewis,2 Lacey E. Dobrolecki,2 Charles Kanos,3 Stephen Gardner,3 Philip Hodge,3 Fred Nelson,3 Michael Lynn,3 Jeff Edenfield,3 Christopher Corless,4 David Schammel,5 Howland E. Crosswell,1 Teresa M. DesRochers1. 1 _KIYATEC, Inc., Greenville, SC;_ 2 _StemMed Cancer, Houston, TX;_ 3 _Greenville Health System, Greenville, SC;_ 4 _Knight Diagnostics Laboratories, Portland, OR;_ 5 _Pathology Associates, Greenville, SC_.

Glioblastoma (GBM) has a median survival of less than 2 years due to intra-tumoral heterogeneity, diffuse infiltrations of adjacent brain tissue, and a lack of effective therapies. Development of more efficacious therapies will require better GBM models for the testing and identification of novel agents. Towards this end, we have successfully developed a GBM 3D tissue model that can provide in vitro, patient-specific compound screening. Stable populations of glioma stem cells (GSC) from 24 of 41 patient samples have been successfully established and cultured long-term with minimal changes. To confirm stemness of the GSC population, we have successfully established a limiting-dilution series within SCID/Bg mice and characterized the resultant tumors. 4 of these lines have been used to establish patient-derived xenograft (PDX) models in mice. The original, primary patient tissue established GSC populations, and the resultant PDX tissues have been characterized by flow cytometry, IHC, RNA expression, NGS, and MGMT methylation status. With the goal of better modeling the patient tumor tissue in vitro, our GSC populations have also been used to establish complex microtumors within the KIYATEC 3DKUBE™ perfusion system, consisting of monoculture GSCs, GSCs co-cultured with human brain endothelial cells (HBECs), and GSCs co-cultured with HBECs and CD14+ peripheral blood mononuclear cells. Our monoculture microtumors consisting of only GSCs show a maintenance of GSC markers Nestin and Sox2 by both IHC and mRNA. Interestingly, when these cells are used to produce PDX, they up-regulate GFAP as a marker of differentiation that is not observed in the neurosphere or monoculture microtumor cultures. We have shown these 3D models to be viable for more than 1 month in perfusion and to be effective models for drug compound screening by dosing the microtumors on a weekly basis with temozolomide (TMZ). We have correlated TMZ response to MGMT methylation as reported both clinically and measured in vitro. Finally, In vitro drug response has been compared to both matched PDX in vivo drug response and the patient's clinical response to TMZ and MGMT methylation. Our data supports that this complex, 3D, patient-derived GBM model can be used to effectively screen, identify and characterize novel treatments of GBM.

#1122

MST4 phosphorylation of ATG4B regulates autophagic activity, tumorigenicity, and radio resistance in glioblastoma.

Tianzhi Huang,1 Chung Kwon Kim,1 Angel A. Alvarez,1 Rajendra P Pangeni,1 Xuechao Wan,1 xiao song,1 Taiping shi,1 Yongyong Yongyong,1 Namratha Sastry,1 Craig Horbinski,1 Songjian Lu,2 Roger Stupp,1 John Kessler,1 Ryo Nishikawa,3 Ichiro Nakano,4 Erik Sulman,5 Xinghua Lu,2 Charles David James,1 Xiao-Ming Yin,6 Bo Hu,1 Shi-Yuan Cheng1. 1 _Northwestern Univ., Chicago, IL;_ 2 _University of Pittsburgh, Pittsburgh, PA;_ 3 _Saitama Medical University International Medical Center, Saitama, Japan;_ 4 _The University of Alabama at Birmingham, Birmingham, AL;_ 5 _The University of Texas M. D. Anderson Cancer Center, Houston, TX;_ 6 _Indiana University, Indianapolis, IN_.

Autophagy-mediated intracellular catabolism sustains the rapid growth of established tumors and tumors in response to multiple stresses including genotoxic/cytotoxic therapies. Here we identify and validate ATG4B, a key regulator that stimulates autophagic process by promoting autophagosome through reversible modification of ATG8 as a novel substrate of mammalian sterile20-like kinase (STK) 26/MST4 that is less known for its function in cellular process (none in autophagy) and unknown authentic substrates in cancer. We show that MST4 phosphorylates ATG4B at serine residue 383, which stimulates ATG4B activity and increases autophagic flux. Inhibition of MST4 or ATG4B activities suppresses autophagic process and the tumorigenicity of glioblastoma (GBM) cells. Furthermore, radiation induces MST4 expression, ATG4B phosphorylation and autophagy. Inhibiting ATG4B in combination with radiotherapy in treating mice with intracranial GBM tumor xenografts markedly slows tumor growth and provides significant survival benefit to animal subjects. This study not only describes a novel regulatory mechanism by which the MST4-ATG4B axis accelerates autophagic process, regulates GBM tumorigenicity, and responses to radiotherapy (RT), but also explores imminent clinical utility of combination of ATG4B inhibition with RT to suppress orthotopic GBM tumor xenografts. This study should prove generalizable to other types of cancer and have positive impacts in advancing our knowledge of cancer biology and designing new cancer treatments.

#1123

TRAF2 and NCK-interacting protein kinase (TNIK) regulates cancer stemness and adipogenesis of osteosarcoma cells.

Toru Hirozane,1 Mari Masuda,2 Naoko Goto,2 Teppei Sugano,2 Naofumi Asano,1 Eisuke Kobayashi,3 Keisuke Horiuchi,1 Hideo Morioka,1 Akira Kawai,3 Masaaki Sawa,4 Morio Matsumoto,1 Masaya Nakamura,1 Tesshi Yamada2. 1 _Keio University School of Medicine, Tokyo, Japan;_ 2 _National Cancer Center Research Institute, Tokyo, Japan;_ 3 _National Cancer Center Hospital, Tokyo, Japan;_ 4 _Carna Biosciences, Inc, Kobe, Japan_.

[Introduction] Osteosarcoma (OS) is a rare malignant bone tumor and affects predominantly adolescents and young adults. There is no molecular targeted therapy for OS and breakthrough intervention has long been awaited for decades. Disturbances in differentiation towards mature osteoblast from mesenchymal stem cell are related to OS pathogenesis. Wnt signaling is a key component in managing this differentiation process. We previously reported that TNIK was a positive regulator of Wnt signaling. To develop the first molecular targeted therapy for OS patients, we focused on Wnt pathway-targeted therapy in OS and initiated this study to demonstrate TNIK as a novel therapeutic target for OS treatment. [Methods] Eleven different osteosarcoma cell lines were used in the present study. Twenty clinical samples were obtained at biopsy and surgical resection from ten OS patients. TNIK expression was assessed by western blotting, immunofluorescence and immunohistochemical staining. The generation of the novel TNIK inhibitor was previously described (Masuda et al., 2016). Cells were cultured with TNIK inhibitor or siTNIK. Cell viability was evaluated by real-time cell analysis system or ATP quantitation assay. The effect of TNIK inhibitor in vivo was also assessed by a mouse xenograft model. RNA-seq was performed using the RNA extracted from vehicle or TNIK inhibitor-treated OS cells. Cancer stem cell (CSC)-like properties were assessed by soft-agar colony-formation assay, limiting dilution assay, ALDH activities and the expression of CSC markers. Adipogenic phenotypes were qualitatively and quantitatively analyzed by Oil red O staining and the expression of adipogenesis markers. [Results] TNIK was identified in all cell lines and most of cases. Immunohistochemistry of OS tissue microarray also revealed that 52 out of 55 OS samples were positive for TNIK protein. TNIK inhibitor suppressed OS cell proliferation both in vitro and in vivo. SiTNIK also inhibited OS cell growth. Global gene expression analysis revealed a significant decrease in the expression of the genes relating to Wnt signaling pathway and maintenance of stem cell pluripotency. Soft-agar colony-formation assay and limiting dilution assay revealed that TNIK inhibitor decreased sphere formation activities in OS cells. Accordingly, TNIK inhibitor significantly suppressed ALDH activities and the expression of the proteins that are involved in the maintenance of CSCs, including NANOG, SOX2, OCT4A and MYC. Concomitantly, TNIK inhibitor drove adipogenic transdifferentiation of OS cells with PPARG activation. TNIK knock-down U2OS cells created by shRNAs showed nearly identical results. [Conclusion] We present that TNIK inhibition promotes adipogenic transdifferentiation of OS cells and eliminates CSCs. Our study thus suggests the feasibility of TNIK as a target for OS treatment.

#1124

Wnt-producing niche cells support cancer stem-like cells in pancreatic ductal adenocarcinoma.

Katherine Wu,1 Olivera Grbovic-Huezo,1 Griffin Hartmann,1 Tyler Jacks,2 Tuomas Tammela1. 1 _Memorial Sloan Kettering Cancer Center, New York, NY;_ 2 _Koch Institute for Integrative Cancer Research, Cambridge, MA_.

Despite modern advances in cancer therapy, the prognosis for pancreatic ductal adenocarcinoma (PDAC) remains dismal, highlighting the need for better understanding of the biologic underpinnings of the disease. Well established as a pathway for growth and regeneration, Wnt signaling maintains subpopulations of cancer cells in a stem-like state, contributing to the heterogeneous composition of tumors. Our previous work demonstrated that Porcupine, an essential enzyme for secretion of paracrine Wnt signals, can be used to mark Wnt-producing cells in normal and tumor tissues. We detected the presence of nuclear beta-catenin, a biomarker of active Wnt-signaling, in a subpopulation of cancer cells adjacent to Porcupine+ niche cells in murine PDAC tissue. Furthermore, genetic inactivation of Porcupine translated to a 25% increase in survival in PDAC-bearing mice. In vitro, we see a marked increase in spheroid formation and proliferation of the stem-like subpopulation over the rest of the primary tumor cells. Ongoing lineage-tracing experiments in an autochthonous murine model of PDAC will determine whether these cancer stem cells are able to produce their Wnt-secreting niche in vivo. Our current efforts are focused on the preclinical evaluation of Wnt inhibitors in the treatment of mouse and human PDAC as single agents or in combination with chemotherapy. Such findings open the door for interrogation of the Wnt pathway as a possible target for improving the clinical outcome of pancreatic cancer.

#1125

Developmental pluripotency associated 4 oncogene interacts with Wnt/β-catenin signaling and stem cells regulatory genes to control pituitary tumor cells invasiveness.

Dipak K. Sarkar, Shaima Jabbar, Omkaram Gangisetty. _Rutgers Univ., New Brunswick, NJ_.

The developmental pluripotency associated 4 (DPPA4) gene has an important role in self-renewal and pluripotency in embryonic stem cells. It is re-expressed in several malignant tumors and is identified as a new pluripotency-related oncogene. We have recently identified elevated expression of DPPA4 in aggressive pituitary tumors induced by estrogen in fetal alcohol exposed rats. In this study, we determined the role of DPPA4 in pituitary tumor growth and invasiveness by employing CRISPR/Cas9 knock-down of DPPA4 and conducting cell proliferation, in vitro soft agar anchorage-independent growth assays, and in vivo tumor formation assay using cell spheres from pituitary tumors of alcohol-fed and control-fed animals. Pituitary tumor cells of fetal alcohol-fed rats and control diet-fed rats were grown in ultra-low attachment plate to develop pituispheres. In vitro and in vivo studies showed pituispheres of alcohol-fed rats had higher cell proliferation rate, increased colony formation in soft agar, and higher tumor development in mice xenografts compared to those in pituispheres of alcohol-fed rats. Pituispheres of alcohol-fed rats also had increased mRNA levels of cell growth, cell stemness and EMT regulatory genes. CRISPR-Cas9 knockdown of DPPA4 in pituispheres reduced the tumor cell ability to proliferate, migrate and form colony in soft agar and grow in mice xenografts. Pituispheres of alcohol-fed rats also showed increased protein levels of Wnt/beta-catenin signaling molecules (Wnt 3, beta-catenin, LPR5, GSK3), stemness regulatory factors (KLF4, Sox2, Nanog) and EMT factors as compared to those in control-fed pituispheres. DPPA4 knockdown reduced the levels of Wnt signaling and stem cells regulatory proteins and EMT factors in alcohol-fed rat pituispheres. These data suggest that DPPA4 is expressed in aggressive pituitary tumors and regulates the growth and invasion of these tumors via activation of Wnt/beta-catenin signaling and stem cells regulatory genes. (This work is supported by a National Institute of Health grant R01 AA11591).

#1126

Exploring the roles of Suppressor of Fused in the postnatal neurogenic niche and tumorigenesis.

Jesse Garcia Castillo, Hector G. Gomez, David Aguilar, Samuel J. Pleasure, Odessa Yabut. _UCSF, San Francisco, CA_.

Isolated brain tumors consist of cells with stem cell features and a tissue microenvironment similar to the neurogenic niche, implying that tumors must require the same niche factors for tumor growth and progression. Thus, a better understanding of how neural stem cell (NSC) behavior is regulated can have significant therapeutic implications in brain cancers. To this end, we examined the roles of Suppressor of Fused (Sufu) in the postnatal neurogenic niche of the forebrain, the ventricular-subventricular zone (V-SVZ), a potential site of origin for forebrain tumors. Given that recent studies showed low expression of Sufu in gliomas, we wondered how conditional deletion of Sufu using the hGFAP-Cre driver (hGFAP-cre/+;Sufu-fl/fl) influences NSC behavior in the V-SVZ. We found that Sufu critically controls NSC proliferation, since loss of Sufu in the hGFAP-cre/+;Sufu-fl/fl mice resulted in abnormal expansion of the V-SVZ. These defects were due to the increased production of DCX+ Type A cells at the expense of transit amplifying Type C and oligodendrocyte precursor cells. Furthermore, many of these cells failed to migrate out of the V-SVZ, where they remained and continued to proliferate. Nevertheless, an increase in apoptotic cells was observed, which likely prevented the formation of brain tumors from loss of Sufu function alone. Indeed, our preliminary studies showed that the combined deletion of Sufu and the tumor suppressor p53 (hGFAP-Cre/+;Sufu-fl/fl;p53-fl/fl) resulted in a more severe and sustained cell expansion phenotype in the V-SVZ. We predict that these genetic conditions predispose NSCs within the V-SVZ to form tumors at later postnatal stages. We are now investigating this and the molecular mechanisms by which Sufu exerted this effect. Of particular focus is the role of Sufu as an inhibitor of the mitogenic Sonic hedgehog (Shh) signaling, which is highly active in various glioblastoma subtypes. Taken together, our findings point to a potential role for Sufu in preventing tumor development in the forebrain. Our studies also provide novel insights on how developmental and oncogenic signals converge to initiate tumorigenesis or tumor progression. We hope that these studies will lead to the identification of critical niche factors that may be targeted to treat brain cancers and improve patient outcome.

#1127

Role of NRF1 regulatory gene-networks in glioblastoma.

Kaumudi Bhawe, Jayanta Das, Changwon Yoo, Deodutta Roy. _Florida International University, Miami, FL_.

Nuclear Respiratory Factor 1 (NRF1), a transcription factor historically known to regulate several mitochondrial genes, has been recently implicated in human cancers. We have previously demonstrated the importance of NRF1 in estrogen driven breast cancer. The NRF1 motif exhibits some of the strongest pioneer activity (pioneer index indicates an increase in chromatin opening activity during developmental process) in mouse embryonic stem cells. Both neurogenesis and synaptogenesis are controlled by NRF1 regulated genes. There is a strong association of NRF1 with human fetal brain and neural development. To begin our study of the effect of NRF1 on glioblastoma pathogenesis, we examined whether NRF1 contributes to the acquisition of multipotent neural stem/progenitor cell (NSPC) properties in adult human brain cells. We generated NRF1 overexpressing stable human brain cerebral cortex astrocytes. Both 17β-estradiol (E2, 100 pg/ml) and NRF1 overexpression increased the percentage of cells co-expressing SOX2 and Nestin by 8 and 20%, respectively as shown by flow cytometry. Nestin is an intermediate filament protein important for guaranteeing neural stem pluripotent stem cell (NSPC) self-renewal while SOX2 is expressed at the earliest development stage of the brain and functions as a marker of neural development. SOX2 also plays a key role in the maintenance of neural stem cell (NSC) properties, including proliferation/survival, self-renewal, and neurogenesis. Multipotency allows NSPCs to differentiate into the neurons, astrocytes or oligodendrocytes. NRF1-induced SOX2+Nestin+ NSPCs expressed the neuronal marker β-III-tubulin, the neural phenotype, and neurosphere formation. NRF1 induced NSPCs were subjected to FACS analysis after FITC Annexin V and Propidium Iodide staining. Dead cells stained positive for both FITC Annexin V and PI and these cells are lowest in NRF1+ (0.42%) compared to vector control (4%). In summary, overexpression of NRF1 suppresses cellular senescence and increases resistance to apoptosis. Both are major barriers to cell reprogramming that may contribute in the generation of glioblastoma stem cells. To investigate the role of NRF1 gene network in glioblastoma, we used Bayesian network (BN) analysis on TCGA based RNA-seq data of 154 glioblastoma patients samples. NRF1 based gene association networks were significantly different in low overall survival (OS) patients compared with high OS patients. Specifically, TNFRSF10B, HGF, EBF4, and PAX5 showed up consistently as first degree Markov Blanket genes of NRF1 in low OS (<6mo) glioblastoma patients but not in the high OS patients. In conclusion, these data provide evidence in support of the role for NRF1 in glioblastoma. Clarifying these NRF1 driven complex mechanisms can be a guiding force in the development of new treatments to slow or prevent the development of glioblastoma.

#1128

Exosomal ID3 is pro-metastatic through guiding NRF1-induced breast cancer stem cells across the blood-brain-barrier.

Jayanta K. Das, Mayur Doke, Alok Deoraj, Quentin Felty, Deodutta Roy. _Florida International University, Miami, FL_.

Nuclear respiratory factor 1 (NRF1) and inhibitor of differentiation protein 3 (ID3) are transcription regulating proteins. Recently we have uncovered a novel oncogenic function of NRF1 in breast cancer development and progression. In this study, we tested the postulate that exposure to PCB153 contributes in ID3 over-expressing endothelial stem cells (EndSCs) chaperoning and guiding of mesenchymal NRF1 breast cancer stem cells (BCSCs) across the blood-brain barrier. BCSCs/EndSCs were subjected to functional gain/loss of ID3 and/or NRF1 to test if PCB153 [1ng/ml] exposure produces NRF/ID3 signals regulating lineage specific BCSCs organ entry. First, we tested whether NRF1 promotes transmigration of breast cancer using a 3D blood-brain barrier (BBB) model consisting of breast tumor initiating cells (BTICs) or MDAMB231 cells, brain endothelial cell layer, and astrocytes. NRF1 overexpression increased the propensity for BTICs and mesenchymal enriched MDAMB231 BCSCs to adhere to brain endothelial cells and migrate across a human BBB model. Increased adhesion of NRF1+ BCSCs with ID3+ EndSCs was detected. ID3 EndSCs promoted the transmigration of BCSCs through the BBB. We found differential effects by endocrine disruptors, namely PCB153 and PCB77. The treatment with PCB153 showed increased growth of NRF1+ BCSCs tumor spheroids. PCB153 also induced the tumor cell adhesion to microvascular endothelium and transendothelial migration of BCSCs. The exosomal ID3 released from endothelial cells helped the mesenchymal NRF1+ BCSCs to cross through blood brain barriers. Xenograft experiments showed that ID3+ brain EndSCs not only supported the growth of estrogen treated NRF1+ BCSC tumorospheroids, but guided them to the brain in zebrafish. These findings show for the first time a key role for ID3 and NRF1 by which specific circulatory EndSCs accompany a particular sub-type of BCSCs to distant metastatic sites where they most likely facilitate the seeding, survival, and proliferation of BCSCs. This knowledge is important for pre-clinical testing of NRF1/ID3 modifying agents that prevent the spread of breast cancer to the brain. This work was, in part, supported by a VA MERIT Review (VA BX001463) grant to DR.

#1129

Sox2 drives ST6Gal-I expression and activity to promote a CSC phenotype in ovarian cancer.

Kaitlyn A. Dorsett, Susan L. Bellis. _Univ. of Alabama at Birmingham, Birmingham, AL_.

This study elucidates ST6Gal-I, a sialyltransferase, as a functional driver of a cancer stem cell (CSC) phenotype regulated by the stem cell transcription factor Sox2. ST6Gal-I is upregulated in 98% of ovarian cancers (OC); it functions to add an α2-6 sialic acid, a large, negatively charged sugar, to N-glycosylated proteins bound for the cell surface. Normal differentiated epithelia have very low expression of ST6Gal-I, however, expression is turned on in stem cell compartments and transformed tissues. Furthermore, our work has shown that ST6Gal-I plays a causal role in conferring hallmark CSC properties including greater tumor-initiating capabilities, promotion of recurrence, and resistance to tumor-associated stressors like chemotherapies, serum deprivation, and hypoxia. Even given this stark upregulation and apparent functional import of ST6Gal-I in cancers, very little work has been done to identify the transcriptional driver of ST6Gal-I expression. We identified that Sox2 and ST6Gal-I are both part of one of the most commonly enriched amplicons in human cancer, amplicon 3q26. We then examined the TCGA database and found that these two genes are co-amplified in 48/73 cancer cohorts, including ovarian cancer. In addition to being genetically co-amplified, Sox2 has been shown to bind to the promoter of ST6Gal-I, though no further analyses have been performed to assess a functional link. Herein we address a novel, glycosylation-dependent mechanism that drives a CSC phenotype. We hypothesize that Sox2, a key stem-associated transcription factor in CSCs, directly induces expression of ST6Gal-I. In our ovarian cancer cell model systems, we forced overexpression or knockdown of Sox2 and found that Sox2 expression directly correlated with ST6Gal-I expression. Overexpression or knockdown of Sox2 resulted in up- or down- regulation of CSC markers Oct4 and Nanog respectively. We then forced ST6Gal-I knockdown to inhibit the Sox2-induced ST6Gal-I upregulation and found that this prevented the enhanced expression of Oct4 and Nanog. Here we show that Sox2 requires ST6Gal-I expression to promote a CSC phenotype.

#1130

The role of TET2 in mammary stem cell fate decision.

Meng-Ju Wu, Chun-Ju Chang. _Purdue University, West Lafayette, IN_.

Epigenetic mechanisms, including DNA methylation, has an essential role in governing the stem cell fate decision. The Ten-Eleven-Translocation (TET) protein family serves as a primary epigenetic regulator responsible for catalyzing the conversion of the modified genomic base 5-methylcytosine into 5-hydroxymethylcytosine to mediate DNA demethylation. It has been shown that loss of TET2 promotes aberrant stem cell self-renewal in hematopoietic malignancy.

Interestingly, our recent study has revealed that expression of TET2 directs the breast cancer stem cell-like population to the differentiation cell fate in vitro. Since aberrant stem cell fate is often associated with tumorigenesis, delineating the role that a critical epigenetic regulator (e.g., TET2) plays to govern the mammary stem cell (MaSC) fate will likely provide new strategies used for breast cancer treatment.

Thus, to determine the physiological and pathological roles of TET2 in the regulation of MaSC fate and mammary gland development using a mouse model with mammary specific deletion of the Tet2 gene (MMTV-Cre; Tet2fl/fl). Our data show that deletion of Tet2 in mouse mammary gland leads to disrupted mammary epithelial morphology, defective lobular-alveolar development, along with aberrant mammary stem cell fate decision, contributing to an expansion of the MaSC population and development of mammary lesions. Together, the data demonstrate that TET2 plays an important role in controlling MaSC fate decision to maintain epithelial homeostasis, and loss of TET2 may contribute to the development of mammary malignancies.

#1131

IRAK1 and MYD88 mediate divergent signaling functions in MLL-AF9 leukemia.

Joseph M. Cannova, Wei Wei, Rafael Gutierrez, Peter Breslin, Jiwang Zhang. _Loyola University Chicago, Chicago, IL_.

Acute myeloid leukemia (AML) is an aggressive hematological malignancy that occurs disproportionately in the elderly population. First line therapy is a regimen of anthracycline and cytarabine treatment or, more recently, demethylating agents. However, elderly AML patients are unable to tolerate aggressive chemotherapy regimens and there is an urgent need for targeted, tolerable therapies. To identify candidate pathways for targeted treatments, a microarray on transcripts for inflammatory signaling was performed on 588 primary patient AML samples. Non-hierarchical clustering revealed that Toll-like receptor (TLR) signaling transcripts are overexpressed in myelomonocytic and monocytic AML subtypes (M4 and M5 AML subtypes, respectively). Myeloid differentiation primary response 88 (MYD88) and downstream IL-1 receptor-associated kinases (IRAKs) were found to be overexpressed in these AML subtypes. MYD88 is an adaptor molecule for IL-1 and most TLR mediated signaling. To determine whether MYD88-dependent signaling is required for leukemogenesis, we derived a CreERT2-LoxP inducible system to delete Myd88 in a murine leukemia model. Because M4 and M5 AML subtypes are enriched for mutations in the mixed lineage leukemia (MLL) gene and overexpressed TLRs, MYD88, and IRAK2, an MLL-AF9 expressing construct was utilized to transform murine progenitor cells into leukemia cells. To study whether Myd88-dependent signaling is required for in vitro colony formation of the leukemic cells, Myd88 knockout (Myd88-/-) MLL-AF9 leukemic cells were subjected to colony-forming assays. Compared to Myd88 wild-type (Myd88fl/fl and Myd88+/+) leukemia cells, reduced colony-forming capacity was observed in the Myd88-/- leukemia cells. Further, reduced proliferation was observed in Myd88-/- cells compared to Myd88+/+ cells. Cell surface staining indicates Myd88-/- cells exhibit a loss of CD117, a marker of myeloid stemness, and a gain of CD11b, a marker of myeloid differentiation, when compared to Myd88+/+ cells, indicating that Myd88 deletion correlates with MLL-AF9 leukemia cell partial differentiation. In vivo transplantation of Myd88-/- cells delays leukemia development compared to Myd88+/+ cells. To determine whether IRAK1, a downstream kinase of MYD88, is also required to prevent partial differentiation, an IRAK1 shRNA knockdown cell line was generated and assayed for partial differentiation. In IRAK1KD MLL-AF9 cells, no partial differentiation was detected. Similarly, application of an IRAK1/4 inhibitor to Myd88+/+ MLL-AF9 cells failed to induce partial differentiation, despite suppressing proliferation. This indicates that MYD88-dependent signaling has functions independent of IRAK1 signaling that prevent partial differentiation in MLL-AF9 leukemia cells. Thus, MYD88 and IRAK1 signaling appear to mediate divergent functions, and both MYD88 and IRAK1 function appear to be required for MLL-AF9 leukemia.

#1132

PAX6 drives cancer cells toward a stem like state via GLI-SOX2 signaling axis in lung adenocarcinoma.

Akira Oki, Mohammad O. Hoque. _Johns Hopkins Univ. School of Medicine, Baltimore, MD_.

PAX6 is an essential transcription factor for embryonic stem cell maintenance, but its contribution to lung cancer stem cells (CSCs) remains unexplored. We demonstrate that PAX6 acts as an oncogene responsible for induction of cancer stemness properties and plays a biological role in tumor initiation and CSC expansion in lung adenocarcinoma (LUAD). Mechanistically, PAX6 activates Hedgehog-GLI signaling in a SMO-independent noncanonical manner, resulting in SOX2 upregulation directly by the binding of GLI to the proximal promoter region of the SOX2 gene. The overexpressed SOX2 enhances the expression of key pluripotent factors (OCT4 and NANOG) and suppresses differentiation lineage factors (HOPX and NKX2-1). Thus, PAX6 drives cancer cells toward a stem-like state via the GLI-SOX2 signaling axis in LUAD. In contrast, in the differentiated non-CSCs that represent most of the tumor cell population, PAX6 is transcriptionally silenced by its promoter methylation. In human lung cancer tissues, the positive linear correlations of PAX6 expression with GLI and SOX2 expression and its negative correlations with HOPX and NKX2-1 expression were observed. Therapeutically, the blockade of the PAX6-GLI-SOX2 signaling axis elicits a long-lasting therapeutic efficacy by limiting CSC expansion following chemotherapy. Our findings provide a rationale for targeting the PAX6-GLI-SOX2 signaling axis with chemotherapy as an effective therapeutic strategy.

#1133

Mitochondria localized EGFRvIII promotes stemness through regulation of mitochondrial dynamics in glioblastoma stem cells.

Yeonhee You,1 Jun Hee Hong,2 Jinlong Yin,2 Young Taek Oh,2 Sung Soo Kim,1 Jong Bae Park1. 1 _National Cancer Center Graduate School of Cancer Science and Policy, Goyang-si, Republic of Korea;_ 2 _National Cancer Center, Goyang-si, Republic of Korea_.

Purpose Changes in mitochondrial morphology have been linked with mitochondrial function and host cell homeostasis in many metabolic diseases. Cancer is increasingly perceived as metabolic disease, so understanding and regulating mitochondrial dynamics is of interest in cancer therapeutics. Recent studies indicate that distinct metabolic profile of cancer is dependent on genes that regulate the fusion and fission of mitochondria. Importantly, high levels of mitochondrial fission activity are common in cancer cell malignancy and in stem cell′s resistance to differentiation. Since these two phenotypes converge into cancer stem cells, investigating mitochondrial fission activity will provide hints to target cancer stem cell in therapeutics. Purpose of this study is to elucidate what regulates mitochondrial dynamics in cancer stem cells to sustain their stemness properties. In GBM, amplification of the oncogenic variant of EGFR, EGFRvIII, is associated with poor patient prognosis and has recently been known as the major contributor for stemness. Thus, we investigated the role of this oncogenic change in mitochondrial dynamics and stemness control in glioblastoma stem cells (GSCs). Methods Patient-derived GSCs were cultured and sorted by FACS based on tetramethylrhodamine ethyl ester (TMRE) dye emission level, which indicates mitochondrial membrane potential and functional activeness. The highest and the lowest TMRE groups each was collected and subject to further experiments. siRNA was used for loss-of-function study and lentivirus containing overexpression vector was used for gain-of-function study. In order to show the effect of EGFRvIII kinase activity inhibition, gefitinib and its derivative were used.

Results High TMRE GSCs showed stronger self-renewal ability in spheroid culture and higher levels of stemness marker Nestin, EGFRvIII, and mitochondrial fission regulator DRP1 expression than low TMRE GSCs. The high TMRE GSCs also showed more fragmented mitochondrial pattern, indicative of active mitochondrial fission. As underlying mechanism of EGFRvIII action on mitochondrial membrane potential, we figured out that EGFRvIII is localized at mitochondria. In order target mitochondria localized EGFRvIII, we used a gefitinib derivative which has mitochondria targeting moiety. This drug reduced mitochondrial membrane potential, self-renewal property, protein levels that regulate mitochondrial dynamics and less fragmented mitochondria, compared to gefitinib treated cells.

Conclusion EGFRvIII promotes self-renewal property of GSCs by translocating to mitochondria and upregulating mitochondrial membrane potential and fission-related protein levels. This finding highlights the role of mitochondrial dynamics as a mediator mechanism between tumor-specific oncogenic change and stem cell self-renewal ability.

#1134

The tumor suppressor activity of miR-300 is detrimental for leukemia development but required for leukemia stem cell maintenance.

Giovannino Silvestri,1 Lorenzo Stramucci,2 Justin Ellis,3 Jason Harb,4 Paolo Neviani,5 Bin Zhang,6 Klara Srutova,7 Gabriel Pineda,8 Catriona Jamieson,8 Bruno Calabretta,9 Fabio Stagno,10 Paolo Vigneri,11 Georgios Nteliopoulos,12 Philippa May,12 Alistar Reid,12 Ramiro Garzon,13 Denis-Claude Roy,14 Martin Guimond,14 Peter Hokland,15 Michael Deininger,16 Garrett Fitzgerald,17 Chris Harman,17 Francesco Dazzi,18 Dragana Milojkovic,12 Jane Apperley,12 Guido Marcucci,6 Jianfei Qi,1 Katerina Machova-Polakova,7 Xiaoxuan Fan,1 Maria Baer,1 Rossana Trotta,1 Danilo Perrotti1. 1 _Greenebaum Comprehensive Cancer Center, University of Maryland School of Medicine, Baltimore, MD;_ 2 _Regina Elena, Rome, Italy;_ 3 _Ohio State University, Columbus, OH;_ 4 _Blood Center of Wisconsin, WI;_ 5 _Norris Comprehensive Cancer Center, CA;_ 6 _City of Hope National Medical Center, CA;_ 7 _University of Prague, Czech Republic;_ 8 _University of California, CA;_ 9 _Thomas Jefferson University, PA;_ 10 _Policlinico - Vittorio Emanuele, Catania, Italy;_ 11 _University of Catania, Italy;_ 12 _Imperial College, United Kingdom;_ 13 _Ohio State University, OH;_ 14 _University of Montreal, Quebec, Canada;_ 15 _Aarthus University Hospital, Denmark;_ 16 _University of Utah, UT;_ 17 _University of Maryland Medical Center, Baltimore, MD;_ 18 _King's College London, London, United Kingdom_.

Inhibition of protein phosphatase 2A (PP2A) tumor suppressor is essential for chronic myelogenous leukemia (CML) stem cell (LSC) maintenance and disease development. Persistence of drug-resistant quiescent LSCs depends on cell-autonomous and bone marrow (BM) signals. Herein, we identified miR-300 as an miRNA inhibited in CD34\+ CML progenitors and during blastic transformation (BC) through the BCR-ABL1-dependent inhibition of C/EBPβ-induced miR-300 transcription. In CML progenitors, ectopic mir-300 expression directly targets the PP2A inhibitory pathway (i.e., Jak2, hnRNPA1, SET) and other factors essential for LSC maintenance and disease progression (e.g., CCND1/2, b-catenin, Myc, Twist-1). In leukemic but not normal CD34+ cells, miR-300 acts as a potent tumor suppressor by inducing cell cycle exit and promoting apoptosis. Conversely, hypoxia-induced BCR-ABL1 inhibition and induction of C/EBPβ were found essential for increased miR-300 levels in quiescent LSCs, although mesenchymal stromal cells (MSC)-derived exosomal miR-300 also contributed to it. Moreover, low O2 levels and MSC-derived exosomes induced quiescence of CD34+ CML cells. Notably, expression of an anti-miR-300 in MSCs prevented exosome-induced CD34+ CML growth arrest. LSCs escaped miR-300-induced apoptosis through the autocrine/paracrine TGFβ1-induced expression of TUG1, a lncRNA acting as an miR-300 sponge. In fact, TUG1 or TGFβ1 inhibition decreased quiescent (CFSEMAX) LSC number. By contrast, miR-300 inhibition did not alter LSC survival/self-renewal, further supporting a role for TUG1 as an miR-300 sponge. Accordingly, TUG1 was markedly induced in CFSEMAX but not dividing CD34+ CML cells. In fact, low levels of ectopic miR-300 induced growth arrest (decreased LTC-IC and CFC/replating activity) without affecting quiescent LSC number. By contrast, high doses of miR-300 but not scramble CpG-ODN impaired LSC survival (LTC-IC) and self-renewal (CFC/replating), induced marked killing of quiescent LSCs and dividing progenitors, and impaired CML engraftment in NRG-SGM3 mice. Such effects were further enhanced when CPG-miR-300 and CPG-anti-TUG1 were combined. By contrast, high CpG-miR-300 levels did not affect normal CD34+ cell survival/self-renewal likely because of high TUG1 expression. Altogether our results indicate that while miR-300 loss is essential for survival/proliferation of leukemic progenitors, increased miR-300 levels are required for LSC maintenance. Thus, induction of TUG1 may occur to preserve LSC survival in the BM endosteal niche where quiescence is induced by MSCs and low O2 levels through abnormal miR-300 induction. Thus, disrupting the miR-300/TUG1 balance may represent a potential therapeutic approach for treatment/eradication of LSC-derived leukemias.

This work is supported in part by NIH-NCI R01CA163800.

#1135

SOX9 regulates cancer stem-like cells and chemotherapy response in non-small cell lung cancer.

Maria Voronkova,1 Sudjit Luanpitpong,2 Yon Rojanasakul1. 1 _West Virginia University, Morgantown, WV;_ 2 _Mahidol University, Bangkok, Thailand_.

Lung cancer is the leading cause of cancer-related death worldwide in both men and women. A major limitation of the lung cancer chemotherapy effectiveness is drug resistance and tumor relapse, which both have been attributed to the acquisition of cancer stem-like cells (CSCs). Thus, it is crucial to identify specific markers and drug targets for this cell population. Our work is focused on the embryonic transcription factor SOX9, which has been implicated in CSC regulation in a number of cancer types, but little is known about its role in non-small cell lung cancer (NSCLC), the predominant type of lung cancer. In addition, the regulatory mechanisms and downstream targets of SOX9 as well as its role in chemoresistance are largely unknown. We hypothesized that SOX9 plays a key role in lung cancer progression and chemoresistance by stimulating cancer stem-like properties. To test this hypothesis, we genetically manipulated SOX9 expression in various NSCLC cell lines and evaluated its effects on CSC formation, biomarkers expression, and drug resistance. Our results showed that SOX9 depletion decreases the number of tumor spheres formed by NSCLC cells, thus supporting its role in CSC regulation in lung cancer. We also showed that SOX9 depletion dramatically downregulates the expression of a functional CSC regulator ALDH1A1. Consistent with this finding, SOX9 overexpression in non-cancer lung epithelial cells promotes tumor sphere formation. Moreover, we noticed that SOX9 expression goes up in response to chemotherapy exposure. Notably, SOX9 knockdown increases cell sensitivity to the chemotherapeutic cisplatin, whereas SOX9 overexpression decreases it. Depletion of SOX9 also increases CSC sensitivity to cisplatin, as demonstrated by the tumor sphere formation assay. Analysis of the Cancer Genome Atlas (TCGA) data showed that high SOX9 expression correlates with poor survival in NSCLC patients. Taken together, our results indicate the essential role of SOX9 in the regulation of CSCs in NSCLC. Since CSCs are a key driver of chemoresistance and relapse, our findings may have important implications in the development of novel therapeutic strategies for chemoresistant and recurrent lung cancer.

#1136

Decrease in maturation of stem cells along the neuroendocrine lineage contributes to stem cell overpopulation that drives human CRC development.

Bruce M. Boman,1 Tao Zhang,1 Shirin Modarai,1 Lynn Opdenacker,1 Fields Jeremy2. 1 _University of Delaware, Newark, DE;_ 2 _CATX Inc, Princeton, NJ_.

Stem cell (SC) overpopulation is thought to drive tumor initiation and progression during colon cancer development, but it is unclear what causes the SC overpopulation. Because most neuroendocrine (NE) cells (NECs) in normal colonic crypts reside within the SC niche at the crypt bottom, we thought that aberrant NECs might be linked to the SC overpopulation. We hypothesized that (i) in normal crypts, SC and NEC populations are anatomically and functionally related, and (ii) during tumorigenesis, changes in these relationships quantitatively correlate with increased SC numbers. We tested this hypothesis - using quantitative immunohistochemical mapping - as a first step towards understanding how interactions between SC and NEC might become altered during colon tumorigenesis. In normal human colonic crypts, most (>80%) cells staining for ALDH1, a colonic SC marker, co-stained for chromogranin-A (CGA) and several other NE markers. Neither ALDH1+ nor CGA+ cells co-stained for MCM2, a marker for proliferating cells. These findings indicate that, in normal colonic crypts, many ALDH1+ cells have NE characteristics. In contrast, the proportion of ALDH1+ cells that co-stained for NE markers progressively decreased (to <20%) during the stepwise progression to cancer (from normal crypts to normal-appearing FAP crypts to adenomatous crypts to colon carcinomas). However, the absolute number of ALDH+ cells progressively increased during this progression, reflecting the extent of the SC overpopulation. These findings suggest that (i) NEC maturation is integral to regulation of SC population size and (ii) a decreased rate of NEC maturation contributes to SC overpopulation during colon tumorigenesis.

#1137

ANO1 confers the maintenance of stemness and radioresistance in glioma stem cells by stabilizing EGFRvIII.

Dae-Hee Lee,1 Hee-Jin Kim,2 Jeong-Yub Kim,2 Jea-Yong Park,3 Myung-Jin Park2. 1 _Korea Univ. Medical Center, Seoul, Republic of Korea;_ 2 _Korea Institute of Radiological and Medical Sciences, Seoul, Republic of Korea;_ 3 _School of Biomedical Science Korea University, Seoul, Republic of Korea_.

Anoctamin-1 (ANO1), also known as TMEM16A, is a Ca2+-activated chloride channel known to mediate a number of physiologic functions such as mediating acute and chronic pain and regulating blood vessels and airway smooth muscle. In particular, ANO1 expression is amplified in a variety of cancers, and overexpression of ANO1 is not favorable for clinical prognosis. Previous studies have reported that ANO1 is associated with tumorigenesis, proliferation, migration, and invasion of cancer cells, but the mechanism is not clear yet. In this study, we examined role of ANO1 in the maintenance of stemness and radio-resistance in glioma stem cells (GSCs). Knockdown of ANO1 with shRNA and treatment of ANO1 specific inhibitors (CaCCAo1 and TAO1) suppressed the expression of Notch intracellular domain, Nestin and EGFRvIII and decreased self-renewal activity and invasion in GSCs. Suppression of ANO1 increased radiation sensitivity by inducing ROS-mediated GSCs death. However, inhibition of chloride channel activity, the main function of ANO1, was found to be unaffected in this event. Interestingly, we found that ANO1 retained stemness by direct binding to EGFRvIII, which is specifically expressed in GSCs, and that the chaperone property of ANO1 function may affect the stability of EGFRvIII. In intracranial mouse model of GSCs, knockdown of ANO1 increased the survival rate of mice by suppressing tumor formation. Taken together, we suggest that ANO1 is a crucial molecule involved in the maintenance of stemness and radiation resistance in GSCs and may be a good therapeutic target for treating glioblastoma.

#1138

LBH, a novel WNT effector in promoting basal-like breast cancer.

Kilan C. Ashad-Bishop, Karoline J. Briegel. _Univ. of Miami, Miami, FL_.

BACKGROUND: Clinically aggressive basal-like breast cancers (BLBC) disproportionally contribute to cancer deaths, but lack effective treatment options due to absence of expression of key therapeutic targets (ER, PR, HER2). New evidence suggests BLBC may originate from luminal breast epithelial cells through luminal-to-basal cell lineage conversion and dedifferentiation. However, the factors that reprogram cell fate and differentiation states during BLBC development remain poorly understood.

Our laboratory has identified a novel WNT/β-Catenin target transcriptional regulator, Limb-Bud-and-Heart (LBH), which is majorly overexpressed in aggressive BLBC harboring WNT hyperactivation. We previously showed in a conditional LBH knockout mouse model in vivo that LBH is a key stem cell and basal lineage regulator in normal mammary gland development. However, it's role in breast cancer remains elusive. Here, we crossed conditional LBH knockout (KO) mice with a mouse model for WNT-driven breast cancer (MMTV-Wnt1) to test if LBH is a critical effector of WNT-driven basaloid breast cancer, and whether its inhibition may affect BLBC tumorigenesis.

RESULTS: In our in vivo mouse model, epithelial-specific LBH inactivation using a Keratin 14/K14-Cre significantly attenuated Wnt1-induced mammary gland hyperplasia and delayed tumor onset. Yet, tumor volumes and burden were not changed, indicating that LBH is not essential for WNT-induced tumorigenesis. However, LBH-deficient MMTV-Wnt1-transgenic tumors exhibited pronounced histopathological differences compared to LBH WT MMTV-Wnt1-transgenic tumors. Whereas Wnt1+;K14CreLbh WT tumors were highly vascularized, disorganized, with mixed basal and luminal cell identity; tumors from Wnt1+;K14CreLbh KO mice were more organized, differentiated, and predominantly luminal Keratin 8 positive. We are currently investigating if the attenuated hyperplasia and tumor onset caused by LBH loss may be due to a deficiency in cancer stem cells in this breast cancer model.

CONCLUSION: Our data indicate that LBH is an essential cell fate regulator downstream of WNT signaling that maintains basal lineage identity and the undifferentiated nature of BLBC. Thus, LBH inhibition may be a novel strategy to reduce the high mortality of BLBC patients through differentiation therapy.

#1139

An shRNA screening reveals that the tumorigenic effect of FBXO11 is inversely correlated with EMT profiles in breast cancer.

Jiyoung Kim, Sofie O. Bagger, Branden B. Hopkinson, René Villadsen, Ole W. Petersen. _Univ. of Copenhagen, Copenhagen N, Denmark_.

Over the last decade a trend to study cancer stem cells (CSCs) has become synonymous to studying epithelial to mesenchymal transition (EMT), and has emerged in the field of breast cancer research. In turn, tumorigenesis of epithelial luminal-like breast cancer has been largely overlooked. Recently however, we and others have provided evidence that tumorigenic capacity is not always restricted to EMT-like cancer cells. Furthermore, the metastatic feature is conveyed by subclones of phenotypically differentiated non-EMT cancer cells. In this study, we aimed to identify the differential tumorigenic mechanisms of more aggressive non-EMT cells against the less aggressive EMT cells in breast cancer. By using loss of function screening, we found that a member of the E3 ubiquitin ligase complexes, FBXO11, facilitates tumor formation of non-EMT like cells by inhibiting the p53/p21 pathway. More importantly, our data showed that roles of FBXO11 in tumorigenesis are highly cell-type dependent. In conclusion, our study empathizes that it is important to understand the complexities of individual tumors in the development of personalized therapy.

#1140

Genes preserving stem cell state in group 3 medulloblastoma brain tumor initiating cells contribute to therapy evasion and relapse.

Sheila Kumari Singh, David Bakhshinyan, Chitra Venugopal, Ashley Adile, Mohini Singh, Maleeha Qazi, Branavan Manoranjan, Michelle Kameda-Smith. _McMaster University, Hamilton, Ontario, Canada_.

Medulloblastoma (MB) is the most common malignant pediatric brain tumor. Of current molecular subgroups, Group 3 patients face the highest incidence of metastatic spread and overall patient survival of less than 50%. Current clinical trials for recurrent MB patients based on genomic profiles of primary, treatment-naïve tumors provide limited clinical benefit, since recurrent metastatic MBs are highly genetically divergent from their primary tumors. By adapting the existing COG (Children's Oncology Group) protocol for children with newly diagnosed high-risk MB to the treatment of immuno-deficient mice intracranially engrafted with human MB brain tumor initiating cells (BTICs), we have characterized the rare treatment-refractory cell population in Group 3 MBs. MB cell populations recovered separately from brains and spines during the course of tumor development and therapy were comprehensively profiled for gene expression analysis, stem cell and molecular features to generate a global, comparative profile of MB cells through therapy to relapse. One of the most intriguing observations from our gene expression data was consistent over-expression in the treatment-refractory cell population of proteins belonging to the Inhibitor of DNA-binding/differentiation (ID) family (transcription factors with a basic helix-loop-helix motif that act as suppressors cellular differentiation), and a longevity-associated protein known as bactericidal/permeability-increasing fold-containing-family-B-member-4 (BPIFB4). This persistent upregulation of genes preserving undifferentiated state and cellular longevity further strengthens the hypothesis of stem-cell like cells driving tumor relapse in MB. Targeting ID1 and BPIFB4 using both knockdown (KD) and knockout (KO) strategies has resulted in decreased self-renewal and tumorigenicity of both primary and recurrent MB cells, further highlighting their potential as novel therapeutic targets in MB. Our differential genomic and gene expression profiles of the "treatment-responsive" tumors against those that fail therapy have successfully contributed to discovery and characterization of novel therapeutic targets for the most aggressive subgroup of MB.

#1141

A novel regulatory gene subnetwork of glioma stem-like cells.

Changwang Deng, Son B. Le, David D. Tran. _University of Florida, Gainesville, FL_.

Glioblastoma (GBM) is the most common and deadliest malignant brain tumor in adults. GBM is enriched in glioma stem-like cells (GSCs) that contribute to tumor initiation and treatment resistance and thus a natural focus for therapeutic development. However, targeting GSCs has been a challenge because of a lack of master regulators specific to GSCs and not to normal brain cells. Therefore, systematic identification of the GSC-specific common regulatory program will improve our understanding of GSCs and provide an opportunity to develop GSC-specific therapeutics that are efficacious and safe. To accomplish these objectives, we have recently developed two innovative tandem computational platforms, GeneRep and nSCORE, which were subsequently experimentally validated. The tandem platforms complement each other and allow us to 1) interrogate large-scale gene expression profiles of GSCs, GBM-differentiated cells, normal neuronal precursor cells (NPCs) and astrocytes (NAs) to extract candidate GSC-specific gene regulatory networks; and 2) functionally rank GSC-specific core master regulators, respectively. A cluster of 8 interrelated master subnetworks emerged, which were functionally grouped into the stemness and cancer pathways, some of which belonged to the Sox, Olig and Myc families. By enforcing the expression of this cluster in NAs, we were able to reprogram these normal cells into GSCs as measured by the neurosphere formation and in vivo tumorigenesis assays, confirming the critical requirement of this cluster for the GSC identity. Furthermore, this network cluster was present and functioned cooperatively to maintain GSCs in several patient-derived GSC lines examined to date. Survival and neurosphere formation rates of GSCs were profoundly suppressed when at least two nodes in this cluster were depleted by siRNA compared to depletion of the individual nodes. Our data establish a novel gene regulatory cluster controlling GSCs and provide a compelling rationale for its therapeutic targeting to reduce tumor recurrence.

#1142

STK31 contributes to lung carcinogenesis through induction of stemness and resistance to EGFR-TKI therapy.

Eunice Yuen-Ting Lau, Isabella Kit-Nam Chin, Alvin Hong-Wai Fong, Victor Wan-San Ma, William Chi-Shing Cho. _Queen Elizabeth Hospital, Hong Kong, Hong Kong_.

Lung adenocarcinoma (LUAD), the most common histological subtype of lung cancer, remains one of the leading causes of cancer-related deaths worldwide. With improving knowledge in the molecular aberrations underlying lung carcinogenesis, a large number of targeted therapies are being developed to better patient's survival. EGFR-activating mutations are one of the most frequent genetic alterations in advanced lung adenocarcinoma, and patients harbouring certain types of EGFR activating mutation confer sensitivity to EGFR tyrosine kinase inhibitors (TKIs). Despite promising initial responses, majority of patients develop resistance to treatment and the mechanism of resistance remains unknown in up to 30% of patients. Recent studies revealed that cancer stem cells (CSCs) endowed with stem cell-like properties confer resistance to EGFR-TKI treatment. Identification of pathways maintaining stemness properties potentially provide novel drug targets to overcome treatment resistance. Protein phosphorylation is the most common form of reversible post-translational modification and is frequently involved in the deregulation of signal transductions associated with cancer development and progression. In this study, we have identified Serine/Threonine Kinase 31 (STK31) to be upregulated in enriched lung CSC populations, as well as in the erlotinib resistant derivatives from two LUAD cell lines. Through analysing The Cancer Genome Atlas (TCGA) data, mRNA expression of STK31 is observed to be higher in LUAD tumor when compared with paired normal tissues (p<0.001). High mRNA expression of STK31 was also found to be associated with poorer disease-free survival (p=0.0324) and overall survival (p=0.0012) in LUAD patients. Functional studies using shRNA based knockdown of STK31 in lung cancer cell lines revealed its regulatory role on cancer and stem cell-like properties in vitro and in vivo including tumorigenicity, self-renewal, drug resistance and metastasis. Repressing the expression of STK31 in erlotinib resistant cells re-sensitized the cells to drug treatment. Further studies will be done to delineate the downstream mechanism by which STK31 regulates lung CSCs and erlotinib resistance.

#1143

Prox1 and Notch mark distinct colorectal cancer stem cell populations.

Jenny Högström,1 Sarika Heino,1 Pauliina Kallio,1 Marianne Lähde,1 Veli-Matti Leppänen,1 Seppo Kaijalainen,1 Diego Balboa,1 Timo Otonkoski,1 Sylvie Robine,2 Zoltan Wiener,1 Kari Alitalo1. 1 _University of Helsinki, Helsinki, Finland;_ 2 _Institut Curie, Paris, France_.

Colorectal cancer (CRC) is one of the leading causes of cancer-related morbidity and mortality. Signaling pathways, such as the Wnt and Notch pathways, are essential for the maintenance and differentiation of wild-type intestinal stem cells. We have shown that the Prox1 transcription promotes expansion of the colorectal cancer stem cell pool and that a subpopulation of the Prox1+ CRC cells display stem cell activity. Because of these results we have further analyzed the role of Prox1 in the regulation of CRC stem cell. Here, we report that Prox1 regulates CRC stem-like cells via bidirectional interaction with Notch1 during CRC initiation and progression. Using genetic in vivo models and ex vivo 3D organoid cultures, we show that Notch inhibition decreases the number of Lgr5+ stem cells whereas it increases expression of the Prox1. Although Notch inhibition led to increased proliferation of the Prox1 positive cells, it did not affect their ability to give rise to differentiated Prox1 negative progeny. Ectopic overexpression of the active fragment of Notch1 suppressed Prox1 expression and inhibited stem cell activity in the CRC cells. On the other hand, the PROX1-NuRD complex suppressed the Notch signaling pathway and Prox1 deletion increased Notch target gene expression and promoter activity, indicating reciprocal regulation between Prox1 and Notch1. Thus, although Prox1 and Notch suppress each other in colorectal cancer cells, Prox1+ cells can function as a stem cell population without the need for Notch pathway activity. 

### Translational Therapeutics in Cancer Models 1

#1144

Building Kras (G12D)/Trp53 null/Pdx1-cre (KPC) mouse allograft tumor model for efficacy evaluation of combinatory immunotherapies.

Yanrui Song, Benqi Liu, Annie Xiaoyu An, Likun Zhang, Jie Cai, Qian Shi, Henry Qixiang Li, Davy Xuesong Ouyang. _Crown Bioscience, Inc., Taicang, Jiangsu, China_.

Pancreas ductal adenocarcinoma (PDAC) has dreadful 5-year survival of 6% and remains one of the most difficult to treat cancers with very few standard-of-care options. Fueled by the clinical success of immune checkpoint inhibitors in other solid tumors, several clinical trials have been conducted in PDAC patients. Unfortunately, those attempts have achieved limited clinical benefits as single-agent treatment. Now investigators are eager to test combinatory therapies of chemotherapies, small-molecule immune modulators, and immune checkpoint antibodies. Highly relevant preclinical models are very much needed for proof-of-principle efficacy evaluation. KPC model, first described by Tuveson and colleagues as LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx1-Cre, and later established in a variety of derivatives by several labs with Trp53 heterozygous or homozygous knockouts, recapitulates human PDAC tumors in many aspects including morphologic small ductal tumors with enriched stromal contents; key features of immune microenvironment of PDAC representing robust inflammatory reactions, like high levels of B cells and macrophages and exclusion of effector T cells; and most importantly, is nonresponsive to a series of chemotherapies and immune checkpoint inhibitors, including PD1 and CTLA4 antibodies. These features make KPC model an ideal model to test combinatory therapies for PDAC. However, parental KPC model is difficult to use for pharmacologic studies due to the spontaneous nature of tumor onset and progression. The compound mutant mice are also costly to breed. We have generated a transplantable KPC tumor model by passaging the primary tumor subcutaneously in the C57BL/6 mice. These KPC allografts retain morphologic similarity to human PDAC. Immune profiling of both subcutaneously and orthotopically grafted tumors demonstrated highly enriched CD45+ lymphocytes, particularly B cells and macrophages, in the tumor. The tumor is also nonresponsive to gemcitabine treatment, in line with the data from parental line. Orthotopically engrafted tumor leads to lethality of the host with the median survival time of 25 days. We are now testing a variety of combinatory therapies with chemotherapies, immune modulators and immune checkpoint antibodies. The results will be presented at the meeting.

#1145

Humanized immune-PDX mouse models for 10 different tumor entities in translational immuno-oncology research.

Maria Stecklum, Annika Wulf-Goldenberg, Magdalena Paterka, Bernadette Brzezicha, Iduna Fichtner, Jens Hoffmann. _EPO Berlin-Buch GmbH, Berlin, Germany_.

We established humanized mouse models by transplantation of human hematopoietic (stem) cells to immunodeficient mice. Engraftment results in the development of a functional human immune system. By co-transplantation of human patient-derived xenografts (PDX) we successfully generated a fully human tumor-immune-cell (TIC) model in mice. These humanized TIC-PDX models for melanoma and lung cancer were further characterized by treatment with immune therapy drugs like the CTLA4 inhibitor ipilimumab (Ipi) and the PD-1 inhibitor nivolumab (Nivo). In our current studies we monitored the effects of the human immune cells and evaluated concepts for combination therapies i.e. with chemotherapy or radiation. We further established new human TIC-PDX models from other tumor entities like lymphoma, pancreatic or breast cancer.

Hematopoietic stem cell engraftment was monitored by regular FACS analysis for human immune cells in the blood. PD-L1 expression on tumors as a target for immunotherapy was determined by FACS and immunohistochemistry. PD-L1 positive and negative PDX from 10 different entities were transplanted on humanized mice and treated with Nivo or Ipi alone or in combination therapy with radiation. Blood and tumor samples were analysed by FACS and immunohistochemistry for immune cell infiltration and activation.

The transplanted stem cells showed engraftment in immunodeficient mice with proliferation and differentiation and established a functional human immune system with T-cells, B-cells, NK-cells, monocytes, and dendritic cells. So far, we have transplanted more than 40 different PDX from 10 different tumor entities on humanized mice. Most of investigated PDX (>70%) successfully engrafted on humanized mice and showed no difference in tumor growth compared to growth on non-humanized mice. However, for a few PDX we observed a delayed tumor growth or a complete rejection. These results suggest an immune reaction of the engrafted human immune cells against the PDX. The tumor engraftment or rejection in the humanized mice seems to be independent from the tumor entity. To evaluate whether engraftment of the PDX in mice with a human immune system is in part enabled by checkpoint mediated immune-tolerance, we have treated over 30 PDX models with Ipi or Nivo alone or in combination. Significant tumor growth delay, observed in most models, accompanied by an increased number of T-cells in the blood and in the tumor, has functionally validated our immune-PDX model. Response to the checkpoint inhibitors seems to correlate with innate immune responsiveness and PD-L1 expression and can be further increased by combination with radiotherapy.

Our humanized immune-PDX models enable appropriate preclinical translational research in tumor immune biology and the evaluation of new therapies and combinations, as well as the identification and validation of biomarkers for immune therapy.

#1146

Early PD-1 inhibition abrogates PBMC engraftment in a triple-humanized (tumor/PBMC/MSC) bioluminescent orthotopic model of pancreatic cancer.

Benjamin G. Cuiffo, Caitlin S. Parello, Chelsea Ritchie, Alexandra Kury, Scott Anderson, Gregory D. Lyng, Stephen T. Sonis. _Biomodels, Watertown, MA_.

Accuracy of context is critical in efforts to improve the translational potential of preclinical oncology models. Pancreatic cancer has high mortality and current models are insufficient for advancing therapeutics. Here we note key observations in the ongoing development of a new pancreatic cancer mouse model designed to enable translational immuno-oncology and/or tumor microenvironment (TME) targeted therapeutics in a "triple-humanized" orthotopic context including: 1) tumor: bioluminescent PD-L1+ BxPC3-Red-FLuc human pancreatic ductal adenocarcinoma (PDAC) cells surgically implanted into the pancreas of aged NSG™ mice; 2) T cells: human peripheral blood mononuclear cells (PBMCs) derived from a healthy donor and shown to respond to PD-1 inhibition; and 3) tumor-tropic stem-like stromal cells: bone-marrow (BM) derived human mesenchymal stem/stromal cells (MSCs) derived from a healthy donor. MSCs are described to home to PDAC stroma where they react to TME and contribute to PDAC progression via pleiotropic therapeutic refractory effects including stromal remodeling favoring effector immunosuppression, angiogenesis and desmoplasia. To assess the role of MSCs in PDAC response to PD-1 blockade, we injected BxPC3-Red-FLuc cells into the pancreas of 14-week NSG™ mice, +/-MSCs; coinjected animals received weekly MSCs bolus injections to simulate continuous migration from BM. Some groups concurrently received PBMCs characterized for aPD-1 response. Ten days after injection with human cells, animals were randomized by tumor radiance into treatment groups administered +/- 150µg anti-human PD-1 mAb (clone: EH12.2H7) (aPD-1) 2x/week. Immunohistochemistry analysis of tumors was performed. Engraftment/characterization of PBMCs was assessed by flow cytometry of PB in-life and spleen at terminus. MSCs alone did not increase primary tumor growth, but PBMC engraftment was increased by administration of MSC. Tumor growth was decreased with PBMC but primary tumor growth was increased in animals receiving PBMC+MSC compared to PBMC alone. The antigen-experienced status of the T cells was not affected by MSC treatment. In contrast, the diversity of engrafting T cells was increased by MSC treatment. Strikingly, aPD-1 treatment completely suppressed PBMC engraftment, regardless of MSC presence. This work details ongoing development of a contextually improved "triple-humanized" mouse model of pancreatic cancer. Key findings agree with recent findings of the importance of timing in aPD-1 treatment in humanized models and clinical applications.

#1147

Developing an individualized human colonic crypt cell culture as an ex vivo model for immunotherapy.

Peh Yean Cheah,1 Yee Syuen Low,1 Michelle Lo,1 See Aik Tang,1 Alphaeus Ng,1 Choong Leong Tang,1 Siew Heng Wong2. 1 _Singapore General Hospital, Singapore, Singapore;_ 2 _J.W. Bioscience, Singapore, Singapore_.

Colorectal Cancer (CRC) is the third leading cause of cancer death worldwide. Majority of mortality is attributable to metastasis to distal organs. Current treatment targeting the ERK/MAPK pathway used monoclonal anti-EGFR antibody after the first line of chemotherapy fails. However these treatments are only effective for a small percentage of patients with wild-type KRAS, and these same patients eventually develop resistance. Small molecule drugs are toxic and have limited success. A combination of antibodies is an attractive alternative. This study aims to investigate whether anti-KRAS antibody can enter live cells in an individualized ex vivo culture system and whether it can bind and inactivate KRAS. The transient culture has advantages over cell line study as it better mimics the human colonic environment and tumors can be compared to their matched normals to partially address the problem of heterogeneity. Samples were obtained from patients' resected tumour and matched mucosa, cultured ex vivo, and immunostaining performed. We show that anti-KRAS antibody is internalized in both the cultured mucosa and tumor crypt cells prior to fixation. Subcellular localization of the internalized antibody in the mucosa cells is similar in the pre-fixed and post-fixed cells. In contrast, the internal cell membrane staining observed in post-fixed tumor cells appear more cytoplasmic and 'punctated' in the pre-fixed tumors, mimicking that of the mucosa cells suggesting that the internalized anti-KRAS may have altered the sub-cellular localization and hence cycling of KRAS. Furthermore, staining with anti-keratin antibody indicates that live tumor cells treated with anti-KRAS has partial reversal of the phenotype to become more like the regular striated pattern of the matched mucosa. Preliminary double labelling of cultured tumours with monoclonal anti-KRAS prior to fixation and polyclonal anti-KRAS post-fixation revealed punctate staining of KRAS in the cytoplasm that were mainly non-overlapping suggesting that the punctate structures are in different compartments.

#1148

Promising preclinical platform for evaluation of immuno-oncology drugs using Hu-PBL-NSG lung cancer models.

Sun Min Lim,1 Kyoung-Ho Pyo,2 Jae Hwan Kim,3 Ji Min Lee,3 Seong Keun Kim,1 Sung Eun Kim,1 Ha Ni Jo,3 Jae Soek Cho,1 Hye Ryun Kim,3 Byoung Chul Cho3. 1 _CHA Bundang Medical Center, Seoul, Republic of Korea;_ 2 _Severance Biomedical Science Institute, Seoul, Republic of Korea;_ 3 _Yonsei Cancer Center, Seoul, Republic of Korea_.

Introduction: With the advance of immunotherapy, treatment of non-small-cell lung cancer (NSCLC) has revolutionized by having anti-PD-1 therapy in front-line setting. In this era of cancer immunotherapy, humanized mouse models which recapitulate human immune system, are needed for predicting immunotherapy response in patients. We established a Hu-PBL-NSG mouse model which can be used as a preclinical testing platform for assessing efficacy of different immunotherapeutic agents.

Methods: Hu-PBL-NSG mouse model was established by engrafting human peripheral blood lymphocytes (PBLs) into NOD-scid (NSG) mice. The 6-7 week-old NSG mouse was transfused with 1 x 107 cells of PBL via tail vein, and human immune cells were characterized after 24 hours, and weekly thereafter. Cytokine array was performed to assess serological similarity between patient and the Hu-PBL-NSG mouse, and microscopic immune cell infiltration was observed in various organs mouse model. Human anti-PD-1 therapy was treated for assessing drug efficacy in patient-derived tumor.

Results: hCD3+hCD45+ T-cells and antigen presenting cells (dendritic cells, macrophages, and MDSC) increased in the serum of Hu-PBL-NSG mouse 24 hour after the transfusion of human PBLs, and CD3+ T cells were observed in lung, liver, kidney, spleen sections. Cytokine arrays of human and Hu-PBL-NSG mouse revealed high similarity of Th1, Th2, Th17-related cytokines. A tumor xenograft was engrafted from an EML4-ALK patient, and Hu-PBL-NSG mouse was sacrificed for histological analyses. hCD3+ T cells were infiltrated within the tumor, and CD11c+ cells, which represent antigen-presenting capability, were seen in spleen, lung, liver and kidney. When anti-PD-1 Ab was treated intraperitoneally, xenograft tumor showed significant reduction in volume after day 6, and increased expression of immune response-related genes on microarray analysis in the tumor. Mostly IFN-gamma and its related gene sets were significantly changed (FDR < 0.25, GSEA).

Conclusion: Hu-PBL-NSG mouse model which highly resembles human immune system was successfully established. This model could be a strong preclinical model for testing efficacy of immunotherapeutic agents, and also for pursuing novel immunotherapy treatment strategies in advanced NSCLC.

#1149

Humanized knock-in mouse models for evaluating in vivo efficacy of immune-oncology drugs targeting inhibitory immune checkpoint molecules.

Baozhu Zhang, Chaoshe Guo, Yuelei Shen, Yanan Guo. _Beijing Biocytogen Co., Ltd, Beijing, China_.

Recently, increasingly attention has been paid to the development of immuno-oncology (IO) drugs, due to the promising clinical results acquired by CTLA-4, PD-1 and PD-L1 antibodies. However, along the IO drug development process, in vivo efficacy models have always been a rate-limiting step. Mouse surrogate antibodies were often used in immune-competent mice with murine syngeneic tumor models to evaluate in vivo efficacy of the IO drugs. However, the efficacy of a surrogate antibody cannot fully represent the human drug in the clinical scenario. In most cases, a human monoclonal antibody does not have mouse cross-reactivity. Therefore, we generated humanized knock-in mouse to evaluate the in vivo efficacy of human IO antibodies. For example, human PD-1 knock-in (B-hPD-1) mice were generated with a chimeric PD-1 receptor, which is recognizable by human PD-1 antibodies, and can be used to test human PD-1 antibody in vivo efficacy. Mouse cancer cell lines including colon cancer MC38, melanoma B16F10 and lung cancer LLC1 were genetically modified with over-expression of hPD-L1 and knock-out of mPD-L1, and used to evaluate the in vivo efficacy of human PD-L1 antibodies. Additionally, more knock-in mice targeting inhibitory immune checkpoint molecules were developed, such as B-hPD-L1, B-hCTLA-4, B-hLAG3, B-hTIM3, B-hTIGIT, B-hBTLA, B-hSIRPa et al,. Humanized knock-in mice, pairing with genetically modified mouse cancer cells, represent a promising in vivo efficacy model for the development of IO drugs, and contribute to the clinical translation of animal studies.

#1150

**Acute myeloid leukemia human/mouse co-clinical trial feasibility study optimized in human transgenic IL-3/GMCSF NOD/Shi-** scid **-IL2rγ** null **mice.**

Neal C. Goodwin,1 Daniel H. Albert,2 Angela M. Davies,1 Jenny Rowe,3 Gerold Feuer,3 Michael Boyiadzis,4 Kathleen A. Dorritie,4 Maria Mancini,1 Regina Gandour-Edwards,5 Warren M. Kati,2 Mark D. McKee,2 Keith F. McDaniel,2 David J. Frost2. 1 _Champions Oncology, Inc., Hackensack, NJ;_ 2 _Abbvie, Inc., North Chicago, IL;_ 3 _HuMurine Technologies, La Verne, CA;_ 4 _University of Pittsburgh, Pittsburgh, PA;_ 5 _University of California Davis, Sacramento, CA_.

Acute myeloid leukemia (AML) co-clinical modeling has been optimized with peripheral blood mononuclear cells (PBMCs) collected from low volume (14 mL) patient samples to establish an algorithm for efficiently co-clinically modeling AML patients. Methods: PBMCs were ficoll gradient purified and viably cryopreserved. Intrahepatic (i.h.) inoculation of AML PBMCs in neonate NOD/Shi-scid-IL2rγnull (NOG) mice and intravenous (i.v.) inoculation in both juvenile NOG mice and juvenile human transgenic IL-3/GMCSF NOD/Shi-scid-IL2rγnull mice (NOG-EXL) were evaluated. Bone marrow (BM) aspirates, splenocytes and PBMCs from mice were evaluated by fluorescence-activated cell sorting (FACS) at 12 weeks post AML inoculation for engraftment as determined by % ratio of human CD33+ cells to total CD45+ cells (human + murine cells). Humerus bones from inoculated animals were also evaluated by human CD33 immunohistochemistry (IHC). Results: Cells from 2/6 AML patient samples (CTG-2224 and CTG-2357) successfully engrafted into neonate mice. Animals were dosed with vehicle, cytarabine, ABBV-075 (clinical trial-staged BET family bromodomain (BD) inhibitor), or ABBV-744 (a preclinical BDII selective inhibitor) and evaluated for tumor burden six weeks post drug treatment initiation. ABBV-075 and ABBV-744 treated animals had lower tumor burden in the CTG-2224 model, 17% (p<0.05) and 4% (p<0.01); respectively. Similar trends, albeit at lower engraftment (10%), were observed for both models in spleen and blood compartments. In an effort to improve engraftment efficiency NOG-EXL mice were evaluated as hosts for engraftment with a patient PBMC sample (CTG-2357) and compared with juvenile NOG mice. Efficiency in the number of mice engrafted (8/8 vs. 3/7) and the extent of BM engraftment (51% vs. 14%) following a 0.5 X 106 PBMC inoculation was improved with the NOG-EXL mice. Furthermore, a patient inoculum (CTG-2241) that had previously shown no engraftment in juvenile NOG mice exhibited a 92% take rate with 93% BM tumor burden in NOG-EXL mice. Discussion: Prior advances in AML modeling show engraftment in IL2rγnul mouse models including i.h. and i.v. inoculation of BM clinical isolates in neonate NSG mice, i.v. inoculation of leukapheresis clinical isolates in juvenile NSG and NSG-SGM3 mice, and i.v. inoculation of BM aspirates in MISTRG mice. Each approach has limitations in terms of tedious neonate mice husbandry, ease of clinical sample collection with respect to clinical patient compliance, and mouse strain availability. Here we examined the feasibility of efficiently engrafting AML regardless of mutation or clinical stage from 14 ml PBMC samples. Results showed that i.v. inoculation of AML clinical samples in juvenile NOG-EXL mice was advantageous. This research is prerequisite for conducting translational co-clinical in vivo pharmacology studies for ABBV-075.

#1151

A unique layered 3D stromal tissue model for in vitro evaluation of immunologic agents and immune cell infiltration into cancer microenvironment.

Rii Morimura,1 Kei Tsukamoto,1 Shiro Kitano,2 Eiji Shinozaki3. 1 _Toppan Printing Co.,Ltd., Saitama, Japan;_ 2 _Osaka University, Saitama, Japan;_ 3 _Cancer Institute Hospital of Japanese Foundation for Cancer Research, Tokyo, Japan_.

Background; The modulation of the immune system is a proven key target for cancer therapy, however, the complex interplay between immune cells and cancer cells in cancer microenvironment remains only partially understood. In addition, the currently available in vitro cancer microenvironment models are not designed to study the immunologic agents and a role of immune cells. Here, we developed a unique layered 3D stromal tissue model comprising an allogeneic immune, stromal and cancer cells.

Method; Human umbilical vein endothelial cells (HUVEC) and normal human dermal fibroblasts (NHDF) were coated with collagen and heparin to construct Extracellular matrix (ECM) nanofilm and cultured to form multi-layers. Lung cancer cells were placed between stromal cells. Furthermore, we added Peripheral Blood Mononuclear Cell (PBMC) and Programmed cell death 1 (PD-1) blockade with Nivolumab and evaluated cytotoxic effects of immune cells on cancer cells and immune cell infiltration by immunohistochemistry.

Result; The co-cultured stromal tissues were multi-layered (approximately 20 layers), and the presence of lung cancer cells between stromal cells was observed. Immunohistochemistry revealed CD8 positive immune cell infiltration into the fibroblast layers. PBMC with Nivolumab treatment significantly decreased the viability of lung cancer cells in this 3D co-culture compared with the results in 2D mono-culture. High level of programmed cell death ligand 1 (PD-L1) expression on lung cancer cell lines correlated with a higher response to PD-1 blockade in vitro as well as clinical trials.

Conclusion; In summary, we present a unique 3D cell culture system based on an allogeneic stromal/cancer/immune cell setting to study immunologic agents and immune cell infiltration in cancer microenvironment. This 3D model has the ability to reflect in vivo conditions closer than 2D model and provide a tool for in vitro immunologic agent testing.

#1152

Therapeutic response to PD1/PDL1 axis in a disseminated C1498 syngeneic tumor model.

Vivek Mahajan, Thi Bui, Ian Belle, Edgar Wood, Lindsay Usher, Elizabeth Rainbolt, Shasta Kidder, Aidan Synnott, David Harris. _Charles River, Morrisville, NC_.

The majority of the leukemia cases are diagnosed in adults, of which, acute myeloid leukemia (AML) and chronic lymphocytic leukemia (CLL) are the most common subtypes. Treatment options include chemotherapy, stem cell transplantation and more recently the use of checkpoint inhibitors such as anti-PD-1 and anti-CTLA-4 to enhance antitumor responses. In this study we developed a disseminated AML disease model using PD-L1+, C1498 tumor cells to evaluate the therapeutic response to immune checkpoint inhibitors, PD-1, PD-L1 and CTLA-4 and to examine the effect of treatment on infiltration of immune cell populations in selected tissues. C1498 cells were transduced with a lentivirus vector construct containing luciferase and GFP reporter genes. Upon establishment of a clonal population with stable reporter expression, C1498-GFP-Luc expressing cells were implanted intravenously into syngeneic albino C57BL/6 mice and growth of the leukemic tumor cells was monitored via in vivo bioluminescence imaging (BLI) using IVIS Spectrum CT®. In vehicle-treated animals tumor growth was progressive and most animals reached endpoint within 30 days. Luciferase expression was widespread with metastatic development in peripheral organs and tissues. An efficacious response to therapy directed against the PD-1/PD-L1 axis was observed, with anti-PD-L1 showing greater overall efficacy than anti-PD-1. Combination of either PD-1/PD-L1 with anti-CTLA-4 further improved the therapeutic efficacy as measured by prolonged survival and reduced tumor growth. To gain mechanistic insights into how these immunotherapies function in suppressing tumor growth we performed additional studies utilizing flow cytometry and immunohistochemistry to assess the composition and phenotype of immune cell infiltrates in hematological and non-hematological tissues. In summary, comprehensive immunological characterization of the C1498 model revealed the importance of PD1 / PD-L1 pathway in immune evasion and provides a rationale for therapeutic intervention with checkpoint blockade.

#1153

**Development and validation of a novel in vitro** **model of the tumor immune microenvironment.**

RUOLAN ZHOU, Rachel Weathered, Luis Alonzo, Renata Mezyk-Kopec, Melody Swartz. _The University of Chicago, Chicago, IL_.

Tumor-associated lymphatics and their connection to the tumor-draining lymph node (TDLN) play important roles in cancer metastasis as well as in modulating the immune microenvironment. They bathe the TDLN in cytokines, exosomes, antigens, and suppressive factors from the tumor microenvironment to alter the anti-tumor immune response. These interactions are difficult to study in vivo, yet currently, these interactions cannot be readily explored in vitro. Here, we developed a perfused paper-based cell culture platform which can recreate the 3-dimensional architecture, stromal component and physiological levels of interstitial flow of the native tissue environment, while keeping the device high-throughput, easy to use, and inexpensive. This platform can sustain the long-term cultures (>3 weeks) of a number of tumor cell lines, which form complex architectures reminiscent of actual tumors, as well as of excised tumor tissue. The platform also supports the function and viability of immune cells (such as T lymphocytes and dendritic cells) and stromal cells (such as lymphatic endothelial cells and fibroblasts). To model the tumor interaction with its TDLN, we created a model in which tumor cells and lymph node stromal cells (LNSC) are cultured in separate but connected compartments under perfusion. To validate this, we have demonstrated preferential tumor migration to preconditioned (vs. unconditioned) LN stroma. The LN stroma compartment also supports co-culture of dendritic cells and T lymphocytes. To validate the modeling of the crosstalk between the tumor and TDLN, we show that tumor conditionioning of the LN primes the LN microenvironment to dampen cytotoxic T cell function. Together, these data highlight the potential of this system for studying complex tumor-host cell interactions in the tumor microenvironment over long periods of time, and paves the way towards an in vitro screening platform for drug screening for drugs that target cells or factors in the tumor microenvironment that serve as barriers to both chemotherapy as well as immunotherapies.

#1154

3D tissue models to predict patient-specific response to immuno-oncology based therapies.

Kathryn M. Appleton,1 Alina Lotstein,1 Qi Guo,1 Ashley Elrod,1 Larry Puls,2 Jeffrey Elder,2 Lillia Holmes,1 Howland E. Crosswell,1 Teresa M. DesRochers1. 1 _KIYATEC, Inc., Greenville, SC;_ 2 _Greenville Health System, Greenville, SC_.

While immuno-oncology based therapies are rapidly becoming the treatment of choice for many tumor types, the assay systems to accurately test them in preclinical development are lacking. This is primarily due to the complexity and function of the immune system and its interaction with tumors in terms of cell types, cellular recruitment and organization, and microenvironment. The ultimate in vitro systems would 1. Combine patient specific tumor cells with autologous immune cells to counteract cytotoxicity due to interactions between allogeneic lymphocytes and tumor cells, 2. Be designed to force recruitment of lymphocytes and macrophages by the tumor cells and their microenvironment, 3. Promote macrophage polarization and lymphocyte activation that recapitulates the patient's tumor by recapitulating the microenvironment, and 4. Result in tumor cell death that correlates to clinical response, biomarker expression (PD-1, PD-L1, CTLA-4, etc.), and therapy mechanism of action. To develop these systems, we have focused our work on numerous areas with 2 presented here, 1. Checkpoint inhibitors, pembrolizumab and Ipilimumab, in solid tumors such as ovarian cancer and melanoma, and 2. Lymphocyte and Macrophage migration, activation, and polarization in breast cancer. For checkpoint inhibitor studies, we have screened primary ovarian cancer tissues, melanoma, and matching lymphocytes for the expression of PD-1, PD-L1, and CTLA-4 by histology and flow cytometry. Both negative and positive tissues have then been utilized in 3D tissue models to examine the effects of drug upon the tumor cells and the other cell types within the model. We have seen correlation between Pembrolizumab binding to lymphocytes and PD-1 expression and shown further correlation to the expression of PD-L1 on the matching tumor cells and drug efficacy. In breast cancer we have used our complex, multi-cell type models to examine macrophage and lymphocyte migration and recruitment into the tumor. We have also examined the polarization of macrophages in these systems and their impact upon tumor cell viability with the tumor cells promoting M2 macrophage polarization and the M2 macrophages promoting tumor cell viability compared to M1 macrophages. Secreted cytokines as measured by multiplex technology have also supported an immune protective environment with M2 macrophages as seen by changes in IFNγ, IL-10, and TNFα compared to the presence of M1 macrophages. Our data to date reveals that these complex 3D in vitro models have the ability to recapitulate in vivo biology and biomarkers correlated drug response. These models can be used to both predict individual patient response to immune-oncology agents and test new agents for efficacy in a preclinical setting.

#1155

**The SRG** TM **rat: A novel SCID rat for humanization studies.**

Fallon K. Noto,1 Bisoye Towobola,1 Angela Arey,1 Goutham Narla,2 Christopher Chengelis,1 Tseten Yeshi1. 1 _Hera BioLabs, Lexington, KY;_ 2 _Case Comprehensive Cancer Center, Cleveland, OH_.

In vivo modeling of human cancer in genetically engineered rodents can provide insights into tumor kinetics, genetics and molecular biology, and allow for the testing of drug efficacy. Over the recent years, studies have demonstrated that immunodeficient mice, such as the NSG, reconstituted with functional human immune cells, such as peripheral blood mononuclear cells (PBMCs), are promising models for immuno-oncology efficacy studies. Immune humanized mice engrafted with human cancer cells show human-specific immune responses when treated with drugs that target immune pathways, such as CTLA-4 and PD-1, leading to the inhibition of tumor growth. These models provide a critical platform to study how the immune system can be engaged to drive anti-cancer efficacy.

Although the NSG and similar immunodeficient mouse strains have been beneficial for human immuno-oncology studies, there are many caveats to performing these studies in mice, including inconsistent tumor kinetics, small tumor size for downstream analyses, limited blood for PK/PD studies due to the small size of the mouse, and graft vs. host disease (GvHD) onset around 4-6 weeks post-engraftment. Humanized rat models would allow for the development of larger tumors and the ability to perform serial blood sampling on a routine basis throughout the course of treatment. We have created a Rag2 null, Il2rg null rat on the Sprague Dawley background (SRGTM) that lacks B, T, and NK cells and supports the growth of multiple human cancer cell lines, including lines that do not engraft or grow well in existing mouse models: HCT-116, H358, and VCaP. The SRGTM rat is also permissive to immune humanization with PBMCs. PBMC-engrafted SRGTM rats have a significant amount of human CD45+ lymphocytes in peripheral blood, of which the majority are T cells, comparable to immune-humanized NSG and NOG mice. Some animals also show significant levels of circulating human B cells. Interestingly, the incidence of GvHD is delayed to 12-14 weeks post-engraftment in the immune humanized SRG™ rat.

We are currently assessing functionality of the PBMC-immune humanized SRG™ rat in immuno-oncology studies as well as characterizing the ability of the SRG™ rat to support the growth of human patient derived xenografts (PDX). The generation of this novel humanized SRG™ rat model could allow for a more permissive host system to test existing and novel immunomodulatory strategies for the treatment of human disease.

#1156

New spontaneous and carcinogen-induced mouse-derived isograft (MDI) tumor models for immune therapeutic approaches.

Peter Jantscheff,1 Janette Beshay,1 Thomas Lemarchand,2 Cynthia Obodozie,1 Christoph Schaechtele,1 Holger Weber1. 1 _ProQinase GmbH, Freiburg, Germany;_ 2 _TPL Path Labs, Freiburg, Germany_.

First findings in the late 1990s and the early 2000s, that blockade of immune checkpoint inhibitors (ICI) by antibodies could induce rejection of established tumors and induce immunity also to secondary exposure with these tumor cells, led again to a stronger focus of experimental studies on syngeneic tumor models in immunocompetent animals. The availability of such models, however, is mainly limited by the small number of genetically-modified (GEM) or long-term passaged cell line-derived tumor models. We here describe establishment and first characterization of a new type of animal tumor models, named Mouse-Derived-Isografts (MDI), from spontaneously appearing or carcinogen-induced syngeneic tumors in both sexes of various mouse strains (C3H/HeN, CBA/J, BALB/C, DBA/2N and C57BL/6N). The tumors were obtained from animals during long-term observation (≥ 1-2 years) of normally fed, otherwise untreated animals (spontaneous - sMDI) or during short-term observation (3-9 month) of once subcutaneously, intramuscularly, intraperitoneally or orally methylcholanthrene (MCA) or methyl-nitroso-urea (MNU) treated animals (carcinogen - cMDI). Criteria to perform a primary necropsy were critical weight loss, bad general conditions or externally observable tumor growth in monitored animals. At this first step, tissues were assessed only macroscopically, and conspicuous ones were transplanted (in PDX-like manner) as small pieces into sex-matched syngeneic animals. If possible tissue/tumor pieces were also frozen and stored in 10% DMSO freezing medium at -80°C. In a second step these re-transplanted, outgrowing tumors were amplified once again in syngeneic mice for freeze storing, and then the models were finally established by re-transplantation and testing outgrowth of the frozen stored tumor pieces in the syngeneic animals. The tumors appear phenotypically stable, as far as routine standard HE stained sections could show. General characteristics of these models are; they are primary spontaneous or carcinogen-induced tumors, of low passage number (<10), propagated as tissue pieces only in mice without tissue culturing, and thus with conserved original tumor characteristics as assessed microscopically and intra-tumoral immune cell populations analyzed by flow cytometry.

#1157

The B-NDG mouse is a perfect tool for immune system humanization and patient derived xenograft transplantation.

Meiling Zhang, Chaoshe Guo, Yuelei Shen, Yanan Guo. _Beijing Biocytogen Co., Ltd, Beijing, China_.

The B-NDG mice (NOD-Prkdcscid Il2rgtm1/Bcgen), independently developed by Biocytogen, display a severe immunodeficiency phenotype, with no mature T, B or functional NK cells, and deficient in cytokine signaling. The B-NDG mouse is one of the best models for reconstitution of human immune system using peripheral blood mononuclear cells (PBMC) or hematopoietic stem cells (CD34+). Human PBMC reconstituted well in B-NDG mice, with the percentage of hCD45+ cells was over 50% in five weeks. To further analyze the subsets of immune cell composition, we found the majority of human cells were CD8+ T lymphocytes. Other than PBMC, human CD34+ hematopoietic stem cells also demonstrated a high-engraftment efficacy in B-NDG mice. Successful engraftment of CD34+ cells develops all major cell types via myeloid and lymphoid lineage to reconstitute functional human immune system. At 10 weeks post CD34+ cell implantation, the percentage of hCD45+ cells is over 30% with detectable human T, B and NK cells. Therefore, the immuno-humanized B-NDG mice are useful tools for studying hematopoiesis, immunology, infectious disease, and ES/iPS cell research. With barely any rejection to human derived cells, the B-NDG mouse is an ideal host for transplantation of PDX (Patient-Derived Xenograft), and retains the high heterogeneity characteristics of tumor tissues. Each PDX model can be considered as a single tumor patient and fully embodies the patient group characteristics. We have a collection of various PDX models including leukemia, breast cancer, gastric, lung, pancreatic, and colon et al. The humanized PDX model by implantation of PDX on the immuno-humanized B-NDG mice will be a promising tool to evaluate the in vivo efficacy of immune checkpoint antibodies in monotherapy or in combination. Therefore, therapeutic efficacy studies using B-NDG based PDX models are more meaningful and predictive in preclinical drug development and discovery.

#1158

Phenotypic screening using AMIDA identifies different drug responses in breast and prostate cancer cell lines in an organotypic cell culture model.

Mervi Toriseva,1 Katja Fagerlund,2 Jesse Mattsson,1 Tiina E. Kähkönen,2 Ilmari Ahonen,3 Malin Åkerfelt,1 Jenni Bernoulli,2 Jussi M. Halleen,2 Matthias Nees,1 Jenni H. Mäki-Jouppila2. 1 _Institute of Biomedicine, University of Turku, Turku, Finland;_ 2 _Pharmatest Services, Turku, Finland;_ 3 _Department of Mathematics and Statistics, University of Turku, Turku, Finland_.

Organotypic 3D cell culture models combined with screening modalities and automated high-content image analyses provide tools to gain a spectrum of biologically relevant information simultaneously from drug responses in tumor cells. This includes information about cell growth, death, differentiation and tumor cell invasion.

In this study, we examined the phenotypic drug responses of prostate cancer cell lines PC-3 and LNCaP, and breast cancer cell lines MDA-MB-231 (SA) and MCF-7 cultured in a miniaturized, imaging-optimized, Matrigel-based organotypic screening platform. We demonstrate the use of the 3D cell culture technology combined with automated morphometric image data analysis software AMIDA, for phenotypic, high-content screening. The emerging tumor organoids were treated with the cytostatic drugs doxorubicin, docetaxel and paclitaxel, a selective inhibitor of matrix metalloproteinase-13 (WAY170523), and with ROCK-inhibitors RKI-1447 and Y-27632. Treatments were conducted at seven different drug concentrations for 4-10 days. At the end point, confocal live cell images were captured and analyzed using AMIDA. Among others, the numerical data representing cell growth (Area) and cell invasion (Appendages) were visualized and used for statistics. EC50 values were calculated based on the Area-parameter derived from AMIDA analysis.

All cell lines initially formed multicellular, round organoids. PC-3 cells formed round and well-differentiated structures but spontaneously converted around day 9 of culture into structures showing massive, string-like collective invasion into the surrounding matrix. A different pattern of cell invasion was observed in MDA-MB-231 organoids, which developed strong and multicellular extensions by day 7-8. Interestingly, both ROCK-inhibitors promoted the invasion of PC-3 cells, as detected by phenotypic analysis with AMIDA. In contrast, Y-27632 reduced the invasion of MDA-MB-231 cells, whereas both ROCK-inhibitors induced cell invasion in MCF-7 cells at high concentration. WAY170523 inhibited the invasion of PC-3 cells but not of MDA-MB-231 cells, pointing to a different mechanism of invasiveness. All cell lines were highly sensitive to doxorubicin, docetaxel and paclitaxel at nanomolar range, as detected by the decrease of cell invasion, reduced organoid size, and increased cell death. The breast cancer cells were more sensitive to taxanes than PC-3 cells.

Organotypic 3D cultures combined with high-content phenotypic analysis with AMIDA software provide a quantitative view of drug effects and enable assessment of differential drug responses on various cell lines.

#1159

Development of uterine leiomyoma 3D in vitro models for high-throughput drug and chemical compound screenings: Towards personalized medicine.

Simona Bramante,1 Vilja Pietiäinen,2 Lassi Paavolainen,2 Annukka Pasanen,3 Netta Mäkinen,1 Hanna-Riikka Heinonen,1 Pirjo Ikonen,4 Oskari Heikinheimo,4 Jari Sjöberg,4 Ralf Bützow,3 Lauri Aaltonen1. 1 _Department of Medical and Clinical Genetics and Genome-Scale Biology Research Program, University of Helsinki, Helsinki, Finland;_ 2 _Institute for Molecular Medicine Finland (FIMM), University of Helsinki, Helsinki, Finland;_ 3 _Department of Pathology and HUSLAB, University of Helsinki and Helsinki University Hospital, Helsinki, Finland;_ 4 _Department of Obstetrics and Gynecology, University of Helsinki and Helsinki University Hospital, Helsinki, Finland_.

Uterine leiomyomas (ULs) are extremely common smooth muscle tumors, occurring in an estimated 77% of women of reproductive age. Although benign, ULs form a major burden to women's health and are the leading cause of hysterectomy worldwide. Recent findings show that ULs can be classified into at least three distinct molecular subclasses, each with a characteristic genetic driver aberration and global gene expression profile: MED12 (mediator complex subunit 12) mutation-positive, HMGA2 (high mobility group AT-hook 2)-overexpressing, FH (fumarate hydratase)-deficient. However, ULs are currently treated without taking into account the possible different subclasses. The aim of this project is to develop and characterize 3D in vitro models of ULs. The models will be used to examine responses to existing treatments in different subclasses of UL and to identify subclass-specific novel lead compounds for drugs. The work is based on the hypothesis that different molecular UL subclasses have distinct sensitivity and resistance patterns to existing treatment options. Therefore, ULs should not be treated as a single entity. The study material consists of UL tissue belonging to different subclasses and myometrium, collected from hysterectomy patients during surgery in Helsinki University Hospital, Finland. Freshly operated samples are processed to initiate primary cell cultures. The primary cells are seeded on Ultra-Low Attachment (ULA) multiwell plates and grown to form 3D spheroids. In case of MED12-mutated tumors, the spheroids are screened for MED12 mutations by Sanger sequencing. MED12-wild type samples and spheroids are analyzed for overexpression of HMGA2 and FH-deficiency. Immunofluorescence staining is performed to characterize different cell types in the spheroid structure. The presence of live/dead cells in spheroids is monitored during the drug treatments in 384-well plates with high-content confocal microscopy. The approach will first focus on the most common subclasses (MED12-mutated and HMGA2-overexpressing tumors) and most common treatment options. Here, we have set up novel patient-derived cell models for ULs. We have characterized myometrium and UL-derived spheroids and show that they retain the characteristics and genetic driver aberration of the original samples. Furthermore, we show they are composed mostly of live cells. Subsequent in vitro high-throughput drug screening will lead to characterization of drug sensitivities and identification of novel lead compounds for drugs. This information is important for rational drug design, where the effects of candidate drugs can be evaluated in light of the molecular class of the respective tumors. This work will lead to new knowledge towards precision medicine, which has potential for scientific breakthrough, since it would avoid exposing the patients to ineffective treatment options.

#1160

The novel osimertinib resistant lung cancer mice model harboring EGFR mutations driven by the SP-C promoter.

Kadoaki Ohashi,1 Hisao Higo,2 Go Makimoto,2 Kenichiro Kudo,3 Kazuya Nishii,2 Kiichiro Ninomiya,2 Hiroe Kayatani,2 Takashi Ninomiya,1 Toshio Kubo,1 Kammei Rai,1 Eiki Ichihara,1 Katsuyuki Hotta,1 Masahiro Tabata,1 Katsuyuki Kiura1. 1 _Okayama university hospital, Okayama, Japan;_ 2 _Okayama University, Okayama, Japan;_ 3 _NHO Iwakuni Medical Center, Okayama, Japan_.

Background: Discovery of driver mutations has drastically changed clinical practice in patients with lung tumors. Management of lung tumors with appropriated oncoprotein inhibitors is a key for longer survival. Osimertinib is an irreversible epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) and the new compound changed the standard treatment for non-small-cell lung cancer (NSCLC) harboring EGFR T790M. We previously established two strains of transgenic lung cancer mice model harboring mice Egfr ex19 deletion(mDEL) or human EGFR L858R(hLR) driven by an SP-C promoter (Ohashi Cancer sci 2008, Ohashi Cancer res 2009). The mice develop EGFR dependent lung adenocarcinoma multifocally, so it detaches to clinical primary lung cancers which usually occur mono-focally. In addition, the tumors develop from type II pneumocyte one after another. The more clinically relevant lung cancer mice model is warranted.

Material and Methods: The lung tumors from each of transgenic mice, mDEL or hLR, were transplanted subcutaneously into C57BL/6J mice. The each of engrafted tumors was able to be serially transplanted and grew in subcutaneous of other C57BL/6J mice. In addition, the tumor engraftment was succeeded in peritoneum by intraperitoneal administration or lung by tail vein injection. Gefitinib (50mg/kg) or Osimertinib (5 or 15 mg/kg) were orally administrated. Results These tumors maintained the EGFR dependency in novel syngenic xenograft models, thus EGFR-TKI, gefitinib inhibited the tumor growth significantly. However, same as human lung tumors, these tumors eventually acquired resistance and re-grew. mDEL tumors got the resistance for five months and hLR tumors acquired the resistance for two months after gefitinib treatment. Interestingly secondary mutation Egfr T790I on mDEL tumors and secondary mutation EGFR T790M on hLR tumors were detected. Strikingly, third-generation EGFR-TKI, osimertinib inhibited the tumor growth in vivo in each of gefitinib-resistant tumors harboring secondary mutations in vivo. Resembling human lung cancer, these tumors again acquired the resistance to osimertinib and re-grew two months after of treatment with osimertinib. The resistant mechanisms are under investigation.

Conclusion: We established clinically relevant novel EGFR-TKI resistant mice model. The strength of the model is 1) syngenic model, 2) the mice have a normal immune system and 3) the model presented a clinically relevant resistant mechanism. We are assessing an alternative treatment including combination therapies or immunotherapy in this mice model. (This work was supported by KAKEN 16K19454, KAKEN 15H04830)

#1161

Interaction between doxorubicin and amniotic membrane stem cells on the canine inflammatory mammary cancer cell line IPC-366.

Jessica Borghesi,1 Sara Caceres,2 Lara C. Mario,1 Rafael Gonçalves Hayashi,1 Laura Peña,2 Angela Alonso-Diez,2 Maria J. Illera,2 Gema Silvan,2 Maria A. Miglino,1 Ana C. Oliveira Carreira,3 Phelipe Oliveira Favaron,1 Juan C. Illera2. 1 _University of Sao Paulo. Veterinary medicine and animal science school, Brazil;_ 2 _University Complutense of Madrid. Veterinary School, Spain;_ 3 _University of Sao Paulo. Medicine school - chemistry institute, Brazil_.

The aim of this study was to evaluate the efficiency of doxorubicin, canine amniotic membrane stem cells (AMC) and the association between them in the triple-negative canine inflammatory mammary cancer cell line IPC-366. Experiments were carried out for 72 hours in 4 groups: IPC-366 untreated cells; cells treated with 10mg/mL of doxorubicin; coculture IPC-366 cells with 105 AMC; and cells treated with doxorubicin+AMC. MTT assays were performed for evaluating proliferation; flow cytometry to analyze hormonal and angiogenesis markers, and anti-inflammatory cytokines; and EIA techniques were used to evaluate secretion of steroid hormones (progesterone (P4); dehydroepiandrosterone (DHEA); androtenedione (A4); 17β-estradiol (E2); estrone sulphate (SO4E1)) in cell culture media. Results showed that cells treated with 10 mg/mL of doxorubicin resulted in a reduction of 71.64% on cell proliferation at 72h of treatment. A reduction in the expression of VEGF and PCNA-3 was observed by flow cyotmetry in treated cells compared to control cells. Differences on IL-10 were found within groups: treatment with doxorubicin resulted in a reduction on IL-10 expression although in AMC group expression remained similar to control group. Estrogen intracellular levels were significantly increased in doxorubicin+AMC group (4.67% vs. 27.1%). Regarding steroid hormone secretion, in AMC treated group any differences in hormone levels were found compared to control group. P4 and E2 secretion resulted in an increase in doxorubicin-treated group although in doxorubicin+AMC group these levels decreased with respect to control group. DHEA secretion was significantly decreased in treated groups compared to control group. Interestingly, A4 and SO4E1 secretion levels showed a significant increase compared to control group. Collectively, these results suggest that combination of doxorubicin and AMC caused a synergy that alters the cancer cell's secretion hormone profile by accumulation of estrogens. These findings attempt to give an approach of the combination of well-known treatments and amniotic stem cells as a canine mammary tumors therapy.

#1162

Castration-resistant prostate cancer bone metastasis model to assess new therapeutics.

Tiina E. Kähkönen,1 Mari I. Suominen,1 Jussi M. Halleen,1 Jenni Bernoulli,1 Pascale Lejeune2. 1 _Pharmatest Services, Turku, Finland;_ 2 _Bayer AG, Berlin, Germany_.

The role of androgens in the regulation of growth of primary prostate cancer is well established but less recognized in the context of advanced bone metastatic disease, which is the cause of high morbidity in patients. At advanced stage, prostate cancer cells typically lose their dependence on endogenous androgens leading to metastatic castration resistant prostate cancer (mCRPC). The aim of the study was to establish a novel mCRPC model resembling clinical aspects of the bone metastatic disease, which could be used for the evaluation of efficacy of new therapies.

To obtain a model for castration resistant growth, 5-6 weeks old NOD.Scid male mice were divided into three study groups. Mice in two study groups were castrated either before inoculation of the cancer cells or at four weeks after inoculation when the tumors were already growing. The mice in one study group were left intact. All mice received an intratibial injection of 2x106 VCaP human prostate cancer cells originally derived from vertebral metastatic site. Tumor growth was followed biweekly for 16 weeks by PSA measurements and X-ray imaging of tumor induced bone changes (lesions). At endpoint, androgen dependent organs were weighed and tumor-bearing tibias were subjected to histological evaluation.

At endpoint, a tumor take of 60%, 40% and 50% was observed in the intact mice and in the mice castrated one week before and four weeks after VCaP cell inoculation, respectively. Tumor take was assessed by PSA, X-ray and histology at endpoint. The PSA levels were higher in the intact mice and in the mice castrated at four weeks compared to the mice castrated before cancer cell inoculation. The PSA levels became detectable between 6-8 weeks after cancer cell inoculation in the intact mice and in the mice castrated at four weeks. In the mice castrated before cancer cell inoculation the PSA levels started to increase very late at 14-16 weeks. VCaP cells induced mainly new bone formation, an osteoblastic bone reaction typical for prostate cancer patients. In castrated mice, fewer bone lesions were observed compared to intact mice. The bone lesion areas quantified from X-ray images were larger in the intact mice compared to the mice castrated before cancer cell inoculation. As expected, the weight of androgen dependent organs was lower in the castrated mice compared to the intact mice.

In conclusion, a model mimicking important clinical aspects of castration resistant prostate cancer bone metastases was established. Tumor take and growth rates indicated that early phases of tumor development into the bone are androgen dependent whereas tumor growth at later stage relies on the bone microenvironment. The results highlight the significance of the tumor microenvironment in establishing clinically relevant preclinical models for drug development.

#1163

**Chemoprevention of testosterone-induced benign prostatic hyperplasia by hexane fraction from seeds of** Annona muricata **(soursop) in rats.**

Oluwatosin A. Adaramoye, Taiwo D. Oladipo, Olubukola O. Akanni, Solomon O. Owumi, Olusoji J. Abiola. _University of Ibadan, Ibadan, Nigeria_.

Alternative medicine is an extensively recognized therapeutic approach for the management of several diseases. Annona muricata commonly called sour sop is traditionally used to manage an array of human diseases, especially cancer. In this study, we hypothesized that hexane fraction from seeds of Annona muricata (HFAM) will ameliorate testosterone propionate (TP)-induced benign prostatic hyperplasia (BPH) in rat model. Castration was performed via the scrotal route under ketamine anesthesia and BPH was induced through daily intraperitoneal injection of TP (3 mg/kg) for 4 weeks after castration. Rats were assigned into six groups of six animals each viz: non-castrated control, castrated control, castrated rats that received TP (BPH group), BPH + HFAM, BPH + HFAM + finasteride, BPH + finasteride. Results showed that BPH rats had 3.1 and 2.4 folds increase in prostatic and organo-somatic weight, respectively. Furthermore, BPH increased the activities of total acid and alkaline phosphatases by 62% and 53%, respectively, and levels of lipid peroxidation products by 48% in the prostate of rats. Also, BPH caused significant increase in the levels of inflammatory markers (Myeloperoxidase and total nitric oxide) in rats. In contrast, there were no significant differences in the activities of prostatic antioxidant enzymes in BPH group relative to others. In BPH group, immuno-histochemistry revealed strong expression of prostatic inducible nitric oxide synthase, Bcl2, beta-catenin, androgen and estrogen receptors proteins, while mild expression of p53 and Bax were observed. Interestingly, treatment of BPH rats with HFAM or HFAM + finasteride significantly attenuated inflammatory indices and induced apoptotic parameters. Overall, hexane fraction from seeds of Annona muricata may be used as a therapeutic agent for BPH via anti-inflammatory effect.

#1164

Pdx model of pleural effusion of lung cancer patient for testing on targeted drug sensitivity and resistance.

Feifei Zhang,1 Zhen Zhou,2 Yuan Long,1 Wenhua Xu,1 Shizhu Zhao,1 Yang Yang,1 Hui Liu,1 Jijun Cheng,1 Shun Lu,2 Danyi Wen1. 1 _LIDE Biotech, Shanghai, China;_ 2 _Shanghai Chest Hospital, Shanghai, China_.

Advanced lung cancer of late stage patients is selectively sensitive to available targeted therapeutics and often develops resistance to early sensitive drug treatment. Surgery tumor tissues are usually not available to establish patient-derived xenograft (PDX) models which is critical to evaluate drug sensitivity and resistance. We applied CD45 magnet beads to enrich the tumor cell population from pleural effusion specimens of advanced lung cancer. Valuable drug-resistant PDX models were successfully established both from pleural effusion and from routine surgery tissues. Genetic profiling of the PDX models revealed TKI drug-associated EGFR mutations (L858R, T790M, Exon19del, C797S). In particular we successfully established a clinically AZD9291-resistant EGFR triple mutant (Ex19del, T790M, C797S) PDX model. In vivo efficacy results with gefitinib, erlotinib and AZD9291 well matched corresponding gene mutations among the models. Interestingly, we successfully induced AZD9291-resistant clones in vivo using an EGFR double mutant (L858R, T790M) PDX model, and NGS revealed mutations in kinases involved in other signaling pathways, indicating potential therapeutic options against resistance emergence. In conclusion, these precious PDX models provide insight into resistance mechanisms as well as serve as models to test potency of many drugs including current investigational TKIs.

#1165

**A tissue-engineered bone mimetic** **in vitro model for monitoring metastatic PCa growth and therapy response.**

Claudia Paindelli,1 Dietmar Hutmacher,2 Peter Friedl,1 Eleonora Dondossola1. 1 _UT MD Anderson Cancer Ctr., Houston, TX;_ 2 _Queensland University of Technology, Australia_.

Despite recent advances in prostate cancer (PCa) treatment, the outcome of metastatic disease remains frequently fatal and the underlying biology poorly understood. Thus, the development of clinically relevant in vitro models to monitor PCa biology in organotypic bone-like environment is critical to uncover mechanisms of therapy resistance and identify more effective treatments. To establish a bone-mimetic culture, we combined the following components: (i) bioactive osteoblasts depositing bone-like calcified extracellular matrix, (ii) complex 3D surface geometries, (iii) multicellular tumor application as spheroids/organoids, and (iv) applicability for live-cell microscopy to monitor the development of lesions over time. Calcified polycaprolactone (PCL) scaffolds were functionalized with bone-derived human mesenchymal stem cells (hMSCs) differentiated to osteoblasts, to generate a 3D niche-like calcified scaffold. PCa spheroids (PC3, C4-2B, patient-derived xenografts) were on-planted and their growth and invasion longitudinally monitored by advanced microscopy. PCa spheroids seeded on the organotypic bone model could be maintained and expanded over weeks, sufficient for monitoring therapy response to docetaxel, a first-line therapy for advanced PCa. The bone mimetic culture further revealed resistance to docetaxel mainly at the invasive edges, through a mechanism depending on the presence of osteoblasts. Thus, this 3D in vitro organotypic model will be suitable for dissecting the physical and molecular PCa cell-osteoblast interaction involved in PCa growth and therapy resistance.

#1166

Identification of predictive markers for clinical efficacy of triple-negative breast cancer chemotherapy.

Jihui Yun,1 Saem Hur,1 Wonyoung Kang,2 Deukchae Na,3 Jeesoo Chae,1 Jinjoo Kang,4 Ju-Hee Kim,5 Jiwoo Lee,5 Woohang Heo,1 Wonshik Han,5 Charles Lee,2 Dong-Young Noh,5 Hyeong-Gon Moon,5 Jong-Il Kim1. 1 _Seoul National University College of Medicine, Seoul, Republic of Korea;_ 2 _The Jackson Laboratory for Genomic Medicine, CT;_ 3 _Ewha Womans University Mokdong Hospital, Seoul, Republic of Korea;_ 4 _Ewha Womans University, Seoul, Republic of Korea;_ 5 _Seoul National University Hospital, Seoul, Republic of Korea_.

Triple Negative Breast Cancer (TNBC) is uncommon type of invasive breast cancer accounting for about 10~20% of all breast cancers. TNBC is characterized by a lack of expression of estrogen receptors (ER), human epidermal growth factor receptor 2 (HER2) and progesterone receptors (PR). Due to this characteristic, common treatments including hormone therapy and drugs targeting these receptors are not effective against TNBC despite more aggressive clinical behavior when compared to other subtypes. Paclitaxel is one of the well-known chemotherapeutic agents used in treatment for patients with breast cancer. However, previous studies have reported that advanced breast cancer treated with paclitaxel often develop resistance to the paclitaxel. To understand molecular characteristics and mechanism associated with molecular adaption in cancer cell when treated with paclitaxel, we performed next generation sequencing (NGS) and in vivo experiment using patient derived xenograft (PDX) models. We established ten PDX models from two cases with TNBC each. Mice were divided into two group consisting of control (n=5) and paclitaxel treatment (n=5). We performed exome and transcriptome sequencing on three of five samples for each group. We didn't find significant differences between control and paclitaxel treatment groups in exome data. In transcriptome data, We identified 14 differentially expressed genes (DEGs) including members of CXCL gene family between control and paclitaxel treatment groups. Among these DEGs, 13 DEGs were up-regulated in paclitaxel treatment groups when compared to control group. We are conducting in vitro experiment for several genes among these 13 DEGs. Furthermore, we will perform transcriptome sequencing on PDX tumor samples of additional cases with TNBC for validation of the results so far achieved.

#1167

A syngeneic MIND model for triple-negative breast cancer: A novel metastatic mouse model for disease progression and therapeutic studies.

Arnab Ghosh,1 Sandipto Sarkar,2 Fariba Behbod,2 Snigdha Banerjee,1 Ossama W. Tawfik,2 Sushanta K. Banerjee1. 1 _VA Medical Ctr. Kansas City, Kansas City, MO;_ 2 _University of Kansas Medical Center, Kansas City, KS_.

Background and Objective: In the era of targeted therapy mouse models have significantly contributed to our understanding of cancer biology. Insight into the molecular pathways has opened new avenues for cancer therapeutic strategies. Majority of the available mouse models depict a portion of the breast cancer progression cascade to be investigated. Some mimic the tumorigenesis at an early stage, some portray the later stage of metastatic process. Additionally, most of the mouse models lack the ability to depict the functional contribution of immune system during metastatic progression because very often they deal with immune-compromised mice. Hence a mouse model that can represent all the stages of breast cancer tumorigenesis and metastasis in native microenvironment in presence of immunity is exceptionally essential. This notion gave us an idea to develop a syngeneic mice model that will epitomize all the breast cancer progression stages and in very short time.

Methods: A modified mouse mammary intraductal (MIND) method was adopted for this study. MVT-1 and 4T1 cells were injected into cleaved nipples of 4th inguinal mammary glands of FVB/N and BALB/c wild-type female mice, respectively. The concentration of cells was 10,000 cells/µL. The final volume did not exceed 5 µL per injection per gland. Tissues were harvested at required time points. Histopathology was confirmed by haematoxylin-eosin staining and was further confirmed by immunohistochemical markers for cell proliferation, epithelial-to-mesenchymal transition (EMT), invasion, intravasation, extravasation and metastasis.

Results and Conclusions: Primary tumor had been established by the end of first week of injection and was confirmed by histopathology and whole mount analysis of 4th inguinal mammary gland. During second week after cell inoculation, cells had gone through EMT and had prepared and started invasion. Invasion was followed by intravasation and metastasis. Microscopic metastatic patches in the lungs were observed during third week and visible patches were observed during fourth week after injection. The main advantage of this model is short time span. Our model becomes more relevant as the origin of primary tumor and metastasis takes place in same animal, closely resembling biologic progression of breast cancer with the ability to depict the functional contribution of immune system during metastatic progression. A syngeneic mice model like ours can recapitulate the successive stages of breast cancer tumor progression and metastasis and could give the entire picture of metastatic process. We believe that our syngeneic mouse model will facilitate "drug-hunters" and basic science researchers towards developing improved breast cancer treatment.

#1168

The "humanized" chicken embryo chorioallantoic membrane (CAM) as a personalized platform for high-throughput (HTP) screening of cancer therapeutics.

Shiran Shapira, Oren Bogin, Nadir Arber. _Tel Aviv Sourasky Medical Ctr., Tel Aviv, Israel_.

Background

Every day, millions of people are taking medications that will not help them. "Personalized medicine," is the tailoring of medical treatment to a single person, shape his response to a particular treatment and aims to better target intervention, maximize benefit and minimize harm. Several techniques are available to grow primary cell cultures from tumors; however, very few have been found to be promising. Novel, cost-effective model that similarly mimic tumor biology and provide faster information on the activity of anticancer therapies could therefore make an important contribution to the advancement of personalized medicine. Chick Chorioallantoic Membrane (CAM) assays have been used to study the process and therapeutics involving angiogenesis, tumor cell invasion and metastasis. The CAM is naturally immuno-deficient and rich in vascularity therefore an ideal system, allowing to generate 3D cancerous "organoids" in a very efficient, reproducible and cost-effective manner and translates basic research to the clinic.

Aim

1) Generate a "personalized medicine" HTP system for a quick and reliable evaluation of the effectiveness of different therapeutic options on of 3D "organoid" tumors; 2) Using the "humanized egg" as a low-cost and highly efficient mimetic of the mouse PDX model ".

Methods

Fertilized eggs were incubated until day 3 (37°C, 75-90% humidity). 2ml of albumin was pulled from the egg to separate the CAM membrane from the egg shell. Then, a small window in the egg shell has been made, and resealed with adhesive tape. On day 7, 3-5x106 tumor cells were transplanted onto the CAM membrane. The cancer cells become a visible tumor after 3 days. Different therapeutics (such as Erbitux, anti-CD24 mAb) were tested. Tumor growth was monitored by "live imaging" device.

Results

The efficiency and reproducibility of human cancer cell lines engraftment has been demonstrated. LV-GFP-Puro plasmid was constructed and used to generate GFP-encoded lentiviruses which then were used to generate a stable colorectal cancer cell line expressing GFP. The CAM tumors were evaluated histopathologicaly and IVIS fluorescent imaging.

Anti-CD24 mAb inhibited tumor growth by ~70%.The presence of the humanized mAb in the engrafted tissue was confirmed by western blot analysis and IHC.

Conclusions

The ability to evaluate ex-vivo cancer tissue response to potential therapeutics in "humanized" settings within days is a very powerful tool.

#1169

Novel renal medullary carcinoma cell lines, UOK353 and UOK360, provide preclinical tools to identify new therapeutics.

Darmood Wei,1 Youfeng Yang,1 Christopher J. Ricketts,1 Cathy D. Vocke,1 Mark W. Ball,1 Carole Sourbier,1 Darawalee Wangsa,1 Danny Wangsa,1 Rajarshi Guha,2 Xiaohu Zhang,2 Paul Meltzer,1 Thomas Reid,1 Craig J. Thomas,2 Maria J. Merino,1 W. Marston Linehan1. 1 _NCI, Bethesda, MD;_ 2 _NCATS, Rockville, MD_.

Renal medullary carcinoma (RMC) is a rare and aggressive disease that predominantly afflicts individuals of African or Mediterranean descent with sickle cell trait. RMC comprises 1% of all renal cell carcinoma diagnoses, and the median overall survival for RMC patients is only 13 months. Patients are typically young (median age-22yo) and male (male:female ratio of 2:1). RMC was first characterized in 1995, and its hallmark SMARCB1 loss was identified in 2008. Due to the low incidence of RMC and the disease's aggressiveness, treatment decisions are often based on case reports. Thus, it is critical to develop preclinical models of RMC to both better understand the pathogenesis of this disease and identify an effective therapy. These RMC cell lines overexpress EZH2, relative to normal renal proximal tubule epithelium, thus making it an attractive druggable target. EZH2 inhibition in RMC tumor spheroids did not result in decreased viability. We have evaluated a potential clinical treatment (bortezomib) found in the literature and validate bortezomib's potency in vitro. Intriguingly, these cells lines not only lack NQO1, an antioxidant enzyme, but also the addition of N-acetyl cysteine, an antioxidant, has a protective effect against bortezomib. Together the data suggest that RMC are highly sensitive to oxidative stress. This evidence is consistent with case reports in the literature that found bortezomib treatment of RMC resulted in positive outcomes.

#1170

NCI-H1975 L858R/T790M/C797S isogenic cell line for next-generation EGFR inhibitor screening.

Feng Hao, Feng He, Wenna Zhang, Zhaoshuai Bai, Jinying Ning. _KYinno Biotechnology Co., Ltd, Beijing, China_.

Non-small-cell lung cancer (NSCLC) is the most common cause of cancer death. Over-activation of EGFR occurs in 13%~23% of NSCLC. The identification of EGFR as a cancer driver gene had led to rapid development of NSCLC targeted therapy, but the mutation of EGFR during treatment can cause drug resistance and cancer relapse. For example, T790M mutation causes resistance to the first generation of EGFR inhibitor Erlotinib and C797S mutation contributes to the resistance to the 3rd EGFR inhibitor AZD9291. There are several human cell lines for T790M mutation research, but there is not yet any primary human cancer cell line with C797S mutation. In the current study, a human isogenic EGFR C797S mutation cell line was generated through introducing nucleotide mutation into the EGFR gene in NCI-H1975 cell line using CRISRP technology, which originally bears L858R and T790M mutations of EGFR. First, the sgRNA sequence around the exon-20 of EGFR gene was screened using CRIPSR plasmid. Second, the donor plasmid with the homology arm containing exon-20 of EGFR and selecting marker was constructed and co-transfected into the parental cell line. Then, the NCI-H1975 C797S isogenic cell line was obtained and confirmed after screening over 100 single clones using the PCR method. The NCI-H1975 C797S isogenic cell line was resistant to AZD9291 but sensitive with EAI045 (the potential 4th generation EGFR inhibitor) combined with cetuximab. These data indicate that our NCI-H1975 L858R/T790M/C797S isogenic line is a useful model for next generation EGFR inhibitor study.

#1170A

Organoids: A new tool for the prediction of breast cancer patients' outcome.

Giuseppina Roscigno,1 Assunta Cirella,1 Alessandra Affinito,1 Francesco Palma,1 Maurizio Albero,1 Martina Messere,1 Martina Gelormini,1 Lorenza Cocca,1 Renato Thomas,2 Gerolama Condorelli1. 1 _Federico II University, Napoli, Italy;_ 2 _Clinica Mediterranea, Napoli, Italy_.

Breast cancer is one of the leading causes of cancer mortality among women. The main causes of death are related to recurrence and metastasis. One major limitation in the breast cancer research field is that there are few in vitro models of breast cancer able to predict clinical patients' outcome. The main reason of this failure is that the present models do not take complexity or heterogeneity into account. Therefore, innovative models are now required to better represent tumor fidelity. To address this issue, we developed an in vitro 3D organoid culture system using primary human breast cancer tissue. Organoids are miniature forms of tissues that exhibit three-dimensional architecture and are able to maintain phenotypical properties cultured in a dish. Therefore, they could be used as models for diseases, including cancer. A major difficulty in the development of such models is to identify in vitro conditions that preserve the breast cancer phenotypes observed in vivo. For this reason, we isolated organoids from breast cancer patients and optimized the organoids growing conditions. We used a specialised medium and matrix (Cultrex Trevigen) required for supporting cell growth as it occurs in the human body. Then, we decided to test whether the obtained organoids could be used as an in vitro prediction tool. First, we performed immunofluorescence assay to assess the preservation of patient architecture (CK14, CK18 and E cadherin) and clinical phenotype (HER2, ER, PR). Then, we used an innovative chemotaxis assay in 3D collagen gels for analysing organoids cell invasion. It is based on a chamber in which a chemoattractant gradient is generated by diffusion through a porous membrane for mimicking blood microenvironment. Then we analysed the morphological changes after chemotherapeutics treatment to correlate the organoids response with patient tumor behaviour. Thus, organoids can be considered as a new tool for studying breast cancer and developing personalized medicine approaches for this disease.

### Tumor Heterogeneity 2

#1171

Quantitative evaluation of hepatobiliary transporter density with microscopic fluorescence imaging using indocyanine green.

Hui Meng, Yuan Gao, Lin Yin, Xibo Ma, Kun Wang, Jie Tian. _Chinese Academy of Sciences, Beijing, China_.

The sodium-dependent taurocholate cotransporter polypeptide (NTCP) has been identified as a predictive marker for post-surgery survival rate of hepatocellular carcinoma (HCC) patients. Previous studies indicated that the expression of NTCP was associated with tumor differentiation. The ability to quantify protein expression is imperative to ensure successful personalized medicine including treatment stratification and therapeutic monitoring. Current standards for transporter analysis are based on immunohistochemistry and immunofluorescence. These measurements are complicated and too time consuming. Here, we endeavored to identify the expression of NTCP by quantitative microscopic fluorescence imaging (MFI) features, that could be used for investigation of tumor heterogeneity. Quantitative image features including the intensity, texture and wavelet texture were extracted from microscopic fluorescence images. Using multiple xenograft tumor models with varying NTCP expression, we determined the NTCP density in each model with MFI using indocyanine green. The MFI-calculated transporter density was correlated with ex vivo immunohistochemistry. Overall, our results argue that MFI provides a promising measure of transporter expression equivalent to immune-staining, with implications for use in investigation of tumor heterogeneity.

#1173

The identification of circulating tumor cells in the cerebrospinal fluid of gastric cancer patients with brain metastasis and the evaluation of intra-patient tumor heterogeneity.

Jang Ho Cho, Sun Young Kim, Kyung Kim, Jusun Kim, Su Jin Lee, Jeeyun Lee, Se Hoon Park, Joon Oh Park, Young Suk Park, Ho Yeong Lim, Won Ki Kang, Seungtae Kim. _Division of Hematology-Oncology, Department of Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Republic of Korea_.

PURPOSE : The purpose of this study was to identify circulating tumor cells (CTC) and evaluate tumor heterogeneity in cerebrospinal fluid (CSF) of advanced gastric cancer (GC) patients with leptomeningeal seeding. METHOD : We used the DEPArray system to identify and align single enabled CTCs in CSF based on multi-parallel fluorescence analysis. CSF were stained with an antibody cocktail recognizing cytokeratin, EpCAM, and HER2. CTC subpopulations of CSF were quantified as both absolute cell count and relative frequency. Based on the stained morphology of each single CSFTCs, five groups (Group A~E) were categorized.

RESULTS : CSF samples were obtained from a HER2 positive GC patient with leptomeningeal seeding. Each single CTC was collected according to the characteristics of morphology and the difference of marker expression. Group A CTC revealed HER2 positive, CK positive and EpCAM positive cells. The ratio of group A cells to total cells was 56.7%. Although the tumor of patient was HER2 positive cancer, HER2 negative CTCs in CSF (Group D + E) accounted for 21.8% of the total cells. Among CTCs we detected in CSF, there was no CTC revealing HER2 positive, CK negative and EpCAM negative.

CONCLUSIONS : DEPArray analysis might be a novel tool to capture CTC in CSF samples and evaluate the intra-patient's tumor heterogeneity. These findings suggested that DEPArray analysis could be used as a useful tool for the precision medicine.

Characterization of HER2 + CSFTCs on gastric cancer patient

---

HER2 | Cytokeratin | EpCam | Cell# | %/total cell | Group

Positive | Positive | Positive | 135 | 56.7 | A

Positive | Positive | Negative | 2 | 0.8 | B

Positive | Negative | Positive | 49 | 20.6 | C

Positive | Negative | Negative | 0 | 0.0

|

Negative | Positive | Positive | 5 | 2.1 | D

Negative | Positive | Negative | 0 | 0.0

|

Negative | Negative | Positive | 47 | 19.7 | E

#1174

Characterization of the immunologic intra-tumor heterogeneity in early stages of non-small cell lung carcinoma using multiplex immunofluorescence and image analysis approaches.

Alejandro Francisco Cruz, Edwin R. Parra, Mei Jiang, Junya Fujimoto, Chi-Wan Chow, Jaime Rodriguez-Canales, Carmen Behrens, Neda Kalhor, Annikka Weissferdt, John Heymach, Stephen Swisher, Boris Sepesi, J. Jack Lee, Cesar Moran, P. Andrew Futreal, Jianjun Zhang, Ignacio I. Wistuba. _The University of Texas MD Anderson Cancer Center, Houston, TX_.

Introduction. Recurrence of non-small cell lung carcinoma (NSCLC) is associated with genetic and epigenetic intra-tumor heterogeneity (ITH). The interaction between malignant cells, stromal cells, and tumor-associated immune-cells (TAICs), such as T-cell lymphocytes (TCLs) and tumor-associated macrophages (TAMs), is important for progression of NSCLC and the characterization of the immunologic ITH might be relevant to predict recurrence in surgically treated patients at early stages of NSCLC. The aim of this study was to characterize the immunologic ITH of primary NSCLC tumors at early stages using image analysis and multiplex immunofluorescence (mIF) approaches.

Material and methods. Eight cases of stage IA and 8 cases of stage IB surgically resected NSCLC (11 adenocarcinomas, ADCs; and 5 squamous-cell carcinomas, SCCs) with a history of early recurrence were selected for this preliminary analysis. Formalin-fixed, paraffin-embedded (FFPE) blocks were obtained and consecutive sections were stained with two panels of mIF for immune profiling, panel 1: pan-cytokeratin (AE1/AE3), PD-L1, PD-1, CD3, CD8, and CD68; panel 2: AE1/AE3, CD3, CD8, granzyme-B (GB), CD45RO, and FOXP3. Three not adjacent, intra-tumor regions (3mm2 each) per case were randomly selected after gridding the whole tumor section. A total of 41 intra-tumor regions were scanned by Vectra multispectral-microscope (PerkinElmer) and analyzed using InForm-software (PerkinElmer). TAICs were quantified in the epithelial and stromal compartments from each intra-tumor region.

Results. The median density of TCLs and TAMs were 1527 cells/mm2 and 635 cells/mm2, respectively, without significant differences between histologic subtypes. TCLs were predominantly concentered in the stromal compartment (median, 2222 cells/mm2) when compared with epithelial compartment (median, 332 cells/mm2). The percentage and density of TCLs and TAMs varied 4 and 8 times, respectively, between cases and regions. Non-cytotoxic T-cells and inactive cytotoxic T-cells were the most prevalent phenotypes. Higher density of TAMs and antigen-experienced TCLs were observed in stage IB than stage IA in the primary tumor of patients with NSCLC.

Conclusion. The characterization of the immunologic ITH of NSCLC is able by mIF and image analysis with FFPE tumor tissue. There is a variability of TAICs densities between regions from the same tumor and different subpopulations were observed. TAMs and exhausted T-cells were more prominent in stage IB (tumor >3cm) suggesting these cells may play an important role in recurrence. Ongoing studies with a larger cohort and comparison with non-recurrent surgically treated patients are warranted. Supported in part by CPRIT RP160668 grant

#1175

The establishment of single colorectal cancer organoid clones derived from regional biopsies for studying tumor heterogeneity.

Myoung-Hyun Song, Ja-Lok Ku. _Korean Cell Line Bank & Laboratory of Cell Biology, Cancer Research Institute at Seoul National University, Seoul, Republic of Korea_.

Nowadays, there is a spotlight on organoid culture, which is cultivated in 3D by using specimens derived from human or another mammalian. In addition, in the process of testing and screening anticancer drugs in vitro, the limitations of 2D culture have been pointed out because homogenous culture condition does not sufficiently represent the real environment in human body. Simultaneously, there has been consensus that 3D models such as spheroids and organoids are more physiologically similar to in vivo system when compared to 2D. In fact, tumor tissue grows in 3D and in heterogenous environment including extra-cellular matrix, immune cells and various lineage of cells differentiated from stem cells. To further comprehend complex in vivo tumor development pathway, new experimental model for mimicking real three-dimensional biopsy has been required and successfully become a part of biological field. Accordingly, organoid culture technic as an emerging next generation ex-vivo model has drawn tremendous attention for its advantages in lots of experimental and therapeutic application. Thus, we have established organoid (3D) lines by isolating tumor epithelium from colon cancer patient tissue and to look into the heterogenous environment of human intestine as well as investigating sensitivity in response to various stimulations such as anti-cancer reagents treatment and irradiation (professors Seung-Yong Jeong and Ji-Won Park contributed this work). And in particular, six to eight single organoids were isolated from the primary passage in each regional biopsy. 4 tumor tissues and 6 to 8 single organoids were obtained from a patient and each biopsy, respectively. In sum, more than 30 organoid lines from one patient were established and cultured to confirm both heterogeneity among regional biopsies and single organoids derived from each biopsy. Each single organoid was successfully isolated by using micromanipulator and micropipette without contamination. Then, we confirmed whether single clones were solely isolated. Furthermore, during the process of expansion of each organoid population, we observed differences in morphology and proliferation rate among organoid lines. However, distinct mechanisms that mainly influences on the tumor heterogeneity are still not fully understood. The technic of single organoid clone isolation could give insights for further studies on investigating heterogeneity among regional tumor biopsies.

#1176

Genomic profiling of canine glioma: Comparative analyses with respect to drivers of human glioma.

Samirkumar B. Amin,1 Juan Emmanuel Martinez-Ledesma,2 Beth Boudreau,3 Hoon Kim,1 Kevin C. Johnson,1 Peter V. Dickinson,4 Rebecca Packer,5 Amanda R. Taylor,6 John H. Rossmeisl,7 Amy Heimberger,2 Jonathan Levine,3 Roel Verhaak1. 1 _The Jackson Laboratory, Farmington, CT;_ 2 _The UT MD Anderson Cancer Center, Houston, TX;_ 3 _Texas A &M College of Veterinary Medicine, College Station, TX; _4 _UC Davis School of Veterinary Medicine, Davis, CA;_ 5 _Colorado State University, Fort Collins, CO;_ 6 _Auburn University College of Veterinary Medicine, Auburn, AL;_ 7 _VA-MD College of Veterinary Medicine, Blacksburg, VA_.

Sporadic glioma occurs in companion dogs at frequencies comparable to humans, despite differences in environmental exposures and age at time of diagnosis. Despite advances in molecular and phenotypic characterization of adult gliomas, accurate prognostication and curative treatment modalities are often limited by lack of animal translational models that can faithfully recapitulate underlying oncogenic processes and treatment response to newer therapies. Studying canine glioma at molecular level has several merits: First, it has a distinct advantage over genetically modified animal models to study natural course of glioma in dogs. Second, breed-specific elevated cancer risk, e.g., short-nosed breeds have higher risk for gliomas and smaller effective population size compared to humans potentially allow better characterization driver elements in the evolving canine glioma. Finally, dogs with the natural mammalian immune system have spontaneous occurrence of glioma, making it an ideal model for preclinical immunotherapy testing. Based on these merits, we performed whole genome, exome, transcriptome and methylation (reduced bisulfite) sequencing on 178 canine tumor and germline samples. As in humans, we show frequently occurring mutations and copy number alterations in canine gliomas in p53 pathway, cell cycle pathway (CDK4, CDKN2A), and receptor tyrosine kinases (EGFR, PDGFRA). We identified R132 mutations in the IDH1 gene reflecting a remarkable and species-agnostic but cancer-specific driving effect. Frequent whole chromosome gains were observed of syntenic region of chromosome 13, harboring human glioma oncogenes PDGFRA and MYC, but human glioma specific changes such as 1p/19q co-deletion and whole chromosome 10 loss/whole chromosome 7 gain were absent. We calculate mutational processes and highlight ones related to DNA damage repair and transcriptional strand bias in driving glioma in both species. Based on recent study, we also estimate relative timing of mutations in driver genes and copy number gains in canine glioma and compare those to human glioma in mapping life history of glioma, i.e., are canine glioma more similar to adult or pediatric human glioma cohort? In addition, transcriptional profiling delineated tumor-associated cell fractions from 22 immune cell types. The monocyte gene signature scored highest among canine glioma transcriptomes, in contrast to macrophages/microglia in human disease. Our results suggest similarities between the canine and human glioma microenvironment and we are validating those using canine immunohistochemistry. In bringing together a large canine glioma genomic and transcriptomic sequencing dataset and comparing to human glioma, our study provides unique new insights into glioma etiology and the chronology of glioma-causing somatic alterations.

#1177

Clonal dissection of pancreatic tumors unmasks functional and genomic heterogeneous long-term self-renewing compartments at the origin of treatment resistance.

Sahil Seth, Chieh-Yuan Li, I-Lin Ho, Denise Corti, Sara Loponte, Luigi Sapio, Edoardo Del Poggetto, Michael Peoples, Tatiana Karpinets, Giannicola Genovese, Andrew Futreal, Giulio Draetta, Alessandro Carugo, Andrea Viale. _UT MD Anderson Cancer Center, Houston, TX_.

Intrinsic and adaptive drug-resistance mechanisms allow human tumors to evade treatment through the demonstrated expansion of treatment-resistant clones. Thus, tumors are complex, dynamic ecosystems wherein populations of cells harboring both founder clones and unique, subclonal mutations coexist and progressively evolve. Modeling this functional heterogeneity of tumors can uncover critical contributions of distinct tumor cell sub-populations toward identifying rational drug combinations. Here, studying clonal evolution of tumor cells derived from human pancreatic tumors, we demonstrate that in vitro adherent cultures and in vivo tumors are maintained by a common set of long-term self-renewing cells that can be used to establish Clonal Replica Tumors (CRTs), large cohorts of animals bearing human tumors with identical clonal composition. Using CRTs to conduct quantitative assessments of clonal dynamics and adaptive responses to therapeutic challenge across different animals over time, we uncovered that the long term self-renewing compartment of pancreatic cancer is represented by a multitude of functionally heterogeneous subpopulations of cells with differential degrees of sensitivity to therapeutics. Consistent with the stem cell hypothesis, although tumors respond to treatments and undergo a transient regression, their clonal complexity at the time of relapse is only partially compromised, implying that many tumorigenic cells survive the treatment and sustain tumor relapse. Moreover, our ability to track the same cell populations in different animals enabled us to demonstrate that the clonal composition of relapsed pancreatic tumors varied across the different drug treatment groups (gemcitabine, MEK1 inhibitor and dual PI3K/mTOR inhibitor), suggesting that the compartment of long-term self-renewing tumorigenic cells is highly functionally diverse in mediating drug resistance to different therapies. Notably, high-throughput isolation and deep characterization of unique clonal lineages isolated through CRTs demonstrated that individual self-renewing populations display a remarkable genetic and molecular heterogeneity that can account for the differential functional responses and adaptation to perturbations. So, our findings portend a model in which the genomic and functional heterogeneity within human tumors is maintained, propagated and recapitulated entirely by distinct pools of long-term self-renewing cells. This concept has important implications for the efficacy of pharmacological combinations, which has historically been ascribed to their synergistic effects to abrogate the emergence of resistance, may instead be linked to the ability of mechanistically unrelated drugs to delay relapse by targeting multiple populations of tumorigenic cells simultaneously.

#1178

Genotype-fitness maps guide targeted therapy combination in lung adenocarcinoma.

Asaf Zviran,1 Patrick Bolan,2 Lisa Brenan,3 Amy Goodale,3 Denisse Rotem,3 Viktor Adalsteinsson,3 Federica Piccioni,4 Cory Johannessen,4 Dan Landau2. 1 _New York Genome Center, New York, NY;_ 2 _Weill Cornell Medicine, New York, NY;_ 3 _Broad Institute, Cambridge, MA;_ 4 _Broad Institute, New York, NY_.

Targeted EGFR inhibition in lung cancer leads to dramatic responses. Nonetheless, disease evolution to resistance is the rule. As this evolutionary process is fueled by intra-tumoral genetic diversity, a comprehensive mapping of clonal fitness is required to inform strategies to overcome resistance.

To model intra-tumoral diversity in vitro, we performed a genome-wide, over-expression genetic perturbation assay in EGFR-driven non-small cell lung cancer (NSCLC) PC9 cells. Our screen covered 17,255 ORFs (open reading frame constructs) representing 12,728 wildtype and mutated genes, and examined the effects of a first-generation EGFR inhibitor (erlotinib), a third-generation EGFR inhibitor (osimertinib) and a MEK inhibitor (binimetinib) on evolutionary selection, alone or in combination. Specifically, to obtain genotype-to-fitness maps for each drug, we measured clonal abundance up to 4 times during the screen and mathematically resolved their growth behavior. Finally, for each drug, we applied multiple doses to obtain dose-fitness relationships, which may impact tumor evolution in patients due to high inter- and intra-tumoral variability in drug delivery. In total, we have performed 78 genome-wide screens to map the evolutionary landscape of PC9 resistance to targeted therapy.

Erlotinib and osimertinib both result in an overwhelming reduction of fitness, as expected from their clinical benefit. Known clinical resistance mechanisms involving ERBB2, PIK3CA, AXL, and BRAF confer a pronounced fitness advantage in the presence of both drugs, while EGFR (T790M) improves fitness only with erlotinib. Novel resistance mechanisms include alternative tyrosine kinases (NTRK, PDGFRB, CSF1R, KIT), KRAS, G-coupled protein receptors, transcription factors (SOX15, FOXA1), and cellular transporters (ABCG2). Notably, we observe significant divergence in dose-fitness relationships between different resistance mechanisms. For example, PIK3CA confers a modest but persistent advantage across the dose range, in contrast to BRAF which results in a fitness advantage only when sufficiently high drug levels are added.

The fitness landscape for binimetinib resistance appeared to be largely orthogonal to that of the EGFR inhibitors, with decreased fitness noted for many tyrosine kinases as well as KRAS mutations. MEK inhibitor resistance results from RAF family member overexpression (ARAF, BRAF, RAF1) and MAPK activation. The orthogonal resistance landscapes of EGFR inhibition and MEK inhibition translate into highly synergistic effects in drug combination, with effective prediction of combinatorial fitness from the single-agent fitness landscapes using generalized linear models. These results validate this approach as a systematic method to address the combinatorial problem of optimizing drug combinations and doses to directly anticipate and address cancer evolution.

#1179

PAM50 subtype admixture in individual breast cancers and the relationship of this intratumoral heterogeneity to clinical variables.

Peter H. Gann,1 Neeraj Kumar,1 Dan Zhao,1 Amit Sethi2. 1 _Univ. of Illinois at Chicago, Chicago, IL;_ 2 _Indian Institute of Technology Bombay, Mumbai, India_.

Classification of breast cancers by PAM50 gene profiling assigns each cancer to one of five intrinsic subtypes. However, individual cancers vary in their adherence to the assigned prototype, and some may exhibit expression patterns that indicate admixture with other subtypes. The prevalence of this intratumoral heterogeneity phenomenon and the relationship of subtype admixture to clinical endpoints has not been extensively studied. We obtained microarray data on 1,460 cases from the public Gene Expression Omnibus and processed the data to normalize and log-transform expression levels and control for batch effects. For each case, we extracted levels for PAM50 genes and computed the Mahalanobis distance in multidimensional space for each case from all five centroids, including the one assigned. This metric obtains the standardized distance from a centroid for a single case while accounting for correlation between genes. We then discriminated pure from admixed cases by setting criteria for distance from the assigned centroid as well as distance from nonassigned centroids. Using the criteria that pure cases are those within the 75th percentile of distance from the assigned centroid, while being greater than the 75th percentile from any other centroid, we found that 59% of basal cases were categorized as pure, versus luminal A 31%, luminal B 35%, HER2 34% and normal 27%. We used t-Distributed Stochastic Neighbor Embedding (t-SNE) plots to visualize these admixtures in two dimensions. This confirmed that basal cancers were more heterogeneous than the other types, and that LumA, LumB and normal were more frequently admixed than other combinations. Pure LumA cases were less likely to be high-grade than LumA cases admixed with LumB, basal, HER2 or null cases not near any other centroid (18.5% vs. 7.2%, P < 0.01). Pure basal cases were more likely to be high-grade than basal cases admixed with any other subtype. Kaplan-Meier curves for metastasis-free survival for pure LumA were very similar to admixed cases for the first 5 years, but then diverged, with worse survival when LumA was admixed with worse subtypes. These results support the hypothesis that subtype admixture, or at least the tendency for some breast cancers to exhibit this form of intratumoral heterogeneity, can be identified and that it should be explored for clinical consequences, including failure to respond to subtype-specific therapy.

#1180

Quantifying melanoma drug resistance and tumor heterogeneity by live cell interferometry.

Kevin A. Leslie,1 Dian Huang,2 Graeme Murray,1 Daniel Guest,1 Irena J. Roy,2 Marco Piva,2 Gatien Moriceau,2 Roger S. Lo,2 Michael A. Teitell,2 Jason C. Reed1. 1 _Virginia Commonwealth University, Richmond, VA;_ 2 _University of California - Los Angeles, Los Angeles, CA_.

Melanoma is the most aggressive type of skin cancer. Tumor heterogeneity and drug resistance are significant obstacles to survival benefits from mutation-targeted therapy. Over 50% of metastatic melanomas harbor V600BRAF mutations. Targeting of MUTBRAF melanoma with BRAF inhibitor monotherapy or the combination of BRAF and MEK inhibitors leads to a high rate of initial responses, but adaptive and acquired resistance frequently leads to clinical relapse. Although the presence of V600BRAF mutations guides the selection of therapy with MAPK inhibitors, methods to track preexisting and adaptive resistance and thereby predict response patterns, or to use this new information to modify therapy, are still lacking. Here, we aimed to develop a rapid, massively parallel tumor cell profiling method, based on Live Cell Interferometry (LCI), to quantify heterogeneous single tumor cell responses and emergent drug resistance. LCI is an ex vivo imaging approach that quantifies changes in total cell biomass, biomass motion, or cell stiffness over time. We report a novel high-throughput screening version of the LCI platform, HSLCI, which rapidly profiles changes in biomass in BRAF inhibitor (BRAFi)-sensitive, parental melanoma cell lines and their isogenic, BRAFi-resistant sub-lines. We show reproducible results from two different HSLCI platforms at two institutions and generate biomass kinetic signatures capable of discriminating between BRAFi-sensitive and -resistant melanoma cells within 24 hours. Our measurements require no fluorescence or dye labeling and are faster than field-standard growth inhibition assays. The accuracy and speed of HSLCI in profiling tumor cell heterogeneity and therapy resistance are promising features of potential tools to guide patient therapeutic selections.

#1181

Structure and evolution of double minutes in a pediatric high-grade glioma.

Ke C. Xu, Liang Ding, Ti-Cheng Chang, Shuoguo Wang, Yong-Dong Wang, Heather Mulder, Ying Shao, John Easton, Jinghui Zhang, Suzanne J. Baker, Gang Wu. _St. Jude Children's Research Hospital, Memphis, TN_.

Double minutes (DMs) are circular extrachromosomal DNA frequently found in brain tumors, often associated with overexpression of oncogenes. To understand the evolution of DMs, we developed a graph-based method to identify simple cycles in a directed graph consisting of highly amplified copy number alteration (CNA) segments and structural variants (SVs) found at the boundaries of each segment. With this method, we presented a case of DM evolution in a pair of diagnosis and relapse whole genome sequencing (WGS) samples from a pediatric high-grade glioma patient, followed by validation with Chromium linked-read sequencing. Using an empirical value of CNA threshold, we extracted 44 and 29 highly amplified CNA segments from the diagnosis and relapse samples respectively. We then identified 29 and 19 unique SVs associated with these CNA segments from the two samples. We constructed five major DMs in the diagnosis sample and five major DMs in the relapse sample, covering all the CNA segments and the SVs. One DM harboring EGFR is identified in both samples. Two of the five relapse DMs are also found in the diagnosis sample but in a trace amount, suggesting a shifting from minor clones in the diagnosis to dominant clones in the relapse. Two major DMs, one carrying MYC and the other carrying CDK6, were only identified in the relapse sample. Analysis of mutations on the DMs revealed that new somatic mutations were acquired on sub-populations of the DMs. Importantly, for the EGFR-carrying DM shared in both samples, we identified an 8-bp deletion at a splicing site of EGFR that has 2% allele frequency in the diagnosis but reached 96% in the relapse. This deletion leads to a mRNA transcription skipping of exon 16 with an 87 fold up-regulation of EGFR compared with other high-grade gliomas, suggesting a possible drug resistance mechanism and positive selection of tumor cells resulting from the mutated DM. We propose an evolutionary model that simultaneously involves a branching model for DM formation and a selection model for mutations on DM. We applied the same methods to the WGS data from two adult glioblastoma patients in TCGA, and found that our proposed model could also explain the DM evolution in these patients. Therefore, our study reveals that DMs follow an evolutionary trajectory independent of linear chromosomes and contribute to tumor heterogeneity.

#1182

Exploring longitudinal intra-tumor heterogeneity in cancer using whole genome sequencing and RNA rescue.

Rahul Parulkar, Steve Benz, Charlie Vaske, Amie Radenbaugh, Christopher Szeto. _NantOmics, CA_.

We present a methodology for confidently characterizing subclonal exonic variants by pairing whole genome sequencing (WGS) with transcriptomics (RNAseq). We demonstrate the utility of this method by studying subclonal population changes in longitudinally collected samples.

Detection of variants specific to minority subclones has ordinarily only been achievable with very high-depth sequencing obtained by whole exome (WES) or targeted sequencing, and most available tools for studying subclonality require WES data as input. Although WGS provides more coverage breadth than WES, identifying subclonal mutations by WGS alone is difficult due to reduced read support for each variant. Detecting minority variants in WGS data is made especially difficult when tumor purity is low and subclonality is high. RNAseq is typically performed at high-depth and may be useful for replacing WES in detecting subclonal variants.

Our method has two steps: 1) Use WGS to establish a low-confidence preliminary set of variants present in the tumor, and 2) bolster the confidence in those variants by observing whether the variants are expressed in the RNAseq. We used this methodology to generate WES-like data from WGS+RNAseq in simulated data, and show that we can correctly identify subclonal populations using publicly available tools designed for WES data. Additionally, we demonstrate using this method to characterize the evolutionary lineage of the tumor population in a patient who was biopsied multiple times throughout several treatments, for whom only WGS and RNAseq data is available. Our proposed method resulted in 60% more confident variants than its WGS-only counterpart.

As tumor tissue availability is low and cost of sequencing is substantial, the choice of which molecular characterizations to perform is difficult. Here we show that paired WGS and RNAseq can be leveraged to infer information typically only obtained from WES, in addition to providing intergenic and expression information.

#1183

Targeted sequencing of cell-free DNA data enables comprehensive profiling of tumor copy number landscape from blood.

Catalin Barbacioru, Eric Collisson, Darya Chudova, Justin Odegaard, Richard Lanman, AmirAli Talasaz. _Guardant Health, Redwood City, CA_.

Background: The evolution of cancer genomes within a single tumor creates mixed cell populations with divergent somatic mutational architectures. Inference of tumor subpopulations has been disproportionately focused on the assessment of somatic point mutations, whereas computational methods targeting copy number alterations (CNA) in targeted sequencing data remain underdeveloped.

Methods: Using a large database of clinical cell-free DNA (cfDNA) sequencing data (Guardant Health, CA), we developed a coverage-based probabilistic model to simultaneously normalize molecular coverage, segment the genome, predict copy number alterations, and estimate the tumor content in cfDNA samples, while accounting for mixtures of cell populations. This model was technically validated using tissue fluorescence in situ hybridization (FISH) data and then applied to a unique set of cfDNA sequencing data from >5,000 normal and clinical samples spanning multiple cancer types, where model predictions were compared to the observed allelic frequencies of somatic driver mutations and heterozygous germline SNPs.

Results: Technical validation against FISH-derived tissue copy number estimates demonstrated high concordance with model estimates. Analysis of clinical samples demonstrated a wide range of copy number architectures, including prevalent copy number neutral loss-of-heterozygosity, large chromosomal deletions, and high focal amplifications, all of which are not easily detected and/or differentiated with standard sequencing analysis approaches on highly-fragmented cfDNA.

Conclusion: Our results show that probabilistic modeling of coverage data generated from targeted cfDNA sequencing can detect and differentiate heterogeneous tumor populations with diverse somatic variations, CNA, and LOH landscape. This method may enable improvements in CNA detection accuracy, sensitivity, and specificity and provides more precise interrogation of tumor fraction and clonal architecture.

#1184

Clonal evolution of diffuse intrinsic pontine glioma.

Scott T. Ryall, Robert Siddaway, Arun Ramani, Andrei Turinsky, Michael Brudno, Cynthia Hawkins. _Hospital for Sick Children, Toronto, Ontario, Canada_.

Diffuse intrinsic pontine gliomas (DIPG) are devastating tumours arising in the pons of children. Despite collaborative efforts, patient prognosis remains dismal with a median survival of 10 months and a 2+ year survival at ~5%. Previous efforts have identified the genetic drivers of DIPG revealing recurrent K27M mutations in histone H3 HIST1H3B or variant H3F3A which have been shown to dysregulate global lysine K27 methylation patterns of the tumour. Additional genetic hits including those in P53 and ACVR1 have also been identified. Previously, we showed that these driver mutations were conserved across all sites of DIPG dissemination, while secondary genetic hits showed subclonal enrichment. Here, we aim to investigate how DIPGs evolve with respect to both genetic (SNVs, CNVs) and non-genetic (epigenetics and transcriptomic) factors. We hypothesized that the genetic evolution of the disease will interplay with both the tumour's epigenetic changes and RNA expression to better explain DIPG development. To date, we have collected a total of 43 samples (range 3-12) from both the primary and disseminated site of 7 DIPG samples with matched normal brain. All samples have been subjected to whole exome sequencing, whole transcriptome sequencing (RNAseq) and MethylationEPIC analysis. The mutation burden across the samples ranged from 1.8-4.7 SNVs/Mb, with the exclusion of 4 sample locations that had acquired a hypermutant phenotype (range: 7.3-36.4 SNVs/Mb). Histone H3 mutations were the most frequent, being detected across all sampling locations in 6 of the 7 patients (5 in H3F3A and 1 in HIST1H3B). P53 mutations or LOH were detected in 4 patients, all of which also harboured a H3F3A mutation. No ACVR1 mutations were detected in this dataset. The final patient harboured MYCN, MYC-PVT1, and ID2 amplifications consistent with the MYCN subtype of DIPG. Clonal evolution analysis revealed distinct tumour heterogeneity in 6/7 samples, with the MYCN driven tumour appearing homogenous throughout all disseminated sites. In the remaining 6 samples, an average of 5 clones (range 4-11) were identified. H3F3A, HIST1H3B and P53 mutations were universally observed in the truncal clone of tumours and maintained throughout all sampling sites. Events driving subclones included PDGFRA amplification, MET amplification, PTEN loss, PIK3R1 mutations and hypermutant driving POLE and POLH mutations. Our preliminary work here provides insight into the genetic evolution of DIPGs. This work suggests that DIPG are heterogeneous in their development, but maintain homogeneity of the key tumour driver events throughout dissemination. However, due to the strong therapeutic potential of subclonal events described here, it remains important that the tumour's genetic complexity is not underestimated. Future goals looks to integrate both epigenetic and transcriptomic data into the evolution of DIPG to provide a clear and concise roadmap of how these tumours develop.

#1185

Synergistic drug combinations promote expansion of partially-resistant subpopulations in computational modeling of cancer heterogeneity and graded plasticity.

Elysia C. Saputra,1 Lu Huang,2 Lisa Tucker-Kellogg1. 1 _Duke-NUS Medical School, Singapore, Singapore;_ 2 _Singapore-MIT Alliance, Singapore, Singapore_.

Introduction: Synergistic drug combinations are widely sought as anti-cancer strategies because they yield higher therapeutic efficacy than additive or antagonistic combinations, enabling reduced doses. The effect of synergistic drugs (versus non-synergistic drugs) on the process of drug resistance evolution is not well understood, particularly for heterogeneous cell populations and for gradual phenotype transitions toward resistance. In this work, we use computational modeling of cancer cell numbers to interrogate the dynamics of resistance evolution during treatment with two-drug combinations.

Methods: We developed computational models of cancer evolution under two-drug combination therapy, to study the development of drug resistance in cancers with varying heterogeneity or graded plasticity of resistance. A large population of cells was simulated to undergo infrequent but stochastic phenotype change over time, subject to a pharmacological dosing model of combination effects.

Results: When synergistic and non-synergistic combinations were administered at equally effective doses in silico, drug resistance evolved more rapidly under the synergistic drugs. When we modeled graded plasticity by simulating multiple levels of drug resistance, results were similar except with additional bottlenecks before fully resistant cells could emerge. Meanwhile, as heterogeneity increased, synergism increased the speed of forming resistant subclones with higher fitness. Because synergistic treatments by nature exploit the simultaneous action of both drugs for their efficacy, the development of partial or single-drug resistance caused a disproportionate escape from drug effects and faster clonal expansion under synergistic therapy, compared with additive or non-synergistic therapies. Our simulations consistently showed that pairs of drugs with less synergism were able to prolong the time until double-resistance arose, provided the comparison combinations were dosed for equal initial efficacy.

Conclusion: We identify dosing criteria whereby synergistic pairs of drugs actually have worse performance than additive or non-synergistic pairs, for delaying the onset of double-drug resistance. Dosing criteria are also shown for graded resistance and variable levels of population heterogeneity. Future work must examine non-heritable contributions to resistance, such as micro-environmental effects. Our theoretical work shows the need for further study of the divergence between short-term and long-term drug efficacy in the presence of clonal selection, and suggests increased attention to non-synergistic drug combinations as therapeutic candidates.

#1186

Intratumoral heterogeneity in small cell carcinoma of the lung: An evolutionary and ecologic process.

Craig D. Peacock, Priyanka Gopal, Aaron Petty, Kevin Rogacki, Mohamed E. Abazeed. _Cleveland Clinic, Cleveland, OH_.

It is increasingly apparent that tumors represent dynamic structures in which cell populations, defined by genetic, epigenetic and non-genetic variation, compete for space and resources, while attempting to evade host defenses, resist therapeutic interventions and colonize new environments. Yet, the extent of this intratumoral genetic heterogeneity and the rules governing this ecosystem remain largely unknown. Using a large inventory of patient-derived xenograft models and an ex vivo culture system that maintains the transcriptional fidelity of the primary tumors, we have interrogated small cell lung cancer (SCLC) as a tractable model system for studying tumor evolution. In the process, we have identified at least three morphologically distinct intratumoral subpopulations across several samples: (i) suspended aggregates of small cells (neuroendocrine or NE), (ii) larger, pleomorphic cells with visible cytoplasm and spindle-like membrane extensions growing as a monolayer (mesenchymal or MS) and (iii) pre-suspension aggregates that are nested above mesenchymal-like cells (proneural or PN). Moreover, we have shown that these subpopulations are functionally distinct as measured by cellular proliferation, migration/invasion ability and sensitivity to chemotherapy/radiation. Intriguingly, distinct inter-tumoral transcriptional states assembled from earlier analyses of SCLC gene expression values across 53 and 71 cell lines and primary tumors, respectively, were preserved in the three intratumoral subpopulations within our examined SCLC lines. The proportion of each well-defined subpopulation varied across samples, suggesting sample-specific fractional setpoints. Finally, we have identified transcription factors that function as central nodes in the gene expression networks of each population and have used computational tools to nominate and functionally confirm biological processes within each population. Namely, we have shown that ASCL1 marks the rapidly proliferating NE population, NEUROD1 marks the primitive PN population and YAP1 marks the slow-growing MS population. We posit that understanding the SCLC ecosystem will provide substantial insight into its biological and clinical behavior and advance therapeutic possibilities in this recalcitrant disease.

#1187

Comparison of intra-tumor heterogeneity and clonal evolution across lung cancer subtypes by multi-region whole genome sequencing.

Tongwu Zhang,1 Joshua Sampson,1 Pier Alberto Bertazzi,2 Angela Pesatori,2 Dario Consonni,2 Bin Zhu,1 Belynda Hicks,1 Xing Hua,1 Jianxin Shi,1 Kevin Brown,1 Stephen Chanock,1 Maria Teresa Landi1. 1 _National Cancer Institute, Bethsda, MD;_ 2 _University of Milan, Milan, Italy_.

Both lung squamous cell carcinoma (LUSC) and lung cancer with neuroendocrine features (LUNE; i.e., small cell carcinoma (SCLC), large cell carcinoma (LCNEC) and other subtypes) are associated with tobacco smoking and have a very poor prognosis. We sought to characterize their clonal architecture and illustrate how the evolutionary patterns differ across these lung cancer subtypes. We performed multi-region whole genome sequencing of 83 samples from 36 LUSC and 67 samples from 23 LUNE patients that had been resected before systemic therapy.

Among somatic copy number alterations, we observed genome doubling intra-tumor heterogeneity (ITH) and massive genomic instability in both subtypes. For somatic nucleotide variants (SNVs), the total mutation burden was higher in LUSC (16.7 average number of mutations/Mb) than LUNE (12.7/Mb), but for SNVs ITH was higher in LUNE. Among driver genes, TP53, CDKN2A, CR1, and NRK in LUSC and TP53, KRAS, KMT2D, and RB1 in LUNE were frequently mutated and were always clonal. Evidence of positive selection was found for TP53, PTEN, KMT2D and KEAP1 in LUSC, and for TP53 and KRAS in LUNE. As expected, the dominant mutation signatures in LUSC clones included signatures 4, 1, and 5, while APOBEC signatures (2 and 13) were predominantly found in sub-clones. In contrast, no evidence of APOBEC signatures were found in LUNE. Among structural variants, LUNE had more events (339.25 average number of events/tumor) than LUSC (206.7/tumor), with considerable ITH in both subtypes, particularly in LUNE. In LUNE, the dominant mechanism responsible for SVs included fork stalling and template switching within the microhomology-mediated break-induced repair (MMBIR). We found recurrent fusion events with hotspots including those previously identified near TTC28/CHEK2 (42% LUSC and 35% LUNE) and LRP1B (42% LUSC). Additional hotspots were found in chromosome bands 20p12.1 (22% LUSC), 1p31.1 (19% LUSC) and 12p11.22 (22% LUNE). We found >10-fold higher transposon insertions (LINE1) in LUSC (71.9 average number of events/tumor) than LUNE (6.3/tumor). Our findings improve our evolutionary understanding of these lung cancer subtypes and warrant further investigations on the mechanisms involved in clonal genomic instability.

#1188

Detecting intratumor heterogeneity of PAM50 subtypes in H&E-stained slides using deep learning.

Mustafa Jaber,1 Bing Song,1 Clive R. Taylor,2 Charles J. Vaske,3 Christopher W. Szeto3. 1 _NantOmics, LLC, Culver City, CA;_ 2 _University of Southern California, Los Angeles, CA;_ 3 _NantOmics, LLC, Santa Cruz, CA_.

We present a methodology for identifying PAM50 intrinsic molecular breast cancer subtypes from only H&E-stained tissue section whole-slide images (WSI) of breast biopsies without using RNA expression data. We then use this system to identify patients presenting multiple subtypes simultaneously (i.e. intra-tumor heterogeneity), and validate the clinical utility of identifying patients with heterogeneous tumors.

Several methods have been proposed to stratify breast cancer subtypes including histological, immunohistochemical, and molecular. Intrinsic molecular subtypes such as PAM50 subgroups demonstrably outperform clinical factors and IHC in prognostic power. Yet molecular subtyping is fundamentally limited in two ways: 1) molecular characterization is relatively expensive and so not ubiquitously performed; and 2) non-single-cell molecular characterization assays the bulk tumor population, making studying intra-tumor heterogeneity difficult. The presented subtyping system uses routinely-gathered H&E stained WSIs to mimic molecular subtyping. Three modules form the proposed WSI-based subtyping system: First, WSIs are broken into multi-scale 400px x 400px patches and converted to descriptive tensors using the Inception-v3 neural net architecture. Next, a subset of cancer-enriched patches is automatically selected to summarize WSI tumor content and used in further analysis. Finally, each patch is assigned a subtype in a 4-way classifier (Basal, HER2-enriched, Luminal A, and Luminal B). Optionally, patient-based subtype classifications can be made by employing a voting mechanism upon the patch-based results.

We demonstrate this subtyping system using publicly available diagnostic WSIs from the TCGA BRCA cohort. We trained on 582 randomly selected patients, then tested subtyping accuracy on a held-out set of 223 patients. The subtype accuracy in held-out samples was 66% (compared to 34% from a random classifier, and 52% based on the majority-class classifier). We focused on 76 patients containing WSI patches with predictions for both Basal and Luminal A, and contrast them to 204 patients with majority Luminal A patches and 82 patients with majority Basal patches. We validated that this mixed-subtype population have outcomes and expression patterns that support a heterogeneous cellular population: The mixed subtype population have intermediate survival times between Luminal A and Basal in Kaplan-Meier analysis, varied hormone-receptor levels, and form a cluster equidistant between Luminal A and Basal in batch analysis.

These results demonstrate using readily-available data to characterize tumor subtypes and sub-populations. Correctly identifying these sub-populations may provide crucial additional information that is lost to bulk-tumor assays.

#1189

Deciphering the impact of immune editing on liver cancer clonal evolution using immunogenomics.

Bojan Losic,1 Amanda J. Craig,1 Sebastiao N. Martins-Filho,1 Carlos Villacorta-Martin,1 Nicholas Akers,1 Xintong Chen,1 Mehmet E. Ahsen,1 Ismail Labgaa,1 Delia D'Avola,1 Sergio A. Lira,1 Glaucia C. Furtado,1 Ashley Stueck,2 Stphen C. Ward,1 Maria I. Fiel,1 Ganesh Gunasekaran,1 Daniela Sia,1 Eric E. Schadt,1 Myron Schwartz,1 Josep M. Llovet,1 Swan Thung,1 Gustavo Stolovitzky,1 Augusto Villanueva1. 1 _Icahn School of Medicine at Mount Sinai, New York, NY;_ 2 _Dalhousie University, Halifax, Nova Scotia, Canada_.

Background and aims: Clonal evolution of a tumor ecosystem depends not only on somatic mutations driving uncontrolled growth, but on a full array of selection pressures, principally immune and resource mediated. We aimed at mapping the spatio-temporal interactions between cancer and immune cells in hepatocellular carcinoma (HCC) by quantifying regional adaptive immune editing, and how this impacts clonal evolution.

Methods: We integrated data from RNAseq, targeted DNA sequencing, SNP array and histological evaluation from multiple regions of 10 HCC treatment-naive surgical specimens (55 samples, median of 4 tumor and 2 non-tumor regions per patient), 6 of whom with Hepatitis B virus infection (HBV). Immune cells were assessed with immunofluorescence for T (CD3) and B (CD19) markers. MAGEA3 was down-regulated in 3 liver cancer cell lines using shRNA and proliferation assessed with the MTS assay. Analyses included: intra-tumoral and HBV differential gene expression, prediction of immunogenicity of expressed mutations (i.e., tumor neoantigens) and HBV antigens, T and B cell receptor sequencing and survival and network analysis on the liver cancer TCGA dataset.

Results & Conclusions: There is a tumor-driven adaptive immune response contributing to HCC heterogeneity, mainly recruited by subclonal mutations as compared to an interplay between clonal mutations and HBV epitopes. Indeed, we found different regional configurations of tumor infiltrating lymphocytes with higher density of Tertiary Lymphoid Structures in areas enriched in highly immunogenic tumor neoantigens. Furthermore, regional differences in gene expression of heterogeneous tumors can capture stronger prognostic signals than best-in-class single biopsy based predictors tested on TCGA LIHC HCC data. This demonstrates that the breadth of molecular states in a single tumor can easily approach that of a large population sample of tumors. We also found evidence that some tumors' spatiotemporal expression profile is directly correlated with HBV expression, including the extreme case where some regions of a HBV infected patient have zero HBV expression, suggesting regional selection of infected clones via resource constraints. Finally we found that some tumoral evolution is dominated by cancer testis antigen (CTA) dysregulation led by MAGEA3/6, which we determined is a key causal driver of a major regulatory subnetwork from a Bayesian gene interaction network we inferred from TCGA LIHC HCC data. Hypothesizing a oncodriver role for MAGEA3, we confirmed that MAGEA3 downregulation has anti-tumoral effects in HCC experimental models. 

## EPIDEMIOLOGY:

### Descriptive Epidemiology of Cancer

#1190

Incidence of gastric cancer in the United States during 1999 to 2013: A 50-state analysis.

Zhensheng Wang, Anam Khan, Maya Balakrishnan, Hashem B. El-Serag, Aaron P. Thrift. _Baylor College of Medicine, Houston, TX_.

Background: The incidence of gastric cancer while declining in many places worldwide is characterized by considerable geographic variability in incidence rates and temporal trends. The United States has large racial, ethnic, and regional variation; we collected data from all 50 states to better characterize recent changes in gastric cancer incidence in the entire United States.

Methods: We extracted data on the annual gastric cancer (ICD-O-3: C16.0-C16.9) incidence rates for the period 1999-2013 from the United Stated Cancer Statistics (USCS) registry, which covers 97% of the population. Both age-adjusted incidence rates and temporal trends of gastric cancer incidence were examined overall, as well as stratified by sociodemographic factors and at the state level. We used Joinpoint regression analysis to compute annual percent change (APC) and average annual percent change (AAPC) and corresponding 95% CIs. Heat maps were created to highlight the temporal trend in age-adjusted incidence rates in each state over three 5-year periods: 1999-2003, 2004-2008, and 2009-2013. As a secondary analysis, we also extracted data from the SEER 13 registries to examine the incidence rates and trends separately for cardia and non-cardia gastric cancers.

Results: Overall gastric cancer incidence decreased from 7.64 per 100,000 in 1999 to 6.55 per 100,000 in 2013, decreasing by 1.02% (AAPC=-1.02; 95% CI, -1.28 to -0.76) annually between 1999 and 2013. However, while overall gastric cancer incidence rates decreased (1999-2008 APC=-1.75, 95% CI, -2.02 to -1.47) and then plateaued (2008-2013 APC=-0.24, 95% CI: -0.89, 0.42) among persons aged ≥50 years, the rates increased among persons aged<50 years (AAPC=0.89, 95% CI: 0.61 to 1.16). Most notable increases were observed among non-Hispanic white females (AAPC=1.46, 95% CI: 0.87 to 2.06) and Hispanic females (AAPC=0.47, 95% CI: 0.10 to 0.84). Based on the analysis of SEER 13 registries, the decreasing trend among persons aged ≥50 years is mainly due to decreasing incidence of non-cardia gastric cancer (AAPC=-2.20,

95% CI, -2.51 to -1.90). Increasing rate of non-cardia cancer helped explain the uprising trend of gastric cancer among aged <50 years (AAPC=0.69, 95% CI: -0.06 to 1.44). States with rapid increase of high risk population groups such as Hispanic females including California and Texas are among the top 5 states with both overall high incidence rate of gastric cancer (1999-2013) and high AAPC (California: 1.77, 95% CI, 0.52 to 2.34; Texas: 1.70, 95% CI, 0.25 to 3.17) within last 5 years (2009-2013) among females aged <50 years.

Conclusions: While gastric cancer, particularly non-cardia, rates have decreased rapidly among persons aged ≥50 years in the United States, the incidence rates among persons aged <50 years have increased. Future studies on explanatory factors for this trend are warranted.

#1191

Differences in time to second primary malignancies: Development among patients with head and neck squamous cell carcinoma.

Eric Adjei Boakye,1 Paula Buchanan,1 Leslie Hinyard,1 Nosayaba Osazuwa-Peters,2 Mario Schootman,3 Jay F. Piccirillo4. 1 _St. Louis Univ. Center for Health Outcomes Research, Saint Louis, MO;_ 2 _St. Louis Univ. Cancer Center, Saint Louis, MO;_ 3 _St. Louis Univ. College for Public Health and Social Justice, Saint Louis, MO;_ 4 _Washington Uni. School of Medicine in St. Louis, Saint Louis, MO_.

Purpose: Second primary malignancies (SPMs) are the leading cause of death in survivors of head and neck squamous cell carcinoma (HNSCC). Patients with HNSCC are at increased risk of SPM. This is concerning since the number of survivors among these patients has been increasing. The human papillomavirus (HPV) has emerged as a distinct risk factor for oropharyngeal squamous cell carcinoma, which has different prognosis from classic tobacco/alcohol-associated HNSCC. We assessed the time to development of SPM in a large U.S. cohort of patients with an index HNSCC, comparing potentially-HPV-associated and non-HPV-associated HNSCC.

Methods: This was a population-based cohort study of 109,512 patients with HNSCC in the Surveillance, Epidemiology, and End Results registry (2000-2014). HNSCC included were for invasive squamous cell carcinoma per the International Classification of Diseases for Oncology, third edition codes. SPM was defined as the first subsequent primary cancer occurring at least 2 months after first cancer diagnosis. Time to SPM development was calculated as the date of diagnosis of SPM minus 2 months after the index diagnosis date. The main exposure was HPV-relatedness, based on whether patients' first HNSCC was potentially-HPV-associated or non-HPV-associated. Adjusted Cox proportional hazards regression model was used to evaluate the differences in the time to development of SPM and HPV-relatedness, adjusting for age at diagnosis, sex, race, marital status, year of diagnosis of first cancer, stage, grade, and treatment.

Results: Among the 109,512 patients with an index HNSCC, 12.3% developed SPM. Median time to SPM development overall was 31 months, for an index potentially-HPV-associated HNSCC patients was 32 months, and for an index non-HPV-associated HNSCC patients was 30 months. Patients diagnosed with an index non-HPV-associated HNSCC had a 36% higher risk of developing SPM (aHR=1.36; 95% CI: 1.38-2.53) compared with those with an index potentially-HPV-associated HNSCC. When sites were broken down further, compared to patients with oropharynx cancer, patients with hypopharynx (aHR=1.63; 95% CI: 1.50-1.77) had the higher risk of developing SPM, followed by oral cavity (aHR=1.40; 95% CI: 1.33-1.47), and larynx (aHR=1.27; 95% CI: 1.21-1.33) cancers. Patients had higher risk of developing SPM if they were 60-69 (aHR=2.49; 95% CI: 2.34-2.66) or ≥70 (aHR=2.82; 95% CI: 2.64-3.02) vs. 18-49 years, male (aHR=1.08; 95% CI: 1.04-1.13), black (aHR=1.23; 95% CI: 1.16-1.30) vs. white, and distant (aHR=1.15; 95% CI: 1.09-1.23) vs. localized tumor.

Conclusion: Patients with an index non-HPV-associated-HNSCC were more likely to develop SPM compared to those with potentially-HPV-associated-HNSCC. Health care professionals may consider this during follow-up of patients with HNSCC.

#1192

Independent and joint use of statins and metformin on overall survival after diagnosis of hepatocellular carcinoma: A SEER-Medicare matched study.

Samuel O. Antwi, Zhuo Li, Kabir Mody, Tushar C. Patel. _Mayo Clinic, Jacksonville, FL_.

Background: Hepatocellular carcinoma (HCC) is a lethal malignancy that currently ranks fourth in cancer-related deaths in the US and is projected to become the third leading cause of cancer death in the US by 2030. Thus, there is a pressing need to identify modifying risk factors to improve survival after diagnosis of HCC. Statins and metformin are among the most commonly prescribed medications in the elderly in the US and there are experimental data suggesting that these medications may improve overall survival after cancer diagnosis; however, results from observational studies have been equivocal. We therefore investigated the potential benefits of pre- and post-diagnosis use of statins and metformin both independently and jointly on overall survival after diagnosis of HCC. Methods: The study was performed using data from the Surveillance, Epidemiology, and End Results (SEER-18) registry linked to Medicare claims data and included 8,922 elderly (≥65 years) patients diagnosed with HCC between 2009-2013. Data on metformin and statins use were extracted from the Medicare Part D Claims file and the patients were grouped as statins only users, metformin only users, both statins and metformin users, or neither (i.e., non-users, referent group). We assessed association of both pre-diagnosis (≥2 years) and post-diagnosis statins and metformin use on risk of death after diagnosis of HCC. Statins users were further categorized into high potency statins users (simvastatin, atorvastatin, and rosuvastatin) or low potency statins users (pravastatin, lovastatin, and fluvastatin) and compared with non-users. Cox proportional hazard models were used to calculate hazard ratios (HRs) and 95% confidence intervals (CIs), adjusting for multiple risk factors, including age sex, race, cancer stage at diagnosis, cancer grade, first course therapy, neighborhood level education and income, Charlson comorbidity index, and diabetes severity index. Results: Compared with non-users, post-diagnosis statins use was independently associated with 27% lower risk of death after diagnosis of HCC (HR=0.73, 95% CI: 0.60-0.88; p-value = 0.002), adjusting for pre-diagnosis statins use and other risk factors. Further analysis showed that the association for post-diagnosis statins use is evident only among high potency statins users (HR=0.72, 95% CI: 0.60-0.88; users versus non-users) but not low potency statins users (HR=0.83, 95% CI: 0.55-1.25; users versus non-users). No association was found for pre-diagnosis statins use or for pre- or post-diagnosis metformin use. Conclusions: This study suggests that post-diagnosis statins use, particularly high potency statins use, may improve overall survival after diagnosis of HCC. Metformin use, both before and after diagnosis of HCC, is not associated with overall survival.

#1193

The impact of rapid changes in reproductive factors on the increased incidence of breast cancer in Korea.

Seho Park,1 Hyunsoon Cho,2 Jee Ye Kim,1 Hyung Seok Park,1 Seung Il Kim,1 Young Up Cho,1 Byeong-Woo Park,1 Graham A. Colditz,3 Yikyung Park3. 1 _Yonsei University College of Medicine, Seoul, Republic of Korea;_ 2 _Graduate School of Cancer Science and Policy, National Cancer Center, Goyang, Republic of Korea;_ 3 _Washington University School of Medicine, St. Louis, MO_.

Introduction The age-standardized incidence rates of Korean breast cancer have significantly increased from 26.1 in 1999 to 63.9 in 2014, which coincides with changes in lifestyle, especially reproductive factors. We examined the contribution of temporal changes in reproductive factors to the increased incidence of breast cancer in Korea.

Methods We estimated the means of age at menarche, number of pregnancy, age at first pregnancy and duration of breastfeeding by birth cohort using the nationwide Korean National Health and Nutrition Examination Survey 2007-2015 (n=20,522 women aged between 20 and 69 years with no history of cancer). Cohorts were divided into a ten-year interval of birth year from 1940s to 1970s. Analyses took into account the complex sample design by incorporating weights, stratification and clustering. The percent increase in breast cancer incidence due to a specific factor was calculated as 100 x (RRd \- 1) (RR, relative risk for a specific factor; and d, difference in the mean of a risk factor among birth cohorts).

Results Weighted proportion of birth cohorts was 12.1% in 1940s (mean age=64.9y), 24.1% in 1950s (56.1y), 31.9% in 1960s (46.8y), and 31.9% in 1970s (36.9y). We observed notable changes in reproductive factors over time: younger menarche, lower number of pregnancies, older age at first pregnancy and shorter duration of breastfeeding. A decrease in the number of pregnancies from 5.0 in 1940s to 2.7 in 1970s cohorts contributed to an 18.3% increase in breast cancer incidence. Shorter duration of lactation, earlier onset of menarche and older age at first pregnancy also contributed to 16.5%, 10.7%, and 7.1% increase in the incidence rates, respectively.

Conclusion We found that significant changes in reproductive factors in Korean women had appreciable impacts on the increase in breast cancer in Korea. Further study is warranted to evaluate the burden of breast cancer due to other modifiable risk factors such as adiposity, alcohol drinking and physical activity.

Table. Changes in risk factors by birth cohorts and their contribution to incidence change

---

Risk factors | Birth cohort | RR (/unit) | Contribution to incidence change (1940s vs. 1970s)

1940s | 1950s | 1960s | 1970s | |

Age at menarche (years) | 16.05 | 15.40 | 14.46 | 13.57 | 0.96 | 10.65%

Number of pregnancy (n) | 4.98 | 3.92 | 3.41 | 2.66 | 0.93 | 18.34%

Age at first pregnancy (years) | 23.80 | 24.53 | 26.06 | 27.42 | 1.1 (/5 years) | 7.14%

Duration of breastfeeding (months) | 50.53 | 27.53 | 13.71 | 12.83 | 0.98 (/5 months) | 16.54%

#1194

U.S. childhood and adolescent cancer survival between 1992 and 2013: An analysis of population-based data.

Xiaoyan Wang, Kimberly J. Johnson. _Washington University in St.Louis, St. Louis, MO_.

Background

Monitoring cancer survival over time and across different demographic groups is an important part of cancer control. In the current study, our objective was to examine U.S. cancer survival patterns in childhood and adolescent cancer patients between different time periods and demographic groups.

Methods

Survival data for primary cancers diagnosed in individuals ≤ 19 years from 1992 to 2014 were obtained from the Surveillance, Epidemiology, and End Results 13 database. Five-year relative survival rates were estimated according to the cohort method using SEER*Stat software. Survival differences were examined for subgroups defined by diagnosis time period (1992-1994, 1995-1999, 2000-2004, 2005-2009), sex (male and female), age at diagnosis (<1, 1-4, 5-9, 10-14, 15-19 years) and race/ethnicity (Non-Hispanic White, Non-Hispanic-Black, Hispanic, and Non-Hispanic Other). The annual percent change (APC) in 5-year relative survival was estimated using Joinpoint software. Finally, period survival analysis was also conducted using SEER*Stat software to provide the most up-to-date five-year relative survival predictions using data from cases diagnosed between 2007 and 2013.

Results

Five-year relative survival increased significantly for cancers overall (75.9% vs. 82.9 for 1992-1994 and 2005-2009 respectively, p<0.001) and for half of the cancer subtypes. The largest improvements were observed for leukemias, myeloproliferative and myelodysplastic disease (+ 11.6%, p<0.001) and lymphomas and reticuloendothelial neoplasms (+5.8%, p<0.001). Significant improvement in relative survival over time was also observed for both sexes with APCs of 0.67 (males) and 0.51 (females), across all age groups with APCs of 0.68 (infants), 0.63 (1-4 years), 0.68 (10-14 years) and 0.65 (15-19 years) except 5-9 years (APC: 0.32), and all racial/ethnic groups with APCs of 0.61 (non-Hispanic White), 0.64 (Non-Hispanic Black), 0.51 (Hispanic), and 0.75 (Non-Hispanic Other). No statistically significant differences in the APCs were observed between sex, age, and race/ethnicity subgroups. However, survival disparities were apparent across age and race/ethnicity subgroups in period analyses, with infant and Hispanic, non-Hispanic Black and non-Hispanic Other cases having lower survival rates than older and non-Hispanic White cases (infant: 77.2% vs 1-4 years: 85.5%, 5-9 years: 84.1%, 10-14 years: 84.2% and 15-19 years: 84.0%; Non-Hispanic Black: 81.6%, Non-Hispanic Other: 81.2%, and Hispanic: 79.8% vs. Non-Hispanic White: 87.5%).

Conclusion

Our results indicate that childhood and adolescent cancer survival continues to improve for all demographic groups. However, persistent inferior survival rates in infants and in minority race/ethnicity groups reinforces the need for continued research that aims to eliminate childhood and adolescent cancer survival inequities.

#1195

Incidence rates of head and neck cancer across Thailand and the USA.

Ilona Argirion,1 Katie Rentschler,1 Patravoot Vatanasapt,2 Hutcha Sriplung,3 Laura S. Rozek1. 1 _University of Michigan, Ann Arbor, MI;_ 2 _Khon Kaen University, Khon Kaen, Thailand;_ 3 _Prince of Songkla University, Songkhla, Thailand_.

Background: Head and neck cancer is the sixth most common cancer in the world, with the largest burden occurring in developing countries. Although the primary risk factors have been well characterized, little is known about the temporal trends in head and neck cancer across Thailand. Using population based registries from three Thai provinces (Songkhla, Lampang and Khon Kaen), we have characterized the age-standardized incidence rates of head and neck cancer by sex and age and compared them to those observed in the United States. Methods: Cancer cases diagnosed between 1990 and 2014 were selected by ICD-10 code directly from the Songkhla, Lampang and Khon Kaen Cancer Registries as well as the Surveillance, Epidemiology, and End Results Program (SEER) for the following sites: oral cavity (00, 03-06), tongue (01-02), pharynx (09-10, 12-14), and larynx (32). The data were analyzed using R software (3.1.1) and Joinpoint Regression Software (4.4.0) to determine age-standardized incidence rates and trends of annual percent change. Incidence rates were standardized using the Segi (1960). Additional exploratory analyses using stratified linear regression models were conducted to assess temporal trends in head and neck cancer across 10-year age groupings. Results: Overall head and neck cancer rates are decreasing across all registries except Khon Kaen, where there is an observed, albeit not statistically significant, increase among males (APC: 0.53, p=0.3). Subsite analyses demonstrate consistent decreases in both larynx and oral cavity cancers, but suggested increases in pharynx and tongue cancers among both genders in the US (APC: 1.9 and 1.6, p<0.05 respectively) as well as pharynx cancer in Khon Kaen males (APC: 2.1, p<0.05). Furthermore, age-stratified APC analyses showed increased incidence in tongue cancer among Khon Kaen females age 30-39, Khon Kaen males age 30-59, Lampang males age 50-59, Songkhla females age 30-49, US females age 30-49 and 70-85+, as well as US males age 40-79. Pharynx cancers were seen to increase in Khon Kaen males age 40-59 and US males age 50-59. Conclusions: Although overall trends in head and neck cancer do seem to be decreasing across both Thailand and the United States, there is reason to believe that the etiological shift affecting both pharynx and tongue cancers in the US may be similarly presenting in Thailand. While disparities across gender and region do still exist, further investigation into the increased rates of early onset cancer is needed.

#1196

Epidemiology of childhood hematologic cancers in Africa: A systematic review of the evidence.

Davies Adeloye,1 Asa Auta,2 Jhonathan P. Dos Santos,3 Michael O. Harhay4. 1 _Covenant University, Ota, Nigeria;_ 2 _University of Central Lancashire, Preston, United Kingdom;_ 3 _University of São Paulo, São Paulo, Brazil;_ 4 _University of Pennsylvania, Philadelphia, PA_.

Background: In developed economies, less than 1% of all cancers occur in children younger than 15 years. Less is known about the distribution of childhood cancers in Africa due to a lack of comprehensive data on pediatric oncology needs and burdens.

Methods: We searched EMBASE, Medline and Global Health to identify population- or hospital-based registry studies on childhood haematological cancers in Africa, with publication date set from January 1980 to December 2016. We applied a Bayesian network meta-analysis modelling approach on individual national or subnational estimates to arrive at a standardized estimate for Africa and African sub-regions.

Results: Our search identified 2717 studies, 35 of which met the inclusion criteria. Most studies were from the Southern and West African countries, and the majority (90%) of the cancer registries were population-based. Fifteen (43%) out of the 35 included studies were of high quality, while the remaining 20 (57%) included studies were of moderate quality. Most of the assessed studies (80%) were considered representative of their target/subnational population, with only 5 (14%) studies representative of their national population. From all sites, the pooled incidence of childhood leukemia in Africa was highest at 16.9 (95% CI: 14.1-19.7) per 1,000,000 child years (cy), with higher incidence in North Africa compared to sub-Saharan Africa (SSA). This was closely followed by non-Hodgkin lymphoma (NHL) at 16.8 (95% CI: 13.6-19.3) and Burkitts lymphoma (BL) at 16.8 (95% CI: 12.7-20.2) per 1,000,000 cy, with higher incidence rates of both cancer types significantly higher in SSA compared to North Africa. The overall pooled incidence of childhood Hodgkin lymphoma (HL) in Africa was the lowest at 15.0 (95% CI: 12.7-17.8) per 100,000 cy. Across all sub-regions, the incidence rates of the four cancer types were highest in East Africa, and consistently increased between 1980 and 2015.

Conclusion: Describing the epidemiology of childhood cancers remains difficult owing to few and limited capacities of cancer registration systems in Africa. Available evidence suggests an increasing incidence, with the East African region contributing highest to the burden of childhood hematological cancers on the continent. To provide evidence-based summaries on paediatric malignancies and guide appropriate response on the continent, there is need to re-organize existing registries and incorporate upcoming registries to comprehensively account for incidence, mortality, survival and relevant details on environmental, genetic and molecular characteristics of childhood cancers across different population groups.

#1197

Soft tissue sarcoma clinical presentation, treatment, and survival in adolescents and young adults compared to older adults: A report from the Scandinavian Sarcoma Group.

Vidal M. Arroyo,1 Philip J. Lupo,1 Beatrice Melin,2 Emelie Styring,3 Olga Zaikova,4 Karin Papworth2. 1 _Baylor College of Medicine, Houston, TX;_ 2 _Umeå University, Umeå, Sweden;_ 3 _Lund University, Lund, Sweden;_ 4 _Oslo University, Oslo, Norway_.

Background: Five-year survival rates for those diagnosed with soft tissue sarcoma (STS) have improved significantly among children and older adults (OAs), but these same trends have not been observed for adolescents and young adults (AYAs). While these disparities could be due to differences in biology or treatment, few studies have evaluated STS occurrence and outcome in AYAs. Therefore, the purpose of this study was to evaluate differences between adolescents and young adults (AYAs) and older adults (OAs) diagnosed with STS by stratifying analysis by: (1) clinical presentation; (2) treatment; and (3) survival.

Methods: Data were obtained from the Scandinavian Sarcoma Group (SSG) Central Register, which includes information on 5,747 patients from Sweden and Norway, diagnosed with a STS during 1986-2011. Variables included: age at diagnosis, metastasis at diagnosis, tumor size, histology, adjuvant treatment, date of death or last follow-up. AYAs were defined as those diagnosed ages 15-39 years. Categorical variables were analyzed using chi-square tests, and continuous variables were analyzed using t-tests. Overall survival (OS) and recurrence-free survival (RFS) were compared between AYAs and OAs using Kaplan-Meier estimates and log-rank tests. All analyses were conducted overall and by common STS subtypes.

Results: Overall and by STS subtype, there were significant differences between AYAs and OAs on presentation, treatment, and survival. The distribution of STS subtypes was different between OAs and AYAs. For example, OAs were more likely to be diagnosed with leiomyosarcoma compared to AYAs (18% vs. 10%, p<0.001), whereas AYAs were more likely to be diagnosed with malignant peripheral nerve sheath tumor (MPNST, 9% vs. 4%, p<0.001). OAs were also more likely to have larger tumors (>5 cm, 67% vs. 52%, p<0.001) and higher malignancy grade (grade IV, 45% vs. 31%, p<0.001). Interestingly, AYAs were more likely to be treated with radiotherapy and chemotherapy compared to OAs (12% vs. 5%, p<0.001). There were also differences within STS subtypes. For example, OAs were more likely to have metastasis compared to AYAs if diagnosed with leiomyosarcoma (18% vs. 10%, p=0.04). In most scenarios AYAs had significantly better OS and RFS compared to OAs, other than for MPNST (OS: p=0.19, RFS: p=0.28).

Conclusions: There were several differences between AYAs and OAs on STS presentation, treatment, and outcome. AYAs not only had differences in terms of STS subtypes but also tumor size and malignancy grade within subtypes. Additional work is needed to characterize the biology underlying these differences, which will inform future treatment strategies for both AYAs and OAs with STS.

#1198

Rare isn't rare: Rare cancers compose over 50% of all U.S. diagnoses.

Katherine Arline, Emily Zeme, Robert L. Treuting, Rodrigo Bravo, Jamie Barber, Christopher Higginson, Jon Goetz, William Siders, Johanne Kaplan. _SHEPHERD Therapeutics, Natick, MA_.

Rare cancers are an understudied and deadly public health problem. Estimates for the percentage of cancer diagnoses that are rare vary depending upon the source cited and the definition of what constitutes a rare cancer. A frequently cited report states that 25% of all adult cancers are rare. However, given the ever-changing landscape of cancer biology and as a result of changes in disease classification, oft-cited percentages underestimate the burden of rare cancers. To address this discrepancy, we reviewed the most recent publicly available data regarding the incidence of all cancers in the United States. Following the NCI definition of a rare disease as one affecting fewer than 15 persons per 100,000 per year, for 2017, a cancer affecting 48,952 or fewer is classified as rare. We considered each cancer individually, acknowledging subtypes based upon the criteria of scientifically accepted definitions, treatments, and histology. We then compiled statistics for each type and subtype. By this method, the total number of estimated new cancer patients in 2017 was 1,685,346, which differed only 0.2% from the Cancer.gov's publicly available estimate of 1,688,780. However, in contrast to previously published data that indicated that 60 of 71 cancer types were rare, we identified a total of 347 cancers, of which 337 were rare. The estimate for rare cancer diagnoses in the U.S. per year was therefore 875,084. Ultimately, 51.9% of all cancer diagnoses are classified as rare cancers by this method. In addition, of 52 major groupings of cancer, 35 (67.3%) disproportionately affect minorities in terms of incidence or overall survival. Seven groupings exclusively or almost exclusively affect women, and 7 additional groups display disproportionate incidence or negative outcomes in women. In addition, while many cancers affect children, and while all cancer in children is rare, at least 55 cancers exclusively or frequently affect children. This analysis clearly shows that the incidence of rare cancer in the United States is widely misunderstood and that cancer in total disproportionately affects historically underserved populations. Treatment options are needed for each form of cancer and should be developed based on a thorough understanding of each disease's biology. In addition, efforts to understand and address the origins of demographic disparities can help reduce disease burden.

#1199

Adult Hispanic males experience a higher incidence of acute lymphoblastic leukemia compared to non-Hispanic Whites.

Manuel R. Espinoza-Gutarra, Edgar Munoz, Dimpy P. Shah, Anand Karnad, Ruben A. Mesa, Amelie G. Ramirez. _UTHSCSA, San Antonio, TX_.

Introduction: Acute leukemia is an important cause of morbidity and mortality with differences in incidence and outcomes between patients of different ethnic backgrounds. We conducted this study to examine the ethnic disparities in incidence of ALL as well as other types of acute leukemia in the Latino population in South Texas.

Methods: Data were obtained from the U.S. Surveillance, Epidemiology and End Results 18 (SEER) Program and the Texas Cancer Registry (TCR) under Limited-Use Data Agreements between sources and authors. Adult patients (15 years or older) were identified in both SEER and TCR databases from 2000 to 2014 and ICD-O-3 codes were used to select specific malignancies: ALL (9727-9729, 9835-9837), acute promyelocytic leukemia (APL) (9866) and non-APL acute myeloid leukemia (AML) (9840, 9861, 9867, 9870-9874, 9891, 9895-9897, 9910, 9920, 9930, 9931). Ethnicity was classified as Hispanic (H) and non-Hispanic White (NHW) per the North American Association of Central Cancer Registry (NAACR) Hispanic Identification Algorithm. Cases were obtained from SEER 18 and TCR, including Texas and the 38 counties of South Texas. Comparisons used the SEER population of NHW as reference. We used SEER*Stat software v 8.3.4 (SEER*Stat, NIH), to generate 2000-2014 average annual age-specific and age-adjusted acute leukemia incidence rates (per 1000000), rate ratios (RR) and 95% confidence interval (CI) for Hispanic and NHW populations in SEER, Texas, and South Texas.

Results: Table 1 shows Hispanic males in South Texas had significantly higher RR for development of ALL (p<0.05), and lower for non-APL AML; there was no statistical difference for APL RR compared to NHW. Males (15-39 and 70-79 years) had higher rates of ALL.

Conclusion: Latinos in South Texas experience a higher incidence of ALL and a lower incidence of non-APL AML compared to NHW. Future studies are needed to identify specific risk factors associated with this higher burden in Hispanic population. | |  | |  | |  | |

|

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

Table 1 | SEER | TCR

|  | Texas | South Texas

|

H | NHW | RR (95%CI) | H | NHW | RR (95%CI) | H | NHW | RR (95%CI)

ALL | 2137 | 4442 | 1.50 (1.42-1.59) | 978 | 985 | 1.43 (1.33-1.53) | 377 | 107 | 1.63 (1.46-1.81)

Non-APL AML | 33980 | 4719 | 0.77 (0.75-0.80) | 2163 | 7593 | 0.41 (0.40-0.43) | 848 | 898 | 0.48 (0.45-0.51)

APL | 770 | 2224 | 1.22 (1.11-1.33) | 309 | 472 | 0.90 (0.80- 1.02) | 109 | 51 | 0.94 (0.77- 1.14)

#1200

25OH vitamin D concentrations are associated with both higher and lower incidence of specific cancers.

Fie J. Vojdeman,1 Christian M. Madsen,2 Darshana Durup,3 Kirsten Frederiksen,4 Anja Olsen,4 Louise Hansen,4 Anne-Marie Heegaard,3 Bent S. Lind,5 Anne Tjønneland,4 Henrik L. Jørgensen,1 Peter Schwarz6. 1 _Bispebjerg Frederiksberg Hospital, Copenhagen NV, Denmark;_ 2 _Herlev Gentofte Hospital, Herlev, Denmark;_ 3 _Copenhagen University, Copenhagen East, Denmark;_ 4 _Danish Cancer Society Research Center, Copenhagen East, Denmark;_ 5 _Amager Hvidovre Hospital, Hvidovre, Denmark;_ 6 _Rigshospitalet, Copenhagen East, Denmark_.

This study examined the association between concentrations of 25OH vitamin D and cancer incidence in individuals from the Capital Region of Denmark who had 25OH vitamin D analyzed in serum via their general practitioner between April 2004 and January 2010 (CopD). CopD data were linked to the Danish Cancer Registry, the National Patient Registry and the Danish Civil Registration System as of December 2014.

CopD consists of 246,858 individuals with 25OH vitamin D analyses. Individuals diagnosed with cancer prior to their 25OH vitamin D analysis (N=29,614) were excluded. The population analyzed in the study (217,244 individuals) had a mean level of 25OH vitamin D of 50.2 nmol/L, a mean age of 49.1 years (SD 20.1), female predominance (65.3%), and low comorbidity burden (Charlson Comorbidity index (CCI) ≥1 in 20.5%).

Cox regression models with time since blood sampling as the underlying time scale adjusted for age in one year intervals, sex, month of sampling, and CCI as strata variables were performed in SAS 9.4 (SAS Institute, Cary, USA). P-values less than 0.05 were considered statistically significant.

The study population experienced a total of 18,359 incident cancers during the follow-up period. Non-melanoma skin cancer was the most frequent form of cancer, followed by breast-, lung-, and prostate cancer (Table). In the adjusted analysis higher levels of 25OH vitamin D in increments of 10 nmol/L were associated with higher incidence of non-melanoma- and melanoma skin cancer, as well as prostate- and hematological cancers, but lower incidence of lung cancer. Excluding those diagnosed with cancer within 5 years of blood sampling, revealed similar results (Table).

In conclusion, higher concentrations of 25OH vitamin D are associated with both higher and lower incidence of cancer depending on cancer type in CopD. Thus, in a population from general practice we find higher 25OH vitamin D concentrations are associated with the incidence of skin-, prostate and hematological cancers.

Hazard ratios (HR) per 10nmol/L increase in 25OH vitamin D.

---

|

Adjusted HR for all | |  | |

HR for cancer after 5+ years | |

|

|

N cases | HR | 95% Cl | p | N cases | HR | 95% Cl | p

Skin cancer | |  | |  | |  | |

Non-melanoma | 5045 | 1.09 | 1.09-1.10 | <0.0001 | 1062 | 1.11 | 1.09-1.13 | <0.0001

Melanoma | 684 | 1.10 | 1.08-1.13 | <0.0001 | 130 | 1.14 | 1.08-1.20 | <0.0001

Sex specific cancers | |  | |  | |  | |

Breast | 2167 | 1.00 | 0.99-1.02 | 0.64 | 455 | 1.02 | 0.99-1.05 | 0.21

Ovary | 254 | 0.99 | 0.94-1.03 | 0.58 | 53 | 0.98 | 0.89-1.08 | 0.68

C. Uteri | 347 | 0.99 | 0.96-1.03 | 0.71 | 90 | 0.99 | 0.92-1.06 | 0.71

Prostate | 1470 | 1.05 | 1.03-1.07 | <0.0001 | 221 | 1.09 | 1.04-1.15 | 0.0002

Tobacco related cancers | |  | |  | |  | |

Lung | 1707 | 0.95 | 0.93-0.97 | <0.0001 | 318 | 0.93 | 0.89-0.98 | 0.003

Colon | 1108 | 0.98 | 0.96-1.00 | 0.10 | 222 | 0.97 | 0.93-1.03 | 0.32

Rectum | 461 | 0.98 | 0.94-1.01 | 0.18 | 88 | 0.98 | 0.91-1.06 | 0.67

Urinary | 1016 | 0.99 | 0.96-1.01 | 0.34 | 213 | 0.98 | 0.93-1.04 | 0.50

Other cancers | |  | |  | |  | |

Hematological | 968 | 1.03 | 1.01-1.06 | 0.004 | 181 | 1.08 | 1.03-1.13 | 0.002

Central Nervous System incl. eye | 689 | 1.00 | 0.98-1.03 | 0.87 | 144 | 1.00 | 0.95-1.06 | 0.94

#1201

Breast cancer risk factors and volumetric breast density in a national breast cancer screening program.

Kirsti V. Hjerkind,1 Merete Ellingjord-Dale,2 Anna L. Johansson,3 Hildegunn S. Aase,4 Solveig R. Hoff,5 Solveig Hofvind,1 Siri Fagerheim,6 Linda Vos,1 Isabel dos Santos Silva,7 Giske Ursin1. 1 _Cancer Registry of Norway, Oslo, Norway;_ 2 _Imperial College London, London, United Kingdom;_ 3 _Karolinska Institutet, Stockholm, Sweden;_ 4 _Haukeland University Hospital, Bergen, Norway;_ 5 _Helse Møre og Romsdal HF, Ålesund, Norway;_ 6 _Stavanger University Hospital, Stavanger, Norway;_ 7 _London School of Hygiene and Tropical Medicine, London, United Kingdom_.

Background: Studying associations between percent or absolute volumetric breast density (VBD) with age and menopausal status, and whether the associations are modified by demographic, lifestyle, reproductive, or hormonal exposures, can uncover underlying biological mechanisms and improve breast cancer risk prediction.

Material and methods: The cohort consisted of women (aged 49-71 years) who participated in the Norwegian Breast Cancer Screening Program (NBCPS) between 2007 and 2014, had information on VBD and completed questionnaires on standard breast cancer risk factors as part of the program (n=46 428). We estimated least squared means of percent and absolute VBD associated with age at mammography, menopausal status, age at menopause, reproductive and hormonal factors (ages at menarche and first birth, number of pregnancies lasting ≥6 months, duration of breastfeeding, oral contraceptives, and menopausal hormone therapy), self-reported height and body mass index (BMI), education, and lifestyle factors (physical activity, alcohol intake, and smoking).

Results: For a 5-year increase in age, the reduction in percent VBD was -0.18% in pre- and perimenopausal and -0.08% in postmenopausal women, and the reduction in absolute VBD was -0.11 cm³ in pre- and perimenopausal and -0.03 cm³ in postmenopausal women (p for interaction by menopausal status <0.001). In multivariate analyses, the associations between demographic, lifestyle, reproductive and hormonal risk factors and percent and absolute VBD were highly significant, however the magnitude of the effects were modest (1-2%), and the range of percent VBD across levels of risk factors rather narrow. The strongest association was with BMI, which was inversely associated with percent VBD, with a threefold higher percent VBD in women with BMI<20 kg/m² than in women with BMI>33 kg/m² (12.9% versus 3.9%). Interestingly, BMI was positively associated with absolute VBD, with 1.5

times higher VBD in women with BMI≥33 kg/cm² (37.9 cm³ versus 58.4 cm³). Models were adjusted for BMI, education, and parity.

Conclusion: This large cohort analysis found percent and absolute VBD to decrease with increasing age both among pre/perimenopausal and postmenopausal women. The rate of decline was larger among pre/perimenopausal women. Percent and absolute VBD are associated with several established breast cancer risk factors, especially BMI, where the direction of the association differed for percent and absolute VBD.

#1202

Spatial analysis of late-stage oropharyngeal cancer diagnosis in Florida: Hierarchical models and geographically-weighted regression.

Juan M. Hincapie-Castillo, Yi Guo, Natalie L. Silver, Hong Xiao. _University of Florida, Gainesville, FL_.

Literature on spatial variation of cancer outcomes in the US remains scarce. Studies have not assessed how late-stage OPC diagnosis varies across geography nor the effect of spatially different socioeconomic factors. Identifying risk factors that have higher impact on certain localities can help inform clinicians and policy-makers. Our study aimed to evaluate spatial differences in stage of diagnosis of oropharyngeal cancer (OPC) in Florida using hierarchical and geographically-weighted regression (GWR) models. We used the Florida Cancer Data System to derive a cohort of 57,082 patients diagnosed with OPC during 1990-2010. The individual-level covariates that informed our models consisted of age at diagnosis, gender, race, married status, health insurance payer, history of cigarette smoking, and year of diagnosis. Patients' addresses were geocoded to obtain a point feature used to map the spatial distribution of individuals using ArcGIS. A hot-spot analysis (Getis-or-Gi) revealed areas of high clustering of patients with late-stage diagnosis. To evaluate spatially-relevant factors for such variation, we fitted a mixed logistic regression model using PROC GLIMMIX in SAS with the individual-level variables in addition to county and census tract-level covariates. We used US Census data to extract records on the percent of residents unemployed, living in poverty, and foreign-born. Variables for the county level extracted from the Area Health Resource File and the Behavioral Risk Factor Data included the percent of residents engaging in heavy drinking, who couldn't visit a dentist due to cost, and who were smokers, a flag for Health Professional Shortage Areas for primary care doctors and dentists, presence of a hospital with oncology services, and a flag for rural designation. The model showed significant higher odds for late-stage OPC diagnosis for non-White patients (OR 1.63, 95%CI 1.48-1.79 for Black and 1.29, 95%CI 1.03-1.62 for other-race), current smoker status (OR 1.45, 95%CI 1.36-1.54), former smoker status (OR 1.36, 95%CI 1.27-1.45), patients without health insurance (OR 1.60, 95%CI 1.41-1.82), patients covered by Medicaid (OR 1.73, 95%CI 1.5-1.97), patients diagnosed with cancer in pharyngeal sites (OR 4.74, 95%CI 4.53-4.95), and higher percentage of residents in census tract living in poverty (OR 1.01, 95%CI 1.00-1.011). The GWR conducted at the county-level revealed areas where coefficients associated with various risk-factors contributed to a higher proportion of late-stage OPC diagnosis. For easier communication of findings, we created density maps that will be presented. Our study empirically showed variation of late-stage OPC diagnosis across Florida and differential effects of spatial socioeconomic factors. Future public health initiatives can use a targeted approach to focus resources in these specific areas more likely to be impacted.

#1203

Incidence of AJCC7 stage III or regionally advanced cutaneous melanoma in the United States.

Ahmad M. Tarhini,1 Sameer R. Ghate,2 Antonio Nakasato,2 Raluca Ionescu-Ittu,3 Sherry Shi,3 Briana Ndife,2 Rebecca Burne,3 François Laliberté,3 Mei Sheng Duh4. 1 _Cleveland Clinic Taussig Cancer Institute, Cleveland, OH;_ 2 _Novartis Pharmaceuticals Corporation, East Hanover, NJ;_ 3 _Analysis Group, Inc, Montreal, Quebec, Canada;_ 4 _Analysis Group, Inc, Boston, MA_.

Background: Incidence of stage III/regional melanoma by patient characteristics can help estimate the number of high-risk patients (pts) potentially eligible for adjuvant treatment in a rapidly evolving field and guide the design of future clinical trials based on real-world data. The study describes the incidence of AJCC7 stage III/regional melanoma in the US (a) over time and (b) by melanoma prognostic factors.

Methods: Pts newly diagnosed with invasive melanoma were identified in the US Surveillance, Epidemiology, and End Results (SEER) cancer registry (years 2010 -2014; n = 106,195). Stage III melanoma was defined based on the AJCC7 classification (n = 7,669); pts classified as AJCC7 stages I/II but with reported evidence of lymphatic metastasis were considered regionally advanced and included (n =169). Annual incidence was defined as the proportion of individuals in the general population who developed stage III/regionally advanced melanoma over a one-year period (the denominator included the full or age/sex-specific population, as appropriate).

Results: During the study period, 7,838 (7.4%) pts were diagnosed with stage III/regional melanoma . The incidence of invasive melanoma has increased from year 2010 to year 2014: from 14.16 to 16.15/100,000 for stages I-IV combined and from 1.25 to 1.50/100,000 for stage III/regional melanoma. Based on the 2014 incidence and the most recent age-specific census population data, it is estimated that 5,109 pts were newly diagnosed in year 2016 with stage III/regional melanoma. In 2014, the incidence of stage III/regional melanoma was higher in elderly than adults and children (incidence per 100,000: 5.16 vs 1.33 and 0.08, respectively) and in males than females (1.79 vs 1.23). Table 1 presents incidence estimates by melanoma characteristics.

Conclusions: The results of this analysis indicate an increase in stage III/regional melanoma incidence from 2010 to 2014 and a high incidence of stage III melanoma with poor prognostic factors.

Table 1.

|

---|---

Stage III1/regional melanoma | Incidence in year 20142

(estimate of newly diagnosed in the US in 20163)

By tumor thickness4

|

≤ 0.8 mm | 0.12/ 100,000 population (n=393)

0.81 - 1mm | 0.04 / 100,000 population (n=143)

1.01 - 2 mm | 0.31/ 100,000 population (n=1,028)

2.01 - 4 mm | 0.29/ 100,000 population (n=990)

> 4 mm | 0.37/ 100,000 population (n=1,270)

By ulceration

|

Yes | 0.54/ 100,000 population (n=1,858)

No | 0.62/ 100,000 population (n=2,099)

Unknown | 0.34/ 100,000 population (n=1,152)

By number mitoses per square mm

|

None | 0.05/ 100,000 population (n=174)

1 - 5 | 0.58/ 100,000 population (n=1,961)

6 - 10 | 0.20/ 100,000 population (n=672)

≥ 11 | 0.17/ 100,000 population (n=588)

Unknown | 0.50/ 100,000 population (n=1,713)

Any LN involvement | 1.40/ 100,000 population (n=4,725)

1 LN | 0.78 / 100,000 population (n=2,622)

2 - 3 LNs | 0.34 / 100,000 population (n=1,155)

> 3 LNs | 0.16/ 100,000 population (n=531)

Unknown number of LNs | 0.12/ 100,000 population (n=416)

Microscopic nodal metastasis5 (regardless of the number of LNs) | 0.81/ 100,000 population (n=2,725)

Satellite / in-transit without LN involvement  | 0.09/100,000 population (n=311)

LN, lymph node; [1] Defined using the American Joint Committee on Cancer (AJCC) 7 edition classification. [2] New cases diagnosed in 2014 (most recent year available in SEER) / 100,000 population; based on mid-year general population in 2014 for the geographical areas covered by the SEER (118,955,244 individuals). [3] Based on age-specific incidence and most recent population estimates (year 2016) from the US Census Bureau (age < 21 years = 86,391,289; age 21- 64 years = 187,492,029; age > 64 years = 49,244,195). [4] "Unknown" not shown if incidence <0.05 /100,000 population. [5] Includes pts identified with clinically occult (microscopic) LN metastases only, pts with isolated tumor cells only in LN, and/or pts who underwent sentinel LN biopsy and were found to have LN metastasis.

#1204

Incidences of malignant primary soft-tissue sarcomas regardless of primary site: A nationwide population-based study in Taiwan.

Giun-Yi Hung,1 Jiun-Lin Horng,2 Chueh-Chuan Yen1. 1 _Taipei Veterans General Hospital, Taipei, Taiwan;_ 2 _Taipei Medical University, Taipei, Taiwan_.

Population-based studies on soft tissue sarcomas (STS) incidence beyond extremities and trunk wall are lacking in Asia. In this study, we aimed to report the incidences of STS arise in any part of the body. The data of primary STS registered in the population-based Taiwan Cancer Registry during 2007 and 2013 was acquired via the Health and Welfare Data Science Center, Taiwan. Histologic subtype-, age-, and gender-specific STS incidence rates of all sites were analyzed. A total of 11393 subjects were identified, with an age-standardized incidence rate of 5.62 (95% CI, 5.51-5.73) per 100,000 person-years. Approximately 30% of STS occurred in soft tissues (ICD-O-3 topography codes of C47, C49), and 70% occurred in all other primary sites (ICD-O-3 topography codes of C00-C80 except C47, C49, C40 and C41). The three most common primary sites other than soft tissues were stomach (15.9%), skin (14.3%) and small intestine (10.5%). Gastrointestinal stromal tumor (GIST) was the most common subtype, accounting for 29.2% of STS, followed by liposarcoma (11.5%) and leiomyosarcoma (9.7%). For overall STS, no significant incidence trend was identified during 2007-2013. However, a significant declining trend in incidence rate of undifferentiated pleomorphic sarcoma was found (annual percentage change, APC, -7.8%). By contrast, a significant upward trend was identified in other specified sarcomas (APC, 7.3%). For the first time, this analysis demonstrated that STS incidence varied by histologic subtype, gender, age, and primary site in Asians. In the future, more population-based studies should be performed in Asians for comparison with the results from Western countries to better clarification if racial variation exists in any subtype-specific incidence rates of STS.

#1205

Long-term survival of children with acute lymphoblastic leukemia, according to treatment regimen, in a regional French population.

Sophie Gauthier,1 Morgane Mounier,1 Stéphanie Girard,1 Claire Briandet,2 Paule Marie Carli,1 Marc Maynadie1. 1 _INSERM U1231 - Registre des Hémopathies Malignes de Côte d'Or, Dijon, France;_ 2 _CHU Dijon, Dijon, France_.

Acute Lymphoblastic Leukemia (ALL) is the main neoplastic disease in children. Treatment is based on chemotherapy associated or not to bone marrow transplantation. The 5-year survival rate in European children is reported as 86% but long term survival data are not available. We investigated the 20 year Overall Survival (OS) and Net survival (NS) of the patients diagnosed in the population of the Côte d'Or area in France. 162 cases were diagnosed in the population of the Côte d'Or area between 1970 and 2015. Cases diagnosed between 1970 and 1979 (n=29) were not formally registered in the registry database but data were available through the unique regional, reference hematology laboratory. All cases were followed until 01 01 2017. The treatment regimens that were used were as follows in chronological order (from the earliest to the most recent); LAL 76 and 80; the FRALLE 83, 87 and 89 until 1993; the FRALLE 93 from 1993 to 2001 and then the FRALLE 2000. The 20-y OS and NS of ALL was respectively 66% and 67%. It was 59% in boys but 75% in girls (p=0.04). No statistical difference was found according to age. According to lineage subtype, the 20-y NS was 82% in B-ALL, 65% in T-ALL and 34% in ALL, not otherwise specified (p<0.001). According to treatment regimen, the 20-y-NS was 51% before the use of the FRALLE 93, compared to 71% for cases treated by the FRALLE 93 regimen and 95% for those treated with the FRALLE 2000 regimen (p<0.001). The survival was 95% irrespective of the time of diagnosis following introduction of the FRALLE 2000 regimen. Data on long term survival of ALL in children are scarce. Our results show 1) a very high 20 year survival rate indicating the efficacy and safety of the treatment regimens used, 2) the utility of randomized clinical trials to define new standards of care.

#1206

Patterns of BRAF testing and treatment in patients with metastatic melanoma presumed BRAF positive.

Sameer Ghate,1 Antonio Nakasato,1 Raluca Ionescu-Ittu,2 Sherry Shi,2 Briana Ndife,1 Rebecca Burne,2 François Laliberté,2 Mei Sheng Duh3. 1 _Novartis Pharmaceuticals Corporation, East Hanover, NJ;_ 2 _Groupe d'analyse, Ltée, Montréal, Quebec, Canada;_ 3 _Analysis Group, Boston, MA_.

Background: BRAF mutation affects approximately half of the patients with metastatic melanoma (MM). The NCCN guidelines recommend first-line (1L) therapy with both immune checkpoint inhibitors (I-O) and BRAF-targeted therapies (TT) for patients with BRAF mutated MM. Using two claims-based databases, the current study investigated (a) patterns of BRAF testing among patients with MM presumed to be BRAF positive and (b) characteristics of patients initiated on 1L treatment with I-O versus TT after a presumably positive BRAF test.

Methods: Adults with MM initiated on I-O and TT in 1L were identified in Truven MarketScan (Q1/2014 - Q3/2016; n = 1,156) and Quintiles IMS Real-World Data Adjudicated Claims database (Q1/2014 - Q2/2016; n = 1,455) databases. Patients were presumed to be BRAF positive if they received TT in at least one line of MM therapy. All patients were required to have ≥ 2 lines of therapy for MM (n = 162 in Truven sample and n = 247 in QuintilesIMS sample).

Results: In the Truven sample, 57% of presumed BRAF positive patients were tested for BRAF mutation between first melanoma diagnosis in the data and 1L initiation; in QuintilesIMS, 64% were tested. Among patients tested for BRAF mutation before 1L initiation, the distribution of I-O vs. TT regimens in 1L was 34% vs. 66% in Truven and 38% vs 62% in QuintilesIMS. Patients with a BRAF test before 1L who were initiated on TT appeared to have more brain metastases, and auto-immune, liver, and renal diseases than those initiated on I-O (Table 1). Of 70 patients in MarketScan and 89 patients in QuintilesIMS not tested before 1L, 11% and 10% respectively were tested between 1L initiation and 2L initiation.

Conclusions: This real-world study in two databases showed that approximately two thirds of those tested for BRAF mutation received TT for BRAF in 1L. Among patients tested for BRAF before 1L initiaiton, TT appeared to be channeled towards sicker patients. Future research should ascertain the physician decision on treatment selection.

Table 1. | |  | |

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

|

Patients tested for BRAF before 1L start, by 1L regimen

|

Truven sample

(n = 92) | QuintilesIMS sample

(n = 158)

I-O

(n= 31) | Targeted

(n= 61) | I-O

(n= 58) | Targeted

(n= 100)

Age at 1L initiation, Median [Q1-Q3] | 57 [47-62] | 57 [47-62] | 51.5 [43-57] | 54 [46-60]

Male, N (%) | 15 (48%) | 40 (66%) | 29 (50%) | 63 (63%)

Comorbidity score (range 0-23, higher values indicate higher risk of death),* Median [Q1-Q3] | 6 [6-7] | 7 [6-8] | 6 [6-7] | 6 [6-8]

Brain metastases, N (%) | 9 (29%) | 28 (46%) | 14 (24%) | 31 (31%)

Comorbid conditions, N (%) | |  | |

Auto-immune disease** | 0 (0%) | 6 (10%) | 3 (5%) | 9 (9%)

Anemia | 5 (16%) | 19 (31%) | 12 (21%) | 20 (20%)

Liver disease | 4 (13%) | 16 (26%) | 9 (16%) | 25 (25%)

Renal disease | 0 (0%) | 8 (13%) | 1 (2%) | 7 (7%)

* Deyo et al. J Clin Epi 1992.

** Ankylosing spondylitis, thyroiditis, celiac disease, Graves' disease, inflammatory bowel disease (including Chron''s disease and ulcerative colitis), multiple sclerosis, psoriasis, psoriatic arthritis, rheumatoid arthritis, systemic lupus erythematosus.

#1207

A systematic literature review of the prevalence of PIK3CA mutations and mutation hotspots in HR+/HER2- metastatic breast cancer.

Lea Mollon,1 Alejandra Aguilar,1 Elizabeth Anderson,1 Joni Dean,1 Lisa Davis,1 Terri Warholak,1 Ayal A. Aizer,2 Emma Platt,3 Aditya Bardiya,2 Derek Tang3. 1 _University of Arizona, Tucson, AZ;_ 2 _Harvard Medical School, Boston, MA;_ 3 _Novartis, Morris Plains, NJ_.

Introduction: Clinical research on the predictive value of PIK3CA mutations in hormone receptor-positive (HR+), human epidermal growth factor 2-negative (HER2–) metastatic breast cancer (mBC) has advanced in recent years. However, knowledge of epidemiological prevalence has not been systematically evaluated. This study aimed to report prevalence of PIK3CA mutation using different biopsy techniques as well as specific hotspot mutations across the available literature.

Methods: A comprehensive search of PubMed/MEDLINE, EMBASE, Cochrane Central, and select conference abstracts was performed by two independent researchers that included, but was not limited to, keywords: "breast neoplasm", "PIK3CA protein", "hormone receptor positive", and "metastases". English-language studies in HR+, HER2– mBC detailing the prevalence of PIK3CA mutations and published between January 1993 through August 2017 were included. Content analysis was employed to quantify collected data elements.

Results: Of 558 studies included for full-text review, 36 met inclusion criteria. Most included studies (n = 18) were observational in nature. A total of 4,247 samples were tested for genetic mutations. Most studies used tissue biopsy samples (n = 33; 89%). Tumor samples accounted for 84.6% of all samples (n = 3597). Liquid biopsies were performed in 4 studies (11%) and accounted for 15.3% of all samples (n = 650). One study reported both liquid and tumor biopsy data. Overall, reported prevalence of the PIK3CA mutation ranged from 13.3% to 61.5%. Median prevalence was 36.4% (25th percentile = 28.6%; 75th percentile = 48.4%). Among studies using tissue biopsies, the majority reported prevalence from 16.7% to 61.5%. Among studies using liquid biopsies, the majority (n = 3) reported prevalence from 43.3% to 46.8%; one other study reported 13.3%. The most commonly tested hotspot mutations were H1047R and E545K. Among studies reporting specific hotspot mutation prevalence (n = 9), the H1047R hotspot mutation prevalence in these studies ranged between 25% and 75% while the E545K prevalence ranged between 11.1% and 50%.

Conclusions: Although discrepancies exist with respect to mutation prevalence estimated across various tissue vs. liquid biopsy techniques, PIK3CA mutations and mutation hotspots (specifically H1047R and E545K) frequently occur in HR+/HER2– mBC.

#1208

Is the decrease in the incidence of large cell carcinoma of the lung due to changes in classification towards adenocarcinoma.

Dana Hashim,1 Ariana Znaor,2 William Travis,3 Paolo Boffetta1. 1 _Icahn School of Medicine at Mount Sinai, New York, NY;_ 2 _International Agency for Research on Cancer, Lyon, France;_ 3 _Memorial Sloan Kettering Cancer Center, New York, NY_.

Background: Data from many populations show a decrease in the incidence of large cell carcinoma (LCC) of the lung while incidence of lung adenocarcinoma (AC) is decreasing to a lower rate or even increasing from the 1990s. We compared the changes in incidence of lung LCC and AC worldwide to test the hypothesis that at least part of the decline in LCC incidence was due to classification of these tumors as AC.

Methods: We analyzed the annual percent change (APC) in the age-standardized rates (ASRs) of lung LCC and AC incidence between 1996 and 2007 in 78 high-quality cancer registries from Europe, Asia, North America, and South America, using linear regression.

Results: Among women, the median APC of LCC incidence was -5.42, and that of AC incidence was -0.96. There was a positive correlation between the two measures (β=0.29; p=0.01). The APC of LCC incidence was negative in 64 populations (82.1%); that of AC incidence was negative in 50 populations (63.3%, p of the χ2 distribution = 0.07). Among women, the median APC for LCC and AC incidence was -5.06 and +2.11, respectively. The correlation between the two measures was 0.17 (p=0.03). The APC of LCC incidence was negative in 59 populations (76.6%); that of AC incidence was negative in 23 populations (29.9%, p of the χ2 distribution = 0.41).

Conclusions: The decrease in LCC may be, in part, due to changes in histologic classification and triaging towards a diagnosis of adenocarcinoma.

#1209

Patterns of treatment with immune check point inhibitors and targeted therapy in patients with metastatic melanoma presumed BRAF V600 positive.

Sameer Ghate,1 Antonio Nakasato,1 Raluca Ionescu-Ittu,2 Sherry Shi,2 Briana Ndife,1 Rebecca Burne,2 François Laliberté,2 Mei Sheng Duh3. 1 _Novartis Pharmaceuticals Corporation, East Hanover, NJ;_ 2 _Groupe d'analyse, Ltée, Montréal, Quebec, Canada;_ 3 _Analysis Group, Inc, Boston, MA_.

Background: Immune check point inhibitors (I-O) and targeted therapies (TT) have changed the treatment landscape for patients with metastatic melanoma (MM), particularly for patients with BRAFV600 (BRAF) mutation who are eligible for both types of treatment after a diagnosis of MM. The aim of the current study was to describe patterns of treatment with I-O and TT in first line (1L) and subsequent lines of therapy for MM in a sample of patients presumed BRAF positive.

Methods: Adults with MM initiated on I-O (ipilimumab, pembrolizumab, nivolumab) and TT (vemurafenib, dabrafenib, trametinib) therapies in 1L were identified in Symphony Health Solutions' Integrated Dataverse (Q1/2014 - Q1/2017; n = 4,196), the largest pharmacy database in the US. Lines of pharmacological therapy were investigated from the first I-O/TT (index date) until the end of the observation period using an algorithm that relies on prescription/administration dates, days of supply and periods without any therapies. Patients were presumed BRAF positive if they received TT in at least one line of MM therapy. All patients in this analysis were required to have ≥ 2 lines of therapy for MM.

Results: Of 366 presumed BRAF patients in the study sample, 110 (30%) and 256 (70%) were initiated on I-O and TT in 1L, respectively. The table below presents treatment patterns in 1L, 2L, and 3L for MM. The distribution of I-O vs TT was 30% vs. 70% in 1L, 25% vs. 57% in 2L, and 41% vs. 39% in 3L (table).

Conclusions: This real-world data study showed dabrafenib+trametinib was the most common treatment for patients with MM presumed BRAF positive, even in the era of I-O availability. During the study period (years 2014-2017), ipilimumab continued to be the most common I-O therapy used in 1L and 2L among presumed BRAF patients.

Table. | |

|

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

Years 2014-2017 | 1L

N=366 | 2L

N=366 | 3L

N=111

Regimens | |

|

I-O, N (%) | 110 (30%) | 103 (28%) | 46 (41%)

Up to third most common I-O regimen, N (%) | Ipilimumab 72 (20%) | Ipilimumab 32 (9%) | Nivolumab 14 (13%)

Nivolumab 13 (4%) | Pembrolizumab 32 (9%) | Pembrolizumab 12 (11%)

Pembrolizumab 13 (4%) | Nivolumab 22 (6%) | Ipilimumab + Nivolumab 10 (9%)

TT, N (%) | 256 (70%) | 207 (57%) | 43 (39%)

Up to third most common TT regimen, N (%) | Dabrafenib+Trametinib 134 (37%) | Dabrafenib+Trametinib 117 (32%) | Dabrafenib+Trametinib 18 (16%)

Vemurafenib 69 (19%) | Vemurafenib 22 (6%) | Vemurafenib 5 (5%)

Dabrafenib 31 (8%) | Dabrafenib 18 (5%) | Trametinib 5 (5%)

|

Vemurafenib + Cobimetinib 18 (5%) | Vemurafenib + Cobimetinib 5 (5%)

Both I-O and TT, N (%) | 0 (by design) | 18 (5%) | 7 (2%)

Other antineoplastic agents, N (%) | 0 (by design) | 38 (10%) | 15 (4%)

Top 3 most frequent treatment sequences | |

|

Patients with ≥2 lines of therapy (n=366 ) | 1L -> 2L (%)

|

TT -> TT (34%)

|

TT -> I-O (25%)

|

I-O -> TT (23%)

|

Patients with ≥3 lines of therapy (n=111) | 1L -> 2L -> 3L (%)

TT -> I-O -> I-O (14%)

I-O -> TT -> I-O (12%)

TT -> TT -> I-O (11%)

1L, 2L, 3L: first, second, third-line of therapy; I-O: immune check point inhibitors; TT, targeted therapy.

#1210

Trend in the incidence of gynecologic malignancies in Federal Teaching Hospital Abakaliki: A 5-year review.

Joseph Agboeze, Robinson Onoh, Matthew Igwe. _Federal Teaching Hospital,Abakaliki, Abakaliki, Nigeria_.

Background: Gynecological malignancies are common and are among the leading causes of cancer-related deaths worldwide Objective: To determine the trend in the incidence of gynaecological malignancies in Federal Teaching Hospital, Abakaliki, Nigeria.

Methods: This was a retrospective study. The study population was the patients diagnosed of gynaecological cancers at the Federal Teaching Hospital, Abakaliki between January, 2012 to December 2015. Data were extracted from case notes of patients, discharge books and the operating theatre record books. The data were analyzed using Epi Info version 7.1.4 and the results expressed in descriptive statistics by simple percentages.

Results: There were 2528 gynaecological admissions during the period. There were 97 women who had genital tract malignancies giving an incidence of 3.8% of gynaecological admissions. Cervical cancer was the most common female genital tract malignancy occurring in 68% of gynaecological cancers, followed by ovarian cancer (11.3%), Endometrial cancer (8.3%), Vulva cancer (6.2%),Choriocarcinoma (4.1%), and the least was leiomyosarcoma (1.0%). The mean age was 54.2 ±11.9 years.The incidences of cervical, ovarian vulva and endometrial cancers showed a steady increase over the period. Choriocarcinoma did not show a significant change in incidence.

Conclusion: This study showed the rising incidences of gynaecological cancers. Cancer of the cervix is the commonest gynaecological malignancy and most patients presented late. Cervical cancer screening programmes, early detection and awareness campaign is highly recommended.

#1211

Ecological model-generated hypothesis for high prostate cancer incidence in African-Americans: TRPV6a gene variant and calcium-ion hypersensitivity.

Constance B. Hilliard. _University of North Texas, Denton, TX_.

INTRODUCTION: This Ecological Model System (EMS) applies an evidence-based hypothesis to a health disparities conundrum posed by low-calcium-consuming African-American males in the sizable medical literature that attributes metastatic Prostate Cancer (PCa) for which this group is at high risk, to calcium over-consumption. This EMS identifies the ethnic-specific TRPV6 calcium ion channel,because of its unusually high calcium absorption capacity relative to the non-African variant, as the culprit. While it allowed the Niger-Kordofanian West African ancestors of black Americans to maintain strong bones on a 200 mg/ca/day diet, it is oncologically maladaptive for U.S. blacks. In America's high calcium, dairy food culture, the TRPV6a variant allows blacks, who are generally lactase non-persistent, to nevertheless absorb more of the mineral than whites and excrete less in their urine. These excess free calcium ions appear to initiate cell proliferation, leading to metastatic PCa, in which the TRPV6 gene becomes over-expressed.

METHODS: To generate an algorithm-driven informative matrix, Medline and JSTOR searches were mined using keywords such as: metastatic prostatic cancer, androgen-castration-resistant, Niger-Kordofanian West Africans, TRPV6, TRPV5-renal excretion, TRPV6 over-expression biomarker. Secondary data sets from (1) "Prostate, Lung, Colorectal, Ovarian Cancer Screening Trial," conducted between 1993 and 2001, 38,349 males (ages 55-74 years); (2) "California Collaborative Prostate Cancer Study", a study of 4321 African-Americans and non-Hispanic Whites was analyzed using multivariable conditional logistic regression. RESULTS: This EMS reveals that at 400 mg/day/ca, metastatic PCa is observed in 17% of Blacks, but 0% of American Whites. 100% of the Black incidence is below 1150 mg/calcium intake, while 59% of White incidence from 1250-2000 mg.

SUMMARY: Because the African TRPV6a calcium ion channel variant is more absorbent of free calcium ions than its non-African counterpart, this Ecological Model shows that carriers of this variant are at increased risk of metastatic PCa when exposed to high calcium food environments.

CONCLUSION: Black hypersensitivity to calcium triggers metastatic PCa caused by high-calcium-absorbing African TRPV6a calcium ion channel variant.

This EMS further hypothesizes that the high susceptibility of Blacks to other TRPV6-expressing cancers (e.g.triple negative breast cancer and colorectal cancer) share the same pathogenesis. This Model can be applied as well to generating hypotheses for STEM basic and translational research regarding Type II diabetes, hypertension, kidney disease.

#1212

Urinary bladder cancer in Sokoto, Northwestern Nigeria: A 10-year histopathological review.

Saddiku Malami Sahabi, Kabiru Abdullahi. _Usmanu Danfodiyo University Teaching Hospital, Sokoto, Nigeria_.

Background: Urinary Bladder Cancer is a common disease worldwide. It ranks ninth in cancer incidence and is the 13th leading cause of cancer death among men and women worldwide. The highest incidence is seen usually in developed countries. In our environment, it is the leading cause of cancer and cancer deaths among males because of endemicity of urinary schistosomiasis. Yet, little is done to curb this preventable risk factor of bladder cancer.

Objective: The objectives of this study were to describe histopathological pattern of urinary bladder cancer in Sokoto, Northwestern Nigeria.

Method: This was a retrospective study of all histologically confirmed cases of bladder cancer seen at the Department of Histopathology, Usmanu Danfodiyo University Teaching Hospital, Sokoto between the years 2006 and 2015. Information was obtained from the database of the Department of Histopathology. The data were validated using Microsoft Excel and exported to SPSS for analysis. The data were analysed for age, sex and histological types using SPSS version 20 software. The results are presented in the form of simple frequency tables and percentages.

Results: A total of 190 patients were seen with histologically confirmed bladder cancer out of the 3933 patients with malignancies between the years 2006 and 2015, constituting 4.8% of all malignancies diagnosed in the same period and comprises 160(84.2%) males and 30(15.8%) females with male to female ratio of 5.3:1. Their mean age was 50.8, SD ±14.9 years and range from 1-90 years. The most common bladder cancer was squamous cell carcinoma with 97 (51.1%) cases. 81 (42.6%) patients had urothelial carcinoma, 5(2.6%) were metastatic carcinoma, 4(2.1%) were sarcomatoid carcinoma, 2(1.1%) were rhabdomyosarcoma and only 1(0.5%) case of a signet ring cell carcinoma was recorded.

Conclusion: Squamous cell carcinoma remained the most common histological type of bladder cancer in our setting followed by urothelial carcinoma. Preventive measures aim at eradicating urinary schistosomiasis, a predominant and known risk factor of bladder cancer in our environment will go a long way in curtailing the burden of this disease among our people.

#1213

Comorbidities and concomitant medication use in small cell lung cancer patients treated with third and later-lines of therapy: An analysis of electronic health records data.

Rui Jiang,1 Scott H. Gulbranson,1 Philip Komarnitsky,1 Fabio A. Lievano,1 Martina M. Koch,2 Eros Papademetriou,3 Jerzy E. Tyczynski1. 1 _Abbvie, North Chicago, IL;_ 2 _AbbVie Stemcentrx, South San Francisco, CA;_ 3 _SmartAnalyst Inc., New York, NY_.

Recent real-world data on comorbidities and concomitant medications in SCLC patients are scarce, particularly for later-lines of treatment. The purpose of this study was to describe comorbidities' pattern and concomitant medication use in SCLC patients who received third- and later-lines of therapy (LOT). We conducted an analysis of 8,291 patients with newly diagnosed SCLC between 01/01/2008 and 09/30/2016 using Optum's electronic health records (EHR) database. The database consists of de-identified EHR data from a network of health care provider organizations in the United States. Of the 8,291 SCLC patients followed longitudinally, 428 received 3rd LOT. 206 LOTs were 4th or later-lines. At start of 3rd LOT, median age was 65, 46% were male, 97% had extensive disease (excluding n=52 with missing staging). The most prevalent comorbidities by the Charlson Comorbidity Index (CCI) category in SCLC patients during 6 months before start of 3rd LOT were metastatic solid tumor, chronic pulmonary disease, cardiovascular disease, and diabetes (top 10 are listed in Table 1). The frequently prescribed concomitant medications in SCLC patients within 6 month after start of 3rd LOT include analgesics, systemic corticosteroids, drugs for gastrointestinal complaints, anxiolytics, respiratory agents, antihypertensives and other cardiovascular agents, antidiabetics, and anti-infectives (top 10 are listed in Table 1). The patterns of comorbidities and concomitant medications were similar between 3rd LOT and 4th\+ LOTs (4th\+ LOT data not shown). In conclusion, this analysis provides unique real-world comorbidity and concomitant medication data in SCLC with 3rd and later-lines of therapy. These patients are mostly prescribed with medications for pain and inflammation due to the ongoing disease. Comorbidity conditions that are common in older adults are also observed in SCLC patients receiving later-lines therapy.

Table 1. Top 10 comorbidities by Charlson Comorbidity Index (CCI) and top 10 concomitant medications

---

CCI | Percentage (%), n=428

Metastatic solid tumor | 76.4

Chronic pulmonary disease | 47.4

Diabetes | 21.5

Mild liver disease | 21.3

Peripheral vascular disease | 16.1

Cerebrovascular disease | 11.2

Renal disease | 7.7

Congestive heart failure | 6.8

Myocardial infarction | 5.1

Diabetes with chronic complications | 4.0

Drug Class

|

Narcotic analgesics | 79.4

Glucocorticoids | 72.2

Intravenous supplies | 69.9

5-hydroxytryptamine-3 antagonists (antiemetic agent) | 69.6

Heparins or glycosaminoglycans | 58.2

Anxiolytics | 57.5

Antidopaminergics (antiemetic agents) | 53.0

Antihistamines (sedating) | 45.6

Acetaminophen and proton pump inhibitors | 44.4

Intravenous nutritional therapy; electrolyte; trace element; metal; vitamin; alone or combinations | 39.7

#1214

Nasopharyngeal carcinoma clinical stage, histologic grade and biomarker status at presentation are usually of poor prognostic characteristics: A clinicopathologic study in a low-resource setting.

Aralola A. Olusanya, Gabriel O. Ogun, Adebolajo Adeyemo. _University College Hospital, Ibadan, Nigeria_.

Introduction: Nasopharyngeal carcinoma (NPC) is the 8th commonest malignancy in men and the commonest head and neck tumor in Ibadan Cancer Registry (IBCR).

Methods: Clinical files and records of all nasopharyngeal carcinoma diagnosed at the Department of Pathology, University College Hospital affiliated with IBCR between January 2007 and December 2016 were reviewed for demographic data, clinical presentation, clinical stage, histologic grade, LMP-1 and EGFR status.

Results: A total of 71 cases of nasopharyngeal carcinoma were diagnosed during the study period, comprising 44 males and 27 females with a male:female ratio of 1.6. Age ranged from 12 years to 80 years, the modal age group was 40-49 and the modal age was 45 years. Sixty-three percent of cases were non-keratinizing undifferentiated tumors, 31% were non-keratinizing differentiated, 4.2% were keratinizing tumors and 1.4% basaloid squamous cell carcinoma. In all, non-keratinizing undifferentiated tumors constituted 64% of tumors in males and 63% in females. Conversely, non-keratinizing differentiated tumor was commoner in males, accounting for 34% of tumors compared to only 26% in females. Also, keratinizing tumor was twice as common in females than males; there was only a single basaloid squamous cell carcinoma recorded. The age group 30-39 years had the highest incidence of non-keratinizing undifferentiated tumor while non-keratinizing differentiated tumor was more common in the 40-49 age group. Patients presented most frequently with stage II disease, which accounted for 62% of cases. About 20% of these patients presented with stage IV disease. Nasal and aural symptoms accounted for the commonest presenting complaint, occurring in 38% of patients. Thirty-six percent presented with nasal symptoms alone while 13% had a combination of nasal, aural and visual symptoms; this group was mainly patients with stage IV disease. LMP-1 positivity was observed in as many as 99% of all cases. EGFR was positive in 94% of cases, indicating that NPCs are usually of poor prognosis in our practice setting at presentation.

Conclusion: In our practice, nasopharyngeal carcinoma is characterized by late-stage presentation, high-grade histologic characteristics and have biomarker indicators of poor prognosis.

#1215

Epidemiology of breast malignancies in sub-Saharan Africa: A ten-year retrospective evaluation of 2,419 patients at a major tertiary institution in Southwestern Nigeria.

Funlayo O. Buraimoh, Abiodun O. Popoola. _Lagos State University College of Medicine, Lagos, Nigeria_.

Background: There is limited information on the epidemiology and clinical presentation of breast cancer in Sub-Saharan Africa. This analysis evaluated the prevalence, characteristics of the disease over a ten-year period, as well as the impact of sociodemographic factors on late breast cancer presentation in Lagos State University Teaching Hospital (LASUTH) Lagos, a major tertiary institution in Nigeria, the most populous country in Africa. Objectives: The aim of this study is to determine the epidemiological variants of all breast cancer cases at LASUTH, 2007- 2017. Materials and Methods: There was no restriction on gender or age groups. Late presentation was defined by symptomatic presentation. Tumors were characterized by histological subtypes. Mean, frequencies and percentages were performed using STATA/IC 12.1 for Windows. Results: A total of 2419 breast cancer patients were seen during the ten-year period. There were more female malignancies (n=2380, 98.4%) compared to male malignancies (n=39, 1.6%). The prevalence of breast cancer was found to be 37.5%. The overall mean age (years) was 47.27, the age range was 19 to 91 years. Increased risk of late presentation (n=1910, 79%). Overall higher presentation were made from christians (n= 1849, 76.4%), married individuals (n=2089, 86.4%), people in unskilled professions such as traders (n=808, 33.4%) and people with a tertiary level of education (n= 239, 12.8%). As the primary level of treatment, most of the patients had mastectomy (n=1533, 63.4%), chemotherapy (n=600, 24.8%) and radiotherapy (n=51, 2.1%). The topography of all reported cases include upper-outer quadrant of breast (n=433, 17.9%), axillary tail of breast (n=381, 15.8%), overlying lesion of breast (n=144, 11.5%), lower-inner quadrant of breast (n=133, 5.5%), upper-inner quadrant of breast (n=92, 3.8%), nipple (n=77, 3.2%), lower-outer quadrant of breast (n= 75, 3.1%) and the central portion of breast (n= 42, 1.7%). The three most common morphologies were Pleomorphic carcinoma (n=717, 29.6%), Infiltrating duct carcinoma (n=630, 26%) and giant cell type of the malignant tumor (322, 13.3%). Conclusion: Our study shows that breast cancers usually present mostly in young women in Nigeria, and shows the influence of culture on late presentation. Therefore we should institute deliberate public health education and counseling in our social institutions such as religious circles that are designed particularly to favour the peculiar demographics of the population to prevent late presentation. In addition, cross-sectional studies of breast cancer tumors in younger women (before the age of 40) should be explored to better understand the disease and further understanding too of breast cancers in African American women.

#1216

Cancer epidemiology research in Vietnam: Current status, challenges and opportunities.

Thuan V. Tran,1 Chi T. Du Tran,2 Huong T. Tran,1 Mai V. Nguyen,2 Mo T. Tran,2 Quang H. Tran,2 Thuy Thi-Van Tuong,2 Martha J. Shrubsole,3 Qiuyin Cai,3 Wei Zheng,3 Xiao-Ou Shu,3 Linh C. Le,2 Paolo Boffetta,4 Hung N. Luu5. 1 _Vietnam National Cancer Hospital, Hanoi, Viet Nam;_ 2 _Vinmec Healthcare System, Hanoi, Viet Nam;_ 3 _Vanderbilt University School of Medicine, Nashville, TN;_ 4 _Mount Sinai School of Medicine, New York, NY;_ 5 _University of South Florida, Tampa, FL_.

Background: Rapid economic growth, industrialization, and widespread adoption of a Western lifestyle in Vietnam have been accompanied by an emerging epidemic of noncommunicable diseases (NCDs), including cancer. We investigated current status, identified challenges and evaluated opportunities for the development of a comprehensive program in cancer epidemiology research in Vietnam.

Methods: We systematically searched OvidMedline, PubMed, and the Cochrane Library for studies published in English on cancer epidemiology in Vietnam prior to November 2017. Data from Vietnam cancer registries were used for descriptive analyses. Technical reports in Vietnamese were also included in our review.

Results: Cancer registry data show that by end of 2013, the total number of new cancer cases was 146,457. The most common cancers, in descending order, among men were lung, gastric, colorectal, liver and esophageal cancers while breast, lung, colorectal, gastric and thyroid cancers were the most common cancers among women. Data from six (6) cancer registries during 2004-2013 show that the incidence of several cancers has increased during the past decade, including colorectal cancer (both genders), lung cancer (men), and breast and thyroid cancers (women). Mortality data are limited due to the unavailability of death registries and/or modes of death ascertainment (i.e., causes of death). There is a scarcity of high-quality research on the risk factors (e.g., lifestyle such as smoking, alcohol consumption, physical activity, and BMI, environmental or genetic factors) for cancer in Vietnam. The majority of prior research consists of hospital-based studies (either case-control or cross-sectional designs) with small sample sizes. Since there is no population-based epidemiologic study on cancer in Vietnam, it is difficult to assess temporal trends and risk factors for cancers. Additional challenges include a lack of studies on infectious agents related to cancers and a paucity of molecular or genetic data in cancer, except for a few publications on HBV, HCV, H. Pylori, or EBV or susceptibility genes, such as BRCA1, BRCA2 for breast cancer or GSTA1, GSTP1, GSTT1 and GSTM1 for gastric cancer.

Conclusions: Currently, there is no population-based epidemiologic research program for cancer in Vietnam due to the lack of a population-based cancer registry, a dearth of well-trained investigators, and the absence of proper research support infrastructure. This, along with the lack of comprehensive etiologic research in Vietnam, prevents the evaluation of the temporal trends of cancer and risk factors for cancer. The cancer registry has provided important data on cancer incidence; however, there are no data on cancer mortality. Data on molecular and genetic epidemiology of cancer are also limited. These challenges shows the need to develop a comprehensive program in cancer control in Vietnam.

#1217

The association between birth order and childhood brain tumors: A systematic review and meta-analysis.

Mai V. Nguyen,1 Mo Thi Tran,1 Chi TDu Tran,1 Quang H. Tran,1 Thuy Thi-Van Tuong,1 Linh C. Le,1 Paolo Boffetta,2 Hung N. Luu3. 1 _Vinmec Healthcare System, Hanoi, Viet Nam;_ 2 _Mount Sinai School of Medicine, New York, NY;_ 3 _University of South Florida, Tampa, FL_.

Background: The incidence of childhood brain tumors (CBT) has increased worldwide, likely resulting from the improvements of early diagnostics. The only known risk factors for CBT are the Li-Fraumeni syndrome and iodization. While both genetic and environmental factors are suggested roles in CBT, birth characteristics, including birth order, maternal age, and mode of delivery, might present the interactions between genetic susceptibility and perinatal environmental causes. Birth order has been hypothesized to play an important role in CBT carcinogenesis due to its possible role as proxy for 1) early infection exposure and 2) hormone levels since first pregnancy differs endocrinologically from later pregnancies. We conducted a systematic review and meta-analysis to clarify the association between birth order and CBT.

Methods: We followed established guidelines to systematically search OvidMedline, PubMed, and the Cochrane Library for English-language studies (both case-control and cohort designs) published prior to November 2017. Meta-analysis provided pooled risk estimates and their 95% confidence intervals (CI) for birth order and CBT. Funnel plots were used to identify sources of heterogeneity and publication bias.

Results: We identified 16 case-control studies with a total sample of 32,439 cases and 166,144 controls. Compared to first birth order, the meta-odds ratio (mOR) for second birth order was 1.04 (95% CI 1.01-1.07), and that for third birth order was 0.98 (95% CI: 0.90-1.06). We also identified 3 cohorts comprising a total of 1,208 cases. The meta-hazard ratio (mHR) for second or higher birth order compared to first birth order was 1.00 (95% CI: 0.96-1.05).

Conclusions: We found no overall association between birth order and CBT in both case-control and cohort study designs; the small association observed for second birth order in case-control studies deserves further consideration. Limited statistical power might have hampered the results of the other analyses. 

### Genetic Contributions to Cancer Epidemiology 2: Next-Generation Sequencing and Familial Cancers

#1218

An eQTL and mQTL study of 147 genetic variants associated with prostate cancer susceptibility.

Xiaoyu Wang,1 Anqi Cheng,2 the PRACTICAL Consortium, Janet L. Stanford,1 James Y. Dai1. 1 _Fred Hutchinson Cancer Research Center, Seattle, WA;_ 2 _University of Washington, Seattle, WA_.

Genome-wide association studies by the PRACTICAL consortium have identified and validated 147 single nucleotide polymorphisms (SNPs) associated with prostate cancer (PCa) risk (Schumacher et al., Nat Genet 2017). However, most of these genetic variants are in noncoding regions, which poses a challenge for understanding the molecular mechanisms underlying these risk loci. Herein we study both expression quantitative trait loci (eQTL) and methylation quantitative trait loci (mQTL) among these PCa risk loci to identify molecular alterations associated with these genetic variants. Data were available for a Fred Hutchinson (FH)-based cohort of patients diagnosed with localized stage PCa, including SNP genotypes (Infinium iCOGS and OncoArray-500K BeadChips) from the PRACTICAL consortium and genome-wide DNA methylation (Infinium Human Methylation450 BeadChip) and gene expression (HumanHT-12 v4 BeadChip) profiling of primary tumor tissues obtained at radical prostatectomy. Missing genotypes were imputed using the October 2014 (Phase 3) release of the 1KGP data as the reference panel. These data were used to identify eQTL (N=355) and mQTL (N=377) in cis-acting associations where the transcripts or CpG sites are located within 2Mb (±1Mb) of the genetic variants. The Matrix eQTL software was used to conduct eQTL and mQTL analyses. In total, 4,300 SNP-transcript pairs and 104,600 SNP-CpG pairs were tested for eQTL and mQTL, respectively, among which we identified 64 eQTL-transcript pairs (37 PCa risk loci and transcripts in 54 genes) and 1,405 mQTL-CpG pairs (116 PCa risk loci and CpGs in 321 genes), after correction for multiple testing (FDR<0.05). The remaining 29 (20%) PCa risk loci were neither eQTLs nor mQTLs. Interestingly, 35 (95%) eQTLs were also mQTLs, while the majority of mQTLs were not eQTLs (Fisher's exact test of independence, p=0.005), suggesting that DNA methylation may have alternative biologic regulatory functions other than directly affecting gene expression. Several of these genes associated with eQTLs such as INCENP, NUDT11, RGS17, ASCL2, C10orf32, MSMB, BHLHA15, and IRX4 were previously found in PCa. Furthermore, we discovered a number of novel genes such as HLA-DRB3 and HLA-DRB4 associated with eQTLs in our data. Further validation using the TCGA-PRAD data is under way. In summary, we identified eQTLs and mQTLs among newly established genetic risk loci for PCa. These results provide new insight into both the genetic and epigenetic mechanisms through which these SNPs may alter susceptibility to PCa.

#1219

Association between MICA polymorphisms, s-MICA levels, and pancreatic cancer risk in a population-based case-control study.

Guillaume C. Onyeaghala,1 John Lane,2 Nathan Pankratz,2 Heather H. Nelson,1 Bharat Thyagarajan,2 Kristin E. Anderson,1 Anna E. Prizment1. 1 _Univ. of Minnesota School of Public Health, Minneapolis, MN;_ 2 _Univ. of Minnesota, Minneapolis, MN_.

Objectives: The immune system has been shown to play an important role in pancreatic cancer. Abnormal cells express the transmembrane major histocompatibility complex class I chain-related gene A (MICA) protein, which is recognized by receptors present on NK and cytotoxic T cells. However, pancreatic tumor cells release MICA protein in soluble form (called s-MICA) from the tumor surface and thus avoid immune surveillance. In our previous study, we showed that a higher serum concentration of the s-MICA protein was associated with increased pancreatic cancer risk. In the current study, we hypothesized that functional variants in the MICA gene are associated with higher circulating s-MICA levels and increased pancreatic cancer risk. We focused on the A5.1 MICA allele, as this allele encodes a MICA protein that is shorter than its normal counterpart and is more easily cleaved from the cell surface.

Methods: MICA alleles and s-MICA levels were measured in 116 cases and 492 controls (n=608) in a population-based case-control study in Minnesota. Allele assignments were based on the number of repeat units in the amplified regions of the MICA gene, and s-MICA levels were measured by enzyme-linked immunoabsorbent assay. To address the non-normal distribution of s-MICA levels, general linear regression with a log link was used to assess mean s-MICA levels across MICA alleles. Unconditional logistic regression was used to calculate the odds ratio (OR) and 95% confidence intervals (CI) for pancreatic cancer after adjustment for age, sex, education, smoking status, alcohol consumption, and diabetes status. The analysis was restricted to Caucasians, who represented 96% of all study participants.

Results: 452 participants (74%) possessed at least one copy of the A5.1 allele. After adjustment for confounders, participants with at least one copy of the MICA A5.1 allele were at an increased risk of pancreatic cancer (multivariable OR=2.00, 95% CI: 1.06-3.79), compared to participants who did not possess any A5.1 allele. Participants who had at least one copy of the A5.1 allele had 2.46 (95%CI: 2.36-2.56) times greater mean s-MICA levels than those without the A5.1 allele. These associations were consistent among pancreatic cancer cases and controls.

Conclusions: Our study supports the role of the MICA A5.1 allele in impaired immune response, which may increase the risk of pancreatic cancer.

#1220

Integrative genomic analysis discovers the causative regulatory mechanisms of a breast cancer-associated genetic variant.

Yi Zhang,1 Mohith Manjunath,1 Shilu Zhang,2 Deborah Chasman,2 Sushmita Roy,2 Jun S. Song1. 1 _University of Illinois at Urbana-Champaign, Urbana, IL;_ 2 _University of Wisconsin-Madison, Madison, WI_.

Genome-wide association studies (GWAS) have identified genetic variants that may significantly modulate breast cancer susceptibility. However, the precise molecular mechanisms behind these associations remain largely unknown; often, it is not even clear whether the GWAS variants are functional themselves or just genetically linked to other functional variants. We here provide an integrated method for identifying functional regulatory variants associated with breast cancer and their target genes by combining the analyses of expression quantitative trait loci (eQTL), a modified version of allele-specific expression (ASE) systematically utilizing haplotype information, transcription factor (TF) binding preference, and epigenetic information. Application of our method to the breast cancer susceptibility region in 5p12 demonstrates that the GWAS risk allele rs4415084-T is correlated with higher expression levels of the protein-coding gene MRPS30 and lncRNA RP11-53O19.1. We propose that an intergenic SNP, in linkage disequilibrium (LD) with the GWAS SNP rs4415084, is the predicted functional SNP. We provide multiple levels of evidence that the risk allele of the predicted functional SNP, in phase with the GWAS risk allele rs4415084-T, creates a GATA3 binding motif within a regulatory element, resulting in differential GATA3 binding and chromatin accessibility, which thereby promote the transcription of MRPS30 and RP11-53O19.1. MRPS30 encodes a member of the mitochondrial ribosomal proteins, implicating the risk SNP's role in modulating mitochondrial activities in breast cancer. Our computational framework can be extended to facilitate the rapid functional characterization of other genetic variants modulating cancer susceptibility and provides an effective way of integrating GWAS results with high-throughput genomic and epigenomic data.

#1221

Chronic lymphocytic leukemia risk variants are associated with alteration of epigenetic states in regulatory regions.

Huihuang Yan, Shulan Tian, Geffen Kleinstern, Susan Slager. _Mayo Clinic, Rochester, MN_.

Chronic lymphocytic leukemia risk variants are associated with alteration of epigenetic state in regulatory regions The vast majority of disease-associated single-nucleotide polymorphisms (SNPs) identified by genome-wide association studies (GWASs) reside in noncoding genomic regions. Functional impact of noncoding casual variants on target gene expression often involves regulatory mechanisms, typically through altering local chromatin accessibility, histone modifications, transcription factor (TF) binding affinity and chromatin interactions. In chronic lymphocytic leukemia (CLL), we and others have identified 41 GWAS risk variants, whose mechanism of action in CLL etiology remains largely unknown. In this study, we aim to decipher the regulatory potential of these variants and to better understand the mechanisms contributing to dysregulation of genes implicated in CLL pathogenesis. We performed ChIP-seq for H3K4me3, H3K4me1, H3K27ac and H3K27me3 in CLL. In addition, we also analyzed over 250 public ChIP-seq, ATAC-seq, DNase-seq, FAIRE-seq, and chromatin interaction data in CLL, B cells and/or lymphoblastoid cell lines. Comparing the 41 index SNPs and the nearby SNPs they associated with in linkage disequilibrium (R2 at least 0.5 in the EUR ethnic group in 1000 Genomes Project) to the above epigenetic data sets revealed overlaps with regulatory regions in 38 of the risk loci, with 28 of the loci overlapping super-enhancers. We used two approaches to elucidate the mechanisms of potential SNP causality. First, we used GATK UnifiedGenotyper to infer genotype for the SNPs that overlapped open chromatin regions and ChIP-seq peaks, by which evidence of imbalance between reference and alternative allele was identified at heterozygous sites. Second, for SNPs located within open chromatin regions or ChIP-seq peaks, we used FIMO program to scan the 100bp sequences centered on the SNPs for significant matches (p=1e-05) to the position weight matrices in five TF binding motif databases; SNPs that had TF motifs altered by the alternative alleles were retained. Together, these two approaches revealed evidence for allele imbalance or TF binding motif alterations at 36 of the 38 CLL risk loci, suggesting the generality of epigenetic regulation in CLL pathogenesis. Further analysis of chromatin interaction, gene expression and expression quantitative trait loci data will likely link the casual SNPs to their target genes.

#1222

Functional single-nucleotide polymorphisms in the TGFb and TGFbRI gene and their association with prostate cancer in Western Mexicans.

Abril Martínez-Rizo,1 Brenda González-Cordova,1 Christopher Peña-George,1 Cuerpo Académico de Investigación Bioquímica (CA-UAN-266), José Navarro-Partida,1 Martha González-Carrillo,2 Jackeline Arvizu-Gómez,1 Adriana Álvarez-Rodríguez3. 1 _Universidad Autónoma de Nayarit, Tepic, Mexico;_ 2 _Instituto Mexicano del Seguro Social, Delegación Nayarit, Tepic, Mexico;_ 3 _Universidad de Guadalajara, Guadalajara, Jalisco, Mexico_.

Background: Prostatic carcinogenesis has been linked to chronic inflammation. Several studies demonstrated the duality of TGFβ during carcinogenesis. This pleiotropic cytokine participates in cell proliferation, migration and survival, epithelial-mesenchymal transition, and apoptosis, among other functions. An increase in its expression is associated with antiproliferative effects, tumor angiogenesis and metastasis of cancer cells. Single-nucleotide polymorphisms (SNPs) of the TGFB1 and TGFBR1 genes have been associated with risk and progression of many cancers.

Aim: To investigate the synergic role T29C (rs1800470) of the TGFβ gene and TGFBR1 rs334348 polymorphisms in risk, progression and severity of prostate cancer.

Materials and Methods: The present cases-control study includes 211 patients with PCa, and as controls 297 patients with benign prostatic hyperplasia (BPH) were obtained. The functional polymorphisms rs1800470 of the TGFβ gene and TGFBR1 rs334348 were detected by real-time PCR using the TaqMan probe system, according to the manufacturer's amplification recommendations. Hardy Weinberg Equilibrium was calculated by chi-square (χ2) test. Odds ratios (ORs) and 95% confidence intervals (95% CIs) for each SNP were calculated. The haplotypes were analyzed using the Haploview program.

Results: The genotype frequencies of the SNPs analyzed in this work were no significant deviation from Hardy-Weinberg Equilibrium. Chi-square analysis established that there was no significant association between prostate cancer risk and TGFβ or TGFBR1 genotypes and alleles. Neither of the polymorphisms was associated with progression or severity of prostate cancer. Furthermore, the GT haplotype revealed protection for development of prostate cancer (OR=0.57, 95% CI (0.36-0.89) p=0.01).

Conclusion: To our knowledge, this research is the first genetic association study to show that GT haplotype confers protection in patients from Western Mexico with PCa.

#1223

Significance of GSTM1 and GSTT1 gene polymorphism to breast cancer susceptibility in Nigerian women.

Olubanke O. Ogunlana,1 Omaghomi Ortiseweyinmi,1 Sadiq Ajoke,1 Omolara Fatiregun,2 Solomon O. Rotimi,1 Bose E. Adegboye,1 Emeka E. Iweala,1 Angie O. Igbinoba-Adebayo1. 1 _Covenant University, Ota, Nigeria;_ 2 _Lagos State University Teaching Hospital, Ikeja, Lagos, Nigeria_.

Human glutathione-S-transferases play a key role in the metabolism of drugs and environmental chemicals. There have been conflicting reports on the association of breast cancer susceptibility with null genotypes of glutathione-S-transferase (GST) classes of mu and theta (GSTM1 and GSTT1). However, this is the first report of the association of null genotypes of GSTs with breast cancer patients from Nigerian population. By multiplex PCR, we examined the null genotypes of GSTM1 and GSTT1 in relation to breast cancer risk in Nigerian women. The case-control study included 56 clinically diagnosed breast cancer patients and age-matched control participants. Odds ratio (OR) and 95% confidence interval (Cl) from conditional logistic regression model were used to estimate the association between GSTM1 and GSTT1 subtypes and breast cancer risk. The frequencies of GSTM1 and GSTT1 null genotypes in breast cancer (BC) patients differed from healthy controls (HC) (61% in BC vs. 39% in HC and 66% in BC vs. 34% in HC for GSTM1 and GSTT1 respectively). GSTM1 and GSTT1 null genotypes and their combinations were associated with increased breast cancer risk [OR = 3.06 (95% Cl 0.98-9.48)], [OR = 14.06 (95% Cl 3.02-70.6)] and [OR = 6.98 (95% Cl = 0.11-16.5)] respectively. The study showed an increased breast cancer risk in patients with GSTT1 homozygous gene deletions with relative risk (RR) value of 5.6 than those with GSTM1 (RR = 1.77). In conclusion, the data from our work provide evidence of increased risk of breast cancer associated with GSTM1 and GSTT1 homozygous gene deletions in women from Nigeria.

#1224

Deep targeted tumor sequencing of colorectal cancer cases to study associations of molecular subtypes with clinical, genetic, and lifestyle risk factors.

Syed Zaidi,1 Amanda Phipps,2 Tabitha Harrison,2 Catherine Grasso,3 Robert Steinfelder,2 Quang Trinh,1 Charles Connolly,2 Barbara Banbury,2 Adilya Rafikova,1 Philipp Hofer,4 Stefanie Brezina,4 Marios Giannakis,5 Xinmeng Jasmine Mu,6 Michael Quist,2 Charles Fuchs,7 Levi Garraway,5 Li Hsu,2 Lincoln Stein,1 Andrea Gsur,4 Shuji Ogino,8 Steven Gallinger,9 Polly Newcomb,2 Peter Campbell,10 Wei Sun,2 Thomas Hudson,1 Ulrike Peters2. 1 _Ontario Institute for Cancer Research, Toronto, Ontario, Canada;_ 2 _Fred Hutchinson Cancer Research Center, Seattle, WA;_ 3 _University of California, Los Angeles, CA;_ 4 _Medical University of Vienna, Vienna, Austria;_ 5 _Dana Farber Cancer Institute, Boston, MA;_ 6 _Broad Institute of MIT and Harvard, Cambridge, MA;_ 7 _Yale Cancer Center, New Haven, CT;_ 8 _Harvard Medical School, Boston, MA;_ 9 _Mount Sinai Hospital, Toronto, Ontario, Canada;_ 10 _American Cancer Society, Atlanta, GA_.

Colorectal cancer (CRC), a common malignancy, is a biologically heterogeneous disease. Next-generation sequencing (NGS) has enabled CRC characterization by identifying somatically mutated genes which now allow us to better define colorectal tumor subtypes (e.g. by mutated pathways). However, the relationship of such CRC subtypes to patient survival and genetic and lifestyle risk factors has not been comprehensively studied.

To identify somatic mutations in CRC cases, we designed a targeted AmpliSeq panel of CRC related genes and genomic regions informed by whole exome sequencing data from ~1,200 CRC cases. The sequencing was conducted on Illumina HiSeq 2500 with a mean coverage of 740x and 240x for DNA extracted from FFPE tumor tissues and matched normal samples, respectively. Strelka, MuTect, VarDict, and Varscan2 were used to identify somatic single nucleotide variants and indels. Sanger sequencing was performed to validate a subset of variants. To date, we have sequenced ~2,400 CRC tumors and matched control tissues from four studies participating in the Colon Cancer Family Registry (CCFR) and Genetics and Epidemiology of Colorectal Cancer Consortium (GECCO). In most tumors, we identified non-silent mutations in genes belonging to the WNT (77%), p53 (44%), IGF2/PI3K (22%), RTK-RAS (47%), and TGF-beta (26%) signaling pathways. Among the 15% of tumors that could be classified as hypermutated, based on the number of mutations, 39% exhibited non-silent mutations in MLH1, MLH3, MSH2, MSH6, and PMS2 and 41% exhibited non-silent mutations in POLE and POLD1.

In a subset of studies with available survival data, we used Cox regression to assess the association of hypermutation status and the presence of non-silencing mutations in key signaling pathways with overall (OS) and disease-specific (DSS) survival. OS and DSS were significantly more favorable in cases with hypermutated vs. non-hypermutated CRC (HR=0.77, 95% CI: 0.60-0.98, p=0.04 and HR=0.35, 95% CI: 0.22-0.57, p=2x10-5, respectively); these associations were most pronounced for POLE/POLD1 mutated hypermutated CRC (HR=0.69, 95% CI: 0.46-1.02, p=0.06, HR=0.21, 95% CI: 0.08-0.56, p=2x10-3, respectively). There was no significant association of mutations in WNT, p53, IGF2/PI3K, RTK-RAS, or TGF-beta pathways with survival (p>0.05).

The comprehensive molecular characterization of this large panel of CRC cases will support further studies of molecular subtypes of CRC with clinical, lifestyle, and environmental factors. A better understanding of molecular mechanisms of CRC will be valuable in developing strategies for prevention, diagnosis, and treatment of this life-threatening disease.

#1225

Rare variants in DNA damage repair genes are associated with male breast cancer predisposition.

Sarah L. Maguire,1 Katarzyna Tomczyk,2 Eleni Perrakis,2 Edward Saunders,2 Daniel Leongamornlert,2 ZSofia Kote-Jarai,2 Rosalind Eeles,2 Montserrat Garcia-Closas,3 Paul Pharoah,4 Douglas Easton,4 Christopher J. Lord,2 Alan Ashworth,5 Anthony Swerdlow,2 Nick Orr1. 1 _Queen's University Belfast, Belfast, United Kingdom;_ 2 _Institute of Cancer Research, London, United Kingdom;_ 3 _National Cancer Institute, Rockville, MD;_ 4 _University of Cambridge, Cambridge, United Kingdom;_ 5 _UCSF, San Francisco, CA_.

Male breast cancer accounts for approximately 1% of all breast cancers diagnosed each year in the UK. Germline mutations in DNA damage response genes are known to be associated with many forms of cancer, including breast cancer. In order to identify low frequency male breast cancer predisposition genes, we performed targeted resequencing of more than 300 genes that were selected on the basis of involvement in DNA maintenance and repair pathways. Using a cost-effective protocol based on an automated library preparation method and a multiplexed hybridization enrichment step, we sequenced 1,029 male breast cancer cases from the UK Breast Cancer Now male breast cancer study (MBCS) and 1,254 controls from the MBCS and the ICR1000 UK exome series. Rare variants localising to 19 genes were significantly associated with risk of male breast cancer. Genes with an excess of rare coding mutations included the known breast cancer susceptibility genes BRCA2, CHEK2 and ATM as well as multiple members of the Fanconi anaemia (FA) pathway. Loss of heterozygosity (LOH) affecting the wildtype allele of putative predisposition genes was observed more than 10% of cases with matched tumor samples. In addition to LOH in BRCA2 mutation carries, somatic losses were observed that affected CCNE1, ATM, PALB2, BARD1, CHEK2, POLL and RAD9A. These data represent the largest analysis of rare variants in male breast cancer to date and have enabled the identification of known and putative novel susceptibility genes.

#1226

Rare germline variants segregating in chronic lymphocytic leukemia (CLL) families.

Alyssa I. Clay-Gilmour,1 Daniel R. O'Brien,1 Sara J. Achenbach,1 Celine M. Vachon,1 Kari G. Chaffee,1 Timothy G. Call,1 Jose F. Leis,2 Aaron D. Norman,1 Brian F. Kabat,1 Sameer A. Parikh,1 Neil E. Kay,1 Esteban Braggio,2 James R. Cerhan,1 Susan L. Slager1. 1 _Mayo Clinic, Rochester, MN;_ 2 _Mayo Clinic, Phoenix, AZ_.

CLL is a highly heritable cancer with first degree relatives of CLL cases having a 7.5-fold increased CLL risk. Genome-wide association studies (GWAS) and linkage studies have been performed to study inherited predisposition; however a larger proportion of heritability to CLL remains unexplained. Rare coding variants might account for the missing heritability information. Inherited loss of function variants in shelterin complex genes (POT1, ACD, TERF1, TINF2, TERF2, TERF2IP- involved in telomere regulation), CDK1 (critical for cell division) and ATM (tumor suppressor gene) have been found to co-segregate in CLL families and be enriched in CLL cases using exome-wide sequencing data. Our study evaluates rare germline variants from these suspect genes segregating in CLL families who are followed at the Mayo Clinic.

Using whole exome sequencing (WES), we sequenced 93 CLL families with at least 2 reported CLL cases consisting of 443 individuals: 160 with CLL, 73 with monoclonal B-cell lymphocytosis (MBL), and 210 relatives. DNA was extracted from buccal cells, coding exons were selectively captured using Agilent 50Mb and SureSelect Human All Exon V4 capture kits; sequencing was performed using Illumina HiSeq 2000. Mayo Clinic's DNASeq pipeline uses Novoalign (initial read alignment), Picard (marking duplicate reads), and the Genome Analysis Toolkit (GATK) for local realignment, recalibration, and variant calling. The variant discovery step leverages GATK's HaplotypeCaller in per sample mode and all of the samples across the cohort are jointly genotyped together. All called variants are evaluated with GATK's Variant Quality Score Recalibration tool and annotated for biological relevance (BIOR). Quality control included removing variants that had <75% call rate across the two capture kits, <8x coverage, or phred score<10, resulting in 317,666 remaining variants. Of these, over 80% of the coding sequence had a median read depth of 23 reads. In our pedigrees, we searched for rare variants within the genes described above. We identified suspect variants with the following criteria: 1) enriched in CLL and MBL samples compared to unaffected samples; 2) multiple affected members with the variant within a family; 3) variants present in all sequenced affecteds within the family; 4) rarely seen in an in-house database of non-cancer controls or 1K Genomes; and 5) predicted to have a functional damaging effect (using SIFT).

We identified three novel rare missense variants, defined as functionally deleterious, which each co-segregated within a CLL family. Specifically, these variants from shelterin complex genes; POT1 (rs116916706), TERF2IP (rs138458227), and TERF2 (rs749171225), met the criteria. This study further highlights telomere dysregulation as a key process in CLL development. Investigating rare variants within CLL pedigrees with WES can help identify germline variants impacting predisposition to familial CLL.

#1227

Somatic genome alterations in cancer as compared to inferred patient ancestry.

Caitlin F. Connelly,1 Jian Carrot-Zhang,2 Philip J. Stephens,1 Garrett M. Frampton1. 1 _Foundation Medicine, Inc., Cambridge, MA;_ 2 _Dana-Farber Cancer Institute, Broad Institute of Harvard and MIT, Cambridge, MA_.

Background

The frequency of targetable somatic genomic alterations is known to differ based on a patient's ancestry; for example, in lung adenocarcinoma EGFR mutations are more common in East Asians than Europeans. Accurately characterizing these differences will help to illuminate differences in the genetic etiology of cancer between populations.

Methods

We inferred estimated population ancestry for more than 100,000 de-identified patients from comprehensive genomic profiling (CGP) of tumor specimens assessed in the course of routine clinical care. More than 40,000 germline single nucleotide polymorphisms (SNPs) are sequenced as part of our CGP assay which were also characterized in the publicly available 1000 Genomes data. We used the 1000 Genomes data to train and validate a classifier using these overlapping SNPs to classify individuals into one of five inferred population groups, estimated to be of predominantly African, European, Central and South American, South Asian, or East Asian ethnic origin. We then performed statistical testing to identify differences in tumor somatic genomic properties between the five inferred ancestry groups for each cancer type.

Results

We found that the inference of population ancestry from tumor sequence data and its comparison to somatic mutations recapitulated known differences between populations. Specifically, in lung adenocarcinoma, EGFR mutations were significantly more common in tumor specimens from individuals with inferred East Asian SNP-inferred ancestry (rate=0.52) compared to individuals with inferred European ancestry (rate=0.17, p < 10-119), and KRAS mutations were more common in tumor specimens from individuals with inferred European ancestry (rate=0.40) than individuals with inferred East Asian ancestry (rate=0.14, p < 10-60). In lung adenocarcinoma, we also found that tumor mutational burden (TMB), a biomarker for immunotherapy response, differed significantly between populations. Tumor specimens from patients with inferred East Asian ancestry had the lowest TMB (4.5 mutations/Mb median, 4.7% >20), then from patients with inferred European ancestry (6.3 median, 12.8% >= 20), and inferred African ancestry the highest (9.0 median, 21.2% >=20). Finally, we found that in glioblastoma, TERT promoter mutations were more common in specimens from individuals with inferred European ancestry (rate=0.69) compared to specimens from individuals with inferred East Asian ancestry (rate=0.44). Based on the statistical power provided by this large cohort, novel ancestry based differences in gene alteration rates will be presented across multiple cancer types.

Conclusions

Clear differences in genomics based on SNP-inferred ancestry were observed. In particular, we found that TMB differs significantly between populations in lung adenocarcinoma, suggesting that the likelihood of individuals benefiting from immunotherapy may differ between populations.

#1228

Comparative genomic analysis of young-onset and late-onset colorectal cancer.

Jingqin Luo,1 Marios Giannakis,2 Graham Colditz,1 Jean Wang,1 William Chapman,1 Adetunji T. Toriola,1 Yoshiko Mito,1 Reiko Nishihara,3 Jonathan Nowak,3 Charles Fuchs,4 Edward Giovannucci,5 Andrew T. Chan,6 Adam Bass,2 Shuji Ogino,2 Ryan Fields,1 Yin Cao1. 1 _Washington University in St. Louis, St Louis, MO;_ 2 _Dana-Farber Cancer Institute, MA;_ 3 _Brigham and Women's Hospital, MA;_ 4 _Yale University, CT;_ 5 _Harvard University, MA;_ 6 _Massachusetts General Hospital, MA_.

Background: Young-onset colorectal cancer, the majority of which are sporadic, may have distinct molecular characteristics and etiologies. However, thus far, the genomic landscape of young-onset CRCs has remained largely uncharacterized. This study aimed to compare somatic mutation differences between young-onset and late-onset CRCs using the AACR GENIE data, a consortium of patients treated at 8 global cancer centers. Methods: Clinical and DNA mutation data on 2,081 CRC samples were downloaded from Sage Bionetworks (Synapse ID: syn7222066). A total of 162 samples from CRC patients who had more than one samples or were likely to have Lynch syndrome were excluded. Young-onset CRCs were defined as cancers arising at age <50 years old while late-onset CRCs were defined as cancers arising at age ≥50 years old. Mutation rate per megabase (Mb) was calculated for each CRC and compared between groups by Wilcoxon rank sum test. The mutation frequency of each gene in young-onset and late-onset CRC was calculated separately for primary CRCs and metastatic CRCs. Mutation frequency differences between young- vs late-onset CRCs were tested by the Fisher's exact test with significance defined at the 5% alpha level. Results: Mutation data on 613 genes were analyzed on 955 primary and 676 metastatic CRCs. Overall, the most frequently mutated genes with a >10% mutation frequency include: TP53 (66.86%), APC (59.46%), KRAS (47.42%), PIK3CA (18.40%), SMAD4 (12.71%) and BRAF (10.06%). The metastatic CRCs were on average slightly older than primary CRCs (mean age=56.96 vs. 55.90, two sample t test P=0.09). Comparing gene mutation between 300 young-onset versus 655 late-onset primary CRCs, mutation rate per Mb was slightly but not significantly higher in the latter (median=3.74 vs. 4.14, P=0.2199). DOT1L (9.82% vs. 1.95%, P=0.0014), IGF1R (6.12% vs. 1.72%, P=0.0057), BRCA1 (5.61% vs. 1.47%, P=0.0069), JUN (6.25% vs. 1.17%, P=0.0104), PTCH1 (7.14% vs. 2.7%, P=0.0151), TSHR (5.36% vs. 1.17%, P=0.0253), EIF1AX (3.06% vs. 0%, P=0.02811), JAK2 (2.0% vs. 0.46%, P=0.0311), EP300 (8.67% vs. 4.41%, P=0.0414), PHOX2B (2.20% vs. 0.27%, P=0.0419), SMC1A (3.57% vs. 0%, P=0.0446) and CSF1R (3.02% vs. 1.03%, P=0.0439) had a significantly higher mutation rate in the young-onset primary CRCs. Meanwhile, KRAS was mutated more frequently in late-onset primary CRCs (44.0% vs. 51.3%, P=0.03679). For metastatic CRCs, NTRK1 was found mutated in 12 (3.80%) out of 316 later-onset metastatic CRCs but none out of 129 young-onset metastatic CRCs (P=0.0223). Meanwhile, CIC was mutated in only 1 out of 103 young-onset metastatic CRCs but 17 (6.44%) out of 264 late-onset metastatic CRCs (P=0.0302) and TP53 had a nearly 8% higher mutation rate in young-onset metastatic CRCs (79.01% vs. 71.19%, P=0.0488). Conclusions: Mutation rates were found significantly higher in many genes among young-onset primary CRCs compared to late-onset primary CRCs.

#1229

Clonal hematopoiesis of indeterminate potential (CHIP), centenarians and age-related cardiovascular risk: Is TET2 the culprit.

Luca Bertamini,1 Claudia Sala,1 Nicola Martinelli,2 Cristina Papayannidis,1 Cristina Giuliani,1 Giovanni Malerba,2 Paolo Garagnani,1 Maria Abbondanza Pantaleo,1 Michele Cavo,1 Oliviero Olivieri,2 Giovanni Martinelli,3 Claudio Franceschi,1 Domenico Girelli2. 1 _University of Bologna, Bologna, Italy;_ 2 _University of Verona, Verona, Italy;_ 3 _Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, Meldola, Italy_.

Background. Clonal hematopoiesis of indeterminate potential (CHIP) is an age-related hematological condition, that has been associated with an increased risk of hematological malignancies and of overall mortality. This excess mortality in subjects with CHIP seems to be related to an increased risk of cardiovascular atherosclerotic diseases (Jaiswal S, NEJM 2014). Our aim was to analyze the prevalence of CHIP in patients with Coronary Artery Disease (CAD) and in a group of selected ultra-centenarians ((age >105 years), since no data hasve been produced until now in such a peculiar population of "super-controls" (Garagnani P, Aging 2013).

Methods. We performed whole-exome sequencing (WES) from peripheral-blood cells DNA in 99 patients with angiographically proven severe CAD from the Verona Heart Study (Girelli D, NEJM 2000;), and 79 semisupercentenarians (age >105 years). The mean coverage varied from 30x to 100x (higher in centenarians). We analyzed somatic mutations predicted to alter function in 6 key genes (TET2, ASXL1, DNMT3A, JAK2, PPM1D, TP53), selected as the most frequently detected in CHIP according to literature.

Results. The prevalence of CHIP in CAD patients was 18.2%, similarly to the increased prevalence detected in other CAD populations (Jaiswal S, NEJM 2017). On the other hand, CHIP was rare in centenarians (prevalence of 2.5%), a result that contradicts the expected exponential increase of CHIP observed after the age of 70 years in previous studies. This result suggested a kind of "survival bias", that indirectly supports the pathogenic role of age-related CHIP. An utterly fascinating finding was in the frequency of driver mutations in CAD subjects with CHIP: the majority (85%) of CAD patients presented mutations in TET2.

Conclusions. Our outcomes add further insights to the nature of CHIP, which could be seen as a preleukemic condition that also leads to an intrinsic odd of cardiovascular events. The Our outcomes add further insights to the recent hypothesis that links CHIP to an increased risk of cardiovascular disease. The study results raise the question over a possible many-sided clinical and biological significance of CHIP that might depend on the driver mutation. In particular, we propose that TET2-driven CHIP is the most responsible for the higher cardiovascular risk, consistently with the pro-atherosclerotic role of this gene in mouse models (Fuster JJ, Science 2017, Jaiswal S, NEJM 2017).

#1230

**Association of common germline variants with** TMPRSS2-ERG **gene fusion status in prostate cancer.**

Indu Kohaar,1 Yongmei Chen,1 Lakshmi Ravindranath,1 Denise Young,1 Amina Ali,1 Qiyuan Li,2 Albert Dobi,1 Inger L. Rosner,1 Isabell Sesterhenn,3 Jennifer Cullen,1 Matthew Freedman,4 Shiv Srivastava,1 Gyorgy Petrovics1. 1 _Center for Prostate Disease Research, Department of Surgery, Uniformed Services University and the Walter Reed National Military Medical Center, Rockville, MD;_ 2 _Xiamen University, Xiamen, China;_ 3 _Joint Pathology Center, Rockville, MD;_ 4 _Dana-Farber Cancer Institute, Boston, MA_.

Introduction and Objectives: Oncogenic activation of ERG resulting from prevalent gene fusions (predominantly as TMPRSS2-ERG) is a key driver event in prostate cancer (CaP) pathogenesis. Our laboratory and others have recently reported that major cancer driver genes, including ERG, show significant racial/ethnic differences in CaP. It is present in two thirds of CaP patients of European Ancestry including Caucasian Americans (CA) but is present at lower frequencies in African Americans (AA), Africans and Asians. Racial differences of CaP associated SNPs have also been extensively described. However, there is limited data on germline association with ERG fusion status. The goal of this study is to identify germline molecular determinants associating with ERG status of CaP. Methods: Blood derived genomic DNA samples were prepared from 270 AA men and 130 CA men treated by radical prostatectomy. ERG status was determined by immuno-histochemistry (IHC) for ERG protein expression. SNP genotyping was performed on the Illumina Golden Gate platform using Infinium Oncoarray SNP chip. Data analysis approaches included association analyses based on EMMAX and imputation analysis by IMPUTE2. SNP genotyping was performed using droplet digital polymerase chain reaction (ddPCR) approach Results: SNP genotyping analysis was performed in 321 patients with 478,299 SNPs. We identified SNPs associated with ERG status using EMMAX analysis. The SNPs most significantly (p <10-5) associated with ERG fusion status included rs6698333, an intron variant of Kruppel-like factor 17 (KLF17) and two SNPs (rs1889877, rs3798999) in the intron of adhesion G protein-coupled receptor B3 (ADGRB3). 4 SNPs (rs10215144, rs3818136, rs9380660 and rs1792695) were found to be significantly (p <10-5) associated with ERG positive phenotype under any tumor foci positive for the fusion. Fine-mapping of SNPs by genotype imputation analysis (IMPUTE2) using the 1000 Genomes reference dataset, found rs34349373 and rs2055272 to be significantly associated (p <10-7). The 2 variants were found to be in strong linkage disequilibrium (LD) in both CA and AA populations with r2 of 1.0 and 0.91 respectively. Imputed SNP rs2055272 was further experimentally evaluated by Taqman based ddPCR SNP genotyping approach. Concordance between Taqman genotypes and imputed genotypes was found to be 98.04%. Association analysis of the SNPs with clinico-pathological features of CaP and functional annotation of the significant SNPs by in silico eQTL based analysis are being performed. Conclusions: This study identified SNPs associated with ERG status of CaP, a major driver oncogene in CaP. Although the biological significance as it relates to ERG status of CaP still needs to be determined, these SNPs, with independent validation, may help as markers in stratifying patients early (even before CaP is detected) for targeted prevention and treatment options.

#1231

Using oral and lung microbiome to assess microbial dysbiosis and inflammatory response to electronic cigarettes and to cigarettes.

Kevin L. Ying,1 Min-Ae Song,1 Daniel Y. Weng,1 Quentin A. Nickerson,1 Joseph P. McElroy,1 David Frankhouser,1 Pearlly S. Yan,1 Ralf Bundschuh,1 Theodore M. Brasky,1 Mark D. Wewers,1 Ewy Mathé,1 Jo L. Freudenheim,2 Peter G. Shields1. 1 _Ohio State Univ., Columbus, OH;_ 2 _University of Buffalo, Buffalo, NY_.

Background: Alteration of the oral microbiome (microbial dysbiosis) with cigarette smoking is well established. However, the effect of electronic cigarettes (e-cigs) use on the oral microbiome is unknown, although there are emerging data that e-cigs induce microbial changes similar to smoking. In smoking-related diseases, such as chronic obstructive pulmonary disease, there are changes in the oral microbiome and in the expression of genes involved in inflammatory pathways. Similar to the oral microbiome, it is feasible that smoking tobacco and e-cig use could also affect the lung microbiome. To the best of our knowledge, there is only one published study investigating smoking tobacco effects on the oral and lung microbiome. No published studies have evaluated concurrent effects of e-cigs in the oral and lung microbiome.

Aims: We hypothesize that microbial dysbiosis and expression of inflammatory cytokines in the oral cavity and lung will differ between smokers and nonsmokers, and that e-cig users will have microbial dysbiosis more similar to smokers. To accomplish this, we propose 1) to examine the association of oral and lung microbiome in nonsmokers, smokers and e-cig users, 2) to determine if the oral microbiome and the lung microbiome differ among these groups, and 3) to determine correlation of the microbiota with host expression of inflammation-related genes.

Methods: A cross-sectional study using bronchoscopy and oral rinse collection of 10 never-smokers, 8 cigarette smokers, and 10 e-cig users was conducted. For each study participant, RNA was extracted from saliva and bronchoalveolar lavage (BAL) samples for total transcriptome analysis using RNA-seq; facilitating this approach allows measurement of bacterial communities and human inflammatory cytokine expression in the same assay. To determine microbial dysbiosis by smoking status, the Mann Whitney U-test and Kruskal-Wallis H-test were used with Bonferroni correction for multiple comparisons. Both effect size (fold change >1.5) and adjusted p-value cutoffs (<0.05) were used to identify statistical significance.

Results: In preliminary analyses we identified 2,257 bacterial strains in saliva samples and 1592 in BAL samples. We found a lack of concordance of highly abundant bacteria in the oral cavity and lungs. The top twenty expressed human genes were associated with RNA splicing, RNA elongation and miRNAs. Comparisons of microbial dysbiosis by smoking status are currently under way.

Conclusion: The composition of the microbiome for saliva is different from that of BAL. Comparison of the metatranscriptome and transcriptome between the lung and oral cavity, as well as between smokers, nonsmokers and e-cigarette users, will allow us to observe how e-cig use compares with cigarette smoking and never smoking in terms of microbial dysbiosis and inflammatory cytokines.

#1232

**Familial mesothelioma: Beyond** BAP1 **.**

Mitchell Cheung,1 Jill A. Ohar,2 Suzanne E. Howard,2 Timothy D. Howard,2 Mary Hesdorffer,3 Joseph R. Testa1. 1 _Fox Chase Cancer Ctr., Philadelphia, PA;_ 2 _Wake Forest University School of Medicine, Winston-Salem, NC;_ 3 _Mesothelioma Applied Research Foundation, Alexandria, VA_.

BAP1 germline mutations are associated with some, but not all, familial malignant mesotheliomas. We sought to identify new genetic risk factors from a large cohort of malignant mesotheliomas cases with a family history of cancers but no germline BAP1 mutation. Whole genome sequencing was performed on 15 samples (14 peripheral blood and 1 MM sample). Promising mutated candidate genes were identified in 12 of 14 BAP1 mutation-negative cases, with 3 different candidate genes mutated in 3 cases, 2 different candidates mutated in 5 cases, and 1 candidate mutated in 4 cases. Candidate genes encoded proteins involved in DNA damage repair, DNA polymerase, DNA and histone methytransferase, as well as mitotic control, helicase, and kinase factors. All mutations were confirmed by Sanger sequencing. Mutations included in/dels predicting frameshifts (4), splice site mutations (6), and missense mutations (14). Notably, in addition to being BAP1 mutation-negative, germline mutations were not identified in any of the genes known to be commonly mutated somatically in malignant mesothelioma, i.e., CDKN2A, NF2, TP53, SETD2, or PBRM1. Sequencing of blood and/or tumor DNA from several available affected members of these families validated co-segregation of mutations in candidate genes with the development of various cancers. Malignant mesothelioma specimens from two members of one family showed somatic loss of heterozygosity of the wild-type allele, indicating biallelic inactivation of the candidate gene—a suspected tumor suppressor locus—in these tumors.

#1233

Reducing sequence artifact in clinical sequencing of treatment-naïve NSCLC patient using molecular barcoding system.

Kei Namba, Shuta Tomida, Yuta Takahashi, Eisuke Kurihara, Yusuke Ogoshi, Takahiro Yosioka, Hidejiro Torigoe, Hiroki Sato, Kazuhiko Shien, Hiromasa Yamamoto, Junichi Soh, Shinichi Toyooka. _Okayama Univ. Graduate School of Medicine, Okayama, Japan_.

Background: Recently the clinical significance of compound mutations in cancer relapse and/or acquired resistance has been reported. Amplicon-based deep sequence enables analysis of tiny amount of input DNA, however, a major problem of these high-throughput DNA sequencing is the high rate (~1%) of errors causing potential sequence artifact. Molecular barcoding system, which has been developed to reduce sequencing artifacts as well as to improve mutation detection accuracy, was applied to analyze the frequency of compound EGFR mutations in early stage non-small-cell lung cancer (NSCLC) patients who undergo surgical resection without any presurgical treatment is less described.

Materials and Methods: From 590 consecutive patients, 64 adenocarcinoma cases who underwent surgical resection for primary lung cancer were analyzed by using amplicon-based targeted sequencing method incorporating molecular barcodes in order to detect genetic alterations of 47 genes including EGFR.

Results: Out of 64 samples, EGFR common mutation profiles of 63 (98.4%) by molecular-barcode sequencing corresponded to those by clinical test. Uncommon EGFR mutations were detected in 7 cases (10.9%). Among the three types of EGFR major mutation, G719X (60%, 3/5) showed a significantly higher incidence of EGFR double mutations than L858R (9.5%, 4/42) or Ex19del (0%, 0/17) (p = 0.0052). Co-mutations of other genes were observed in 20 EGFR-mutated cases. TP53 mutations were frequently detected in younger age (p = 0.0066) and pStage II-III cases (p = 0.042).

Conclusion: Amplicon sequencing incorporating molecular barcoding system is a feasible approach to characterize predictive or prognostic mutations in early stage treatment naïve NSCLC patients, revealing those case who harbor EGFR G719X mutation have a significantly higher incidence of EGFR double mutations, likely to have worse prognosis.

#1234

Somatic mutations of FAT1 in oral cancer are associated with tumor progression and survival.

Chung Ji Liu,1 Shu-Chun Lin,2 Li-Han Lin1. 1 _Mackay Memorial Hospital, Taipei, Taiwan;_ 2 _National Yang Ming University, Taipei, Taiwan_.

In recent years, the incidence and mortality rates of oral squamous cell carcinoma (OSCC) have increased worldwide. Therefore, understanding genomic alterations in OSCC carcinogenesis is crucial for appropriate diagnosis and therapy. Procadherin FAT1, which encodes 4588 amino acid residues, regulates complex mechanisms to promote oncogenesis or suppression of malignancies. Multiplex polymerase chain reaction (PCR)-based next-generation sequencing (NGS) revealed FAT1 somatic mutations. The clinicopathologic implications of FAT1 in OSCC were investigated using expression assays, and the functional role of FAT1 in HNSCC pathogenesis was determined using ectopic expression and knockdown experiments. Approximately 29% of patients with OSCC harbored damaging FAT1 mutations. Each type of mutation (missense, nonsense, and frameshift mutations) accounted for nearly one-third of deleterious mutations. FAT1 mutations correlated with lower FAT1 expression in tumors. The knockdown of the endogenous expression of FAT1 and exogenous expression of crucial FAT1 domains unequivocally indicated that FAT1 suppressed the migration and invasion capability of OSCC cells. Functional analysis suggested that nonsense mutations in FAT1 result in the loss of the suppression of tumor progression. FAT1 mutations and downregulation defined nodal involvement, lymphovascular permeation, and tumor recurrence. In addition, FAT1 mutations and downregulation are independent predictors of poor disease-free survival in patients with HNSCC. This study is the first to perform multiplex PCR-based NGS to indicate marked nonsynonymous FAT1 mutations in OSCC, which are prognostic indicators. The gene analysis strategy proposed for detecting FAT1 mutations may be a valid method for mutation screening.

#1235

Repeated whole-exome sequencing for cell-free tumor DNA profiling of colon cancer patients: Searching for mechanisms of acquired chemoresistance.

Veronika Vymetalkova,1 Barbara Pardini,2 Katerina Jiraskova,1 Marjketa Urbanova,1 Miroslav Levy,3 Veronika Veskrnova,3 Pavel Vodicka,1 Alessio Naccarati2. 1 _Institute of Experimental Medicine, Prague, Czech Republic;_ 2 _IIIGM Italian Institute for Genomic Medicine, Torino, Italy;_ 3 _Thomayer Hospital, Prague, Czech Republic_.

Colorectal cancer (CRC) is a worldwide health burden with nearly 1.2 million new cases expected each year globally. When CRC is identified early, there is a five-year survival rate of about 90% but it drops to almost 12% once there are distant metastases. Drug resistance is a limiting factor of the efficacy of chemotherapy in CRC treatment. Significant efforts are currently being employed to discriminate patients who will benefit or not from chemotherapy. In fact, there is no current 'gold standard' to differentiate responders from non-responders. In the present work, we focused on good responders (those who benefit from the chemotherapy, not having side effects like toxicity, no relapses and having a complete response with no residual cancer) and non-/bad responders (patients with acquired chemoresistance and lack any response, or developing toxicity). The main aim was to identify markers/mutations in plasma cell-free DNA that could be associated with good/bad responders and chemoresistance. Paired plasma samples from colon cancer patients collected before and after therapy and the relative cell-free tumor DNA (ctDNA) was profiled by a whole exome sequencing approach in order to identify novel acquired mutations. Plasma ctDNA was isolated by QIAamp Circulating Nucleic Acid Kit. The SureSelectXT HS Reagent Kit was used for library preparation and libraries were sequenced by the HiSeq2500 system. Analysis was performed on nine patients (stage II and III) treated with the same 5-FU based therapy. The first sampling was collected at the time of diagnosis (i.e., active disease), and the second after 6-9 months depending on patient` s conditions (i.e., covering the tumor resection, administration of adjuvant chemotherapy). After follow up, three patients were classified as good responders and six as non/poor responders. The bioinformatics analysis is currently running. The number of total reads exceeded 990x106. Total length of all exons was 221,924,089bp. After trimming, the average read length was 113bp. Alignment to human reference genome (hg38) is > 99.8%. Coverage of exons ranges from 17 to 34. Results of the study will be presented during the meeting.

Supported by grant AZV MZ 17-30920A, 15-26535A and GACR 17-16857S.

#1236

**Insight into genetic susceptibility to** BRCA **-negative male breast cancer by multigene panel testing: Results from a multicenter study in Italy.**

Piera Rizzolo,1 Veronica Zelli,1 Valentina Silvestri,1 Virginia Valentini,1 Alessandro Spinelli,2 Maria Grazia Tibiletti,3 Antonio Russo,4 Liliana Varesco,5 Giuseppe Giannini,1 Daniele Calistri,6 Laura Cortesi,7 Alessandra Viel,8 Marco Montagna,9 Paolo Peterlongo,10 Paolo Radice,11 Domenico Palli,12 Laura Ottini1. 1 _Sapienza University of Rome, Rome, Italy;_ 2 _IRCCS-Burlo, Trieste, Italy;_ 3 _Circolo Hospital, Varese, Italy;_ 4 _University of Palermo, Palermo, Italy;_ 5 _IRCCS AOU San Martino – IST, Genova, Italy;_ 6 _IRST- Meldola, Forlì, Italy;_ 7 _University of Modena and Reggio Emilia, Modena, Italy;_ 8 _CRO Aviano, National Cancer Institute, Aviano, Italy;_ 9 _Veneto Institute of Oncology IOV - IRCCS, Padova, Italy;_ 10 _IFOM, FIRC, Milano, Italy;_ 11 _IRCCS-INT, Milano, Italy;_ 12 _ISPO, Firenze, Italy_.

Background: Male breast cancer (MBC) is a rare disease, representing less than 1% of all breast cancers (BC). Although rare, MBC remains a substantial cause for morbidity and mortality in men. MBC etiology appears to be largely associated with genetic factors. Inherited mutations in BRCA2, PALB2 and BRCA1 genes, predispose to MBC and account for about 15% of all cases. Thus, a fraction of MBC cases remains to be assigned to specific genetic factors. Here, we aimed at investigating the genetic component of BRCA1/2 mutation negative MBC cases and at identifying germline mutations that could further explain MBC genetic susceptibility.

Materials and methods: We performed a genomic screening of a well-characterized series of 502 BRCA1/2 mutation negative Italian MBC cases by a comprehensive multi-gene custom panel of 50 cancer-related genes, using MiniSeq platform (Illumina). We also compared the main clinical-pathologic characteristics of MBCs in mutation carriers and non-carriers using Fisher exact test and t-test, where appropriate. All statistical analyses were performed with the R software (www.r-project.org).

Results: Overall 6% of BRCA1/2 mutation negative MBC cases were found to carry a pathogenic variant in the genes analyzed. In particular, PALB2 and ATM were the most frequently altered genes (1.1% and 0.7%, respectively). Mutations in known/proposed BC genes, such as BARD1, BLM, BRIP1, CASP8, CHEK2, FANCM, NBN, NF1, RAD50, RAD51C and RAD51D, as well as in genes considered not closely related to BC predisposition, such as APC, EPCAM and MUTYH, were also identified. Intriguingly, the same NBN mutation was identified in three unrelated MBC cases. Mutation carriers were more likely to have a personal history of cancer in addition to MBC (p=0.0045) and family history of cancer other than breast and ovarian cancer (p=0.0004).

Conclusions: This study may help to gain more insight into the genetic susceptibility of MBC. Our results support the central role of PALB2 in MBC susceptibility, point to a relevant role of ATM and confirm a low impact of CHEK2 mutations on MBC predisposition in the Italian population. Considering that pathogenic mutations were found only in a fraction of the genes analyzed, our data indicate that the identification of the more appropriate genes for the genomic screening of MBC cases is essential in order to develop a comprehensive and specific MBC susceptibility panel with implications for clinical management and counselling of patients and their families. Moreover, our results suggest that multigene testing approach may benefit appropriately selected patients, especially those with a personal or family history of cancer other than breast/ovarian cancer.

Study supported by AIRC (IG 16933) to L.O.

#1237

Cancer risk in myotonic dystrophy type I: First evidence of a role for disease severity.

Rotana Alsaggaf,1 Diane Marie M. St. George,2 Ruth M. Pfeiffer,1 Youjin Wang,1 Min Zhan,2 Kathryn R. Wagner,3 Mark H. Greene,1 Sania Amr,2 Shahinaz M. Gadalla1. 1 _National Cancer Institute, NIH, Rockville, MD;_ 2 _University of Maryland, Baltimore, Baltimore, MD;_ 3 _Kennedy Krieger Institute, Baltimore, MD_.

Background. Myotonic Dystrophy type 1 (DM1) is the first inherited nucleotide repeat disorder found to increase cancer susceptibility. The molecular mechanism underlying DM-related carcinogenesis is unknown. We hypothesized that cancer risk in DM1 is modified by disease severity. In this study, we evaluated the effect of age at DM1 diagnosis, a known proxy for disease severity, on the association between DM1 and cancer. Methods. Utilizing the United Kingdom Clinical Practice Research Datalink from 1988-2016, we identified 927 DM1-affected and 13,085 DM1-free individuals, matched on birth year, gender, clinic, and registration year. Follow-up started at the later of age at DM1 diagnosis/selection, practice registration, or study start date (January 1, 1988). We used Cox regression models to compare the risks of organ-specific cancers for DM1 patients and their matched cohort. Analyses were further stratified by age at DM1 diagnosis (0-10 years: congenital/childhood; 11-40 years: classic; and >40 years: late-onset) to assess effect modification. The baseline hazards were stratified on the matched sets, and models were adjusted for average number of primary care visits per year. Results. Of the 927 DM1 patients, 41 (4%) developed cancer, compared with 708 (5%) of the 13,085 DM1-free cohort (mean age at cancer= 56 versus 61, respectively). No cancers were observed in patients with congenital/childhood DM1 versus 1% of DM1-free subjects (p=0.6). Patients with classic DM1 were at elevated risk of cancers overall (HR=1.8; 95%CI=1.1-2.9); cancers of the thyroid (HR=15.9; 95%CI=2.4-103.6), uterus (HR=26.8; 95%CI=2.3-309.3), and cutaneous melanoma (HR=6.0; 95%CI=1.2-28.8) accounted for the excess. In late-onset DM1 patients, a reduced risk was observed (HR=0.5; 95%CI=0.3-0.9), possibly driven by hematological malignancies (DM1=0 cases, DM1-free=54 cases; p=0.02). Conclusions. Our results suggest that excess cancer risk in DM1 patients occurs mainly in those with the classic form, driven by previously-reported DM1 site-specific cancers. The lack of cancers in congenital/childhood DM1 patients is likely due to competing mortality (mean age at death=38 years). The significant difference in cancer risk between classic and late-onset DM1 patients provides the first evidence that more severe disease increases DM1-related cancer susceptibility. Validation is warranted; if confirmed, these novel findings may guide clinical management and scientific planning for investigating the underlying molecular mechanisms in DM-related carcinogenesis.

#1238

International comparison of cancer risks for Lynch syndrome.

Mark A. Jenkins, Jeanette Reece, Grant Lee, Aung K. Win, International Mismatch Repair Consortium. _University of Melbourne, Melbourne, Australia_.

Aims: The International Mismatch Repair Consortium (IMRC) was formed in 2010 and comprises major worldwide consortia involved in the research and/or clinical treatment of Lynch syndrome (cancer predisposition caused by inherited mutations in mismatch repair (MMR) genes: MLH1, MSH2, MSH6, PMS2 and EPCAM); http://www.sphinx.org.au/imrc. The aim of this study was to determine cancer risks by geographic region.

Methods: For each cancer, we estimated the age-specific cumulative risks (penetrance) and the hazard ratios compared to the average population risk. We used a modified segregation analysis and adjusted for any ascertainment of families given this is an analysis of prospective data. We estimated penetrance by: cancer sex, mismatch repair gene, type of mutation, and geographic region.

Results: 43 investigators representing 53 different sites in 23 countries have submitted pedigree data. In total IMRC consists of 6,436 Lynch syndrome families (North America = 2,482, South America = 101, Europe = 3,307, Asia = 216, Australasia = 330). Of these, 2,214 were MLH1, 2,717 were MSH2, 1,118 were MSH6, 558 were PMS2, and 44 were EPCAM. The total number of distinct variants were 1,270 and the maximum size of families was 333 relatives. Of the distinct MMR variants submitted to the IMRC, 89% were classified as Class 4 (likely pathogenic) or 5 (pathogenic) by the InSiGHT Variant classification criteria (LOVD). Penetrance analyses were conducted only for the pathogenic or likely pathogenic variants. Preliminary analyses suggest that the risk of colorectal cancer to age 70 is highest for carriers in Australasia and North America and lowest for carriers in South America and Asia (Table 1).

Conclusion: Preliminary results suggest that cancer risks for people with Lynch syndrome differ by geographic region, which is consistent with environmental modifiers for the disease and might justify region-specific screening guidelines.

Risk (% to age 70) and hazard ratio (HR) of colorectal cancer by MMR gene and region

---

Gene | Region | HR (95% CI) | Risk Males | Risk Females

MLH1 | Europe | 19 (14-26) | 50% | 32%

MLH1 | Nth America | 32 (26-38) | 61% | 48%

MLH1 | Australasia | 32 (22-46) | 68% | 55%

MLH1 | Sth America | 6 (2-22) | 12% | 10%

MLH1 | Asia | 7 (5-15) | 39% | 24%

MSH2 | Europe | 14 (11-16) | 39% | 24%

MSH2 | Nth America | 33 (28-40) | 61% | 50%

MSH2 | Australasia | 36 (24-54) | 73% | 59%

MSH2 | Sth America | 70 (10-490) | 82% | 75%

MSH2 | Asia | 8 (5-17) | 21% | 15%

#1239

Risk of pancreatic cancer is increased among first-degree relatives of pancreatic cancer probands who have non-O ABO blood type.

Aarti Kolluri,1 Samuel O. Antwi,2 Sarah E. Fagan,3 Kari G. Chaffee,1 Brendan T. Broderick,1 William R. Bamlet,1 Ann L. Oberg,1 Robert R. McWilliams,1 Gloria M. Petersen1. 1 _Mayo Clinic, Rochester, MN;_ 2 _Mayo Clinic, Jacksonville, FL;_ 3 _National Institutes of Health, Bethesda, MD_.

Background: Pancreatic cancer (PC) ranks among the top causes of cancer mortality. Risk stratification is an important step toward prevention and early detection. Prior work has shown that ABO blood type (non-O) and first-degree family history of PC are associated with increased PC risk. Individuals with first-degree family history of PC have a 2- to 3-fold increased risk, but it is unclear if ABO blood type plays a role in the familial aggregation of PC. We investigated PC risk among first-degree relatives (FDRs) of PC probands based on the probands' serologic blood type (O vs non-O).

Methods: A total of 702 probands sequentially enrolled in the Mayo Clinic Pancreas Research Registry from 2000-2016 with serologic ABO blood type provided information on family history of cancer for 5,178 FDRs. FDRs were grouped based on their related probands' blood type as type O (n=242; 1,768 FDRs) vs non-O (n=460; 3,410 FDRs). Standardized incidence ratios (SIRs) and 95% confidence intervals (CIs) were calculated by comparing the number of PC cases observed among FDRs to those expected using data from the Surveillance, Epidemiology and End Results (SEER) Program as the reference population. SIRs for PC risk were calculated separately for FDRs of probands with type O vs FDRs of probands with non-O blood, and stratified by sex of FDRs and relationship to proband (parent, sibling, or offspring).

Results: Compared to the SEER reference, PC risk was nearly 2-fold higher among FDRs of PC probands (SIR=1.76; 95%CI: 1.35-2.26). When stratified by probands' ABO type, risk for PC was higher among FDRs of probands with non-O blood (SIR=1.94; 95%CI: 1.41-2.60), compared to FDRs of probands with O blood (SIR=1.42; 95%CI: 0.83-2.28). Parents of probands with non-O blood had increased PC risk (SIR=3.28; 95%CI: 2.18-4.74, n=920), but not parents of probands with O blood (SIR=1.75; 95%CI: 0.75-3.45, n=484), p-value for between-group difference=0.10. Among FDRs of probands with O blood, female FDRs had increased PC risk (SIR=2.12; 95%CI: 1.06-3.79), but male FDRs did not (SIR=0.86; 95%CI: 0.31-1.88). Higher PC risk was observed among female FDRs of probands with non-O blood (SIR=2.28; 95%CI: 1.44-3.41) than male FDRs of probands with non-O blood (SIR=1.62; 95%CI: 1.00-2.48). Mothers of probands with O blood had increased PC risk (SIR=2.89; 95%CI: 1.05-6.28) compared to fathers (SIR=0.79; 95%CI: 0.09-2.84). Both mothers (SIR=3.60; 95%CI: 1.96-6.03) and fathers (SIR=2.95; 95%CI: 1.61-4.95) of probands with non-O blood had increased PC risk. No major findings were observed among probands' siblings or offspring.

Conclusions: PC risk is greater among FDRs of PC probands with non-O blood than among FDRs of probands with type O. PC risk is higher among parents of probands with non-O blood, and female FDRs and mothers of probands with non-O blood. Possible clustering of non-O blood type in families may contribute to increased PC risk.

#1240

Comprehensive analysis of germline variants in Mexican patients with hereditary breast and ovarian cancer susceptibility.

Felipe Vaca-Paniagua,1 Rosalía Quezada-Urban,1 Clara E. Díaz-Velásquez,1 Rina Gitler,2 María P. Rojo-Castillo,2 Max Sirota-Toporek,2 Andrea Figueroa-Morales,2 Oscar Moreno-García,2 Lizbeth García Esquivel,2 Gabriela Torres-Mejía,3 Michael Dean,4 Ivan Delgado-Enciso,5 Héctor Ochoa-Díaz-López,5 Fernando Rodriguez-León,6 Virginia Jan,7 Victor H. Hugo Garzón-Barrientos,8 Pablo Ruiz-Flores,9 Perla K. Espino-Silva,9 Jorge Haro-Santa Cruz,9 Héctor Martínez-Gregorio,1 Ernesto Rojas-Jiménez,1 Rosa M. Álvarez-Gómez,10 Luis A. Herrera,10 Isabelle Romieu,11 Luis I. Terrazas,1 Yolanda I. Chirino,1 Cecilia Frecha,12 Javier Oliver,12 Sandra Perdomo13. 1 _Facultad de Estudios Superiores Iztacala, México, Mexico;_ 2 _Fundación ALMA, México, Mexico;_ 3 _Instituto Nacional de Salud Pública, Cuernavaca Morelos, Mexico;_ 4 _National Cancer Institute, DC;_ 5 _Instituto Estatal de Cancerología de Colima, Colima, Mexico;_ 6 _El Colegio de la Frontera Sur (ECOSUR),, San Cristóbal de Las Casas, Chiapas,, Mexico;_ 7 _Hospital de Especialidades Vida Mejor, ISSTECH, Textla Gutiérrez, Chiapas, Mexico;_ 8 _Hospital General de Chilpancingo, Chilpancigo, Guerrero, Mexico;_ 9 _Universidad Autónoma de Coahuila, Coahuila, Mexico;_ 10 _Instituto Nacional de Cancerología, México, Mexico;_ 11 _International Agency for Research on Cancer, Lyon, France;_ 12 _Hospital Italiano, Buenos Aires, Argentina;_ 13 _Universidad El Bosque, Mexico_.

Hereditary breast and ovarian cancer syndrome (HBOC) is an autosomal dominant disease that represents approximately 5-10% of all patients with breast cancer. This syndrome is mainly associated to high-risk pathogenic alleles in BRCA1 and BRCA2 genes, but only for 25% of HBOC cases. This work aimed to find new pathogenic alleles in a panel of 143 cancer-predisposing genes in 300 Mexican cancer patients with suspicion of HBOC and 31 non-cancer patients with a severe family history of cancer, using massive parallel sequencing. We found 15% (45/300) patients with pathogenic variants in the group of cancer patients; 12% (35/300) harbored variants with unknown clinical significance (VUS) and 73% (220/300) were negative. The non-cancer group had a 32% (10/31) of patients with pathogenic variants, 3% (1/31) had VUS and 65% (20/31) were negative. Moreover, the most recurrent mutation was the Mexican founder deletion of exons 9-12 in BRCA1, found in 5 of 16 cancer patients with alterations in this gene. Private or rare VUS variants with potential impact at protein level were found in 22 genes, being CHEK2 the one with most VUS (6/39). Noteworthy, our results show for the first time in the Mexican population an equal contribution of pathogenic alleles in other susceptibility cancer genes (50%) as in BRCA1/2 (50%). This highlights the high locus heterogeneity of HBOC and the necessity of expanding genetic tests for this disease to broader gene panels. Further studies need to be conducted to define the clinical impact of the pathogenic alleles and VUS identified.

#1241

Profile of BRCA1/BRCA2 mutations in Russian ovarian cancer population detected by NGS and MLPA analysis: Interim results of OVATAR study.

Alexandra Tyulyandina,1 Vera Gorbunova,1 Svetlana Khokhlova,1 Larisa Kolomiets,2 Maksim Filipenko,3 Evgeny Imyanitov,4 Irina Demidova,5 Yuri Moliaka,6 Nadezhda Cherdyntseva,2 Dmitriy Vodolajskiy,7 Ludmila Lyubchenko,1 Sergei Tjulandin,1 Ilya Tsimafeyeu,8 Olga Vedrova,9 Vera Karaseva,9 Sergei Andreev,9 Tatiana Kekeeva10. 1 _Russian Cancer Research Centre, Moscow, Russian Federation;_ 2 _Tomsk Cancer Research Institute, Tomsk, Russian Federation;_ 3 _Institute of Chemical Biology and Fundamental Medicine, Novosibirsk, Russian Federation;_ 4 _NN Petrov Institute of Oncology, Russian Federation;_ 5 _Moscow Oncological Hospital #62, Moscow, Russian Federation;_ 6 _Krasnodar Oncological Hospital, Russian Federation;_ 7 _Rostov-on-Don Scientific Research Institute of Oncology, Russian Federation;_ 8 _Russian Society of Clinical Oncology, Moscow, Russian Federation;_ 9 _AstraZeneca, Moscow, Russian Federation;_ 10 _Research Centre for Medical Genetics, Moscow, Russian Federation_.

Background: This study is a first attempt to determine frequency of gBRCAm and share of sBRCAm in Russian ovarian cancer (OC) cancer patients (pts) using next-generation sequencing (NGS) and multiplex ligation-dependent probe amplification (MLPA). Russian population is known to have a sizable proportion of "frequent" germline mutations in BRCA genes, with occurrence in >2% of all BRCAm cases.

Methods: 498 pts with primary serous and endometrioid OC were enrolled in noninterventional study OVATAR (NCT02122588). NGS testing of BRCAm in genomic DNA (gDNA) from leukocytes and primary tumor tissue was performed. MLPA assay for large rearrangements (LGR) was used on gDNA from leukocytes.

Results: Interim analysis includes pairs of tumor and blood samples from 400 pts. The total rate of BRCA1/2 mutations was 35% (140/400 pts) including 29.8% (119/400) of germline mutations (gBRCAm) and 5.2% (21/400) of somatic mutations. Alterations reported hereby were either classified as deleterious/pathogenic in public databases, or identified as "likely pathogenic" (e.g., loss-of-function). VUS were not included. Frequent gBRCAm were detected in 49.3% of gBRCAm cases (69/140). BRCAm were counted as rare: in 30.7% (43/140) pts, including LGR in 3.6% (5/140) pts. sBRCAm: in 15% (21/140) pts. Although previously counted as frequent, 6174delT in BRCA2 was not detected. 4 pts carried pathogenic germline BRCA2 c.T5286G:p.Y1762* nonsense mutation, with prevalence 2.9% among BRCAm carriers, which makes it the new and only potential "hot-spot" in BRCA2 gene. Large deletions comprise 5% of all BRCAm and mostly occur in BRCA1 gene.

Conclusion: The overall rate of both somatic and germline BRCA variations in Russian OC population is in line with global data, with high percent of 8 frequent gBRCAm (49.3%). Use of MLPA is limited by blood samples with low rate of germline LGR. NGS is becoming a method of choice to hit both small variations and LGR in BRCA genes. | |  | |

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

gene/mutation | # of pts (n=140) and % of BRCAm

gBRCAm | Frequent mutations n=69 (49,3%) | BRCA1

5382insC | 37 | 26,4%

4154delA | 7 | 5,0%

2080delA | 6 | 4,3%

C61G | 5 | 3,6%

185delAG | 4 | 2,9%

3819del5 | 3 | 2,1%

3875del4 | 3 | 2,1%

BRCA2

T5286G (c.T5286G:p.Y1762*) | 4 | 2,9%

Rare mutations n=43 (30,7%) | BRCA1 | 24 | 17,1%

BRCA2 | 19 | 13,6%

Exons deletions

n=7 (5%) | BRCA1 | 6 | 4,3%

BRCA2 | 1 | 0,7%

sBRCAm | n=21 (15%) | BRCA1 | 13 | 9,3%

BRCA2 | 8 | 5,7%

#1242

Differences of cytochrome P450 1B1 polymorphisms on prostate cancer risk between races.

Taku Kato,1 Yutaka Hashimoto,1 Ryan Wong,2 Yozo Mitsui,1 Shigekatsu Maekawa,1 Soichiro Yamamura,1 Shahana Majid,1 Sharanjot Saini,1 Varahram Shahryari,2 Laura Tabatabai,1 Hiroaki Shiina,3 Rajvir Dahiya,1 Takashi Deguchi,4 Yuichiro Tanaka1. 1 _Veterans Affairs Medical Center and University of California, San Francisco, San Francisco, CA;_ 2 _Veterans Affairs Medical Center, San Francisco, San Francisco, CA;_ 3 _Shimane University, Izumo, Japan;_ 4 _Gifu University, Gifu, Japan_.

Prostate cancer is the most common cancer among men in the United States but when comparing between races, rates are much higher in African-Americans (AfA) compared to Caucasians (Cau). Reasons for this discrepancy are unknown but studies have shown that polymorphisms of cytochrome P450 1B1 (CYP1B1), a gene that activates carcinogens, to be involved in prostate cancer pathogenesis. In this study, how race influences CYP1B1 polymorphisms and prostate cancer risk were investigated. Blood genomic DNA from a Cau (469 controls, 500 prostate cancer), AfA (152 controls, 218 cases) and Japanese (JPN) (131 controls, 177 cases) population were obtained. Eight polymorphic sites of the promoter or 5'-untranslated region of CYP1B1 (rs2551188 G to A, rs2567206 C to T, rs2567207 T to C, rs162556 T to C, rs10175368 G to A, rs163090 T to A, rs162330 T to G, and rs162331 A to G) were analyzed using Taqman genotyping assays and sequence-specific PCR, and genotyping results tabulated. Also, CYP1B1 protein expression was determined in 52 Cau benign prostatic hyperplasia specimens and levels categorized by polymorphic sites. Racial differences for prostate cancer risk due to CYP1B1 polymorphisms were observed. In Cau, minor alleles rs2551188 A, rs2567206 T and rs10175368 A showed increased risk for cancer (P<0.05). Linkage was observed between rs2551188-rs2567206-rs2567207-rs10175368 and the major G-C-T-G haplotype was reduced in cancer cases (P<0.001). Interestingly when analyzing prostatic tissue specimens, minor genotypes of rs2551188 (P<0.05), rs2567206 (P<0.05) and rs10175368 (Ptrend=0.065) displayed higher levels and major G-C-T-G haplotype lower levels (P<0.05) of CYP1B1 protein expression, which were consistent with genotyping results. For AfA, minor rs2551188 A allele decreased risk for prostate cancer (P<0.001). Rs163090-rs162331 were linked and the minor A-G haplotype displayed a reduced prostate cancer risk (P<0.05). Among JPN, minor rs2567206 T allele also reduced cancer risk (P<0.05). Two sets of linkages, rs2551188-rs2567206-rs2567207-rs162556-rs10175368 and rs163090-rs162330-rs162331 were observed and the minor A-T-C-T-A and major A-T-A haplotypes respectively, showed a decreased risk for cancer (P<0.01). These results suggest a differential effect of CYP1B1 polymorphisms for prostate cancer risk between races and these results are important in understanding its role in this disease.

#1243

Elucidating the molecular pathogenesis of familial glioma.

Daniel I. Jacobs,1 Kazutaka Fukumura,2 Matthew N. Bainbridge,3 Georgina N. Armstrong,1 Donna M. Muzny,1 Beatrice Melin,4 Jason T. Huse,2 Melissa L. Bondy1. 1 _Baylor College of Medicine, Houston, TX;_ 2 _University of Texas MD Anderson Cancer Center, Houston, TX;_ 3 _Rady Children's Hospital, San Diego, CA;_ 4 _Umeå University, Umeå, Sweden_.

INTRODUCTION: In recent years, the molecular characterization of sporadically arising diffuse gliomas has identified recurrent driving alterations and delineated molecularly and clinically distinct subclasses of disease. However, less is known about the molecular nature of gliomas that are familial in origin. To address this question, we integrated germline and somatic genomic data to characterize the molecular pathogenesis of 20 tumors arising in unrelated individuals with a family history of glioma collected through the Gliogene International Consortium. METHODS: FFPE tumor specimens were sectioned and reviewed to localize neoplastic tissue for DNA extraction. Library preparation, exome plus targeted capture, and paired-end sequencing on the Illumina HiSeq 2000 platform was performed at the Baylor College of Medicine Human Genome Sequencing Center. Single-nucleotide variants and indels were called with respect to germline DNA sequencing data for each case using MuTect2. Copy number profiling was performed on the Illumina HumanOmniExpress BeadChip and analyzed using GenomeStudio v2.0. Genotypes at known glioma risk polymorphisms were determined from germline DNA profiled on the Illumina Infinium OncoArray and rare, predicted deleterious germline mutations were identified from germline whole-exome sequencing data. RESULTS: Tumor exome sequencing was completed at an average read depth of 116X and we detected a median of 54 non-silent somatic mutations per tumor across the 20 tumors profiled. All three molecular subtypes of sporadic glioma were represented, including IDH-mutant, 1p/19q codeleted (n=3), IDH-mutant, 1p/19q intact (n=7), and IDH-wildtype tumors (n=10). Characteristic subtype-specific mutations and copy number alterations (e.g., TP53 and ATRX mutations among IDH-mutant, 1p/19q intact tumors) were observed, and the frequencies of recurrent alterations were comparable to sporadic glioma cases analyzed by The Cancer Genome Atlas. Notably, all 20 cases had alterations in genes regulating telomere length; 17 had acquired mutations in ATRX or the TERT promoter as typically seen in sporadic glioma, while three instead had germline mutations in telomere shelterin complex genes POT1 or TERF2. Frequencies of known common glioma risk alleles were similar to those among sporadic cases and correlations between risk alleles and specific somatic mutations were not observed. CONCLUSIONS: This study illustrates: 1) the complementarity of inherited and acquired alterations in driving gliomagenesis in some individuals with a familial predisposition to the disease; and 2) that the molecular characteristics of familial tumors profiled largely recapitulate what is known about sporadic glioma. In the majority of cases, the source of germline genetic susceptibility is not known but does not appear to be conferred by common risk polymorphisms.

#1244

BHD-associated kidney cancer exhibits unique molecular characteristics and a wide variety of mutations in chromatin remodeling genes.

Ryosuke Jikuya,1 Mitsuko Furuya,1 Masaya Baba,2 Hiroyuki Aburatani,3 Adam R. Metwalli,4 Laura S. Schmidt,4 W. Marston Linehan,4 Masahiro Yao,1 Hisashi Hasumi1. 1 _Yokohama City University, Yokohama, Japan;_ 2 _Kumamoto University, Kumamoto, Japan;_ 3 _Research Center for Advanced Science and Technology, The University of Tokyo, Tokyo, Japan;_ 4 _Center for Cancer Research, National Cancer Institute, National Institute of Health, Bethesda, MD_.

Background

Birt-Hogg-Dubé (BHD) syndrome is a hereditary kidney cancer syndrome, which predisposes patients to develop cutaneous fibrofolliculomas, pulmonary cysts and renal tumors with various types of histology including chromophobe renal cell carcinoma (chRCC), hybrid oncocytic/chromophobe tumor (HOCT), clear cell renal cell carcinoma (ccRCC), papillary renal cell carcinoma (pRCC) and oncocytoma. The responsible gene FLCN is a tumor suppressor for kidney cancer which plays an important role in energy homeostasis through the regulation of mitochondrial oxidative metabolism. In previous studies, we demonstrated that kidney-targeted Flcn knockout mice develops hyperproliferative polycystic kidney; however, this mouse model dies at three weeks of age before developing kidney cancer, suggesting that mutations in addition to FLCN are necessary for the development of renal neoplasms. To understand the molecular mechanisms by which alterations in metabolism under FLCN-deficiency lead to kidney cancer development, we conducted whole-exome sequencing analysis as well as metabolite analysis of BHD-associated kidney cancer.

Results

Whole-exome sequencing analysis of BHD-associated kidney cancer revealed that copy number variations (CNV) of BHD-associated kidney cancer are considerably different from those already reported in sporadic cases. We observed less CNV in chRCC, HOCT and oncocytoma compared to ccRCC and pRCC, which might reflect the indolent nature of chRCC, HOCT and oncocytoma. Somatic mutation analysis revealed that very few genes are commonly mutated in BHD-associated kidney cancer; however, chromatin remodeling genes were frequently mutated in BHD-associated kidney cancer (17/29 tumors, 59%), suggesting that alterations in epigenome regulation might drive kidney cancer development in cooperation with FLCN mutations. Metabolite analysis of BHD-associated kidney cancer revealed the metabolic reprogramming towards mitochondrial oxidative metabolism and upregulated lipolysis, which may fuel FLCN-deficient tumor cells to acquire a growth advantage driving tumor progression. Redox regulations including pentose phosphate pathway and glutathione synthetic pathway were upregulated in BHD-associated kidney cancer, which may protect FLCN-deficient tumor cell from reactive oxygen species produced from abundant mitochondria with increased respiratory capacity.

Conclusions

BHD-associated kidney cancer displays unique molecular characteristics which are completely different from sporadic kidney cancer. These data provides mechanistic insight into tumorigenesis under FLCN deficiency as well as a foundation for the development of novel therapeutics for kidney cancer.

Funded in part under NCI,NIH contract HHSN261200800001.

#1245

Identification of germline variants in cancer susceptibility genes in patients with hereditary breast and ovarian cancer syndrome by massive parallel sequencing in Argentinean population.

Alejandra Franco,1 Fernando Orti,1 Sandra Perdomo,2 Cecilia Riggi,1 Cecilia Frecha,1 Javier Oliver1. 1 _Hospital Italiano, Buenos Aires, Argentina;_ 2 _Universidad el Bosque, Bogota, Colombia_.

Breast cancer is the most frequent cancer and ovarian cancer the seventh most frequent cancer among females worldwide, representing approximately 25% and 4% of all cancers. In Argentina, breast cancer and Ovarian cancer cause 6163 and 1332 deaths per year, respectively. Both breast and ovarian cancers are heterogeneous diseases composed of different tumor types with distinctive features and behaviors. The main risk factors for breast and ovarian cancer include age, family history, and genetics.The genetic components of both of the diseases have been well established, contributing to up to 10% of all breast cancer cases and 15% of all ovarian cancer cases.The two major susceptibility genes for both diseases are BRCA1 and BRCA2, and several other susceptibility genes have been identified. However, in the majority of high-risk breast and/or ovarian cancer (HBOC) families, the genetic predisposition factors remain unidentified, making the genetic counseling of these families challenging. The aim of the current study is to utilize an NGS approach to identify genetic factors that predispose individuals to hereditary breast and/or ovarian cancer (HBOC) in the high-risk Argentinian BRCA1/2 mutation-negative HBOC patients.16 patients were analyzed, the DNA was extracted from a blood samples and was subjected to a targeted massively parallel sequencing platform comprising 141 cancer predisposition genes. We detected in these patient 6 pathogenic variants in SBDS, FANCA, MC1R, WRN, MSR1 and SDHD genes. The effect of these variants are of type: non-synonymous (67%) and stop codon gains (33%). We also found 6 variants of uncertain significance (VUS) in APC, RAD50, SDHB, FANCD2, MLH1 and CDH1 genes.In conclusion, the identification of new variants could help to discover new genes associated to HBOC in the population of Argentina.These findings provide an excellent premise for further studies and could be utilized in the design of more efficient clinical management strategies for HBOC.

#1246

A family history of CRC confers protection against other cancers in a cohort of patients at risk for CRC.

Martin Tobi,1 Fadi Antaki,2 MaryAnn Rambus,2 Michael Lawson,3 Daniel Ezekwudo1. 1 _Saginaw VA Medical Ctr., Saginaw, MI;_ 2 _Detroit VA Medical Ctr., Detroit, MI;_ 3 _Kaiser Permanente Medical Ctr., Sacramento, CA_.

Risk for colorectal cancer (CRC) confers susceptibility to other cancers such as small bowel, breast, prostate, and female genital. However, these cancer family histories (F/H) have not been studied systematically.

Methods: 1,948 patients at increased CRC risk were followed to determine if the biomarker outcome was related to personal cancer prevalence or a cancer F/H by risk questionnaire which categorized patients as: Group 0 (no family history of cancer), Group 1 (F/H of breast cancer), Group 2 (F/H CRC), and Group 3 (P/H uterine/ovarian cancers).

Results: Demographics (age, sex, race, obesity) and NSAID use were similar in all groups and 88% were males. The proportion of cancer incidence in Group 1 is statistically greater [RR 1.36 (1.08-1.72) p = 0.014] compared to group 0 (Figure). Conversely, group 2 had statistically less cancer [RR 0.72 [0.52-0.99], p=0.045]. There was no significance difference between Group 0 and Group 3. About half the tumors in this older group of Veterans were prostatic. There was a wide variety of other cancers. In the entire cohort of the 5 tumors the most common tumor was prostate (n=222) followed by lung (n=61); CRC (n=53); bladder (n=23) and breast (n=20).

Conclusions: These differences may be explained by the effect of exosomes which play a major role in CRC and other cancers. Exosomes are nano-lipid particles of 30-100 nm released into the extracellular compartment. The ability of exosomes to facilitate cell-cell communication could be used to inhibit tumorigenic pathways. Exosomes may be endogenous in the case of CRC cell release or exogenous, found in breast milk containing microRNA moieties relevant to inhibition of carcinogenesis of cancers. Many of these mutated genes may inhibit tumorigenic process of cancers thus conferring protection against incipient cancers thus explaining our observation in Group 2. A commonality between CRC and endometrial cancer genetics potentially explains the reduced cancer prevalence in Groups 2 & 3. Breast cancer does not share BRCA "founder genes" with CRC and in contrast to CRC, K-ras mutations are not major events and thus F/H does not confer protection. This hypothesis remains to be proven but in the interim, timely screening for common cancers in patients with a family history of breast cancer may be effective.

#1247

Hereditary paraganglioma-pheochromocytoma syndrome: Patterns of presentation in a Brazilian oncogenetics clinic.

Diogo C. Soares,1 Ana Milena G. Camacho,1 Maria Isabel Achatz,2 Maria Nirvana C. Formiga1. 1 _A.C. Camargo Cancer Center, Sao Paulo, Brazil;_ 2 _National Cancer Institute, Bethesda, MD_.

Introductory Sentence: More than 35% of pheochromocytomas and paragangliomas (PPGL) are thought to occur due to an underlying genetic predisposition, which explain approximately 30% of familial cases. Methods: We analyzed data of four families with clinical and molecular diagnosis of PPGL followed at Oncogenetics Department of A.C. Camargo Cancer Center, from 2010 to 2017. Results: The proband of Family 1 is a 40-year-old woman with glomus jugulare paraganglioma and confirmed SDHB mutation. Her two children have the same mutation in SDHB gene and her son had the diagnosis of retroperitoneal paraganglioma at the age 13. The brother of the index case had a pheochromocytoma and her nephew had an abdominal paraganglioma at the age of 8. In the Family 2 the index case is a 33-year-old woman with paraganglioma of the carotid body bilateral and had a molecular diagnosis with SDHB mutation. There are no other paraganglioma cases in the members of this family. Family 3 initiated counseling in our clinics with a woman with paraganglioma of the carotid body bilateral at 25-year-old with recurrence at 35-year-old and it was found in genetic test a SDHD mutation. She had two cousins with bilateral cervical paraganglioma, but without genetic testing. Our fourth family: Man with pheochromocytoma at 28-year-old and mutation in the SDHD gene. A son with metastatic phaeochromocytoma in the retroperitoneum, mediastinum and lung at 11-year-old and bilateral carotid paraganglioma at 12-year-old. Sister of the index case with glomus jugulare paraganglioma at the age of 38. Conclusions: Although PPGL are rare tumors, it is estimated that up to 30-40% of cases are hereditary, based on this it is relevant to raise the PPGL syndrome suspicion and refer a paraganglioma or pheochromocytoma patient to a cancer genetic counseling. There are clear follow-up guidelines for the early detection of these tumors in carriers and the genotype-phenotype correlation can direct the diagnosis and in some cases the management of these patients.

## PREVENTION RESEARCH:

### Molecular Mechanisms and Targets for Cancer Prevention

#1248

Gut microbiome and metabolomic profiling reveal preventive effects of American ginseng on inflammation-associated colon cancer.

Chong-Zhi Wang, Haiqiang Yao, Jin-Yi Wan, Jinxiang Zeng, Clara Sava-Segal, Chun-Su Yuan. _University of Chicago, Chicago, IL_.

Inflammatory bowel disease is a significant public health issue in America. This gut disease is also a risk factor for the initiation and development of colon cancer. As an anti-inflammatory botanical, American ginseng has been shown to play a role in suppressing colitis, reducing inflammation, and preventing cancer. However, the effects of ginseng on the enteric microbiome and on specific molecular profiles have yet to be explored. Using an azoxymethane (AOM)/dextran sulfate sodium (DSS)-induced gut inflammation and tumorigenesis mouse model, the effects of oral American ginseng (15 and 30 mg/kg/day) on colitis and colon cancer were determined. After the establishment of a 16S rRNA illumina library from fecal samples, MiSeq sequencing was carried out to reveal the microbial population. A metabolomic analysis was conducted to detect endogenous metabolite changes for potential biomarkers. The results showed that in the acute phase, chemically-induced colitis was significantly reduced by ginseng in a dose-dependent manner, which was supported by histology evaluation. In the chronic phase, obvious colon carcinogenesis was observed in the model group. Ginseng treatment significantly reduced colon tumor multiplicity, which was consistent with pro-inflammatory cytokine level changes. For the fecal microbial communities, greater colitis severity was negatively associated with alpha diversity. With beta diversity, AOM/DSS induced an obvious separation between the control and model groups; however, the plots of the ginseng treatment group were very close to those of the untreated control group. Chemically induced colitis and colon tumorigenesis were correlated with an augment of Bacteroidaceae and Porphyromonadaceae, while the proportion of these families was diminished by ginseng treatment. Our results suggest that the colon cancer preventive effects of ginseng are mediated through an enteric microbiome population-shift recovery and dysbiosis restoration. In both acute and chronic phases, serum metabolomic analysis showed that the plots of the model group were clearly separated from the normal control group, while ginseng treatment, especially in high-dose group, pushed the plots back towards the control group. Compared to model group, ginseng treatment appeared to restore the levels of the observed key endogenous metabolites including glutamine, 6-P-glucose, EPA and oleic acid, which were related to inflammatory responses and energy balance regulations. In summary, we used a platform combining metabolomic and microbiome data to interpret the efficacies of ginseng. With further verification, microbiome profiles and selective endogenous small molecules could be used as biomarkers to elucidate the effects of American ginseng on colitis-associated colon cancer. (This work was supported in part by the NIH/NCCAM grants AT004418 and AT005362).

#1249

Animal model in the prevention of alcoholic pancreatitis.

Supriya Srinivasan,1 Tulasigeri Totiger,1 Michael VanSaun,1 Fanuel Messaggio,1 Chanjuan Shi,2 Austin Dosch,1 Eric Nestler,3 Nipun Merchant,1 Nagaraj Nagathihalli1. 1 _Department of Surgery, University of Miami Miller School of Medicine, Sylvester Comprehensive Cancer Center, Miami, FL;_ 2 _Department of Pathology, Vanderbilt Medical Center, Nashville, TN;_ 3 _Icahn School of Medicine at Mount Sinai, New York, NY_.

Background: Alcohol abuse is a major risk factor for pancreatitis. Alcohol addiction-induced molecular pathogenesis of pancreatitis remains obscure, and no current effective treatment exists. Therefore, approaches to investigate pathogenesis, prevention and cellular mechanisms by which alcohol causes pancreatitis are necessary for establishing therapeutics. Our efforts demonstrate that alcohol induces activation of a major intracellular transcriptional factor, cyclic AMP response element-binding protein (CREB). We further investigated the functional role of CREB in alcohol-induced pathogenesis of pancreatitis using cellular and genetic mouse models of pancreas.

Materials and Methods: Human tissue microarrays were immunostained to determine the significance of pCREB expression among pancreatic tissues obtained from normal and chronic pancreatitis. Rat acinar cell line AR42J and mouse PSCs (mPSCs) were exposed to alcohol (50 mMol/L). Inducible Ptf1aCreERTM knockin mice and Ptf1aCreERTM;CREBfl/fl (iPC) mice were fed with Lieber Decarli diet alcohol or regular diet for 14 weeks with or without caerulein (50 µg/kg). Mice were then euthanized 24 hours after the last caerulein injection, and pancreas tissues were processed for morphometric analysis (necrosis, vacuolization, hemorrhage, edema and inflammation) and immunohistochemical analysis of amylase, trichrome blue, SMA, collagen 1, fibronectin and pCREB expression. To determine whether alcohol accelerated morbidity in mice, we evaluated pathogenesis of chronic pancreatitis by analyzing acinar atrophy and pancreatic fibrosis.

Results: Expression of pCREB was significantly higher (p <0.001) in chronic pancreatitis vs. normal patient tissues, confirming the role of activated CREB in pancreatitis. Activated CREB levels were very high in alcohol-fed Ptf1aCreERTM mice when compared with control diet-fed mice. Pancreatic sections from alcohol-fed mice challenged with caerulein revealed significantly higher score of acinar cell vacuolization and necrosis, inflammatory infiltrate and hemorrhage compared with minimal lesions in control diet-fed animals receiving caerulein. Pancreatic sections from alcohol-fed Ptf1aCreERTM animals showed higher score of histologic injury, extracellular matrix deposition, collagen deposition and increased pancreatic fibrosis when compared with control-fed mice. iPC mice showed decrease in the pathogenesis of chronic pancreatitis when compared to Ptf1aCreERTM mice with alcohol.

Conclusion: CREB is overexpressed in pancreatitis and alcohol activates CREB, which then drives pathogenesis of pancreatitis. Severity of pancreatitis in response to alcohol is diminished in the absence of CREB. Therefore, we conclude that targeting CREB represents a promising treatment for alcohol-induced pancreatitis.

#1250

Evaluation of the prevention by pioglitazone/metformin of ER-positive and ER-negative mammary cancers occurring in rodents on a high-fat diet.

Barbara K. Dunn,1 Chen Suen,1 Clinton Grubbs2. 1 _National Cancer Inst., Rockville, MD;_ 2 _University of Alabama, Birmingham, AL_.

Both metformin and pioglitazone have demonstrated cancer chemopreventive properties. Actoplus/Met, a combination of the two agents, is currently used in the clinic with an additive effect on improving insulin resistance, a proposed risk factor for certain cancers. Based on this prior knowledge, we tested pioglitazone and metformin individually and in combination for preventive efficacy in both rat (ER-positive) and mouse (ER-negative) mammary cancer models, using high-fat and high-fructose diets.

Pioglitazone in both rats and mice at doses as high as 20 mg/kg BW/day, 7X/week was not toxic, although the high-fat diets resulted in weight gain.

Rats: Pioglitazone alone in rats receiving a high-fat diet led to a 2% increase in mammary cancer multiplicity at a high dose (20 mg/kg BW/day) and a 21% decrease in multiplicity at a low dose (10 mg/kg BW/day). In rats fed a high-fructose diet, the combination of pioglitazone and metformin caused a 20% decrease in multiplicity, in contrast to pioglitazone alone which led to a 10% decrease in mammary cancer number. In rats fed a high-fat diet, the combination caused a 24% increase versus a 1.0% decrease in cancer multiplicity with pioglitazone alone.

Mice: Pioglitazone alone at a high or low dose (20 or 10 mg/kg BW/day) in mice fed a high-fat diet resulted in a 28% and 47% reduction in mammary cancer multiplicity, respectively. In mice fed a high-fructose diet in a combination study, pioglitazone alone caused a 7% decrease in mammary cancer whereas the combination of pioglitazone with metformin showed no effect on mammary cancer reduction.

Metformin alone in rats on a high-fat diet caused a 35% decrease in mammary cancer multiplicity at 150 mg/kg BW/day but a 9% decrease at 300 mg/kg BW/day. Metformin alone in rats on a high-fructose diet caused an 11% increase but the combination with pioglitazone caused a 20% decrease in cancer multiplicity. When rats were given a high-fat diet, metformin alone caused a 14% decrease and in combination with pioglitazone caused a 24% decrease in ER-positive mammary cancers.

The conclusions are: (1) Pioglitazone is not active as a chemopreventive agent when given alone or in combination with metformin in rats treated with MNU on a high-fat or a high-fructose diet. (2) Pioglitazone is not active as a chemopreventive agent when given alone or in combination with metformin in MMTV/Neu mice treated with DMBA on a

high-fat or a high-fructose diet. (3) Metformin is not active as a chemopreventive agent when given alone or in combination with pioglitazone in rats treated with MNU on a high-fat or a high-fructose diet. (4) Metformin showed some chemopreventive efficacy when given alone to MMTV/Neu mice receiving DMBA and on a high-fat diet. Efficacy was also observed with metformin alone and in combination with pioglitazone in mice receiving a high-fructose diet.

#1251

**Fisetin for the management of** PIK3CA **-mutant colorectal cancer.**

Naghma Khan, Farah Jajeh, Devon Miller, Rachel Van Doorn, Emily Lauren Eberhardt, Richard B. Halberg, Hasan Mukhtar. _Univ. of Wisconsin-Madison, Madison, WI_.

Colorectal cancer (CRC) is the third most common cause of cancer-related deaths in the United States and is one of the most frequent and deadliest cancers worldwide. The relative survival rate is very poor for patients with metastatic CRC, regardless of the progress in chemotherapy. 5-Fluorouracil (5-FU) is widely used chemotherapeutic drug for the treatment of colon cancer, and the efficacious tumor therapy is generally obstructed by the progression of tumor resistance mechanisms. PIK3CA is one of the most frequently mutated genes in CRC, as about 15-20% of advanced CRC harbor activating mutations in PIK3CA exon 9 and/or exon 20. Therefore, modeling the effects of this type of mutation in the mammalian colon is significant. Fisetin (3,3',4',7-tetrahydroxyflavone), a naturally occurring flavonoid, is present in several fruits and vegetables. We have earlier shown that treatment of PIK3CA-mutant colon cancer cells with fisetin and 5-FU caused induction of apoptosis, decrease in the expression of PI3K, phosphorylation of Akt, phosphorylation of mTOR and its target proteins with an increase in the phosphorylation of AMPKα. Based on these exciting results, we determined the effect of the treatment of combination of fisetin and 5-FU on the multiplicity of colonic tumors and on the regression of invasive adenocarcinomas in FC13K1ApcMin/+ mice. In this in-vivo model of colon cancer, synergy exists between the loss of the tumor suppressor APC and the presence of a dominant active PI3K. The tumors form as adenomas, progress to invasive adenocarcinomas and eventually metastasize to regional lymph nodes, as observed in human patients. The anti-tumorigenic properties of fisetin were tested by treating these mice either early when mice had no or few visible colon polyps and late when mice had developed cancers. The treatment regime was the same for both groups. The only difference was age when treatment initiated; the early group started treatment between 25 and 30 days, whereas late group started treatment between 45 and 50 days. In the early treatment groups, fewer fisetin-treated mice developed tumors than controls and fisetin-treated animals bearing tumors developed fewer colon tumors than controls. We also found that 5-FU suppressed growth of tumors and the effects of fisetin and 5-FU are additive. At the end of the experiment, as compared to control group, there was 27% decrease in tumor counts on treatment of animals with 5-FU, 36% decrease with fisetin and 49% decrease on treatment with combination of fisetin and 5-FU. There was also decrease in tumor incidence on treatment of animals with fisetin and combination of fisetin and 5-FU. Treatment with fisetin, 5-FU, or the combination did not appear to affect tumorigenesis when given late. Interestingly, the activation of PI3K appears to make 5-FU less effective at this stage. Our data suggest that fisetin could be used as a preventive agent as well as an adjuvant with 5-FU for the prevention of PIK3CA-mutant CRC.

#1252

RNA-Seq transcriptome analyses of human LNCaP prostate cancer cells treated with a novel bis-aspirinyl selenazolidine AS-10.

Sangyub Kim, Deepkamal N. Karelia, Yuka Imamura, Srinivasa Ramisetti, Cheng Jiang, Shantu Amin, Arun K. Sharma, Junxuan Lu. _Penn State College of Medicine, Hershey, PA_.

Purpose: Our goal is to develop novel selenium-containing aspirin compound(s) for the chemoprevention of prostate cancer. Our current studies have shown significant inhibitory effects of bis-aspirinyl selenazolidine AS-10 on androgen receptor (AR) signaling, cell proliferation and survival (apoptosis) of human prostate cancer LNCaP cells in the single micromolar range of continued exposure. Immunoblot analyses of the AS-10 exposed cells showed the upregulation or activation of DNA damage response (DDR) protein p53 and p21Cip1 as well as DNA double strand break marker histone H2A.X associated with the apoptotic metrics. The objectives of the current work were: 1) to cross-validate involvement of transcriptional action of AS-10 to regulate key molecules related to AR signaling, cell cycle arrest, DDR and apoptosis responses, and 2) to discover potential transcriptome signatures and pathway networks induced by AS-10 that help to delineate its mechanisms of action.

Experimental Design: RNA-seq was performed for LNCaP cells that were treated with AS-10 at 5 and 10 μM, with DMSO as a control for 3, 6 or 12 h. The sequencing results were analyzed by Ingenuity Pathway Analysis (IPA) software with several bioinformatic approaches, including functional enrichment analysis, gene-gene interaction network analysis and upstream regulator analysis.

Results: A total of 12055 genes were positively aligned. At AS-10 exposure concentration of 5 and 10 μM, 2535 and 4556 genes were affected by two-fold, respectively (p<0.01). For targeted analyses of mRNA abundance changes of AR and its best known transcriptional target prostate specific antigen (PSA/KLK3), AS-10 decreased them in synchrony with their protein changes.

For un-biased profiling, IPA functional enrichment analysis revealed that many of the differentially expressed genes were associated with cell cycle-associated functions, cell death, and P53 signaling. For gene-gene network interactions, the top ranked networks from all doses and treatment time-points revealed significant links with cancer, organismal injury and abnormalities, cellular development, growth and proliferation, cell death and survival. At AS-10 exposure concentration of 5μM, MYC and PSA were initially targeted, followed by AR and P53. At AS-10 exposure concentration of 10μM, MYC and AR were initially targeted, followed by AR. As expected, higher concentration of AS-10 induced the genes related to cell death earlier than lower level. Consistent with RNA-seq data, our protein assay showed that AS-10 10μM induced cell death at 12 hr.

Conclusions: The RNA-seq data on AR/PSA and p53/p21 identify transcriptional as well as post-transcriptional regulation by AS-10 to affect their protein levels in the LNCaP cells. The transcriptome profiling approaches reveal comprehensive network interactions and cross-talks to contribute to cell cycle arrest and apoptosis.

#1253

Dissecting the signaling cascades involved in growth inhibition and apoptosis of prostate cancer cells exposed to a novel bis-aspirinyl selenazolidine AS-10.

Deepkamal N. Karelia, Sangyub Kim, Srinivasa Ramisetti, Cheng Jiang, Shantu Amin, Arun K. Sharma, Junxuan Lu. _Penn State Univ. College of Medicine, Hershey, PA_.

Aspirin has been recommended for lowering risk of colorectal cancer, in addition to its cardiovascular health benefit at low dose (baby Aspirin) and over-the-counter pain medication. Through an innovative research program focused on enhancing its anticancer activity by incorporation of selenium, we identified bis-aspirinyl selenazolidine (AS-10) as a novel promising lead compound against different cancer cell types in the NCI-60 cancer cell line panel, including prostate cancer (PCa) (e.g., EC50 in range of 1.7 to 2.5 μM for PCa cells compared with aspirin in the millimolar range). We have earlier found that AS-10 inhibited androgen receptor (AR) signaling in LNCaP cells and induced caspase-mediated apoptosis along with activation of DNA damage response (DDR) proteins such as P53 and its canonical target P21Cip1 and the DNA double strand break marker protein phospho-H2A.X. The objective of the present work is to analyze the signaling pathways underlying the growth arrest and apoptotic effects of AS-10. In time course studies, AS-10 treatment decreased the expression of AR and its downstream target prostate specific antigen (PSA) as early as 3 h in LNCaP cells. AS-10 inhibited the progression of LNCaP cells from G1 into S phase, far ahead of an increase in apoptotic sub-G0/G1 cells. At the protein level, AS-10 treatment decreased the expression of proliferative oncoprotein c-Myc and anti-apoptotic molecules such as Mcl-1, Bcl-xL and survivin. Temporally, AS-10 increased P53 and P21Cip1 expression and PARP cleavage ahead of increased phospho-H2A.X which might simply mark apoptotically fragmented DNA. By si-RNA transfection, knocking down of P53 or P21Cip1 in LNCaP cells increased cell death induced by AS-10, indicating an anti-apoptotic role of this regulator-target pair. These data suggest that suppression of AR signaling and prosurvival proteins by AS-10 may contribute to cell cycle arrest and apoptosis, subject to P53 status.

Additionally, aspirin has been known to acetylate histones and other proteins. Because AS-10 contains two aspirin moieties, we hypothesize that the combination of AS-10 with a histone deacetylates (HDAC) inhibitor (Vorinostat) may further increase overall acetylation and synergize to induce PCa cell death. Our results demonstrate that the combination of AS-10 and Vorinostat induced more than additive cell death response.

In summary, AS-10 represents a promising potential chemopreventive agent for prostate carcinogenesis, alone or in combination with other drugs, through multiple molecular targets and cellular pathways and merits efficacy assessment in animal models.

#1254

Metformin's potential as a breast cancer preventative agent by altering epigenetic patterns.

Caitriona Tyndall,1 Charlene Lam,1 Marc Gunter,2 James Flanagan1. 1 _Imperial College London, London, United Kingdom;_ 2 _International Agency for Research on Cancer, Lyon, France_.

Metformin is one of the most commonly prescribed Type II Diabetes Mellitus (T2DM) drugs and has been shown in diabetic long-term users to reduce the likelihood of breast cancer by 56%. Breast cancer is the most commonly diagnosed cancer in the UK accounting for 55,000 new cases per year. It has been estimated that as much as 27% of cases could be prevented based on current knowledge. This study aims to investigate the novel hypothesis that metformin acts as a preventative agent by altering epigenetic patterns in non-cancerous breast epithelial cells and the T2DM environment, namely hyperinsulinemia and hyperglycaemia, potentially modulates this.

Crystal Violet cell viability assay and Western Blot for p-AMPK, a known target for metformin, were performed to assess the response of two non-cancerous breast epithelial cells lines, MCF10A and MCF12A, to metformin in a range of insulin and glucose concentrations. Western blot was performed to assess changes in histone post-translational modifications (PTMs), such as H2K27ac and H3K27me3, following 3-days treatment with metformin at 2.5mM and 5mM. Additionally, we conducted an Illumina MethylationEPIC Array on MCF10A and MCF12A cell lines treated for 3-days with 2.5mM and 5mM metformin. Pyrosequencing assay was used to further validate interesting CpG sites.

Our findings suggest non-cancerous breast epithelial cells, MCF10A and MCF12A cell lines, are sensitive to metformin following 3 and 7-days treatment. Further to this, our data suggests the T2DM environment impacts the effectiveness of metformin where low glucose and high insulin significantly increase metformin effectiveness in MCF10A and MCF12 cell lines following 3-day treatment, respectively. Metformin treatment leads to the activation of p-AMPK in normal breast epithelial cells. Analysis of Illumina MethylationEPIC array suggests metformin in hyperglycaemic and hyperinsulinemic conditions induces differential DNA methylation changes in MCF10A and MCF12A non-cancerous breast epithelial cell lines and the effects are dose dependent. Of the 850,000 probes, 13 CpG sites in MCF10A and 5 CpG sites in MCF12A showed significant changes in methylation following metformin treatment (p<0.001).

In summary, this study has shown a potential mechanism of metformin action through altering of epigenetic patterns in non-cancerous breast epithelial cells. Future work aims to further explore epigenetic changes induced by metformin in the context of the T2DM environment and whether these changes could potentially predict reduced breast cancer risk.

#1255

Delta-tocopherol induced endoplasmic reticulum stress causes autophagic degradation of ER and cell death in bladder cancer models.

Christopher A. Blair,1 Hanze Hu,1 Tim Huynh,1 Maggie Wu,1 Chung S. Yang,2 Xiaolin Zi1. 1 _UC Irvine, Irvine, CA;_ 2 _Rutgers University, Piscataway, NJ_.

Vitamin E has been the subject of numerous basic and clinical studies, which have demonstrated a range of results that necessitate the further analysis of its individual components. We demonstrate that of the major tocopherol components of Vitamin E, Delta tocopherol (δ-T) is significantly more effective than the more common Alpha- and Gamma- tocopherols at inducing bladder cancer cell death in vitro and reduces tumor growth and burden in vivo. As a major site of protein synthesis and folding the endoplasmic reticulum (ER) is highly sensitive to stress induced by increased unfolded protein load, which induces both pro-survival and ultimately pro-apoptotic responses via the different branches of the unfolded protein response (UPR). δ-T induces the UPR in human bladder cancer cells and ultimately results in cell death, inducing both apoptosis and autophagic cell death. Analysis of bladder cancer cells treated with δ-T revealed strong evidence of UPR activation in the form of upregulation and activation of ER stress sensors PERK and IRE1α, and the upregulation of folding chaperone GRP78 and transcription factors ATF4 and CHOP. These changes in turn induced downstream effects including Death Receptor 5 (DR5)-mediated apoptosis and visible induction of autophagy. In addition to the observation of autophagosome formation, induction of autophagy was further confirmed by upregulation of protein expression of components of the autophagic machinery LC3B and Atg family members. Electron Microscope imaging of treated cells revealed the presence of ER-whorls within the large autophagosomes which were themselves visible under conventional light microscopy, which has to our knowledge not been previously observed in cancer models. This observation is supported by the ER stress-based mechanism of action exhibited by δ-T on bladder cancer cells, which recapitulates the observations of the few previous reports of "ER-phagy" described in yeast models. ER stress induced expansion of the ER is believed to aid in the improvement of stressed cells' protein folding capacity, and selective degradation of the ER-whorls formed in this manner by a previously undescribed mechanism of autophagy was first described in yeast and remains unexplored in human cancer models. These findings may provide new insights into δ-T's mechanism of action and inform new approaches to the development of tocopherols as novel chemopreventive agents.

#1256

Lynch syndrome premalignancy upregulates immune checkpoints independently from neoantigen and mutational rates.

Kyle Chang. _MD Anderson Cancer Center, Houston, TX_.

Colorectal carcinomas in Lynch syndrome (LS) patients arise in a background of mismatch repair deficiency that leads to the accumulation of high numbers of mutations and neoantigens, thus causing infiltration by abundant tumor-infiltrating lymphocytes, a unique immune profile with upregulation of immune checkpoints, and response to immunotherapy. However, there is still a knowledge gap with regards to the level of immune activation and the assessment of mutation and neoantigen rates in LS premalignancy. We performed whole transcriptomic analysis using next-generation sequencing in a total of 28 colorectal polyps (26 tubular adenomas and 2 hyperplastic polyps) from 21 patients diagnosed with LS (n=11) and FAP (n=10) as comparators and a model of mismatch repair proficient colorectal premalignancy.Overall, LS polyps presented with low mutational and neoantigen rates but displayed a strinking immune activation profile characterized by CD4 T-cells, proinflammatory (TNF, IL12) and checkpoint molecules (LAG-3 and PD-L1). This immune profile was independent of mutational rate, neoantigen formation, and MMR status. In addition, we identified a small subset of LS polyps with high mutational and neoantigen rates that were secondary to accumulation of indels enriched for deregulation of DNA damage repair pathways (ATM and BRCA1 signaling). Our findings challenge the canonical model, based on the observations made in carcinomas, that emphasizes a dependency of immune activation on the acquisition of high levels of mutations and neoantigens, thus opening the door to the implementation of immune checkpoint inhibitors and vaccines for cancer prevention in Lynch syndrome.

#1257

**Human endogenous retrovirus type K (HERV-K)** env **protein as a vaccine target for HERV-K+ cancer prevention.**

Feng Wang-Johanning, Jia Li, Ming Li, Gary L. Johanning, Albert Lee, Tianhe Huang. _SRI International, Menlo Park, CA_.

Some tumors, including BC tumors, are poorly responsive or develop resistance to therapy, which creates an impetus to identify novel strategies that potentiate immunotherapy in unresponsive cancers. Vaccines against human BC have shown only limited success for prevention of BC, with the exception of yellow fever vaccine 17D (YFV), which is closely homologous to HERV-K. In order to check immune response in animal models, murine mammary tumor cells (4T1) or melanoma cells (B16F10) were employed to express a full-length HERV-K Env protein obtained from a BC patient with invasive ductal carcinoma (IDC) by stably transfecting with pLVXKenv [full-length HERV-K env, expressing extracellular surface (SU) and transmembrane (TM) domains] or pLVX vector (control). To determine whether the SU or TM has cancer-preventive effects when dosed as an HERV-K antigen vaccine, C57BL/6 female mice were immunized with cyclic dinucleotides (CDN:15 µg) and HERV-K SU (KSU), TM [harboring the HERV-K immunosuppressive domain (ISD)], or GST protein (25 µg) on day 1 and boosted on days 14 and 28. Mice were challenged with B16F10pLVX or B16F10pLVXKenv (3 X 105 cells) 18 days after the last boost of immunization. The effect of tumor challenge on tumor growth and weight was compared. Increased weight of pLVXKenv relative to pLVX control cells was observed in mice immunized with GST (2-fold increased weight). In contrast, we observed reduced weight of pLVXKenv relative to pLVX tumors in mice immunized with KSU (50% reduced weight), showing the protective effect of KSU vaccination. This protective effect disappeared in mice immunized with the TM (1.65-fold increased tumor weight), indicating that the ISD of TM may prevent an immune response to the vaccine. Increased anti-HERV-K antibody titers were demonstrated in mice immunized with KSU than with KTM. Significantly increased anti-KSU antibody titers were demonstrated in mice immunized with KSU than with GST. Interestingly, significantly increased anti-KTM antibody titers were demonstrated in mice inoculated with pLVXKenv than with pLVX. Lower percentages of CD3+CD4+, CD3+CD8+, and CD3-NK T cells from tumor tissues were observed in mice immunized with TM or SU protein and challenged with B16F10Kenv cells. Significantly increased proliferation of CD4 or CD8 T cells was demonstrated in mice immunized with KSU protein and challenged with B16F10Kenv cells than after challenge with B16F10pLVX cells. In contrast, significantly decreased proliferation of CD4 and CD8 T cells was demonstrated in mice immunized with TM and challenged with B16F10Kenv cells than with B16F10pLVX cells, reflecting immunosuppression induced by the TM vaccine. In conclusion, HERV-K SU protein, but not TM protein, can be used for cancer prevention and immunotherapy for HERV-K positive cancers. Also, TM blockade may reduce immunosuppression.

#1258

MMP23B expression and protein levels in blood and urine are associated with bladder cancer risk.

Barbara Pardini,1 Alessandra Allione,1 Clara Viberti,1 Giuliana Giribaldi,2 Stefano Turini,2 Cornelia Di Gaetano,1 Simonetta Guarrera,1 Francesca Cordero,2 Marco Oderda,3 Marco Allasia,3 Paolo Gontero,3 Carlotta Sacerdote,3 Alessio Naccarati,1 Paolo Vineis,1 Giuseppe Matullo1. 1 _Italian Institute for Genomic Medicine IIGM, Torino, Italy;_ 2 _University of Turin, Torino, Italy;_ 3 _Città della Salute e della Scienza, Torino, Italy_.

Urothelial bladder cancer (UBC) represents a public health problem because of its elevated incidence and relapse rate. After prostate cancer, UBC is the most frequent malignancy of the urinary tract with a higher incidence in men and smokers.

To date, there are no suitable biomarkers for an early diagnosis or detection of relapse/progression. In the attempt to improve diagnostic accuracy and to overcome the disadvantages of current diagnostic strategies, biomarkers found in easily accessible biofluids, such as blood or urine, represent a non-invasive and promising approach.

The first aim of this study was the identification of reliable biomarkers of UBC risk starting with whole gene expression profile in white blood cells from 66 UBC cases and 70 controls. We observed a lower expression of MMP23B and its relative pseudogene MMP23A in UBC compared to controls (LogFC=-0.37, FDR adjusted p-value=0.02 and LogFC=-0.23, FDR adjusted p-value=0.03, respectively, adjusted for age, smoking, and batch). Thus, we investigated MMP23B protein levels both in plasma (49 controls and 53 UBC) and in urine (57 controls and 59 UBC) samples, to confirm gene expression observations. Unexpectedly, both western blot evidences and ELISA quantification showed increased MMP23B levels in UBC cases respect to controls, reaching a statistical significance in urine (mean UBC=1797.70pg/ml, mean controls=1075.73pg/ml, p-value=0.02). A positive trend of association of MMP23 levels was observed also for grade and risk. As the lack of correlation between mRNA and protein levels could be due to a post-transcriptional crosstalk mediated by microRNAs (miRNAs), we investigated the expression levels of miRNAs targeting MMP23B

gene in urine of UBC patients and controls. We identified 5 differentially expressed miRNAs targeting MMP23B 3'UTR in UBC cases and controls.

The present outcomes indicate a potential role of MMP23B as non-invasive UBC biomarkers. Moreover, we reported the first evidence of MMP23B secretion both in plasma and urine, suggesting a role of this poorly characterized metalloproteinase in UBC. Further analyses are needed to better elucidate the mechanism of regulation of MMP23B expression by miRNAs present in UBC urine.

#1259

Loss of TFF1 promotes growth and chemotherapeutic resistance in gastric cancer.

Zheng Chen,1 Zheng Li,2 Mohammed Soutto,1 Weizhi Wang,2 Shoumin Zhu,1 Alejandro Corvalan,3 Zekuan Xu,2 Wael El-Rifai1. 1 _University of Miami, Miami, FL;_ 2 _The First Affiliated Hospital of Nanjing Medical University, Nanjing, China;_ 3 _Pontifical Catholic University of Chile, Chile_.

Background: Downregulation or loss of TFF1 expression occurs in more than half of human gastric adenocarcinomas through gene deletions, mutation, and a loss of heterozygosity or hypermethylation. Our previous studies have shown that Tff1 knockout (KO) in mice induces gastric lesions from low- grade dysplasia (LGD), high- grade dysplasia (HGD) to adenocarcinoma. BRD2, a family member of BET proteins, promotes aberrant gene expression in a variety of malignant tumors. In this study, with the analysis of Tff1 KO mice and human gastric cancer tissue samples, we discovered that loss of Tff1 promotes gastric cancer proliferation and drug resistance through upregulating BRD2 expression level.

Methods and Results: Integration Next Generation Sequencing data analysis in both Tff1 KO mice and human gastric cancer tissue samples demonstrated that miR-143-3p was significantly down-regulated in both mice and human gastric cancer samples (P<0.05). Our findings were further validated by RT-PCR in Tff1 KO mouse gastric legions (LGD and adenocarcinoma) and 3 different cohorts of human gastric cancer tissue samples from USA, Chile, and China. Using different gastric cancer cell models we further confirmed the decrease of miR-143-3p regulated by loss of TFF1. Next, we wanted to find out the downstream effector of miR-143-3p down-regulation. Western blot data showed that BRD2 protein level was regulated by miR-143-3p in 4 gastric cancer cell lines. BRD2 3'UTR luciferase reporter assay further confirmed that miR-143-3p decreased BRD2 protein expression through directly binding to its 3'UTR sites. These data suggested that, for the first time, BRD2 is a direct downstream target of miR-143-3p. At the meantime, the reconstitution of TFF1 in gastric cancer cells up-regulated miR-143-3p expression level, which in turn, decreased the protein expression levels of BRD2, MYC and BCL-2. Using western blot, we showed the synergistic effect of BRD2 inhibitor and CDDP chemotherapy in human gastric cancer cells. Data from 320 human gastric cancer patients demonstrated that high BRD2 expression level in gastric cancer tissues significantly decreased the overall patient survival rate (P<0.0001).

Conclusion: This study unveils, for the first time, loss of TFF1 promotes BRD2 activation in gastric cancer through decreasing miR-143-3p. This axis presents novel therapeutic opportunities by using approaches that reconstitute miR-143-3p or utilizing the recently developed clinical trials in human gastric cancer.

#1260

Mechanism of skin cancer chemoprevention by dietary grape: A global proteomics approach.

Charlotte A. Mintie, Chandra K. Singh, Mary A. Ndiaye, Gregory A. Barrett-Wilt, Nihal Ahmad. _University of Wisconsin-Madison, Madison, WI_.

Non-melanoma skin cancer (NMSC), composed mainly of squamous cell carcinomas (SCC) and basal cell carcinomas (BCC), is the most commonly diagnosed form of cancer in the United States, with more than 5 million cases diagnosed each year. Ultraviolet (UV) radiation, a major component of sunlight, is the primary contributing factor in the development of NMSCs. Based on research in the past few decades, the naturally occurring dietary antioxidants have shown considerable promise towards preventing or delaying the process of carcinogenesis. We have previously demonstrated that dietary grape powder (GP) imparts considerable chemopreventive effects against UVB exposure-mediated skin carcinogenesis in SKH-1 hairless mice (AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5263). Here, we extended our study to determine the molecular mechanisms associated with the observed chemopreventive response of grapes. We employed a quantitative proteomics approach to identify the mechanism of observed protective effects of GP against UVB exposure-mediated skin carcinogenesis. The trypsin-digested protein extracts from UVB exposure-mediated skin tumors excised from control and 5% GP-fed mice were labeled using TMT10plex isobaric mass tagging reagents, fractionated, and analyzed by high-resolution Orbitrap LC-MS/MS. Post-acquisition analysis was performed using MaxQuant and Perseus computational software. We found that approximately 2600 total proteins were modulated by GP consumption. In our first set of data reduction, we used cut-off parameters of a p-value less than 0.05, 3 unique peptides, and greater than 1.2 fold change. A low fold change threshold was selected due to ratio compression, a known phenomenon in isobaric mass tagging which leads to underestimation of quantitative ratios. The resulting subset of 239 proteins were then subjected to Ingenuity Pathway Analysis (IPA), which identified Acute Phase Response (APR), a systemic response to trauma which is linked to poor cancer prognosis when prolonged, as a pathway affected by GP. We observed modulations in nine APR proteins (APCS, HP, RBP1, FGB, FGA, CRABP2, C1S, HPX, IL36G, AMBP). Application of a more stringent data filter (permutation FDR q-value less than 0.07) exposed a subset of 20 proteins. When analyzed by IPA, the results suggested that GP affects signaling proteins associated with the 20s proteasome (PSMA6, PSMA3, and PSMB7) and 19s proteasome (UCHL5), both components of the 26s proteasome signaling, which plays an important role in cancer. Further studies are underway to validate our proteomics data.

#1261

Detection of autophagy in gastric mucosae of Chilean individuals residing in a geographic region characterized by high rates of gastric cancer-related death.

Alejandro Corvalan,1 Hernan Pulgar,2 Claudio Cruzat,2 Nicole Cespedes,2 Francisco Ruiz,3 Rodrigo Prado,3 Nelson E. Brown2. 1 _Pontifical Catholic University of Chile, Santiago, Chile;_ 2 _Univ. of Talca (Chile), Talca, Chile;_ 3 _Hospital of Curico, Curico, Chile_.

Gastric cancer is the fourth most common cancer worldwide. In Chile, gastric cancer is the first cause of cancer-related deaths, affecting most prominently the southern provinces. Therefore, the elucidation of molecular pathways that might be exploited to improve prognosis and therapies remains an important challenge. There is emerging evidence pointing to the role of autophagy in tumorigenesis. Autophagy, a lysosome-mediated catabolic process, represents a prosurvival process implemented by neoplastic cells in response to stressful stimuli. So far, markers of autophagy have been used as prognosis determinants in early stages of gastric cancer, or have been associated to recurrence and reduced survival in patients with advanced disease. However, the role of autophagy in precursor lesions (chronic gastritis, metaplasia, dysplasia) has not been explored. Our goal was to examine the presence of autophagy (cleaved-LC3A) and proliferation (Ki-67) markers in gastric biopsies obtained from healthy individuals residing in a Chilean geographic zone characterized by a high gastric cancer-related mortality. Toward this goal, 165 asymptomatic individuals, aged 40 to 70, from the Molina area in the south of Chile were subjected to endoscopy and biopsy sampling according to the Operative Link on Gastritis Assessment (OLGA) staging system. Each tissue section was processed for immunohistochemical detection of cleaved-LC3A and Ki67, and the results were correlated with the histopathological findings and the presence or absence of Helicobacter pylori. Our preliminary analyses indicate that 60% of individuals harbor at least one out of five biopsies with the highest score values for cleaved-LC3A staining. Most of these high-scored samples involved the gastric antrum (73.3%) and were also positive for the Ki67 proliferation marker (63.3%). Of note, tissue samples showing the highest score values for cleaved-LC3A staining occurred almost always (93.3%) in the context of chronic gastritis or a combination of chronic gastritis and intestinal metaplasia, even though only 56.7% of cleaved-LC3A positive tissues showed signs of Helicobacter pylori infection. These results seem to indicate that autophagy may allow gastric epithelial cells to circumvent inflammatory stress, highlighting this process as a potential target for the development of anti-cancer or preventive therapies.Grant Support: FIC-30388034-0, CONICYT-FONDAP 115130011, Fondecyt 1151411, PIA-Gastric cancer UTALCA

#1262

Type-2 cGMP-dependent protein kinase has a tumor-suppressive role in the colon epithelium.

Bianca N. Islam,1 Sarah K. Sharman,1 Yali Hou,1 Justin Ashby,1 Rui Wang,2 Subbaramiah Sridhar,1 Ravindra Kolhe,1 Darren D. Browning1. 1 _Augusta University, Augusta, GA;_ 2 _University of Texas MD Anderson Cancer Center, Houston, TN_.

The cGMP level plays a central role in regulating homeostasis in the colon epithelium and is emerging as a potential target for colon cancer prevention. The signaling components downstream of cGMP, and the tumor suppressive mechanism remain poorly understood. The present study has examined the expression of cGMP-dependent protein kinase isoforms (PKG1, PKG2) in normal intestinal mucosa and in colon cancer from human specimens. Immunohistochemical analysis detected PKG1 in supportive cells in the lamina propria and in smooth-muscle tissue, but not in the epithelium of normal intestine and colon. In contrast, PKG2 was detected exclusively in the epithelium. In colon cancer, PKG1 was restricted to the stroma where it colocalized with vimentin, whereas PKG2 was only detected in the tumor epithelium where it colocalized with cytokeratin. This pattern of PKG isoform expression was similar in the mouse intestine and colon, where only PKG2 was detected in purified colonic crypts. PKG2 knockout (KO) mice were used interrogate possible anti-carcinogenic roles in the colon epithelium. When subjected to the AOM/DSS model of colon carcinogenesis, the PKG2 KO animals produced significantly more polyps than wild type controls (1.75-fold, p=0.0037). While the polyps from PKG2 KO animals were slightly smaller than those from wild type, the trend was not significant (p=0.13). Wild type and PKG2 KO mice responded equally to DSS treatment, but the PKG2-deficient animals exhibited crypt hyperplasia and increased apoptosis in luminal epithelium relative to wild type animals. Taken together these findings suggest that PKG2 has a tumor suppressive role in the colon epithelium, and that reducing the proliferative compartment may be part of the mechanism.

#1263

TGF-ß inhibition as a novel strategy to prevent breast cancer brain metastases.

Mark V. Pinti. _West Virginia University, Morgantown, WV_.

Background: Incidence of breast cancer brain metastases (BCBM) is on the rise due to improved management of systemic disease and ranges from 15-20% in women with disseminated breast cancer. Common chemotherapeutic strategies have limited efficacy for brain metastases due to the presence of the blood brain barrier (BBB). Prevention of metastasis after diagnosis of primary breast tumors may lead to new treatment strategies. TGF-ß is a biomolecule in the metastatic pathway and inhibition of its expression can be achieved by treatment with the antihypertensive agent Losartan. Herein we hypothesized that treatment with Losartan will decrease metastatic burden and improve survival in a preclinical model BCBM.

Methods: Athymic female nude mice were separated into four groups as follows: Vehicle, (saline, 50µL/day), SB431542 (Inhibitor, 5mg/kg/day), Low Dose Losartan (10mg/kg/day) and High Dose Losartan (25mg/kg/day). On day 7 mice were intracardially injected with 175,000 cells of either JIMT-1-Br-LUC-GFP (HER2+) or MDA-MB-231Br-LUC (TNBC) breast cancer cell lines. Tumor burden was monitored once weekly utilizing bioluminescent imaging. Mice were allowed to progress until neurological symptoms arose. Following euthanasia, brains were extracted, sectioned and analyzed to determine number of metastatic lesions and size.

Results: With regard to survival, no significance was shown in the JIMT-1 (HER2+) metastatic model. The MDA-MB-231Br (TNBC) model demonstrated an increase in survival in all treatment groups compared to vehicle. End time-point analyses showed no difference in metastases number in either model, however, the low dose treatment group in the MDA-MB-231Br model had a smaller brain burden of metastases than high dose treatment as well as inhibitor treatment.

Conclusions: Losartan reduced metastatic seeding in the MDA-MB-231Br (TNBC) brain metastasis model, but had little to no effect in the JIMT-1 (HER2+) model.

#1264

Commensal microbiota is involved in anti-PD-1 mAb-mediated antitumor activity in a preclinical tumor model.

Fei Chen, Jian Ding, Xiangchao Gu, Juan Zhang, wenqing Yang, Qian Shi. _CrownBio, Taicang, Jiangsu Province, China_.

Background: A number of studies have shown that the gut microbiome may influence anti-tumor immune responses via innate and adaptive immunity, and the recent work in mice has highlighted the key roles of the gut microbiota in mediating tumor responses to chemotherapeutic agents and immunotherapies targeting PD-L1, PD-1 or CTLA-4. Multiple studies have demonstrated that the gut microbiome can markedly influence the outcome of PD-1 blockade in mice and patients and several commensal have been proved to be associated with favorable clinical outcome. Considering complexity of intestinal micro-organisms, a larger panel or wider spectrum of microbiota species maybe involved in this process.

Methods: Since compositional differences in the microbiome may influence cancer development and responses to therapies, we sought to determine whether intestinal bacterial composition and abundances of the tumor bearing mice were associated with a specific treatment outcome of immunotherapy. To test this, we analyzed and compared an enrichment of intestinal microbiota of CT26 syngeneic tumor bearing mice before and after a series of anti-PD-1 mAb treatment were given.

Results The results demonstrating that some strains were robustly enriched and this enrichment was associated with tumor response to anti-PD 1 therapy, while as other strains were down regulated upon the checkpoint blockade.

Conclusions: Upon further characterization and confirmation, these intriguing findings could lead to identification of one or more microbiota strains that could have biomarker potentials or therapeutic values for cancer immunotherapy.

#1265

Protective efficacy of gut microbiome and microbial metabolites after rice bran consumption against colon tumorigenesis.

Akhilendra K. Maurya,1 Sushil Kumar,1 Rama Kant,1 Dileep Kumar,1 Bupinder Raina,1 Anita Yadav,1 Elizabeth P. Ryan,2 Komal Raina1. 1 _University of Colorado Denver, Skaggs School of Pharmacy and Pharmaceutical Sciences, Aurora, CO;_ 2 _Colorado State University, Department of Environmental and Radiological Health Sciences, Fort Collins, CO_.

Colorectal cancer (CRC) is the 2nd leading cause of cancer-related deaths in US. Therefore, practical interventions that reduce incidence of adenomatous polyps and/or prevent their progression to CRC are urgently needed. In this regard, several studies have reported the chemopreventive potential of dietary rice bran [a wasted 'by-product' of white rice (Oryza sativa) processing] against CRC. Given that high heat can stabilize rice-bran for safe human consumption, our research efforts are evaluating 'stabilized-rice bran', as a functional food for CRC control and prevention. Rice bran has been previously shown to increase colonization of native gut probiotics and undergo microbiota fermentation. However, little is known about rice bran constituents as prebiotics, microbial substrates that promote healthy gut microbiota, and how these microbes produce novel, bioactive microbial metabolites. In this regard, our studies show that native gut probiotic - fermented rice bran extracts have strong in vitro activity against CRC cells and significantly decrease their colonosphere formation (a stem cell characteristic). Rice bran (fermented and non-fermented) also strongly inhibits TNF alpha-induced NF-kappa B activation and interleukin (IL)- 4-induced expression of CD44, which along with its variant form (CD44 v3-v6) plays an essential role in the adhesion and infiltration of inflammatory cells in CRC. Notably, even the stool extracts from rice bran-fed CRC survivors showed increased inhibitory effects on CRC cell viability compared to the stools collected from the same individuals prior to rice bran consumption. Next, extensive in vivo investigations were performed to determine the relative efficacy of dietary rice bran-modified gut microbiota and microbiota-fermented rice bran on inflammation associated-colon tumorigenesis. First, Bifidobacterium Longum -fermented (fermented with selected rice bran responsive microbe that was induced during rice bran feeding clinical trials) and non-fermented dietary rice bran were fed to conventionally-raised or germ-free mice and evaluated for their comparative efficacy against azoxymethane (AOM)/ dextran sodium sulphate (DSS)-induced CRC. In second set of studies, we 'humanized' gut of germ-free mice with stool inoculum from rice bran feeding human clinical trials and evaluated their efficacy against AOM/DSS-induced CRC. In conventional mice study, both the rice bran diets showed protective efficacy against CRC. Interestingly, germ-free mice having undergone transfaunation with rice-bran modified stool inoculum showed significantly less cancerous lesions compared to mice humanized with control stool inoculums, indicating that rice bran has the potential to re-structure the 'high risk' gut microflora of CRC patients towards the colonization of populations with protective effects against CRC growth and progression.

#1266

Downregulation of PD-L1 by NSAID administration augments the effects of a multi-antigen vaccine for the prevention of adenomatous polyps in APC(Min/+) mice.

Mary L. Disis,1 Lauren R. Corulli,1 Clinton Grubbs,2 Ronald A. Lubet,3 Paul Cowan,1 Ekram Gad1. 1 _University of Washington, Seattle, WA;_ 2 _University of Alabama, Birmingham, AL;_ 3 _National Cancer Institute, Bethesda, MD_.

Introduction: We have shown that treatment of APC(Min/+) mice with NSAIDs will inhibit development of adenomatous polyps and induce significant levels of polyp-infiltrating CD8 T-cells. We sought to identify the mechanism of NSAID induced immune stimulation and questioned whether administration of NSAIDs concurrent with vaccination could further reduce polyp formation.

Methods: PD-L1 expression and T-cell infiltrates were assessed by IHC and flow cytometry. MC38 and RKO (murine/human colorectal carcinoma cell lines) were treated with naproxen (200uM-1000 uM) and harvested at 24, 48 and 72h. At 4-6 weeks, APC(Min/+) mice were given a multi-antigen peptide vaccine (COX2, CDC25B, EGFR) with CFA/IFA or adjuvant alone. Two groups received 400ppm naproxen orally 7d/wk for 18 weeks. Polyps were quantified at ≤24 weeks. Spleens and polyps were collected for IFN-gamma ELISPOT, flow cytometry, and IHC.

Results: Polyps from the APC(Min/+) mouse and both cancer cell lines highly express PD-L1. PD-L1 expression was significantly decreased in MC38 (p<0.01) and RKO (p<0.01) as compared to control after incubation with naproxen at all doses. The inhibitory effect of NSAIDs on PD-L1 expression was both time and dose dependent. We evaluated the in vivo effect of combination immunoprevention in the APC(Min/+). Animals receiving vaccine alone showed a 33% inhibition of polyp formation while naproxen alone showed 54% inhibition (p<0.0001) compared to adjuvant alone. Combination treatment demonstrated significantly greater inhibition of polyps than either modality (p<0.001), 81% inhibition vs. adjuvant. Antigen specific T-cells could be detected at higher levels than control antigen in both the vaccine alone (p=0.0001) and combination-treated animals (p<0.0001). The antigen specific responses seen in combination treated animals were nearly two-fold that of vaccine alone (p=0.008). No antigen specific immunity was detected in naproxen and adjuvant only treated mice. The magnitude of the immune response was significantly correlated with lower polyp counts, with a Pearson correlation coefficient of -0.55 (p=0.0014). Detailed immunologic analysis of tumors will be presented.

Conclusions: NSAIDs, via modulation of PD-L1, synergize with vaccines to increase immunogenicity and enhance influx of polyp infiltrating lymphocytes. This synergy results in superior prevention of polyp formation compared to treatment with NSAID or vaccine alone and has high potential for clinical application.

#1267

A novel mitochondria-based targeting to restore therapeutic responsiveness in cisplatin- and geftinib-resistant human lung cancer cells.

Liyuan Yin,1 Gina Chia-Yi Chu,1 Ruoxiang Wang,1 Anisha Madhav,1 Lijuan Yin,1 Neil Bhowmick,1 Haiyen E. Zhau,1 Qinghua Zhou,2 Leland W.K. Chung1. 1 _Cedars-Sinai Medical Center, Los Angeles, CA;_ 2 _Sichuan Lung Cancer Institute,Sichuan Lung Cancer Center, Chengdu, China_.

BACKGROUND: Cisplatin and tyrosine kinase inhibitors (TKIs) are recommended to treat non-small-cell lung cancer (NSCLC). However, ubiquitous acquired drug resistance in NSCLC patients diminishes their therapeutic efficacy. Overcome cisplatin and TKIs resistance is an unmet medical need. DZ-191, a novel synthetic statin derivative, is capable of targeting specifically tumor cells, bypassing the liver. We observed this agent inhibited tumor cell growth in vitro, tumor growth in vivo and accelerated tumor regression in mice.

METHODS: Cell viability was examined by crystal violet assay in human NSCLC A549, A549DDP, H1650, and H1975 cells. DZ-191-induced apoptosis in NSCLC cells was detected by Annexin V-FITC/PI staining followed by FACS and confirmed with western blot of apoptosis-associated proteins. Mitochondrial membrane potential was determined by rhodamine-123 followed by flow cytometry. H1650 tumor growth in mice was assessed and autophagy markers were analyzed by western blotting.

RESULTS: Treatment with DZ-191 alone inhibited the growth of A549, H1650 and H1975 NSCLC cells, with IC50 values in the range of 8±1μM. In the cisplatin-resistant A549DDP cells, DZ-191 exposure at the IC10, markedly sensitized the cisplatin-resistant A549DDP cells by lowering its IC50 value from 87.6±1.0 to 13.6±1.2 μM. We observed DZ-191, but not statins, significantly increased the apoptosis of A549DDP and H1650 cells. These results are consistent with the observation that DZ191, but not statins, accumulates in mitochondria and lysosomes, resulting in depolarized mitochondrial membrane potential, decreased autophagy and lysosomal protein degradation. We observed decreased autophagy markers, assessed by decreased LC3 conversion and increased p62 accumulation. Combining with our RNA-seq data, DZ191 could induce NSCLC cell death by reducing the removal of damaged mitochondria through mitophagy. Furthermore, we observed DZ-191, but not statins, could resensitize anti-tumor responses of geftinib in a TKI-resistant NSCLC tumor xenografts in nude mice.

CONCLUSIONS: These results demonstrated that DZ-191 is superior to statins in inhibiting and resensitizing the anti-tumor responses of cisplatin- and geftinib-resistant NSCLC cells by targeting mitochondria-mediated autophagy and downstream lysosome-related protein degradation. DZ-191 can be employed as a promising sensitizer to overcome cisplatin- and geftinib-resistance in NSCLC patients.

Key words: DZ-191, statins, cisplatin- geftinib-resistance, autophagy, NSCLC, mitochondria, lysosomes

This work is support in part by US NCI P01 CA098912 and a Board of Governors Chair

of Cancer Research fund from Cedars-Sinai Medical Center to LWK Chung and China National 863 Lung Cancer Research Grant (2012AA02A502) and International Technology Targeted Therapeutics Grant (2016YEE0103400) to Q. Zhou

#1268

Anti-PD-1 monotherapy outperforms multiple immunotherapy combination strategies in an oral cancer prevention mouse model.

Jose Augusto Monteiro de Oliveira Novaes, Marlese A. Pisegna, Alissa Poteete, Fahao Zhang, John V. Heymach, William N. William. _MD Anderson, Houston, TX_.

Oral premalignant lesion (OPL) expression of PD-L1 is associated with increased cancer risk. These data suggest that immune evasion is already present at the OPL stage and warrant an evaluation of immune modulatory therapies for cancer prevention. We conducted this preclinical study in a carcinogen-induced mouse model of oral cancer to evaluate the efficacy of multiple immunomodulatory strategies, given at a time point when only OPLs are present, to reduce the incidence of oral squamous cell carcinoma (OSCC).

We treated C57BL/6 mice with the carcinogen 4-nitroquinoline 1-oxide (4-NQO) for 8 weeks in drinking water. Eight weeks after discontinuing 4-NQO treatment, a group of mice (N=6) was sacrificed for assessment of invasive and non-invasive tongue lesions. A second group of mice was treated with either anti-PD-1 (N=40), anti-CTLA-4 (N=20), anti-OX40 (N=20), anti-PD-1 + anti-CTLA-4 (N=20), anti-PD-1 + anti-OX40 (N=20) antibodies, or IgG (N=40) for a total of 3 doses every 3 days, initiating 8 weeks after the cessation of 4-NQO. Antibodies used in combination were given on the same day . Mice were sacrificed 56 days after the last dose of treatment. We assessed serial H&E-stained cross sections of the tongues harvested at that same time point for development of OPLs and cancer and measured the OSCC area of each tongue using aperio imagescope software. Mann-Whitney and Fisher's exact tests were used for statistical comparisons between groups.

Eight weeks after 4NQO cessation, 100% of mice developed tongue hyperplasia or dysplasia and invasive lesions were not identified, indicating this to be an ideal time point to initiate treatment strategies addressing OPLs. Tongues from mice treated with anti-PD-1 antibody displayed a decrease in the mean OSCC area when compared with mice treated with IgG (mean 3.53 mm2 versus 6.62 mm2, respectively; p= 0.018). At this time point, invasive oral cancers had developed in 75% versus 50% of IgG and anti-PD-1 treated mice, respectively (p=0.03). There were also non-significant decreases in the mean OSCC area in tongues from mice treated with anti-CTLA-4 (mean = 5.07 mm2), anti-OX40 (mean = 3.79 mm2) and anti-PD-1 + anti-CTLA-4 (mean = 3.86 mm2) antibodies when compared to mice treated with IgG (mean = 6.62 mm2) control. In the group treated with anti-PD-1 + anti-OX40 combination therapy, there was an increase in the mean OSCC area when compared to anti-PD-1 monotherapy (mean 3.53 mm2 versus 9.03 mm2), respectively (p= 0.01).

Short-term anti-PD-1 immune checkpoint inhibitor therapy in the context of OPLs led to a reduction in the incidence of oral cancer. When comparing the mean area of OSCC in each group, anti-PD-1 monotherapy was superior to IgG and all other treatment strategies. Paradoxically, combining anti-OX40 and anti-PD-1 antibodies created an antagonizing effect on the therapy and increased total tumor burden when compared with anti-PD-1 monotherapy.

#1269

Diosgenin, a naturally occurring steroidal saponin, prevents colon cancer in animal models of hereditary and sporadic CRC.

Venkateshwar Madka,1 Jayadev Raju,2 Gopal Pathuri,1 Snigdha Smriti,1 Anh Bao,1 Yuting Zhang,1 Nicole Stratton,1 Mudassir Farooqui,1 Stanley Lightfoot,1 Adam S. Asch,1 Chinthalapally V. Rao1. 1 _Univ. of Oklahoma Health Sciences Ctr., Oklahoma City, OK;_ 2 _Health Canada, Ottawa, Ontario, Canada_.

Colorectal Cancer (CRC) affects more than 1.35 million people worldwide annually. In the US it is anticipated ~140,000 new cases and 50,000 deaths for the year 2017. In spite of existing preventive measures, recent data suggest an increase in the number of CRC incidences in the younger age groups (30 to 55 years) urging the need for better preventives. In the present study we have studied, Diosgenin against colon cancer in APCmin/+ mice and azoxymethane (AOM)-induced rat CRC models. Diosgenin is a steroid saponin with proven diverse medicinal properties, including anti-diabetic, antiobesity and antiinflammatory, and anticancer properties. APCmin/+ mice, a hereditary polyposis mouse model, were bred in-house and starting at 6 weeks age they were given AIN7-6A diets containing diosgenin (0, 500 and 1,000 ppm) for 14 weeks. At 20-wks of age all mice were euthanized and intestines, both small and large, were analyzed for tumors. In the sporadic CRC model, colon tumors were induced in the male F344 rats by two weekly s.c. injections of AOM (15mg/kg BW) followed by diosgenin administration (0 and 1,000ppm) for 38 weeks. Dietary diosgenin showed significant suppression of colon tumor incidence and multiplicity in both models. In APCmin/+ mice both small intestinal polyps (SIP) and colon tumors (CT) were suppressed in a dose-dependent manner with a significant effect in the high-dose treatment. Diosgenin administered female mice had 33% (25±4.1; Mean±SE, p<0.044) and 54% (17.1+3.8; p<0.003) inhibition of SIP as compared to control mice (37.3±5.6); in male mice there was a greater inhibitory effect (p<0.0001) with 53% (14.1±1.8;) and 65% (10.7±1.7; p<0.0001) suppression of the SIP at 500 and 1,000 ppm respectively, as compared to control mice (30.4±2.3). Colon tumor multiplicity was also inhibited by 65% (0.45±0.24; p<0.05) and 50% (0.5±0.18; p>0.05) with high dose in both male and female mice as compared to their respective controls (1.3±0.20 and 1.0±0.25). Importantly, diosgenin showed a similar inhibitory effect on AOM-induced colon adenocarcinoma incidence and multiplicity in the rat model. Administration of diosgenin significantly suppressed both invasive and non-invasive colon adenocarcinoma incidence by 60% (p<0.005) and multiplicity by 65 % (p<0.0001). HPLC analysis of the intestinal contents of diosgenin suggested that more than 50% reached the colon in its active form. Biomarker analysis suggested that the strong chemopreventive effects are due to the suppression of proliferation (PCNA) and pro-inflammatory proteins (COX-2 and iNOS) with an increase in apoptosis (Casp-3). Based on these results from the preclinical models representing both hereditary and sporadic CRC patients, it is highly imperative to conclude that diosgenin is a potential chemopreventive agent for colon cancer prevention which needs to be further investigated in the clinic. (Endowed Chair Funds from CVR/ASA)

#1270

Potential metabolic and molecular mechanisms of black raspberry-mediated oral cancer chemoprevention.

Steve Oghumu, Thomas J. Knobloch, Logan C. Weghorst, Lei Bruschweiler-Li, Cheng Wang, Rafael Bruschweiler, Christopher M. Weghorst. _Ohio State University, Columbus, OH_.

Oral cancer accounts for about 50,000 new cases and 10,000 new deaths in the U.S. every year. This amounts to about one person every hour every day. To combat this public health challenge, there is a need to identify agents that prevent oral cancer development and fully characterize their mechanisms of action. Preclinical and clinical studies demonstrate the ability of black raspberries (BRBs) to inhibit oral carcinogenesis. We recently showed that in an experimental model of rat oral carcinogenesis using the carcinogen 4 nitroquinoline-1-oxide (4NQO), BRBs reduce oral lesion incidence and multiplicity. However, understanding how the bioactive compounds in BRBs drive the metabolic and molecular pathways that lead to oral cancer chemoprevention remains unclear. In this study, we determined the potential metabolic and molecular mechanisms associated with BRB-mediated chemoprevention of oral carcinogenesis using the well-established carcinogen-induced rat oral cancer model. Male F344 rats were divided into 4 groups: sentinel group, 4NQO only group, 4NQO + 5% BRB group, and 4NQO + 10% BRB group. 4NQO was administered in drinking water (20ug/mL) for 10 weeks after which regular drinking water was provided for 6 weeks. BRBs were incorporated into rat diets at 5% or 10% concentrations and fed to the rat treatment groups for the last 6 weeks after 4NQO administration. Sentinel animals did not receive 4NQO or BRB. At terminal sacrifice, urine samples from all groups were harvested and analyzed by NMR-based metabolomics. We identified metabolites in rat urine samples by "Complex Mixture Analysis by NMR" (COLMAR) followed by 2D 13C-1H HSQC NMR quantitative analysis. RNA was extracted from rat tongue samples and used for RNA sequencing analysis. 4NQO only administered rats showed highest overall intensity, which was about 2.5 times higher than 4NQO + BRB treated rats. 171 metabolites were identified in urine samples of 4NQO only administered rats while 101 and 90 metabolites were identified in 4NQO administered rats that were treated with 10% and 5% BRB, respectively. About 31 "unique" metabolites were identified in 4NQO only administered rat urine samples but were absent in BRB treated groups. Fourteen additional metabolites were increased and 11 metabolites were decreased following BRB treatment of 4NQO administered rats compared to rats administered with 4NQO only. Our metabolomic and transcriptomic analyses reveal distinct molecular and metabolic markers and pathways that drive oral carcinogenesis and BRB-mediated oral chemoprevention, which can potentially be exploited in oral cancer treatment strategies.

#1271

Restricted feeding of a high-fat diet to the dark phase of the day attenuates mammary tumorigenesis in MMTV-PyMT mice.

Sneha Sundaram, Lin Yan. _Grand Forks Human Nutrition Research Center, ARS, USDA, Grand Forks, ND_.

Obesity is a risk factor for breast cancer. Chronic overeating during the rest phase of the day leads to metabolic dysfunction and obesity. Lifestyle changes including restriction of meal times may be successful weight loss strategies. In the present study, we tested the hypothesis that restricted feeding of a high-fat diet to the dark phase (the active phase) of the day reduces diet-enhanced mammary tumorigenesis in an MMTV-PyMT transgenic mouse model. Mice (female) were fed the AIN93G diet (16% of energy from soybean oil) or the high-fat diet (45% of energy from soybean oil) with or without restricted feeding to the dark phase (12 hours) over eight weeks of the study. The body fat mass of the restricted feeding group was lower than the high-fat group but similar to the AIN93G control group. The mammary tumor latency was 5.8, 7.0, and 6.4 weeks for the control, high-fat and high-fat restricted groups, respectively. Restricted feeding reduced mammary tumor progression by 58% compared to the unrestricted feeding of the high-fat diet. Compared to the AIN93 diet, the high-fat diet significantly increased plasma concentrations of plasminogen activator inhibitor-1, monocyte chemoattractant protein-1, resistin, leptin, insulin, angiopoietin-2, hepatocyte growth factor and vascular endothelial growth factor. The restricted feeding reduced concentrations of the aforementioned variables in plasma, except resistin, leptin, and insulin. In summary, restricted feeding of the high-fat diet to the dark phase reduces diet-enhanced mammary tumorigenesis. This protection of the restricted feeding against breast tumorigenesis may be associated with its actions in attenuating adipogenesis and production of related inflammatory cytokines and angiogenic factors.

#1272

Docosahexaenoic acid supplementation enhances the response of PC-3 prostate cancer cells to enzalutamide.

Irvin V. Ma,1 Andrew R. Cooper,1 Ying Hu,2 Arianne G. Sorreta,1 Palvinder K. Bains,1 Prem Kumar,1 Ronald S. Pardini1. 1 _University of Nevada, Reno, Reno, NV;_ 2 _Beijing Chest Hospital, Beijing, China_.

Prostate cancer is currently the second leading cause of death from cancer among men in the United States. Initial treatments generally focus on hormone therapy to inhibit the production of androgens, but most prostate cancers eventually become castration resistant and no longer respond to androgen-suppression therapy. The recent development of drugs that inhibit androgen binding to the androgen receptor (AR) have proven effective in treating castration-resistant prostate cancer (CRPC). Studies in our laboratory have shown that docosahexaenoic acid (DHA; C22:6 n-3), a long chain omega-3 polyunsaturated fatty acid (PUFA), inhibits tumorigenesis in both androgen-dependent (LNCaP) and androgen-independent (PC-3, DU145) prostate cancer cell lines in culture. Moreover, in vivo studies with n-3 PUFA supplementation have been shown to significantly reduce tumor growth of PC-3 and DU145 compared to corn oil diets rich in linoleic acid, (C18:2, n-6). With evidence suggesting that n-3 PUFA dietary supplementation can inhibit tumorigenesis in CRPC, the current study investigated the impact of DHA supplementation in combination with enzalutamide in PC-3 tumor cells. Enzalutamide is a Food and Drug Administration-approved treatment for patients with metastatic CRPC. Enzalutamide acts by interrupting the binding of androgens to the AR, which influences the AR signaling pathway--a target of interest in CRPC treatment. Cell viability was determined by measuring intracellular ATP in PC-3 cells treated with DHA and enzalutamide alone and in combination. Cell viability was reduced with the combination of DHA and enzalutamide compared to enzalutamide or DHA alone, suggesting that DHA enrichment can augment enzalutamide therapy with PC-3 cells in vitro. Studies have also shown that combinatorial treatments with enzalutamide and HIF-1α inhibitors result in synergistic inhibition of CRPC tumor proliferation, and our laboratory has shown that DHA inhibits HIF-1α by decreasing intracellular ATP in breast and lung cancer cells in vitro. These results serve as a prelude to a pilot clinical trial to evaluate the effects of nutritional supplementation with high levels of omega-3 fatty acids on enzalutamide inhibition of CRPC.

#1273

Effect of a Western diet on colitis-associated colon tumor formation in BALB/c mice.

Charlotte Groeschel,1 Samawansha Tennakoon,1 Abhishek Aggarwal,2 Enikoe Kallay1. 1 _Medical Univ. of Vienna, Vienna, Austria;_ 2 _Stanford University, Stanford, CA_.

Patients with inflammatory bowel disease (IBD) are at increased risk for inflammation associated colorectal cancer (CAC). The incidence of IBD is increasing in the Western industrialized world. Epidemiological studies suggest that a diet, high in fat but low in vitamin D, calcium, fibre and methyl donors is a risk factor for both, IBD and colorectal cancer (CRC). It has been shown previously that a "New Western Diet", caused development of sporadic colonic tumours in mice.

Our study was performed to evaluate whether a Western Diet (WD) aggravates azoxymethane (AOM) /dextran sodium sulfate salt (DSS)-induced colitis-associated carcinogenesis in mice, and if a switch to the standard purified mouse diet AIN93G after cancer initiation is still able to ameliorate or delay the onset of the disease. Female BALB/c mice received either the WD (0.5 mg/g calcium, 2% fibre, 21 % butter fat, 0.23 μg/g folic acid) or AIN93G standard diet for 5 weeks until AOM was administered at a dose of 12.5 μg/kg intraperitoneally, followed by 3 cycles of DSS (2.5%). In one group the WD was switched to AIN93G diet one day before first administration of DSS. The mice were euthanized 80 days after AOM injection. One part of the colon, small intestine, kidney and liver were frozen in liquid nitrogen, the rest formalin fixed and paraffin embedded for mRNA and immunhistochemical analysis. Feeding constantly the WD shortened the colon (p<0.05) and increased the number (p<0.05) and size (p<0.01) of aberrant colonic crypt foci. The switch to the AIN93G diet ameliorated this effect, leading to reduced colitis and tumor promotion comparable with the AIN93G group. In colon ascendens of the WD group, Ki67 protein levels (p<0.001) and relative mRNA levels of the Wnt target genes c-myc (p<0.05) and axin2 (p<0.001) were reduced while mRNA levels of the vitamin D catabolizing enzyme cyp24a1 (p<0.001) and inos (p<0.05) were increased when compared with the AIN93G group.

A healthy diet protects colonic mucosa against AOM/DSS-treatment and is an effective chemopreventive strategy to reduce chronic colonic inflammation and tumorigenesis.

This project is funded by the Vienna Science and Technology Fund and the Austrian Research Fund.

#1274

Effects of diet on lung adenoma carcinogenesis in metformin treated A/J mice.

Vannesa Souksavong, Beverly R. Wuertz, Donna Seabloom, Frank G. Ondrey. _Univ. of Minnesota, Minneapolis, MN_.

There has been considerable interest in the effects of biguanides, utilized to treat type 2 diabetes, on human carcinogenesis. Several population studies established diabetes patients treated with metformin and other type 2 diabetes agents experience decreased cancers in several organ systems. However, the effects are not universal in all organ systems and metabolically complex. In the present study, we utilized a dosing regimen of dietary metformin in benzo[a]pyrene treated mice known to reduce lung adenoma formation in A/J mice by approximately 50% over controls. For the duration of this study we used several animal diets commonly employed for mouse chemoprevention experiments with and without 850 mg/kg/day metformin added to the diet. These included AIN93M, AIN76A, D62 (Dr. Lee Wattenberg's chemoprevention diet), a modification of D62 with added maltodextrin and sucrose, and Teklad 4% fat pellets from Envigo. At the conclusion of the study, animals were sacrificed and surface adenoma counts were performed on fresh lungs from each group of animals. We found 850 mg/kg/day metformin for 16 weeks reduced adenoma formation by 35-60% in the diets we employed for the study. Interestingly, the Teklad diet which contains dehulled soybean meal had significantly reduced tumor formation in the control animals, demonstrating some animal diets provide chemoprevention efficacy independent of any pharmaceutical effects. There was no observed toxicity of metformin in the animals; however we have observed significant differences in the average end weights of animals treated with metformin. We conclude metformin is an efficacious agent in lung adenoma chemoprevention and similar reductions in adenoma formation occurred in several of the diets commonly employed for chemoprevention studies in B[a]P treated mice. However, attention to diet formulation is vital due to potential chemoprevention and metabolic effects independent of metformin treatment.

#1275

A role for FGF2 in visceral adiposity-associated mammary epithelial transformation.

Vanessa Benham, Debrup Chakraborty, Blair Bullard, Jamie J. Bernard. _Michigan State Univ., East Lansing, MI_.

Obesity is a leading risk factor for post-menopausal breast cancer, and this is concerning as 38% of Americans are obese. Furthermore, 40% of cancer diagnoses in 2014 were associated with overweight/obesity. Despite the epidemiologic link between obesity and breast cancer risk, the underlying mechanism responsible is unknown. We recently published that VAT releases FGF2 and stimulates the transformation of skin epithelial cells. As obesity is differentially associated with many cancer types, this mechanistic link could be translational to other cancers. Specifically, FGF2 and FGFR1 are implicated in breast cancer progression, but their role in tumor formation is unknown. Therefore, I hypothesize that VAT-derived FGF2 plays a translational role in promoting mammary tumor formation.

#1276

Differentiation of human colon adenomas in an enteroid culture: A treatment comparison.

Shannon D. McClintock, Justin A. Colacino, Durga Attili, Michael K. Dame, Areeba H. Rizvi, Aliah Richter, Anusha R. Reddy, Venkatesha Basrur, D. Kim Turgeon, James Varani, Muhammad N. Aslam. _University of Michigan, Ann Arbor, MI_.

Introduction: Previous murine studies have demonstrated that dietary Aquamin®, a calcium-rich, multi-mineral natural product, suppressed colon polyp formation and transition to invasive tumors more effectively than calcium alone when provided over the lifespan of the animals.

Methods: In the present study, we compared Aquamin® to calcium for modulation of growth and differentiation in human colon adenomas in enteroid culture. Adenoma growth and differentiation were assessed at the light and electron microscopic levels and by immunostaining. Image quantitation of immunohistochemical markers was performed using Aperio Imagescope to compare these interventions. A proteomic assessment was done to compare protein expression patterns in treated cultures as compared to cultures maintained under control conditions (calcium 0.15 mM). Enteroids established from normal colonic tissue were examined in parallel.

Results: Both calcium and Aquamin® provided at 1.5 mM calcium fostered differentiation in the adenoma enteroid cultures as compared to control, but Aquamin® was more effective. Even at a concentration providing only 0.15 mM calcium, Aquamin® induced differentiation in some individual enteroids. Aquamin® was more effective than calcium in inducing upregulation of proteins. There were 35 proteins upregulated with Aquamin as compared to 20 with calcium 1.5mM at 1.8-fold change or above (in 3 colonic adenomas) with less than 2% FDR. Both calcium and Aquamin® induced differences in the expression pattern of proteins known to be involved in differentiation (table). In addition, several keratins and histones, as well as merlin and Olfactomedin-4 were upregulated.

Conclusion: These findings support that i) calcium (1.5 mM) has the capacity to modulate growth and differentiation in large human colon adenomas and ii) the additional trace elements provided along with calcium in Aquamin® can have effects on proliferation and differentiation at lower levels than observed with calcium at 1.5mM. | |  | |

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

Expression of Immunohistochemical Markers in enteroid cultures of Human Adenomas (Fold-Changes)

IHC Markers | Calcium 0.15mM | Aquamin 0.15mM | Calcium 1.5mM | Aquamin 1.5mM

Ki67 | 1 | 0.75 | 0.59* | 0.49*

CK20 | 1 | 2.13* | 2.36* | 2.35*

E-Cadherin | 1 | 1.05 | 1.57* | 1.57*

NF2 (Merlin) | 1 | 2.44*# | 1.01 | 2.56*#

Occludin | 1 | 1.23* | 1.25* | 1.29*

*reflects significance as compared to Ca 0.15mM. #reflects significance as compared to Ca 1.5mM

#1277

3-Isothiocyanato-1-propene as biomodulatory agent in ameliorating phenobarbital induced alterations in profile of detoxifying enzymes in different metabolic sites of Wistar rats.

Saroj Arora, Sakshi Bhushan, Adarsh Pal Vig, Pardeep Kaur, Robin .. _Guru Nanak Dev University, Amritsar, India_.

Backgound: Human exposure to xenobiotics is responsible for increased susceptibility to genetic and epigenetic changes. These changes are further accountable for disruption of pathways involved in cell proliferation, apoptosis, differentiation and senescence. Among array of plant products, that play an imperative role against such adverse catastrophes, the naturally occurring isothiocyanate known as 3-Isothiocyanato-1-propene (AITC) possesses abundant biological activities of therapeutic interest.

Purpose of the study: Hence, considering these facts, the present study was planned to analyse four metabolic sites viz., liver, kidneys, stomach and lungs for protective activities of AITC against phenobarbital induced damage in Wistar rats.

Methodology: The rats were divided into eight groups and treatments were given intraperitoneally for five days followed by sacrificing of animals on sixth day. The organs were homogenized and analysed for biochemical parameters and histopathology.

Results: AITC in all the metabolic sites attenuated the levels of cytochrome dependent oxygenases, phase II detoxifying enzymes (DT-diaphorase, γ-glutamyltranspeptidase and glutathione-S-transferase), cascade of antioxidant enzymes (superoxide dismutase, catalase, ascorbate peroxidase, guaiacol peroxidase), membrane bound ATPases (Na+/K+-ATPase, Mg2+-ATPase, Ca2+-ATPase) and serum marker enzymes (SGOT, SGPT, direct bilirubin, indirect bilirubin, alkaline phosphastase, creatinine, blood urea nitrogen). Futhermore, the different doses of AITC (50, 100 and 150 µM/kg bw) resumed the necroinflammatory score by improving the piecemeal necrosis, focal lytic necrosis and portal inflammation.

Conclusion: The maximum efficacy of amelioration by AITC was observed in liver due to the fact that amount of these enzymes in metabolic sites other than liver is quantitatively low. Hence, AITC emerged as potent candidate for ameliorating the harmful effects of non genotoxic carcinogen (phenobarbital) by regulating its metabolism efficiently in rats. Owing to its useful functions, AITC can act as an important bioprotective agent. 

## BIOINFORMATICS AND SYSTEMS BIOLOGY:

### Application of Bioinformatics to Cancer Biology 1

#1278

Deciphering intratumor heterogeneity at single-cell resolution in multiple myeloma.

Ali Mahdipour-Shirayeh, Rodger E. Tiedemann. _University Health Network, Toronto, Ontario, Canada_.

Single-cell RNA sequencing (scRNA-seq) is a powerful tool for the examination of transcriptional heterogeneity in cancer cells. MM is a mature B cell neoplasm that, despite advances in therapeutic strategies, remains incurable and over-represented in cancer death rates. The absence of cure in this disease, despite deep treatment responses, suggests that rare tumor cells are able to escape conventional therapy to cause relapse. We are using 10X Genomics 3' scRNAseq to study intratumor heterogeneity in multiple myeloma (MM). To characterize heterogeneity among MM cancer cells within a mixed population of tumor and nontumor cells, tumor cells must first be identified as such. As most single-nucleotide variations (SNVs) are subclonal in MM and are not captured by the 10X 3' platform, identification of SNVs is not a feasible solution; moreover, as normal cells can express oncogenes while senescent cancer cells may suppress oncogenes, reliance on a pure gene expression approach is unjustified. In contrast, many chromosomal copy number variations (CNVs) in MM arise early in disease biology and are clonal. We therefore developed methods to infer chromosomal CNVs across the genome of single cells. We successfully verified the karyotype predictions from our inferred-CNV (iCNV) analysis of single cells on several tumor samples by fluorescent in situ hybridization (FISH) studies and by whole-exome sequencing (WES) of pooled tumor cells. Using our iCNV data we developed a tumor likeness score that is applied to single cells and that represents the similarity of individual cells to the average tumor cell karyotype. Testing of our iCNV and tumor score methods on separate healthy control and tumor samples confirms that single cells can be assigned to tumor or nontumor compartments with extreme accuracy. These assignments correlate well with the use of clonal immunoglobulin IGH and IGL restriction as an alternate method to detect tumor cells; however, an iCNV tumor score approach has the advantage that it can readily be applied to non-B cell and non-T cell populations, unlike IG approaches. Using these methods we have studied 10 primary bone marrow samples from MM patients, separating clonal MM cells from nontumor plasma cells and B cells. Intraclonal heterogeneity of (20,000+) MM cells in gene expression, cell cycle, differentiation and CNV will be presented. With these data and analyses we aim to achieve a better understanding of tumor cell heterogeneity and cellular drug-resistance mechanisms in MM in order that curative therapeutic strategies can be developed.

#1279

Passenger mutation landscape in cancer genomes.

Sushant Kumar,1 Jonathan Warrel,1 Patrick Mcgillivray,1 William Meyerson,1 Shantao Li,1 Leonidas Salichos,1 Arif Harmanci,1 Alexander Fundichely,2 Calvin Chan,3 Carl Herrmann,4 Morten Nielsen,5 Lucas Lochovsky,1 Yan Zhang,6 Xiaotong Li,1 Ekta Khurana,2 Gad Getz,7 Mark Gerstein1. 1 _Yale University, New Haven, CT;_ 2 _Weill Cornell Medical Colege, New York, NY;_ 3 _Heidelberg University, Germany;_ 4 _German Cancer Research Center (DKFZ), Germany;_ 5 _Aarhus University, Denmark;_ 6 _Ohio State University, Columbus, CT;_ 7 _Massachusetts General Hospital, Boston, MA_.

Cancer progression is an evolutionary process during which thousands of somatic variants are accumulated within an individual. In the classic view of cancer progression, a handful of driver variants are thought to give a positive selection advantage to the cancer cell. The remaining variants, termed passengers, represent the overwhelming majority of the variants in cancer genomes, and their functional consequences are poorly understood. Furthermore, the bulk of these passengers fall within noncoding regions of the genome, making these the main product of whole-genome sequencing of tumors. In this work, we explore the functional landscape of passenger variants in various cancer cohorts by leveraging extensive pan-cancer variant calls from ~2500 uniformly processed whole cancer genomes. More specifically, we integrate their annotations and predicted functional impact scores to quantify the overall burdening of various elements in cancer genomes. We show that disruption of genetic regulatory elements in the noncoding genome correlates with altered gene expression. Furthermore, we also show how overall functional burdening of various genomic elements correlate with patient survival time, and tumor clonality. Finally, we observe statistical signals consistent with the notion that aggregated subsets of passenger variants - particularly those we predict to be functionally impactful- might confer weak selective effects.

#1280

Chromosome-scale haplotyping enables comprehensive discovery of cancer rearrangements and germline-related susceptibility mutations.

Stephanie U. Greer,1 Billy T. Lau,1 Lincoln D. Nadauld,2 Hanlee P. Ji1. 1 _Stanford Univ., Stanford, CA;_ 2 _Intermountain Healthcare, Salt Lake City, UT_.

To understand the complexity of cancer genomes, one needs to define both the structure and order of genomic variants relative to their respective parental chromosome. This process is commonly referred to as phasing and generates haplotypes with group of variants that originated from one parental chromosome or the other. Importantly, genetic variants such as rearrangements are difficult to detect and phase with conventional next-generation sequencing; short reads do not contain long-range genomic information for phasing. Recently, we demonstrated a new technology called linked-read sequencing, which uses droplet barcoding to tag high-molecular-weight (HMW) DNA in order to reconstruct genomic haplotypes and SVs in the range of megabases. Using single nanograms of DNA, we determined the structure of normal and cancer genomes at single-molecule resolution. In this study, we developed a method to generate haplotypes spanning entire chromosome arms and even across centromeric regions. The method is based on leveraging identity-by-descent (IBD) principles in haplotype-resolved sequence data. Taking advantage of shared haplotypes between an individual and a single parent, we exploited the fact that any single chromosomal homologue consists of a contiguous segment from one parent regardless of any recombination events that occurred during meiosis. By determining which of the two homologous segments in a haplotyped segment belongs to a parent, we can then stitch together the haplotypes that are physically contiguous in the child. Using phased haplotypes generated from linked-read sequencing, we improve on the experimentally generated haplotypes by at least an order of magnitude. As a control, we performed this type of analysis on a series of control genomes with comprehensive haplotype information. We achieved near-perfect concordance in the comprehensive phasing of these control samples regardless of which parental genome was used. We performed this analysis on linked-read whole-genome sequence data derived from familial cohorts with an inherited predisposition to cancer of unknown origin. For example, in one family predisposed to cholangiocarcinoma, we resolved the haplotype structure of entire chromosomes and segregated entire segments belonging to affected and unaffected individuals. A similar process was applied to the cancer samples originating from affected family members. The joint information was used to identify potential candidate driver genes for this cancer type. Overall, we demonstrated the utility of linked-read analysis and whole-chromosome haplotypes in discovering cancer genes.

#1281

Genomic structure variation in large screening for pediatric sarcoma therapy.

Lijun Cheng,1 Pooja Chandra,1 Limei Wang,1 Karen Pollok,1 Pankita Pandya,1 Mary Murray,1 Jacquelyn Carter,1 Michael Ferguson,1 Mohammad Reza,1 Mashall Mark,1 Lang Li,2 Jamie Renbarger1. 1 _Indiana University, Indianapolis, IN;_ 2 _Ohio State University, Columbus, OH_.

Complex chromosomal aberrations such as amplification and deletion of DNA copy number are frequently seen in sarcoma. Fifty-five DNA structure variation has been listed as standard clinical diagnosis for sarcoma by standard of National Comprehensive Cancer Network (NCCN). However, copy number variation (CNV) as a biomarker of drug treatment for pediatrics sarcoma is still unclear, especially for relapsed and high recurrent patients of pediatric sarcoma. The paper aims to detect the prognosis biomarkers for rhabdomyosarcoma, Ewing's sarcoma (ES), and osteosarcoma based on copy number variation for 128 FDA-approved cancer drugs systematically. The 182 copy number variation (CNV) profiles from clinical sarcoma patients across three types of sarcoma, including osteosarcoma, rhabdomyosarcoma and ES, are observed. Cox survival regression model is used to select significant cancer-related CNVs systematically by correlation with overall survival analysis. 532 significant common CNVs and 782 specific CNVs for different types of sarcoma are observed by p-value <0.01. By comparing with healthy persons, COSMIC database, 947 cancer cells and our INHOUSE tumor datasets, 32 out of 532 CNVs for specific sarcoma types are validated. This systematic CNV comparison makes it possible to map DNA copy number changes and identify chromosomal regions containing "target genes" responsible for tumor development and/or progression. Integrating large-scale drug screening with copy number variation analysis on 38 sarcoma cancer cells, 95 out of 532 are confirmed as drugs resistance and sensitive biomarkers for different types of sarcoma. 15 chemotherapies show strong signals connecting with 42 biomarkers, including genes MYC, RAD21, and RB1 amplification. By bridging molecular biomarkers between tumors and sarcoma cell lines, 5 optimum drugs associated with 14 enzyme biomarkers are recommended for pediatric rhabdomyosarcoma, ES, and osteosarcoma treatment respectively. The research not only detects copy number variations both of sarcoma cell lines and tumors, but also provides novel insights into drug biomarkers based on copy number amplification or deletion for bone sarcoma and soft tissue sarcoma, respectively.

#1282

Activity of APOBEC3A, APOBEC3B, REV1, UNG, and FHIT is associated with drug sensitivity in specific cancer subtypes.

Suleyman Vural, Julia Krushkal, Richard Simon. _NCI/NIH, Rockville, MD_.

We used gene expression information and mutational signature analysis of cell lines from a diverse range of cancerous tissues to examine the activity of two members of the APOBEC family of cytidine deaminases, APOBEC3A and APOBEC3B, and three additional genes. APOBEC3B is known to increase the mutation load in many cancers, generating kataegis clusters of closely spaced, single strand-specific DNA substitutions with a characteristic hypermutation signature. Some studies also suggested that APOBEC3A, REV1, UNG, and FHIT may also participate in hypermutation processes associated with APOBEC activity. We investigated how the activities of these five genes may affect the abundance of APOBEC-like signatures and whether they may be associated with sensitivity of cancer cells to treatment in different cancer categories. We analyzed several data sources for 1,408 cell lines from 26 cancer types available from the Cancer Cell Line Encyclopedia (CCLE) and the Genomics of Drug Sensitivity in Cancer (GDSC) resources. These data included whole exome sequencing (WES) data, microarray gene expression information, and drug sensitivity information. We examined correlations of the abundance of APOBEC-associated motifs and WES-wide mutation loads with APOBEC3A, APOBEC3B, REV1, UNG, and FHIT gene expression, as well as their association with cell line chemosensitivity to 255 antitumor drugs in multiple cancer subtypes. We were able to confirm several previously reported expression and chemosensitivity associations and found additional correlations, which may be clinically important. Sensitivity to JQ1, JNK inhibitors AS601245 and VIII, BMS-509744, bicalutamide, and several other agents was correlated with candidate gene expression levels or with abundance of APOBEC-like motif clusters in specific cancer categories such as pancreatic, breast and non-small cell lung cancer cell lines. For example, we observed a strong negative correlation between APOBEC3A expression in pancreatic cell lines and sensitivity to JQ1, a BET inhibitor, which had been reported to inhibit pancreatic cancer cells in vitro and in vivo. In glioma cell lines, APOBEC3B expression was significantly negatively correlated with sensitivity to a CDK9 inhibitor THZ-2-49 and HSP90 inhibitors AUY922 and 17-AAG (tanespimycin). We observed a strong correlation between the combined length of kataegis clusters and chemoresistance to bicalutamide, a nonsteroidal antiandrogen drug, in breast cancer cell lines. Our findings suggest that associations of sensitivity to drug treatment with activities of APOBEC3A/B, REV1, UNG, and FHIT and with the rates of APOBEC-associated mutagenic processes may vary among different cancer categories.

#1283

Integrative genome-scale analysis of TCGA dataset for features of tumor immunity.

Binchen Mao,1 Sheng Guo,1 Henry Li,1 Xinzhong Wang,2 Ying Pan2. 1 _Crown Bioscience, Inc., Santa Clara, CA;_ 2 _Cstone Pharmaceuticals, Shanghai, China_.

Background: Only a small portion of patients respond to immune checkpoint inhibitors (ICI), and the response rate varies among different cancer types. While an inflammatory tumor microenvironment (TME) is often believed to be critical for positive ICI response, the inflammatory status of different cancers and the molecular differences between the inflamed and non-inflamed tumors are yet to be elucidated.

Methods: We performed an integrative genome-scale analysis of TCGA dataset, using 9 emerging predictive biomarkers of ICI treatment to evaluate cancer immunity. The biomarkers include tumor mutation burden (TMB), innate anti-PD1 resistance (IPRES), immune infiltrated score (IIS), T-cell infiltrated score (TIS), cytolytic activity index (CYT), antigen presentation machine (APM), angiogenesis, and IFN-𝛾;;;; 18-gene (IFN18). We first analyzed the correlation between the biomarkers, then combined the representative biomarkers to classify tumors into 'inflamed' and 'non-inflamed' subtypes for comparison to identify prognostic significance of 'inflamed' tumor and associated genomic features.

Results: A total of 9338 tumors from 24 types of cancer were analyzed. Of all analyzed cancer types, non-small cell lung carcinoma and melanoma have the highest TMB, which was consistent with previous report. Renal cell carcinoma, despite the moderate TMB, has the highest cytolytic activity as determined by CYT, which is consistent with the descent response rate to ICI (25~46%). TMB was not strongly correlated with immunity aspects reflected by other biomarkers in most cancers, and IPRES was actually positively associated with T-cell inflammatory status in some cancers including bladder cancer. The IFN-18 signature is highly associated with the antigen presentation activity and T cell cytolytic activity as reflected by the APM and CYT signature respectively. The IIS and TIS are largely overlapping. Taking together, an integrated signature (IFN18+IIS) might have more broad predictive power and was therefore used to identify the "inflamed" (IFN18highIIShigh) and "non-inflamed" (IFN18lowIISlow) tumors in 5 cancer types. We found that patients with more inflamed TME have significantly longer overall survival in lung adenocarcinoma, liver hepatocellular carcinoma, colon adenocarcinoma and head and neck squamous cell carcinoma. Genes involved in cell cycle are generally downregulated in "inflamed" tumors. Genes involved in DNA repair are mostly down-regulated in lung squamous cell carcinoma, and genes involved in chromatin organization are down-regulated in liver hepatocellular carcinoma.

Conclusions: Our findings suggest that a high TMB does not necessarily associate with inflammation, and IPRES has limited value as a pan-cancer predictive biomarker. These findings will contribute to better understanding of the most up-to-date predictive biomarkers and the application of ICI in different cancers.

#1284

How can you interpret gene lists from -omics experiments.

Jeffrey V. Wong,1 Augustin Luna,2 Emek Demir,3 Igor Rodchenkov,1 Özgün Babur,3 Chris Sander,2 Gary D. Bader1. 1 _University of Toronto, Toronto, Ontario, Canada;_ 2 _Harvard Medical School, Boston, MA;_ 3 _Oregon Health & Science University, Portland, OR_.

Understanding the mechanisms responsible for a cellular behaviour often begins with observations of genes and gene products. Depending on the type of experiment, the number of resulting genes can be small, but increasingly, researchers are faced with many thousands of measurements, as in the case of transcriptomic or protein-DNA binding observations. Here, we describe ways to pair experimental results consisting of one or more genes with analysis tools with the overall aim being to make results more biologically interpretable. In certain cases, experimental approaches such as screens for essential genes can generate one or a few 'genes of interest' and there is a desire to understand their relationship to one another as well as discover links to additional, interesting genes. To this end, 'GeneMANIA' is a web tool that accepts gene names and returns a network visualization of related genes based on similarity in expression, localization, protein domains and those involved in physical interactions. Likewise, 'PCViz' is a web tool that displays a network of interactions drawn from Pathway Commons, a web resource for pathway and interaction knowledge. In cases where experiments generate a lengthy list of genes, for instance, transcriptomic measurements, there is a desire to understand their relevance to a phenotype of interest. Pathway enrichment analysis methods aim to summarize gene lists as pathways, which have a closer link to cell function. An online 'Guide' by Pathway Commons includes workflows that illustrate how to chain together software tools to identify pathways from the corresponding gene-level data then organize and summarize the pathway-level results in an interactive visualization known as an Enrichment Map. For those wishing to drill-down to individual pathways, Pathway Commons offers a set of web apps, including 'Search' that enables users to query by keyword and visualize ranked search results. Ongoing development of web apps aims to enhance the accessibility to pathways and integrate support for analysis and visualization of experimental data. The full complement of data, tools and resources offered by Pathway Commons in support of pathway analysis are described.

#1285

**Deep mutagenesis analysis on** BRCA1 **variants in** **high throughput** **DNA repair assays.**

Mariame Diabate, Alexandrea Adamovich, Tapahsama Banerjee, Michael Freitas, Jeffrey Parvin. _The Ohio State University, Columbus, OH_.

Genetic screening of Hereditary Breast and Ovarian Cancer (HBOC)-associated genes can aid in prevention of the onset of cancer by increased surveillance or preventative surgery if the genetic information is clear. Often, the genetic information is ambiguous since missense substitutions may be common in aggregate but individually these missense variants occur too infrequently for genetic segregation to be conclusive and are known as Variants of Unknown Significance (VUS). One commonly screened HBOC gene is BRCA1, with mutations found in 40-45% of hereditary breast cancer cases. The BRCA1 protein has the critical tumor suppressive function mediating homology-directed repair (HDR), which is used for the repair of double-stranded breaks (DSB) in DNA. Clinical mutant databases, such as ClinVar, contain the clinical classification of BRCA1, but, unfortunately, in many of these databases, most of the variants are classified as VUS or have conflicting data. We suggest that laboratory-based functional analysis can augment the genetic and clinical data, enabling the understanding of whether a specific variant is benign or pathogenic and predisposing to breast and ovarian cancer. We have developed a high-throughput method for analyzing the function of individual missense variants of BRCA1 in the DNA repair process. The goal is to have functional analysis for every possible amino acid substitution in BRCA1, and this will be publicly available for geneticists to consider when evaluating the likelihood of cancer predisposition an individual with a VUS in BRCA1. We have completed analysis of the amino-terminal RING domain, and we will present new results for the carboxy-terminal BRCT domain. We develop a computational pipeline that starts with sequence data from DNA repair assays to obtain a score for each variant. This pipeline will include statistical programs to evaluate the confidence level for these results, which will then be compared to the information provided in the ClinVar database. In summary, this work describes a computational framework for deconvoluting sequencing results of a high-throughput approach to measure the functional impact of each possible missense variant in the BRCT domain of BRCA1.

#1286

Analytical validation of an integrated next-generation sequencing pan-cancer liquid biopsy approach for detection of microsatellite instability.

Andrew Georgiadis, Derrick Wood, Derek Murphy, Sonya Parpart-Li, David Riley, Naomi Sengamalay, Marian Novak, Sian Jones, Samuel A. Angiuoli, Mark Sausen. _Personal Genome Diagnostics, Baltimore, MD_.

Failure of the DNA mismatch repair (MMR) pathway during DNA replication in cancer leads to the increased accumulation of somatic mutations. The molecular hallmark of MMR deficiency is microsatellite instability (MSI), which presents as polymorphism of tandem nucleotide repeat lengths ubiquitously distributed throughout the genome. Furthermore, the presence of MMR-deficiency or MSI across solid tumors has been recently demonstrated to be a robust biomarker for immunotherapy response with checkpoint inhibition. We have previously developed technology to detect MSI in tumor tissue through hybrid-capture based targeted next-generation sequencing analyses of a 125 gene panel, with probes capturing mononucleotide repeat markers. Using a set of 32 MSI and 27 microsatellite stable (MSS) formalin-fixed paraffin-embedded tumor tissue specimens, we obtained 100% sensitivity and 100% specificity with a limit of detection of 20% tumor purity. The clinical importance of MSI status now requires a fast, objective, highly sensitive screening method, particularly in late-stage patients where tumor material may not be readily obtained. However, to extend this approach to a liquid biopsy panel requires technological advances to overcome the inherent challenges associated with low circulating tumor DNA (ctDNA) levels that are compounded by polymerase slippage in mononucleotide repeat regions during PCR amplification. To overcome these limitations, we applied a multifactorial error correction approach together with a novel peak finding algorithm to more accurately identify the specific mononucleotide sequences in cell-free DNA (cfDNA) analyses. By eliminating a significant majority of sequencing errors and polymerase slippage artifacts, we were able to reduce background error rates by > 90%. Combined with implementation of a distribution modeling and a peak finding algorithm, we were able to accurately sequence the mononucleotide tracts to minimize false discovery rates for cfDNA analyses. To evaluate the performance of this approach in clinical samples, for a cohort of plasma samples obtained from 13 late-stage clinical cases with matched tumor tissue data available (9 MSI and 4 MSS) together with 66 healthy donor plasma samples, we obtained 89% sensitivity and 100% specificity, with a lowest reported mutant allele fraction of 0.4%. Taken together, this integrated next-generation sequencing liquid biopsy approach is valuable for the determination of MSI status to inform immunotherapy treatment.

#1287

Exploring somatic DNA structural alteration and aberrant genomic interactions in cancer through GenomePaint.

Yu Liu,1 Xin Zhou,1 Jian Wang,1 Ying Shao,1 John Easton,1 Mark W. Zimmerman,2 Brian J. Abraham,3 A. Thomas Look,2 Jinghui Zhang1. 1 _St. Jude Children's Research Hospital, Memphis, TN;_ 2 _Dana-Farber Cancer Institute, Boston, MA;_ 3 _Whitehead Institute for Biomedical Research, Cambridge, MA_.

Somatic structural variations (SV) play an important role in tumorigenesis as they may cause oncogenic gene fusions or transcriptional activation of oncogenes by introducing aberrant promoter-enhancer interactions. Evaluating the oncogenic implications of SVs is challenging, especially for SVs in noncoding regions, which requires integrating data from 1) whole-genome sequencing (WGS), 2) RNA-seq from the tumor sample and series patient samples with same tumor type to compare with, 3) epigenetic profiling (ChIP-seq), and 4) genome-wide chromosome conformation capture studies such as Hi-C. To facilitate the discovery of oncogenic SVs, we developed GenomePaint, an interactive browser to integrate and visualize somatic SVs and copy number alterations (CNAs) analyzed by WGS, and gene expression data analyzed by RNA-seq in >2,000 pediatric cancers, as well as ChIP-seq and Hi-C data from pediatric tumors and cell lines. The genomic profiling of patient samples was generated by the St Jude/Washington University Pediatric Cancer Genome Project and the NCI-TARGET project, and includes all major subtypes of pediatric leukemia, solid tumors and brain tumors. A global view of the entire patient cohort using GenomePaint revealed SV hotspots (e.g. within first intron of TP53 in osterosarcoma) and recurrent CNAs (e.g. TAL1 deletion in T-ALL) in each cancer type. Each SV was integrated with results on gene expression, ChIP-seq and Hi-C, on the rearranged chromosome. Our discovery of the mechanism underlying aberrant MYC overexpression, in a subset of neuroblastomas (NBL) lacking MYCN amplification, proves the power of this tool in facilitating the discovery of oncogenic SV drivers. The sample with the highest MYC expression in this cohort harbors an SV between chr 8 and 4, with breakpoint on chr 8 located 50 Kb downstream of MYC. Similar SVs were detected in multiple NBL cell lines by WGS, which also express high levels of MYC. By analyzing Hi-C data from these cell lines, a new topologically associating domain (TAD) extending beyond the SV breakpoint was observed along the chimeric chromosome. This new TAD shows an aberrant interaction of a super-enhancer with a broad H3K27ac peak associated with HAND2/FBXO8 on chr 4 and the MYC promoter, demonstrating the rewired regulatory architecture introduced by SV as the driver for MYC dysregulation. In conclusion, our new GenomePaint interactive browser facilitates the analysis of SVs, CNAs and gene expression in concert studies by WGS and Hi-C in pediatric tumors. Coding mutations are rarely identified in many types of childhood tumors, even in the presence of pronounced chromosomal SVs, indicating new approaches are needed to unveil oncogenic mechanisms. GenomePaint provides an integrative analysis tool to evaluate the pathogenic basis underlying the often complex SVs in noncoding genome and function as oncogenic drivers in a large fraction of pediatric malignancies.

#1288

A comprehensive investigation of factors impacting the accuracy of mutation detection using next generation sequencing technology.

Wenming Xiao,1 Yongmei Zhao,2 Somatic Mutation Working Group, SEQC2 Consortium. 1 _FDA/NCTR, Jefferson, AR;_ 2 _Leidos, Frederick, MD_.

Next generation sequencing (NGS) allows researchers to survey DNA mutations in tumor cells at very large scales with affordable cost. However, distinguishing tumor cell sub-population mutations from errors due to NGS platform biases, sample preservation effects, assay protocols and suboptimal or incongruous bioinformatics analysis methods is currently complex and difficult. For NGS to be successfully leveraged in a clinical translational manner, increased ability to discriminate between true mutations and artifacts is paramount.

To address this urgent unmet clinical need, we systematically investigated somatic mutation events in paired cell lines (breast cancer and normal) with multiple NGS platforms and protocols to fully characterize factors that affect accuracy, specificity and sensitivity of mutation detection. We applied machine learning algorithms to define high confidence mutation calls with data sets from 8 NGS platforms combined with 9 bioinformatic analysis pipelines. With "ground truth" established using multiple orthogonal sequencing platforms, we evaluated the performance of mutation detection under various circumstances, including: fresh cell vs FFPE, amount of DNA, library preparation, sequencing platform, tumor purity, and analysis algorithm. Moreover, we performed whole exome sequencing (WES) and whole genome sequencing (WGS) at 6 sequencing sites in parallel, which allowed us to assess the reproducibility of NGS runs on the same biological samples.

By analyzing DNA variant detection recall and precision from data sets derived from various conditions, we identified statistical variance components in overall DNA variant detection framework as well as specific practices that will likely lead to the false identification of variants. By concomitantly examining the effects between sample processing and bioinformatics pipelines, here we recommend best practices for mutation detection using NGS technology and establish quality metrics for cancer studies involving NGS technology.

#1289

Crosstalk between methylation and alternative splicing in cancer.

Dongwook Kim, Seonggyun Han, Younghee Lee. _Unversity of Utah, Salt Lake City, UT_.

Background: DNA methylation in promoter regions leads to transcriptional silencing, such as of tumor suppressor genes in cancer. However, the functions of DNA methylation in the gene body (intragenic, i.e., exons and introns) have not yet been completely elucidated. Recently, hyper-methylation has been implicated in enhancing exon recognition by recruiting methyl-CpG-binding protein (MECP2) to hyper-methylated sites. In this study, we examined intragenic methylation in splicing regulatory elements (SREs: exonic splicing enhancer (ESE), exon splicing silencer (ESS), intronic splicing enhancer (ISE)) as a mechanism to understand how epigenetic factors contribute to alternative splicing events in cancer.

Hypothesis: Methylation patterns of SREs are involved in directing alternative splicing events in cancer.

Methods: We developed a splicing decision model to identify actionable methylation loci potentially affecting splicing events (i.e., exon skipping) among CpG sites in putative intragenic SRE regions. We used DNA methylation and RNA-Seq data for breast and lung cancer samples and normal cases from The Cancer Genome Atlas (TCGA). We additionally used hyper- and hypo-methylation data published in Selamat et al. (2013) as an independent lung cancer dataset. To identify regions hyper- and hypo-methylated in cancer, we performed an unpaired t-test for normally distributed data and a Mann-Whitney-Wilcox test for non-normally distributed data for differential methylation status (beta value) between cancer and normal cases. Multiple testing was accounted for by controlling the false discovery rate (p< 0.05 with FDR< 0.1).

Results: We investigated whether specific patterns of methylation were enriched in SRE regions affecting exon skipping. We found that for breast cancer samples, differential methylation status was enriched in SRE regions but not in non-SRE regions. Particularly, hypo-methylation in cancer showed a greater enrichment in ESE regions with putative exon skipping events. Similar results were observed in the both the TCGA lung cancer cases and an independent lung cancer dataset. We calculated GC contents and the methylation frequencies at each position of the hexameric sequence of SREs and further investigated whether certain SREs are prevalent to be with methylation or not. These data suggest that hypo-methylation in SRE regions may cause failure to recruit MECP2, therefore leading to exon skipping.

Conclusion: Our study suggests that intragenic methylation status is important for splicing regulation and may improve our understanding of how intragenic epigenetic markers play a role in gene regulation by affecting alternative splicing.

#1290

Expanding the CIViC variant to complex combinations of regions in the cancer genome.

Arpad Danos, Kilannin Krysiak, Alex Wagner, Susanna Kiwala, Joshua McMichael, Adam Coffman, Erica Barnell, Yang-Yang Feng, Benjamin Ainscough, Cody Ramirez, Malachi Griffith, Obi Griffith. _Washington University in Saint Louis, Saint Louis, MO_.

The Clinical Interpretations of Variants in Cancer (CIViC) database was designed as a fully open-access resource specifically focusing on the subset of cancer variants with well-defined clinical information associated to them and targeting a broad user base in cancer, including clinicians, researchers and patient advocates. CIViC follows a crowd-sourced curation model with expert moderation, and emphasizes transparency in that all changes, approvals, and discussion of content are tracked and displayed in the interface. Evidence items (EIDs) make up the fundamental unit of CIViC. EIDs connect a predictive, prognostic, diagnostic or predisposing clinical significance to a variant in the context of a specific disease, and each EID links back to specific published and peer-reviewed evidence. Multiple EIDs of high value can be used to make clinical assertions. While the CIViC variant is intentionally broad and can capture specific SNVs and Indels, while also admitting umbrella variants such as "EGFR mutation," it is also anchored to a single gene entity (pulled directly from the NCBI Entrez database). Two drawbacks result from this strict association. First, clinical information associated with multiple genes is difficult to implement in knowledgebases like CIViC. In cases where two variants co-occur on the same gene, such as the erlotinib-sensitizing EGFR L858R and resistance mutation T790M, CIViC allows a "L858R and T790M" variant to be created under the EGFR gene. In other cases, such as FLT3 internal tandem duplication and DNMT3A mutation in AML, there is no CIViC variant that can capture this combination. In addition, non-gene entities important in cancer such as viral oncogene, microsatellite instability, or loss of chromosome region are not possible to represent. To address this, we are preparing CIViC V2, which will be implemented via modification of the existing database and web interface. The architecture around the EID as fundamental unit of CIViC will remain unchanged, but the CIViC variant will be re-envisioned, introducing the concepts of region and genotype. A variant will link to one or more regions, and region will be drawn from a list of types—genome, chromosome, rearrangement, gene and pathogen—which can be expanded when needed. The notion of complex genotype will be realized as a combination of variants drawn from multiple regions in either an additive manner (HER2 overexpression AND PIK3CA mutation) or employing more complex variant combinations using OR and NOT. Multicomponent genotypes will point back to EID collections associated with each individual component, while also enabling users to write EIDs unique to that genotype, which can, in turn, be used to create genotype clinical assertions. This new scheme will be presented via refactoring of the UI, and allow for a highly flexible concept of region and genotype reflecting the growing understanding of the combinatorial nature of variants in cancer.

#1291

Master regulator analysis of paragangliomas carrying SDH, VHL, or MAML3 genetic alterations.

John A. Smestad, Louis J. Maher. _Mayo Clinic, Rochester, MN_.

Background: Succinate dehydrogenase (SDH) loss and mastermind-like 3 (MAML3) translocation are two clinically important genetic alterations that occur in subtypes of human paraganglioma and pheochromocytoma (PPGL) neuroendocrine tumors that correlate with increased rates of metastasis. Although hypotheses exist for how global activation of pseudohypoxia and epigenomic hypermethylation are driven via dioxygenase poisoning in the context of SDH loss, it remains unclear how these defects drive tumorigenesis. Additionally, MAML3 translocation has recently been identified as a genetic alteration in PGL and is poorly understood. We hypothesize that a key to understanding the tumorigenic basis for these genetic alterations is the deconvolution of the gene expression profiles in these tumors and identification of the transcription factors responsible for the observed transcriptional changes.

Methods: In the present work, we leverage publicly-available human tumor gene expression profiling experiments (N=179) to reconstruct a PPGL tumor-specific transcriptional network. Inferred network structure was subsequently validated by examining the frequency of known transcription factor DNA-binding motifs in the inferred regulons. We subsequently use the inferred transcriptional networks and known gene expression signatures for specific PPGL subtypes to perform a master regulator analysis nominating specific transcription factors that may control observed oncogenic patterns of gene expression. The findings of the master regulator analysis were confirmed by duplication of the analysis on a second PPGL-specific inferred transcriptional network (N=188 specimens).

Results: A small number of master regulator transcription factors are likely drivers of the observed subtype-specific gene expression patterns in SDH loss- and MAML3 translocation-positive PPGL. Unexpectedly, hypoxia-inducible transcription factors were not identified as master regulators in SDH-loss tumors. Additionally, analysis of the MAML3 translocation subtype master regulators revealed that a single transcription factor, IRX4, may control 37% of the deferentially expressed genes in this highly malignant tumor subtype.

Conclusions: These observations from unbiased analyses suggest a basis for future development of targeted therapies for specific PPGL molecular subtypes.

#1292

Methods of improving accuracy of neoantigen identification for therapeutic and diagnostic use in immuno-oncology.

Sean Michael Boyle, Ravi Alla, Ryan Wang, Eric Levy, Gabor Bartha, Jason Harris, Robert McCord, Rena McClory, John West, Richard Chen. _Personalis, Inc., CA_.

Background:Neoantigens are increasingly critical in immuno-oncology as a therapeutic target for neoantigen-based personalized cancer vaccines and as a potential biomarker for immunotherapy response. However, the methods for identifying which neoepitopes are more likely to provoke an immune response remains an important challenge for improving both the effectiveness of neoantigen-based vaccines and enabling the potential use of neoantigens as a biomarker in immunotherapy.

Methods:We sought to improve overall neoantigen identification performance by systematically improving critical components of our ACE ImmunoID assays and neoantigen pipeline. Personalis' Accuracy and Content Enhanced (ACE) technology was developed to fill critical gaps in conventional exome and transcriptome sequencing that can lead to missed neoantigens. To improve MHC-epitope binding prediction, we trained neural networks on mass spectrometry derived MHC-epitope binding data. This is in contrast to other MHC binding algorithms that have been primarily trained using in vitro competitive binding data, which suffer from having not been processed, loaded, nor shuttled natively into the HLA binding domain. HLA typing, a key input into the neoantigen prediction algorithms, was improved through exome augmentation of the HLA region with an optimized HLA typing algorithm. Other enhancements include RNA based somatic variant calling, peptide phasing, transcript isoform estimation, and identification of indel and fusion derived neoepitopes.

Results:Our ACE augmented exome demonstrates high sensitivity and specificity for SNVs, indels, and fusions at MAF >=10%. These are all variant types that result in putative neoantigens. Further, we show that our augmented ACE transcriptome can achieve high sensitivity for RNA derived variants and can be an important filter for putative neoantigens. When compared with commercially available MHC binding algorithms for specific HLA alleles, our MHC binding prediction algorithm consistently achieves a higher overall sensitivity and specificity than other tools. For example, our MHC class I-epitope binding prediction algorithm demonstrated an aggregative precision value of 0.88 across HLA alleles, as opposed to 0.50 for other widely used tools. To assess overall HLA-typing performance, we performed a blinded clinical HLA typing validation demonstrating 98% and 95% concordance with Class I and II HLA results (respectively) from clinical testing. We also show instances where peptide phasing, SNP, indel and fusion-derived neoepitopes are important for more accurate and comprehensive neoantigen identification.

#1293

A high through approach to delineate protein complexes in adenocarcinoma tissues.

Sofia Lage Vickers,1 Juan Bizzotto,1 Emiliano Ortiz,1 Alejandra Paez,1 Nicolás Anselmino,1 Javier Brandani,1 Mercedes Abbate,1 Javier Cotignola,1 Maria Pía Valacco,2 Elba Vazquez,1 Geraldine Gueron1. 1 _Department of Biological Chemistry, FCEN, University of Buenos Aires, IQUIBICEN-CONICET, Buenos Aires, Argentina, Capital Federal, Argentina;_ 2 _CEQUIBIEM - IQUIBICEN, FCEN, University of Buenos Aires, Argentina, Capital Federal, Argentina_.

Although it is well known that prostate cancer (PCa) is a progressive disease involving multiple gene alterations, little is known at the proteome level. Most of the functional information of the cancer-associated genes relies in the proteome, an exceptionally complex biological system involving several proteins that function through dynamic protein-protein interactions and post-translational modifications. To identify potential PCa protein biomarkers, we carried out an in depth proteomic analysis (ESI-MS/MS) using human PCa and BPH tissue. Samples were obtained using phase-transfer surfactant-aided extraction/tryptic digestion of formalin-fixed and paraffin-embedded sections mounted on microscope slides. We identified 1331 and 1239 proteins in PCa and BPH tissue proteomes respectively. 71 proteins were present in at least 50% of PCa samples and not in BPH samples, while 122 proteins where present in at least 50% of BPH samples and not in carcinoma samples. In order to prioritize candidate markers for PCa, we compared the differential protein expression based on normalized spectral counts between tissue samples. We set as cut-offs proteins that were found with a minimum of three peptides within our PCa proteomes. This filter resulted in the selection of 11 proteins. The list contained proteins that were previously studied in the context of prostate cancer progression, including SSBP1, GDF15, NDRG1, C4A & APOE, thus providing further confirmation for the robustness of our quantification method. Bioinformatics analysis (Oncomine) showed that the proteins aforementioned were significant up-regulated (fold change >1.5, P≤0.05) in prostate adenocarcinoma vs. normal prostate gland. Whole exome analysis (cBioportal), revealed amplification as the most frequent genetic alteration and RNASeq data also confirmed a significant up-regulation for these proteins (P≤0.05).

To contextualize the proteomic data, we utilized protein complexes information from CORUM database. Utilizing biological complexes as a cluster vector, we calculate a Proteomics Signature Profile (PSP) for each sample based on the hit rates of its reported proteins, against the cluster vector. We identified 25 complexes differentially expressed between PCa and BPH. Among those, the top ranked cluster upregulated in PCa was EIF3 (p-value= 4,37x10-5), a protein complex that functions during the initiation phase of eukaryotic translation, composed of EIF3B, EIF3J and EIF3I.

We hereby report and offer a new set of biomarkers in addition to the existing diagnostic tests that could significantly improve sensitivity and specificity in PCa diagnosis.

#1294

Preoperative prediction of microvascular invasion in HCC using radiomics on multisequence gadoxetic acid-enhanced MR images.

Jingwei Wei, Dongsheng Gu, Di Dong, Shuaitong Zhang, Yushen Jin, Jie Tian. _Chinese Academy of Sciences, Institute of Automation, Beijing, China_.

Micro-vascular invasion (MVI) is underscored as a judicious risk factor for early-stage recurrence and poor prognosis of hepatocellular carcinoma (HCC). Preoperative knowledge of MVI would assist in therapy decision making beforehand, thus prolong the survival of HCC patients. However, current diagnosis of MVI could only be obtained by pathological confirmation after the surgery or through punctures, which hinders timely therapeutic regime plan or causes needless suffering for the patients. In this study, we aimed to perform a preoperative prediction on MVI using multi-sequence Radiomics on gadoxetic acid-enhanced MR images, along with multi-variable clinical factor analysis. A cohort of 208 patients diagnosed with HCC was enrolled with institutional review board approval. Radiomic features including shape and size, intensity, textural and wavelet features were extracted on segmented region of interest. The least absolute regression modeling was used to select the most effective radiomic features which had potential for MVI identification. We finally utilized logistic regression modelling to generate the single radiomics signature for each sequence with Akaike information criteria as the stopping rule. Gadoxetic acid-enhanced hepatobiliary phase (HBP) T1-weighted image and HBP T1 map turned out to be the best-performed sequences with AUC over 0.7 on both training and validation cohorts. The fusion radiomics signature incorporating effective features from these two sequences achieved the optimal prediction with AUC of 0.895 on the training cohort and 0.837 on the validation cohort. Adding the pertinent clinical and radiological factors: AFP, irregular tumor border and arterial peritumoral enhancement, the final combined model could successfully predict the MVI with AUC of 0.943 on the training cohort and 0.861 on the validation cohort. Our study revealed the archetypal radiomic features related MVI and highlighted the fusion radiomics signature as a powerful imaging marker for MVI preoperative prediction. Radiomics combined with traditional clinic-radiological information would no doubt improve the clinical decision making in HCC therapy.

#1295

Single cell RNA sequencing of lung adenocarcinoma reveals heterogeneity of immune response-related genes.

Keyue Ma,1 Alexandra A. Schonnesen,1 S. Gail Eckhardt,2 Ning Jiang1. 1 _University of Texas at Austin, Austin, TX;_ 2 _LIVESTRONG Cancer Institutes, Austin, TX_.

Immunotherapies have been the most promising approaches to treating cancers, while the partial responses in multiple clinical trials suggest the significance of characterizing intertumoral and intratumoral heterogeneity for better prognosis and decision-making in treatment. The lack of intratumoral characterization of immune response-related genes in cancer cells, however, hinders the further development of metrics to select and predict immunotherapies. Therefore, we applied single cell RNA-seq data from lung adenocarcinoma patients to identify intratumoral heterogeneity of immune response-related genes and demonstrated its potential effect on immunotherapy efficacy. We found IFNγ signaling pathway genes are heterogeneously expressed and co-regulated in cancer single cells, including MHC class II molecules (MHCII), a set of favorable prognosis markers. The upregulation of MHCII is also mutually exclusive with upregulation of cell cycle pathways, which are associated with unfavorable prognosis. Moreover, analysis of two candidates of cancer vaccination approaches, neoantigens and cancer testis antigens, revealed their ectopic expression in single cells. These analyses provide the rationale of applying combinatorial therapies to prevent tumor escape and basis for future development of prognosis metrics based on intratumoral heterogeneity.

#1296

CanPathPro—development of a platform for predictive pathway modelling using genetically engineered mouse models.

Christoph Wierling,1 Yann Herault,2 Jos Jonkers,3 Aspasia Ploubidou,4 Lucien Frappart,5 Jan Hasenauer,6 Julio Banga,7 Oliver Rinner,8 Valeriya Naumova,9 David Koubi,10 Bodo Lange1. 1 _Alacris Theranostics, Germany;_ 2 _PHENOMIN Mouse Clinical Institute, France;_ 3 _Netherlands Cancer Institute, Netherlands;_ 4 _Leibniz Institute on Aging - Fritz Lipmann Institute, Germany;_ 5 _Leibniz Institute on Aging – Fritz Lipmann Institute, Germany;_ 6 _Helmholtz Zentrum München GmbH, Germany;_ 7 _Spanish National Research Council, Spain;_ 8 _Biognosys AG, Switzerland;_ 9 _Simula Research Laboratory AS, Norway;_ 10 _Finovatis SAS, France_.

Omics technologies are generating complex molecular datasets that are exponentially increasing the cancer knowledge base and opening up new therapeutic possibilities. However, current approaches to analysing such data are often confined to statistical and pattern recognition techniques, or at best modelling of a single cellular signalling pathway, rather than the complex cross-talks of pathways that determine cancer onset and progression and response to therapy. New solutions to optimally exploit this wealth of data for basic research, better treatment and stratification of patients, as well as more efficient targeted drug development are required. CanPathPro (www.canpathpro.eu), an EU Horizon 2020 project, is addressing the challenge of predictive modelling of biological data by developing and refining bioinformatic and experimental tools for the evaluation and control of systems biology modelling predictions. Components comprise highly defined mouse and organotypic experimental systems, next-generation sequencing, SWATH-based proteomics and a systems biology computational model for data integration, visualisation and predictive modelling. Within CanPathPro, genetically engineered mouse models are used to follow the temporal changes occurring during cancer development, including the histology of the tumour, the genome and transcriptome using next-generation sequencing and the (phospho-)proteome using SWATH technology. The systems biology computational model is optimised in an iterative fashion through perturbation experiments of tumor-tissue-derived cell lines and organoids, permitting the validation of pathway and parameter information. In this way, CanPathPro takes a unique approach combining classic cancer research with omics data and systems biology tools, to develop and validate a new biotechnological application: a combined systems and experimental biology platform for generating and testing cancer signalling hypotheses in biomedical research. The CanPathPro-generated platform will enable in silico identification of cancer signalling networks critical for tumour development and will allow users to predict activation status of individual pathways, following integration of user (or public) data sets in the pathway models. The innovative approach taken by CanPathPro is set to have broad and significant impact on diverse areas, from cancer research and personalised medicine to drug discovery and development, and ultimately improving outcomes for cancer patients.

#1297

Studying tumor metabolic reprogramming through integration of metabolomics and transcriptomics.

Weiruo Zhang, Sylvia Plevritis. _Stanford University, Stanford, CA_.

Metabolic reprogramming is a cancer hallmark important to tumor initiation, progression and drug response. However, our knowledge of the tumor metabolome is limited since only a small number of metabolites can be reliably measured at a time. To enhance our understanding of tumor metabolism, we integrate metabolomics with transcriptomics to reveal potential metabolism-related transcriptional drivers. Our novel method, METRI (for MEtabolomic-TRanscriptomic Integration), identifies (1) metabolic alterations between two conditions, before and after treatment in our case and (2) interactions between signaling pathways and metabolic alterations. First, to identify metabolic alterations, METRI builds a matrix of gene and metabolite expressions for each metabolic pathway obtained from Kyoto Encyclopedia of Genes and Genomes. METRI computes a distance before and after treatment, weighted by the pathway coherence, where coherence is defined as the average absolute correlations across the genes and metabolites, resulting in a score for pathway ranking. Bootstrap is employed to obtain a p-value to assess each pathway's ranking significance. Second, to build an interaction network between metabolic alterations and signaling pathways, METRI identifies signaling genes correlated with each metabolic pathway and obtains enriched signaling pathways of each altered metabolic pathway. We applied METRI to two public non-small cell lung cancer datasets (GSE49644 and GSE17708/ST000010) to study the metabolic alterations associated with the Epithelial-Mesenchymal Transition (EMT), a phenotypic change related to cancer invasion and drug resistance. Both studies generated metabolomic and transcriptomic data and induced EMT with TGF-β treatment on cell lines (A549, HCC827 and NCI-H358) with three replicates for each condition. Using METRI, we found glycerophospholipid metabolism (p-value=0.03), purine metabolism (p-value=0.03), pyrimidine metabolism (p-value=0.04), aminosugars metabolism (p-value=0.04) and sphingolipid metabolism (p-value=0.04) were consistently altered after EMT induction in both datasets. In particular, we identified that several genes of the hexosamine biosynthesis pathway, a major pathway producing aminosugars for protein glycosylation, were upregulated in EMT. In addition, we identified that MAPK (p-value=0.004) and TGF-β (p-value=0.01) signaling pathways were associated with aminosugars metabolism. We also identified p53 signaling (p-value=0.002) was associated with sphingolipid metabolism, which plays a critical role in cancer cell adhesion and migration. In summary, METRI is a new method for data integration to study metabolic alterations and signaling-metabolism interactions and has the potential to reveal new insights to cancer biology and suggest novel targets for therapeutic intervention.

#1298

Mapping the comprehensive landscape of missense-mutation neoantigens across the human genome.

HoJoon Lee, Stephanie U. Greer, Hanlee P. Ji. _Stanford University, Stanford, CA_.

Specific somatic mutations in cancer genomes result in amino acid alterations and derivative novel immunogenic peptides called neoantigens. The MHC molecule mediates the immunologic presentation of these cancer mutation-derived peptides. Based on a limited number of polymorphisms, the configuration of the MHC molecule is one of the major factors in determining whether mutated peptides can elicit immune response or not. To provide insight in the full diversity of potential neoantigens across the entire human genome, we identified all possible amino acid substitutions and estimated their potential binding affinities to the 86 MHC genotypes. As a first step, we evaluated every possible nucleotide changes at every single position using exons and related alternative transcripts as defined by the Consensus Coding Sequence (CCDS) resource. We evaluated 159 million (M) possible nucleotide changes among ~31,000 autosomal transcripts from ~ 19,000 genes. Overall, this comprised a genome sequence "space" of 53 M nucleotides. Among this coding regions space, we evaluated 123 M candidate nonsynonmous mutations. Among these mutations, we identified short mutated peptides of nine amino acids in length across this mutation space. We estimated the binding affinity of all mutated peptides across 86 MHG polymorphisms. Based on the predicted binding affinity, 0.6% ~ 1.0 % were found to be potential strong binders to the one of MHC molecules while 1.7% ~ 2.5% of them are weak binder to the one of MHC molecules. HLA-A0201 had the high number of potential candidate neoantigens. The KRAS cancer had candidate epitopes that binds to >50 different MHC molecules include KRAS D12V, which is very hot spot, that make epitopes that binds to 30 MHC molecules. In addition, we mapped the frequencies of missense mutations observed in TCGA and COSMIC to these candidate neoantigens. For releasing the results of our study, we are developing the "Immunogenomic Explorer" available at http://genomeportal.stanford.edu/immune. This portal enables users to explore the potential immunogenic epitopes from genes of their interest such as KRAS. Users can search by HLA genotypes as well. This web interface allows researchers to address important questions regarding their immunotherapy or cancer vaccine.

#1299

Circular RNA expression is abundant and correlated to aggressiveness in early-stage bladder cancer.

Trine Line H. Okholm,1 Morten M. Nielsen,1 Mark P. Hamilton,2 Lise-Lotte Christensen,1 Søren Vang,1 Jakob Hedegaard,1 Thomas B. Hansen,3 Jørgen Kjems,3 Lars Dyrskjøt,1 Jakob S. Pedersen1. 1 _Aarhus University Hospital, Aarhus N, Denmark;_ 2 _Baylor College of Medicine, Houston, TX;_ 3 _Aarhus University, Aarhus C, Denmark_.

Background: Due to their stability, specificity, and accessibility, circular RNAs (circRNAs) may represent an attractive new class of biomarkers in early-stage bladder cancer (BC) and possess regulatory functions.

Experimental procedures: We characterize circRNA transcripts using whole transcriptome RNA-Seq data from 457 non-muscle-invasive bladder cancer (NMIBC) samples (348 Ta and 109 T1). We identify backsplice-spanning reads by using a modified version of the find_circ pipeline with an increased filtering stringency on both anchor sequences. Same pipeline is used to identify circRNAs in publicly available tissue samples obtained from ENCODE (n = 113) and in locally generated RNA-Seq data from unfractionated BC cell lines (n = 8) as well as from the nucleic and cytoplasmic fractions of three BC cell lines.

Results and limitations: Here, we identify more than 15,000 unique circRNAs supported by at least two reads in at least two different samples. We show that a set of highly expressed circRNAs have conserved core splice sites, are likely to be surrounded by inverted homologous Alu repeats, and are enriched with Synonymous Constraint Elements as well as microRNA target sites. Moreover, we identify 113 abundant circRNAs that are differentially expressed between high and low-risk tumor subtypes. Analysis of progression-free survival reveals 13 circRNAs that are associated with BC progression independently of the linear transcript and parent gene. We point to circHIPK3 and circCDYL as important candidates because they possess strong clinical and biological associations. The progression-free survival analyses reveal a significantly lower risk of progression for patients with high circHIPK3 and circCDYL expression levels compared to patients with low levels. Correspondingly, both circRNA candidates are found at higher levels in non-malignant BC cell lines than metastatic BC cell lines. We are currently conducting knockdown and overexpression studies in BC cell lines to reveal their biological role. Future studies should address whether circRNAs that correlate with BC progression are present in urine and plasma samples, and importantly, validation in independent cohorts should be performed in order to confirm their clinical relevance.

Conclusions: We demonstrate that abundant circRNAs possess key biological characteristics, distinguishing them from low-expressed circRNAs and non-circularized exons, and suggest that circRNAs might serve as a new class of prognostic biomarkers in NMIBC.

#1300

Selective and mechanistic sources of recurrent rearrangements across the cancer genome.

Ofer Shapira,1 Jeremiah Wala,1 Yilong Li,2 David Craft,3 Steven Schumacher,1 Marcin Imielinski,4 James Haber,5 Nicola Roberts,2 Xiaotong Yao,4 Chip stewart,6 Cheng-Zhong Zhang,1 Jose Tubio,7 YoungSeok Ju,2 Peter Campbell,2 Joachim Weischenfeldt,8 Rameen Beroukhim1. 1 _Dana Farber Cancer Institute, Boston, MA;_ 2 _Wellcome Trust Sanger Institute, Hinxton, United Kingdom;_ 3 _Harvard Medical School, Boston, MA;_ 4 _New York Genome Center, New York, NY;_ 5 _Brandeis University, Waltham, MA;_ 6 _Broad Institute, Boston, MA;_ 7 _University of Vigo, Vigo, Spain;_ 8 _University of Copenhagen, Copenhagen, Denmark_.

Cancer cells can acquire profound alterations to the structure of their genomes, including rearrangements that fuse distant DNA breakpoints. We identify both mechanistic and selective factors that determine where rearrangements tend to recur in cancer, using whole-genome sequencing data from 2,693 tumor-normal pairs from 30 histologic subtypes. These data were analyzed through the International Cancer Genome Consortium (ICGC). We observe substantial variation in the density of rearrangements across the genome. After accounting for these variations, we discover recurrent events that are not explained by mechanistic factors alone. These include both significantly recurrent breakpoints (SRBs) and significantly recurrent juxtapositions (SRJs) between pairs of loci brought together by a rearrangement. We identify SRBs at 52 loci. Many of the SRBs indicate novel and potentially functionally relevant events. This included tightly clustered deletions near the BRD4 promoter, which were significantly enriched for rearrangements in ovarian and breast adenocarcinomas. Another SRB cluster at 10p15, was associated with greater than two-fold upregulation of three AKR1C genes in lung squamous and liver cancers. All breakpoints coincided with a cluster of lineage-specific enhancers, suggesting that rearrangements may alter promoter-enhancer interactions at the locus to activate gene expression. Considering both loci fused by a rearrangement, we observe different signatures of rearrangements resembling either single breaks followed by strand invasion or two separate breaks that become joined. Accounting for these signatures, we identify 90 SRJs. Among the 30 most significant SRJs, 14 correspond to known oncogenic SRJs. The sixteen remaining clusters include five with a known driver gene (MDM2, EGFR, TERT, ROS1, ERCC5). The majority of SRJs are tumor-type specific and tend to involve genes with tissue-specific expression. SRJs were frequently associated with disruption of topology-associated domains, juxtaposition of enhancer elements, and increased expression of neighboring genes. Lastly, we find that currently we only have sufficient power to detect SRJs that recur in rates greater than 5-10% within each tissue type. However, we know that events that recur at lower rates can be biologically and clinically significant (e.g., ALK-EML4 fusions that recur at a rate of 1-3% in lung adenocarcinomas). At current sample numbers, we appear to be discovering a new novel rearrangement for every 25 cancer samples we sequence--a remarkable return on investment.

#1301

Using the evolutionary ages of genes to understand hallmark features of cancer.

David L. Goode, Anna S. Trigos, Anthony T. Papenfuss, Richard B. Pearson. _Peter MacCallum Cancer Centre, Melbourne, Australia_.

The evolutionary age of a gene is closely tied to its role in cancer. As organisms evolved into multicellular forms, new pathways emerged to control core cellular processes formed in unicellular ancestors such as cell division, DNA replication and energy metabolism. Tumors rely on many of these core processes, at the same time as they suppress key pathways linked to the emergence of multicellularity. This suggests the guiding principles behind many hallmarks of cancer could be better understood in an evolutionary context, by investigating interactions between unicellular and multicellular genes and how they are disrupted in cancer. We have found the evolutionary histories of genes are associated with their expression and regulation (Trigos et al., PNAS 2017), the selection and response to genetic alterations, as well as the rewiring of transcriptional networks across 7 tumor types. Our results reveal different transcriptional and mutational patterns in cancer between genes with different evolutionary ages, with selection for genetic alterations in early metazoan genes leading to the transcriptional rewiring of the regulation between unicellular and multicellular genes, driving carcinogenesis. Examples of how this approach can help prioritize genes for drug treatment and inform novel treatment strategies will be given.

#1302

OCTAD: An open cancer therapeutic discovery workspace in the era of precision medicine.

Bin Chen. _UCSF, San Francisco, CA_.

Rapidly decreasing costs of RNA sequencing have enabled large-scale profiling of cancer tumor samples with precisely defined clinical and molecular features (e.g., low-grade IDH1 mutant glioma). Identifying drugs targeting a specific subset of cancer patients, particularly those who do not respond to conventional treatments, is critically important for translational research. Many studies have demonstrated the utility of a systems-based approach that connects cancers to efficacious drugs through gene expression signatures to prioritize drugs from a large drug library. From our previous work on liver cancer, Ewing's sarcoma, and basal cell carcinoma, we have shown that the success of this approach is made possible by critical procedures, such as quality control of tumor samples, selection of appropriate reference tissues, evaluation of disease signatures, and weighting cancer cell lines. There is a plethora of relevant datasets and analysis modules that are publicly available, yet are isolated in distinct silos, making it tedious to implement this approach in translational research. As such, we present the current protocol, which we envision as a best practice to prioritize drugs for further experimental evaluation, primarily based on open transcriptomic datasets and the free open-source R language and Bioconductor packages. In this project, we retrieved patient tumor samples based on specified clinical and/or molecular features from the Genomic Data Commons Data Portal using an API. We then created a gene expression signature for these samples through employing normalized RNA-seq counts processed in the UCSC Xena project, where all RNA-seq samples from TCGA, TARGET, and GTEx were aligned and normalized using the same pipeline. We evaluated the quality of samples based on their purity and correlation with cancer cell lines. The reference tissue samples were selected based on their profile similarity with GTEx samples. We evaluated each disease signature via a cross-validation approach. We then created drug signatures using a similar procedure from large-scale, open-access platforms, namely the LINCS L1000 library, which consists of over 20,000 compounds. Our pipeline can then compute and assess the reversal potency between the disease signature and each drug signature. The drugs that present high reversal potency are prioritized as drug hits. Finally, we performed enrichment analysis of drug hits to identify compelling enriched targets and pathways. For our pilot study, we use IDH1 mutant oligodendroglioma as a case study, where the efficacy of over 300 LINCS compounds was measured in three relevant cell lines. We have shown that our prediction corroborates with the experimental data significantly. We envision that this open workspace will help efficiently identify new therapeutic candidates for other cancers.

#1303

Combination cancer therapy can confer benefit via patient to patient variability without drug additivity or synergy.

Adam C. Palmer, Peter K. Sorger. _Harvard Medical School, Boston, MA_.

Combination cancer therapies aim to improve the probability and magnitude of therapeutic responses and reduce the likelihood of acquired resistance in an individual patient. However, drugs are tested in clinical trials on genetically diverse patient populations. We show here that patient-to-patient variability (between-tumor heterogeneity) and 'independent drug action' are sufficient to explain the superiority of many FDA-approved drug combinations in the absence of drug synergy or additivity (pharmacological interaction). In a combination exhibiting independent drug action each patient benefits solely from the drug to which his or her tumor is most sensitive, with no added benefit from other drugs. This result is based on retrospective analyses of human clinical trials, re-analysis of a database of mice carrying patient-derived xenografts, and first principles computational modeling of drug responses in a heterogeneous population of tumors. This analysis identifies occasional cases of truly synergistic drug effects in clinical data. However, when drug combinations exhibit additivity or synergy in pre-clinical models, patient-to-patient variability and low cross resistance often make independent action the dominant mechanism in clinical populations. This insight represents a different way to interpret clinical trial data, and a different way to design new sequential or combination treatment regimens.

#1304

Whole-exome analysis of 1,135 melanomas reveals new significantly mutated cancer genes and copy number alterations.

Jake R. Conway,1 David Liu,2 Stephanie Wankowicz,2 Amaro Taylor-Weiner,1 Felix Dietlein,2 Eliezer Van Allen2. 1 _Harvard Medical School, Boston, MA;_ 2 _Dana Farber Cancer Institute, Boston, MA_.

Introduction: Identifying driver mutations and significantly mutated genes (SMGs) is critical to characterize therapeutic targets and understand biological subtypes. Past studies in melanoma have identified ~20 SMGs and 4 genomic subtypes (BRAF, NRAS, and NF1, and triple wildtype). Critically, these studies have been powered to detect SMGs only at a frequency of >~10%. However, clinically targetable drivers can occur at much lower frequencies (e.g. ALK-rearrangmenet in lung cancer). Thus, we hypothesized that a uniform meta-analysis of all publicly available melanoma whole exome sequences would provide power to identify clinically SMGs not previously associated with melanoma. Methods: We assembled and uniformly analyzed a cohort of 1,135 tumor and matched germline primary and metastatic non-uveal melanoma whole exomes. All samples were realigned to hg19 using the Picard best practice realignment pipeline. These samples passed several QC metrics that included coverage (DepthOfCoverage, 50X tumor and 20X normal), cross sample contamination (ContEst, < 5%), tumor purity (FACETS, > 20%), and tumor-in-normal (deTiN, < 30%). Mutational significance analysis was performed using MutSigCV2 followed by filtering via biological priors using OncoKB. CNA profiles were generated using GATK4 best practices pipeline. Results: We identified 77 SMGs (MutSig q < 0.1) that were implicated in either melanoma or other cancer types according to OncoKB's curated list of 1,018 cancer genes, some of which that have not been considered SMGs or previously reported in melanoma. Novel genes were involved in immune response [B2M (1.7%) and PTPRC (12.1%)], DNA damage checkpoints [MDC1 (7.5%)], negative regulation of the MAPK pathway [CBL (4.6%)], MYC suppression [MGA (9.4%)], and the PI3K/AKT pathway [PIK3CA (3.4%) and PIK3CB (3.5%)]. Within the "triple-wt" subset of melanomas (n=212), we identified additional SMGs including IDH1, SF3B1, CTNNB1, and MAPK2K1 present in 3-5% of tumors, suggesting additional potential therapeutic targets. Copy number profiles of the cohort were consistent with previous findings. By increasing the resolution from cytobands to 1Mb regions, we identified a 1Mb region in chromosome 3 (chr3:138Mb-139Mb) that was enriched in amplifications (29.2%). Genes in this region include MRAS, PI3KCB, FOXL2, and FAIM. Conclusion: Through assembly and uniform analysis of the largest melanoma whole exome cohort to date, we identified significantly mutated known cancer genes not previously associated with the disease, and identified additional drivers within an incompletely characterized subtype of melanoma. This also enabled us to find genome regions enriched in copy number alterations. Taken together, our findings may inform the additional classification of melanoma and identification of novel drivers to shed light on biological subtypes and identify potential therapeutic targets.

#1305

A comprehensive investigation of immune infiltrates in hepatocellular carcinoma.

Sangho Yoon. _Hanyang University, Seoul, Republic of Korea_.

Hepatocellular carcinoma (HCC), a major form of primary liver cancer, is one of the leading causes of cancer-related death, and is caused by a myriad of risk factors from chronic inflammation by viral infection to genetic defects. Recent investigations shed light on the importance and role of immune cells in tumor microenvironment; however, since a majority of studies have focused only on tumor sites, the role of immune cells in tumor-surrounding (or precancerous) area with chronic inflammation was not investigated in HCC in detail. For the treatment of HCC patients, liver transplant (total hepatectomy) has been reported to offer much longer survival and better outcome compared to patients who had local tumor resection (partial hepatectomy). Nonetheless, the significance of tumor-infiltrating immune cells (TICs) established in patients who had suffered from chronic inflammation is still vague. Here, we collected total 122 Korean HCC samples, followed by high-throughput RNA sequencing (RNA-seq), from various premalignant stages—fibrosis and dysplastic nodules, tumors to noncacerous normal liver tissues—enhanced with public RNA-seq datasets including HCC from TCGA. Decomposing immune cell types using the RNA-seq data revealed that TICs are dynamically changed with showing no or weak correlations in the normal liver tissues, while being intertwined into dense and strong correlation networks as the disease progresses. Intriguingly, patients who had total hepatectomy showed different composition of immune cells and longer disease-free survival compared to the partial hepatectomy group; however, the presence of regulatory T cells (Tregs) before the transplantation dampened this outcome in the total hepatectomy patients. Tregs in the total hepatectomy group were found to be specific to tumor sites, and were enriched upon tumor progression, while pretreatment, such as embolization, increased the presence of Tregs. Besides the specific immune cells in tumor sites, the composition of certain types of immune cells in tumor-surrounding inflamed tissues was also important for predicting patients' outcomes in HCC. Particularly, alternatively activated macrophages (M2-type) have more predictive power in the patients' outcome when they were inferred from the tumor-surrounding inflamed samples. Our study not only provides a dynamic map of tumor-surrounding and -infiltrating immune cells, but also offers understanding about immune cell networks in the progression of HCC.

#1306

Density-based mutation clustering in 3D space.

Amila Weerasinghe,1 Sohini Sengupta,1 Adam D. Scott,1 Maththew H. Bailey,1 Michael C. Wendl,1 Ken Chen,2 Gordon Mills,2 Li Ding1. 1 _Washington University in St. Louis, St. Louis, MO;_ 2 _The University of Texas MD Anderson Cancer Center, Houston, TX_.

Cancer driver mutation and gene discovery has been a major challenge in cancer research. Several computational tools focus on clustering mutations on 3D protein structures to identify cancer drivers, including HotSpot3D, CLUMPS, HotMAPS, 3DHotSpots.org and e-Driver. While other tools provide a single snapshot of mutation clusters on the 3D structure, HotSpot3D utilizes a unique density-based clustering module capable of providing a full dynamical profile of clusters with varying densities. The density module (DM) is capable of detecting subtle variation in the density of mutations in 3D structures with little computation time and complexity. DM gives users the ability to detect the bridging effect, where more dense subclusters form a less dense supercluster due to a small number of mutations in between the subclusters connecting them. In addition to clustering based on physical density of mutations, DM is capable of clustering by additional biologic properties, including mutation recurrence and pathogenicity. Moreover, the underlying OPTICS algorithm utilized in the DM is inherently a "fuzzy" clustering algorithm, allowing slight variation of clusters based on the start point of the clustering algorithm from one run to another. Therefore, the DM provides a cluster membership probability measure for each mutation, giving the user the ability to choose the preferred stringency. Additionally, DM allows users to visualize the density clusters using Pymol. Since the whole dynamical set of clusters is produced by a single run, the visualization is performed so that one can reveal other clusters with higher densities by zooming in and out. Using a curated set of experimentally validated oncogenic mutations, we evaluated the performance of DM in detecting functionally activating mutations; our tool performed well in identifying driver mutations at high density thresholds. Moreover, the DM was able to identify mutations clustering at high density in BRAF, PIK3CA and KEAP1, which were not detected by other 3D clustering tools and sequence-based tools (SIFT, PolyPhen2, CHASM, etc.). The DM was also better at detecting clusters along the interface of protein-protein complexes (e.g., BRAF-KEAP1) compared to other 3D tools. In summary, HotSpot3D-DM allows for dynamical clustering, improved visualization, and identifies novel driver mutations missed by previous tools.

#1307

Effect of tumor purity on somatic mutation detection using next-generation sequencing technology: A benchmarking study.

Wendell Jones,1 Wenming Xiao,2 Somatic Mutation Working Group, SEQC2 Consortium. 1 _Q2 Solutions/EA Genomics, Morrisville, NC;_ 2 _FDA/NCTR, Jefferson, AR_.

Next generation sequencing (NGS) is becoming an essential tool for clinical cancer care. Whole genome sequencing (WGS), whole exome sequencing (WES), and targeted gene sequencing (TGS) have emerged as important tools and are critical for advancing precision oncology to meaningfully impact patient lives. To enable this goal, we must first thoroughly characterize the complexities of using NGS to identify somatic mutations in samples with tumor heterogeneity and variable tissue sample purity.

Here, we applied both experiment and in silico approaches to determine the effects of tumor heterogeneity on somatic mutation detection. First, we systematically investigated somatic mutation events in paired cell lines (breast carcinoma and matched normal) using a myriad of NGS platforms and protocols. Then, we applied machine learning algorithms to define high confidence mutation calls with datasets from the combination of all platforms and pipelines. Finally, we performed whole genome sequencing from tumor DNA samples titrated with matched normal cells to create a gradient of tumor purities from 100%, 75%, 50%, 20%, 10%, 5%, and 0%. We also created a series of in silico mixes by pooling reads from tumor and normal cell lines at respective ratios.

With somatic variation "ground truth" established by multiple orthogonal sequencing platforms and multiple analysis tools, we evaluated performance of individual software tools for tumor purity assessment and for mutation detection. Our experimental design allowed us to systematically evaluate accuracy, specificity and sensitivity of mutation detection across a range of tumor purities, as well as conduct a head-to-head comparison of in silico approaches versus experimental approaches.

This comprehensive study will help to clarify our understanding of experimental and analytical challenges that arise when confronted with tumor heterogeneity in patient care clinical decisions. In addition, data generated in this study will be used as a benchmark resource for the community to develop and improve tools to meet clinical challenges.

### Integrative Cancer Biology 2

#1308

Post translational crosstalk networks identify strategies to overcome EMT-mediated resistance to EGFR inhibitors.

Guolin Zhang,1 Karen Ross,2 Bin Fang,1 Jun-Min Zhou,1 Paul A. Stewart,1 Emma Adhikari,1 Eric A. Welsh,1 Xuefeng Wang,1 John M. Koomen,1 Cathy H. Wu,3 Eric B. Haura1. 1 _Moffitt Cancer Center, TAMPA, FL;_ 2 _Georgetown University Medical Center Washington, Washington, DC;_ 3 _University of Delaware, Newark, DE_.

Epithelial-mesenchymal transition (EMT) mediates intrinsic and acquired resistance to epidermal growth factor receptor (EGFR) inhibitors. This becomes a major hurdle in lung cancer treatment due to the lack of effective therapeutic strategies. We hypothesized that decoding the EMT signaling network could provide insights into the specific combinatorial logic associated with EMT signaling and identify new therapeutic strategies to combat EGFRi resistance. To test this hypothesis, we applied sequential enrichment of post-translational modifications (SEPTM) proteomics to analyze proteomes of expressed proteins and multiple post-translational modifications (PTM) including phosphorylation, ubiquitination, and acetylation in erlotinib sensitive cells (HCC4006) and matched erlotinib resistant cells after EMT (HCC4006ER). We conducted integrative informatics to characterize EMT associated proteins, PTMs, pathways, cross-talk among PTMs and signaling networks from our data. We used siRNA and small molecules to functionally interrogate our results by assaying cell viability and migration. We identified 6,641 proteins, 2,418 unique pSTY sites, 784 unique UbK-sites and 713 unique AcK-sites respectively. We found 377 proteins increased and 1377 proteins decreased (p<0.05, fold>2) in HCC4006ER cells compared to parent HCC4006 cells. We constructed an EMT signaling network, composed of 206 proteins with PTM changes including pSTY-sites (141 increase, 191 decrease), UbK-sites (29 increase, 32 decrease) and AcK-sites (14 increase, 46 decrease). Of 206 differentially modified proteins, 88 proteins are reported to be associated with EMT. Pathway analysis enriched 284 pathways from this EMT signaling network. We identified small molecule inhibitors associated with various pathways and tested for their effects on resistant cells. Inhibitors targeting 17 pathways and 3 major transcription factors were found to have effects on H4006ER viability, with inhibitors targeting DDR1, WNT and CDK signaling pathways demonstrating the most impact. Using RNAi, we found that that loss-of-function of 8 of 88 EMT-associated proteins (TAGLN2, STMN1, FYN, HNRNPA2B1, DDR1, INPPL1, OSMR and PRKAR2A) decreased HCC4006ER cell viability. Finally, integrative informatics revealed cross-talk among PTMs within EMT signaling network. From this analysis, we found that inhibiting GLI induced transcription sensitizes H4006ER cells to both EGFR inhibitor and Casein Kinase inhibitor. Collectively, SEPTM proteomics allows decoding the complex interplay in PTM modulation associated with EMT-mediated resistance. Our results suggest DDR1 as a potential actionable target for EMT driven resistance, which can serve as an example for combinatorial targeting of EMT proteins and signaling pathways as a strategy for overcoming EMT-mediated drug resistance.

#1309

Network as a biomarker to predict drug candidates: Mapping driver dysregulated target networks onto pharmacologic data-derived drug networks identifies cardiac glycosides as the potential treatment of Group 3 medulloblastomas.

Lei Huang,1 Sarah Garrett Injac,2 Xiaonan Li,2 Adesina Adekunle,2 Hong Zhao,1 Ching Lau,3 Stephen Wong1. 1 _Houston Methodist Research Institute, Houston, TX;_ 2 _Texas Children's Hospital, Houston, TX;_ 3 _The University of Connecticut School of Medicine, Hartford, CT_.

Background: Medulloblastoma is a heterogeneous group of tumors that consists of four subtypes with distinct genomic signatures. Two of these subgroups are defined by a single dysfunctional signaling pathway, WNT and SHH respectively, which has raised the prospect of taking a rational target based approach to the development of new therapies. Conversely, the other 2 sub-types, Groups 3 and 4, which compose 60-65% of total medulloblastoma cases, are associated with much more complicated genetic, epigenetic and genomic contents and display significant intragroup heterogeneity. In addition, Group 3 are associated with the worst prognosis among all the subgroups and are frequently metastatic at presentation which makes the need for novel approaches to drug discovery for these tumors particularly acute. Results: We developed a computational systems biology method that incorporates novel algorithms for driver signaling network identification (DSNI) and drug functional network-(DFN) based drug repositioning to integrate multiple types of genomics profiles for Group 3 MB patients with human cancer signaling pathways resources and gene expression profiles of 1,309 drugs in CMAP with drug structure information and effects. By applying the DSNI-DFN method on Groups 3 MB data we identified five members of the cardiac glycoside family, a group of Na/K pump inhibitors best known for their role in the treatment of heart failure, as potential inhibitors of driver dysregulated network of the Groups 3 MB. We were subsequently able to validate this finding in cell culture where all 5 compounds lead to significant growth inhibition of Group 3 derived medulloblastoma cell lines. Members of the cardiac glycoside family showed efficacy in vivo in animal models inhibiting proliferation. Digoxin significantly prolonged survival in both PDOX models of Group 3 medulloblastoma (ICb-2055) at a dose which resulted in plasma trough levels similar to those targeted in cardiac patients receiving digoxin. Digoxin-treated mice (n=10) had a median survival of 180 days compared to a median survival of 102 in untreated controls (n=8) (P<0.0001). Furthermore, in the Group 3 model digoxin showed survival extension comparable to ionizing radiation which represents a major component of the current standard care for medulloblastoma. Taken together, these data demonstrate that digoxin has a strong in vivo anti-tumor effect against preformed PDOX tumors derived from Groups 3 medulloblastoma. Conclusions: Our findings represent both an exciting potential new therapy for Group 3 medulloblastoma and validation of an approach that can be applied to identify driver dysregulated networks and predict drugs for a wide variety of genomically complex tumors.

#1310

Systematic evaluation of gene networks for discovery of disease genes.

Justin K. Huang, Daniel E. Carlin, Michael K. Yu, Wei Zhang, Jason F. Kreisberg, Pablo Tamayo, Trey Ideker. _University of California, San Diego, La Jolla, CA_.

Gene networks are rapidly growing in size and number, raising the question of which networks are most appropriate for a particular application. Here, we evaluate 21 human genome-wide interaction networks for their ability to recover gene sets associated with 446 different diseases and 9 cancer hallmarks. While all networks have some ability in these recovery tasks, we observe a wide range of performance with STRING, GeneMANIA and GIANT networks having the best performance overall. A general tendency is that performance scales with network size, suggesting that new interaction discovery currently outweighs the detrimental effects of false positives. Correcting for size, we find that the DIP network provides the highest efficiency (value per interaction). Based on these results we create a parsimonious composite network with both high efficiency and absolute performance, which outperforms any single resource. This work provides a benchmark for selection of molecular networks in human disease research.

#1311

Integrating network biology and polypharmacology to reveal TAMS/CXCL-1 as key mediator of XIAOPI formula preventing breast cancer metastasis.

Neng Wang, Zhiyu Wang, Yifeng Zheng, Shengqi Wang, Fengxue Zhang, Yi Lin. _The Research Centre for Integrative Medicine, Guangdong Provincial Academy of Chinese Medical Sciences, Guangzhou University of Chinese Medicine, Guangzhou, China_.

Purpose: Network pharmacology has become a powerful means of understanding the mechanisms underlying the action of Chinese herbs in cancer treatment. This study aims to validate the preventive effects and molecular mechanisms of a clinical prescription XIAOPI formula against breast cancer.

Experimental Design: The anti-metastasis ability of XIAOPI was validated by MMTV-PyMT mice and in vitro cell models. A network pharmacology-based method, which combined large-scale text-mining, drug-likeness filtering, target prediction and network analysis, was conducted to predict TAMS/CXCL-1 as key mediator of XIAOPI formula preventing breast cancer metastasis, further supported by cytokine array analysis in vitro and in vivo.

Results: In vivo breast cancer xenograft data showed that XIAOPI delayed breast cancer development and efficiently inhibited lung metastasis, accompanied by prolonged survival benefits and decreased cancer stem cell subpopulations. However, similar phenomenon were not observed in a cell model. The herb-ingredient-target network analysis further identified a total of 81 genes closely correlated with the breast cancer chemoprevention effects of XIAOPI. Cytokine array analysis further validated CXCL-1 as the key target of XIAOPI both in vitro and in vivo. Evaluation of the mechanism demonstrated that CXCL-1 administration significantly abrogated the metastatic inhibition effects of XIAOPI on breast cancer migration, invasion, stem cells subpopulations, epithelial-mesenchymal transition(EMT), or mammosphere formation abilities.

Conclusions: Overall, our study not only provides experimental evidence and molecular mechanisms that may facilitate the safe and effective use of herbal medicine for the prevention of breast cancer growth or metastasis, and may lead to CXCL-1-based therapeutic strategies for mammary malignancies.

#1312

Predicting ligand-dependent tumors from multi-dimensional signaling features.

Helge Hass,1 Kristina Masson,1 Sibylle Wohlgemuth,1 Violette Paragas,1 John E. Allen,1 Mark Sevecka,1 Emily Pace,1 Jens Timmer,2 Joerg Stelling,3 Gavin MacBeath,1 Birgit Schoeberl,1 Andreas Raue1. 1 _Merrimack Pharmaceuticals, Cambridge, MA;_ 2 _University of Freiburg, Germany;_ 3 _ETH Zuerich, Switzerland_.

Receptor tyrosine kinases (RTKs) are high-affinity cell surface receptors for growth factors that are frequently deregulated in cancer. Signaling through these receptors has been associated with increased cancer cell proliferation and resistance to cytotoxic therapies. To block this detrimental signaling, many companies are developing inhibitory antibodies against various RTKs. A key challenge in clinical studies is the optimal stratification of patients who may benefit from these therapies. For an RTK targeted antibody, the detection of the respective growth factor in the tumor microenvironment may be an important bio-marker. Beyond the physical presence of the growth factor, the decision whether a cancer cell will respond to growth factor-induced signals is governed by complex intra-cellular signaling networks. We compared different approaches to predict cellular responses and will highlight a hybrid approach that combines mechanistic modeling based on ordinary differential equations with a machine learning algorithm. The models are trained on in vitro drug response screens and then applied to predict response in patient samples. The mechanistic models are trained on quantitative data from signal transduction studies as well as RNAseq data for cellular characterization. Using the hybrid approach, a correlation between growth factor expression in the tumor microenvironment and its predicted response was identified. This supports the hypothesis of addiction of tumors to growth factors abundant in the tumor microenvironment, and might enable more robust patient stratification in the future.

#1313

A combined protein-protein interaction and genetic interaction map defines new and critical Kras effectors in NSCLC.

Peter K. Jackson. _Stanford University School of Medicine, Stanford, CA_.

Despite intensive study, no drugs in clinical use specifically target KRAS-mutant tumors. Uncharacterized feedback mechanisms and parallel pathways have stymied the treatment of KRAS-mutant tumors with Raf and PI3K inhibitors, and the KRas protein itself does not easily accommodate binding of small-molecule inhibitors. These challenges demand more systematic and quantitative characterization of the physical and genetic relationships between Ras regulators and effectors. Thus we used tandem affinity purification of Kras, Hras and Nras, their activated alleles and key proteins with known regulatory (GEFs, GAPs) or effector (Raf, RalGDS) roles in both 293 cells and A549 NSCLC cells to generate a high-confidence protein-protein interaction (PPI) network. This map of 220 proteins and 1,400 physical interactions was used to design an sgRNA library with 10 guides/gene. This library was screened in Cas9-expressing A549 cells and grown for 14 days before analysis for dropout or enhanced representation of sgRNAs. Approximately 120 genes showed positive or negative growth effects. PPIs and genetic interactions (GIs) were cross-referenced with public PPI data and TCGA patient data to assemble a combined physical PPI and genetic map informed by cancer mutations. This map suggests many hypotheses for PPIs critical for growth control. This set was used to construct a sgRNA library covering 120 genes of probable relevance to the Ras pathway with ~60 "safe harbor" control sgRNAs. This library was screened in a two-cassette sgRNA system testing 14K pairwise genetic effects to identify quantitative changes in growth in A549 and H23 NSCLC lines. This screen showed >100 genetic interactions, which in conjunction with PPIs identify coupling between the Raf/MEK/ERK kinase, Ral and Rap GTPase, RNA processing and cell adhesion pathways. The screen identified new candidate effector pathways for cell adhesion, RNA processing, Rap GTPase regulation and protein processing, including the RADIL, RGL and RIN Kras effectors. Validation focused on using the synthetic lethal interactions observed in the sgRNA screen to predict drug combinations showing drug synergy in A549 and H23 cells. Using 11-point dose titrations and isobologram analysis of drug combinations, we see strong synergy among PI3 kinase, Raf and Erk inhibitors in these cells. Using the recently described Kras G12C inhibitor, expressed in H23 cells, we have validated that sgRNA deletion of the the key Kras effector for specific pathway,s including cell adhesion (RADIL), growth signaling (RAF) and endocytosis/ macropinocytosis (RIN), is affected and that use of the Kras inhibitor ARS-853 shows much-reduced effects on specific Kras effector pathways in cells deleted for these effectors. These systematic data underscore the limitations of our current understanding of Kras-driven cancers, revealing new genetic vulnerabilities and target candidates.

#1314

A prostate cancer androgen receptor variant gene network with prognostic and therapeutic value identified using integrated systems analyses.

Maria J. Martinez, Fiorella Magani, Eric R. Bray, Valeria Copello, Ning Zhao, Stephanie Peacock, David J. Wiley, Gennaro D'Urso, Kerry L. Burnstein. _University of Miami Miller School of Medicine, Miami, FL_.

Constitutively active androgen receptor (AR) splice variants such as AR-V7 represent a well-established mechanism of disease progression to castration-resistant prostate cancer (CRPC), the incurable form of PC. These AR variants lack the carboxyl-terminal ligand binding domain, the target of all approved drugs against AR. Consequently, a significant challenge is to identify novel therapeutic strategies that are effective against AR variants-expressing prostate tumors without requiring direct targeting of these variants, which is challenging due in part to the unstructured nature of the AR amino terminus. We used a multi-pronged, unbiased systems biology approach to identify downstream AR-V7-regulated hub genes that both drive cancer progression and feedback to enhance AR-V7 activity. Such genes likely encode prognostic markers as well as potential therapeutic targets acting within the AR-V7 network. Our systems biology approach utilized: 1) clinically relevant gene sets upregulated during human PC progression obtained by Weighted Gene-Co-expression Network Analysis (WGCNA); 2) an AR-V7 functional interactome from a high-throughput synthetic genetic array (SGA) screening in the yeast, Schizosaccharomyces pombe; and 3) PC cell AR-V7 transcriptome. We identified seven genes that were upregulated in human PC, functionally interacted with AR-V7 and were AR-V7 targets in PC. This gene set, composed of cell cycle and mitosis-regulating genes, not only encoded select proteins previously linked to PC, such as Cyclin B1 and Topoisomerase 2A (CCNB1 and TOP2A), but also proteins with little or no prior association with AR-V7 or PC. The seven genes comprised a signature that strongly correlated with patient Gleason score and predicted disease free-survival in large independent PC patient cohorts. In contrast, this gene set was not associated with survival metrics in other human cancers. Individual depletion of the expression of these genes decreased ligand-independent AR transcriptional activity and reduced CRPC cell proliferation. Furthermore, individual knock down of the members of the gene set affected the expression of the other members of the set indicative of the interrelationship of these genes. Consistent with CRPC dependence on these interrelated mitotic genes, combinatorial targeting of two of its members: TOP2A and CCNB1, led to a synergistic and selective inhibition of AR-driven CRPC cell proliferation but had no effect on non-tumorigenic prostate epithelial cells. This unbiased and novel gene discovery strategy, which is broadly applicable to other cancers, identified clinically-relevant interacting gene hubs forming the basis for both prognostic use and rational, combinatorial therapy for CRPC.

#1315

Biophysics of polyploidal cancer cells in an aging stroma.

Michelle Dawson, Botai Xuan, Deepraj Ghosh. _Brown University, Providence, RI_.

Senescence is a potent tumor-suppressive mechanism that irreversibly arrests the growth of damaged cells. However, senescent cells that accumulate in tissues eventually develop a senescence-associated secretory phenotype (SASP) that alters the microenvironment to promote cancer. Paracrine factors in the SASP may also contribute to the formation of rare giant polyploidal cancer cells (GPCCs). A single-cell mechanical approach was used to profile cytoskeletal and nuclear mechanics, morphology, motility, and adhesion for breast cancer cells treated with conditioned media from senescent fibroblasts. Our study showed that a small but significant population of MDA-MB-231 breast cancer cells (less than 5%) treated with conditioned media from senescent LF-1 fibroblasts develop an enlarged morphology, chromosomal instability, and polyploidy, a phenotype associated with GPCCs. Although GPCCs are highly invasive and chemoresistant, little is known about their biophysical properties. First, we developed a method for identifying the small subpopulation of GPCCs in a heterogeneous population of cancer cells based on increased nuclear area and confirmed that GPCCs are more resistant to paclitaxel than normal-size MDA-MB-231 cells (NCCs). We then compared critical biophysical properties of NCCs and GPCCs, including cytoskeletal and nuclear mechanics, cell and nuclear morphology, motility, and adhesion. Cells were stained for cytoskeletal proteins actin, tubulin, and vinculin. Cytoskeletal organization was dramatically altered in GPCCs compared to NCCs. GPCCs displayed more disorganized microtubule structure, dense actin stress fibers, and mature focal adhesions. Intracellular particle tracking microrheology was used to measure cytoskeletal and nuclear mechanics. These studies demonstrated that although GPCCs are thought to be highly invasive cancer cells, they are inherently stiffer than NCCs, in terms of both their cytoskeletal and nuclear mechanics. This was surprising since more invasive cancer cells are often more compliant than less invasive cancer cells. This result may be in part to the ability for GPCCs to behave like activated stromal cells that stiffen in the tumor; we confirmed that GPCCs display similar adhesive behavior as activated stromal cells. To determine how mechanics correlates with cell migration, we used time-lapse nuclear tracking to measure cell motility. The average cell speed was higher for NCCs than for GPCCs; however, GPCCs moved longer distances over time because their motion was more directional. These findings highlight the unusual biophysical behavior of GPCCs. To develop pharmacologic tools that target GPCCs, it is imperative to understand their biophysical properties.

#1316

Vegetal scaffold as radiobiology model to study radiation cancer response.

Jerome Lacombe, Ryan Zenhausern, Frederic Zenhausern. _University of Arizona, Phoenix, AZ_.

Current in vitro tumor models have issues in accuracy in that the 2D structures and (often rare) cell co-culture technologies that exist, lack many features or characteristics found in vivo. The use of decellularized plant structures recellularized with human cells, aims to overcome these issues by taking advantage of their natural 3D structure. By using this approach on spinach leaves as a 3D scaffold, we have developed a new model that may be used as a new tumor model for radiobiology research. Spinach leaves were decellularized following serial chemical treatments with hexanes, SDS, Triton-X100 and bleach. In order to characterize the efficiency of the decellularization process, the rigidity of the leaves was assessed by Atomic Force Microscope (AFM) and DNA and protein quantification. Human prostate (PC3) and breast (MCF7) cancer cells were then seeded onto leaf. Seeding efficiency was assessed by optical microscopy and viability and proliferation ability were tested by MTT assay. In order to evaluate if the cells were biologically active, we then assessed radiation response. Extra cell-seeded leaves were irradiated and the expression of radiation-responsive genes were assessed in MCF7 cells. Additionally, DNA damage levels in PC3 cells were evaluated by γ-H2AX foci measurement using fluorescence microscopy. The decellularization process was successful, showing a protein content of 0.31 μg/mg tissue compared to the fresh leaf at 14.4 μg/mg tissue. The DNA quantity was similarly disparate between fresh and decellularized leaves. Microscopy showed that PC3 and MCF7 cells were well attached to the decellularized leaf surface after 24h incubation. Mechanical testing with AFM confirmed attachment by measuring Young's modulus values of 2.81, 88 and 197 MPa for decellularized, recellularized and fresh leaves respectively. Viability assays confirmed that cells were alive and able to proliferate. The gene expression assay showed changes in expression levels between 2D cell culture and cells seeded on leaves both at basal state and after 5Gy-irradiation in MCF7 cells. Finally, γ-H2AX immunofluorescent imaging showed DNA damage repairs are induced 1 hour after 5Gy of X-ray irradiation in PC3 cells and are effective up to 24h. Plant can be decellularized in order to create a 3D scaffold that may act as a support for cell seeding. Interestingly, radiation response can be measured on this new model and even show significant difference with standard 2D cell culture. Together, these results suggest that this approach is a new promising 3D cellular model for radiation research. However, additional studies are required to compare this model with in vivo response in order to clearly assess if this model is more suitable to mimick in vivo tumor/microenvironment than 2D standard model.

#1317

Multi-scale mapping of the physical and functional architecture of the cancer cell.

Fan Zheng,1 Michael K. Yu,1 Minkyu Kim,2 Keiichiro Ono,1 Mitchell Flagg,1 Jason F. Kreisberg,1 Nevan Krogan,2 Trey Ideker1. 1 _University of California, San Diego, La Jolla, CA;_ 2 _University of California, San Francisco, San Francisco, CA_.

Cancer is governed by modular systems of genes, the composition and organization of which remains poorly understood. Here, we integrate physical and functional networks from a wide range of molecular studies to assemble a comprehensive multi-scale map of human cancer cell biology. This map consists of a hierarchical catalog of protein complexes, signaling pathways and inter-pathway crosstalk implicated in cancer, and it suggests many uncharacterized functional modules as intriguing hypotheses for further validation. Analysis of the pattern of somatic mutations in The Cancer Genome Atlas (TCGA) reveals that these mutations target systems of varying scales above the level of individual genes. The map also provides a platform to integrate and interpret new 'omics data; we integrate new protein-protein interactions identified using AP-MS in multiple breast cancer cell lines, revealing how different functional modules are rewired in cancer cells. A general model browsing tool has been created to visualize and navigate these hierarchical cancer maps. This multi-scale mapping approach elucidates the molecular heterogeneity of cancer, connects tumor genotypes to phenotypes and, ultimately, enables a platform for cancer precision medicine.

#1318

Mapping tumoral and immune heterogeneity in PD-1 responsive glioblastoma multiforme.

Bojan Losic, Raymund Yong, Nicholas Akers, Ilaria Laface, Nadejda Tsankova, Robert Sebra, Sacha Gnjatic, Adilia Hormigo. _Icahn School of Medicine at Mount Sinai, New York, NY_.

Background and aims: Clonal evolution and the immune microenvironment in glioblastoma multiforme (GBM) harbor critical clues to its well-known but poorly understood treatment resistance, and to assessing the potential benefit of checkpoint inhibition. We created a tumor-immune interaction map by computing the mutational and neo-epitope tumoral burden, along with the magnitude and clonality of infiltrating lymphocytes, from initial and recurrent tumor of a single patient who benefited from PD-1 blockade. The question of how this particular GBM evolution trajectory compares to a typical GBM is addressed by projection of mutational and other signatures onto a causal gene-gene interaction network we derived from the TCGA GBM cohort.

Methods: We compiled samples from a 57- year-old patient who presented with a seizure and had a resection of a large temporal GBM followed by radiotherapy with concurrent temozolomide, with subsequent re-resection for recurrent disease. Starting two weeks after the final resection the patient received 26 cycles of a PD-1 checkpoint inhibitor simultaneous with 20 cycles of bevacizumab, a VEGF-A inhibitor, for a total survival of 25 months after diagnosis. Samples from the primary and three spatial sectors of the recurrence were submitted for RNA sequencing. Data analyses included intra-tumoral gene expression including MHC-I and MHC-II allele-specific expression, expressed relative mutation and (class I and class II) neo-epitope prediction, T and B cell receptor sequencing, and survival and molecular analysis on the GBM TCGA dataset.

Results and Conclusions: We quantified the evolving relative mutational and neo-epitope burden of a GBM, emphasizing the differences between class I and class II neo-epitope spectra and resulting adaptive immune recruitment between the original and recurrent tumor regions. We find no evidence of hypermutation in the recurrence, a relative absence of immune infiltration in all recurrences despite the expression of a number of neo-epitope generating mutations (including a novel clonal mutation of EGFR), and compelling evidence of epigenetically driven aberrations in one recurrence sector. Hypothesizing significant molecular heterogeneity as a result of immune editing and treatment, we derived a tumor-recurrence molecular evolution trajectory and projected it onto a Bayesian inference network we derived from the TCGA GBM cohort. We quantified intra-tumoral heterogeneity, derived a patient survival signature, and also identified immunogenic mutated gene regulatory subnetworks that could be potentially targeted by any induced robust immune response.

#1319

Edge-based integration of multi-omics data gives more accurate prognostic prediction in pancreatic cancer.

Yidi Sun, Chen Li, Rong Zeng. _Shanghai Institute of Biochemistry and Cell Biology, Shanghai, China_.

Pancreatic cancer (PC) is the fourth leading cause of cancer death worldwide, and predicted to be the second within a decade. Five-year survival of PC is less than 5% with a median survival just a couple of months. Therefore, a better understanding of the molecular pathology of PC is an urgent need to achieve advances in clinical treatment for patients. Genomic analyses previously have revealed heterogeneous landscapes of mutation, copy number variation, structural variation and gene expression in pancreatic cancer. While clinical evidence for this proposition is limited and the signaling and biological effects of genomic variations are not routinely determined in human tumors even though they are rationally considered to be drug targets. Luckily, with recent advances in mass spectrometry (MS), comprehensive proteomic and lipidomics analyses provide a potentially valuable approach to validate genomic findings and discover targeted treatments for pancreatic cancer patients.

We generated high-throughput proteomic and lipidomic data for pancreatic cancer patients aged from 40 to 80 years old with matched normal samples using TMT-labeling quantitative proteomic method and target lipidomic method. We found promising protein and lipid biomarkers closely related with the clinical outcome of pancreatic cancer patients. Taking signaling and biologically pathways into consideration, we used an edge-based method to distinguish prognostics, where edge features were constructed based on the correlation between each pair of molecules. We then performed feature selection and prognostic classification for both the original expression dataset and the transformed edge dataset and found the edge-based features can give a more accuracy for prognostic prediction, suggesting the promising role of protein and lipid edge biomarkers for clinical utility in pancreatic cancer.

Our preliminary results highlight the usefulness of edge-based integration of proteomics and lipidomics data. To explain the mechanisms in the development of pancreatic cancer, we are going to integrate genomic variations from whole genome sequencing (WGS) into our current progress for further analysis. In conclusion, this initial work demonstrated a strategy that may enable more accurate prediction of the survival of pancreatic cancer by integrating multi-omics data from genomics, proteomics and lipidomics. In principle, we anticipate the declaration of possible mechanisms enabling the treatment of pancreatic cancer patients in the near future.

#1320

An integrative and interactive colorectal cancer biomarker database.

Xueli Zhang,1 Xiao-Feng Sun,2 Yang Cao,1 Benchen Ye,3 Qiliang Peng,4 Xingyun Liu,3 Bairong Shen,3 Hong Zhang1. 1 _Medical Sciences, Orebro, Sweden;_ 2 _Clinical and Experimental Medicine, Sweden;_ 3 _Centre for System Biology, China;_ 4 _Radiotherapy & Oncology, China_.

We created a colorectal cancer (CRC) biomarker database (CBD) with credible content as a powerful and time-saving tool to provide better and accurate information concerning CRC biomarkers. CRC biomarker database (http://sysbio.suda.edu.cn/CBD/) was established based on 870 identified CRC biomarkers and their relevant information from 1115 original articles in PubMed published from 1986 to 2017. In this version of the CBD, CRC biomarker data was collected, sorted, displayed, and analysed. The CBD with the credible contents as a powerful and time-saving tool can provide more comprehensive and accurate information for further CRC biomarker researches. The CBD was constructed under MySQL server. HEML, PHP and JavaScript languages have been used to implement the web interface. The Apache was selected as HTTP server. All of these web operations were implemented under the Windows system. The CBD was concluded the multiple individual biomarker information and categorised into the biological category, source and application of biomarkers; the experiment methods, results, authors and publication resources; the research region, the patient's age, gender, race, the number of tumours, tumour location, and stage. The key words for searching in PubMed are: (((biomarker[Title/Abstract] OR marker[Title/Abstract]) OR indicator[Title/Abstract]) OR predictor[Title/Abstract]) AND ((colorectal cancer[Title/Abstract] OR rectal cancer[Title/Abstract]) OR bowel cancer[Title/Abstract]). The CBD can provide a professional platform to researchers who are interested in CRC research to communicate, exchange their research ideas and further design high quality research in CRC. They can submit their new findings to our database via th submission page, and communicate with us in the CBD.

#1321

Mutational landscape of adherens junctions in skin cutaneous melanoma.

Praveen Kumar Korla, Daniel Esguerra Gracilla, Brian Yu-Ting Kuo, Jim Jinn-Chyuan Sheu. _NSYSU, Kaousiung, Taiwan_.

Cadherin proteins regulate cell-cell contact through calcium-dependent manner, thus play pivotal roles in morphogenetic and differentiation processes during tissue development. Reprograming in cadherin profiles contributes to epithelial-mesenchymal transition (EMT), leading to disorganized lining of epithelial tissues and cancer metastasis. To elucidate genetic impacts of classical cadherins on melanoma development, mutation profiles were analyzed using data from the TCGA databank. Among 20 classical cadherins, CDH6 showed the highest mutation frequency (11.3 %), and most of which were G to A or C to T nucleotide substitutions, a signature for UV-caused DNA damage. Two groups of unique hotspots were found on the fifth cadherin domain (D5) (S524L and S526F) and cytoplasmic cadherin domain (CDH-C) (E662K and E722K), respectively. Protein interactome and expression co-occurrence analyses placed CDH6 at the center of core cadherin network and showed strong heterophilic interactions of CDH6 with CDH4, CDH9, and CDH12. Multiple sequence alignment indicated the involvement of those hotspots in the conserved regions, which are responsible for heterophilic interaction on D5 or catenin (alpha, beta, and gamma) binding on CDH-C. Molecular modeling defined alterations in protein structures of the D5 and CDH-C domains caused by the hotspot mutations, which may influence cell-cell contact and the down-stream signaling. In sum, our data suggest CDH6 as the key player to modulate adherens junctions during the development of melanoma.

#1322

A systems-level approach identifies novel links between iron and fatty acid metabolism in high-grade serous ovarian cancer.

Anna Konstorum,1 Miranda L. Lynch,2 Suzy V. Torti,1 Frank M. Torti,1 Reinhard C. Laubenbacher1. 1 _UConn Health, Farmington, CT;_ 2 _Roswell Park Cancer Institute, Buffalo, NY_.

The low survival rate for high-grade serous ovarian cancer (HGSOC) motivates novel approaches to identify causative and therapeutically targetable factors involved in HGSOC initiation and progression. Cancer cells are known to sequester iron, which can potentiate cancer progression via mechanisms that have not yet been completely elucidated. In order to uncover novel, and potentially therapeutically tractable links, between iron and HGSOC, we developed an algorithm to identify perturbed regulatory pathways containing differentially expressed iron-related genes in microarray data from clinical sources and an experimental model of HGSOC. Used in tandem with over-representation analysis for iron-related genes, the algorithm led us to uncover an iron dependence of fatty acid import and synthesis pathways, which are upregulated in HGSOC and other cancers, and to develop a network synthesizing the relationship between iron, fatty acid metabolism, and HGSOC. We use the network to derive specific hypotheses of mechanisms by which iron impacts fatty acid metabolic pathways to promote tumorigenesis. We have thus shown that a systems-level approach to identifying novel regulatory links between iron and HGSOC has yielded a previously unappreciated association between iron and fatty acid metabolism that may be exploited for therapeutic potential. This work has been supported in part by NIH grants F32CA214030 to AK, R01CA188025 to SVT, and RO1CA171101 to FMT.

#1322A

**Computational study of the effects of non-synonymous mutations in human** UBE3A **gene present in virus-associated cancers.**

Claudia Machicado, Maria Pia Soto. _Universidad Peruana Cayetano Heredia, Lima, Peru_.

UBE3A gene encodes the ubiquitin ligase E6 associated protein (E6AP), an enzyme that catalyzes the high-risk human papilloma virus (HPV) E6-mediated ubiquitination of p53. Such modification in p53 contributes to the neoplastic progression of cells infected by HPV-16/18 that leads to cervical cancer (CC). The hijacking of E6AP also occurs by Hepatitis C virus (HCV) that targets Rb protein thus conducting to hepatocellular carcinoma (HCC). We hypothesized that certain non-synonymous (missense) mutations of E6AP drives to elevated degradation rates of either p53 or Rb by increasing its enzymatic activity. This study aimed to predict changes in: (i) functional activity in E6AP mutants, and (b) E6AP affinity to viral oncoproteins and p53/Rb. A subset of three virus-associated cancers including cervical carcinoma (CC), hepatocellular carcinoma (HCC) and oral squamous carcinoma (OSC) were analyzed. The Cancer Genome Atlas (TCGA) dataset as well as Biomuta were mined and missense mutations in human UBE3A gene were retrieved. To explore the impairment of protein function and stability across the UBE3A gene mutants, machine learning approaches (i.e. Polyphen, I-Mutant 3.0, Panther and Provean) and NetSurfP were applied. The 3D structures of E6AP variants were modelled and docking simulations with viral oncoproteins and p53/Rb were performed using PyDock. Our results showed 22 mutations in UBE3A gene that were present across CC, HCC and OSC, with most of variants (60%) appearing in HCC. UBE3A variants were different across the three tumor types analyzed. Seven of 22 UBE3A variants were predicted damaging or deleterious substitutions. Five damaging mutations affected the HECT domain responsible for the enzyme activity. Such corresponded to E743K and P850A, existing in CC, as well as M589L, E754K, and Q576*, found in HCC. Two predicted damaging mutations were located within the HCV binding site including G492V (found in CC) as well as F477C (present in OSC). No mutation involved the highly conserved LXXL domain implicated in E6 binding or the E6AP trimerization residues. The predicted E6AP-E6 complexes revealed that LXXLL domain in E6AP participates in E6 binding, both in wild-type and mutants. Interestingly, E6AP E743K mutant showed increased complex stability in comparison with wild-type. In conclusion, most of E6AP mutations (68%) existing in CC, HCC and OSC are likely to be neutral whereas some others (32%) are potentially damaging variants and likely to increase E6AP activity. E6AP-dependent ubiquitylation of p53 represents an important opportunity to discover novel entities that inhibit protein-protein interactions thus avoiding p53 degradation.

## MOLECULAR AND CELLULAR BIOLOGY / GENETICS:

### Autophagy

#1323

A p53-dependent switch between cytoprotective and nonprotective autophagy influences tumor cell sensitivity to cisplatin.

Jingwen Xu,1 Tareq Saleh,2 Nipa Patel,2 Yingliang Wu,1 Santiago Lima,2 David Gewirtz2. 1 _Shenyang Pharmaceutical University, China;_ 2 _Virginia Commonwealth University, Richmond, VA_.

Autophagy is a multi-step, homeostatic process through which cells turnover dysfunctional organelles and proteins. Previous work in our laboratory has identified multiple functional roles of autophagy, specifically cytoprotective, cytotoxic, cytostatic and nonprotective, when subjected to radiation and chemotherapy. Furthermore, we have reported on the existence of an autophagic switch between one functional form (generally cytoprotective) to the other forms. To further explore the autophagic switch, we utilized p53 wild type (p53 +/+) and p53 null (p53 -/-) H460 non-small cell lung cancer (NSCLC) cell lines exposed to cisplatin. The extent of autophagy was similar in both cell lines, but the rate and extent of apoptosis was higher in the p53 +/+ cells, which showed greater cisplatin sensitivity. Pharmacological (chloroquine, 3-Methyladenine) and genetic (ATG5 silencing) inhibition of autophagy failed to sensitize the p53 +/+ H460 cells to cisplatin, suggesting that autophagy has a nonprotective role. In contrast, autophagy inhibition (CQ, 3-MA, or shATG5) increased sensitivity to cisplatin in the p53 -/- H460 cells, indicative of a cytoprotective function of autophagy. These studies support the premise that cytoprotective autophagy may confer resistance to cisplatin. Moreover, we confirm the pivotal role of p53 in determining the function of autophagy induced by cisplatin. Finally, we provide an additional example of a switch between the nonprotective and protective forms of autophagy in p53 wild type and p53 null H460 non-small cell lung cancer (NSCLC) cells, respectively.

#1324

TRPM2 promotes leukemia proliferation and survival through modulation of ROS and autophagy.

Shu-Jen Chen,1 Lei Bao,1 Kerry Keefer,1 Longgui Chen,1 JuFang Wang,2 Xue-Qian Zhang,2 Santhanam Shanmughapriya,2 Iwona Hirschler-Laszkiewicz,1 Muniswamy Madesh,2 Joseph Y. Cheung,2 Hong-Gang Wang,1 Barbara A. Miller1. 1 _Penn State Univ. College of Medicine, Hershey, PA;_ 2 _Temple University, Philadelphia, PA_.

Acute myeloid leukemia (AML) is a heterogeneous group of leukemias that arise in hematopoietic precursors. Understanding the mechanisms involved in AML proliferation and survival is key to development of new therapies. Transient receptor potential melastatin 2 (TRPM2), a calcium permeable ion channel, modulates cell proliferation, survival and oxidative stress-induced cell death and is highly expressed in many cancer cells including AML. To investigate the role of TRPM2 in AML, we depleted the TRPM2 gene in the human AML cell line U937 using clustered regularly interspaced short palindromic repeats/Cas9 (CRISPR) technology. RT-PCR, western blot, and patch clamp analyses confirmed that TRPM2 was depleted in U937 knockout (KO) cells. Cell proliferation and viability measured by XTT and trypan blue exclusion assays were significantly reduced in KO cells after doxorubicin treatment. Expression of wild type TRPM2 (TRPM2-L) restored cell growth and cell survival to doxorubicin-treated TRPM2 KO cells, whereas the TRPM2 channel pore mutant (E960D) or empty vector did not. In a xenograft mouse model, mice were injected with TRPM2 KO or control U937 cells which express luciferase. Mice injected with TRPM2 KO U937 cells showed significantly reduced leukemia growth and enhanced sensitivity to doxorubicin. To examine the mechanisms, mitochondrial ROS were measured with confocal microscopy and MitoSOX Red. ROS were significantly elevated in TRPM2 KO cells compared to scrambled control, and further increased when KO cells were treated with doxorubicin. ATP production, oxygen consumption rate (OCR), and mitochondrial Ca2+ uptake were significantly reduced and mitochondrial membrane potential (∆Ψ) significantly increased in TRPM2 KO cells compared to scrambled control cells treated with doxorubicin, demonstrating dysfunctional mitochondria. Western blot analysis showed that expressions of HIF1/2α, FOXO3a and Nrf2 were downregulated in TRPM2 KO cells, further contributing to the increase in ROS. Expression of autophagy proteins ULK1, Atg7 and Atg5 was significantly reduced and mitochondrial protein Tom20 significantly increased in TRPM2 KO cells, suggesting that depletion of TRPM2 significantly reduced autophagy in AML cells. Reconstitution of wild type TRPM2 but not E960D restored ULK1 expression. In conclusion, depletion of TRPM2 significantly inhibits AML proliferation and increases sensitivity to doxorubicin by reducing mitochondrial function, increasing ROS, and impairing autophagy. TRPM2 contributes significantly to AML survival and may be a novel therapeutic target in leukemia therapy.

#1325

DIRAS3 (ARHI) is required for amino acid-mediated autophagy and nutrient deprivation in dormant ovarian cancers.

Margie Nicole Sutton, Gilbert Y. Huang, Jinhua Zhou, Zhen Lu, Robert C. Bast. _UT MD Anderson Cancer Ctr., Houston, TX_.

The persistence of dormant, drug-resistant cancer cells after cytoreductive surgery and combination chemotherapy is a major challenge that contributes to poor outcomes for ovarian cancer patients. Despite normalization of CA125 and negative imaging following primary treatment, "second look" surgery can detect small, quiescent, poorly vascularized nodules of persistent ovarian cancer in ~50% of patients. After positive second look operations, persistent ovarian cancer may take months or years to become clinically evident, consistent with tumor dormancy. Autophagy is one mechanism by which persistent, potentially dormant cancer cells could survive in a nutrient poor environment. Autophagy or "self-eating" is a catabolic process by which organelles and misfolded proteins are degraded and recycled to provide energy that could protect dormant cancer cells from acute starvation in the face of an inadequate blood supply. Autophagy can be induced by multiple mechanisms. Our group has found that, in contrast to primary ovarian cancers, the cancer cells in >80% of positive second look biopsies exhibit autophagy and express DIRAS3 (ARHI). DIRAS3 is an imprinted tumor suppressor gene that is downregulated in >60% of primary ovarian cancers by multiple mechanisms including loss of heterozygosity, CpG promoter methylation, transcriptional repression (E2F1/4) and miRNA regulation (miR-221/222). Re-expression of DIRAS3 prevents ovarian cancer growth, inhibits motility, induces autophagy and establishes tumor dormancy in xenografts. When DIRAS3 is upregulated in human ovarian carcinoma cell lines from a doxycycline-inducible promoter in nu/nu murine xenografts cancer cells remain dormant until DIRAS3 is downregulated permitting cell cycling, vascularization and progressive growth. Autophagy is required for survival of dormant xenografts in that treatment with chloroquine, a functional inhibitor of autophagy, markedly delays outgrowth of ovarian cancer xenografts when DIRAS3 is downregulated. Mechanisms by which DIRAS3 is upregulated and autophagy induced in positive second look specimens remains poorly understood. We have found that amino acid deprivation upregulates DIRAS3 and induces autophagy. Knockdown of DIRAS3 markedly decreases induction of autophagy following nutrient deprivation. We have identified that amino acid deprivation results in decreased E2F1/E2F4 expression and transcriptional upregulation of DIRAS3, but changes in promoter methylation or miRNA regulation are not observed. Knockdown of E2F1 or E2F4, both genetically and pharmacologically increase DIRAS3 expression and autophagy. Taken together these results suggest that nutrient deprivation results in transcriptional upregulation of DIRAS3-mediated autophagy and thus likely presents a mechanism by which DIRAS3 is upregulated in dormant, second look ovarian cancer tumor specimens.

#1326

Nonprotective autophagy fails to confer resistance to radiation.

Jingwen Xu,1 Emmanuel Kenneth Cudjoe,2 Tareq Saleh,2 Nipa Patel,2 Moureq Alotaibi,3 Yingliang Wu,1 Santiago Lima,2 David Gewirtz2. 1 _Shenyang Pharmaceutical University, China;_ 2 _Virginia Commonwealth University, Richmond, VA;_ 3 _King Saud University, Riyadh, Saudi Arabia_.

Radiation remains a predominant treatment option for advanced, inoperable lung cancer, however, resistance represents a major barrier against effective therapy. The exact mechanisms through which cancer cells exhibit resistance to radiotherapy still remains unresolved. Traditionally, radiation-induced autophagy is considered as a mechanism of resistance which attenuates the antitumor effects of radiotherapy i.e., autophagy plays a cytoprotective role on cell survival. To examine this concept further, we employed H460 non-small cell lung cancer (NSCLC) cells that were either p53 wild-type (p53 +/+) or p53 null (p53 -/-) to study the contribution of autophagy to radiation sensitivity. While autophagy was markedly higher in p53 +/+ cells (determined by p62 degradation, LC3B conversion, and acidic vesicle formation) the p53 +/+ cells were actually more radiosensitive than their counterpart H460 p53 -/- cells, which showed lower levels of radiation-induced autophagy. Despite modest radiosensitization by chloroquine, alternative pharmacological (3-methly adenine) and genetic (ATG5 silencing) inhibition of autophagy failed to radiosensitize either p53 +/+ or p53 -/- cells, indicating that radiation induced autophagy is not cytoprotective in function in these experimental models. Furthermore, secretome analysis (nLC-MS/MS) of p53 +/+ H460 cells showed no significant association with radioresistance-related proteins in comparison with p53 -/- H460 cells. The rate and extent of apoptosis was quite low and similar in the two cells lines, essentially ruling out apoptotic cell death as the basis for differential radiation sensitivity. Finally, Senescence was more pronounced in the p53 +/+ cells compared to the p53 -/- cells, which may contribute to the greater radiation sensitivity in the p53 +/+ cells. However, the most relevant finding in this work was that when autophagy is the nonprotective form, it does not confer inherent radiation resistance in tumor cells.

#1327

Autophagy facilitates tumor promotion in PyMT tumors through up-regulation of Pparg.

Takako Okamoto, Song Chen, Syn Yeo, Jun-Lin Guan. _University of Cincinnati, CIncinnati, OH_.

Autophagy genes have been reported to have either tumor promoting or inhibition functions in various cancers, but it is convoluting because of autophagy-independent functions of the respective autophagy genes. We previously reported that suppression of Fip200, an essential autophagy gene, inhibited tumor growth of PyMT driven mammary tumors. To dissect the specific autophagy-dependent function of FIP200 in these tumors, we recently established Fip200 Knock-in PyMT tumor mice with MMTV-Cre (cKI), allowing for conditional inhibition of autophagy in PyMT driven tumors without perturbing the non-autophagy functions of FIP200 . Compared to wild type (WT) tumors, tumor initiation, growth and metastasis were significantly suppressed in both cKI tumors and conditional Fip200 deleted tumors (cKO). Microarray analysis (GSEA) showed that adipogenesis related genes were significantly enriched in WT tumors compared to cKO tumors. The mRNA expression and protein expression of Pparg, one of the master regulators of adipogenesis, were significantly increased with autophagy induction under starvation condition (especially Glucose starvation) in PyMT isogenic cells compared to Fip200 KO and KI cells. Many Pparg target genes were down-regulated in autophagy deficient tumors. Among these, CD36, a free fatty acid (FFA) transporter, was also decreased compared to WT tumors. This suggests that FFA may have important roles as an energy source for PyMT tumor cells to survive under starvation conditions. In conclusion, our results indicate that tumor promotion of PyMT driven tumors was dependent on canonical autophagy. Our future studies will address the detailed mechanism of autophagy dependent tumor growth through metabolic regulation by Pparg.

#1328

Cadmium-induced endoplasmic reticulum stress causes defective autophagy in human prostate carcinogenesis.

Venkatesh Kolluru, Ashish Tyagi, Balaji Chandrasekaran, Murali K. Ankem, Chendil Damodaran. _University of Louisville, Louisville, KY_.

Introduction: Exposure to cadmium (Cd) is associated with a spectrum of human pathogenesis including the prostate cancer (CaP). A clear dose-response relation between Cd-exposure and CaP have been reported in men exposed to Cd. However, the molecular mechanism underlying the malignant cell transformation following Cd exposure is yet to be determined. One of the possible mechanisms is that Cd causes endoplasmic reticulum (ER) stress, which further induces defective autophagy that plays a cytoprotective role in response to the misfolded and unfolded proteins that are formed during cellular transformation. Hence, the goal of this study is to investigate the underlying mechanism of how Cd causes malignant cell transformation (from normal to cancer cells) and on the development of tumorigenesis by the Cd-transformed cells.

Methods: Normal prostate epithelial cells (RWPE-1) and Cd (10µM)-transforming prostate epithelial cells and cadmium-transformed prostate epithelial cells (CTPE) were utilized. Overexpression and/or silence ER-sensors, EGFR, and p62 were performed in above mention cell lines and subjected to cell viability, apoptosis, autophagy functional studies and Western blot analyses. For statistical analysis, data were analyzed using Student's 't' test with a p-value less than 0.05 considered significant.

Results: Our preliminary results suggest that during cellular transformation, Cd exposure induced ER-stress, which triggered the phosphorylation of stress transducers including protein kinase R-like ER Kinase (PERK) and e1F2-α (eukaryotic translation initiation factor 2A-alpha). Phosphorylation resulted in the activation of ATF4 (Activating Transcription Factor 4) and autophagy induction thus enhancing protection of Cd-damaged cells. Further, inhibition of stress inducers (ATF4) or p62 by siRNA blocked the Cd-induced defective autophagy resulted in growth inhibition in transforming cells. Interestingly, in Cd-transformed cells, blocking EGFR activation by siRNA or pharmacological inhibitors significantly inhibited the growth, but not in the transforming cells suggesting that EGFR activation plays a critical role only after cellular transformation. Further, xenograft tumor tissues generated by Cd-transformed cells expressed high levels of ATF-4, EGFR, p62 and LC3B in correlation with in vitro findings. Moreover, increased expression of the proteins (ATF-4, EGFR, p62, and LC3B) in human CaP specimens correlates with Gleason sum in comparison with benign prostatic hyperplasia and "normal" adjacent tissues.

Conclusions: The results suggest that ER stress responsible for the defective autophagy in Cd-induced transformation. This study highlights the better understanding of the complex interrelationship among prostate cancer phenotypes and the molecular, cellular, biochemical, and pathological changes associated with Cd and prostate cancer.

#1329

Combination treatment with CB-839 and romidepsin induces apoptosis and suppresses cell viability in preclinical models of chondrosarcoma.

Tahir N. Sheikh, Parag P. Patwardhan, Gary K. Schwartz. _Columbia Univ. Medical Ctr, New York, NY_.

Chondrosarcomas are the second most frequently occurring type of bone malignancy, and account for approximately 25% of all bone sarcomas. They are often highly aggressive neoplasms that rapidly progress and eventually recur and give distant metastases. They are largely considered to be resistant to conventional chemotherapy and radiotherapy. Several studies have reported that mTORC1 stimulates glutamine metabolism and increased mitogenic signaling through activation of the mTORC pathway, coupled with deregulation of the Cyclin D/retinoblastoma (Rb) pathway is a common feature of certain malignancies. In the present study, we hypothesized that concurrent inhibition of glutamine metabolism and histone acetylation could result in decreased viability of chondrosarcoma cells. To test this, we used a panel of chondrosarcoma cell lines including IDH wild type (CH2879), IDH1 mutant (JJ012) and IDH2 mutant (SW1353) cell line. Results from our in vitro proliferation assay showed that combination of sub-IC50 concentrations of the CB-839 (Glutaminase inhibitor) and Romidepsin (HDAC inhibitor) resulted in decreased cell viability of all the three chondrosarcoma cell lines when compared to either of the two drugs alone. Western blot analysis showed induction of cleaved Poly-ADP Ribose Polymerase (PARP) as well as cleaved caspase 3, known markers of apoptosis and down regulation of mTORC1 targets such as phospho-S6 and Phospho-p44/42 MAPK such as ERK1/2. Combination treatment also resulted in increased induction of histone acetylation (AcH3). Cell cycle analysis showed induction of sub-G1 population phase in combination treatment. Previous reports have shown that CpG methylation is a critical event in transcriptional repression of hyper methylated genes in cancer. To this end, we show that combination treatment with CB-839 and Romidepsin results in down regulation of (H3K9) methylation. Taken together, our data strongly suggests that combination treatment with CB-839 (Glutaminase inhibitor) and Romidepsin (HDAC inhibitor) is a novel treatment approach and merits evaluation in the treatment of chondrosarcoma.

#1330

Role of epigenetic remodeling in sensitizing triple-negative breast cancer cells to treatment through enhanced chemotherapy-induced autophagy.

Liliya Tyutyunyk, Joseph Landry, David Gewirtz, Nga Dao. _Virginia Commonwealth University, Richmond, VA_.

Dysregulation of the epigenome is implicated in initiation and progression of variety of cancers and their acquired resistance to chemotherapy. As such targeting epigenetic regulators has the potential to modulate cancer cell biology and their sensitivity to chemotherapy and/or radiation. Our studies demonstrates that silencing of the epigenetic regulator Nucleosome Remodeling Factor (NURF) sensitizes breast tumor cells to chemotherapy and enhances the anti-tumor immune response. A screen of a variety of chemotherapeutic agents shows that NURF KD cells are selectively sensitized to Topo II inhibitors, which includes doxorubicin. NURF KD increases DNA damage (gamma H2AX staining) and autophagy (acridine orange staining) in breast tumor cells exposed to doxorubicin (Dox) and enhances growth inhibition as well as suppressing the ability of the cells to recover proliferative capacity. Increased autophagy as well as breast tumor sensitization to doxorubicin were observed using a small molecule inhibitor of NURF, suggesting that NURF can also be targeted pharmacologically. Our studies suggest that enhanced autophagy may be a primary contributor to chemo sensitivity in NURF KD cells. Studies are currently underway to confirm the role of autophagy in chemosensitization in- vitro and enhanced immune response in-vivo using autophagy deficient cells (through ATG silencing). Doxorubicin produces profound DNA damage and promotes immunogenic cell death enhanced by NURF KD. Increased cell autonomous antitumor effects by doxorubicin in concert with increased cell non-autonomous antigenicity could help to achieve tumor regression, reduce metastasis, and possibly promote long term remission in breast cancer.

#1331

Inhibition of mitochondrial reprogramming regulated c-Src in triple-negative breast cancer activates autophagy-mediated survival mechanism.

Kwang Hwa Jung,1 Jun Hyoung Park,1 Tirupataiah Sirupangi,1 Dongya Jia,2 Shivanand Pudakalakatti,3 Nishant Gandhi,4 Jessica Elswood,5 Vasanta Putluri,1 Chad J. Creighton,1 Weston Porter,5 Michael T. Lewis,1 Xi Chen,1 Nagireddy Putluri,1 Pratip K. Bhattacharya,3 Lee-Jun C. Wong,1 Gokul M. Das,4 Benny A. Kaipparettu1. 1 _Baylor College of Medicine, Houston, TX;_ 2 _Rice University, Houston, TX;_ 3 _MD Anderson Cancer Center, Houston, TX;_ 4 _Roswell Park Cancer Institute, Buffalo, NY;_ 5 _Texas A &M University, College Station, TX_.

c-Src is a proto-oncogene involved in signaling that culminates in the control of multiple biological functions. Src is also one of the most frequently upregulated pathways in triple negative breast cancer (TNBC). Dysregulation of Src has been detected in TNBC and is strongly associated with tumor metastasis and poor prognosis. However, even after promising preclinical studies, Src inhibitors did not show major clinical advantage in unselected TNBC populations. Thus, understanding the mechanism of drug resistance to Src inhibition has major clinical significance in TNBC patients. The full activation of Src signature depends on the autophosphorylation at Y419 that allows the substrate to gain access. We have previously published that metastatic TNBC has high energy-dependency to mitochondrial fatty acid beta-oxidation (FAO) and FAO activate Src by inducing autophosphorylation at Y419. However, our recent analysis suggests that as observed with the Src inhibitors, treatment with FAO inhibitors only attenuate the TNBC tumor growth, but do not result in complete regression. Evaluation of their drug resistance mechanism revealed that while short-term inhibition of FAO or Src induces autophagic and apoptotic cell deaths, long-term inhibition results in autophagy-mediated drug resistance and survival. Studies using p53 knocked out TNBC cells confirmed that the autophagy-mediated resistance to Src inhibition is independent of their p53 status. Further analyses suggest that FAO and Src inhibitors increase the phosphorylation of ERK1/2 in TNBC. Treatment with MAPK/ERK inhibitors abolished the FAO or Src inhibitor-mediated autophagy activation. Validation of in vitro findings using in vivo TNBC patient-derived xenograft (PDX) models confirmed that Src inhibition enhances ERK1/2 activity and induces autophagy in TNBC. Overall, our results suggest that long-term FAO or Src inhibition results in ERK-mediated autophagy activation and therapeutic resistance in TNBC. This finding will have major therapeutic impact in the management of currently non-targetable aggressive TNBC.

#1332

Autophagic clearance of protein aggregates is impaired in cancer cells with dysfunctional mitochondria.

Thomas Biel, Ashutosh Rao. _FDA-CDER, Silver Spring, MD_.

Mitophagy and aggrephagy are selective types of autophagy that degrade damaged mitochondria and protein aggregates, respectively. Dysfunctional mitochondria are known to undergo mitophagy and cause protein aggregation. How a cell can simultaneously coordinate the removal of dysfunctional mitochondria and protein aggregates via autophagy is unknown. Here, two agents, Mitoquinone (MitoQ) and MitoApocynin (MitoApo), known to induce mitochondrial dysfunction and mitophagy in MDA-MB-231 cells were used to investigate the accumulation of protein aggregates and the mechanisms that contribute to selective autophagy. Using a protein aggregate dye and a poly-ubiquitinated antibody, carbonyl cyanide m-chlorophenyl hydrazine (CCCP), MitoQ, and MitoApo were identified as agents that caused an accumulation of poly-ubiquitinated protein aggregates in cancer cells by FACS analysis and confocal imaging. Microflow imaging of the cellular lysates confirmed a relative accumulation of large protein (>460 kDa) complexes. To establish the presence of aggrephagy, the aggregates were colocalized with LC3. Wild type and ATG7 knockout MEF cells were used to test whether protein aggregates accumulated in an autophagy-dependent manner. Our data currently demonstrates the kinetics of autophagic clearance of drug-induced protein aggregation and dysfunctional mitochondria.

#1333

Inactivation of NBR1 enhances sensitivity to rapamycin on human urothelial cancer cell lines through AMPK/ULK1-mediated autophagy.

Myeong Joo Kim, Min Ji Cho, In Ho Chang, Young Mi Whang. _Chung-Ang Unviersity, Seoul, Republic of Korea_.

Rapamycin has been highly evaluated in clinical therapeutic intervention for cancer patients as a specific inhibitor of the mammalian target of rapamycin (mTOR) kinase. Rapamycin also stimulates autophagy and mitophagy to remove damaged cells as a regulator of general autophagy. Previously, we identified increased expression of mRNA levels of NBR1 (autophagy cargo receptor) by inhibiting mTOR signaling in human urothelial cancer cell lines. Therefore, we investigated whether loss of NBR1 sensitizes human urothelial cancer cells to autophagic stimulation and stress-induced mitochondrial insults by treatment with rapamycin. NBR1-deficient cells exhibited enhanced sensitivity to rapamycin that was associated with increased autophagy and mitochondrial defects. Loss of NBR1 expression altered the cellular response to rapamycin treatment, resulting in impaired ATP homeostasis and an increase in reactive oxygen species. Moreover, rapamycin treatment also induced autophagy activity through AMPK activation in NBR1-deficient cells, which was involved in blocked mTOR signaling and activated unc-51 like autophagy activating kinase 1 (ULK1). NBR1-deficient cells exhibited profound mitochondrial dysfunction in response to rapamycin treatment as evidenced by Δψm collapse, ATP depletion, ROS accumulation and apoptosis activation. Therefore, our findings provide a rationale for rapamycin treatment of NBR1-deficient human urothelial cancer through induction of autophagy activity by regulating the AMPK/mTOR signaling pathway, indicating that NBR1 can be a potential candidate for the treatment of human urothelial cancer. (NRF-2015R1A1A1A0500110 to I.H.C., NRF-2016R1D1A1B03933826 to Y.M.W., and the Korea Health Technology R&D Project HI17C0710 to C.I.H.)

#1334

Withaferin A induces cytoprotective autophagy in hepatocellular cancer cells and concomitant autophagic inhibition augments the efficacy of Withaferin A in hepatocellular carcinoma.

Sumit Siddharth,1 Nethaji Muniraj,1 Neeraj K. Saxena,2 Dipali Sharma1. 1 _Johns Hopkins School of Medicine, Baltimore, MD;_ 2 _National Cancer Institute, Baltimore, MD_.

Background and Aim: Hepatocellular carcinoma (HCC) is the fifth most common cancer in the world and the third most common cause of cancer-related deaths. While multiple chemotherapeutic and targeted agents have been developed for other cancers, Sorafenib, an oral multikinase inhibitor, is the only approved agent for the treatment of advanced HCC. Clearly, it is imperative to develop novel effective therapeutic strategies for HCC to improve long-term survival of HCC patients. We found Withaferin A (WFA), a bioactive alkaloid derived from Withania somnifera, as an effective agent for HCC inhibition. Interestingly, we observed that WFA also induces autophagy in HCC cells. Autophagy can be cancer inducing or cancer inhibiting at the functional level. We aim to systemically investigate the anti-HCC efficacy of WFA, investigate the functional impact of autophagy and explore the efficacy of combined regimens of WFA and autophagic inhibitors.

Methods: Using in vitro and in vivo models, we established that WFA inhibits liver tumorigenesis. tfLC3B, LC3B-GFP/Lysotracker and LC3B/Rab7 were used to examine the fusion of autophagosomes with lysosomes. Cathepsin-D activation and DQ-BSA assays were conducted to analyze the protein degradation activity in lysosomes. Multiple cell based assays and Chou-Talalay synergy quantification assays were conducted to analyze the synergistic effect of WFA with autophagic inhibitors. IHC staining was used to study autophagic markers in vivo.

Results: WFA inhibited anchorage dependent cell growth in vitro and reduced the HCC tumor burden in vivo. In addition to inducing apoptotic cell death, WFA induced cleavage of Light Chain 3B (LC3B) and increased the expression of autophagic proteins (ATG5, ATG7 and Beclin1) both in vitro and in vivo. The redistribution of LC3B-GFP from cytosol to autophagosome demonstrated enhanced autophagosome formation in WFA treated cells. The fusion of autophagosome with lysosome was observed in WFA-treated HCC cells indicating autolysosome formation. WFA treated HCC cells exhibited elevated lysosomal degradation activity. Inhibiting autophagy either by blocking autophagosome formation or elevating lysosomal pH (CQ and BAF) enhanced WFA-induced growth inhibition and apoptosis indicating the presence of cytoprotective autophagy. Combination index analysis of WFA and CQ showed synergism and increased efficacy as compared to either monotherapy. These in vitro findings were corroborated with in vivo experiments.

Conclusion: Our results reveal that WFA inhibits hepatocellular carcinoma by apoptotic induction but the efficacy of WFA is somewhat abrogated by the concomitant induction of cytoprotective autophagy. Our preclinical findings present evidence to support WFA and chloroquine as an effective combination treatment regimen for HCC.

#1335

Withaferin A induces nonprotective autophagy in a STK11-independent manner and mediates breast cancer inhibition via energetic impairment.

Nethaji Muniraj,1 Arumugam Nagalingam,1 Neeraj K. Saxena,2 Dipali Sharma1. 1 _Johns Hopkins Sidney Kimmel Comp. Cancer Ctr., Baltimore, MD;_ 2 _National Cancer Institute, Rockville, MD_.

Background and Aim: Cancer cells undergo cytoprotective autophagy and evade chemotherapy therefore many clinical trials are investigating the efficacy of autophagy inhibition in combination with chemotherapy. At the functional level, autophagic process can be cytoprotective, cytotoxic, cytostatic or nonprotective. We investigated strategies to convert cellular autophagic response to non-protective autophagy which does not interfere with therapeutic regimens exploiting bioactive molecules.

Methods: Utilizing in vivo xenograft models, we established that Withaferin A (WA), a bioactive molecule from Withania Somnifera inhibits breast tumorigenesis. Autophagy studies were conducted utilizing immunoblot, RT-PCR, and immunofluorescence analyses for autophagy markers, transmission electron microscopy and confocal imaging. The fusion of autophagosome and lysosome was examined by using GFP-LC3/LysoTracker-red and GFP-LC3/mCherryRAB7A. Protein degradation activity of lysosomes and ATP levels were examined by DQ-BSA assay, Cathepsin activity and quantitative ATP assay.

Results: WA inhibited growth and induced apoptosis in breast cancer cells resulting in inhibition of breast carcinogenesis in vivo. Although WA increased tumor suppressor LKB1 which is known to be involved in autophagy, WA-mediated increased cleavage of Light Chain 3 type II (LC3-II) and punctated LC3-II staining was LKB1-independent. The redistribution of EGFP-LC3 from cytosol to autophagosome indicated increased formation of autophagosomes in WA-treated cells. However, WA-induced increased autophagosome-formation was not mediated by increased activation of autophagy by upstream processes but was due to blockade of lysosomal-degradation as evident by higher level of sequestosome 1 (SQSTM1/p62) and decreased turnover of LC3. WA was found to be a potent lysosomal deacidification agent capable of blocking autophagic flux. Accordingly, inhibiting autophagy by blocking formation of autophagosomes or elevating lysosomal pH did not interfere with WA-mediated growth-inhibition. WA blocked autophagic flux decreasing recycling of cellular fuels leading to reduced energy supply. Investigating this alternative mechanism, we discovered that indeed, WA reduced ATP levels and increased phosphorylation of AMP-activated protein kinase (AMPK). Modulating substrates for tricarboxylic acid (TCA) cycle with methyl pyruvate protected WA-treated cells while 2DG potentiated WA-induced cell death.

Conclusion: Our results indicate that WA induces a non-protective autophagy and blocks energy fuels in cancer cells by reducing ATP levels and inhibiting lysosomal acidification hence offering a three-pronged approach to facilitate cancer cell death. WA might be a useful strategic addition to chemotherapy regimens to evade cytoprotective effects of autophagy.

#1336

**Autophagy inhibition reverses EMT and reduces 3D growth of pancreatic adenocarcinoma cells** in vitro **and** in vivo **.**

Jane B. Pearce, Ciara H. O'Flanagan, Stephen D. Hursting. _UNC Chapel Hill, Chapel Hill, NC_.

Pancreatic cancer is the 4th most fatal cancer in US men and women, with 5-year survival rates under 5%. Obesity is one risk factor known to increase pancreatic cancer risk by facilitating tumorigenesis and metabolic processes associated with pancreatic tumor growth and progression. Autophagy is the catabolic process of recycling cellular organelles to provide energy to the cell and is often induced when cells become stressed. Autophagy plays a critical role in cancer growth and metastasis, and in many cancers autophagy inhibition decreases the size of tumors in in vivo models. Our current studies investigate the link between the autophagy inhibition and the invasiveness of pancreatic cancer. To understand the role of autophagy on invasion, EMT and 3D growth in pancreatic cancer, Kras-driven Panc02 mouse pancreatic cancer cells were transduced using CRISPR/Cas9 to cleave the gene encoding autophagy related protein 5 (Atg5). Atg5 deletion resulted in striking morphological alterations, including a more epithelial-like appearance. In addition, Atg5 deletion resulted in increased expression of the epithelial marker E-Cadherin, concurrent with decreased expression of mesenchymal markers N-Cadherin and Snail. Furthermore, Atg5 deletion resulted in significant decreased invasiveness and ability to form colonies under anchorage-independent conditions, with no significant difference in 2D cell growth. Orthotopic, intrapancreatic injection of these cells into C57BL/6 mice resulted in reduced tumor take and growth. Taken together, these results for the first time show that autophagy inhibition results in reduced EMT and 3D growth in pancreatic adenocarcinoma and highlight autophagy's role in pancreatic adenocarcinoma progression.

#1337

MEK1:MAPK1/2 targeting attenuates pro-survival autophagy and enhances antiestrogen-induced apoptosis in breast cancer cells via a cathepsin L-dependent mechanism.

Annie Liu,1 Carol Joseph,1 Jesse Wayson,1 Timothy Summers,1 Haifeng Cai,2 Patricia V. Schoenlein1. 1 _Augusta Univ, Augusta, GA;_ 2 _He bei Uion University, Tang Shan City, China_.

Antiestrogen resistance is still a major clinical challenge preventing the eradication of estrogen receptor positive (ER+) breast cancer. A major goal of our laboratory is to identify targeted therapies that can be combined with antiestrogen treatment to block the emergence of antiestrogen resistant breast cancer. Toward this goal, we previously determined that the catabolic process of autophagy facilitates the emergence of antiestrogen resistance (Sammadar et al., Molecular Cancer Ther. 9, 2008) and identified cathepsin L as a key lysosomal protease required for antiestrogen induced pro-survival autophagy ER+ MCF-7cells (Periyasamy-Thandavan et al., Autophagy. 6:19-35, 2010). Thus, we hypothesize that blocking cathepsin L expression / activity is one approach to targeting pro-survival autophagy during antiestrogen treatment of breast cancer. In support of this hypothesis, we now show that antiestrogen resistant TR5 cells show increased levels of active cathepsin L compared to the levels in the parent antiestrogen sensitive MCF-7 cells. The upregulation of cathepsin L expression is consistent with the fact that TR5 cells utilized autophagy to survive long-term 4-hydroxytamoxifen (4-OHT) selection. Withdrawal of 4-OHT selection during TR5 passage does not lead to a reduction in cathepsin L levels, suggesting that the increased expression of cathepsin L is a stable genetic or epigenetic alteration. Elevated expression of active (phosphorylated) MAPK1/2 is also present in TR5 cells and targeting MEK1/MAPK1/2 with the selective inhibitor U0126 consistently reduced the levels of active cathepsin L. We further established a key role for MEK1/MAPK1/2 in the regulation of cathepsin L in the parent MCF-7 cells by performing cathepsin L activity assays. Cells treated with 4-OHT showed increased cathepsin L activity compared to E2-treated control cells; whereas, cathepsin L activity in cells treated with 4-OHT + UO126 was significantly reduced compared to the levels in 4-OHT-treated cells. This reduction in cathepsin L by U0126-mediated blockade of MEK1/MAPK1/2 correlated directly to "impaired" autophagic flux and increased BimEL-dependent apoptosis in the antiestrogen-treated cell populations and was mediated, at least in part, via transcriptional regulation of cathepsin L as determined by quantitative PCR. Further, MEK1/MAPK1/2 up-regulation of cathepsin L is selective, as cathepsin B activity is not reduced by MEK1 targeting. These studies provide evidence that the targeting MEK1/MAPK1/2 in combination with antiestrogen treatment has the potential to reduce cathepsin-L mediated pro-survival autophagy in ER+ breast cancer.

#1338

Citrobacter rodentium **-induced autophagy protects cancer stem cells to facilitate tumor development and progression in the colons of** Apc1638N/+ **mice.**

Badal C. Roy,1 Ishfaq Ahmed,1 Venkatakrishna R. Jala,2 Bodduluri Haribabu,2 Shrikant Anant,1 Shahid Umar1. 1 _Kansas University Medical Center, Kansas City, KS;_ 2 _University of Louisville, Louisville, KY_.

Background: Colon cancer stem cells (CCSCs) play crucial roles in tumorigenesis, chemotherapy resistance, tumor recurrence and cancer metastasis. Autophagy, a lysosomal degradation and recycling process implicated in cancer progression and therapy resistance, plays an important role in host defense against enteric pathogen, Citrobacter rodentium (CR), and controls CR-induced infectious colitis. However, the role of CR infection in autophagy induction and CCSCs-driven neoplastic transformation has not been studied.

Aim: To examine whether CR-induced autophagy protects CCSCs to promote colon cancer development and progression.

Methods: Both male and female Apc1638N/+ mice from NCI repository were either infected with CR (109CFUs) and additionally given AOM (@10mg/Kg bw) or treated with a combination of AOM+DSS (7 days @ 3%). ApcMin/+ and BLT1-/-;ApcMin/+ mice were used as additional models mimicking FAP in humans. Apc++ mice were used as controls. Standard histologic, biochemical and molecular approaches were implemented.

Results: CR infection induced significant autophagy in Apc++ mice with formation of autophagosomes and increases in LC3B and Beclin-1 staining at 12-27 days post-infection. While the frequency of tumor development in the colons of uninfected mice was lower, CR infection-induced tumor incidence in the colon was associated with increases in Ki-67, β-catenin, EZH2 and CCSC marker Dclk1/AcTub levels, respectively. In response to AOM treatment of CR-infected mice, we discovered colons to respond better compared to small intestine, with increases in Ki-67 and EZH2 staining although increases in Dclk1/AcTub levels were less pronounced compared to AOM-treated alone. When Apc1638N/+ mice were subjected to AOM/DSS treatment, colonic tumors exhibited more pronounced changes in Ki-67, EZH2 and Dclk1/AcTub staining than tumors of the small intestine. These tumors were also highly inflamed as was revealed by infiltration of F4/80+ macrophages and CD3+ lymphocytes. Interestingly, CD3 co-localized with β-catenin in these tumors, indicating contribution of Wnt signaling in the tumorigenic process. Both intestinal and colonic tumors also stained positive for migrating cancer stem cell markers CD110 and CDCP1, respectively. Colonic tumors additionally exhibited stromal positivity, suggesting local spread. Further investigation of tumors from AOM/DSS-treated Apc1638N/+ mice revealed significant autophagy wherein Dclk1/AcTub+ cells co-localized with Atg7 and p62, suggesting that autophagy may be protecting CCSCs. Both ApcMin/+ mice in response to CR infection and BLT1-/-;ApcMin/+ mice exhibited similar increases and co-localization of Dclk1/AcTub+ cells within the tumors with autophagic markers.

Conclusion: CR-induced autophagy may protect CCSCs to promote colon cancer development and progression.

#1339

Development of a novel autophagy-inducing multikinase inhibitor for the treatment of castration-resistant prostate cancer.

Yuanyuan Qiao. _Univ. of Michigan Medical School, Ann Arbor, MI_.

Inhibition of the androgen axis has revolutionized the treatment of metastatic castration-resistant prostate cancer (mCRPC). However, mCRPC continues to be a leading cause of cancer-related deaths in men. Here, we characterized a phase I-cleared multikinase inhibitor, ESK981, in prostate cancer preclinical models. ESK981 was markedly more potent in vitro than other kinase inhibitors, including cabozantinib and crizotinib, that have been evaluated clinically in prostate cancer. Surprisingly, we observed that ESK981 was a potent inducer of cellular vacuolization that was associated with an autophagic response. ESK981-induced autophagy could be blocked by either the depletion of the autophagy vesicle formation gene, atg5, or by autophagy inhibitors such as bafilomycin. ESK981 was further shown to induce autophagy in yeast, which is an evolutionarily conserved core cellular pathway. Moreover, ESK981 triggered prostate cancer cells to release chemokines such as CXCL10 into the cell secretome, thus suggesting that ESK981 may stimulate immune surveillance while exerting its tumor inhibitory effect. Together, ESK981 is a novel and potent autophagy-inducing multikinase inhibitor that effectively inhibits CRPC growth through the activation of the autophagic cascade.

#1340

Assessing autophagic flux in cell culture models with a novel plate-based assay.

Dan F. Lazar,1 Ryan W. Kessens,2 Amani A. Gillette,2 Braeden L. Butler,1 Christopher T. Eggers,1 Brock F. Binkowski,1 Gediminas Vidugiris,1 Michael R. Slater,1 Dongping Ma,1 James J. Cali1. 1 _Promega Corporation, Madison, WI;_ 2 _University of Wisconsin, Madison, WI_.

The importance of autophagy in normal cell health and diseased states, including cancer, immunology and inflammation, has become quite clear. Given the growing interest in screening for autophagy modulators, we have developed a homogeneous plate-based assay to measure autophagic flux in cell culture models using NanoLuc® Binary Technology (NanoBiT™). LC3B (Atg8) protein was tagged on its N-terminus with an 11 amino acid peptide, (HiBiT), as well as an intervening "spacer" sequence. When expressed at low-to-moderate levels in mammalian cell lines, this LC3-based reporter is subject to degradation by the autophagic pathway. Changes in reporter levels following cell treatment are determined by addition of a lytic reagent containing Large BiT (LgBiT), which rapidly associates with HiBiT to produce a bright, luciferase activity in the presence of added substrate. In stable U2OS reporter cells, mTORC inhibitor treatment decreased assay signal by 25% (rapamycin) to 60% (PP242, AZD8055), consistent with compound stimulation of autophagic flux and degradation of the autophagy reporter. In contrast, treatment with autophagy inhibitors (bafilomycin A1, chloroquine) produced a 70-80% increase in assay signal, consistent with accumulation of autophagy reporter following blockade of basal autophagy. Similar results were obtained in HEK293 cells stably expressing the reporter, as well as in cells in which autophagy reporter was transiently expressed via simple, one-step transduction with BacMam viral particles. Mechanism of action of autophagy modulators was confirmed through blockade of effects by 50nM bafilomycin A1 cotreatment. Multiplex with a cell necrosis detection agent allowed for same-well determination of cytotoxic effects that might undermine analysis of a compound's discrete effects on autophagic flux. When assayed in 384-well plates with automation, U2OS and HEK293 autophagy reporter cells produced Z' values of 0.6-0.7 in response to autophagy induction (PP242), and Z' values of 0.7-0.8 by subsequent blockade of autophagy (PP242 + bafilomycin A1). Therefore, using this novel plate-based assay system for the determination of autophagic flux, it is possible to screen test agents and quantitatively assess their effects in cell culture models.

#1341

The non-canonical autophagy network active during retinoic acid therapy in acute myeloid leukemia.

Mario P. Tschan. _Univ. of Bern, Bern, Switzerland_.

Acute myeloid leukemia (AML) is characterized by a differentiation block of hematopoietic precursors at different stages of myeloid development. Acute promyelocytic leukemia (APL) patients with the chromosomal translocation t(15;17) are successfully treated with all-trans retinoic acid (ATRA) and low dose chemotherapy or arsenic trioxide allowing for neutrophil differentiation. Unfortunately, non-APL cells are not responding significantly to ATRA. Macroautophagy (hereafter autophagy) is key for RA-induced granulocytic differentiation of APL cells as well as for healthy neutrophils. Autophagy is a proteolytic self-degradation and recycling process characterized by the formation of double-membraned vesicles, so called autophagosomes, which engulf cytoplasmic contents such as protein aggregates or defective organelles. Deciphering the particular autophagy pathway active during APL differentiation is a prerequisite to develop novel "autophagy-differentiation therapies" to expand this therapeutic approach to additional AML subgroups not responding to current ATRA therapy. Using an RNAi screening approach during neutrophil differentiation of APL cells, we found that key autophagy-related (ATG) genes such as ULK1, VPS34, WIPIs, ATG3, ATG4, ATG5, ATG7, and GATE-16/LC3B are required for ATRA-induced autophagy and differentiation, whereas the master ATG genes Beclin1 and ATG16L1 are not. This is in sharp contrast to non-canonical, starvation-induced autophagy. Interestingly, we identified ATG16L2, an ATG16L1 homolog with no function in regular autophagy, as a key player in ATRA-induced autophagy. Furthermore, we described additional genes such as DAPK2 and MAP1S with new functions in ATRA-induced autophagy. In line with the function of particular ATG genes in neutrophil differentiation, we show that transcription of several ATG genes is directly regulated by the myeloid transcription factor PU.1. Lastly, preliminary results indicate improved neutrophil differentiation of APL and AML cells when combining ATRA with an autophagy inducer. In conclusion, we provide strong evidence for a non-canonical autophagy pathway operative during neutrophil differentiation of APL cells possibly providing novel drug targets to allow for differentiation therapy in additional AML subtypes.

#1342

Mitochondrial dysfunction activates lysosomal-dependent mitophagy selectively in cancer cells.

Thomas Biel, Ashutosh Rao. _FDA-CDER, Silver Spring, MD_.

Mitochondria-targeted drugs are designed to accumulate in the mitochondrion to modulate metabolism. Triphenylphosphonium (TPP) conjugated agents are a class of mitochondria-targeted drugs that induce mitochondrial dysfunction in cancer cells. Mitochondrial dysfunction is known factor that facilitates the onset of mitophagy. However, the activation of mitophagy to remove drug-induced mitochondrial damage is unknown. Here, we subjected breast MDA-MB-231 cancer cells and MCF-12A noncancerous cells to a series of mitochondria-targeted redox-active agents (MTA) that contain TPP to investigate the onset and activation of autophagy, mitophagy and mitochondrial dysfunction. Collectively, we identified three different MTAs from the series that sequentially damage the mitochondria, enhance autophagic flux, and activate mitophagy selectively in MDA-MB-231 cells. Furthermore, we generated stably expressing mt-mKeima MDA-MB-231 and MCF-12A cell lines to establish a mitophagy flux assay for screening new agents. FACS analysis of mt-mKeima, a pH-sensitive fluorophore, revealed MTAs induced lysosomal dependent mitochondrial degradation and the presence of bafilomycin, a lysosomal inhibitor, suppressed MTA-induced mitophagy. To confirm MTAs induced mitochondrial autophagy, we identified mitochondria located within autophagosomes using confocal microscopy, an endogenous autophagy-related protein complex by coimmunoprecipitation and mitochondrial PINK1 accumulation using immunoblot. These in vitro studies were translated into an in vivo rat SST-2 xenograph breast cancer model to demonstrate that mitochondrial PINK1 accumulates in rat tumor tissue following MitoQ treatment. These data suggest that mitochondria-targeted agents selectively caused mitochondrial depolarization, PINK1 accumulation and mitophagy in MDA-MB-231 cancer cells as compared to MCF-12A noncancerous cells.

#1343

Induction of autophagy following TTFields application serves as a survival mechanism mediated by AMPK activation.

Anna Shteingauz, Yaara Porat, Moshe Giladi, Roza Schneiderman, Tali Voloshin, Mijal Munster, Eilon Kirson, Uri Weinberg, Yoram Palti. _NovoCure, Haifa, Israel_.

Tumor treating fields (TTFields) are an approved treatment modality for patients with glioblastoma. TTFields are delivered via noninvasive application of low-intensity, intermediate-frequency, alternating electric fields. Previous studies have shown that TTFields lead to increased granularity which is often associated with autophagy. In this study we evaluated the effect of TTFields on the induction of autophagy in glioma and NSCLC cells. Cells were treated with TTFields using the inovitro system. Cellular granularity was evaluated using flow cytometry. Autophagy was monitored by quantifying levels of lipidated Microtubule Associated Protein Light Chain 3 (LC3-II), in the presence and absence of the lysomotropic agent and autophagy inhibitor chloroquine (CQ), using immunoblotting and immunofluorescence microscopy. Transmission Electron Microscopy (TEM) was used to visualize autophagosome-like structures. Western blot analysis was utilized to evaluate autophagy regulatory activity of the mammalian target of rapamycin (mTOR) through p70 S6 kinase1 (S6K1) and AMP activated protein kinase (AMPK) and its downstream target ULK-1. To determine if AMPK is responsible for TTFields stimulated autophagy pool of siRNAs was used to deplete siAMPK from U87-MG cells. To evaluate involvement of autophagy in cell fate after TTFields treatment we produced glioma cell lines depleted from ATG7 by shATG7 infection. Flow cytometry analysis demonstrated that TTFields application leads to a significant increase in cellular granularity in all tested cell lines. Significant elevation in LC3-II levels was observed in treated cells using fluorescence microscopy, where punctate distribution of LC3-II was observed. TEM micrographs demonstrated the presence of autophagy typical, autophagosome-like structures, in TTFields treated cells. . Evidence of increased autophagic flux following TTFields application was also detected using immunoblotting analysis in the presence of CQ. Western blot analysis of cells after TTFields treatment revealed stimulation of AMPK signaling as well as activation of p70. Depletion of AMPK from U87-MG cells resulted in reduction of autophagy as reflected by LC3-II levels and enhancement of TTFields cytotoxicity. ER stress in treated cells has been evident by immunoblotting, showing increased levels of ER stress marker GRP78. Combination of TTFields with CQ resulted in a significant dose dependent reduction of cell growth compared with TTFields treatment alone. Cells with ATG7 depletion showed similar results. Combined, these results suggest that cells upregulate autophagy in response to ER stress induced by TTFields application and that AMPK may serve as a key regulator of this process.

#1344

Autophagy contributes to therapeutic resistance in head and neck cancer.

Jaimee Eckers, Justin Skiba, Gopika Senthilkumar, Kwang P. Nickel, Adam D. Swick, Randall J. Kimple. _Univ. of Wisconsin-Madison, Madison, WI_.

Background: In the curative setting for head and neck cancer (HNC) a common treatment is radiation combined with cetuximab, an antibody therapeutic targeting EGFR. Despite decades of research into improved treatments, therapeutic resistance remains a major challenge for this malignancy, with roughly 40% of patients developing recurrent disease. Recent evidence has suggested that autophagy, a cellular stress response, may be an additional contributor to therapy resistance, by protecting HNC cells from the cytotoxic effects of radiotherapy and the growth inhibitory effects of anti-EGFR treatment. The mechanism of radiation-induced autophagy is under current investigation.

Methods: Cell lines were source from commercial sources, cultured under recommended conditions, and identity confirmed by short tandem repeat testing. Induction of autophagy was detected by immunoblot flux assays for LC3 and p62, immunofluorescent staining of autophagic vesicles, LC3 reporter flux assay, and flow cytometry using acridine orange. The effect of autophagy inhibition was tested using clonogenic survival assays. Induction of apoptosis was analyzed by immunoblot against cleaved caspase and PARP and via AnnexinV staining.

Results: We evaluated a panel of both human papillomavirus (HPV) positive and negative HNC cell lines for autophagic response to both cetuximab (CTX) treatment and ionizing radiation (XRT). Flux assays revealed that both CTX and XRT treatment induced autophagy in a time- and dose-dependent manner. Immunofluorescent staining of LC3 to identify autophagic vesicles showed that a relatively small fraction of the total cell population is able to induce this response. Flow cytometry analysis demonstrated that autophagic cells were largely non-apoptoic. For example, in the UM-SCC47 cell line treated with CTX for 48 h, flow cytometry for autophagy (20.8%), apoptosis (13.7%) or dual staining (5.4%) suggests a cytoprotective role for autophagy. The addition of the ULK1 inhibitor, SBI-0206965) to CTX and XRT induced apoptosis as shown by caspase activity and AnnexinV staining and reduced clonogenic cell survival.

Conclusions: These preclinical studies have established the proof of concept for the cytoprotective effect of autophagy in response to anticancer treatments including EGFR inhibition and radiotherapy in HNC. Further, we have identified the addition of specific autophagy inhibitors to standard treatments as a potential strategy to overcome this mechanism of resistance.

#1345

ATM kinase activity is dispensable in mitochondrial autophagy.

Aloke Kumar Sarkar, Varsha Gandhi. _The University of Texas MD Anderson Cancer Center, Houston, TX_.

Ataxia telangiectasia mutated (ATM), a critical DNA damage sensor with protein kinase activity, is frequently deleted or mutated in human cancers including mantle cell lymphoma (MCL). Loss of ATM protein is associated with accumulation of nonfunctional mitochondria and defective mitophagy, in both murine thymocytes and in non-cancer cells, however, the molecular mechanism of ATM kinase in cancer cell mitophagy is unknown. Here, we provide evidence that mitophagy in B-Cell Lymphoma including MCL (Granta-519, Jeko-1 and Mino), HeLa and other cancer cell lines is dependent on ATM but independent of its kinase function. Routine ATM kinase activity was performed by FACS analysis following either neocarzinostatin or IR-induced PE-ATMSer1981 and FITC-H2AXSer139 phosphorylations status and reconfirmed by immunoblot assay by probing with phospho-ATMSer1981and ATM targets Kap1Ser824 or Smc1Ser966 phosphorylations. Stable knockdown of ATM in ATM proficient MCL cell lines conferred resistance to mitophagy and was associated with reduced ATP production, oxygen consumption, and increased mitochondrial ROS. ATM protein interacts with the E3 ubiquitin ligase Parkin in a kinase-independent manner in multiple cancer cell lines including MCL and HeLa, while stable knockdown of ATM in HeLa cells (Kd HeLa) resulted in proteasomal degradation of GFP-Parkin which was rescued by the proteasome inhibitor, MG132 suggesting that ATM-Parkin interaction is important for Parkin stability. Confocal analysis revealed the presence of extra-nuclear ATM in both cytoplasm and mitochondrial compartments in HeLa cells and CCCP-induced mitophagy resulted in significantly higher mitochondrial ATM-Tom20 colocalization compared to DMSO control. Moreover both cell fractionation and confocal analysis revealed more GFP-Parkin translocation and loss of Tom20 expression in the mitochondrial fraction in WT cells following CCCP treatment. In contrast, mitochondrial GFP-Parkin expression was undetectable in Kd HeLa cells indicating a specific defect in Parkin translocation in the absence of ATM during CCCP-induced mitophagy. Further, confocal analysis with anti-DNA antibody indicated significantly higher mass of mitochondrial nucleoids in Kd HeLa cells compared to WT controls suggesting that loss of ATM promotes mtDNA accumulation. Neither loss of ATM kinase activity in primary B cell lymphomas nor inhibition of ATM kinase by KU60019 in MCL, non-tumor A-T fibroblasts and HeLa cell lines rescued FCCP or CCCP-induced mitophagy signifying that ATM catalytic activity is dispensable for mitophagy. Malignant primary B-cell lymphomas including MCL without detectable ATM, Parkin, Pink1, and Parkin-Ubser65 phosphorylation were resistant to mitophagy. This data provides the first molecular evidence of the role of ATM kinase function in mitophagy in cancer.

#1346

Lycorine Promotes Autophagy and Apoptosis via TCRP1/Akt/mTOR Axis Inactivation in Human Hepatocellular Carcinoma.

Yuhaiyang Yuhaiyang Yuhaiyang. _Tianjin University of Traditional Chinese Medicine, Tianjin, China_.

Lycorine is a multi-functional bioactive compound, and it possesses potential anti-cancer activities. However, little is known about the underlying mechanism. In this research we have found that lycorine significantly induces the apoptotic and autophagic capacities of hepatocellular carcinoma (HCC) cells in vitro and in vivo. Treatment with specific autophagy inhibitor (3-methyladenine/Bafilomycin A1) or knockdown of LC-3B/Atg5 by siRNA drastically enhances the apoptotic cell death effect by facilitating the switch from autophagy to apoptosis. Molecular validation mechanistically demonstrates that lycorine-induced apoptosis and autophagy in HCC cells is associated with decreased protein levels of tongue cancer resistance-associated protein 1 (TCRP1), and we further find that inhibition of TCRP1 decreases phosphorylation level of Akt and represses Akt/mTOR signaling. Finally, lycorine-induced apoptosis and autophagy suppress the growth of xenograft hepatocellular tumors without remarkable toxicity. Our results elucidate a novel molecular mechanism whereby lycorine promotes apoptosis and autophagy through TCRP1/Akt/mTOR pathway in HCC. Our results reveal that lycorine might be a potential therapeutic agent for the treatment of HCC.

#1347

Autophagy inhibitor, chloroquine synergize oxaliplatin by modulating activity of cytosolic HMGB1 in pancreatic cancer.

Seonmin Lee, Kyu-Pyo Kim, Changhoon Yoo, Danbi Kim, Chorong Kim, Heejung Chae, Jihoon Kang, Baek-Yeol Ryoo. _Asan Medical Center, Seoul, Republic of Korea_.

Normal 0 false false false EN-US KO X-NONE /* Style Definitions */ table.MsoNormalTable {mso-style-name:"Table Normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-parent:""; mso-padding-alt:0cm 5.4pt 0cm 5.4pt; mso-para-margin-top:0cm; mso-para-margin-right:0cm; mso-para-margin-bottom:10.0pt; mso-para-margin-left:0cm; text-align:justify; text-justify:inter-ideograph; line-height:115%; mso-pagination:widow-orphan; font-size:10.0pt; mso-bidi-font-size:11.0pt; font-family:"맑은 고딕"; mso-ascii-font-family:"맑은 고딕"; mso-ascii-theme-font:minor-latin; mso-fareast-font-family:"맑은 고딕"; mso-fareast-theme-font:minor-fareast; mso-hansi-font-family:"맑은 고딕"; mso-hansi-theme-font:minor-latin; mso-font-kerning:1.0pt;} Autophagy is a regulated, destructive catabolic mechanism to degrade cellular organelles and macromolecules. In cancer, the role of autophagy in tumor cell survival or death is still controversial because of each tumor type has a heterogeneity on metabolic status and microenvironment. In this study, we showed that autophagy inhibitor, chloroquine (CQ), increased sensitivity to oxaliplatin in pancreatic cancer cell lines. The autophagy inhibitor, chloroquine effectively inhibited autophagic flux which is important for maintenance of pancreatic cancer cell metabolism and drug resistance. Treatment of oxaliplatin after blockade of autophagy increased chromatin instability and facilitated loss of nuclear HMGB1. In consequence the cells turned into apoptotic cell death. The drug combination effect of chloroquine and oxaliplatin shows that inhibition of autophagy by chloroquine carry on dual role in pancreatic cancer cell death. First, chloroquine occurs cytosolic redox imbalance based on increase mitochondrial ROS (mtROS) following rupture of autolysosome. Second, the cytosolic redox imbalance occurred by chloroquine induces failure of autophagy by incapacitation of cytosolic HMGB1. Here we suggest that autophagy inhibition by chloroquine synergize oxaliplatin by modulating activity of cytosolic HMGB1. Also we suggest HMGB1 as a new target for therapeutic agents against pancreatic cancer<!--EndFragment-->

#1348

Parkin-mediated mitophagy regulates 5-fluorouracil-induced chemoresistance in breast cancer.

Chandan Kanta Das,1 Bikash Chandra Jena,1 Subhayan Das,1 Aditya Parekh,1 Goutam Dey,1 Rashmi Bharti,1 Deblina Bharadwaj,1 Sujit Kumar Bhutia,2 Donat Kögel,3 Mahitosh Mandal1. 1 _IIT Kharagpur, Kharagpur, India;_ 2 _National Institute of Technology, Rourkela, Rourkela, India;_ 3 _Experimental Neurosurgery, Neuroscience Centre, Frankfurt am Main, Germany_.

Introduction Resistance to therapeutics is one of the major concerns for treatment failure in breast cancer. The use of antimetabolite, 5 Fluorouracil (5-FU) based combinational therapies have been an ideal treatment blueprint for different types of solid tumors including breast cancer. However, the efficacy of 5-FU based treatment is limited due to development of resistance. Recently mitophagy has evolved as an important cell survival mechanisms in response to different stress stimuli associated with cancer treatments. Mitophagy is the selective form of autophagy that removes damaged mitochondria. Although mitophagy has dual role in context to cell survival and cell death, still a large number of evidences suggests their prosurvival role with respect to chemoresistance. Here we investigated the role of mitophagy in 5-FU induced breast cancer chemoresistance.

Methodology Chemoresistant cell lines were established by continuous exposure to increasing concentration of 5-FU to breast cancer cell lines (MDA-MB-231 and MDA-MB-468). Chemoresistant cells were characterized by MTT assay, caspase assay and western blot. Induction of autophagy was confirmed by GFP LC3 transfection and western blot. Further prosurvival autophagy was asserted through FACS by using autophagy inhibitor chloroquine (CQ) and stable ATG5 lentiviral knockdown. Mitophagy was confirmed using MTDR (detects mitochondrial mass) and TMRM (detects mitochondrial membrane potential) through FACS. Further immunofluorescence and western blot were used to confirm key mitophagy markers (TOM20 and Porin) with CQ treatments. Involvement of parkin mediated mitophagy was later confirmed by cytosolic/mitochondrial fractionation assay and by siRNA knockdown.

Results Characterization study reveals chemoresistant cells were more apoptosis resistant compared to their parental counterparts.The levels of the autophagy markers LC3-II, p62, ATG5 and Beclin-1 were elevated in chemoresistant cells while mitochondrial mass and mitochondrial membrane potential were reduced. Treatment of CQ and ATG5 knockdown significantly augment 5-FU sensitivity in chemoresistant cells. Simultaneously CQ treatment increased TOM20 and porin expression revealing induction of mitophagy in chemoresistant cells. Further cell fractionation assay and siRNA knockdown of Parkin confirm its mitochondrial localization and prosurvival function in 5-FU resistant breast cancer respectively. some important in vitro findings were also confirmed in xenograft breast cancer model.

Conclusion Here we noticed that chemoresistant breast cancer cells exhibit greater apoptosis resistance to 5-FU compared to parental cells along with enhanced cytoprotective mitophagy, also underscoring the involvement of parkin mediated induction of mitophagy in chemoresistant breast cancer.

#1349

Functional and mechanistic characterization of PRMT6 regulated autophagy in hepatocellular carcinoma.

Noélia Che, Man Tong, Kai Yu Ng, Phillis WF Kau, Xin Yuan Guan, Michael SY Huen, Stephanie Ma. _Univ. of Hong Kong Faculty of Medicine, Hong Kong, Hong Kong_.

Hepatocellular carcinoma (HCC) is a major health burden worldwide. Tumor recurrence and therapy resistance represent major obstacles in the treatment of the disease, with their mechanism of action largely uncharacterized. Autophagy is a critical survival factor for cancer cells, whereby it maintains cellular homeostasis including degradation of damaged organelles and unwanted proteins as well as the support of cellular biosynthesis in response to environmental stress, preventing cells from undergoing apoptosis. Our group has recently reported on the regulatory role of protein arginine methyltransferase 6 (PRMT6) down-regulation in maintenance of HCC, particularly potentiating resistance to sorafenib and chemotherapy. By tandem affinity purification/mass spectrometry analysis of cells with PRMT6 stably overexpressed, we identified a number of autophagy related proteins as potential interacting partners of PRMT6, suggesting a possible role of PRMT6 in modulating autophagy in HCC. Treatment of HCC cells with stress induced conditions, including sorafenib, nutrient deprivation and hypoxic condition, resulted in a marked reduction of PRMT6 expression, concomitant with elevated levels of LC3BII. Further, under these various stimuli, knockdown of PRMT6 in HCC cells would also result in enhanced expression of LC3BII, accumulation of GFP-LC3B positive puncta staining as well as an increase in autophagosome formation. Consistently, treatment of HCC cells with PRMT6 overexpressed would leading to an opposing effect. Further studies by immunoprecipitation analysis validated PRMT6 to physically interact with AMBRA1 (activating molecule in BECN1-regulated autophagy protein 1), a well-known autophagy initiative component, which takes part in autophagy vesicle nucleation, a critical step for autophagic initiation. Our findings suggest PRMT6 down-regulation in HCC tumors to promote tumorigenicity through autophagic flux de-regulation. Targeting the mechanisms of stress response may provide novel therapeutic insights for the disease.

#1350

Inhibiting VPS34 suppresses Wnt/beta-catenin signaling and promotes chemotherapeutic efficacy in colon cancer cells.

Balawant Kumar, Rizwan Ahmad, Sandeep Rana, Amarnath Natarajan, Punita Dhawan, Amar B. Singh. _University of Nebraska Medical Center, Omaha, NE_.

A significant obstacle in the successful management of colon cancer (CRC) patients remains intrinsic or acquired drug resistance. Addressing these obstacles remain priority for reducing CRC-associated deaths. Autophagy is a survival-promoting program and upregulated in cancers to sustain metabolism and survive microenvironmental stress. In preclinical cancer models, inhibition of autophagy can restore chemosensitivity and enhance tumor cell death. However, clinical applicability of the currently available autophagy inhibitors is problematic due to potential toxicity as these inhibitors primarily interfere with lysosomal trafficking program, implicated in essential cellular processes. To overcome this limitation, we examined the effects of inhibiting VPS34, a class III PI-3 Kinase implicated in autophagy initiation, to overcome resistance to therapy by inhibiting autophagy. Notably, #36-077, the inhibitor used in our studies was synthesized based on the recently described novel VPS34 inhibitor, SAR405. Using colon cancer cells and colon tumoroids from APCmin mice, we confirmed marked upregulation of autophagy. Tumoroids exposure to #36-077 inhibited autophagy (LC-3I/II and P-62 expression) and induced apoptosis (Cleaved caspase-3 expression), and thus confirmed survival role for autophagy in CRC. We further examined efficacy of using #36-077 for adjuvant therapy. Combinatorial treatment efficacy of #36-077with 5-Fluorouracil (5-FU) and Erlotinib (anti-EGFR therapy) was tested. SW480 (highly tumorigenic) and HCT116 (Highly Metastatic) cells were used. MTT-assay helped determine cell viability. We found synergy between #36-077 and 5-FU in the killing CRC cells, and the EC50 was 4.9µm, 4.7µm and 2.40µm for 5-FU, #36-077 and 5-FU+#36-077, respectively. Combinatorial use of the #36-077 also boosted the cancer cell killing effects of Erlotinib, and the EC50 was 25.05µm ,5.59µm and 4.09 µm for Erlotinib, #36-077 and Erlotinib+#36-077, respectively. In further studies, we focused on the β-catenin regulation due to its role in promoting Wnt-signaling and autophagy. Indeed, in both, colon cancer cells and tumoroids, combinatorial treatment with 5FU+#36-077 resulted in sharp increases in phospho-β-catenin (Ser33) expression. Of note, phosphorylation of β-catenin at Ser33 by GSK-3β leads to its destabilization and degradation. TOP-FLASH reporter analysis (p<0.001 vs control) further confirmed significant inhibition of Wnt-signaling in cells subjected to combinatorial treatment. In accordance, in cells subjected to combinatorial treatment, Survivin expression (a Wnt-signaling target; p<0.001 vs control) was markedly suppressed while cleaved caspse-3 expression (p<0.05 vs control) was upregulated. Taken together, our data provide strong rationale for the therapeutic targeting of VPS34 in improving therapeutic efficacy of anti-CRC treatments.

#1351

Inhibition of autophagy enhances dihydroartemisinin (DHA)-induced cytotoxicity through DR5 upregulation.

Chen Mei-Chuan. _Taipei Medical University, Taipei, Taiwan_.

Artemisinin (Qinghaosu) comes originally from Qinghao has been used against Plasmodium falciparum malaria for a long time. Dihydroartemisinin, an analogue of artemisinin, is also be listed by WHO to exhibit antimalarial and anticancer properties.

In our study, DHA exhibits antiproliferative activity in several solid tumors, especially against colorectal cancer (CRC) HCT116 cells. Our current results show DHA suppresses cell proliferation and induces caspase-dependent apoptosis without inducing necrosis-like cell death in HCT116 cells. In addition, DHA interferes autophagy pathway by upregulating of LC3 II formation in HCT116 cells. Inhibition of autophagy with either autophagy inhibitor 3-methyladenine (3MA) or KD of ATG5 potentiated DHA-induced cell death. We found DHA increased expression level of DR5 while had no effect on DR4 expression level on surface of tumor cells. Further, cell-surface DR5 expression was significantly elevated in DHA-treated ATG5-KD cells. These findings suggest that inhibition of the autophagy enhances DHA-induced cytotoxicity through DR5 upregulation.

Key words: Apooptsis, DR5 (Death Receptor 5), autophagy, colorectal cancer

### DNA Repair and Damage Response

#1352

Inactivation of factors of DNA double-strand break repair by homologous recombination or non-homologous end-joining leads to frequent catastrophic genomic events in murine and human tumors.

Manasi Ratnaparkhe,1 John Wong,1 Pei-Chi Wei,2 Mario Hlevnjak,1 Paul Northcott,3 David T. Jones,1 Marcel Kool,1 Anna Jauch,4 Agata Pastorczak,5 Andrey Korshunov,4 Rajiv Kumar,1 Susanna M. Downing,6 Stefan M. Pfister,1 Marc Zapatka,1 Peter J. McKinnon,6 Frederick W. Alt,2 Peter Lichter,1 Aurelie Ernst1. 1 _German Cancer Research Center, Heidelberg, Germany;_ 2 _Boston Children's Hospital and Harvard Medical School, Boston, MA;_ 3 _St Jude Children's Research Hospital, Memphis, TN;_ 4 _Heidelberg University, Heidelberg, Germany;_ 5 _Lodz University, Lodz, Poland;_ 6 _St Jude Children's Hospital, Memphis, TN_.

Chromothripsis and chromoanasynthesis are two forms of genomic instability leading to complex genomic rearrangements that affect one or very few chromosomes. These one-off catastrophic events play a role in numerous tumor entities as well as in some congenital diseases. The availability of murine models recapitulating both phenomena would substantially facilitate the investigation of the mechanistic aspects underlying catastrophic genomic events.

Homologous recombination repair (HR) and canonical Non-Homologous-End-Joining (cNHEJ) represent the two major processes for DNA double-strand break repair in mammalian cells. Conditional inactivation of key factors of either of these two pathways, such as Brca2 for HR and Xrcc4 or Lig4 for cNHEJ in nestin-expressing or Emx1-expressing murine neural progenitor cells leads to medulloblastomas and gliomas in a p53-deficient background. We showed by whole-genome sequencing that these tumors frequently display chromothripsis or chromoanasynthesis (33 to 73% of the analyzed tumors, n= 27) and that catastrophic rearrangements drive tumor development.

FISH analysis identified a link between chromoanasynthesis and increased numerical and structural aberrations and with the presence of marker chromosomes. In addition, amplifications of c-Myc and n-Myc likely facilitate catastrophic events. Detailed analysis of the microhomologies at the breakpoint junctions on the chromosomes affected by complex genomic rearrangements identified cNHEJ and alternative end-joining as likely repair processes involved in chromothripsis and chromoanasynthesis.

Treatment of cells derived from the mouse tumors with inhibitors of HR and/or alternative end-joining (e.g. RAD51 and PARP inhibitors, respectively) in combination with DNA damage revealed the dependence of these tumor cells on specific repair processes and showed that these DNA repair deficiencies can be utilized for synthetic lethality approaches.

Comparison of the mouse tumors with whole-genome sequencing data from human medulloblastomas (n=68) and gliomas (n=32) identified an association between chromothripsis and deficiencies in repair processes, by analyzing copy-number level aberrations affecting repair factors and mutational signatures of DNA double-strand break repair defects. This link between DNA repair deficiency and chromothripsis was further confirmed in additional tumor entities such as breast cancer (n=356) and melanoma (n=69).

In analogy to the clinical use of PARP inhibitors in the context of BRCA-deficient breast cancer, our findings point towards therapeutic opportunities to target DNA repair defects in tumors with complex genomic rearrangements.

#1353

Role of TLK1B/NEK1 axis in DNA DSB repair: Implications for prostate cancer progression.

Ishita Ghosh, Arrigo DeBenedetti, Gulshan Sunavala. _LSU Health Science Center, Shreveport, LA_.

Defects in DNA Damage Response and Repair are linked to the majority of cancers, including Prostate Cancer (PCa). Common genotoxic stresses that cause damage to the DNA, or replication fork collapse are generated when PCa cells face Androgen Deprivation Therapy (ADT), and must be bypassed to allow continuous replication and division. Tousled like kinase (TLK1) splice variant TLK1B is implicated in DNA damage repair pathways and is translationally increased following various stresses, including the DDR. We found that the expression of TLK1B is rapidly increased following a shift of LNCaP cells to charcoal-stripped serum (ADT), via an activation of mTOR and phosphorylation of 4EBP1 (inhibitor of the translation factor eIF4E). We recently uncovered the existence of the important DDR axis TLK1B> NEK1> ATR>Chk1.(1). TLK1 phosphorylates NEK1 at T141 and activates its autophosphorylation of Y315. A defect in DNA repair in NEK1-deficient cells is suggested by persistence of DNA double strand breaks (DSB) after low dose ionizing radiation (IR).(2). Cells repair DSB either via HR or NHEJ. We investigated whether TLK1 phosphorylation is essential for NEK1 activity in HR repair. siRNA-mediated NEK1 deficient cells have decreased kinetics of Rad54-S572 phosphorylation and persistent Rad51 foci when induced with Doxorubicin that creates DSB.(3). We tested a NEK1-T141A overexpressing (Hek293) mutant for its effect on Rad54-S572 phosphorylation (pRad54) during a time course of recovery from Doxorubicin. There was no significant delay in pRad54 kinetics compared to the control, unless endogenous (WT) NEK1 was knocked down, in which case we observed a significant delay in pRad54 in NEK1 mutant cells. When DSBs occur during DNA damage, Rad51 becomes localized at DSB repair foci. Delay in Rad54 phosphorylation results in persistent Rad51 foci since aspects of HR subsequent to Rad51 filament formation and strand invasion are impaired. We observed increased Rad51 foci that persisted in NEK1-T141A mutants following two hours of Doxorubicin treatment and 14 hours of recovery. A quantitative approach to monitor HR in NEK1-T141A mutants was obtained with the generation of a stable DR-GFP recombination reporter substrate (4) in HSG cell line. Transfection with the megabase cutter endonuclease I-SceI, creates a DSB in a defective double GFP cassette which would generate a functional GFP gene conversion product. Results indicate that approximately 14-17 % cells from parent population undergoes HR in WT, NEK1 overexpressing, and NEK1-T141A mutants. We would expect statistically significant conclusion from improvising GFP reporter experiments with cells in which endogenous Nek1 and Rad54 is knocked down, consistent with the results obtained with pRad54. Another approach would be inhibiting TLK1B interaction with NEK1 to block HR repair which is thought to be critical in Prostate Cancer progression to Castration Resistant Prostate Cancer.

#1354

TonEBP mediates PCNA polyubiquitination on DNA damage sites via interaction with SHPRH and USP1.

Hyunje Kang, Hyun Park, Eun Jin Yoo, Jun Ho Lee, Soo Youn Choi, Whaseon Lee-Kwon, Kyoo-young Lee, Jin-Hoe Hur, Jeong Kon Seo, Kyungjae Myung, Hyug Moo Kwon. _Ulsan National Institute of Science and Technology, Ulsan, Republic of Korea_.

PCNA ubiquitination is important for DNA damage tolerance in eukaryotic replication. While PCNA monoubiquitination activates a mutagenic translesion synthesis pathway, polyubiquitination activates an error-free template switching for DNA damage bypass. Even though PCNA polyubiquitination is critical for the maintenance of genomic integrity, underlying mechanisms are poorly understood. Here, we report that TonEBP (tonicity-responsive enhancer binding protein) is involved the PCNA polyubiquitination in response to DNA damage. TonEBP sequentially recruited an E3 ubiquitin ligase SHPRH followed by a deubiquitinase USP1 into DNA damaged sites, which showed direct correlation with dynamics of PCNA polyubiquitination. The Rel-homology domain (RHD) of TonEBP, which encircles DNA, was required for the interaction with SHRPH and USP1. Consistent with its importance for DNA damage bypass, knockdown or deletion of RHD in TonEBP increased sensitivity to methyl methanesulfonate. Collectively, TonEBP is a key player in the post-replication DNA repair.

#1355

Mutation signatures including APOBEC in cancer cell lines.

Reuben S. Harris. _Univ. of Minnesota, Minneapolis, MN_.

Background: Multiple endogenous and exogenous sources of DNA damage contribute to the overall mutation burden in cancer, with distinct and overlapping combinations contributing to each cancer type. Many mutation sources result in diagnostic mutation signatures, which can be deduced from tumor genomic DNA sequences. Examples include spontaneous hydrolytic deamination of methyl-cytosine bases in CG motifs (AGEING signature) and C-to-T and C-to-G mutations in 5'-TC(A/T) motifs (APOBEC signature).

Methods: The deconstructSigs R package was used to analyze single base substitution mutation signatures in over 1000 cancer cell lines. Two additional approaches were used to analyze the APOBEC mutation signature.

Results: Most cell lines show evidence for multiple mutation signatures. For instance, the AGEING signature, which is the largest source of mutation in most primary tumors, predominates in the majority of cancer cell lines. The APOBEC mutation signature is enriched in cancer cell lines from breast, lung, head/neck,

bladder, and cervical cancers, where this signature also comprises a large fraction of all mutations.

Conclusions: The single base substitution mutation signatures of cancer cell lines often reflect those of the original tumors from which they are derived. Cancer cell lines with enrichments for distinct mutation signatures such as APOBEC have the potential to become model systems for fundamental research on the underlying mechanisms and for advancing clinical strategies to target these processes and, ultimately, constrain tumor evolvability.

#1356

NSMCE2 enables rescue of collapsed replication forks to prevent mitotic DNA damage.

Kelvin W. Pond, Christelle DeRenty, Mary K. Yagle, Nathan Ellis. _University of Arizona, Tucson, AZ_.

Background: Replication forks that are persistently blocked cannot restart, even after DNA damage is repaired. These forks are termed collapsed forks. In order to fully duplicate the genome, collapsed forks must be rescued by activation of nearby dormant origins of replication. Homologous recombination (HR) is essential in collapsed-fork rescue. Cells deficient in the SUMO E3 ligase NSMCE2 exhibit mitotic defects, are sensitive to DNA damaging agents, and have defects in HR. Thus, NSMCE2 regulates HR during replication stress, but the molecular mechanisms are poorly understood. In yeasts, hypomorphic NSMCE2 (MMS21) alleles accumulate HR intermediates during replication stress. We therefore hypothesized that NSMCE2 regulates RAD51 function during collapsed fork rescue.

Results: It was previously shown that sumoylation of the BLM helicase results in recruitment of RAD51 to stalled forks. Consistent with data in yeast, we found that BLM sumolyation is dependent on NSMCE2. However, contrary to our expectation, we found that the amount of RAD51 protein that accumulated at collapsed forks was over two times greater in NSMCE2-deficient cells than in normal cells. In contrast, the levels of BLM, RPA, single-stranded DNA, and γH2AX at stalled forks is reduced by half. Consistent with the low levels of γH2AX, the double-strand breaks and sister chromatid exchanges that accumulate during collapsed fork rescue were also greatly diminished in NSMCE2-deficient cells. Thus, despite the over-accumulation of RAD51 to sites of collapsed replication forks, cells are unable to perform HR efficiently, indicating that RAD51 is unable to complete its function there. In NSMCE2-deficient cells, the hyper-accumulated RAD51 at collapsed forks persists into mitosis where excess under-replicated DNA causes mitotic DNA damage.

Conclusions: The hyper-accumulation of RAD51 at stalled forks we observed in NSMCE2-deficient cells suggests that NSMCE2 is required for the remodeling of collapsed forks that normally leads to the rescue of collapsed forks, namely, the unloading of RAD51, DNA breakage, repair by HR, and completion of DNA synthesis. We suggest that the excess accumulation of RAD51 that is observed in a substantial number of cancers is not sufficient to demonstrate that the cells are HR proficient. The identification of NSMCE2 as a controller of HR-mediated fork rescue also highlights NSMCE2's potential as a new therapeutic target for combinatorial therapy of HR-dependent cancers.

#1357

Functional characterization of FANCD2 in esophageal squamous cell carcinoma.

Lisa Chan Lei,1 Valen Zhuoyou Yu,1 Lvwen Ning,1 Josephine Mun-Yee Ko,1 Li Dong Wang,2 Maria Li Lung1. 1 _The University of Hong Kong, Hong Kong, Hong Kong;_ 2 _Zhengzhou University, Zhengzhou, China_.

Introduction:

Esophageal squamous cell carcinoma (ESCC) has an especially high incidence in Northern China, where there is evidence for a significant familial association. We performed targeted next-generation sequencing (NGS) analysis on familial ESCC germline samples compared to non-cancer controls from the same high-risk region and compiled a list of candidate cancer predisposition genes. Interestingly, genes related to the Fanconi Anemia (FA) - BRCA pathway are enriched in the list. Among these FA-BRCA genes, Fanconi anemia complementation group D2 (FANCD2) was one of the top candidates, as it also had a high frequency of somatic mutations in ESCC tumor specimens. Therefore, we aim to characterize the role of FANCD2 in tumor development and explore its translational value.

Methods:

We knocked out the FANCD2 gene in ESCC cell lines using the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technique to evaluate its potential oncogenic function in ESCC. Cell proliferation was measured by a MTT 2D clonogenic assay in vitro. Subcutaneous injection of the FANCD2 knockout ESCC cells into BALB/c-nude mice in vivo was performed to assess its functional impact on tumorigenesis. The single cell gel electrophoresis/comet assay was used to investigate the genome stability.

Results:

The FANCD2 knockout efficiency was confirmed by western blotting. Surprisingly, in vitro functional analyses showed that ESCC cells with FANCD2 knockout survive, with a greatly reduced growth rate and colony-forming ability. Consistent with the in vitro data, ESCC cells with FANCD2 knockout form significantly smaller subcutaneous tumors in nude mice. By applying the comet assay to examine the genome integrity, ESCC cells with FANCD2 knockout show significantly greater damage to the genome.

Conclusion:

These results suggest that FANCD2 plays an important role in supporting ESCC tumor growth. We attribute this to its core function in DNA repair ability and genome integrity maintenance.

Acknowledgement:

We acknowledge the grant support from the Hong Kong Research Grants Council Collaborative

Research Fund (C7031.15G to M.L.L.).

#1358

Translational regulation of DNA repair: The mechanism under cap-independent translational regulation of RPA2.

Jia-Jia Cui, Lei-Yun Wang, Ao-Xiang Guo, Ji-Ye Yin. _Department of Clinical Pharmacology, Xiangya Hospital, Central South University, Changsha 410008; P., changsha, China_.

Purpose: Maintenance of genome integrity is critical for any living cells. A variety of endogenous and exogenous factors can cause DNA damage. RPA2 (Replication protein A2) is a kind of single-stranded DNA binding protein complexes and play an important role in the DNA repair. Internal ribosome entry site (IRES) element is a RNA sequence with a complex structure, the activation of RPA2 IRES element can cause its abnormal expression and finally effects DNA repair pathway. IRES is regulated by eukaryotic translation initiation factors (eIFs) and IRES trans-acting factors (ITAFs). UNR (upstream of N-ras), as one of the important IRES trans-acting factors, and eIF3a (Eukaryotic initiation factor 3a) as one of the important eIFs play important roles in the regulation of RPA2 protein expression via IRES elements. We want to explore the role and potential mechanism of translational regulation of UNR and eIF3a in DNA repair. Methods: In the current study, biotin pull down assay was taken to investigate the interaction between RPA2 IRES and UNR. Western blot and qPCR were used to detect protein and mRNA level respectively. CO-IP assay were conducted for interaction of eIF3a with UNR. Immunofluorescence assay was taken to investigate the intracellular localization of UNR and eIF3a, as well as the regulation of UNR on DNA double strand break repair pathway. GST pull down assay was carried out to further identify the interaction between two proteins. Electrophoretic mobility shift assay was carried out to explore the domains of UNR and eIF3a that bind to RPA2 IRES element. Dual-luciferase reporter assay, comet assay and immunofluorescence assay were used to investigate the DNA repair pathway activity. Results: UNR and eIF3a translationally regulate RPA2 by combining with RPA2 IRES. It was found that the expression of RPA2 was down-regulated by UNR with no change on mRNA level. We further found that UNR combined with RPA2 IRES via CSD1 domain. And no domains of eIF3a bind to RPA2 IRES RNA.Meanwhile, the location of UNR and eIF3a was consistent. A series of assays indicate that CSD1 domain of UNR bind to PCI domain of. RPA2 play an important role in the DNA repair. We found that UNR down-regulated double-strand break repair (DSB) activity and nucleotide excision repair (NER) activity. Conclusion: UNR and eIF3a can regulate the expression of RPA2 on translational level, and further regulate the activity of DNA repair, which may reveal the potential mechanism of translational regulation and DNA repair.

#1359

Somatic involution of pathogenic BRCA1 germline mutations.

Vassiliki Kotoula,1 Efterpi Demiri,2 Florentia Fostira,2 Eleni Vrettou,2 Kyriaki Papadopoulou,2 Ioannis Tikas,3 Konstantinos Papazisis,2 Thomas Zaramboukas,2 Asimina Asimaki-Vlachopoulou,1 Spyridon Miliaras,2 Elena Fountzilas,2 Ananias Ananiadis,2 Sofia Chrisafi,2 Christos Poulios,2 Ioannis Natsiopoulos,2 Aris Tsiftsoglou,2 George Fountzilas2. 1 _Aristotle Univ. School of Medicine, Thessaloniki, Greece;_ 2 _Hellenic Cooperative Oncology Group, Greece;_ 3 _Aristotle Univ. School of Medicine, Greece_.

There is currently a wealth of data on the tumor genomic contexture from BRCA1/2 carriers, particularly breast and ovarian carcinomas. By contrast, little attention has as yet been paid to the genomic status of cancer-related normal tissues from these individuals. Here, we investigated the status of pathogenic BRCA1 germline mutations (GM) in breast (B) and gynecologic (GYN) tissues.

Methods: We examined 121 DNA samples (48 B; 36 GYN; 37 tumors) from an equal number of paraffin blocks obtained upon prophylactic or debulking surgery from 44 BRCA1/2 carriers (mean age 38 yrs, range 24-62; 43 BRCA1 carriers). Six women had never had cancer manifestation (CM). At the time of surgery, 32 were cancer-free but had received neo- or adjuvant chemotherapy, and 6 had concurrent cancer without prior treatment. Following multimethod DNA quality control, mutation validation and sample identity match to exclude false negatives, we interrogated GM presence in tissues in comparison to clinicopathologic data and tumor genotypes (60-gene panel; mean read depth over 800).

Results: In 19 samples from 13 BRCA1 carriers, including 13 normal B/GYN and 6 tumors, the germline mutation was present at frequencies lower than 5% (observed with the integrated genome viewer) up to 12%, or it was undetectable with Sanger sequencing and multiplex PCR. This condition, termed GM-loss, was present in 13 GYN, 12 of which in the histologically normal tube, and in only 3 B with fibrocystic disease (p=0.0210). It was also present in one ovarian thecoma but it was absent in usual or atypical hyperplasia in B. GM-loss was observed in 9/55 normal tissues from breast cancer and in 4/6 normal tissues from ovarian cancer patients, while it was absent in the normal tissues from women without CM (p=0.0002). GM-loss mostly affected the BRCA1 BRCT functional domain (p<0.0001) and concerned large deletions (10/17 samples), small indels (5/32; 15.6%) and less so single-nucleotide substitutions (4/66; 6.0%; p<0.0001). Normal tissue GM-loss was rather localized, e.g., in 10 patients with multiple samples it was present in only one B or fallopian tube. It was, however, related to the status of concurrent ovarian tumors in 3/6 cases. In the normal tube of these patients, next to the low-frequency GM a somatic pathogenic BRCA1 mutation was present at 25-41% allelic frequency; the same was observed in one breast tumor after neoadjuvant chemotherapy. Three of the replaced GMs were indels, two corresponded to the well-known BRCA1 p.Q1777fs, and all novel mutations were missense.

Conclusions: We observed BRCA1 GM-loss in normal tissues, breast and mostly fallopian tube, in about 30% of the carriers in the present cohort, all with previous or concurrent cancer. The phenomenon seems analogous to the GM reversion described in tumors but, as shown, it may also occur in the absence of prior treatment. Its origin and impact on cancer dynamics and therapeutic approaches seem worth further pursuing with functional studies.

#1360

A PARylation biosensor genetic screen to identify novel PARylation targets.

Dragomir B. Krastev,1 Stephen J. Pettitt,1 James Campbell,1 Alan Ashworth,2 Christopher J. Lord1. 1 _Institute of Cancer Research, London, United Kingdom;_ 2 _Helen Diller Family Comprehensive Cancer Centre, San Francisco, CA_.

Poly(ADP-ribosylation) (PARylation) is a reversible post-translational protein modification that regulates a number of cellular processes. It is generated by a family of PAR polymerases (PARP), of which PARP1 has recently been exploited as a target in ovarian cancer carrying BRCA1/2 mutations. In addition, tankyrase (PARP5A and B), with its regulation of Wnt signalling, telomeres and the mitotic apparatus, has attracted much effort in the development of targeted therapies. All this necessitates the identification of PARylation targets and their mode of regulation by the modification. Genetically encoded fluorescent biosensor that can report the PARylation state of a target protein has the potential to report the modulation of the PARylation target in living cells. We used PAR-binding PBZ domains fused to a non-fluorescent fragment of a GFP molecule in combination with a protein of interest fused to the complementing fragment of GFP. When this protein is PARylated, it is recognized by the PBZ domain, which brings in proximity the two GFP fragments, and restores the GFP fluorescence. Known targets of PARylation, e.g. TERF1 and AXIN1, showed specific spatial patterns of PARylation validating our approach. In order to identify novel PARylation targets, we have designed a genetic screen based on the biosensor. We introduced the GFP fragment in a transposon, which was randomly integrated in cellular genome, thus generating cellular libraries of thousands of tagged genes. The introduction of the PAR binding probe lead to GFP fluorescence in clones that harbour putative PARylation targets. We developed a next generation sequencing approach to isolate the transposon integration sites and annotated the target genes. Reassuringly, we have identified NPM1, a well-known PARylation target, as a strongly scoring gene. We focused on one of novel identified hits (CTIF). We showed that CTIF is PARylated in vivo in a tankyrase-dependent manner. Furthermore, the biosensor revealed that this PARylation is predominant in a specific cellular compartment surrounding the centrosome. Functional studies have implicated this protein the regulation of the centrosomal satellites. Collectively, we are presenting a new approach to identify PARylated proteins and study their patterns of PARylation and regulation in vivo.

#1361

Micro RNA-200C is one of the important Fanconi Anemia (FA) pathway downstream regulators in lung cancer.

Wenrui Duan,1 Shirley Tang,2 Li Gao,1 Kathleen Dotts,2 Andrew Fink,2 Arjun Kalvala,2 Brittany Aguila,2 Miguel A. Villalona-Calero3. 1 _Florida International University, Miami, FL;_ 2 _The Ohio State University, Columbus, OH;_ 3 _Miami Cancer Institute, Miami, FL_.

The Fanconi Anemia (FA) pathway is essential for human cells to maintain integrity following DNA damage. This pathway is involved in the endogenous repair of double stranded DNA breaks and homologous recombination as well as repair of DNA cross-linking caused by exogenous agents. Cancers with defective FA pathway are expected to be more sensitive to cross-link based therapy, or to treatments in which additional repair mechanisms are targeted. We have recently reported the detection of 22% of NSCLC to be FA functionally inactive by Fanconi Anemia Triple Staining Immunofluorescence (FATSI) test. Studies have shown involvement of certain micro RNA (miRNA) as regulatory elements in the development of lung cancer. We set out to evaluate potential involvement of miRNAs in the regulation of the Fanconi Anemia (FA) pathway. Using Nanostring counter miRNA array we screened 734 different miRNA expression in two FA defective lung cancer cells and matched control cells along with two FA pathway deficient lung tumors and matched non-tumor lung tissue samples. Selected miRNA expression were validated with real-time PCR analysis. miRNA target gene expression was analyzed through AmpliSeq RNA gene expression analysis. Among 734 different miRNAs, a cluster of microRNAs were found to be up-regulated including an important cancer related micro RNA, miR-200C. Nanostring data showed that miR-200C was increased 7.5 fold on average in the FA defective lung cancer cells as compared to control cell. An average of 22 fold increase in miR-200C was detected in the FA defective lung tumor tissues comparing to matching non-tumor tissues. AmpliSeq analysis showed the ZEB1(zinc finger E-box binding homeobox 1) mRNA expression was down regulated in10 out 10 lung tumors (100%) comparing to non-tumor tissues, and 9 out of 10 samples (90%) showed reduction in ZEB2 expression. MiRNA-200C has been reported as a negative regulator of epithelial-mesenchymal transition (EMT) and inhibiting cell migration and invasion by promoting the upregulation of E-cadherin through targeting ZEB1 and ZEB2 transcription factors. Our findings indicate that the FA pathway regulates downstream genes through regulation of miRNAs in lung cancer. MiR-200C appears to be one of the most important FA downstream regulators in lung cancer. Validation with a larger sample size will be needed to confirm our findings.

#1363

Regulation of BRCA1 by SIRT2.

Elizabeth Minten, Hui Zhang, Chunyang Li, PamelaSara Head, David Yu. _Emory University, Atlanta, GA_.

In our everyday lives, we are constantly being exposed to DNA damaging agents, both from internal and external sources. The most deleterious type of DNA damage a cell can suffer is the DNA double-stranded break (DSB). Breast Cancer 1 (BRCA1) is a protein necessary for the proper repair of resultant DNA DSBs through the homologous recombination (HR) pathway. Defects in BRCA1 have been linked to different types of cancer in both men and women, including breast, ovarian, and pancreatic cancer. However, the regulation of BRCA1 is not yet well understood, and in many cases, how defects in this pathway lead to an increased risk of developing cancer is also unknown, making preventative care and treatment of resulting cancers more difficult. Our lab has discovered that SIRT2, a histone deacetylase and putative human tumor suppressor, plays a crucial role in the DNA damage response (DDR) and repair of DNA DSBs. We have shown that depletion of SIRT2 impairs HR and increases cell sensitivity to IR in a deacetylase-dependent manner. A mass spectrometry analysis showed SIRT2 interacts with a number of proteins involved in DDR, including BRCA1. We validated the interaction between SIRT2 and BRCA1 and also found SIRT2 deacetylates BRCA1 both in vitro and in cells. Depletion of SIRT2 and subsequent deacetylation of BRCA1 decreases BRCA1 protein levels in cells and thus impairs HR. Our results show SIRT2 is a novel regulator of BRCA1 and is critical for the repair of DNA DSBs through HR. These findings provide insight into how dysregulation of SIRT2 or BRCA1 can result in genomic instability and lead to the development of cancer.

#1364

**Tip60 dependent DNA homologous recombination repair is impaired in** VHL **-deficient clear cell renal cell carcinoma.**

Lijun Zhou, Patrick G. Pilie, Christine B. Peterson, Xian-de Liu, Xuesong Zhang, Eric Jonasch. _MD Anderson, Houston, TX_.

Introduction: Clear cell renal cell carcinoma (ccRCC) displays genomic instability across all tumor stages, indicative of increased replicative stress and defects in DNA damage response (DDR) pathways including homologous repair (HR); however ccRCC does not display mutations in canonical DDR genes. We hypothesized that biallelic VHL loss is sufficient to cause HR deficiency (HRD) in ccRCC via direct regulation of DDR pathways. Experimental Procedures: We performed whole-exome (WES) sequencing of 15 small ccRCC tumors. We performed in silico genomic, transcriptomic, and proteomic analysis of tumors in KIRC TCGA to assess for HRD, using published HRD signatures. We assessed HR efficiency as a product of biallelic VHL loss in engineered cell line models. We studied the VHL-dependent DNA damage repair molecular mechanisms with cell line models, by HR and nonhomologous end joining (NHEJ) using reporter gene assays. We then assessed the status of TIP60 activation in Vhl deficient murine embryo fibroblasts, isogenic VHL deficient and proficient cell lines by determining DSB induced tip60 acetylation and foci formation. DSB induced HR and NHEJ activation were analyzed by assessing the induction of marker proteins including γH2AX, RAD51, and 53BP1. To examine VHL-protein interaction, green-fluorescent protein (GFP)-trap with GFP-tagged VHL and co-immunoprecipitation with specific antibodies were used. Results: 15/15 early stage ccRCC had biallelic VHL mutations and approximately 100 additional mutations per tumor, but no driver mutations typically associated with HRD. Nonetheless, in silico analysis showed 67% of KIRC TCGA displayed an HRD gene signature, and this signature was significantly higher in stage I disease (p=2.21e-08). Patients with VHL-mutated tumors were more commonly HRD than HRI (p=0.03), with frameshift/nonsense variants in VHL more likely to result in HRD than missense variants (p=0.02). Multivariate analysis showed HRD predicted for better overall survival compared to an HR intact (HRI) state (p<0.0001). Using Vhl knockout and VHL overexpressing cell models we demonstrated that VHL interacts with the acetyltransferase Tip60, and Tip60 is required for VHL-dependent HR but not for nonhomologous end joining (NHEJ). We also observed that during genotoxic stress, VHL-associated-Tip60 dissociated from histone H3 and increased its association with H4, consistent with VHL having a regulatory role on histone H4. Conclusions: Early stage, VHL-deficient ccRCC shows a strong HRD signature in the absence of additional driver mutations associated with DDR. Biallelic loss of VHL is sufficient to cause HRD by impairing Tip60 mediated DDR complex activation. Our findings provide a mechanistic explanation for the DDR defect in ccRCC, and provide a foundation for treatment strategies that target specific elements of the DDR pathway in VHL deficient ccRCC.

#1365

NR1D1 enhances reactive oxygen species-induced DNA damage by inhibition of PARP1.

Na-Lee Ka, Tae-Young Na, Mi-Ock Lee. _Seoul National University, Seoul, Republic of Korea_.

Breast cancer is the most commonly diagnosed cancer in women worldwide. Cancer cells produce higher levels of intracellular reactive oxygen species (ROS) than their normal counterparts, because of their accelerated metabolism, mitochondrial dysfunction, and antioxidant deficit. The oxidative stress in cancer cells may provide clinical benefits, which can be associated with a better response to anticancer therapies. Therefore, identifying the regulatory pathway of oxidative stress in cancer cells is important in the development of therapeutic targets that enhance sensitivity to ROS-generating anticancer therapies. In this study, we identified that nuclear receptor subfamily 1, group D, member 1 (NR1D1; Rev-erbα) inhibited DNA repair of ROS-induced DNA damage in breast cancer cells. NR1D1 interacted with poly(ADP-ribose) polymerase 1 (PARP1) and subsequently inhibited catalytic activity of PARP1. NR1D1 enhanced accumulation of oxidative DNA damage, which increased sensitivity of breast cancer cells to the ROS-induced cell death. Our findings suggest that NR1D1 may provide better therapeutic options for breast cancer treatment, especially in those patients treated with ROS-inducing chemotherapeutic agents.

#1366

MBD4 **guards against DNA damage from methylcytosine deamination.**

Edward Chew,1 Mathijs A. Sanders,2 Christoffer Flensburg,1 Annelieke Zeilemaker,2 Sarah E. Miller,1 Adil S. al Hinai,2 Ashish Bajel,3 Bram Luiken,2 Melissa Rijken,2 Tamara Mclennan,1 Remco M. Hoogenboezem,2 François G. Kavelaars,2 Marnie E. Blewitt,1 Eric M. Bindels,2 Warren S. Alexander,1 Bob Löwenberg,2 Andrew W. Roberts,1 Ian J. Majewski,1 Peter J. Valk2. 1 _Walter & Eliza Hall Institute of Medical Research, Parkville, Australia; _2 _Erasmus University Medical Center, Rotterdam, Netherlands;_ 3 _Royal Melbourne Hospital, Parkville, Australia_.

Spontaneous methylcytosine (5mC) deamination is a common source of cytosine to thymine (C>T) mutations. These mutations accumulate over time, serving as a "molecular clock" that tracks cellular age. MBD4 is a thymine glycosylase that recognises sites of 5mC deamination and initiates base excision repair.

Three patients (of whom 2 are siblings) with germline MBD4 loss of function (LOF) mutations developed acute myeloid leukemia (AML) at a young age (<35 years old). Whole exome (WES) and whole genome sequencing (WGS) showed an elevated C>T mutation rate ~33 times the average for AML. The mutations occurred nearly exclusively at CG dinucleotides. Reduced representation bisulfite sequencing showed the mutations occurred at previously methylated cytosines. The 3 AMLs had somatic C>T mutations in the same driver genes (DNMT3A and either IDH1 or IDH2), suggesting the methylation damage charts a recurrent path towards malignancy in the hematopoietic system.

MBD4 is inactivated in other cancers, although this appears to be a rare event. Nine of 10,638 cancers in the TCGA database had MBD4 LOF mutations. In a uveal melanoma and a glioblastoma multiforme, there was also loss of heterozygosity of the wild-type MBD4. These 2 cancers with MBD4 deficiency exhibit the same mutational signature, with a high C>T mutation rate at CG dinucleotides.

To verify the link between MBD4 and the 5mC damage signature, Mbd4-/- and Mbd4+/+ mouse bone marrow were cultured in semi-solid agar and WGS was performed on individual myeloid progenitor colonies. Mbd4-/- myeloid progenitor colonies displayed the same increase in C>T mutations at CG dinucleotides. This highlights the importance of MBD4 across species.

Mbd4 deficient mice offer a model system to investigate mutation acquisition and cancer pathogenesis. Work on the interaction of MBD4 with other leukaemia initiating genes is underway.

#1367

BRCA1 and BARD1 protein interactions that are required for DNA repair function.

Aleksandra Adamovich, Margaret Wingo, Tapahsama Banerjee, Miranda Gardner, Michael Freitas, Jeffrey Parvin. _Ohio State University, Columbus, OH_.

Breast and ovarian cancers are prevalent among women, and hereditary breast and ovarian cancers (HBOCs) have been associated with germline mutations in genes such as BRCA1 and BARD1. BRCA1 and BARD1 form an obligate heterodimer, and the BRCA1/BARD1 complex is required for tumor suppression functions. Our lab has tested hundreds of BARD1 and BRCA1 variants and identified many that are deficient in homologous recombination, which we have shown to accurately predict cancer predisposition in the clinic. Several of these functionally defective BRCA1 mutants map to a pocket of amino acids on the surface of the protein that does not have any known binding partners. Repair-deficient mutations are also present on the surface of the BARD1 protein in domains that are not known to be associated with DNA repair. We hypothesize that the DNA repair deficiencies mediated by these BRCA1 and BARD1 mutants are due to differences in protein binding when compared to wild-type protein. We have found that BRCA1 mutants in this protein pocket do not phosphorylate and do not localize to the nucleus following DNA damage, both of which are characteristic of the DNA damage response. However, BARD1 repair-deficient mutants still bind phosphorylated BRCA1, indicating that their deficiencies are not due to loss of BRCA1 function. To investigate protein interaction differences we are creating fusion proteins of wild-type and mutant BRCA1 with BioID2, which will biotinylate proteins that bind to BRCA1. We will then be able to identify, via avidin purification and mass spectrometry, protein interactions that are present in the wild-type but absent in the mutant BRCA1. Novel proteins that we identify will be tested for DNA repair and tumor suppressor function. This information will allow us to better understand both BARD1 and BRCA1 function and the mechanism of homologous recombination.

#1368

Variable penetrance of mammary tumors in mouse models of Li-Fraumeni syndrome is linked to replication-associated repair.

Prabin Dhangada Majhi,1 Nicholas B. Griner,1 Shannon Compton,1 Jeffrey Kane,1 Trevor Baptiste,1 Jacob Mayfield,1 Ellen Dickinson,1 Evan Savage,2 Karen Dunphy,1 Kim Obermeier,3 Lisa Wiesmüller,3 D Joseph Jerry1. 1 _Univ. of Massachusetts Amherst, Amherst, MA;_ 2 _Genome Explorations, Memphis, TN;_ 3 _Ulm University, Ulm, Germany_.

Background: The p53 tumor suppressor gene plays a key role in sporadic and inherited breast cancers. Spontaneous mammary tumors develop in ~55% of female BALB/c-Trp53+/- mice providing a model for breast cancer in Li-Fraumeni Syndrome. In contrast, C57BL/6J-Trp53+/- females are devoid of mammary tumors. This strain difference offers a dramatic example of variable penetrance and tools to define genes contributing to the risk of mammary tumors.

Methods: Genome-wide linkage analysis of the mammary tumor incidence was performed in F1 and N2 backcross mice. The C57BL/6J alleles of SM1 locus were introgressed into the BALB/c genetic background through 10 backcrosses to generate SM1-Trp53+/- mice. Radiation sensitivity, DNA double-strand break repair, processitivity of DNA replication and expression of DNA repair genes in SM1-Trp53+/- mice were compared with BALB/c-Trp53+/- and C57BL/6J-Trp53+/-.

Results: Genome-wide linkage analysis identified a major modifier locus on chromosome 7 (designated SM1) and another on chromosome 2 with an overall LOD score of 6.1 using a multigenic model of inheritance. The SM1 locus does not undergo loss of heterozygosity in mammary tumors consistent with dominant-acting risk alleles from BALB/c mice. Mammary epithelial cells (MMEC) of SM1 mice, having C57BL/6 alleles of SM1 in BALB/c background, were sensitive to radiation as were BALB/c MMEC. Functional assays of DNA double-strand break repair (1) demonstrate error-prone repair through the single strand annealing (SSA) pathway level in SM1 embryo fibroblasts (MEF) was similar to that in C57BL/6 MEF, which was 2.8-fold higher in BALB/c-Trp53+/- MEF and 2.5-fold higher in [BALB.B6]F1-Trp53+/-. Similarly, DNA fiber assays showed that processivity of DNA replication was significantly decreased in BALB/c and F1 MEF with median fork length (MFL) 6.4µM but increased in C57BL/6 and complemented in SM1 MEF (MFL ~8.2µM). In addition, gene expression profile of at least 5 DNA repair proteins (Ercc5, Rev1, Rev3l, Trex2, Rdm1) that are differentially expressed in C57BL/6 and BALB/c were complemented by SM1 locus.

Conclusions: Gene(s) within the 20Mb SM1 locus impair movement of replications forks. Stalling of replication may precipitate fork collapse and DNA double strand breaks resulting in the loss of heterozygosity for Trp53 through recombination observed in 90% of mammary tumors.

References: 1. Böhringer et al., 0ncogene 10: , 2013. 32(48):5458-70. (PMID: 23435420)

Supported by grants to DJJ (National Cancer Institute, R01CA105452)

#1369

Analysis of association between homologous recombination deficiency and tumor mutational burden in solid tumors.

Hua Li,1 Liqun Wu,2 Tao Shou,3 Bo Jiang,4 Li Zhuang,5 Kunsong Li,6 Xiang Tan,7 Chao Guo,8 Weidong Guo,9 Yan Guan,10 Zheng Chen,11 Xiao Ding,12 Jun Guo,13 Zhenfang Du,14 Qiang Cui,15 Yue Che,15 Ming Yao15. 1 _The Third Affiliated Hospital,Sun Yat-Sen University, China;_ 2 _The Affiliated Hospital of Qingdao University, China;_ 3 _First People's Hospital of Yunnan Province, China;_ 4 _Yunnan Tumor Hospital, China;_ 5 _The Third Affiliated Hospital of Kunming Medical University,Yunnan Tumour Hospital, China;_ 6 _Guangdong Second Provincial General Hospital, China;_ 7 _First Affiliated Hospital of Guangxi Medical University, China;_ 8 _People's Hospital of Hunan Province, China;_ 9 _OrigiMed Inc.; The Affiliated Hospital of Qingdao University, China;_ 10 _Shandong Cancer Hospital Affiliated to Shandong University, Shandong Academy of Medical Sciences, China;_ 11 _Shandong Provincial Hospital Affiliated to Shandong University, Shanghai, China;_ 12 _Shandong Provincial Hospital Affiliated to Shandong University, China;_ 13 _Xingtai People's Hospital, China;_ 14 _Vanderbilt-Ingram Cancer Ctr., Nashville, TN;_ 15 _OrigiMed Inc., Shanghai, China_.

Background: Multiple types of tumors exhibit DNA repair dysfunction via homologous recombination deficiency (HRD) due to genetic and epigenetic alterations of HR pathway genes, including BRCA1/2, PALB2, ATM, ATR, CHK1/2, BARD1, BIRP1, MRE11A, RAD51 family and FANC family. Some studies showed that tumors with homologous recombination deficiency might result in more mutations than tumors with non-deficient HR. Accumulating evidence suggests that tumor mutational burden (TMB) evaluated by comprehensive genomic profiling was associated with the responses of immune checkpoint inhibitors in solid tumors.

Methods: FFPE tumor samples of 777 Chinese patients were collected for next-generation sequencing (NGS)-based targeted panel assay across multiple types of tumors, including lung, breast, soft tissue, gastrointestinal and gynecology cancers. There were 471 males (60.6%) and 306 females (39.4%) with a median age of 58 years old. Genomic alterations of HR-related genes and TMB values were assessed by next-generation sequencing assay, including single base substitution, short and long insertions/deletions, copy number variations, gene fusions and rearrangements.

Results: In total, 25% of the 777 solid tumor patients had at least one genomic alteration of the HR genes (N=197, HRD group). The top mutant HR genes included BRCA1/2 (28.8%), FANC family (27.6%), ATM (27.4%), ATR (11.2%), RAD51 family (8%), BARD1 (7.6%), PALB2 (7.1%), BIRP1 (5.6%), CHK1/2 (5.0%) and MRE11A (4.6%). TMB was 10.4 muts/Mb in the HRD group compared to 6.4 muts/Mb in the rest of the patients (p<0.01). In addition, 43.2% of the patients in the HRD group had TMB-high values, which was higher than in the non-HRD group at 22.8% (P<0.01). High TMB values were more commonly detected in the colorectal cancer patients.

Conclusions: Our study revealed that the solid tumors harboring HR gene mutation were more likely to have higher TMB values. BRCA1/2, FANC family and ATM were the most common mutant genes. CRC was the dominant cancer type showing high TMB values associated with HRD. Further analysis is warranted to investigate the association.

#1370

Association of the ATM mRNA, protein expression and the somatic mutation counts in human cancers and its impact on overall survival in breast and gastric cancer.

Koung Jin Suh,1 Kwangsoo Kim,1 Seongmin Choi,1 Kyung-Hun Lee,1 Jin Won Kim,2 Keun-Wook Lee,2 Jee Hyun Kim,2 Seongyeong Kim,3 Ahrum Min,3 Tae-Yong Kim,1 Seock-Ah Im1. 1 _Seoul National University Hospital, Seoul, Republic of Korea;_ 2 _Seoul National University Bundang Hospital, Seongnam-si, Republic of Korea;_ 3 _Seoul National University, Seoul, Republic of Korea_.

Background: ATM is a tumor suppressor gene, and involves in DNA damage repair and cell cycle checkpoint activation. We aim to investigate the relationship between mRNA and protein expression levels of ATM gene and the somatic mutation count using whole exome sequencing data derived from TCGA database in various cancer types, including breast and stomach cancer.

Materials and Methods: mRNA expression, protein expression, and somatic mutation data of 22 cancer types were collected from the TCGA data portal in April 2017, and their association with overall survival (OS) was analyzed. Considering the heterogeneous nature of the tumors, subgroups for breast cancer (luminal A, luminal B, HER2, and triple-negative) and for stomach cancer (microsatellite-instability [MSI] status) were applied. To validate the survival outcome in an independent cohort, MTCI Breast Cancer Survival Analysis Tool (http://glados.ucd.ie/BreastMark/) was used for survival analysis in breast cancer.

Results: Out of 22 cancer types from the TCGA database, we found that mRNA expression levels of ATM exhibited significant inverse correlation with somatic mutation counts in 4 cancer types (adenoid cystic carcinoma, breast cancer, stomach cancer, and thyroid carcinoma). Tumors with higher somatic mutation counts above the median value had shorter OS compared to those with lower somatic mutation count (p=0.001) in these cancer types. mRNA expression and protein level of ATM showed positive correlation in breast and stomach cancer (p<0.001 and p=0.001, respectively). Regarding the subgroups of breast cancer, low ATM mRNA expression (the bottom 25% expression level) was associated with high somatic mutation count (p=0.023), and trends to shorter OS (median 114 months vs. 123 months, p=0.071) in luminal A subtype. Median OS was significantly shorter in tumors with low ATM mRNA compared to high in the MTCI validation cohort (n=609, HR 0.54, 95% CI 0.33-0.86, p=0.009). In contrast, ATM mRNA expression was not associated with somatic mutation count in TNBC, and the high somatic mutation count above the median was associated with longer OS (median not reached vs. 114 months, p=0.037). In stomach cancer, ATM mRNA expression level was not significantly associated with OS. However, MSI-high tumors had higher somatic mutation counts and lower ATM mRNA expression level compared to microsatellite stable (MSS) or MSI-low tumors, and low ATM mRNA level was associated with trend to better survival in MSI-high tumors (median OS 55 months vs. 31 months, p=0.136).

Conclusion: These results suggest that the ATM mRNA expression is associated with somatic mutation count and prognosis in some types of cancers. However, these associations need to be assessed in the contexts of molecular subtypes and MSI status. Further studies are warranted to confirm the findings in our study.

#1371

Establishment of a cell-based model system to study the biological functions of RECQ1 and its cancer-risk associated variants.

Swetha Parvathaneni, Xing Lu, Sudha Sharma. _Howard University, Washington, DC_.

RECQ1 belongs to the family of RecQ helicases that are DNA unwinding enzymes with

important roles in genome maintenance. RECQ1 is frequently upregulated in a variety of cancers

and RECQ1's function as well as expression levels correlate with patient survival and response to

anti-cancer therapy. Germline mutations in catalytic domains of RECQ1 increase an individual's

risk of developing breast cancer. Till date a RECQ1 knockout in humans is not reported and the

main goal of the study was to establish an in vitro cell-based model system to investigate the

biological functions of RECQ1 and its cancer risk associated variants in isogenic cell lines. We

utilized CRISPR-Cas9 genome editing technique to specifically knockout RECQ1 in MDA-MB-

231 breast cancer cell line. Disease associated point mutations in RECQ1 were introduced by site-

directed mutagenesis and stable cell lines expressing either the wild-type RECQ1 or RECQ1

variants were established by complementation of the knockout line with vectors encoding either

RECQ1 wild-type or RECQ1 variants. Our results demonstrate successful generation of an in vitro

cell-based model system and RECQ1-knockout MDA-MB-231 cells displayed reduced cell

growth and proliferation, increased DNA damage accumulation and increased sensitivity to

camptothecin as compared to the MDA-MB-231 cells expressing wild-type RECQ1. Growth and

proliferation defects of RECQ1 knockout could be rescued by reintroducing wild-type RECQ1,

whereas breast cancer risk associated RECQ1 variants failed to rescue cellular defects of RECQ1-

loss. As compared to RECQ1 wild-type MDA-MB-231 cells, stable cells expressing RECQ1

cancer-risk associated variants displayed reduced cell growth and proliferation, accumulated

endogenous DNA damage and were more sensitive to camptothecin treatment. Collectively, our

results demonstrate that the catalytic functions of RECQ1 are important for maintaining cell

growth and proliferation and resolve replication stress induced by camptothecin in MDA-MB-231

cells. Our ongoing experiments are investigating the detailed mechanism of RECQ1's role in

response to replication stress and its implications in therapeutic response.

Funding: This work was funded by the NIGMS/NIH grant SC1GM093999 to Sudha Sharma.

We thank Drs. Ritu Chaudhary, Xiaoling Li and Ashish Lal (NCI) for their help and support.

#1372

Novel role of the actin-binding factor Profilin-1 in DNA replication.

Cuige Zhu,1 Sun-Joong Kim,1 Sean Holohan,2 Anna Rogers,3 Jieya Shao1. 1 _Washington University School of Medicine in St. Louis, St.Louis, MO;_ 2 _University College of London, London, United Kingdom;_ 3 _University of Minnesota, Minneapolis, MN_.

Introduction: Proper control of DNA replication is important for genome stability of all mitotic cells, and its deregulation leads to many forms of cancer. While the basic replication machinery and core regulatory factors have been thoroughly studied, new functional regulators continue to emerge. Here we present evidence for a novel function of the actin-binding protein profilin-1 (Pfn1) in DNA replication under both normal and stressed conditions. Pfn1 is essential for actin assembly, cell proliferation and survival, yet showing paradoxical antitumor activities for various types of cancer (breast, bladder, pancreatic, liver). It contains a unique binding site for poly-L-proline (PLP) motifs (XP≥5; X=A, L, I, G, S). Though predominantly cytoplasmic, it is also present in the cellular nucleus with poorly understood functions.

Results: We find by chromatin fractionation that Pfn1 knockdown in the untransformed breast epithelial MCF-10A cells significantly inhibits the DNA-binding of many proteins in the replisome complex (e.g. PCNA, MCM-7, RFC, RPA, and DNA polymerase δ and ε) without affecting their cellular level. The knockdown specificity is confirmed by the phenotypic rescue via a Pfn1 construct containing RNAi-resistant silent mutations. In contrast, Pfn1 overexpression increases the DNA-binding of these proteins, and this requires its ability to bind PLPs since a PLP-binding defective Pfn1 mutant (S137D) shows no effect. Interestingly, Pfn1 undergoes robust nuclear translocation upon hydroxyurea (HU)-induced replication stress, and this occurs in various cell lines including MCF-10A and the breast cancer MCF-7 and MDA-MB-231 cells. Consistent with a role of Pfn1 in cellular response to stalled replication forks, its knockdown in HU-treated MCF-10A cells decreases ATR-dependent signaling (p-ATR, p-CHK1, p-P53, p-CDC2, p-RPA) but increases cellular survival, suggesting possible cell cycle checkpoint bypass and evasion of apoptotic cell death in the absence of Pfn1.

Conclusions: By uncovering the effects of Pfn1 on DNA replication factors and cellular response to replication stress, we provide for the first time the compelling evidence for a "moonlighting" nuclear function of this well-known actin-binding protein that has been implicated in many forms of cancer. Our data suggest that nuclear Pfn1 is required for normal DNA replication, at least partially through its ability to secure the replisome complex at replication forks. Upon replication stress, more Pfn1 is translocated into the nucleus to facilitate ATR activation and proper DNA damage response (checkpoint activation, DNA repair, and apoptosis) which likely results from Pfn1's ability to stabilize the replication forks and the various associated proteins. Future work will be directed towards understanding the mechanistic details of Pfn1 involvement in DNA replication and translating this novel knowledge into anticancer treatments.

#1373

**Circular RNAs generated from the** BRCA1 **pseudogene** **regulates the DNA damage response through SERBP1 RNA-binding protein.**

Yoo J. Han,1 Jing Zhang,1 Jennifer M. Mason,2 Toshio F. Yoshimatsu,1 Xinxin Du,1 Ian Hurley,1 Danny E. Kim,1 Aparna Anantharaman,3 Laia P. Brunet,4 Aleix Prat,4 John Kwon,5 Kannanganattu V. Prasanth,3 Olufunmilayo I. Olopade1. 1 _University of Chicago, Chicago, IL;_ 2 _Clemson University, Clemson, SC;_ 3 _University of Illinois at Urbana-Champaign, IL;_ 4 _August Pi i Sunyer Biomedical Research Institute, Spain;_ 5 _University of Texas Southwestern, TX_.

The genomic region encompassing the BRCA1 gene includes the BRCA1P1 pseudogene (BRCA1P1) within ~170kb at chromosome 17q21. The homology and proximity of BRCA1 and BRCA1P1 create a hot spot for recombination, resulting in a large genomic rearrangement found in some families with breast and ovarian cancers. While the significant role of BRCA1 in regulating the DNA damage response (DDR) is well defined, little is known about the biological properties of the BRCA1P1 pseudogene. To functionally annotate BRCA1P1, we conducted a CRISPR-Cas 9 knockout of the pseudogene, as well as knockdown of the expression using antisense oligonucleotides. Both knockout and knockdown of the pseudogene result in increases in apoptosis and sensitivity to DNA damaging drugs. Depletion of BRCA1P1 accumulates spontaneous DNA damage foci and leads to replication fork stalling. Mechanical studies reveal that the BRCA1P1 generates circular RNAs, which are retained in nuclei. Mass spectrometry followed by chromatin isolation by RNA purification (ChIRP) identifies a specific association of the circular RNAs with SERBP1 RNA-binding protein, which was confirmed by ChIRP-Western. Consequently, BRCA1P1 knockout decreased SERBP1 protein expression in the nuclei. Based on the previous report that SERBP1 binds to mRNAs of DDR proteins and controls their translation, we propose a mechanism of BRCA1P1-driven regulation of DDR protein expression through SERBP1. Our data indicate that the BRCA1P1 pseudogene plays a role in regulating replication fork progression and genomic instability in breast cancer cells through an interaction with SERBP1 RNA-binding protein.

#1374

Modeling the CRISPR/Cas9 structural complex with sgRNA and DNA.

Anita Wary. _York Community High School, Elmhurst, IL_.

The ability of genome editing tool to correct genetic disease, or edit the tumor causing gene, will not only benefit human race, but also plants and animal kingdom. The CRISPR/Cas9 system can be used to target specific genomic loci by single guide RNAs (sgRNAs). Using publicly available literature, we have modeled the structure of Streptococcus pyogenes Cas9 molecule in complex with sgRNA and its target DNA at 2.5 A° resolution. The crystal structure determination revealed a two-lobed architecture made of: (a) target recognition and (b) nuclease lobes, positioning the sgRNA:DNA hetero-duplex in a positively charged groove at their interface. While the recognition lobe interacts with the sgRNA and DNA, the nuclease lobe contains the HNH and RuvC nuclease domains that are responsible for cleavage of the complementary and non-complementary target DNA strands, respectively. The nuclease lobe also harbors a carboxyl-terminal domain to provide additional specificity for interaction with the protospacer adjacent motif (PAM) DNA sequence. This structural analyses show the molecular mechanism of RNA-guided DNA targeting by Cas9, thus providing the CRIPSR/Cas9 genetic machinery its ability to edit specific DNA sequence. (Key Ref: Nishimasu H et al., Cell. 2014;156:935-49)

#1375

High mobility group box proteins modulate chemotherapeutic DNA damage repair processing in human cells.

Anirban Mukherjee, Wade A. Reh, Karen M. Vasquez. _The University of Texas at Austin, Austin, TX_.

Many anticancer chemotherapeutic agents induce DNA interstrand crosslinks (ICLs) and DNA double-strand breaks (DSBs) leading to cytotoxicity; however, the mechanisms involved in processing such damage is not completely understood. Thus, a better understanding of the processing of ICLs and DSBs will assist in the identification of novel pharmacological targets, and improved drug design, particularly in chemo-resistant populations. We discovered that the High Mobility Group Box proteins (HMGBs), a family of non-histone architectural proteins, modulate DNA lesion processing in human cancer cells and significantly alter cell survival. We have shown that one of the HMGB family members, HMGB1, binds with high affinity to ICLs targeted to specific sites using triplex-forming oligonucleotides (TFOs), and modulates the repair of ICLs as a co-factor of the nucleotide excision repair (NER) mechanism. We found that other HMGB family members, HMGB2 and HMGB3, which share sequence and structural homology with HMGB1, also modulate DNA repair. Based on our preliminary results, we hypothesize that the HMGB proteins modulate lesion processing via unique and distinct mechanisms, and promote the repair of chemotherapeutic and carcinogenic lesions. In our studies site-specific ICLs were formed by incubating DNA substrates with psoralen-conjugated TFOs followed by UVA irradiation (TFO-ICLs) and used in mutagenesis studies in human cells. Protein recruitment to the damaged site was determined by chromatin immuno-precipitation (ChIP). Non-homologous end-joining (NHEJ) and homologous recombination (HR) were studied via fluorescence activated cell sorting of fluorescent reporter genes in human cells. Cellular localization was visualized by immunofluorescence microscopy. Chromosomal aberrations were determined from metaphase spreads using light microscopy. In response to ICLs, siRNA-mediated depletion of HMGB proteins increased mutagenesis, suggesting involvement in error-free DNA damage repair. Interestingly, analysis of the mutants indicated an increase in single nucleotide mutations in the absence of HMGB1, whereas depletion of HMGB2 or HMGB3 predominantly resulted in small deletions, indicating their involvement in different repair pathways. Specifically, HMGB2 or HMGB3 depletion reduced ICL-induced single-strand annealing (a sub-pathway of HR) events. Interestingly, we found that both HMGB1 and HMGB2 forms distinct foci after damage induction. HMGB2 depletion stimulated NHEJ events, while depletion of HMGB2 or HMGB3 reduced DSB-induced HR events. Finally, depletion of HMGB proteins increased chromosomal aberrations in cancer cells treated with ICL- and DSB-inducing agents. Our novel findings suggest that the HMGB proteins are involved in processing of carcinogenic and chemotherapeutic lesions in human cells via distinct repair mechanisms.

#1376

Effect of gold nanoparticle on radiation induced DNA damage in MCF7 breast cancer cells.

Branislava Janic,1 Fangchao Liu,2 Kevin Bobbitt,1 Stephen Brown,1 Guangzhao Mao,2 Indrin J. Chetty,1 Benjamin Movsas,1 Ning Winston Wen1. 1 _Henry Ford Hospital, Detroit, MI;_ 2 _Wayne State University, Detroit, MI_.

Nanoparticles (NPs) are 1-100 nm constructs explored for their application in cancer diagnosis and treatment. Exposure of cells to ionizing irradiation leads to DNA damage, with DNA double strand breaks (DSBs) being the most toxic that can lead to apoptosis. The presence of gold in NPs enhances radiation damage and DSBs, the latter measured by the levels of phosphorylated DNA histone protein H2AX (γH2AX). The goal of this study was to decipher biological mechanisms of NP radio-sensitization. We analyzed the effects of two different sizes of gold nanoparticles (AuNP) on DSBs in MCF-7 breast cancer cells by assessing H2AX phosphorylation at three photon energies. Cells were incubated with either 4 nm or 14 nm AuNP and irradiated with 2, 4 or 8 Gy using 2.5 FFF MV (60 MU/min), 6 MV (600 MU/min) or 10 FFF MV (2400 MU/min) X-rays. Percent of cells positive for γH2AX was determined by flow cytometry. Live cells (100000) were gated using forward (FSC; cell size) versus side scatter (SSC; complexity) characteristics (gate P1). Cells positive for γH2AX were further gated to identify subpopulations exhibiting brighter (gate P2) or dimmer (gate P3) fluorescence intensity. Treatment with 4 nm AuNP resulted in significantly more γH2AX positive cells after irradiation at all three energies, compared to their respective controls. When treated at 6 MV energy, increases in the percentage of cells positive for H2AX phosphorylation was detected at all three doses (2, 4 and 8 Gy). Increased phosphorylation was accompanied by an increase of cells in the P2 gate accompanied by an increase in their FCS and SSC characteristics, consistent with activation. In cells treated with 10 MV the effect was most pronounced at 4Gy dose, while 2 and 8 Gy resulted in a slight increase in the percentage of γH2AX positive cells. Nevertheless, these cells still exhibited prominent increases within the P2 gate and FCS vs SSC characteristics, compared to their irradiated controls. In cells treated with 2.5 MV energy similar results were observed using 4 and 8 Gy, while no effect was detected with 2 Gy. Data from the experiments using 14 nm size AuNP were less clear and require further investigation. The difference may be due to the localization of the smaller NPs within the nucleus and therefore cause more DNA damage thereby greater H2AX phosphorylation. We hypothesize that the NP radio-sensitization mechanism involves directing cells towards apoptosis by enhancing DNA damage and interfering with DNA repair. Hence, γH2AX positive cells exhibiting bright fluorescence (P2) and an increase in FCS and SSC identified even in the conditions without significant increase in the percent of total γH2AX positive cells, may reflect cells primed for apoptosis. Future studies are planned to elucidate the exact intracellular mechanisms of NP biological radio-sensitization effect.

### Epigenetic Therapy

#1377

Immune regulation of the tumor microenvironment by EZH2: Epigenetic priming for immune oncology combinations.

Justin P. Lucas,1 Sharon Yang,2 Fang Wang,2 Pei Pei Kung,3 Shikhar Sharma,3 Joseph M. Wu,4 Kim Arndt,2 Daniel C. Rowe,2 Robert Rollins2. 1 _Pfizer/New York Medical College, Pearl River, NY;_ 2 _Pfizer, Pearl River, NY;_ 3 _Pfizer, La Jolla, CA;_ 4 _New York Medical College, Valhalla, NY_.

EZH2 plays diverse roles in epigenetic control of carcinogenesis. While small molecule inhibitors of EZH2 have demonstrated signal agent activity in genetically defined tumor types, combination strategies may be required to fully unlock the potential of this novel class of epigenetic drugs. To this end, we are currently exploring the ability of EZH2 to control immune regulatory mechanisms in the context of the tumor microenvironment. We hypothesize that treatment of melanoma tumors with an EZH2 inhibitor will lead to global changes in immune regulatory gene expression that would prime the immune system and augment the response to checkpoint inhibitor monoclonal antibody therapies. Through the use of various experimental techniques including Mass Spectrometry, RNA Seq, Western, FACs, and IHC, we demonstrate significant changes in immune response mechanisms after EZH2i treatment in vitro and in vivo. These changes include synergistic up-regulation of the IFN-γ response genes, such as PD-L1 on the tumor and immune cells, increased expression of ICAM-1 resulting in greater influx of tumor infiltrating T lymphocytes, enrichment of a central memory phenotype in T lymphocytes, and an enhancement of the CD4 TH1 and CD8 cytotoxic cytokine response. In the B16F10 syngeneic model we were able to show that priming the tumor cells and immune system with EZH2i, followed by IO combinations with PD-L1 and CTLA4, significantly reduced the tumors ability to proliferate, over either of the single agents and establishes a central memory to B16 tumor antigens, preventing any further challenge in animals previously treated. This data suggests that inhibitors of EZH2 catalytic activity could potentially be used in combination with immuno-therapies to potentiate their clinical activity.

#1378

Low-dose demethylation therapy for the treatment of cisplatin-resistant testicular cancer.

Andrea Corbet,1 Costantine Albany,2 Emmanuel Bikorimana,1 Ema Khan,1 Jennifer Rodriguez,1 Brock C. Christensen,3 George Sandusky,2 Lawrence H. Einhorn,2 Sarah J. Freemantle,1 Michael J. Spinella1. 1 _University of Illinois, Urbana, IL;_ 2 _Indiana University School of Medicine, Indianapolis, IN;_ 3 _Dartmouth Medical School, Hanover, NH_.

Testicular germ cell tumors (TGCTs) are the most common cancers of young males. A portion of TGCT patients are refractory to cisplatin. Only 30% of patients refractory to cisplatin respond to salvage therapies while to remainder die from progressive disease. Embryonal carcinoma (EC) are the stem cells of TGCTs. We have found that EC cells were highly sensitive to the DNA methyltransferase inhibitor, 5-aza deoxycytidine (5-aza). As an initial step in bringing demethylation therapy to the clinic for TGCT patients, we evaluated the effects of the clinically optimized, second generation demethylating agent guadecitabine (SGI-110) on EC cells in an animal model of cisplatin refractory testicular cancer. EC cells were exquisitely sensitive to guadecitabine and the hypersensitivity was dependent on high levels of DNA methyltransferase 3B. Guadecitabine mediated transcriptional reprogramming of EC cells included induction of p53 targets and repression of pluripotency genes. As a single agent, guadecitabine completely abolished progression and induced complete regression of cisplatin resistant EC xenografts even at doses well below those required to impact somatic solid tumors. Low dose guadecitabine also sensitized refractory EC cells to cisplatin in vivo. Genome-wide analysis indicated that in vivo antitumor activity was associated with activation of p53 and immune-related pathways and the antitumor effects of guadecitabine were dependent on p53, a gene rarely mutated in TGCTs. Together, these preclinical findings provided the rationale for our recently initiated and promising phase I clinical trial using SGI-110 to treat cisplatin refractory TGCT patients. We discuss our recent genome-wide molecular studies aimed to identify potential mechanism(s) to account for the hypersensitivity of TGCTs to 5-aza including promoter demethylation, p53 activation and dsRNA MDA5/MAVS/IRF7 viral mimicry. We also discuss preliminary findings from our ongoing trial. Our findings suggest that guadecitabine alone or in combination with cisplatin is a promising strategy to treat refractory TGCT patients.

#1379

INCB059872, a novel FAD-directed LSD1 Inhibitor, is active in prostate cancer models and impacts prostate cancer stem-like cells.

Gianluca Civenni,1 Giada Zoppi,1 Ramiro Vazquez,1 Dhreeraj Shinde,1 Alyssa Paganoni,1 Aleksandra Kokanovic,1 Sang Hyun Lee,2 Bruce Ruggeri,2 Giuseppina M. Carbone,1 Carlo V. Catapano1. 1 _Institute of Oncology Research (IOR), Bellinzona, Switzerland;_ 2 _Incyte Corporation, Wilmington, DE_.

Cancer of the prostate is one of the most common malignancies and the second leading cause of cancer death in men in developed countries. There is increasing evidence that cancer stem-like cells (CSCs) are implicated in CRPC disease progression and treatment resistance. Transcriptional, epigenetic and metabolic reprogramming are key features for the acquisition and maintenance of stem-like properties. Understanding the factors regulating self-renewal and survival of prostate CSCs may offer novel targets for innovative therapeutic strategies. LSD1/KDM1A is a lysine demethylase for histone and non-histone proteins and functions as transcriptional corepressor or coactivator depending on the binding partners and substrates. LSD1 is a key epigenetic modifier controlling the fate of pluripotent stem cells in several tissues. Overexpression of LSD1 is found in many human cancers and has been linked to tumorigenic and CSC-like features. The involvement of LSD1 in stem cell pluripotency and differentiation suggests that LSD1 inhibitors, such as INCB059872, might be useful to target the prostate CSC subpopulation. Here, we examined the effects of INCB059872 on the growth properties, self-renewal and tumorigenic capability of prostate CSCs derived from human cell lines and the Pb-Cre4;Ptenflox/flox;Rosa26ERG/ERG (ERG/PTEN) mice, which develop highly invasive prostate adenocarcinomas. In ex vivo tumor-sphere assays, INCB059872 significantly suppressed the growth of tumor-initiating stem-like cells isolated from prostatic tumors generated in ERG/PTEN mice. Furthermore, treatment with INCB059872 inhibited colony and tumor-sphere formation by human prostate cancer cells. Conversely, the effects on proliferation and viability of bulk tumor cells were limited and required long-term exposure to the drug. These effects were observed in multiple prostate cancer cell lines, irrespective of the distinctive genetic features and AR status. Importantly, genetic knockdown of LSD1 by siRNAs and shRNAs recapitulated the effects of INCB059872. Both transient and stable knockdown of LSD1 had limited effects on proliferation and viability of bulk tumor cells, whereas they significantly reduced growth of colony and tumor-sphere forming stem-like cancer cells. Furthermore, LSD1 knockdown drastically reduced tumor growth and tumorigenic stem-like cells in subcutaneous xenografts of human prostate cancer cells in nude mice. These results support the hypothesis that LSD1 has a major role in sustaining the stem-like and tumorigenic subpopulation in prostate tumors and its inhibition by chemical or genetic approaches prevents survival and self-renewal capability of prostate CSCs. These data suggest that INCB059872 could be a valid addition to the current treatment strategies for prostate cancer. INCB059872 is currently in phase1/2 clinical studies

#1380

Towards peptidomimetics to target DOT1L recruitment in MLL-AF9 leukemia.

Sierrah Marie Grigsby,1 Jennifer Chase,1 Bridget Waas,1 Ann Friedman,1 Lei Du,2 Aihong Yao,2 James Ropa,1 Justin Serio,1 Andrew Muntean,1 Ivan Maillard,1 Haying Sun,2 Zaneta Nikolovska-Coleska1. 1 _University of Michigan, Ann Arbor, MI;_ 2 _China Pharmaceutical University, Tongjiaxiang, China_.

Leukemias harboring rearrangements of mixed-lineage leukemia gene (MLL1) are associated with poor clinical outcomes, and new therapeutic approaches are needed. Rearrangement of the MLL1 gene generates fusion oncoproteins which drive the high expression of the clustered homeobox (HOX) genes and induce leukemic transformation. Genome-wide histone methylation studies have revealed that the abnormal expression of MLL1 fusion target genes is associated with high levels of histone H3 lysine 79 (H3K79) methylation. Recruitment of DOT1L (disruptor of telomeric silencing 1-like), a unique histone methyltransferase that catalyzes methylation of H3K79, proved to be essential for the transforming activity of multiple MLL fusion proteins. We have mapped the binding site to a short segment of 10 amino acids in DOT1L and shown that DOT1L mutants lacking these residues did not support transformation by MLL-AF9. We hypothesized that by targeting the AF9-DOT1L protein-protein interactions (PPIs), we would selectively kill MLL-AF9 cells without effecting DOT1L role in normal hematopoiesis. Using established DOT1Lf/f MLL-AF9 with reintroduced WT-DOT1L, DOT1L missing 10aa AF9-binding domain (D10), DOT1L with a point mutation in the AF9-binding domain (I867A) and enzymatically inactive DOT1L (RCR), we were able to demonstrate that by disrupting the AF9-DOT1L PPIs, although we can inhibit leukemogenesis similarly to enzymatic inhibition, this interaction is not essential for normal hematopoiesis. Based on our initial studies to map the DOT1L interaction site and in conjunction with utilizing reported NMR structures of the AF9-DOT1L complex, we investigated the nature of the interactions and the minimum length of the peptide. Using different natural and unnatural amino acids, we successfully designed a 7mer peptide with KD of 10 nM and 25 nM against AF9 and ENL, respectively, showing similar potency as the originally identified and validated 10mer peptide. These results lay the groundwork for further optimization of the 7mer peptide towards developing DOT1L peptidomimetics with improved potency and cellular activity, to further validate the PPIs between DOT1L and MLL-fusion proteins as a potential therapeutic target for MLL rearranged leukemia.

#1381

Targeting histone acetyltransferases to reprogram high C-MYC expressing cancers.

Elodie M. Da Costa,1 Gregory Armaos,1 Annie Beaudry,1 Chantal Richer,1 Maxime Caron,1 Pascal St-Onge,1 Jeffrey Johnson,2 Nevan Krogan,3 Yuka Sai,4 Michael Downey,4 Daniel Sinnett,1 Serge McGraw,1 Noël J. Raynal1. 1 _CHU Sainte-Justine Hospital Research Center, Montreal, Quebec, Canada;_ 2 _University of California San Francisco, San Franscisco, CA;_ 3 _University of California San Francisco, San Francisco, CA;_ 4 _Ottawa Institute of Systems Biology, Ottawa, Ontario, Canada_.

In cancer, epigenetic modifications are strongly altered and are responsible for gene expression aberrations. In a drug screening initiative, we recently reported that proscillaridin, a cardiac glycoside (CG), exhibits unsuspected epigenetic and anticancer activities. To understand CG's epigenetic mechanisms of action, we performed RNA sequencing analysis, which showed proscillaridin effects on global gene expressions in acute lymphoblastic leukemia cells (MOLT-4). Genes associated with apoptosis and cell differentiation were upregulated whereas master transcription factors and oncogenic pathway genes were downregulated. Mechanistic studies revealed that proscillaridin decreased histone 3 acetylation, which correlated with histone acetyltransferase (KATs) downregulation (CBP, P300, TIP60, GCN5 and MOZ). Acetylome studies by mass spectrometry showed an acetylation loss in chromatin regulators, the oncogene C-MYC and its associated proteins. Proscillaridin induced C-MYC transcript and protein degradation. Moreover, in a panel of cancer cell lines, we measured that cancer cells sensitivity to proscillaridin treatment was positively correlated with C-MYC protein levels. Conversely, proscillaridin did not affect C-MYC protein level in low C-MYC expressing cancer cell lines. For the first time, we showed that CGs target histone acetyltransferases and C-MYC oncogene in high C-MYC expressing cancers. We propose that CGs can be repurposed as new epigenetic drugs in high C-MYC expressing cancers.

#1382

Novel dual inhibitors of LSD1-HDAC6/8 for treatment of cancer.

Sivanandhan Dhanalakshmi, Sridharan Rajagopal, Sreekala Nair, Chandru Gajendran, Dimpy Ghosh, Subramanyam Janardhan Tantry, Purushottam Dewang, Mahanandeesha Hallur, M Kannan, Srinatha KC, Damodara Kuntrapaku, M Dilipkumar, Radha Sharma, S Meghashree, D P. Kumar, Mohd Zainuddin, A B. Vinod, Sriram Rajagopal. _Jubilant Biosys Ltd., Bangalore, India_.

LSD1 is an FAD-dependent amine oxidase that removes methyl groups from mono- or dimethylated histone H3 lysine 4 (H3-K4) as a part of CoREST repressor complex. Recent studies have shown that there is crosstalk between two components of CoREST complex, LSD1 and HDAC, which provides an advantage to cancer cell proliferation and survival by regulating key signaling pathways. Accordingly, combined inhibition of LSD1 and HDAC has been shown to be more efficacious in inhibiting the growth of glioblastoma, AML and breast cancer. Therefore, dual inhibitors targeting both LSD1 and HDAC could be useful in treating several cancers effectively without enhancing systemic toxicity mediated by administration of multiple drugs. To test the hypothesis, we have developed a set of molecules that either have LSD1 activity alone or dual activity on LSD1 and HDAC. Computational chemistry approaches were used to design LSD1-HDAC dual inhibitors. In vitro LSD1 potency was assessed by TR-FRET assay and in vitro HDAC activity by fluorimetric HDAC activity assay. Western blotting and RT qPCR were used to assess biomarkers of LSD1 and HDAC inhibition. Alamar blue cytotoxicity assay was used to assess cell proliferation. Multiple compounds from this series show an in vitro potency of less than 0.05 μM against LSD1 with more than 100-fold selectivity against MAOs. The dual inhibitors showed stronger activity on HDAC6/8. JBI-097, one such dual molecule with LSD1/HDAC/6/8 selectivity, showed an IC50 of 0.007uM on LSD1 and an IC50 of 0.06 and 0.1uM on HDAC6 and HDAC8, respectively, with about 100-fold selectivity against other HDAC isoforms. JBI-097 showed strong antiproliferative activity on leukemia and multiple myeloma cell lines. In MM.1S cell line, JBI-097 showed stronger potency when compared to LSD1 inhibitor GSK2879552 and HDAC6 inhibitor ACY1215. In cell-based and in vivo target engagement studies there was a concomitant increase in CD11b, CD86 and GFI1b and tubulin acetylation levels. JBI-097 was more efficacious in inhibiting the growth of HEL92.1.7 xenograft by oral administration when compared to IP administration of an HDAC inhibitor. In MM.1S tumor model, JBI-097 showed stronger effect in inhibiting tumor growth when compared to LSD1 inhibitor and HDAC6 inhibitor used as single agents. Further, combination with topotecan resulted in complete tumor growth inhibition of MM.1S xenograft. The data obtained demonstrate that it is feasible to design dual LSD1-HDAC6/8 inhibitors that retain individual activity and potently inhibit cell proliferation, and such inhibitors could serve as powerful therapeutic agents for cancer. Further understanding of mechanisms to identify the right tool compound is in progress.

#1383

A CRISPR-Cas9 tiling screen to identify functional domains within DNMT1 and/or DNMT3B that can be targeted for therapeutic intervention in AML.

Balpreet Bhogal,1 Barbara Weir,1 Ramona Crescenzo,2 Min Chul Kwon,2 Ulrike Philippar,2 Ricardo Attar,1 Glenn Cowley,1 David Pocalyko1. 1 _Janssen Research and Development LLC, Spring House, PA;_ 2 _Janssen Research and Development LLC, Beerse, Belgium_.

DNA methylation is an epigenetic mechanism that regulates gene expression during many stages of development, including genomic imprinting, stem cell regulation, and X-chromosome inactivation. Moreover, aberrant DNA methylation patterns, characterized by genome-wide hypomethylation and promoter-specific hypermethylation, are a prominent feature of cancer. Methylation of DNA at the 5-position of cytosines is mediated by the DNA methyltransferase (DNMT) protein family, which regulates both maintenance methylation (DNMT1) and de novo methylation (DNMT3A and DNMT3B). Loss-of-function mutations of DNMT3A have been identified in hematological malignancies including acute myeloid leukemia (AML), where DNMT3A is mutated in approximately 25% of known cases.

Published reports suggest the existence of a synthetic lethal interaction between DNMT3A and DNMT1/3B. To further study this potential genetic interaction, we are performing a CRISPR-Cas9 tiling screen to identify functional domains within DNMT1 and/or DNMT3B that are synthetic lethal with DNMT3A. We generated a lentiviral library containing 777 and 421 single guide RNAs (sgRNAs) that tile the coding region of DNMT1 and DNMT3B, respectively and performed viability screens in AML cell lines that are either wild-type or mutant for DNMT3A. This screen was designed to identify in-frame alterations within functional domains that lead to effects on cell viability. Next generation sequencing of sgRNAs identified three functional domains of DNMT1 which, when mutated, leads to decreases in cell viability. Current efforts are focused on verifying the essentiality of these functional domains using CRISPR-Cas9-based approaches as well as mutagenesis by integrated tiles (MITE)-seq analyses.

#1384

The epigenetic modifier EZH2 represses antitumor immunity in melanoma.

Jessamy Tiffen, Elena Shklovskaya, Stuart Gallagher, Dilini Gunatilake, Peter Hersey. _The Centenary Institute, Sydney, Australia_.

Despite major advances in immuno- and targeted therapies for metastatic melanoma, not all patients respond to such treatments and the development of therapy resistance remains a key obstacle. Novel targets are required and the epigenetic modifier EZH2 may represent such a solution. EZH2 methylates histones causing chromatin compaction and silencing of gene expression. Aberrant EZH2 in cancer results in the silencing of hundreds of tumor-suppressor genes that would normally constrain cancer growth, and 26% of patients in the Australian Melanoma Genome Project (AMGP) display such abnormal EZH2. Using ChIP-seq and microarrays to reveal downstream EZH2 target genes, we identified many tumor-suppressor genes including some that play a role in the antitumor immune response. Interestingly, xenografts in immune-deficient NOD SCID mice showed no significant inhibition in tumor growth when treated with an EZH2 inhibitor. However, growth inhibition was achieved when the same experiment was performed in nude mice, which retain some mediators of innate immunity. The effect of EZH2 overexpression in melanoma on immune-mediated rejection was studied in an inducible in vivo model. Subcutaneous B16 tumors expressing model antigen were initially rejected but relapsed in the majority of immunodeficient mice transferred with CD4 T cells. Long-term (120-day) survival was reduced from 40% to 22% if the tumors also overexpressed EZH2. Our studies suggest that aberrant EZH2 in melanoma drives aggressive disease via suppression of genes that would normally trigger the antitumor immune response. The progression of several preclinical, small-molecule inhibitors of EZH2 remains promising.

#1385

Galangin is an epigenetic modulator or BRCA1 and induces estrogen receptor alpha in triple negative breast cancer cells.

Micah G. Donovan, Ornella I. Selmin, Donato F. Romagnolo. _University of Arizona, Tucson, AZ_.

The aryl hydrocarbon receptor (AhR) is a driver of breast cancer progression and its overexpression is often observed in triple negative breast cancer (TNBC). Activation of AhR results in epigenetic silencing of the breast cancer 1 gene (BRCA1), a necessary factor in the transcriptional activation of estrogen receptor-alpha (ERα). The purpose of this study was to examine the capacity for galangin (GAL; 3,5,7-trihydroxy-2-phenylchromen-4-one), a natural AhR antagonist, to mimic the effects of synthetic AhR antagonists alpha-naphthoflavone (αNF, 2-phenylbenzo[h]chromen-4-one) and 3'-methoxy-4'-nitroflavone (MNF; 2-(3-methoxy-4-nitrophenyl)chromen-4-one) in regards to counteracting epigenetic silencing of BRCA1 and restoring ERα protein expression. We utilized TNBC cells with hypermethylated BRCA1 and constitutively active AhR (HCC38 cells) for these experiments. After 72h in phenol-red free media, cells were treated for up to 72h against a range of GAL, αNF, and MNF concentrations in the presence or absence of estradiol (E2). Transcript levels of AhR and its downstream target genes CYP1A1 and CYP1B1 were determined by real-time polymerase chain reaction (RT-PCR). Protein levels of BRCA1, ERα, and AhR were determined by Western blot. Treatment with GAL results in stabilization of BRCA1 and ERα protein levels, similarly to αNF and MNF. To determine potential mechanisms of these observations, we are currently conducting experiments to analyze changes in BRCA1 promoter methylation using methylation-specific RT-PCR (MSP) and DNA methyltransferase 1 (DNMT1) protein levels by Western blot. These data suggest the naturally occurring flavanoid GAL may be an effective compound for modulating epigenetic repression of BRCA1 and restoring ERα levels, which may provide options for anti-hormonal therapy in ERα-negative breast cancers.

#1386

Safety and tolerability of guadecitabine (SGI-110) plus cisplatin in patients with platinum refractory germ cell tumors (GCT): A phase 1 study.

Costantine Albany,1 Neda S. Hashemi,1 Fang Fang,2 James Lowder,3 Lawrence Einhorn,1 Kenneth Nephew2. 1 _Indiana University, Indianapolis, IN;_ 2 _Indiana University, Bloomington, IN;_ 3 _Astex Pharmaceuticals, Inc, Pleasanton, CA_.

BACKGROUND: Guadecitabine (SGI-110) is a novel hypomethylating dinucleotide of decitabine and deoxyguanosine resistant to degradation by cytidine deaminase. Germ cell tumor including platinum-resistant embryonal carcinoma cells lines are extremely sensitive to hypomethylating agents and low dose decitabine can restore cisplatin sensitivity in cell lines. We aimed to assess the safety and clinical activity of guadecitabine in combination with cisplatin in patients with platinum-refractory GCT.

METHODS: In this open-label, phase 1 study, patients with GCT refractory to or had relapsed after platinum-based treatment were treated with subcutaneous (SQ) guadecitabine, once-daily for 5 consecutive days, followed by cisplatin on day 8 in a 28-day treatment cycle. A modified toxicity probability interval (mTPI) dose-escalation design was used in which we treated patients with guadecitabine doses of 30-45 mg/m2 plus cisplatin 100 mg/m2 up to 6 cycles until progression or intolerable toxicity. The primary objective was to assess safety and tolerability of guadecitabine in combination with cisplatin, determine the maximum tolerated and identify the recommended phase 2 dose of guadecitabine. Safety analyses included all patients who received at least one dose of guadecitabine. Pharmacodynamic analyses to determine the biologically effective dose included all patients for whom samples were available. This study is registered with ClinicalTrials.gov, number NCT02429466.

FINDINGS: Between 2015 and 2017, we enrolled and treated 10 patients. Grade 3 or 4 adverse events were febrile neutropenia, thrombocytopenia, anemia, and diarrhea. The most common serious adverse events were febrile neutropenia and thrombocytopenia. Guadecitabine 45 mg/m2 was associated with prolonged SAE and felt to be unsafe to continue. The maximum tolerated dose was 30 mg/m2 daily × 5. Two patients achieved a complete response to treatment lasting more than 6 months.

INTERPRETATION: Guadecitabine SQ at 30 mg/m2 daily × 5 is well tolerated and is clinically and biologically active in patients with platinum-refractory GCT. The study is still ongoing.

#1387

HER2-positive breast cancer cell lines are particularly sensitive to the novel KDM5 inhibitor GS-701644: Definition of a gene expression model predicting sensitivity to the agent.

Enrico Garattini. _Istituto di Ricerche Farmacologiche Mario Negri, Milano, Italy_.

BACKGROUND

Histone methylation controls DNA transcription via modulation of the chromatin structure. Histone methyl-transferases catalyze the transfer of methyl groups to lysine/arginine residues of histone proteins. The demethylation process is controlled by demethylases including members of the KDM family. Alterations in the profiles of histone methylation are observed in the neoplastic cell and pharmacological targeting of this epigenetic process may result in anti-tumor effects.

RESULTS

GS-701644 is a novel inhibitor of KDM5 histone demethylases. We evaluated the growth inhibitory effects of GS-701644 in 42 breast cancer cell lines. The concentration-dependent effects of GS-701644 (0.001-10 μM) demonstrated that HER2-positivity was a primary determinant of GS-701644 sensitivity. GS-701644 sensitivity was also associated with enrichment in estrogen-receptor-positive (ER\+ ) cell lines.

Given the sensitivity of HER2-positive (HER2+) cell lines, we evaluated the anti-proliferative action of GS-701644 in combination with the two HER2 targeting drugs, trastuzumab and lapatinib. We exposed HER2+ BT-474 and SK-BR-3 cells to increasing concentrations of GS-701644 and trastuzumab or lapatinib alone or in combination. When BT-474 and SK-BR-3 cells were challenged with trastuzumab or lapatinib alone, a dose-dependent growth inhibition was observed. If the two cells were treated with combinations of GS-701644 and trastuzumab or lapatinib, strong synergistic interactions were demonstrated. In vivo, GS-701644 showed single-agent activity in xenografts of HER2+ BT-474 cells.

We determined the basal levels of KDM5A, KDM5B, and KDM5C proteins across our panel of cell lines to look for correlations with sensitivity to GS-701644. No significant association was observed between the amounts of these proteins and sensitivity to GS-701644, promoting a search for other molecular determinants of sensitivity. To achieve this, we analyzed RNA-seq data associated with each breast cancer cell line using a machine learning strategy to define a predictive model of GS-701644 sensitivity. This resulted in the identification of 15 genes whose expression was directly or inversely associated with GS-701644 sensitivity. This gene-expression model predicted responsiveness of cross-validated samples and maintained performance in the test set. Application of the model to the breast cancer cases of public datasets correctly predicted sensitivity to GS-701644 in a large fraction of HER2+ tumors.

CONCLUSIONS

GS-701644 has potential as a single agent, or in combination with standard-of-care agents, in the treatment of HER2+ breast cancer. The gene-expression model predicting sensitivity to GS-701644 represents a useful diagnostic tool for the selection of patients who may benefit from treatments based on the KDM5 inhibitor.

#1388

Pharmacologic targeting of DNA methylation blocks breast cancer growth and metastasis.

Niaz Mahmood, Ani Arakelian, William J. Muller, Moshe Szyf, Shafaat A. Rabbani. _McGill University, Montreal, Quebec, Canada_.

Abnormal DNA methylation is a hallmark of cancer which orchestrates changes in gene transcription seen in cancer. Both hypermethylation-mediated inactivation of tumor suppressor genes and hypomethylation-mediated activation of pro-metastatic genes are common attributes of cancer cells which make the methylome an attractive anti-cancer therapeutic target. Interestingly, in contrast to genetic changes, DNA methylation-mediated epigenetic changes are potentially reversible by either dietary supplements or therapeutic strategies. Consequently, a wide variety of epigenetic drugs primarily targeting DNA hypermethylation has been shown to be effective in preclinical and clinical settings. Pioneering works done by us have shown that treatment of various human cell lines (breast, prostate, osteosarcoma) with a methylating agent S-adenosyl methionine (SAM) can block tumor growth and metastasis in vivo. At the molecular level, SAM treatment induces hypermethylation of promoters of key pro-metastatic genes; and thereby inhibits their expression. However, the anti-cancer effect of SAM has never been examined. Based on the heterogeneity of tumor cells which are at different stages of tumor invasiveness, we hypothesized that treatment with demethylating (Decitabine) and methylating agents (SAM) would collectively lead to the activation of tumor suppressor genes and suppression of pro-metastatic genes to block cancer growth and metastasis.

In the current study, we first investigated the effects of Decitabine and SAM alone and in combination to prevent breast cancer development, growth, and metastasis using the MDA-MB-231 xenograft model of breast cancer. Our data showed that treatment with Decitabine and SAM resulted in a significant delay in the progression of mammary tumors in experimental animals compared to controls, effects which were significantly more pronounced when Decitabine and SAM were administered in a combination setting. Gene expression analysis of the cancer cells revealed that SAM-treatment repressed the expression of several key genes involved in cancer progression. In addition, immunohistochemical analysis of primary tumors revealed that the combination treatment (Decitabine, SAM) reduced the number of Ki67 positive cells as well reduced the expression of angiogenesis marker CD-31. Further studies examining the effects of combined therapy on genome-wide gene expression changes as well as any potential side effects on animal behavior and toxicity will be presented and discussed.

Results from this study will provide compelling evidence and rationale for the initiation of clinical trials with SAM alone as a monotherapy and in combination with currently approved epigenetic drugs (Decitabine) to reduce breast cancer-associated morbidity and mortality.

#1389

Inhibition of the histone H3K27 demethylase UTX enhances tumor cell radiosensitivity.

Barbara H. Rath, Isabella Waung, Kevin Camphausen, Philip Tofilon. _National Cancer Institute, Bethesda, MD_.

The processes mediating the repair of DNA double strand breaks (DSBs) are critical determinants of radiosensitivity and provide a source of potential targets for tumor radiosensitization. Among the events required for efficient DSB repair are a variety of post-translational histone modifications including methylation. Because trimethylation of histone H3 on lysine 27 (H3K27me3) has been associated with chromatin condensation, which can influence DSB repair, we determined the effects of radiation on H3K27me3 levels in three in three human tumor cell lines: U251; MDA-MB-231 and A549, and two normal cell lines. Irradiation (10Gy) of tumor cells resulted in a rapid loss of H3K27me3. According to immunoblot analysis, tumor cells had significant higher levels of the histone demethylase UTX compared to JMJD3, suggesting a more prominent role for UTX in the demethylation of H3K27me3 after irradiation. The rapid loss of H3K27me3 was prevented by the siRNA-mediated knockdown UTX and enhanced the radiosensitivity of each tumor cell line, whereas the knockdown of JMJD3 had no effect. To investigate whether inhibition of UTX could serve as a potential target to target to radiosensitize tumor cells, we focused on GSKJ4 a H3K27 demethylase inhibitor. GSKJ4 treatment (4 μM) of tumor cells was found to block the radiation-induced decrease of H3K27me3. Further, based on clonogenic survival analysis addition of GSKJ4 immediately prior to irradiation significantly enhanced the radiosensitivity of tumor cells but not that of normal cells. To begin to investigate the mechanism responsible for this radiosensitization tumor cells were irradiated (10Gy), treated with GSKJ4 and collected at 0.5-24h later for neutral comet assay, a measure of DNA double strand breaks (DSBs). GSKJ4 had no effect on the initial comet-tail moment, yet significantly increased the comet tail-moment up to 24h after radiation, suggestive of an inhibition of DSB repair. Analysis of γH2AX nuclear foci, a surrogate marker for DNA DSBs 16h after irradiation (6Gy) and treatment of tumor cells with GSKJ4 (4μM) increased the percentage of cells expressing γH2AX as compared to radiation alone suggesting that GSKJ4 inhibits DSB repair. Thus, data generated from the neutral comet and γH2AX assays suggest that the inhibition of H3K27m3 demethylation mediated by GSKJ4 inhibits the repair of radiation-induced DSBs, which could then account for the observed radiosensitization. Consistent with in vitro results radiation reduced the H3K27me3 methylation in mice bearing leg tumor xenografts. Treatment of mice bearing leg tumor xenografts with GSKJ4 and radiation significantly enhanced radiation-induce tumor growth delay. These data suggest that H3K27me3 demethylation contributes to DSB repair in tumor cells and that UTX, the demethylase responsible, provides a target for selective tumor cell radiosensitization in vivo.

#1390

**Honokiol, a bioactive component from** Magnolia **plant, promotes DNA demethylation and reactivates silenced tumor suppressors in pancreatic cancer cells through TET-dependent mechanism.**

Ram Prasad,1 Santosh Katiyar2. 1 _University of Alabama at Birmingham, Birmingham, AL;_ 2 _Morehouse School of Medicine, Atlanta, GA_.

Pancreatic cancer is the fourth leading cause of cancer-related deaths in the United States. American Cancer Society estimated that in 2017, approximately 53,670 new cases of pancreatic cancer will be diagnosed and 43,090 deaths will occur. In addition to genetic changes, epigenetic alterations are commonly associated with cancers including pancreatic cancer. Studies have shown that DNA hypermethylation contributes to tumor growth by inactivating tumor suppressors. DNA methyltransferases (DNMTs) regulate DNA methylation, but mechanisms underlying DNA demethylation have not fully elucidated and have enormous translational potential. Ten-eleven translocations (TETs) have been shown to convert 5-methylcytosine (5-mC) into 5-hydroxymethylcytosine (5-hmC) and eventually reactivates tumor suppressors. Bioactive components from plants have shown their efficacy as potential chemopreventive and therapeutic agents. Here, we investigated the effect of honokiol on DNA methylation using in vitro cell culture model. We first checked the expression levels of global DNA methylation in pancreatic cancer cell lines (PANC-1, AsPC-1) and compared with normal human pancreas duct epithelial cells. Our data revealed that the levels of global DNA methylation were higher in pancreatic cancer cell lines. Treatment of AsPC-1 and PANC-1 cells with honokiol for five days resulted in significant decrease in global DNA methylation levels (60-80%, p<0.001) compared to non-honokiol treated cells. To detect the comparative levels of 5-mC/5-hmC in AsPC-1 and PANC-1 cells immunofluorescence staining was used. Honokiol treatment increases 5-hmC levels in pancreatic cancer cells compared to non-honokiol-treated cells. The number of 5-hmC positive cells were significantly higher in honokiol-treated AsPC-1 (12-61%; p<0.01-0.001) and PANC-1 (42-77%; p<0.001) cells in a dose-dependent manner compared to non-honokiol treated control group as verified by flow cytometry. Honokiol treatment also significantly increases the levels of TET activity and protein expression in cancer cell lines. We also found that concomitant treatment of cancer cells with cycloheximide (proteins synthesis blocking agent) prevented honokiol-induced TET expression and formation of 5-hmC and therefore suggest that honokiol promotes 5-hmC formation (i.e., demethylation) through TET activation. Ascorbate, an inducer of TETs, also promotes 5-hmC formation in pancreatic cancer cells. Additionally, honokiol treatment reactivates tumor suppressor genes/proteins levels, such as p16INK4a and RASSF1A in cancer cells. Together, this study provides evidence that honokiol acts as a DNA demethylating agent and is able to reactivate an epigenetically silenced tumor suppressors and thus inhibits the growth of pancreatic cancer cells.

#1391

Establishment and validation of a high-content imaging assay with a 14-day incubation for the testing of epigenetic target-based therapeutics.

Jesse J. Parry, Vanessa L. Norman, Charles R. Wageman, Lee R. Cavedine, Timothy J. Sindelar, Alyssa M. Croff, Steven M. Garner, Brogan A. Epkins, Natiya E. Robinson, Kristin C. Dempsey, Usha Warrior, Alastair J. King. _Eurofins Panlabs, Inc., St. Charles, MO_.

Given the importance and significance of targeting mutations and epigenetic modification in cancer therapy, we sought to expand on our ability to test drugs with epigenetic modulatory abilities by increasing the exposure time of drugs against cell lines by the OncoPanel™ cell-based profiling service to 14 days. Using historic internal control data, generated in OncoPanel™ cell proliferation assays for 3-, 5-, and 10-day exposures, we were able to calculate the theoretical number of doublings in 14 days for each cell line. This information was used to calculate the seeding density at the time of cell plating that would provide the same endpoint per well density for a select number of cell lines from the panel. Due to the issue of increasing coefficient of variation (CV) with decreasing starting number of cells per well, we eliminated cell lines that had a calculated seeding density of <15 cells per well. For cell line validation, cells were plated at the calculated theoretical density for each cell line in 384-well plates and incubated overnight. A time zero (T0) plate was also seeded for each cell line to allow calculation of the number of doublings in the assay. Test compounds (staurosporine, entinostat, and vorinostat) were added to the assay plates over a range of 10 concentrations, in triplicate, using acoustic liquid transfer. The assay plates were incubated continuously for 7 days, upon which the growth media were removed and replaced with fresh media, followed by re-dosing with compounds. Assay plates were then incubated continuously for an additional 7 days, after which the cells were fixed and stained to allow high-content, fluorescent imaging of nuclei. Data were analyzed as the relative cell count, where the measured fluorescence intensity was transformed to percent of control, as compared with a vehicle-treated control. Cellular response parameters were calculated, using nonlinear regression to a sigmoidal single-site dose response model. The criteria for each cell line to pass quality control included a vehicle CV of <30%, endpoint well confluence, tightness of curve-fit, and an empirically-determined doubling number that was comparable with the calculated number of doublings. Comparison of the doubling numbers was striking and illustrated very high accuracy, adding to the consistency of the assay's performance. In summary, we have validated 110 human cancer cell lines, comprising 16 tissue types, in the 14-day OncoPanel™ proliferation assay, and show comparative analysis of each compound's activity profile from 3-, 5-, 10-, and 14-day incubations. The extended incubation time is highly relevant and useful for testing epigenetic target modulators, which may require a longer-term exposure to show full efficacy, with a greater potential therapeutic outcome.

#1392

Preclinical In vivo evaluation of efficacy, pharmacokinetics and pharmacodynamics of novel PRMT5 inhibitors in multiple tumor models.

Dinesh Chikkanna, Sunil Kumar Panigrahi, Sujatha Rajagopalan, Srinivasa Raju Sammeta, Darshan Chawla, Pavithra S, Samiulla D.S, Angelene Prasanna, Priyabrata Chand, Kiran Aithal, Sai Sudheer Marri, Naveen Kumar, Srinivasa Rao Ganipisetty, Raju Mutyala, Kasieswara Rao, Thomas Antony, Girish Daginakatte, Anirudha Lakshminarasimhan, Mohan R, Narasihmarao K, Shekar Chelur, Chetan Pandit, Susanta Samajdar, Murali Ramachandra. _Aurigene Discovery Technologies Limited, Bangalore, India_.

PRMT5 is a typical type II methyltransferase, transferring two methyl groups to arginine, leading to symmetric dimethylation of the substrate. It can symmetrically methylate histones H2AR3, H3R2, H3R8, and H4R3 and can also methylate many non-histone proteins contributing to tumorigenesis by regulating cell cycle progression, DNA repair, cell growth, apoptosis, and inflammation. Overexpression of PRMT5 is reported in several human malignancies including lymphoma, glioma, melanoma, lung, breast, ovarian, and prostate cancers. Elevated levels correlate with poor prognosis in NSCLC, ovarian cancers, and GBM. Therefore, PRMT5 is considered an attractive target for cancer therapy. We sought to discover and develop PRMT5 inhibitors with the "best-in-class" profile with an emphasis on improved permeability for their potential use in solid tumors. Utilizing structure-guided drug design and SAR-based approaches, we have optimized two chemical series of substrate competitive PRMT5 inhibitors. Determination of co-crystal structures with several de novo designed hits aided in the identification of lead compounds that exhibited potent inhibition of PRMT5. Lead compounds AU-574 and AU-755 were highly active in inhibiting proliferation of a number of cell lines derived from solid tumors that correlated well with cellular H4R3Me2s inhibition, confirming the mechanism. Lead compounds exhibited desirable drug-like properties including solubility, permeability, lack of CYP inhibition, and pharmacokinetic exposure. In xenograft models of Z-138 (lymphoma) and H-358 (lung cancer), treatment with lead compounds resulted in significant tumor growth inhibition while correlating with tumor drug levels and modulation of H4R3Me2s as the pharmacodynamic effect. In summary, we have identified PRMT5 inhibitors with "best-in-class" drug-like properties including optimized permeability and antitumor efficacy. Evaluation of these lead compounds in in vitro selectivity screening and in toxicity studies in higher species is currently under way.

#1393

JARID1 family inhibitor recovers sensitivity of drug-tolerant subpopulation in lung cancer cell lines.

Shin Ariga, Ichiro Kinoshita, Junko Kikuchi, Yasushi Shimizu, Hirotoshi Dosaka-Akita. _Hokkaido University Graduate School of Medicine, Sapporo, Japan_.

Epigenetic alteration contributes to tumor initiation, progression and invasion. Epigenetic machinery has become a major focus for the new development of molecular targeted cancer therapeutics. H3K4 trimethylation (H3K4me3) is an epigenetic mark which exist surrounding the transcription start site and have been presumed to be associated with cancer stem like cells. JARID1 family is demethylase of H3K4me2/me3. Especially, JARID1a and JARID1b have been suggested to have oncogenic properties in several cancers, including lung cancer. Here we examined the effects of a JARID1 family inhibitor against human lung cancer cell lines. 2-4(4-methylphenyl)-1,2-benzisothiazol-3(2H)-one (PBIT) was screened from more than 15000 small molecules as a JARID1 family inhibitor by Sayegh et al. First, we confirmed that PBIT increased H3K4me3 levels in time-dependent and dose-dependent manners in non-small cell lung cancer cell lines. PBIT inhibited cell growth in a dose-dependent manner with the IC50 value of about 10 µM to 30 µM. Next, we detected drug-tolerant persisters (DTPs) from each cell line by exposing them to very high concentration of the drug that is effective to each of the cell lines (gefitinib 10µ for PC9, PC3 and HCC827; AZD9291 5µM for H1975; CDDP 100µM for H226; paclitaxel 500nM for H460, A549 and H1299). DTPs detected were less than 1% in each cell line after 9-day exposure. These small subpopulations showed increased expression of JARID1a and/or JARID1b. Likewise, side population cells, which are considered to contain cancer stem like cells, showed increased expression of JARID1a and decreased H3K4me3 levels. PBIT in combination with anticancer drugs reduced colony formation of DTPs from all cell lines whose DTPs formed colonies. The DTPs showed decreased H3K4me3 levels and increased expression of cancer stem cell marker Oct-4, while these changes were abrogated by subsequent PBIT treatment. These results indicate that PBIT, a JARID1 family inhibitor, recovers sensitivity of drug-resistant lung cancer cells.

#1394

Resistance to BET inhibitor leads to new therapeutic vulnerabilities in castration resistant prostate cancer.

Aishwarya S. Pawar, Irfan A. Asangani. _University of Pennsylvania, Philadelphia, PA_.

BRD4 plays a major role in the transcription networks orchestrated by androgen receptor (AR) in castration resistant prostate cancer (CRPC) cells. Bromodomain and extraterminal protein (BET) inhibitors displace BRD4 protein from chromatin, resulting in the inhibition of oncogenic transcriptional programs. Several BET inhibitors (BETi) are in phase I clinical trials for a variety of malignancies including CRPC. Here we describe a general mechanism of acquired resistance to BETi due to modulation of cellular pathways that are amenable to targeted therapies in CRPC cells. BETi resistant CRPC cells displayed cross resistance to a variety of BETi in the absence of gatekeeper mutations or persistent drug pump activation. Resistant cells exhibited reduced chromatin bound BRD4, and were less sensitive to Proteolysis Targeting Chimeric (PROTAC) -mediated degradation or genetic knockdown, suggesting a BRD4-independent transcription program. Transcriptomic analysis revealed reactivation of AR-signaling due to CDK9-mediated serine-81 phosphorylation of AR, with a consequent increase in sensitivity to CDK9 inhibitors and anti-androgens in BETi resistant cells. Additionally, increased DNA damage associated with PRC2-mediated transcriptional silencing of DNA damage response (DDR) genes was observed due to the loss of BRD4 from their proximal promoter regions in the resistant cells, leading to PARP inhibitor sensitivity. Collectively, our results identify the therapeutic limitation of BETi as a monotherapy in CRPC. However, data showing the reactivation of AR-signaling and increased DNA damage in the BETi resistant cells provide unique opportunities for combination therapies in managing CRPC.

#1395

HDAC6 and DNMT inhibition affect immunogenicity of ovarian cancer cells: A rationale for combining epigenetic and immune therapy in ovarian cancer.

Aneil P. Srivastava, Sara M. Moufarrij, Melissa Hadley, Sarah Chisholm, Micael Lopez-Acevedo, Alejandro Villagra, Katherine B. Chiappinelli. _The George Washington University, Washington, DC_.

Background: Therapies that activate the immune system to fight cancer have shown robust responses in solid tumors. However, most patients, including those with ovarian cancer, do not respond to these therapies alone. Drugs that inhibit epigenetic modifiers increase immune signaling from cancer cells. Epigenetic modifiers DNA methyltransferase inhibitors (DNMTi) and selective histone deacetylase inhibitors (HDACi),in particular selective HDAC6i, modulate immune-related pathways involved in anti-tumor immune responses. HDAC6i downregulate immunosuppressive ligands PD-L1 and PD-L2 via dephosphorylating pSTAT3 and upregulate tumor associated antigens (TAA) and antigen presentation machinery. Similarly, DNMTi activate anti-viral signaling via expression of Endogenous Retroviruses (ERVs) to trigger the type I interferon response, upregulate tumor antigen processing and presentation, and stimulate pro-inflammatory cytokines. The aim of our study is to test if the combination of epigenetic modulators Nexturastat A (Next A), a selective HDAC6i, and 5-azacytidine (AZA), a DNMTi, can be safely used to increase an immune response in ovarian cancer. We hypothesize that these drugs will enhance tumor immunity alone and when combined with immune checkpoint blockades targeting PD-1. Results: HDAC enzymes are differentially expressed in A2780, HEY, OVCAR3, SKOV3, and TYKNu human ovarian cancer cell lines. HDAC1 and HDAC2 proteins were similarly expressed in HEY and SKOV3 whereas HDAC6 was expressed at lower levels in HEY and TYKNu but at higher levels in SKOV3, OVCAR3, and A2780. As previously reported we believe this may be due to the presence of the chromatin modifier ARID1A mutation in the SKOV3 and A2780 cell lines. The upregulation of HDAC6 also correlated with a higher IC50 for NextA treatment in those particular cell lines. Further immunoblots showed that PD-L1, a marker of poor prognosis in ovarian cancer, decreased after treatment with NextA and even more in combination with AZA. Additionally, DNMT1, the known target of AZA, was decreased after treatment with AZA and NextA, both independently and in combination, a finding that has not been previously reported. Conclusions: As shown previously, HDAC6 enzyme levels are higher in cell lines with ARID1A mutations. DNMT1 was decreased after treatment with AZA, as expected, but surprisingly also after treatment with NextA. PD-L1 decreased after treatment with NextA and even more so when combined with AZA. We thus believe that combining these two epigenetic modifiers will lead to an additive effect on immune signaling through stimulation of antiviral signaling (DNMTi), which can upregulate the immunosuppressive ligand PD-L1, which is then reduced by HDAC6 inhibition. We are currently testing the combination of both epigenetic modifiers with anti-PD-1 in an immunocompetent mouse model of ovarian cancer.

#1396

Pathway-directed DNA demethylation as an epithelial to mesenchymal targeting strategy.

HAO HUANG,1 Qing Yu,2 Maooj Kandpal,3 Ramana V. Davuluri,3 Daniela Matei1. 1 _Division of Gynecologic Oncology, Feinberg School of Medicine, Northwestern University, Chicago, IL;_ 2 _Department of Medicine, Indiana University School of Medicine, Indianapolis, IL;_ 3 _Division of Health and Biomedical Informatics, Department of Preventive Medicine, Feinberg School of Medicine, Northwestern University, Chicago, IL_.

It has been proposed that by silencing tumor suppressor genes, DNA methylation plays an important function in cancer initiation and progression. DNA hypomethylating agents are being developed as anti-cancer agents. However, this approach causes unrestricted hypomethylation, which could potentially activate tumor-promoting mechanisms. Therefore a strategy capable of inducing hypomethylation of selected genes is needed. Towards this goal, we designed a strategy to induce demethylation of a coordinated gene program by using Ten-eleven translocation (TET1) dioxygenase, which converts 5 -methylcytosine to 5-hydroxymethylcytosine. We constructed a fusion gene TET1-DBD containing the catalytic domain of TET1 and the DNA binding domain of Snail, the transcription factor that regulates epithelial-to-mesenchymal transition (EMT). We hypothesized that this fusion gene will bind to promoters targeted by Snail, induce hypomethylation, reactivate gene expression, and block EMT. To test this hypothesis, we used TET1-DBD and measured induction of EMT in an ovarian (SKOV3) and breast cancer (MCF10A) model driven by TGF-β. Genome-wide DNA methylation was assessed by Reduced Representation Bisulfite Sequencing (RRBS) to measure the downstream targets of TET1-DBD. TET1-DBD increased mRNA and protein expression levels of E-cadherin in transfected cells and prevented TGF-β induced expression of fibronectin and vimentin in MCF10A and SKOV3 cells. RRBS analysis demonstrated hypomethylation of genes targeted by Snail-DBD. Our results suggest that TET1-DBD selectively binds to and hypomethylates promoters of genes targeted by Snail, resulting in inhibition of the EMT program. We propose a novel approach to induce selective promoter hypomethylation and to block key oncogenic processes by using TET1 fused to a transcription factor "carrier".

#1397

The regulation of DNMT3B expression by lncRNA-NCRMS in cancer.

Wan-xin Peng, Yin-yuan Mo. _Cancer Institute, University of Mississippi Medical Center, Jackson, MS_.

5-aza-2'-deoxycytidine (decitabine; DAC) is an inhibitor of DNA methyltransferases which catalyze the transfer of a methyl group to DNA. It is well known that aberrant hypermethylation occurs in many types of tumors and often contributes to suppression of important genes such as tumor suppressor genes. Reactivation of the methylated genes by DAC has been shown to induce cytotoxicity and cell cycle dynamics. Increasing evidence indicates that lncRNAs may play critical roles in cancer development through epigenetic regulation of gene expression. However, little is known whether and how lncRNAs are involved in regulation of methylation pathways. In present study, we applies the CRISPR/Cas9-based synergistic activation mediator (SAM) system to a DAC-induced cytotoxicity model to identify potential lncRNAs capable of conferring the resistance to DAC in breast cancer. This screen identified several potential lncRNA candidates and among them is NCRMS which is capable of promoting resistance to DAC. We further confirmed that ectopic expression of NCRMS causes resistance to DAC whereas knockout of NCRMS increases the sensitivity to DAC. Mechanistically, NCRMS functions as a positive regulator of DNA (cytosine-5-)-methyltransferase 3 beta (DNMT3B). Further characterizations demonstrated that NCRMS promotes the stability of DNMT3B mRNA. Together, these results suggest that NCRMS may serve as a potential target for cancer therapy. Studies are underway to delineate the detailed mechanism of how NCRMS regulates the stability of DNMT3B mRNA.

#1398

Super-enhancers: Possible target in pancreatic cancer for therapeutic approaches.

Chandrayee Ghosh,1 Sumedha Gunewardena,1 Prasad Dandawate,1 Santanu Paul,1 Ossama Tawfik,1 Cameron West,2 Shrikant Anant,1 Animesh Dhar1. 1 _University of Kansas Medical Center, Kansas City, KS;_ 2 _Genzada Pharamceuticals, Sterling, KS_.

Introduction: Super enhancers (SE) are distinctive areas of the genome that are densely bound by numerous transcription factors and play a pivotal role in the cell identity, tissue specification, and maintenance. Although the exact functions of super-enhancers are not yet well understood, these regions are known for driving high-level transcription. Studies have established most of the oncogenes playing important role in cancer are driven by SEs. It is likely that the peculiar pancreatic-specific tumor phenotype is a consequence of oncogenes hacking the resident tissue regenerative program, thus interfering with super enhancer-driven repair networks might exert a disproportionately disruptive effect on tumor versus normal pancreatic tissue.

The hypothesis of this study is that the tumor cells in PDAC acquire super-enhancers at key oncogenes during tumor development, which drive higher levels of transcription of these genes than in healthy cells. Acquired super-enhancers may thus be biomarkers that could be useful for diagnosis and therapeutic intervention. Thus drugs which could affect super enhancers in cancer may have therapeutic effect.

Methods: In this study, we have used ChIP-Seq techniques and bioinformatics analysis following qPCR to identify SE in PDAC cell lines and in pancreatic cancer tissues. Immunohistochemistry, proliferation assays, western blot, RNAseq and CETSA (cellular thermal shift assay) have been performed for understanding the role of enhancers and SE in PDAC.

Results: H3K27ac marks were identified at enhancer region of numerous genes that act as SE in PDAC. The most prominent super-enhancers identified, based on a high level of H3K27ac marks were associated with c-MYC, MED1, OCT-4, NANOG and SOX2 genes. Differential association of SE in non-cancerous pancreatic cells, cancerous and metastatic cell lines of PDAC have been implicated in comparison normal and PDAC cells. In this study, we found that GZ17-6.02, combinations of natural compounds, affects acetylation of some of the major SE related genes and at a higher dose, a complete reduction in acetylation marks was seen in embryonic stem cell transcription factors. The mRNA sequencing data after GZ17-6.02 treatment also shows a reduction in transcription of, major transcription factors, SHH pathway genes, and stem cell markers both in vitro and in vivo pancreatic cancer orthotopic models. Cancer cells are more sensitive to lower doses of GZ17-6.02 than normal cell; hence normal cells are expected to have less side effects.

Conclusions: There is no systematic study showing an association of SE with pancreatic cancer so far. Hence, in the present study, we have established that several super-enhancer marks can be targeted by combination of natural compounds, GZ17-6.02, in PDAC. This study concludes that super-enhancers can be an important therapeutic target for PDAC.

### Genotype/Phenotype Correlations

#1399

Passenger deletion vulnerability in glioblastoma.

Nicole Oatman,1 Kakajan Komurov,1 Priyanka Arora,2 Pankaj Desai,2 Nupur Dasgupta,1 Kwangmin Choi,1 Biplab Dasgupta1. 1 _Cincinnati Children's Hospital, Cincinnati, OH;_ 2 _University of Cincinnati, Cincinnati, OH_.

Glioblastoma multiforme (GBM) is the most common, aggressive and lethal primary brain tumor in humans. Despite aggressive therapy the median survival of GBM patients is approximately 15 months. Monoallelic loss of PTEN occurs in ~70% of GBM. Through experimental validation studies in patient-derived primary GBM cell lines, we discovered that the lipogenic gene SCD undergoes unintended co-deletion as a passenger to PTEN, resulting in hemizygous loss of SCD in a subset of GBM. Additional in silico, genetic and biochemical analysis lead to the identification of two distinct PTEN-deleted subgroups: one with hemizygous co-deletion of PTEN and SCD (hereafter SCD-expressing), and another with very little or no PTEN/SCD expression due to PTEN mutation/homozygous loss and epigenetic suppression of the remaining SCD allele (hereafter SCD-non-expressing). SCD is an integral membrane protein of the endoplasmic reticulum (ER), involved in mediating the rate-limiting step of unsaturated fatty acid biosynthesis. SCD catalyzes the desaturation of the saturated fatty acids stearic (C18:0) and palmitic acids (C16:0) to the monounsaturated fatty acids oleic (C18:1) and palmitoleic acids (C16:1), respectively. We showed that the SCD-expressing lines are highly sensitive to SCD inhibition in the absence of dietary oleate and that the SCD-non-expressing lines are resistant to SCD inhibition. In vivo, we showed that mice treated with the SCD inhibitor survive longer than mice treated with vehicle. We also showed that SCD-expressing GBM lines acquire inhibitor resistance upon long-term exposure to the inhibitor in vitro. To understand the mechanism of resistance, we performed Reverse Phase Protein Array (RRPA) and RNA-Seq. Results illustrated the up-regulation of SCD among other genes. To identify the gene/s that provide SCD inhibitor resistance, we are currently performing a targeted shRNA screen of the highly expressed genes that we determined through RPPA and RNA-Seq.

#1400

The function of Brca1 and Brca2 mutations in breast and fallopian tube epithelial cells varies by location.

Justyna E. Kanska, Kruttika Dabke, Zachary Schwartz, Norma I. Rodriquez-Malave, Simon Knott, Simon Gayther. _Cedars-Sinai Medical Ctr., Los Angeles, CA_.

The identification of BRCA1 and BRCA2 mutations has greatly improved risk prediction and prevention strategies for ovarian and breast cancer. However, understanding the variation in cancer risks attributed to different BRCA1 and BRCA2 mutations has proved challenging, because these mutations are functionally recessive at the cellular level. Here, we aimed to identify the functional consequences of different BRCA1 and BRCA2 mutants by creating hundreads of truncating mutations throughout the coding sequence of each gene using a CRISPR/Cas9 screen in precursor cell types for Basal-like Breast Cancer (BLBC) and High-Grade Serous Ovarian Cancer (HGSOC). First, we created partially transformed models of breast and fallopian tube epithelial cells (MCF10A and FT282, respectively) via an overexpression of a hot-spot p53 mutation (R175H). TP53 is frequently mutated in BLBC and HGSOC. Next, we ectopically expressed S. pyogenes Cas9 enabling cells to be amenable to CRISPR/Cas9 screens. We then generated truncating mutations in MCF10Ap53 and FT282p53 cells in two regions of BRCA1 (exon 11 and 16) and two regions of BRCA2 (exon 9 and 13) using CRISPR/Cas9 technology. These regions were selected because they are associated with different risks of ovarian and breast cancer in patients. We also performed knockout of the olfactory receptor gene OR10A4 as a negative control. Modified cells were evaluated for a range of phenotypes including proliferation, anchorage-independent growth, chemoresistance, genomic instability and aneuploidy. MCF10Ap53 and FT282p53 cells engineered to carry BRCA1 or BRCA2 truncating mutations exhibit differences in proliferation, drug sensitivity, DNA damage repair capability and aneuploidy, compared to parental cells and cells carrying the OR10A4 mutation. We also detected phenotypic differences between different BRCA1 and BRCA2 mutants, consistently with the differences in breast and ovarian cancer risks associated with these mutation locations. To functionally study the broad spectrum of BRCA1 and BRCA2 mutants in a single assay, we designed barcoded libraries of lentiviral sgRNA plasmids to generate mutations spanning the entire coding sequences of BRCA1 and BRCA2 genes using CRISPR/Cas9 screen. Each plasmid in these libraries contains two sgRNAs targeting one PAM site and a unique barcode, which allows a robust and unbiased quantification of cells enriched for a given phenotype. In summary, these sgRNA libraries will map the functional differences between approximately 500 mutations spanning BRCA1 and 650 mutations spanning BRCA2 gene. Performing CRISPR/Cas9 screens both in MCF10Ap53 and FT282p53 cells will allow us to relate these functional differences to genetic epidemiological observations demonstrating the different breast and ovarian cancer risks for each gene depending on mutation location.

#1402

Silencing of B4GALT1 inversely regulates malignant phenotypes of hepatocellular carcinoma cells.

Po-Da Chen, Ai-An Chang, Yao-Ming Wu, Min-Chuan Huang. _National Taiwan University, Taipei, Taiwan_.

Identifying molecular targets for hepatocellular carcinoma (HCC) is an urgent need to overcome the treatment failure and death from cancer cell invasion and metastasis. Beta-1,4-galactosyltransferase 1 (B4GALT1) has been documented as the key enzyme in morphogenesis and cellular adhesion, for it adds galactose to N-acetylglucosamine residues of glycoprotein carbohydrate chains; whereas abnormal B4GALT1 expression is associated with tumor progress in many cancers. However, functional roles of B4GALT1 in HCC remains unclear. With clinical correlation, the immunohistochemistry confirmed that lower expression of B4GALT1 in cancer tissue is associated with higher ratio of vascular invasion and worse overall survival. Consistently, stable knockdown of B4GALT1 regarding HCC cell lines promoted cell migration, and invasion; as the overexpressed B4GALT1 inhibited cell migration and invasion of HCC cell lines. Moreover, our data suggested that down-regulated B4GALT1 modified LEL lectin, and interfered hepatocyte growth factor receptor (HGFR), Epidermal growth factor receptor (EGFR), and Axl signaling. Given that, B4GALT1 is sufficient to inversely regulate malignant behaviors of HCC cells with a significant correlation between gene expression and clinical prognosis, implying that B4GALT1 played a crucial role in cancer progress of HCC.

#1403

Modeling of Abi1 loss using spheroid cultures to investigate mechanisms of prostate tumorigenesis.

Disharee Das,1 Ming Chen,2 Claudia Mondragon,1 Dawn Post,1 Alexander Nappi,1 Heidi Hehnly,1 Anita Hryniewicz-Jankowska,1 Pier Paolo Pandolfi,2 Gennady Bratslavsky,1 Leszek Kotula1. 1 _SUNY Upstate Medical Univ., Syracuse, NY;_ 2 _Beth Israel Deaconess Medical Center, Boston, MA_.

American Cancer Society predicts over 160,000 new prostate cancer cases in 2017, which amounts to about 20 percent of all cancer diagnoses in men. Despite several treatment options being available, poor prognosis of high-risk and metastasis remains a major concern for prostate cancer (PCa) patients. Therefore, it is important to understand molecular mechanisms of PCa progression to aggressive disease. Our previous research identified Abelson interactor 1 (Abi1), a member of WAVE complex, as a tumor suppressor in human prostate. Not only did we find mutations and deletions in the gene in prostate tumor patient samples, but WAVE complex levels were also found to be inversely correlated to disease recurrence and castration resistance. Moreover, Abi1 is downregulated in majority of prostate cancer cell lines, and patient organoid cultures derived from metastatic bone and lymph node biopsies. These data point to the clinical significance of defining Abi1-dependent tumor suppression mechanisms. To understand these mechanisms we generated CRISPR-mediated Abi1 KO in the RWPE-1 non-tumor human prostate cell line. When plated in 3D cultures in matrigel, we observed striking phenotypic differences in the appearance and behavior of the Abi1 KO spheroids compared to the parental cells. While the parental RWPE-1 grew into spherical organoids with tight cell-cell boundaries, the Abi1 KO cells grew as loose irregular-shaped spheres, with many cells migrating out of the organoids. Hence, the loss of Abi1 in these non-tumor cells lead to gain of an invasive phenotype. Western Blots of Abi1-KO cells demonstrated concurrent disruption of WAVE complex and deregulation of WAVE complex protein levels. Moreover, cell-cell adhesion proteins such as E-cadherin and β-catenin showed modest decrease and/or a substantial loss of membrane localization. Rescue experiments of Abi1 re-expression in the KO cells reversed the phenotype indicating that the observed phenotype is Abi1-dependent. Analyses of RNA sequencing of cells obtained from 3D cultures demonstrated differences in expression signatures characteristic of altered cell-cell and cell-matrix adhesion pathways and indicated upregulation of pathways associated with the invasive phenotype of cancer cells. We propose that Abi1 loss and WAVE complex deregulation represents key mediators of invasive phenotype in PCa. [Supported by NCI R01 CA161018 and NYS Department of Health Prostate Cancer Hypothesis Development RFA #1410200115]

#1404

Role of catechol-O-methyltransferase gene in prostate cancer.

Shigekatsu Maekawa,1 Taku Kato,1 Yutaka Hashimoto,1 Marisa Shiina,1 Ryan K. Wong,1 Varahram Shahryari,1 Soichiro Yamamura,1 Sahana Majid,1 Sharanjot Saini,1 Laura Z. Tabatabai,1 Yukio Homma,2 Rajvir Dahiya,1 Yuichiro Tanaka1. 1 _UCSF VA Medical Ctr., San Francisco, CA;_ 2 _Japanese Red Cross Medical Center, Tokyo, Japan_.

Prostate cancer is one of the most common malignancies and ranks the second most common cause of cancer‐related deaths in men in the United States. Catechol-O-methyltransferase (COMT) is a phase II enzyme that detoxifies various catechol compounds that are reactive toward DNA and damaging to the cell. Studies have shown COMT to play a protective role against cancers such as renal and breast, but their effect on prostate is not well understood. In this study, the biological properties and function of COMT in prostate cancer were studied. Expression of COMT was initially measured in normal/benign and cancerous prostate tissues by immunohistochemistry, and cell lines by real‐time PCR and western blotting. Cancerous cells displaying the lowest levels was then transfected with COMT. Gene effect on various cellular properties such as cell proliferation, migration, invasion and apoptosis, as well as growth in athymic nude mice were determined. COMT protein expression was lower in cancer regions compared to benign and normal regions of prostate tissues. Cancerous DU145 and DuPro cells also had reduced mRNA levels of COMT but with undetectable protein levels. Interestingly, re‐expressing COMT in DU145 and DuPro cells led to decreased cell proliferation, migration, wound healing ability and invasion, and increased apoptosis compared to vector control. COMT also inhibited cell tumor formation in animal models. As a possible target, the TNFRSF11B gene was upregulated due to COMT. These results demonstrate COMT to protect against prostate cancer progression and to have a functional role by affecting apoptosis. COMT may thus be a potential biomarker or therapeutic target for prostate cancer.

#1405

PAX2 induces tubular structure in ovarian cancer cells.

Kholoud Alwosaibai. _King Fahad Specilaist Hospital, Ottawa, Ontario, Canada_.

In adult tissues, PAX2 protein is expressed in normal oviductal epithelial cells (OVE) but not in normal ovarian surface epithelial cells (OSE). Recent studies reported that PAX2 is expressed in serous ovarian carcinoma cases but the role of PAX2 in enhancing ovarian cancer is unrevealed yet. The aim of this study is to understand the biological consequences after Pax2 overexpression in mouse ovarian surface epithelial (MOSE) cells. We found that Pax2 overexpression in MOSE cells induced the formation of vascular channels both in vitro and in vivo, which indicate a possible contribution of PAX2 to ovarian cancer progression by increasing the vascular channels to supply nutrients to the tumor cells.

#1406

EGF+61 A>G polymorphism is not a lung cancer risk: A case-control study in a large Brazilian population.

Ana C. Laus, Flavia E. de Paula, Marcos A. Lima, Carolina D. Carlos, Izabela N. Gomes, Pedro R. de Marchi, Luciano S. Viana, Cristovam Scapulatempo Neto, Rui M. Reis. _Barretos Cancer Hospital, Barretos, Brazil_.

Lung cancer is a malignancy with high incidence and mortality worldwide, being in Brazil the first most lethal cancer in men and second in women. Epidermal growth factor (EGF) and its receptor (EGFR) play a central role in lung carcinogenesis, once EGF/EGFR interaction activates several intracellular pathways that control cellular growth, proliferation, differentiation, migration and apoptosis. It has been described the association between a single nucleotide polymorphism (SNP) in EGF promoter region (EGF+61 A>G - rs4444903) and cancer susceptibility to distinct tumors. In lung cancer, the results are still scarce and unclear, with different reports showing discrepant results. Therefore, the aim of this study is to evaluate the risk of lung cancer development associated with the EGF+61 A>G SNP in the Brazilian population. For that, 669 lung cancer patients and 1104 controls were analyzed. Following DNA isolation from both cases (FFPE or blood) and controls (blood), the EGF+61 A>G genotype was assessed by PCR-RFLP in FFPE samples, and TaqMan genotyping assay for blood's DNAs. As expected, uni- and multivariate analyses, showed that tobacco consumption (p<0.001; OR=11.47; 95% CI 8.44-15.58 for smokers and p<0.001; OR 3.06; 95% CI 2.28-4.10 for ex-smokers) and age (p<0.001; OR=3.74; 95% CI 1.2.95-4.74 for >65 years) were important risk factors for lung cancer. Both patients and controls were in Hardy-Weinberg equilibrium. The genotype frequencies observed in lung cancer patients were 27.4% of AA, 47.4% of AG and 25.3% of GG, and for controls were 25.3% of AA, 51.6% of AG and 23.1% of GG. The allele frequencies were 51.1% of A and 48.9% of G in patients for both cases and controls. No significant differences for the three genotypes (AA, AG and GG - model 1) were observed between cases and controls (p=0.116; AG genotype: OR=0.80; 95% CI 0.61-1.05 and GG genotype: OR=1.03; 95% CI 0.75-1.42). We further grouped AG and GG and compared with the AA genotype (model 2), as well grouped AA and AG, compared to GG genotype (model 3), and no significant differences were found (model 2 - p=0.3; OR=0.87; 95% CI 0.67-1.12; model 3 - p=0.197; OR= 1.19; 95% CI 0.91-1.55). Moreover, disease-free and overall survivals were calculated considering the 3 models, and no significant differences were observed. In conclusion, the present study suggest that EGF+61 A>G polymorphism is not a risk factor for lung cancer in Brazilian population.

#1407

Telomere length and melanoma survival.

Sivaramakrishna Rachakonda,1 Nalini Srinivas,1 Zaida Garcia-Casado,2 Celia Requena,2 Eduardo Nagore,2 Rajiv Kumar1. 1 _German Cancer Research Ctr., Heidelberg, Germany;_ 2 _Instituto Valenciano de Oncologia, Valencia, Spain_.

Telomeres at chromosomal ends are comprised of multiple short repeat sequences. In humans TTAGGG repeats account for 10-15 kb of double-stranded telomeres that end in single stranded G-rich tails of about 150-200 nucleotides. Telomeres in somatic cells undergo gradual shortening due to inherent limitations of DNA replication and limited levels of specialized enzyme telomerase. We previously reported an association between increased telomere length and risk of melanoma. In this study based on 1019 stage I and II cutaneous melanoma patients, we determined the effect of telomere length on patient survival. The telomere length was measured in DNA from peripheral blood cells using monochrome multiplex quantitative real-time PCR where telomere repeat copy number (T) was compared to a single-copy gene, albumin (S) and results presented as relative telomere length based on T/S ratio. A univariate Cox regression showed increased patient survival with increased telomere length (HR 0.65, 95% CI 0.42-1.00, P 0.05). With every decrease in 1 unit of telomere length the estimated odds of survival decreased by an approximate factor of 1.54 (95%CI 1.00-2.38). A model based on median distribution showed short rather than long telomere associated with poor survival with hazard ratio (HR) of 2.05 (95% confidence interval (CI) 1.33-3.16; log rank P 0.001); the model after adjustment with confounding factors including age at diagnosis showed a HR of 1.50 (95%CI 0.96-2.34). Stratification of patients based on median age showed statistically significant effect of decreased telomere length on poor survival in patients below the median age (HR 2.27 95%CI 1.09-4.27) but not in patients above the median age (HR 1.42 95%CI 0.82-2.46). Distribution of the patients into quantiles based on age showed the largest effect of decreased telomere length in patients younger than 30 years (HR 3.90 95%CI 1.28-11.93); the effect decreased with increasing age. Three single nucleotide polymorphisms, rs131178082, rs7726159 and rs6060627, previously shown to be associated with telomere length, were used to construct a weighted genetic score and paradoxically, the patients carrying the alleles associated with increased telomere length showed poor survival with shorter telomeres (HR 2.94 95%CI 1.54-5.62). Multivariate analysis that included, age, sex, outdoor life style, presence of actinic keratosis, number of nevi, tumor location, tumor stage, Breslow thickness and tumor ulceration showed a statistically significant association between composite genetic score and poor survival (HR 2.68 95%CI 1.24-5.80). Thus, the results from the study show that in contrast to association between melanoma risk and increased telomere length, the decreased telomere length associates with poor patient survival. The adverse effect on survival due to decreased telomere length was more pronounced in young rather than old patients and on the patients carrying genotypes for longer telomeres.

#1408

SF3B1 mutations induce proteome remodeling, metabolic reprogramming, and a novel kind of tumor surface antigen in human cells.

William B. Dalton, Daniel Shinn, Noel Walsh, Eric Christenson, Taylor Groginski, Dhanashree Kelkar, Anil Magugundu, Arun Patil, Daniel Zabransky, Arielle Medford, Justin Lee, Alex Cole, Josh Donaldson, Amy DeZern, Karen Cravero, David Chu, Natasha Hunter, Akhilesh Pandey, Josh Lauring, Ben Park. _Johns Hopkins University, Baltimore, MD_.

Mutations in the SF3B1 gene occur in multiple cancers, creating a neomorphic spliceosome protein that induces aberrant mRNA splicing in thousands of genes. Little is known about the downstream oncogenic or therapeutic consequences of this missplicing. To study this, we engineered knockins of SF3B1 mutation in untransformed breast epithelial cells, as well as "reverse" knockins in which SF3B1-mutant uveal melanoma cells were corrected to wild type. Transcriptomic analysis of these isogenic pairs showed cryptic splicing induced by mutant SF3B1, and SILAC proteomics demonstrated a broad reshaping of the proteome dominated by downregulation of misspliced genes. Interestingly, pathway analysis revealed a suppression of proteins involved in energy metabolism. This included mitochondrial complex III, which rescue experiments showed was downregulated through missplicing of its assembly factor UQCC1, and PHGDH, a serine synthesis enzyme likewise misspliced and downregulated. Metabolomic analysis also showed evidence of metabolic reprogramming by mutant SF3B1, including suppression of serine, glycine, and aspartate levels. Because serine and glycine (SG) starvation can have anticancer activity in vivo—and because PHGDH expression modulates this effect—we tested SG starvation on SF3B1-mutant cells. Growth of our SF3B1-mutant isogenic cells was differentially suppressed by SG starvation, and two independent AML cell lines with endogenous SF3B1 mutation demonstrated significant cell death upon SG starvation, in contrast to multiple AML cell lines wild type for SF3B1. These data suggest impaired serine synthesis may be a therapeutic vulnerability in SF3B1-mutant cancers. In addition to downregulation of genes induced by mutant SF3B1, our proteomic data also revealed a novel cryptic protein that was highly enriched in SF3B1-mutant cells. This protein, CD98, is a surface membrane protein, and the cryptic peptide change occurred in its extracellular domain. Additional transcriptome analysis revealed eleven more candidate surface proteins with mutant SF3B1-induced cryptic isoforms containing in-frame peptide changes in extracellular domains, and overexpression studies demonstrated at least two of these cryptic proteins can traffic to the cell surface. We have named these putative proteins MASAs, for Missplicing-Associated Surface Antigens, and we believe they represent a novel kind of tumor surface antigen that may be targetable with antibody-based immunotherapeutics. In summary, our data provide evidence that SF3B1 mutations induce proteome alterations, metabolic reprogramming, and a new kind of tumor surface antigen in human cells.

#1409

p53 negatively regulates keratin 17 expression in oral squamous cell carcinoma.

Mayu Enaka, Masako Nakanishi, Yasuteru Muragaki. _Wakayama Medical University of Medicine, Wakayama City, Japan_.

Background: Cytokeratin 17 (K17) has been shown to promote tumorigenesis and aggressiveness in oral squamous cell carcinomas (OSCCs) and other various carcinomas. Although p53 reportedly induces K17 transcription by irradiation, the correlation between p53 and K17 in OSCCs remains unknown. Methods: Human OSCC cell lines SAS and Ca9-22 were examined by Western blot, immunohistochemistry, ChIP assays, and overexpression of wild-type p53. Biopsy specimens from patients with OSCC were used for immunohistochemistry.Results: Western blot and immunohistochemistry showed that K17 expression negatively correlated with p53 expression. In biopsy specimens, positive staining for K17 was observed in OSCC cells that were negative for p53 in a mutually exclusive manner. SAS cells, which have higher K17 expression and almost negative p53 expression, showed more aggressive characteristics than Ca9-22 cells. A mutation to cause a premature stop codon was identified in the TP53 gene in SAS cells by DNA sequencing, whereas no mutation in the TP53 gene was identified in Ca9-22 cells. In addition, ChIP analysis showed that p53 bound specifically to the promoter region of the K17 gene. Furthermore, overexpression of wild-type p53 suppressed K17 expression in SAS cells. Conclusion: p53 acts as a direct transcriptional repressor of K17 in OSCCs. The interaction between p53 and K17 may regulate aggressiveness of the carcinoma cells.

#1410

**Pedigree analysis equally identifies cases of pancreatic cancer in families with** BRCA1 **and** BRCA2 **mutations.**

Evelin Schrock,1 Karl Hackmann,1 Franziska Kuhlee,1 Arne Jahn,1 Johannes Wagner,1 Anne-Karin Kahlert,1 Joseph Porrmann,1 Andreas Tzschach,1 Daniela Aust,2 Gustavo Baretton,2 Karin Kast,3 Pauline Wimberger,3 Michael Laniado,4 Christoph Kahlert,5 Thilo Welsch,5 Jürgen Weitz,5 Barbara Klink,1 Andreas Rump,1 Laura Gieldon1. 1 _Institut für Klinische Genetik, Medizinische Fakultät Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany;_ 2 _Institut für Pathologie, Universitätsklinikum Carl Gustav Carus an der Technischen Universität Dresden, Dresden, Germany;_ 3 _Klinik und Poliklinik für Frauenheilkunde und Geburtshilfe, Universitätsklinikum Carl Gustav Carus an der Technischen Universität Dresden, Dresden, Germany;_ 4 _Klinik und Poliklinik für Nuklearmedizin, Universitätsklinikum Carl Gustav Carus an der Technischen Universität Dresden, Dresden, Germany;_ 5 _Klinik und Poliklinik für Viszeral-, Thorax- und Gefäßchirurgie, Universitätsklinikum Carl Gustav Carus an der Technischen Universität Dresden, Dresden, Germany_.

Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer death in western countries, although lifetime risk is estimated at only 1.3% in the general population. BRCA2 mutations, however, have been attributed with a lifetime risk for PDAC development of 5‐10%, while the risk for PDAC development in BRCA1 mutation carriers has been the subject of an ongoing scientific discussion, with current estimates of an increased PDAC risk of only 2‐4‐fold.

We retrospectively analyzed 180 pedigrees for occurrence of PDAC. All families were counselled for hereditary breast and ovarian cancer (HBOC) within three years (2015 to 2017) at the Center for Hereditary Breast and Ovarian Cancer in Dresden. The pedigrees of 111 families with a pathogenic

BRCA1 germline mutation and of 69 families with a pathogenic BRCA2 germline mutation were available for analysis. Whenever possible genetic testing (NGS or targeted sequencing) was performed using blood and/or tumor tissue of affected individuals. 14 "BRCA1 families" were identified to have at least one family member affected by PDAC (12.6% of families). Three of the patients were available for testing and carrier status could be confirmed in all of them. According to formal genetic risk calculation 6 of the PDAC patients had a mutation carrier risk of 50%, 5 of 25% and 1 of 12.5%. Age of onset (AO) of the disease was known in 13 of the families and the mean AO, calculated for these patients, was 58.7 years. Interestingly, two female Mutation carriers had both breast‐ and pancreatic cancer and one family with a BRCA1 mutation presented

with two sisters with PDAC. Tumor tissue was available from one of them and sequencing indicated loss‐of‐heterozygosity, supporting the assumption that the BRCA1 mutation might have been causative for tumorigenesis.

Of the "BRCA2 families" 10 (14,5%) were identified to have one family member each with PDAC. To date, however, none of them was available for genetic testing. 3 of the patients had a mutation carrier risk calculated at 50%, 5 at 25%, 1 at 12.5% and 1 at 6.25%. The mean AO was calculated to be 60.1 years. One female PDAC patient additionally had breast cancer and one of the male patients also suffered from prostate cancer. While the number of families represented in this retrospective study is limited, our observations indicate that BRCA1 mutations might be underdiagnosed in PDAC, especially in patients with an earlier AO. Extensive genetic analyses of PDAC patients will be necessary in order to elucidate this presumption and could also indicate whether additional genetic risk modifiers play a role in PAC development in BRCA1/2 families. 

### High-Throughput Sequencing 2

#1411

Proteogenomic analysis of the syngeneic mouse gastric stem cell-like cancer cell line.

Hanna Yang, Hark Kyun Kim. _National Cancer Center, Goyang-si, Gyeonggi-do*, Republic of Korea_.

Gastric cancer is the third main cause of cancer deaths worldwide. To understand molecular mechanism of gastric cancer incidence and metastasis, we generated NCC-S1M lacking Smad4, Trp53 and Cdh1, which is the unique syngeneic gastric cancer cell line transplant model in the scientific society. NCC-S1M showed cancer stem cell-like features and strong tumor-initiating potential. Using proteogenomic analysis of NCC-S1M, we elucidated the specific signaling pathways that control gastric cancer stem cell-like properties and immune checkpoint. Cd274, Ccne1 and Il1rl1 were overexpressed and their protein levels were higher than normal gastric tissues. Cd274 encodes PD-L1, which is one of the important immune checkpoint proteins. The growth rate of NCC-S1M allograft was reduced after treatment with anti-PD-1. When Smad4 was ectopically expressed in NCC-S1M, Cd274 expression was decreased. In addition, TGF-β treatment induced reduction in Cd274 expression of Smad4 overexpressed NCC-S1M cells even more comparing with normal Smad4 overexpressed NCC-S1M. The knockdown of Il1rl1 in NCC-S1M reduced tumorigenicity and in vivo chemoresistance. We demonstrated that NCC-S1M focally amplified Ccne1 gene through array comparative genomic hybridization. Ccne1 knocked down NCC-S1M showed reduced metastatic potential. The proteogenomic characterization of NCC-S1M explains that Smad4 is important for PD-L1 immune evasion and that Il1rl1 has a significant role in cancer stem cell-like properties. Furthermore, it could imply that Ccne1 regulates metastasis.

#1412

Comprehensive characterization of clock genes in human cancers.

Youqiong Ye,1 Yu Xiang,1 Joseph Takahashi,2 Gordon Mills,3 Seung-Hee Yoo,1 Leng Han1. 1 _UTHealth, Houston, TX;_ 2 _Howard Hughes Medical Institute, Dallas, TX;_ 3 _The University of Texas MD Anderson Cancer Center, Houston, TX_.

Cancer chronotherapy has been considered to optimize the balance of anti-tumor effects and toxicity. To develop cancer chronotherapy, the first crucial step is to understand the significant roles of clock genes in cancer. Circadian machinery is controlled by two main transcription auto-regulatory feedback loops, which including 14 core circadian genes. Core clock genes have at least one paralog gene may be functionally redundant in circadian regulation. Dysregulation of circadian genes promotes tumorigenesis through mechanisms that include the cell cycle, DNA damage and metabolism. However, there has been no comprehensive analysis to characterize the roles of circadian genes and circadian rhythms in tumorigenesis and to assess their potential utility in cancer chronotherapy. We determined genetic and epigenetic alterations of clock genes in human cancer, and explore clock gene clinical effect through integrative analysis, based on The Cancer Genome Atlas (TCGA) profiles contained DNA, RNA, protein, epigenetic levels and clinical data. Our results suggested several clock genes may function as oncogene, such as ARNTL2, NR1D1, and NPAS2, while several other clock genes may function as tumor suppressors, such as PERs, CRYs, RORs. We revealed disruption and reprogramming of circadian rhythms across patient samples by showing significantly reduced number of genes correlated to clock genes. We further showed that transcriptional dysregulation of clock genes is strongly associated with patient survival, tumor stage and subtype. Taken together, our results provide the comprehensive analysis of clock system across different cancer types and expected outcomes will lay the groundwork for the future development of cancer chronotherapy.

#1413

Automation of a barcoded ctDNA sequencing assay based on hybrid capture on two robotic pipetting platforms enables processing of large numbers of liquid biopsy samples.

Maria Lange, Sandra Siemann, Corinna Woestmann, Sylvie McNamara, Mirjam Feldkamp, Anja Blueher, Bernd Hinzmann, Andre Rosenthal. _Roche Sequencing Solutions, Potsdam, Germany_.

Introduction: Today's ctDNA sequencing assays are either lab-developed tests (LDTs) and offered as centralized service by CLIA labs, or distributed kits (Qiagen, Roche Avenio ctDNA kit) that require manual processing of samples. The AVENIO ctDNA assays (RUO, Roche Molecular Solutions, Pleasanton CA, USA) are based on the hybrid capture technology that utilizes molecular barcoding and detects four mutation classes (SNV, IDels, CNVs, Fusions). The current manual presequencing workflow requires three full working days. With the recent progress in sequencing of longitudinal liquid biopsy samples there is a clear need for automation of the workflow. Here, we present two automated workflows on the Perkin Elmer NGS Sciclone and a modified Hamilton Star.

Methods: Batches of 24 samples of 50ng genomic DNA and 24 samples of 50ng DNA from Horizon cell line blends (HD70, HD731, HD733, HD734) were tested in microtiter plates using manual and automated workflows for comparison. AVENIO cfDNA extraction kit and MagnaPure 96 (MP96) were used for manual and automated extractions of 2 mL of plasma. The AVENIO ctDNA assay was used for manual processing of library prep and target enrichment according to the published instructions for use. Two pipetting robots were used for an automated version of the AVENIO ctDNA workflow. The Perkin Elmer Sciclone has a 96 pipetting head and can process 96 samples in 2.5 days. The modified Hamilton Star has a 8 pipetting head and can process 24 samples in 3 days. After library prep and target enrichment, pools of 12 samples were run on the NextSeq. Sequencing QC parameters were analyzed with focus on on-target rate, duplication rate, deduped depth and insert sizes.

Results: An automated workflow for the commercially available AVENIO ctDNA assay was successfully established on both the Perkin Elmer Sciclone and modified Hamilton Star. The main sequencing QC parameters were very similar between Sciclone and modified Star. There was no difference in the somatic calls for the HD cell lines from 5% AF (HD730, 731) down to 2% AF (HD733) and 1.3% AF (HD734) for the manual workflow compared to the two automated workflows. Finally, two sets of 24x 2mL plasma samples from 24 patients with metastatic CRC were processed using (a) the manual cfDNA extraction and AVENIO ctDNA workflow, and (b) automated cfDNA extraction (MP96) and automated workflow on the Sciclone. No major difference was observed between the sequencing QC parameters.

Conclusions: We have successfully automated the AVENIO ctDNA workflow on both the Perkin Elmer Sciclone and a modified Hamilton Star. Performance characteristics are very similar on both platforms with respect to on-target and duplication rates, deduped depth and insert sizes. The Sciclone is a widely available pipetting robotic liquid handler and can support processing between 8-192 samples per week with one operator.

#1414

Germline cancer predisposition gene mutations among patients with early onset rectal cancer.

Caroline Beltrami,1 Luisa Matos Alvim,1 Bruna Elisa Kupper,1 Annabeth Petersen,2 Mads Jørgensen,2 Samuel Aguiar Junior,1 Silvia Regina Rogatto2. 1 _A C Camargo Cancer Center, São Paulo, Brazil;_ 2 _Institute of Regional Health Research - University of Southern Denmark, Vejle, Denmark_.

Background: Although the incidence rates of rectal cancer (ReCa) have been increasing, the mechanisms or mutations involved in the development of the disease are poorly explored, especially in younger patients. To date, rectal tumors from young patients show molecular, clinical and pathological differences compared with colorectal cancer from older patients. Few studies have reported the presence of a hereditary factor involved in the risk of developing ReCa in a set of patients even in the absence of family history of cancer.

Patients and Method: Twenty-seven ReCa younger patients (<40 years old) were screened for germline alterations in DNA from blood samples. The family history and clinical-pathological findings were assessed from the medical records from all subjects. DNA library was performed from SureSelectQXT Library Prep kit (Agilent, Santa Clara, CA) and next-generation sequencing was applied using a multigenic panel with 105 cancer-related genes (SureSelectXT Custom Panel; Agilent, Santa Clara). The generated sequences were aligned to human genome (hg19) and reported as pathogenic, likely pathogenic, variant of uncertain significance (VUS), benign and likely benign according to the American College of Medical Genetics and Genomics guidelines. Sanger sequencing was used to confirm the pathogenic variants.

Results: Two patients carried out high penetrance genes associated with colorectal cancer, MUTYH biallelic and BMPR1A. MTHFR and MUTYH variants were found in two patients. POT1 (melanoma predisposition), MEN1 (high penetrance) and ATM (moderate penetrance) variants were found in three additional ReCa patients. Three patients carried out likely pathogenic varaints (BMPR1A, CHEK2 and RAD51D genes). Four of ten patients with pathogenic variants had no family history of cancer.

Conclusion: Pathogenic (7/27) and likely pathogenic (3/27) variants were found in 37% of ReCa patients younger than 40 years old affecting genes with high or moderate penetrance. Our data suggest that genetic testing using a multigene panel and genetic counseling should be offered for younger patients with rectal cancer.

#1415

Target sequencing of tumor driver genes in lung adenocarcinoma tissues.

Jiawei Zhu, Lin Feng, Ting Xiao. _National Cancer Center/Cancer Hospital, CAMS, PUMC, Beijing, China_.

Background and objective As the main histological subtype of lung cancer, lung adenocarcinoma (ADC) contributes to a cancer-related death in the word. With the development of high-throughput genomic analysis, whole-genome sequencing provides a new opportunity to identify genetic mutations that drive the origination, growth, and spread of cancer. The aim of this study is to detect the mutated genes in lung adenocarcinoma tissues and to analyze its clinical significance, based on 483 tumor driver gene sequence of ADC samples.

Methods We obtained 40 pairs of samples from ADC tumors and matched normal adjacent tissue, which included female (58%), male (43%), people over 60 years old (63%), stage I (40%), stage II (20%), stage III (38%), and stage IV (3%). Based on the Illumina HiSeq2500 sequencing technology platform, small fragment library was built, high coverage of the target area was captured by NovoP products (Agilent sure select XT Custom 0.5-2.9Mb kit, contains 483 tumor related target gene probe). DNA samples was examined by Paired-End sequencing; sequencing data of 40 cases of tumor samples was obtained. We analyzed the sequencing results of lung adenocarcinoma tumor and matched normal adjacent tissues by using bioinformatics analysis methods, and sought for somatic mutations in lung adenocarcinoma tumor and matched normal adjacent tissues.

Results There were 31 ADC samples had reliable exons mutations in the target area, totally 142 (102 genes) mutation positions, including non-synonymous mutations (65%), synonymous mutations (26%), and terminating translation mutation (8%). Totally 116 mutation positions (81.69%) were located in the target capture area. Gene mutations were more frequent in smoking and aged patients. Top ten mutation frequency genes were TP53, EGFR, PIK3CA, ATN1, ATXN1, ABL1, ALK, EGF, EPHA3, and KRAS. And compared to The Cancer Genome Atlas (TCGA) data, the mutation frequency of EGFR was significantly higher in our research.

Conclusion These data provided evidence for the further determination of the molecular mechanism and molecular typing of lung adenocarcinoma. Mutation frequency of TP53, EGFR and PIK3CA in lung adenocarcinoma tissue was the highest.

#1416

Pan-cancer mutual vulnerability and viability.

Huwate Yeerna,1 William Kim,2 William C. Hahn,3 Jill Mesirov,1 Pablo Tamayo1. 1 _University of California, San Diego, La Jolla, CA;_ 2 _Dana-Farber Cancer Institute, Cambridge, MA;_ 3 _Harvard University, Cambridge, MA_.

We used a skew-distribution-based statistical methodology to fit parametric models of cell-viability profiles from genome-wide RNAi-gene knockdowns. We identify the subset of genes with the highest degree of skewness that represent most oncogenes and tumor suppressors. We decomposed those genes using matrix decomposition in order to define mutual vulnerabilities. The resulting matrix decomposition components represent coordinated patterns of gene dependency shared by many genes in many cell lines across cancer types. We identify the most salient mutual vulnerabilities, generate a comprehensive catalog of them, and annotate them based on their association with a variety of genomic features including: gene, protein, and pathway expression, mutational landscape, copy number alterations, promoter methylations, metabolites, miRNAs, and drug sensitivities. We also generate an Onco-GPS (Oncogenic Positioning System) Map that provides a summary and visual representation of the mutual vulnerabilities and their components, and allow us to compare them with their counterparts in Pan-Cancer transcriptional and drug sensitivity spaces.

#1417

Investigating the functional impacts of single-nucleotide variants in anaplastic large cell lymphoma.

Hugo Larose,1 Shahid A. Mian,2 Edem Nuglozeh,2 Feroze M. Fazaludeen,3 Ahmed M. Elmouna,2 Ibraheem Ashankyty,2 Ming-Qing Du,1 Gerald Hofler,4 Sarka Pospisilova,5 Wilhem Woessmann,6 Christine Damm-Welk,6 Alina Fedorova,7 Laurence Lamant,8 Michaela Schlederer,9 Olaf Merkel,9 Lukas Kenner,9 Suzanne D. Turner1. 1 _University of Cambridge, Cambridge, United Kingdom;_ 2 _University of Ha'il, Ha'il, Saudi Arabia;_ 3 _University of Eastern Finland, Kuopio, Finland;_ 4 _Medical University of Graz, Graz, Austria;_ 5 _CEITEC, Brno, Czech Republic;_ 6 _Justus-Liebig University, Giessen, Germany;_ 7 _Belarusian Centre for Paediatric Oncology, Minsk, Belarus;_ 8 _Institut Universitaire du Cancer Toulouse, Toulouse, France;_ 9 _Medical University of Vienna, Vienna, Austria_.

In order to understand the biology of Anaplastic Large Cell Lymphoma (ALCL) and to develop biomarkers, we performed Whole-Exome Sequencing (WES) of DNA extracted from patient tumor samples (of at least 90% tumor cell content; n=31), and are exploring the functional implications of the mutations detected. Bioinformatics processing involved variant calling using software Pindel1 and CaVeman, while annotation was done using Annovar2. Single nucleotide polymorphisms (SNPs) and single nucleotide variants (SNVs) with an allele frequency in excedent of 0.1% (as determined by the dbSNP database, build 150), and variants not predicted to be damaging (ie with a low variant effect prediction - VEP - score) were filtered out.

Pathway analysis using the Panther database3 revealed that our variants were enriched in genes of the Wnt signalling pathway and of the signalling pathway of the Nicotinic acetylcholine receptor. 54 of our hits, single nucleotide variants or Insertions/Deletions (Indels) were identified as being common to at least 20% of all our sequenced samples. In 8.6% of patient samples (n=71) and 1 of 4 ALCL cell lines, a novel SNV in Notch1 was detected and investigated further for its functional consequences. Inhibition of Notch1 with several Gamma-Secretase Inhibitors or shRNA led to a significant decrease in cell growth concomitant with an increase in cell death. Furthermore, co-treatment with the ALK/cMet/ROS inhibitor Crizotinib together with Notch inhibitors led to additive effects on cell death. Our genomics study indicates an important role for Notch1 in the biology of ALCL, although the exact functional implications of the SNV detected is as yet unclear.

References

1 Ye K, Schulz MH, Long Q, Apweiler R, Ning Z. Pindel: A pattern growth approach to detect break points of large deletions and medium sized insertions from paired-end short reads. Bioinformatics 2009; 25: 2865-2871.

2 Wang K, Li M, Hakonarson H. ANNOVAR: Functional annotation of genetic variants from high-throughput sequencing data. Nucleic Acids Res 2010; 38: 1-7.

3 Mi H, Huang X, Muruganujan A, Tang H, Mills C, Kang D et al. PANTHER version 11: Expanded annotation data from Gene Ontology and Reactome pathways, and data analysis tool enhancements. Nucleic Acids Res 2017; 45: D183-D189.

#1418

Identification of recurrent regulatory mutations in breast cancer.

Kelsy C. Cotto,1 Arpad Danos,1 Robert Lesurf,2 Morag Park,3 Malachi Griffith,1 Obi L. Griffith1. 1 _Washington University in St. Louis, Saint Louis, MO;_ 2 _Ontario Institute for Cancer Research, Toronto, Ontario, Canada;_ 3 _McGill University, Montreal, Quebec, Canada_.

Since the identification of recurrent TERT promoter mutations in melanoma resulting in increased TERT expression, there has been increased interest in identifying recurrent regulatory non-coding mutations (Horn et al. 2013, Huang et al. 2013). Several studies have attempted pan-cancer analyses in order to identify these types of mutations, but often the results suffer from low coverage of regulatory regions or do not extend to breast cancer. While some breast cancer specific studies have identified some significantly mutated promoters and lncRNAs, they have often failed to incorporate transcriptome data to assess the impact and relevance of mutations on the expression of genes within tumors (Nik-Zainal et al. 2016). In order to address this, we assembled and generated a data set consisting of 458 breast cancer cases with matched tumor/normal pairs. This cohort consists of 22.4% luminal A, 19% luminal B, 16.4% HER2-enriched, 21% basal-like, 0.8% normal-like, and 20.4% unknown with regards to molecular subtype. This is important due to different breast cancer subtypes having dissimilar phenotypes and varying rates of gene coding mutations. This data set has a mix of whole genome, exome, transcriptome, and custom capture sequencing. We designed a custom capture reagent that covers regions assembled from regulatory databases, 5' untranslated regions, 500 bases upstream and downstream of transcription start sites, and 50,000 bases upstream and downstream of 178 genes that have been implicated as being important in breast cancer (Lesurf et al. 2016). While this custom capture region is similar in size to an exome, it has advantages over whole genome and exome sequencing, particularly with respect to coverage in GC-rich promoter regions. With these data, we predict that we will be able to identify novel, regulatory coding and non-coding drivers of breast cancer that would not be discovered without integrated analysis of the DNA- and RNA-seq data for each tumor. Instrument data were processed using the McDonnell Genome Institute somatic variant calling pipeline that includes 5 SNV callers and 3 indel callers. We then used these steps to filter variants: min. 20x coverage in both the tumor and normal sample, min. 2.5% tumor variant allele frequency, min. 3 variant supporting reads in the tumor sample, max. 10% variant allele frequency in the normal sample. We also filtered against gnomAD and a panel of normals. Rheinbay et al. 2017 identified recurrently mutated promoter regions for nine genes: TBC1D12, ZNF143, ALDOA, NEAT1, RMRP, CITED2, FOXA1, CTNNB1, LEPROTL1. Our preliminary analysis has revealed that we also see mutations within these regions. We plan to present on the significance of mutations within these previously seen regions, based on recurrence and transcriptome changes, as well as novel recurrent regulatory regions that our analysis reveals, particularly with respect to molecular subtype.

#1419

Dissection of single-cell transcriptional and translational regulation by digital mRNA and protein quantification.

Gretchen Lam, Eleen Shum, Christina Chang, Hemi Shah, Devon Jensen, James Ghadiali, Jody Martin, David Rosenfeld, Christina H. Fan. _BD Biosciences, Menlo Park, CA_.

The immune system consists of complex gene regulatory networks that allow a rapid transition of different cellular states during an immune response. Cell-surface marker analysis using flow cytometry or single cell RNA-seq has allowed characterization of immune subpopulations and a greater understanding of the complexity of immune cells. However, restrictions on protein-only or RNA-only analysis can greatly limit the understanding of how genes are regulated in cells. For example, many cell surface markers - such as CD4 in T cells - has thousands of protein copies per cell using antigen density calculations, yet is fueled by a small number of mRNA transcripts per CD4+ T cell. Moreover, conventional whole-transcriptome analysis of mRNA can further mute the expression detection of CD4 mRNA in T cells due to the abundance of housekeeping ribosomal genes. To bridge the understanding of protein and mRNA expression differences, we used Ab-Seq on BD RhapsodyTM platform to provide digital quantification of both protein and mRNA expression level in single cells. An oligo-conjugated antibody panel against immune-relevant cell-surface markers was created and used for this multi-omic gene expression profiling effort. This approach is coupled with mRNA analysis using the BD Rhapsody Immune Response Panel, a targeted method of RNA-seq that allows a higher sensitivity of immune markers than conventional whole transcriptome assays. We studied the dynamics of early T cell activation in vitro to understand this response on transcriptional, post-transcriptional, and translational levels. Different time points following anti-CD3 and anti-CD28 treatment were collected and multiplexed on to BD Rhapsody cartridge for single cell capture and analysis. Using Ab-Seq on BD Rhapsody, we were able to detect the difference in mRNA and protein levels of crucial markers, allowing us to dissect the intricate gene regulatory pathways during an immune response in a single cell level.

#1420

**Prevalence of** PALB2 **mutations in an unselected cohort of breast cancer patients and unaffected individuals from Malaysia and Singapore.**

Patsy P. Ng,1 Wei Xiong Wen,1 Eldarina Wijaya,1 Jamie Allen,2 Joanna Lim,1 Shao Yan Lau,1 Brennan Decker,2 Karen Pooley,2 Leila Dorling,2 Craig Luccarini,2 Caroline Baynes,2 Don Conroy,2 Patricia Harrington,2 Shivaani Mariapun,1 Siti Norhidayu Hasan,1 Daphne Shin-Chin Lee,1 Sheau Yee Lee,1 Sook Yee Yoon,1 Cheng Har Yip,3 Nur Aishah Taib,4 Weang Kee Ho,5 Mikael Hartman,6 Antonis C Antoniou,2 Alison M Dunning,2 Douglas F Easton,2 Soo Hwang Teo1. 1 _Cancer Research Malaysia, Subang Jaya, Selangor, Malaysia;_ 2 _University of Cambridge, Cambridge, United Kingdom;_ 3 _Sime Darby Medical Centre, Subang Jaya, Selangor, Malaysia;_ 4 _University Malaya Medical Centre, Kuala Lumpur, Malaysia;_ 5 _The University of Nottingham Malaysia Campus, Semenyih, Malaysia;_ 6 _National University Hospital, Singapore, Singapore_.

Background: Rare variants such as protein truncating and splice-junction variants in PALB2 have been found to confer increased risk to breast cancer. However, previous studies have only investigated the prevalence of mutation carriers in individuals selected on the basis of earlier age of diagnosis or on family history of breast cancer. In this study, we sought to determine the prevalence of PALB2 in an unselected hospital-based multi-ethnic cohort of breast cancer cases and healthy women from Malaysia and Singapore.

Method: Amplicon-based targeted sequencing of the PALB2 gene which include all coding exons and splice site junctions was performed to identify germline alterations in an unselected cohort of 5021 affected and 5192 healthy individuals recruited from multiple centres. Associations between pathogenic (protein truncating) variants and breast cancer risk were evaluated using logistic regression and a Fisher's exact test.

Results: Truncating variants in PALB2 were associated with increased risk of breast cancer with an estimated OR=6.61 (95% CI 3.27 to 13.37, p<0.0001). In total, there were 31 unique protein truncating variants identified in 66 individuals (57 cases [1.1%], 9 controls [0.2%]). The majority of these truncating variants were rare; of the 31 unique variants identified, 18 (58%) were found only in 1 individual. The common truncating variants in our cohort were PALB2 c.2968G>T (p.Glu990Ter, rs876659036) found in 5 cases and 2 controls; PALB2 c.1037_1041delAAGAA (p.Lys346Thrfs, rs587776410) found in 4 cases and PALB2 c.1059delA (p.Lys353Asnfs, rs730881872) found in 4 cases. Of the PALB2 carriers, 24% developed ER+/HER2- disease, 14.0% developed ER+/Her2+ or ER-/Her2+ disease and 10.5% developed triple negative breast cancer.

Conclusions: We found that 1.1% of breast cancer patients and 0.2% of unaffected individuals carry a pathogenic mutation in PALB2. To the best of our knowledge, this is the first large population-based case control study that was able to estimate the breast cancer risk associated with truncating mutations in PALB2 gene in a multi-ethnic population in South East Asia.

#1421

Flexible tools for the development and performance verification of customized target enrichment panels.

Cynthia L. Hendrickson,1 Evan Mauceli,1 Andrew Barry,2 Kruti Patel,1 Catherine Huang,3 Scott Adams,1 Bharathi Anekella3. 1 _Directed Genomics, Ipswich, MA;_ 2 _New England Biolabs, Ipswich, MA;_ 3 _Seracare Life Sciences, Gaithersburg, MD_.

Implementation of next generation sequencing for variant identification and discovery presents difficulties both in the selection of genomic loci for inclusion in a panel, as well as the ability to effectively verify the performance of the assay based on the content selected. Balancing the relevance of genomic content with the practical hurdles of developing specific assays necessitates new approaches for the application of next generation sequencing toward precision medicine that are aimed at providing the flexibility of content to efficiently provide data capable of testing fundamental biological hypotheses.

NEBNext Direct Custom Ready Panels overcome these obstacles by enabling the rapid development and deployment of customized target enrichment panels. This offering allows researchers to select from a large library of genes for which baits have been developed and optimized to produce sequencing data with extremely high specificity and coverage uniformity. These panels are effective in targeting a wide range of content, from highly focused, single-gene panels, to comprehensive panels comprised of hundreds of genes.

In a similar approach for the verification and validation of new panels, SeraCare has developed the VariantFlex™ system, which is comprised of a broad library of mutations to develop custom controls and reference materials for NGS-based assays. These biosynthetic constructs contain variants such as SNVs, indel, frameshift and fusion variants that can be used to formulate controls across a range of variant allele frequencies, providing the necessary content and flexibility to verify assay performance.

Here we will present data demonstrating the combined utility of these offerings to assess target enrichment panel performance with a range of genomic content, panel sizes and variant allele frequencies.

#1422

NEBNext Direct® Custom Ready myeloid genes for NGS target enrichment.

Scott M. Adams,1 Kruti M. Patel,1 Amy B. Emerman,1 Sarah K. Bowman,1 Charles D. Elfe,1 Evan Mauceli,1 Andrew Barry,2 Theodore Davis,2 Cynthia L. Hendrickson1. 1 _Directed Genomics, Ipswich, MA;_ 2 _New England BioLabs, Ipswich, MA_.

Comprehensive genetic evaluation of patients with blood cancer can be imperative for identifying and classifying myeloid malignancies in a patient. Since liquid tumors comprise of highly variable percentages of normal cells or multiple tumor clones, this type of screening is critical for early stage disease patients where a cytogenetic profile informs diagnosis or disease stratification, prognosis, and treatment options. For patients with late-stage disease, genetic testing can assist in choosing options for alternative treatments, including targeted therapies. Thus, next generation sequencing technologies are needed that can provide the sensitivity and specificity to identify these genetic complexities. Here we introduce a selection of over 50 genes from the NEBNext Direct® Custom Ready gene catalog that are known to be involved in myeloid malignancies. The NEBNext Direct® technology uses a novel approach to selectively enrich nucleic acid targets, and rapid customized panels can be generated by combining one to several hundred genes from the Custom Ready catalog. Unlike alternative hybridization methods, NEBNext Direct® does not require upfront library preparation. The approach rapidly hybridizes both strands of genomic DNA to biotinylated baits, captures the targets on streptavidin beads, enzymatically removes off-target sequence, and directly converts captured molecules into Illumina-compatible sequencing libraries in a single day protocol. A 12-base unique molecular identifier (UMI) tags each individual molecule prior to the final PCR amplification to enable identification of PCR duplicates, and an 8-base barcode is added to each library during PCR for high-throughput pooling. With 100ng of DNA input the panel provides highly uniform coverage and approximately 95% of sequence reads map to the targeted myeloid genes. The NEBNext Direct® Custom Ready myeloid genes demonstrate a high-throughput and cost-effective approach for the rapid and sensitive analysis of exon coding regions (~200kb) for over 50 oncogenic genes important in the pathogenesis of these types of blood disorders. These gene mutations are powerful prognostic markers that can be used as a guide for effective patient management.

#1423

Integrative analysis predicts lncRNA regulating gene alternative splicing in breast cancer.

Yunyun Zhou. _Univ. of Mississippi Medical Center, Oxford, MS_.

Background: Non-coding region occupies 98% of the whole human genome and plays a

regulatory role for protein-coding genes. About 95% of the protein-coding genes undergo

alternative splicing, however, limited understandings for the complexity of alternative splicing

(AS) mechanisms. Recent studies reported that some aberrant alternative splicing are

associated with cancer development. Cancer-specific AS could be regulated by long non-

coding RNAs (lncRNAs) directly or indirectly through other intermediate molecules . LncRNAs

regulate gene splicing either by binding to their splicing factor proteins or competing with

miRNAs to influence their targeted genes. Therefore, it is critically important to develop an

effective computational framework capturing the lncRNAs regulatory mechanism in the process

of AS from heterogeneous molecular relationships.

Method: To model the complicated regulation mechanism in splicing event, we developed a

integrative analysis framework based on machine learning algorithms. We firstly identified

breast cancer-specific lncRNAs and AS genes by edgeR differential gene analysis from TCGA

RNAseq tumor (n=1101) /normal (n=139) samples. Then we built co-expression network based

on Spearman correlation pairwise distances from these cancer-specific lncRNAs and AS. We

ranked top lncRNAs regulating alternative splicing in tumorigenesis by an optimized random walk

multi-graphic method from the integrative networks of co-expression network, publically curated

epigenetic network (e.g. ENCODE) and protein-protein interaction network (e.g. STRING). Linear

regression analysis further refined individual candidate lncRNA regulation relating to their most

correlated target splicing genes. The secondary structure of binding lncRNAs to potential splicing

genes was predicted by support vector machine (SVM) algorithm to confirm the most conserved

targets.

Results: We identified 496 lncRNAs and 418 coding genes with alternative splicing isoforms

associated with breast cancer. Pathway analysis predicted the functions of 14 candidate

lncRNAs potentially regulating AS, are associated with cell migration, cell cycle progression,

and more. We detected a high confidence lncRNA MALAT1 that regulates PKM gene

alternative splicing, exclusion of either exon 9 or 10, in breast cancer tumorigenesis, which

confirms the predictive strength of our proposed method.

Conclusion: Understanding cancer-specific splicing machinery is therapeutically crucial in

targeting the molecules that can influence the splicing process. We developed a network-based

integrative framework to predict lncRNA regulations on breast cancer-specific alternative splicing

as the potential therapeutic targets.

#1424

Elucidating the microRNA-203 specific biological processes in glioblastoma cells from high-throughput RNA-sequencing.

Bhavesh K. Ahir, Herbert H. Engelhard, Sajani S. Lakka. _The University of Illinois at Chicago, Chicago, IL_.

Glioblastoma (GBM) is the most common primary malignant intracranial adult brain tumor. Allelic deletion on chromosome 14q play an essential role in GBM pathogenesis, and this chromosome 14q site was thought to harbor multiple tumor suppressor gene associated with GBM, a region that also encodes microRNA-203 (miR-203). This study was conducted to identify gene expression profile changes associated with mir-203 expression by high-throughput RNA sequencing. Enrichment analyses for gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis revealed that mir-203 expression had a strong, negative effect on a number of fundamental and interconnected biological processes involved in cell growth and proliferation. The biological processes mostly influenced were p53 signaling pathway, FoxO signaling pathway, DNA replication, cell cycle, MAPK signaling pathway and Apoptosis. In total, 847 upregulated and 345 downregulated differentially expressed genes (DEGs) were identified in control versus miR203 expressing glioma cells. After GO enrichment, the downregulated DEGs and miR-203 predicted target genes such as BCL-2, SPARC, PDGFA, CREB were found to be mainly enriched in cell cycle regulation and apoptosis processes, whereas the upregulated DEGs, such as CCND1, E2F1 were involved in the DNA replication and cell cycle regulation of gene expression. We also demonstrate that mir-203 expression suppressed BCL-2 protein and mRNA by western blotting and qRT-PCR analysis. Moreover, co-transfection experiments using a luciferase-based reporter assay demonstrated that mir-203 directly regulated BCL-2 expression and BCL-2 overexpression suppressed mir-203 mediated cell death. Knowledge gained through this profiling study can facilitate targeted therapeutic interventions.

#1425

A multi-enzyme DNA repair mix improves library quality and sequencing accuracy in FFPE tumor samples.

Pingfang Liu, Margaret Heider, Chen Song, Lixin Chen, Laurence Ettwiller, Lauren Higgins, Eileen Dimalanta, Theodore Davis, Thomas Evans. _New England Biolabs, Inc., Ipswich, MA_.

Next-generation sequencing (NGS) methods are used extensively to profile mutations present in diseased human tissues. These genomic approaches hold great promise for personalized medicine but sequencing accuracy is essential for proper patient diagnosis and determining a treatment plan. A common source of DNA for genomic profiling is formalin-fixed, paraffin-embedded (FFPE) tissue samples obtained from patient biopsy. FFPE DNA poses important challenges for preparing NGS libraries including low input amounts and poor DNA quality, resulting from extensive fixation- and storage-induced DNA damage. Additionally, these damage-induced sequencing artifacts raise the background level of mutations, making it difficult to discern true, low frequency, disease-causing variants from noise. We previously showed that a major fraction of somatic mutations described in publicly available datasets are due to such sequencing artifacts (Chen et al., Science 2017). Furthermore, we showed that enzymatic repair of DNA before library preparation improves the library quality and reduces background noise. We developed a second-generation DNA repair enzyme mix (V2) that efficiently repairs the most prevalent damage types found in FFPE DNA and further improves the quality and yield of NGS libraries. Additionally, we tested the efficacy of the V2 repair mix in improving sequencing accuracy for FFPE DNA samples obtained from different cancer tissues. We performed target enrichment on a panel of 151 cancer genes, deep sequenced, and performed variant analysis. For a subset of variants, we further validated our results using a droplet digital PCR (ddPCR) assay. Both methods showed that the V2 repair mix did not alter the overall frequency of variants identified, thus it did not introduce bias, but significantly improved the sequencing accuracy by reducing the number of false variant calls. Therefore, enzymatic repair is a critical first step in preparing FFPE DNA sequencing libraries, allowing more sensitive and robust detection of low frequency, disease variants.

#1426

Targeted sequencing of colorectal cancer in search for prognostic biomarkers.

Yoshikage Inoue,1 Nobuyuki Kakiuchi,1 Kenichi Yoshida,1 Yusuke Shiozawa,2 Kenichi Chiba,2 Tetsuichi Yoshizato,1 Yuichi Shiraishi,2 Satoru Miyano,2 Satoshi Nagayama,3 Yoshiharu Sakai,1 Seishi Ogawa1. 1 _Kyoto University, Kyoto, Japan;_ 2 _University of Tokyo, Tokyo, Japan;_ 3 _The Cancer Institute Hospital of JFCR, Tokyo, Japan_.

Introduction

Extensive genetic studies of recent years using next generation sequencing have clarified a comprehensive registry of genetic alteration in colorectal cancer (CRC). However, the clinical impacts of these genetic alterations have not fully been investigated, except for a better response to immune checkpoint blockade and favorable prognosis in patients with microsatellite instability (MSI) cancer. In this study, we investigated the effects of common genetic alterations on survival in CRC patients using large-scale mutation profiling.

Methods

We performed targeted-capture sequencing to common driver genes (n=128) in CRC in a total of 544 patients with stage I (n = 36), II (n = 236) and III (n = 272) CRC. All patients were treated at a single institute between 2004 and 2016 and clinically well-annotated. Genome-wide copy numbers, fusion genes and MSI were also interrogated using baits for 1,605 SNPs, 7 introns and 18 microsatellites, respectively. RT-PCR was performed for the detection of PTPRK-RSPO3 fusion gene.

Results

The median age at diagnosis was 65 (32-95) with a median observation period of 1250 days. Of the 156 patients with recurrence, 15, 61 and 80 patients had stage I, II and III diseases, respectively. APC, TP53, KRAS, and PIK3CA represented the most frequently mutated, while common focal copy number alterations involved PTEN, SMAD4, SMAD2, FGFR1 and IGF2. Novel fusion genes including PTPRK-RSPO3 were detected in only 7 (1.5%) samples. Fifty-five patients had more than 20 mutations, comprising a group of hypermutated CRC. Among these, 44 were thought to have MSI based on the microsatellite analysis, while the remaining 11 were explained by POLE mutations affecting the proof reading domain. CRCs with POLE mutations were diagnosed earlier in life, with the median age of 49 (35-66). The frequency of CRCs with MSI was significantly lower than previous reports from the western population. In contrast to mutation frequency, copy number alterations were infrequent, which most hypermutated tumors showed Genomic Instability Index (GII) below 0.2. FBXW7 mutation positively affected tumor free survival. BRAF mutation, SMAD4 mutation, or existence of fusion genes did not have any effect on prognosis. In accordance with previous reports, only 4 patients (9.1%) with MSI had recurrence, confirming a better prognosis of MSI-positive patients. Similarly, only one of the 11 patients (9.1%) with POLE mutation had recurrence. The low relapse rate was also observed among an additional cohort of 819 patients, which included 6 POLE mutated cases, none of whom had recurrence, suggesting that POLE mutation was associated with a favorable prognosis.

Conclusions

We revealed the landscape of driver mutations in patients with stage I, II and III CRC with their implication in clinical outcomes. In addition to FBXW7 mutations, the MSI status and somatic POLE mutations are significantly associated with a better prognosis.

#1427

Comprehensive investigation of false mutation discoveries in FFPE samples.

Thomas M. Blomquist,1 Wenming Xiao,2 Somatic Mutation Working Group of the SEQC2 Consortium. 1 _Univ. of Toledo, Waterville, OH;_ 2 _NCTR/FDA, Jefferson, AR_.

Next generation sequencing (NGS) has emerged as a primary tool for "precision" medicine, especially in the rapidly evolving field of cancer care. However NGS-based somatic variant calling in a clinical oncology setting is susceptible to numerous sources of technical error impeding optimal patient management. One important source of error in variant reporting is formalin fixation and paraffin embedding (FFPE). Of note, FFPE-derived errors are commonly present at clinically actionable loci and can occur at allelic frequencies (5-65%) that meet treatment criteria. Compounding this problem, degree of FFPE error is highly susceptible to inter-specimen and inter-laboratory variations. In this context, static variant calling thresholds to control for "general FFPE-effect" are likely inadequate; resulting in both False-Negatives (ignoring true somatic variation) and False-Positives (reporting technical error as truth). Despite the importance of fresh tissue for molecular studies, routine tissue histology (derived from FFPE) continues to be the gold standard for cancer staging/grading. And, as emphasis on smaller sample sizes becomes the norm, routine histology will be prioritized in many scenarios. Thus, the need to understand how molecular testing performs in FFPE tissue is a driving imperative for optimal cancer care.

In this study, our aim was to develop and evaluate "best" quality control practices that would minimize False-Positive or -Negative somatic variant calling in FFPE specimens. Reference samples (paired breast carcinoma and "normal" lymphoblast cell-lines) from the FDA-led Sequencing Quality Control Phase II (SEQC2) consortium were used. These specimens were subjected to varying FFPE conditions (n=8) mimicking the pathology laboratory setting. For each condition, multiple replicates of whole genome and exome sequencing (WGS/WES) were performed with ~100X coverage across the entire human genome. Observed somatic variation frequencies for each genomic position for each FFPE condition were compared to ground truth and quality control measures derived from matched fresh materials using numerous statistical models.

Applying rigorous analytical strategies, we aim to identify genomic regions that serve as markers (internal control regions) for the full range of FFPE-effect, develop QC measures and statistics to define lower limit of detection for variant calling for each genomic position, and provide general guidance for the community on how to accurately report mutations in FFPE processed clinical cancer specimens.

#1428

Application of ONCO/REVEAL Lung & Colon Cancer Panel in CRC samples and effect of DNA damage in FFPE samples.

Jianfeng Xiao, Jiajie Tang, Xiangzhi Liu, Xiaoyan Tian. _Shanghai Zhengu Biotech Ltd., Shanghai, China_.

Introduction:

The ONCO/REVEAL Lung&Colon Caner Panel (LC103, Pillar Biosciences Inc.) interrogates regions in 22 genes that are frequently mutated in NSCLC and CRC. To evaluate the performance of ONCO/REVEAL Lung&Colon Cancer Panel, we assessed a cohort of 207 colorectal cancer FFPE samples collected by a top-tier hospital in Shanghai between 2015 and 2016. Among them, 27 FFPE samples showed abnormally high numbers of low frequency variants (<2%) and were further investigated to assess the effect of DNA damage in somatic variant detection.

Materials and Methods:

DNA library preparation and sequencing: 10-20 ng of FFPE DNA (Quantitated by Qubit, Life Tech.) was used to prepare libraries using the LC103 panel. All libraries were subsequently sequenced on Illumina MiSeq sequencer.

FFPE DNA repair: DNA extracted from 27 FFPE samples with a high number of low VAF (<2%) variants was treated with NEBNext FFPE DNA Repair Mix prior to library preparation. FFPE and DNA repaired FFPE were compared to analyze the effect of DNA damage in variant detection. Matched fresh frozen tumor tissue samples were also tested.

Data analysis: PiVATTM (Pillar Biosciences Inc.) was used for data analysis. PCR errors and sequencing errors are reduced to be well below 1% VAF through the PiVAT error correction algorithm.

Results:

100% successful rate of library preparation and sequencing: All of 207 FFPE samples yielded high quality sequencing data that detected mutant alleles at frequencies as low as 1%.

Variant detection: A total of 414 somatic variants, including SNV and small indels, were identified above 2% VAF in 193 out of 207 samples. TP53 (38%), KRAS (24%), PIK3CA (12%), FBXW7 (9%) and PTEN (4%) were the most frequently mutated genes. CNVs were identified in EGFR, MET, ERBB2, KRAS and FGFR1 genes.

The effect of DNA damage: The majority of detected variants between 1% and 2% VAF, are not known hotspot mutations. C:G>T:A mutations account for 73.8% of variants between 1% and 2% VAF. DNA repair by NEBNext FFPE DNA Repair Mix or other UDG enzymes reduced 1-2% VAF calls significantly, indicating that these variants are false positive calls. However, DNA repair enzymes used in our study could not eliminate false positive calls completely. Other mechanisms are suspected to contribute to the remaining low frequency calls.

Variant between 2% and 5% VAF: In total, 33 somatic variants were detected at 2-5% VAF. There are many clinically actionable mutations or common driver mutations, including KRAS G12C (2.10%), KRAS G12D (2.21%), KRAS Q61L (2.74%), BRAF G469E (2.51%), PIK3CA E545K (2.08%, 2.39%, 4.78%), PIK3CA E545G (4.61%), PIK3CA Q546K (4.51%), PIK3CA H1047R (2.80%).

Conclusions:

The ONCO/REVEAL Lung&Colon Caner Panel is a robust and sensitive NGS Assay for the detection of somatic variants. DNA damage confounds variant identification in FFPE samples between 1 and 5% frequency.

#1429

MYH8 R1292X: A novel mutation in relapsed AML induces EMT features and poor prognosis.

Hyejoo Park,1 Daeyoon Kim,1 Dongchan Kim,1 Yungyeong Park,1 Youngil Koh,2 Sung-Soo Yoon2. 1 _Seoul National University, Seoul, Republic of Korea;_ 2 _Seoul National University Hospital, Seoul, Republic of Korea_.

Introduction: Recent advances in tumor genomic analysis have led to the discovery of NPM1, FLT3, CEBPA, MLL, NRAS, and RUNX1 alterations as the cause of AML. Nevertheless, there is a limit to the treatment and clarification of AML, and research for the identification of novel genetic alterations that cause AML is actively underway.

Materials and methods: In this study, we performed Whole exome sequencing (WES) with 53 AML patient's samples and conducted targeted re-sequencing using 391 AML patient's samples based on locus of somatic mutation that were found by WES. For functional validation of novel oncogenic mutations, we used CRISPR-Cas9 system to generate knock-in (KI) cell line. For characterization of mutant cells, we performed proliferation assay, cell cycle assay, adhesion assay, and wound healing assay. Epithelial to mesenchymal transition (EMT) markers were checked by western blotting.

Results: Using WES and targeted resequencing, we could identify MYH8 R1292X novel mutation as recurrent potentially oncogenic mutation. Additional validation using separate AML cohort revealed MYH8 R1292X variants in four AML patients, suggesting that MYH8 R1292X is potential oncogenic mutation. In functional validation using KI cell line, we could not find change in morphology of KI cells. However, there was a difference in proliferation – the rate of proliferation was faster in KI cells than in cells without mutation. In the cell cycle assay, the mutant cells showed more S phase DNA than the non-mutant cells. Wound healing assay showed that the mutant cells had higher migration ability and lowered the ability of adhesion in comparison. PCR and western blot showed that EMT markers except vimentin increased in mutant cells.Survival analysis based on TCGA data showed that both the overall survival and the disease-free survival curves were significantly different according to MYH8 alterations.

Conclusion: Taken together, we conclude that the novel alteration MYH8 R1292X is associated with recurrent AML and poor prognosis by increasing migration, and inducing an increase in EMT markers.

#1430

Improved sample indexing for high fidelity demultiplexing to increase confidence in low frequency variant calling.

Jonathan C. Irish, Jordan RoseFigura, Sukhinder K. Sandhu, Bita Carrion, Laurie Kurihara, Vladimir Makarov. _Swift Biosciences, Inc., Ann Arbor, MI_.

Due to recent scientific advances, there is a growing demand for new indices with high fidelity to eliminate errors associated with certain index combinations and sequencing chemistries. These errors are due to insufficient edit distances (the minimum number of changes required to transform one index sequence to the other) between index sequences or index hopping. Recent publications have highlighted how sequencing reads are misassigned due to "index hopping" on Illumina patterned flow cells, such as the recently launched Illumina Novaseq which can generate billions of reads (>100 exomes) in a single run. This misassignment canlead to false positives in ultra-sensitive assays where low frequency variants or nucleic acid species are monitored. We have observed misassignment due to insufficient edit distance among Illumina TruSeq HT i7 indices at a frequency up to 1.5%. Therefore, we developed 968nt i7 indices that can be paired with the existing TruSeq HT i5 indices to achieve 768 high throughput dual combinations, which were validated using a novel method on both Illumina's 2- and 4-channel technologies as both single and dual indices. This method involved thepreparation of 96 libraries with unique, non-overlapping inserts to facilitate tracking of index misassignment. This allowed us to assess not only which index has misassigned library molecules, but also pinpoint the origin and rate of misassignment within a single run. With our 96 i7 indices, misassignment was observed at rates <0.1%. For even higher fidelity de-multiplexing, we have paired our 96 indices in a non-tandem manner for single use in both the i5 and i7 positions, known as Unique Dual Indices (UDIs). The use of UDIs further eliminates index read errors that misassign reads, enabling increased confidence in calling low frequency variants. UDIs can also eliminate PCR-induced chimerisms, which can significantly improve data from a variety of assays. We are validating 96 new indices as UDIs for avoidance of index hopping and for eliminating PCR-induced chimerism during multiplexed library amplification.

### Metabolic Pathways

#1431

Autophagic degradation of the endoplasmic reticulum leads to reduced phospholipid biosynthesis in mutant isocitrate dehydrogenase 1 gliomas.

Pavithra Viswanath, Russell O. Pieper, Joanna J. Phillips, Sabrina M. Ronen. _University of California San Francisco, San Francisco, CA_.

Introduction: Cancer cells up-regulate phospholipid biosynthesis in order to meet the demands of uncontrolled cell proliferation. Unusually, mutant isocitrate dehydrogenase 1 (IDHmut) gliomas down-regulate levels of the phospholipid precursors phosphocholine and phosphoethanolamine relative to wild-type IDH1 (IDHwt) gliomas. The goal of this study was therefore to investigate whether the phospholipids phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdE) are also down-regulated in IDHmut gliomas.

Methods: Immortalized normal human astrocytes and U87 cells expressing IDHwt or IDHmut enzyme were studied. Steady-state phospholipid levels were measured by 31P-magnetic resonance spectroscopy (MRS) of cell extracts. De novo phospholipid biosynthesis was quantified by 13C-MRS of cells labeled with 56μM [1,2-13C]-choline and [1,2-13C]-ethanolamine. ER area was measured by STORM imaging of calnexin fluorescence.

Results: Steady-state PtdCho and PtdE levels and de novo phospholipid biosynthesis were significantly reduced in IDHmut cells relative to IDHwt. Concomitantly, the activities of CTP:PC cytidylyltransferase (CCT) and CTP:PE cytidylyltransferase (ECT), the rate-limiting enzymes for phospholipid biosynthesis, were significantly reduced in IDHmut cells. CCT and ECT are localized to the endoplasmic reticulum (ER), the site of phospholipid biosynthesis. We therefore measured ER area by super-resolution STORM imaging and found a significant reduction in IDHmut cells relative to IDHwt while cell size remained unchanged. Autophagy of the ER (ER-phagy) can regulate ER area and we found that IDHmut cells showed increased expression of FAM134b, the ER-phagy receptor and higher autophagic flux. These results suggest that IDHmut cells undergo ER-phagy, leading to reduced phospholipid biosynthesis. Silencing FAM134b or the autophagy proteins Atg5 and Atg7 restored ER area and phospholipid biosynthesis and

abrogated clonogenicity of IDHmut cells, linking ER-phagy to reduced phospholipid biosynthesis and pointing to a therapeutic opportunity. In the absence of specific ER-phagy inhibitors, we used the late-stage autophagy inhibitor chloroquine (CQ) to test proof-of-principle and found that CQ significantly inhibited orthotopic IDHmut glioma growth in vivo. Importantly, we observed ER-phagy and down-regulation of phospholipid levels in IDHmut glioma patient biopsies, highlighting the translational validity of our findings.

Conclusions: We find that down-regulation of phospholipid biosynthesis is a unique metabolic outcome of ER-phagy in IDHmut gliomas. While autophagy has been linked to glucose and glutamine metabolism, this study is the first to link autophagy to phospholipid biosynthesis. Importantly, our study identifies a rationale for the development of novel therapies targeting ER-phagy in IDHmut gliomas.

#1432

Deranged tyrosine metabolism drives tumorigenesis in liver cancer.

Man Tong,1 Tin Lok Wong,1 Steve Tin-Chi Luk,1 Noelia Che,1 Xin Yuan Guan,1 Yun Fei Yuan,2 Terence Kin-Wah Lee,3 Stephanie Ma1. 1 _Univ. of Hong Kong, Faculty of Medicine, Pokfulam, Hong Kong;_ 2 _Sun Yat-Sen University Cancer Center, Guanzhou, China;_ 3 _The Hong Kong Polytechnic University, Kowloon, Hong Kong_.

Tyrosine, like other amino acids, is the building block for proteins as well as an alternative energy source for cellular functions. Liver is the major organ where tyrosine degradation takes place to produce intermediates or precursors for gluconeogenesis and ketogenesis. In normal liver, tyrosine can be converted to fumarate and acetoacetate through multiple steps of enzymatic reactions. In patients with liver cancer, an upregulation of serum tyrosine levels has been previously reported, suggesting a deregulated tyrosine catabolism in liver cancer. In this study, we observed a significant down-regulation of the five enzymes involved in tyrosine catabolism (TAT, HPD, HGD, GSTZ1 and FAH) in tumor samples compared with normal liver samples from TCGA database. Further, downregulation of the first three enzymes significantly correlates with overall and disease-free survival in liver cancer patients. Downregulation of these enzymes is also confirmed in a separate liver cancer patient transcriptome cohort of Asian ethnicity, as well as at the proteomic level by both Western blot and immunohistochemistry assays. Our group has previously reported that downregulation of the first tyrosine catabolizing enzyme TAT (tyrosine aminotransferase) could promote liver tumorigenesis. Here, we further investigate if reprogrammed tyrosine metabolism could promote cancer development by modulating gene expression and activity of the second enzyme HPD (4-hydroxyphenylpyruvate dioxygenase) in the tyrosine catabolism pathway. We found blocking HPD gene expression or its enzymatic activity could promote cell proliferation and tumor formation in liver cancer. This could be partially attributed to enhanced mitochondrial respiratory capacity accompanied with reduction in ROS level, NADP/NADPH ratio and DNA damage. Conversely, overexpression of HPD led to an opposing effect. Taken together, this study reveals an unreported alteration of tyrosine metabolism which drives cancer development in liver cancer.

#1433

Estrogen-related receptor alpha (ERRα) functions to promote prostate cancer cell stemness via its transcriptional regulation of ZIP1 and ACON2 to enhance oxidative phosphorylation.

Taiyang Ma, Zhenyu Xu, Leung Franky Chan. _The Chinese University of Hong Kong, Hong Kong, Hong Kong_.

Accumulating evidences suggest that cancer stem-like cells (CSCs) play critical roles in cancer relapse and metastasis, likely due to their strong self-renewal capacity and chemo-radiotherapy resistance. Recent advances also indicate that the energy metabolism status of the CSCs differs greatly from the bulk cancer cells, and that such energy metabolism shift may favor the maintenance of cancer cell stemness. Estrogen-related receptor alpha (ERRα, NR3B1) is a key energy metabolism regulator. In this study, we aim to characterize its role in the energy metabolism regulation in prostate cancer stem-like cells (PCSCs). By analyzing the oxygen consumption and extracellular acidification rates using the Seahorse XF Analyzer, we revealed that the PCSCs, isolated from prostate cancer cell lines (LNCaP, DU145 and PC3) by low attachment 3D-culture, exhibited lower aerobic glycolysis but higher oxidative phosphorylation status. Expression analyses by qPCR and Western blot showed that the isolated PCSCs expressed higher levels of ERRα. Further functional studies showed that overexpression of ERRα could promote the oxidative phosphorylation but lower the glycolysis status, and also enhance the 3-D spheroid formation capacity (stemness) of prostate cancer cells (LNCaP, DU145 and PC3), whereas its knockdown could reverse the effects. Furthermore, we identified by combined chromatin immunoprecipitation (ChIP) and PCR analysis that ERRα could directly regulate two key energy metabolic genes ACON2 (a rate limiting enzyme in TCA cycle) and ZIP1 (a zinc transporter) in PCSCs. Western blot analysis showed that ERRα could up-regulate the ACON2 but repress the ZIP1 expression, whereas its knockdown reverse the expressions of these two proteins in prostate cancer cells. Together, our results show that ERRα can promote the stemness of prostate cancer cells and enhance the cellular energy metabolism towards oxidative phosphorylation in PCSCs via its transcriptional regulation of ACON2 and ZIP1 genes.This study is supported by a General Research Fund from the Research Grants Council of Hong Kong (project code 14107116).

#1434

LSR promotes lipid metabolism via beta oxidation and contributes cell viability in epithelial ovarian cancer.

Kosuke Hiramatsu,1 Satoshi Serada,1 Minoru Fujimoto,1 Shinya Matsuzaki,2 Yutaka Ueda,2 Kiyoshi Yoshino,2 Tadashi Kimura,2 Tetsuji Naka1. 1 _Kochi university, Kochi, Japan;_ 2 _Osaka university, Osaka, Japan_.

Background:Previously, we identified lipolysis-stimulated lipoprotein receptor (LSR) as a new target of epithelial ovarian cancer (EOC), and we reported inhibitory effect of our newly developed monoclonal antibody (mAb) against LSR-positive ovarian cancer cells in vitro and in vivo. We also demonstrated that LSR took in triglyceride-rich protein and promoted cell proliferation, however, LSR function in EOC cells is not investigated enough. Therefore, we aimed to reveal the function of LSR in EOC cells focusing on lipid metabolism including beta oxidation and subsequent TCA cycle.

Material and Method: Using LSR-positive ovarian cancer cell lines (OVCAR3 and RMG-I), we investigated activation of beta-oxidation by LSR after administration of VLDL which is one of the triglyceride-rich protein. Moreover, we constructed LSR-knockdown EOC cells and investigated the effect of VLDL administration in these cells. In addition, we showed inhibitory effect of our anti-LSR mAb against lipid metabolism. Expression of beta-oxidation related proteins were analyzed by western blot analysis. Production of acetyl CoA and ATP were measured using each assay kit. Cell viability was analyzed by WST-8 assay and thymidine incorporation assay.

Results: In LSR-positive EOC cells, LSR promoted uptake of VLDL in glucose restricted environment compared with LSR-knockdown cells (p < 0.05) and VLDL administration promoted acetyl CoA and ATP production (p < 0.05), suggesting that beta-oxidation, TCA cycle and electron transport system were activated by fatty acid from VLDL in LSR-positive cells. Moreover, in LSR-positive EOC cells, VLDL administration increased viable cells compared with LSR-knockdown cells (p < 0.05), however, DNA synthesis was not promoted (p < 0.05). These results showed that VLDL uptake via LSR promoted lipid metabolism and contributed cell viability. Finally, our anti-LSR mAb inhibited these processes.

Conclusion: In glucose restricted environment, LSR takes in triglyceride-rich protein, activates lipid metabolism via beta-oxidation and contributes cell viability in LSR-positive EOC cells.

#1435

Investigation of phosphoserine aminotransferase 1: Its role in breast cancer progression.

Stephanie Metcalf, Traci Kruer, James Wittliff, Carolyn Klinge, Brian F. Clem. _Univ. of Louisville, Louisville, KY_.

Metastasis and endocrine resistance are two factors that complicate therapeutic intervention in breast cancer patients and lead to poorer overall survival. Metastasis is known to be responsible for 90% of cancer related deaths, and is especially prevalent in triple negative breast cancer (TNBC); while endocrine resistance can affect up to 50% of patients diagnosed with estrogen receptor positive breast cancer (ER+BC). Phosphoserine aminotransferase 1 (PSAT1) catalyzes the second step within de novo serine biosynthesis and increased expression of enzymes in this pathway have been linked to progression of breast cancer and poor clinical outcomes. Within our preliminary retrospective analysis of human breast cancer patients, we identified an inverse association with elevated transcript levels and poorer distant metastasis free survival, which, coupled to a previously reported correlation of PSAT1 with response to endocrine therapy in patients with ER+BC, we postulate that PSAT1 contributes to breast cancer progression through promotion of metastasis and/or endocrine resistance. To initially determine relevance for PSAT1, immunohistochemistry was performed to determine PSAT1 expression in human breast cancer patients. We found that PSAT1 expression is increased through breast cancer progression, with highest levels observed within metastatic conditions. To investigate the metastatic contribution of PSAT1, we silenced PSAT1 expression within the triple negative breast cancer cell line (TNBC), MDA-MB-231. While suppression of PSAT1 had no effect on proliferation, there was a significant decrease in the motility and invasion of these cells. In addition, decreased PSAT1 substantially inhibited tumor nodule formation following tail-vein injections of MDA-MB-231 cells in vivo. To investigate PSAT1's role in endocrine resistance, we used parental MCF-7 cells and an endocrine-resistant derivative cell lines and found that resistant cells exhibited higher PSAT1 expression compared to parental MCF-7 cells. Lastly, suppression of PSAT1 trended to sensitize the resistant cells to 4-hydroxytamoxifen treatment. Taken together, these data indicate that PSAT1 may contribute to the progression of human breast cancer via either metastasis or endocrine resistance or both and could potentially serve as a viable target for new therapies.

#1436

Beta-caryophyllene regulates lipid biosynthesis in breast cancer cells.

Mam Y. Mboge,1 Adam P. Bullock,1 Riley O'Dennell,1 John V. Matthias,1 Julie A. Davila,1 Christopher J. Frost,2 Susan C. Frost1. 1 _Univ. of Florida, Gainesville, FL;_ 2 _University of Louisville, Louisville, KY_.

The search for therapeutic anti-cancer drugs has spanned both synthetic and natural products approaches, and notable success has been achieved from unique chemistries produced by plants. Early studies in E.coli showed that cyclic hydrocarbons, including terpenes, interact directly with biological membranes. Accumulation of hydrocarbons results in membrane swelling and increased membrane fluidity, both signs of cell stress. At biological temperatures, membrane fluidity is controlled by the saturation state of the acyl chains of fatty acids (primarily in phospholipids) and cholesterol content. Changes in either of these parameters leads to membrane remodeling which can affect membrane function. Terpenes are, themselves, precursors to complex sterols across all kingdoms of life. Beta-caryophylene (BCP), a bicyclic sesquiterpene, induces cell death across a variety of cancer cell types, although the mechanism(s) by which this occurs is not completely known. Our data show that BCP induces membrane permeability in breast cancer lines representing both ER-positive and triple negative phenotypes (TNBC), as measured by lactate dehydrogenase release from cells. We sought to understand this phenomenon by looking at changes in the transcriptome of treated cells compared to controls. We chose to examine cells exposed to hypoxia, rationalizing that this condition, in vivo, creates an aggressive phenotype, is associated with TNBC and drug-resistant (recurrent) breast cancers, and is an independent prognosticator for poor patient outcome. We isolated high quality RNA (RIN>9) at the University of Florida. The Genomics Core at the University of Louisville prepared libraries and performed the sequencing run (Illumina NextSeq 500). This generated over 144 million 75bp reads that aligned to the human genome (96.3% alignment rate), or approximately 24 million reads per sample. As a first approach in data analysis, we selected differential expression based on the FDR adjusted p-values (q-values) <0.05 (as determined by CuffDiff), and analyzed this gene set for pathway involvement using the reactome data base. Reactome also assesses FDR values for pathway analysis, and we selected pathway FDR <0.05 providing the highest level of specificity. This search modality revealed an orchestrated up-regulation of lipid metabolism, including that of cholesterol biosynthesis. This is consistent with BCP-induced membrane remodeling. We have validated these results, not by PCR, but through protein expression and metabolic assays. Because these events occur at concentrations that are sub-cytotoxic, this potentially reveals a new mechanism underlying the development of drug resistance.

#1437

De novo fatty acid synthesis-driven sphingolipid metabolism promotes metastatic potential of colorectal cancer.

Naser Jafari, James Drury, Andrew J. Morris, Fredrick O. Onono, Payton D. Stevens, Tianyan Gao, Eun Y. Lee, Heidi L. Weiss, B Mark Evers, Yekaterina Zaytseva. _Univ. of Kentucky, Lexington, KY_.

Colorectal cancer (CRC) is the second leading cause of cancer-related deaths in the USA. Development of metastasis is the most common cause of death in these patients. Fatty acid synthase (FASN) and Sphingosine Kinases 1 and 2 (SPHK1 and 2) are overexpressed in many cancers, including CRC. However, the contribution of FASN-mediated upregulation of sphingolipid metabolism to CRC metastasis and potential of these pathways as targets for therapeutic intervention remains unknown. The purpose of this study was to determine (i) expression of FASN, SPHK1 and SPHK2 in human CRC tissues, (ii) the effect of upregulation of FASN on sphingolipid metabolism and (iii) functional significance of the FASN/SPHK axis in advanced CRC.

Methods: Expression of FASN, SPHK1 and SPHK2 was assessed in a CRC tumor microarray (matched normal colon and tumor; 56 cases) by immunohistochemistry. Sphingolipids were measured by mass spectrometry. Primary CRC cells were established from CRC patient-derived xenograft (PDX) tumors and treated with TVB-3664, a novel FASN inhibitor, or FTY-720, an S1P mimetic that inhibits SPHKs and S1P receptors. Cellular proliferation was measured using a cell counter. Migration capabilities of cells were assessed by live cell imaging using Nikon BioStation. Invadopodia and focal adhesions were assessed by total internal reflection fluorescence microscopy. Tumor tissues were implanted into 6 to 8-week-old NOD scid gamma mice to establish PDX models. Expression of proteins involved in adhesion, migration and invasion were assessed by western blot.

Results: SPHK1 and SPHK2 were overexpressed in CRC as compared to normal mucosa and expression of FASN correlates with expression of SPHK2 (Spearman's r=0.27894, p=0.0374). Furthermore, FASN and SPHKs co-localized within invadopodia of primary CRC cells. Moreover, FASN inhibition decreased expression of SPHK2 and the level of Sphingosine-1-phosphate (S1P) in primary and established CRC cells. Inhibition of de novo lipogenesis using TVB-3664 or FTY-720 significantly inhibited proliferation, migration, focal adhesion formation and gelatin degradation ability of primary CRC cells. Inhibition of the FASN/SPHK/S1P axis was accompanied by a decrease in activation of p-MET, p-FAK, and p-Paxilin in vitro and in vivo. S1P treatment rescued FASN-mediated inhibition of these proteins suggesting that FASN promotes metastatic properties of CRC cells, in part, through an increase in sphingolipid metabolism.

Conclusion: Upregulation of the FASN/SPHK/S1P axis promotes CRC progression by enhancing cellular proliferation, adhesion and migration. Therefore, this study provides a strong rationale for further investigation of the interconnection of de novo lipogenesis and sphingolipid metabolism that would potentially lead to identification of new therapeutic targets and strategies for CRC.

#1438

Kynurenine pathway enzyme AADAT promotes breast cancer progression and immunosuppression in triple-negative breast cancer.

Franklin Gu, Susmita Samanta, Lin Tian, Chandra Ambati, Salil Bhowmik, Preeti Purwaha, Weijie Zhang, Benny Kaipparettu, Balasubramaniam Karanam, Arundhati Rao, Nagireddy Putluri, Xiang Zhang, Arun Sreekumar. _Baylor College of Medicine, Houston, TX_.

Triple-negative breast cancer (TNBC) is an aggressive and heterogeneous disease that presents a clinical challenge due to a lack of targeted therapies. Recently, increased levels of tumor-infiltrating lymphocytes (TILs) in TNBC have been associated with improved clinical outcomes. These findings suggest that elucidation of the molecular mechanisms through which TNBC modulates immunity can identify novel therapeutic targets. Using targeted mass spectrometry based metabolomics, we identified increased levels of the tryptophan catabolic metabolite kynurenine in TNBC tumors. Interestingly, tissue microarray analysis also found that the expression of AADAT, an enzyme involved in kynurenine metabolism, is negatively correlated with CD8 TILs in TNBC tumors, suggesting that AADAT mediates immune suppression. Genetic knockdown of AADAT in human TNBC cell lines inhibited cell proliferation and invasion in vitro and in vivo in immunodeficient mouse models. In an immunocompetent mouse model, genetic knockdowns of AADAT in mouse TNBC cell line inhibited tumor take and T cell infiltration into tumors. Gene set expression analysis (GSEA) revealed that breast cancer tumors with high AADAT expression are enriched in hypoxia-related concepts. Our genetic knockdown models of AADAT showed decreased transcriptional activity of hypoxia-inducible factor-1 (HIF-1), the primary transcriptional factor involved in hypoxic adaptation. Furthermore, expression of the immune checkpoint pathway ligand PD-L1, a known HIF-1 target, was also decreased in TNBC cells when AADAT expression was genetically inhibited. Taken together, these results indicate that kynurenine metabolism by the enzyme AADAT promotes breast cancer progression and immunosuppression.

#1439

Asparagine stimulates growth and endoplasmic reticulum stress response in a fumarate hydratase-deficient renal cell carcinoma cell line.

Rony Panarsky, Youfeng Yang, W. Marston Linehan, Jeffrey A. Moscow. _National Cancer Institute, Bethesda, MD_.

Hereditary leiomyomatosis and renal cell carcinoma (HLRCC) is a syndrome characterized by leiomyomas and an aggressive form of papillary kidney cancer. The genetic driver of HLRCC is a loss-of-function mutation of the Krebs cycle enzyme fumarate hydratase (FH), resulting in increased levels of the Krebs cycle intermediate fumarate, and resulting impaired oxidative phosphorylation. as well as in stabilization of HIF1α protein, which is the final common pathway of many different types of renal cell carcinomas (RCCs). To better understand the energy sources utilized by FH-deficient cells, we examined the nutrient requirements of FH-deficient RCC UOK262 (UOK262D) cell line in comparison to UOK262 cells transfected with an FH transgene (UOK262R). Experiments with selective addition of nonessential amino acids (NEAA) from the media revealed that both UOK262D and UOK262R cell lines required asparagine (A), but not other NEAA, for growth after 96 hours of incubation in the absence of glutamine (G). A (100 uM) and G (2 mM) demonstrated an approximately equal effect on the growth rate of both FH deficient and repleted cells, which was only observed after a minimum of 96 hrs of incubation. The growth effects of A and G were additive in both cell lines. While G increased the oxygen consumption rate (OCR) and decreased glycolysis (ECAR) in both cell lines, addition of A had no additive effect on both parameters, suggesting that G is used for energy production and A is required for biomass production. Similar effects of A and G, alone and in combination, on proliferation were seen in FH deficient and repleted UOK268 cell lines and in the SDH deficient and repleted UOK269 cells. Both A and G activated the mTOR pathway in both UOK262D and UOK262R cell lines as indicated by increased phosphorylation of mTOR, S6K and S6RP at 48h. However, mTOR pathway activation was not observed after A and/or G exposure in UOK268 or UOK269 pairs of cell lines. To determine the genomic response of UOK262 cell lines to A and/or G, we conducted RNASeq analysis in cells incubated for 96 hours to A, G, both or neither. In UOK262D cells, A+G, but not A or G individually, increased RNA levels of many genes associated with endoplasmic reticulum stress response. These gene expression changes were not seen in the FH repleted cell line UOK262R incubated in A+G. Therefore, although the growth effects of A+G in UOK262R and UOK262D cells were similar, and the effects on mTOR signaling pathway effects were similar between the two cell lines, the RNASeq experiment revealed a fundamentally different response to the combination of the 2 amino acids in the FH deficient cells vs. the FH repleted cells. In summary, we have identified asparagine as a key nutrient in HLRCC, establishing it as a potential target for a metabolically rational treatment approach.

This research was supported by the Intramural Research Program of the National Cancer Institute, NIH.

#1440

Vitamin D uptake and metabolism in breast cancer tumors: Differential expression of megalin, VDR, CYP27B1 and CYP24A.

Gabriela Valarezo,1 Victoria Ortega-Hernández,1 Gonzalo Escobar-Massú,1 Wanda Fernández,2 María Paz Marzolo,1 Pilar Carvallo1. 1 _Pontificia Universidad Católica de Chile, Santiago, Chile;_ 2 _Hospital Clínico San Borja Arriarán, Santiago, Chile_.

Diverse studies have concluded that Vitamin D plays a main role of in breast cancer risk. The active metabolite of vitamin D, is a pleiotropic hormone that regulates proliferation, survival, and differentiation of many cell types, through its nuclear receptor VDR. Clinical and epidemiological studies have described the association of decreased serum concentrations of 25-hydroxy-vitamin D with a poor prognosis in patients with breast cancer. The metabolism of vitamin D is meticulously regulated through a complex process, involving the vitamin D-activating enzyme CYP27B1, responsible for the final hydroxylation step to form the active metabolite and CYP24A1, the key enzyme in the inactivation of this metabolite. It is also known that VDR is central for vitamin D-mediated transcription regulation. For all these processes it is necessary that vitamin D enters the cell. In the kidney, internalization of the circulating vitamin D is accomplished by megalin, a membrane receptor for vitamin D, which has not been defined in mammary tissue. Until today reports published in relation to the mechanism of vitamin D uptake and its metabolism are scarce in breast cancer tumors. Our aim was to quantify the expression and to determine the localization of: megalin, VDR, CYP27B1 and CYP24A1 enzymes in breast cancer tumor tissues and normal breast tissue. For this purpose, tissue microarrays of formalin-fixed and paraffin-embedded tumor samples and normal breast tissues were analyzed through immunohistochemistry. This study showed that megalin is localized apically in epithelial cells of normal breast tissue, as previously described in the kidney. On the other hand, in breast cancer tumors megalin expression was diminished. Interestingly, we found that megalin had a different localization in two breast cancer tumor subtypes. In luminal breast cancer tumors, megalin was localized mainly in the cellular membrane whereas in triple negative tumors we observed a cytoplasmic localization. For VDR, CYP27B1 and CYP24A1 our results showed that all these proteins are expressed in normal breast tissues. VDR and CYP24A1 have a heterogeneous expression in relation to tumor subtypes and these are highly expressed in all luminal breast cancer tumors, whereas in triple negative breast cancer subtype its expression was low. Our results suggest that exists an alteration in classical metabolic pathway of vitamin D in the tumor subtypes analyzed of breast cancer. With these results we can suggest that in luminal tumors, which have the better prognosis compared with other subtypes, tumor cells still to respond to vitamin D, while in triple negative subtype, the tumor cells reduce their ability to: uptake, catabolize the active metabolite and respond to vitamin D favoring tumor progression.

#1441

MYC expression promotes lipid metabolism and metabolic plasticity in human mammary epithelial cells.

Jessica C. Casciano,1 Adam Cohen-Nowak,1 Johan Vande Voorde,2 Qifeng Zhang,3 Susan Chalmers,4 Mairi Sandison,4 Ann Hedley,2 Tony McBryan,2 Thomas Beer,1 Hsin-Yao Tang,1 David W. Speicher,1 Peter Adams,5 Xiufeng Liu,6 Richard Schlegel,6 John McCarron,4 Michael J. Wakelam,3 Eyal Gottlieb,2 Zachary T. Schug1. 1 _The Wistar Institute, Philadelphia, PA;_ 2 _The Beatson Institute, Glasgow, United Kingdom;_ 3 _The Babraham Institute, Cambridge, United Kingdom;_ 4 _Strathclyde Institute of Pharmacy and Biomedical Sciences, Glasgow, United Kingdom;_ 5 _Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA;_ 6 _Georgetown University Medical Center, Washington, DC_.

MYC is one of the most commonly mutated and highly amplified oncogenes in human breast cancer. MYC amplifications occur most frequently in triple-negative breast cancers (TNBCs). TNBCs can be divided into two molecular subtypes: basal-like and claudin-low breast cancers. These cancers tend to be extremely aggressive and are strongly associated with disease recurrence, poor prognosis and high mortality. In particular, claudin-low tumors are classified by a loss of tight junctions and cell-to-cell contacts and an enrichment for genes associated with an epithelial-to-mesenchymal transition (EMT) and mammary stem cells (also known as tumor-initiating cells). Despite the high level of disease severity, there are no targeted therapies for claudin-low TNBCs. To address this unmet need, we utilized human mammary epithelial cells (HuMECs) that express oncogenic levels of MYC and a mutant MYC (T58A) to characterize the behavioral and metabolic changes that occur during the formation of MYC-driven breast cancers. We found that MYC regulates the expression of genes associated with cell stemness, EMT, lipid metabolism, and calcium (Ca2+) signaling and that the expression of this gene signature promotes cell growth, survival, migration, and metabolic plasticity. The gene signature of MYC-expressing HuMECs highly correlates with the gene signature of claudin-low breast cancers, therefore highlighting the relevance of our HuMEC model to human claudin-low breast cancer. We found the major drivers underlying the MYC-dependent changes in cell behavior to be stimulation of Ca2+ signaling and strong activation of lipid metabolism. Ca2+ signaling is stimulated through the MYC-dependent repression of Ca2+ efflux mechanisms; elevated cytosolic Ca2+ then consequently stimulates a Ca2+/calmodulin kinase kinase 2 (CAMKK2)/AMPK signaling axis that activates fatty acid scavenging and transport, as well as β-oxidation. Enhanced lipid metabolism thereby provides the necessary biomass (fatty acids) for phospholipid biosynthesis and energy (ATP) to support the metabolically demanding processes of cell growth, proliferation, and migration. In all, our findings provide a strong rationale for targeting lipid metabolism and the Ca2+/CAMKK2/AMPK signaling axis in MYC-driven, and potentially claudin-low, breast cancers.

#1442

Novel linkage of thymidine catabolism and the glycolytic pathway in human cancer cells.

Sho Tabata,1 Masatatsu Yamamoto,2 Hisatsugu Goto,3 Yasuhiko Nishioka,3 Masaru Tomita,1 Tomoyoshi Soga,1 Tatsuhiko Furukawa,2 Shin-ichi Akiyama4. 1 _Institute for Advanced Biosciences, Keio University, Tsuruoka, Japan;_ 2 _Graduate School Medical and Dental Sciences, Kagoshima University, Kagoshima, Japan;_ 3 _Tokushima University Graduate School, Tokushima, Japan;_ 4 _Clinical Research Center, National Kyushu Cancer Center, Fukuoka, Japan_.

Thymidine phosphorylase (TP), a rate-limiting enzyme in the thymidine catabolic pathway, catalyzes the reversible conversion of thymidine to thymine and 2-deoxy-D-ribose 1-phosphate (DR1P). We previously demonstrated that TP is identical to an anigogenic factor, platelet-derived endothelial cell growth factor (PD-ECGF). Although many reports indicate that TP has angiogenic activity and plays a pivotal role in tumor progression, the mechanism of the TP functions is not fully understood. Here, we find that TP-mediated thymidine catabolism can supply the carbon source in the glycolytic pathway and thus contribute to cell survival under conditions of nutrient deprivation. In certain procaryotes, phosphopentosemutase converts thymidine-derived DR1P to 2-deoxy-D-ribose 5-phosphate (DR5P), which is then converted to glyceraldehyde 3-phosphate (GAP) and acetaldehyde by deoxyriboaldorase (DERA). Given that GAP is an intermediate in the glycolytic pathway and the pentose phosphate pathway (PPP), we hypothesized that the activation of thymidine catabolism by TP could supply the carbon source for those pathways. However, the thymidine catabolic pathway in mammalian cells remains unclarified. We used 13C-labeled thymidine (13C5-thymidine) as a tracer and examined whether 13C5-thymidine-derived metabolites enter the glycolytic pathway and the PPP in TP-expressing cancer cells using capillary electrophoresis time-of-flight mass spectrometry. Thymidine was converted to metabolites, including glucose 6-phosphate, lactate, 5-phospho-α-ribose 1-diphosphate, and serine, via the glycolytic pathway both in vitro and in vivo. These thymidine-derived metabolites were required for the survival of cells under low-glucose conditions. Furthermore, enhanced thymidine catabolism was observed in human gastric cancer. These findings suggest that the thymidine catabolism plays an important role in the survival and growth of cancer cells in a microenvironment where glucose is insufficient.

#1443

Overexpression of CD36 promotes tumorigenesis in colorectal cancer.

James M. Drury, Naser Jafari, B Mark Evers, Yekaterina Y. Zaytseva. _Univ. of Kentucky, Lexington, KY_.

Fatty acid translocase (CD36), a multifunctional glycoprotein, has an important role in fatty acid metabolism as a fatty acid receptor and transporter. The presence of CD36 positive metastasis-initiating cells correlates with a poorer prognosis in glioblastoma and oral carcinoma. Fatty acid synthase (FASN), a critical enzyme involved in de novo lipogenesis, is upregulated and associated with poorer prognosis in many cancers including colorectal cancer (CRC). The role of CD36 in primary and metastatic CRC as well as its relation to de novo fatty acid synthesis is not understood. The purpose of our study was: (i) to determine the role of CD36 in primary and metastatic CRC, and (ii) to delineate the association of CD36 expression with FASN as a possible mechanism of resistance to FASN inhibition.

METHODS. Expression of CD36 and FASN was assessed in a CRC tumor microarray (matched normal colon and primary tumor; 56 cases) as well as matched normal colon, primary and metastatic tumors (liver [n=12] and lung metastasis [n=5]) by immunohistochemistry. CD36 expression was analyzed in control and FASN shRNA knockout CRC cells and tissues from APC/FASN/CRE mouse models by western blot. Cell proliferation was assessed in primary CRC cells established from patient derived xenografts (PDX) treated in combination with Sulfo-N-succinimidyl oleate (SSO), an irreversible inhibitor of CD36, and FASN inhibitor TVB-3664. CD36 expression levels in primary and metastatic PDX derived CRC cells were analyzed via western blot and immunofluorescence imaging.

RESULTS. CD36 is overexpressed in primary tumors as compared to normal colon mucosa and its expression positively correlates with expression of FASN. Cell proliferation was significantly reduced when CD36 was inhibited by SSO and a further reduction in cell proliferation was observed when SSO treatment was combined with TVB-3664. Treatment with SSO induced apoptotic markers such as cleaved capspase-3 and decreased survivin. Western blot analysis of primary and metastatic CRC cells showed an upregulation of CD36 expression in the metastatic CRC cells. Additionally, FASN shRNA knockdown of FASN in CRC cells and Cre recombinase-mediated intestinal deletion of FASN in an APC/FASN/CRE mouse model led to an induction of CD36 expression. Immunofluorescence imaging of primary CRC treated with TVB-3664 showed an upregulation of membrane bound CD36.

CONCLUSION. CD36 upregulation is associated with CRC progression and inhibition of CD36 decreases proliferation and survival of primary CRC cells. Correlation between expression of CD36 and FASN suggests an interconnection between CD36 and de novo lipid synthesis. Furthermore, a decrease in FASN expression is associated with an induction of CD36, suggesting a possible mechanism of resistance to FASN inhibition. Better understanding the role of CD36 may provide new therapeutic approaches for treatment of CRC patients.

#1444

RhoC regulation of inflammatory breast cancer metabolism.

Laura E. Goo, Joel A. Yates, Liwei Bao, Zhifen Wu, Sofia D. Merajver. _University of Michigan, Ann Arbor, MI_.

Inflammatory breast cancer (IBC) is an extremely aggressive and lethal type of cancer due to its ability to rapidly metastasize. Our previous reports have shown that the metabolic characteristics of IBC are altered from those of normal breast epithelial cells. Specifically, the levels of the metabolite N-acetylaspartate (NAA) are very high in the IBC-derived triple negative cell line SUM149 relative to the triple negative cancer cell line MDA-MB-231. Recent studies have also correlated high levels of tumoral NAA with significantly lower survival rates in ovarian cancer.

N-acetylaspartate (NAA) is the second most abundant metabolite in the brain, only exceeded by glutamate. NAA is synthesized from aspartate and acetyl-CoA by the enzyme Asp-NAT, which is encoded for by the gene NAT8L. The catabolism of NAA to aspartate and acetate is carried out by the enzyme aspartoacyclase (ASPA). In the central nervous system (CNS), altered levels of NAA have been historically used as a marker for neurodegenerative diseases such as Canavan disease, a fatal genetic disorder caused by a deleterious mutation in the ASPA gene. However, the fundamental role of NAA, especially in the context of cancer remains elusive.

We seek to understand and describe the role of NAA in the adapted metabolic pathways of inflammatory breast cancer that enable proliferation and metastasis of the disease. Our previous work revealed that the pro-metastatic small GTPase RhoC acts as a regulator of NAA metabolism in SUM149 cells. Knockdown of RhoC using shRNA significantly decreases NAT8L expression. RhoC and RhoA knockout cell lines have been generated using CRIPSR-Cas9 in both SUM149 and MDA-MB-231 cells to further distinguish the relationship between NAA and RhoC in these different cell lines. We have conducted metabolic studies to determine the relative abundance of common metabolites, including NAA, throughout glycolysis, the pentose-phosphate pathway, and the tricarboxylic acid cycle. We have performed similar metabolomics using xenograft mice implanted with these CRISPR knockout cell lines to determine in vivo recapitulation of the NAA phenotypes. Our work ultimately hopes to identify promising therapeutic pathways of NAA as well as further characterize NAA's role as a potential oncometabolite.

#1445

Metastasis-resistant fatty liver induced by a choline-deficient diet.

Miki Nakamura,1 Atsushi Suetsugu,1 Tomoyuki Satake,1 Kosuke Hasegawa,1 Masahito Shimizu,1 Shigetoyo Saji,1 Hisataka Moriwaki,1 Robert M. Hoffman2. 1 _Gifu Univ. Graduate School of Medicine, Gifu, Japan;_ 2 _AntiCancer,Inc., San Diego, CA_.

Background: Fatty liver disease is increasing worldwide. The differences of the liver metastasis between fatty liver and normal liver remain unclear. We established the EL4-RFP lymphoma models in mice with fatty liver induced by a choline-deficient-diet (CDD) comparing the frequency of liver-metastasis of this tumor in mice with fatty and normal livers.

Material and method: C57BL/6-GFP transgenic mice were fed with a CDD in order to establish a fatty liver model. EL4-RFP malignant lymphoma cells were injected in the spleen of fatty-liver mice and normal mice. Using the Olympus SZX7 microscope and the Olympus FV1000 confocal microscope, metastases in mice with fatty liver or normal liver were imaged.

Results: EL4-RFP tumors were observed to metastasize to the liver and bone marrow as well as form ascites. Fewer metastases in the fatty liver were observed than in the normal liver. Similarly, fewer cancer-associated fibroblasts (CAFs) were observed in the fatty liver.

Conclusion: The relative metastatic resistance of the fatty liver may be due to the reduced number of CAFs in the fatty liver. The fatty liver may not be a suitable environment for CAFs which may be due to re-programmed methionine metabolism which may have occurred during the time the animals were on the CDD.

#1446

Glycogen accumulation in aggressive breast cancers under hypoxia.

Megan Ann Altemus, Joel A. Yates, ZhiFen Wu, LiWei Bao, Sofia D. Merajver. _Univ. of Michigan, Ann Arbor, MI_.

Cancers utilize diverse metabolic pathways to increase survival and proliferation; therefore, the identification of specific metabolic alterations in aggressive triple-negative and inflammatory breast cancers is a compelling avenue for the development of new treatments. The ability to survive and grow in hypoxia is necessary for breast cancer cells in the center of the tumor mass or in the lymphatic system and other organ environments. Glucose reserves are typically stored in liver and muscle cells as the polysaccharide glycogen. Elevated levels of glycogen have been observed in various cancers, and recent studies have suggested that glycogen metabolism is important for promoting cancer cell survival. We hypothesize that aggressive breast cancers modulate glycogen metabolism to provide nutritional flexibility for proliferation and invasion during cancer progression. To test our hypothesis, we investigated the regulation of glycogen levels in breast cancer and normal-like cells in atmospheric and 1% oxygen. The glycogen content of six breast cancer and one normal-like cell line was measured under normoxic and hypoxic conditions. All cell lines had increased glycogen accumulation under hypoxia, but to varying degrees that did not correlate with receptor status. Expression levels of various genes involved in glycogen synthesis and degradation were determined by qRT-PCR, with no consensus expression pattern between all cell lines. Several key alterations were noted in synthesis and degradation enzymes. We will present an integrative picture of glycogen synthesis and degradation in breast cancer cells. Studies are ongoing to determine the mechanisms through which these changes occur and whether the accumulation of glycogen confers an advantage for continued survival and growth of cancer.

#1447

Aberrant tryptophan catabolism marked by high kynureninase expression contributes to immunosuppression and poor outcome in lung adenocarcinoma.

Edwin J. Ostrin, Johannes F. Fahrmann, Ichidai Tanaka, Muge Celiktas, Clemente Aguilar, Mitzi Aguilar, Jennifer B. Dennison, Eunice Murage, Satyendra C. Tripathi, Oliver Delgado, Hong Wang, Jaime Rodriguez-Canales, Carmen Behrens, Ignacio I. Wistuba, Ayumu Taguchi, Samir M. Hanash. _MD Anderson Cancer Center, Houston, TX_.

Low tryptophan levels promote tumor immune evasion by suppressing immune function. Elucidation of the immunomodulatory effects of altered tryptophan catabolism has relevance to cancer immunotherapy. Previous studies have largely focused on the role of the rate-limiting enzyme indoleamine 2,3-dioxygenase 1 (IDO1) and tryptophan depletion in the microenvironment. However, IDO1 levels are highly heterogeneous in different cancers, and numerous other downstream tryptophan catabolites have also been shown to have immunomodulatory activity. We surveyed the proteomes of 123 cancer cell lines for regulators of tryptophan metabolism which revealed overexpression of kynureninase (KYNU) in KEAP1 mutant lung adenocarcinomas. Functional analysis indicated that KYNU expression is regulated through nuclear factor, erythroid 2-like (Nrf2) activation. KYNU-high cell lines exhibited increased secretion of KYNU-derived anthranilate/3-hydroxyanthranilate. Ex-vivo studies using isolated PBMCs demonstrated that 3-hydroxyanthranilate reduced CD8+ T-cell viability. KYNU mRNA expression in three independent datasets of lung adenocarcinomas and additionally KYNU protein expression in lung adenocarcinoma tissue microarrays revealed that high KYNU expression predicts poor survival and disease recurrence. Our findings indicate that KYNU promotes immune suppression and is an independent prognostic marker for lung adenocarcinoma.

#1448

Integrated molecular analysis reveals novel SPARC-regulated metabolic programming in ovarian cancer.

Christine Naczki, Bincy John, Chirayu Patel, Ashlyn Lafferty, Alia Ghoneum, Hesham Afify, Amanda Davis, Guangxu Jin, Neveen Said. _Wake Forest Health Sciences, Winston Salem, NC_.

We have reported a tumor suppressor effect of SPARC in ovarian cancer as both host and tumor SPARC are implicated in anti-proliferative, anti-adhesive effects as well as normalization of the peritoneal tumor microenvironment. Herein, we extend our studies using a syngeneic model of peritoneal ovarian carcinomatosis to gain comprehensive insight on the effect of SPARC on metabolic programming of the ovarian cancer ecosystem. We performed comprehensive metabolomic and transcriptomic profiling of micro-dissected ID8 omental tumors that developed after intra-peritoneal injection in SP-/- and SP+/+ mice. Integrated analysis revealed that the accelerated growth of ID8 tumors in SP-/- mice was associated with metabolic programming of cancer cells with up-regulation of the genes involved in glycolysis, fatty acid oxidation, and oxidative phosphorylation. Tumors also exhibited perturbed redox homeostasis, as well as mitochondrial and ribosomal biogenesis. Comparative analysis of the syngeneic tumors with human high grade serous ovarian cancer (HGSC) revealed upregulation of the same genes involved in metabolic programming with inverse correlation with SPARC transcript expression, implying a role of stromal-SPARC in the metabolic programming of HGSC. Bio-energetic studies revealed that SPARC inhibits basal glycolysis, glycolytic reserve, oxygen consumption rate and mitochondrial ATP synthesis. To the best of our knowledge this is the first study that characterizes the metabolic programming of the ovarian cancer ecosystem by host-SPARC.

#1449

Aberrant expression of protein kinase D1 influences metabolic reconditioning in pancreatic cancer.

Sonam Kumari, Sheema Khan, Murali M. Yallapu, Subhash C. Chauhan, Meena Jaggi. _University of Tennessee Health Science Center, Memphis, TN_.

Objective: Pancreatic cancer is one of the deadliest diseases, and the fourth most common cause of cancer-related deaths in the United States. Protein Kinase D1 (PKD1), a serine-threonine kinase, is an important modulator of several kinase signal transduction pathways. Although, PKD1 is known to be involved in pancreatic cancer pathogenesis, the underlying signaling mechanisms are largely unknown. Therefore, we investigated how PKD1 contributes to pancreatic tumor growth and progression. Our studies suggest a novel role of PKD1 in regulating glucose metabolism in pancreatic cancer, which drives pancreatic tumorigenesis and progression.

Methods: Glucose and Lactate assays were performed in pancreatic cancer cells following PKD1 transfection. Cell culture media was collected after 48 hrs to measure the amount of glucose consumption and L-lactate production on PKD1 expression in pancreatic cancer cells using in vitro assay kits (Cayman Chemicals). Immunoblotting and qRT-PCR assays were performed to assess the expression of protein and mRNA levels, respectively, of key signaling molecules in glucose metabolism in pancreatic cancer cells. Cell proliferation and colony forming assays were performed to determine the effect of PKD1 on cell proliferation and survival under both normoxic and hypoxic conditions. In vitro functional assays for investigating migration and invasion were performed using boyden chamber and Matrigel assays, respectively, under normoxic and hypoxic conditions.

Results: Our results show that PKD1 leads to altered glucose metabolism in pancreatic cancer cells. We observed increased amount of L-lactate production and glucose consumption on PKD1 expression in cells. This indicates the alterations in glucose metabolism associated with PKD1 expression in cells. These alterations were accompanied with enhanced expression of both protein and mRNA levels of Glut-1, HIF-1α, and KRAS, which are associated with glucose metabolism. These events were observed in both normoxic and hypoxic conditions, indicating that the PKD1 modulated events are independent of oxygen tension. Furthermore, we observed enhanced invasion and migration of pancreatic cancer cells on PKD1 expression, which was further increased by addition of lactate (an end product of aerobic glycolysis). Altogether, our studies indicate a role of PKD1 as a key regulator of the glucose metabolism and promoter of pancreatic cancer oncogenesis.

Conclusion: These results suggest that PKD1 plays an important role in metabolic reprogramming of pancreatic cancer cell metabolism to induce cancer growth and enhanced cellular invasion and motility. This is a first study that suggests the involvement of PKD1 in metabolic remodeling of pancreatic cancer cells leading to enhanced tumor growth and progression.

#1450

**Intratumoral** CYP27A1 **expression in relation to cholesterol synthesis and vitamin D signaling and its association with lethal prostate cancer.**

Nabeela A. Khan,1 Konrad H. Stopsack,1 Emma H. Allott,2 Travis A. Gerke,3 Edward L. Giovannucci,4 Lorelei A. Mucci,4 Philip W. Kantoff1. 1 _Memorial Sloan Kettering Cancer Center, New York, NY;_ 2 _University of North Carolina, Chapel Hill, NC;_ 3 _Moffitt Cancer Center, Tampa, FL;_ 4 _Harvard T.H. Chan School of Public Health, Boston, MA_.

Background: Higher intratumoral cholesterol synthesis is associated with a worse prognosis in prostate cancer. A recent study identified the vitamin D-regulated enzyme CYP27A1, which converts cholesterol to 27-hydroxycholesterol, to negatively affect cholesterol synthesis and to be associated with biochemical recurrence. We hypothesized that low CYP27A1 expression occurs in patients with low vitamin D signaling and high intratumoral cholesterol synthesis, and that low CYP27A1 expression is associated with higher risk of lethal prostate cancer.

Methods: We studied 404 prostate cancer patients in the prospective Health Professionals Follow-up Study (HPFS) and the Physicians' Health Study (PHS) cohorts. After centralized histopathologic review, we measured tumor expression of CYP27A1, SQLE (as a measure of cholesterol synthesis), and CYP24A1 (as a proxy of vitamin D signaling) using mRNA expression profiling. In subgroups, prediagnostic plasma levels of 25-hydroxyvitamin D (25(OH)D, n = 132) and tumor protein expression of the vitamin D receptor (VDR, n = 300) were also available. Using logistic regression, we estimated odds ratios (ORs) for lethal prostate cancer, defined as prostate cancer mortality or metastases, in contrast to non-lethal disease without metastases after at least eight years of follow up.

Results: CYP27A1 expression was weakly positively correlated with expression of the vitamin D target gene CYP24A1 (r = 0.17; p < 0.001) but did not differ by plasma 25(OH)D (p-trend = 0.59) or by VDR expression (p-trend = 0.26). CYP27A1 expression was lower in tumors with higher expression of the second rate-limiting enzyme of cholesterol synthesis, SQLE (r = -0.21; p < 0.001). Tumors with higher Gleason grade had lower CYP27A1 expression (HPFS, p < 0.001; PHS, p = 0.004). Higher CYP27A1 was associated with lower risk of lethal cancer in HPFS (OR for highest vs. lowest quintile of expression, 0.31; 95% CI, 0.13 to 0.73; p-trend = 0.007) and in PHS (OR, 0.10; 95% CI, 0.01 to 0.88; p-trend = 0.043) in univariable models. Combining the cohorts and adjusting for baseline clinical characteristics and SQLE, the OR was 0.30 (95% CI, 0.12 to 0.72; p-trend = 0.009). CYP27A1 was less strongly associated with lethal disease when additionally adjusting for Gleason grade (OR, 0.48; 95% CI, 0.18 to 1.31; p-trend = 0.21).

Conclusion: Low CYP27A1 expression is associated with higher cholesterol synthesis and a higher risk of progression to lethal disease among prostate cancer patients, potentially partly due to its association with higher Gleason grade. We found little evidence that intratumoral CYP27A1 expression is associated with circulating 25(OH)D. Our results highlight the importance of the regulation of cholesterol metabolism in prostate cancer progression.

#1451

A novel lipid-centrosomal axis in prostate cancer is regulated by PEDF.

Francesca Nardi,1 Philip Fitchev,1 Omar E. Franco,1 Jelena Ivanisevic,1 Adrian Scheibler,1 Simon W. Hayward,1 Yuan Ji,1 Charles B. Brendler,1 Michael A. Welte,2 Susan E. Crawford1. 1 _NorthShore University Research Institute, Affiliate of University of Chicago, Evanston, IL;_ 2 _University of Rochester, Rochester, NY_.

Background: Prostate tumor cells undergo metabolic adaptations and revert to lipid stores to meet the energy demands of their high proliferative capacity. To fuel progression, cancer cells accumulate triacylglycerol in cytoplasmic lipid droplets (LDs) and amplify cell cycle regulators such as centrosomes and non-centrosomal microtubule organizing centers (ncMTOCs). LDs not only store lipid, they also can carry cargo using microtubules to facilitate movement of proteins to various cellular locations. It is not known whether crosstalk exists between lipid storage organelles and non-membrane bound centrosomes in prostate cancer cells or stromal cancer associated fibroblasts (CAFs). We postulated that MTOCs have a metabolic sensing function in prostate cancer cells and a lipid-enriched microenvironment facilitates a PEDF-dependent metabolic switch that directly promotes centrosomal amplification. Methods: Prostate cancer cell lines, PC-3 and LNCap, normal fibroblasts and CAFs derived from human prostate cancer specimens were analyzed for TAG regulating proteins, PEDF, adipose triglyceride lipase (ATGL), comparative gene index-58 and GO/G1 switch gene 2 by western blot and immunofluorescence (IF). Centrosomes were stained with matrix proteins, pericentrin and γ-tubulin and quantified by IF. Cells were treated with recombinant PEDF or a diacylglycerol transferase I (DGAT1) inhibitor. Lipid storage was assessed in human prostate tumor tissue from various Gleason scores (n=120) by quantifying LD density using stains for neutral lipid. Results: Normal prostate fibroblasts expressed high levels of PEDF and ATGL whereas only minimal expression of these proteins was detected in CAFs and prostate cancer cells. Cytoplasmic LD density in human prostate tumors increased progressively with disease grade. A plasticity in centrosomal amplification in CAFs and cancer cells was discovered when restoration of PEDF normalized the centrosomal number. A new lipid-centrosomal signaling axis emerged when use of a DGAT1 inhibitor to block lipogenesis suppressed LD density and concurrently reduced the centrosomal or ncMTOC number. A collaborative interaction between LDs and MTOCs was noted when LDs were found to carry centrosomal proteins, pericentrin and γ-tubulin, on their surface and lipolytic regulator PEDF co-localized with pericentrin. Conclusions: These results suggest that prostate CAFs are simultaneously keeping pace with their tumor cell partners by making both centrosomal and pro-lipogenic metabolic adaptations. Normalization of MTOCs by restoring PEDF or by blocking lipogenesis in prostate cancer cells or CAFs highlight a previously unrecognized plasticity in centrosomal biology. These data suggest that lipid-laden CAFs and tumors cells can modulate MTOC distribution and number by signaling through a new PEDF-dependent lipid-centrosomal axis.

#1452

The receptor tyrosine kinase EphA2 promotes glutamine metabolism in tumors by activating the transcriptional coactivators YAP and TAZ.

Deanna N. Edwards,1 Verra M. Ngwa,2 Shan Wang,1 Eileen Shiuan,2 Dana Brantley-Sieders,1 Laura Kim,2 Albert B. Reynolds,2 Jin Chen1. 1 _Vanderbilt University Medical Center, Nashville, TN;_ 2 _Vanderbilt University, Nashville, TN_.

Malignant tumors reprogram cellular metabolism to support cancer cell proliferation and survival. Although most cancers depend on a high rate of aerobic glycolysis, many cancer cells also display addiction to glutamine. Glutamine transporters and glutaminase activity are critical for glutamine metabolism in tumor cells. We found that the receptor tyrosine kinase EphA2 activated the TEAD family transcriptional coactivators YAP and TAZ (YAP/TAZ), likely in a ligand-independent manner, to promote glutamine metabolism in cells and mouse models of HER2-positive breast cancer. Overexpression of EphA2 induced the nuclear accumulation of YAP and TAZ and increased the expression of YAP/TAZ target genes. Inhibition of the GTPase Rho or the kinase ROCK abolished EphA2-dependent YAP/TAZ nuclear localization. Silencing YAP or TAZ substantially reduced the amount of intracellular glutamate through decreased expression of SLC1A5 and GLS, respectively, genes that encode proteins that promote glutamine uptake and metabolism. The regulatory DNA elements of both SLC1A5 and GLS contain TEAD binding sites and were bound by TEAD4 in an EphA2-dependent manner. In patient breast cancer tissues, EphA2 expression positively correlated with that of YAP and TAZ, as well as that of GLS and SLC1A5. Although high expression or EphA2 predicted enhanced metastatic potential and poor patient survival, it also rendered HER2-positive breast cancer cells more sensitive to glutaminase inhibition. The findings define a previously unknown mechanism of EphA2-mediated glutaminolysis through YAP/TAZ activation in HER2-positive breast cancer and identify therapeutic targets in patients.

#1453

SLC25A32 sustains cancer cell proliferation by regulating flavin adenine dinucleotide (FAD) metabolism.

Valeria Santoro,1 Ilya Kovalenko,2 Kim Vriens,3 Stefan Christen,3 Andreas Bernthaler,1 Andrea Haegebarth,1 Sarah-Maria Fendt,3 Sven Christian1. 1 _Bayer AG, Berlin, Germany;_ 2 _University of Michigan, Cancer Center, Ann Arbor, MI;_ 3 _VIB Center for Cancer Biology, Leuven, Belgium_.

SLC25A32 is a member of the solute carrier 25 family of mitochondrial transporters. SLC25A32 transports tetrahydrofolate (THF) as well as FAD into mitochondria and regulates mitochondrial one-carbon metabolism and redox balance. While it is known that cancer cells require one-carbon and FAD-dependent mitochondrial metabolism for generation of nucleotides, lipids and for maintenance of redox homeostasis to sustain growth, the role of SLC25A32 in cancer cell survival remains unexplored.

Our results indicate that the SLC25A32 gene is highly amplified in different tumors and that its amplification correlates with increased mRNA expression and with reduced patient survival. SiRNA-mediated knock-down and CRISPR-mediated knock-out of SLC25A32 in cancer cells of different origins, resulted in the identification of sensitive and resistant cells to SLC25A32 inhibition. Mechanistically, tracing of deuterated serine upon SLC25A32 knock-down did not affect the mitochondrial/cytosolic folate flux as measured by Liquid Chromatography coupled Mass Spectrometry (LC-MS). Instead, SLC25A32 inhibition resulted in respiratory chain dysfunction at the FAD-dependent complex II enzyme, induction of Reactive Oxygen

Species (ROS) and depletion of reduced glutathione (GSH), impairing cancer cell proliferation. Moreover, Buthionine sulfoximine (BSO) treatment further sensitized cells to ROS-mediated inhibition of cell proliferation upon SLC25A32 knock-down. Treatment of cells with the FAD precursor riboflavin and with GSH rescued cancer cell proliferation upon SLC25A32 down-regulation.

Our results indicate that inhibition of mitochondrial FAD transport by targeting SLC25A32 has potential clinical applications as a single agent or in combination with approved cancer drugs that lead to increased oxidative stress and reduced tumor growth.

#1454

Glutamate dehydrogenase 1 mediates metabolic reprogramming and promotes tumor metastasis through CamKK2-AMPK signaling in LKB1-deficient lung cancer.

Sumin Kang, Lingtao Jin, Chaoyun Pan, Jaemoo Chun. _Emory Univ. Winship Cancer Inst., Atlanta, GA_.

Although elevated glutaminolysis has been demonstrated in cancer cells, the precise mechanism by which glutaminolysis is exploited to promote tumor metastasis has not been elucidated. Lung cancer frequently metastasizes and is the leading cause of cancer-related death worldwide. Loss of LKB1 is associated with increased metastasis and poor prognosis in lung cancer, but the development of targeted agents in LKB1-deficient lung cancer is still in its infancy. Our recent study reveal that glutamate dehydrogenase 1, upregulated upon detachment via the transcription factor pleomorphic adenoma gene 1 (PLAG1), provides anti-anoikis and pro-metastatic signals in LKB1-deficient lung cancer. Mechanistically, the GDH1 product alpha-KG activates CamKK2 by enhancing its substrate AMPK binding and triggers CamKK2-mediated AMPK signaling, which contributes to energy production that confers anoikis resistance. The effect of GDH1 on AMPK is evident in LKB1-deficient lung cancer, where AMPK activation predominantly depends on CamKK2. Targeting GDH1 with the small molecule GDH1 inhibitor R162 led to attenuated anoikis resistance and tumor metastasis in an LKB1-deficient lung patient-derived xenograft model. Correlation studies between GDH1 signaling and metastatic cancer progression in lung cancer patients further validated the clinical relevance of our finding. Our study provides insight into the molecular mechanism by which GDH1-mediated metabolic reprogramming of glutaminolysis mediates lung cancer metastasis. This knowledge could be leveraged for the development of effective therapeutic strategies for patients with LKB1-deficient lung cancer.

#1455

FAK overexpression upregulates glucose metabolism in glioblastoma.

Kiran Kumar Velpula, Michael Pajor, Maheedhara R. Guda, Collin M. Labak, Simon Park, Andrew J. Tsung. _Univ. of Illinois College of Med. at Peoria, Peoria, IL_.

Glioblastoma (GBM) is the most aggressive brain cancer, afflicting nearly 1 in 6 adult patients with brain tumors. Currently, no effective curative treatment for this CNS neoplasm exists. The high prevalence and dismal prognosis of GBM increases the need to identify novel therapeutic targets. Previous research has suggested that increased expression of focal adhesion kinase (FAK) may play a role in tumor metabolism. Our preliminary data using datamining approach of the TCGA and Oncomine™ databases showed that mRNA levels of FAK correlate with expression levels of the glucose transporter GLUT1. Subsequent mass-spectrometric and immunoprecipitation experiments provided evidence that FAK binds to GLUT1, while immunohistochemistry and immunocytochemistry experiments confirmed their interaction. Western blot analysis using shRNA to silence FAK demonstrated a significant decrease in GLUT1 expression. Cells treated with the same FAK shRNA showed decreased metabolites from anaerobic glycolysis, instead relying on oxygen-dependent metabolism utilizing oxidative phosphorylation for their energy supply. Collectively, we propose that FAK is a viable target in modulating glucose uptake and metabolism in GBM.

#1456

Integrin beta4-induced mitophagy promotes the lactate production of cancer-associated fibroblasts in breast cancer.

Jin Sol Sung, Suki Kang, Joo Hyun Lee, Seong Gyeong Mun, Baek Gil Kim, Nam Hoon Cho. _Yonsei University College of Medicine, Seoul, Republic of Korea_.

Integrin beta4 neoexpression is induced in cancer-associated fibroblasts (CAFs) by some triple negative breast cancer cells (MDA-MB-231 and BT-20) through a contact-dependent manner. Integrin beta4 expression is well known to contribute to the migration and survival of cancer cells. However, there is very little known about the role of integrin beta4 expression in CAFs, especially in the aspect of tumor progression. In our study, we found that the exogenous expression of integrin beta4 increased the lactate production of CAFs. The observation made us to hypothesize that integrin beta4 neoexpression is associated with the mitophagy, a cause for aerobic glycolysis, in CAFs. To confirm the assumption, the expression alteration of mitophagy-related genes was first screened in the CAF transfected with integrin beta4 overexpression plasmid. BCL2 interacting protein 3 like (BNIP3L) and microtubule associated protein 1 light chain 3 alpha-II (LC3-II) were significantly upregulated in the integrin beta4 overexpressing CAFs, which was reversed both in the knockdown of integrin beta4 expression and the treatment with an integrin beta4 inhibitor. Autophagosome maturation and lysosomal fusion were also increased in the integrin beta4 overexpressing CAFs compared to the control. Phosphorylation level of c-Jun was upregulated by integrin beta4 overexpression in CAFs and was able to bind to the putative promoter regions of BNIP3L and LC3-II. Taken together, these results demonstrate that integrin beta4 neoexpression induces upregulation of BNIP3L and LC3-II ultimately leading to mitophagy in CAFs, contributing to cancer metabolism via mitophagy-induced lactate production, namely reverse Warburg effect.

#1457

SCAP/SREBP-1 regulates lipid metabolism reprogramming in cancer cell.

chunming cheng, feng geng, xiang cheng, arnab chakravarti, deliang guo. _The Ohio State University Comprehensive Cancer Center, columbus, OH_.

Warburg initial observation shows tumorigenesis is associated with an increased glucose consumption. Recently, accumulating evidence show that lipid metabolism is reprogrammed in cancers. Elevated lipogenesis which contributes to the development of malignant phenotypes is known to meet requirements for cancer cell rapid growth. However, how they interact to promote cancer growth remain unclear.

Through biochemistry and molecular biology assays, we find that EGFR signaling, by increasing glucose uptake, promotes N-glycosylation of sterol regulatory element-binding protein (SREBP) cleavage-activating protein (SCAP) and consequent activation of SREBP-1, an ER-bound transcription factor with central roles in lipid metabolism. N-glycosylation promotes SCAP stabilization and dissociates from Insig-1, allowing movement of SCAP/SREBP to the Golgi and consequent SREBP activation. Blocking SCAP N-glycosylation ameliorates EGFRvIII-driven glioblastoma growth in xenograft mouse model.

These results show glucose consumption and lipogenesis are linked as reciprocal metabolic cross-talk in tumorigenesis, and targeting SCAP and its N-glycosylation may provide a promising means of more effective cancer therapy.

#1458

TRPM2 modulates neuroblastoma cell survival through Pyk2, CREB and MCU activation.

Iwona Malgorzata Hirschler-Laszkiewicz,1 Shu-jen Chen,1 Lei Bao,1 JuFang Wang,2 Xue-Qian Zhang,2 Santhanam Shanmughapriya,2 Kerry Keefer,1 Muniswamy Madesh,2 Joseph Y. Cheung,2 Barbara A. Miller1. 1 _Penn State Univ., Hershey, PA;_ 2 _Temple University, Philadelphia, PA_.

Transient Receptor Potential Melastatin subfamily member 2 (TRPM2) is highly expressed in many cancers including neuroblastoma. Proline-rich tyrosine kinase 2 (Pyk2) is calcium-sensitive non-receptor tyrosine kinase which enhances cell survival and is activated by calcium influx through TRPM2. Pyk2 also modulates mitochondrial calcium uptake through phosphorylation of the mitochondrial calcium uniporter (MCU), impacting mitochondrial function, and through its effects on CREB, which regulates transcription of MCU. The purpose of this work was to investigate the mechanisms by which TRPM2 regulates cancer cell survival in order to develop new therapeutic strategies based on inhibition of TRPM2. Two models of TRPM2 inhibition were utilized: 1) reducing TRPM2 function in SH-SY5Y cells by expression of the dominant negative construct TRPM2-S, a short isoform of TRPM2, and 2) depletion of TRPM2 expression with CRISPR/Cas9 technology. Treatment with Doxorubicin (Doxo) was used as a model of oxidative stress. Doxorubicin reduced growth of cells in which TRPM2 was inhibited significantly more than control cells. Inhibition of TRPM2 also reduced Pyk2 and CREB expression and activation. Using shRNA technology we showed that Pyk2 mediated reduced expression and activation of CREB and activation of STAT3 in TRPM2 expressing cells. Fractionation experiments showed that depletion of TRPM2 reduced phosphorylation of whole cell and mitochondrial Src and Pyk2 and expression of mitochondrial CREB and MCU. Src has an important role in Pyk2 phosphorylation. In TRPM2 depleted cells, mitochondrial Ca uptake was significantly reduced since both peak mitochondrial calcium uniporter currents and amount of calcium transported (current-time integral) measured in mitoplasts were lower. Wt Pyk2 but not the phospho-deficient mutant Y402F and wt TRPM2 but not the TRPM2 Ca-impermeant mutant E960D, restored viability after Doxo treatment of SH-SY5Y cells in which TRPM2 was inhibited. In conclusion, these results suggest that TRPM2 inhibition leads to reduced viability in neuroblastoma through the Src-Pyk2-CREB-MCU pathway, which plays a key role in regulating mitochondrial function and cellular bioenergetics.

### Mitosis and Telomeres

#1459

A mechanism counteracting micronucleation for maintenance of genomic integrity.

Matthias Samwer,1 Maximilian W. Schneider,2 Rudolf Hoefler,2 Philipp Schmalhorst,3 Julian Jude,4 Johannes Zuber,4 Daniel W. Gerlich2. 1 _Boehringer Ingelheim RCV, Vienna, Austria;_ 2 _Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna, Austria;_ 3 _Institute of Science and Technology Austria (IST Austria), Klosterneuburg, Austria;_ 4 _Research Institute of Molecular Pathology (IMP), Vienna, Austria_.

A hallmark of eukaryotic cells is that they store all their chromosomes in a single nucleus. This is important for the maintenance of genomic integrity, as individual chromosomes packaged into separate micronuclei are prone to massive DNA damage. Micronuclei are characteristic for cancer cells and thought to drive the evolution of cancer genomes. However, the mechanisms normally promoting the packaging of all chromosomes into a single nucleus have remained largely elusive. Animal cells undergo an open mitosis, in which the cell disassembles its nucleus to release a set of individualized chromosomes. At the end of mitosis, cells reassemble a single nucleus around a complete set of chromosomes, utilizing endoplasmic reticulum-derived membranes. How cells restrict nuclear envelope assembly to the surface around the set of anaphase chromosomes is unclear. It has been proposed that the mitotic spindle may help anaphase chromosomes form a continuous surface by bringing them into proximity. Using live cell imaging, we found that the mitotic spindle is not required to form a single nucleus in human cells. We then performed an RNAi screen for spindle-independent factors that are required to shape a single nucleus and identified the protein barrier-to-autointegration factor (BAF). BAF has been previously suggested to contribute to nuclear assembly by linking chromatin to nuclear envelope membranes containing "LEM" domain (Lap2/Emerin/Man1) proteins. However, by RNAi and mutational analysis, we unexpectedly discovered that BAF's LEM-binding activity was dispensable for nuclear assembly in human cells. Instead, we found that BAF's ability to dimerize and cross-bridge distant DNA segments was essential to shape a single nucleus. By probing purified chromatin and recombinant BAF proteins with atomic force microscopy, we found that BAF forms a rigid shell at the chromatin surface, depending on its DNA-DNA cross-bridging activity. In cells, BAF restricted membranes to the surface formed by the set of anaphase chromosomes, thereby preventing the formation of micronuclei. Thus, BAF regulates chromosome mechanics to shape a single nucleus during mitotic exit.

#1460

Stathmin regulates mammary gland morphogenesis and tumorigenesis.

Ilenia Segatto, Mara De Marco Zompit, Gian Luca Rampioni Vinciguerra, Francesca Citron, Sara D'Andrea, Tiziana Perin, Gustavo Baldassarre, Barbara Belletti. _National Cancer Institute, CRO of Aviano, Aviano, Italy_.

Microtubule (MT) dynamics is essential to provide a correct apico-basal polarization of epithelial cells, by regulating the movement of vesicles and proteins within the cell and by ensuring a correct orientation of the mitotic spindle during cell division. Little is known about the role of the MT-dynamics and of MT-regulating proteins during development of the mammary gland, a dynamic organ that displays a complex and finely organized apico-basal architecture. Stathmin, a MT-destabilizing protein, is often overexpressed in breast cancer (BC). By studying knockout (STM KO) mice we observed that STM KO females were unable to nurse their offspring. This phenotype was accompanied by defective development of the mammary gland, particularly evident during pregnancy and post-partum phases. Our results showed that stathmin absence mainly impinged on two key features of the mammary epithelial organization, polarity and proliferation. In vitro, normal mammary epithelial cells silenced for stathmin were not able to form organized acini in 3D-matrices. In vivo, when stathmin was KO, many molecules involved in mammary gland functions were not properly localized, indicating a disruption of the apico-basal polarity. Furthermore, loss of stathmin strongly decreased proliferation and induced disoriented positioning of the mitotic spindle in dividing epithelial cells, preventing the correct orientation of the daughter cells. Loss of polarity and deregulated proliferation are tightly linked to tumorigenesis. We therefore examined the role of stathmin also in the context of HER2-driven tumorigenesis. To this end, we used a transgenic mouse model expressing a constitutively active form of HER2 (Δ16HER2), which is an alternatively spliced, very aggressive form of HER2, and intercrossed them with STM WT and KO mice. We analyzed the role of stathmin in both early and late stages of tumorigenesis. STM KO mice displayed a significantly reduced number of pre-neoplastic foci and this phenotype was maintained also in late stages of tumorigenesis. In WT mice, pre-neoplastic lesions expressed high level of stathmin compared with normal tissue supporting the idea that stathmin played an active role in tumor initiation. Analysis of proliferation in tumors revealed that loss of stathmin strongly decreased the mitotic rate of Δ16HER2-transformed cells. Furthermore, we assessed that stathmin plays a cell autonomous function in tumor initiation. Our study has shed new light in the field of mammary gland development and of breast cancer biology. Collectively, our data indicate that loss of stathmin by altering the dynamics of microtubules, causes a profound disorganization of the normal mammary gland architecture and a delay in initiation of the tumorigenic process. We have uncovered new functions of stathmin that could have important implications in BC, also in consideration that several microtubules acting drugs are currently employed in the therapy of BC patients

#1461

Targeting mitotic kinases in breast cancers in Latinas.

Yainyrette Rivera-Rivera,1 Mihaela Marina,2 Jamie King,3 Miyoung Lee,4 Harold I. Saavedra1. 1 _Ponce Health Sciences University, Ponce, Puerto Rico;_ 2 _Meditech Media, Atlanta, GA;_ 3 _Emory University, Atlanta, GA;_ 4 _Aflac Cancer and Blood Disorders Center, Atlanta, GA_.

African Americans and Latino women (Latinas) in the US have higher risks of developing molecular subtypes of breast cancer that associate with poor-prognosis, including hormone receptor-negative (HR-): triple-negative (ER-PR-Her2-), and Her2-positive breast cancer relative to whites. These women also present with higher-stage breast tumors relative to whites. We specifically study Puerto Rican women, a population in which breast cancers are the primary cause of cancer-related death, and who display a higher frequency of HR- breast tumors than other Latinas. We propose that chromosome instability (CIN) is a principal contributor to poor prognosis of breast cancers in African Americans and Latinas, since HR- breast cancers accumulate the highest CIN of all breast cancer subtypes. CIN in cancers correlates with poor clinical outcomes and centrosome amplification (CA) -defined as the presence of 3 or more centrosomes within a cell-, and defective mitosis are two principal drivers of CIN. Albeit CA correlates with invasion, metastasis, poor clinical outcomes, and HR- subtypes, molecular mechanisms responsible for CA in breast cancers are still unknown. We have identified MPS1 and NEK2 as centrosomes/mitotic kinases that drive CA/CIN in HR- breast cancer cells, and that can potentially be targeted to curb the malignant behavior of the most aggressive breast cancer subtypes affecting ethnic minorities. MPS1 and NEK2 are upregulated in HR- breast tumors and are co-overexpressed in several predictive signatures of poor prognosis subtypes and metastasis. Our preliminary data indicated that these kinases are overexpressed in over 60% breast cancers from Puerto Rican women and that they are exclusively co-upregulated in basal breast cancers. Our preliminary data also showed that these two mitotic kinases can significantly influence epithelial-to-mesenchymal transition (EMT) and invasion -activities never described for mitotic kinases. Also, although inactivating NEK2 suppresses S phase, inhibition of MPS1 reduces survival of breast cancer cells. Because inhibitors are available for MPS1 and NEK2, it would be feasible to target these kinases to combat metastatic HR- breast cancers. Our work will allow the identification of pathways influenced by mitotic regulators in breast cancer cells driving invasion and metastasis and will provide potential prognostic markers for high-risk breast tumors in various ethnic/racial groups, including African American and Latinas (including Puerto Rican women).

#1462

The oncogene MYC induces chromosomal instability through dysregulating mitotic progression evoking a dependency on TPX2.

Julia Rohrberg, Alexandra Corella, Moufida Taileb, Marie-Lena Jokisch, Roman Camarda, Sanjeev Balakrishnan, Alicia Zhou, Andrei Goga. _UCSF, San Francisco, CA_.

More than 70% of common solid tumors are aneuploid. Aneuploidy has been implicated in tumorigenesis for decades. A direct cause of aneuploidy is chromosomal instability (CIN) that fuels tumor evolution by providing a heterogeneous subpopulation of cells that contributes to the lethal outcome of cancer, metastasis, therapeutic failure, and drug resistance. The oncogene c-MYC is overexpressed in many cancers and has been shown to induce structural and numerical chromosomal aberrations and aneuploidy. While the role of MYC in overcoming the G1/S checkpoint is well established, the role of MYC in altering mitotic dynamics and eliciting CIN remains poorly understood. Here, we identify a direct influence of increased MYC expression on mitotic progression and spindle assembly. We observe MYC-dependent microtubule nucleation and polymerization defects in mitosis. MYC overexpression induces altered spindle morphology promoting chromosome segregation defects resulting in micronuclei formation, CIN and aneuploidy. Importantly, attenuation of MYC expression reverses the mitotic defects observed, even in established tumor cell lines, implicating an ongoing role for MYC in the persistence of a CIN phenotype in tumors. Gene expression data reveal that MYC regulates numerous genes involved in mitosis, kinetochore function and microtubule behavior. We performed a screen to identify microtubule regulators required for the survival of MYC overexpressing cells. MYC overexpressing cells are reliant on high TPX2 expression, a master microtubule regulator. High TPX2 expression is permissive for mitotic spindle assembly and chromosome segregation in MYC high cells, while TPX2 depletion attenuates cell viability and tumor growth in vivo. Our data suggest that TPX2 is a transcriptional target of MYC. We postulate that high levels of TPX2 are required to tolerate mitotic stress induced by MYC. TPX2 compensates for impaired mitotic progression and prevents the development of physiologically intolerable levels of CIN. High levels of TPX2 might be required for tumors with oncogene-induced mitotic stress to faithfully proceed through mitosis providing an explanation for the abundant upregulation of TPX2 in cancer. Thus, we describe a conditional role for MYC in altering spindle assembly and dynamics, resulting in a dependency on TPX2 for tumor survival.

#1463

Efficacy of Tumor Treating Fields (TTFields) and Aurora B kinase inhibitor.

Dietmar Krex,1 Achim Temme,1 Rosa S Schneiderman,,2 Einav Zeevi,2 Karnit Gotlib,2 Tali Voloshin,2 Moshe Giladi,2 Adrian Kinzel,3 Eilon D Kirson,2 Uri Weinberg,2 Yoram Plati2. 1 _University Hospital Carl Gustav Carus, Dresden, Germany;_ 2 _Novocure, Israel;_ 3 _Novocure, Germany_.

Tumor Treating Fields (TTFields) are an effective antineoplastic treatment modality delivered via noninvasive application of low-intensity, intermediate-frequency, alternating electric fields. TTFields is approved for the treatment of both newly diagnosed and recurrent glioblastoma. A promising approach to enhance the efficiency of TTFields is the use of drugs that extend metaphase-anaphase transition and telophase. Specifically, inhibitors or drugs interfering with components of the chromosomal passenger complex, in particular affecting Aurora B kinase, are potential candidates for combinatorial use with TTFields. The goal of the present study is to test the hypothesis that TTFields effect on tumor cells can be exaggerated by an additional inhibition of cytokinesis through chemical inhibition of Aurora B kinase. Efficacy of the combined treatment of TTFields and Aurora B kinase inhibitors (AZD1152) was tested in 3 different glioma cell lines: U87-MG, U87-MGshP53 and U-251. TTFields (1.6 V/cm RMS, 200 kHz) were applied for 72 hours using the inovitro system. AZD1152 was added to the media in concentrations of up to 100 nmol/L. Cell counts, cell cycle and clonogenic potential were determined at the end of treatment. Formation of multinuclear cells was determined using microscopic images of cells stained with crystal violet. The combined treatment of TTFields and AZD1152 led to a significant reduction in the number of U251, U-87 MG and U-87 MGshP53 cells (2-way ANOVA, p<0.001 in all three cell lines) as compared to each treatment alone. The overall effect taking into account not just the cytotoxic effect at the end of treatment, but also the clonogenic potential, demonstrated a significant reduction in U87-MG, U87-MGshP53 and U-251 cells (2-way ANOVA, p<0.001 in all 3 cell lines) as compared to each treatment alone. Microscopy images of U87-MG and U87-MGshP53 cells stained with crystal violet after treatment revealed high prevalence of multinuclear cells in cells exposed to TTFields and AZD1152 (25nM) as compared to cells treated with AZD1152 (25nM) alone. Cells treated with TTFields and higher doses of AZD1152 (50-100nM) demonstrated increased rates of pyknosis. The results presented in this work demonstrate that the combination of TTFields and AZD1152 can be an effective treatment against glioma cells. Based on the above, there is a strong rationale to continue exploring the potential of combining TTFields and AZD1152 in the clinical settings.

#1464

Telomere DNA damage links benign prostatic hypertrophy, intraepithelial neoplasia, and prostate cancer.

Jianchun Wu, David L. Crowe. _Univ. of Illinois Cancer Ctr., Chicago, IL_.

Telomere DNA damage has been demonstrated in benign prostatic hypertrophy (BPH), which is associated with prostate epithelial cell senescence. Telomere DNA damage is the most frequently observed genetic alteration in prostatic intraepithelial neoplasia, and is associated with poor clinical outcomes in prostate cancer. Gene expression database analysis revealed decreased TRF2 expression during malignant progression of the prostate gland. We reasoned that reduced TRF2 expression in prostate epithelium, by activating the telomere DNA damage response, would allow us to model both benign and malignant prostate disease. TRF2 expression was deleted in prostate epithelium using conditional null mutant mice. TRF2 expression in prostate glands and tumors was determined by quantitative reverse transcription polymerase chain reaction. Histopathology was analyzed by hematoxylin and eosin staining. Telomere DNA damage response was determined by 53BP1 localization at telomeres, and by ATM/Chk2/p53 western blotting. Androgen receptor, smooth muscle actin, keratin expression, and proliferation index were determined by immunohistochemistry. Cellular senescence was determined by histone H3K9me3 immunofluorescence microscopy. Apoptotic cells were determined by TUNEL analysis. Telomerase activity was determined by telomere repeat amplification protocol. Alternative lengthening of telomeres was determined by PML localization at telomeres and telomere circular DNA analysis. Genomic instability was determined by metaphase telomere fluorescence in situ hybridization. Sca1+/CD49f+ prostate cancer stem cells were sorted by flow cytometry and transplanted subcutaneously with urogenital sinus mesenchyme cells in Matrigel. Prostate glands with reduced epithelial TRF2 expression developed age- and p53-dependent hypertrophy, senescence, ductal dilation, and smooth muscle hyperplasia similar to human BPH. Prostate tumors with reduced TRF2 expression were classified as high grade androgen receptor negative luminal adenocarcinomas which exhibited decreased latency, increased proliferation, and distant metastases. Prostate cancer stem cells with reduced TRF2 expression were highly tumorigenic and maintained telomeres both by telomerase and alternative lengthening (ALT). Telomerase inhibition in prostate tumors with reduced TRF2 expression produced significant reduction in prostate tumor incidence by halting progression at intraepithelial neoplasia (PIN). These lesions were highly differentiated, exhibited low proliferation index, and high apoptotic cell fraction. Prostate tumors with reduced TRF2 expression and telomerase inhibition failed to metastasize and did not exhibit ALT. Our results demonstrate that the telomere DNA damage response links BPH, PIN, and prostate cancer and may be therapeutically manipulated to prevent prostate cancer progression.

#1465

Clinicopathologic characteristics of TZAP mutation and expression in hepatocellular carcinomas.

Jae-Ho Lee, Keon Uk Park. _Keimyung Univ. School of Medicine, Daegu, Republic of Korea_.

The zinc finger protein ZBTB48 is a telomere-associated factor and renamed it as telomeric zinc finger-associated protein (TZAP). It binds preferentially to long telomeres competing with TRF1 and TRF2. However, its expression in cancers has not been performed. Methods: In the present study, we analyzed TZAP mutation and expression in 123 hepatocellular carcinomas (HCC) and its association with telomere length and TERT expression was also investigated. TZAP mutations (c.1272G>A, L424L) was found in 10.6% (13/123) and TZAP expression level was not different between HCC and paired non-cancerous tissues. There was no association between TZAP mutation and TZAP expression (p = 0.53). TZAP mutation did not have any clinical and prognostic values in HCC. However, TZAP expression was positively associated with TERT expression (29.4% vs. 80.0%, P = 0.047). And, TZAP expression tended to induce poorer survival result (overall survival, χ2= 2.83, P = 0.092; disease-free survival, χ2= 3.59, p = 0.058). TCGA data also showed a positive correlation between TZAP and TERT expression levels (r = 0.839, p < 0.001). However, TCGA survival analysis showed no prognostic value of TZAP expression in HCC (p = 0.576). This result suggested that TZAP expression appears to be a possible prognosis marker dependently with TERT expression in HCC.

#1466

DAXX localizes ATRX to suppress alternative lengthening of telomeres in osteosarcoma.

Sarah Faith Clatterbuck Soper,1 Kathryn E. Yost,2 Robert L. Walker,1 Marbin A. Pineda,1 Yuelin J. Zhu,1 Joshua J. Waterfall,3 Paul S. Meltzer1. 1 _National Cancer Institute, Bethesda, MD;_ 2 _Stanford University, CA;_ 3 _Institut Curie, Paris, France_.

To maintain genome stability, proliferating cells must add telomere sequence to counteract the chromosome end replication problem. In normal cells, telomeres are lengthened through the action of the enzyme telomerase. In about 10-15% of tumors, however, telomeres are lengthened through a telomerase-independent mechanism known as Alternative Lengthening of Telomeres or ALT. Many tumors that use ALT have poor prognoses, so ALT represents an appealing therapeutic target. It has been previously observed that ALT tumors frequently carry mutations in ATRX, which partners with the protein DAXX in a chromatin remodeling complex that deposits histone variant H3.3. How these mutations facilitate the ALT pathway is not well understood. Previous work in our lab identified an ALT-positive osteosarcoma cell line, G292, in which ATRX is wild-type but DAXX has undergone a fusion event with the non-canonical kinesin KIFC3. The DAXX-KIFC3 fusion leads to a loss of DAXX function, and inducible restoration of wild-type DAXX reversibly abrogates ALT in this cell line. We observe that expression of wild-type DAXX results in localization of ATRX to PML bodies, increased occupancy of ATRX at telomeric chromatin, and higher levels of histone H3.3 at telomeres. We conclude that full-length DAXX is required for the functional localization of ATRX to telomeres. Leveraging this our inducible system, we continue to probe the role of the ATRX/DAXX complex in suppressing ALT.

#1467

Isolation and characterization of cancer cells containing ultrabright telomere DNA foci associated with alternative lengthening of telomeres (ALT): A novel utility for combined telomere-specific FISH and flow cytometry (Flow FISH).

Jacqueline A. Brosnan-Cashman, Christopher M. Heaphy, Alan K. Meeker. _Johns Hopkins University School of Medicine, Baltimore, MD_.

Telomeres consist of many kilobases of repeated TTAGGG sequences at the ends of chromosomes, protected by a sequence-specific protein cap. Telomeres shorten with each cell division and ultimately become critically short; due to their extensive proliferation, cancer cells must find a way to offset this telomere loss. Several cancer subtypes types, including neuroblastomas and sarcomas, use a telomerase-independent strategy for telomere maintenance, alternative lengthening of telomeres (ALT). The molecular event(s) through which ALT occurs in cancer remain poorly understood. Increased knowledge of these mechanism(s) is critical to our ability to effectively treat ALT(+) cancers. ALT(+) cancers harbor several hallmarks, including telomere DNA that manifests as unique, ultrabright foci by telomere-specific in situ hybridization (FISH) in histologic cancer specimens. Despite the fact that ALT-associated telomere DNA foci (ATDFs) are a reliable marker for ALT occurring in a cancer, not all cells within the cancer display them. Still, ATDFs are predicted to be the nodes for a recombination-based telomere elongation process in ALT, so may be critical for the ALT process. Despite their potential importance to the biology of ALT(+) cancers, cells containing ATDFs have not been specifically isolated and characterized. In order to study the ATDF biomarker, we have performed telomere-specific FISH on ALT(+) cancer cells in suspension, followed by isolation of the ATDF(+) cells by flow cytometry. Our approach is modified from a well-validated clinical assay ("Flow FISH") used for measuring telomere lengths in blood. Ours is the first known application of Flow FISH to solid tumor cells. We have successfully and reproducibly isolated pure populations of ATDF(+) and ATDF(-) cells from two well-characterized ALT(+) cancer cell lines: SAOS2 (an osteosarcoma cell line) and SK-N-FI (a neuroblastoma cell line). Thus, we have overcome the technical barriers that are necessary to study ATDF(+) cancer cells in order to understand this putative biomarker and its mechanistic role in ALT-mediated telomere maintenance. Work is ongoing to study ATDF(+) versus ATDF(-) ALT-positive cancer cells through gene-expression profiling and proteomic analysis to 1) determine the existence of actionable pathways that will allow for therapeutic targeting of ATDF(+) cells, and 2) examine the mechanistic link of ATDFs to the ALT process. Therefore, for the first time, we have developed an approach to effectively and specifically study the population of cancer cells containing an ALT-specific biomarker. Through this ongoing work on multiple cancer types, we will unlock critical information about the global ALT mechanism, which will provide novel, actionable targets for ALT(+) cancers.

#1468

ZBTB48 is both a vertebrate telomere-binding protein and a transcriptional activator.

Arne Jahn,1 Grishma Rane,2 Maciej Paszkowski-Rogacz,1 Sergi Sayols,3 Alina Bluhm,3 Chung-Ting Han,3 Irena Draškovič,4 Arturo Londoño,4 Alan Prem Kumar,2 Frank Buchholz,1 Falk Butter,3 Dennis Kappei2. 1 _Medical Systems Biology, UCC, University Hospital and Faculty of Medicine Carl Gustav Carus, TU Dresden, Dresden, Germany;_ 2 _Cancer Science Institute of Singapore, National University of Singapore, Singapore, Singapore;_ 3 _Institute of Molecular Biology (IMB) gGmbH, Mainz, Germany;_ 4 _Telomeres & Cancer Laboratory, Paris, France_.

Telomeres consist of repetitive TTAGGG repeats and together with the shelterin complex protect the ends of linear chromosomes from DNA repair mechanisms. Due to telomere shortening with every cell division proliferating cells need to ensure telomere maintenance by telomerase or in about 15% of cancer cells via the recombination based alternative lengthening of telomeres. Recently ZBTB48 was identified as a novel telomere-associated factor throughout the vertebrate lineage. Here we show that ZBTB48 binds directly to telomeres in vitro and in vivo, independently of the telomere maintenance mechanism. Loss of the protein leads to longer telomeres indicating a role as negative regulator of telomere length. Furthermore RNAseq, ChIPseq and quantitative expression proteomics demonstrate a function as transcriptional activator for a small set of target genes, including mitochondrial fission process 1 (MTFP1). This regulatory role links its telomeric function to mitochondrial metabolism and might play a role in cancer development. In summary ZBTB48 is a novel telomere binding protein which regulates telomere length and transcriptional activity.

#1469

T-oligo mediates DNA damage responses by modulating telomere associated proteins and telomerase.

Nabiha Haleema Khan,1 Zachary Schrank,2 Joseph Kellen,2 Sanjana Singh,1 Chike Osude,1 Neelu Puri,1 Gagan Chhabra3. 1 _University of Illinois College of Medicine, Rockford, IL;_ 2 _Augustana College, Rock Island, IL;_ 3 _University of Wisconsin, WI_.

T-oligo, a guanine-rich oligonucleotide (GRO) homologous to the 3ʹ overhang of telomeres, elicits potent DNA-damage responses (DDRs) in cancer cells. However, the detailed molecular mechanism of T-oligo in cancer cells remains elusive. Recent studies confirmed that T-oligo can form intermolecular G-quadruplexes (G4), which are stabilized by hydrogen bonding of guanine residues, and cause disruption of the protective shelterin complex of telomeres. We hypothesize that single-stranded (SS) T-oligo and G4 T-oligo may modulate the shelterin proteins TRF2 and POT2 and thus induce DDRs. In this study, we utilized a pull-down assay using T-oligo, showing that T-oligo is co-localized with TRF2 and POT1 and indicating that T-oligo may interact with these telomeric proteins. We further investigated the modulation of these proteins by western blotting, showing that T-oligo treatment upregulates TRF2 by 2.2 and 3.0-fold (p<0.01) at 48h and 72h, respectively, and POT1 by 3.0-fold (p<0.02) both at 48h and 72h in melanoma cells (MM-AN). Immunofluorescence studies confirmed upregulation of TRF2 (2.4-fold) and POT1 (2.0-fold). Additionally we found that T-oligo can co-localize with telomere binding proteins TRF2 (88.4±4.5%) and POT1 (84.5±8%) using immunofluorescence.

Using qRT-PCR, we found that T-oligo inhibited mRNA expression of hTERT, a catalytic subunit of telomerase, by 50%. It has been suggested that JNK activation may lead to downregulation of hTERT, hence we investigated the effect of the JNK inhibitor SP600125 on hTERT expression and found that treatment with SP600125 in presence of T-oligo partially reversed the downregulation of hTERT. We found a 16% decrease in hTERT expression in comparison to 50% reduction by T-oligo treatment alone. Recently, it has been reported that the novel drug 6-Thio-2'-Deoxyguanosine (6-thio-dG), a nucleoside analogue of the approved drug 6-thioguanine, inhibits growth of cancer cells by its incorporation into the telomere via telomerase followed by subsequent shelterin disruption. Furthermore, this study aims to investigate the potential of 6-thio-dG in combination with T-oligo as an anticancer therapeutic. We first studied the concentrations of 6-thio-dG that are effective in inhibiting the growth of melanoma cells. We found that 1.25μM and 2.5μM 6-thio-dG significantly inhibited growth of melanoma cells by 1.6 and 3.8-fold, respectively (p<0.01). However, treatment of melanoma cells in combination with both 10μM T-oligo and 6-thio-dG (1.25μM or 2.5μM) did not significantly inhibit cell growth in comparison to T-oligo alone. T-oligo may downregulate hTERT, which is required for telomerase activity, and/or disrupt the shelterin complex, both of which are necessary for 6-thio-dG mediated inhibition of growth of melanoma cells. These results indicate that T-oligo and 6-thio-dG may induce their effects by a similar mechanism of action.

#1470

Relative telomere length in tumor tissue and adjacent mucosa of colorectal cancer patients.

Michal Kroupa,1 Vaclav Liska,1 Krishna Rachakonda,2 Marketa Urbanova,3 Michaela Schneiderova,4 Katerina Jiraskova,3 Ondrej Vycital,1 Ludmila Vodickova,5 Rajiv Kumar,2 Pavel Vodicka5. 1 _Faculty of Medicine in Pilsen, Charles University, Pilsen, Czech Republic;_ 2 _German Cancer Research Center, Heidelberg, Germany;_ 3 _First Faculty of Medicine, Charles University, Prague, Czech Republic;_ 4 _General University Hospital in Prague, Prague, Czech Republic;_ 5 _Institute of Experimental Medicine, The Czech Academy of Sciences, Prague, Czech Republic_.

Colorectal cancer (CRC) represents a major public health problem, being the fourth leading cause of cancer related deaths mainly due to the late diagnosis and a lack of proper prognostic markers. According to the current knowledge, shortening of telomeres is important contributor in cancer onset. Telomeres, repetitive TTAGGG sequences and boud proteins, are structures at the end of all linear eukaryotic chromosomes. Telomeres are double-edged sword regarding to cancer. On one hand, they serve as a potent tumor-supressor mechanism limiting the cells ability of uncontroled growth. However, on the other hand, shortening of telomeres can lead to the formation of anaphase bridges, initiation of breakage-fusion-bridges cycles and eventually to the induction of chromosome instability phenotype. Telomeric regions are composed of microsatellite repeats and DNA mismatch repair (MMR) defects should directly affect telomere length. Studies looking into the relationship between microsatellite instability (MSI) and telomere length have been sparse. We hypothesize that CRC patients with MSI should have better prognosis, longer survival, also due to the MMR defects in telomere regions replication failure. The aim of our present study was to investigate relative telomere length (RTL) in tumor tissue and adjacent mucosa of 650 CRC patients; it is the largest study on RTL in target tissue so far. Moreover, we measured RTL in peripheral blood lymphocytes obtained from 150 cancer patients. Tumor and adjacent non-affected tissue samples were obtained from patients at surgical resection. RTL was measured using monochrome multiplex quantitative PCR method. Despite the analysis is still running, our preliminary results showed significantly shorter RTL in tumor tissues as compared to adjacent mucosa. The shortest RTL was discovered in right, ascendent part of colon (0.68±0.59, 25.5% of cases), followed by left, descendent colon (0.77±0.47, 37.6%) and the longest RTL was in rectal tumors (0.92±0.69, 36.9%). We recorded gradually increasing RTL ratio (RTL in tumor tissue/ RTL in adjacent mucosa) in later stages according to TNM staging system. Interestingly, patients with RTL ratio > 1 had also worse survival. RTL of CRC tissue and adjacent mucosa may provide a valuable prognostic tool in patients treatment management. This study was supported by GACR 15-14789S and by the National Sustainability Program I (NPU I) Nr. LO1503 provided by the Ministry of Education Youth and Sports of the Czech Republic

### Navigating the Cancer Chromatin Landscape

#1471

Genomic drivers differ across 33 tumor types, but factors regulating hallmarks of cancer are similar.

Christopher L. Plaisier. _Arizona State University, Tempe, AZ_.

The unique complement of genes expressed in a given cell type modifies the response to a specific somatic mutation. Thus, each tumor type would present with a different set of mutations that modulate the activity of common factors regulating genes which influence the cancer phenotypes (e.g. the hallmarks of cancer). We assessed the validity of this hypothesis by applying the SYstems Genetics Network AnaLysis (SYGNAL) pipeline to the ~10,000 multi-omic profiles across 33 tumor types from The Cancer Genome Atlas (TCGA). The SYGNAL pipeline synthesizes correlative approaches with mechanistic knowledge to construct a gene regulatory network. A basis of the SYGNAL approach is the discovery of biclusters: gene modules that are both co-expressed and co-regulated over subsets of tumors, by specific transcription factors (TFs) and miRNAs. On top of the biclusters we discover potential mechanistic influences of somatic mutations modulating the activity of these regulators. We discovered 284 somatically mutated genes modulating the activity of 187 transcriptional regulators (175 TFs and 12 miRNAs) which regulated 7,786 significantly co-expressed biclusters that were significantly associated with patient survival. The discovery of 1.5 fold more somatic mutations than transcriptional regulators is consistent with our hypothesis that mutations will be unique to tumor types while the regulation of the genes involved in the hallmarks of cancer will be common across tumor types. We also find that only 15% of causal flows (mutation->regulator->gene) are observed across multiple tumor types. We observed only four causal flows across three tumor types (NFE2L2->BCL6B->HSPG2 for BLCA, ESCA, and HNSC tumor types; TP53->ERG->MTX1 for BRCA, LUAD, and STAD tumor types; TP53->ZNF511->NAA38 for LUAD, PRAD, and READ tumor types; and TP53->AEBP2->THOC6 for COAD, KICH, and READ tumor types). There were no causal flows observed across more than three tumor types. Using our top-down systems approach we are able to catalog the impact of somatic mutations on the transcriptional regulatory network across 33 tumor types. We are working to replicate the co-expression and survival relationships we discovered in independent cohorts for each tumor type. In addition, we are working to identify potential therapeutic targets at both the regulator and gene level. Future studies will use predictive models based on our detailed transcriptional regulatory networks to identify transcriptional regulators driving cancer phenotypes that won't have general toxicity in normal cells.

#1472

The role of the BAF complex in Wnt signaling-mediated transcriptional regulation in colorectal cancer.

Madhobi Sen, Feda H. Hamdan, Xin Wang, Jacobe Rapp, Steven A. Johnsen. _University Medical Center, Göttingen, Göttingen, Germany_.

Recent genome- and exome-wide sequencing studies have revealed a close association between the epigenome and the pathogenesis of cancer. Not only were chromatin regulators found to be commonly mutated in a wide range of cancers, but a class of these regulators, the ATP-dependent chromatin remodelers, especially subunits of the mammalian BAF complex, were among the most frequently mutated group of genes across cancer types, mutated in over 20% of cancers. Among these subunits, loss of function mutations in ARID1A (AT-rich interactive domain-containing protein 1A) are most frequent across cancer types including 10% of colorectal cancer (CRC). Very interestingly, a recent publication described the pivotal role of the loss of ARID1A in driving CRC wherein its inactivation alone led to the formation of invasive adenocarcinomas in mice. However, surprisingly, in contrast to the expected tumor suppressive role of ARID1A in CRC, we observe that the knockout (KO) of ARID1A in CRC cell lines leads to impaired proliferation. Moreover, subcutaneous xenografts in SCID/SHO mice using ARID1A KO CRC cells did not form more aggressive tumors than their wild type counterparts. One of the most commonly occurring mutations in CRC is in the APC gene, which leads to hyperactive Wnt signaling. Thus CRC progression is typically highly dependent on this pathway. Notably, it is reported that loss of ARID1A in the context of APC mutations results in diminished tumor formation in mice and increased differentiation. This led us to hypothesize that ARID1A is required for Wnt signaling-mediated transcriptional (de)regulation in CRC. To uncover this further, we utilized several publically available ChIP-seq, RNA-seq and ATAC-seq datasets as well as our own ChIP-seq data for ARID1A in the HCT116 cell line. We observe a substantial co-localization of the BAF complex with TCF7L2, a downstream effector of the Wnt signaling pathway, suggesting cooperation between these factors. Further, these sites are also co-occupied by AP1 transcription factors. A crosstalk between the TCF7L2 and AP1 factors has been reported in intestinal tumors. From data available in the ARID1A KO system, we observe a downregulation of potential target genes which are co-occupied at neighboring sites by these factors. Therefore, we suggest that ARID1A plays an important role in the regulation of colorectal cancer relevant genes that are targets of TCF7L2/AP1, thus facilitating a pathway that is most commonly hijacked in colorectal cancer.

#1473

**Role of acetyltransferases CBP and p300 in de novo** **fatty acid synthesis in colorectal cancer.**

Aaron Waddell, Iqbal Mahmud, Daiqing Liao. _University of Florida, Gainesville, FL_.

CBP/p300 are two paralogous lysine acetyltransferases that acetylate protein substrates including histones, and serve as transcriptional co-activators for numerous signaling pathways involved in tumorigenesis and cancer progression. Recently, evidence has emerged that p300 regulates the transcription of genes involved in lipogenesis in cancers. Dysregulated lipogenesis and increased de novo production of fatty acids (FA) represent a major metabolic shift in cancer. De novo FA synthesis in cancer is suggested to be a source of lipids for membrane biogenesis, a reservoir of energy, and a source of pro-survival signaling molecules. Therefore, de novo FA synthesis has been linked to survival and increased proliferation of cancer cells, representing a potential area for therapeutic intervention. While emerging evidence indicates CBP/p300 may play a key role in regulating lipogenesis, little is known about the mechanism of how these proteins control lipid production in different cancers. Colorectal cancer (CRC) is a commonly diagnosed human cancer and is a major cause of cancer-related mortality. Key regulators for de novo FA synthesis, such as FASN, have been reported to be elevated in colorectal cancer. We analyzed publically available data sets and report that in colon adenocarcinoma, the mRNA levels of CBP/p300 positively correlate with the expression of genes involved in lipogenesis, such as FASN, SREBP1 and SREBP2. Furthermore, our analysis shows that EP300 expression is negatively correlated with patient survival. Strikingly, our preliminary data suggests that HDAC inhibitors may regulate p300 activity and could serve as a therapeutic approach for suppressing lipogenesis. We are currently utilizing small molecule inhibitors, genetic manipulation of the CREBBP/EP300 genes, and biochemical experiments to investigate the role of CBP/p300 in de novo FA synthesis in colorectal cancer. We hypothesize that inhibition of CBP/p300 could be an attractive therapeutic option for inhibiting tumor growth through downregulating de novo FA synthesis in colorectal cancer. (Supported by grants from James and Esther King Biomedical Research Program and Bankhead-Coly Cancer Research Program, Florida Department of Health.)

#1474

Identifying novel chromatin binding sites of nucleolin protein in cancer cells.

Guanhui Wu, Buket Onel, Danzhou Yang. _Purdue University, West Lafayette, IN_.

Nucleolin is an important regulator of cell proliferation and has been reported to play a role in c-Myc transcriptional regulation. Elevated expression levels of nucleolin have been shown in a number of malignant cells, but how nucleolin contributes to cancer development and progression remains unknown. Recently, G-quadruplexes (G4s) have been shown in vivo to form in G-rich sequences with biological importance. Nucleolin has been reported to bind to G-quadruplexes for its biological functions. In particular, G4s have been shown to form in the MYC promoter and inhibit MYC transcription; nucleolin was found to display a remarkable binding affinity to the c-Myc promoter G4 over its best-known RNA target. CX-3543, the first-in-class G-quadruplex-targeting small molecule, was shown to induce nucleolin redistribution from the cell nucleolus to the cell nucleoplasm and apoptosis in cancer cells, which was suggested to act through the c-Myc promoter G-quadruplex. Therefore, unbiased analysis of the genomic binding sites of nucleolin in human chromatin is pivotal in understanding nucleolin's biological functions. Using chromatin immunoprecipitation (ChIP) assay, we demonstrated that nucleolin binds to the c-Myc NHE III1 region in vivo. Using an anti-nucleolin antibody, the isolated DNA from the immunoprecipitated DNA-protein complexes from HeLa cells yielded numerous c-Myc promoter sequences that were determined by real-time PCR, suggesting the direct interaction between nucleolin and the c-Myc promoter G4-forming region in vivo. G4-interactive ligands have been shown to facilitate the formation of G4 structures in cells. To elucidate the association of nucleolin with G-quadruplexes in vivo, we monitored cellular localization of nucleolin before and after G4 ligand treatments and observed a rapid cellular translocation of nucleolin from the cell nucleolus to the cell nucleoplasm. Using ChIP coupled with next-generation sequencing analysis (ChIP-seq), we identified several binding sites of nucleolin in human chromatin. The determined chromatin binding sites of nucleolin will provide insights into the biological functions of nucleolin and its relationship with G-quadruplexes in cancer cells.

#1475

Transcription of BUB1 is regulated by the CDE and CHR elements through the cell cycle.

Fernando Luna, Marco A. Andonegui, Fernanda Cisneros, Alfredo Cantu, Luis A. Herrera. _National Cancer Institute of Mexico, Mexico City, Mexico_.

To ensure the accuracy of chromosome segregation during cell division, eukaryotic cells have developed a surveillance system, the mitotic checkpoint, which delays anaphase onset until all kinetochores are properly attached to microtubules emanating from opposite spindle poles. One of the core components of this checkpoint is Bub1 protein. Bub1 protein is encoded in the BUB1 gene, which is known to have a low basal levels of transcripts during interphase and increases its transcriptional activity in G2 and mitosis. However, the mechanisms involved in the regulation of BUB1 are not fully understood. Some of the possible components that participate in the transcriptional regulation of BUB1 are the cell cycle-dependent element (CDE) and the cell cycle homology region (CHR) element. The CDE/CHR are DNA sequences of 6 nucleotides, rich in CGs, located at proximal promoter regions of genes, and recruit repressive and activation factors during the initial and late phases of cell cycle, respectively. For instance, the promoters of CDC25C, CCNA2 and CDK1 genes contain the CDE/CHR elements and have high expression in G2 and mitosis. Thus, the aim of this study was to identify the mechanisms involved in the transcriptional regulation of BUB1 in a cell cycle dependent manner, using RT-qPCR, immunoblotting, flow cytometry, luciferase reporter gene assay, Methyl Sensitive-PCR. In this study, we unravel the mechanisms involved in the transcriptional regulation of BUB1 through the cell cycle. We found that it's expressed in a cell cycle-dependent manner in HeLa cells, being highly expressed during G2/M, followed by a rapid decline of both the mRNA and protein levels at the G1 phase. Also, we identified that the CDE/CHR elements are located at the BUB1 promoter and participate in its repression during the early phases of cell cycle, and his activation in G2 and mitosis. Finally, we found that the BUB1 promoter is located inside a CpG island which is mainly unmethylated in cancer cell lines regardless the cell type, indicating that DNA methylation might not be a determinant factor involved in its transcription. However, it's still unclear whether DNA methylation can affect BUB1 transcriptional activity through the cell cycle, particularly the methylation at specific CpG sites, such as those contained in the CDE and CHR elements. In summary, BUB1 transcription is regulated in a cell cycle dependent manner by the CDE/CHR elements located at its promoter. Also, DNA methylation is not a determinant factor implicated in its transcriptional activity.

Nevertheless, whether BUB1 transcriptional deregulation can contribute towards the development of a cancer phenotype is still unclear. Thus, this study contributes to the understanding of the mechanisms implicated on the transcriptional deregulation of cell cycle genes, such as BUB1.

#1476

Landscape of fusion RNAs generated by cis-spliced adjacent genes in cancer and normal physiology.

Fujun Qin. _Univ. of Virginia, Charlottesville, VA_.

The fusion RNA, SLC45A3-ELK4, was found to be a product of cis-splicing between the two adjacent genes (cis-SAGe). Previously, we used LNCaP, a prostate cancer cell line as a model, and identified 16 additional cis-SAGe events by silencing transcription factor CTCF and paired-end RNA sequencing. One out of the 16 fusion RNAs, D2HGDH-GAL3ST2, is more frequently seen in cancer samples, and seems to be enriched in the African American group. It is more frequently detected in late stage cancer, suggesting a role in cancer progression.

By studying the features associated with the 16 fusions, we developed a set of rules: 1) the parental genes are same-strand-neighboring genes, 2) the distance of between the genes is within 30kb; 3) the 5' genes are actively transcribing; and 4) the fusion RNAs tend to have the second-to-last exon in the 5' genes joined to the second exon in the 3' genes. To investigate the cis-SAGe fusion RNAs at global, we first randomly selected 20 neighboring genes in the genome, and detected four fusion events using these rules in prostate cancer and non-cancerous cells.

To further understand the cis-SAGe fusions in human tissues and cells, we then performed, curated and analyzed nearly 300 RNA-seq libraries covering 30 different non-neoplastic human tissues and cells. More than 10,000 fusion transcripts were found. We focused on the neighboring fusions and performed analysis. Appling our cis-SAGe rules, we identified around 1486 cis-SAGe fusion candidates. A few sequence motifs were found enriched close to the fusion junction sites. We performed functional experiments on a few widely expressed fusions, such as ADCK4-NUMBL, CLN6-CAML4, DUS4L-BCAP29, and found that silencing them resulted in dramatic reduction in moral cell growth and /or motility.

#1477

The interactomes of H3.1 and H3.3 reveal novel interactions, and associations with histone chaperones.

Scott Milos,1 Robert Siddaway,1 Sanja Pajovic,1 Eric Campos,1 Brian Raught,2 Cynthia Hawkins1. 1 _The Hospital for Sick Children, Toronto, Ontario, Canada;_ 2 _Princess Margaret Cancer Centre, Toronto, Ontario, Canada_.

Pediatric high grade gliomas are incurable brain tumors with very high mortality rates. Recent genomic studies have uncovered unique driver mutations involving histone genes encoding either H3.1 or H3.3. Histone H3 interacts with diverse variety of cellular machinery which regulates chromatin structure and function, transcription, and DNA replication. Previous efforts using IP-mass spectroscopy have revealed a great deal about H3.1 and H3.3 biology and the different interaction networks between them, including the chaperones that transport histones around the cell and incorporate them into chromatin. For example HIRA specifically deposits H3.3 into active chromatin, while CAF-1 has been shown to deposit H3.1 during DNA replication. However, these studies typically use high salt extraction, which disrupts all but the most stable protein-protein interactions. In order to more fully characterise the interactome of histone H3.1 and H3.3 in an unbiased fashion and provide insight into their potentially different roles in pediatric brain tumors, we employed proximity dependent biotinylation (BioID). We generated Flp-In HEK293 cells expressing FLAG-BirA*, alone or fused to H3.1 or H3.3, under the control of a doxycycline-inducible promoter. BirA* is a highly promiscuous biotin ligase that biotinylates proteins within a 20 nm radius. Cells were induced with doxycycline and biotin for 24 hours. SDS-based lysis and streptavidin pulldowns followed by mass-spectrometry analysis were used to identify proteins interacting with FLAG-BirA*-H3.1/H3.3 but not FLAG-BirA* alone. We validated our results by affinity purification followed by western blotting, and proximity ligation assays. FLAG-BirA*-H3 displays normal cellular localisation and is incorporated into DNA in nucleosomes of the same stability as endogenous H3-containing nucleosomes. Furthermore, chromatin stability is not changed in the presence of FLAG-BirA*-H3, showing that FLAG-BirA* fusion does not affect normal histone functions. Comparison with mass spectroscopy data indentified many previously described interactors, as well as 465 interactors not previously identified by affinity purification-mass spectroscopy, suggesting novel histone functions. The interactomes of H3.1 and H3.3 were strikingly similar, however key difference were identified. As expected, the histone chaperones NASP and ASF1 were similarly enriched in both H3 proteins, while HIRA was enriched with H3.3 vs. H3.1. Surprisingly, however, CAF-1 was not enriched in H3.1 vs. H3.3 suggesting CAF-1 may also serve as a chaperone for H3.3. Our results suggest BioID is a useful tool for unbiased interactome characterization, including proteins such as histones that require harsh extraction methods. Identification and understanding of histone H3.1 and H3.3 interactors will further aid in uncovering what role histone mutations may play in cancer initiation.

#1478

Gene-selective recruitment of NuRD drives chromatin reprogramming in cancer cells.

Adone Mohd-Sarip,1 Diana Zatreanu,2 Jeroen A. Demmers,3 C Peter Verrijzer3. 1 _Queen's University Belfast, CCRCB, Belfast, United Kingdom;_ 2 _The Francis Crick Institute, London, United Kingdom;_ 3 _Erasmus University Medical Center, Rotterdam, Netherlands_.

ATP-dependent chromatin remodelers are frequently mutated in cancers. However, the molecular basis of the association between mutations in specific remodeler subunits and particular types of cancer is poorly understood. To understand how remodelers are targeted in a cell-type specific manner, we aim to explore the mechanisms of the Nucleosome Remodeling and Deacetylase (NURD) complex during cancer progression. We recently showed that DOC1-dependent recruitment of NuRD reveals antagonism with SWI/SNF during epithelial-mesenchymal transition in oral cancer cells (Mohd-Sarip et al., 2017). It is instructive to compare the function of DOC1 in oral squamous cell carcinomas (OSCCs) with that of the SWI/SNF subunit SMARCB1/hSNF5 in malignant rhabdoid tumors (MRTs). MRTs are an extremely aggressive pediatric cancer caused by the loss of SMARCB1 (Kia et al., 2008). Although the loss of DOC1 in OSCCs or that of SMARCB1 in MRTs generates opposite epigenetic states of their target genes, in both cases, this is caused by failed remodeler recruitment. The loss of a single subunit, such as DOC1 or SMARCB1, does not abrogate all other remodeler functions. We suggest that subunit-dependent gene selection is a major cause of the association between the loss of specific remodeler subunits and particular types of cancer. Our results emphasize that gene control involves a dynamic equilibrium between opposing chromatin modulating enzymes rather than a static chromatin state. Disturbances in this balance can initiate a cascade of chromatin reprogramming events that drives oncogenesis. Such an intertwined system of epigenetic regulation suggests therapeutic strategies aimed at restoring the balance between antagonistic activities. Prompted by these findings, we will present an inclusive view of the protein regulatory network in order to gain a wholesome understanding of the molecular transactions and dynamic composition of NuRD complexes in specific cancer types. These were identified by immunopurifications using in-house antibodies followed by mass spectrometry. Our proteomic studies of the NuRD complex in flies (Reddy et al., 2010) and HeLa nuclear extracts suggest that mammalian NuRD share the same subunits with flies. Upon closer inspection, we found unanticipated novel interacting partners of NuRD and that they are altered in specific cancers namely prostate, pancreatic and ovarian. This will be followed by confirmation of the interactomes using cell-based and biochemistry assays in combination with genome-wide approaches, as well as potentially leading to the characterization of novel targets of NuRD. These findings will represent the types of multiprotein NuRD-like complexes that can form during cancer progression and how they are targeted to chromatin.

#1479

Hypoxia upregulates the long noncoding RNA MALAT1 through increased chromatin looping.

Joshua Kenneth Stone, Jung-Hyun Kim, Ming Tan, Eun-Young Erin Ahn. _Univ. of South Alabama Mitchell Cancer Inst., Mobile, AL_.

Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is a long noncoding RNA that is overexpressed in many solid tumors and promotes proliferation, angiogenesis, drug resistance, invasion and metastasis. MALAT1 contributes to oncogenesis through increased expression and splicing of cell cycle and motility-associated transcripts. While the role of MALAT1 in cancer progression is well understood, the mechanisms underlying MALAT1 overexpression in cancer are poorly characterized. Hypoxia is a known contributor to MALAT1 upregulation in multiple cancers, but the exact molecular mechanism is unknown. Here, we report that hypoxia causes changes in long-range chromatin interactions at the MALAT1 locus in breast cancer and upregulates MALAT1 in a hypoxia inducible factor (HIF)-independent manner.To determine the mechanism of hypoxic-mediated upregulation, we first cultured a panel of cell lines including one non-tumorigenic, immortalized mammary cell line (MCF10A) and eight breast cancer cell lines in normoxia (21% O2) or hypoxia (1% O2) for 24 hours. This demonstrated MALAT1 was upregulated under hypoxia in only the cancer cell lines. Chromatin immunoprecipitation and qPCR (ChIP-qPCR) demonstrated both HIF-1α and HIF-2α enrichment increased at the MALAT1 promoter in the cancer lines MCF7 and MDA-MB-231 under hypoxia, but HIF knockdown had no effect on MALAT1 expression. Through an extensive genomic data analysis, we then identified two putative enhancer elements at -21kb and -11kb upstream of the transcription start site and one downstream of the transcription termination site (+9kb enhancer). ChIP-qPCR showed an increased in active promoter mark H3K4me3 at the promoter, active enhancer mark H3K4me1 at the +9 and -11kb enhancers, and increase in RNA polymerase II occupancy at the promoter, +9, -11, and -21kb enhancers under hypoxia. To investigate whether these enhancers regulate MALAT1 expression through long-range chromatin interactions, we performed chromosome conformation capture and qPCR (3C-qPCR). The results demonstrated a hypoxic-specific increase in chromatin interaction between the enhancer elements and promoter in the cancer lines MCF7 and MDA-MB-231, which were notably absent in the non-tumorigenic line MCF10A. In conclusion, our results demonstrate a novel mechanism of MALAT1 upregulation in hypoxia specific to cancer cells. Increased chromatin looping and contacts between enhancer elements allow upregulation of MALAT1, which does not depend on HIF, suggesting other cancer-specific factor(s) guide loop formation or take advantage of this novel structure for transcription initiation.

#1480

Overexpression of histone H3.1 induced cell malignant transformation and mutation of H3.1 C96/110A further induced chromatin instability.

Yusha Zhu, Hong Sun, Max Costa. _New York University, Tuxedo Park, NY_.

Histones are major proteins in chromatin and are important for chromatin structure and gene expression. Canonical histones are defined as a group of replication-dependent histone proteins and are mainly synthesized during S phase of the cell cycle. Their mRNAs are devoid of poly-adenylated tails, but end up with a stem loop structure, which can be targeted by proteins such as stem-loop binding proteins (SLBP) for processing, exportation, and degradation. In our previous studies, arsenic exposure was found to be associated with reduced levels of SLBP and accumulation of poly-adenylated mRNA of canonical histone H3.1, and ultimately led to malignant transformation of cells. Histone H3.1 and H3.2 are canonical histones and they differ by only one amino acid, Ser96 on H3.2 compared with Cys96 on H3.1, and both of them have a Cys110. Histone H3 is the only histone that contains cysteine. Cysteine (C) is a unique amino acid that is rarely used by nature and it has a reactive thiol side chain that can easily form dimers containing disulfide bridge when oxidized. Here it is hypothesized that cysteine is vital for the structure and stability of chromatin. To test this hypothesis, we generated an H3.1 mutant H3.1 C96/110A, where two cysteine residues were replaced by alanine (A). The wild-type H3.1 and H3.1 C96/110A were exogenously expressed in the Beas-2B cells. Interestingly, Beas-2B cells ectopically expressing poly-adenylated H3.1 and H3.1 C96/110A showed decreased proliferation rate with cell cycle arrest at S phase, and cells also acquired cancer cell properties such as increased anchorage-independent growth along with migration and invasion ability. It was confirmed that H3.1 C96/110A could be incorporated into chromatin, resulting in increased chromatin instability and DNA damage. In addition, the RNA-seq assay revealed a total of 678 genes that were oppositely expressed between H3.1 and H3.1 C96/110A ectopically expressed cells. These findings emphasize the importance of a balance among histone variants in the cell and an essential role of cysteines in histone H3.1.

#1481

Selective protein expression in tumor cells controlled by progression elevated gene-3 promoter.

Ruben Pando, Tej Shrestha, Marla Pyle, Deryl Troyer. _Kansas State University, Manhattan, KS_.

The progression elevated gene-3 (PEG-3) promoter is a rodent gene that has shown strong activity in tumor progression. Demonstrating tumor specificity and activity in at least five different human cancers, PEG-3 is considered a novel promoter for tumor-specific gene expression. Understanding how PEG-3 controls expression of reporter proteins is of biomedical significance because of the role it could play in new therapeutic strategies against certain types of cancer. In this study, we aim to use the PEG-3 promoter to observe the expression of firefly luciferase 2 (luc 2), a bioluminescent reporter protein, in the transfection of mammalian cells. Ultimately, we plan to use PEG-3 to express a therapeutic gene, cytosine deaminase, in cancer specific areas of breast, lung, and ovary.

#1482

SON represses RUNX1 expression and impairs megakaryocytic differentiation in Down syndrome acute megakaryoblastic leukemia (DS-AMKL).

Lana Vukadin, Eun young Park, Jung Hyun Kim, Erin Eun Young Ahn. _University of South Alabama, Mobile, AL_.

Individuals with Down syndrome (DS) are highly predisposed to develop hematopoietic malignancies, especially acute megakaryoblastic leukemia (DS-AMKL). Generally, genes located on chromosome 21 tend to be increased in Down syndrome patients, but how these individual genes contribute to development of DS-AMKL is still unclear. SON, a gene located on chromosome 21, is upregulated in Down syndrome patients, and it has been reported that DS-AMKL cells have higher level of SON expression. The SON protein functions as a splicing factor and a transcription regulator in leukemia but the role of SON upregulation in DS-AMKL pathogenesis is still not fully elucidated. In addition, runt-related transcription factor 1 (RUNX1) is also located on chromosome 21, but its expression level is downregulated in DS-AMKL patients despite the increased gene dosage. We show that SON represses RUNX1 expression and blocks terminal megakaryocytic differentiation in DS-AMKL. Knockdown of SON in DS-AMKL cell lines leads to morphologic changes and increased cell adhesion. Moreover, cell surface marker analyses demonstrate that the depletion of SON induces increased CD41+ CD61+ populations as well as polyploidy cells, indicating megakaryocytic differentiation. Consistent with these results, SON deletion causes upregulation of RUNX1, which is a critical factor in the megakaryocytic differentiation. Furthermore, our data show that knockdown of SON increases several megakaryocytic transcription factors and decreases erythropoietic transcription factors, facilitating terminal megakaryocytic differentiation of leukemic blasts. Mechanistically, SON binds to the proximal promoter region as well as two intronic enhancer regions upstream of the proximal promoter at the RUNX1 gene locus, and suppresses transcription of RUNX1 by lowering the level of H3K4me3. Taken together, we propose that the increased genomic dosage of SON in Down syndrome impairs terminal differentiation of megakaryo-erythroblasts into mature megakaryocytes through binding to the RUNX1 promoter/enhancer to repress its transcription, which contributes to the higher incidence of AMKL in Down syndrome patients. Our results reveal a novel connection of SON to DS-AMKL pathogenesis and provide clinical importance of SON upregulation in DS. These findings suggest that SON could be a promising therapeutic target for DS-AMKL patients.

#1483

Targeting oncogenic enhancer activity with a small-molecule CBP/P300 bromodomain inhibitor.

Ryan Raisner, Russell Bainer, Emily Chan, Jesse Garcia, Mark Merchant, Karen Gascoigne. _Genentech, Inc., South San Francisco, CA_.

Transcriptional networks are often dramatically altered in cancer, this dysregulation occurring at both the genetic and epigenetic level. Recently much attention has been given to the role of enhancer-driven gene expression in cancer, with the observation that numerous key oncogenes are driven by super-enhancers. This opens up the possibility for therapeutic intervention with small molecules that modulate enhancer activity, leading to control of expression of otherwise un-drugable oncogenes. CBP/P300 are histone acetyl transferases (HATS) that are critical mediators of enhancer activity. Here we use a novel small molecule inhibitor of the CBP/P300 bromodomain to specifically probe the role of these HATS at enhancers, and more broadly evaluate the consequences of enhancer disruption on cancer cell growth in vitro and in vivo.

#1485

Allelic specificity of immunoglobulin heavy chain (IGH) translocation in B-cell acute lymphoblastic leukemia (B-ALL) unveiled by long-read sequencing.

Liqing Tian,1 Beisi Xu,1 Ying Shao,1 Xiaotu Ma,1 Yongjin Li,1 Scott Newman,1 Xin Zhou,1 Meredith Ashby,2 Ting Hon,2 Tyson Clark,2 Elizabeth Tseng,2 John Easton,1 Jinghui Zhang1. 1 _St. Jude Children's Research Hospital, Memphis, TN;_ 2 _Pacific Biosciences, Menlo Park, CA_.

Translocation that juxtaposes the IGH@ enhancer (Eμ) to a proto-oncogene such as CRLF2, EPOR and DUX4 is a common oncogenic mechanism that drives B-ALL. B-cells achieve immunoglobulin monospecificity by inactivating one of the IGH alleles during development. We used a combination of long and short read sequencing technologies to investigate the interplay between this allelic exclusion and IGH@ translocation.

We compared the allelic specificity of an IGH-DUX4 fusion and full-length IGH (Igμ) in the Nalm6 B-ALL cell line - known to carry an IGH-DUX4 translocation - using whole-genome sequencing (WGS) and RNA-seq with both long-read (i.e. the PacBio Iso-Seq method and Chromium WGS) and short-read technologies (Illumina total stranded RNA and WGS). This was accomplished by calibrating allelic imbalance of heterozygous single nucleotide polymorphisms (SNPs), which were identified by WGS, in the transcriptome, epigenome and 3-D genome structure.

Using the Chromium WGS data, we assembled two haplotypes spanning a 23 Kb DNA segment near the IGH-DUX4 fusion region. One of the haplotypes harbored the DUX4-fusion and the other represented the wild-type allele. By mapping long-read Iso-Seq transcripts against the assembled haplotypes, we found that DUX4 and Igμ were transcribed from different haplotypes. Notably, allelic imbalance in RNA-seq, ATAC-seq, and H3K27ac ChIP-seq showed that DUX4 was transcribed at much lower level than Igμ (<10%).

Genome-wide Hi-C data showed that DUX4 was regulated by Eμ, the native enhancer of Igμ. Deep sequencing of targeted chromosome conformation capture (Capture-C) revealed that the contact intensity of DUX4 and Eμ is 14-fold lower than that of the Igμ promoter and Eμ. The weaker enhancer-promoter interaction at the DUX4-fusion haplotype may explain the lower expression of DUX4 compared to Igμ. Furthermore, heterozgyous SNPs in the Igμ promoter region were uniformly mono-allelic in Capture-C, confirming that Igμ was transcribed only from one haplotype.

Our analysis of Nalm6 shown that, counterintuitively, the IGH-DUX4 fusion occurred on the haplotype that does not express Igμ and the fusion gene exhibited lower expression compared to Igμ. Epigenetic regulation at IGH locus is a plausible explanation as we observed weaker interaction of Eμ to DUX4 than that of Eμ to Igμ. Further examination of published RNA-seq data from 72 B-ALL patient samples that harbor IGH@ translocation showed that the majority had higher (median 6.2 fold, range 2.2-57.1) IGH expression than their partner gene (28 of 32 IGH-DUX4, 16 out of 16 IGH-EPOR and 17 out of 24 IGH-CRLF2). This suggests that the majority of IGH@ translocations in B-ALL may have occurred in the haplotype with allelic exclusion.

#1486

Cxcr4/Sdf-1 expression in Her2/Neu breast cancer biopsies and its relation to adjuvant treatment.

Natalia C. Ayala, Melina N. Lorenzini Campos, Mariana M. Gomez Pescie, Angel E. Alsina, Jorge R. Zimerman, Maria C. Zimmermann. _Universidad Nacional del Nordeste, Corrientes, Argentina_.

Background: Chemokines have a fundamental role in inflammation and in cancer progression. Chemokine receptor 4 (CXCR4) is a transmembrane receptor that belongs to the CXC chemokine receptor family and is the only known receptor for the Stromal Cell derived Factor-1(SDF-1). Many retrospective studies have documented that the expression of various chemokine receptors, particularly CXCR4, was associated with a poor prognosis in patients with breast cancer. In the present study, we proposed an SDF-1/CXCR4 induced expression in HER2/neu positive breast cancer biopsies. We, additionally, study its relation to patient's treatment, prior and post adjuvant therapy. Methods: Immunohistochemical staining of her2/neu and ki64 were analyzed in mammary tumor biopsies. Reduction mammoplasty of normal patients were also studied as normal controls. Stained hematoxilyn and eosin slides where previously revised to confirm histological diagnostic and tumoral classification based on established criteria. Additional information considering biological markers such as estrogen receptor (ER), progesterone receptor (PR) expression were also observed in histopathological reports. Quantitative PCR was performed by amplifying a conserved region of the CXCR-4 and SDF-1 genes, and their correlation to Her2/neu expression and adjuvant therapy were analyzed. Statistical analysis was assess using appropriate parametric and nonparametric tests. Results: Her2/neu breast tumor specimens expressed SDF-1 at varying levels and differed upon tumor classification. Expression of the receptor correlates mainly with the tumor type and degree. Quantitative PCR showed low constitutive CXCR4 expression in normal breast tissue that increased in breast tumor tissue, particularly in those expressing her2/neu. Variations upon treatment correspond to those of tumor progression to malignancies and recurrence. Conclusions: We established a correlation between the expression of SDF-1/CXCR4 and the tumoral characteristics prior and post adjuvant treatment. A more careful examination of experimental/clinical data may help to explain the mechanism(s) involving the SDF-1/CXCR4 signaling axis in the breast cancer pathophysiology.

#1487

Investigation of triple-negative breast cancer tumor conversion through high-content screening approaches.

Albane Gaudeau,1 Véronique Lamamy,1 Anne-Laure Jaskowiak,1 Xavier Scerri,1 Benjamin Chanrion,1 Thierry Dorval,1 Elaine Del Nery,2 Franck Perez,2 Jacques Camonis,2 Jean-Philippe Stéphan1. 1 _Servier Research Institute, Croissy-sur-Seine, France;_ 2 _Institut Curie, Paris, France_.

Among Breast Cancer (BC) classification, Triple-Negative (TN) form is the most aggressive, lacking efficient and specific treatment. Our project aims at investigating the possibility to convert TNBC tumor cell lines in Estrogen Receptor (ER), Progesterone Receptor (PR) and/or Human Epidermal growth factor Receptor 2 (HER2)-positive cells using various modalities (siRNA, CRISPR-Cas9, small molecules). Our approach consists in optimizing a multi-parametric High-Content Screening (HCS) relying on phenotypic read-outs such as expression of TNBC positive and negative markers, cell behavior and viability. This poster will present our initial results, validation and future plans. This thesis project fits within a context of drug discovery, innovation, comparison of technologies and understanding of key biological processes and pathways, carried out in partnership between Servier Research Institute and Institut Curie.

#1488

Inter-individual variation in hTERT regulation pathway genes in normal bronchial epithelial cells.

Rose Zolondek, Daniel J. Craig, Erin L. Crawford, James C. Willey. _University of Toledo, Toledo, OH_.

Background: Genome-wide association studies (GWAS) and candidate gene studies suggest that telomere maintenance genes participate in etiology of lung cancer, idiopathic pulmonary fibrosis, and COPD. Telomeres are nucleoprotein structures located at the end of chromosomes that are maintained by telomerase encoded by hTERT. Telomerase is active in cancers, has the ability to aid in rapid cell division, and is dysfunctional in chronic conditions induced by inflammation as such as diabetes, renal failure, and COPD. hTERT is a known downstream target of β-catenin and WNT signaling. However, little is known regarding expression levels of these genes in normal bronchial epithelial cells (NBEC). The purpose of this study is to investigate inter-individual variation in regulation of β-catenin and WNT in NBEC, and if present, identify variants in regulatory region of human telomerase reverse transcriptase (hTERT) that could be used as candidate biomarkers for lung cancer risk. Methods: NBEC specimens from subjects with COPD (n=5) or without COPD (n=9) were obtained by bronchoscopy brush under IRB approved protocol. β-catenin, and WNT4 were measured by competitive multiplex-RT-PCR. Results: Both β-catenin and WNT4 displayed significant inter-individual variation as measured by Intra-Class Correlation (ICC) analysis (β-catenin ICC = 0.47, 95% Confidence Interval (0.2, 0.55), p < 0.05; WNT4 ICC = 0.60, 95% Confidence Interval (0.35, 0.85), p < 0.05). In patients with COPD, WNT4 expression was significantly higher in current smokers compared to former smokers (p<0.05). Conclusions: Inter-individual variation in NBEC expression of β-catenin and/or WNT4 was observed and it is reasonable to test the hypothesis that these genes and related pathway genes may contribute to variation in regulation of hTERT in NBEC and risk for lung cancer, COPD, and/or other lung diseases.

#1489

Differential gene expression profiles between normal cervical tissue and high-grade squamous intraepithelial lesion expressing p16 and high-risk HPV E6/E7 mRNA.

Ines Benedetti, Lia Barrios, Niradiz Reyes. _Universidad de Cartagena, Cartagena, Colombia_.

Background:

HPV is necessary but not sufficient for development of uterine cervix carcinoma. There are genetic factors that influence the progression of premalignant lesions. Gene expression profile differences between healthy and premalignant lesion could contribute to identify potential biomarkers for each step of cervical tumor progression.

Design:

A female patient with an initial diagnosis of low grade squamous intraepithelial lesion (SIL) was invited to participate in the study. During follow-up a high grade SIL was observed by colposcopy, and two samples were obtained from the lesion: one sample was processed for histopathologic diagnosis, and additionally stained by IHC with p16 antibody; the other sample, along with a matched sample of healthy tissue taken distantly from the lesion were stored in a nucleic acids preservation reagent for subsequent analysis of gene expression. Differences in gene expression between healthy tissue and high grade SIL tissue were assessed in triplicate using the cDNA gene expression Illumina microarray Human-HT-12-V4. A statistical p value was computed comparing expression levels from the tissue lesion and healthy tissue. Genes with a fold-change ≥ 2 and a p value <0.05 were regarded as differentially expressed. Additionally, two cervix swabs were obtained for detection of HPV DNA by PCR, and for assessment of high risk HPV E6/E7 mRNA expression by RT-PCR.

Results:

Histopathological evaluation of the lesion confirmed a high grade squamous intraepithelial lesion, with positive p16 staining. HPV-18 DNA was detected and E6/E7 HPV 18 mRNA expression was confirmed. Gene expression analysis detected 33 overexpressed genes, including TMEM45A, SPRR2B, KRT16 and RBP1, and 30 underexpressed genes, including SPARCL1, GCNT3, TMPRSS11b

and ESR1, in high grade SIL compared to healthy uterine cervical tissue. Overexpressed genes belonged to important cellular pathways, such as queratinization, defensins, cytoskeletal signaling, cellular adhesion and extracellular matrix remodeling. Underexpressed genes belonged to metabolic pathways, cell trafficking, and chemokine signaling, among others.

Conclusions:

Microarray analysis identified differential expression between healthy cervical tissue and high grade SIL with confirmed HPV-18 transforming infection. Differentially expressed genes belonged to important cellular pathways. Further studies are required to evaluate the usefulness of these genes as risk markers for progression.

#1490

Molecular function of the read-through transcript PRR5-ARHGAP8.

Jasmeen K. Sethi, Oscar G. Wong, Esther S. Wong, Annie N. Cheung. _The University of Hong Kong, Hong Kong_.

Background: Ovarian carcinoma is one of the most fatal malignancies in females. From the analysis of RNA-seq we have previously conducted to study the transcriptome of ovarian carcinoma upon knockdown of transcription factor hepatic nuclear factor 1β (HNF-1β), PRR5-ARHGAP8 was identified as the only read-through transcript not targeted for the nonsense-mediated decay pathway. Read-through transcription refers to the splicing of the intergenic region from two adjacent genes resulting in a chimeric transcript comprising of one or more exons from both genes. Although several of such transcripts are found in the transcriptome, their significance remains an enigma. Our aim was to ascertain the differential expression of the read-through transcript PRR5-ARHGAP8 in ovarian cancers and to determine its molecular functions.

Materials & Methods: The deregulation of PRR5-ARHGAP8 was validated by RT-qPCR in several ovarian carcinoma cell lines. The expression of this fusion transcript and the individual genes were studied in immortalised human ovarian surface epithelial (HOSE) cell lines, ovarian carcinoma cell lines, and matched tumour and non-tumour pairs from frozen patient samples. Overexpression of this transcript was achieved by transient transfection in HEK293T cells to study its effect on proliferation using MTT assays. RNA and protein lysates were also obtained at several time points to evaluate the pathways affected by RT-qPCR and western blot, respectively.

Results: There was an approximate 3- to 9-fold upregulation of PRR5-ARHGAP8 transcript with a decrease in the expression of PRR5 and ARHGAP8 transcripts in OVMANA, OVISE and KK ovarian carcinoma cell lines upon the knockdown of HNF-1β. Moreover, higher transcript levels of PRR5, ARHGAP8 and PRR5-ARHGAP8 were observed in ovarian carcinoma cell lines compared to HOSE cell lines and in ovarian cancer samples compared to the non-tumour counterparts. Overexpression of PRR5-ARHGAP8 in HEK293T cells resulted in the decrease of phosphorylated Akt protein expression and a reduction of cell viability. RT-qPCR analysis showed a decrease in E-cadherin and an increase in vimentin expression upon PRR5-ARHGAP8 overexpression.

Discussion: The present data suggests PRR5-ARHGAP8 is highly abundant in ovarian carcinomas and may mediate its effects on proliferation via the MAPK pathway and on migration by regulating the epithelial to mesenchymal transition. Overall, this study provides novel insights into the biological relevance of read-through transcription in carcinogenesis.

#1491

Expression of osteopontin splicing isoforms in childhood B-cell precursor acute lymphoblastic leukemia.

Ana Clara Santos da Fonseca Bastos,1 Caroline Barbieri Blunck Blunck,1 Luciana Bueno Ferreira,1 Maria do Socorro Pombo-de-Oliveira,1 Mariana Emerenciano,1 Etel R. Gimba2. 1 _INCA, Rio de Janeiro, Brazil;_ 2 _UFF/INCA, Rio de Janeiro, Brazil_.

Osteopontin (OPN) suffers alternative splicing generating three OPN splicing isoforms (OPN-SI), named OPNa, OPNb and OPNc. However, OPN-SI roles in hematological malignancies (HMs) are still under investigation. Detection of specific gene rearrangements allows the identification of relevant prognostic subgroups of childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Patients harbouring the ETV6-RUNX1 rearrangement are classified as a good prognostic subgroup, while those with the KMT2A-AFF1 fusion are included into poor prognostic subgroup. This work aimed to analyze the expression levels of OPN-SI in BCP-ALL cell lines and patient samples. We tested 4 BCP-ALL cell lines: 207, ALL-PO, RS4;11 and REH. Patient samples included 28 samples from patients harbouring the ETV6-RUNX1 fusion and 26 samples presenting the KMT2A-AFF1 rearrangement. We found that these three OPN-SIs are expressed in all BCP-ALL cell lines and in 70% of both patient prognostic subgroups tested. Both 207 and REH cell lines displayed higher OPNc and OPNb levels than OPNa. We found that OPNc is expressed in higher levels than OPNb and OPNa in unfavorable prognostic subgroup and is also overexpressed in relation to OPNc levels in relation to the ETV6-RUNX1 fusion the comparative subgroup. Interestingly, we found that higher OPNc transcriptional levels are associated with poor prognostic features, such as central nervous system (CNS), initial white blood cell counting and high risk of relapse. In conclusion, we found that the three tested OPN-SI are expressed in most tested BCP-ALL cell lines and BCP-ALL human samples. Since higher OPNc levels are associated to prognostic features, our data provide early evidence that these OPN-SIs could specifically contribute to step BCP-ALL progression. Further work should investigate their putative applications as additional risk-stratification and prognostic markers for BCP-ALL, as well as, their roles on modulating HM progression.

#1492

High-efficiency, strand-specific, low-bias library preparation method for transcriptome profiling of low-input RNA.

Anetta Nowosielska-Vecchietti. _New England Biolabs, Ipswich, MA_.

RNA Sequencing (RNA-seq) is a powerful technique for transcriptome analysis. It is widely used for gene expression analysis, detection of mutations, fusion transcripts, alternative splicing, and the study of post transcriptional modifications. RNA-seq is emerging as a powerful tool for molecular diagnostics as well, providing insights into the biological pathways and molecular mechanisms associated with disease progression including cancer. Recent improvements in next generation sequencing technologies (NGS) and sample barcoding strategies have resulted in significant cost reduction. As RNA-seq is increasingly adopted for molecular diagnostics, the quality and reproducibility of library preparation methods become more important. In addition, demand for library preparation methods that produce successful NGS libraries from very low input RNA or precious clinical samples is increasing.

To achieve these goals, we have developed an RNA-seq library preparation method which can be used across a wide range of input RNA (5-1000 ng total RNA). GC content analysis, gene body coverage and gene expression correlation show that these important parameters remain consistent across varying inputs, even though input amounts vary by two orders of magnitude. As a result, our method has increased sensitivity and specificity for low-abundance transcripts, and reduced PCR duplicates and sequence bias, delivering high quality strand-specific data. Our method is compatible with poly A-tail enriched and ribosomal RNA depleted samples as well as RNA extracted from FFPE (formaldehyde fixed paraffin embedded) tissue samples.

#1493

Lysyl oxidase expression is associated with early recurrence and poor survival in hepatocellular carcinoma.

Naoki Umezaki, Shigeki Nakagawa, Takanobu Yamao, Masayo Tsukamoto, Kota Arima, Tatsunori Miyata, Hirohisa Okabe, Katsunori Imai, Yo-ichi Yamashita, Hidetoshi Nitta, Akira Chikamoto, Hideo Baba. _Kumamoto University, Kumamoto, Japan_.

Background: Hepatectomy is one of the an established one of a curative treatment for hepatocellular carcinoma (HCC). However, HCC has is a disease that high recurrence rates even after curative hepatectomy. We searched the gene for the prediction of the HCC recurrence from intrahepatic metastasis.

Methods: For the gene selection, we used public data base (GSE10141) and searched genes with hHigh early recurrence rate and higher expression genes in tumor area compared with background liver. We detected Lysyl oxidase. As a validation cohort, From 2004 to 2012, 149 patients underwent hepatectomy at Kumamoto University Hospital from 2004 to 2012 were enrolled. The expression of target gene was evaluated by real time PCR. Upper 25% patients were considered as the high expression group, and the other 75% patients were considered as the low expression group. The immunehistochemical (IHC) staining was evaluated with the intensity and area of immunoreactivity of LOX and were scored each factors from 0 to 3. Low expression of LOX group was for scores from 0 to 5. The ones with scores from 6 to 9 were defined as high expression of LOX.

Result: As a first step toward discovering the targets genes, we investigated using GSE 10141, which is a microarray database of cancerous part and background liver tissue of hepatocellular carcinoma cases to select genes useful as drug therapy targets. We extracted LOX as a genes with high hazard ratio (HR > 3) forof early recurrence to select genes useful as prevention of early recurrence. In real time PCR, LOX high expression group had a significantly high recurrence rate (2 years recurrence rate was 64.3% VS 42.7%, p = 0.024) and poor survival rate (3 years rate was 62.7% VS 82.0%, p = 0.0441) than LOX low expression group. Moreover, in the comparisons of clinicopathological factors between LOX high and low expression, there was significantly more Vp positive patients in the LOX high expression group (p = 0.004). Multivariate Analysis demonstrated that Tumor size larger than 50 mm (HR, 2.20; p = 0.002), LOX high expression (HR, 1.73; p = 0.04) were independent risk factors for early recurrence. In immunohistochemistry, LOX high expression group had a significantly high recurrence rate (2 years recurrence rate was 58.5% VS 42.3%, p = 0.004) and poor survival rate (3 years rate was 65.1% VS 88.8%, p = 0.004) than LOX low expression. In addition the comparisons of clinicopathological factors between LOX high and low expression, there was significantly more Ig positive patients in the LOX high expression group (p = 0.04). Multivariate Analysis demonstrated that PIVKA-II score higher than 100 (HR, 1.84; p = 0.03), multiple tumors tumor more than 2 pieces (HR, 2.11; p = 0.009), LOX high expression (HR, 1.90; p = 0.014) were independent risk factors for early recurrence.

Conclusion: LOX high expression is associated with early recurrence and poor survival in HCC.

#1494

SOX18: A novel master regulator of high-grade serous ovarian tumorigenesis.

Annie Y. Liu,1 Kevin C. Vavra,1 Rosario I. Corona,1 Forough Abassi,1 Marcos Fonseca,2 Felipe Segato,2 Matthew L. Freedman,3 Simon A. Gayther,1 Houtan Noushmehr,2 Kate Lawrenson1. 1 _Cedars-Sinai Medical Center, Los Angeles, CA;_ 2 _University of Sao Paulo, Sao Paulo, Brazil;_ 3 _Dana-Farber Cancer Institute, Boston, MA_.

High-grade serous ovarian cancer (HGSOC) is the most common and lethal subtype of ovarian cancer. Recent data indicate that fallopian tube secretory epithelial cells (FTSECs) represent the cell of origin for HGSOC. While many studies have characterized molecular features associated with HGSOC biology, the master transcription factors (TFs) that drive disease development are not known. To identify master TFs for HGSOC, we first performed an integrative analysis of gene expression profiles from 73 FTSEC samples and 394 HGSOCs from The Cancer Genome Atlas (TCGA) project. We then integrated these data with chromatin immunoprecipitation sequencing (ChIP-seq) analyses performed on primary HGSOC samples to select active chromatin regions marked by H3k27ac and to identify super-enhancer (SE) regions. These analyses identified overexpressed TFs that coincide with tumor-specific SEs, a hallmark of master TFs. We identified multiple putative master TFs including SOX18, which was highly overexpressed in HGSOCs relative to normal FTSECs (log2 fold change in expression = 3.5, p = 2.5x10-19). SOX18 is a TF that regulates development of blood and lymphatic vessels, but its association with HGSOC development has not been reported before. We examined the expression of SOX18 in ~8,000 tumors representing 17 tumor types from TCGA. SOX18 expression was significantly elevated in HGSOC relative to any other tumor types, suggesting it is highly specific to this cancer. We found that SOX18 is highly expressed in the HGSOC cell lines UWB1.289, Kuramochi, and EFO21, and we then performed lentiviral-mediated shRNA knockdown of SOX18 in these cell lines to establish the effects of SOX18 depletion on neoplastic phenotypes. In ongoing experiments, we are performing gene expression profiling and H3k27ac ChIP-seq analysis after SOX18 knockdown to identify SOX18 target genes and characterize the landscape of SOX18 binding sites in HGSOC. These studies will likely identify novel molecular biomarkers that may represent much-needed therapeutic targets for HGSOC.

#1496

**Different effects of lncRNA** NDRG1-OT1 **_v4 fragments on regulating NDRG1 transcription in breast cancer cells under hypoxia.**

Chingching Yeh, Mong-Hsun Tsai, Eric Chuang, Liang-Chuan Lai. _College of Medicine, National Taiwan University, Taipei, Taiwan_.

Hypoxia is a crucial factor in aggressiveness of solid tumor by driving multiple signaling pathways. Recent researchers indicated that long non-coding RNA (lncRNA) could promote or inhibit tumor aggressiveness by regulating gene expression. Previous studies in our laboratory found the expression of lncRNA NDRG1-OT1_v4 significantly increased under hypoxia by next-generation sequencing (NGS). Moreover, it was discovered that the NDRG1-OT1_v4 inhibited NDRG1 at both mRNA and protein levels of NDRG1. At the protein level, NDRG1-OT1_v4 improved NDRG1 degradation via ubiquitin-mediated proteolysis pathway. However, the repressive mechanism of NDRG1 at RNA level was still unknown. In this studies, we found that NDRG1-OT1_v4 decreased the NDRG1 promoter activities when we overexpressed NDRG1-OT1_v4 under hypoxia. The fragment (150-263 nt) of NDRG1-OT1_v4 repressed NDRG1 promoter activity significantly by increasing the binding affinity of hnRNPA1. On the other hand, another fragment (264-392 nt) of NDRG1-OT1_v4 improved NDRG1 promoter activity by recruiting HIF-1 alpha. In conclusion, we found a novel mechanism that different fragments of same lncRNA could cause opposite effects on the identical target gene. Keywords: Hypoxia, LncRNA, NDRG1-OT1_v4, NDRG1, HIF-1 alpha, hnRNPA1

#1497

A PRC2 independent role of EZH2 in androgen receptor signaling in prostate cancer.

Yongik Lee, Jung a Kim, Zhao Changsheng, Xiadong Lu, Ka Wing Fong, Jindan Yu. _Northwestern University, Chicago, IL_.

Prostate cancer is the most frequently diagnosed cancer and third most frequent cause of cancer deaths in US males. Prostate cancer patients have benefitted from androgen deprivation therapy and small molecular inhibitors targeting androgen receptor (AR). However, 30% of patients have primary resistance to both forms of treatment with a majority of patients eventually developing resistance to these therapies and progressing from androgen dependent prostate cancer (ADPC) to castration resistant prostate cancer (CRPC). Unfortunately, there are no effective therapeutic options available for CRPC patients, so novel and more effective therapeutic strategies for CRPC are urgently needed. Enhancer of Zeste 2 (EZH2) is a well-characterized oncogene that is one of the most highly upregulated genes in aggressive PCa and suggested as a prognostic biomarker. EZH2 is a part of polycomb repressive complex2 (PRC2) with embryonic ectoderm development (EED) and suppressor of zeste 12 (SUZ12) to function as a repressor through the SET domain, especially Histidine 689 to methylate H3K27. Recently, some studies suggest a context-dependent or PRC2 independent function of EZH2, but new functional role of EZH2 in cancer that are yet to be delineated. In the present study, we demonstrated EZH2 enhances AR signaling through direct AR promoter occupancy at exon1, independent of histone methyltransferase activity. EZH2 has a dual role as a transcriptional activator mediated by AR and an epigenetic silencer mediated by H3K27me3. Therefore, simultaneously targeting EZH2 and AR by drug inhibitors, EPZ and enzalutamide (Enz), respectively, leads to synergistic cell growth inhibition. Taken together, our data reveal a dual function of EZH2 in PCa as a transcriptional activator and an epigenetic silencer. We believe that this study provide not only a novel function of EZH2 in the regulating AR signaling pathway but also provide novel therapeutic strategies for advanced prostate cancer which currently has no therapeutic options available.

#1498

The role of tissues specific super-enhancers in mediating the genetic risk of ovarian cancer.

Kevin C. Vavra,1 Rosario I. Corona,1 Ji-Heui Seo,2 Simon G. Coetzee,1 Janet M. Lee,1 Matthew L. Freedman,2 Paul D. Pharoah,3 Dennis J. Hazelett,1 Simon A. Gayther,1 Kate Lawrenson1. 1 _Cedars-Sinai Medical Center, Los Angeles, CA;_ 2 _Dana-Farber Cancer Institute, Boston, MA;_ 3 _University of Cambridge, Cambridge, United Kingdom_.

Super-enhancers demarcate regions of master regulators involved in cellular differentiation and tumorigenesis. While many trait-associated variants discovered through genome wide association studies are enriched in regulatory elements, the role of super-enhancers in disease susceptibility is less well understood. We evaluated the role of super-enhancers, defined by H3K27ac chromatin immunoprecipitation sequencing (ChIP-seq) analysis of epithelial ovarian cancers (EOC) and precursor cells, in mediating the genetic risk of EOC. We characterized super-enhancer-risk SNP intersections to identify a series of risk-associated loci where multiple candidate causal alleles overlap with ovarian super-enhancers (approximately 33% of candidate causal loci). In particular, a risk locus at 3q25 marked by SNP rs7651446, (odds ratio = 1.59, p = 1.5x10-34), is tagged by 79 candidate causal risk SNPs, more than half of which coincide with super-enhancers detected in EOC precursor cells (ovarian and fallopian tube epithelia) but not in ovarian cancer cell lines or primary tumors suggesting that loss of activity of this super-enhancer is associated with EOC development.

We hypothesized that SNPs at the 3q25 locus modulate activity of super-enhancers that regulate in cis the expression of genes involved in the identity of normal tissue precursors, and that these genes are downregulated during tumorigenesis. We tested the effect of decreasing the binding affinity of BRD4, a protein that binds super-enhancer regions, using a small molecule inhibitor (JQ1). We measured the expression of genes at this locus following JQ1 treatment in EOC precursor cells, identifying three candidate genes, LEKR1, SSR3, and TiPARP, that show decreased expression due to BRD4 binding inhibition. For SSR3 and TIPARP, these expression level changes mirrored the differential expression observed between normal precursor cells and primary high grade serous ovarian cancer. We then established stable shRNA-mediated knockdown models for SSR3 and TIPARP in normal EOC precursor cell lines to establish a role for these genes in ovarian cancer development. In summary, we have identified a potential role for super-enhancers at a subset of EOC risk loci, and present a functional pipeline for identification of the target gene/genes and evaluation of their role in neoplastic transformation. 

### Novel Biomarkers and Drivers of the Cancer Transcriptome

#1499

Vitamin D promotes differentiation in patient-derived organoids in benign and cancer prostate models.

Tara N. McCray, Giovani Lugli, Larisa Nonn. _University of Illinois Chicago, Chicago, IL_.

Deficiency in the prohormone vitamin D is associated with increased risk of lethal prostate cancer (PCa) and less-differentiated, more aggressive tumors. As cancer progresses there is aberrant expression of vitamin D metabolic genes causing dysregulation of the vitamin D pathway, indicating its role in disease. Vitamin D has many chemopreventive actions in vitro, including promotion of differentiation and apoptosis. The ability for vitamin D to drive differentiation has been explored in various cell types and cancer lines, but its mechanism of action in the prostate is not well defined. Our lab found that vitamin D treatment globally upregulates microRNAs (miRs) in prostate cell lines, primary cells and patient samples. Temporal regulation of miRs is critical for differentiation and levels of mature miRs are globally lower in PCa. Given the relationship between vitamin D and microRNAs, and their influence over differentiation, we hypothesize that vitamin D promotes differentiation in the prostate in a microRNA-dependent manner and that this process is disrupted during cancer. Here we examined organoid differentiation in the presence of 1,25D, the active metabolite of vitamin D. Organoids were grown in matrigel from human primary prostate epithelial progenitor cells. Differentiation was assessed by organoid size and branching, and by expression of epithelial markers. Organoids grown in the presence of 1,25D were strikingly larger and formed more complex branching structures than those treated with control. Whole mount immunofluorescence revealed expression of basal and luminal epithelial cells in the organoids. Flow-sorted organoids had a greater composition of basal cells when grown in the presence of 1,25D. RT-qPCR analysis of the flow-sorted epithelial populations revealed an enhancement in the WNT pathway with 1,25D, this pathway is critical in prostate development and dysregulated in cancer. The role of miRNAs was determined by reducing global miRNA levels via knockdown of Drosha, an essential miRNA-processing protein. Knockdown abrogated the effect of 1,25D, as evidenced by reduced size, round shape, and protein expression. Together our findings indicate a role of 1,25D in normal prostate development and branching, a phenotype that is lost when mature miR levels are reduced. Ongoing studies examine these phenotypes in PCa organoids.

#1500

The role of FUBP1 in the hematopoietic system and leukemia.

Marlene Steiner,1 Van T. Hoang,1 Jasmin Yillah,1 Katharina Gerlach,1 Jörn Lausen,2 Hans-Michael Kvasnicka,3 Thomas Oellerich,4 Hanibal Bohnenberger,5 Daniela Krause,1 Martin Zörnig1. 1 _Georg Speyer Haus, Frankfurt, Germany;_ 2 _Goethe University and German Red Cross Blood Service, Frankfurt, Germany;_ 3 _Goethe University, Frankfurt, Germany;_ 4 _University Hospital, Frankfurt, Germany;_ 5 _University Medical Center, Göttingen, Germany_.

The transcriptional regulator FUBP1 (Far Upstream Element Binding Protein 1) acts as an oncoprotein in hepatocellular carcinoma (HCC) and is important for hematopoietic stem cell (HSC) self-renewal and erythroid maturation in mice. In this study, we investigated the transcriptional network by which FUBP1 controls hematopoiesis and elucidated the relevance of FUBP1 for human erythropoiesis. Furthermore, we shed light on the role of FUBP1 in leukemia initiating cells. Searching for upstream-regulators of FUBP1, we identified E-boxes as potential TAL1 binding sites in the FUBP1 promoter. Indeed, we demonstrated the regulation of FUBP1 expression by TAL1 in human primary CD34+ donor cells. In chromatin immunoprecipitation (ChIP) experiments, the binding of TAL1 to the FUBP1 promoter increased during erythroid differentiation, correlating with up-regulated FUBP1 and TAL1 expression. Activation of the FUBP1 promoter by TAL1 binding was confirmed in luciferase assays. We observed a reduction in erythroid colony-forming units and glycophorin A positive cells derived from erythroid differentiated human CD34+ cells upon knockdown of FUBP1, supporting the hypothesis that FUBP1 is required for efficient erythropoiesis. In the transduction/transplantation leukemia mouse models for BCR-ABL1+ CML and MLL-AF9+ AML, we observed that Fubp1 knockdown resulted in reduced total cell and progenitor cell numbers. In CML, Fubp1 knockdown cells showed lower cell cycle activity and increased apoptosis. Consistently, CML and AML mice transplanted with Fubp1 knockdown cells survived longer than control mice that received transduced bone marrow expressing wildtype FUBP1 levels. Furthermore, pharmacological treatment of AML mice with the FUBP1 inhibitor irinotecan prolonged their survival significantly as a single drug or in combination with Ara-C. Analysis of FUBP1 expression in bone sections derived from CML and AML patients, and from healthy donors by immunohistochemistry showed no increased FUBP1 expression in leukemic samples, but we noticed a shorter overall survival in those AML patients with strong FUBP1 expression. In CML patients, FUBP1 levels correlate with the disease stage. Thus, elevated expression of FUBP1 might be an indicator for the aggressiveness of leukemia. Our data identify TAL1 as an FUBP1 upstream-regulator and confirm the importance of FUBP1 for HSC self-renewal and erythroid maturation, not only in murine but also in human cells. Furthermore, FUBP1 acts as an oncogenic factor in leukemia. Our findings might provide important evidence for the potential use of FUBP1 in clinical settings, e.g. as a molecular target for the treatment of leukemia patients and as a modulator for the production of red cells.

#1501

Developing novel strategy for the treatment of acute myeloid leukemia by targeting retinoic acid signaling pathways.

Metis Hasipek, Dale Grabowski, James G. Phillips, Yihong Guan, Hetty Carraway, Jaroslaw Maciejewski, Babal K. Jha. _Cleveland Clinic Foundation, Cleveland, OH_.

Retinoic acid (RA), the active metabolite of vitamin A, influences biological processes by activating the retinoic acid receptor (RAR). RARs are ligand-controlled transcription factors that function as heterodimers with retinoid X receptors (RXRs) to regulate cell growth and survival. The success of RAR modulation in the treatment of acute promyelocytic leukaemia (APL) particularly by the use of all-trans retinoic acid (atRA) has stimulated considerable interest in the development of small molecules that can modulate RAR and RXR. Recent studies have demonstrated that RA can also activate the peroxisome proliferator-activated receptor β/δ (PPARβ/δ). In the aqueous intracellular milieu, RA is transported by the cellular retinoid-binding protein CRABP-II, or by the fatty-acid-binding protein FABP5, depending on the ratio of FABP5 to CRABP-II. In cells expressing high CRABP-II and low FABP5, RA activates the RAR, whereas in the presence of the reverse ratio, RA activates PPAR β/δ. These two different mode of RA delivery due to different ratio of these two cargos leads to opposite cellular outcomes. Cells harboring high level of CRABP-II, RA is delivered to RAR leading to apoptosis, growth arrest, and anticancer activity. However, when FABP5 expression is high RA is delivered to PPAR β/δ resulting in survival, proliferation, and tumor growth. In both cases, retinoid X receptor (RXR) is the indispensable partner of the nuclear receptor involved. The analysis of TCGA data set revealed that a certain class of AML patients have low level of CRABP-II and high level of FABP5, that in part explain the inability of at-RA to induce terminal differentiation in AML cells. To test our hypothesis, we screened AML and APL patient bone marrow cells and found that a number of AML patients bone marrow have high FABP5 and low CRABP-II protein levels while the atRA responding APL patients has opposite ratio determined by western blotting. Therefore, low CRABP-II and high FABP5 levels in a subset of AML patients lead to the activation of pro-survival PPAR β/δ pathway that promotes proliferation and opposes the differentiation. We also analyzed different AML cell lines for mRNA expression using qRT-PCR and protein by western blotting in using highly specific antibodies against FABP5 and CRABP-II. High FABP5 levels were observed in the majority of the AML cell lines. Efficacy of novel small molecule FABP5 inhibitor as a single agent and in combination with atRA was evaluated in HL-60 cells. Here, we demonstrate that a small molecule inhibitor of FABP5 synergizes with atRA and induces the differentiation in AML cells.

#1502

PDEF inhibits prostate cancer progression by promoting luminal differentiation.

Fengtian Wang,1 Sweaty Koul,1 Hari K. Koul2. 1 _Louisiana State University Health Sciences Center-Shreveport, Overton Brooks VA Medical Center, Shreveport, LA;_ 2 _Louisiana State University Health Sciences Center-Shreveport, Overton Brooks VA Medical Center, Feist Weiller Cancer Center, Shreveport, LA_.

Background: The emergence of metastatic castration-resistant prostate cancer (PCa) is accompanied by the loss of prostate luminal cell identity. Cell identity is defined by key transcription factors by association with enhancer and super-enhancer elements. Our previous studies have shown that Sam Pointed Domain Ets Transcription Factor a.k.a. Prostate-Derived Ets Factor (SPDEF/PDEF), inhibits tumor metastasis in vivo. Recently, PDEF has been reported as one of the super enhancer related transcription factors in the luminal PCa cell line LNCaP and observed to be highly expressed in prostate luminal cells. We propose that PDEF functions as a putative tumor metastasis suppressor and inhibits tumor progression by restoring luminal cell identity. The present study was designed to investigate the role of PDEF in metastatic PCa progression.

Methods: PC3 and DU145 cells were stably transfected with PDEF/control pBABE retroviral vectors. Global gene expression changes were probed using Affymetrix microarray. Microarray was analyzed with GSEA. qRT-PCR was performed to confirm the differential gene expression profile and immunohistochemistry, immunofluorescence and immunoblot were performed to visualize protein expression. ChIP-seq data were extracted from SRP002475 and aligned with Bowtie. Peaks were identified by MACS2. Clinical data were extracted from GSE16560, GSE21034 and TCGA database. Statistical analysis was performed with GraphPad and R.

Results: Analysis of our microarray studies revealed that PDEF expression resulted in the negative enrichment of metastasis-related gene sets and the positive enrichment of gene sets involved in luminal differentiation. Especially, PDEF restores canonical AR signaling in PC3 cells. Confirmed with qPCR, IB and IF, PDEF also inhibits the expression of neuroendocrine-related genes, stemness-related genes while promoting the expression of prostate luminal differentiation-related genes. Furthermore, PDEF ChIP-seq analysis revealed a novel PDEF binding site at the putative promoter region of prostate luminal cell marker cytokeratin 18 while no significant peaks were identified within the regulatory elements of EMT related transcription factors. Our data suggest that PDEF promotes the expression of cytokeratin 18 in vitro and in vivo. Knockdown of cytokeratin 18 by siRNA increases the expression of Twist1 while decreases the expression of E-Cadherin while PDEF expression level is not affected. Moreover, data analysis from multiple clinical prostate cancer cohorts suggests the loss of PDEF is associated with tumor metastasis, tumor progression, and poor survival.

Conclusions: PDEF expression leads to luminal differentiation phenotype in advanced prostate cancer cells. PDEF inhibits cell migration and metastasis in part by down-regulating EMT related transcription factors in part through directly promoting the expression of cytokeratin 18.

#1503

Arsenic trioxide inhibits proliferation of acute leukemia cells through reduced expression of E2F1, cyclin E, and activation of retinoblastoma protein.

Sanjay Kumar, Paul Tchounwou. _Jackson State Univ., Jackson, MS_.

Arsenic trioxide (ATO) is has been used successfully in the treatment of all age group of acute promyelocytic leukemia (APL) patients in both induction and consolidation therapy. It inhibits growth APL cells through various mechanism including oxidative stress, cell cycle regulation, and apoptosis. However, detail molecular mechanism ATO-induced APL cell proliferation inhibition mostly poorly known. E2F1, a transcriptional factor widely involved proliferation and differention of many types of cancer cells through providing signal to accumulation p53, cell cycle regulation, and apoptosis. We hypothesized that ATO inhibits proliferation and differentiation of APL cells through reduced expression of E2 F1, cyclin E and stimulation of retinoblastoma and AMPKα2 expressions in APL cells. To test hypothesis, we used APL cell lines and after treatment with different concentration (2,4,6, and 8μg/ml) ATO, checked expression level of E2F1, cyclin A, cyclin E, Akt, retinoblastoma (Rb D20 & H1) and AMPKα2 by western blotting, immunocytochemistry, and confocal imaging. We found that it reduced expression significantly of E2F1, cyclin E by modulating expression profile of retinoblastoma (Rb D20 & H1), Akt and AMPKα2 proteins in APL cells concentration dependent manner. Our data showed that E2F1 associated with p53 and its association may involve ATO -induced cell cycle regulation and apoptosis. On the basis of these findings, we conclude that ATO inhibits APL cells proliferation by reducing E2F1, cyclin E expression through stimulation of retinoblastoma (Rb D20 & H1), Akt , and AMPKα2 proteins. It is novel mode of action of ATO in APL cells and may help in the designing of new anti-leukemic drugs for treatment of APL patients more efficiently and quickly.

Keywords: Arsenic trioxide, E2F1, cyclin E, Rb proteins, AMPKα2, and APL cells.

Acknowledgements: This research was financially supported by National Institutes of Health NCRR Grant No. 5G12RR013459 and MIMHD Grant No. 8G12MD007581, through the RCMI-Center for Environmental Health at Jackson State University.

#1504

Opposing roles of FoxM1 and the hepatic specification genes FoxA1/A2 dictate differentiation state of liver cancer cells.

Vaibhav Chand, Akshay Pandey, Dragana Kopanja, Grace Guzman, Pradip Raychaudhuri. _University of Illinois at Chicago, Chicago, IL_.

The pro-proliferation transcription factor FoxM1 is over-expressed mainly in high-grade cancers. However, the significance of its over-expression in cancers, including poorly differentiated hepatocellular carcinoma (HCC), is not known. Here we provide evidence for a causal role of the over-expressed FoxM1 in driving high-grade progression of HCC. Immunohistochemical staining of human HCC specimens revealed opposite expression patterns of FoxM1 and the liver differentiation genes FoxA1/A2. Moreover, using a transgenic mouse model for oncogenic Ras-driven HCC as well as cell-based studies, we provide in vivo genetic evidence for a direct repression of FoxA1/A2 by FoxM1. Interestingly, FoxM1 represses these differentiation genes mainly in G1 phase, a phase in the cell cycle in which cells can undergo differentiation. Moreover, repression of FoxA1/A2 in G1 phase is important, as these genes are capable of inhibiting expression of the pluripotency genes that are mainly expressed in S/G2 phases. Also, we show that FoxA1/A2 inhibit expression of FoxM1 by inhibiting binding of FoxM1 to its own promoter and thus blocking its auto-activation. Our observations identify a significant new mechanism in which FoxM1 and the FoxA genes play opposing roles that determine the differentiation state of the HCC cells.

#1505

GATA3 expression is associated with poor differentiation of osteosarcoma cells.

Koji Hiraoka. _Kurume University School of Medicine, Kurume, Japan_.

Objective: GATA3, a transcriptional factor promoting differentiation of Th2 cells, was expressed in breast cancer cells or bladder cancer cells. We reported that Gata3 expression in soft tissue sarcoma is an independent factor for poor prognosis. However, the role of GATA3 in osteosarcoma is unknown. We investigated whether GATA3 expression is associated with cell differentiation or clinical prognosis of osteosarcoma. Methods: We examined whether decreased GATA3 expression in MG63 osteosarcoma cells by induction of siRNA GATA3 using electroporation affects cell differentiation. We also evaluated GATA3 expression in biopsy samples of 31 osteosarcoma cases using immunohistochemical analysis, and statistically compared clinicopathological characteristics of GATA3-positive and GATA3-negative cases. In particular, the correlation between osteoid formation and GATA3 expression was examined. Result: GATA3 expression was observed in MG63 cells by Western blotting and real-time PCR. Decreased GATA3 expression caused by induction of siRNA promoted the expression of the osteoblast differentiation markers; osteopontin, osteocalcin and RUNX2; in MG63 cells, while ALP and DMP-1, pre-osteocyte markers, had not increased. The expression of p21, one of the differentiation markers of osteosarcoma, is also elevated by decreased GATA3 expression. GATA3 significantly expressed in osteosarcoma cases with less osteoid formation (P<0.001), although no significant correlation between GATA3 expression and clinical prognosis was observed. Conclusion: These data indicated that GATA3 expression might be associated with poor differentiation of osteosarcoma cells. GATA3 may be a target to treat for osteosarcoma through promotion of cell differentiation.

#1506

Characterization of fibronectin type III domain containing 3B (FNDC3B) as a novel fusion partner of retinoic acid receptor alpha in granulocytic differentiation.

Zhe Wand, Chi Keung Cheng, Terry Wong, Kin Mang Lau, Thomas S.k. Wan, Kam Tong Leung, Margaret Heung-Ling Ng. _Chinese University of Hong Kong, Hong Kong, Hong Kong_.

Acute promyelocytic leukemia (APL) is characterized by the promyelocytic leukemia-retinoic acid receptor alpha (PML-RARA) fusion. Recently, we identified fibronectin type III domain containing 3B (FNDC3B) as a novel RARA fusion partner resulting from the t(3;17)(q26;q21) translocation in a variant APL patient. Unlike other RARA fusion partners, FNDC3B appears as the only partner involved in granulocytic differentiation but the molecular mechanisms underlying its actions remain to be characterized. FNDC3B mRNA expression in normal and malignant hematopoietic cells was determined by quantitative RT-PCR. Transcriptional control of FNDC3B was studied by reporter gene assays, site-directed mutagenesis and chromatin immunoprecipitation (ChIP) assays. FNDC3B-regulated pathways were identified by transcriptome analyses. Using BloodSpot, we observed that FNDC3B expression was progressively up-regulated as human hematopoietic stem cells (HSCs) differentiate along the granulocytic lineage, with the highest level in terminally differentiated polymorphonuclear cells. Concordantly, we demonstrated lower expression of FNDC3B in human cord blood CD34\+ HSCs than HL-60 promyelocytes. The specific involvement of FNDC3B in granulocytic differentiation was further suggested by higher FNDC3B expression and promoter activity in human promyelocytic than monocytic cell lines. Mutational analyses indicated that two putative CCAAT-enhancer binding protein epsilon (CEBPE) and one Sp1 binding sites were critical for FNDC3B promoter activity in HL-60 cells. The specific association of CEBPE and Sp1 with the FNDC3B promoter could be confirmed by ChIP assays. Knockdown of CEBPE in HL60 cells by small interfering RNA (siRNA) reduced FNDC3B expression and promoter activity. Furthermore, we observed a strong positive correlation between CEBPE and FNDC3B expression in the Cancer Genome Atlas (TCGA)-acute myeloid leukemia (AML) dataset (n=173, r=0.66, P<0.0001). Collectively, these findings indicate CEBPE, which is critical in terminal granulopoiesis, as an important factor controlling FNDC3B transcription in granulocytic cells. Using RNA-seq, we found that FNDC3B knockdown in NB4 promyelocytes was associated with preferential downregulation of genes encoding granule and endoplasmic reticulum (ER) structural/functional proteins. Concordantly, DAVID functional annotation of genes that are positively correlated with FNDC3B in the TCGA-AML cohort revealed enrichment of Gene Ontology terms related to ER functions. Together, these findings suggest that FNDC3B mediates its functions primarily by targeting the ER/granule pathway, which is important for granulocytic maturation. In summary, our data indicate FNDC3B as a novel CEBPE target in the myeloid transcriptional hierarchy and may provide new clues in abnormal granulopoiesis.

#1507

Multiple mechanisms of Myb gene activation revealed through transcriptome analysis of salivary gland adenoid cystic carcinoma.

Candace Frerich,1 Kathryn Brayer,1 Brandon Painter,2 Scott Ness1. 1 _University of New Mexico HSC, Albuquerque, NM;_ 2 _University of New Mexico, Albuquerque, NM_.

Adenoid cystic carcinoma (ACC) of the salivary gland is a rare head and neck malignancy with poor long-term prognosis. Hallmark chromosomal translocations result in MYB to NFIB (t(6;9)) and MYBL1 to NFIB (t(8;9)) gene fusions. Gene rearrangement and aberrant expression of the MYB and MYBL1 genes, along with the encoded transcription factors, are the oncogenic driver in the majority of ACC tumors. Previous RNA-sequencing (RNA-seq) data collected from FFPE fixed ACC tumors illustrated MYB and MYBL1 elicit similar gene expression profiles in ACC, suggesting these transcription factors drive ACC oncogenes via similar mechanisms. Using these RNA-seq data we have identified EN1 and SOX4 as important Myb regulated genes and demonstrated activation of their promoters in reporter gene assays. We hope to utilize these reporters in high-throughput screens to identify new drug candidates for ACC. We have utilized RNA-seq data to identify an additional mechanism leading to MYB overexpression that appears unique to ACC tumors. In these tumors transcription of the MYB gene is aberrantly activated through use of an alternative promoter located within the first intron of the gene. Transcripts originating from this promoter have been confirmed in an ACC tumor via 5'RACE and the resulting Myb protein has a small N-terminal truncation eliminating the epitope recognized by a common Myb antibody. The alternative promoter is active and regulated in a cell type specific manner in reporter gene assays. Previous studies have shown chromosomal translocations juxtapose NFIB enhancers to activate MYB gene transcription. ChIP and 3C assays in fresh frozen ACC tumors will be utilized to test the hypothesis that NFIB enhancers are specifically recruited to the alternative MYB promoter, thereby activating gene transcription. Thus, multiple mechanisms lead to the aberrant over-expression and truncation of MYB and drives ACC oncogenesis.

#1508

**Cloning, expression and purification of** Cionasavignyi **polypeptide CS5931 and its antitumor activity.**

Yangyang Zhang, Honglin Lai, Weixiao An, Shousong Cao, Xiukun Lin. _Southwest Medical University, Luzhou, China_.

Previous study in our laboratory has shown that a novel polypeptide CS5931 (molecular weight 5931 Da) derived from Ciona savignyi possesses potent cytotoxicity against a variety of human cancer cells including cervical carcinoma Hela cells, colorectal carcinoma HCT-116 and RKO cells, lung carcinoma A549 cells, and leukemia HL60 cells ,with IC50 values of 4.17, 2.50, 5.54, 4.69 and 4.98uM,respectively. This result suggested that HCT116 cells are the most sensitive to the treatment of CS5931. We also found that CS5931 markedly induced apoptosis in a dose-dependent manner in HCT116 cells by Annexin V-FITC/PI double staining analysis. However, its content in Ciona savignyi is very low thus developing a novel method for increase of the production of the polypeptide is promising. GRN polypeptides typically consist of 56-58 amino acids with six disulfide bonds and four beta-sheets to make the GRN polypeptide structurally compact and complex so large-scale preparation of natural polypeptide CS5931 is very difficult. Therefore, recombinant expression of CS5931 by genetic engineering is one of the effective ways to solve this problem. In the present study, we cloned a full length cDNA of CS5931 precursor by rapid amplification of cDNA ends (RACE) and obtained the amino acid sequence of CS5931. We further made the polypeptide expression with high efficiency in E. coli. The expressed protein was purified through affinity chromatography. The gene fragment coding CS5931 was successfully cloned and then linked into prokaryotic expression carrier pET28a(+) and pPICZαa, respectively. Two expression vectors pET28a(+)-CS5931 and pPICZαa-CS5931 were transformed into E. coli and Pichia pastoris, respectively. Then recombinant polypeptide CS5931 were successfully induced with high expression . To investigate the expression and anticancer effect of polypeptide CS5931 in different vectors and strains, we purified the expressed product developed suitable methods for CS5931 great expression while maintaining its potent anticancer activity and identified the role of CS5931 target proteins. Moreover, the purified polypeptide native CS5931 and recombinant CS5931 equally display great antitumor activity against HT29 cells. Our studies indicate that we have successfully cloned, expressed and purified Ciona savignyi polypeptide CS5931 and demonstrated its potent antitumor activity against colorectal cancer cells. Therefore, CS5931 may be developed as a novel anticancer agent in the treatment of colorectal cancer clinically.

#1509

Cachexia: Leveraging transcriptomics to identify potential therapeutics.

Matthew Ung, Kevin D. Fowler, Jason M. Funt, Renan Escalante-Chong, Gregory Koytiger, Sarah Kolitz, Rebecca L. Kusko, Benjamin Zeskind. _Immuneering, Cambridge, MA_.

Cachexia is a common cause of morbidity across multiple cancer types, yet remains largely under-studied especially in the realm of transcriptomics. An essential component of cancer cachexia is the induced transition of adipocytes from white to brown, which increases energetic inefficiency and contributes to systemic loss of adipose tissue. This is likely caused by the tumor secreting thermo-stimulatory factors into the extracellular space.

Here, we integrate publicly-available data to pinpoint actionable gene targets that enable cancer cells to induce fat "browning". In particular, we identify genes differentially expressed between Lewis lung carcinoma subclones that can induce thermogenesis in primary adipocytes at high or low levels in vitro (GSE57797). We then evaluate whether the expression of these thermo-stimulatory genes predict patient survival in an independent gene expression dataset of primary tumors from advanced stage gastric cancer patients (GSE15459). Finally, we leverage drug gene expression data from CMAP and LINCS to identify molecules that reverse the thermo-stimulatory signature.

Our analysis reveals that the thermo-stimulatory signature predicts prognosis in patients with advanced gastric cancer (p=0.005), with the low thermo-stimulatory group exhibiting improved prognosis. The thermo-stimulatory signature is most strongly reversed by expression signatures from inhibitors of MEK (MAP2K1), including pd-0325901, AZD-8330 (ARRY-704), CI-1040, trametinib, and selumetinib. Interestingly, we observe a time-dependent effect of these MEK inhibitors, with shorter durations of exposure consistently reversing thermo-stimulatory signatures more effectively than longer durations of exposure.

Analysis of cachexia-related transcriptomic data reveals key marker genes and pathways which can be used to optimize MEK inhibitors for cachexia. Results from this study support the development of MEK inhibitors to treat cachexia, and suggest that careful optimization of pharmacokinetics will be important for success in the clinic.

#1511

Differential expression analysis and biomarker identification in five salivary gland carcinoma types.

Kathryn J. Brayer,1 Yoshitsugu Mitani,2 Adel El-Naggar,2 Scott A. Ness1. 1 _University of New Mexico Comprehensive Cancer Center, Albuquerque, NM;_ 2 _The University of Texas, MD Anderson Cancer Center, Houston, TX_.

Significant morphologic overlap can exist between distinct salivary gland neoplasms, making differential diagnosis challenging, even with the use of new immunohistochemistry stains and FISH probes (Griffith et al., Arch Pathol Lab Med 2017). RNA-sequencing (RNAseq) technology is increasingly being used as a tool for identify diagnostic molecular signatures in a wide variety of diseases (Byron et al., Nat Rev Genet 2016). Furthermore, transcriptomics data are being used to develop biomarkers that can be used in drug discovery assays. Using an unbiased RNAseq analysis pipeline (Brayer et al., Cancer Discov 2016; Brown et al., PLos One 2017), we developed molecular profiles for 126 salivary gland tumors with accompanying clinical data--68 adenoid cystic carcinoma (ACC), 23 acinic cell carcinoma (Acinic), 5 basal cell adenoma (BCA), 20 basal cell adenocarcinoma (BCAC), and 10 secretory carcinoma (SC)--and have identified distinct gene expression patterns for each neoplasm. Additionally, a detailed analysis within each cancer type has revealed heterogeneous gene expression patterns, with altered signaling pathways, suggesting molecular subtypes exist. In both ACC and Acinic samples we identified unique molecular signatures found in patients with poor overall survival. Transcripts within each molecular signature that are independent of gender, age, and cancer stage, but correlated with clinical outcome data, are being identified for use as diagnostic or prognostic biomarkers and for use in drug discovery assays. Preliminary results to identify biomarkers for these neoplasms will also be presented.

#1512

Regulation of the CDC42 signaling pathway by IKZF1 in T-cell acute lymphoblastic leukemia.

Mario Soliman,1 Chunhua Song,1 Jonathon L. Payne,2 Zheng Ge,1 Chandrika Gowda,1 Yali Ding,1 Kimberly J. Payne,2 Sinisa Dovat1. 1 _Pennsylvania State University College of Medicine, Hershey, PA;_ 2 _Loma Linda University School of Medicine, Loma Linda, CA_.

The IKZF1 gene encodes Ikaros—a DNA-binding, Kruppel-like zinc finger protein that functions as a tumor suppressor in T-cell acute lymphoblastic leukemia (T-ALL). Ikaros regulates transcription of a large number of genes that are involved in control of cellular proliferation. However, the molecular mechanisms through which Ikaros regulates proliferation of T-ALL cells are still unknown. The use of quantitative chromatin immunoprecipitation (qChIP) showed that Ikaros binds to the promoter regions of CDC42 in vivo in primary T-ALL patient samples as well as in T-ALL cell lines. Ikaros overexpression in B-ALL cells via retroviral transduction results in decreased transcription of CDC42, as evidenced by qRT-PCR and Western blot. The luciferase reporter assay showed that Ikaros represses CDC42 transcription by directly binding to its promoter. The shRNA-mediated knock-down of Ikaros in T-ALL resulted in increased expression of CDC42. These results suggest that Ikaros functions as a transcriptional repressor of CDC42 in T-ALL. Next, we studied the upstream signaling pathways that regulate Ikaros-mediated control of CDC42 transcription in T-ALL. Because Ikaros activity in leukemia is regulated via direct phosphorylation by Casein Kinase II (CK2), we tested the effect of CK2 inhibition on Ikaros-mediated regulation of CDC42 expression. Treatment of T-ALL cells with a specific CK2 inhibitor, CX-4945, resulted in reduced expression of CDC42 in T-ALL cells. This was associated with an increase in DNA-binding of Ikaros to the promoter of the CDC42 gene. Ikaros knock-down restored high expression of CDC42 in T-ALL cells that were treated with CK2 inhibitors. These data suggest that transcriptional repression of the CDC42 gene by Ikaros is impaired in T-ALL due to increased CK2 activity and reduced Ikaros DNA-binding affinity toward the CDC42 gene promoter. Inhibition of CK2 restores Ikaros function and results in transcriptional repression of the CDC42 gene. In conclusion, our results suggest that Ikaros regulates CDC42 signaling pathway via transcriptional repression of the small GTPase, CDC42, in T-ALL. Ikaros-mediated repression of CDC42 in T-ALL is negatively regulated by CK2. These results suggest that regulation of CDC42 expression is one of the mechanisms through which Ikaros regulates cellular proliferation in T-ALL.

#1513

Epigenetic regulation of CD117 expression in B-cell acute lymphoblastic leukemia by Ikaros and histone deacetylase HDAC1.

Shriya Kane,1 Jonathon L. Payne,2 Mario Soliman,1 Chandrika Gowda,1 Meixan Xiang,1 Chunhua Song,1 Kimberly J. Payne,2 Sinisa Dovat1. 1 _Pennsylvania State University College of Medicine, Hershey, PA;_ 2 _Loma Linda University School of Medicine, Loma Linda, CA_.

The type III receptor tyrosine kinase, CD117, functions as a receptor for stem cell factor (SCF) and is encoded by the c-kit gene. During hematopoiesis, CD117 is normally expressed in hematopoietic stem cells, multipotent progenitors, common lymphoid progenitors, and early-stage thymocytes. Overexpression and/or activating mutations of c-kit have been demonstrated in acute myeloid leukemia (AML), early T-cell precursor acute lymphoblastic leukemia (ETP-ALL), and B-cell acute lymphoblastic leukemia (B-ALL). It has been suggested that increased expression of CD117 is associated with stem-like phenotype and worse clinical outcomes in AML and T-ALL. The regulation of expression of c-kit in leukemia is still largely unknown. Here we report that transcription of c-kit in B-ALL is regulated by the Ikaros tumor suppressor protein and histone deacetylase HDAC1. Global genome-wide binding studies using ChIP-seq, demonstrate the occupancy of both Ikaros and HDAC1 at the promoter of the c-kit gene in B-ALL cells. Ikaros and HDAC1 binding to the c-kit promoter was confirmed by quantitative chromatin immunoprecipitation (qChIP). Overexpression of Ikaros via retroviral transduction results in reduced transcription of c-kit in B-ALL cells. Consistent with this, Ikaros knock-down with shRNA results in increased transcription of c-kit in B-ALL. These data suggest that Ikaros represses transcription of c-kit. Ikaros overexpression was associated with increased HDAC1 occupancy while Ikaros knock-down resulted in reduced HDAC1 binding to the promoter of the c-kit gene. We tested whether Ikaros-mediated transcriptional repression of c-kit requires HDAC1 activity. Results showed that inhibition of HDAC1 activity with a pan-histone deacetylase inhibitor, trichostatin (TSA), or a specific HDAC1 inhibitor, MS-275, abolishes Ikaros' ability to repress c-kit transcription in luciferase reporter assays. Molecular inhibition of HDAC1 with shRNA confirmed that HDAC1 activity is essential for Ikaros-mediated transcriptional repression of c-kit. A serial qChIP assay spanning the c-kit promoter was used to analyze the epigenetic changes that are associated with Ikaros and HDAC1 binding at the c-kit promoter. Results showed that increased Ikaros and HDAC1 occupancy at the c-kit promoter in B-ALL cells results in enrichment for the markers of the repressive chromatin, H3K9me3 and H3K27me3, as well as reduced occupancy of H3K9ac, a marker of active chromatin. In conclusion, the presented results show that the expression of c-kit in B-ALL is regulated at the transcriptional level by Ikaros and HDAC1 via chromatin remodeling. These data provide a novel insight into the role of Ikaros in both tumor suppression and transcriptional regulation of gene expression in B-cell acute lymphoblastic leukemia.

#1514

Role of polycomb proteins in neuroendocrine prostate cancer.

Zachary Connelly, Shu Yang, Anthony Blankenship, Xiuping Yu. _Louisiana State Univ. Health Sciences Ctr., Shreveport, LA_.

Prostate cancer (PCa) is the most common non-skin cancer and the 3rd leading cause of cancer-related death in American men. Androgen ablation therapy has been the gold standard for treating advanced stage PCa since the 1960s. Initially, the PCa patients respond to hormone ablation very positively. However, over time these tumors almost always become resistant to androgen ablation therapy, and tumors begin to grow again. Recent studies found after failure of the new drugs that block androgen action, PCa has increased neuroendocrine phenotype. Understanding the mechanisms through which PCa cells gain neuroendocrine phenotype is critical for the development of novel therapeutics. Polycomb repressive complex are involved in early embryo development and carcinogenesis in several types of cancer including PCa. In this study, we evaluated the involvement of Polycomb proteins in neuroendocrine PCa. Using immunohistochemistry staining, we examined the expression of Polycomb protein EZH2 in advanced stage PCa. We found that EZH2 is highly expressed in both human and mouse neuroendocrine PCa. Also, we found when PCa cells are induced to undergo neuroendocrine differentiation, multiple Polycomb genes are induced. These data suggest that Polycomb genes play a role in the development of neuroendocrine PCa.

#1515

NCOA3 promotes tumor growth by activating telomerase reverse transcriptase and predicts poor prognosis in hepatocellular carcinoma.

Miao Chen,1 Wenbin Li,1 Yixin Li,1 Changlin Zhang,1 Ge Qin,1 Qian Long,1 Dingbo Shi,1 Wei Guo,2 Shuihan Shi,3 Kefang Zhang,3 Wuguo Deng1. 1 _Sun Yat-Sen University, Guangzhou, China;_ 2 _Dalian Medical University, Dalian, China;_ 3 _Global Life Care Institute, Guangzhou, China_.

Hepatocellular carcinoma (HCC) is one of the most common cancers and a leading cause of cancer death worldwide. The rising incidence and mortality rate of HCC in most countries are due to an increased rate of chronic Hepatitis C virus infection, alcohol abuse and obesity. The molecular mechanisms leading to the development of HCC are complicated, consist of various genetic and epigenetic changes, and are associated with altered signaling pathways. Among these molecular events, elevated expression of human telomerase reverse transcriptase (TERT) is an important alteration in HCC and a hallmark of human cancers. TERT is highly expressed in HCC and positively correlated with the development of HCC. However, the detailed mechanism of TERT activation in HCC remains largely unclear. Here, we identified nuclear receptor coactivator-3 (NCOA3) as a TERT promoter-binding protein, which specifically bound to the -234~-144 region of the TERT promoter. Overexpression of NCOA3 enhanced TERT promoter activity and expression, and promoted HCC cell growth. Moreover, we demonstrated that NCOA3 interacted with the transcription factor SP1 to promote TERT expression. The clinical data showed that the expression of NCOA3 and TERT were positively correlated and elevated in HCC tissues, and HCC patients with high level of NCOA3/TERT had poor prognosis. Collectively, our study revealed that NCOA3 bound to the TERT promoter and activated TERT expression to promote HCC growth, indicating that the NCOA3/TERT signaling pathway could be a potential therapeutic target for HCC. [Funding support: This work was supported by the funds from the National Natural Science Foundation of China (81772925, 81472178, 81702761), the State "973 Program" of China (2014CB542005), and the Natural Science Foundation of Guangdong Province (2016A03031100, 2015A030313018, 2017A030313615)].

#1516

BAT-1 up-regulation decreases cell invasion and migration in vitro.

Aileen M. Garcia-Vargas,1 Maria Sanchez,2 Magaly Martinez-Ferrer1. 1 _University of Puerto Rico Medical Sciences Campus, San Juan, PR;_ 2 _University of Puerto Rico Comprehensive Cancer Center, San Juan, PR_.

Prostate cancer is the most common cancer in men in the United States and is the third cause of cancer related deaths in men. In Puerto Rico, prostate cancer is the most common type of cancer and the leading cause of cancer death in men. Approximately one out of seven men will be diagnosed with prostate cancer in his lifetime. The current available biomarkers are unable to predict malignant outcomes such as recurrence. Thus, there is a critical demand for the development of innovative diagnostic and prognostic tools for the management of prostate cancer. In the current study, we evaluated the biological role of BAT-1 in prostate cancer. Preliminary data from patients who had prostate cancer recurrence identified that HLA-B associated transcript 1 (BAT-1) was down-regulated in patients with prostate cancer recurrence when compared with non-recurrent patients. We up-regulated BAT-1 in androgen independent PC3 and androgen dependent 22RV1 prostate cancer cell lines using a BAT-1 recombinant protein. In vitro assays were performed to measure proliferation, migration and invasion. Proliferation assays using MTS showed no significant change in BAT-1 recombinant protein concentrations ranging from 0.25-2.0μg/mL at 12, 24 and 48hrs for PC3 and 22RV1 cells. Wound healing assay showed that cells treated with BAT-1 recombinant protein at 0.25μg/mL and 2.0μg/mL significantly decreased migration at 6, 12hrs and 24hrs in PC3 cells when compared to control. Boyden assay showed that cells treated with BAT-1 recombinant protein at 0.25μg/mL and 2.0μg/mL significantly decreased invasion in PC3 and 22RV1 cells at 24hrs when compared to control. Our results showed that BAT-1 up-regulation decreased migration and cell motility. Cells did not show a significant change in proliferation. These results were expected due to previous data showing that BAT-1 down-regulation increased cell migration and invasion, suggesting that BAT-1 expression promotes aggressiveness in prostate cancer recurrence. A possible mechanism of action for BAT1 is the modulation of cell migration and invasion.

#1517

Retinoic acid-dependent profiles of gene expression in sensitive and resistant breast cancer cell lines.

Marco Bolis. _Istituto di Ricerche Farmacologiche, Milano, Italy_.

BACKGROUND

Breast cancer is a collection of diseases which can be divided in various subgroups according to their profile of gene expression and positivity to estrogen receptor (ER), progesterone receptor (PR) and the tyrosine kinase receptor, HER2. All-trans retinoic acid (ATRA) is a cyto-differentiating agent and it is used in the treatment of acute promyelocytic leukemia with remarkable results. Given its unusual mechanism of action and low level of toxicity, ATRA has raised interest also for the treatment of solid tumors, including breast cancer. Pre-clinical data support the idea that ATRA is a promising agent in the treatment and chemoprevention of certain subgroups of breast cancer, with particular reference to ER+ and HER2+ tumors characterized by co-amplification of the retinoic acid receptor alpha gene. There is also a low proportion of triple-negative breast cancers which show sensitivity to this unusual anti-tumor agent.

RESULTS

In previous studies, we profiled a large panel of breast cancer cells (>50 lines), representative of the disease heterogeneity, for their sensitivity to the anti-proliferative action of ATRA (Bolis M. et al. Ann Oncol. 2017, 28:611; Centritto F et al.. EMBO Mol Med. 2015, 7:950). To identify gene-networks and gene pathways involved in the anti-proliferative action of ATRA, we performed total RNA-sequencing experiments in a panel of 16 sensitive and resistant cells (8 luminal and 8 triple-negative lines) before and after treatment with the retinoid (1.0 microM) for 48 hours. We identified gene-networks whose expression is selectively modulated by ATRA in retinoid-sensitive luminal and triple-negative cell lines as well as other gene-networks which are commonly regulated in both cell groups. Among the networks stimulated by ATRA, the group of genes involved in interferon-responses is of particular interest, as it indicates that the retinoid exerts a strong and specific immuno-modulatory action in sensitive breast cancer cell lines. A second pathway of relevance is represented by genes involved in the EMT (epithelial-to-mesenchymal transition) process which are down-regulated by ATRA in sensitive luminal cells. We are evaluating the functional significance of specific elements of these gene-networks for the anti-tumor/-metastatic action of ATRA with the use of silencing and over-expression approaches.

CONCLUSIONS

The results obtained in our cellular models provide insights into the molecular mechanisms underlying the anti-tumor action of ATRA in breast cancer. In addition, the sequencing data led to the identification of ATRA-dependent pathways and gene-networks with significance for the anti-tumor activity of the retinoid. Finally, the approach provides information as to potential new molecular targets for the design of rational therapeutic combinations based on ATRA for the treatment and secondary chemo-prevention of certain types of breast cancer.

#1518

Gene expression of trefoil proteins, gastrokines and mucins in Indian gastric cancer patients.

Dipjit Basak,1 Pronoy Kanti Mondal,1 Brishti Mondal,1 Ranajoy Ghosh,2 Aniket Halder,2 Gopal Krishna Dhali,2 Abhijit Chowdhury,2 Indranil Mukhopadhyay,1 Shalini Datta1. 1 _Indian Statistical Institute, Kolkata, India;_ 2 _Institute of Postgraduate Medical Education and Research, Kolkata, India_.

Background: Gastric cancer is the (GC) third most cause of cancer related mortality worldwide. Histopathologically, GC is sub classified into two major histopathological subtypes- diffuse and intestinal. The trefoil factor family - TFF1, TFF2 and TFF3 is known to play a pivotal role in gastrointestinal mucosal defence and repair. Gastrokines (GKN1, GKN2) are stomach specific secretary proteins which play fundamental roles in regulating gastric epithelial homeostasis. Similarly, secreted mucins are large O-glycosylated proteins that participate in the protection/defence of underlying mucosa in normal adults. The loss of expression of trefoil proteins, gastrokines and several mucins in retinoblastoma and colorectal cancer are reported in recent studies.

Aims: The molecular underpinnings and gene expression of Indian GC patients are largely unknown. In this study, we evaluated the gene expression of TFF1, TFF2, TFF3, GKN1, GKN2, MUC3A, MUC5AC, MUC13 in gastric cancer patients from India. Additionally, we wanted to see whether these gene expressions are different in two histopathological groups.

Materials and Methods: Endoscopic biopsy tissue from gastric tumour and adjacent normal regions were collected with ethical approval and informed consent from 42 patients . After histopathological confirmation by two pathologists, total RNA were isolated from tumour and corresponding normal tissue and gene expression was measured by qRT-PCR. Statistical analysis was performed by R package.

Result and Discussion: We identified that TFF1, TFF2, TFF3, GKN1, GKN2 are significantly differentially expressed in gastric tumour tissue with respect to adjacent normal tissue whereas mucins had no significant deregulation. TFF1, TFF2, GKN1 and GKN2 are found to be down regulated in most tumour tissues compared to adjacent normal. Only GKN2 expression is significantly different among diffuse and intestinal subtypes . Our statistical analysis also revealed that gene expression of TFF1, TFF2, GKN1 and GKN2 are highly correlated with the gene expression of MUC5AC. Further study with larger patient sample is required to fully understand the complex interplay of trefoil, gastrokines and mucins in GC.

#1519

TonEBP promotes hepatocellular carcinogenesis, recurrence, and metastasis.

Jun Ho Lee,1 Jae Hee Suh,2 Soo Youn Choi,1 Hyun Je Kang,1 Hwan Hee Lee,1 Whaseon Lee-Kwon,1 Jiyoung Park,1 Kyungjae Myung,1 Neung Hwa Park,2 Hyug Moo Kwon1. 1 _Ulsan National Inst. of Science & Technology, Ulsan, Republic of Korea; _2 _Ulsan University Hospital, Ulsan, Republic of Korea_.

Introduction: Hepatocellular carcinoma (HCC) is a common cancer with high rate of recurrence and mortality. Diverse etiological agents and wide heterogeneity in individual tumors impede effective and personalized treatment. Tonicity-responsive enhancer-binding protein (TonEBP) is a transcriptional cofactor for the expression of pro-inflammatory genes. Although inflammation is intimately associated with the pathogenesis of HCC, the role of TonEBP is unknown. We aimed to identify function of TonEBP in HCC.

Methods: Tumors with surrounding hepatic tissues were obtained from 296 patients with HCC who received completion resection. TonEBP expression was analyzed by qRT-PCR and immunohistochemical analyses of tissue microarrays. Mice with TonEBP haplo-deficiency, and hepatocyte- and myeloid-specific TonEBP deletion were used along with HCC and hepatocyte cell lines.

Results: TonEBP expression is higher in tumors than in adjacent non-tumor tissues in 92.6% of 296 patients with HCC regardless of etiology associated and DEN-induced mouse HCC. Hepatic induction of TonEBP is mediated by a fall in the miR-223 abundance. The TonEBP expression in tumors and adjacent non-tumor tissues predicts recurrence, metastasis, and death in multivariate analyses. Univariate analysis of two layers of patients showed that higher TonEBP expression was significantly associated with bigger tumor, advanced tumor grade, and vascular invasion. TonEBP promotes HCC initiation and growth via oxidative stress and inflammation in various mouse models of HCC. The association between TonEBP and inflammation was confirmed from analysis of the RNA-seq dataset from TCGA. TonEBP-dependent stimulation of tumor growth was dependent on COX-2. TonEBP drives the expression of COX-2 by stimulating the promoter in association with transcription factor YY1 and histone acetyltransferase p300. TonEBP is required for the recruitment of both YY1 and p300 to the promoter in situ. The interaction between TonEBP and YY1 is mediated by RHD and spacer domain. TonEBP deficiency resulted in reduced COX-2 expression leading to reduced production of prostaglandin E2 in various animal models of HCC and acute liver injury. In addition, self-renewal of hepatic cancer stem cells (hCSCs) contributing to recurrence was driven by TonEBP in association with ERCC1. In mouse models of HCC and acute liver injury, three common sites of TonEBP action in response to diverse etiological agents leading to tumorigenesis and tumor progression were found: cell injury and inflammation, induction by oxidative stress, and stimulation of the COX-2 promoter

Conclusions: TonEBP is a key component of the common pathway in tumorigenesis and tumor progression of HCC in response to diverse etiological insults. TonEBP is involved in multiple steps along the pathway rendering it an attractive therapeutic target as well as a prognostic biomarker.

#1520

Regulation of stemness by perturbation of OCT4-PP1 axis reduces malignancy of embryonal carcinoma.

Bomin Song, Hyonchol Jang. _National Cancer Center, Goyang-si, Republic of Korea_.

OCT4 (encoded by pou5f1) is a master regulator of pluripotency and closely related to tumor malignancies. Its associations with cancer drug-resistance, recurrence and poor prognosis of the patients has been widely reported. As OCT4 transcriptional activity is directly and indirectly involved in the maintenance of cancer stemness, inhibition of OCT4 activity could be a strategy to reduced cancer malignancies by eliminating stemness. In this study, we investigated a way to regulate the stemness of embryonal carcinoma (EC) by perturbation of posttranslational modification of OCT4. First, to confirm the role of OCT4 in the maintenance of stemness in ECs, we generated the cells which are downregulated in OCT4 doxycycline-dependently. Down-regulation of OCT4 caused weaker alkaline phosphates staining, reduced tumor sphere formation, and reduced expression of stemness marker proteins. Also, the cells underwent retardation of proliferation, cell-cycle arrest in G1 phase, and eventual apoptosis by OCT4 downregulation. The most altered genes in ECs by OCT4 downregulation were networked to signaling pathways regulating pluripotent stem cells, Hippo signaling pathways and transcriptional misregulation in cancer. Second, to regulate OCT4 transcriptional activity, we used phosphorylation of serine 236th (S236) on OCT4. ECs cells, with which their endogenous OCT4 was substituted with an OCT4 phosphorylation-mimicking mutant (Serine 236 to aspartate; S236D), showed similar phenotype to those cells that were depleted in OCT4. Chemical inhibition or knockdown of PP1 (protein phosphatase 1), which remove the phosphorylation, induced accumulation of S236 phosphorylation, and also caused similar phenotype to those cells that were depleted in OCT4. Finally, to evaluate whether regulation of OCT4 can reduce tumor malignancy, we tested whether drug-tolerant persister cancer cells could be reduced by regulation of OCT4. Co-treatment of inhibits PP1 activity with cisplatin significantly reduced drug-resistant colonies compared with single treatment of cisplatin. These results suggested that stemness of ECs could be regulated by OCT4 posttranslational modifications and regulation of cancer stemness could reduce tumor malignancy.

#1521

Role of Trim28 in prostate cancer.

Ka-Wing Fong, Jonathan Zhao, Bin Zheng, Jindan Yu. _Northwestern Univ., Chicago, IL_.

TRIM28 is an essential gene during embryonic development, which is involved in a wide range of biologic processes. From the pathologic viewpoint, TRIM28 overexpression was proved to broadly exist in pancreatic, liver, breast, glioma and lung cancer, and often expression of TRIM28 is correlated with clinical stage. Functional assays have demonstrated that TRIM28 promotes cancer cell growth and metastasis, further supporting TRIM28 as a promising target for therapeutic intervention. For past decade, TRIM28 is considered to be a transcriptional co-repressor due to TRIM28-dependent recruitment of histone modifiers such as CHD1/NuRD complex and SETDB1 for histone methylation and heterochromatin formation to achieve gene silencing. Surprisingly, transcriptome study revealed that TRIM28 can activate the expression of its target genes as much as it represses, hypothesizing the main role of TRIM28 may not lie in transcriptional repression. In addition, recent findings demonstrated that TRIM28 interacts with 7SK snRNP complex, which facilitates P-TEFb kinase module recruitment and productive transcription elongation at inducible genes. This evidence implicated that TRIM28 may form distinct complexes to induce or repress gene expression in context-dependent manner. On top of that, it remains unclear about the role of TRIM28 in prostate cancer. Our preliminary study revealed that TRIM28 is upregulated in castration-resistant prostate cancer (CRPC). TRIM28 knockdown markedly decreased prostate cancer cell proliferation, clonogenicity as well as CRPC-xenograft tumor growth. Moreover, proteomic analysis in LNCaP prostate cancer cells revealed that TRIM28 associates with a number of transcription co-activators. Gene expression profiling showed that androgen signaling is positively regulated by TRIM28. We proposed that TRIM28 forms the transcription activation complexes that modulate gene expression of various oncogenic pathway and promote PCa progression.

### Targeting the Cell Cycle: Mechanism and Therapy

#1522

The CDK4/6 inhibitor G1T38 enhances response to targeted therapies in preclinical models of non-small cell lung cancer.

Jessica A. Sorrentino, Daniel M. Freed, John E. Bisi, Jay C. Strum, Patrick J. Roberts. _G1 Therapeutics, Research Triangle Park, NC_.

The recent FDA approvals of palbociclib, ribociclib, and abemaciclib in breast cancer validate cyclin-dependent kinases 4 and 6 (CDK4/6) as key therapeutic targets, and warrant investigations in other tumor types with frequently observed alterations in the CDK4/6 pathway. Towards this end, we assessed the preclinical antitumor efficacy of G1T38, an oral, potent, and selective small-molecule CDK4/6 inhibitor in clinical development, in models of non-small cell lung cancer (NSCLC). Importantly, preclinical studies have shown that G1T38 has a differentiated and potential best-in-class profile since it can be dosed continuously without causing severe neutropenia, which could result in better tumor efficacy. Initial efficacy screening of G1T38 in 60 patient-derived NSCLC xenografts revealed significant tumor growth inhibition (TGI), primarily in lung adenocarcinomas harboring frequently observed oncogenic alterations in KRAS, EGFR, BRAF, and ALK. In vitro analyses show that CDK4/6 inhibition enhances the antiproliferative effect of inhibitors targeting these oncogenes (or their signaling pathways), suggesting a possible role for G1T38 in augmenting response and/or delaying acquired resistance to agents currently utilized in the clinic. These results were validated in vivo in mouse NSCLC xenograft models with defined oncogenic alterations, including EGFR mutations. For example, in H1975 xenografts harboring EGFR L858R/T790M (the T790M mutation is responsible for over 50% of cases of acquired resistance to first-generation EGFR inhibitors), combination of G1T38 with erlotinib resulted in 77% TGI after 18 days--a significant improvement over either therapy alone. Moreover, in H1975 xenografts, G1T38 delayed resistance to the second-generation EGFR inhibitor afatinib, and upon development of resistance to afatinib monotherapy, addition of G1T38 to afatanib treatment resulted in stabilization of tumor growth. G1T38 also augmented the response of EGFR-mutant NSCLC xenografts to the third-generation EGFR inhibitor osimertinib, significantly enhancing TGI compared to either therapy alone. As a result, a safety, pharmacokinetics, and efficacy study of G1T38 + osimertinib (G1T38-03) is scheduled for initiation in 1Q18 in patients with EGFR-mutant, T790M-positive NSCLC. Additional efforts are ongoing to investigate the combination of G1T38 with targeted therapies in NSCLC murine models harboring alterations in KRAS, ALK, and BRAF. Together, our results suggest a compelling rationale for utilizing G1T38 as a backbone for multiple targeted therapy combination regimens in NSCLC.

#1523

Repression of IRF4 and CYTIP unleashes anti-tumor interferon response in CDK4 inhibitor therapy in mantle cell lymphoma.

Maurizio Di Liberto, Peter Martin, Xiangao Huang, Priyanka Vijay, David Chiron, Scott Ely, Christopher Mason, Olivier Elemento, John Leonard, Selina Chen-Kiang. _Weill Cornell Medical College, New York, NY_.

Inhibition of CDK4/6 has emerged as an effective cancer therapy, but the underlying mechanism remains obscure. We have addressed this question in mantle cell lymphoma (MCL), a Non-Hodgkin's B cell lymphoma that remains incurable due to the development of drug resistance. As dysregulation of CDK4 (CDK6 is silenced) and cyclin D1 underlies unrestrained proliferation of MCL cells, targeting CDK4 also represents a rational approach to MCL therapy.

Previously we have demonstrated in preclinical studies that sustained inhibition of CDK4 with palbociclib led to prolonged early G1 arrest (pG1) that both prevented proliferation of MCL cells and reprogrammed them for killing by diverse agents. On this basis, we hypothesized that CDK4 inhibition may prolong and deepen the clinical response to its partner drug in combination therapy. Indeed, in a phase I clinical trial of palbociclib plus ibrutinib (BTK inhibitor) in recurrent MCL (n=28), complete response was observed in 43% of patients as compared with 23% in response to ibrutinib alone. Moreover, the responses were rapid and durable; only 4 of the 18 responding-patients have progressed in the 3.5 years since the trial opened.

To address how CDK4 inhibition enhances therapy vulnerability, in a separate phase I clinical trial palbociclib was administered to MCL patients for 12 days to induce pG1; bortezomib was given in pG1 and again after synchronous S phase reentry (pG1-S). At the optimal dose combination only one of 6 patients progressed and complete remission in one patient continues into its 6th year. Longitudinal integrative RNA and exome-sequencing (WTS/WES) of MCL cells isolated from lymph node biopsies at baseline, in pG1 (day 8) and in pG1-S (day 21) of individual patients showed that inhibition of CDK4 induced pG1 in all patients initially, regardless of the subsequent clinical response.

However, induction of pG1 led to a striking difference in cellular gene expression in pG1 (day 8/day 1) in MCL cells of responders (R) and non-responders (NR). There were 2041 genes upregulated and 1483 genes downregulated 1.5-fold or greater in R (N=4, EdgeR, FDR 0.05), but only 39 genes upregulated and 401 genes dowregulated in NR (N=3). Moreover, 5 genes were down regulated in R and upregulated in NR. Gene set enrichment analysis together with loss and gain of function studies revealed that among them, loss of IRF4 in R led to de-repression of IRF7 and induction of the interferon response. This in turn resulted in TRAIL mediated apoptosis due to repression of CYTIP downstream of IRF4 repression.

In summary, by longitudinal functional genomics of purified MCL cells and functional studies, we have discovered that CDK4 inhibition reprograms MCL for therapy vulnerability through induction of anti-tumor IFN response, and identified CYTIP and IRF4 as biomarkers that discriminate sensitivity from resistance to therapeutic targeting of CDK4.

#1524

A first-in-class CDK4 inhibitor shows excellent in vitro and in vivo efficacy against ovarian cancer.

Laychiluh B. Mekonnen,1 Solomon Zeleke,1 Jimma Lenjisa,1 Mingfeng Yu,1 Benjamin Noll,1 Gary Heinemann,1 Robert Milne,1 Hugo Albrecht,1 Martin K. Oehler,2 Shudong Wang1. 1 _University of South Australia, Adelaide, Australia;_ 2 _Royal Adelaide Hospital, Adelaide, Australia_.

Ovarian cancer is one of the most common malignancies, accounting for more deaths than any other cancer of the female reproductive system. Cyclin-dependent kinases 4 and 6 (CDK4/6) are fundamental drivers of the cell cycle and are required for the initiation and progression of various cancers. Over-expression of CDK4/6 &/or cyclin D1 is commonly observed in ovarian cancer and is associated with an aggressive phenotype and poor prognosis. Patients with advanced ovarian cancer whose tumor demonstrated Rb-positive & a low expression of p16INK4a have the worst clinical outcome, but these patients are likely to gain benefit from CDK4/6 inhibition. Here, we describe the preclinical development of 2-94; a potent, highly selective and orally bioavailable CDK4 inhibitor for the treatment of ovarian cancer. Specifically, 2-94 exhibits high selectivity for CDK4/D1 (Ki = 2 nM) over CDK6/D3 (Ki = 279 nM). Consistent with a CDK4-targeted mechanism, 2-94 potently inhibited proliferation of Rb proficient ovarian cancer cells. It induced G1 cell cycle arrest by reducing Rb phosphorylation at S780, S795 and S807/11 residues in a dose dependent manner. Apoptosis was induced with evident increase of caspase 3/7 activity and reduction of Bcl-2 protein levels in A2780 cells. In addition, 2-94 has shown synergistic activity with mTOR, MEK, PI3K and PARP inhibitors. In contrast, no apparent cell cycle effect, senescence or apoptosis was observed in human bone marrow mononuclear (hBMNCs) cells by 2-94 treatment unlike palbociclib and ribociclib which increased the G1 population of hBMNCs, suggesting the low toxicity of 2-94 compared to clinical compounds. Consistent with its favourable pharmacokinetic properties and high oral bioavailability, 2-94 delayed tumor growth and significantly increased the survival of A2780 tumor bearing nude mice by oral administration. 2-94 was more efficacious when compared to palbociclib, resulting in a T/C = 22% for 2-94 vs T/C = 31% for palbociclib. Moreover, 2-94 showed little or no effect on lymphocyte and neutrophil counts whilst palbociclib caused their reduction. This lower effect of 2-94 on the neutrophils and lymphocytes might be due to its lesser effect on CDK6-cyclin D3 compared to palbociclib. Both 2-94 and palbociclib didn't cause histopathological changes in the bone marrow, intestine, liver, heart and kidney of the mice. Pharmacodynamical studies confirmed that 2-94 reduced the Rb phosphorylation at S780, S795 and S807/811 in the tumour models. In conclusion, 2-94 is a first-in-class CDK4 inhibitor with high selectivity against a panel of 360 human kinases. This selectivity can be taken as a reason for the observed better safety of 2-94 compared to palbociclib. The favourable oral pharmacokinetics, robust antitumor efficacy and excellent safety profiles make 2-94 a highly attractive candidate for development towards the clinic.

#1525

SY-1365, a selective CDK7 inhibitor, exhibits potent antitumor activity against ovarian cancer models in vitro and in vivo.

Panagiotis A. Konstantinopoulos,1 Graeme Hodgson,2 Nisha Rajagopal,2 Liv Johannessen,2 Joyce F. Liu,1 Paul T. Kirschmeier,1 Shan Zhou,1 Cam Anh Tran,1 David Orlando,2 Christian Fritz,2 Emmanuelle di Tomaso,2 Ursula A. Matulonis1. 1 _Dana Farber Cancer Institute, Boston, MA;_ 2 _Syros Pharmaceuticals, Inc., Cambridge, MA_.

Introduction: CDK7, a key regulator of transcription and cell cycle progression, has been implicated in the pathogenesis of ovarian carcinoma. SY-1365, a potent and selective inhibitor of CDK7, was developed to exploit tumor dependencies driven by CDK7 and is in phase 1 clinical development. Here we report, for the first time, on the activity of SY-1365 in preclinical models of ovarian carcinoma and on potential biomarkers of SY-1365 response.

Methods: SY-1365 growth rate inhibition (GR) curves were determined in 314 solid tumor cell lines, including 22 ovarian carcinoma lines. Cell lines were clustered into cytotoxic and cytostatic response groups and assessed for RNA expression to identify markers associated with SY-1365 sensitivity. In vivo, SY-1365 activity was evaluated at the maximum tolerated dose in 17 independent ovarian PDX models derived from heavily pretreated patients; a signal-finding design was used in which tumor growth in SY-1365 treated mice (n=2-3 per PDX model) was compared to vehicle-treated mice or historical growth in untreated mice.

Results: SY-1365 induced cytotoxicity (GR <0) in 61% (190/314) of all tumor lines at <50nM. In ovarian carcinoma lines, 36% (8/22) were cytotoxic at <50nM, with deep responses (GRmax < -0.5) seen in 23% (5/22). Lower expression of BCL2L1, which encodes the mitochondrial apoptosis antagonist BCLXL, was the most predictive marker of sensitivity across all lines (accuracy = 70%; FDR=0.006). Reasoning that low BCL2L1 may be a marker of dependence on other mitochondrial apoptosis antagonists (MCL1 and/or BCL2) for survival, we evaluated expression of BCL2, BCLXL, and MCL1 protein at baseline and post-SY-1365 treatment in ovarian carcinoma lines. As with BCL2L1 RNA, BCLXL expression was lower at baseline in cytotoxic- versus cytostatic-responsive lines. In addition, SY-1365 induced MCL1 downregulation in cytotoxic, but not in cytostatic lines. In vivo, SY-1365 induced responses in 52% (9/17) of ovarian carcinoma PDX models. Of the 9 responders, 2 exhibited 40-60% tumor growth inhibition (TGI), 7 exhibited ≥ 100% TGI. As in cell lines, low BCLXL, in combination with low BCL2, was associated with SY-1365 response in PDXs. In addition, 7/9 responsive models had alterations in the RB signaling pathway including loss of RB1 expression (n=3), high CCNE1 expression (n=3), or CDKN2A loss (n=1).

Conclusions: SY-1365 induces cytotoxicity in ovarian carcinoma cell lines and induces robust responses in heavily pretreated (including PARP-inhibitor and platinum-resistant) ovarian PDX models. Alterations in expression of mitochondrial apoptosis antagonists and RB pathway regulators are associated with SY-1365 response and support exploration of these as predictive biomarkers of SY-1365 clinical activity in ovarian carcinoma. SY-1365 is currently being assessed in a phase 1 trial in adult patients with advanced solid tumors (NCT03134638).

#1526

WEE1 inhibition suppresses esophageal adenocarcinoma tumor growth both in vitro and in vivo.

Mylan Blomquist, Vashti M. Carson, Ross M. Bremner, Timothy G. Whitsett, Landon J. Inge. _St. Joseph's Hospital and Medical Center, Phoenix, AZ_.

Esophageal adenocarcinoma (EAC) has seen a 400% increase in incidence over the past 30 years. The 5-year survival rate is under 20% due to ineffective therapeutics and a lack of actionable oncogenic drivers, necessitating novel therapeutic avenues in this disease. Genomic analysis indicates that a subset of EAC tumors have alterations in the G2/M cell cycle check point and DNA damage response governed by mutations in TP53 or other hits in this pathway, suggesting targeting the G2/M pathway might be a viable option in EAC. We and others have demonstrated that the WEE1 inhibitor AZD1775 is effective both in vitro and in vivo against tumors with DNA damage response alterations, and this drug is currently in clinical trials across a number of tumor types. We hypothesized that inhibition of WEE1 would induce DNA damage and cell death in EAC tumors, and provide a rational therapeutic avenue against this deadly disease. Across multiple EAC cell lines, AZD1775 suppressed tumor cell growth either as a monotherapy or in combination with DNA-damaging therapies. The growth suppression was accompanied by persistent DNA damage as determined by γH2AX and the induction of apoptosis. FLO-1 cells, responsive to monotherapy AZD1775, showed a dramatic reduction in G2 cells 24 hours post AZD1775 exposure with concomitant increases in cells in G0 and S phases of the cycle; while SK-GT-4, resistant to monotherapy AZD1775, showed an initial reduction in cells in G2 but recovered to normal levels 24 hours post-exposure. To assess AZD1775 in vivo, we generated a patient-derived xenograft (PDX) model of an EAC tumor excised at our institution. Combinations of AZD1775 with radiation or cisplatin significantly reduced in vivo tumor growth compared to vehicle, and the combination of AZD1775 with cisplatin was as effective as standard-of-care treatment (cisplatin + docetaxel + radiation), a therapeutic avenue plagued by toxicity in humans. Exposure to AZD1775 in combination with cisplatin showed suppression of phospho-CDC2 (target-hit) and induction of γH2AX in vivo compared to vehicle. Towards understanding a mechanism for lack of response of certain EAC cell lines to AZD1775 monotherapy, we explored the PI3K pathway that has been previously implicated in AZD1775 resistance. SK-GT-4 cells, which are unresponsive to AZD1775 monotherapy, demonstrated elevated phospho-AKT and phospho-p70S6K protein levels compared to other cell lines and were found more sensitive to PI3K or MTOR inhibition, suggesting a dependence on this pathway for tumor cell survival. Collectively, our data suggest that AZD1775, both alone and in combination with standard-of-care DNA damage, may be an effective therapeutic strategy for those EAC tumors dependent on the G2/M checkpoint.

#1527

Splicing factors determine breast cancer cell mitosis through control of sister chromatid cohesion.

Esmee Koedoot,1 John Martens,2 Sylvia E. Le Dévédec,1 Bob van de Water1. 1 _Leiden University, Leiden, Netherlands;_ 2 _Erasmus MC, Rotterdam, Netherlands_.

Breast cancer is the mostly diagnosed cancer in women worldwide and is also the leading cause of death from cancer in women, mainly due to metastasis formation. Accumulating evidence suggests that RNA splicing is critical in breast cancer progression. To investigate the role of every single component of the splicing machinery (the spliceosome) in this process, we systematically evaluated the effect of siRNA-mediated knockdown of 244 splicing factors on proliferation in two highly proliferative breast cancer cell lines MDA-MB-231 and Hs578T. Hits of the primary screen were validated with single siRNAs and separated based on their effects on migration and proliferation respectively. Knockdown of nine validated proliferation hits, amongst which was SF3B1, a known breast cancer driver gene, resulted for all nine hits in a distinct poly-lobed nuclear phenotype in both cell types, accompanied by increased DNA content and higher number of cells in G1-S transition. Increased levels of phospho-Histone H3 and lack of metaphase alignment suggested a defect in mitosis, which was confirmed by decreased RNA expression of sister chromatid cohesion factors MAU2, ESPL1 and SMC1A and interestingly, increased CDCA5 RNA levels. Upon splicing factor knockdown, CDCA5 intron 1 was retained resulting in decreased CDCA5 protein levels, inaccurate metaphase alignment and finally cell death. To identify the direct partners of the splicing factors that control mitosis, we coupled pull-down of GFP fused splicing factors with mass spectrometry. Seven out of the nine splicing factors that were identified in our screen causing poly-lobed nuclear phenotypes, resided in the same protein complex together with proteins involved in snRNP assembly and some mitosis-related factors. In conclusion, we have identified several splicing factors that are critically determining breast cancer cell proliferation through modulating the expression of chromatid cohesion factors and thereby mediating the successful metaphase alignment and ultimate mitosis.

## CLINICAL RESEARCH:

### Adoptive Cell Therapy 1

#1530

A multifunctional role for adjuvant anti-4-1BB therapy in augmenting antitumor responses by CAR T cells.

Sherly Mardiana, Liza B. John, Michael H. Kershaw, Paul A. Beavis, Phillip K. Darcy. _PETER MACCALLUM CANCER CENTRE, MELBOURNE, Australia_.

Adoptive immunotherapy utilising chimeric antigen receptor (CAR) T cells has demonstrated high success rates in hematological cancers, with up to 90% complete remission being reported in patients with acute lymphoblastic leukemia (ALL). However, this success is not currently transferrable to solid malignancies due in part to the immunosuppressive tumor microenvironment limiting CAR T cell responses. Given that activation of the 4-1BB (CD137) pathway using an agonistic α-4-1BB antibody is known to provide strong co-stimulatory signals for augmenting and diversifying T cell responses, we hypothesized that the combination of α-4-1BB and CAR T cell therapy would result in improved anti-tumor responses. Using a human-Her2 self-antigen mouse model, we found that α-4-1BB significantly enhanced CAR T cell efficacy directed against the Her2 antigen in two different established solid tumor settings. Investigation into the mechanism revealed increased expression of IFNγ and the proliferation marker Ki67 by tumor-infiltrating CAR T cells when combined with α-4-1BB. The importance of IFNγ was demonstrated in vivo where synergistic effects of the combination therapy were abrogated when IFNγ was depleted. Strikingly, we also found that α-4-1BB significantly reduced host immunosuppressive cells at the tumor site including regulatory T cells (Tregs) and myeloid derived suppressor cells (MDSCs), correlating with an increased therapeutic response. Investigation in immunocompromised RAG -/- mice showed reduced synergistic effects following the combination therapy, suggesting some contribution by host T cells. Furthermore, we found increased frequency of endogenous T cells and dendritic cells in the tumor-draining lymph nodes, supporting potential engagement of host immune cells. We therefore conclude α-4-1BB has a multifunctional role for enhancing CAR T cell responses including engagement of endogenous immune cells, and this combination approach has high translational potential given that CAR T cell therapy has recently been FDA approved for pediatric ALL, and that α-4-1BB is currently being tested in clinical trials against various types of cancer.

#1531

A bispecific chimeric-antigen receptor T-cell factory for triple-negative breast cancer.

Emily G. Kuiper,1 Leo L. Chan,2 Matthew R. Chang,1 Quan K. Zhu,1 Wayne A. Marasco1. 1 _Dana-Farber Cancer Institute, Boston, MA;_ 2 _Nexcelom Bioscience, Lawrence, MA_.

Triple-negative breast cancer (TNBC) is a highly aggressive subtype of breast cancer with poor clinical prognosis. While treated with chemotherapies, the high incidence of relapse signifies the need for novel, targeted therapies. Immune therapies offer an exciting therapeutic option for TNBC. Our goal is to engineer a chimeric-antigen receptor (CAR) T-cell factory, a CAR T-cell that secretes immune-modulating antibodies, for TNBC.

Chimeric-antigen receptor (CAR) T-cell therapies redirect a patient's T-cells to kill tumor cells by the exogenous expression of a CAR. A CAR is a membrane spanning fusion protein that links the antigen recognition domain of an antibody to the intracellular signaling domains of the T-cell receptor and co-receptor. Solid tumors offer unique challenges for CAR-T therapies. Unlike blood cancers, tumor-associated target proteins are overexpressed between the tumor and healthy tissue resulting in on-target/off-tumor T-cell killing of healthy tissues. Furthermore, immune repression in the tumor microenvironment (TME) limits the activation of CAR-T cells towards killing the tumor.

We hypothesize that a bispecific CAR targeting two antigens on TNBC will mitigate on-target/off-tumor T-cell killing and that the secretion of a checkpoint blockade antibody will remove repression in the tumor microenvironment. Following local immune restoration, the CAR-T cells and other cells in the TME will work synergistically to shrink and clear tumors. Our current work evaluates human single-chain variable fragments (scFvs) to serve as CAR-targeting moieties. We are evaluating the efficacy of scFvs to specifically kill target cells in vitro. This work will define lead scFvs used for engineering of our bispecific CAR. Future work will evaluate this therapy in humanized mouse models of TNBC.

#1532

Canine CAR T cells therapy for mammary carcinomas in dogs.

Xavier E. Ramos Cardona, Sulma Mohammed, Philip S. Low, Yong G. Lee. _Purdue University, West Lafayette, IN_.

About 63,410 cases of DCIS will be diagnosed in 2017 in USA alone. DCIS is treated with mastectomy/lumpectomy with adjuvant radio- or hormonal therapy with the goal of reducing the risk of IBC and death. However, aggressive treatment of DCIS with radiotherapy after lumpectomy was shown to increase mortality, suggesting that local therapy alone is insufficient to treat DCIS and thus new approaches are needed. The presence of tumor infiltrating lymphocytes (TIL) in IBC has correlated positively with survival, thus providing the rationale for implementing immunotherapeutic strategies, such as adaptive T-cell therapies. One recent and exciting approach is the use of chimeric antigen receptor (CAR) T-cells, which express genetically engineered receptors that allow the modified T-cells to target a tumor-associated antigen. First, second, and third generation CAR T-cells have been developed and have shown promise in treating many malignancies, but in solid tumors has been hindered by many limitations. To overcome these limitations, we designed a genetically engineered universal CAR T-cell that must be activated and targeted by a small molecule adaptor before it can kill cancer cells. In this study, we plan to design canine CAR T-cells and test it functionality in vitro.

#1533

CD4+ T cells reprogram tumor metabolism and drive oxidative stress-induced tumor destruction.

Gang Zhou, Tsadik Habtetsion, Zhi-Chun Ding. _Augusta University, Augusta, GA_.

The inhibitory effects of cancer on T cell metabolism have been well established, but the metabolic impact of immunotherapy on tumor cells is poorly understood. Here, we developed a CD4+ T cell-based adoptive immunotherapy protocol that was curative for mice with implanted colorectal cancer. By conducting metabolic profiling on resected tumors, we show that adoptive immunotherapy profoundly altered tumor metabolism, resulting in glutathione depletion and accumulation of reactive oxygen species (ROS) along with increased oxidative DNA damages. We further demonstrate that T cell-derived TNFα can synergize with chemotherapy to drive heightened oxidative stress and tumor cell death. Reduction of oxidative stress, by neutralizing TNFα or scavenging ROS, antagonized the therapeutic effects of adoptive T-cell therapy. Conversely, provision of pro-oxidants after chemotherapy can partially recapitulate the antitumor effect of T-cell transfer. Furthermore, T cell-induced oxidative stress in tumor was observed in several different tumor models. These findings imply that tumor redox modulation represents an important mechanism underlying the efficacy of adoptive immunotherapy.

#1534

Analysis of CAR 41-BB versus CD28 co-stimulatory domains exposes emergence of extramedullary disease in acute myeloid leukemia.

Lila Yang,1 Samiksha Tarun,2 Christopher D. Chien,1 Mark E. Kohler,1 Haiying Qin,1 Terry J. Fry1. 1 _National Institutes of Health, Washington, DC;_ 2 _Saint Louis University School of Medicine, Saint Louis, MO_.

Phase I CD19 and CD22 targeting Chimeric Antigen Receptor (CAR) T cell trials have shown tremendous results against acute lymphoid leukemia (ALL). Across these trials there were variations in the types of costimulatory molecules included in the CAR constructs and this lead to the discovery that CAR T cells with CD28 result in earlier potency and activation with decreased persistence, while CARs with 4-1BB show increased expansion and persistence. There is great interest in translating these results in ALL trials into other diseases such as acute myeloid leukemia (AML). It is unclear which costimulatory domain in a CAR will be most effective in treating AML. To look at how co-stimulatory domains impact CAR functionality in AML, we developed CD33 targeting CD28 and 4-1BB CARs.

In vitro testing of the constructs revealed that CD33 CD28 CAR consistently produced more IL2 and Interferon-gamma than CD33 4-1BB across multiple AML cell lines. To translate these findings in vivo, xenograft models were injected with Molm 14 AML cells and treated with either CD33 CD28 or CD33 4-1BB CAR T cells. By bioluminescence imaging, CD33 CD28 treated mice had no detectable disease while CD33 4-1BB treated mice were ridden with leukemia. Combined, the in vitro and in vivo results suggest that the co-stimulatory domain does play a critical role in CAR T cell functionality and may improve CAR potency. To confirm the presence of AML in mice detected by bioluminescence, flow cytometry was performed on tissues from mock and CD33 4-1BB treated mice. No leukemia was found in the bone marrow of mock T cell treated mice. In contrast, CD33 4-1BB treated animals were clear of any leukemia in the bone marrow, suggesting the presence of extra medullary disease (EMD). The development of EMD in the less potent CD33 4-1BB CAR treated mice suggests that CAR immune pressure may be potent enough to clear primary sites of leukemia such as the bone marrow, but unable to eliminate disease in secondary tissues that AML can seed. This is not surprising since treatment of AML with chemotherapy often leads to the development of extramedullary disease in the form of chloromas.

To further investigate the effects of these two factors, we moved onto another AML model, THP1, that regularly presents with EMD even in the absence of CAR pressure. With CD33 CD28 against THP1, there was clearance in compartments bone marrow, however CD33 CD28 CAR was not able to prevent the development of EMD. These experiments suggest that although the CD28 costimulatory domain is more potent than 4-1BB in Molm14, the potency of CD28 is still not able to overcome EMD in all models. Using further studies of different AML models, we will continue to tease apart the contribution the impact of immune pressure from CARs, the natural progression of AML models, and effect of CAR potency on leukemia distribution.

#1535

T Cells engineered to express the IL-13Rα2 specific CAR and a PD-L1 specific switch receptor show increased anti-tumor activity in the Glioblastoma tumor microenvironment.

Abel De Castro, Megan E. Keys, Jonathan Arroyo, Megan E. Prosser. _Azusa Pacific University, Azusa, CA_.

Glioblastoma multiforme (GBM) is an aggressive brain cancer with poor prognosis with traditional treatments such as chemotherapy and radiotherapy, which often leave lasting and pervasive damage to the individual. GBM cells upregulate the surface protein interleukin 13 receptor subunit alpha-2 (IL13Rα2), which can be targeted as a novel immunotherapeutic marker for the immune response to be initiated. In this study, IL13Rα2 is targeted using a first generation chimeric antigen receptor (CAR) that contains an intracellular zeta chain domain. However, a limitation to this approach is that the tumor microenvironment exhausts the T cells that interact with the tumor through a receptor-mediated response. Programmed Death Ligand 1 (PD-L1) is a molecule that is expressed on cancerous cells that binds to Programmed Death 1 (PD-1) on T cells. When the ligand binds to PD-1 on T cells, there is decreased T cell proliferation, cytokine production, and anti-tumor activity. This problem can be avoided via creation of a switch receptor with a PD-1 extracellular domain fused to transmembrane and intracellular costimulatory CD28, which converts tumor mediated inhibition into stimulation upon ligation of PD-L1. It is hypothesized that the incorporation of additional costimulatory domains will enhance the stimulatory effects of the switch receptor. Engineered T cells expressing varied switch receptors in conjunction with the IL13Rα2-specific CAR have been created and have been assessed in vitro as a basis for glioblastoma treatment. Tests performed include cytokine secretion and T-cell proliferation assays. Results of these preliminary studies suggest that the the inclusion of additional costimulatory domains in the switch receptor design may be advantageous in enhancing anti-tumor responses in the GBM microenvironment.

#1536

**Enhancing CAR T cell activity by linking IL-12 expression to the endogenous** PDCD1 **promoter.**

Baeckseung Lee, Wai-Hang Leung, Jasdeep Mann, Kyle Havens, Joel Gay, Richard A. Morgan, Alexander Astrakhan. _bluebiridbio, Seattle, WA_.

Chimeric antigen receptor (CAR) T cells have shown great promise in treating certain late stage hematological malignancies. While very encouraging, current CAR T cell therapies have not shown the same level of success in targeting solid tumors and alternative approaches are required to achieve clinical efficacy in solid tumor patients. We describe a combinatorial approach whereby targeted gene deletion and transgene insertion occur simultaneously resulting in more potent CAR T cells for solid tumor applications.

Immunomodulatory cytokines can stimulate vigorous antitumor responses and are candidates for increasing CAR T cell efficacy in solid tumors. However, the clinical application of cytokine therapy has been limited by systemic toxicity, particularly for strong effector cytokines such as IL-12. Limiting IL-12 expression to within the tumor microenvironment may reduce unwanted toxicity while enhancing CAR T cell functionality.

Immune checkpoint gene programed cell death 1 (PDCD1) is a regulator of T cell functionality that is highly upregulated following T cell activation, with antibody and nuclease-mediated inactivation of the PD-1 signaling pathway having been shown to enhance CAR T cell functionality. Here, we used megaTAL genome editing/homology directed repair (HDR) to place an IL-12 transgene under the control of the PDCD1 promoter, linking IL-12 production with CAR T cell activation as well as eliminating PD-1 expression. CAR expression was combined with site specific transgene expression as follows: lentiviral vector-engineered T cells were treated with a PDCD1-specific megaTAL and transduced with adeno associated virus-6 (AAV6) containing a promoter-less IL-12 transgene flanked by PDCD1 homology regions. We observed highly efficient HDR, with inducible IL-12 expression from the endogenous PDCD1 promoter being dependent on T cell activation. Minimal IL-12 production was detected under resting conditions, whereas PMA/Ionomycin or co-culture with CAR+ target cell lines resulted in higher IL-12 secretion. Expression of IL-12 under the PDCD1 promoter enhanced CAR T cell cytokine production and cytotoxicity, especially under conditions of repeated antigen exposure.

In summary, we describe a novel genome editing strategy to enhance CAR T cell functionality. Using HDR, we were able to engineer CAR T cells to simultaneously disrupt the PDCD1 gene and place a potentially therapeutic transgene under inducible transcriptional control. The IL-12/CAR T cells exhibited activation-dependent IL-12 production and enhanced cytokine and cytotoxicity responses against tumor cells in vitro. HDR may represent a promising approach to enhance CAR T cell functionality in solid tumor applications.

#1537

Anti-CD19 ARTEMIS™ T cells prevent excessive inflammatory cytokine release, including IL-6, in a co-culture model of CRS.

Hong Liu,1 Lucas H. Horan,1 Stephan A. Grupp,2 David M. Barrett,2 Cheng Liu1. 1 _Eureka Therapeutics, Inc., Emeryville, CA;_ 2 _Children's Hospital of Philadelphia, Philadelphia, PA_.

We designed the ARTEMISTM T-cell signaling platform to be a safer ACT therapy by relying on endogenous T-cell signaling factors. We show evidence that the ARTEMISTM receptor disentangles efficacy from CRS and holds the potential to be a clinically safer therapy by preventing ACT-triggered CRS. Despite the remarkable curative potential of anti-CD19-CAR T cells the therapy continues to trigger a potentially life threatening cytokine release syndrome (CRS) that is characterized by excessive concentrations of inflammatory cytokines. We reasoned that by redesigning the T-cell activating antigen receptor to require signaling through the endogenous CD3 complex the T-cell could better regulate its cellular responses and thus reduce the risk of CRS when used as an adoptive T-cell transfer (ACT) therapy. Through protein engineering, we developed the ARTEMIS™ signaling platform which when coupled with Eureka's human anti-CD19 antibody, ET190L1, and expressed on primary T-cells, results in a significant reduction of cytokine release during antigen specific T-cell activation. When CAR vs. ARTEMIS™ comparisons were tested in vitro, ET190L1-ARTEMISTM T-cells and ET190L1-CD28z-CAR T-cells specifically lysed multiple CD19+ leukemia and lymphoma cell lines with similar potencies. However, during the 16 hour in vitro killing assays, ARTEMIS™ T-cells secreted demonstrably less IL-2, IFN-γ, GM-CSF, and TNFα as compared to CAR T-cells. ARTEMISTM T-cells also accumulated less PD-1, LAG3, and TIM3 on their surface during culturing and following in vitro killing, indicating a diminished propensity for exhaustion compared to corresponding CAR T-cells. The ability of the anti-IL6 antibody tocilizumab to mitigate toxicity in patients that develop severe CRS in the clinic suggests that limiting IL-6 expression could improve the safety of adoptive T-cell therapies. However, T-cells are not efficient expressors of IL-6 and the vast majority of elevated IL-6 is produced by monocytes in response to activated CAR T-cells. To assay the effects of T-cell cytotoxicity on upregulation of cytokine expression within monocytes as a model for CRS, we co-cultured T-cells with CD19-positive ALL tumor cells on one side of a transwell and monocytes on the other side. After tumor cell lysis, RNA expression levels were measured from both T-cells and monocytes. Using ET190L1-ARTEMIS™ T-cells to kill NALM-6 tumor cells resulted in dramatically reduced levels of IL-6 and IL-5 mRNA upregulation within monocytes compared to that observed with CAR transduced T-cells. When tested in vivo against CD19+ xenografts mouse models, intravenous administration of ET190L1-ARTEMISTM T-cells caused efficient tumor regression. In agreement with the in vitro data, mice treated with ARTEMISTM T-cells released significantly lower levels of cytokines in their blood at 24 hours post dosing than mice treated with CAR T-cells.

#1538

Analysis of the paired TCRα- and β-V(D)J full-length chains of single-cell sequence from human naïve and antigen-experienced T cells.

Fei Wang,1 Qing Zhou,1 Cheng-chi Chao2. 1 _BGI-Shenzhen, ShenZhen, China;_ 2 _Complete Genomics, Inc., Mountain View, CA_.

The unbiased and paired characterization of T-cell receptor (TCR) alpha and beta chains is critical to understand the TCR repertoire and adaptive immunity. However, the direct cloning of the single human full-length TCRα- and TCRβ-chains has very few reports. We present a new methodology enabling capture of natively paired, full-length rearranged T-cell receptor (TCR) sequences at single-cell level of human T cells. More than 50% paired TCRα- and β- chains were captured in 100 single naïve human CD8+ T cells using 5'SMART and 3' TCRαβ constant region nested gene-specific primers (GSP). Furthermore, we sequenced the TCR of the specific T cells that were stimulated by representative tumor-associated antigens (TAA), virus-related antigens (VRA) and neoantigens. The highest proportion of TCR subtypes were extracted in the TCR abundance ranking by IMGT blast. Finally, we obtained the paired, full-length V(D)J TCR from melanoma antigen Melan A epitope 27-35 that was TRAV(12-2)J(47), TRBV(7-6)J(2-1), and the TCR from antigen EBV LMP2-FLY that was TRAV(26-1)J(7), TRBV(27)J(2-1). These results suggest that the method provides accurate identification of the paired, V(D)J rearrangements for each individual human naïve and antigen-experienced T cells. The information could be used in the construction of an engineered T-cell receptor for cancer immunotherapy and infectious diseases.

#1539

In vivo study of natural killer cell cytotoxicity against cholangiocarcinoma in a nude mouse model.

Inhye Jung,1 DoHee Kim,1 Yong Yoon Chung,2 Seung Woo Park,1 Da Kyung Yoo2. 1 _Yonsei University College of Medicine, Seoul, Republic of Korea;_ 2 _3SMTbio Co., Seoul, Republic of Korea_.

NK cell is one of the lymphocytes clinically used for various cancer types. Cytotoxicity of NK cell to cholangiocarcinoma (CC), however, has not been studied yet, nor has NK cell therapy against CC been clinically applied. In this study, relevance of NK cell therapy for antitumor efficacy against CC was preclinically investigated. Human HuCCT-1 cells, an intrahepatic CC cell line, were xenografted into nude mice. The HuCCT-1 tumor-bearing nude mice then received multiple infusions of ex vivo expanded human NK cell (SMT01) and were evaluated for in vivo cytotoxic activity of the NK cells. Dosage-dependent safety and toxicity of SMT01 was evaluated at three different dose levels. The mice well tolerated the maximum dose of SMT01. All of the three dosage groups did not show any significant adverse event. To evaluate in vivo activity of SMT01 against CC, we administered 5 infusions of SMT01 into the HuCCT-1 tumor-bearing nude mice. A standard chemotherapy as a positive control was also included. The result showed that SMT01 infusions resulted in significant inhibition of the CC tumor growth compared to the negative control (normal saline infusion), suggesting that NK cell contains in vivo cytolytic activity against cholangiocarcinoma. When body weights of the nude mice were measured, however, the body weight of the nude mice administered with chemotherapy was found to be maintained as lowest among all treatment groups while all the NK cell infusion groups well maintained the body weight during the treatment, suggesting beneficial effect of NK cell therapy in relevance of quality of life. IHC experiments and TUNEL assay using the tumor tissue sections clearly supported the cytotoxicity results of SMT01 against CC. We found that a robust infiltration of the infused NK cells was located along the loose tissue where feeding vessels entered. TUNEL-positive tumor cells were mainly localized at the vicinity of populated NK cells. In conclusion, our in vivo study demonstrates that NK cell also contains cytolytic activity against cholangiocarcinoma. Further investigation of the NK cell-based immunotherapy can be useful to determine cancer therapeutics for the specific tumor.

#1540

Allogeneic chimeric antigen receptor T cells targeting B cell maturation antigen.

Henia Dar, Daniel Henderson, Zinkal Padalia, Ashley Porras, Dakai Mu, Kyungah Maeng, Seshi Police, Demetrios Kalaitzidis, Jonathan Terrett, Jason Sagert. _CRISPR Therapeutics, Cambridge, MA_.

B cell maturation antigen (BCMA) is a tumor necrosis factor family cell surface receptor that binds B cell activating factor (BAFF) and a proliferation inducing ligand (APRIL) and is involved in the long-term survival of B cells. BCMA has been shown to be expressed broadly on monoclonal gammopathy of undetermined significance (MGUS) and multiple myeloma (MM) cells and for this reason has been pursued as a potential antigen for the treatment of MM. Indeed, clinical studies evaluating autologous chimeric antigen receptor T cells (CAR-T cells) targeting BCMA for the treatment of MM have shown promising efficacy with >80% response rates. However, logistical challenges will potentially limit the number of patients that have access to autologous therapies. Using the CRISPR/CAS9 system, we generated allogeneic CAR-T cells targeting BCMA by disrupting the beta-2-microglobulin (B2M) and T cell receptor alpha constant (TRAC) genes and inserting an anti-BCMA CAR into the TRAC locus. This results in allogeneic CAR-T cells lacking the major histocompatibility complex (MHC) and endogenous T cell receptor (TCR) expression while being potently cytotoxic towards cells expressing BCMA. These data show proof-of-concept for an "off the shelf" CAR-T therapy targeting BCMA.

#1541

Directed, unbiased mapping of lentiviral integrations with next generation sequencing in CAR-T cells.

Timothy G. Johnstone, Rajagopal Chari, David Koppstein, Ronald J. Hause, Rafael Ponce, Adrian W. Briggs, Francois Vigneault. _Juno Therapeutics, Seattle, WA_.

During manufacturing of an exemplary CD19-directed CAR-T cell product, patient T cells were transduced with a viral vector encoding the CD19-directed, 41BBζ endodomain-containing chimeric antigen receptor (CAR) construct, enabling permanent genetic modification of the target T cells. Lentiviral transduction with a self-inactivating (SIN) vector can offer certain advantages in safety and consistency over other DNA delivery methods. Although the integration patterns of wildtype HIV-1 have been well-characterized and shown to have a low risk of insertional transformation, the addition of an artificial construct may have the potential to affect integration patterns. Existing techniques for viral integration mapping, such as ligation-mediated polymerase chain reaction (LM-PCR), have limitations precluding comprehensive, unbiased, high-throughput insertion site mapping. To characterize insertional events in engineered CAR-T cells, we developed an unbiased NGS assay and computational pipeline capable of characterizing viral integration events genome-wide. The method leverages Tn5 transposase, coupled with a long terminal repeat-specific PCR, to map integration events down to nulceotide resolution. This assay identified over 150,000 high-confidence integration events across 23 patient-derived CAR-T cell products. Analysis of these sites revealed a similar integration preference to lentiviruses described in the literature: driven by GC content, local nucleotide content, chromatin status, and transcriptional activity, with no indication of preferential integration at oncogenic loci. In conclusion, in this assay a CD19-directed, CAR-containing lentivirus exhibited a low risk integration profile with no significant evidence of construct-specific risk factors. Furthermore, the findings suggest that the NGS assay may be useful for more efficient, cost-effective, and sensitive measurement of CAR-T integration events for a range of constructs.

#1542

Safety and efficacy of high purity and activity NK cells therapy in combination with IgG1 antibody in patients with gastric or colorectal cancer: A phase I clinical trial.

Takeshi Ishikawa,1 Tetsuya Okayama,1 Naoyuki Sakamoto,1 Mitsuko Ideno,2 Kaname Oka,1 Tatsuji Enoki,2 Junichi Mineno,2 Hideyuki Konishi,1 Satoshi Kokura,1 Yuji Naito,1 Yoshito Itoh1. 1 _Kyoto Prefectural University of Medicine, Kyoto, Japan;_ 2 _Takara Bio Inc, Kusatsu, Japan_.

Background; Natural killer (NK) cells exhibit strong cytotoxic activity against tumor cells, and produce numerous cytokines resulting in the activation of the adoptive immune systems. Thus, NK cell is considered ideal cell for adoptive cancer immunotherapy. But the difficulty of obtaining large numbers of NK cells that are safe to administer deters its clinical use. We successfully generated large numbers of activated NK cells from small quantities of blood and also determined that the expanded cells were safe to administer in a monotherapy (J Transl Med (2015) 13:277). Since NK cells play a key role in antibody-dependent cellular cytotoxicity (ADCC), administration of NK cells would be expected to enhance the efficacy of IgG1 monoclonal antibodies by augmenting ADCC. This phase I clinical trial evaluated the safety, toxicity, and immunological response of the adoptive NK therapy in combination with IgG1 antibodies.

Patients and Methods; Patients with unresectable advanced gastric or colorectal cancer who have administered or have planned to administer IgG1 antibody (i.e. trastuzumab for HER2 positive gastric cancer or cetuximab for RAS-wild colorectal cancer). NK cells were expanded from PBMCs with the same method in previous clinical trial, briefly ex vivo by stimulating PBMCs with OK432, IL-2, and modified FN-CH296-induced T cells. Expanded NK cells were intravenously injected 3 days after IgG1 antibody administration in a dose-escalating manner (dose 0.5 × 109, 1.0 × 109, 2.0 × 109 cells/injection, three patients/one cohort), every 3 weeks for 3 cycles. We evaluated the safety and efficacy of the combination therapy. To assess the immunological response, immunomonitoring was performed.

Results; Nine eligible patients were enrolled. NK cell populations expanded with our system were confirmed as being enriched in NK cells (median 92.9%) with high expression of NKG2D(97.6%) and CD16(69.6%). This combination therapy was well tolerated with no severe adverse events. Among six evaluable patients, 4 presented SD, 2 presented PD. Of the four SD patients, 3 showed an overall decrease in tumor size after combination therapy. In immunomonitoring analysis, whole blood IFN-gamma production level was increased and the proportion of Treg (Treg/CD4) in peripheral blood was decreased after the combination therapy.

Conclusions; This is the first clinical trial of the combination of adoptive NK cell therapy and IgG1 monoclonal antibodies. Our data provide evidence of good tolerability and preliminary anti-tumor activity for this combination therapy. Moreover, immunological monitoring revealed that the combination therapy induced Th1-type immune response and reduced peripheral Tregs. However, given the limited activity observed in this clinical trial, additional studies are necessary to develop these combination approaches.

#1543

Upregulation of mannose 6-phosphate receptor (MPR) has a potential to increase the efficacy of combined therapy for melanoma patients.

Cigdem Atay,1 Sergio Lavilla-Alonso,2 Dmitry Gabrilovich1. 1 _The Wistar Institute, Philadelphia, PA;_ 2 _In vivo Pharmacology Group, Janssen Oncology Research & Development Pharmaceutical Companies of Johnson & Johnson, Beerse, Belgium_.

BRAF -targeting therapy demonstrates impressive clinical response in patients with metastatic melanoma. However, patients quickly develop resistance associated with rapid tumor progression. Therefore, additional therapeutic approaches are necessary. Our group has previously shown that transient induction of cation-independent mannose 6-phosphate receptor (MPR) is important for the antitumor effect of chemo-immunotherapy. In this study, we aimed to decipher the role of MPR during possible combination therapy of BRAF inhibitor with adoptive T-cell therapy of BRAF inhibitor-resistant melanoma. For this aim, we have used PLX4720 as a BRAFV600E inhibitor and observed that in vitro PLX4720 treatment of human V600E-positive melanoma cells, including WM-35, SK-Mel-624 and WM983B, resulted in a dose-dependent upregulation of MPR on the cell surface. Importantly, increased levels of MPR on the cell surface induced the uptake of recombinant Granzyme B, which is a major mechanism of cell killing by cytotoxic T cells (CTL). We found that PLX4720 treatment rendered WM35 cells more prone to lysis by HLA-matching tumor-infiltrating lymphocytes (TILs) and increased cell killing is not due to PLX4720 treatment itself but increased levels of MPR on the cell surface. In order to prove this, we generated MPR-overexpressing WM35 cell line and showed that upregulation of MPR, independently of PLX4720 treatment, sensitizes melanoma cells for T cell killing in vitro. We further showed that PLX4720 treatment leads to upregulation of MPR in vivo by a mouse model bearing xenograft WM35-derived subcutaneous tumors as well. According to our data, 3 days of PLX4720 treatment is enough to induce MPR in tumors and this effect continues up to 6 days after 5 days treatment ends. Unexpectedly, we found that PLX4720 caused upregulation of MPR in resistant cell lines and that those cell lines became more sensitive to cytotoxic effect of TILs. Thus, our data suggest that adoptive T-cell therapy can be potentially effective in melanoma resistant to BRAF inhibitor. (This study is supported by NIH grant P50 CA168536.)

### Biomarker Discovery 2

#1544

Subcutaneous adiposity is an independent predictor in metastases patients.

Ngan M. Tsang. _Chang Gung Memorial Hospital, Taoyuan, Taiwan_.

Background: The prognostic significance of muscle and adipose tissue in cancer patients provide an appropriate assessment of body composition in recent years. We investigated the independent prognostic significance of visceral and subcutaneous adipose on survival in metastases patients. Methods: We measured visceral adipose tissue index (VATI), subcutaneous adipose tissue index (SATI), and muscle index in 959 metastases patients via computed tomography at L3 level. Univariate and multivariate cox proportional hazards model were used to determine mortality hazard ratio (HR) and were presented in corresponding 95% CI. Result: SATI and gender are independent predictors among patients with metastases disease. High SATI linked to lower mortality (HR:0.749; 95% CI: 0.640-0.877; p<0.001) and longer overall survival (8.38 months; 95% CI, 0.10-137.42) in compared to low SATI patients (4.44 months; 95% CI, 0.03-108.36). Conclusion: The body composition appeared to be true determinants of the prognosis in metastases patients. The research provides an understanding of the association between subcutaneous adipose and the overall survival in metastases patients.

#1546

Low-molecular-weight protein tyrosine phosphatase is a useful biomarker for predicting postoperative biochemical recurrence in RM0 prostate cancer.

Hirofumi Kurose,1 Kosuke Ueda,2 Reiichiro Kondo,2 Kiyoaki Nishihara,2 Makoto Nakiri,2 Mitsunori Matsuo,2 Shigetaka Suekane,2 Sakiko Sanada,2 Sachiko Ogasawara,2 Tsukasa Igawa,2 Hirohisa Yano2. 1 _Kurume University School of Medicine, Kurume, Japan;_ 2 _Kurume University, Kurume, Japan_.

Objective: Low-molecular-weight protein tyrosine phosphatase (LMPTP) has a molecular weight of 18 kDa. Studies have shown that the elevated expression of this enzyme is associated with poor prognosis of certain cancers. For prostate cancer, positive surgical margins are considered an important predictor of such recurrences. However, biochemical recurrence is observed even with a negative surgical margin (RM0), and some cases require salvage therapy. Few reports have identified useful biomarkers to predict these cases. In this study, we investigated whether LMPTP expression level could be a biomarker of these recurrences in RM0 prostate cancer.

Methods: The subjects were 119 patients who underwent a total prostatectomy with a negative resection margin, at our hospital, between 2007 and 2011. Immunostaining of 2 TMA samples per patient was performed to evaluate LMPTP expression levels. Expression was classified as negative, 0; weak, 1; moderate, 2; or strong, 3 by two pathologists. Each patient was given a total expression score consisting of 13 levels from 0 to 12. By constructing a ROC curve, LMPTP expressions were categorized into either a high expression group (score: ≥5) or a low expression group (score: ≤4). We then examined the relationship of LMPTP expression level with other pathological observations and biochemical recurrence. Cancer survival analysis was performed using the Kaplan-Meier method, log-rank test, and Cox's proportional hazards model. The threshold for statistical significance was set at p < 0.05.

Results: Evaluation of immunostained samples by the two pathologists were highly reliable, with an Intraclass correlation (ICC) score for two distinct measurements of 0.77 and 0.98, respectively. 68 patients (57.1%) were categorized in the LMPTP high expression group; and 51 patients (42.9%), in the low expression group. The log-rank test revealed that early biochemical recurrence was observed in the high LMPTP expression group (p = 0.0011). In addition, both the univariate and multivariate analyses revealed that high LMPTP expression level was an independent prognostic factor for biochemical recurrence. Furthermore, in the RM0 cases, high LMPTP expression level (HR:3.62, p = 0.023) was shown to be a better prognostic factor than Gleason score (GS; p = 0.406). Lastly, high LMPTP expression level strongly correlated with the Ki-67 proliferation marker (p < 0.0001), suggesting that LMPTP is expressed in tumor cells with high proliferative capacity.

Conclusion: This study suggests that immunostaining of LMPTP is a better predictor of biochemical recurrence than GS in cases of RM0 total prostatectomy. This biomarker can be evaluated with a single immunostaining with high reproducibility, and can therefore be a clinically applicable tool in postoperative follow-ups and as a predictor of recurrence that require salvage therapy.

#1547

Expression of epithelial-to-mesenchymal transition markers (EMT) in treated pancreatic duct adenocarcinoma.

Minhua Wang, Jeannelyn S. Estrella, Matthew H. Katz, Asif Rashid, Jeffrey E. Lee, Anirban Maitra, Robert A. Wolff, Gauri R. Varadhachary, Huamin Wang. _University of Texas, MD Anderson Cancer Center, Houston, TX_.

Background: Pancreatic duct adenocarcinoma (PDAC) is an aggressive cancer with poor prognosis. Epithelial to mesenchymal transition (EMT) plays an important role in the progression, metastasis and chemoresistance of PDAC. However the expression of EMT markers and their clinical significance in PDAC patients who received neoadjuvant therapy are not clear.

Methods: One hundred and twenty cases were included in this study. All patients received neoadjuvant chemoradiation therapy and underwent surgical resection at our institution from 1999 to 2007. Expressions of EMT markers, including Zeb-1, E-cadherin, vimentin and N-cadherin, were evaluated by immunohistochemistry using tissue microarrays. The staining for Zeb-1 was categorized as positive (≥10% nuclear staining in tumor cells) and negative (<10% nuclear staining in tumor cells); the staining for E-cadherin was categorized as low (negative or <50% membranous staining) or high (≥50% membranous staining); the staining for vimentin and N-cadherin was categorized negative (<10% cytoplasmic staining) or positive (≥10% cytoplasmic staining). The expression results were correlated with clinicopathologic parameters and survival. All the statistical analyses were carried out with the SPSS software.

Results: Among 120 cases, 45 (37.5%) were positive for Zeb-1, 25 (20.8 %) were E-cadherin-low, 14 (11.7%) were positive for vimentin, and 2 (1.7%) were positive for N-cadherin. There was negative correlation between the expression of E-cadherin and vimentin (p=0.03). E-cadherin-low and positive vimentin expression correlated with poor differentiation (p=0.02 and p=0.004, respectively). However, no correlations between the EMT markers with other clinical pathologic parameters were found (p>0.05). The median overall survival (OS) and disease-free survival (DFS) were 35.3 ± 2.8 months and 15.9 ± 3.6 months, respectively, in vimentin-negative group compared to 16.1 ± 1.1 months (p=0.03) and 7.0 ± 1.1 months (p=0.02), respectively, in vimentin-positive group. There were no correlation between the expression of Zeb-1, E-cadherin or N-cadherin and survival (P>0.05). In multivariate analysis, expression of vimentin was an independent predictor of shorter OS [HR (95% CI): 2.57 (1.34-4.93), p=0.004] and DFS [HR (95% CI): 2.80 (1.45-5.43), p=0.002].

Conclusion: Our results show that EMT markers are frequently expressed in treated PDAC. Expression of vimentin is a prognostic biomarker for both OS and DFS in patients with PDAC who received neoadjuvent therapy and surgery.

#1548

Genetic biomarkers predict clinical response and survival in myelodysplasia.

Lynette Chee,1 David Ritchie,1 Jessica Chung,2 Daniel Park,2 Mandy Ludford-Menting,1 Jane Ripley,1 Melita Kenealy,3 Rachel Koldej1. 1 _The Royal Melbourne Hospital, Victoria, Australia;_ 2 _The University of Melbourne, Victoria, Australia;_ 3 _Cabrini Hospital, Victoria, Australia_.

Background: Hypomethylating agents (HMA) used in higher-risk myelodysplastic syndromes (MDS) improve survival but HMA-failure has a poor prognosis. Abnormal bone marrow (BM) colony-forming units (CFUs) persist in treated MDS patients despite achievement of complete remission, suggesting persistent abnormal stem cell function. We aim to identify genetic biomarkers following treatment with Azacitidine ± Thalidomide or Lenalidomide that predict clinical outcomes in MDS.

Methods: BM cells from patients enrolled in ALLG MDS3 and MDS4 clinical trials at baseline and after 4 cycles of treatment (C4) were grown in Methocult for 14 days. CFUs were pooled at baseline; C4 macroscopically normal and abnormal colonies were harvested separately. mRNA expression was quantified using the Nanostring nCounter PanCancer Pathways panel. Clinical outcomes analysed were: (1) clinical benefit at 12 months (haematological improvement or better as per IWG criteria) (2) best response achieved. Genes expressed above background level in ≥25% of samples were included for statistical analyses, resulting in 516 genes across 56 samples from 23 different patients. R limma package was used for differential expression analysis. Patients were weighted using limma's voomWithQualityWeights function. Moderated t-tests with empirical Bayes were done to identify differentially expressed genes. For testing between colonies, a log-fold-change cut-off of 0.5 was used with limma's treat function. P-values were adjusted for multiple hypothesis testing.

Results: 98 genes exhibited significantly different expression (p <0.05) when comparing C4 normal to baseline colonies and 118 genes were differentially expressed between C4 normal to C4 abnormal colonies. Key results are summarised: (1) Within C4 normal colonies - RFC3 (p=0.04) and LTBP1 (p=0.04) were upregulated in patients with clinical benefit at 12 months (2) Within C4 abnormal colonies - FN1 (p=0.03) was upregulated in those failing to achieve at least a partial response (PR) (3) Comparing C4 normal colonies to baseline: a. The top 5 differentially expressed genes: MAPK12, PLAU, FGFR1, IL10 and FLNA (p<0.001) were downregulated in the C4 normal group; b. In addition, for patients with clinical benefit, MYD88 and PIK3R5 were downregulated (p<0.001) while; c. In patients who achieved at least a PR as best response, NFKB1, SYK and TGFBR2 were downregulated (p<0.001). d. Gene ontology analysis revealed upregulated genes involved in plasma membrane and cytokine production were over-represented at baseline while; e. KEGG pathway analysis showed upregulated genes involved in the cytokine receptor, Notch and NF-kB signalling pathways.

Conclusion: We identified changes in gene expression following treatment in MDS that predict outcomes in response and clinical benefit. These genetic biomarkers require further validation and could define early markers of resistance for investigation of novel therapies.

#1549

LAPTM5 in clear cell renal cell carcinoma.

Hyewon Ryu,1 Solbi Kim,2 Nayoung Kim,2 Jin-Man Kim,3 Hyo Jin Lee4. 1 _Department of Internal Medicine, Infection Control Convergence Research Center, Chungnam National University College of Medicine, Daejeon, Republic of Korea;_ 2 _Infection Control Convergence Research Center, Department of Medical Science, Chungnam National University College of Medicine, Daejeon, Republic of Korea;_ 3 _Infection Control Convergence Research Center, Department of Pathology, Chungnam National University College of Medicine, Daejeon, Republic of Korea;_ 4 _Department of Internal Medicine, Infection Control Convergence, Chungnam National University College of Medicine, ResearchCancer Research Institute, Chungnam National University, Daejeon, Republic of Korea_.

Lysosomal-associated multispanning membrane protein 5 (LAPTM5) is membrane protein that mostly expressed in immune cells and hematopoietic cells. Recently, it has been demonstrated that LAPTM5 expression levels are decreased in several cancer cells by inducing regression of Hela cells and neuroblastomas. However, its exact role in cancer has yet to be defined. Furthermore, the clinical effect of LAPTM5 expression in cancer has not been reported so far. Thus, we investigated the association between LAPTM5 expression and its clinicopathological characteristics in 112 cases of clear cell renal cell carcinoma using tissue microarrays by immunohistochemical staining. Tumors were classified into four grades based on the staining intensity: 0 = no staining, +1 = weak, +2 = intermediate, and +3 = strong. The staining intensity was classified into LAPTM5-negative group or LAPTM5-positive groups. (0; negative, +1 ~ +3; positive). Of all 112 cases, 9 cases showed LAPTM5-negative, while 93 cases showed LAPTM5-positive: +1 (35 cases), +2 (28 cases), and +3 (30 cases). LAPTM5-positivity was significantly associated with decreased tumor recurrence. In addition, recurrence-free survival (RFS) (p = 0.010), cancer-specific survival (CSS) (p = 0.022), and overall survival (OS) (p = 0.022) were significantly decreased in the LAPTM5-negative group. Univariate analysis indicated that tumor size (HR = 3.44; 95% CI, 1.000-11.836), Fuhrman nuclear grade (HR = 1.863; 95% CI, 1.169-2.971), and negative LAPTM5 expression (HR = 3.173; 95% CI, 1.249-8.064) significantly affected the RFS of clear cell renal cell carcinoma patients. Moreover, CSS and OS were significantly affected by Fuhrman nuclear grade (HR = 2.177; 95% CI, 1.261-3.757 and HR = 1.673, 95% CI, 1.062-2.635, respectively) and negative LAPTM5 expression (HR = 3.398; 95% CI, 1.110-10.405 and HR = 2.826; 95% CI, 1.136-7.031, respectively). Multivariate analysis showed that a high Fuhrman nuclear grade and negative LAPTM5 expression were significant independent risk factors. Based on these results, loss of LAPTM5 was associated with poor oncological outcomes; this suggests that LAPTM5 might be a useful prognostic marker in patients with clear cell renal cell carcinoma.

#1550

Limb-bud and heart inhibits the proliferation and metastasis of human lung adenocarcinoma cells and predicts survival outcome.

Mingming Deng,1 Ruoxi Yu,2 Zhi Li,2 Xiaofang Che,2 Huicong Song,2 Ximing Wang,1 Zhe Zhang,3 Qingjie Lv,3 Shuo Wang,2 Xiujuan Qu,2 Yunpeng Liu,2 Ye Zhang,2 Xuejun Hu1. 1 _Department of Respiratory and Infectious Disease of Geriatrics, the First Hospital of China Medical University, Shenyang, China;_ 2 _Department of Medical Oncology, the First Hospital of China Medical University, Shenyang, China;_ 3 _Department of Pathology, Shengjing Hospital of China Medical University, Shenyang, China_.

Background/Aims: Limb-bud and heart (LBH) is an important transcription cofactor involved in embryonic development. But, the role of LBH in human cancer remains less understood. The aim of this study was to explore the role of LBH in lung cancer, and its correlation with clinical characteristics and prognosis of lung cancer patients, especially those with lung adenocarcinoma (LUAD).

Methods: TCGA, GEO (GSE19188), Kaplan-Meier Plotter, and our own tissue microarray (TMA) containing 41 lung cancer tissues and paired normal tissues were used to compare the differences of LBH expression, and the relation with clinical characteristics. Another 70 LUAD tissues with patient follow-up records were used to explore the correlation of clinical characteristics and prognosis by Kaplan-Meier and Cox-regression analysis. Cell viability assay, wound healing assay, colony formation assay, transwell assay, and flow cytometry were used to explore the biological function of LBH on LUAD cells. In addition, bioinformatics analyses (GO pathway and Gene Set Enrichment Analysis) and Western blotting were carried out to determine the potential LBH pathways.

Results: LBH was significantly down-regulated in lung cancer tissues, and correlated with prognosis and clinical characteristics of lung cancer patients based on public database and TMA. Importantly, the expression level of LBH significantly correlated to depth of invasion (P=0.048), lymph node status (P=0.031), and TNM stage (P=0.007) of LUAD patients. Kaplan-Meier survival analysis revealed that LBH-negative was associated with the poor overall survival (OS) of LUAD patients (P=0.004). Cox univariate and multivariate regression analysis showed that LBH expression status was a favorable independent prognostic factor(hazard ratio [HR]=0.12, 95% confidence interval [CI]: 0.06-0.89, P=0.039).Knockdown of LBH accelerated LUAD cell proliferation, migration, and invasion. Furthermore, bioinformatics analyses indicated LBH was significantly related to the cell adhesion pathway. Western blotting confirmed that LBH could regulate several proteins of the integrin family (integrin-α1, integrin-α2, integrin-α4, integrin-αv and integrin-β4), but didn't influence EMT-related proteins.

Conclusion: Our data suggest that LBH plays an important role in lung cancer. Importantly, LBH is an independent prognostic factor in LUAD, and can inhibit the proliferation and metastasis of LUAD cells. LBH may be a potential prognostic biomarker in LUAD patients.

#1551

Determination of the origin of oral squamous cell carcinoma by microarray analysis.

Makoto Kinouchi,1 Sayaka Izumi,2 Daisuke Uchida,1 Nobuyuki Kuribayashi,1 Yuske Komiyama,1 Shuji Tsuchida,1 Atsushi Fujita,1 Hitoshi Kawamata1. 1 _Dokkyo Medical University School of Medicine, Tochigi, Japan;_ 2 _Kamma Memorial Hospital, Tochigi, Japan_.

More than 90% of oral cancer is histopathologically squamous cell carcinoma (SCC). According to its clinical behavior and histopathological features, we hypothesize that oral SCC can originate from either oral squamous epithelium or the minor salivary glands. In this study, we attempted to clarify the possibility of some oral SCC originating from the minor salivary glands, and to investigate whether these tumors showed particularly aggressive biological behavior. The mRNA expression profile of samples obtained from 6 patients with SCC of the oral floor (5 men, 1 woman; mean age 62.7 years) was analyzed by microarray containing 32,878 genes. Clustering analysis of the expression level of the 845 genes that were differentially expressed in normal oral squamous epithelium and normal salivary glands revealed that the 6 samples can be divided into 2 groups. The expression profile in four cases was similar to that of normal oral squamous epithelium, and in two cases was similar to that of normal salivary glands. Furthermore, we identified nine genes that reveal the origin of the oral SCC. Subsequently, we examined the expression levels of these 9 marker genes by reverse transcriptase-polymerase chain reaction to determine the origin of 66 oral SCCs. Twelve of the 66 oral SCCs were considered to originate from minor salivary glands, and these tumors showed high metastatic potential (p<0.05) and poor event free survival rate (p<0.05). In conclusion, determination of the origin of the oral SCC is helpful when making a treatment plan for patients with oral SCC.

#1552

SATB1 **is a novel prognostic factor in esophageal cancer patients and promotes migration and invasion of esophageal cancer cells.**

Yusuke Tsuruda,1 Ryunosuke Kogo,2 Shuhei Ito,1 Akihiro Kitagawa,1 Atsushi Fujii,1 Shotaro Kuramitsu,1 Dai Simizu,1 Yosuke Kuroda,1 Hidetoshi Eguchi,1 Tkaaki Masuda,1 Shoji Natsugoe,3 Masaki Mori,4 Koshi Mimori1. 1 _Kyushu University Beppu Hospital, Beppu, Japan;_ 2 _Kyushu University, Fukuoka, Japan;_ 3 _Kagoshima University, Kagoshima, Japan;_ 4 _Osaka University, Osaka, Japan_.

Introduction: Esophageal squamous cell carcinoma (ESCC) is a highly aggressive malignancy with extremely poor overall survival. There is no available molecular marker in clinical application until now. SATB1(special AT-rich binding protein 1) tether genomic DNA in nucleolus and hold three-dimensional structure of genomic DNA. We evaluated SATB1 expression using clinical samples from patients of ESCC and analyzed the association with clinicopathological factors and prognosis to identify novel prognostic marker in ESCC. In addition, we investigated the function of SATB1 using ESCC cells. We focused on matrix metalloproteinase protein (MMP3), which has a SATB1 binding site in its upstream.

Materials and Methods: We accumulated 71 samples of ESCC patients who underwent primary tumor resection. The relative expression levels of SATB1 were obtained by normalizing the amount of SATB1 mRNA with respect to GAPDH mRNA as an endogenous control for each sample. We divided 71 ESCC samples into 2 groups, which are SATB1 low expression group (n=35) and SATB1 high expression group (n=36) according to the median of SATB1 expression level, and performed the comparison between two groups. In order to elucidate biological significance, we generated SATB1 overexpression stable transfectants in KYSE150, and performed migration and invasion assays. Next, we knockdowned SATB1 expression of KYSE410, which are ESCC lines, using SATB1 siRNA and evaluated the capability of migration and invasion. Moreover, to validate whether SATB1 altered MMP3 genomic locus, we analyzed comparative genomic hybridization (CGH) array obtained from 54 esophageal cancer samples. We examined MMP3 expression in SATB1 overexpression cells using qRT-PCR.

Result: The SATB1 high expression group showed greater tumor depth than the low expression group (P=0.003). In the overall survival curve, patients in the SATB1 high expression group had a significant poorer prognosis than those in the SATB1 low expression group (P=0.013). Multivariate analysis showed that the level of SATB1 expression was an independent prognostic predictor (OR; 1.56, 95%CI; 1.04 - 2.44, P = 0.043). SATB1 transfectants significantly increased the capability of migration and invasion compared to mock (P<0.05). KYSE410 cells transfected with SATB1 siRNA significantly decreased the capability of migration and invasion compared to control cells (P<0.05). Genomic alterations in SATB1 locus were strongly correlated with those in MMP3 locus (P<0.0001, ρ=0.5156). Overexpression of SATB1 showed the elevated MMP3 level compared to mock (P<0.05).

Conclusion: We indicated that SATB1 is novel prognostic factor in ESCC and that SATB1 promote migration and invasion of ESCC cells. SATB1 may be a promising new candidate for targeted therapies for ESCC.

#1553

Carcinoembryonic antigen (CEA) expression is regulated by mutant KAS in mucinous colon cancers.

Murali Kuracha, Venkatesh Govindarajan, Brian W. Loggie, Peter Thomas. _Creighton Univ. School of Medicine, Omaha, NE_.

Purpose: Carcinoembryonic antigen (CEA/CEACAM5) is a marker and prognostic indicator for colorectal cancers (CRCs). CEA overexpression is also a major causative factor in the development of liver metastasis from a significant fraction of colorectal cancers (CRC) including mucinous adenocarcinomas (MCAs). Very little is known about the molecular mechanisms that regulate the expression of CEA in cancers in general and in CRCs in particular. This study tests the hypothesis that mutant KRAS, a frequently mutated gene in CRCs, regulates CEA expression in CRCs.

Methods: Mutant KRAS expression was reduced in two high CEA-expressing mucinous CRC cell lines, LS174T (KRAS G12D) and RW7213 (KRAS G12C), by short hairpin RNA (shRNA)-mediated knockdown using a lentiviral vector. Inducible and reversible knockdown of KRAS was achieved by addition or withdrawal of doxycycline. Alterations in activation of ERK-and PI3K-pathway effectors downstream of KRAS were assessed by Western blots. Two small-molecule inhibitors, pictilisib (GDC0941) and cobimetinib (GDC0973), were used to inhibit PI3K and MEK respectively.

Results: Knockdown of mutant KRAS in CRC cell lines reduced CEA expression. However, knockdown of wild-type RAS did not change CEA expression. Restoration of mutant KRAS reestablished CEA expression in both cell lines. This recovery of CEA expression was blocked by addition of the MEK inhibitor, cobimetinib or the PI3K inhibitor, picstilisib. Combined treatment with both inhibitors was more effective in reducing CEA expression than single-agent treatment.

Conclusions: Our results suggest that mutant KRAS, through engagement of MEK-ERK and PI3K-AKT signaling pathways, regulates CEA expression in CRCs. CEA has been implicated in the development of lung and liver metastasis from CRCs, by reacting with stromal cells and altering the tumor microenvironment. Our results therefore, suggest a potential vulnerability that could be exploited for antimetastatic therapy.

#1554

High stromal fibrillar collagen predicts poor survival in surgical non-small cell lung cancer patients.

Jordi Alcaraz,1 Laura Millares,2 Josep-Lluís Carrasco,3 Iuliana-Cristiana Benchea,3 Francisco-Javier Fernández,3 Esther Barreiro,4 Anabel Martínez,4 Julio Sánchez,5 María Ángeles González-Castro,6 Ana Blanco,6 Rosa Sánchez-Gil,6 Mireia Serra,7 Ramon Rami-Porta,7 Jaume Sauleda,8 Esther Fernández,9 Rosario Melchor,10 Luis Seijo,10 Leonardo de Esteban,11 Eduard Monsó2. 1 _Univ. of Barcelona-CIBERES, Barcelona, Spain;_ 2 _Fundació Parc Taulí-CIBERES, Sabadell, Spain;_ 3 _Univ. of Barcelona, Barcelona, Spain;_ 4 _IMIM-Hospital del Mar-CIBERES, Barcelona, Spain;_ 5 _Hospital San Pedro de Alcántara-CIBERES, Cáceres, Spain;_ 6 _Hospital Virgen Del Rocío-CIBERES, Sevilla, Spain;_ 7 _Hospital Mutua de Terrassa-CIBERES, Barcelona, Spain;_ 8 _Hospital Son Espases-CIBERES, Mallorca, Spain;_ 9 _Hospital Germans Trias i Pujol-CIBERES, Badalona, Spain;_ 10 _Fundación Jímenez Díaz-CIBERES, Madrid, Spain;_ 11 _Hospital Joan XXIII-CIBERES, Tarragona, Spain_.

A major hallmark of non-small cell lung cancer (NSCLC) and other solid tumors is a desmoplastic tumor stroma rich in activated fibroblasts and fibrillar collagens. Previous studies have revealed that these tumor-associated fibroblasts contribute to virtually all steps of tumor progression and even therapy resistance. However, our knowledge of the prognostic value of markers of activated fibroblasts in NSCLC is very scarce. To fill this gap of knowledge we conducted a retrospective multicentric study of the prognostic value of the standard markers of activated fibroblasts α-SMA and fibrillar collagens by quantitative image analysis of α-SMA immunostaining and picrosirius red (PSR) staining, respectively. For this purpose tissue microarrays from a cohort of 222 patients with early-stage (I-IIp) NSCLC recruited in Spain for the International Association for the Study of Lung Cancer TNM-16 staging project were analyzed. A percentage of positive staining area/image was obtained for each marker and patient. Kaplan-Meier analysis revealed that PSR was associated with poor survival when using data obtained from both bright-field (p = 0.04) and polarized microscopy (p = 0.002), whereas α-SMA was not (p = 0.7). Remarkably, the prognostic value of PSR was independent of TNM stage, and patients with PSR above the cut-offs exhibited a 55% (for bright-field; HR=1.55, 95%CI (1.0-2.4)) and 109% (for polarized microscopy; HR=2.09, 95%CI (1.3-3.2)) increased risk of 3 year mortality. These results reveal that the content of stromal fibrillar collagens, assessed from image analysis of picrosirius red staining (particularly using polarized microscopy) is associated with adverse prognosis independently of the tumor stage. This new prognostic biomarker may help predict which patients subject to therapeutic surgery are at higher risk of progression and death. Moreover it supports that antifibrotic drugs aiming to target stromal fibrillar collagens may be an effective therapeutic approach to improve survival in NSCLC.

#1555

A gene signature associated with PTEN activation defines good outcomes in intermediate-risk prostate cancer cases.

Chee Wee Ong, Pamela Maxwell, Muhammad A. Alvi, Stephen McQuaid, Ian G. Mills, Manuel Salto-Tellez, David J. Waugh. _Movember FASTMAN Centre of Excellence, Centre for Cancer Research and Cell Biology, Queen's University Belfast, Belfast, United Kingdom_.

Background: Accurate identification of Gleason score (GS) 3+4 prostate cancer patients with low risk of disease progression is an unmet challenge in treatment decision making. We investigated the potential of using integrative genomic profiling to improve stratification of GS 3+4 prostate cancer patients to aid decision making.

Methods: High-throughput gene expression, methylation and mutational analysis were performed on a subset of 28 GS 3+4 cases selected from a cohort (N = 69) of intermediate risk prostate cancer cases. Preselection was based on immunohistochemistry (IHC) expression of 6 major drivers (PTEN, MYC, RB1, TP53, AURKA and AR) of aggressive disease in prostate cancer. Fuzzy clustering and unsupervised hierarchical clustering were utilized to determine molecular subgroups. Genes with high expression differences between subgroups were identified through group difference statistics. A prognostic index based on Cox proportional hazard model was generated for individual and combinations of differentially expressed genes and was tested in GS 3+4 cases (total N = 215, median follow-up = 65.8 months) extracted from 3 prostate cancer datasets available at Gene Expression Omnibus (GSE21032, GSE16560 and GSE40272). Hazard risk (HR) and 95% confidence interval (CI) of association with outcomes (recurrence and overall survival) were analyzed using Cox modelling and log-rank analysis.

Results: We found that GS 3+4 prostate cancer cases could be classified as "complex" (differential expression of more than one driver) or "simple" (differential expression of only one) based on IHC analysis. Focusing on the "simple" cases, gene expression and methylation PTEN expression were tightly correlated and not observed in other drivers (R = 0.41, P = 0.03). Group difference statistics showed 35 genes were highly expressed in PTEN-high subgroup and were evaluated individually and in combination for prognostic value in 3 independent cohorts. The prognostic potential of the combinatorial signature was observed in all 3 cohorts: GSE21032 (HR: 6.95, 95% CI: 2.73-17.54, P < 0.001), GSE40272 (HR: 6.40, 95% CI: 2.28-17.96, P < 0.001) and GSE16560 (HR: 1.77, 95% CI, 1.29-2.41; P = 0.003) datasets. Individual gene-by-gene analysis showed that 4 genes (ACTA2, ACTG2, MYH11 and TPM2) were prognostic (all P < 0.05) in all 3 cohorts. Compared to the combination of 35 genes (AUC = 0.728), the prognostic potential of combining these 4 genes was still of significant value (AUC = 0.689).

Conclusion: This study shows that by using IHC as an upfront stratifier of intermediate risk prostate cancers, it is possible to identify, through subsequent differential gene expression profiling, a geneset with prognostic power across multiple cohorts. This strategy has not been used previously and the signature, subject to further validation, has the potential to impact on treatment decisions in GS 3+4 prostate cancer patients.

#1556

Does surgical treatment affect serum levels of vascular endothelial growth factor (VEGF) in orofacial tumors.

Aimuamwosa Obarisiagbon,1 Peter Egbor,1 Ozoemene Ndubuisi Obuekwe,1 Osagie Akpata,1 Akin Ojo,1 Nosayaba Osazuwa-Peters2. 1 _University of Benin Teaching Hospital, Benin City, Nigeria;_ 2 _St. Louis University School of Medicine, St. Louis, MO_.

Introduction: Angiogenesis, the generation of new vessels, is regulated by several growth factors. The most important of them is vascular endothelial growth factor (VEGF), which is closely related to tumor progression and prognosis. To my knowledge no study in sub-Saharan Africa has evaluated the level of VEGF following surgery in African patients. This study therefore aims to evaluate the pattern and clinical implication of VEGF expression in benign and malignant neoplasms and determine the effect of surgical intervention on the levels of circulating VEGF.

Methods: This case-control study was carried out at the University of Benin Teaching Hospital (UBTH), Benin City, Nigeria, between October 2015 and February 2017. Cases were drawn from among histologically confirmed orofacial tumors, while controls were from consecutive, ambulatory dental patients, matched for age and sex. Calculated sample size for minimum effect was 77 for both cases and controls. Blood samples were taken from cases and controls, pre- and 1-month post-surgery, allowed to clot and serum aliquots were stored at -600 C till analyses. Post-surgery tissue was processed and stained with anti-VEGF antibody for immunohistochemical analysis. Serum VEGF was analyzed using Sandwich ELISA, and mean levels as well as median were measured. Spearman correlation was estimated, t-test measured significance (at p ≤ 0.05), and one-way ANOVA measured between group differences.

Results: Following 12% attrition, 81 subjects were retained in the study (55 cases [20 malignant, 35 benign] and 26 controls). The most common malignant neoplasms were head and neck squamous cell carcinoma (35%) and polymorphous low-grade adenocarcinoma (20%), while the benign tumors were mostly unicystic ameloblastoma (42.9%) and solid multicystic ameloblastoma (31.4%). Before surgical treatment, there were significantly different serum levels of VEGF in benign (48.11 ± 25.19 pg/ml) vs. malignant cases (1065.00 ± 412.14 pg/ml), and compared with controls (45.42 ± 29.83 pg/ml), p <0.001. Also, the level of circulating VEGF correlated strongly and positively with tumor tissue expression in malignant cases (r = 0.674, p <0.001). After definitive surgical intervention, there was a significant reduction in the level of serum VEGF in both benign (45.50 ± 24.71 pg/mL) and malignant cases (51.22 ± 16.84 pg/mL), p <0.001. One-way ANOVA revealed no significant differences between cases and controls post-surgery.

Conclusion: There were elevated levels of serum VEGF in benign and malignant orofacial neoplasia, which correlate positively with the expression of VEGF in tumor tissue. Surgery caused a significant reduction of circulating VEGF to normal levels irrespective of age, gender, previous medical history, perceived duration of the lesion and lymph node involvement.

#1557

Analysis of angiogenic and stromal biomarkers in a large malignant mesothelioma cohort.

Puey Ling Chia,1 Prudence Russell,2 Carmel Murone,1 Marzena Walkiewicz,1 Ulf Eriksson,3 Andrew Scott,1 Thomas John1. 1 _Olivia Newton-John Cancer Research Institute, Heidelberg, Australia;_ 2 _St Vincent's Hospital, Melbourne, Australia;_ 3 _Karolinska Institute, Sweden_.

Background:

Malignant mesothelioma (MM) is an aggressive malignancy of the pleura and other serosal membranes originating from mesothelial cells. Agents targeting vascular endothelial growth factor (VEGF) receptor such as bevacizumab; and multi-kinase inhibitors such as nintedanib [angiokinase inhibitor of VEGF, platelet-derived growth factor (PDGF) receptor and fibroblast growth factor receptor (FGFR)] have recently demonstrated efficacy in MM. In the setting of these new therapeutic agents, it is important to evaluate VEGF, PDGF and CD31 (angiogenesis marker) in MM to assess their associated prognostic implications.

Methods:

Tissue microarrays (TMAs) were created from formalin-fixed, paraffin-embedded tissue samples obtained from 326 patients who underwent surgical resection or biopsy for MM between 1988 and 2014. The expression of PDGF-C, VEGF and CD31 were analysed by immunohistochemical (IHC) staining. The H-score method assigned a score of 0-300 to each sample, based on the percentage of cells stained at different intensities viewed at various magnifications by a pathologist and an investigator. The discriminatory threshold was set for each IHC stain (usually the median score) and the samples were classified as low (below median score) or high expression (above median score) for each of stain. CD31 was evaluated via Chalkley's method (objective method of measuring vascularity) to evaluate microvessel density. We evaluated the association between expression of the biomarkers, clinicopathological factors and outcomes, in patients with MM.

Results:

The histological subtypes comprised of 203/325 (62.5%) epithelioid; 72/325 (22.2%) biphasic; 42/325 (12.9%) sarcomatoid, or indeterminate. The median age was 67 (range 24-88) with Male: Female ratio of 266: 53. CD31 high (≥5) was seen in only 31/302 (10.3%) irrespective of histology (13/31 (42%) epithelioid; 10/31 (32%) sarcomatoid; 7/31 (23%) biphasic; 1/31 indeterminate). PDGF-C high IHC (≥150) was seen in 203 /310 (65%) of all samples but was higher in epithelioid histology (129/203 (64%) epithelioid; 45/203 (22%) biphasic and 28/203 (13.8%) sarcomatoid; 1/203 indeterminate). VEGF high (≥200) was seen in 219/322 (68%) of all MM and was also higher in epithelioid histology [143/209 (68%)]. There was no association of VEGF IHC with survival nor differences between histological subtypes. There was a non-significant trend towards poorer survival in epithelioid tumours with increased PDGFC expression (HR 0.7928; 95% CI 0.5958 to 1.055, P=0.0434). High CD31 score was associated with significantly poorer survival (OS 12 vs 8.6 months; HR 0.48; 95% CI 0.2873 to 0.7941, P=0.0044). Of the 31 patients with high CD31 scores; 23/31 (74%) were also high for PDGF-C and 20/31 (64%) with high VEGF scores.

Conclusions:

High PDGF-C expression and CD31 scores are associated with poor survival in MM. Abrogating these pathways may have prognostic implications.

#1558

STAG2 as a biomarker for prediction of recurrence in papillary non-muscle invasive bladder cancer.

Alana Lelo,1 Brent T. Harris,1 Deborah L. Berry,1 Krysta Chaldekas,1 Anagha Kumar,2 David Solomon,3 Jeffry Simko,3 Pritish Bhattacharyya,4 Ciaran Mannion,4 Jung-Sik Kim,1 George Philips,5 Todd Waldman1. 1 _Georgetown University, Washington, DC;_ 2 _Medstar Health Research Institute, Washington, DC;_ 3 _University of California San Francisco School of Medicine, San Francisco, CA;_ 4 _Hackensack University Medical Center, Hackensack, NJ;_ 5 _Medstar Washington Hospital Center, Washington, DC_.

Purpose: Bladder cancer is the sixth most common human cancer. Most cases are early-stage tumors known as papillary non-muscle invasive bladder cancer (NMIBC). After resection, 60-70% of NMIBCs recur locally, and ~15% progress to muscle invasion and metastasis. There is an unmet need for a biomarker to stratify tumors on their likelihood of recurrence. Such a biomarker could make it possible to identify those patients most likely to benefit from adjuvant intravesical therapy such as BCG. Furthermore, lower-risk patients could become eligible for less frequent post-resection surveillance by cystoscopy than the extremely intensive current standard of care. We recently identified STAG2 as among the most commonly mutated genes in NMIBC and provided initial evidence that STAG2 mutant tumors may recur less frequently than STAG2 wild-type tumors. Here we report a STAG2 biomarker study using an independent cohort of clinically annotated papillary NMIBC tumors.

Experimental Design: We identified all cases of papillary NMIBC treated at the Lombardi Comprehensive Cancer Center prior to 2013. STAG2 immunohistochemistry was performed and correlated with time to recurrence. This assay is particularly robust because (i) it utilizes a monoclonal antibody whose epitope is in the extreme carboxyl-terminus of STAG2, and ~85% of tumor-derived mutations of STAG2 are truncating; (ii) STAG2 is on the X-chromosome, so only a single mutation is required for complete gene inactivation; and (iii) STAG2 is among the most abundant proteins in the human proteome.

Results: 52% of NMIBC tumors with STAG2 expression recurred, whereas only 25% of NMIBC tumors with loss of STAG2 expression recurred (p=0.017). Multivariate analysis identified STAG2 as the only significant independent predictor of recurrence (p=0.050). The relative risk of recurrence for patients with STAG2-expressing tumors was 2.408 times the risk for patients with STAG2-negative tumors.

Conclusions: Here we demonstrate that a simple, robust assay for identifying STAG2-mutant tumors stratifies patients into two groups: (i) patients with retention of STAG2 expression (wild-type), ~52% of whom will recur; and (ii) patients with loss of STAG2 expression, of whom only ~25% will recur. Of note, STAG2 expression status, unlike pathologic grade--the current standard--was a significant predictor of recurrence. This information could help physicians make more informed decisions about the need for adjuvant immunotherapy/chemotherapy and also help inform the frequency of post-resection surveillance needed for individual patients. In conclusion, STAG2 is a simple, powerful, and potentially useful biomarker for prediction of recurrence in papillary NMIBC.

#1559

Detection and retrieval of multiple myeloma cells with single-cell molecular confirmation from peripheral blood by multiparameter immunofluorescence, automated scanning, and image analysis.

Lance U'Ren,1 Nolan Ericson,1 Daniel Campton,1 Arturo Ramirez,1 C. Anthony Blau,2 Eric Kaldljian1. 1 _RareCyte, Inc., Seattle, WA;_ 2 _University of Washington, Seattle, WA_.

Background: The detection of multiple myeloma (MM) cells is currently performed by analysis of bone marrow aspirates. Detection of MM cells from the peripheral blood would allow for less invasive and more frequent assessments to monitor residual disease. Using the RareCyte platform, we developed a 5-parameter assay to visualize and retrieve MM cells from peripheral blood and applied it to MM patient samples under an IRB-approved protocol. We also used this to identify plasma cells from the peripheral blood of healthy volunteer donors.

Materials and Methods: Healthy volunteer or MM patient peripheral blood was collected into RareCyte blood collection tubes. RPMI8226 cells spiked into normal donor blood samples were used as a model for MM cell assay development. Buffy coats isolated from 7.5mL of blood by AccuCyte® separation were spread onto slides. Slides were stained with a 5-marker panel that included antibodies to CD138, CD38, CD56, CD45, and a nuclear dye on the Leica Bond Rx auto-stainer. Slides were scanned with CyteFinder® and MM cells identified by positive CD138 and CD56 staining and negative CD45 staining. Identified MM cells and WBC control cells were mechanically retrieved with the integrated CytePicker module, whole genome amplified, and Sanger sequencing was performed for specific mutations in four genes (IDH1, NRAS, PHLDA1, RPH3AL) identified by genomic analysis of a bone marrow sample. Plasma cells from normal donors were identified by co-expression of CD138 and CD38.

Results: In a pretreatment MM sample, the 5-parameter assay identified 55 MM cells per mL of whole blood. In isolated single cells with at least one successful sequencing reaction, 0 of 13 WBCs contained mutations in at least one of the genes. Mutations were found in 9 of 15 MM cells (defined as CD138+, CD56+, CD38-, and CD45-). In healthy volunteer samples, 5 plasma cells (defined as CD138+, CD38+, and CD45 dim/negative) were identified per mL of whole blood.

Conclusions: The 5-parameter assay successfully detected rare cells with an MM phenotype from the peripheral blood of an MM patient. Individually retrieved MM cells were demonstrated to contain mutations specific to the patient's tumor, confirming that the cells identified were MM cells. This approach may allow for less invasive assessment of MM and monitoring of residual disease.

#1560

Real-time monitoring of solid tumor progression by circulating stromal cells from early- to late-stage disease.

Daniel L. Adams,1 Raymond Bergan,2 Martin J. Edelman,3 Stuart S. Martin,4 Rena Lapidus,4 Saranya Chumsri,5 Cha-Mei Tang,6 Steven H. Lin7. 1 _Creatv MicroTech, Inc., Monmouth Junction, NJ;_ 2 _Oregon Health and Science University, Portland, OR;_ 3 _Fox Chase Cancer Center, Philadelphia, PA;_ 4 _University of Maryland, Baltimore, MD;_ 5 _Mayo Clinic Cancer Center, Jacksonville, FL;_ 6 _Creatv MicroTech, Inc., Potomac, MD;_ 7 _MD Anderson Cancer Center, Houston, TX_.

Background: Blood-based biomarkers (PSA, CEA, CA125) are used to track real-time progression of disease in parallel with imaging. However, while numerous blood biomarkers exist, they are specific to cancer type (i.e., PSA to prostate and CEA to colon) and may not appear in all diseased individuals. Recently, cancer-associated macrophage-like cells (CAMLs), a circulating stromal cell subtype, were identified in various solid cancer types, which were observed increasing in size and in hyperploidy during progressive disease. To assess whether CAML enlargement is a biomarker of progression/response, we tracked CAML growth/shrinkage in a pilot study of patients (n=34). Blood was drawn from patients with lung, prostate, or breast cancers over a 3-month period, baseline through 2 treatment cycles, followed by continued monitoring for 2 years. These data suggest that morphologic assessment of CAMLs (growth/hyperploidy) appear to parallel cancer progression, or response to treatment, in multiple solid tumors.

Methods: A prospective multi-institutional study used anonymized peripheral blood samples from 34 cancer patients undergoing therapy [stage I (n=2), II (n=3), III (n=8) & IV (n=21)] with breast (n=10), lung (n=16), & prostate (n=8). Samples were taken prior to therapy (BL), at 1 month (FU1) follow-up and a 3-month (FU2) follow-up, after induction of therapy. Blood was processed by the CellSieve™ microfiltration technique at 4 institutions and stained for cytokeratin 8, 18 and 19, CD14 and CD45. After identification and quantification, CAMLs were measured based on their hyperploidy and cell size.

Results: CAMLs were found in 97% of cancer patients at BL, 97% at FU1 and 91% of FU2. Over the 2-year follow-up, 7 patients showed no signs of clinical disease progression, while 27 patients had observable progression. Of the 7 patients who did not progress, only two had CAMLs of ≥50µm at BL and at FU1, but whose CAMLs shrunk to <50 µm by FU2. Of the 27 patients who progressed, 22 patients had ≥50µm CAMLs at all time points, while 5 patients had small <50µm CAMLs at BL. Interestingly, CAMLs in these 5 patients had enlarged to ≥50µm by the FU2 time point.

Conclusions: We show that increased polynucleation and CAML enlargement indicate shorter progression-free survival in a number of cancer types after baseline. By monitoring CAML changes over time for the 34 individual patients, we demonstrated correlation of ongoing progression, or response, in tumors to the enlargement or shrinkage in CAMLs at follow-up time points from treatment induction. This pilot study suggests that CAMLs have the potential to monitor the progression/regression of malignancy in real time and suggests the need for larger validation studies.

#1561

Increased phenotypic depth for automated visual identification of biomarkers on circulating tumor cells by cocktailing epithelial markers EpCAM and cytokeratin.

Yao Sun,1 Arturo Ramirez,1 Daniel Campton,1 Tanisha Mojica,2 Alisa Clein,3 Celestia Higano,3 Daniel Sabath,3 Eric Kaldjian1. 1 _RareCyte, Inc., Seattle, WA;_ 2 _Seattle Cancer Care Alliance, Seattle, WA;_ 3 _University of Washington, Seattle, WA_.

Background: Canonical methods for CTC identification (such as CellSearch®) have typically relied on co-expression of surface (EpCAM) and cytoplasmic (cytokeratin, CK) epithelial markers to separate them from white blood cells. RareCyte has developed AccuCyte®-CyteFinder®, a platform for automated visual identification and retrieval of rare cells in blood by immunofluorescence (IF). For CTC detection, EpCAM and CK are placed in individual fluorescence channels, allowing for identification of CTCs that may be EpCAM(-) /CK(+) as well as EpCAM(+)/CK(-). Recently we have developed technology that increases the number of markers from 4 to 6 to allow for biomarker phenotypic analysis in the additional two channels. Given that our platform does not require co-expression of EpCAM and CK, we hypothesized that we could gain a third biomarker channel by combining detection reagents for EpCAM and CK into a cocktail within a single channel. We tested feasibility of epithelial marker cocktailing with an assay to detect androgen receptor (AR) in prostate CTCs.

Methods: Normal human whole blood samples were spiked with prostate cancer lines PC3, LNCaP, and 22RV1 as model CTCs (mCTCs). Blood samples from University of Washington patients with advanced cancer were collected under an IRB-approved protocol. Blood was processed onto microscope slides and stained on an automated stainer using a CTC assay for detection of AR in which anti-cytokeratin and anti-EpCAM antibodies were combined in a reagent cocktail into a single channel; the CD45 WBC marker and a nuclear dye were included in the assay. After development using spike-in models, the assay was applied to a patient sample.

Results: No CTCs were identified in non-spiked normal donor blood samples. In spike-in models, the epithelial cocktail assay performed as well as the non-cocktail assay in identifying CTCs. AR fluorescence signal was present in LNCaP and 22RV1 mCTCs (which express AR) and absent in PC3 mCTCs (which do not express AR), confirming specificity of AR staining. When the assay was applied to a blood sample from a prostate cancer patient with known high CTC count, 160 CTCs per mL were identified; of these, 114 were observed to be AR-positive (71%).

Conclusions: Combining the epithelial markers cytokeratin and EpCAM into a single-wavelength cocktail is feasible for IF identification of CTCs, allowing an additional channel for phenotypic investigation. In a patient with advanced prostate cancer with high CTC count, there was high incidence (71%) of AR expression in identified CTCs.

#1562

Clusters of circulating tumor cells were selectively isolated in the blood of 12/12 epithelial ovarian cancer patients using facile gravity-flow-based filtration method adapted to clinical use.

Anne Meunier,1 Sara Kheireddine,1 J. Alejandro Hernández-Castro,1 Benjamin Péant,2 Diane Provencher,2 Anne-Marie Mes-Masson,2 Teodor Veres,3 David Juncker1. 1 _McGill University, Montreal, Quebec, Canada;_ 2 _Université de Montréal, Montreal, Quebec, Canada;_ 3 _National Research Council of Canada, Boucherville, Quebec, Canada_.

Background

The presence of circulating tumor cells (CTCs) in blood is correlated with disease progression in many cancers. Their prognosis value in ovarian cancer is still under debate.1 CTCs are heterogeneous in size and marker expression, and sub-populations with various metastatic potentials have been identified. CTC clusters, although rare and difficult to isolate, have emerged as a possible driver of metastasis owing to ~50-time higher metastatic potential than single CTCs.2 Only few methods have emerged to capture clusters, and are often complex and cumbersome, limiting our understanding of the role of clusters in metastasis. Here, we present a new filtration method for the selective capture of CTC clusters from blood and found clusters in 12/12 epithelial ovarian cancer (EOC) patients.

Method

Cluster capture was performed by filtration using a 3D printed cartridge3 and filters4 with pore diameters of 8, 10, 12, 15, 20 or 28 μm. We developed a gravity-driven process, generating reduced shear stress, and optimized capture using blood (1:6, v/v, in PBS) spiked with OV-90 and OVCAR-3 ovarian cancer single cells and clusters. Blood samples from 12 EOC patients were filtered. Clusters can be stained and imaged on the filter, or released for downstream analysis.

Results

Using the gravity-setup, we were able to selectively capture clusters with good integrity and with a rate that outperforms other technologies to the best of our knowledge. Viable CTC clusters, with 2 to >100 cells, were captured from 12/12 EOC patients. Their size distribution was surprisingly similar between patients. Small clusters (2-3 cells) were the most frequent, and this frequency decreased as their size increased.

The molecular characterization of the captured clusters revealed a low and localized, heterogeneous expression of EpCAM (epithelial cell adhesion molecule), in combination with a widespread expression of c-MET (hepatocyte growth factor receptor) in all patients, suggesting a mesenchymal-like profile.

Conclusion

Using the gravity-filtration setup, CTC clusters were captured from the blood of all patients tested, suggesting that clusters are much more widespread than anticipated, and are in fact the norm rather than the exception. The cluster size distribution was conserved between patients with small clusters dominating, and some rare, very large clusters. Cluster staining revealed a mesenchymal profile, in agreement with a higher metastatic potential. Together, these results suggest that clusters should significantly contribute to disease progression, a hypothesis, which may be explored using our facile and selective method.

References

1. Y. Zhou, et al. PLoS ONE 2015, 10, e0130873.

2. N. Aceto, et al. Cell 2014, 158, 1110.

3. A. Meunier, et al. Anal. Chem. 2016, 88, 8510.

4. J. A. Hernandez-Castro, et al. LOC 2017, 17, 1960

#1563

Characterization of two new recombinant rabbit anti-PDL1 IHC staining in bladder cancer, NSCLC, and melanoma with immune cell markers.

Rachel M. Gonzalez,1 Wei Fu,1 Evelin Logis,1 Emma Ding,2 Casey Chen,2 Xiaojie Li,2 Sonia Merritt,2 Mingjuan Liu,2 Amy Zhang,1 Yiran Wang,2 Guangli Wang,2 Donghui Ma1. 1 _BioCheck Inc, Rockville, MD;_ 2 _OriGene Technologies Inc, Rockville, MD_.

Published studies show tumor cells overexpress PD-L1 to escape the host immune defense by binding to PD-1 on surveilling T-cells causing the T-cells to shut down. Immunotherapies disrupting PD-1/PD-L1 signaling interactions have great success in treating PD-L1 positive tumors. Immunohistochemistry (IHC) is an important diagnostic tool currently used to determine the expression level of PD-L1 in tumor and immune cells. The FDA approved PD-L1 clones (SP142 and 28-8) for IHC present different staining patterns when evaluated on the same tumor tissue. In this study, we assessed both the immune cells and tumor cells IHC positive stain of PD-L1. To do this we evaluated multiple immune cell markers with multiple PD-L1 antibodies. IHC screens were done with 5 PD-L1 antibodies, 2 recombinant rabbit monoclonal antibodies (clone OR-5E3 and OR-5H8), the mouse monoclonal antibody (clone UMAB229), and the FDA approved clones (SP142 and 28-8). The immune cell markers used were CD3, CD8A, CD20, CD68, and FOXP3. NSCLC, Bladder Cancer, and Melanoma immune cells, as indicated by the CD3, CD8A, CD20, CD68 and FOXP3 staining, generated different distribution pattern in the three tumor types. The five PD-L1 antibodies did show variation in detection of both the immune and tumor cells. For example, PD-L1 clones OR-5E3 and OR-5H8 stained tumor cells stronger and picked up weak expression of PD-L1 better than clone SP142, suggesting the two antibodies have higher affinity. The mouse monoclonal anti-PDL1 clone UMAB229 picked up strong and weak staining similar or better than clones OR-5H8 and picked up the immune cells similar to SP-142. The five PD-L1 antibodies were made from different antigens which may contribute to their sensitivity and specificity to detect PD-L1 in tumor cells. This study suggests the new generation of PD-L1 antibodies may make a better tool for diagnostic screens.

#1564

Detecting low allele frequency somatic mutations using targeted amplicon sequencing and a novel double strand molecular barcoding scheme.

Li Jacey Zhang, Lucie Siu Lee, Yang Lily Liu, Jeff Liu, Tao Chen, Guoying Liu, Zhitong Liu. _Paragon Genomics, Hayward, CA_.

Using next generation sequencing (NGS) with a noise reduction mechanism to detect somatic variants of 0.1% minor allele frequency or lower has attracted increasing interest for cancer research and diagnostics. Molecular barcoding is a technology that aims to reduce the noise and errors caused by PCR and sequencing artifacts, and improves mutation detection accuracy. Here, we report a novel multiplex PCR-based, ultra-sensitive molecular barcoding technology that drastically reduces false positive calls, additionally, barcoded sequencing reads can be assigned back to both the forward and reverse strands of the original DNA fragment, thus further remove variant call noise. While providing a simple and fast workflow, this technology does not have the base bias associated with capture hybridization. It achieves an error rate of 4 X 10-7 nucleotides, and detects allele frequencies at 0.1%.

#1565

Identification of genetic and epigenetic biomarkers for prediction of chemoresistance in the treatment of high-risk advanced retinoblastoma.

Wenping Song, Rongguang Shao, Liang Li. _Institute of Medical Biotechnology, PUMC, Beijing, China_.

Background: Retinoblastoma (RB) is the most common pediatric retinal tumor initiated by biallelic inactivation of the retinoblastoma gene (RB1). Currently, chemotherapy with carboplatin, vincristine, and etoposide followed by optic enucleation is the most commonly used for retinoblastoma patients in China. The recurrence and metastasis of high-risk advanced RB is still a major obstacle for successful chemotherapy with very low survival rate only 26% in China, mainly due to chemotherapeutic resistance. The aim of the study is to identify genetic/epigenetic biomarkers with regarding to the prediction of outcome of chemotherapy for high-risk advanced RB, and to functionally characterize for mechanisms of candidate genes or potential biomarkers. Methods: Total of 41 pediatric patients with D- or E-stage retinoblastoma according to International Intraocular Retinoblastoma Classification (IIRC) staging standard were collected in the present study. Four complete remission (CR) and four recurrence patients due to their typical symptoms were selected to compare differences in profiles of whole-exome and genomic DNA methylation with high throughput next-generation sequencing technology by using their peripheral blood samples and to identify candidates in an integrative analysis of omics datasets. Subsequently, functional validation study was performed by using a human retinoblastoma Y79 cell line for those relevant candidate genes. Specifically, Y79 cells were knockdown or overexpressed with specific siRNA or expression vectors, respectively, followed by treatments with a diluted concentrations of carboplatin, vincristine, and etoposide in Cell Counting Kit (CCK-8) assay. Results: There were significantly different profiles of whole-exome and genomic DNA methylation in the integrative analysis. Total of 556 SNPs and 35 Indels were discriminated from whole-exome sequencing data, and 721 DNA methylation sites were detected with significant differences in RRBS assay between the datasets of those recurrence and CR patients. One variant (rs140693) in the exon 3 of MBD4 gene was identified in all those 4 recurrence patients and down-regulation of this gene by its specific siRNAs significantly altered the resistance of Y79 cells to all those above three drugs, as well as the cell growth rate, suggesting that the candidate variant/gene might be functional to impact on multiple drug resistances to carboplatin, vincristine and etoposide. Further functional studies would be required for characterization of its action mechanisms. Conclusion: Our study had identified novel genetic/epigenetic biomarkers to predict resistance against chemotherapeutics for high-risk advanced RB. It would be helpful as the first step toward precise medicine to explore intrinsic mechanism of chemoresistance and develop potential therapeutic targets for RB in future.

#1566

Establishment of analytical performance features of two immunohistochemistry assays for PD-L1 expression in lung cancer.

Lei Zhang,1 Claire Begot,2 Robert McGee,3 Steven M. Anderson4. 1 _Covance, Singapore, Singapore;_ 2 _Covance, Geneva, Switzerland;_ 3 _Covance, Indianapolis, IN;_ 4 _Covance, Durham, NC_.

Therapies that target PD-1/PD-L1 and overcome checkpoint inhibition have shown great promise in the treatment of a variety of cancers, with PD-L1 expression demonstrated as a biomarker of therapy response in a variety of tumor types. There are multiple IHC assays that are used to measure PD-L1 expression in formalin-fixed, paraffin-embedded tissues (FFPE) with significant variability in primary antibody, assay platform, scoring criteria, and assay cut-offs. This situation creates potential confusion in applications of PD-L1 immunohistochemistry (IHC) assays for the appropriate intended use. In this study we evaluated two assays (Dako pharmDx PD-L1 28-8 assay and Ventana PD-L1 SP263) in 45 non-small cell lung cancer samples encompassing predominately squamous cell carcinomas (35) and adenocarcinomas (10). PD-L1 protein expression, measured by the percentage of tumor cell showing positivity for PD-L1 staining, has been compared between two assays. PD-L1 results were interpreted by using various assay cutoffs. Correlation of PD-L1 expression measured by positivity in percentage of tumor cell staining with each of the two assays achieved a significant concordance (r2 = 0.91). Similar trends in percentage of positive of tumor cells were demonstrated across a broad dynamic range of staining. Cutoffs that have been established in clinical studies and referenced in the respective assay package inserts (>1% positivity percentage of tumor cells for 28-8 assay and >25% positivity percentage of tumor cells for the SP263 assay) were used as the reference, for interpreting the test results as positive or negative. When these established cutoffs were applied, a low-to-medium concordance between two assays has been demonstrated (r2 = 0.58). When the same cutoff (1% or 25%) was applied, the correlation has been improved markedly (r2 = 0.73 or 0.95 respectively). In this sample set the 25% cutoff produced the more consistent data when comparing the two methods. Changes in the classification of PD-L1 status was also observed for both Dako pharmDx PD-L1 28-8 assay (29%, 13/45) and Ventana PD-L1 SP263 (22%, 10/45) assays when the established cutoff was replaced. The findings of this study are consistent with others such as those from Blueprint phase I study. This comparison study showed that the two assays (Dako pharmDx PD-L1 28-8 assay and Ventana PD-L1 SP263) were analytically comparable. Further investigation is warranted to explore both the analytical and clinical performance of the various PD-L1 assays across multiple assay cut-points.

#1567

Ku70, Ku80 and Rab5C as biomarkers for radiotherapy resistance in rectal tumors during neoadjuvant therapy.

Vilma Regina Martins,1 Antuani Rafael Baptistella,1 Petrus Paulo Silva,1 Edson Kuatelela Cassinela,1 Fabio Marchi,1 Adriana Fp Leme,2 Maria Dirlei Begnami,1 Samuel Aguiar1. 1 _A.C. Camargo Cancer Center, Sao Paulo, Brazil;_ 2 _Brazilian Biosciences National Laboratory – LNBio, Campinas, Brazil_.

Rectal cancer is the second most common cancer in the large intestine, corresponding to one-third of the total cases of colorectal cancer. The neoadjuvant chemoradiation in rectal cancer treatment reduces the risk of local recurrence, but pathologic complete response is achieved only in 10-40% of patients. The response to neoadjuvant treatment is poorly understated in rectal tumors as well as the mechanisms associated with chemoradiation resistance. In order to identify the players involved with the therapy resistance, we performed proteomic analysis of biopsies from stage II and III rectal adenocarcinoma patients before neoadjuvant treatment and compared with residual tumor tissue removed by surgery after neoadjuvant therapy. Ku70, Ku80 and Rab5C showed a significant increased expression after the chemoradiation treatment. To better understand the mechanism associated with therapy resistance, we generated an ionizing radiation-resistant rectal adenocarcinoma cell line. These cells and also their secreted exosomes exhibit overexpression of the same three proteins identified in tissue samples from patients submitted to chemoradiation. Using this in vitro model, we identified that Rab5C modulates EGFR internalization promoted by irradiation, which was also essential to regulate the expression of the DNA repair proteins Ku70 and Ku80, and cell resistance to ionizing radiation. Taken together, these findings indicate that EGFR and Rab5C are potential targets to sensitize rectal cancer cells to neoadjuvant treatment. In addition, the presence of Ku70, Ku80 and Rab5C in circulating exosomes can be explored as biomarkers to follow resistance to radiotherapy in patients under neoadjunat therapy for rectal tumors.

#1568

Advancing convergence and innovation in cancer research: National Cancer Institute Center for Strategic Scientific Initiatives (CSSI).

Sarah G. Elder, Jerry S. Lee, Michelle A. Berny-Lang, Sean E. Hanlon. _National Cancer Institute, Bethesda, MD_.

Since 2003, the National Cancer Institute's Center for Strategic Scientific Initiatives (CSSI) has worked to develop the resources and infrastructures investigators need to surmount roadblocks in cancer research. To this end, CSSI manages programs that promote technology development and cross-disciplinary collaboration and provide support for investigators in nascent and challenging research fields. This support includes funding opportunities, shared reagent and database resources, and assistance in the development of standards and protocols. CSSI also provides a network of partners in industry and government that can help NCI-funded researchers advance their technologies toward commercialization and translation. This presentation will highlight technologies including single-cell isolation and analysis techniques that have been supported through various CSSI mechanisms from proof of concept to translation into the clinic.

#1569

Circulating tumor cells and circulating tumor microemboli in the context of gastric adenocarcinoma.

Emne Ali Abdallah, Alexcia Camila Braun, Bianca Troncarelli Flores, Lais Lie Senda, Maria Dirlei Ferreira de Souza Begnami, Felipe José Coimbra, Emmanuel Dias-Neto, Diana Nunes, Wilson Luiz Costa Junior, Ludmilla T. Chinen. _ACCamargo Cancer Center, São Paulo, Brazil_.

Background: Whereas the pathological aspects of Gastric Adenocarcinomas (GAC) are well defined, there is still an important lack of biomarkers predictive of its occurrence, aggressiveness and response to neoadjuvant treatment. Nowadays, it is known that isolated or clustered Circulating Tumor Cells (respectively CTCs or CTMs, for Microemboli) can be found in the blood of patients many months before tumor detection. In this sense, the identification of CTCs/CTMs in GAC patients has a potential value as biomarkers for tumor growth and invasiveness.

Objective: to study the predictive value of CTCs and CTMs from GAC patients, evaluating their presence and correlation with HER2-status and clinical-pathological characteristics.

Methods: Blood was prospectively collected from 72 GAC patients (62.5% men) recruited at the Department of Abdominal Surgery of the A.C.Camargo Cancer Center, São Paulo, Brazil, from March 2016 to March 2017, after the signature of informed consent forms previously approved by the institutional review board. Blood was first collected before the first neoadjuvant cycle (at diagnosis) and also before adjuvant treatment (diagnosis of metastasis). The samples were processed and filtered in ISET (Rarecells, France) allowing the isolation and quantification of CTCs and CTMs, and immunocytochemistry (ICC) with anti-HER2.

Results: The most common tumor status were T1b (15/20.8%) and N1 (14/19.4%) and diffuse tumors (Lauren's) was the most common subtype (37/51.4%); 55 patients had non-metastatic disease (76.4%) at moment of inclusion. CTCs were found in 61/72 patients (84.7%) with a median of 2.5 CTCs/mL (0-39.6 CTCs/mL). We found more CTC/mL in non-metastatic disease (2.8 CTCs/mL versus 0.9 CTCs/mL, p<0.001). CTMs were found in 28 patients (38.9%) and we also found more CTCs/mL CTM-positive cases (p<0.001). Among non-metastatic disease, 23/55 patients had CTM (41.8%) and in the metastatic scenario, 5/16 patients had CTM (31.3%). Besides finding no differences in the amount of CTCs between histological subtypes (intestinal versus diffuse), we observed more CTMs on diffuse GAC subtype compared to intestinal (69.6% x 30.4%). HER-2 status was evaluated in CTCs and primary tumor derived from 40 patients, with a level of concordance of 67.5%.

Conclusions: CTCs were more prevalent in patients with initial disease, reinforcing their possible role in the growth of the primary tumor maybe in a self-seeding mechanism. Also, the good HER2-correlation found between primary tumors and CTCs suggest the use of the later as surrogates of the primary tumor for determining targeted therapies with anti-HER2 antibodies.

#1570

Loss of mismatch repair protein MSH6 sensitizes diffuse-type gastric cancer cells to DNA-PKcs inhibitor KU60648.

Vaidehi Krishnan, Wen Min Lau, Eileen Teng, Jin Wei Tan, Haresh Sankar, Debbie Xiu En Lim, Crystal Kok Yan Pang, Ming Teh, Jimmy Bok Yan So, Patrick Tan, Yoshiaki Ito, Shing Leng Chan. _National Univ. of Singapore, Singapore, Singapore_.

Diffuse-type gastric cancer (DGC) is a histologic subtype characterized by poor cellular cohesiveness, increased metastasis and poor prognosis. As preclinical models of DGC that reliably predict clinical activity of novel compounds are lacking, we generated patient-derived xenograft (PDX) models using malignant ascites from DGC patients that closely recapitulated the primary cancer. To uncover novel therapeutic targets, we applied the concept of synthetic lethality and asked if DGCs require DNA repair proteins for survival. Major DNA repair kinases ATM, ATR and DNA-PKcs in established DGC cell lines and ex vivo cell lines from PDXs (GAGA3, GAGA6 and GAS24) were inhibited using small-molecule inhibitors KU55933, VE-821 and KU60648, respectively. Interestingly, most diffuse-type gastric cells exhibited elevated lethality when DNA-PKcs, a central kinase that regulates the non-homologous end joining pathway (NHEJ), was inhibited by KU60648 as compared to the nontransformed gastric epithelial line, HFE-145. We confirmed that genetic depletion of DNA-PKcs by siRNA resulted in cell death in two PDX lines GAS24 and GAGA6 cells but not in HFE-145 cells, suggesting the dependence of these cells on DNA-PKcs for survival. Two mechanisms appear to sensitize DGC cells to DNA-PKcs inhibition; firstly, impaired homologous recombination repair in GAS24 and GAGA6 as shown by compromised ability to resolve radiation-induced γH2AX foci, and secondly, transcriptional downregulation of the mismatch repair protein, MSH6, in GAGA3 and GAGA6. Knockdown of MSH6 in GAS24 cells enhanced their sensitivity to KU60648, suggesting a functional link between MSH6 and DNA-PK function. Lastly, to develop MSH6 as a biomarker for predicting DNA-PKcs sensitivity, we analyzed MSH6 levels by immunohistochemical staining in formalin-fixed, paraffin-embedded (FFPE) tissue sample sections derived from PDXs. In these preliminary studies, MSH6 expression was lower with high heterogeneity in GAGA3 and GAGA6 compared to GAS24. In this study, we show for the first time the downregulation of DNA mismatch protein MSH6 in sporadic diffuse gastric cancer, and its potential as a therapeutic predictor for DNA-PKcs inhibition treatment.

#1571

**Benign lipomatous lesions and liposarcomas have metabolic differences as identified in a high-resolution** 1 **H and** 31 **P MRS pilot study.**

Santosh K. Bharti, Brett Shannon, Adam Levin, Carol D. Morris, Laura Fayad, Zaver M. Bhujwalla. _The Johns Hopkins University School of Medicine, Baltimore, MD_.

Adipocytic tumors present a spectrum of neoplastic disease including benign lipomas, atypical lipomatous tumors (ALTs), and malignant liposarcomas. Liposarcomas are the most common soft tissue sarcoma, accounting for approximately 20% of all adult soft tissue sarcomas [1]. Some liposarcomas are suspected to arise through dedifferentiation of ALTs, a process that is not well understood. Pleomorphic liposarcomas are high-grade, aggressive tumors with high metastatic potential and overall 5-year survival rate of 20-30% [3]. Distinguishing high-grade liposarcomas from benign and atypical lipomatous tumors can be a diagnostic challenge with implications for surgical and clinical management. The rate of misdiagnosis is approximately 30–40% following radiological detection [4], and 7–17% on histological evaluation [5].

De-identified human surgical samples were collected from the Dept. of Orthopaedic Surgery, JHU, School of Medicine Baltimore, MD. One tissue specimen from a benign pleomorphic lipoma and two tissue specimens from a pleomorphic liposarcoma were examined using 1H and 31P magnetic resonance spectroscopy (MRS) on 750 MHz MR spectrometer. Dual phase solvent extraction was performed to obtain aqueous and lipid phase spectra.

Metabolites were quantified and changes were reported in terms of percentage change compared to levels in pleomorphic lipoma set to 100%. Depletion in branched chain amino acids (-10%), tyrosine (-15%), phenylalanine (-25%), lactate (-45%), pyruvate (-35-50%), acetate (-5-25%), and elevation in alanine (40%), succinate (35-60%), glycine (400-500%), histidine (15-25%), glucose (100%), glutamine (50%), glutamate (75%), and myo-inositol (300%) were observed in pleomorphic liposarcoma. 31P MRS of the aqueous phase revealed high levels of phosphocholine in the pleomorphic liposarcoma as compared to the lipoma (not detected). MRS analysis of the lipid phase identified depletion in lipid (-65%, CH3 peak at 0.9 ppm was quantified) and an increase in phosphatidylethanolamine (75%) and phosphatidylcholine (30%). Visible variations in TAG glycerol backbone (4.2-4.3ppm) and -CH=CH- peaks from polyunsaturated fatty acids at 2.8ppm and 5.3ppm were also observed. We are currently analyzing additional samples from several types of sarcomas to expand this study. Our preliminary data support investigating the use of 1H and 31P MRS of liposarcomas for differentiation between subtypes.

References

1.Dei Tos AP: Histopathology 2014, 64(1):38-52; 2.Nassif NA et al: F1000Research 2016, 5:2907; 3.Downes KA et al: Modn path Inc 0000, 14(3):179-184; 4.Brisson M et al: Skeletal Radiology 2013, 42(5):635-647; 5.Hasegawa T et al: Human Pathology 2002, 33(1):111-115.

#1572

The value of p16 or HPV as predictive marker of anti-EGFR treatment in recurrent/metastatic HNSCC.

Joo Han Lim, Soon-Sun Hong, Jin-Chul Kim, Moon Hee Lee, Jinhyun Cho. _Inha Univ. School of Medicine, Incheon, Republic of Korea_.

Introduction: The anti-epidermal growth factor receptor (EGFR) agent was approved and has been a mainstay of treatment of recurrent/metastatic head and neck squamous cell cancers (HNSCC). However, it is unclear whether human papilloma virus (HPV) modulates the response to targeting the EGFR. Some subset analysis of the randomized phase III study is confusing and recent The Cancer Genome Atlas data showed that EGFR gene is rarely abnormal in HPV-positive HNSCC tumors while it is frequently altered in HPV-negative HNSCC tumors. The purpose of our meta-analysis is to evaluate the effects of anti-EGFR agents regarding HPV status.

Materials and Methods: We conducted a pooled analysis to evaluate the association of p16 expression status with outcomes in patients with recurrent/metastatic HNSCC receiving palliative anti-EGFR treatment with or without chemotherapy. A literature search of PubMed, EMBASE, and meeting abstracts of the American Society of Clinical Oncology and European Society of Medical Oncology through December 2017 was conducted.

Results: A total of seven trials met the selection criteria. The total number of patients was 700 (554 with the HPV-negative group and 146 with the HPV-positive group). Anti-EGFR treatment was associated with higher response rate in p16-negative patients compared with in p16-positive patients (OR 2.18; 95% CI, 1.18-3.99, p=0.01).

Conclusion: A meta-analysis of trials with anti-EGFR agents in p16-negative RMHNSCC patients versus in p16-positive recurrent/metastatic HNSCC patients yielded that p16 status could be a predictive biomarker for anti-EGFR treatment in recurrent/metastatic HNSCC. Prospective study is warranted to determine the value of p16 or HPV as a predictive marker of anti-EGFR treatment in recurrent/metastatic HNSCC.

#1573

A clinical biomarker assay to quantitate MIP-1a in human serum.

Li Chin Wong, Oitak Wong, Omar F. Laterza, Xuemei Zhao. _Merck & Co., Inc., Kenilworth, NJ_.

Macrophage inflammatory protein 1-alpha (MIP-1alpha, MIP-1α, CCL3) is a cytokine that is involved in the acute inflammatory state in recruitment and activation of polymorphonuclear leukocytes through binding to the receptors CCR1, CCR4 and CCR5. MIP-1α is expressed by macrophages, dendritic cells and lymphocytes. Crucial in immune response against infection and inflammation, MIP-1α induces the synthesis and release of IL-6, IL-10 and TNFα. As a proinflammatory chemokine, MIP-1α is primarily associated with cell adhesion and migration of monocytes to inflammatory tissues. In addition, MIP-1 α inhibits the proliferation of hematopoietic stem cells in vitro and in vivo. Expression levels of MIP-1 α have provided important information in the progression of diseases such as multiple myeloma, lung cancer, multiple sclerosis, HIV infection, allergic asthma and sepsis. 

### Liquid Biopsy 2

#1574

KRAS genotyping by digital PCR combining melting curve analysis.

Junko Tanaka, Yuzuru Shimazaki, Tatsuo Nakagawa, Akiko Shiratori, Masao Kamahori, Takahide Yokoi, Kunio Harada, Yoshinobu Kohara. _Hitachi, Ltd., Tokyo, Japan_.

Background

ctDNA is a remarkable liquid biopsy for cancer diagnosis. Highly sensitive quantification method is required to measure a tiny amount of ctDNA. Digital PCR has been developed as a method that can quantify nucleic acids more sensitively than real-time PCR does. However, the digital PCR has large fluctuation in the fluorescence intensity of the droplets or chambers resulting in lower accuracy. Main cause is probably due to the insufficient PCR in the small partitions. In this study, we have proposed a new measurement method combined a digital PCR with melting curve analysis using molecular beacons to solve above mentioned problems, and applied it to KRAS genotyping.

Methods

Molecular beacons, which have hydrophobic moiety in the stem, were designed for detecting KRAS mutation. The digital PCR with combination of asymmetric PCR was performed using the molecular beacons in the droplets. After the PCR, the fluorescence of the droplets was observed with a microscope while changing the temperature. A melting curve was prepared from the change in fluorescence intensity of the droplet, and the melting temperature (Tm) was calculated from the differential melting curve.

Results

The melting curve analysis for the KRAS mutation was performed in the droplets where the asymmetric PCR was performed using molecular beacons with hydrophobic stem, which improved signal-to-noise ratio of melting curves. The use of molecular beacons with hydrophobic stem can keep a background fluorescence at a constant value even at high temperature. The change in fluorescence intensity of PCR solution using molecular beacons with hydrophobic stem during the measurement was one-tenth of that using molecular beacon without hydrophobic stem. The asymmetric PCR enabled us to increase the amount of the PCR products hybridized with molecular beacons, resulting in the increase in the fluorescence intensity. The KRAS genotyping of wild-type (WT) and G12D mutant was conducted by the melting curve analysis with a combination of the asymmetric PCR with molecular beacons. The results showed that the peaks of the distributions of the Tm values of DNA in the droplets were 77.9°C for WT and 74.0°C for G12D mutant, which indicates that the WT and the mutant could be successfully discriminated by the proposed method.

Conclusion

We have proposed a new measurement method combining digital PCR with asymmetric PCR using molecular beacons and melting curve analysis. The genotyping of KRAS mutation was successfully performed by the proposed method. We are planning to prove the concept of this method for the clinical specimens in the future.

#1575

Circulating tumor DNA as a tool for predicting response to targeted therapy in gastrointestinal malignancies.

Jaime L. Schneider, Aparna Parikh, Mehlika Rethinam, Brandon Nadres, Emily Van Seventer, Heather Shahzade, Ryan B. Corcoran. _Massachusetts General Hospital, Boston, MA_.

Background: Patients with gastrointestinal (GI) malignancies undergoing treatment are surveyed for disease relapse with radiographic imaging and measurement of standard tumor markers. However, there is a need for more sensitive techniques to detect early cancer progression. Measurement of circulating tumor DNA (ctDNA) is emerging as an important diagnostic tool that can provide real-time information about disease response during treatment. In this study, we evaluated whether dynamic monitoring of ctDNA may predict cancer progression earlier than standard methods in patients with GI malignancies undergoing targeted therapy. Methods: Tumor and blood specimens were obtained from patients treated at the Massachusetts General Hospital (MGH) under IRB-approved studies. Tumor biopsies were performed at the time of diagnosis and were subjected to the MGH standard molecular diagnostics panel for 104 known cancer genes. Blood samples for ctDNA analysis were collected at baseline at the start of targeted therapy, and one more more driver mutations were followed longitudinaly at two week intervals starting one month after treatment. ctDNA was extracted from plasma using QIAGEN-based protocols and amplified by digital droplet PCR (ddPCR) using primers for tumor-specific point mutations. Tumor markers and CT scans were part of routine clinical care and procured using standard procedures at MGH. RECIST measurements, if available, were obtained in a blinded fashion using the Tumor Imaging Metrics Core. Results: We studied 44 patients with GI malignancies undergoing treatment with targeted therapies. Represented cancer types included colorectal (53%), gastroesophageal (21%), biliary (20%), and pancreatic cancers (6%). We correlated the % change in ctDNA mutant allele fraction (MAF) or standard tumor markers 4 weeks after treatment initiation with radiographic response by RECIST. Tumor-specific MAF decreased by 88% in patients who went on to show partial response (PR) or stable disease (SD), compared to an increase by 51% in patients shown to have progressive disease (PD) (p=1.8x10-5). All patients who achieved PR, except one, had a reduction in ctDNA levels by at least 93% or more. In contrast, standard tumor markers did not predict response as there was no significant difference in the % change at 4 weeks in the PR/SD group compared to PD (p=0.64). Conclusions: Real-time monitoring of patient-specific tumor mutations by ctDNA analysis has the potential to predict response to targeted therapy earlier than standard surveillance mechanisms. Integration of liquid biopsies into medical decision-making may allow for earlier identification of cancer progression and consequent modification of therapeutic approach. However, more studies are needed to determine whether incorporation of ctDNA analysis into clinical practice would impact patient outcomes.

#1576

Clonal diversity revealed by morphoproteomic and copy number profiles of single prostate cancer cells at diagnosis.

Paymaneh D. Malihi,1 Michael Morikado,1 Lisa Welter,1 Anders Carlsson,1 Carmen Ruiz Velasco,1 Anand Kolatkar,1 Mariam Rodriguez-Lee,1 James Hicks,1 Peter Kuhn,1 Sandy T. Liu,2 Eric T. Miller,2 Radu M. Cadaneanu,2 Beatrice S. Knudsen,2 Michael S. Lewis,2 Isla P. Garraway2. 1 _University of Southern California, Los Angeles, CA;_ 2 _University of California, Los Angeles, Los Angeles, CA_.

Tumor heterogeneity is prevalent in both treatment-naïve and end-stage metastatic castration-resistant prostate cancer (PCa), and may contribute to the broad range of clinical presentation, treatment response, and disease progression. To characterize molecular heterogeneity associated with de novo metastatic PCa, multiplatform single cell profiling was performed using High Definition Single Cell Analysis (HD-SCA). HD-SCA enabled morphoproteomic and morphogenomic profiling of single cells from touch preparations of tissue cores (prostate and bone marrow biopsies) as well as liquid samples (peripheral blood and bone marrow aspirate). Morphology, nuclear features, copy number alterations, and protein expression were analyzed. Tumor cells isolated from prostate tissue touch preparation (PTTP) and bone marrow touch preparation (BMTP) as well as metastatic tumor cells (MTCs) isolated from bone marrow aspirate were characterized by morphology and cytokeratin expression. Although peripheral blood was examined, circulating tumor cells were not definitively observed. Targeted proteomics of PTTP, BMTP, and MTCs revealed cell lineage and luminal prostate epithelial differentiation associated with PCa, including co-expression of EpCAM, PSA, and PSMA. Androgen receptor expression was highest in MTCs. Hallmark PCa copy number alterations, including PTEN and ETV6 deletions and NCOA2 amplification, were observed in cells within the primary tumor and bone marrow biopsy samples. Genomic landscape of MTCs revealed to be a mix of both primary and bone metastatic tissue. This multiplatform analysis of single cells reveals several clonal origins of metastatic PCa in a newly diagnosed, untreated patient with polymetastatic disease. This case demonstrates that real-time molecular profiling of cells collected through prostate and bone marrow biopsies is feasible and has the potential to elucidate the origin and evolution of metastatic tumor cells. Altogether, biological and genomic data obtained through longitudinal biopsies can be used to reveal the properties of PCa and can impact clinical management.

#1577

Correlation of exosomes and CTCs in non-small cell lung cancer patients with disease stage.

Zujun Que,1 Yi Jiang,1 Fangfang Qian,1 Zhi Qiao,2 Hua Xiao,3 Lingshuang Liu,4 Hegen Li,4 Jianhui Tian1. 1 _Oncology Inst. of Traditional Chinese Medicine, Shanghai, China;_ 2 _Shanghai Jiao Tong University, Shanghai, China;_ 3 _School of Life Science and Biotechnology, Shanghai Jiao Tong University, Shanghai, China;_ 4 _Longhua Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai, China_.

Lung cancer is the most common cancer, which also leading cause of global cancer mortality. Metastasis is a complex multi-step process that represents the most deadly aspect of cancer. One key step in metastasis is the entry of circulating tumor cells (CTCs) into secondary or distant organ sites to become disseminated tumor cells for subsequent metastasis. Exosomes, a subset of extracellular vesicles (EVs), function as a mode of intercellular communication and molecular transfer. Tumor cells are avid producers of exosomes, and tumour-derived exosomes uptaken by organ-specific cells prepare the pre-metastatic niche. The aim of this pilot study is to investigate the relationship between the count of circulating tumor cells in peripheral blood and the clinical stage of lung cancer, and the difference between normal (healthy) person exosomes and cancer patients exosomes in the rate of exosomes release and the content (most notably proteins). A total of 140 patients participated in the project and twelve blood samples from healthy volunteers were also collected in the Longhua Hospital Affiliated to Shanghai University of Traditional Chinese Medicine. After obtaining the informed consent, blood samples (5 ml) were stored in the ACD anticoagulant tube. CTCs was counted by immunomagnetic beads negative enrichment technique combined with immunofluorescence staining of chromosome in situ hybridization. The correlation between the count of CTCs and the clinical stage was analyzed. Exosomes were isolated by Density Gradient (DG) centrifugation. Exosomes concentration and size was analyzed by Nanoparticle Tracking Analysis (NTA), and protein was analyzed by HPLC-CHIP-MS/MS. In 114 patients, the positive rate of CTCs was 93.86% (in 3.2mL blood CTCs≥1, 107/114). The count of CTCs was negatively correlated with clinical stage (rs=-0.279, p=0.003), which suggest that CTCs is highly expressed in early lung cancer patients. Exosomes test results showed that there was a significant difference between lung cancer patients and healthy person. The size of exosome in healthy person was significantly smaller and the concentration was significantly higher when compared with lung cancer patients (p<0.05). In addition, the mass spectrometry results showed that the control group had 135 proteins in the exosomes, while the patient group contained 167

proteins, which 89 proteins were shared with each other. These results suggest that CTCs plays an important role in the pathogenesis of lung cancer and should be a target for early postoperative metastasis. The analysis of exosomes in NSCLC could represent a noninvasive test for assess disease progression.

#1578

NanoVelcro CTC Purification Systems for expressional analysis of circulating tumor cells from prostate cancer patients.

Yu Jen Jan,1 Jie-Fu Chen,1 Sungyong You,1 Junhee Yoon,1 Nu Yao,1 Shirley Cheng,1 Michael R. Freeman,1 Hsian-Rong Tseng,2 Edwin M. Posadas1. 1 _Cedars-Sinai Medical Center, Los Angeles, CA;_ 2 _University of California, Los Angeles, Los Angeles, CA_.

Background: Circulating tumor cells (CTCs) are blood borne tumor cells shed from all present disease sites, including primary and metastatic tumors. Researchers have been exploring the use of CTCs to identify important transcriptomic features such as androgen receptor splicing variants in prostate cancer (PCa). Newer transcriptomic profiles such as the prostate cancer classification system (PCS) have been developed that may be useful clinically. Over the past decade, we have been developing the NanoVelcro CTC purification system which includes 3 different stimuli-responsive strategies (temperature-dependent, glycan-stimulated, click chemistry-mediated) to capture and release viable CTCs with intact RNA. The purified CTCs can then be subjected to transcriptomic analysis such as quantitative PCR (qPCR), Droplet Digital PCR (ddPCR) and NanoString's nCounter system. In this study, we benchmarked the efficiency of these platforms for purification of CTCs from blood specimens and the feasibility for detection of PCa-related RNA signatures from purified CTCs.

Methods: NanoVelcro CTC purification system is combined with qPCR and ddPCR platform to detect PCa-specific RNA targets such as full length AR (AR-FL), AR splicing variant 7 (AR-V7), KLK3 (prostate-specific antigen, PSA), FOLH1 (prostate-specific membrane antigen, PSMA), and long-noncoding RNA SChLAP1 (second chromosome locus associated with prostate-1) in purified CTCs from patients' blood samples. We also developed a modified PCS panel of 30 genes adapted to the nCounter platform and tested purified CTCs from PCa patient blood samples and performed differential expression analysis for PCa disease profiling.

Results: PCa-related RNA signals were detected in 16/17 CTC(+) PCa patients, including 3 of 4 non-metastatic patients. We also tested the gene expressions of the modified PCS panel using 32 patient blood samples. Upon performing clustering of samples based on the RNA signatures, we found the differential expression of CTC-target genes grouped patients in a manner which strongly related to drug response and clinical status.

Conclusions: With the evolution of our NanoVelcro CTC purification system, we have shown the capability of purifying CTCs with high efficiency, while retaining cell viability and molecular integrity, allowing for detection of PCa-specific RNA signatures from CTCs. Our non-invasive, blood-based assay will be useful for detecting and continuous monitoring molecular alterations related to disease evolvement, addressing an unmet need for prostate cancer clinical care.

#1579

IMI's CANCER-ID: Implications of liquid biopsy technology standardization for its clinical use.

Klaus Pantel,1 Leon W. Terstappen,2 Barbara Baggiani,3 Thomas Krahn,4 Catherine Panabières,5 Evi Lianidou,6 Bjørn Naume,7 Françoise Farace,8 Thomas Schlange4. 1 _University Cancer Center Hamburg/Eppendorf, Hamburg, Germany;_ 2 _University Twente, Enschede, Netherlands;_ 3 _Menarini/Silicon Biosystems, Bologna, Italy;_ 4 _Bayer AG, Wuppertal, Germany;_ 5 _University Medical Centre of Montpellier, Montpellier, France;_ 6 _National and Kapodistrian University of Athens, Athens, Greece;_ 7 _University of Oslo, Oslo, Norway;_ 8 _Institute Gustave Roussy, Villejuif, France_.

Liquid biopsies offer the opportunity to improve the diagnosis and timely adaption of therapy options to the progression and heterogeneity of cancer. The CANCER-ID (www.cancer-id.eu) consortium was established in early 2015 as part of the Innovative Medicines Initiative (IMI) and has currently 37 partners from 14 countries, including the USA and Singapore. The goal of the CANCER-ID program is to foster implementation of liquid biopsy technologies in multicenter clinical studies and the potential regulatory approval of devices or protocols in the field. Within the project best practice protocols for using technologies aiming at the enrichment, isolation and analysis of Circulating Tumor Cells (CTCs), circulating free tumor DNA (ctDNA) and microRNAs (miRNAs) are being developed and benchmarking data for the performance of different technologies are collected. To this end defined standards for the different biomarker classes are crucial and their introduction and design is being supported by CANCER-ID. Another integral part of our work is the development of integrated workflows from patient sample collection, pre-analytical sample handling, sample and bioinformatics analyses down to the actionable information guiding patient selection and personalized treatment.

CANCER-ID partners are currently planning clinical studies to assess the clinical utility of analyzing PD-L1 expression on CTCs in their core indications Non-Small Cell Lung Cancer (NSCLC) and metastatic breast cancer. While the benefit of immune checkpoint inhibition has been impressively demonstrated in NSCLC, most advanced breast cancer patients do not benefit from targeting PD-1 with the exception of the triple-negative disease type. The liquid biopsy workflows established by the CANCER-ID consortium will be used to correlate CTC PD-L1 expression in patients treated with PD-1 blocking antibodies with clinical response and in parallel perform mutational load analysis in blood. The aim will be to attempt to develop a liquid biopsy-based rationale for patient selection guiding effective immune checkpoint therapies. The study plan and current status of this multicenter effort will be presented.

This work is supported by IMI JU & EFPIA (grand no. 115749, CANCER-ID). Samples from patients and healthy volunteers, respectively, were collected under signed informed consent.

#1580

Separation of circulating biomarkers by nanoDLD lab-on-a-chip technology.

Stacey M. Gifford, Benjamin H. Wunsch, Joshua T. Smith, Navneet Dogra, Sungcheol Kim, Gustavo Stolovitzky. _IBM Research, Yorktown Heights, NY_.

Efficient isolation of circulating biomarkers is key for enabling liquid biopsies for cancer diagnosis and prognosis. Circulating biomarkers such as exosomes and cell-free DNA hold promise for non-invasive early cancer diagnosis, determining cancer stage and prognosis, as well as monitoring treatment progression and the development of drug resistance. However, isolation of these biomarkers has proven challenging in the clinic. Exosomes range in size from approximately 30 to 150 nm and the current gold standard isolation method includes large-scale ultracentrifugation, which is time-consuming and lacks reproducibility. Circulating cell-free DNA is found in a variety of sizes and states and while clinical applications for prenatal testing have emerged, applications in oncology remain elusive in part due to high background and challenges in isolating relevant molecules. We have developed a lab-on-a-chip technology based on deterministic lateral displacement at the nanoscale (nanoDLD) which separates and concentrates particles as small as 20 nm from smaller particles in continuous flow. Analysis of nanoDLD isolation of exosomes and DNA show improved resolution, recovery, and concentration compared to standard techniques. Current efforts to scale nanoDLD technology to clinically relevant volumes will provide a reproducible and automated tool for isolation, study, and clinical use of these important non-invasive biomarkers.

#1581

Circulating cell-free DNA for metastatic cervical cancer detection, genotyping and monitoring.

Zhigang Kang, Sanja Stevanović, Christian Hinrichs, Laing Cao. _National Cancer Inst., Bethesda, MD_.

Purpose: Circulating cell-free (ccf) human papillomavirus (HPV) DNA is a unique tumor marker for metastatic cervical cancers. We developed a method to genotype and quantify circulating HPV DNA in patients with HPV16- or HPV18-positive metastatic cervical cancer for disease monitoring and treatment-related decision making.

Patients and Methods: In this retrospective study, HPV ccfDNA was measured in serum samples from metastatic cervical cancer patients by duplex digital droplet (dd) PCR. Nine patients had received tumor-infiltrating lymphocyte (TIL) immunotherapy. ccfDNA data were aligned with the tumor HPV data, drug treatment, and clinical outcome.

Results: In blinded tests, HPV ccfDNA was detected in 19/19 (100%) patients with HPV-positive metastatic cervical cancer but not in any of the 45 healthy blood donors. The HPV genotype harbored in the patients' tumors was correctly identified in 87/87 (100%) sequential patient serum samples. In three patients who experienced objective responses after TIL immunotherapy, a transient HPV ccfDNA peak was detected 2-3 days after TIL infusion. Furthermore, a persistent clearance of HPV ccfDNA was observed in only two patients with a complete response (CR) after TIL immunotherapy.

Conclusions: HPV ccfDNA represents a promising tumor marker for non-invasive HPV genotyping and may be used in selecting patients for HPV type-specific T-cell-based immunotherapies. It may also have value in detecting the anti-tumor activities of therapeutic agents and in the long-term follow-up of cervical cancer patients in remission.

#1582

The coupling of MDSCs with a computational neural network (NN) to detect solid tumors.

George A. Dominguez,1 Kristen Maslar,1 Alexander Polo,2 Cyrus Sholevar,1 Anthony Campisi,1 John Roop,1 Dmitry Gabrilovich,2 Frank Rauscher,2 Amit Kumar1. 1 _ITUS Corporation, CA;_ 2 _The Wistar Institute, Philadelphia, PA_.

The goal of this study was to create a non-invasive cancer detection assay that analyzes flow cytometry data in an objective method. By using an artificial neural network (NN), we were able to distinguish between cancer patients (CPs) and benign/healthy donors (HDs) based upon the flow cytometry profiles of MDSCs and other leukocytes. Myeloid-derived suppressor cells (MDSCs) are known to be key contributors in supporting tumor progression and tumor escape through their ability to suppress anti-tumor responses mediated through T cell and natural killer (NK) cell activity. Several studies have demonstrated their utility as indicators of tumor progression and possible predictors of clinical outcomes, but there is significant overlap with healthy individuals preventing discrete and accurate calls. We used standard multiparametric flow cytometry techniques to immunophenotype MDSCs and other leukocytes found in the peripheral blood of 65 biopsy-confirmed CPs with solid tumors and 84 HDs. A series of NNs utilizing pattern recognition computational algorithms are then created using three data sets: 1) the training set - this 'teaches' the two output categories of cancer and not cancer, 2) the validation set - this uses backpropagation to improve the accuracy of the trained networks, and 3) the testing set - this is used to rank the trained networks against each other. Finally, a naïve testing set is then used to determine the overall sensitivity and specificity for the top-ranking networks. Using traditional flow cytometry gating methods to analyze MDSCs as a biomarker for cancer detection, it is difficult to achieve high sensitivity and specificity due to the substantial overlap with healthy individuals. Here, we incorporated a standard 12 marker flow cytometry assay with NN technology to achieve a sensitivity of 92% and a specificity of 89%. Pairing the advanced analytical capabilities of our NN with surface biomarker based analysis of MDSCs and certain leukocytes measured in peripheral blood has enabled us the ability to objectively identify patterns indicative for the existence of a solid tumor.

#1583

**Clinical significance of** PIK3CA **,** AKT1 **, and** ESR1 **mutation in plasma cell-free DNA from estrogen receptor-positive breast cancer patients.**

Takashi Takeshita,1 Mai Tomiguchi,1 Aiko Sueta,2 Mutsuko Yamamoto-Ibusuki,1 Yutaka Yamamoto,2 Hirotaka Iwase2. 1 _Kumamoto University Hospital, Kumamoto, Japan;_ 2 _Kumamoto University, Kumamoto, Japan_.

Background: In endocrine therapy (ET) resistance mechanisms, PI3K / AKT pathway abnormality and ESR1 mutation are drawing attention. In recent large-scale clinical trials, it was revealed that identification of these genetic abnormalities in cell-free DNA (cfDNA) was useful for rapid assessment and monitoring of the therapeutic effect in ET-resistant breast cancer (BC). However, the frequency of them per treatment line and its clinical significance have not been verified. Here we studied the clinical significance of PIK3CA, AKT1, and ESR1 mutation according to the treatment line in estrogen receptor (ER)-positive BC.

METHODS: From 2003 to 2017, a total of 251 plasma specimens were collected from 128 patients with ER positive BC treated at our hospital. The breakdown were 133 plasma samples from 73 primary BC (PBC) patients and 118 plasma samples from 68 metastatic BC (MBC) patients. CfDNA was extracted from 500 μL of plasma. The hotspot of PIK3CA, AKT1, and ESR1 mutation in plasma cfDNA was verified using multiplex digital PCR method.

RESULTS: In the PBC patient group, PIK3CA mutation was recognized at 15.1%, AKT1 mutation at 1.4%, and ESR1 mutation at 2.7%. The presence or absence of PIK3CA mutation did not affect clinical outcome. In the MBC patient group, the frequency of PIK3CA mutation increased from 16% to 32% and that of ESR1 mutation increased from 23% to 41.9% as the treatment line advanced. Furthermore, we examined the time to treatment failure (TTF) by dividing into early treatment line and late treatment line. In the early treatment group, patients with PIK3CA mutation had significantly shorter TTF (P = 0.035). However, the presence or absence of ESR1 mutation did not affect TTF. On the other hand, in the late treatment group, patients with ESR1 mutation had significantly shorter TTF (P = 0.048). However, the presence or absence of PIK3CA mutation did not affect TTF. Since AKT1 mutation was rare in both PBC patients and MBC patients, its clinical significance was unknown.

CONCLUSION: We showed clinical significance of verification of PIK3CA, AKT1, and ESR1 mutation in cfDNA according to treatment line in ER positive BC patients.

#1584

Comparison of circulating tumor cell (CTC) capture/identification methods and NanoString evaluation of gene expression in CTCs and cell-free circulating tumor mRNA (cctmRNA) in patients with metastatic lung cancer.

Yanis Boumber,1 Charu Aggarwal,2 Catherine Thrash-Bingham,1 Patricia Fittipaldi,1 Polina Guseva,3 Ramillya Vlasenkova,3 Chandra Rao,4 Jianming Pei,1 Brian L. Egleston,1 Hossein Borghaei,1 Massimo Cristofanilli,5 Ranee Mehra,6 Ilya Serebriiskii,1 R. Katherine Alpaugh1. 1 _Fox Chase Cancer Center, Philadelphia, PA;_ 2 _University of Pennsylvania, Philadelphia, PA;_ 3 _Kazan Federal University, Kazan, Russian Federation;_ 4 _Janssen Diagnostics LLC, Huntingdon Valley, PA;_ 5 _Robert H. Lurie Cancer Center of Northwestern University, Chicago, IL;_ 6 _Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, MD_.

Circulating tumor cells (CTC) and cell free circulating tumor mRNA (cctmRNA) can serve as biomarkers for prognosis and treatment response in lung and other cancers. A barrier to CTC use in some cancers is the low numbers of CTC captured, and the low percent of patients with detectable CTCs. Past comparisons of non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) patients have shown larger cohorts of patients with detectable CTCs, and higher CTC numbers, for SCLCs. We compared methods for CTC capture/identification, and profiled mRNA from both the CTCs and cell free plasma RNA (cctmRNA) for to identify tumor-associated biomarkers. Peripheral blood was collected from 10 SCLC and 10 NSCLC patients prior to therapy, and from 4 normal controls. Capture using the standard EpCAM CellSearch reagent (unicapture) was run in parallel with EpCAM plus Her2, EGFR and MUC-1 combined ferrofluid (quadcapture) plus the use of anti-cytokeratin 7 (CK7) to assist in CTC identification. Unicapture detected CTCs in 40% of NSCLC and 60% of SCLC; whereas, quadcapture/CK7 identified 20% CTCs in NSCLC and 80% in SCLC. Overall, the quadcapture did not improve CTC capture efficiency. Exploratory NanoString analysis was performed on cell-free plasma from these patients and the ferrofluid isolated CTCs. Cancer-specific biomarkers were detected in mRNA from plasma and CTCs by NanoString included CCL5, CLU and SRGN. In summary, quadcapture ferrofluid reagent did not improve the capture efficiency, although the addition of CK7 may have the potential to identify additional CTCs. Nanostring analysis of cctmRNA and CTCs mRNA suggested potential new lung cancer biomarkers that require validation in larger studies.

#1585

Evaluation of circulating free DNA in non-metastatic breast cancer.

Jhajaira M. Araujo,1 Jaime Ponce,1 Alexis Murillo,2 Pamela Rebaza,1 Pierina Danos,2 Alfredo Aguilar,1 Ricardo Fujita,2 Henry L. Gomez,1 Joseph A. Pinto,1 Jose Buleje2. 1 _Oncosalud-AUNA, Lima, Peru;_ 2 _Universidad de San Martín de Porres, Lima, Peru_.

Background Circulating free DNA (cfDNA) is becoming an important tool in diagnosis and prognosis in several types of cancer. In this study we evaluated the association between cfDNA concentrations in peripheral blood with tumoral features of non-metastatic breast cancer patients.

Methods cfDNA was extracted from plasma of peripheral blood of patients with non-metastatic breast cancer and healthy controls. In patients, blood samples were collected before and after surgery, while in controls samples were collected after their results of breast cancer screening. Detection of copy number of PUM1 and RNaseP genes were performed in a QuantStudio® 3D Digital PCR System and quantification was obtained with QuantStudio® 3D AnalysisSuite™ Cloud Software. Raw values were compared among the different clinicopathologic features.

Results In total 26 patients and 25 controls were included. There were significant differences between controls and patients for levels counts of PUM1 and RNaseP genes (p<0.0001 in both cases). In patients, median concentrations before and after surgery for PUM1 were 10.6 copies/uL (range: 2.2-16.3) vs. 7.6 copies/uL (range: 1.3-12.8), respectively (P=0.04) and for RNaseP were 6.6 copies/uL (range: 0.9-20.0) vs. 5.3 copies/uL (range: 0.5-16.0), respectively (P=0.103). Hormonal tumors produced more cfDNA in terms of RNaseP at diagnosis (7.9 vs. 5.6, P=0.049). When we compared patients according to their hormonal status with controls, RNaseP was able to identified positive hormonal receptors patients (P<0.0001). There were no significant differences between other clinicopathologic features. There was a significant decrease after surgery of RNaseP levels in node negative tumors (P=0.049) and PUM1 levels in >10mm tumors (P=0.041).

Conclusions Our results suggest that PUM1 is a better diagnostic biomarker for non-metastatic breast cancer because RNaseP loss sensitivity in negative hormone receptor cases. Hormonal tumors produced more circulating free DNA. RNaseP could be used to monitor node negative patients and PUM1 to patients with >10mm tumors.

#1586

Evaluation of AXL expression on circulating tumor cells from EGFR mutated lung cancer patients who have relapsed after the EGFR TKI treatment.

Young Hun Kim,1 Myoung Shin Kim,1 Jun Sup Lee,1 Hyun Kyung Lee,1 Jae Hyuk Lee,1 Young Woong Sohn,1 Koichi Tazaki,2 Kenji Nakamaru,2 Kenichi Wakita,2 Byung Hee Jeon,1 Seokhyung Kim,3 Se-Hoon Lee3. 1 _Cytogen, Inc., Seoul, Republic of Korea;_ 2 _Daiichi Sankyo Co., Ltd, Tokyo, Japan;_ 3 _Samsung Medical Center, Seoul, Republic of Korea_.

Background: The EGFR (epidermal growth factor receptor) TKI (tyrosine kinase inhibitor) has become the standard treatment in lung cancer patients with EGFR mutations. When these patients are treated with EGFR TKI, 80~90% of the patients show responses to the drug. However the tumor begins to progress again following the development of resistance about 1 year later on average. About a half of resistance mechanisms are caused by the additional mutation of the EGFR gene (T790M), and the other half of the resistance mechanisms are caused by various mechanisms, with one of them being the overexpression of the AXL protein. Confirming AXL overexpression in circulating tumor cells (CTCs) can be an alternative method for tissue biopsy, but almost no research has been conducted on this so far. Here, we evaluate the AXL expression in CTC in case resistance occurs after the EGFR inhibitor treatment.

Materials and Methods: The blood samples (10 ml) were collected from 10 EGFR TKI treated and relapsed lung cancer patients (TKI group) and 10 non-treated patients (control group). The blood samples were processed through Cytogen protocol to enrich CTCs. The enriched cells were immunofluorescent stained for CTC markers (EpCAM or Vimentin), AXL and WBC marker (CD45). The immunofluorescent stained cells were analyzed for each markers using Image Analysis program.

Result: CTCs were detected by CTC markers in all patients from TKI group (range 0-210) and control group (range 1-42). AXL positive CTCs were detected in 9 out of 10 cases from TKI group and in all cases from control group. Interestingly, the quantification of AXL staining intensity in CTCs showed that AXL overexpression (3 fold or greater than baseline) was in 50% of TKI group and 20 % of control group.

Conclusion: We have tested a new approach to evaluate the AXL overexpression in tumor cells of EGFR TKI resistant patients, and showed the feasibility of the this method. Most of lung cancer patients who undergo biopsy require hospitalization and some degree of complication due to biopsy is inevitable. Here we suggested that the blood-based method as a good alternative to the tissue biopsy in patients with tolerance.

#1587

Detection of PD-L1 and PD-1 positive circulating tumor cells (CTCs) in non-small cell lung cancer (NSCLC) patients treated with nivolumab.

Galatea Kallergi,1 Anastasios Koutsopoulos,1 Eleni Lagoudaki,1 Despoina Agouraki,1 Eleni-Kiriaki Vetsika,1 Anastasia Voumvouraki,1 Stuart S. Martin,2 Filippos Koinis,1 Christos Stournaras,1 Vassilis Georgoulias,1 Athanasios Kotsakis1. 1 _Univ. of Crete, Medical School, Heraklion, Greece;_ 2 _University of Maryland, School of Medicine, Marlene and Stewart Greenebaum Cancer Center, Baltimore, MD_.

Introduction: Circulating tumor cells (CTCs) are considered as a "liquid biopsy" that allows the assessment of tumor changes over time. Tumor cells may escape from the immune system through the activation of PD-1/PD-L1 axis. Targeting these molecules with monoclonal antibodies has shown encouraging results at many types of cancers, including NSCLC. In the current study we investigated the expression of PD-1/PD-L1 molecules on the CTCs isolated from NSCLC patients treated with Nivolumab.

Methods: CTCs were isolated based on their size using the ISET platform from 27 patients before treatment, after 1 cycle and 3 cycles. CTCs were detected with Giemsa staining and immunofluorescence (IF) experiments, using either pancytokeratin (A45-B/B3) (CK7)/PD-1/CD45 or (A45-B/B3)(CK7)/PD-L1/CD45 combination of antibodies and analysis with the ARIOL system. Spiking experiments using the NSCLC cell lines: H460, H1299, HCC827 and SKMES in normal blood were used to evaluate the detection method.

Results: Giemsa evaluation in Nivolumab-treated patients at baseline (25 evaluable samples), after the 1st (9 evaluable samples) and the 3rd (8 evaluable patients) cycle of treatment showed that CTCs could be detected in 48% (12/25), 33.3% (3/9) and 50% (4/8) of patients, respectively. IF could also reveal the presence of CK-positive cells in 44.4% (12/27), 22% (2/9) and 75% (6/8) patients, respectively. PD-1 (+) CTCs were detected in 33.3% (4/12) of patients at baseline, in, 0% after the 1 and 16.7% (1/6) of patients after the 3rd cycle. The same percentages were identified for PD-L1 expression in the same cohort of patients. The expression of PD-1 at baseline was associated with poorer OS (p=0.022) and PFS (p=0.011), while the expression of PD-L1 was associated with shorter PFS (p=0.011). Multivariate analysis revealed that the presence of CK-positive cells is an independent prognostic factor for OS (p=0.028)

Conclusion: Nivolumab reduced the number of PD-1- and PD-L1-expressing CTCs in advanced NSCLC patients. Furthermore the expression of both markers at baseline is associated with the clinical outcome.

#1588

Liquid biopsy analyses of cell-free circulating tumor DNA as predictive and prognostic biomarker for colorectal cancer patients with metastatic disease.

Iris van 't Erve,1 Jillian Phallen,2 Karen Bolhuis,3 Joost Huiskens,3 Nicole C. van Grieken,4 Veerle Coupé,4 Annegien Broeks,1 Daan van den Broek,1 Alessandro Leal,2 Victor E. Velculescu,2 Cornelis J. Punt,3 Gerrit A. Meijer,1 Remond J. Fijneman1. 1 _Netherlands Cancer Institute, Amsterdam, Netherlands;_ 2 _Johns Hopkins Kimmel Cancer Center, Baltimore, MD;_ 3 _Academic Medical Center (AMC), Amsterdam, Netherlands;_ 4 _VU University Medical Center, Amsterdam, Netherlands_.

Background: Most colorectal cancer (CRC) deaths are caused by metastatic disease (mCRC). Therapeutic approaches include treatment with monoclonal antibodies (mAbs) against EGFR and VEGF. There is an urgent clinical need to stratify mCRC patients for optimal treatment. Cell-free circulating tumor DNA (ctDNA) derived from blood plasma is expected to improve stratification by early detection of therapy resistance and disease progression.

Aim: The general aim of this study is to advance towards clinical implementation of ctDNA-based tests as molecular biomarkers to improve disease management of mCRC patients. We will investigate added value of liquid biopsy ctDNA-based gene mutation analyses compared to: 1) tissue-based tests for RAS mutation status as determined in standard clinical care setting; and 2) monitoring of disease progression by computed tomography (CT) imaging.

Methods: CAIRO5 is a multicenter, randomized, phase 3 clinical trial of the Dutch Colorectal Cancer Group (DCCG) and includes patients with initially unresectable, liver-only mCRC, as confirmed by a central panel of liver surgeons/radiologists based on CT imaging. This study involves nation-wide longitudinal collection of liquid biopsies (blood samples) using cell-save tubes and CT imaging from up to 564 patients. Hotspot mutations in ctDNA will be analyzed by droplet digital PCR (ddPCR), and mutations in a panel of genes by targeted error correction sequencing (TEC-seq). Clinical, imaging, biobanking, and molecular data will be collected using standardized data fields and data formats and integrated for querying and viewing in tranSMART, making use of the national Health-RI research IT infrastructure.

Results: The nation-wide multi-center logistics for longitudinal blood sample collection and plasma processing has been established, with participation of more than 40 Dutch hospitals. At present (Nov 2017), over 220 patients have been included from whom more than 550 blood samples and 330 CT images were obtained. Proof of concept for the validity of this workflow was obtained by successful subjection of 11 plasma samples to ctDNA mutation analysis by TEC-seq (Phallen et al., 2017).

Discussion: Implementation of ctDNA-based tests as molecular biomarkers to improve disease management of mCRC patients requires collection of information from large, well-defined studies with longitudinal patient follow-up. This translational research project will provide the data that are needed to determine cost-effectiveness analysis of ctDNA mutation analyses by health technology assessment, yielding recommendations for clinical implementation of ctDNA applications.

#1589

**Presence of PD-L1** + **tumor cells in tumor tissue and blood significantly associated with T/N stage and lymphovascular invasion in colorectal cancer patients.**

Ju-Yu Tseng,1 Chwen-Cheng Chen,2 Yen-Ru Chen,1 Chia-Ying Lee,1 Chun-Chi Lin,3 Shin-Hang Wang,1 Tzu-Chao Hung,1 Hong-Ling Wang,1 Yi Chung,1 Yen-Lun Tseng,1 Mu-Yi Chen,1 Jeng-Kai Jiang3. 1 _MiCareo, Taipei, Taiwan;_ 2 _JN Biopharma Consulting, Taipei, Taiwan;_ 3 _Taipei Venture General Hospital, Taipei, Taiwan_.

Background:

PD-L1 assessment on tumor tissue is considered as an important predictive marker for anti-PD-1 treatments and has been approved by FDA as companion diagnostics for immunocheckpoint antagonists. Nonetheless, it remains challenging because of the dynamic nature and heterogeneity of PD-L1 expression, and the availability of tumor tissue. Measurement of circulating tumor cells (CTC) serves a form of liquid biopsy and tumor biomarker due to its non-invasiveness and real-time assessment. Here, we analyzed and compared PD-L1 expression in CTCs and tumor cells harvested from surgical specimen from colorectal cancer (CRC) patients.

Methods:

Mesenteric vein blood (MVB) samples were collected into K2EDTA-containing vacationer tubes and subsequently performed CTC enumeration and PD-L1 analysis by MiSelect R system. Tumor tissues were homogenized via collagenase enzyme digestion to single cell suspension before subjected to MiSelect R analysis. Tumor cells and CTCs are defined as EpCAM+, cytokeratin\+ and CD45\- with intact nuclei. PD-L1 analysis was performed by anti-PD-L1 primary antibody (clone 28-8). Tumor cells, CTCs and PD-L1+ tumor cells were counted and compared among different clinicopathological parameters.

Results:

MVB sample were collected intraoperatively from 116 CRC patients across various stages (stage I: 24, II: 38, III:42 and IV:12). CTC presence correlated with T stage and CEA level (P = 0.018 and 0.049, respectively); furthermore, PD-L1+ CTC presence significantly correlated with clinical stage, N stage, microscopic blood vascular and lymphatic vascular invasion (P = 0.0017, 0.013, 0.014 and 0.004, respectively). The percentage of PD-L1+ tumor cells showed positive correlation between tumor tissue and CTC, and both correlated with tumor stage.

Conclusion:

PD-L1+ cell was detectable in both tumor tissue and CTC in CRC patients and its percentage increased as disease progressed, which might reflect escape of tumor cell from immune surveillance. The percentage of PD-L1 expression on CTCs correlated with that in paired tumor tissues. On the other hand, the expression of PD-L1 on CTC was significantly associated with blood and lymphatic vessel invasion, which might be one of the potential mechanisms for local and distant metastasis of cancer. Further investigation is warranted to better understand the clinical significances of PD-L1-expression on CTCs and its potential utility as prognostic biomarker for

cancer immunotherapy.

#1590

Personalized circulating tumor DNA analysis to monitor colorectal cancer.

Thomas Reinert,1 Tenna V. Henriksen,1 Mads H. Rasmussen,1 Himanshu Sethi,2 Shruti Sharma,2 Hsin-Ta Wu,2 Dina Hafez,2 Prashanthi Natarajan,1 Scott Dashner,1 Mustafa Balcioglu,1 Ann Nguyen,1 Derrick Renner,1 Bernhard Zimmermann,2 Lene H- Iversen,1 Mogens R. Madsen,3 Cheng-Ho Jimmy Lin,2 Claus L. Andersen1. 1 _Aarhus University Hospital, Aarhus, Denmark;_ 2 _Natera Inc, San Carlos, CA;_ 3 _Regional Hospital Herning, Herning, Denmark_.

Early detection of disease recurrence has shown to improve survival in patients with early-stage colorectal cancer (CRC) (Pita-Fernandez et al., 2015). Detection of circulating tumor DNA (ctDNA) postoperatively in patients with stage II colon cancer provides direct evidence of residual disease and identifies patients at high risk of recurrence (Tie et al., 2016). Previous studies have performed ctDNA analysis to monitor tumor burden in early-stage CRC using small gene panel sequencing or digital droplet PCR (ddPCR) assays to detect specific variants (Tie et al., 2016, Reinert et al., 2016). Building upon these previous studies, here we use a personalized multiplex ctDNA technology measuring 16 mutations specific to each patient's tumor respectively to assess minimal residual disease postoperatively and to monitor treatment response in CRC. Our study includes 130 patients diagnosed with stage I-III CRC treated with intended curative surgery. Of the 130 patients, 80 received adjuvant chemotherapy for a duration of six months. For 119 patients, one pre-operative and one post-operative plasma sample were available. In addition, longitudinally-collected plasma samples were available from 78 of 130 patients for early detection of disease recurrence and to determine their relationship with imaging and carcinoembryonic antigen (CEA) during a 3-year post-operative surveillance period.

To identify patient-specific tumor mutation signatures, whole-exome sequencing of CRC tissue and germline DNA is used. Patient specific multiplex-PCR assay panels are designed to target 16 tumor-specific single-nucleotide variants (SNVs) in plasma. Targeted sequencing is then performed on plasma samples collected pre- and post-operative and during adjuvant therapy. We then correlate ctDNA status to the clinical outcome, including CEA and radiographic imaging, for each patient. Data collection is ongoing and results will be presented at the AACR 2018 meeting.

Conclusion. This study provides a novel methodology to detect minimal residual disease postoperatively and to monitor treatment efficacy in CRC using a personalized multiplex-PCR approach. The performance of this patient-specific ctDNA technology will be compared to the current standard of care for monitoring disease burden.

References:

S. Pita-Fernàndez, et al. Ann. Oncol. 26, 644–656 (2015).

Tie J, et al. Sci Transl Med. 2016 Jul 6;8(346):346ra92.

Reinert T. Gut. 2016 Apr;65(4):625-34.

#1591

Cell-free DNA quantification reflects survival in non-small cell lung cancer patients with low metabolic tumor burden.

Myung Han Hyun, Eun Sung Lee, Jae Seon Eo, Jae Sook Sung, Eun Joo Kang, Yoon Jee Choi, Kyong Hwa Park, Sang Won Shin, Sung Yong Lee, Yeul Hong Kim. _Korea Univ. Hospital, Seoul, Republic of Korea_.

We aimed to assess the role of tumor biological behavior/aggressiveness measured by cell-free DNA (cfDNA) quantification according to metabolic tumor burden measured by FDG-PET/CT scan to predict survival of advanced non-small cell lung cancer (NSCLC). Newly-diagnosed eighty-two NSCLC patients with baseline cfDNA quantification and positron emission tomography/computed tomography (PET/CT) scan were prospectively enrolled. The cfDNA level was quantified by Agilent High Sensitivity DNA kit. Metabolic tumor burden was measured by metabolic tumor volume (MTV) and total lesion glycolysis (TLG) from PET/CT scan. The optimal cut-off value for cfDNA quantification, MTV, and TLG were found using X-tile analysis. The high-cfDNA quantification is associated with poor overall survival (OS) (hazard ratio=2.71, 95% confidential interval=1.33-5.52; p=0.006) than that of low. After categorized by metabolic tumor burden, however, the patients with high-MTV showed poor prognosis regardless of cfDNA quantification (low-cfDNA quantification vs high-cfDNA quantification, median OS = 3.9 months vs 7.0 months; p value > 0.05). On the other hands, the patients with high-cfDNA quantification showed poor prognosis in patients with low-MTV (low-cfDNA quantification vs high-cfDNA quantification, median OS = 52.8 months vs 22.9 months; p values < 0.001). According to baseline TLG level, the role of cfDNA quantification to predict survival was similar to MTV. Although cfDNA quantification independent prognostic factors for OS, the significance was disappeared when adjusted by MTV or TLG. This finding provides novel insights that the cfDNA quantification reflect survival for selected patients with low metabolic tumor burden in NSCLC.

#1592

Bladder cancer circulating tumor cell concentration fluctuates in the absence of systemic therapy or surgery.

Stephanie A. Glavaris, Heather J. Chalfin, Megan H. Fong, Eric Christenson, A. Karim Ahmed, Emily A. Caruso, John Shin, Michael Johnson, Liang Dong, Michael A. Gorin, Trinity J. Bivalacqua, Kenneth J. Pienta, Noah Hahn, David McConkey. _Johns Hopkins University, Baltimore, MD_.

Background: "Liquid biopsies" such as circulating tumor cells (CTCs) aim to monitor response to therapy or provide a noninvasive measure of disease progression. In bladder cancer, it is not known to what extent CTC counts fluctuate over time during the same treatment phase (in the absence of systemic therapy or surgery). Understanding the baseline variation in CTC counts is a crucial step towards defining what constitutes a meaningful change in CTC detection. Here, we collected peripheral blood (PB) samples from metastatic bladder cancer patients with replicates from the same date and at multiple time points over different days.

Methods: 3 replicate PB samples (A, B, C) were collected on Day 1 from 5 metastatic bladder cancer patients. On Days 2 and 3, PB was collected from 80% (n=4/5) patients. The median CTC count of the 3 replicates from Day 1 was used. Each PB was processed with the AccuCyte® system to isolate and transfer the buffy coat onto 8 slides. Slides were stained with DAPI (nuclear), anti-pan-cytokeratin and anti-EpCAM (epithelial), and anti-CD45/CD66b/CD34/CD14/CD11b (white blood cell) antibodies. Slides were scanned and analyzed using the CyteFinder® system, an assay validated to have high recovery (greater than 80%) and sensitivity for detecting individual CTCs (DAPI positive, epithelial-marker positive, and white blood cell marker negative).

Results: For replicate samples, the range of CTC counts/7.5 mL PB for Patient 1 was 70 (A=43, B=102, C=32), Patient 2 was 5 (A=6, B=2, C=1), Patient 3 was 1 (A=1, B=0, C=0), Patient 4 was 2 (A=2, B=4, C=2), and Patient 5 was 1 (A=1, B=1, C=0). The median of the ranges (70, 5, 1, 2, 1) was 2 CTCs/7.5 mL PB. For PB collected on different days, the range of CTC counts for Patient 1 was 192 (43, 170, 235), Patient 2 was 11 (2, 13, 5), Patient 3 was 3 (0, 3, 3), and Patient 4 was 4 (2, 5, 6). The median of the ranges (192, 11, 3, 4) was 7 CTCs/7.5 mL PB.

Conclusion: While the median fluctuations of 2 and 7 CTCs/7.5 mL PB were low, in certain cases we did detect considerable variation in CTC count from sample to sample and from day to day. Ongoing work is necessary to study the natural fluctuation in CTC counts and the heterogeneity of blood CTC concentration from moment to moment. Given the many challenges of CTC detection, ctDNA may emerge as the preferred liquid biopsy in bladder cancer.

#1593

Multicenter evaluation of miRNA extraction technologies for the development of a clinically relevant miRNA analysis workflow.

Martin H. Neumann,1 Francesca Di Pasquale,2 Markus Sprenger-Haussels,2 Jonathan M. Shaffer,3 Martin Schlumpberger,2 Fay Betsou,4 Wim Ammerlaan,4 Taija af Hallström,5 Evi Lianidou,6 Robert Sjöback,7 Sebastian Bender,8 Thomas Schlange1. 1 _Bayer AG, Wuppertal, Germany;_ 2 _Qiagen GmbH, Hilden, Germany;_ 3 _Qiagen Sciences, Frederick, MD;_ 4 _IBBL, Dudelange, Luxembourg;_ 5 _Orion Oyi, Espoo, Finland;_ 6 _University of Athens, Athens, Greece;_ 7 _TATAA Biocenter, Gothenburg, Sweden;_ 8 _Bayer AG, Berlin, Germany_.

MicroRNAs (miRNAs) are small noncoding RNAs that interfere with and regulate different biological pathways. Therefore, dysregulation of miRNAs could lead to disease initiation and progression, particularly in various types of cancer. In human body fluids, miRNAs can be found as circulating cell-free miRNAs (cfmiRNAs, often associated with RNA-binding proteins) as well as exosomal miRNAs (exomiRNAs), both serving as potentially valuable biomarkers to evaluate patient's disease progression, or to monitor treatment efficacy. Various methods for the extraction and analysis of miRNAs, cfmiRNAs or exomiRNAs, are commercially available. Here, we describe the efforts of the Innovative Medicines Initiative (IMI) consortium CANCER-ID (www.cancer-id.eu), a public-private partnership of experts from academia, biotech and pharmaceutical industry, in generating comparative data using different RNA/miRNA extraction technologies in a multicenter ring study.

To address clinical relevance in a later stage of the project, K2EDTA plasma samples from 20 healthy donors were collected in a centralized way with respective informed consent. Five different RNA/miRNA extraction methods and two extraction methods specific for exomiRNAs were selected for comparison: mirVana (ThermoFisher Scientific), miRCURY RNA Isolation Kit (Exiqon), miRNeasy Serum/Plasma kit (QIAGEN), miRNeasy Advanced Serum/Plasma kit (QIAGEN), Plasma/Serum RNA Purification mini kit (Norgen), exoRNeasy Serum/Plasma (QIAGEN) and ultracentrifugation. The eluates were sent to one CANCER-ID partner, centrally performing RT-qPCR analysis on six miRNAs, as well as miRNA massive parallel sequencing.

After extraction using the miRCURY RNA isolation kit and miRNeasy Advanced Serum/Plasma kit, highest miRNA-seq calls (mean: 2x10^6) and lowest RT-qPCR Cq-values (mean: 26.6) for six miRNAs were observed with both extraction methods. Samples from patients and healthy volunteers, respectively, were collected under signed informed consent.

Based on the results of the evaluation of the different miRNA extraction technologies a single extraction technology will be selected for the next iteration of ring studies, the comparison of different analysis platforms and the integration of downstream workflows. First results of these ring studies will be presented.

This work is supported by IMI JU & EFPIA (grand no. 115749).

#1594

A fast and sensitive workflow to screen therapy-relevant mutations in circulating tumor cells and quantification of cancer-associated extracellular vesicle-derived mRNA in plasma of cancer patients.

Martin H. Neumann,1 Sebastian Bender,2 Thomas Krahn,1 Thomas Schlange1. 1 _Bayer AG, Wuppertal, Germany;_ 2 _Bayer AG, Berlin, Germany_.

Liquid biopsy can be considered a test that detects rare cells or nucleic acids circulating in blood or in other biofluids. The concept of liquid biopsy aims to monitor the status of a disease or treatment efficacy in a simple, fast, cost efficient way and at any point in time with acceptable risk and burden for the patient. However, the volumes of available blood samples can be quite limited and rare cells as well as circulating nucleic acids are normally present in low abundance. Therefore, detection of rare events or the quantification of limited material requires robust, highly sensitive technologies like droplet digital PCR (ddPCR).

Here, a novel workflow is presented combining the effective enrichment of rare cells using the Parsortix microfluidic filtration system with the analysis by ddPCR. Ten or 100 cells of NSCLC cell line NCI-H441 were spiked into a suspension of 100k NCI-H1563 cells or healthy EDTA blood. The resulting cell suspensions were processed using the microfluidic device Parsortix to enrich cell factions by size and deformability. Enriched cell fractions were harvested or lysed directly in the Parsortix cassette. Genomic DNA was prepared from all cell fractions. NCI-H441 cells are heterozygous for KRAS G12V and TP53 R158L mutations, while NCI-H1563 cells do not contain the mutations. Ten cells or less were enough to detect KRAS G12V or TP53 R158L mutations in a standard blood sample (7-10 ml). This combined workflow enables quantification of circulating tumor cells and analysis for therapy relevant point mutations in a fast, EpCAM independent manner.

Additionally, extracellular vesicle derived mRNA (EVmRNA) isolated from either cell culture supernatant or plasma from cancer patients was analyzed to demonstrate the suitability of ddPCR for assessing specific EVmRNA as biomarkers in different cancer types. In 13 NSCLC and 17 metastatic breast cancer plasma samples the levels of epithelial and oncogene EVmRNA, including EpCAM, ERBB2 or FGFR3 were determined and compared to healthy control plasma. Levels of potentially cancer-associated EVmRNA were only detectable in plasma from cancer patients, but not in healthy controls. Samples from patients and healthy volunteers, respectively, were collected under signed informed consent.

We demonstrated, that ddPCR allows the verification of therapy relevant point mutations in CTCs has the potential to be used as a screening approach. Moreover, despite the small amounts of mRNA in extracellular vesicles from patient blood, using ddPCR differential mRNA levels can be determined in patient plasma and enable and expand the scope of biomarker analysis from EVs.

This work is supported by IMI JU & EFPIA (grand no. 115749).

#1595

Detection of tumor-derived DNA in urine cell-free DNA of pre-operative renal cell carcinoma patients.

Selena Lin,1 Jennifer A. Linehan,1 Ruby Kuang,1 Selvi Guharaj,2 Timothy G. Wilson,1 Dave SB Hoon1. 1 _John Wayne Cancer Institute, Santa Monica, CA;_ 2 _Bio-rad Laboratories, MA_.

The current clinical approaches lack reliable methods (i.e. imaging and serum biomarkers) to identify patients with renal cell carcinoma (RCC) resulting in late detection, where the 5-year survival rate is poor (<8%). Thus, there is an urgent need for better minimally-invasive screening and diagnostic blood/urine assays for earlier detection. Given the logistical ease of collecting urine, urinary cell-free DNA (cfDNA) represents an advantageous source to detect tumor biomarkers and permit for routine/repeat sampling. We previously demonstrated urine cfDNA contains tumor-derived DNA mutations in liver cancer, distal of the kidney filtration barrier. However to date, urinary cfDNA as a source of RCC biomarkers remains unexplored. We hypothesize due to the direct contact of urinary flow-through with the kidney organ that urinary cfDNA is enriched for RCC-derived DNA mutations. To test this, we evaluated the feasibility of detecting tumor-derived DNA mutations utilizing a hotspot 50-cancer gene panel droplet next generation sequencing (dNGS, Raindance Technologies) assay in a pilot cohort of 42 specimens obtained from 13 unique RCC patients and 3 controls (2 RCC cell-lines and 1 normal DNA). The specimens evaluated include matched tumor formalin-fix paraffin-embedded (FPPE) DNA (n=11), urine cfDNA (n=15), and peripheral blood lymphocytes (PBL) DNA (n=13), serving as a germline control. 150ng of genomic DNA from FFPE or PBLs and ≥5ng of urine cfDNA was used for the dNGS assay. An average of 5.5E10 reads were obtained of which 86% were on-target for all 42 specimens providing an average 3.9E3x coverage. Initial dNGS variant analysis utilizing a conservative 5% variant frequency (VF%) cut-off was performed in 11 unique RCC patients containing matched FFPE-urine cfDNA-PBL, of which five patients also contained post-operative urine. 100% of tumor FFPEs (n=11) contained detectable single nucleotide variants (SNVs) (1-10 SNVs) upon germline variant removal. 80% of matched urine cfDNA contained ≥1 tumor-concordant SNV. Additionally, 37.5% of patients contained previously reported somatic mutations (i.e. PIK3CA T1025T, RET L769L, TP53 H233Y, etc) that were tumor-urine concordant. Interestingly, three patients harbored known somatic mutations that were not detected in the matched tumor and three post-operative urine cfDNA specimens contained tumor-derived SNVs despite surgical resection. Ongoing follow-up will reveal if urine cfDNA is a suitable source for identifying tumor heterogeneity and/or residual disease in RCC patients. Furthermore, evaluation of matched plasma is ongoing. Overall, this proof-of-concept study demonstrates feasibility of utilizing urinary cfDNA as a noninvasive source to detect RCC-related biomarkers and provides a potential platform to study and discover novel RCC biomarkers for earlier detection of new/recurrent RCCs.

#1596

Feasibility of liquid biopsy using extracellular vesicle-derived DNA isolated from bronchoalveolar lavage fluid and plasma in addition to tissue rebiopsy to detect T790M mutation in NSCLC patients with acquired resistance to EGFR-TKIs.

Jong Sik Lee, Hee Joung Kim, In Ae Kim, Jae Young Hur, Kye Young Lee. _Lung Cancer Center, Konkuk University Medical Center and Department of Pulmonary Medicine, Konkuk University School of Medicine, Seoul, Republic of Korea_.

Introduction: Tissue rebiopsy has limited value for detecting T790M mutation to prescribe the third-generation EGFR-TKIs. Liquid biopsy using cell-free DNA is being adopted but it plays a complementary role because of low sensitivity. Recently, extracellular vesicles (EV) are proven to contain double-stranded DNA and liquid biopsy using EV DNA is highly promising owing to its intact stability. We aimed to investigate the feasibility of liquid biopsy using EV DNA isolated from bronchoalveolar lavage fluid (BALF) and plasma in addition to tissue rebiopsy to detect T790M mutation in NSCLC patients who developed acquired resistance to EGFR-TKIs

Methods: From October 2014 to December 2016, we enrolled 30 patients who had disease progression after previous treatment with EGFR-TKIs. Rebiopsy was performed in 23 patients and liquid biopsy was done in 16 BALF and 6 plasma samples. Sixteen BALF samples were obtained in 9 T790M-negative cases by rebiopsy and 7 patients ineligible for rebiopsy. Plasma testing was done in 6 BALF T790M-negative cases. EV were isolated from BALF and plasma by ultracentrifuge method. After DNA extraction from the isolated EV, EGFR genotyping to detect T790M mutation was done by peptide nucleic acid (PNA)-clamping method. We retrospectively reviewed patient characteristics, types of tumor rebiopsy procedure, and responses to third generation EGFR-TKIs.

Results: Thirty patients who experienced progression to EGFR-TKIs were evaluated. Tumor rebiopsy was not possible in 7 patients. Out of 23 patients who performed tumor rebiopsy, 22 patients achieved adequate tissue specimen for evaluation, among whom 5 patients conducted rebiopsy twice (4 patients) or three times (1 patient), but 1 patient did not get evaluable tumor cells. Twenty two evaluable tumor rebiopsy revealed 54.5% (12/22) of T790M positivity. Among 17 patients who showed T790M negativity for tumor rebiopsy (n=10) or were ineligible for tumor rebiopsy (n=7), BAL was done in 16 patients and EGFR genotyping using BALF EV DNA showed 31.3% (5/16) of T790M positivity. In 11 patients with T790M negativity from EGFR genotyping using BALF EV DNA, 6 available plasma samples were tested for EGFR genotyping using plasma EV DNA and 3 patients revealed T790M positivity (3/6 = 50%). Finally, we found 8 more T790M positive patients by liquid biopsy (BALF EV DNA = 5, plasma EV DNA = 3) in addition to standard tissue rebiopsy (n=12).

Conclusion: Liquid biopsy using EV DNA isolated from BALF and plasma could be an alternative and promising method to detect T790M mutation in NSCLC patients with acquired resistance to EGFR-TKIs.

#1597

Increased circulating tumor cell (CTC) after systemic therapy is associated with younger age in stage III/IV breast cancer patients.

Qiang Zhang, Lorenzo Gerratana, Lisa Flaum, Youbin Zhang, William Gradishar, Leonidas Platanias, Massimo Cristofanilli. _Northwestern Univ., Chicago, IL_.

Introduction: Breast cancers (BCa) in younger women are more likely to be fast-growing, higher grade and hormone receptor-negative. As such these tumors tend to be associated with a worse prognosis even after systemic treatments with surgery, chemotherapy and radiation. The mechanisms that contribute to these tumors' aggressiveness remain largely unknown. Surveillance of circulating tumor cells (CTCs) may help to understand BCa prognosis and predict treatment benefit, especially for women with metastatic disease. Herein, we report a new finding of the correlation between age and the changes of CTC after the systemic therapies.

Methods: 160 whole blood samples (7.5ml/each) were collected from 100 patients with stage III/IV BCa at baseline, before and after systemic therapy. CTC enrichment and enumeration were performed in FDA approved semi-automated fluorescence CELLTRACKS ANALYZERII® System (Janssen Diagnostics) by using CELLSEARCH® CXC Kit (Cell Search). CTCs were confirmed using Anti-CK-PE, CD25, and EpCaM antibody and DAPI nuclear stain. Anti-CD45-APC antibodies were used to identify leukocytes. The CTCs were classified based on morphology and correct phenotype as CK+, EpCAM+, DAPI+ and CD25-. Database of CTCs was generated and linked with clinical database. Student test was used for statistics.

Results: Among 100 subjects who met inclusion criteria, 54 subjects (68%) at least 1 detectable CTCs in the baseline blood drawn prior to treatment. Of those, 37 patients had ≥ 5 CTCs, 17 patients had 1-5 CTCs, and 46 patients had no CTCs. There was no significant difference on the average age between these CTCs groups, which are 54.89, 53.52 and 53.30 respectively (P>0.05). Among the initial cohort, 42 subjects had at least 2 blood draws before and after systemic therapies respectively. Among these 42 patients, 11 had more CTCs after systemic therapy [Group 1 (26%), the increased range of CTC is from 1 to 756] and 17 patients had no CTCs before or after therapy [Group 2 (40%)]. There were 14 patients who had significantly fewer CTCs after systemic therapy [Group 3 (34%), the decreased range of CTC is from -1 to -23]. There was a significant difference in the average ages of each group (48.45, 56.00 and 57.28 years respectively; P=0.02). The results indicated that younger age (less than 50 years old) is associated with persistent of higher CTCs after systemic therapy likely reflecting decreased benefit from systemic therapy.

Conclusion: Our original findings suggest age-related differences in CTCs response after systemic therapies for advanced BCa. These findings may indicate that younger patients may be at higher risk of developing more distant metastases as suggested by the increased numbers of CTCs after the systemic therapies. We propose that a combination of baseline CTCs detection and age be considered as a potential new criteria for selecting the systemic therapies for BCa patients.

#1598

EGFR mutational detection in vortex-enriched CTCs, ctDNA, and comparison to tumor tissue in non-small cell lung cancer (NSCLC) patients.

Elodie Sollier-Christen,1 Haiyan E. Liu,1 Meghah Vuppalapaty,1 Michael Chiu,1 James Che,1 Charles Wilkerson,1 Nasim Barzanian,1 Steve Crouse,1 James Carroll,2 Melissa Matsumoto,3 Edward B. Garon,4 Jonathan W. Goldman4. 1 _Vortex Biosciences Inc, Menlo Park, CA;_ 2 _UCLA Hematology Oncology, Santa Monica, CA;_ 3 _UCLA, Los Angeles, CA;_ 4 _David Geffen School of Medicine at UCLA, Los Angeles, CA_.

Background Lung cancer is the leading cause of cancer-related mortality worldwide and 85% cases are NSCLC. Epidermal growth factor receptor (EGFR) mutations occur in 10-30% of NSCLC patients1. EGFR tyrosine kinase inhibitor (TKI) therapies, based on the evaluation of EGFR mutation, have shown dramatic clinical benefits. EGFR assays are mainly performed on tumor biopsies, which carry risks and expense and are not always successful1. In order to identify the development of secondary EGFR mutations, which cause resistance to 1st and 2nd generation TKI's and an indication for therapy with a 3rd generation drug, effective and non-invasive monitoring is needed. Liquid biopsies containing circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA), allow such monitoring over the course of the therapy2. The Vortex Biosciences' VTX-1 Liquid Biopsy System enables the label-free capture of CTCs from blood samples, with high CTC recovery, purity and compatibility with downstream genomic assays on CTCs and plasma3,4. Our combined CTC and ctDNA EGFR assay was applied to blood samples from NSCLC patients. Method ctEGFR kit (EntroGen) was selected and validated for the detection of EGFR major mutations in a single PCR reaction, for both ctDNA and CTCs. Several blood collection tubes (BCT: EDTA, CellSave, LBGard and Streck) were tested using spiked cells, considering CTC recovery, DNA yield, and EGFR profiling. 20 blood samples were studied from 15 NSCLC patients. Plasma was extracted for ctDNA assay. CTCs were isolated from the plasma-depleted-blood using the VTX-1, immunostained and enumerated5. EGFR mutations were then detected in CTC+ctDNA, and compared to the tissue results. Results The VTX-1 provided a similar CTC recovery from plasma-depleted-blood and whole blood, enabling CTC+ctDNA EGFR profiling from the same tube of blood. Among the BCTs tested, LBGard obtained the best CTC capture and EGFR mutation detection performance. 10/15 patients showed the same mutations between tissue and CTC+ctDNA. For one patient with two blood draws without mutation in tissue, no mutation was detected in the first draw, while a T790M mutation was identified 6 months later. For another patient, an Exon19 deletion was detected in the ctDNA+CTC but not in the tissue, and a repeat draw confirmed the result. 4/15 patients had mutations in tissue that were missed in CTC+ctDNA. Conclusion Performing EGFR mutation analysis on the combination of ctDNA and VTX-1-collected CTCs may offer an improved sensitivity of detection over analysis of only the ctDNA, and will be potentially a useful tool for monitoring treatment and medication guidance of NSCLC patients. [1] Calabuig-Fariñas et al. Transl Lung Cancer Res. 2016. [2] Sundaresan TK et al. Clin Cancer Res. 2016. [3] Kidess-Sigal E et al. Oncotarget 2016. [4] Haiyan E L et al. npj Genomic Medicine (2017) 2:34. [5] Che J. et al., Oncotarget (2016)

#1599

Capture of tumor cells in blood with "Universal CTC-chip".

Kazue Yoneda, Taiji Kuwata, Fumihiro Tanaka. _Univ. of Occupational And Environmental Health, Kitakyushu, Japan_.

Background: Circulating tumor cells (CTCs) are not only a surrogate of distant metastasis, but also useful for material of liquid biopsy. "Universal" CTC-chip is a polymeric microfluidic device that can capture CTCs according to their surface marker from whole blood. We have examined the capture efficiency of lung cancer and mesothelioma cells in the blood with CTC-chip coated with antibodies against various surface marker.

Methods: Lung cancer cell lines (PC-9) or mesothelioma cell lines (ACC-MESO-4, MSTO-211H) were used in this study. Cells were CFSE-labeled and added in the blood of healthy donor. A polymeric CTC-chip was coated with two steps: base- and capture-antibody. As capture antibody, we used anti-EpCAM, podoplanin, and EGFR antibody. After flowing these samples, capture efficiency was calculated (number of captured cells/number of flowed cells).

Results: Capture efficiency of PC-9, MESO-4, and METO-211H was 100.0%, 9.5% and 9.1% (EpCAM-chip; anti-EpCAM antibody clone HEA125); 5.8%, 85.9% and 9.0 % (podoplanin-chip; anti-podoplanin antibody clone E1); 38.6, 112.5%, 11.7% (podoplanin-chip; anti-podoplanin antibody clone NZ-1); 30.2%, 21.8%, 38.5% (EGFR-chip; anti-EGFR antibody clone 528), respectively.

Conclusion: Using "Universal CTC-chip" with various antibodies, it was possible to capture nonepithelial tumor cells such as mesothelioma cells.

### Molecular Diagnostics 2: Laboratory Correlates for Targeted Agents and Preclinical and Clinical Trials

#1600

Expanded multiplexing capability in brightfield immunohistochemistry utilizing multiple chromogen chemistries and detection enzymes.

Esteban Roberts, William Day, Brian D. Kelly, Nathan W. Polaske, Julia Ashworth-Sharpe, Donald Johnson, Mark Lefever, Jerry Kosmeder, Hongjun Zhang, Jian Zhang, Tsu-Shuen Tsao, Mike Farrell, Joerg Bredno, Robert Ochs, Larry Morrison. _Ventana Medical Systems, Tucson, AZ_.

Introduction

Immunohistochemistry (IHC) is a valuable means to examine the distribution of protein expression at the subcellular level throughout human tissues while retaining morphology of the cells and tissue. Given the large and growing number of established protein biomarkers and the often very limited amount of tissue provided by tumor biopsy, the ability to evaluate multiple biomarkers on a single-slide mounted specimen via multiplexed IHC is of growing importance. However, due to the limited number of conventional chromogens, their broad absorbance spectra, and deposition chemistry based on only two enzymes, brightfield multiplexing has been typically limited to only two protein targets.

Methods

To expand multiplexing capability, our project objective was to reduce the antibody incubation, detection, and enzyme deactivation steps to produce high-level multiplexed chromogenic assays with short run times and minimal exposure to potentially destructive enzyme deactivation reagents. We utilized unmodified and tagged primary antibodies with secondary anti-primary and anti-tag antibodies conjugated to several enzymes employing orthogonal enzymatic detection systems. Peroxidase-catalyzed tyramide signal amplification (TSA) and quinone methide signal amplification (QMSA) chemistry adapted to two different hydrolase enzymes provided several compatible chromogens with relatively narrow absorbance bands and good spectral separation. All assays were fully automated and performed on the Benchmark XT platform.

Results

Multiplex IHC staining was demonstrated using combinations of TSA and QMSA, with the former catalyzed by horseradish peroxidase (HRP) and the latter established to work successfully with two different hydrolase enzymes: alkaline phosphatase (AP) and neuraminidase. Three- and four-chromogen multiplex assays were demonstrated on a variety of targets including breast cancer (Her2, ER, PR, Ki-67) and immune cell targets (CD3, CD8, cytokeratin, FOXP3, PD-L1) on a variety of tumor and tonsil tissues. Use of both tagged and untagged primary antibodies and up to three enzymes allow reduction of antibody removal and enzyme deactivation steps, significantly reducing assay time. Quantitative comparisons of multiplex staining to 'gold standard' DAB staining on serial sections verified the multiplex assay performance.

Conclusion

We have expanded on the current HRP and AP brightfield IHC detection technologies by utilizing TSA- and QMSA-based chromogens and introducing the new neuraminidase detection chemistry. These detection chemistries combined with tagged and untagged primary antibodies provided up to four-chromogen brightfield multiplex IHC with verified assay performance.

#1601

Clinical studies to treat without surgery for breast cancer cases who achieved clinical complete response by neoadjuvant chemotherapy.

Takashi Ishikawa,1 Mariko Asaoka,1 Kazutaka Narui,2 Akimitsu Yamada,2 Nobuyasu Suganuma,3 Takashi Chishima,4 Takahiko Kawate,1 Kana Miyahara,1 Kazuaki Takabe,5 Yasushi Ichikawa,6 Eiichi Sato1. 1 _Tokyo Medical University, Tokyo, Japan;_ 2 _Yokohama City University Medical Center, Yokohama, Japan;_ 3 _Kanagawa Cancer Center, Yokohama, Japan;_ 4 _Yokohama Rosai Hospital, Yokohama, Japan;_ 5 _Roswell Park Cancer Institute, Baffalo, NY;_ 6 _Yokohama City University, Yokohama, Japan_.

Introduction: The pathological complete response (pCR) rate is approaching to 50% particularly in patients with hormone-receptor negative disease by neoadjuvant chemotherapy (NAC). If the disease is not recurred locally in cases with pCR and pCR could be accurately diagnosed preoperatively, it is possible to treat some population of patients without surgery after NAC. We conducted two studies to examine: 1) outcomes of patients who had achieved pCR by NAC with a special attention to local recurrence. 2) diagnostic accuracy of pCR with core needle biopsy (CNB) for cases obtained clinical complete response (cCR) by NAC.

Methods: 1) Clinical outcomes were retrospectively investigated in 395 patients who achieved pCR from 1599 patients with primary operable breast cancer treated by NAC (overall pCR rate of 24.7%: 395/1599). The association of clinic-pathological factors with recurrence was investigated. 2) Ultrasound-guided CNB was performed prospectively for 86 cases who achieved cCR by MRI after NAC. The concordance of pathological results between CNB and surgical specimens was examined. The pathological diagnosis was categorized as no carcinoma (pCR), carcinoma in situ (CIS) and invasive carcinoma (INV).

Results: 1) 395 pCR cases consisted of 50 Luminal type (pCR rate: 7.2%), 98 Luminal-HER2 type (32.1%), 116 HER2 type (52.5%), and 131 triple negative (TN) type (34.2%). The median follow-up was 41 months. Recurrent diseases including local recurrence or distant metastasis were found in 5.80% (23/395). According to subtypes, these were 2.00% (1/50) for Luminal type, 4.08% (4/98) for Luminal-HER2 type, 10.3% (12/116) for HER2 type, and 4.58% (6/131) for TN type. Local recurrence was found in 1.2% of cases (5/395). Risk factors of recurrence were clinical stage before NAC and nodal status after NAC in the univariate analysis, and only clinical stage remained statistically significant in the multivariate analysis. 2) Pathology of surgical specimen revealed pCR in 41 cases (48%), CIS in 17 cases (20%) and INV in 28 cases (32%). Discordant cases: pCR in CNB, but CIS and INV in surgical specimens were found in 24 cases (14 CIS and 10 INV). The false negative rate (FNR) and accuracy of CNB to predict INV were 22.2% and 88.4%, respectively.

Conclusion: Except HER2 type, recurrence was not frequent particularly in cases with an early clinical stage. Local recurrence was rarely observed in any subtype. However, ultrasound-guided CNB for cCR cases diagnosed by MRI was not reliable enough to predict pCR. Thus, imaging diagnosis and biopsy methods need to be improved to achieve 10% of FNR and 90% of accuracy for predicting pCR and to proceed to an observational study without performing surgery.

#1602

Urine cell-free DNA as early biomarker in immunotherapy response in NSCLC.

Anthonie J. van der Wekken, Arja ter Elst, Anneke Miedema, Thijo J. Hiltermann, Harry J. Goen, Ed Schuuring. _University Medical Center Groningen, Groningen, Netherlands_.

Introduction: Immunotherapy shows a durable tumor response in approximately 20% of advanced NSCLC patients. Imaging by CT is used to evaluate tumor response. PD-L1 immunohistochemistry is generally used to predict survival advantages, however with low predictive value. Therefore, it is important that alternative predictive biomarkers are investigated. Minimal invasive techniques may be a solution. The role of circulating cell-free DNA (cfDNA) was found to be an early biomarker for tumor response in KRAS positive Non-Small Cell Lung Cancer (NSCLC) patients treated with immunotherapy (N=6). In this study we will determine the role of urine cell-free DNA (ucfDNA) in an extended cohort. The non-invasive method of urine collection makes it an excellent source for mutation detection and follow-up.

Methods: In an ongoing study KRAS positive patients treated with immunotherapy, are being tested for cfDNA at baseline (before treatment, 1, 2, 4, 6 and thereafter every 12 weeks until disease progression) with a KRAS G12-13 or Q61H digital droplet PCR (ddPCR) screening assay (BioRad). In a pilot study, urine from these patients is collected in a tube with 5 ml of STRECK reagent at baseline and at 6 weeks.

Results: In 16/27 patients with KRAS positive advanced NSCLC, the mutation was detected in plasma at baseline and subsequent evaluation time points. Urine cfDNA as a predictive marker seems promising and results will be reported and presented at the AACR meeting.

Conclusion: Among patients with advanced NSCLC treated with immunotherapy plasma and urine cfDNA are collected and will be presented as non-invasive early biomarker for tumor response evaluation.

#1603

Patient derived tumor organoids identify actionable targets in heavily pretreated metastatic breast cancer patients.

Michael J. Churchill,1 Franz X. Schaub,1 Hallie A. Swan,1 Rachele Rosati,2 Fengting Yan,3 Reid C. Shaw,2 Kay E. Gurley,3 Robert L. Diaz,1 Shalini C. Pereira,1 Carla Grandori,1 Christopher J. Kemp,3 Vijayakrishna K. Gadi3. 1 _SEngine Precision Medicine, Seattle, WA;_ 2 _Cure First, Seattle, WA;_ 3 _Fred Hutchinson Cancer Research Center, Seattle, WA_.

Recurrent metastatic breast cancer is difficult to treat and patients often exhaust standard-of-care regimens. P.A.R.I.S. is a CLIA certified high throughput chemosensitivity test developed by SEngine Precision Medicine. (Seattle, WA USA) Tumor cells derived from patient breast tissue are expanded as mammospheres, and challenged with a library of 123 clinically actionable, targeted drugs. These results are integrated with patient genomic data and reported to the oncologist to prioritize treatment options. To date, we have performed over 100 such drug screens using low passage patient-derived tumor cells in 70 independent replicate pairs. Of 18 viable primary breast tumor samples received to date, we have successfully derived organotypic cultures in 13 cases. (72%) Of these, 7 were challenged to the full 123 agent library. Three of the seven cases were HER2 positive and demonstrated clear sensitivity to HER2 kinase inhibitors. Overall, patients can be separated into two groups, characterized by families of drug sensitivities. Patients in group one respond preferentially to inhibitors of signaling pathways such as IGF1R, ALK, FGFR and EGFR. Patients in the second group responded to therapies targeting epigenetic modifiers, cell cycle control and apoptosis.

Presented here is a premenopausal female, diagnosed in 2010 as pT2N0M0 (stage IIA), ER+ PR+ HER2+. This patient underwent surgery and received adjuvant chemotherapy, followed by tamoxifen. Upon progression to metastatic disease, biopsy of her recurrent lesion showed loss of ER and PR expression, but remained positive for HER-2 amplification. Sequencing of this biopsy exposed a PIK3CA G106R mutation and amplification of unknown significance. Tumor cells were isolated from ascites, cultured and the P.A.R.I.S. test was performed. Patient derived tumor cells were sensitive to HER2 targeting agents, concordant with the patient's HER2 amplification. Of these, the non-reversible inhibitor neratinib exhibited a significantly enhanced effect and achieved maximum growth inhibition of 80% over the duration of the assay. Two different AKT inhibitors, afuresertib and AZD5363, demonstrated activity in the patient cells consistent with the activating PIK3CA mutation. Also consistent with the loss of ER/PR expression, the patient derived tumor cells no longer responded to anti-estrogen therapies nor to chemotherapies the patient had been refractory to prior: cyclophosphamide, docetaxel, doxorubicin, gemcitabine, and paclitaxel.The PARIS assay is a robust ex vivo screening platform to objectively quantify patient specific sensitivities to a panel of 123 clinically actionable oncology drugs. We show here concordance with both genomic data and prior treatments the patient received in the course of disease however the same approach may be used to successfully identify unique sensitivities in the absence of established biomarkers.

#1604

Highly sensitive digital detection of circulating DNA cancer mutations using synthetic genome standards.

Christina M. Wood-Bouwens,1 Robert P. St.Onge,2 Hanlee P. Ji1. 1 _Stanford University, Stanford, CA;_ 2 _Stanford Genome Technology Center, Stanford, CA_.

With the emergence of precision cancer medicine, there is an increasing need for new longitudinal diagnostic tests to evaluate patients at the initial diagnosis, during treatment, and for routine monitoring. As a general solution, we developed a single-color digital PCR assay technology that detects and quantifies circulating DNA cancer mutations collected from the plasma of cancer patients. The assay has a sensitivity of 0.1% mutation allelic fraction and can be designed for nearly any cancer mutation. In addition, this assay demonstrates design flexibility, low cost, and robust performance, making it a powerful approach for longitudinal monitoring of cancer patients during treatment.

Positive controls are a critical component for highly sensitive detection of circulating DNA cancer mutations. However, there are minimal resources for providing standard curves. Commercially available cancer cell lines cover a very limited number of cancer mutations. Amplicons or oligonucleotides pose a high risk of contamination due to the sheer number of molecules generated, an important consideration when working with rare molecular species such as cell-free DNA.

To address this challenge, we developed a novel technology that enables a single copy of synthetic human genome sequence to be introduced into a non-mammalian eukaryotic genome. The system highly-scalable and capable of producing thousands of unique clones which can be archived for future use, accessed individually, or combined to create customizable target pools on-demand. These custom designed synthetic positive controls are therefore readily renewable, require no amplification to obtain, and most importantly allow precise control over the number of molar equivalents of each target in a reaction. Using these synthetic controls, we simulate low mutation-allele fractions to validate the specificity and sensitivity of our assay.

As a proof of concept, we have designed approximately 400 cancer mutation controls in driver genes such as KRAS and EGFR, and developed a design process to generate multiplex digital PCR assays targeting these driver genes. We will validate the digital PCR assays with standard bulk PCR and will validate the template by comparing to results generated using cancer cell line derived DNA. In the era of circulating DNA diagnostics, these genome engineered synthetic controls meet a critical need for reliable and customizable positive controls.

#1605

Personalization of cancer treatments with a CLIA-certified high-complexity and high-throughput drug sensitivity test.

Hallie A. Swan,1 Michael J. Churchill,1 Rachele Rosati,2 Franz X. Schaub,1 Reid C. Shaw,2 Roland M. Watt,1 Eduardo Mendez,3 Christopher J. Kemp,4 Vijayakrishna K. Gadi,4 Stephanie A. Murphy,1 Brady Bernard,5 Robert L. Diaz,1 Shalini C. Pereira,1 Carla Grandori1. 1 _SEngine Precision Medicine, Seattle, WA;_ 2 _Cure First, Seattle, WA;_ 3 _University of Washington, Seattle, WA;_ 4 _Fred Hutchinson Cancer Research Center, Seattle, WA;_ 5 _Institute for Systems Biology, Portland, OR_.

In June 2017, SEngine Precision Medicine (Seattle, WA USA) obtained CLIA certification for the first high-complexity and high throughput organotypic chemosensitivity test, P.A.R.I.S., thus opening its doors to oncologists and pharmaceutical companies that may consider personalization of cancer treatments in their practice or in the clinical trial setting. Just as Paris slayed the invulnerable Achilles by aiming specifically at its weaknesses, the PARIS test challenges tumor cells with a library of 137 clinically actionable targeted drugs to identify sensitivity and resistance patterns unique to each individual cancer in an unbiased manner. Here we will present an overview of the approach that has led to CLIA certification, including novel cell culture techniques and analysis metrics. We will also highlight insights gained by comparative analysis across a multitude of cancer samples. SEngine has derived and expanded primary cancer cells from a cohort of over 100 patient derived specimens. Patients include many who have been heavily pre-treated with prior chemotherapy and several with rare cancers, including adrenocortical carcinoma, neuroblastoma, leiomyosarcoma and cholangiocarcinoma. Standard operating procedures have been established for transport of specimens as well as primary cultures in three dimensional organotypic conditions. Our analysis pipeline evaluates the multi-dose response of a given patient's tumor to each drug in our library and compares those responses to all prior patients. This establishes not only functional sensitivity but also the uniqueness of the patient's response. We calculate the SPM score, a proprietary ranking metric weighing both the sensitivity and uniqueness of the response. The results from the P.A.R.I.S. test are highly reproducible (Spearman rank correlation coefficients: 0.95, 0.88 technical and biological replicates respectively). We perform bi-annual proficiency validation of our platform with another CLIA-certified laboratory. Ten samples tested demonstrated greater than 75% concordance between responses. The data obtained are visualized by an in-house application suite (SEngine Medicine APP), consisting of interactive charts, statistical analysis, and reactive reports which can be shared with oncologists and investigators. A cohort of 50 samples was employed to demonstrate sensitivities or resistances concordant with those predicted by the patient's genomic aberrations and/or prior clinical responses.In summary, we envision that the P.A.R.I.S. test will be essential for patient's selection while developing novel targeted drugs, providing crucial functional information much needed to decode the complexity of individual cancer genomics.

#1606

Clinical routine massive parallel sequencing highlights clinicopathological associations of the RAS/RAF mutational module in metastatic colorectal cancer and reveals high frequency of concomitant RAS/RAF mutations.

Edoardo Isnaldi,1 Anna Garuti,1 Gabriella Cirmena,1 Stefano Scabini,2 Federica Grillo,2 Luca Mastracci,2 Lorenzo Ferrando,1 Roberto Murialdo,2 Maurizio Gallo,1 Christine Desmedt,3 Roberto Fiocca,2 Emanuele Romairone,2 Alberto Ballestrero,1 Gabriele Zoppoli1. 1 _University of Genova, Genova, Italy;_ 2 _Ospedale Policlinico San Martino, Genova, Italy;_ 3 _Institut Jules Bordet, Brussels, Belgium_.

Background: Over the past few years, massive parallel sequencing (MPS) has become reliable and cost-effective, and its use in clinical practice has become a reality. A relevant role for MPS is the prediction of response to anti-EGFR agents in metastatic colorectal cancer (mCRC), where multiple exons from KRAS, NRAS, and BRAF must be sequenced simultaneously.Patients and methods: We optimized a 14-amplicon panel to assess by MPS the exonic regions of KRAS, NRAS, BRAF, and PIK3CA from formalin-fixed, paraffin-embedded specimen-extracted DNA. We analyzed the samples of a monocentric, prospective, consecutive cohort of patients affected by mCRC collected for diagnostic and research purposes at the time of diagnosis in our referral basin.Results: Over two years, we evaluated 219 mCRC specimens. We observed an unexpected, statistically significant association of RAS mutations with sex, young age, and tumor site (right colon). We further demonstrated, by transversal validation using digital polymerase chain reaction, that concomitant mutations in the KRAS/BRAF/NRAS module are not infrequent in mCRC. As anticipated by large, whole-genome studies, RAS and PIK3CA tended to be concurrently mutated. We corroborated associations such as the higher prevalence of BRAF mutations in right mCRCtumors, in concomitance with microsatellite instability. Finally, survival analysis showed that BRAF mutants had a shorter progression-free survival compared to KRAS/NRAS mutant patients and wildtype ones upon I line treatment.Conclusions: To our knowledge, this is the first monocentric, consecutive, prospectively accrued clinical mCRC cancer cohort tested on a clinical routine basis by MPS for KRAS, NRAS, BRAF, and PIK3CA. Our study has highlighted in clinical practice findings such as the concomitance of mutations in the RAS/RAF module, the co-occurrence of RAS and PIK3CA mutations, and possible associations of gender with specific mutations. These results need clinical follow-up in the context of treatment to assess their translational relevance.

#1607

**An** ERBB2 **follow-on companion diagnostic for clinical care of patients with breast cancer.**

Wai-ki Yip, Joel Skoletsky, Pei Ma, Jun Luo, Coren Milbury, Christine Burns, John Truesdell, Julia Elvin, Geoff Otto, Doron Lipson, Jeffrey Ross, Vincent Miller, Philip Stephens, Michael Doherty, Christine Vietz, James X. Sun, Yali Li. _Foundation Medicine, Inc, Cambridge, MA_.

Introduction: Patients with HER2-positive breast cancer may benefit from targeted therapies including trastuzumab and pertuzumab. While FDA has approved companion diagnostics (CDx) using FISH or IHC for identifying ERBB2 amplifications, molecular diagnostic testing in breast cancer is rapidly evolving towards comprehensive genomic profiling (CGP) due to an increasing number of biomarkers. However, clinical validity of ERBB2 testing in CGP has not yet been demonstrated in an FDA-approved manner. We present the first ERBB2 test using NGS as a follow-on CDx, which is part of FoundationOne CDx (F1CDx). Methods: Clinical validity was established against the FDA-approved HER2 FISH test, using retrospective breast cancer patient samples. For each sample, two tests using the FISH assay were performed, and clinical validity was established such that the F1CDx result is statistically non-inferior to the performance between two runs of FISH. Concordance was calculated on samples that agree between the two FISH tests. Results: Results are shown in Table 1. An exploratory analysis including low-level ERBB2 amplifications (CN=4; ploidy+2) detected by F1CDx demonstrated an improved PPA of 93.8%. A non-inferiority test demonstrated a margin of 8.0%. Further analysis showed that the discordance is most significant among samples with a low FISH ratio. In samples that are FISH-positive but F1CDx-negative, a second test of FISH only yields a FISH-FISH concordance of only 55%, suggesting significantly decreased FISH-FISH reproducibility. Conclusion: We presented a follow-on CDx for ERBB2. The F1CDx platform provides a single assay that identifies cancer patients who may be eligible to receive FDA-approved targeted therapeutics, conserves tissue by avoiding serial testing and can serve as clinical trial assay for studies requiring a molecular biomarker for eligibility. The data demonstrating clinical and analytical validity of ERBB2 may accelerate the use of CGP for routine clinical use.

Table 1. The ERBB2 companion diagnostic in FoundationOne CDx

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Companion diagnostic | ERBB2 amplification

Indicated use | Trastuzumab, ado-trastuzumab-emtansine, or pertuzumab in breast cancer

Approved CDx comparator | HER2 FISH PharmDx® Kit (Dako Denmark A/S)

Positive percent agreement (PPA) | 89.4% (101/113)

Negative percent agreement (NPA) | 98.4% (180/183)

Median unique sequence coverage | >500X

Precision | 100%

Limit of Detection | 19.7% tumor content

#1608

Pharmacologic screening identifies synthetic lethality interactions with Chk1 inhibitors in basal-like breast and ovarian cancer.

Eva M. Galan-Moya,1 Ana Alcaraz-Sanabria,2 Cristina Nieto-Jimenez,2 Veronica Corrales-Sanchez,2 Miriam Nuncia-Cantarero,1 Juan C. Montero,3 Atanasio Pandiella,3 Alberto Ocaña2. 1 _University of Castilla-La Mancha, Albacete, Spain;_ 2 _Albacete University Hospital, Albacete, Spain;_ 3 _Cancer Research Center, Salamanca, Spain_.

Basal-like breast cancer and ovarian cancer are considered as unmet entities as patient outcome in advanced stages is short, even receiving the most appropriate treatment. These tumors are characterized by a high grade of genetic instability and a notable impairment of DNA repair processes. Thus, in response to DNA damage, they depend on checkpoint arrest proteins, such as Chek1, to stop division. Inactivation of Chk1 permits cell cycle progression without a proper DNA repair, therefore increasing genetic instability and aneuploidy and eventually leading to cell death. Chk1 inhibitors are currently in clinical development and, therefore, the identification of chemotherapies that could be used as clinical partners to potentiate their effect is a main goal. OVCAR3 and IGROV ovarian cancer cells as well as the basal-like MDA-MB-231 and HS578T cell lines were used to screen for agents that may increase the antiproliferative properties of Chk1 inhibitors. Combination of DNA-damaging agents, like platinum compounds or gemcitabine, with the Chk1 inhibitors LY2603618 and SAR020106 had a synergistic effect, not observed when combined with agents targeting mitosis, like taxotere, eribulin or vinorelbine. A similar antiproliferative effect was observed for PARP inhibitors such as olaparib, reinforcing the concept that inhibition of Chk1 allows cells to enter apoptosis when a relevant genetic instability is present. Clonogenic assays and 3D colony formation studies confirmed the effect observed on proliferation also on invasion and long-term survival. Analyses of cell cycle, cell death and intracellular mediators were performed by flow cytometry and Western blot. The effect of the association of LY2603618 with cisplatin and gemcitabine did not change the cell cycle. However, combination of both agents produced a marked induction of apoptosis. Experiments evaluating caspase activity demonstrated that the principal mechanism associated with the induction of apoptosis was caspase dependent. We also explored combination of agents that have a clear translation to the clinics including double combinations with chemotherapies like cisplatin and gemcitabine. We observed that these triple combinations were more effective than double or single agents. Finally, we wanted to evaluate the antiproliferative effect when adding LY2603618 to olaparib in addition to a DNA-damaging agent. Of note, we observed a higher effect than single-agent olaparib or the chemotherapy. Interestingly, inhibition of Chk1 also reverted the effect of cisplatin in platinum-resistant cells. Our findings support the clinical development of Chk1 inhibitors in combination with DNA-damaging agents in basal-like and ovarian cancer.

#1609

Simple and rapid high-plex analysis of RNA and protein from low-frequency sorted T cells.

Kit Fuhrman,1 Brian Birditt,1 SuFey Ong,1 Alyssa Rosenbloom,1 Douglas Hinderfeld,1 Gary Geiss,1 Miguel Tam,2 Christina Bailey1. 1 _Nanostring Technologies, Inc., Seattle, WA;_ 2 _BioLegend, Seattle, WA_.

The immune system is increasingly becoming a direct target for cancer therapeutics. These cancer immunotherapy efforts require deep biologic characterization to better understand and characterize rare or low-frequency immune cells, necessitating next-generation methods to expand or complement current flow cytometry or similar methods. Recent advances in flow and mass cytometry have resulted in an improved understanding of immune system heterogeneity. These technologies, however, remain limited in the number of parameters and types of analytes that can be examined in a single sample. To obtain high-plex multi-omic data from rare cells, we utilized a novel workflow, integrating fluorescence-activated cell sorting (FACS) with the NanoString nCounter® Vantage 3D™ RNA:Protein Immune Cell Profiling Assay to deeply characterize multiple flow-sorted immune cell populations. This enabled us to simultaneously interrogate 30 cell surface proteins and 770 immune-related mRNA starting from a heterogenous cell suspension. We employed antibody-conjugated panels from BioLegend that were designed to minimize interference between fluorescently tagged and NanoString oligo-tagged antibodies. To streamline this workflow, cells were co-stained with both BioLegend and NanoString antibodies in a single 30+ antibody stain. Multiple T-cell populations were sorted and profiled from PBMC, including memory and naïve Tregs and CD8 T cells with and without stimulation, allowing for analysis of differentially regulated genes and proteins across samples. Co-stain with both fluorescently and oligo-tagged antibodies did not alter the phenotypic profiles of immune cells, validating the design of the compatible panels used to sort Tregs and CD8 T cells. Demonstrating the value of this workflow in analyzing rare populations, cells were titrated to determine the sensitivity of the workflow, producing concordant protein data from only 500 cells. High-plex RNA data were obtained from 5,000 cells without the requirement for additional molecular biology methods, such as amplification or sequencing library construction, reducing potential for technical biases. Data analysis and visualization was accomplished with nSolver™ and Cytobank software packages to rapidly analyze this high-plex data. Our analysis highlighted the population diversity and activation of key pathways between these different T-cell subtypes. Focused analysis revealed discordance between mRNA and protein levels in key immuno-oncology target genes, including PD-L1. These experiments illustrate the "3D Flow™ Analysis" method as ideally suited for incorporation into cell-sorting workflows, simultaneously producing high-plex, multi-omic data from multiple rare immune cell populations.

#1610

Expression of genes of the uPA system from LCM-procured breast carcinoma cells and their relationships with clinical outcomes.

Seth B. Sereff,1 Michael W. Daniels,2 James L. Wittliff1. 1 _Univ. of Louisville, Louisville, KY;_ 2 _Univ. of Colorado, Aurora, CO_.

Introduction: The urokinase plasminogen activator (uPA), its receptor uPAR and serine protease inhibitors PAI-1 or PAI-2 play a key role in tissue membrane remodeling and invasion of basement membranes by induction of a fibrinolytic pathway. Earlier studies reported that uPA and PAI-1 protein levels assist in prediction of breast cancer response to chemotherapy. Our goal is to develop molecular signatures of candidate genes and identify novel relationships with these four protein biomarkers that demonstrate clinical utility for assessment of breast carcinoma outcomes. Methods: The retrospective study used de-identified tissue biopsies from primary breast cancers on which biomarker and clinical outcomes were stored in an IRB-approved Database. ELISA analyses of uPA, uPAR and PAI-1 performed using IMUBIND kits (American Diagnostica Inc.) used cutoff values previously reported. Estrogen (ER) and progestin receptor (PR) assays were performed either by EIA (Abbott Labs) or by radioligand binding (NEN/DuPont). Relative expression levels of 22,000 genes were determined by microarray using RNA extracted and amplified from Laser-Capture Microdissection (LCM) procured breast carcinoma cells. Univariable and multivariable Cox regression analyses, Kaplan-Meier plots, Violin and scatter grams were performed by R Studio version 3.4.1. External validation of gene subsets derived were performed with SurvExpress. Results: uPA and PAI-1 protein content of a carcinoma were predictive of overall survival (OS). Examination of biomarker gene expression by Violin plots revealed that either ER- or PR- breast cancers expressed elevated levels of UPA, UPAR and PAI2 compared to that of ER+ or PR+ carcinoma cells. Scatter grams suggested an inverse relationship between ER/PR protein levels and expression of UPA, UPAR and PAI2. Univariable Cox regression analyses indicated that PAI2 expression was associated with progression-free survival (PFS) and OS while UPA and PAI1 expression was only associated with OS with a p value < 0.3 (selected as the discovery limit). When carcinomas were sorted by biomarker levels, qPCR expression of RERG and NQO-1 were elevated in uPA- lesions while CD34 and EDG-1 were elevated in uPAR- cancers. However, ERBB4 expression was elevated in PAI-1+ carcinomas. Multivariate Cox regression, performed with backward conditional selection using microarray data with ER or PR status revealed clinically relevant genes for PFS and OS. SurvExpress, an online tool, validated gene subsets externally. Conclusions: Use of LCM-procured breast carcinoma cells in a nondestructive manner uncovered relationships between gene expression and either uPA, uPAR, PAI-1 or PAI-2 protein content of a lesion to reveal candidates for predicting clinical outcomes. Certain of these gene subsets appear related to patient prognosis when considered with ER/PR status of the carcinomas.

#1611

High quality next generation sequencing results for breast cancer using dual-mode biopsy tissue preservation.

Deborah A. Dillon,1 Elizabeth P. Garcia,1 Michele Baltay,1 Krishan Taneja,1 Teri Bowman,1 Eva C. Gombos,1 Tom Gal,1 Stuart G. Silverman,1 Erez Nevo,2 Shoshan Nevo2. 1 _Brigham and Women's Hospital, Boston, MA;_ 2 _Robin Medical, Inc., Baltimore, MD_.

Introduction: Formalin-fixed paraffin-embedded (FFPE) tissue is commonly used for molecular analysis, including for next generation sequencing (NGS). In an effort to improve the quality of molecular results in small tissue samples, we developed a novel biopsy transport device that splits the biopsy sample longitudinally for dual-mode preservation by freezing (for biomolecular analysis) and formalin (for the evaluation of histologic features without freezing artifact). In this feasibility study we compared NGS results from samples acquired by the new device with those obtained using standard biopsy handling procedures.

Methods: Following informed consent, biopsy samples were obtained ex-vivo by a breast imaging radiologist under ultrasound guidance from invasive cancers in two mastectomy specimens using a standard biopsy needle (Mission 14G, CR Bard). A reference sample (REF) was placed directly into 10% neutral buffered formalin. An experimental sample (EXP) was placed in the new device, ink marked on one end for orientation and sectioned longitudinally. After sectioning, one half-sample was formalin fixed and one half was frozen and then stored at -80 degrees until further processing. The REF samples and one half of the EXP samples were fixed for 24 hours then processed routinely into FFPE blocks. DNA was extracted from the REF sample and the frozen EXP half-sample and evaluated using NGS Oncopanel (targeted exome sequencing, Illumina) at 25ng, 50ng and 100ng input template.

Results: The formalin-fixed EXP halves maintained high quality of tissue histology with minimal artifacts. The number of unique, aligned, high-quality reads was on average 1.85-fold greater in the EXP specimens relative to the paired REF samples for all three template amounts in both tumors that were analyzed (range 1.58-fold to 2.29-fold; p=0.0016). More than twice as many high-quality reads were present in the EXP specimens for Tier 2 and Tier 3 mutations (TP53, PIK3CA and GATA3) at the lowest template amounts (average 2.40-fold at 25ng). Copy number reads showed lower cutoff scores (a measure of signal variability across segments) in EXP samples than in REF samples (p=0.0052).

Conclusions: Dual-mode preservation of core biopsy samples by freezing and formalin can be done with the new device and it provides equivalent quality of histology sections. Preservation of biopsy samples by freezing rather than formalin improved the quality of NGS evaluation in all samples down to 25ng input template, both for single variant analysis and for copy number detection. Longitudinal sectioning of the tissue samples allows for assessment of tumor purity and for macrodissection of the oriented frozen half-sample as needed. These improvements allow for greater confidence in variant and copy number calls with lower input template amounts.

#1612

Molecular analysis of clinically actionable gene alterations in clear cell carcinoma of the ovary using next-generation sequencing and immunohistochemical CNV analysis.

Hiroshi Asano,1 Yutaka Hatanaka,1 Kanako C. Hatanaka,1 Asami Okumura,1 Toraji Amano,1 Takashi Mitamura,2 Tatsuya Kato,1 Yosuke Konno,1 Daisuke Endo,1 Kei Ihira,1 Ayako Nozaki,1 Yoshihiro Matsuno,1 Hidemichi Watari,1 Noriaki Sakuragi2. 1 _Hokkaido University Hospital, Sapporo, Japan;_ 2 _Hokkaido University, Sapporo, Japan_.

Background: Ovarian clear cell carcinoma (OCCC) is more prevalent in Japan than western countries, exhibits chemoresistant phenotype and poor survival. Although several mutations in OCCC have been reported, sensitive and rapid detection about actionable gene alterations in formalin-fixed-paraffin embedded (FFPE) tissue is required for clinical usage. In this study, we aimed to investigate major eight actionable mutations using next-generaion sequencing (NGS), and CNV-associated overexpression and phosphorylation related to their pathway using immunohistochemistry (IHC) in OCCC.

Methods: Sixty-nine consecutive patients diagnosed of OCCC in our hospital were included. SNV analyses of FFPE tissues from the primary lesions were investigated using the newly-developed GeneReader NGS platform and the Actionable Insight Tumour Panel (Qiagen) with high availability to old FFPE sample, and IHC was performed using tissue microarray (TMA).

Results: Median (range) follow-up period was 46 (0-270) months. In FIGO2014 system, 43 patients (62%) were in stage I, 3 (4%) in stage II, 19 (28%) in stage III, and 4 (6%) in stage IV. Genomic DNAs could be extracted and be analyzed in NGS from 61 FFPE specimens (88%), of which median storage period was 116 (22-269) months. Somatic gene mutations were detected in seven genes of 40/61 patients; PIK3CA, 33 (54%); KRAS, 12 (20%); ERBB3, 4 (6.6%); EGFR, 4 (6.6%); BRAF, 3 (4.9%); RAF1, 1 (1.6%); ERBB2, 1 (1.6%). In comparison with Cosmic database and previous reports, PIK3CA, KRAS and BRAF mutation were detected more frequently in our cohort, that may be associated with higher performance of our deep sequencing, or with racial features of Japanese OCCC population. PIK3CA mutation was not correlated with prognosis in overall survival (OS), progression-free survival (PFS) and OS after recurrence. However, in the patients with KRAS mutation, median OS after recurrence was 5.5 months, which was significantly worse than that without the mutation (31 months, p value = 0.0274), although OS and PFS were not significantly different. KRAS mutation was positively correlated with MET, HER2 and HER3 overexpression and AKT phosphorylation in IHC.

Conclusion: We could more frequently detect several actionable gene mutations even in old FFPE specimens, which may warrant the use of deep sequencing analysis about actionable gene mutations.

#1613

Enhanced sensitivity HERmark® assay: A sensitive and quantitative immunoassay superior to immunohistochemistry for the measurement of low HER2 protein levels in formalin-fixed, paraffin-embedded samples.

Gerald Wallweber, Roy Ravanera, Paul Theobald, Weidong Huang, Christos Petropoulos. _Monogram Biosciences, Laboratory Corporation of America® Holdings, South San Francisco, CA_.

Introduction: Breast cancers that express low levels of HER2 that are not considered HER2-positive disease by current HER2 classification standards are the targeted treatment populations for many new and promising HER2-targeted therapies, e.g. HER2 antibody-drug conjugates, tyrosine kinase inhibitors and vaccines. HER2 status is routinely determined by using immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). These technologies may have limited utility or lack the sensitivity required to adequately stratify low HER2 protein expression. Following upon the successes of the HERmark Breast Cancer Assay, we have developed the Enhanced Sensitivity HERmark Assay (ESHA) to quantify low HER2 protein expression in formalin-fixed, paraffin-embedded (FFPE) samples. The ESHA format relies on dual-antibody binding for the proximity-dependent release of a fluorescent reporter, which is measured with high sensitivity and reproducibility via capillary electrophoresis to accurately quantify HER2 protein expression.

Methods: The HERmark and HERmark ESHA assays utilize HER2 mAb8 conjugated to a light-releasable VeraTag® reporter and HER2 mAb15 conjugated to a photosensitizer. Low HER2 expressing cancer cell lines were used to optimize assay conditions that improved signal strength and extended the lower end of the dynamic range of the HERmark assay. FFPE cancer cell lines and breast tumors were utilized to evaluate assay background, reproducibility, and to compare HER2 assessments based on HERmark, HERmark ESHA and HER2 IHC.

Results: The ESHA format expanded the HERmark assay lower limit of quantification by more than 10-fold, and increased the relative fluorescent signal by an average of 16-fold, which also resulted in a decrease in assay variation. ESHA measurements of HER2 protein in a panel of FFPE cell lines correlated with HER2 gene expression levels as reported in the cancer cell line encyclopedia (https://portals.broadinstitute.org/ccle, R2 = 0.857). ESHA measurements of HER2 protein correlated well with conventional IHC scores, along with the added ability to quantitatively distinguish HER2 protein expression levels.

Conclusions: We have developed an Enhanced Sensitivity HERmark Assay for the quantification of low HER2 protein expression in formalin-fixed, paraffin-embedded tissue samples. This assay can be a particularly useful tool for the development of novel HER2-targeting therapies in patient populations that are not considered HER2 positive by conventional HER2 classification standards. Improving the stratification of HER2 expression may better define, and perhaps expand, the number of patients that are more likely to benefit from HER2-targeted therapy.

#1614

**Alternative splicing of** NTRK **and** ROS1 **fusion transcripts promotes the expression of oncogenic fusions targeted by entrectinib.**

Ian M. Silverman, Thuy Nguyen, Adam Baer, Heather A. Ely, Marissa Chen, Kristen M. Smith, Jennifer Lamoureux, Jason Christiansen, Robert Shoemaker. _Ignyta, San Diego, CA_.

Oncogenic kinase fusions, including those involving NTRK (NTRK1/2/3) and ROS1, are potent oncogenes found in a variety of cancer types. In contrast to single nucleotide variants, insertions and deletions, oncogenic fusion breakpoints are highly variable, creating a significant challenge for diagnostic detection. The most widespread next generation sequencing (NGS) assays used to detect oncogenic fusions are DNA-based and require algorithms to predict whether the fusion is in-frame and oncogenic. In contrast, RNA-based NGS tests directly sequence the expressed fusion transcripts, allowing precise determination of coding and oncogenic potential.

As part of the STARTRK-2 clinical trial for entrectinib, a CNS-active, potent, and selective inhibitor of TRK and ROS1, we screened tumors from patients for oncogenic fusions involving NTRK and ROS1 using the Trailblaze Pharos® assay. Trailblaze Pharos® is an RNA-based NGS assay that detects fusions without a priori knowledge of the rearrangement partner. We observed multiple fusion transcript isoforms expressed in 33% of NTRK and ROS1 fusion positive samples, with 12% of cases showing ≥ 3 distinct isoforms. Furthermore, 73% of multi-isoform fusions expressed both in-frame and out-of-frame fusion transcripts. Importantly, we observed cases where alternative splicing was required for expression of an in-frame fusion. These results demonstrate that alternative splicing of NTRK and ROS1 fusion transcripts is common in tumors and increases the likelihood of an in-frame and oncogenic fusion being expressed.

We also performed total RNA Sequencing on five cell line models driven by oncogenic kinase fusions, including; KM-12 (TPM3-NTRK1), M0-91 (ETV6-NTRK3), IMS-M2 (ETV6-NTRK3), HCC-78 (SLC34A2-ROS1) and U-118 MG (GOPC-ROS1). We detected multiple fusion transcript isoforms in 3 of 5 (60%) models. In KM-12 cells, we observed three distinct TPM3-NTRK1 isoforms, two of which were in-frame. A third isoform included the 3' untranslated region of TPM3, spliced to the middle of NTRK1 exon 8, and was out-of-frame. In both M0-91 and HCC-78 cells, two in-frame fusion transcript isoforms were observed, although one isoform in M0-91 lacked the kinase domain and was predicted to be non-functional.

In this study, we uncovered widespread alternative splicing of fusion transcripts in tumors from patients and cell lines harboring NTRK and ROS1 fusions. In some cases, this alternative splicing was required for expression of an in-frame fusion. These findings have important implications, especially for DNA-based assays, which do not interrogate post-transcriptional processing of fusions, and thus have limited ability to predict mixed splicing populations. A more detailed understanding of alternative splicing of oncogenic kinase fusions will improve the identification of actionable fusions and ultimately lead to better outcomes for patients.

#1615

First-line CDK4/6 inhibitor treatment for HR+, HER2-negative metastatic breast cancer (MBC).

Kent F. Hoskins,1 Paul Richards,2 Kari Wisinski3. 1 _University of Illinois at Chicago, Chicago, IL;_ 2 _DBA Blue Ridge Cancer Care, Salem, VA;_ 3 _University of Wisconsin Carbone Cancer Center, Madison, WI_.

Introduction: Endocrine therapy (ET) is a standard first-line (1L) therapy for hormone receptor-positive (HR+), human epidermal growth factor receptor 2-negative (HER2-) MBC, but treatment resistance eventually emerges. Dysregulation of cyclin-dependent kinases 4 and 6 (CDK4/6) is implicated in ET resistance; thus, combined ET and CDK4/6 inhibition is a rational approach to prolong disease control and delay cytotoxic therapy. We summarize available Phase 3 data of CDK4/6 inhibitors (CDK4/6i) + ET for the 1L treatment of HR+, HER2- MBC, and highlight ongoing studies.

Methods: PubMed was searched for: "advanced breast cancer" AND ("first" OR "initial" OR "previously untreated") AND (ribociclib OR palbociclib OR abemaciclib). Results were manually filtered for Phase 3 studies of 1L treatment. Abstracts at AACR, ASCO, ESMO, and SABCS from 2016-2017 were also considered; clinicaltrials.gov was searched for ongoing trials.

Results: PubMed yielded 42 results, with 5 relevant after filtering; key congresses resulted in 34 abstracts. Four randomized Phase 3 trials investigating 1L CDK4/6i + ET reported primary data and subgroup analyses. PALOMA-2 (palbociclib [PAL] + letrozole [LET]), MONALEESA-2 (ribociclib [RIB] + LET), and MONARCH-3 (abemaciclib [ABE] + nonsteroidal [NS] aromatase inhibitor [AI]) included postmenopausal women with HR+, HER2- MBC. Across the studies, median progression-free survival (PFS) with ET alone was 14.5-16.0 months, and was significantly prolonged with addition of a CDK4/6i: PAL 24.8 months (HR 0.58), RIB 25.3 months (HR 0.57), and ABE not reached (HR 0.54), all p<0.001. To date, no study has reported an overall survival advantage. PFS data from relevant subgroup analyses and comparison of study populations will be presented. Most common adverse events (>50% in any study) were neutropenia (PAL all-grade 80%/Grade 3 or 4 66%; RIB 74%/59%; ABE 41%/21%), diarrhea (PAL 26%/1%; RIB 35%/1%; ABE 81%/10%), nausea (PAL 35%/<1%; RIB 52%/2%; ABE 39%/1%), and infections (PAL not reported/4%; RIB 50%/4%; ABE 39%/5%). The MONALEESA-7 trial of RIB + tamoxifen/NSAI + goserelin, the only study in premenopausal women with ovarian suppression, met its PFS primary endpoint. Ongoing studies of CDK4/6i + 1L ET include: PAL + LET in postmenopausal Asian women (PALOMA-4), PAL + tamoxifen (NCT02668666) and RIB + LET (CompLEEment-1) in men and pre/postmenopausal women, PAL + AI followed by PAL + fulvestrant (vs continued PAL + AI) after emergence of ESR1 mutations (confer resistance to AIs) and before disease progression (PADA-1), and assessment of mechanisms of resistance to RIB + LET (NCT03050398).

Conclusion: Phase 3 trials support CDK4/6 inhibition + ET for the 1L treatment of women with HR+, HER2- MBC. Ongoing trials are evaluating the safety and efficacy of CDK4/6i with other ET, in more diverse populations, continuation of CDK4/6 inhibition after first progression, and mechanisms of resistance.

#1616

Novel caged hapten proximity assay platform enables detection of oncogenic signaling-associated complexes in the clinical pathology laboratory setting.

Brian D. Kelly,1 Nathan W. Polaske,1 Yuri Belosludtsev,1 Matthew A. Smith,2 Theresa Boyle,2 Y. Ann Chen,2 Eric B. Haura2. 1 _Ventana Medical Systems, Tucson, AZ;_ 2 _Moffitt Cancer Center, Tampa Bay, FL_.

Assays measuring protein complexes represent a novel class of protein-based biomarkers. Using proximity-based readouts, we and others have previously shown utility in monitoring signaling-associated complexes to predict response to targeted therapeutics in lung cancer. However, fluorescence-based proximity assay readouts are difficult to implement in a clinical setting and are not conducive to typical pathology laboratory workflow. Here, we provide evidence of a novel proximity-based technology platform that utilizes a "caged hapten" proximity readout followed by chromogenic immunohistochemistry (IHC) detection. We have synthesized haptens modified with a phosphorylated quinone methide precursor. This enzyme- labile caging group abrogates antibody binding; however, upon exposure to alkaline phosphatase (AP) the native hapten is regenerated. These caged haptens may be applied in a proximity assay format by the use of a first antibody labeled with caged haptens that can be uncaged by AP conjugated to the second antibody. Only when the two epitopes of interest are in close proximity to one another will the AP interact with the caged hapten and uncage it. The native hapten, which represents the protein complex population, can then be visualized by an anti-hapten antibody conjugated to horseradish peroxidase (HRP) followed by 3,3'-diaminobenzidine (DAB) detection. These reagents and assays are fully automated on the VENTANA DISCOVERY ULTRA platform. We demonstrate detection of β-Catenin:E-Cadherin, EGFR:GRB2 and MET:GRB2 complexes in formalin-fixed paraffin embedded (FFPE) lung cancer cell lines, patient-derived xenografts (PDX) and non-small cell lung cancer (NSCLC) patient specimens. Assay specificity was confirmed through technical controls involving reagent omission experiments, and biologically by treatment with small molecule kinase inhibitors that disrupt kinase:adaptor interactions. We revealed heterogeneous EGFR and MET signaling in MET-amplified PDX specimens, replicating previously-published findings. For EGFR:GRB2, we developed a pathologist-guided ordinal scoring system and evaluated inter-rater concordance using a tissue microarray containing 120 NSCLC cores. Kappa statistics and inter-rater correlations reveal excellent correlation among multiple raters.Collectively, these data support the adaptation of proximity-based approaches in clinical pathology settings. The use of caged hapten proximity assays on VENTANA automated slide stainers provides a novel and robust platform to evaluate protein complexes as potential biomarkers that may be incorporated as molecular correlates in ongoing early phase clinical trials. This approach also facilitates analysis of a large number of slides in a reproducible assay format enabling retrospective evaluation of tumor specimens from completed trials.

#1617

Utility of a solid-tumor NGS panel in the differential diagnosis of composite neoplasms with divergent phenotypes.

Andres M. Acosta, Mohamed Rizwan H. Al Rasheed, Dipti Panchal, Magdalena Rogozinska, Frederick G. Behm, Gayatry Mohapatra. _University of Illinois at Chicago, Chicago, IL_.

Introduction: Composite neoplasms (CN) are rare, diagnostically challenging lesions that require differentiating between three possibilities: clonal tumors with divergent phenotypes (mixed tumors, MT), collision of two synchronous adjacent tumors (CT) and tumor-to-tumor metastasis (TTM). To make this distinction, pathologists have relied on morphology, immunohistochemistry and limited molecular techniques such as single-gene sequencing. In this study, we performed next-generation sequencing (NGS) on 4 CN to illustrate the diagnostic utility of NGS-based approach in these rare tumors.

Materials and Methods: Of the 4 CN included in the study, 2 had been diagnosed as MT containing phenotypically different cell populations (mixed adenoneuroendocrine carcinoma of the gallbladder and metastatic papillary thyroid carcinoma with squamous dedifferentiation), while the remaining 2 had been interpreted as TTM (esophageal adenocarcinoma to lung adenocarcinoma and small cell carcinoma of the lung to meningeal melanoma). Pathology diagnoses were made using clinical, histologic and immunophenotypic information. Manual dissection of the tumor components was performed on formalin-fixed, paraffin-embedded tissue sections for extraction of DNA and RNA. NGS was performed using the Oncomine Comprehensive Panel on Ion S5XL sequencer. Ion Reporter variant caller pipeline was used for data analysis.

Results: Sequencing results confirmed the histopathologic diagnosis in all cases (Table 1). Importantly, comparison of our results with data from TCGA studies allowed a meaningful interpretation of the genetic aberrations found, and shed light on the biology of these lesions. | |  | |  | |  | |

|

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

Table 1 – Integration of immunophenotypic and massively parallel sequencing data from 4 composite neoplasms

N | Diagnosis | Components | IHC | Gene | Type of aberration | Variant effect (AA change) | Interpretation | CN

|

1 | Mixed adenoneuroendocrine carcinoma | Adenocarcinoma | CK20 -/p53 -/p63 -/CK7 +/ CDX2 + | ERBB2 | CNV | \-- | Amplification | 8.57

|

|  | |  | CCNE1 | CNV | \-- | Amplification | 16.7

|

|  | Large cell neuroendocrine carcinoma | CK20 -/p53 -/p63 -/CK7 +/ CDX2 +/Chromo +/Synapto + | CCND1 | CNV | \-- | Amplification | 4.63

|

|  | |  | ATM | CNV | \-- | Deletion | 0.39

|

|  | |  | TP53 | CNV | \-- | Deletion | 0.56

|

|  | |  | CCNE1 | CNV | \-- | Amplification | 40

|

2 | Metastatic papillary thyroid carcinoma with squamous dedifferentiation | Papillary thyroid carcinoma | CK 5/6 -/p40 -/PAX8 +/

TTF1 +/TG + | BRAF | Mutation (c.1799T>A) | Missense (p.Val600Glu) | Deleterious (GOF) | \--

|

|  | Squamous cell carcinoma | CK5/6 +/p40 +/PAX8 +/TTF1 -/TG - | BRAF | Mutation (c.1799T>A) | Missense (p.Val600Glu) | Deleterious (GOF) | \--

|

|  | |  | PIK3CA | Mutation (c.1633G>A) | Missense (p.Glu545Lys) | Deleterious (GOF) | \--

|

3 | Esophageal adenocarcinoma

metastatic to lung adenocarcinoma | Low grade adenocarcinoma (lung) | TTF1 +/HER2 - | CDKN2A | CNV | \-- | Deletion | 0.76

|

|  | |  | CDKN2A | Mutation (c.170C>T) | Missense (p.Ala57Val) | Deleterious (LOF) | \--

|

|  | |  | KRAS | Mutation (c.35G>T) | Missense (p.Gly12Val) | Deleterious (GOF) | \--

|

|  | |  | STK11 | Mutation (c.580G>T) | Missense (p.Asp194Tyr) | Deleterious (LOF) | \--

|

|  | High grade adenocarcinoma (lung) | TTF1 -/HER2 + | CDKN2A | Mutation (c.170C>T) | Missense (p.Ala57Val) | Deleterious (LOF) | \--

|

|  | |  | KRAS | Mutation (c.35G>T) | Missense (p.Gly12Val) | Deleterious (GOF) | \--

|

|  | |  | STK11 | Mutation (c.580G>T) | Missense (p.Asp194Tyr) | Deleterious (LOF) | \--

|

|  | High grade adenocarcinoma (esophagus) | \-- | CDKN2A | Mutation (c.170C>T) | Missense (p.Ala57Val) | Deleterious (LOF) | \--

|

|  | |  | KRAS | CNV | \-- | Amplification | 22.01

|

|  | |  | TP53 | Mutation (c.614A>G) | Missense (p.Tyr205Cys) | Deleterious (LOF) | \--

|

4 | Small cell carcinoma of the lung combined metastatic to meningeal melanoma | Small cell carcinoma | TTF1 +/Synapto +/Chromo +/AE1+3 +/CAM 5.2 +/MelanA -/HMB 45 - | RB1 | Mutation (c.1183C>T) | Nonsense | Deleterious (LOF) | |

|  | |  | GAS6 | CNV | \-- | Amplification | 6.14

|

|  | |  | TP53 | Mutation (c.473G>T) | Missense (p.Arg158Leu) | Deleterious (LOF) | |

|  | Melanoma | TTF1 -/Synapto -/Chromo -/AE1+3 -/CAM 5.2 -/Melan-A +/Pan-M +/MITF + | NRAS | Mutation (c.35G>A) | Missense (p.Gly12Asp) | Deleterious (GOF) | |

|  | |  | TERT | CNV | \-- | Amplification | 21.34

|

Conclusion: This study illustrates the diagnostic utility of NGS in tumors with more than a single histologic component. Additionally, it demonstrates a potential role for NGS in the detection of clinically actionable targets in a group of neoplasms that lack standardized treatment.

#1618

**Simultaneous isolation of exosomes and cfDNA from liquid biopsies using universal kit based on SubX-Matrix** TM **technology.**

Andrei G. Malykh,1 Anastasia Malek,2 Anna Lokshin,3 Vladimir Evtushenko4. 1 _Capital Biosciences, Inc., Gaithersburg, MD;_ 2 _N.N. Petrov Institute of Oncology, St. Petersburg, Russian Federation;_ 3 _University of Pittsburgh Cancer Institute, Pittsburgh, PA;_ 4 _Russian State Center of Radiology and Surgery Technologies, St. Petersburg, Russian Federation_.

In 2016 we have developed novel approach for isolation of circulating cell-free DNA employing the proprietary bi-functional substance SubX® that tightly binds DNA and forms sandwich [cfDNA- SubX®-magnetic particles]. These sandwich aggregates are easily separated from bio-liquid with magnet followed by cfDNA isolation by speedy protocol. Our approach does not require Proteinase K digestion and 2-3-fold dilution of bio-liquid with lysis/binding buffer in contrast to all commercially available kits. Our kit has been evaluated in various research and clinical labs for cfDNA isolation from serum, plasma and urine of patients with different forms of cancer. SubX®-approach also allows for isolation of cfDNA from large volumes of urine samples without its concentration. We demonstrated linear increase of the DNA yield with liquid biopsy volume. Another unique property of the SubX® is that both ends of the molecule display lipid binding groups. This feature allows each molecule of SubX® to anchor two exosomes (i.e. dimerize). Excess of SubX® molecules in the solution results in aggregation of up to 10-15 exosomes and formation of micron-size particles that are easily precipitated in a brief 14K x g (or 30 min 2K x g) centrifugation step. A specially designed buffer allows for reconstitution of the pelleted exosomes back to monomer format for downstream applications. Flow cytometry with specific surface marker CD9 confirmed that SubX®-aggregated vesicles are exosomes. Analysis of membrane-bound protein markers and microRNA profile of exosomes isolated from plasma of prostate cancer patients will be presented. Difference in SubX® binding strength for cfDNA and exosomes allows for consequent isolation from the [cfDNA/exosomes- SubX®-magnetic particles] pellet first exosomes, then cfDNA. Eventually, we combined both approaches in one kit for isolation of cfDNA and exosomes from the same sample. This simultaneous isolation protocol is simple and fast, it allows to employ minimal volume of valuable bio samples as well as to reduce labor and cost. The kit was validated in serum and urine samples from patients with ovarian and pancreatic cancer. Exosome nature of isolated vesicles was confirmed by three independents methods - light scattering, immunological and electron microscopy. Thus, one could analyze both genetic (DNA and RNA) as well as protein markers present in the same liquid biopsy sample.

#1619

Personalized medicine: A CLIA-certified high-throughput drug screening platform for ovarian cancer.

Hallie A. Swan,1 Rachele Rosati,2 Caroline Bridgwater,1 Michael J. Churchill,1 Roland M. Watt,1 Reid C. Shaw,2 Stephanie A. Murphy,1 Robert L. Diaz,1 Shalini C. Pereira,1 Franz X. Schaub,1 Carla Grandori1. 1 _SEngine Precision Medicine, Seattle, WA;_ 2 _Cure First, Seattle, WA_.

Background:

Metastatic disease in ovarian cancer is difficult to treat and patients often exhaust standard-of-care regimens. To get a better understanding of potential treatments, genomic data is used in some cases. However, this only points to therapeutic options in a minority of cases highlighting an urgent need to develop assays to identify potential therapies. Current functional tests are limited in the number of tested drugs. Here we present the first CLIA certified high-throughput functional assay employing organoid cultures derived from primary patient specimens to directly aid oncologists for personalized treatment selection (P.A.R.I.S. Assay, SEngine Precision Medicine, Seattle, WA).

Experimental Procedures:

Organoids are treated for 6 days with a library of 123 clinically relevant drugs. Compounds are evaluated at a multi-dose response curve and ranked by SPM score, which weights both the sensitivity (degree of cell death) and specificity, which compares the patient's tumor cells response to the drug relative to all prior patients. The results are further integrated with genomic data and reported to the clinician to highlight treatment options. SEngine has performed >150 drug screens and established high reproducibility including multiple ovarian cancer cases from either ascites, surgical or core biopsies.

Results:

Here we present two n-of-one patient studies. Patient 1 is a 48 year old woman with positive family history who was diagnosed with late stage serous ovarian cancer. SEngine generated cancer organoids and performed high-throughput screening with a panel of 123 drugs. Sensitivities to PARP inhibitors prompted SEngine to advise for germline testing which revealed a BRCA1 and TP53 mutation. The cells were also uniquely sensitive to paclitaxel and the combination with carboplatin resulted in remission indicating concordance with clinical data. In addition, highlighting the importance of functional screening, a unique response to a sub-group of EGFR inhibitors was identified, of potential consideration in case of recurrence.

The second case also has a family history of ovarian cancer, but no BRCA mutations were detected. Genomic data indicated FGF6 and FGF23 amplification which directly corresponded to a unique sensitivity to one FGFR inhibitor (AZD-4547). The organoids were also resistant to PARP inhibitors, consistent with the absence of BRCA mutations.

Impact:

We developed a robust ex vivo screening platform to objectively quantify patient specific sensitivity to a panel of 123 oncology drugs. SEngine is compiling a registry capturing clinical data, outcome following the P.A.R.I.S. test as well as genomic data. The power of high throughput technology and organoid isolation will enable the rapid selection of optimal individualized therapies as single agents or in combination.

#1620

ATR inhibition for the treatment of ATM-deficient gastric cancer.

Mu-Yan Cai,1 Hunter D. Reavis,1 Jie-Wei Chen,2 Chirag Ganesa,1 Bose Kochupurakkal,1 Geoffrey I. Shapiro,1 Alan D. D'Andrea1. 1 _Dana-Farber Cancer Institute, Boston, MA;_ 2 _Sun Yat-Sen University Cancer Center, Guangzhou, China_.

ATM and ATR are central kinases in the cellular response to DNA damage and play a crucial role in maintaining genome integrity. Inactivation of ATM has been observed in multiple tumor types and pre-clinical studies suggest that inhibition of ATR is synthetic-lethal in ATM-deficient tumors. More than 10% of gastric cancers carry potentially deleterious mutations in ATM and patients with ATM-deficient gastric cancers could potentially benefit from ATR inhibitor-based therapy. This study focuses on establishing the critical parameters for clinical development of ATR inhibitors in treating gastric cancer. First, we tested the cytotoxic response to ATR inhibition of ATM-deficient and ATM-complemented derivative of human fibroblasts. Next, we utilized a panel of gastric cancer cell lines that represent the spectrum of ATM mutations and ATM expression levels to further confirm the correlation between reduced ATM activity and the cytotoxic effects of ATR inhibition. Third, we evaluated the effect of a series of missense and truncating mutations in ATM that were identified in The Cancer Genome Atlas (TCGA) project. Finally, we developed an immunohistochemistry (IHC) assay to assess the expression level of ATM in gastric tumor samples. Our results confirmed that ATM-deficient human fibroblasts were acutely sensitive to ATR inhibition compared to ATM-complemented cells. Acute sensitivity to ATR inhibition was also observed in gastric cancer cell lines that carry deleterious mutations in ATM and in those that express very low levels of ATM. Complementation experiments with mutant versions of the ATM ORF using ATM-deficient human fibroblasts identified deleterious mutations in ATM that sensitize to ATR inhibition. The IHC assay was applied to a cohort of 208 primary gastric cancers. ATM expression was undetectable in approximately 11% of the samples; an additional 26% expressed very low levels. Our findings suggest that ATR inhibition may elicit favorable responses in gastric cancer patients with tumors harboring deleterious ATM mutations or very low levels of ATM expression. Targeted exon-sequencing methods that are used widely combined with IHC for ATM may be useful for patient selection.

#1621

Radotinib, a medicine for chronic myeloid leukemia, induces cell death of mantle cell lymphoma cells.

Sook-Kyoung Heo, Eui-Kyu Noh, Yoo Kyung Jeong, Jeong Yi Kim, Yunsuk Choi, Jaekyung Cheon, SuJin Koh, Jin Ho Baek, Young Joo Min, Jae-Cheol Jo. _Ulsan University Hospital, Ulsan, Republic of Korea_.

Background: Radotinib is a medicine for the treatment of some types of cancer. It is approved in South Korea for use as a second-line treatment of chronic myeloid leukemia (CML). Its mechanism of action involves inhibition of the tyrosine kinase Bcr-Abl and of platelet-derived growth factor receptor (PDGFR). It has been little known the effects of radotinib on mantle cell lymphoma cells (MCL). Methods: First, we examined cytotoxicity of radotinib on MCL cell line, MAVER-1 and REC-1. Also, cytotoxicity of radotinib on both cell lines which have a different genetic back ground. Annexin V positive cell, caspas-3 and -9 activities, cell cycle distribution and mitochondrial membrane potential (MMP) were observed by analyzed with flow cytometric analysis. And diverse signaling pathways were investigated by Western blotting in MCL cells.

Results: Interestingly, radotinib caused cell death of MCL cells. And radotinib induces G1-phase arrest in MAVER-1 and REC-1 cells. And it also inhibited the expression of CDK2, CDK4, and cyclin D1, D3 and E. Therefore, radotinib induces G1-phase arrest via suppression of CDK2, CDK4, and cyclin D1, D3 and E. Generally, the intrinsic apoptotic pathway involves mitochondrial activation and caspase and phosphatidylserine externalization. Radotinib induced Annexin V positive cells, and caspase pathway activation including caspase-3, -7 and -9. And its treatment remarkably decreased MMP in MCL cells at 72 h. As well as we observed that cytochrome c accumulated dose dependently in the cytosol of radotinib-treated MAVER-1 and REC-1 cells. Moreover, radotinib decreased the expression of Bcl-xL and Bcl-2, and increased the expression of Bax in MCL cells. These results indicate that radotinib induces cell death of MCL cells by induction of G1-phase arrest and mitochondrial-dependent apoptosis. Furthermore, the expression of p-AKT, AKT and ERK was significantly reduced by radotinib in MCL cells. Conclusion: Radotinib may play an important role as a candidate or chemosensitizer for treatment agent in MCL. These data indicate that radotinib has a potential for anti-cancer therapy in MCL.

#1622

In vivo study of dasatinib in combination with veliparib plus carboplatin in triple-negative breast cancer xenograft models.

Yuliang Sun, Xiaoqian Lin, Jennifer Carlson, Pradip De, Nandini Dey, Casey Williams, Brian Leyland-Jones. _Avera Cancer Institute, Sioux Falls, SD_.

Background: Recently, poly ADP ribose polymerase (PARP) inhibitors have shown potential to increase the sensitivity of platinum-based agents in tumor cells and provide therapeutic benefit for patients with advanced malignancies in BRCA or DNA repair pathway genes altered or WT condition (Basourakos et al., 2017; Gampenrieder et al., 2017). Src, a non-receptor tyrosine kinase, is involved in the regulation of cell proliferation, survival and apoptotic ability of cancer cells (Tryfonopoulos et al., 2011; Pusztai et al., 2014). Dasatinib (D) is a short-acting dual ABL/SRC family tyrosine kinase inhibitor (TKI), which has displayed anti-proliferative and anti-metastatic effectiveness against TNBC in both preclinical and clinical studies (Finn et al., 2011).

Our current study examines whether combinational inhibition of Src pathway (D) would exhibit greater antitumor activity of PARP inhibitor [veliparib (V)] plus carboplatin (C) in TNBC xenograft models with different genetic back grounds.

Methodology: a) We utilized MDA-MB-231, MDA-MB-468 and SUM149PT cells for in vivo study. b) TNBC cells were implanted subcutaneously into immune-compromised mice (NCr Nu/Nu, Taconic) to generate tumor xenografts. c) We evaluated the effects on tumor growth inhibition of D (12.5 mg/kg, i.p., Qd for 4 weeks) / V (25 mg/kg, oral, Bid for 5 days) / C (40mg/kg, i.v., once) as single agent or in combinations (Chan et al., 2012; De et al., 2014). d) For IHC studies, tumor tissue sections were stained with respective antibodies for proliferation, cell survival, angiogenesis and apoptosis, including: phospho-Src, Ki-67, phospho-S6 RP, phospho-Akt, phospho-ERK, CD31, cyclin D1 and cleaved caspase3. e) Student's t test was used for data analyses.

Results: We observed that 1) MDA-MB-231(KRAS/BRAF mutated) xenograft model: D alone or combined with V or C or in triple combination inhibited tumor growth, the best tumor inhibition result was induced by triple combination (p < 0.004); 2) MDA-MB-468 (PTEN null) xenograft model: D alone or combined with V or C or in triple combination significantly inhibited

tumor growth, the best tumor inhibition result was observed by triple combination (p < 0.00006); 3) SUM149PT (BRCA1 mutated, PTEN null) xenograft model: D alone or combined with V or C or in triple combination inhibited tumor growth, and triple combination, as well as D plus C, showed greater (p < 0.0025) than other combinations / single agents; 4) Single agent of D / V / C was well tolerated with less than 10% mean body weight loss compared with control groups in all models.

Conclusion: Our in vivo study suggests that D may enhance the antitumor activity of PARP inhibitor plus standard cytotoxic agent in TNBC xenograft tumors with different genetic backgrounds. Immunohistochemical studies of xenograft tumor samples are ongoing, the results of which will be presented at the meeting.

#1623

Inter-laboratory harmonization of next generation sequencing somatic mutation assays for cancer response prediction.

James C. Willey,1 Thomas M. Blomquist,1 Erin L. Crawford,1 Joshua Xu,2 Leihong Wu,2 Thomas Morrison3. 1 _Univ. of Toledo, Toledo, OH;_ 2 _National Center for Toxicological Research, FDA, Jefferson, AR;_ 3 _Accugenomics, Inc., Toledo, OH_.

Background: The potential for next generation sequencing (NGS) analysis in personalized diagnosis and treatment of cancer is advancing rapidly. Measurement of mutation fraction less than 5% may be clinically relevant in analysis of circulating tumor DNA and/or small cytologic samples. However, due to uncertainty introduced by variation in sample quality, the mutation fraction lower limit of detection (LOD) reported by clinical laboratories does not typically extend beyond this 5% threshold. Even with a more conservative LOD criterion, (e.g., 20% mutation fraction), poor harmony may result from inter-laboratory variation in skills to assess sample quality as well as specimen characteristics that are not evident even to expert clinical pathologists. Thus, a critical unmet need is methods that harmonize reporting of mutation fraction measured by NGS by accurately estimating specimen-specific confidence limits for each measurement. Methods: We designed a mixture of spike-in competitive internal standards for tumor somatic mutations in 32 currently actionable genes that enable quality-controlled measurement by targeted NGS using either PCR-amplicon or hybrid-capture enrichment. We also developed a data analysis pipeline suitable to separate target and internal standard sequencing reads prior to analysis of target copy number and sequencing errors. Through participation in the FDA-led SEquencing Quality Control Project Phase 2 (SEQC2), where a set of highly characterized cell line mixtures was developed to assess cross-site reproducibility and mutation detection accuracy of multiple targeted sequencing platforms, we aim to comprehensively evaluate the utility of the spike-in mixture with various targeted NGS panels to measure 95% confidence limits around each somatic mutation fraction measurement. Results: In pilot studies, combining the mixture of internal standards with specimens prior to NGS library preparation enabled reliable measurement of 95% confidence limits around each somatic mutation measurement. Further, using the internal standards to control for specimen adequacy enabled reliable measurement of actionable mutation fraction at as low as 0.01% in suitable specimens. Conclusion: We expect that the full cross-site SEQC2 study will provide further support for the role of competitive spike-in mixtures to provide reliable 95% confidence limits around each measurement of somatic mutation fraction in a clinical specimen. Disclaimer: The views expressed here are those of the authors only and do not necessarily express the views/policies of the FDA.

#1624

Reliability of immune biomarker assessment in breast cancer: A report on interobserver variability from studies at a single institution.

Cinzia Solinas, Gert Van den Eynden, Alexandre De Wind, François Richard, Anaïs Boisson, Soizic Garaud, Karen Willard-Gallo. _Institut Jules Bordet, Bruxelles, Belgium_.

Reproducible evaluation of immune infiltration parameters is needed for the development of reliable biomarkers in the era of cancer immunotherapy. This study investigated inter-observer reproducibility when evaluating tumor-infiltrating lymphocytes (TIL), tertiary lymphoid structures (TLS), PD-1 and PD-L1 positivity on immunohistochemically-stained (IHC) breast cancer (BC) tissues, including detailed analysis of spatial positioning in the tumor microenvironment and expression on individual positive cells. Retrospectively collected archival BC samples (N=441) were dual IHC-stained for CD3 (pan T cells) plus CD20 (pan B cells) for TIL and TLS assessment with a consecutive tissue section dual-IHC stained for PD-1 and PD-L1. The 882 tissue sections were independently read by two immuno-pathologists blinded to the clinical data for specific studies between 2014 and 2017. Five independent study series were used for the current analysis: 1) samples of BC relapses, 2) residual disease in breast after neodjuvant treatment, and 3) three independent series of untreated primary tumors. Intraclass correlation coefficients for evaluations are shown in the table below: | Relapses | Residual disease | Untreated primary tumors

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

|

Series 1: N=88 | Series 2: N=20 | Series 3: N=85 | Series 4: N=113 | Series 5: N=135

|

2014 | 2014 | 2014-2015 | 2016 | 2017

|

ICC | ICC | ICC | ICC | ICC

%Stromal TIL | 0.85 [0.76-0.90] | 0.71 [0.27-0.89] | 0.79 [0.68-0.87] | 0.74 [0.62-0.82] | 0.77 [0.68-0.83]

%Intratumoral TIL | 0.40 [0.09-0.61] | 0.63 [0.08-0.86] | 0.73 [0.58-0.82] | 0.87 [0.81-0.91] | 0.64 [0.51-0.74]

%Global TIL | 0.87 [0.80-0.92] | 0.79 [0.47-0.92] | 0.83 [0.74-0.89] | 0.80 [0.72-0.86] | 0.81 [0.73-0.86]

%CD3 | 0.83 [0.74-0.89] | 0.71 [0.28-0.89] | 0.81 [0.70-0.88] | 0.72 [0.60-0.80] | 0.73 [0.63-0.81]

%CD20 | 0.74 [0.62-0.81] | 0.81 [0.52-0.92] | 0.76 [0.63-0.84] | 0.78 [0.68-0.84] | 0.78 [0.70-0.84]

TLS | 0.80 [0.70-0.87] | 0.78 [0.45-0.91] | 0.83 [0.74-0.89] | 0.95 [0.93-0.97] | 0.97 [0.96-0.98]

%PD-L1 tumor cells | 0.78 [0.66-0.86] | 0.56 [-0.12-0.82] | 0.82 [0.73-0.89] | 0.62 [0.45-0.74] | 0.45 [0.25-0.61]

%PD-L1 stromal cells | 0.61 [0.40-0.74] | -1.56 [-1.53-0.60] | 0.43 [0.12-0.63] | 0.61 [0.44-0.73] | 0.31 [0.05-0.5]

%PD-L1 immune cells | 0.53 [0.29-0.70] | 0.94 [0.84-0.97] | 0.29 [-0.09-0.54] | 0.38 [0.10-0.57] | 0.10 [-0.25-0.35]

%PD-1 TLS | -0.00 [-0.54-0.35] | NA | 0.72 [0.57-0.82] | 0.57 [0.35-0.72] | 0.76 [0.62-0.84]

Cohen's κ coefficients reveal poor agreement (κ<0.4) for assessment of the categorical global PD-1 and PD-L1 variables (cut-off >1%). Sporadically, fair agreement (κ>0.4) was observed for PD-L1 expressed by immune and tumor cells. Globally, the inter-observer agreement remained constant overtime. Poor reproducibility for categorical global PD-1 and global PD-L1 assessment was observed. On the contrary, pathologists have excellent reproducibility for assessing global TIL and TLS, good/excellent reproducibility for PD-1 in TLS (primary tumors) and for PD-L1 expression on tumor cells which make these of great interest for clinical use.

#1625

Clinical biomarkers of survival in renal cell carcinoma patients treated with sorafenib.

Daniel James Crona,1 Andrew D. Skol,2 Veli-Matti Leppänen,3 Dylan M. Glubb,4 Amy S. Etheridge,1 Eleanor Hilliard,5 Carol Peña,6 Yuri K. Peterson,5 Nancy Klauber-DeMore,5 Kari K. Alitalo,3 Federico Innocenti1. 1 _Univ. of North Carolina, Chapel Hill, NC;_ 2 _University of Chicago, Chicago, IL;_ 3 _University of Helsink, Helsinki, Finland;_ 4 _QIMR Berghofer Medical Research Institute, Brisbane, Australia;_ 5 _Medical University of South Carolina, Charleston, SC;_ 6 _Bayer HealthCare Pharmaceuticals, Montville, NJ_.

Purpose: To discover genetic determinants of overall survival (OS) in metastatic renal cell carcinoma (mRCC) patients treated with sorafenib.

Patients and Methods: Information for 11,117 germline DNA variants from 56 genes was obtained, using both Illumina GoldenGate genotyping and imputation, from 295 patients originally enrolled in the phase III TARGET trial (155 sorafenib and 140 placebo patients). OS was this genotyping study's primary endpoint, and associations between variants and OS were tested using multivariate Cox regression. Associations were considered significant if P<0.05 and FDR q<0.1. Bioinformatic resources helped prioritize variants for functional validation. Haploreg v4 and RegulomeDB were used to help mine ENCODE data, and SIFT/PolyPhen-2 predicted functional effects of nonsynonymous variants. Dual reporter gene luciferase assays assessed variant effects on promoter activity. Isogenic cell lines were created, and direct cell proliferation assays elucidated variant effects on cell proliferation/cytotoxicity, while endothelial branch formation assays assessed variant effects on endothelial tube formation. Western blotting evaluated nonsynonymous variant effects on receptor phosphorylation, and pulse-chase experiments assessed variant effects on post-translational processing of proteins.

Results: Five intragenic variants significantly associated with OS. Among patients in the sorafenib arm, VEGFA rs1885657 (HR=17.3, 95% CI 5.7-52.7; P=1.4x10-4), ITGAV rs3816375 (HR=5.9, 95% CI 2.1-16.4; P=4.9x10-4), and WWOX rs8047917 (HR=4.1, 95% CI 1.9-8.8; P=3.3x10-4) associated with shorter OS. Among patients from both arms, FLT4 rs307826 (HR=13.8, 95% CI 3.0-62.6; P=1.2x10-4) and VEGFA rs3024987 (HR=3.0, 95% CI 1.7-5.4; P=8.3x10-5) associated with shorter OS. No significant associations were discovered in the placebo arm. Bioinformatic evidence was used to prioritize rs1885657, rs58159269 (in complete linkage disequilibrium with rs1885657), and rs307826. rs1885657 and rs58159269 increased luciferase activity in human endothelial (TIME, LPEC) and mRCC (Caki-1) cells, and rs58159269 increased cell proliferation/reduced sorafenib cytotoxicity, and reduced endothelial tube formation in TIME cells. rs307826 increased VEGFR-3 phosphorylation, and led to post-translational VEGFR-3 processing differences in HUVECs.

Conclusions: Regulatory VEGFA and FLT4 variants associated with OS in mRCC patients induce cellular alterations that affect angiogenesis and sorafenib activity. These are novel and potentially impactful clinical biomarkers for mRCC and sorafenib, and their relevance to other multikinase inhibitors used in mRCC should be tested.

#1626

Sensitive detection of MET exon 14 skipping by RT-qPCR and next generation sequencing.

Mukund Patel, Honey Polur, Patrick Hurban. _Q2 Lab Solutions, Morrisville, NC_.

The MET receptor tyrosine kinase (MET) gene is a proto-oncogene whose abnormal activation can trigger tumor growth, angiogenesis, and metastasis. Numerous mutations have been identified that lead to over-activation of the gene, including well characterized amplification and somatic mutations. Mutations that lead to the skipping of MET exon 14 are likely to be highly predictive of response to MET inhibition. Exon 14 contains a motif that is required for the efficient recruitment of the ubiquitin ligase, CBL, which targets MET for degradation. Loss of exon 14 leads to prolonged stability of the MET protein which causes increased signaling upon stimulation by hepatocyte growth factor. Over 200 distinct mutations have been identified that cause MET exon 14 skipping ranging from single nucleotide mutations to large insertion/deletions. Due to this complexity, detecting MET exon 14 skipping mutations using DNA-based NGS hybridization capture methods is challenging. RNA-based testing can circumvent the complexity of DNA-based changes. We developed a RT-qPCR assay that detects MET exon 14 skipping independent of the causal mutation. Analytical validation using RNA admixtures demonstrated 1% sensitivity of detecting MET exon 14 mutant transcripts. 100 FFPE lung adenocarcinomas were screened with the RT-qPCR assay. Of 100, two samples (2%) were identified as positive for MET exon 14 skipping. The two positive samples along with three negative samples were orthogonally confirmed using the Illumina TruSight Tumor 170 hybridization capture-based targeted panel assay that simultaneously analyzes DNA and RNA. Overall, the number of samples identified positive for MET exon 14 by RT-qPCR test is in line with the frequency observed from other studies in lung adenocarcinomas of about ~3%. The assay can detect as little as 1% transcripts with MET exon 14 skipping suggesting that we have developed an accurate and sensitive RNA-based assay for use in clinical trials.

#1627

Custom VariantFlex technology allows rapid design and testing of reference materials for tumor genotyping assays.

Catherine Huang,1 Ram Santhanam,2 Dan Brudzewsky,2 Trevor Brown,2 Russell Garlick,2 Bharathi Anekella1. 1 _Seracare Life Sciences, Gaithersburg, MD;_ 2 _Seracare Life Sciences, Milford, MA_.

Introduction: Development and validation of tumor genotyping assays requires highly characterized reference materials for effective assessment of accuracy, precision, reproducibility and limits of detection. Highly multiplexed reference materials can facilitate and expedite these studies; however, there is not a single reference material that is appropriate for all assays. Traditional cancer cell line methods or genetic engineering methods can take 6 - 9 months, so more flexible solutions for reference materials are needed. Here we describe the development of VariantFlex technology, which enables a laboratory to configure a reference material to contain the variants of interest at the allele frequency of relevance to their studies in rapid time. Methods: A library of biosynthetic constructs was generated that contains ~140 somatic cancer DNA variants (SNVs, small and large indels and tandem duplications), ~27 RNA fusions, and 6 very large gene region constructs for copy number gains. TaqMan® digital PCR assays were developed to precisely quantify the variant allele fraction, fusion RNA concentration or copy number gain. As an example, biosynthetic constructs bearing multiple SNVs in TP53, PIK3CA and EGFR, large deletions in EGFR, and the structural variants NCOA4-RET and TPR-ALK, were selected from the VariantFlex library and spiked into DNA extracted from the highly characterized GM24385 cell line. The mixture was processed to obtain ctDNA-like profile, encapsulated to provide stability, and then diluted in artificial plasma to generate a customized reference material for liquid biopsy. Results: The ctDNA reference material was quantified by digital PCR and performance was verified on hybrid capture and amplicon based NGS assays. Variant allele frequency as low as 0.125% VAF was accurately targeted (range for 0.125% target was +/- 0.025% by digital PCR). The VariantFlex technology has been validated to work with amplified NGS workflows, hybrid-capture based NGS assays as well as non-NGS assays such as qPCR and array based methods. Conclusions: This study shows how a large library of cancer-related variants can facilitate rapid production of customized reference materials. The flexibility to include exactly the variants of interest, at the critical allele frequencies, and in the most patient-like format will facilitate assay development and validation.

#1628

The molecular landscape of sarcoma can inform selection of personalized chemotherapy.

Sheeno P. Thyparambil,1 Shankar Sellappan,1 Dongyao Yan,1 Fabiola Cecchi,1 Antoine Italiano,2 Todd A. Hembrough1. 1 _NantOmics, Rockville, MD;_ 2 _Institut Bergonie, Bordeaux, France_.

Background: Chemotherapy represents the cornerstone of treatment for patients with advanced sarcomas. Anthracyclines are the first line of treatment; however, other agents such as gemcitabine, topotecan, taxanes and temozolomide have shown clinical activity in sarcoma patients. While no biomarker for chemotherapy has been approved for clinical use, a growing body of literature has identified tumor molecular characteristics predictive of response or resistance to chemotherapy. These include expression of the proteins ERCC1 (platinum), TUBB3 (taxane), TOP2A (anthracyclines), TOP1 (irinotecan, topotecan), hENT1/RRM1 (gemcitabine) and MGMT (temozolomide). Our goal was to assess the landscape of chemotherapy biomarkers in several sarcoma subtypes to identify potentially beneficial treatment regimens.

Methods: Formalin-fixed, paraffin-embedded tumor tissues from clinical samples of sarcoma representing 5 subtypes were microdissected and assayed with the GPS Cancer molecular test. The test combines whole-genome sequencing, RNA-seq, and proteomic expression analysis of 30 biomarker proteins using mass spectrometry.

Results: Seventy-five sarcoma samples were characterized (Table 1). Expression of protein biomarkers varied by sarcoma subtype. Of interest, approximately two-thirds of leiomyosarcomas lacked resistance markers for platinum (ERCC1) and taxanes (TUBB3). Of osteosarcomas, 93% expressed the response marker for gemcitabine (hENT1), while only 14% expressed the protein marker of gemcitabine resistance (RRM1). The vast majority of Ewing's sarcomas expressed MGMT, a marker of temozolomide resistance.

Conclusions: In clinical sarcoma samples, proteomic analysis can identify distinct patterns of expression that are predictive of response or resistance to chemotherapies. Additional analysis is in progress; correlation between expression of chemotherapeutic biomarkers and clinical outcomes in a subset of sarcoma patients will be presented. | |  | |  | |

|

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

Table 1. Proportions of sarcoma patients (N=75) with tumor expression of chemotherapeutic biomarker proteins

|

Markers of response (agent) | Markers of resistance (agent)

Sarcoma subtype | TOP1(topotecan, irinotecan) | TOP2A(doxorubicin) | hENT1(gemcitabine) | ERCC1(platinum) | TUBB3(taxane) | RRM1(gemcitabine) | MGMT(temozolomide)

Leiomyosarcoma (n=25) | 4 | 13 | 32 | 28 | 32 | 16 | 58

Liposarcoma (n=13) | 15 | 0 | 46 | 54 | 62 | 16 | 67

Ewing's sarcoma (n=12) | 33 | 8 | 33 | 83 | 50 | 42 | 88

Osteosarcoma (n=14) | 7 | 7 | 93 | 46 | 57 | 14 | 67

Rhabdomyosarcoma (n=11) | 36 | 27 | 82 | 64 | 45 | 64 | 88

Tumor expression is defined as quantitated protein above the threshold determined to be indicative of clinical response/resistance. The thresholds (in attomoles per microgram of total tumor protein) are as follows: TOP1: 1340, TOP2A: 1570, hENT1: 100, ERCC1: 75, TUBB3: 850, RRM1: 700, MGMT: 200.

### Pediatric Cancer

#1629

Targeting resistance mechanisms to CDK4/6 inhibitors in Ewing sarcoma with an IGF1R inhibitor drug combination strategy.

Lillian M. Guenther,1 Neekesh V. Dharia,1 Linda Ross,1 Amy S. Conway,1 Amanda L. Robichaud,1 Alanna J. Church,2 Rajarshi Guha,3 Mindy I. Davis,3 Gabriela Alexe,1 Jaume Mora,4 Federica Piccioni,5 Kimberly Stegmaier1. 1 _Dana-Farber Cancer Institute, Boston, MA;_ 2 _Boston Children's Hospital, Boston, MA;_ 3 _National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, MD;_ 4 _Hospital Sant Joan de Déu, Barcelona, Spain;_ 5 _Broad Institute, Cambridge, MA_.

Novel targeted drug combinations have transformed the care of subsets of patients with cancer. In pediatric malignancies, however, where tumor genomes are typically simple with infrequent targetable mutations, there have been few new FDA-approved targeted drugs. The CDK4/6 pathway, which has been extensively validated as a target in estrogen receptor-positive breast cancer, recently emerged as a dependency in Ewing sarcoma, an aggressive pediatric bone tumor. Given the heightened efficacy of CDK4/6 inhibitors (CDK4/6i) in combination with other targeted drugs in breast cancer, as well as the known propensity of resistance to emerge with single agent targeted cancer therapy, we aimed to identify CDK4/6i resistance mechanisms and biologically relevant drug combinations in Ewing.

To identify candidate resistance mechanisms, we performed a genome-wide lentiviral open-reading frame (ORF) rescue screen in two Ewing cell lines sensitive to the CDK4/6 inhibitors palbociclib and ribociclib and looked for genes that confer resistance to these drugs. This revealed IGF1R as a gene whose overexpression promoted drug escape. In parallel to the ORF screen, we established resistance to CDK4/6i in Ewing sarcoma cell lines by chronic ribociclib exposure. We found elevated levels of phospho-IGF1R in resistant cells by immunoblotting, also supporting that this pathway's activation is relevant to acquired resistance. We performed CRISPR knockout of IGF1R in these drug resistant cells and demonstrated rescue of sensitivity to CDK4/6i. Furthermore, resistant lines maintained high sensitivity to an IGF1R inhibitor.

Concurrently, in a small molecule screen, an IGF1R inhibitor scored as synergistic with CDK4/6i in Ewing cells. We thus tested ribociclib and the IGF1R tool compound AEW541 in a panel of six Ewing cell lines in vitro. We observed strong synergy utilizing the Chou-Talalay method. This finding was next validated in an in vivo cell line xenograft mouse model, where we demonstrated prolonged survival and decreased tumor volumes with combination drug treatment. In order to investigate mechanism of CDK4/6-IGF1R inhibitor synergy, we used proteomic analysis by reverse phase protein array (RPPA) in single and combined drug treated Ewing cells. RPPA revealed enhanced suppression of the PI3K/mTOR pathway in the combination, a central cancer cell signaling axis. Taken together, these results suggest that IGF1R activation is an escape mechanism to CDK4/6i in Ewing and that dual targeting of CDK4/6 and IGF1R provides a candidate synergistic drug combination for this disease.

#1630

FLT3 chimeric antigen receptor T cell therapy induces B to T cell lineage switch in infant acute lymphoblastic leukemia.

Christopher D. Chien,1 Lila Yang,1 Sang M. Nguyen,2 Christopher T. Sauter,3 Kazusa Ishii,1 Feng Shen,3 Sarah K. Tasian,3 Terry J. Fry1. 1 _NIH, Bethesda, MD;_ 2 _University of California Riverside School of Medicine, Riverside, CA;_ 3 _University of Pennsylvania, Philadelphia, PA_.

Though childhood acute lymphoblastic leukemia (ALL) is highly treatable, there remain subsets of pediatric ALL with very poor prognoses. Infant ALL, found in children under the age of 1, is difficult to treat due to the scarcity of cases impeding the ability of even the largest pediatric oncology centers from gaining experience in treating the disease, the more aggressive initial clinical presentation, as well as the inability for these young patients to tolerate toxicities associated with chemotherapeutic regimens and procedures. Despite being only 5% of total ALL cases, 80% of infant ALL cases are marked by mixed lineage leukemia (MLL/KMT2A) rearrangements. In KMT2A rearranged (KMT2Ar) ALL, FLT3 is the most differentially expressed gene that distinguishes KMT2Ar ALL from non-KMT2Ar ALL making FLT3 an attractive target for infant ALL. CD19 and CD22 targeting chimeric antigen receptor (CAR) T cell therapy has demonstrated outstanding responses in phase 1 clinical trials for relapsed/refractory B ALL, leading to tremendous interest in testing other CAR targets. Here we explore FLT3 CAR as a potential treatment for B ALL and the unexpected finding that FLT3 CAR T cells induce lineage switch of an infant ALL from a B to T cell phenotype. We generated a FLT3-targeting CAR consisting of a FLT3 binding domain derived from a well-characterized anti-human FLT3 antibody coupled to 4-1BB costimulatory and CD3-zeta activation domains. In vitro studies confirmed that human T cells expressing the FLT3 CAR produced interferon-gamma and interleukin-2 after co-culture with KMT2Ar B ALL SEM and infant B ALL KOPN8. FLT3 CAR T cells eliminated ALL in vivo in NOD-SCID-IL2Rγc-/- (NSG) mice engrafted with high FLT3 expressing SEM. KOPN8, which expresses lower levels of FLT3 when treated with FLT3 CAR T cells showed an initial clearance of leukemia in NSG mice, however relapsed with ALL that no longer expressed FLT3 or B cell marker CD19. Interestingly, this loss of FLT3 and CD19 happened concurrently with gain of T cell markers (CD3+ and either CD4+ or CD8+). The durability of this T cell phenotype was transient because when T lineage switched KOPN8 was cultured ex vivo without immune pressure, the KOPN8 cells reverted to the parental B ALL phenotype (FLT3+, CD19+, CD3 neg, CD4 neg, CD8 neg) suggesting that the ability to lineage switch is not a selection of a clone that genetically does not express B cell markers while expressing T cell markers, but rather a potential epigenetic mechanism driving the cell lineage change. Contrary to reports from CD19 CAR treated KMT2Ar B ALL that switched to a myeloid phenotype, these cells did not upregulate myeloid markers (CD33, CD11b). Taken together these data imply that lineage switch driven by CAR T cell immune pressure may cause different types of lineage switch based on the target of the CAR. Furthermore, using CAR T cell immunotherapy we may be able to interrogate the biology of leukemia.

#1631

T cell dysfunction in pediatric cancer patients at diagnosis and after chemotherapy can limit chimeric antigen receptor potential.

Rajat K. Das, Julie Storm, David M. Barrett. _Children's Hospital of Philadelphia, Philadelphia, PA_.

Cellular therapy using engineered T cells has demonstrated clinical efficacy in the treatment of hematologic malignancies. Many patients, however, have T cells that result in a poor clinical product that either fails manufacture or does not proliferate in the patient. We have prospectively characterized peripheral blood T cells from 157 pediatric cancer patients and identified key metabolic changes associated with CAR T cell potential. We collected peripheral blood samples from 157 pediatric patients with acute lymphoblastic leukemia (ALL), non-Hodgkin Lymphomas (NHL) , neuroblastoma, osteosarcoma, rhabdomyosarcoma, Wilms, Hodgkins, chronic myelogenous leukemia and Ewings sarcoma at diagnosis and after each cycle of chemotherapy. We depleted the adherent cells from this collection, quantified the CD3+ population using flow cytometry, and expanded these T cells using CD3/CD28 stimulatory beads as in CAR T cell manufacturing. We noted very poor CAR T cell potential in all tumor types (<30% pass) except ALL (80% pass) and Wilms tumor (50% pass) in the pre-chemotherapy samples. We noted a decline in CAR T cell potential with cumulative chemotherapy in all cases, though this was particularly significant in children less than 3yo. Nanostring RNA profiling of metabolic pathways revealed a bias towards glycolysis and away from fatty acids in the poor performing samples. In vitro assays of chemotherapy exposure reveal that certain classes are especially toxic to Naïve T cells versus memory T cells, and that this correlates with poor potential as CAR T cells. In particular, cyclophosphamide and doxorubicin containing regimens severely depleted CAR T cell potential in patients. These chemotherapy agents were associated with mitochondrial dysfunction both in vitro and in patient T cells after in vivo chemotherapy. Oxygen consumption rate (OCR) analysis also reveals a poor spare respiratory capacity (SRC) in the T cell samples that go on to perform poorly as CAR T cells. Enforced use of fatty acids such as palmitate helped restore the SRC in chemotherapy exposed T cells.

These data represent the most detailed analysis of T cell number, phenotype and metabolic function from pediatric cancer patients. Low abundance of Naïve T cells (whether pre-chemotherapy or post), reliance on glycolytic pathways and poor SRC define patients with poor potential to have an active CAR T cell product made. This data raises important questions for the development of CAR T cell therapies for cancers other than ALL in pediatrics, as inherent T cell dysfunction and more intensive chemotherapy regimens in solid tumors may represent new barriers to efficacy.

#1632

Suppression of EWS-FLI1 transcription using a combination therapy of mithramycin and cyclin-dependent kinase 9 inhibition.

Guillermo Flores,1 Joel Everett,2 Brandon Oswald,1 Natasha Caplen,3 Lee Helman,3 Zachary Madaj,1 Patrick Grohar1. 1 _Van Andel Institute, Grand Rapids, MI;_ 2 _Vanderbilt University School of Medicine, Nashville, TN;_ 3 _National Cancer Institute, Bethesda, MD_.

Background: Ewing sarcoma (ES) is a pediatric soft tissue tumor with a poor prognosis. ES is dependent on the presence and activity of the oncogenic transcription factor, EWS-FLI1, that dysregulates gene expression at hundreds of targets. We have previously identified mithramycin (MMA) as a potent inhibitor of EWS-FLI1 driven transcription. However, only sub-therapeutic concentrations have been achieved in pediatric patients. To maximize inhibition, we conducted an siRNA screen to identify gene targets that potentiated the effects of MMA in ES. Knock-down of several transcriptionally related genes significantly sensitized ES cells to the effects of MMA. We next developed a drug matrix screening platform to identify commercially available transcriptional inhibitors that also potentiated the effects of MMA on EWS-FLI1 driven transcription and ES cell viability. PHA-767491, a cyclin-dependent kinase 9 inhibitor (CDK9i), reverses the effect on EWS-FLI1 on several targets at both the mRNA and protein level across multiple ES cell lines when combined with MMA.

Methods: We utilize matrix drug screening to identify that PHA-767491 synergizes with MMA in terms of a reduction in cell viability as measured by MTS and cell growth as measured by time-lapse microscopy. We use RT-qPCR to measure changes in gene expression across multiple well characterized targets of EWS-FLI1 driven transcription. We use western blot analysis to measure changes at the protein level for these targets as well. We then use an orthotopic xenograft mouse model to measure changes in tumor size after administration of our MMA-CDK9i combination therapy.

Results: Our combination of MMA and PHA-767491 displays strong synergy as measured by Bliss-Independence. Multiple ES cell lines become unviable, with minimal effect on non-ES cells, and this effect is stable after removal of the compounds. This synergy is recapitulated as a reversal of EWS-FLI1 driven transcription across multiple targets at both the mRNA and protein levels. We are currently evaluating the combination therapy in our animal model comparing MMA-CDK9i to control or either agent alone. Importantly our MMA-CDK9i combination uses drug concentrations that are clinically achievable.

Conclusions: We describe an MMA-CDK9i combination that displays excellent activity against EWS-FLI1 driven transcription. We confirmed this using multiple independent assays in both in vitro and in vivo models. We complete this work with the hope that it can eventually be translated to patients.

#1633

Chemical proteomics identifies druggable proteins in ALK-driven neuroblastomas.

Smita Matkar,1 Renata Sano,1 Colleen Larmour,1 Kateryna Krytska,1 Gabriela M. Witek,1 Mark Gerelus,1 Tim J. Stuhlmiller,2 Gary L. Johnson,3 Yael P. Mosse1. 1 _Children's Hospital of Philadelphia, Philadelphia, PA;_ 2 _SPYRYX Biosciences, Apex, NC;_ 3 _University of North Carolina, Chapel Hill, NC_.

The discovery of mutations in the ALK oncogene as the genetic etiology of familial neuroblastoma (NB) and as somatically acquired in 14% of patients with the most aggressive form of the disease has positioned ALK as the major tractable oncogene product for targeted therapy in NB. ALK tyrosine kinase domain mutations are found mostly at three hotspots (F1174, F1245 and R1275), with the F1174* and F1245* mutations conferring broad resistance to crizotinib. We have recently demonstrated that lorlatinib, a novel ATP-competitive macrocyclic ALK inhibitor, overcomes de novo resistance and exerts unprecedented activity as a single agent in patient-derived xenografts (PDXs) harboring F1174L or F1245C mutations, while also inducing durable responses in R1275Q xenografts. The objective of our work was to identify differences in reprogramming of the kinome upon ALK inhibition with crizotinib and lorlatinib. We hypothesize that identification of the targets through which lorlatinib mediates its superior anti-tumor effects will provide insights into the mechanisms by which full-length ALK inhibition abrogates tumorigenesis, activates compensatory pathways, and uncover novel targets to overcome resistance to ALK inhibition. We used Multiplexed Inhibitor Beads coupled with Mass Spectrometry (MIB/MS) to quantitatively measure kinase activity dynamics on a proteomic scale. Three PDX models harboring F1174L (COG-N-453x) or the other two most-common mutations, R1275Q (NB1643) and R1245C (Felix) were treated with either crizotinib (100 mg/kg/day) or lorlatinib (10 mg/kg/day) for 2.5 days or 6.5 days prior to MIB/MS analysis. Lorlatinib more potently inhibited ALK and preferentially inhibited a series of other tyrosine kinases, correlating with the superior efficacy of lorlatinib in pre-clinical studies. Moreover, a number of kinases involved in G2/M cell cycle transition including PLK1, CHEK1 and aurora kinases were significantly inhibited by lorlatinib, proposing that this multi-kinase inhibition is responsible for the robust anti-tumor activity observed in vivo with lorlatinib. We interrogated the role of SHP2, a ubiquitously expressed SH2 domain-containing tyrosine phosphatase recently implicated in the proliferation of receptor-tyrosine kinase driven cancers and shown to have a direct role in the G2/M checkpoint. Inhibition of ALK by treatment with lorlatinib as well as shRNA knockdown of ALK in SY5Y (ALK F1174L, PTPN11 T507K) and NB1643 NB cell lines showed decreased levels of pSHP2. Depletion of SHP2 using CRISPR constructs significantly inhibited cell proliferation in SY5Y, suggesting that SHP2 plays a crucial role in ALK driven NB. Additionally, knockdown of ALK in SY5Y downregulated PLK1 expression and arrested cells in G2/M phase. Whether SHP2 is regulated primarily downstream of ALK or jointly through alternative or compensatory signaling networks warrants further investigation.

#1634

BRCA-like phenotype constitutes hallmark of osteosarcoma.

Michal Kovac,1 Sebastian Ribi,1 Claudia Blattmann,2 Michaela Nathrath,3 Daniel Baumhoer1. 1 _The Institute of Pathology, Basel, Switzerland;_ 2 _Klinikum Stuttgart Olgahospital, Stuttgart, Germany;_ 3 _Klinikum Kassel, Kassel, Germany_.

Background: Osteosarcomas are aggressive bone tumours with a high degree of genetic heterogeneity, which limits treatment options in patients who do not respond to standard chemotherapy.

Methods: We sequenced 104 exomes and low-coverage genomes from 14 time-series consisting of chemotherapy-naïve biopsies, resection specimens, locally recurring tumours and/or distant metastases. We then identified cancer driver mutations, signatures of mutation processes and reconciled clonal structures of tumours with phylogenetic trees, thus building and evolutionary history of each tumour. Where possible, we searched for the evidence of selection acting on the tumours and molecular hallmarks that could be therapeutically exploited.

Results: Interrogating genomic data from time series, we identified pathogenic mutations in at least 10 cancer genes, but there was no universal cancer driver event present in the majority of tumours at any given time point. However, >80% of osteosarcomas exhibited specific combination of single-base substitutions, LOH and large-scale genome instability characteristic of BRCA1/2-deficient tumours. In osteosarcoma, "BRCAness" developed recurrently through independent BRCA1/2 mutations and mutations in their binding partners such as PALB2 or CHEK2. We therefore investigated the sensitivity of a panel of osteosarcoma cell lines to the poly(ADP)-ribose polymerase inhibitor talazoparib. In combination with temozolomide, talazoparib led to the to good response of osteosarcoma cell lines, activation of BAX and BAK, loss of mitochondrial membrane potential, DNA fragmentation and caspase-dependent cell death.

Conclusion: The acquisition of a BRCA-like phenotype in osteosarcoma has implications for the development of novel treatment strategies using PARP inhibitors alone, or together with standard chemotherapy.

### Special Populations / Biostatistics in Clinical Trials

#1637

The evolution of patient navigation research models to reduce breast cancer disparities: Case studies of patient navigation dissemination and implementation in Chicago.

Melissa A. Simon,1 Laura S. Tom,1 Julia R. Trosman,2 Christine B. Weldon,2 Catherine A. O'Brian,1 Marcie C. Kirschner,1 XinQi Dong3. 1 _Northwestern University Feinberg School of Medicine, Chicago, IL;_ 2 _Center for Business Models in Healthcare, Chicago, IL;_ 3 _Rush University Medical Center, Chicago, IL_.

Background: Patient navigation for breast cancer seeks to diminish social, economic, cultural and medical system barriers to timely quality care. In the context of unmet patient needs, growing complexity of health care delivery systems, and significant local implementation challenges, patient navigation models must continue to evolve in order to advance context-appropriate implementation and sustainment of patient navigation into routine practice. We present five case studies in Chicago demonstrating evolving patient navigation models and in each case study, we explore challenges and opportunities related to advancing successful implementation of patient navigation interventions across the breast cancer care continuum.

Methods: Case studies include: (1) a community-level adaptation of patient navigation in Chicago's Chinatown; (2) a community navigation program in Chicago's racially/ethnically diverse North Lawndale and Humboldt Park; (3) a county-wide dissemination of navigation in suburban DuPage County; (4) a state-wide scaling of a patient navigation model to the Illinois Department of Health and Family Services; and (5) a 4R patient navigation oncology model (4R = Right Information / Care / Patient / Time) grounded in systems engineering and design that leverages collaborative care plans and iterative examination and improvement of processes to address challenges of breast cancer care planning, team-based delivery and patient engagement.

Results: Case studies in the evolution of patient navigation research programs show how identified gaps in care were remedied pragmatically in the course of each study. We show how patient navigation implementation efforts have been aided with evolving models incorporating the local implementation context, characteristics of program implementers, and structural, macro-level factors involving care delivery systems and public policy. Results highlight approaches for supporting organizational learning to enhance implementation and sustainability of navigation interventions; for furthering task clarification and coordination to optimize integration of patient navigation into cancer care delivery settings; and for tackling the impact of existing local, state, and national policy initiatives on patient navigation.

Conclusions: Reducing breast cancer burden, especially among underserved populations, warrants adaptive, ongoing improvement of patient navigation models. Through iterative intervention development, evaluation, and refinement, patient navigation can move beyond serving as a temporary bandage for health systems fragmentation and toward an essential role in systems change for optimizing cancer care delivery.

#1638

Is short interval colonoscopy at the discretion of the gastroenterologist.

Hassan Brim,1 Abhimanyu Tushir,1 Shashidhar Manchegowda,1 Negin Farsi,1 Edward L. Lee,1 Laiyemo O. Adeyinka,1 Seyed M. Nouraie,2 Hassan Ashktorab1. 1 _Howard University, DC;_ 2 _University of Pittsburgh, PA_.

BACKGROUND: Gastroenterologists do not always follow guidelines for performing colonoscopies. Short interval colonoscopies are recommended at the discretion of gastroenterologists depending on various factors. We here evaluated such factors in an African American patient population.

AIM: To assess factors contributing to short interval (within 3 months) colonoscopies.

METHODS: Data was retrieved retrospectively from Howard University Hospital database (Jan. 2010 to Dec. 2016). African American patients who had repeated colonoscopies within a period of 3 months were selected for this study. We analyzed patients' demographics, family history, polyp characteristics, and reasons for repeat colonoscopy along with latest follow-up findings.

RESULTS: There were 36 patients who underwent two colonoscopies within 3 months, 13 females and 23 males with mean age of 61 years. Out of these, 7 patients (19.4%) had poor bowel preparation. For the remaining 29 patients, the reason for second colonoscopy were partial removal and suspicion of partial removal (61.1%), difficult access (16.7%), and follow-up for Crohn's disease (2.8%). Follow-up of these 36 patients after their last colonoscopy revealed that 8 patients (22.2%) had colon cancer/resection, 4 other patients (11.1%) had cancer of other organs. Of the remaining, 6 (16.6%) had future colon polyp resection, 3 (8.3%) had normal screening colonoscopy and 4 (11.1%) visited the hospital for reasons unrelated to gastroenterology while 11 patients (30.6%) had no follow up.

CONCLUSION: Even though there are no recommendations to repeat colonoscopy within 3 months, endoscopists perform such colonoscopies at their own discretion based on factors like high risk individuals, poor bowel preparation, multiple lesions, type of lesions and resection clearance. Of interest, a significant number of patients in our study had colon cancer/resection on eventual follow-ups.

#1639

Androgen receptor and ALDH1 expression among internationally diverse patient populations.

Evelyn M. Jiagge,1 Aisha Jibri,2 Jessica Bensenhaver,3 Max Wicha,1 Baffour Awuah,4 Sofia Merajver,1 Lisa Newman3. 1 _Univ. of Michigan Health System, Ann Arbor, MI;_ 2 _Paul's Hospital in Addis Ababa, Addis Ababa, Ethiopia;_ 3 _Henry Ford Health System, Detroit, MI;_ 4 _Komfo Anokye Teaching Hospital, Kumasi, Ghana_.

Background: Population-based incidence rates of breast cancers that are negative for the estrogen receptor (ER), progesterone receptor (PR), HER2/neu triple-negative breast cancer (TNBC) are higher among African American (AA) compared to White American (WA) women, and several studies suggest that TNBC prevalence is increased among selected populations of African patients. The colonial-era transatlantic slave trade resulted in shared ancestry between contemporary AA and Gh populations. The extent to which associations between TNBC are related to East African versus West African ancestry, and whether these associations extend to expression of additional hormone receptors such as androgen receptor (AR) and stem cell markers such as ALDH1, is uncertain, but this research may explain breast cancer disparities between domestic communities within the United States as well as between international population subsets.

Methods: We utilized immunohistochemistry to evaluate ER, PR, HER2/neu, AR and ALDH1 expression among White American (n=153), African American (n=76), Ethiopian (Eth)/East African (n=90), and Ghanaian (Gh)/West African (n=286) breast cancers through an IRB-approved international research program.

Results: Mean age at breast cancer diagnosis was 43, 49, 60, and 57 years for the Eth, Gh, AA, and WA patients, respectively. Frequency of TNBC was significantly higher for the AA and Gh patients (41% and 54%, respectively) compared to the WA and Eth patients (23% and 15%, respectively; p<0.001). These associations were unchanged when limited to patients age 50 years and younger (47% and 49% for AA and Gh, respectively, versus 18% and 16% for WA and Eth, respectively; p<0.001). Frequency of ALDH1 positivity was also higher for the AA and Gh tumors (32% and 36%, respectively) compared to the WA and Eth tumors (23% and 17%, respectively; p=0.007). Significant differences were also observed for distribution of AR positivity, which was 71%, 55%, 42%, and 50% for the WA, AA, Gh, and Eth cases, respectively (p=0.008). Frequency of ALDH1 expression was numerically higher in the AA, Gh, and Eth TNBC cases compared to the WA TNBC cases, but this was not statistically significant (33%, 41%, and 31%, respectively, versus 18%; p=0.47).

Conclusions: Extent of African ancestry appears to be associated with particular breast cancer phenotypes. West African ancestry correlates with increased risk of TNBC and breast cancers that are positive for ALDH1.

#1640

Class switch or identical regimen as a second-line chemotherapy for patients with metastatic gastric adenocarcinoma: A retrospective analysis of two medical centers in Taiwan.

Li-Yuan Bai,1 Yung-Chia Kuo,2 Jen-Shi Chen,2 Wen-Chi Chou,2 Chang-Fang Chiu,1 Yu-Min Liao3. 1 _China Medical University Hospital, Taichung, Taiwan;_ 2 _Linkou Chang Gung Memorial Hospital, Taiwan;_ 3 _Taipei Medical University Hospital, Taiwan_.

Introduction: Background: Chemotherapy with a fluoropyrimidine with or without a platinum is currently the standard care for metastatic gastric cancer (GC). Randomized trials have established the role of either a taxane- or an irinotecan-containing regimen as second line chemotherapy. However, medical insurance systems don't permit the reimbursement of both taxane and irinotecan in some countries, including Taiwan. Patients who fail the first line chemotherapy in Taiwan usually receive a regimen containing a fluoropyrimidine / platinum different from the prior regimen, or a self-paid regimen containing paclitaxel, docetaxel or irinotecan. The impact of selecting an identical vs. a class switch chemotherapy on the outcomes of patients is not known.

Procedures: The medical records of patients who were diagnosed with metastatic gastric adenocarcinoma from 2008 to 2012 and had received a second line chemotherapy after a first line regimen of fluoropyrimidine (5-fluorouracil, UFT, capecitabine or S-1), and/or platinum (cisplatin, carboplatin or oxaliplatin) were retrospectively obtained from two medical centers in Taiwan. In a second line situation, we defined an identical regimen as a regimen containing another fluoropyrimidine with or without a platinum; and a class switch regimen as a regimen containing paclitaxel, docetaxel or irinotecan. The characteristic and clinical outcomes of the two groups of patients were compared. Kaplan-Meier curves were used to demonstrate the progression free survival (PFS) and overall survival (OS) function of each treatment group. All reported p values were two-sided with the alpha set at a significance of 0.05.

Findings: A total of 201 patients met the study criteria. Patients in the identical regimen group were older (p=0.001) and were less likely to stop first line treatment due to disease progression (PD; p=0.026) compared to the class switch group. Although the response rates (CR+PR) were similar (7.8% vs. 8.3%), toxicity occurred more frequently in identical group than in class switch group by 5%. The survival outcomes measures for the identical regimen group and class switch group included 9 and 6.8 weeks (p=0.949) median PFS since second line treatment, 16 and 12 weeks (p=0.585) median OS since second line treatment, and 48 and 58 weeks (p=0.457) median OS since disease diagnosis, respectively.

Conclusions: Patients receiving an identical chemotherapy after failure of a fluoropyrimidine with/without a platinum regimen have similar PFS and OS compared to patients receiving a class switch chemotherapy for GC. A higher percentage of PD as the reason to stop first line therapy was noted in patients receiving a class switch chemotherapy and might influence the assessment of a class switch regimen. Further prospective clinical trials are needed to clarify this potential confounding factor.

#1641

Utilization of palliative therapies among Hispanics with stage IV non-small cell lung cancer.

Narjust Duma, Urshila Durani, Julian Molina, Timothy J. Moynihan. _Mayo Clinic, Rochester, MN_.

Background: Racial disparities in the treatment of lung cancer are well documented. However, research in disparities in palliative care is limited. Early integration of palliative care in advanced non-small cell lung cancer (NSCLC) has been proven to improve quality of life and overall survival in this subset of patients. We proposed to study the use of palliative treatments for stage IV NSCLC among Hispanic patients (pts). Methods: Using the National Cancer Database (NCDB), we identified all Hispanic pts (self-reported) diagnosed with stage IV NSCLC from 2005 to 2013. Cases with incomplete data, unknown life/death status or classified as Hispanic by surname only were excluded. Hispanics were grouped based on place of origin. Pearson chi-square tests were used to estimate differences in categorical data; predictors of palliative care referral/use were determined by logistic regression analysis. Results: 10,441 pts were included. The median age was 66 years. Regarding place of origin: 15.5% of pts were from Mexico, 8% from South/Central America, 7% from Cuba, 6% from Puerto Rico, 2.4% from the Dominican Republic and 57.6% were no-otherwise specified. When all Hispanic pts were included, 3.5% received surgery, 45% radiation and 52.9% chemotherapy. Overall, 2.2% of pts received a referral for palliative pain management +/- other palliative therapies. When divided by place of origin, Dominican pts had the highest percentage of pain management referrals at 5.5% (p<0.01) followed by Puerto Rican patients (2.4%). On the other hand, only 1.3% of pts from South/Central America were referred to pain management. In multivariate analysis, Dominican Republic origin (OR: 3.30, 95%CI: 1.69-6.44, p<0.01), bone metastasis (OR: 1.98, 95%CI: 1.17-3.3, p<0.01) and a Charlson comorbidity index ≥2 (OR: 2.07, 95%CI: 1.11-3.85, p<0.02) were significant predictors of receiving a pain management referral. We observed an increased number of pain management referrals over time with 1.4% of Hispanic patients getting a referral in 2004 vs. 2.9% in 2013 (p<0.02). Conclusions: We observed that only a small percentage of Hispanic pts with metastatic NSCLC cancer are receiving referral for palliative care/pain management. Several cultural beliefs and barriers may play a role in these findings. Providers should offer early referrals to pain management/palliative care to all patients with metastatic NSCLC independent of their race or ethnicity.

#1642

**Alectinib in treatment-naïve anaplastic lymphoma kinase-positive (** ALK **+) metastatic non-small-cell lung cancer (mNSCLC): Systematic literature review (SLR) and network meta-analysis (NMA).**

Anna Steenrod,1 Michelle Orme,2 Katherine S. MacGilchrist,3 Rachel Rosenthal,1 Juliane Schaefer,1 Vlatka Smoljanovic,1 Emmanuel Mitry,1 Parneet K. Cheema4. 1 _F. Hoffmann-La Roche Ltd, Basel, Switzerland;_ 2 _Icera Consulting Ltd, Swindon, United Kingdom;_ 3 _Epidemica Ltd., Bicester, United Kingdom;_ 4 _William Osler Health System, University of Toronto, Toronto, Ontario, Canada_.

Background: The standard of care for treatment-naïve ALK+ mNSCLC patients (pts) is an ALK tyrosine kinase inhibitor (TKI). Pts treated with crizotinib generally experience disease progression within 1 year, often within the central nervous system (CNS). As such, there is a high unmet need for CNS-active ALK TKIs. Phase III data from 2 ALK TKIs, alectinib and ceritinib, showed improved efficacy vs crizotinib and chemotherapy, respectively; however, no studies have directly compared these TKIs. We report a NMA to compare the efficacy/safety of these therapies.

Methods: A SLR was conducted to identify ALK+ mNSCLC randomized controlled trials (RCTs) in treatment-naïve pts. Efficacy/safety endpoints were extracted and the feasibility of using the data in a NMA was assessed. Data were analyzed using a fixed effect analysis in WinBUGs following NICE guidelines.

Results: 1194 citations were identified, of which 4 RCTs were in treatment-naïve pts (ALEX [NCT02075840], ASCEND-4 [NCT01828099], PROFILE 1014 [NCT01154140] and PROFILE 1029 [NCT01639001]). All were open-label, phase III RCTs and pts were similar in terms of age, ECOG PS, disease stage, sex and smoking status. More pts had baseline CNS metastasis (mets) in ALEX (40%) vs the other trials (26-32%). PROFILE 1029 was conducted in East Asian pts and only included in a sensitivity analysis. Chemotherapy arms differed in that pemetrexed maintenance was included in ASCEND-4 but not in the PROFILE studies. Key efficacy/safety results are shown in the Table. Data from the sensitivity analyses were similar.

Conclusion: Alectinib significantly improved progression-free survival compared with other treatments in the ITT population including in pts with baseline CNS mets. Alectinib showed significantly fewer grade 3-4 AEs than ceritinib. The difference in chemotherapy arms of the 2 trials may have impacted the NMA efficacy results.

Table. NMA results in treatment-naïve ALK+ mNSCLC patients

---

Comparison | Efficacy/Safety Endpoints

PFS by IRC* HR (95% CrI) | Subgroup CNS Mets at Baseline* PFS by IRC HR (95% CrI) | OS* HR (95% CrI) | Grade 3 or 4 AEs§OR (95% CrI)

Alectinib vs chemotherapy | 0.23

(0.15-0.34) | 0.21

(0.10-0.44) | 0.63

(0.34-1.15) | 0.81

(0.44-1.52)

Alectinib vs crizotinib | 0.50

(0.36-0.70) | 0.37

(0.22-0.63) | 0.76

(0.49-1.20) | 0.65

(0.41-1.04)

Alectinib vs ceritinib | 0.41

(0.25-0.67) | 0.31

(0.13-0.71) | 0.85

(0.42-1.73) | 0.36

(0.17-0.79)

Crizotinib vs chemotherapy | 0.45

(0.35-0.58) | 0.57

(0.35-0.93) | 0.82

(0.54-1.24) | 1.24

(0.81-1.91)

Ceritinib vs chemotherapy | 0.55

(0.42-0.72) | 0.70

(0.44-1.11) | 0.73

(0.50-1.06) | 2.25

(1.42-3.61)

Ceritinib vs crizotinib | 1.22

(0.84-1.79) | 1.22

(0.62-2.43) | 0.90

(0.52-1.57) | 1.82

(0.96-3.44)

Statistically significant differences indicated in bold based on 95% CrI; *Hazard ratio <1 indicates lower hazard (higher likelihood of PFS or OS) compared with control; §Odds ratio <1 indicates lower odds of an event compared with control. AEs, adverse events; CNS, central nervous system; CrI, credible interval; HR, hazard ratio; IRC, independent review committee; Mets, metastases; OR, odds ratio; OS, overall survival; PFS, progression-free survival

#1643

Leveraging tumor size and time to death from bevacizumab (BEV) historical data to predict overall survival in ovarian cancer patients treated with vanucizumab (VAN).

Alexandre Sostelly,1 Kevin Smart,2 Felix Jaminion,1 Christophe Boetsch,1 Francois Mercier1. 1 _F. Hoffman La Roche Ltd, Basel, Switzerland;_ 2 _F. Hoffman La Roche Ltd, Welwyn, United Kingdom_.

VAN is a bispecific IgG-like antibody targeting both VEGF-A and Ang-2, two key factors in tumor angiogenesis pathway that was investigated as monotherapy in a single arm study in platinum-resistant ovarian cancer (PROC) patients. A modeling approach working with tumor size data was applied to predict and compare overall survival (OS) of VAN + chemotherapy (CT) against BEV + CT. For VAN, individual tumor size data from 41 patients enrolled in study NCT01688206 were available. Primary endpoint of the study was objective response rate; survival data were not collected. For CT and BEV +CT, historical tumor size data were obtained from AURELIA, a randomized phase 3 study designed to compare progression free survival (PFS) in patients treated with CT alone or in combination with BEV. In AURELIA, patients were followed for OS after treatment discontinuation. In both studies, sum of longest diameters (SLD) were measured on CT/MRI scans collected every 6 to 8 weeks, as per RECIST 1.1. Non-linear tumor kinetics (TK) models accounting for the dynamics of tumor growth, drug effect and resistance to the drug effect were used to fit SLD following each treatment (VAN, CT and BEV+CT). Several tumor metrics summarizing the individual TK were derived: early tumor shrinkage at week 6 (ETS6), tumor size at baseline (TS0), maximum shrinkage (MaxSh) and time to tumor growth (TTG). As no VAN+CT data were available, individual tumor responses were simulated assuming an additive drug effect from CT on VAN. The median TTG was prolonged by 4 weeks and median MaxSh was increased by 7% for V+CT as compared to BEV+CT. To correlate tumor metrics to OS, a time-to-event model was fitted to the OS data from the AURELIA study. Covariates including ECOG, FIGO score at baseline, histological grade and subtype, presence of ascites, CA-125 at baseline, TS0, ETS6 and TTG were tested as prognostic factors in the survival model. In the survival model, two sets of covariates were found significant: those related to disease severity (ECOG, FIGO, presence of ascites) and those describing the key features of TK (ETS6, TTG, TS0). Treatment group was not retained in the final model making the model drug independent. Assuming the same survival model and a similar mechanism for VAN and BEV, the BEV+CT TK metrics were replaced by the VAN+CT TK metrics in the survival model. Based on this assumption, the median OS with VAN+CT was predicted to be 2 months longer compared to BEV+CT. This approach shows the benefit of using TK modeling, when drugs show similar mechanism of action, in early phase to predict potential outcome of drug combination, not yet tested in patients. Leveraging historical data for the development of survival model integrating TK metrics can be used to inform the expected OS outcome in phase 3 and can provide a quantitative tool to evaluate the chance of drug success.

#1644

Progression-free survival (PFS) as a surrogate for overall survival (OS).

David J. Stewart,1 Dominick Bosse,2 John Hilton,1 Glenwood Goss,1 Michael Fung-Kee-Fung,1 Derek Jonker1. 1 _Ottawa Hospital Regional Cancer Ctr., Ottawa, Ontario, Canada;_ 2 _Harvard University, Boston, MA_.

Background: PFS is criticized as being not reliably predictive of OS, and statistically significant gains may be clinically insignificant. However, PFS has the advantage as an endpoint of needing shorter follow up/fewer patients and is not rendered falsely negative by crossover (CO) and long post progression survival (PPS). Absolute PFS gain may be a surrogate for absolute OS gain (Ann Oncol 2016;27:373).

Method: We extracted PFS/OS data from randomized drug comparisons with >200 incurable solid tumor patients published 01/2007-06/12/2017 in J Clinical Oncology and New England J Medicine. For comparisons with CO unspecified or in <20% of patients, and p<0.05 for PFS or OS, we used nonlinear regression analysis (NLRA) of PFS/OS curves (omitting curve sections with <~10 residual patients) to determine PFS/OS half-lives (t1/2s: time to progression/death in half the remaining patients). We calculated ΔmPFS and ΔmOS (experimental minus control arm medians) and ΔPFSt1/2 and ΔOSt1/2 (experimental minus control arm t1/2s).

Results: 249 trials and 287 across-arm comparisons were evaluable. The table presents ΔPFS/ΔOS associations for studies with PFS p<0.05 and CO<20% or unspecified. | |

|

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

PFS change | n | No. (%) of comparisons with associated change in OS (ΔmOS for ΔmPFS or ΔOSt1/2 for ΔPFSt1/2)

|  | ΔOS <2 months | ΔOS > 2 months

ΔmPFS: <1.5 months | 32 | 24 (75%) | 8 (25%)

>1.5 months | 47 | 22 (47%) | 25 (53%)

|  | |

ΔPFSt1/2: <1.5 months | 35 | 31 (89%) | 4 (11%)

>1.5 months | 62 | 10 (16%) | 52 (84%)

>2 months | 41 | 4 (10%) | 37 (90%)

|  | ΔOS <3 months | ΔOS >3 months

ΔPFSt1/2: >3 months | 26 | 3 (12%) | 23 (88%)

|  | |

|  | ΔOS <4 months | ΔOS >4 months

ΔPFSt1/2: >4 months | 13 | 1 (8%) | 12 (92%)

Concordance of ΔOS with ΔPFS for ΔPFS < vs >1.5 months better for t1/2s than medians, Fisher's exact test p=0.0004, and correlation of ΔOS with ΔPFS was also better for t1/2s (R2=0.60, p<0.0001, slope = 1.51) than for medians (R2=0.37, p<0.0001, slope = 0.88)

To assess the risk of ΔPFS under-predicting ΔOS, we then assessed all comparisons, irrespective of CO and PFS significance. Of 287 comparisons, 13 (5%) had ΔOS >2m with p<0.05 but ΔPFS <1.5m or ΔPFS p>0.05. In 7/13, PFS curves fit NLRA 2-phase decay models (suggesting 2 distinct subpopulations), vs 25/100 with PFS 2-phase decay in other comparisons (Fisher's exact test p=0.047).

Conclusions: Demonstrating ΔPFSt1/2 >1.5-2.0 months with p<.05 indicates an 84-90% probability of a ΔOSt1/2 gain >2 months, and could be a useful ΔOSt1/2 surrogate. This endpoint could mitigate impact of CO and PPS while making trials faster/cheaper. T1/2s are superior to medians. If PFS curves fit NLRA 2 phase models in phase II trials one should collect full phase III OS data (censored at CO) to reduce risk of a false negative.

#1645

Innovative discovery of therapeutic plateletpheresis in patients with thrombocytosis using the Fresenius COM.TEC.

Yanxia Jin,1 Guolin Yuan,2 Fuling Zhou1. 1 _Zhongnan Hospital, Wuhan, China;_ 2 _Xiangyang Central Hospital, Xiangyang, China_.

Plateletpheresis is an important therapeutic method for treatment of patients with myeloproliferative neoplasms (MPN) presenting with thrombocytosis. This study analysed the collection efficiency of plateletpheresis (CEPP) for 94 patients with thrombocytosis who underwent plateletpheresis by Fresenius COM.TEC machine, especially analysed the CEPP for enrolled patients with PLT count at 500-1000 × 109/l. The results indicated that this device could significantly decreased PLT count, and with mean CEPP of 24.23±17.42% that ranges from 3.18% to 78.46%. Further analysis shows that the PLT counts were 500-800 × 109/l for patients, which CEPP was above 42.1% with a lower pooled WBC and RBC. Paired t-test suggested that the red blood cell (RBC) counts, PLT counts, white blood cell (WBC) counts, monocyte counts and haematocrit (HCT) significantly decreased after plateletpheresis. Bivariate correlation and multiple logistic regression analysis showed that PLT count groups, hemoglobin (HGB) before apheresis were greatly correlated with CEPP. We suggest that the device is effective at reducing platelet counts with acceptable efficiency and it is available for plateletpheresis when the PLT counts about 500-800 × 109/l for patients with higher HGB pre-apheresis in female patients.

## CANCER CHEMISTRY:

### Target Based Drug Discovery

#1646

Discovery and characterization of BAY-6035, a novel benzodiazepine-based SMYD3 inhibitor.

Stefan Gradl,1 Holger Steuber,1 Jörg Weiske,1 Norbert Schmees,1 Stephan Siegel,1 Detlef Stoeckigt,1 Clara D. Christ,1 Fengling Li,2 Shawna Organ,2 Dalia Barsyte-Lovejoy,2 Magdalena M. Szewczyk,2 Steven Kennedy,2 Viacheslav Trush,2 Masoud Vedadi,2 Cheryl H. Arrowsmith,2 Peter J. Brown,2 Manfred Husemann,1 Amaury E. Fernandez-Montalvan,1 Volker Badock,1 Marcus Bauser,1 Andrea Haegebarth,1 Ingo V. Hartung,1 Carlo Stresemann1. 1 _Bayer AG, Berlin, Germany;_ 2 _Structural Genomics Consortium, Toronto, Ontario, Canada_.

SMYD3 (SET and MYND domain-containing protein 3) is a protein lysine methyltransferase (PKMT) which was initially described as H3K4 methyltransferase involved in transcriptional regulation. SMYD3 has recently been reported to methylate and regulate several non-histone cancer relevant proteins such as mitogen-activated protein kinase kinase kinase 2 (MAP3K2), vascular endothelial growth factor receptor 1 (VEGFR1), and the human epidermal growth factor receptor 2 (HER2). In addition overexpression of SMYD3 has been linked to poor prognosis in certain cancers, thus supporting a possible oncogenic role for SMYD3 and making it an attractive target for anticancer drug development. Here we report the discovery of a novel potent and selective SMYD3 inhibitor series. We performed a thermal shift assay based (TSA) high throughput screening followed by extensive biophysical validation resulting in identification of a benzodiazepine-based SMYD3 inhibitor series. The co-crystallization structures revealed that this series binds to the substrate binding site and occupies the hydrophobic pocket for lysine binding using an unprecedented hydrogen bond pattern. The competitive behavior of the inhibitor in biochemical assays was consistent with the binding mode observed in the crystal structure. Further optimization generated BAY-6035, which showed improved nanomolar potency and was selective against kinases and other PKMTs. Furthermore, BAY-6035 specifically inhibited methylation of MAP3K2 by SMYD3 in a cellular assay with similar potency. In summary, BAY-6035 is a novel selective and potent SMYD3 inhibitor probe and will foster the exploration of the biologic role of SMYD3 in diseased and non-diseased tissues.

#1647

Discovery of AZD0364, a potent and selective oral inhibitor of ERK1/2 that is efficacious in both monotherapy and combination therapy in models of NSCLC.

Iain Simpson,1 Mark J. Anderton,1 David M. Andrews,1 Jason Breed,1 Emma Davies,1 Judit E. Debreczeni,1 Vikki Flemington,1 Francis D. Gibbons,2 Mark A. Graham,1 Philip Hopcroft,1 Tina Howard,1 Julian Hudson,1 Clifford D. Jones,1 Christopher Jones,1 Nicola Lindsay,1 J Elizabeth Pease,1 Philip Rawlins,1 Karen Roberts,1 Steve Swallow,1 Steve St-Gallay,1 Michael E. Tonge,1 Richard A. Ward1. 1 _AstraZeneca, Cambridge, United Kingdom;_ 2 _AstraZeneca, Boston, MA_.

The RAS/MAPK pathway is a major driver in oncogenesis and is dysregulated in approximately 30% of human cancers, primarily by mutations in BRAF or RAS genes. The extracellular-signal-regulated kinases (ERK1 and ERK2) serve as key central nodes within this pathway. The feasibility of targeting the RAS/MAPK pathway has been demonstrated by the initial clinical responses observed to BRAF and MEK inhibitors in BRAF V600E/K metastatic melanoma, however resistance frequently develops by reactivation of the pathway. Direct targeting of ERK1/2, may provide another therapeutic option in tumours with mutations in BRAF or RAS genes. Importantly, ERK1/2 inhibition may have clinical utility in overcoming acquired resistance to RAF and MEK inhibitors where RAS/MAPK pathway reactivation has occurred, such as relapsed BRAF V600E/K melanoma. Starting from our published work,1 we will describe for the first time, a scaffold hopping approach leading to the identification of AZD0364, a pre-clinical ERK1/2 inhibitor candidate drug. Driven by conformational modelling and structure-based design, and by utilising novel sulfamidate ring opening chemistry, a high lipophilicity efficiency core was identified. Structure based, multi-parameter based optimisation of this improved core ultimately led to AZD0364. AZD0364 exhibits high cellular potency against a direct downstream substrate on the MAPK pathway (e.g. inhibition of phospho-p90RSK1 in BRAFV600E mutant A375 cells, IC50 = 6 nM). The molecule is a highly selective kinase inhibitor (10/329 kinases tested are inhibited at >50% at a 1 µM) and has long residence time on the protein (as determined by SPR on human unphosphorylated-ERK2: pKd = 10; t1/2 = 277 mins). The good in vitro potency and selectivity is complemented by excellent physico-chemical properties (maximum absorbable dose estimated to be >4 g) and good oral pharmacokinetics across species, leading to a low predicted dose to man. In xenograft models, AZD0364 inhibits phospho-p90RSK1 in tumors in a dose-dependent manner. AZD0364 induces regressions in the KRAS mutant NSCLC Calu 6 xenograft model. AZD0364 can also be combined safely and effectively with the MEK1/2 inhibitor selumetinib in KRAS mutant NSCLC xenograft models. 1Richard A. Ward et. al. Structure-Guided Discovery of Potent and Selective Inhibitors of ERK1/2 from a Modestly Active and Promiscuous Chemical Start Point, J. Med. Chem. 2017, 60, 3438−3450.

#1648

Discovery and evolution of orally bioavailable selective estrogen receptor degraders for ER+ breast cancer: From GDC-0810 to GDC-0927.

Mehmet Kahraman, Steven P. Govek, Johnny Y. Nagasawa, Andiliy Lai, Celine Bonnefous, Karensa Douglas, John Sensintaffar, Nhin Lu, KyoungJin Lee, Anna Aparicio, Josh Kaufman, Jing Qian, Gang Shao, Rene Prudente, James D. Joseph, Beatrice Darimont, Daniel Brigham, Richard Heyman, Peter J. Rix, Jeffrey H. Hager, Nicholas D. Smith, Robert A. Blake, Jae Chang, Edna Choo, Anneleen Daemen, Lori S. Friedman, Jane Guan, Steven Hartman, Ellen Ingalla, James R. Kiefer, Tracy Kleinheinz, Sharada Labadie, Ciara Metcalfe, Vidhi Mody, Michelle Nannini, Deepak Sampath, Amy Young, Maia Vinogradova, Wei Zhou, Jun Liang, Xiaojing Wang. _Genentech Inc, South San Francisco, CA_.

Breast cancer is the most frequently diagnosed cancer among women and remains the second leading cause of cancer death in women. An estimated 70% of all breast cancers express estrogen receptor alpha (ERα); and endocrine therapies have validated ERα as a target for the treatment of breast cancer. Despite effective endocrine therapies, many patients eventually relapse and become resistant to standard of care treatments. Endocrine resistant tumors often remain dependent on ERα for growth and survival, as evidenced by their sensitivity to the selective estrogen receptor degrader (SERD), fulvestrant. However, fulvestrant may be limited in achieving maximal target occupancy due to pharmaceutical and pharmacokinetics properties which necessitates intramuscular route of administration. Consequently, SERDs with superior drug-like properties were sought to allow consistent and rapid achievement of maximal therapeutic exposure. GDC-0810 and GDC-0927 as first and second generation orally bioavailable SERDs were discovered through a prospective lead optimization on ERα degradation. The evolution from GDC-0810 to GDC-0927 will be described and provides new insights into ERα biology and biochemistry. By shifting away from the acrylic acid moiety in GDC-0810, GDC-0927 achieved increased potency and more consistent, complete suppression of ER signaling. Co-crystal structures of both GDC-0810 and GDC-0927 with ERα will be shared. Subsequent optimization of GDC-0927 resulting in improved pharmacokinetic properties will also be highlighted.

#1649

Impact of linker and conjugation site on tubulysin M ADC stability and in vivo activity.

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

The tubulysins are a potent class of microtubule-disrupting agents consisting of natural products and designed analogues that have become a compelling cytotoxic payload for drug-targeting applications. This is due in large part to their high cytotoxic potency on cancer cells, including those that express transporters conferring the multidrug resistance (MDR+) phenotype. All potent tubulysin natural products contain an acetoxy group at the C11-position in the central tubuvaline residue. Deacetylation results in >100-fold loss of cellular and biochemical potency, underscoring the importance of this structural element. Recently, we developed quaternary ammonium linker systems to conjugate tertiary amine-containing payloads, and applied them to the tubulysins. In this work, protease- and β-glucuronidase-cleavable quaternary ammonium linkers of tubulysin M were evaluated as ADC payloads. In vitro, both dipeptide and glucuronide linker systems provided ADCs with comparable biologic properties. However, in vivo, linker chemistry and conjugation site were important parameters impacting acetate group stability. Higher degrees of stability translated to greater activity in xenograft models. ADCs were prepared with both dipeptide and glucuronide linkers as heterogeneous ADCs loaded at an average of 4-drugs/Ab (DAR 4), and as homogeneous ADCs loaded at 2-drugs/Ab (DAR 2) at the engineered S239C sites. Increased in vivo acetate stability was observed for glucuronide-based conjugates relative to the dipeptide analogue. Further stabilization was achieved by conjugating at the S239C sites (DAR 2) relative to native antibody cysteines (DAR 4). Tubulysin M acetate stabilization by both linker and conjugation site selection resulted in increased ADC potency in preclinical models and, combined, serves as an enabling strategy for antibody-mediated tubulysin M drug delivery.

#1650

Discovery of AZD4573, a potent and selective inhibitor of CDK9 that enables transient target engagement for the treatment of hematologic malignancies.

Bernard Barlaam,1 Chris De Savi,2 Lisa Drew,2 Andrew D. Ferguson,2 Douglas Ferguson,2 Chungang Gu,2 Janet Hawkins,1 Alexander W. Hird,2 Michelle L. Lamb,2 Nichole O'Connell,2 Kurt Pike,1 Theresa Proia,2 Maryann San Martin,2 Melissa M. Vasbinder,2 Jeff Varnes,2 Jianyan Wang,2 Wenlin Shao2. 1 _AstraZeneca, Cambridge, United Kingdom;_ 2 _AstraZeneca, Waltham, MA_.

Cyclin-dependent kinase 9 (CDK9) is a serine/threonine kinase that regulates elongation of transcription through phosphorylation of RNA polymerase II at serine 2 (p-Ser2-RNAPII). Transient inhibition of CDK9 results in reduced protein levels for genes that have short half-lives of transcripts and proteins, thus presenting a potential therapeutic opportunity in tumors dependent upon oncogenes fitting such criteria. One example is Mcl-1, an anti-apoptotic protein that plays a key role in cancer cell survival. A potent and selective CDK9 inhibitor having appropriate physical properties and pharmacokinetics (intravenous administration and short t1/2) would enable short yet tuneable target engagement, allowing high flexibility in order to optimize the efficacy / tolerability balance in the clinic. We previously reported the identification of AZ5576 from an amidopyridine series, as a potent, highly selective and orally bioavailable preclinical inhibitor of CDK9. Here we report further optimization of this series with a focus on pharmacokinetic and physicochemical properties suitable for an intravenous agent with short target engagement. We discuss the Structure Activity Relationships (SAR) and Structure Property Relationships (SPR) in this series, specifically increasing human metabolic clearance (in order to achieve short half-life) and solubility whilst improving potency. This work led to the identification of AZD4573, a potent inhibitor of CDK9 (IC50 of <0.004 μM) with fast-off binding kinetics (t1/2 16 min) and high selectivity versus other kinases, including other CDK family kinases. AZD4573 exhibits a short half-life in multiple preclinical species (less than one hour in rat, dog and monkey) and good solubility for intravenous administration. Short-term treatment with AZD4573 led to a rapid dose- and time-dependent decrease in cellular pSer2-RNAPII, resulting in activation of caspase 3 and cell apoptosis in a broad range of haematological cancer cell lines (e.g. caspase activation EC50 0.0137 μM in an acute myeloid leukemia model MV4-11). Correspondingly, in vivo efficacy was demonstrated in xenograft models derived from multiple haematological tumours (e.g. regression at 15 mg/kg twice weekly in MV4-11 xenografts). These results support AZD4573 as a clinical candidate for the treatment of haematological malignancies (first disclosure of the structure at this meeting).

1 Cidado J et al, AZ5576, a novel, potent and selective CDK9 inhibitor, induces rapid cell death and achieves efficacy in multiple preclinical hematological models, AACR poster presentation, 3572 (2016)

#1651

In vitro and in vivo profile of the preclinical candidate and MPS1 kinase inhibitor CCT289346.

Hannah Woodward, Paolo Innocenti, Kwai-Ming J. Cheung, Sébastien Naud, Amir Faisal, Grace W. Mak, Angela Hayes, Lisa O'Fee, Harry Saville, Alexis De Haven Brandon, Jennie Roberts, Gary Box, Melanie Valenti, Alan T. Heneley, Katie Walsh, Rosemary Burke, Suzanne A. Eccles, Florence I. Raynaud, Rob L. van Montfort, Julian Blagg, Spiros Linardopoulos, Swen Hoelder. _Institute of Cancer Research, London, United Kingdom_.

The mitotic kinase MPS1 (also known as TTK) is one of the main components of the spindle assembly checkpoint. MPS1 is required for chromosome alignment and kinetochore-microtubule error correction. Cancer cells are dependent on MPS1 to cope with chromosomal instability resulting from aberrant numbers of chromosomes. Moreover, MPS1 has been found to be deregulated in a large number of tumor types. MPS1 kinase inhibitors induce cancer cells to prematurely exit mitosis with incorrectly attached and unaligned chromosomes, causing severe chromosome mis-segregation, aneuploidy and cell death. These data stimulated us to pursue MPS1 as a cancer target. Extensive work by us and other groups has shown that MPS1 inhibitors are effective against a variety of cancers, particularly when used in combination with other drugs, such as paclitaxel. Here we disclose CCT289346, an MPS1 inhibitor currently completing late stage preclinical development. We describe the final stages of chemical optimisation and the data driven selection and nomination of CCT289346 as our preclinical candidate. We report key in vitro and in vivo preclinical data such as kinase profiling, PK in mouse, rat and dog, PK/PD relationship and efficacy in different in vivo models.

#1652

Development of a potent class of small molecule inhibitors of the MDM2-p53 protein-protein interaction.

Lynsey Fazal,1 Maria Ahn,1 Luke Bevan,1 Ildiko Buck,1 Juan Castro,1 Gianni Chessari,1 Ben Cons,1 Keisha Hearn,1 Steven Howard,1 Chris Johnson,1 Judith Reeks,1 Emiliano Tamanini,1 Neil Thompson,1 Hugh Walton,1 Pamela Williams,1 Ruth H. Bawn,2 Tim J. Blackburn,2 Celine Cano,2 Sarah J. Cully,2 Bernard Golding,2 Roger Griffin,2 Karen Haggerty,2 Ian Hardcastle,2 Herbie Newell,2 Martin Noble,2 Huw Thomas,2 Elaine Willmore,2 Yan Zhao,2 Steve Wedge2. 1 _Astex Therapeutics, Cambridge, United Kingdom;_ 2 _Nothern Institute for Cancer Research, Newcastle, United Kingdom_.

In response to cellular stress, the p53 tumor suppressor is activated to modulate cell cycle progression, DNA repair, and cell death. The activity of p53 is tightly regulated by MDM2, an E3 ubiquitin ligase that targets p53 for proteasomal degradation. Inhibition of the MDM2-p53 interaction in tumors carrying wild-type p53 can therefore reactivate p53 and elicit an anti-cancer effect. Small molecule inhibitors of the MDM2-p53 interaction remains a promising strategy for cancer therapy and a number of these compounds are in clinical development.

An isoindolinone series, identified by the Northern Institute for Cancer Research (NICR), has been used as a starting point for the development of potent MDM2-p53 inhibitors. Structure based drug design was applied during lead optimisation to gain potency whilst also focusing on stabilizing the main metabolically labile position and reducing lipophilicity. This approach led to potent compounds with EC50 <1 nM against MDM2 in cell-free ELISA assays and EC50 <30 nM for p53 induction in SJSA-1 osteosarcoma cells. Further analyses of the compounds demonstrated an increase in the levels of p53 and p53 transcriptional targets as a result of inhibiting the MDM2-p53 interaction. Using three pairs of isogenic cell lines, the compounds were shown to be specific for cell lines with wild-type p53. Key compounds were also characterized in pharmacokinetic and pharmacodynamic studies in mice bearing the SJSA-1 tumor xenograft where they displayed strong induction of p53, 3 hours post oral administration, together with an increase in the expression of p53 target genes p21 and MDM2. These potent MDM2-p53 inhibitors have also shown significant in vivo efficacy in the SJSA-1 xenograft model at well tolerated oral doses. Thus, promising lead compounds were identified, meriting further optimization of the series.

#1653

Design and synthesis of basic amino selective estrogen receptor degraders (BA-SERDs) for treatment-resistant breast cancer.

Yunlong Lu, Gregory R. Thatcher. _University of Illinois at Chicago, Chicago, IL_.

About 1 in 8 United States women (about 12%) will develop invasive breast cancer during their lifespan. Approximately 40,000 women are expected to die from breast cancer each year. The largest single breast cancer subtype is defined as estrogen receptor alpha (ERα; ESR1) positive breast cancer (75%), wherein ERα played a critical role in cancer cell survival, proliferation and metastasis. The dysregulation of ER signaling formed the most important molecular event in the ER positive (ER+) breast cancer. Endocrine therapy (ET) had been successfully employed on such breast cancer patients and most of them had substantial reduced risks of breast cancer recurrence and mortality in the initial 5-10 years. However, the development of resistance to ET has the greatest impact on clinical use. The emergence of resistance is inevitable over time and will eventually become metastatic disease after long term of ET. Recent studies confirmed treatment failure of SERMs and AIs in ER+ metastatic settings, whereas ERα remains engaged in the advanced disease and contributes to disease pathogenesis. Fortunately, the treatment resistant breast cancer patients remain responsive to fulvestrant (ICI182,780) which has been described as a "pure antagonist" and caused ERα degradation as a SERD. Nonetheless, the pharmaceutical liabilities of fulvestrant including poor solubility and pharmacokinetics (PK) resulted in poor compliance. The clinical development of orally bioavailable SERDs i.e. GDC-0810, AZD9496, RAD1901 is needed to overcome these shortcomings. Herein, our lab identified a novel class of BA-SERDs to circumvent drug resistance.

#1654

Chemical and structure-guided optimization of BAX trigger site activators for cancer therapy.

Denis E. Reyna, Felix Kopp, Evripidis Gavathiotis. _Albert Einstein College of Medicine, Bronx, NY_.

Cancer cells evade mitochondrial apoptosis most commonly by over-expressing anti-apoptotic BCL-2 proteins that suppress the activation of pro-apoptotic proteins. Pro-apoptotic BAX is a critical BCL-2 family effector protein that upon death stimuli is activated and induces mitochondrial outer membrane permeabilization that leads to cell death. The vast majority of cancer cells express wild-type BAX, therefore, direct BAX activation represents a potential therapeutic strategy in cancer. We previously presented the discovery of BTSA1 as a selective BAX activator with anti-leukemic activity and provided proof-of-concept for BAX as a druggable target in acute myeloid leukemia (AML) (Reyna et al, Cancer Cell, 2017). Here, we investigated chemical optimization of BTSA1 core scaffold to rationally optimize the potency and efficacy of the BAX trigger site activators in AML models. We designed compounds using a structural model of BTSA1 bound to the BAX trigger site that also fits with the previous established pharmacophore model. Synthesized compounds were evaluated for binding potency using a fluorescence polarization binding assay that measures compound competition of the fluorescein-label stapled BIM BH3 peptide from the BAX trigger site. Compounds with improved binding activity were evaluated for their potency in biochemical assays of BAX activation using isolated liposomal and mitochondrial membrane assays. Improved binding to BAX of BTSA1 derivatives correlated with better activation capacity. Moreover, compounds were evaluated for their efficacy in induction of BAX-mediated cell death in AML cell lines including Venetoclax-resistant cells. We have identified compounds with similar or superior cellular efficacy compared to BTSA1. Compounds were evaluated for ADME and in vivo pharmacokinetic profiles and these are compared to efficacy and pharmacodynamics data using in vivo AML xenograft models. Our work highlights our strategy for chemical and structure-guided optimization of BTSA1 demonstrating the potential for improved therapeutic induction of apoptosis in AML by BAX Trigger Site Activators.

#1655

Discovery and development of IACS-010759, a novel inhibitor of Complex I currently in phase I studies to exploit oxidative phosphorylation dependency in acute myeloid leukemia and solid tumors.

Maria Emilia Di Francesco,1 Joseph R. Marszalek,1 Timothy McAfoos,1 Christopher L. Carroll,1 Zhijun Kang,1 Gang Liu,1 Jay P. Theroff,1 Jennifer P. Bardenhager,1 Madhavi L. Bandi,1 Jennifer R. Molina,1 Sonal Gera,1 Marina Protopopova,1 Yuting Sun,1 Mary K. Geck Do,1 Ningping Feng,1 Jason P. Gay,1 Florian Muller,2 Marina Konopleva,3 Funda Meric-Bernstam,4 Carlo Toniatti,1 Timothy P. Heffernan,1 Giulio F. Draetta,1 Philip Jones1. 1 _Institute for Applied Cancer Science and Center for Co-Clinical Trials, University of Texas MD Anderson Cancer Center, Houston, TX;_ 2 _Department of Cancer Imaging Systems, University of Texas MD Anderson Cancer Center, Houston, TX;_ 3 _Department of Leukemia, University of Texas MD Anderson Cancer Center, Houston, TX;_ 4 _Department of Investigational Cancer Therapeutics, University of Texas MD Anderson Cancer Center, Houston, TX_.

Over the past few years we and others reported that specific populations of tumor cells including AML, subsets of lymphoma, glioblastoma, triple negative breast cancer (TNBC), melanoma and pancreatic ductal adenocarcinoma (PDAC) are highly dependent upon oxidative phosphorylation (OXPHOS) to meet their energy and biomass needs. Inhibition of OXPHOS in the context of these dependent tumor populations represents therefore an exciting therapeutic opportunity. Through an extensive medicinal chemistry campaign we discovered IACS-010759, a potent, selective small molecule inhibitor of complex I of the mitochondria electron transport chain that possesses excellent pharmacokinetic (PK) and pharmacologic properties, making it suitable for clinical development. We advanced IACS-010759 through IND studies and have recently initiated Phase I studies in patients with relapsed/refractory acute myeloid leukemia (AML) and advanced solid tumors and lymphomas (NCT02882321 and NCT03291938). In this presentation we will describe the identification of a novel series of Complex I inhibitors and their optimization into the clinical candidate compound IACS-010759. Several challenges were successfully overcome, including the optimization of the pharmacokinetic profile and the identification of inhibitors with minimal activity shift across preclinical species, thus enabling a thorough evaluation of the efficacy and toxicology profile. Aspects of the extensive translational research conducted to elucidate the mechanism of action of IACS-010759 and to position it into the clinic will be discussed, including the compelling pharmacological response observed in multiple PDX models of primary AML, and PDX xenograft models of lymphoma, TNBC, glioblastoma, melanoma and PDAC. The observed response was associated with robust pharmacodynamic read-out as assessed by modulation of oxygen consumption rate (OCR), aspartate and specific transcriptional changes. The presentation will also cover the preclinical development activities which resulted in IACS-010759 advancing into on-going phase 1 evaluation in AML and solid tumors.

#1656

First-in-class, fluorinated folate receptor specific agents that target tumor cells via inhibition of serine hydroxymethyltransferase 2 (SHMT2) and 5-aminoimidazole-4-carboxamide formyltransferase (AICARFTase).

Md Junayed Nayeen,1 Khushbu Shah,1 Aleem Gangjee,1 Aamod Dekhne,2 Zhanjun Hou,2 Larry H. Matherly2. 1 _Duquesne University, Pittsburgh, PA;_ 2 _Wayne State University School of Medicine, Detroit, MI_.

Tumor-specific folate receptor (FR) targeting over the ubiquitous reduced folate carrier (RFC) allows for specific tumor targeting without the attending dose-limiting toxicity of all clinically used cytotoxic agents. In an attempt to provide selectivity for FRα and/or β, folic acid has been conjugated with a variety of cytotoxic payloads to alleviate dose-limiting toxicities since folic acid is not transported via RFC (the major tissue folate transporter) to any significant extent. These conjugates have been unsuccessful in clinical trials likely in part due to the instability of the linkers used for conjugation. Premature cleavage results in the cytotoxic component being released prior to tumor penetration, thus precluding any possibility of selective tumor targeting. We designed novel pyrrolo[3,2-d]pyrimidine compounds in which both the targeting component and the cytotoxic component are in the same molecule. These compounds do not require any conjugation or linkers and hence do not suffer any premature cleavage. In this report, we synthesized and evaluated a "first-in-class" series of fluorinated analogues that target FRα/β and have limited transport via RFC, thus providing selectivity for tumors expressing FRα and/or FRβ. Two lead molecules, AGF347 and AGF355, were potent inhibitors of KB human tumor cell proliferation in vitro (IC50 = 1.05 nM and 6.25 nM respectively). Based on protection experiments with glycine, thymidine, adenosine and 5-aminoimidazole-4-carboxamide (AICA), AGF347 and AGF355 were identified as dual inhibitors of serine hydroxymethyltransferase 2 (SHMT2), a key enzyme in mitochondrial one-carbon metabolism and reported oncodriver, and AICA ribonucleotide formyltransferase (AICARFTase), the 2nd folate-dependent step in de novo purine biosynthesis. These compounds are currently being evaluated in preclinical studies as potential candidates for clinical trials.

#1657

Structure-activity relationship studies and biological evaluation of novel maytansinoids, a class of highly selective tubulin inhibitors.

Friederike I. Nollmann, Patricia Perez Galan, Javier Garcia Fernandez, Heidi K. Walter, Johannes P. Magnusson, Federico Medda, Felix Kratz, Stephan D. Koester, Khalid Abu Ajaj, Lara Pes, Serghei Chercheja, Anna Warnecke. _CytRx Corporation, Freiburg, Germany_.

Introduction and objectives: Maytansine and its analogs (DM1 and DM4) are potent microtubule-targeting compounds that inhibit proliferation of cells during mitosis.1 Unfortunately, their narrow therapeutic window prevents a clinical application of these molecules. So far only T-DM1, an antibody-maytansinoid conjugate targeting the HER2 receptor, has been approved for the treatment of resistant breast cancer. Previous work on maytansinoids showed that their potent cytotoxic activity is related to the nature of the substituent at the C3 acyloxy side chain. In order to harness the potential of compounds of the maytansinoid family while diminishing dose-limiting side effects, we synthesized a library of novel analogs which can be attached to serum albumin in vivo through an acid-sensitive linker ensuring release of the active maytansinoid at the tumor site. All novel maytansine analogs contain a keto moiety as an attachment point for the linker and differ in their substitution pattern at C3. The analogs we studied can be classified into those containing an amino acid spacer between C3 and the keto group (maytansine-type) and those obtained through direct esterification at the C3-OH-position with various carboxylic acids (ansamitocin-type).

Methods: All the newly synthesized molecules were screened for their in vitro cytotoxicity against 11 cancer cell lines. In order to explore the influence of the maytansine functional groups at C3, we designed novel analogs with different amino acid spacers at this position. We evaluated the influence of the chain length, the degree of steric hindrance, the need of the chiral center and the influence of the N-methyl on the cytotoxicity as well as stability in murine and human blood plasma. Moreover, we studied the structure-activity relationship of the ansamitocin-type of analogs including the influence of the chain length, the degree of steric hindrance and the effect of introducing different heteroatoms in the alkyl chain.

Result and conclusion: A total of 32 new maytansinoid analogs were synthesized, and seven of them were found to be more potent than the parent drug maytansine in inhibiting the growth of human cancer cells in vitro. Clear SARs were identified for both classes of compounds. Based on these studies, lead compounds have been selected for creating albumin-binding derivatives and their further in vivo evaluation.2

1) W. C. Widdison et al, J. Med. Chem., 49: 4392-4408 (2006); K. A. Poon et al, Toxicology and Applied Pharmacology, 273: 298-313 (2013); H. L. Perez et al, Drug Discovery Today, 19:869-881 (2014)

2) Abstract "In vivo efficacy of novel acid-sensitive albumin-binding Maytansinoid-based prodrugs in human cancer xenograft models in nude mice"

#1658

**5N-substituted pyrrolo[3,2** -d **]pyrimidines: Tumor-targeted agents with first-in-class dual inhibition of serine hydroxymethyl transferase 2 and 5-amino-4-imidazolecarboxamide ribonucleotide formyl transferase enzymes.**

Khushbu Shah,1 Aamod Dekhne,2 Zhanjun Hou,2 Larry Matherly,2 Aleem Gangjee1. 1 _Duquesne Univ., Pittsburgh, PA;_ 2 _Wayne State University, Detroit, MI_.

Design, modeling and synthesis of pyrrolo[3,2-d]pyrimidine analogs were carried out based on the concept of dual mechanisms-of-action for achieving maximal antitumor efficacy. Tumor-targeted delivery via agents that are selectively transported by the folate receptor (FRα/β) and/or PCFT, overexpressed in tumor cells such as epithelial ovarian cancer and non-small cell lung cancer, over the ubiquitously expressed reduced folate carrier (RFC) would afford tumor selectivity. Gangjee, Matherly and coworkers previously reported 5-substituted pyrrolo[2,3-d]pyrimidines as inhibitors of de novo purine nucleotide biosynthesis glycinamide ribonucleotide formyltransferase and 5-aminoimidazole-4-carboxamide (AICA) ribonucleotide formyltransferase (AICARFTase). To synthesize potential dual inhibitors of de novo purine biosynthesis at cytosolic AICARFTase and mitochondrial one-carbon metabolism at serine hydroxymethyl transferase (SHMT) 2, we generated structural hybrids of these inhibitors and 5,10-methylenetetrahydrofolate (the SHMT2 cofactor), 5-substituted pyrrolo[3,2-d]pyrimidine analogs AGF291, AGF299, AGF300, AGF318, AGF320, and AGF331. Molecular modeling of these compounds utilizing X-ray crystal structures of the transporters (FRα/β) and enzyme targets (AICARFTase, SHMT2) predicted excellent activities. The influence of bridge lengths and the aromatic moieties of these novel compounds were evaluated by testing their anti-proliferative activities in Chinese hamster ovary cells expressing human FRα or PCFT, and in FRα- and/or PCFT expressing nasopharyngeal carcinoma (KB) and lung cancer (H460) cells. All the pyrrolo[3,2-d]pyrimidine analogs were inhibitory toward FR- and/or PCFT-expressing cells. Compared to its pyrrolo[2,3-d]pyrimidine analog, AGF300 increased inhibition of cells via FR uptake from IC50 8.6 nM to 2.1 nM (by 4-fold) and decreased uptake through RFC from 56.5 nM to 516 nM (by 10-fold), suggesting a FR-targeted delivery of the agent. Toward H460 xenografts in SCID mice, AGF291 was efficacious. Protection studies with thymidine, glycine, adenosine and AICA and radiotracer studies with [3-14C]serine identified the likely intracellular targets of the pyrrolo[3,2-d]pyrimidine analogs as SHMT2 and AICARFTase. Mitochondrial one-carbon metabolism originating with SHMT2 is a critical source of reducing equivalents and one-carbon units for cytosolic biosynthesis and SHMT2 has been implicated as an oncodriver in several tumor types. While inhibition of SHMT2 was the subject of exploratory studies, our novel pyrrolo[3,2-d]pyrimidine series are "first-in-class" in terms of their in vitro and in vivo antitumor efficacies attributable to dual targeting SHMT2 and AICARFTase.

#1659

C8-linked benzofused pyrrolobenzodiazepine (PBD) hybrid molecules inhibit NF-κB activity in hematologic cancer cells.

David B. Corcoran,1 Thomas Lewis,2 Paul J. Jackson,1 Christopher D. Fegan,2 Chris Pepper,3 David E. Thurston,1 Khondaker Miraz Rahman1. 1 _King's College London, London, United Kingdom;_ 2 _University of Cardiff, Cardiff, United Kingdom;_ 3 _University of Sussex, Brighton, United Kingdom_.

The pyrrolo[2,1-c][1,4]benzodiazepines (PBDs) are a well-known class of sequence-selective covalent-binding DNA-interactive agents that fit perfectly in the minor groove due to their C11a(S)-chirality that provides a right-handed isohelical twist. The most effective synthetic modifications to PBD cores have involved the conjugation of two DNA-interactive moieties via their C8-positions to create PBD dimers capable of cross-linking duplex DNA which significantly enhances cytotoxicity. Examples of PBD dimers are currently being used as cytotoxic payloads for Antibody-Drug Conjugates (ADCs). Other strategies have included the addition of a polyheterocyclic chain to the C8-position of the PBD core which improves DNA binding affinity and sequence-selectivity, and cytotoxicity. A library of C8-conjugated PBDs has been designed and synthesized, with the C8-polyheterocyclic chain varying in structure to produce molecules with the potential to bind to different transcription factor binding sites. Given that NF-κB plays a pivotal role in the initiation and maintenance of many haematological and solid cancers, we evaluated these NF-κB-targeting PBDs in cancer cell lines and primary leukaemia cells. The compounds tested had low nanomolar LD50 values (i.e., 2.65 to 7.8 nM) in multiple myeloma cell lines and primary chronic lymphocytic leukaemia (CLL) cells; normal age-matched B-cells and T-cells were 2.4-fold and 4.6-fold less susceptible to their cytotoxic effects, respectively. In the multiple myeloma JJN3 cell line, the lead compound (DC-1-192) showed significant inhibition of DNA-binding of the p65, p50 and Rel B sub-units of the NF-κB transcription factor after just 4h exposure, demonstrating potent dual inhibitory properties in both the canonical and non-canonical NF-κB pathways. RNA-sequencing confirmed gene-set enrichment for NF-κB pathway genes although, as anticipated, other pathways were also affected. Importantly, the PBDs evaluated here (including the lead molecule DC-1-192) showed synergism with the two clinically-useful agents, bortezomib and ibrutinib, in JJN3 cells and primary CLL cells, respectively. These data provide a strong rationale for combining agents based on DC-1-192 with these approved drugs for the more effective treatment of the indicated haematological cancers.

The observations reported here add significantly to current understanding of the SAR of PBD monomer structures, and have also identified a lead agent (i.e., DC-1-192) for pre-clinical development for the treatment of CLL and multiple myeloma.

#1660

Unraveling isoform selectivities of a broad class of HDAC inhibitors with QM/MM molecular dynamics simulations.

Jung-Hsin Lin. _Academia Sinica, Taipei, Taiwan_.

Therapeutics that have been used by large populations and showed limited or little adverse effects can be good candidates for cancer chemo-prevention. As an example of such therapeutics, statins, i.e., HMG-CA reductase inhibitors, have been widely prescribed for lowering the blood cholesterol level and for treating cardiovascular diseases. In 2008, we discovered that statins are moderate inhibitors of histone deacetylases (HDACs), which provides an epigenetic mechanism for their efficacy in cancer prevention. HDACs have been long recognized for as an important class of therapeutic targets, and these enzymes utilize a Zn2+ ion at its active site for catalysis. Subsequently, in our 2013 J. Med. Chem. article, we showed that we are able to strengthen this epigenetic mechanism and designed more potent and effective therapeutic agents for cancer prevention and treatment, including cancer metastasis, and published in Clin. Cancer Res. in 2016. To investigate the structure-activity relationships of a broad class of HDAC inhibitors with distinct chemical moieties, we have conducted QM/MM molecular dynamics simulations of several systems of HDAC isoforms bound with various ligands. In contrast to molecular dynamics simulations with classical force fields, which always rendered over-coordinated configurations of the Zn2+ at the active site, with the coordination numbers (CN) to be either 6 or 7, in our QM/MM molecular dynamics simulations, the CNs of Zn2+ remain 4 or 5. With the structural and energetics analysis from these QM/MM molecular dynamics simulations, we are able to delineate the origins of isoform-selectivity of different HDAC inhibitors, including the aforementioned HMGR-HDAC dual-action inhibitors that we recently developed.

#1661

**Discovery of potent Abelson kinase inhibitors from the scaffold of pyrano[2,3-** d **]pyrimidin-7-one.**

Zhixing Wu, Boqiao Fu, Changjiang Qiao, Ashila Nagaraju, Dai Lu. _Texas A &M Univ. Health Science Ctr., Kingsville, TX_.

Tyrosine kinases regulate cell proliferation, survival, invasion and angiogenesis during tumor initiation and progression. The Abelson (ABL) family of protein kinases comprises ABL1 and ABL2, which link diverse extracellular stimuli to signaling pathways that control cell growth, survival, invasion, adhesion and migration. Inhibition of ABLs has been implied in many type of hematopoietic malignancies and solid tumors. The compound PD173955 is a potent ABL kinase inhibitor. It was developed from the scaffold pyrido[2,3-d]pyrimidin-7-one by Parke-Davis (Pfizer). To discover novel ABL inhibitors, we investigated the scaffold pyrano[2,3-d]pyrimidin-7-one, which is structurally analogous to pyrido[2,3-d]pyrimidin-7-one. This novel scaffold has not been extensively studied in synthetic chemistry and pharmacology. The compound LDK1504 generated from this scaffold is a close analog of PD173955. It was synthesized as a prototypical compound from the pyrano[2,3-d]pyrimidin-7-one scaffold. In the enzymatic assays, LDK1504 was demonstrated with an inhibitory activity comparable to PD173955 for ABL1 kinase. It can inhibit the ABL1 kinase with an IC50 of 18.7 nM. Further optimization leads to the identification of LDK1512 (Kd = 0.38 nM; IC50 = 1.27 nM) against ABL1. The preparation of this class of compounds was achieved through a facile synthesis of 6-bromo-2-(methylthio)-7H-pyrano[2,3-d]pyrimidin-7-one followed by Suzuki coupling reaction and derivatization of the C-2 position of the pyrimidine ring. In this presentation, our synthesis, preliminary SAR of this class of compounds, and kinase selectivity profiling will be discussed.

#1662

Synthesis and biologic evaluation of diethylbenzaldehyde analogues to probe functional activity of aldehyde dehydrogenase activity in cancer.

Ali Ibrahim,1 Maria Sadiq,1 Daniela Presa,1 Ioannis Ntafoulis,1 Ahmad Alkawi,1 Fiona Frame,2 Norman J. Maitland,2 Zoe Cournia,3 Paul M. Loadman,1 Jan Moreb,4 Klaus Pors1. 1 _Univ. of Bradford Inst. of Cancer Therapeutics, Bradford, United Kingdom;_ 2 _University of York, York, United Kingdom;_ 3 _Academy of Athens, Athens, Greece;_ 4 _University of Florida, Gainsville, FL_.

The human aldehyde dehydrogenases (ALDHs) play a major role in detoxifying highly reactive aldehydes into carboxylic acids. Deregulation of ALDHs has implications in a number of cancers. They play an important role as a cancer stem cell (CSC) marker due to high activity found in CSCs, while high expression is also known to lead to resistance to chemotherapeutic drugs. Although the exact roles of ALDHs are not fully understood, emerging information indicates several isoforms from the ALDH1 family, ALDH3A1 and ALDH7A1, play key roles in several cancer types. To probe the role of ALDH functional activity, we here report on the perturbation of ALDH expression and function using diethylbenzaldehyde (DEAB) analogues. The H1299 lung cancer cell line has negligible amount of ALDH activity, and ALDH-expressing variants were used to study the novel DEAB analogues. H1299 wild-type cells (WT) and cells expressing RFP were used as controls. Expression of the proteins was confirmed by Western blots and ALDH enzymatic activity assay. We used cell culture, colorimetric MTT assay, aldefluor assay and in vitro treatment with DEAB analogues. ALDH-expressing lysates were used to obtain biochemical inhibition data of the DEAB analogues using propionaldehyde as substrate with NAD+ as a co-factor. DEAB has been shown to covalently bind to ALDH7A1, and computational modeling was employed to understand how chemical modifications to DEAB affected the binding mode. Selected ALDH-affinic probe compounds were tested in a panel of colon, lung and prostate cancer cell lines including docetaxel-resistant variant and patient samples to investigate if DEAB analogues had an effect on cell viability as single agents or in combination with docetaxel. In conclusion, the novel DEAB analogues aid us to understand ALDH functional activity and provide a platform for discovering more selective ALDH inhibitors

#1663

Identification and characterization of a small-molecule inhibitor of the human uracil-DNA glycosylase for use in combination with DNA damage-based anticancer therapies.

Mya T. Nguyen, Yan Yan, Yuriy Fedorvo, John Pink, Drew Adams, Stanton Gerson. _Case Western Reserve University, Cleveland, OH_.

Our research aims to develop therapeutically active inhibitors of the human uracil-DNA glycosylase (UNG). UNG, a highly conserved enzyme, plays a pivotal role in preventing abnormal bases, uracil and 5-fluorouracil, from incorporation into DNA as a result of treatment with chemotherapeutic drugs, floxuridine (5-FdU) and pemetrexed (PEM). Our previous publication reports that the depletion of UNG by shRNA causes uracil and 5-FU incorporation into DNA following 5-FdU or PEM exposure, and highly sensitizes certain human cancer cells to these chemotherapeutic drugs. These data suggest that UNG specific inhibitors could improve the anticancer effect of 5-FdU or PEM. However, a few known inhibitors have not been shown to function at therapeutic concentrations in human cells. Therefore, in order to identify a novel, small-molecule compound targeting the human UNG enzymatic activity, we have previously optimized a biochemically high-throughput screening (HTS) assay that measures the removal of uracil from a DNA hairpin based on the un-quenching of fluorescence. This assay has been successfully employed in a pharmacologically active compound collection screen (>3,000 small-molecules+ approved drugs). In 384-well plate runs using control wells in which enzyme is not added, the average assay provides signal to background ratios of 3.0 to 4.0. Typical Z factors range from 0.6-0.7, indicating a robust assay. We have identified five bioactive compounds with IC50 values < 1uM and have selected a nanoMolar inhibitor of the human UNG enzyme termed, UNGi-A. By using an in vitro DNA glycosylase activity assay, this compound displays a significant inhibitory activity towards the purified human UNG enzyme over time, in a dose-dependent manner. Further characterization shows a significant effect of this inhibitor on UNG activity in cellular extract that expresses endogenous UNG from DLD1 human colon cancer cell line. The result suggests that UNGi-A compound diminishes UNG activity in cell extracts. In addition, thermal shift assay (TSA) data suggest that UNGi-A binds directly to the human UNG enzyme. Together, this compound shows potential activity that blocks the human UNG activity and we are currently evaluating the effect of UNGi-A in combination with 5-FdU on UNG activity in human cancer cells.

#1664

4-Bromophenylhydrazinyl benzenesulfonylphenylureas inhibit indoleamine 2,3-dioxygenase in vitro and in vivo.

Shau-Hua Ueng, Ching-Chuan Kuo, Shu-Yu Lin, Teng-Kuang Yeh, Jen-Shin Song, Ming-Shiu Hung, Chiung-Tong Chen. _National Health Research Institutes, Miaoli County, Taiwan_.

Indoleamine 2,3-dioxygenase has been considered as a promising anti-cancer drug target. Several pharmaceutical companies, including Pfizer, Merck, and Bristol-Myers Squibb, are known to be in pursuit of IDO inhibitors, and Incyte recently reported good results in the phase II clinical trial of the IDO inhibitor Epacadostat. In previous work, we developed a series of IDO inhibitors based on a high-throughput screening core structure, sulfonylhydrazide. Further, we developed the 4-bromophenylhydrazinyl benzenesulfonylphenylurea, as a potent IDO inhibitor with an IC50 value within 100 nM in inhibiting IDO in vitro. This compound demonstrated a 30% reduction in tumor weight in a murine CT26 syngeneic model on day 18 with 100 mg/kg oral administration twice daily, indicating that the 4-bromophenylhydrazinyl benzenesulfonylphenylurea may have potential for further investigation in the development of anti-tumor drugs.

#1665

AZ304, a novel dual BRAF inhibitor, exerts antitumor activity in colorectal cancers independent of BRAF status.

Rui Ma,1 Ling Xu,1 Xiaofang Che,1 Xiujuan Qu,1 Lisa Drew,2 Minhui Shen,2 Tony Cheung,2 Yunpeng Liu1. 1 _China Medical Univ. Hospital, Shenyang, China;_ 2 _Oncology iMED, AstraZeneca R &D, Armenia_.

Background: BRAF mutation is associated with poor clinical outcome of malignant tumors and mediates resistance to chemotherapy and targeted therapy. We aimed to determine whether wild type and V600E mutant BRAF colorectal cancers exhibit distinct sensitivities to dual BRAF inhibitor, AZ304.

Methods: We measured kinase activity by AlphaScreen assay. MTT assays, colony-formation assays, and Western blot were performed to evaluate the in vitro anti-tumor effects of AZ304. In vivo efficacy was investigated by xenograft study and Immunohistochemistry.

Results: AZ304 exhibited potent inhibitory activity against both wild type and V600E mutant forms of the serine/threonine-protein kinase BRAF with enzyme IC50values of 79nM and 38nM, respectively. By suppressing the ERK phosphorylation, AZ304 led to effective cell proliferation inhibition against a panel of human cancer cell lines with different BRAF and RAS generic status. In selected colorectal cancer cell lines, AZ304 significantly inhibited cell growth in vitro and in vivo, regardless of BRAF genetic status. Additionally, combination with the EGFR inhibitor Cetuximab enhanced the potency of AZ304 independent of BRAF mutational status.

Conclusions: The BRAF inhibitor, AZ304, had a broad spectrum of antitumor activities and the combination with Cetuximab significantly enhanced AZ304 potency against colorectal cancers in vitro and in vivo.

#1666

Novel glucose transporter inhibitors decrease glioblastoma growth and glucose uptake.

Catherine J. Libby,1 Sixue Zhang,2 Gloria A. Benavides,1 Yanjie Li,1 Matthew Redmann,1 Anh N. Tran,1 Arphaxad Otamias,1 Victor Darley-Usmar,1 Marek Napierala,1 Jianhua Zhang,1 Wei Zhang,2 Anita Hjelmeland1. 1 _University of Alabama at Birmingham, Birmingham, AL;_ 2 _Southern Research, Birmingham, AL_.

Glioblastoma multiform (GBM) is the most common primary malignant adult brain tumor and one of the deadliest cancers. Patients with GBM typically undergo surgical resection, radiation and chemotherapy with temozolomide. Complete tumor resection is virtually impossible due to tumor location and the highly infiltrative nature of GBM, which leads to disease recurrence near the original tumor site. As such, these patients have a dramatically decreased life expectancy. Therapeutic development to prolong survival has been hampered by a high degree of inter- and intra-tumoral heterogeneity. Contributing to tumor heterogeneity is a subset of highly tumorigenic cells, termed brain tumor initiating cells (BTICs), that are able to self-renew and can be highly invasive and therapy resistant. BTICs are often enriched in perinecrotic regions, a GBM hallmark, where they can survive under nutrient restriction via increased glucose transporter 3 expression (GLUT3). GLUT3 has a 5-fold greater capacity for glucose transport than the other major glucose transporter isoform in the brain, GLUT1, and is typically restricted to neurons, testis, preimplantation embryos, and stem cells. GLUT3 expression is elevated in many solid tumor types, including GBM, and correlates with poor patient prognosis. Previously, we have shown that knockdown of GLUT3 in BTICs significantly inhibits their growth both in vitro and in vivo, indicating GLUT3 is a possible target for therapeutic intervention. Using structure based virtual screening, we have identified novel GLUT inhibitors that preferentially decrease the growth and glucose uptake of BTICs with minimal toxicity to non-malignant cells in vitro. Preliminary in vivo assessment of these compounds has not indicated toxicity. Our goal is to identify a potential new therapeutic option targeting metabolic reprogramming for the treatment of glioblastoma, as well as other tumor types.

#1667

Design and development of a parthenolide-combretastatin hybrid drug (VA-11) with highly potent antimicrotubule and NF-kB inhibitory activities.

Viswanath Arutla, Surendra R. Punganuru, Hanumantharao Madala, Kalkunte S. Srivenugopal. _Texas Tech Univ. Health Sciences Ctr., Amarillo, TX_.

Given the molecular complexity of cancers, their extreme heterogeneity and innate or acquired resistance to anticancer drugs, there is an increasing need for hybrid drugs that can effectively and simultaneously impact two or more targets triggering multiple cytocidal events in synergy to obtain superior chemo-sensitivities and prevent tumor resistance. Therefore, we developed a hybrid drug by combining the NF-kb inhibiting and oxidative stress-inducing parthenolide, a sesquiterpene lactone derived from the plant feverfew with the combretastatin A4 (CA4), a potent antimicrotubule and anti-angiogenesis agent. These hybrid compounds were synthesized by incorporating the α-methylene-γ-butyrolactone, an active component of parthenolide into the CA4 by replacing the unstable cis-double bond. The structural design of these hybrid compounds preserved the CA4 nucleus in the cis-configuration and prevented its isomerization into its biologically inactive trans-form. VA-11 was the highly potent among the different compounds prepared in specifically killing the tumor cells of various human cancer types at nano molar range. These included the colon (HT29, HCT116, SW640), breast (MDA-MB-231, SK-BR-3, MCF7), lung (A549, H1299, H460), brain (SF188, GBM10, GBM6, DAOY, T98G, U87MG) and pancreas (MIA-PaCa-2, PANC-1, BxPC-3). Particularly significant was that VA-11 was 10 times more potent than CA4 against the HT29 colon cancer cells. Various biochemical and immunocytochemical assays revealed that VA-11 retained the microtubule-disrupting effects of CA4, including microtubule depolymerization, the formation of aberrant mitotic spindles, and G2/M phase arrest. The hybrid drug also inhibited the polymerization of purified tubulins in vitro. Furthermore, VA-11 potently inhibited the formation of tubes in three-dimensional cultures of the HUVEC (Human umbilical Vein Cells), drastically decreasing the tube length and junctions at 1 μM concentration. The cancer cell migration /invasion, determined by transwell assays were also inhibited, and the inhibition was accompanied by increased E-cadherin levels through NF-κB/Snail pathway. Further, VA-11 inhibited the NF-κB activation, IκBα degradation, and IκB Kinase complex activity in HT29 cells tested in this study. VA-11 pretreatment significantly inhibited the IL-8 secretion induced by the cytokines such as LPS and/or TNF. These studies confirmed that VA-11 inhibits IκB kinase, resulting in stabilization of cytoplasmic IκBα, which in turn leads to inhibition of NF-κB translocation and attenuation of subsequent inflammatory responses and drug resistance. In conclusion, our results indicate that VA-11 represents a new prototype chemo drug with multiple mechanisms of action. Such hybrid anticancer drugs merit further development (supported by CPRIT grants RP130266 and RP170207 to KSS).

#1668

NHWD-870, a novel BET family bromodomain inhibitor targeting BRD4, proved to be effective and promising for treatment of small cell lung cancer.

Yongchang Zhang, Fang Wu, Nong Yang. _Hunan Cancer Hospital, Changsha, China_.

Background, Small molecule inhibitors targeting bromodomain and extraterminal domain (BET) protein is promising for cancer therapy. Previous study show that BRD4 was unregulated in different kind of cancer, with leukemia included, and BETi (BET inhibitors), such as JQ1(+) show evidence to inhibit the proliferation of cancer in vitro and vivo. However, there is no powerful data to evaluate the expression of BRD4, phosphorylation of BRD4 (p-BRD4) and the effectiveness of BETi in small cell lung cancer (SCLC). According to our previous study, BRD4 was highly expressed in 34 SCLC patients (70%) and correlated with poor prognosis in.

Method, More than 120 tissue samples and survival data of SCLC patients in Hunan Cancer Hospital were collected. Immunohistochemistry was conducted to evaluate the expression of BRD4, p-BRD4 and the prognosis of these SCLC patients. Based on our previous finding, we design and modify series small molecular compound with carbocycles shape named NHWDs. We use the MTT in SCLC cell lines to evaluate the activity and selected the best. And then potential pathway was demonstrated with Array and Western blot. Finally, the activity of small compounds free base and HCL formation were conducted by xenograft and Patient Derived Xenograft (PDX) with or without double Platinum chemotherapy patients of etoposide and cisplatin resistant.

Result, In 120 SCLC formalin-fixed specimens, BRD4 and p-BRD4 was highly expressed in SCLC and correlated with the poor prognosis of SCLC patients. All the compounds, free base and HCL formation which design and modify by ourselves were potent and selective BET family bromodomain inhibitor, not only binds bromodomains of BRD2/3/4/T, but the p-BRD4. Compare with JQ1(+) and other molecular compounds, NHWD-870 free base and NHWD-870-HCL have the best effectiveness and powerful anti-cancer in SCLC, and the IC50 was 1.579nM. NHWD-870 exhibited robust single agent activity in cell viability assay across cell lines in vitro and xenografts of SCLC in vivo by downregulating MYC. Consistent with its broad spectrum of activities in invo, NHWD-870 and NHWD-870-HCL have potent tumor suppressive efficacies in PDX model of SCLC. Monotherapy and combining with antiangiogenesis, such as bevacizumab and apatinib, NHWD-870 and NHWD-870-HCL proved to be more effective in SCLC cell lines and PDX in patients who show resistant to double Platinum chemotherapy with etoposide and cisplatin.

Conclusion, NHWD-870 shows to be a powerful anti-cancer drug in SCLC. Further basic and clinical research should be conducted from bench to beside.

#1669

Identification of mutant KRas-Raf-1 binding disruptors.

Rajanikanth Vangipurapu,1 Liwei Chen,1 Perry Kennedy,1 Karen C. Morrison,2 Paul J. Hergenrother,3 Said M. Sebti1. 1 _Moffitt Cancer Center, Tampa, FL;_ 2 _Cal Recycle, CA;_ 3 _University of Illinois, Chicago, IL_.

Mutant KRas is a major driver of human cancer, yet currently no clinically approved therapies that directly target mutant KRas are available. In order to identify novel drugs that bind either mutant KRas or its effector Raf-1, we used alpha-screen and GST-pull down assays to identify disruptors of KRas G12D binding to the Ras-binding domain of Raf-1. From a novel, natural product-inspired chemical library called Complexity To Diversity (CTD), we have identified a series of 5 hits that belong to a family of gibberellic acid-derived compounds and 2 hits from a family of pleuromutilin-derived compounds. At present we are using Differential Scanning Fluorimeter (DSF) and Isothermal calorimetry (ITC) assays to determine whether these hits bind to KRas G12D or Raf-1 RBD. These studies may lead to identifying chemical probes that can be developed to target mutant KRas-driven cancers.

#1670

Discovery of novel water-soluble derivatives of mebendazole as selective CLK1/4 kinase inhibitors and their anticancer cancer activity.

Lijun Sun,1 Jae Eun Cheong,2 Michela Zaffagni,3 Kun Zhou,3 Bruce Zetter4. 1 _Beth Israel Deaconess Medical Ctr. Harvard Medical School, Boston, MA;_ 2 _Beth Israel Deaconess Medical Ctr., Boston, MA;_ 3 _Boston Children's Hospital, Boston, MA;_ 4 _Boston Children's Hospital, Harvard Medical School, Boston, MA_.

Metastatic cancers remain clinically challenging and account for more than 90% of all cancer deaths. Drugs used to treat advanced metastatic cancers often generate drug resistance and relapse. Therefore, there is a critical need for novel therapeutic approaches for patients with advanced stage cancers that do not respond to any currently available anticancer therapies. Mebendazole and structurally related benzimidazole analogues, which are FDA approved compounds used to treat helminthic infections in the gastrointestinal track, are effective in inhibiting in vitro cancer cell proliferation. Unfortunately, their therapeutic applications in metastatic cancer are limited by their extremely low solubility and poor bioavailability. Further, the mechanism of the anticancer activities of this class of compounds is poorly defined. Here, we report the design and synthesis of water-soluble benzimidazoles as novel anticancer agents. Among them, the novel oxetanyl substituted compound, OBD9 (Methyl (5-(4-(methyl(oxetan-3-yl)amino)benzoyl)-1H-benzo[d]imidazol-2-yl)carbamate), demonstrated potent cytotoxicity towards a variety of highly aggressive cancer lines including prostate, lung, and ovarian cancers (IC50: 0.9-3.8 μM). In the NCI60 cancer cell panel screen, OBD9 broadly inhibited the proliferation of leukemia, melanoma, and breast and colon cancers. The aqueous solubility of OBD9 achieved 361 μM vs <1μM for mebendazole. In a mouse xenograft model of the highly metastatic human prostate cancer PC3MLN4, OBD9 (30 mg/kg/day, three times/week for two weeks) significantly inhibited the growth of established tumors (treatment-to-control ratio: 0.36) without noticeable toxicity. We performed broad kinase screening (KINOMEscan, DiscoverX) to explore the mechanism of action and discovered OBD9 as a potent and highly selective inhibitor of Cdc-like kinase 1 and 4 (CLK1 and 4). The Selectivity Score of OBD9 at 10 μM concentration in the 403 non-mutant kinases is 0.002; and its IC50 is 1.5 and 1.2 μM for CLK1 and CLK4, respectively. CLKs are nuclear serine/threonine (S/T) kinases that regulate gene splicing and are frequently over activated in cancers. Our results suggest that OBD9 impedes cancer growth at least in part by inhibiting CLK1/4.

#1671

Identification and characterization of MAM03055A, a novel bivalent sigma-2 receptor agonist.

Cheri Z. Liu,1 Marco Mottinelli,2 Hilary E. Nicholson,1 Eric Zhong,1 Christopher R. McCurdy,2 Wayne D. Bowen1. 1 _Brown University, Providence, RI;_ 2 _University of Florida, Gainesville, FL_.

Sigma-2 receptors are highly expressed in various types of cancer and mediate dual effects on cancer cell viability. Agonists induce programmed cell death by a number of pathways including apoptosis and autophagy. Other classes of sigma-2 receptor ligands activate pathways related to glycolytic metabolism and hypoxia. CM572 is a 6-isothiocyanato benzoxazolone derivative that binds irreversibly and selectively to sigma-2 receptors, presumably through attack of the isothiocyanate moiety by a receptor nucleophile. It is a partial agonist, inducing dose-dependent cell death. We observed by LC/MS analysis, that upon storage in DMSO solution at -20oC over several months, CM572 degrades into a mixture of compounds that appear to retain both receptor binding activity and ability to induce cell death. The degradation was eventually convergent to one main compound (CM572-HN). CM572-HN was identified as a dimeric molecule based on the isotopic fingerprint of the high-resolution mass spectrum. Similarly, a degradation study by 1H NMR of a solution of CM572 in deuterated DMSO showed convergence to one major compound. The degradation site was narrowed down to the isothiocyanate group, as only the signals for its neighboring protons were shifted in the degradation product. We suspected hydrolysis, the final degradation step thus being the corresponding amine. It was then hypothesized that the kinetics of degradation of CM572 towards the corresponding 6-substituted amine (CM571) was slower than that of the nucleophilic attack of CM571 on the isothiocyanate group of CM572. Hence, CM571 and CM572 were synthesized and then reacted together to produce, in fact, a single compound (MAM03055A), with mass identical to one originally identified in the stock solution and 1H NMR spectra fitting those of the compound originally obtained during the NMR degradation study. Thus the novel compound is essentially a thiourea-linked dimer of the amine, CM571. Receptor binding assays revealed that MAM03055A (MAM) exhibited selectivity for sigma-2 receptors over sigma-1, with sigma-1 Ki = 2,829 nM and sigma-2 Ki = 56.7 nM, resulting in 50-fold selectivity for sigma-2 receptors. MAM induced dose-dependent cell death in human SK-N-SH neuroblastoma cells, with an EC50 = 9.24 ± 1.15 µM for a 24h incubation. Interestingly, unlike other sigma-2 agonists that give 100% cell kill, MAM reached a maximum of only 80%, even at a concentration of 100 µM. The true EC50 in this case was therefore around 2 µM. Thus, MAM behaved like a partial agonist. The binding affinities and cytotoxic activity of the dimer resembles that of CM572 (isothiocyanate; EC50 = 7.6 µM), but is very different from CM571 (amine), which has high affinity at both subtypes and stimulates metabolism. Bivalent sigma-2 ligands may thus have interesting properties that deserve further exploration. (*C. L. and M. M. contributed equally as first authors)

#1672

Preclinical evaluation of WXFL1024H251, a potent second-generation HSP90 inhibitor.

Xiaobing yan,1 Charles Z. Ding,1 Lihong Hu,1 Wei Huang,1 Shuhui Chen,2 Dongdong Wu,2 Jiahu Wu,2 Guoqiang Ma,2 Qiyao Zhang,2 Rui Gao,2 Yusong Zhu,2 Tiantian Dong,3 Yizhong Zhu,3 Caixia Yan,3 Yan Yan3. 1 _WuXi AppTex, Wuhan, China;_ 2 _WuXi AppTex, Shanghai, China;_ 3 _Chia Tai Tianqing Pharmaceutical Group Co. Ltd., Nanjing, China_.

Objective: Heat Shock Protein 90 (Hsp90) is a molecular chaperone that mediates the post-translational stability of its protein substrates, many of which are onco-proteins. Hsp90 is emerging as an important target for cancer therapy because its inactivation may abrogate multiple signaling pathways simultaneously, irrespective of the mutational status of proteins involved. We disclose here our clinical candidate WXFL1024H251, a novel second-generation small-molecule Hsp90 inhibitor with a chemical structure unrelated to the first generation, the ansamycin family of Hsp90 inhibitors.

Method: The anti-proliferative activity of WXFL1024H251 was evaluated in a variety of cancer cell lines in a 96-hour viability assay. QW, Q2W or TIW intravenous administration of at doses of 5~50 mg/kg was used to evaluate the in vivo anti-tumor activity of WXFL1024H251 in BT474 cell line-derived xenograft (CDX) breast cancer model. Effects of WXFL1024H251 on HSP90 function were assessed by western blot analysis of PD markers HSP70, Her2 and p-Her2 in the BT474 tumors.

Result: A panel of 20 cancer cell lines were screened for sensitivity to WXFL1024H251. Proliferation of 2 cancer cell lines A549 and BT474 were potently inhibited by WXFL1024H251, with IC50 of 0.007 μM and 0.009 μM, respectively. A549 cell line has KRAS mutation and medium to high EGFR expression. BT474 is an ER+ and ERBB2+ breast cancer cell line. In the BT474 tumor model, 3 week treatment with WXFL1024H251 showed good antitumor efficacy with T/C values of 36% at 7.5 mg/kg Q2W and 45% at 10 mg/kg QW; toxicity were acceptable with both dosing regiments.

Conclusion: These preclinical studies showed good antitumor efficacy of WXFL1024H251, which was associated with client protein maturation and HSP70 up-regulation in the tumor tissue. WXFL1024H251 may represent a novel promising clinical antitumor agent for treating breast cancer and other cancer types with high expression of HSP90.

#1673

Ras degrading small molecules inhibit the transformation of colorectal cancer cells independently of beta-catenin signaling.

Jeong-Ha Hwang, Eun Ji Ro, Wookjin Shin, Kang-Yell Choi. _Yonsei University, Seoul, Republic of Korea_.

Development of drugs controlling Ras is an emerging issue in cancer therapy; however, no clinically applicable drug is currently available. On the basis of the mechanism of Ras protein stability regulation via the Wnt/β-catenin signaling pathway, we previously identified small molecules inhibiting the proliferation and transformation of various colorectal cancer (CRC) cells via degradation of both β-catenin and Ras. Due to the absence of Ras degradation in cells expressing non-degradable mutation in β-catenin, and the need to determine an alternative mechanism for Ras degradation, we established a cell-based system to screen compounds that degrade Ras independently of the Wnt/β-catenin signaling pathway. A cell-based high-content screening (HCS) system monitoring the level of EGFP-K-RasG12V was established using HCT-116 cells harboring a non-degradable mutant β-catenin (ΔS45). Through HCS of a chemical library composed of 10,000 compounds, and by subsequent characterization of candidate compounds, we identified several compounds that degraded Ras without affecting β-catenin levels. KY7749, the most effective compound, inhibited the proliferation and transformation of CRC cells, including those resistant to the EGFR monoclonal antibody, cetuximab, due to KRAS mutations. Small molecules that degrade Ras independently of β-catenin would be a potential treatments for cancers caused by aberrant EGFR, Ras, and β-catenin.

#1674

Novel direct AMPK activator suppresses non-small cell lung cancer through inhibition of lipid metabolism.

Xi Chen, Chun Xie, Xing Xing Fan, Ze Bo Jiang, Vincent Wong, Jia Hui Xu, Xiao Jun Yao, Liang Liu, Elaine L. Leung. _Macau University of Science and Technology, Macau, Macao_.

Lung cancer is the leading cause of cancer mortality. While non-small cell lung cancer (NSCLC) is the most common histological type of lung cancer and dominates almost 85% of all lung cancer cases. Conventional therapy, such as surgery, chemotherapy, or radiotherapy, scarify normal cells during the curing progress are becoming unfavorable. To avoid harmful side-effect on normal cell, target-based therapy is the mainstream of modern cancer therapy. For example, gefitinib is a clinically-marketed drug which acts as tyrosine kinase inhibitor to inhibit epidermal growth factor receptor (EGFR) activating signaling pathway. For the NSCLC patients who harbor activating substitution from leucine to arginine at amino acid 858 (L858R) point mutation and in-frame exon 19 deletion on EGFR, response well to gefitinib and these two common mutation are used a biomarkers for gefitinib prescription. However, gefitinib-resistance commonly happens due to further substitution mutation from threonine to methionine at amino acid position 790 (T790M) occurred after one year or less gefitinib treatment. Therefore, developing new treatment strategy to tackle gefitinib-resistance is urgently. Abnormal energy metabolism is accepted as new cancer hallmark. Recently, a metabolism rate-limiting enzyme 5'-adenosine menophosphate-activated protein kinase (AMPK) has become a promising anti-cancer target. In this study, we have applied computational docking to perform in silico virtual screening, and we have identified a novel direct AMPK agonist, D561-0775 from a compound library. Using enzyme activation assay, we demonstrated that D561-0775 activates directly on AMPK α subunit. In addition, D561-0775 exhibited significant inhibitory effect on gefitinib-resistant NSCLC cell lines but is less cytotoxicity on normal lung fibroblast cells. D561-0775 also activates AMPK/mTOR signaling pathway, resulting in induction of apoptosis, cell cycle arrest, suppressing glycolysis and cholesterol synthesis in gefitinib-resistant H1975 lung cancer cells. Taken together, D561-0775 has provided clue on a new chemical structure that could be developed as cancer drug for gefitinib-resistant NSCLC patients through inhibition lipid metabolism by directly targeting at AMPK directly. Acknowledgments: This work was supported by Macau Science and Technology Development Fund (Project No: 021/2013/A1, 074/2011/A3 & 082/2013/A3)

#1675

Design and synthesis of novel tyrosine kinase enzyme inhibitors and evaluating their anti-colorectal cancer activity.

Hanan A. Assiri,1 Hawazen A. BinMahfouz,1 Fahad A. Alabbasi,1 Marc Devocelle,2 Ibrahim M. El-deeb,3 Ahmed M. Al-Abd4. 1 _King Abdulaziz University, Jeddah, Saudi Arabia;_ 2 _Royal College of Surgeons in Ireland, Dublin, Ireland;_ 3 _Royal College of Surgeons in Ireland-Medical University of Bahrain, Manama, Bahrain;_ 4 _National Research Ctr. of Egypt, Cairo, Egypt_.

Targeting tyrosine kinases is attracting considerable attention for cancer therapeutics, nonetheless colorectal cancer. Herein we rationally designed and synthesized a group of 4-(3-methoxy-5-methylphenyl)-5-(pyridine-4-yl)pyrimidine-2-amine derivatives as potential inhibitors for tyrosine kinase. Synthesized compounds share a general structure and vary in substituents at position-2 of the terminal pyridine ring. The hydroxy-analogue of each methoxy derivative was also prepared to evaluate the effect of the methoxy group, at the phenyl ring in position-4 of the pyrimidine moiety, on activity. Compounds 1 (2-chloropyridyl and methoxyphenyl), 2 (2-chloropyridyl and hydroxyphenyl), 3 (2-(pyridine-3-yl)pyridyl and methoxyphenyl), 4 (2-(pyridine-3-yl)pyridyl and hydroxyphenyl), 5 (2-phenylpyridyl and hydroxyphenyl), 6 (2-(4-cyanophenyl)pyridyl and hydroxyphenyl) and 7 (2-(4-acetylphenyl)pyridyl and hydroxyphenyl) showed 29% to 79% tyrosine kinase inhibitory activity. Yet, cytotoxicity of all compounds were assessed against colorectal cancer cells (HT29 and HCT116) using SRB assay. Any compound (at 10 μM concentration) induced considerable viability drop was further assessed using detailed dose response curve and its cytotoxic parameters were calculated. Compounds 8 (2-(4-(N,N-dimethylamino)phenyl)pyridyl and hydroxyphenyl), 5, 6 and 7 showed considerable cytotoxic activities against HCT116 cells with IC50's of 9.18, 10.37, 9.47 and 16.60 μM, respectively. In addition, compounds 9 (2-chloropyridyl, methoxyphenyl and free 2-amino), 2, 3, 4, 5 and 6 showed considerable cytotoxicity against HT29 cells with IC50's of 9.31, 7.48, 11.26, 6.37, 9.89 and 7.14 μM, respectively. The mechanisms of cell killing effect of compounds with considerable anti-proliferative/cytotoxic activity were further assessed using cell cycle analysis (DNA content flowcytometry) and Annexin-V/FITC apoptosis assessment. Compound 2 induced antiproliferative effect which might be attributed to induction of cell cycle arrest at the G0/G1 phase; cell population in G0/G1 phase after treatment with compound 2 was 59.7±0.7% compared to 53.4±1.6% for control untreated cells. Compounds 4, 5, 6 and 7 induced significant cell cycle arrest at G2/M phase with cell populations equals 25.1±0.6%, 24.8±0.4%, 24.0±0.7% and 25.9±0.1%, respectively, compared to 19.3±1.6% for control untreated cells. Cell cycle arrest induced by compounds 1, 2, 3, 4, 6 and 7 resulted in significant apoptosis in colorectal cancer cells. In conclusion, this group of tyrosine kinase inhibitors showed promising anti-proliferative/cytotoxic effects in colorectal cancer cell lines attributed to cell cycle arrest. Further studies for the different cell cycle check point proteins and kinases are undergoing to clarify the exact underlying mechanism of cell killing attributed to each one of these compounds. 

## IMMUNOLOGY:

### Immune Checkpoints 1

#1676

Involvement of B cells in the efficacy of anti-PD-L1 antibodies: Suppression of B cells by cancer stem cells.

Leiming Xia,1 FEI LIAO,1 Jing Zhang,1 Ming Lin,1 Ronald Herbst,2 Elaine Hurt,2 Alfred Chang,1 Max Wicha,1 Qiao Li1. 1 _Univ. of Michigan, Ann Arbor, MI;_ 2 _MedImmune LLC, Gaithersburg, MD_.

We reported that adoptive transfer of tumor reactive B cells could mediate tumor regression, and that anti-PD-L1 administration significantly augments the therapeutic efficacy of cancer stem cell (CSC) vaccine. However, whether B cells are involved in the anti-tumor immunity mediated by anti-PD-L1 has not been identified. In the present study, we used two murine models: breast tumor 4T1 and colon cancer CT26. Tumor-bearing mice were administered with anti-CD20 mAb (MB20.11) to deplete B cells prior to anti-PD-L1 mAb administration. Depletion of B cells as compared to the control resulted in more aggressive tumor growth, and the anti-tumor efficacy of anti-PD-L1 was significantly reduced (p=0.041 and p=0.0011 for 4T1 and CT26 respectively), indicating the involvement of host B cells in the function of anti-PD-L1. Mechanistically, the administration of anti-PD-L1 partially recovered the humoral immune response, suggesting that the blockage of PD-L1/PD1 pathway could contribute to the rescue of B cell immune function. We detected higher PD-L1 expression on ALDHhigh CSCs than on ALDHlow non-CSCs or on unsorted tumor cells. On the other hand, we detected elevated expression of PD-1 on activated B cells. While the suppressive effect of CSCs on T cells has well been recognized, the interactions between CSCs and B cells is unknown. We hypothesize that CSCs may suppress B cells either directly via the PD-L1/PD1 immune check point interaction or indirectly via CSC suppression on Th cells. To test this hypothesis, we co-cultured purified B cells with ALDHhigh CSCs with or without the addition of anti-PD-L1 mAb. We found that the reduction of IgG secretion of B cells suppressed by CSCs could be rescued by anti-PD-L1 in a dose-dependent manner. These experiments indicate that CSCs can directly suppress the IgG production by B cells via PD-L1/PD-1 interaction. Moreover, when we co-cultured purified B cells plus purified CD4+ Th cells with ALDHhigh CSCs with or without the addition of anti-PD-L1 mAb, we found that the IgG production was further and significantly (p=0.0155) reduced as compared to the co-culture of CSCs and B cells alone. The addition of anti-PD-L1 to the cultures rescued IgG production by T/B/CSC co-cultures to a greater (p=0.0429) extent than in just B/CSC co-culture. These results indicate that CSCs can also indirectly suppress IgG production by B cells via the PD-L1/PD-1 interaction with Th cells. Together, this study demonstrates that the anti-tumor function of anti-PD-L1 mAb involves host B cells, and that CSCs can both directly and indirectly inhibit B cell function through the PD-L1/PD-1 axis on both B cells and Th cells, respectively.

#1677

Differential efficacy of PD-1 targeted immunomodulation in preclinical models of primary and bone metastatic triple-negative breast cancer.

Tiina E. Kähkönen,1 Mari I. Suominen,1 Jussi M. Halleen,1 Teppo Haapaniemi,2 Azusa Tanaka,3 Michael Seiler,3 Jenni Bernoulli1. 1 _Pharmatest Services, Turku, Finland;_ 2 _BioSiteHisto Ltd., Tampere, Finland;_ 3 _Taconic Biosciences, Hudson, NY_.

Immuno-oncology (IO) has provided groundbreaking results in cancer treatment. Triple-negative breast cancer (TNBC) tumors attract immune cells, and the presence of tumor-infiltrating lymphocytes (TILs) is linked to improved survival. Programmed cell death 1 (PD-1) is expressed by TILs and its ligand (PD-L1) by TNBC cells, and targeting of PD-1 has shown promising results in treatment of patients with primary TNBC. High frequency of bone metastases is typical in TNBC patients. As immune regulation is different in bone than in other organs it is essential to study the efficacy of IO therapies in bone microenvironment. The aim of this study was to assess the efficacy of anti-PD-1 therapy (pembrolizumab, Keytruda) in the growth of primary and bone metastatic TNBC in preclinical models.

MDA-MB-231(SA)-luc human TNBC cells were inoculated into the mammary fat pad (orthotopic model) or tibia bone marrow (bone model) of female huNOG mice engrafted with CD34+ cells from two different donors. Treatments with pembrolizumab or human IgG4 isotype control (5 mg/kg, i.p., Q5D, n=8) were started 3 days after the inoculations. Tumor growth was monitored by bioluminescence imaging (BLI), orthotopic tumor volume by caliper, and tumor induced changes in bone by X-ray imaging for 21-24 days. Tumor samples were processed to immunohistochemical (IHC) analysis of TILs, PD-1 and PD-L1. Bone volume was analyzed ex vivo by micro-computed tomography, and serum TRACP5b values were determined as a marker of osteoclast number.

Tumor growth at treatment start was confirmed by BLI. Pembrolizumab decreased tumor growth in the orthotopic model. 37.5% of the mice had partial response and 12.5% had tumor rejection. The mice exhibited donor-related differences in efficacy but the overall response was similar. IHC showed low to moderate PD-L1 expression in the TNBC tumors (tumor proportion score 1-49%) and moderate number of CD4+ and CD8+ TILs. No PD-1 expression was observed in pembrolizumab treated mice due to antibody blocking of the epitope. In the bone model, pembrolizumab had no effect in tumor growth or any bone parameters studied. PD-L1 expression was comparable in orthotopic and bone tumors. In bone tumors, CD4+ cells were dominant and CD8+ and PD-1+ cells were rarely observed.

In orthotopic model the response rate was similar to what has been observed in TNBC patients, but the bone metastatic growth could not be inhibited by the PD-1 blockage. Bone marrow has a unique microenvironment and immune cell compartment compared to any other organ, and it is known to be involved in immunosurveillance and to favour tumor cells to become immune evasive. The lack of efficacy of immunotherapy in bone compared to orthotopic tumor may be due to these differences. These results highlight the importance of using both orthotopic and metastasis models in preclinical oncology studies.

#1678

Integrated genomics and histology based studies of triple negative breast cancer identify ICOS as potential target for therapeutic intervention.

Heather A. Hirsch,1 Tong Zi,1 Rachel Fontana,1 Yun Wu,2 Jason Reeves,1 Alexander Needham,1 Edward Stack,1 David Lee,1 Emma Lees,1 Deborah A. Law,1 Elizabeth Trehu,1 Elizabeth A. Mittendorf2. 1 _Jounce Therapeutics, Cambridge, MA;_ 2 _M.D. Anderson Cancer Center, Houston, TX_.

ICOS (Inducible CO-Stimulator of T cells) is a co-stimulatory molecule expressed primarily on T lymphocytes. ICOS was prioritized as a target of interest based on nonclinical and clinical data that identified ICOS as a potentially key molecule in providing optimal anti-tumor benefit following anti-CTLA-4 therapy. We are developing JTX-2011, an ICOS agonist antibody that is designed to generate an anti-tumor immune response through stimulation of T effector cells and preferential reduction of intratumoral T regulatory (Treg) cells. In preclinical mouse tumor models, efficacy of an ICOS agonist was greatest in tumors with the highest levels of intra-tumoral ICOS, suggesting a potential predictive biomarker approach for clinical development. In assessing ICOS expression across multiple tumor types at both the RNA and protein level, we have identified triple negative breast cancer (TNBC) as a potential indication for an ICOS-targeted immunotherapy approach.

Integrated analysis of RNA, DNA and clinical data from the Cancer Genome Atlas (TCGA) was performed to understand the context in which ICOS is expressed. Additionally, ICOS levels were assessed by IHC in human tumor samples from an orthogonal data set. IHC and RNA analyses revealed a dynamic range of ICOS expression across indications and identified a subpopulation of breast cancer tumors enriched for high ICOS expression. Further analysis of both IHC and RNA data sets revealed that the triple negative subtype has higher enrichment of ICOS expression than other breast cancer subtypes. ICOS levels were correlated to gene signatures of immune infiltrate as well as other clinical attributes and molecular markers. There was a correlation between ICOS, ICOS signature, PD-L1 and IFNγ signatures. We then assessed TNBC samples obtained pre and post-neoadjuvant treatment to further understand the impact of chemotherapy on the tumor microenvironment. This included analysis of ICOS and PD-L1 protein expression as well as assessment of tumor-infiltrating immune cell subsets. While the distribution of certain immune cell subsets differed in pre and post-treatment samples, the expression of ICOS remained consistent. Based on these data, a TNBC cohort, enriched for the ICOS IHC biomarker, is included in the Phase 2 portion of the ICONIC study that is designed to assess the potential for a combination of JTX-2011 with a PD-1 checkpoint in this difficult to treat patient population.

#1679

Interference of PD-1 and PD-L1 interaction with small-molecule inhibitors enhances the efficacy of tumor-specific CTLs.

Priscilla S. Redd,1 Chunwan Lu,1 David Ostrov,2 Iryna Lebedyeva,1 Kebin Liu1. 1 _Augusta University, Augusta, GA;_ 2 _University of Florida, Gainesville, FL_.

Programmed cell death-1 (PD-1) is an immunosuppressive receptor expressed on T cells upon activation. Once interacting with its ligands, programmed death ligand-1 (PD-L1), the PD-1:PD-L pathway curbs T cell activation and thereby serves as a natural protection mechanism against autoimmunity. However, this pathway has been hijacked by cancer cells in order to evade the immune system. In fact, PD-L1 is commonly overexpressed on the surface of tumor cells as well as other cells in the tumor microenvironment, such as myeloid-derived suppressor cells. This interface has already been recognized as an effective target for cancer therapy. Blocking the PD-1:PD-L axis would unleash T cells from suppression to maintain their effector function against tumor cells. At least two anti-PD-1 and three anti-PD-L1 blocking antibodies have been approved for human cancer immunotherapy. However, these drugs are only successful in a small number of cancers. Therefore, developing inhibitors that block this axis more effectively and have a wider scale of impact is necessary. We used a molecular docking model and performed virtual chemical library screening to identify small molecule inhibitors of PD-1 and PD-L1 interactions. To this end, we have identified 175 PD-L1 small molecule inhibitors. Functional assays were established with the 4T1 murine breast cancer cell line and a tumor-specific murine T cell line in the presence or absence of the PD-L1 small molecule inhibitors to test if these small molecule PD-L1 inhibitors can increase T cell killing of tumor cells. Cell viability assays were also done to test the toxicity of these inhibitors on tumor cells in the absence of T cells. From these studies, we have identified ten novel PD-L1 small molecule inhibitors that can significantly increase the lytic activity of the tumor-specific cytotoxic T lymphocytes (CTLs) to kill 4T1 tumor cells. These ten PD-L1 small molecule inhibitors show little to no cytotoxicity to 4T1 tumor cells in the absence of the antigen-specific T cells, suggesting that these small molecule inhibitors themselves are not cytotoxic, and therefore are potentially safe as therapeutic agents. These proof of concept studies indicate that these small molecule inhibitors are potentially potent agents for blocking PD-1 and PD-L1 interaction in cancer immunotherapy.

#1680

The immune checkpoint metabolome and its relationship with choline metabolism.

Jesus Pacheco-Torres, Marie-France Penet, Yelena Mironchik, Sridhar Nimmagadda, Balaji Krishnamachary, Zaver M. Bhujwalla. _The Johns Hopkins University School of Medicine, Baltimore, MD_.

Introduction: Immune checkpoint inhibition has emerged as an exciting treatment option for several cancers. New insights into the role of immune checkpoints in cellular metabolism can be used to optimize their effectiveness in treatment. Programmed cell death-1 ligand (PD-L1) is an immune checkpoint overexpressed in cancers that has been successfully exploited for immune therapy. Here we investigated the relationship between the aberrant choline metabolism observed in most cancers and PD-L1 expression in triple-negative human MDA-MB-231 breast cancer cells, and characterized the metabolic effects of PD-L1 downregulation.

Methods: Experiments were performed with MDA-MB-231 cells transiently transfected with small interfering RNA (siRNA) against either luciferase (control siRNA), choline kinase-α (Chk-α), or PD-L1, following standard protocols. Total RNA was isolated, complementary cDNA synthesized, and quantitative real-time PCR performed using IQ SYBR Green supermix and gene-specific primers. Cell extracts were obtained using a dual-phase extraction method and analyzed by high resolution 1H magnetic resonance spectroscopy. 3 to 6 independent experiments were performed for each group.

Results: Silencing of Chk-α resulted in a significant increase of PD-L1 mRNA expression and silencing of PD-L1 resulted in a significant increase of Chk-α mRNA expression. These increases were not observed when both PD-L1 and Chk-α were silenced. Transfection with control siRNA did not alter Chk-α mRNA, but induced a small increase of PD-L1 mRNA compared to untreated cells. Transfection of cells with Chk-α and PD-L1 siRNA resulted in a significant decreases of Chk-α and PD-L1 mRNA. Consistent with the mRNA results, a significant increase of phosphocholine (PC) was observed in spectra obtained from cells transfected with PD-L1 siRNA compared to cells transfected with control siRNA, confirming that Chk-α increased in these cells. A significant increase of glutamate, glutathione, alanine and lactate was also observed in cells transfected with PD-L1 siRNA. As expected, cells transfected with a combination of Chk-α and PD-L1 siRNA showed a significant decrease of PC due to downregulation of Chk-α.

Discussion: Our data have identified, for the first time, the inverse association between PD-L1 and Chk-α expression following downregulation. These data suggest that treatments that decrease Chk-α and PC could result in cancer cells escaping immune surveillance through increased expression of PD-L1. On the other hand, these cells may become more susceptible to checkpoint inhibitors such as anti-PD-L1 or anti-PD-1 antibodies. PD-L1 downregulation also significantly increased glutathione that acts as a major antioxidant. Our ongoing studies are characterizing the effects of anti-PD-L1 antibody treatment on Chk-α expression.

Supported by NIH R35CA209960 and R01CA82337, and the Martin Escudero Foundation and Emerson Foundation.

#1681

Simultaneous measurement and significance of PD-1, LAG-3 and TIM-3 expression in human solid tumors.

Micaela Morgado,1 Ila Datar,1 Jun Wang,2 Miguel F. Sanmamed,3 Kristen McEachern,4 David Jenkins,4 Lieping Chen,5 Daniel Carvajal-Hausdorf,6 David L. Rimm,7 Roy S. Herbst,8 Kurt A. Schalper9. 1 _Yale, New Haven, CT;_ 2 _Jun Wang, New Haven, CT;_ 3 _Miguel Sanmamed, New Haven, CT;_ 4 _Tesaro, Waltham, MA;_ 5 _Lieping Chen, New Haven, CT;_ 6 _Clinica Alemana, Santiago, Chile;_ 7 _David Rimm, New Haven, CT;_ 8 _Roy Herbst, New Haven, CT;_ 9 _Kurt Schalper, New Haven, CT_.

Introduction: T-cells recognizing tumor antigens can express diverse immune inhibitory receptors such as PD-1, LAG-3 and TIM-3. Blockade of PD-1 induces prominent clinical benefit in patients with melanoma, lung, bladder and gastric carcinomas. However, the relative expression and significance of immune inhibitory receptors across different tumors is unknown.

Methods: Using multiplexed quantitative immunofluorescence (QIF) we measured the levels of CD3 (rabbit polyclonal, Dako), PD-1 (EH33, CST), LAG-3 (17B4, Abcam) and TIM-3 (D5D5R, CST) in >1,300 human malignancies including 119 triple negative breast carcinomas (TNBC), 225 ovarian carcinomas, 382 melanomas, 259 colorectal tumors, 229 bladder urothelial carcinomas and 130 gastric malignancies. The cohorts included FFPE tumor tissues and were studied using tissue microarrays (TMAs). The targets were measured in all cells of the preparation using fluorescence co-localization with DAPI and specifically in CD3-positive T-lymphocytes. Associations between the markers across tumors and with survival were studied.

Results: PD-1, LAG-3, and TIM-3 were expressed predominantly in T-cells and showed a positive association with each other. The highest levels were detected in gastric adenocarcinomas followed by urothelial bladder tumors, ovarian malignancies, melanoma, TNBC and colorectal carcinomas. Gastric and bladder tumors showed comparably high levels of all three markers and were significantly higher than the other tumor types (P<0.001). TNBC and colorectal carcinomas showed comparably lower expression. The levels of PD-1, LAG-3 and TIM-3 were positively associated with CD3+ T-cell infiltration in all tumors except in gastric cancer. Melanoma showed the highest CD3 levels and gastric tumors had the lowest. Elevated T-cell PD-1 was significantly associated with longer overall survival in melanoma and colorectal cancer; and elevated LAG-3 was associated with better survival only in melanoma. The levels of TIM-3 were not associated with survival.

Conclusion: PD-1, LAG-3 and TIM-3 are differentially expressed in human solid tumors and show limited prognostic value. Gastric and bladder carcinomas show the highest levels of the targets, while TNBC and colorectal cancer show the lowest. Despite having the highest T-cell infiltration, melanomas show intermediate levels of the immune inhibitory receptors. These data demonstrate the differences in the immune composition of human solid tumors and could be used for optimal design and interpretation of clinical trials.

#1682

Combined treatment of aldose reductase inhibitor, fidarestat, and anti-PD1 antibodies synergistically prevents T cell- and NK cell-mediated colon cancer growth in murine models.

Ashish Saxena, Himangshu Sonowal, Satish K. Srivastava, Kota V. Ramana. _UT Medical Branch, Galveston, TX_.

Immune checkpoint inhibitors such as anti-programmed death 1 (PD1) and anti-PD-L1 monoclonal antibodies though have great potential for cancer immunotherapy, the acquired drug resistance and unwanted immune side effects limit their use. However, the combination therapy using conventional drugs and immune checkpoint inhibitors not only synergistically increase anti-tumor responses but it also reduces immune-associated adverse events. In our earlier studies, we have shown that inhibition of polyol pathway enzyme, aldose reductase (AR) prevents colon cancer growth and metastasis in culture as well as in mouse models. In this study, we investigated whether fidarestat, a potent AR inhibitor that has already gone through Phase-III clinical trial for diabetic neuropathy and found to safe for human use, can synergize with anti PD-1 to increase antitumor activity and decrease immune-associated adverse effects. Mice bearing syngeneic CT26 colon carcinoma tumors were treated with anti PD-1 alone or in combination with fidarestat, and the tumor growth was monitored. Our results indicate that fidarestat in combination with anti-PD1 synergistically increases tumor regression within 28 days of treatment. Further, a combination of fidarestat and anti-PD1 significantly increased the CD8+ T cells and natural killer (NK) cells and decreased the Myeloid-derived suppressor cells (MDSCs) at tumor milieu. Fidarestat plus anti-PD1 also increased CD8+ T cell and NK cell effector functions as determined by IFNg and granzyme B. Systemically, combination therapy also increased the number of CD8+ T cells and NK cells and decreased MDSCs in the mouse spleens. Further, the effector functions of CD8+ T cells and NK cells were increased with the combination treatment in mice spleens. Moreover, the levels of various inflammatory cytokines and chemokines in the serum and liver were significantly less in fidarestat plus anti-PD1 -treated mice as compared to anti-PD1 alone -treated mice indicating that fidarestat rescues immune-mediated adverse events in various organs. Thus, our results suggest that by regulating the number as well as effector function of CD8+ T cells, NK cells and MDSCs, AR inhibitor in combination with anti-PD1 would not only regress the tumor growth but would also provide a long-lasting specific immunity against colon cancer, which would decrease the chances of tumor recurrence.

#1683

A comparative evaluation of immunotherapy responses in murine colon carcinoma.

Sheri R. Barnes, Sumithra Urs, Mary Anne Meade, David Draper, Hillary Evens, Daniel Saims, Scott Wise, Maryland Rosenfeld Franklin. _MI Bioresearch, Ann Arbor, MI_.

With the drive in oncology to develop molecules targeting the immune system, there is a need for reliable and well-characterized preclinical models. To this end, the impact of checkpoint inhibitors, such as anti-PD-1 and anti-PD-L1, has been extensively investigated preclinically, and the landscape of available models varies greatly in response to these molecules. With several recent checkpoint inhibitor-based drug approvals, the focus of immuno-oncology drug development has shifted to immunometabolism targets and costimulatory molecules, particularly with combination strategies. Comparative preclinical tumor model validation with respect to these targets is generally lacking; however, the same level of granularity as was afforded anti-PD-1 and anti-PD-L1 would be needed with these molecules to allow confidence in designing preclinical proof of concept studies for future clinical development.

To better understand the utility of these agents in preclinical development, we examined the response of two murine colon carcinoma models, CT26 and MC38, against a panel of costimulatory molecules and epacadostat, an IDO inhibitor, using in vivo tumor growth delay studies. Our data demonstrate that CT26 tumors are sensitive to the costimulatory molecules, anti-GITR, anti-CD137, and anti-OX40, resulting in 6/10, 7/10, and 2/10 complete responses (CRs), respectively. In contrast, the response of MC38 tumors to these agents was modest with no CRs and times to evaluation size (TES) extending only 1.4 to 6.9 days beyond control. Epacadostat treatment did not impact growth of CT26 or MC38, with TES's extended by 4.4 or 2.3 days, respectively, compared to control. Interestingly, addition of epacadostat did not improve the response of anti-PD-1 in either model.

To determine whether the changes in response could be related to differences in immune infiltration of these tumors, immunophenotyping from untreated CT26 and MC38 tumors was evaluated. We found that immune cell populations also highlighted potential differences in these models. CT26 tumors were well infiltrated with a mean of 25.2% CD45+ cells, with 54.8% of CD3+ cells being CD8+ T cells. Comparatively, MC38 tumors were weakly infiltrated with CD45+ cells (5.3%) with 35.9% of CD3+ cells being CD8+ T cells. Macrophage and MSDC infiltrates, as a percent of CD11b+ cells, in CT26 tumors were generally double that found in MC38 tumors.

Our results indicate that CT26 is more sensitive to the costimulatory molecules tested than MC38. Further, epacadostat as monotherapy or in combination with anti-PD-1 did not elicit clinically meaningful tumor growth delay in either model. Stark differences were also seen in overall lymphocyte infiltrate and composition between CT26 and MC38. The differences in baseline immune infiltrate and efficacy allow us to determine which models and immunotherapies are best suited for rational drug combination approaches.

#1684

Selective SIRPa blockade potentiates dendritic cell antigen cross-presentation and triggers memory T-cell antitumor responses.

Vanessa Gauttier,1 Sabrina Pengam,1 Justine Durand,2 Aurore Morello,1 Sophie Conchon,2 Bernard Vanhove,1 Nicolas Poirier1. 1 _OSE Immunotherapeutics, Nantes, France;_ 2 _INSERM UMR1064, Nantes, France_.

Targeting immune checkpoints of the adaptive immunity has shown great therapeutic efficacy to fight cancers, but in a limited proportion of patients. Myeloid cells represent a major immune cell type in many solid tumors, and are often associated with a poor outcome. Interaction of SIRPalpha (SIRPa), expressed by myeloid cells, with the ubiquitous receptor CD47 is an important immune checkpoint of the innate response, involved in the regulation of macrophages and neutrophils functions including phagocytosis. Targeting both adaptive and innate immune cells represents a promising therapeutic strategy against cancer. Here we evaluated the impact of a SIRPa checkpoint inhibitor on adaptive immune responses both in rodents and human settings.

Antagonist anti-SIRPa monoclonal antibody (mAbs) was evaluated in vivo in combination with adaptive immune checkpoint inhibitors (anti-PD-L1 mAb) or costimulatory agent (anti-4-1BB mAb) in an orthotopic hepatocellular carcinoma (HCC) model in immunocompetent mice. Whereas monotherapies have shown a modest clinical effect, combination with SIRPa blockade dramatically enhanced the overall survival with up to 70% of mice in complete remission (p < 0.0001 in both combination: n=15 with anti-41BB and n=11 with anti-PDL1 combination). These cured mice showed robust memory immune response since a second tumor challenge (performed up to one month after treatment withdrawal) was rejected in all mice (p<0.01 n=20). In addition, adaptive transfer of T lymphocytes from 4-1BB combo or sera from PD-L1 combo cured mice in naïve untreated mice protected them from orthotopic HCC development (n=5/6 with T-cell from 4-1BB combo; n=5/5 with sera from PD-L1 combo). Tumor infiltrates analyzed by flow cytometry showed enrichment in effector CD8 T cells with the 4-1BB therapy whereas PD-L1 combination led to an accumulation of memory CD4 T cells. Tumor transcriptional analysis using Nanostring technology revealed higher dendritic cells (DCs) and T cells (mainly TH1) immune signature with reduced exhaustion signature. Finally, we found in vitro that selective blockade of SIRPa during antigen processing by mouse dendritic cells (with ovalbumin protein and TCR-transgenic OT-1 T-cell) or human dendritic cells (with melan-A 25-mer long peptide and human antigen-specific T-cell clone from melanoma patients) significantly increases T-cell activation and cytokine (i.e. IFNg) secretion.

In conclusion, we showed that selective SIRPa antagonist increased dendritic cell tumor-antigen cross-presentation and generated robust antitumor memory response in combination with adaptive immunotherapies.

#1685

Genomics based studies of gastric tumors identify ICOS as potential target for therapeutic intervention.

Heather A. Hirsch, Jason Reeves, Tong Zi, Alexander Needham, Edward Stack, David Lee, Emma Lees, Deborah A. Law, Elizabeth Trehu. _Jounce Therapeutics, Inc., Cambridge, MA_.

JTX-2011, an ICOS agonist antibody, is the first clinical program to emerge from Jounce's Translational Science Platform, which couples the choice of target mechanism to potential predictive biomarkers of response. ICOS (Inducible T cell CO-Stimulator), a co-stimulatory molecule expressed primarily on T lymphocytes, was prioritized as a target based on preclinical and clinical data suggesting that it plays an important role in the immune response to cancer. Fundamental data from our preclinical studies shows that tumor reduction occurs only in animals when a certain percentage of ICOS positive immune cells are resident within the tumor or in combination with a PD-1 inhibitor. Thus ICOS expression is a key element of our biomarker-driven approach in the ICONIC clinical trial. Based on this biomarker approach, we have identified gastric cancer as a cancer of potential interest for an ICOS-targeted immunotherapy approach.

Gastric adenocarcinoma was identified as a tumor of interest based on the integrated analysis of RNA, DNA and clinical data from the Cancer Genome Atlas (TCGA). This analysis was performed within stomach adenocarcinoma (STAD) to understand the context in which ICOS is expressed. ICOS levels were correlated to gene signatures of immune infiltrate as well as other clinical attributes and molecular markers. ICOS and PD-L1 levels were assessed by IHC in human tumor samples and in biopsies from ICONIC participants.IHC and RNA analyses reveals a dynamic range of ICOS expression across gastric adenocarcinoma tumors, with high prevalence in both EBV+ and MSI-H tumors as well as a subset of EBV-/MSS tumors. While there is a correlation between ICOS, ICOS signature, PD-L1 and IFNγ signatures, RNA analysis indicates that a subpopulation of gastric tumors with lower levels of PD-L1 expression may be ICOS positive. Integrative analysis of tumors identifies gastric cancer as an attractive indication for exploration of JTX-2011 plus a PD-1 inhibitor based on the relatively high frequency of ICOS expression within this tumor type. Since ICOS expression has a dynamic range of expression, an ICOS IHC biomarker is being used to enrich for patients in a gastric cancer cohort, as well as other tumor-specific cohorts in the Phase 2 portion of the ICONIC clinical study.

#1686

TSC2 enhances antitumor immunity and potentiates PD-1 and CTLA-4 blockade.

Heng-Jia Liu,1 Patrick Lizotte,2 Heng Du,1 Maria Speranza,3 Spencer Vaughan,4 Nicola Alesi,1 Kwok-Kin Wong,5 Gordon Freeman,3 Arlene Sharpe,1 Elizabeth Henske1. 1 _Brigham and Women's Hospital/Harvard Medical School, Boston, MA;_ 2 _Belfer Center for Applied Cancer Science, Boston, MA;_ 3 _Dana-Farber Cancer Institute, Boston, MA;_ 4 _Brigham and Women's Hospital, Boston, MA;_ 5 _New York University Langone Medical Center, New York, NY_.

Tuberous sclerosis complex (TSC) is an incurable multisystem genetic disease characterized by mTORC1-hyperactive hamartomatous tumors of brain, kidney, and lung. TSC is caused by mutations in the two TSC1 or TSC2 tumor-suppressor genes. mTORC1 inhibitors lead to partial antitumor responses in TSC, with tumor regrowth upon treatment cessation. Understanding the interaction between T cells and tumor cells has led to the development of successful checkpoint blockade immunotherapy targeting PD-1, programmed cell death-ligand 1 (PD-L1), and CTLA-4. These therapies have proven to be highly effective in treating several malignancies, including non-small cell lung cancer (NSCLC), renal cell carcinoma (RCC), melanoma, bladder cancer and Hodgkin's lymphoma. It is currently unknown whether benign tumors, such as those arising in TSC, will also respond to checkpoint blockade therapy. Using multiparametric flow cytometry and immunohistochemistry of human angiomyolipomas and pulmonary LAM, we discovered that PD-1 is highly expressed on tumor-infiltrating T cells. Using immunocompetent preclinical models of TSC, we found that dual blockade of PD-1 and CTLA-4 inhibited the growth of TSC2-deficient tumors by 77% (p < 0.0001). This was associated with increased tumor-infiltrating CD8+ and CD4+ T cells and decreased Tregs, G-MDSCs and regulatory CD11b+ DCs. Strikingly, we discovered that complete regression of established tumors can be achieved in 37% of the mice using anti-PD-1 monotherapy and in 62% of the mice using combined anti-PD-1 and anti-CTLA-4 therapy. We also assessed a treatment regimen of rapamycin followed by PD-1 blockade and found that this sequential treatment delays regrowth of TSC2-deficient tumors. Furthermore, TSC2 re-expression in TSC2-deficient tumors promoted antitumor responses via increasing T cells and decreasing CD11b+Ly6G+Ly6Cmed G-MDSCs and CD11b+ DCs infiltration into tumors. Importantly, we found that the potency of dual PD-1 and CTLA-4 blockade is significantly enhanced by TSC2 re-expression. Our data indicate that PD-1 is upregulated on T cells in TSC-associated tumors and that dual blockade of PD-1 and CTLA-4 pathways is effective in delaying tumor growth and improving long-term survival, with long-term complete tumor responses. Therefore, PD-1 and/or CTLA-4 blockade may represent a promising dual immunotherapy for patients with TSC-associated tumors and women with the sporadic form of LAM. Our data may have relevance for the many sporadic human tumors with mTORC1 hyperactivation (~50% of all human malignancies) and also for other hamartomatous tumor syndromes such as neurofibromatosis. Furthermore, the specific role of intratumoral TSC2 in antitumor T cell responses that we discovered may help to elucidate the response to immunotherapy in sporadic human malignancies with mutations in the TSC genes, which include a subset of bladder cancer and renal cell carcinoma.

#1687

Checkpoint inhibitor signatures across endometrial cancer histologies.

Amanda Ramos,1 Sarah Fortin,1 Victoria Melchert,1 David Jenkins,2 Whitfield Growdon,1 Darrell Borger1. 1 _Massachusetts General Hospital, Boston, MA;_ 2 _Tesaro Inc, Waltham, MA_.

Background: Therapies targeting immune checkpoints have revolutionized cancer treatment. However, biomarkers that effectively stratify patients to these therapies are required. Endometrial carcinomas consist of several histologies and a subset harbor hypermutation/microsatellite instability (MSI) that has been associated with response to PD-1 inhibition. The purpose of this study was to investigate the immune checkpoint landscape across endometrial cancer subtypes and ascertain whether more complex signatures indicate directions for combination immune therapies in efforts to improve patient response.

Methods: With IRB approval, 60 diagnostic endometrial tumor samples were obtained from 2013-2017 consisting of MSI low grade endometrioid (n=11), microsatellite stable (MSS) low grade endometrioid (n=11), high grade endometrioid (n=16), carcinosarcoma (n=11), and uterine serous carcinoma (n=11) histologies. Immune RNA expression signatures were evaluated using a Luminex platform that evaluated 142 gene targets, including inflammation mediators, immune checkpoints, immune cell type and immune cell polarity markers. Expression of RNA for genes encoding CTLA-4, PD-1, PD-L1, LAG-3, and TIM-3 were quantified and correlated with clinical outcomes utilizing parametric and non-parametric testing. These signatures will be further investigated with multispectral imaging, including proximity analysis.

Results: Cohort characteristics included 32 patients with stage 1A, 9 patients with stage 1B, 6 with stage II, 8 with stage III and 5 with stage IV disease. The median age at diagnosis was 66 years. RNA expression for CTLA-4, PD-1, PD-L1, LAG-3, and TIM-3 were compared across the various endometrial carcinoma histologies. While there were no significant differences noted between carcinosarcoma and serous carcinoma, levels of TIM-3 and CTLA-4 expression were significantly elevated in endometrioid carcinoma (P<0.01, P<0.001 respectively). When evaluating expression within endometrioid subtypes, high grade tumors were independently associated with elevated checkpoint expression (LAG-3 (P=0.04), PD-L1 (P=0.04), and CTLA-4 (P=0.04)), as was the presence of MSI (TIM-3 (P=0.02), PD-L1 (P=0.01), and CTLA-4 (P=0.014). When both grade and MSI were factored together, PD-1 was significantly elevated in MSI high grade versus MSI low grade cancers (P=0.04). RNA expression levels for PD-L1 and PD-L2 were also significantly elevated in endometrioid cancers. Preliminary analysis suggests that recurrent tumors demonstrate altered levels of immune checkpoint signatures compared to those that do not.

Conclusion: A complex landscape of regulatory immune receptors was found across endometrial cancer histologies, with particular relevance in endometrioid carcinomas that was grade and MSI-dependent. These data suggest directions for combined immune blockade therapy in this cancer type.

#1688

Enhancement of immune checkpoint PD-1 blockade efficacy in ovarian cancer.

Maureen Drakes,1 Swati Mehrotra,1 Ronald Potkul,1 Yueying Liu,2 M. Sharon Stack,2 Patrick Stiff1. 1 _Loyola University Chicago, Maywood, IL;_ 2 _University of Notre Dame, IN_.

Introduction

Advanced stage ovarian cancer is often asymptomatic and only twenty percent of patients with this diagnosis have a survival duration of five years.

Immune checkpoint molecule programmed cell death-1 (PD-1) and its ligand, PD-L1, limit T cell immune responses, and thus are immunosuppressive. Clinical trials using anti-PD-1 or anti-PD-L1 antibody treatment has resulted in objective response rates (ORR) in only 10-15% of ovarian cancer patients. Our ultimate goal is to improve this outcome.

Recepteur d'origine nantais (RON) is a c-Met related tyrosine kinase which binds to macrophage stimulatory protein (MSP). RON is expressed by myeloid suppressor cells and tumor cells. Inhibition of RON/ MSP ligation upregulates STAT-1 and IL-12 in macrophages, driving IFN-γ production in CD8+T cells and might potentiate the efficacy of anti-PD-1 antibody treatment.

Objectives and Methods

We hypothesize that combination therapy of anti-PD-1 blocking antibody which restores T cell proliferative and cytotoxic functions, combined with therapy targeting other suppressive pathways, will concomitantly overcome multiple immune suppressive mechanisms, and prevent ovarian cancer in mice.

In a pilot study, ovarian cancer was induced in female C57BL/6 (H-2Kb) mice (Jackson Laboratories) at 8 weeks old, by intraperitoneal (I.P.) injection of 1 x 106 ID8-RFP ovarian tumor cells in 5 mice/ group. We determined the efficacy of anti-PD-1 antibody (RMP1-14, BioXCell; 200ug/ dose, 0.5 ml vol. I.P., 4 doses) given alone or with a RON inhibitor, BMS-777607 (each dose 50 mg/ kg body weight, 20 doses orally over 5 weeks; Selleck Chemicals). Mice were euthanized at about day 70 when control mice with disease had extended abdomens.

Results

Treatment of mice with an anti-PD-1 blocking antibody combined with a RON inhibitor resulted in disease improvement in 5/5 mice. The average volume of ascites recovered from 5 combination treated mice was 1.62 ml (2/5 mice had no ascites) compared with 6.64 ml from the corresponding control (a 4-fold reduction in ascites with combination treatment). There was an average 2 fold reduction in ascites volume in anti-PD-1 antibody treated mice in comparison with IgG controls (4.14 ml versus 8.62 ml). BMS-777607 treatment (vs vehicle) did not significantly alter ascites volume.

Flow cytometry evaluation of spleen cells showed that in the combination treated mouse group, there was an increase in the average percentage of CD3 (combined treatment group 26.7 vs control 8.5%), CD4 (10.6 vs control 5.0%) and CD8 T cells (10.6 vs control 2.7%). We are investigating changes in immune responses genes in RNA of intestines and livers.

Conclusions

Anti-PD-1 antibody treatment is effective in treating ovarian cancer in mice, but treatment with this agent combined with RON/ c-Met inhibitor, BMS-777607 is superior to either single therapy.

Combination treatment with these 2 agents holds promise as a novel therapeutic approach for ovarian cancer.

#1689

Ovarian tumor-induced CISH expression is an immune checkpoint for NK cells.

Amy K. Stasik, Aparna Yellapa, Pincas Bitterman, Sameer Sharma, Sanjib Basu, Animesh Barua. _Rush University Medical Center, Chicago, IL_.

Background: Non-specificity of symptoms in early stages of ovarian cancer (OVCA), lack of an early detection test and inefficient chemotherapy regimens lead to frequent recurrence and high mortality rates. OVCA differs from other malignancies as it disseminates by diffusion in the peritoneal cavity. Local immune function is critical and NK cells provide a first line of defense against a developing tumor. Tumors escape NK cell recognition by cleaving off ligands (MICA/B) for NK cell receptors (NKG2D) from their surface. However, preventing the cleaving of ligands does not improve tumor recognition by NK cells to desired levels, suggesting the existence of an additional inhibitory mechanism. Cytokines, including IL-15, play important roles in the proliferation and activation of NK cells. Nevertheless, longstanding exposure to cytokines induces exhaustion of NK cells through expression of CISH (Cytokine-inducible SH2-containing protein). The goal of this study was to examine whether NK cells in ovarian tumors express CISH and whether CISH expression is associated with tumor progression. Materials and methods: This study was conducted in an exploratory design using normal ovaries from postmenopausal women (60-80 years old, n=10), malignant ovarian tumors at early and late stages (n=12 from each stage, 3 from each of 4 histological OVCA types). Localization of CISH-expressing NK cells and expression of GRP78, a marker of cellular stress, were examined by immunohistochemistry (IHC) using paraffin sections. Representative samples were used for immunoblotting (WB) and gene expression studies. Results: Intense staining for CISH was observed for NK cells with occasional staining of malignant cells. Compared with normal ovaries, the population of CISH-expressing NK cells was greater in OVCA at early stage and increased further at late stages. The population of CISH-expressing NK cells was higher in serous OVCA at early stage as compared to other histological types at early stages. However, significant differences were not observed in the population of CISH-expressing NK cells among different histological types of OVCA at late stages. Immunoblotting showed stronger bands at ~30kDa for CISH for malignant tumors at early and late stages. Expression of GRP78 was significantly greater in ovarian tumors as compared with normal ovaries and increased further in OVCA at late stages. These results suggest the presence of cellular stress in ovarian malignant tumors and in their microenvironment are conducive to express a marker of exhaustion (CISH) by NK cells, which may suppress their anti-tumor functions. Thus, CISH may be an immune checkpoint for NK cells. Conclusion: The results of this study suggest that ovarian tumor development and progression is associated with increased expression of CISH by malignant cells and NK cells. Tumor-induced CISH expression may be associated with decreased anti-tumor function of NK cells. Support: NIH CA187309

#1690

NKG2A immune checkpoint blockade potentiates cetuximab induced ADCC in head and neck cancer preclinical model.

Caroline Soulas,1 Ana Lalanne,2 Cécile Bonnafous,1 Caroline Hoffman,2 Elodie Bonnet,1 Arnaud Dujardin,1 Violette Breso,1 Mathieu Bléry,1 Olivier Lantz,2 Romain Remark,1 Eric Vivier,3 Pascale Andre1. 1 _Innate Pharma, Marseille, France;_ 2 _Institut Curie, Paris, France;_ 3 _Centre d'Immunologie de Marseille Luminy, Marseille, France_.

Monalizumab (IPH2201) is a first-in-class humanized IgG4 targeting NKG2A (Natural Killer Group 2A), which is expressed as a heterodimer with CD94 at the surface of subsets of NK (Natural Killer) cells, γδ T cells and tumor infiltrating CD8+ T cells. This inhibitory receptor binds to HLA-E (Human Leukocytes Antigen-E) molecules that are frequently up-regulated on human cancer cells, preventing from killing by NKG2A+ immune cells. HLA-E is expressed on most of the patients with Head and Neck Squamous Cell Cancer (HNSCC). Monalizumab blocks the binding of CD94/NKG2A to HLA-E, reducing inhibitory signaling thereby unleashing NK and T cell responses. High expression of EGFR occurs in most epithelial malignancies and particularly in HNSCC and is associated with poor prognosis. The anti-EGFR monoclonal antibody cetuximab (Ctx) is thought to act through blocking oncogenic signaling and by inducing Fcγ receptor-mediated antibody dependent cell cytotoxicity (ADCC) which involves human NK cells. We investigated ex vivo and in vitro the rationale of combining monalizumab with Ctx in the treatment of oral cancers. We first analyzed formalin-fixed paraffin-embedded samples of HNSCC patients by immunohistochemistry (n=65). We confirmed that HLA-E was expressed on carcinoma cells in all patients. We also observed that most of the tumors were highly infiltrated by CD8+ T cells both in the tumor beds and in the stroma and that the majority of tumors were also infiltrated by NKp46+ NK cells in higher numbers in the non-metastatic tumors. Interestingly, HNSCC was found as being one of the tumor types with the highest NKp46+ cell density as compared with renal cell carcinoma, non-small cell lung cancer, colorectal cancer or pancreatic tumors. Moreover CD94+ lymphocytes were detected in the stroma and tumor beds in close contact to tumor cells in about half of the patients. Using multicolor flow cytometry, NK cells and CD8+ T cells from tumor and non-involved adjacent tissues, metastatic lymph nodes and peripheral blood from patients relapsing post-chemotherapy were characterized for expression of activating and inhibitory receptors. Interestingly, NKG2A expression was higher on tumor infiltrating NK cells (CD56dim or CD56bright) and CD8+ T cell compared to the one observed in non-involved adjacent tissue, metastatic lymph node or blood. In vitro, monalizumab increased CD107 mobilization and CD137 upregulation on NKG2A+ NK cells in response to HNSCC cell lines with endogenous HLA-E expression and enhanced Ctx-mediated ADCC in a dose dependent manner. Altogether, these data support the rationale for investigating monalizumab in HNSCC patients and in combination with cetuximab in clinical trials (NCT02643550).

#1691

Clinical characteristics influencing survival in stage-IV non-small cell lung cancer treated with nivolumab: A single-institutional experience.

Abdul Rafeh Naqash, Chipman R. Stroud, Li V. Yang, Mahvish Muzaffar, Paul R. Walker. _East carolina university, Greenville, NC_.

Background: Immune-checkpoint blockade (ICB) using antibodies directed against the programmed cell death-1 (PD-1) axis has revolutionized the management of non-small cell lung cancer (NSCLC). Based on results from the Checkmate 017 and 057, nivolumab, an anti-PD-1 antibody, has emerged as the standard of care in the second line setting for advanced NSCLC. However, the lack of uniform responses as noted in daily practice suggests clinical heterogeneity. Hence, it is essential to identify characteristics that may predict inferior efficacy to ICB. Methods: Retrospectively we identified 61 stage IV NSCLC patients treated with nivolumab from April 2015 to March 2017 after progressing on a platinum doublet. These patients were part of an ongoing institutionally approved prospective biomarker-based study. Follow up cutoff for survival analysis was set on October 1, 2017. Overall survival after immunotherapy (OSI) was defined as the time from ICB initiation to last follow up or death. Cox regression was used to assess the correlation of clinical and biochemical factors influencing OSI. Results: The median age was 63 years with predominant histology being adenocarcinoma in 65.6 %. A majority of patients were of Caucasian ethnicity (68.9%) and male gender (60.7%). Skeletal involvement (54.1 %), liver (27.9 %) and brain (26.2 %) were the most common metastatic compartments. Simultaneous involvement of greater than 1 metastatic compartment at ICB initiation was observed in 54.1% of patients. Approximately 67.3 % patients were either former/never smoker at the time of nivolumab initiation. The median OSI for our NSCLC cohort treated with nivolumab was 6.3 months. On adjusted Cox univariate analysis of the clinically relevant covariates, baseline factors present at anti-PD-1 initiation that were associated with inferior OSI were age greater than 65 [p= 0.036; 2.37 (1.05-5.30)], brain metastasis [p=0.014; 3.30 (1.25-7.37)] and greater than 1 metastatic compartment involvement [P=0.041; 2.78 (1.04-7.44). Using adjusted multivariate Cox regression model with backward elimination, all three factors were noted to be independently associated with inferior OSI.Conclusions: Our study identified subgroups with certain baseline clinical features that tend to behave poorly despite ICB. This suggests that improving outcomes in such subgroups may require exploring strategies involving combination ICB or other novel targeted therapies. Also, better identification of such subgroups via larger datasets may help in the appropriate designation of patients for optimally tailored ICB and thus assist in improving outcomes in NSCLC.

#1692

The impact of neutrophil/lymphocyte ratio as the predictive marker to anti-PD-1 antibody treatment in NSCLC patients.

Keiko Tanimura,1 Tadaaki Yamada,1 Nobuyo Tamiya,1 Yoshiko Kaneko,1 Takayuki Takeda,2 Junji Uchino,1 Yutaka Kubota,3 Koichi Takayama1. 1 _Kyoto Prefectural University of Medicine, Kyoto-city, Japan;_ 2 _Uji-Tokushukai Medical Center, Kyoto, Japan;_ 3 _Japanese Red Cross Kyoto Daini Hospital, Kyoto-city, Japan_.

Background: Anti-Programmed death 1 (PD-1) antibody which enhances anti-tumor activity of cytotoxic-T-lymphocytes by blockade of PD-1/PD-L1 pathway has demonstrated improvement of survival in the patients with advanced non-small-cell lung cancer (NSCLC). PD-1 and its ligand PD-L1 is also known as key players in the formation of tumor microenvironment, closely related with inflammatory cytokines. Here, we retrospectively analyzed the peripheral blood cells as a surrogate marker for predicting outcome of immune checkpoint inhibitors (ICI). Method: Total of 78 cases treated with anti-PD-1 antibodies, nivolumab or pembrolizumab from February 2016 to September 2017 were registered retrospectively. We investigated the differential leukocyte counts, C-reactive protein (CRP) and other peripheral blood parameter on the treatment progress with ICI, and evaluated correlation between response to ICI and peripheral blood cells. Result: Out of 78 cases, 56 cases (77%) were treated with nivolumab, 18 cases (23%) were treated with pembrolizumab, and 4 cases (5%) have treatment history of both agents. Median follow up period was 5.7 months (range: 0.4-18.7 months). Median progression free survival (PFS) and median overall survival (OS) were 2.4 and 8.7 months, respectively. Overall response rate was 21.8%. Of some parameters in peripheral blood cells, higher absolute neutrophil count (ANC), CRP at baseline were associated with shorter OS. Furthermore, we determined neutrophil / lymphocyte ratio (NLR), platelet / lymphocyte ratio (PLR), lymphocyte / monocyte ratio (LMR) and prognostic nutritional index (PNI), which were known as predictive biomarkers in various tumor type. In patients with longer PFS for ICI treatment, ANC, NLR and CRP remained low level until disease progression, whereas these markers increased during ICI treatment in the patients with shorter PFS for ICI. Conclusion: Our retrospective analysis showed that ANC, NLR and CRP may predict response and acquisition of resistance to anti-PD-1 therapy in NSCLC patients. These results suggest that peripheral blood findings might be promising as a predictive biomarker of ICI treatment.

#1693

Prediction of the efficacy of nivolumab using resting energy expenditure in metastatic non-small cell lung cancer (mNSCLC) patients.

Claire Gervais,1 Pascaline Boudou-Rouquette,2 Anne Jouinot,1 Jeanne Chapron,1 Jennifer Arrondeau,1 Marco Alifano,1 Frédérique Giraud,1 Olivier Huillard,1 Jérôme Alexandre,1 Clara Vazeille,1 Jean-Philippe Durand,1 Karen Leroy,1 Marie-Pierre Revel,1 Jean-Pascal de Bandt,1 Luc Cynober,1 Diane Damotte,1 Audrey Lupo-Mansuet,1 François Goldwasser1. 1 _Cochin Hospital, CERTIM, AP-HP, Paris Descartes University, Paris, France;_ 2 _Cochin Hospital, CERTIM, AP-HP, Paris Descartes University, PARIS, France_.

Background: Immune evasion and deregulation of energy metabolism play a pivotal role in cancer progression. Immunosuppression in the tumor microenvironment can be based on the mutual metabolic requirements of immune and tumor cells. We evaluated the value of resting energy expenditure (REE) as a predictor of outcome, in mNSCLC patients under Nivolumab, an immune checkpoint inhibitor.

Methods: We studied the relation between REE, clinical and biological markers of cachexia and inflammation, and response to Nivolumab in 82 consecutive mNSCLC patients. Efficacy was assessed every 2 months according to Response Evaluation Criteria in Solid Tumors (RECIST) criteria. REE was measured using indirect calorimetry, before the initiation of Nivolumab. According to their REE and with the use of Boothby's standard, patients were categorized as hypermetabolic, normometabolic and hypometabolic. Body mass index (BMI), performance status (PS), C-reactive protein (CRP), albumin, Neutrophil Lymphocyte R Ratio (NLR) and PD-L1 tumor expression were also recorded.

Results: Patients characteristics were: 62% males, median age of 65 years (range 37-78), 61% PS 0-1, median BMI of 24 kg.m-² (range 17-39), 78% nonsquamous NSCLC. The analysis of REE was available for 69 out of 82 consecutive pts: 37.7% were hypermetabolic, 47.8% were normometabolic, and 14.5% were hypometabolic. In univariate analysis, hypometabolism was a strong predictive marker of disease progression (Table 1), with positive and negative predictive values of 0.80 and 0.52 respectively. In multivariate analysis, independent parameters associated with disease progression were baseline hypometabolism (vs normometabolism: OR 1.77 [1.31-2.39] p= 0.0004) and albumin (per 1pt increase: OR 0.96 [0.94-0.99] p= 0.005).

Conclusion: Rest energy expenditure assessed by calorimetry appears as a biomarker of nivolumab clinical activity independently of PD1/PDL1 status.

Table 1

---

Disease control (best response) n (%) | <85% calculated REE n=10 (14.5%) Hypometabolic | 85-115% calculated REE n=33 (47.8%) Normometabolic | >115% calculated REE n=26 (37.7%) Hypermetabolic | univariate OR (Hypometabolic vs normometabolic) | p

Progression | 8 (80%) | 11 (33%) | 17 (65%) | 8 [1.4-44.2] | 0.0007

Disease control rate | 2 (20%) | 22 (67%) | 9 (35%) | |

#1694

Computational analysis of multiplexed immunohistochemistry for understanding immune profiles in clinical biopsies.

Joseph S. Krueger, Brooke Hirsch, Mandy Bell, Karen Ryall, Nick Landis, G David Young, Kile McFadden, Ben Landis. _Flagship Biosciences, Westminster, CO_.

Using immunohistochemistry (IHC) to examine immune infiltrates in tissue biopsies to support exploratory investigations for immuno-oncology drug development is critical for understanding the abundance and spatial relationships of different immune cell types and how they may change with drug treatment. Multiplex IHC approaches are utilized to examine these types of pharmacodynamic responses because of the limited tissue available from repeat needle core biopsies and the need to visualize multiple biomarkers in the same tissue section. While several different methods of fluorescent multiplexing approaches exist, the complexity of these fluorescent assays limits the ability to develop and validate bespoke assays to meet the needs of hypothesis-driven research which aims to elucidate predictors of clinical response. Furthermore, these methodologies present known challenges for design control processes and regulatory approval as companion diagnostics, preventing wide use beyond exploratory research settings.

In contrast, development and analytical validation of chromogenic IHC assays enables an agile and bespoke approach to IHC assay development which can support all phases drug development, including direct translations of these methods into companion diagnostic building approaches. However, investigating multiple biomarkers using chromogenic assays presents its own challenges, as there is a more limited repertoire of chromagens than fluorophores, which have significant spectral overlap in wavelength absorbance and optical density. As such, a multiplex chromogenic IHC assay requires specialized performance specifications as well as sophisticated interpretation methods to ensure accurate interpretations.

While these limitations often create too great a challenge for pathologists to interpret, computational analysis of tissue using Flagship's cTA™ platform resolves these challenges and enables drug developers to rely on chromogenic IHC approaches to meet the needs of drug and diagnostic development. To demonstrate the application of this method, we developed several chromogenic multiplex IHC assays which capture critical immune profiles for immuno-oncology studies and interpreted the endpoints using Flagship's cTA™ approach. These assays/interpretations provide quantitative data about: 1) General immune profile (CD8/CD68/FoxP3); 2) T-cell ratio (CD3/CD8); 3)T-cell activation (CD8/Ki67); and 4) T-cell suppression (CD8/FoxP3). Using this approach, we demonstrate how Flagship's cTA™ platform enables investigation of complex biological questions using chromogenic assays, which cannot be achieved by relying on traditional manual pathology interpretation.

#1695

A multi-institutional study to evaluate automated scoring of immunohistochemistry slides for assessment of programmed death-ligand 1 (PD-L1) expression in non-small cell lung cancer.

Clive Taylor,1 Anagha P. Jadhav,2 Abhi Gholap,3 Gurunath Kamble,2 Jiaoti Huang,4 Allen Gown,5 Isha Doshi,3 David Rimm6. 1 _University of Southern California, Los Angeles, CA;_ 2 _OptraSCAN, Pune, India;_ 3 _OptraSCAN, Sunnyvale, CA;_ 4 _Duke University School of Medicine, Durham, NC;_ 5 _PhenoPath, Seattle, WA;_ 6 _Yale School of Medicine, New Haven, CT_.

Purpose: Assessment of PD-L1 expression is a critical part of patient management for immunotherapy. However, studies have shown that pathologist-based analysis lacks reproducibility, especially for immune cell expression. The purpose of this study was to validate and to assess reproducibility of the automated Optra image analysis for PD-L1 IHC for both tumor cells and immune cells.

Experimental Design: We compared conventional pathologists' scores (3 board-certified pathologists active in routine signout of these cases) for both tumor and immune cell positivity separately, using 22c3 antibody on the Dako Link 48 platform for PD-L1 expression in non-small cell lung carcinoma (NSCLC). We examined interpathologist PD-L1 expression scoring variability for both, tumor and immune cells using ordinal tumor proportion scores for tumor cells and continuous percentage positive scores. The cohort included 230 NSCLCs obtained from Yale School of Medicine, Department of Pathology archives. We assessed interpretation first by pathologists and secondly by the Optra PD-L1 image analysis scores for both tumor and immune cells. The Intra Class Correlation (ICC) for each pathologist was measured to assess variability between pathologists in scoring both tumor and immune cells. The concordance between pathologists using digital manual reads of PD-L1 staining percentages and Optra PD-L1 image analysis quantitative scores was then assessed using the Lin's concordance correlation coefficient for both tumor and immune cells.

Results: Intraclass correlation coefficients to evaluate the correlation between pathologists for tumor cell (ICC = 0.750) showed an excellent concordance but lower concordance for immune cell scoring (ICC = 0.4). To compare the pathologist scores to the Optra automated system, the scores from the 3 pathologists were averaged to produce a single conventional read score. The Lin's concordance correlation coefficient between the conventional read and the machine score was 0.83 for tumor cells and 0.6 for immune cell population in intra- and peritumoral compartments. This is considered excellent agreement for tumor cells and good concordance for immune cells.

Conclusion: The interpathologist assessment seen in this study is similar to previously reported studies where agreement is higher in tumor cells than immune cells. The Optra PD-L1 image analysis showed concordance with the pathologists' average scores that were comparable to interpathologist scores. This suggests promise for automated assessment of PD-L1 in NSCLC. These results justify similar studies with immunotherapy-treated patients with known outcomes.

#1696

Glycogen synthase kinase (GSK-3) inactivation downregulates PD-1 and synergizes with PD-1/PL1 and CTLA-4 blockade in cancer immunotherapy.

Christopher E. Rudd. _Centre de Recherche Hopital Maissoneuve-Rosemont, Montreal, Quebec, Canada_.

Immune checkpoint blockade (ICB) of negative co-receptors on T-cells such as cytotoxic T-cell antigen-4 (CTLA-4) and programmed cell death-1 (PD-1) is a promising approach for the treatment of cancer. Despite this success, the poor prognosis for most patients continues to highlight a need for developing novel clinical interventions. In this context, we identified the enzyme glycogen synthase kinase-3 (GSK-3) as the major regulator of PD-1 expression on T-cells (Taylor et al., 2016 Immunity). We have shown that small molecule inhibitors (SMIs) of GSK-3 are as effective as anti-PD-1 in controlling the growth of B16 melanoma, or EL4 lymphoma, in primary tumor and metastatic settings (Taylor et al., 2017 Can Res; Krueger and Rudd, Immunity 2017). At the same time, the fact that GSK-3 inactivation up-regulates transcription factor T-bet which upregulates interferon gamma (IFNγ) and granzyme B (GZMB) suggested that GSK-3 inactivation might provide extra value beyond of its down-regulation of PD-1. Here, we show that GSK-3 SMIs can synergize with anti-PD-1 and anti-CTLA-4 to eliminate B16 solid tumors that are otherwise resistant to anti-PD-1, or anti-CTLA-4 inhibition alone. At a cellular level, GSK-3 inactivation preferentially down-regulated PD-1 on CD8+ tumor infiltrating T-cells, while at a molecular level, GSK-3 SMIs inhibited PD-1 transcription, increased granzyme B (GZMB)/ interferon-gamma1 (IFNγ) transcription and directly acted upon glycogen synthase to skew metabolism towards greater glycolysis. We further showed that the inactivation of GSK-3α/β through siRNA or by SMIs substituted for CD28 co-stimulation in the potentiation of cytotoxic CD8+ CTL function. This data supports a model where GSK-3 mediates the dependency of anti-PD-1 immunotherapy on the expression of CD28. Overall, our data show that GSK-3 inactivation is an immune-sensitizer which assists the immune system to overcome tumor resistance to immune check-point blockade.

#1698

Loss of MHC-I expression is associated with altered immune microenvironment and decreased response to PD-1 checkpoint inhibition.

Cecile Gstalder, Priya Pancholi, Dorota Sadowicz, Rizwan Haq. _Dana-Farber Cancer Institute, Boston, MA_.

Cancer immunotherapies, such as PD-1 inhibitors, achieve durable responses in a subset of cancer patients, however, a significant proportion of patients fail to respond to treatment. Defects in antigen presentation, such as the loss of MHC-I expression, are a major mechanism of resistance to PD-1 inhibitors. Developing new therapeutic strategies that overcome the lack of antigen presentation will improve cancer treatment and patient survival. To develop a pre-clinical model to evaluate therapies that could overcome resistance, we have generated a unique PD-1 blockade sensitive, syngeneic immunocompetent melanoma mouse model. We generated defects in antigen presentation using the CRISPR/Cas9 system, creating B2m-deficient melanoma cell lines. We demonstrated that B2m is necessary for IFN-gamma induced MHC-I surface expression. Unlike control tumors, we found that B2m-deficient tumors were resistant to PD-1 blockade, leading to decreased mouse overall survival. Using multi-parameter flow cytometry, we showed an increase in NK cell, regulatory T cell and dendritic cell infiltration in B2m-deficient tumors compared to controls, while the other immune cell population were unchanged. Since NK cell activity is negatively regulated by MHC-I, our findings suggest that additional factors are required to further invigorate infiltrating NK cells and to trigger a NK cell-mediated anti-tumor response. We are currently evaluating new approaches targeting NK cells and regulatory T cells on B2m-deficient tumors. This pre-clinical model could enable the development of a therapeutic strategy serving to improve the treatment of tumors lacking antigen presentation.

#1699

Treatment with agonist anti-GITR antibody after chemotherapy enhances tumor immunity.

Allison Betof Warner,1 Daniel Hirschhorn,1 Adam Cohen,1 Cailian Liu,1 Walter Newman,2 Cyndi Sirard,2 Jedd Wolchok,1 Taha Merghoub1. 1 _Memorial Sloan Kettering Cancer Center, New York, NY;_ 2 _Leap Therapeutics, Cambridge, MA_.

Stimulation of glucocorticoid-induced tumor necrosis factor receptor (GITR) has been shown to enhance antitumor immunity by stimulating effector CD4+ and CD8+ T cells and attenuating suppression and depleting by CD4+Foxp3+ regulatory T cells (Treg). However, GITR monotherapy does not effectively control tumor growth. We hypothesize that cyclophosphamide (CTX), a cytotoxic chemotherapeutic agent with key immunomodulatory properties, can enhance the potency of GITR engagement anti-tumor effects. Further, we hypothesize that CTX can induce neoantigen formation by increasing tumor mutation burden. In this study, we administered high-dose CTX (250 mg/kg) followed the next day by an agonist anti-GITR antibody (DTA-1) to C56BL/6 mice bearing poorly immunogenic B16 melanoma tumors. We found that the combination therapy can cause regression of established tumors whereas the single agents did not. Mechanistically, we found that CTX administration results in tumor antigen release, which enhances the antitumor T cell response after treatment with DTA-1. This effect is lost when lower doses of CTX are given or CTX is administered prior to tumor inoculation, suggesting that tumor cell death and cross-presentation of tumor antigens to T cells is necessary. Furthermore, Treg cells are an important target of combination therapy. Intratumoral Treg levels were substantially reduced in treated mice, which greatly increased the ratio of T effector/Treg in the tumor. In conclusion, CTX and agonist anti-GITR antibody slows tumor progression and can induce regression of palpable, poorly immunogenic B16 melanoma tumors, likely by enhancing cross-priming and increasing the T effector/Treg ratio in the tumor. Thus, this combination warrants further investigation as a novel immunotherapy approach and provides rational for exploring combination of GITR agonist antibodies with chemotherapeutic treatments in clinical settings.

#1700

Metabolic adaptations confer immunotherapy resistance in melanoma.

Ashvin R. Jaiswal, Shivanand Pudakalakatti, Dutta Prasanta, Arthur Liu, Todd Bartkowiak, Casey Ager, Michael A. Davies, Richard E. Davis, Jennifer Wargo, Pratip K. Bhattacharya, David S. Hong, Michael A. Curran. _The University of Texas MD Anderson Cancer Center, Houston, TX_.

Background: Despite the success of T-cell checkpoint blockade antibodies in treating an array of cancers, the majority of patients still fail to respond to these therapies, or respond transiently and then relapse. The molecular mechanisms that drive lack of response to checkpoint blockade, whether pre-existing or evolved on therapy, remain unclear.

Materials and Methods: To address this critical gap in clinical knowledge, we established a mouse model of melanoma designed to elucidate the molecular mechanisms underlying immunotherapy resistance. Through multiple in vivo passages, we selected a B16 melanoma tumor line that evolved complete resistance to combination blockade of CTLA-4, PD-1, and PD-L1, which cures ~75% of mice of the parental tumor. Using gene expression analysis, proteomics, and immunogenomics, we determined the adaptations engaged by this melanoma to become completely immunotherapy resistant. NMR spectroscopy, Seahorse XF Analysis, flow cytometry, confocal microscopy and Western blot analysis provided further insight into the mechanisms driving checkpoint blockade resistance.

Results: Acquisition of immunotherapy resistance by these melanomas was driven by acquisition of a hypermetabolic state through coordinate upregulation of both glycolysis and oxidative phosphorylation (OxPhos). Depletion of essential nutrients from the tumor microenvironment by these resistant tumors functionally compromises infiltrating T cells to an extent that cannot be rescued by even triple checkpoint blockade. The glycolysis and OxPhos-associated genes most highly induced by these resistant melanomas were capable of conferring enhanced resistance to checkpoint blockade when reintroduced monogenically into the parental tumor. We have validated upregulation of these pathways in a unique cohort of melanoma patients who failed dual checkpoint blockade. Additionally, we employed a novel MRI imaging technique to visualize metabolic changes acquired by resistant tumors in live mice. Clinical application of this technique could provide a much-needed noninvasive tool to predict immunotherapeutic sensitivity of patient cancers.

Conclusion: Acquisition of a hypermetabolic state by melanoma tumor cells cripples T cells in the microenvironment and confers complete resistance to checkpoint blockade.

#1701

EPL-1410, a novel fused heterocycle based orally active dual inhibitor of IDO1/TDO2, as a potential immune-oncology therapeutic.

Srinivas Gullapalli, Abhijit Roychowdhury, Tushar Khaladkar, Sangameshaver Sawargave, Ravindra Janrao, Vijay Kalhapure, Ganesh Urunkar, Jayanarayan Kulathingal, Ramamohan Reddy Lekkala, Sonali Bhadra, Pankaj Kumar Singh, Krishna Anand Putta, Sonali Manwatkar, Prashant Surve, Mukund Keshav Gurjar. _Emcure Pharmaceuticals, PUNE, India_.

Many tumors use tryptophan (Trp)-catabolizing enzymes such as Indoleamine/Tryptophan-2,3-dioxygenases (IDO1/TDO2) to induce an immunosuppressive environment by depleting Trp, resulting in excessive generation of kynurenine (Kyn) pathway metabolites. IDO1 inhibitors in combination with immune-checkpoint inhibitors are gaining success in multiple cancer clinical trials. This, along with accruing evidences in literature, further strengthens the dual inhibition of IDO1/TDO2 as a highly effective therapeutic prospect.

Herein, we describe the discovery of fused heterocycle based analogue EPL-1410 as a novel orally active dual inhibitor of IDO1/TDO2. In the biochemical and IFNγ stimulated HeLa cell based assays, EPL-1410 inhibited human IDO1 with IC(50) values of 320 nM and 23.6 nM respectively. EPL-1410 also inhibits human TDO2 with an IC(50) of 516 nM in the biochemical assay. Further studies of EPL-1410 in various 'in vitro ADMET' screens (e.g., Metabolic stability, plasma protein binding, hCYP inhibition, hERG, Cerep Safety Screen 44) provided an optimal metabolic stability, free drug concentration fraction and safety profile necessary to qualify it as a lead candidate. The pharmacokinetic profile of EPL-1410 showed an excellent absolute oral bioavailability (%F~100%) in mice and rats. In addition, the characterization of EPL-1410 in a battery of DMPK studies including PK profile in higher species and preliminary safety data will be discussed. EPL-1410 showed a significant dose dependent pharmacological efficacy in reducing the tumor volume in mouse syngeneic cancer models of CT-26 colon carcinoma and B16F10 melanoma. A good correlation of efficacy to biomarker (Kyn/Trp) ratio was observed in plasma, tumor draining lymph node and tumor tissue with no treatment related adverse clinical signs and body weight reduction at all doses of EPL-1410 tested.

The data presented will establish EPL-1410 as a novel, orally active and potent dual inhibitor of IDO1/TDO2 with desired pharmaceutical properties for further development as a potential immuno-oncology therapeutic intervention.

#1702

Assessment of pharmacology and toxicology of anti-CTLA-4 antibody (ADU-1604) in non-human primates and evaluation of local anti-CTLA-4 application.

Maaike Hendriks,1 Joost Kreijtz,1 Paul Vink,1 David Lutje Hulsik,1 Imke Lodewijks,1 Astrid Bertens,1 Jos van de Crommert,1 Maurice Habraken,1 Wout Janssen,1 Judith Stammen-Vogelzang,1 Olivia Gardner,2 Weiwen Deng,2 Meredith Leong,2 Sarah McWhirter,2 Hans van Eenennaam,1 Andrea van Elsas1. 1 _Aduro Biotech Europe, Oss, Netherlands;_ 2 _Aduro Biotech Inc, Berkeley, CA_.

The immune checkpoint CTLA-4 can be targeted with antibodies that are applied as single agent therapy or in combination with other therapies including surgery, radiation and chemotherapy. Anti-CTLA-4 antibodies may also augment other immunotherapies. Indeed, in syngeneic mouse tumor models anti-CTLA-4 strongly enhanced anti-tumor efficacy of live, attenuated double-deleted Listeria monocytogenes (LADD) and of the STING pathway activator ADU-S100. Anti-CTLA-4 therapy has shown clinical activity and durable responses in advanced cancers, but is also associated with severe immune-related adverse events (irAEs), although manageable and reversible. Local anti-CTLA4 application could limit the induction of irAEs while retaining its capacity to induce and enhance tumor-specific T cell responses.

Here we present the results from the preclinical development of ADU-1604, a novel hIgG1 anti-CTLA-4 antibody. ADU-1604 binds a unique epitope on human CTLA-4 and was extensively characterized in vitro demonstrating potent CTLA-4 binding and blockade of CD80 and CD86 and its capability to enhance human T cell responses. As the antibody cross-recognizes CTLA-4 of cynomolgus monkeys this species was selected for the preclinical studies. Male animals received a single intravenous dose of the antibody to assess its pharmacokinetics and pharmacodynamics. ADU-1604 was well tolerated and showed an acceptable half-life. The antibody also enhanced the T cell-dependent antibody response in hepatitis B surface antigen immunized animals. After the single dose PK study a GLP 4-week repeat-dose toxicity study was conducted in cynomolgus monkeys administered 5 doses of ADU-1604 intravenously once a week followed by an 8-week recovery period. In general, ADU-1604 was well tolerated at up to 30 mg/kg/dose. Histopathology data is being processed and recovery data is pending.

Proof of concept for the local administration of anti-CTLA-4 antibodies was conducted in syngeneic mouse models with the mouse CTLA-4 antibody 9D9 and in NSCLC humanized PDX models using ADU-1604 in the context of CD45+ human immune cells. These models demonstrated anti-tumor activity of systemically applied ADU-1604, comparable to a reference anti-CTLA-4 antibody. Intratumoral application of ADU-1604 in these models also induced tumor growth inhibition

In conclusion, the preclinical development package warrants clinical investigation of ADU-1604 as monotherapy and for future combinations with other Aduro proprietary platforms including STING agonists and (p)LADD. Currently ADU-1604 is progressing through regulatory filing in preparation of clinical development.

#1703

**The HDAC6 inhibitor Nexturastat A improves** in vivo **PD-1 immune blockade.**

Tessa Knox,1 Eva Sahakian,2 Debarati Banik,1 Melissa Hadley,1 Erica Palmer,1 Jennifer Kim,1 John Powers,2 Sarthak Shah,1 Alexa Lowe,1 Javier Pinilla,2 Eduardo Sotomayor,1 Norman Lee,1 Alan Kozikowski,3 Alejandro V. Villagra1. 1 _George Washington Univ., Washington, DC;_ 2 _H. Lee Moffitt, Tampa, FL;_ 3 _Starwise, Madison, WI_.

The role of HDACs in cell biology, initially limited to their effects upon histones, now encompasses more complex regulatory functions that vary with their tissue expression, cellular compartmentalization and stage of cellular differentiation. Several recent studies have shown that selective HDAC inhibitors (HDACi) are able to impair in vivo tumor growth. Therefore, there is an emerging interest in the understanding of the molecular mechanisms mediating these anti-tumor properties. In this context, a number of recent publications have demonstrated that the selective inhibition of specific HDACs enhances tumor immunogenicity in a wide variety of tumors, thereby preventing tumor escape and improving immune surveillance. Our group has focused on HDAC6, and shown that both the genetic abrogation and pharmacological inhibition of this HDAC modulates the expression of a variety of immune-regulatory proteins in the tumor microenvironment, including PD-L1, PD-L2, MHC class I, B7-H4 and TRAIL-R1. In particular, we have previously demonstrated that both pharmacological inhibition and/or genetic abrogation of HDAC6 plays a critical role in the immune check point blockade by down-regulating the expression of PD-L1. Moreover, we have also shown that in vivo inhibition of HDAC6 reduces tumor growth in B16 and SM1 murine melanoma models within syngeneic immunocompetent hosts. Additionally, we have found that the combination of the HDAC6i Nexturastat A (NextA) and checkpoint PD-1 immune blockade therapy results in an important improvement in anti-tumor immune responses as evidenced by the reduction of tumor growth when compared to treatment with individual stand-alone agents. Moreover, our data has shown an increase in CD8+, NK+ cell infiltration of tumors treated with the combination therapy versus either standalone treatment. Further analysis of in vivo immune tumor infiltration evidenced that the treatment with NextA importantly diminished the macrophage M2 phenotype in the tumor microenvironment. An outcome that was also observed in combination with PD-1 immune blockade. Lastly, we have found that the anti-PD-1/NextA in vivo combination therapy reduces the expression of PD-L1, PD-L2 and other negative check-point pathways. However, the HDAC6 inhibition has a minimal effect over the expression of their respective counterparts in T cells (i.e. PD-1). Overall, we have found that HDAC6i could be used as a potential adjuvant in ongoing therapeutic options involving immune check-point blockade.

#1705

Systemic analysis of innate responses to anti-PD1/PD-L1 treatment in a melanoma model.

Rodrigo Gassen,1 Alana Welm,2 Cristina Bonorino3. 1 _PUCRS, Porto Alegre, Brazil;_ 2 _University of Utah, Salt Lake City, CA;_ 3 _UCSD, La Jolla, CA_.

Response to anti-PD1 therapy for tumors is often thought to target T cells, directly and exclusively. However, while some patients, even with metastasis, respond completely, most patients do not respond to treatment as predicted. The mechanisms of response ad resistance to anti-PD-1 therapy need to be elucidated, in order to optimize treatment and prevent unnecessary adverse effects. Most innate cells, especially in tumor bearing individuals, express PD-1, not only in the tumor microenvironment, but systemically. Among those are macrophages and dendritic cells. The implications of PD-1 expression by these cells in their immune function are still unknown, and so are the signaling pathways activated by agonists or antagonists of this receptor. Although recent data suggest that PD-1+ tumor associated macrophages (TAMs) promote advanced tumor growth, little is known of the kinetics of PD-1 expression by innate cells during tumor progression. Using an immunocompetent murine melanoma model, we characterized PD-1 and PD-L1 expression in different innate cells, in lymphoid organs, blood, and tumor microenvironment. In NSG mice, using PDX grafts of triple negative breast cancers, we determined that such expression is independent of lymphocytes and NK cells. Treatment with anti-PD-1 mobilized an early (7 days) wave of DC migration that can be seen in the blood, preceding a CD4+ T cell mobilization that peaked ad day 14 after treatment. These early responses correlated with CD8+ T cell infiltration to the tumor microenvironment as well as tumor regression. PD-1 expression and migration in the blood was different among the DC subpopulations, and anti-PD-1 treatment enhanced survival as well as activation of dendritic cells. These findings are relevant to understanding the different outcomes of immunotherapy with anti-PD-1 and anti-PD-L1. 

### Immune Response to Therapies 2

#1706

Inhibition of fibulin-3 reverses macrophage polarization in glioblastoma and increases anti-tumor inflammatory responses.

Sharon L. Longo, Prajna Behera, Mariano S. Viapiano, Mohan Sobhana Nandhu. _SUNY Upstate Medical University, Syracuse, NY_.

Glioblastomas (GBMs) are the most common primary tumors of the Central Nervous System and one of the most aggressive and difficult to treat forms of cancer. Our laboratory was the first to identify the extracellular protein fibulin-3 (EFEMP1) in GBMs and demonstrate the pro-tumoral functions of this protein in solid tumors. We have recently developed a function-blocking antibody ("mAb428.2") against fibulin-3, which inhibits the enhancing effect of this protein on ADAM17 activity and NFkB signaling (Nandhu et al., Clin Cancer Res, 2017). Using mAb428.2 we observed a significant anti-tumor effect and prolongation of survival in nude mice carrying patient-derived GBM xenografts implanted subQ or intracranially (antibody treatment: 8x 30 mg/kg, q24h, IV). Interestingly, reduction of tumor growth during mAb428.2 treatment was accompanied with significant tumor necrosis and increased infiltration of Iba1-positive tumor-associated macrophages (TAMs) both in subQ and orthotopic tumors. Remarkably, TAMs in mAb428.2-treated GBMs showed very low co-expression of Arginase-I, a bona-fide marker of M2 ("tumor promoting") TAM polarization. Further analysis of mouse-specific gene expression in the tumor mass and in recovered macrophages (CD11b-positive cells) showed that TAMs in mAb428.2-treated GBMs had significantly increased expression of inflammatory cytokines (IFN-gamma, IL-1b, IL-10) and downregulation of M2 markers (CD163, CD206, ARG1), suggesting that our anti-fibulin-3 treatment prevented the expected M2 polarization of TAMs. Accordingly, mAb428.2-treated tumors showed drastically decreased expression of CSF-1a, an M2-inducing cytokine that may be regulated by fibulin-3. Co-culture experiments of PMA-activated U937 macrophages with GBM stem-like cells did not affect tumor cell viability when a control IgG1 was added to the cultures. However, addition of mAb428.2 caused significant decrease of GBM cell viability, suggesting that anti-fibulin-3 treatment enables TAMs to attack tumor cells. Taken together, our results strongly suggest that anti-fibulin-3 and possibly other anti-ECM approaches may induce a marked inflammatory reaction driven by innate immune cells, potentiating anti-tumor therapy.

#1707

Tumor treating fields exert cellular and immunologic effects.

Eric T. Wong, Joshua Timmons, Kenneth D. Swanson. _Beth Israel Deaconess Medical Center, Boston, MA_.

While there is clear clinical evidence to support the therapeutic efficacy of low dose alternating electric fields (TTFields) in treating glioblastoma, their intracellular targets and mechanism of action remains poorly defined. Historically, TTFields have been thought to operate by inducing extensive plasma membrane blebbing during the entry into anaphase resulting in aberrant mitotic exit and cell death. However, we have found that cells exposed to TTFields exhibited increased granularity and evidence of endoplasmic reticulum stress and genotoxic stress suggesting other mechanisms to explain their clinical efficacy. Cells exposed to TTFields exhibited reductions in levels of culture media acidification and analysis of intracellular metabolism showed evidence for an increase in mitochondrial respiration and glutaminolysis pathway. Our previous analysis of data from the pivotal phase III EF-11 clinical trial showed that increased corticosteroid use was strongly correlated with poor outcomes in TTFields-treated patients, possibly by suppressing immune effector function required for to drive tumor regression. TTFields drives the cell surface expression of immune stimulatory stress marker proteins calreticulin, and the secretion of the Alarmin/DAMP protein, HMGB1 which are together suggest that TTFields can stimulate immunogenic cell death. Co-culture of bone marrow derived macrophages with TTFields-treated CT26 cells resulted in the up-regulation of cell surface activation markers in the macrophage population that was blocked by a monoclonal antibody directed against HMGB1. Further, supernatants from these co-cultures contained increased levels of pro-inflammatory cytokines and chemokines and a reduction of anti-inflammatory cytokines associated with the presence of the TTFields-treated cells relative to co-cultures containing naïve tumor cells. Further, in vivo, engrafted Lewis lung carcinoma tumors exhibited increased levels of granzyme positive cells following TTFields treatment. Together, these data suggest that TTFields affect cancer cells during interphase and this insult leads to alterations in cancer cell metabolism and increased tumor cell immunogenicity and thus act to drive increased immune recognition and tumor rejection. In patients, these effects are likely to be opposed by the immune suppressive functions within the tumor microenvironment and corticosteroid use. Therefore, this model of TTFields action may provide a therapeutically rational base to devise improved TTFields treatment strategies from.

#1708

Nanofluidic drug-eluting seed for sustained intratumoral immunotherapy in triple negative breast cancer.

Corrine Ying Xuan Chua,1 Priya Jain,1 Marco Folci,1 Andrea Ballerini,1 Jessica Rhudy,1 April Gilbert,1 Shail Shaibala,2 Giacomo Bruno,1 Carly S. Filgueira,1 Cassian Yee,2 E. Brian Butler,1 Alessandro Grattoni1. 1 _Houston Methodist Research Institute, Houston, TX;_ 2 _University of Texas MD Anderson Cancer Center, Houston, TX_.

Conventional systemic immunotherapy administration often results in insufficient anti-tumor immune response and adverse side effects. Delivering immunotherapeutics intratumorally could maximize tumor exposure, elicit robust anti-tumor immune response, and minimize toxicity. To fulfill the unmet clinical need for sustained local drug delivery and to avoid repeated intratumoral injections, we developed a nanofluidic-based device for intratumoral drug delivery called the nanofluidic drug-eluting seed (NDES). The NDES is inserted intratumorally using a minimally invasive trocar method similar to brachytherapy seed insertion and offers a clinical advantage of intratumoral drug elution. Drug diffusion from the NDES is regulated by physical and electrostatic nanoconfinement, thereby resulting in constant and sustained immunotherapeutic delivery without the need for repeated injections or clinician intervention. In this study, the NDES was utilized to deliver immunotherapeutics intratumorally in the 4T1 orthotopic murine mammary carcinoma model, which recapitulates triple negative breast cancer. We demonstrated that NDES-mediated intratumoral release of agonist immunotherapeutic monoclonal antibodies, OX40 and CD40, resulted in potentiation of local and systemic anti-tumor immune response and significant inhibition of tumor growth compared to control mice. Further, mice treated with NDES-CD40 demonstrated minimal liver damage compared to systemically treated mice. Collectively, our study highlights the NDES as an effective platform for sustained intratumoral immunotherapeutic delivery. The potential clinical impact is tremendous given that the NDES is applicable to a broad spectrum of drugs and solid tumors.

#1709

**Enhanced proliferation and effector functions of human immune cells in response to immune checkpoint therapy of MDA-MB-231 tumor bearing huNOG mice engrafted with human CD34** + **cells.**

Thi Bui, Karyn McArtan Shinn, Emily G. O'Koren, Ashleigh Derrick, Beverly Godfrey, Ian Belle, Jason Davis, Aidan Synnott, Anya Avrutskaya, Paula L. Miliani De Marval. _Charles River Discovery Services, Morrisville, NC_.

Given the recent success of cancer immunotherapies, well characterized pre-clinical models are needed to enable drug discovery efforts. The recently developed humanized mouse models composed of various types of immune cells offer a unique tool to evaluate aspects of the human immune system in response to immunotherapy. MDA-MB231 cells express high levels of the T-inhibitory molecule PD-L1 making this breast cancer line an ideal candidate for testing the efficacy of the immune-checkpoint inhibitors pembrolizumab (anti-PD-1) and ipilimumab (anti-CTLA-4) in the CD34+ huNOG humanized mouse strain. We analyzed the distribution of CD4 and CD8 T lymphocytes, Treg cells, NK cells and B cells in blood, spleen and tumor samples from MDA-MB231 tumor bearing huNOG humanized treated-mice via flow cytometry. We found that combination therapy enhanced the effector functions of CD4+ and CD8+ T cells associated with increased expression of IFN-gamma and Granzyme B. Supporting this data, we observed an increase in Ki-67+ cells which correlates with a decrease in PD-1 expression in response to pembrolizumab therapy associated with activation of T lymphocytes. In summary, these results demonstrate the potential of humanized mouse models to evaluate the anti-tumor activity of human immune cells in a preclinical setting in response to human specific immune checkpoint targeted therapies.

#1710

DKN-01, a therapeutic DKK1 neutralizing antibody, has immune modulatory activity in nonclinical tumor models.

Michael Haas,1 Heidi Heath,1 Yinyuan Wu,2 Xinjun Zhang,2 Cynthia Sirard,1 Xi He,2 Walter Newman,1 Michael Kagey1. 1 _Leap Therapeutics, Cambridge, MA;_ 2 _Harvard Medical School, Boston, MA_.

Wnt signaling is a fundamental pathway that is dysregulated in oncology. The Wnt signaling modulator DKK1 is expressed in a variety of tumor types and elevated levels frequently correlate with poor survival. DKK1 promotes tumor growth by stimulating proliferation, metastasis, and angiogenesis, and has been implicated in contributing to an immune suppressive tumor microenvironment. DKN-01 is a humanized monoclonal therapeutic antibody that binds DKK1 with high affinity and selectivity. It is currently being evaluated clinically as a monotherapy and in combination in a variety of solid tumors. Here we describe further characterization of the mechanism of action of DKN-01 and demonstrate immune mediated anti-tumor activity in nonclinical models. A murine version of DKN-01 (mDKN-01) has efficacy in a syngeneic melanoma B16 tumor model. However, mDKN-01 is unable to impede B16 tumor growth in NSG immunodeficient mice, indicating that a functioning immune system is required for antibody activity. Furthermore, preliminary data suggest that mDKN-01 is targeting a myeloid derived suppressor cell population in the tumor microenvironment. These data support an immune mediated mechanism of action of DKN-01 and provide a rationale for clinical development in combination with immunotherapy agents. The first clinical study evaluating DKN-01 in combination with pembrolizumab has initiated enrollment in patients with relapse/refractory esophagogastric malignancies.

#1711

Immunoprofiling of precision-cut cancer tissue slices (PCCTS) as a tool for cancer immunotherapy.

Nicole Grabinski,1 Kristina Bernoth,1 Aljoscha Leusmann,2 Carolin Fleischhauer-Biermann,2 Dorte Wendt,2 Mirja Piller,1 Moiken Petersen,1 Nicole Lange,1 Anna Tiedemann,1 Monika Schoeppler,1 Hartmut Juhl,1 Gerd Helftenbein,2 Andrea Miegel,2 Frank Schnieders,2 Kerstin David1. 1 _Indivumed GmbH, Hamburg, Germany;_ 2 _Provecs Medical GmbH, Hamburg, Germany_.

The recent advances in immunotherapies, such as immune checkpoint modulators, bispecific antibodies, and adoptive T-cell transfer, opens new opportunities for the treatment of cancer. Having this broad spectrum of new therapeutic agents available, the demand for predictive and robust preclinical models to minimize translational failures in immuno-oncology is increasing. Indivumed has successfully implemented a model of Precision Cut Cancer Tissue slices (PCCTS) derived from viable human tumor tissue for different applications such as chemotherapeutic agents, small molecules and antibodies. In this study, we investigated the effects of OKT3® on cancer tissue slices especially in respect of gene expression changes and cytokine release.

Fresh tumor pieces from three colorectal cancer patients were cut into 400 µm slices using a Krumdieck Tissue Slicer®. PCCTS were incubated for 4 h and 24 h with and without OKT3®, (Muromonab), a therapeutic antibody against CD3. After defined time points, slices were frozen, and the supernatants were collected. In addition, a second set of slices was formalin fixed and paraffin embedded for immunohistochemical analysis of T-cells. RNA isolated from frozen slices was used for library preparation with the TruSeq Stranded mRNA Library Preparation Kit followed by sequencing on a NextSeq 500 device from Illumina. The results generated by different software tools showed a background of gene expression changes caused by cultivation. On the other hand, a very specific up-regulation of genes was detectable after OKT3® treatment. Especially an induction of genes involved in immune related functions, such as IFN gamma and IL-12 signaling was seen. The analysis of cytokines in the supernatants using a ten-plex panel form Meso Scale Discovery confirmed the activation of T-cells after OKT3® treatment. In all three patients a significant increase of IFN gamma, IL-2, and TNF alpha was measured, comparable to a so called "cytokine storm" that is described in literature. The intensity of the effects correlated to the amount of tumor infiltrating T-cells, quantified by anti-CD3 staining in tissue sections.

This model of PCCTS represents a unique opportunity to test immune-modulatory compounds in a fully human, patient derived model that is close to the in vivo situation. The data of this study shows that profiling of the immune response by RNA-Seq can be successfully performed. In addition to gene expression changes that occur after treatment, deconvolution based approaches to quantify tumor Infiltrating lymphocytes (TILs) from RNA-Seq data will be a key parameter to personalize the treatment of patients.

#1712

Assessing tumor mutation load using an NGS-based, routine-friendly target gene panel.

Jose Costa,1 Joana Reis,1 Margarida Fernandes,2 Rafaela Silva,1 Luis Cirnes,1 Ruchi Chaudhary,3 Fatima Carneiro,4 Jose C. Machado1. 1 _IPATIMUP, Porto, Portugal;_ 2 _Centro Hospitalar São João, Porto, Portugal;_ 3 _Thermo Fisher Scientific, Carlsbad, CA;_ 4 _Medical Faculty of Porto, Porto, Portugal_.

In several cancer models, it was shown that tumor mutation load correlates with response to immune checkpoint blockade therapy. However, whole exome sequencing of FFPE samples is not ideal for implementation on molecular pathology laboratories. In colorectal cancer (CRC), mutation load is correlated with microsatellite instability (MSI), which can therefore be used as surrogate marker for mutation load. In this study, we aimed at determining whether a target NGS-based mutation panel can be used for routine testing of mutation load in cancer, by assessing its performance in identifying MSI+ and MSI- CRC samples. The Oncomine Tumor Mutation Load Assay, which targets 409 genes corresponding to 1.7Mb of sequencing data, was used. It is a two-pool panel, optimized for FFPE samples, and only requires 20 ng of DNA in total. A cohort of 16 CRC samples was used. These included six cases MSI+, known to present a hypermutable phenotype caused by the loss of DNA mismatch repair activity, and 10 MSI- samples, which typically have a lower rate of somatic mutation. DNA was isolated from 10 uM slide cuts using the MagMAX FFPE DNA/RNA Ultra Kit. A multiplex of eight samples per 540 sequencing chip was sequenced using an Ion S5 XL system. In our data set, we observed an average of 67.2 mutations per Mb for the MSI+ samples and an average of 29.7 mutations per Mb for the MSI- samples (p=0.004). Using a cut-off of 40.0 mutations per Mb, we were able to classify correctly every sample as MSI+ or MSI-. Interestingly, we observed a tri-modal distribution of the samples, suggesting there might be a sub-group of MSI+ CRC cases that show a level of mutation load comparable to that of MSI- CRC cases. It remains to be seen whether these MSI+, low mutation load CRC cases are non-responders to treatment with immunotherapy. A validation cohort of CRC cases is currently being analyzed to validate our findings. In this study, we demonstrate it is viable to use a target and routine-amenable NGS-based sequencing gene panel to identify CRC samples with different levels of mutation load. It remains to be seen whether mutation load is a better predictor of response to immunotherapy than MSI status in CRC. This approach also needs to be validated in different tumor types.

#1713

Immunomodulatory effect of statins on Tregs within human colorectal cancer is determined by the stage of disease.

Belal Al-Husein. _Jordan University of Science and Technology, Irbid, Jordan_.

Colorectal cancer (CRC) is a public health problem worldwide and in Jordan. Statins are best seller cholesterol lowering agents. Beyond their cholesterol-lowering effects, statins use has been reported to reduce the risk of several malignances including CRC. This study aimed to assess the effect of statins on CRC risk by studying cellular infiltration of Tregs into CRC tissues and their effect on Transforming growth factor beta 1 (TGF-β1) level and angiogenesis. A total 47 specimens (25 statins users, and 22 statins nonusers) of CRC were used. Immunohistochemical analysis was performed to study Tregs infiltration by studying their specific marker fork head transcription factor (FOXP3), and angiogenesis by studying CD31 positive blood vessels. TGF-β level was measured using commercial ELISA kits. Results revealed that in case of statins use was associated with more Tregs infiltration, more angiogenesis but no difference in TGF-β1 content in tumor tissue. When further categorized analysis according to stage of disease, angiogenesis effect was more pronounced in early disease and wasn't significant in advanced disease.In addition, more Tregs infiltration was significantly noticed in advanced disease but not in early disease. Same stage-dependence wasn't notices with TGF-β1. Since statins are associated with reduced risk of death in colorectal cancer patients, we could suggest some immunologic mechanism for that. Tregs infiltration into tumor and reduction of inflammation at tumor site might reduce cancer aggressiveness in advanced disease. In advanced disease, however, reduction of angiogenesis would be the cause of tumor aggressiveness. These results suggest that statins might be used in prophylaxis or treatment of CRC. Further studies are needed to evaluate the effect of various types and doses of statins.

#1714

A multifactorial model of T-cell expansion and durable clinical benefit in response to a PD-L1 inhibitor.

Mark D. Leiserson,1 Vasilis Syrgkanis,2 Amy I. Gilson,3 Miroslav Dudik,4 Samuel A. Funt,5 Alexandra Snyder,6 Lester Mackey2. 1 _University of Maryland, College Park, MD;_ 2 _Microsoft Research, Cambridge, MA;_ 3 _California Council on Science and Technology, Sacramento, CA;_ 4 _Microsoft Research, New York, NY;_ 5 _Memorial Sloan Kettering Cancer Center, New York, NY;_ 6 _Adaptive Biotechnologies, Seattle, WA_.

There is an urgent need to improve prediction of patient response to checkpoint inhibitor immunotherapy. Checkpoint inhibitors have achieved unprecedented successes in treating late-stage cancers, but response rates vary across patients, and known biomarkers for response such as high mutation load are not predictive for every patient. Predicting response to checkpoint inhibitors is a critical priority for avoiding adverse responses, identifying additional patients who may benefit, and accelerating the development of new treatments. A key challenge for predicting response is modeling features of the immune system and cancer simultaneously. Recently, clinicians have begun to collect a wealth of molecular tumor and immune system data before and during immunotherapy, but researchers have yet to model how molecular and clinical features interact to affect response. To address this challenge, we developed multifactorial models for response to checkpoint inhibitors. Our approach is based off of the elastic net--a machine learning method for regression that automatically selects informative features from the data--and models clinical, tumor, and immune system features simultaneously. We applied our model to data from Snyder et al. (PLoS Medicine, 2017), who measured mutations and gene expression in the tumor and T-cell receptor (TCR) sequences in the tumor and blood in 29 urothelial cancers treated with anti-PD-L1. Rather than model the clinical response of each patient directly, we modeled the response of each patient's immune system and used the predicted immune responses to stratify patients based on expected clinical benefit. By modeling the immune response, we have the advantage of predicting fine-grained, molecular measurements that are predictive of clinical response. Our models predict immune response with high accuracy, and that predicted immune response is associated with durable clinical benefit (DCB). In held-out patients, our model explains 79% of the variance of the log number of T-cell clones in the tumor that expand in the blood post-therapy. The magnitude of the predicted expansion is associated with DCB, as 100% of held-out patients with DCB have predicted scores above the median score of held-out patients without DCB. Notably, mutation load alone did not demonstrate significant association with DCB. We also evaluated the importance of the tumor, immune system, and clinical features to our model. Our model requires all three feature classes to make accurate predictions and can explain at most 23% of the held-out patient variance when at least one of the classes is removed. Taken together, these results show that models for immune response may be useful for predicting clinical response to immunotherapy and that noninvasive TCR sequencing in the blood may be an effective way to monitor patient response.

#1715

**Treatment-dependent effects of androgen receptor signaling suppression on immune modulation in mouse** Pten **-deficient prostate cancer.**

Marco A. De Velasco,1 Yurie Kura,1 Naomi Ando,1 Kazuko Sakai,1 Barry R. Davies,2 Youngsoo Kim,3 A. Robert MacLeod,3 Masahiro Nozawa,1 Kazuhiro Yoshimura,1 Kazuhiro Yoshikawa,4 Kazuto Nishio,1 Hirotsugu Uemura1. 1 _Kindai University Faculty of Medicine, Osaka-Sayama, Japan;_ 2 _Strategy, Oncology, IMED Biotech Unit, AstraZeneca, United Kingdom;_ 3 _Ionis Pharmaceuticals, CA;_ 4 _Aichi Medical University, Japan_.

Androgen-deprivation therapy (ADT) is the mainstay treatment for advanced metastatic prostate cancer. Treatments targeting androgen receptor (AR) signaling by androgen withdrawal or AR antagonists have been implemented in clinical practice, and clinical trials evaluating treatment combinations with immunotherapy are ongoing. However, AR expression is not limited to prostate epithelial cells and is expressed in several cell types including stromal and immune cells. Preclinical studies have shown that ADT can positively or negatively influence antitumor immunity depending on the treatment approach. To gain insights into the influence of ADT on tumor immunity, we compared surgical castration (Cast), AR-antisense oligonucleotide (ISIS581088, ISI), nonsteroidal antiandrogen (apalutamide, Apa) and hormonal antiandrogen (chlormadinone acetate, CMA) treatments in a mouse model of Pten-deficient prostate cancer. Conditional PSACre/Ptenf/f mice were treated for eight weeks and the antitumor activity and effects on immune organs were assessed. Compared to control mice, treatments with Cast, ISI, Apa and CMA significantly reduced tumor growth by 69.8%, 65.0%, 37.8%, and 40.8%, respectively, P<0.001. A significant enlargement of the thymus was seen in Cast (125 %) and Apa (45.8%)-treated mice whereas significant involution occurred after ISI (-73.3%) and CMA (-60.0%) treatments. Enlargement of spleen was noted in Cast (201.2%, P=0.009) ISI (96.3%, P=0.004) and Apa (55%, P=0.256)-treated mice. Draining lymph nodes were significantly larger in mice treated with ISI (48%, P=0.027), whereas their size decreased after treatment with Apa (-31.6%) and CMA (-15.4%). Focused gene expression profiling of immune and AR responsive genes by qRT-PCR array was performed on prostate tumor samples. Functional enrichment analysis of differentially expressed genes indicated distinct patterns of expression between treatments. Overall, the expression signatures of Cast and ISI were more closely concordant than those of Apa and CMA. Notably, genes associated with abnormal immune tolerance were enriched in Cast, Apa and CMA treatment groups. Genes associated with abnormal T-cell proliferation and PD-1 signaling were enriched in castrated mice. Our study shows that the AR signaling axis influences immune modulation and its effects on tumor immunity vary greatly depending on the pharmacologic approach for AR signaling inhibition. Rational combinations of ADT and immunotherapy will have to be carefully characterized and optimized in order to achieve their full therapeutic potential.

#1716

**The PARP inhibitor rucaparib activates the STING pathway and enhances antitumor responses of immune checkpoint inhibitors in** BRCA **deficient syngeneic models.**

Minh Nguyen, Liliane Robillard, Kevin K. Lin, Thomas C. Harding, Andrew D. Simmons. _Clovis Oncology, San Francisco, CA_.

Background: The poly(ADP ribose) polymerase (PARP) inhibitor rucaparib effectively kills homologous recombination (HR) deficient cells through impeding DNA repair that leads to DNA damage, apoptosis, and cell death. Detection of cytosolic DNA by the stimulator of interferon genes (STING) pathway mediates type I interferon (IFN) production and activates the immune system. Following rucaparib treatment, the accumulation of damaged DNA in HR impaired tumors may elicit an immune response through STING signaling, and enhance rucaparib activity as a single agent or in combination with immune checkpoint blockade. To test this hypothesis, rucaparib efficacy and mechanism of action were evaluated using BRCA deficient syngeneic ovarian tumor models.

Results: Single agent rucaparib showed potent antitumor activity in the BRCAmut BrKras and ID8B3.15 models. In the BrKras model, rucaparib treatment resulted in complete regression and prevented tumor formation upon re-challenge. However, this efficacy was abolished with anti-CD8 but not anti-CD4 depletion. CD8 tumor infiltrating lymphocytes (TILs) but not CD4 TILs increased with rucaparib exposure, and expression profiling of rucaparib treated tumors showed activation of the type I IFN pathway. In vitro PCR assays identified several targets that were upregulated upon rucaparib treatment including Ccl5 and Cxcl10. Notably, increased Ccl5 and Cxcl10 levels were observed in BRCAmut cells but not in BRCAwt cells, and knockdown of the STING pathway genes MB21D1, IRF3, TBK1, and STING (TMEM173) abrogated rucaparib induction of Ccl5 and Cxcl10. Furthermore, rucaparib showed transcriptional activation of IFN type I signaling in BRCAmut reporter cells expressing the IFN stimulated response element consensus sequence driving luciferase expression, but required 6-fold higher concentrations in BRCAwt reporter cells. Similarly, a 10-fold higher rucaparib dose was needed to inhibit proliferation of BRCAwt cells compared to BRCAmut cells in a cell viability assay. Consistent with the in vitro results, rucaparib combined with anti-programmed death 1 or anti-programmed death ligand 1 therapy improved the survival and augmented antitumor responses in BRCA deficient syngeneic tumor models.

Conclusions: Rucaparib treatment in HR deficient cells, and at a higher concentration in HR proficient cells, triggers type I IFN signaling through the STING pathway, which participates in the single agent efficacy of rucaparib and enhances the combination of rucaparib and checkpoint inhibitors in syngeneic models. These findings provide further evidence supporting the rationale for combining rucaparib with checkpoint therapy for the treatment of patients with HR defective cancers.

#1718

Tumor immune gene profile before and after various targeted therapies in NSCLC PDXs.

Elodie Montaudon,1 Sophie Vacher,2 Wilfrid Richer,3 Ahmed Dahmani,1 Caroline Guibaudet,1 Olivier Déas,4 Stefano Cairo,4 Ludmilla De Plater,1 Marine Dubois,3 Fariba Némati,1 Christine Sedlik,3 Jean-Gabriel Judde,4 Nicolas Girard,5 Ivan Bieche,2 Eliane Piaggio,3 Didier Decaudin1. 1 _Institut Curie, Laboratory of Preclinical Investigation (LIP), Paris, France;_ 2 _Institut Curie, Genetics Department, Paris, France;_ 3 _Institut Curie, PSL Research University, INSERM U932, Paris, France;_ 4 _XenTech, Evry, France;_ 5 _Institut Curie, Department of Medical Oncology, Paris, France_.

Introduction: The blockade of immune checkpoints with antibodies directed against CTLA-4, PD-1 and PD-L1 molecules, is now entering in the current management of non-small cell lung cancer (NSCLC) patients; those tumors are heterogeneous regarding driver oncogenes, which predict the efficacy of targeted therapies (TT). In this context, our study aimed to define the relationship between conventional TT used in NSCLC and the dynamic expression of immune genes.

Experimental procedure: Eight different NSCLC Patient-Derived Xenografts (PDXs) with defined mutations, were grafted in immunodeficient mice which were then treated by various TT (Table). About 3 tumors per group were collected at the end of the in vivo experiments. The expression of human and mouse genes, using species-specific primers was determined by qPCR and normalized to the expression of the human or mouse Total Binding Protein gene. Basal gene expression, predictive markers of response and resistance to tested therapies, as well as pharmacodynamics (PD) markers were analyzed.

Results: Basal immune gene expression analysis revealed various immune profiles in this panel of NSCLC PDXs, with clustering of both models and genes. Predictive and pharmacodynamics markers underlined the impact of human HLA class I and II gene expression in the response to erlotinib and everolimus (HLA-A, HLA-DRA, and HLA-DRB); of B7-H3 (CD276), PDL1 (CD274) and Tigit (PVRIG) in the response to erlotinib; and of TNFSF7 (CD70) and TNFSF9 in the response to BKM120. PD markers showed that PDL1, B7-H3, and Tigit expression was highly impacted by everolimus, erlotinib, and BKM120 treatments, respectively. Complete gene expression results can be presented at the meeting.

Conclusions: Using NSCLC PDXs, we have identified specific tumor immune profiles and a set of genes involved in the response to TT. Moreover, PD analysis of immune markers reveals potential combinations of TT with immune checkpoints blockade that may have important translational value. | |  | |  | |  | |

|

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

NSCLC PDXs | LCF4 | LCF9 | LCF12 | LCF15 | LCF25 | LCF29 | ML1 | ML5

Histology | Adenocarcinoma | X | X | X | X | X | X | |

X

|

Large cells | |  | |  | |  | X

|

MUTATIONS | EGFR | |  | X | |  | X | |

|

KRAS | |  | |

X | X | |

|

|

Pi3KCA | |  | X | |  | |

X | X

TREATMENTS (Targets)

Cetuximab (1) | EGFR | X | X | X | |  | X | |

Erlotinib (2) | EGFR | X | X | X | |  | X | |

Afatinib (3) | EGFR/HER2 | X | X | X | |  | X | |

BKM120 (4) | Pi3K | X | X | X | X | X | X | X | X

Everolimus (5) | mTORC1 | |  | |

X | X | |

X | X

Selumetinib (6) | MEK1/2 | X | X | X | X | X | X | X | X

1 + 2 | / | X | X | X | |  | X | |

1 + 3 | / | X | X | X | |  | X | |

1 + 4 | / | |  | X | |  | |

|

2 + 4 | / | |  | X | |  | |

|

3 + 4 | / | |  | X | |  | |

|

5 + 6 | / | |  | |

X | X | |

X | X

#1719

Superior efficacy of neoadjuvant compared to adjuvant immune checkpoint blockade in non-small cell lung cancer.

Tina Cascone, Haifa Hamdi, Fahao Zhang, Alissa Poteete, Lerong Li, Courtney W. Hudgens, Leila J. Williams, Qiuyu Wu, Jayanthi Gudikote, Weiyi Peng, Patrick Hwu, Jing Wang, Michael Tetzlaff, William N. William, John V. Heymach. _MD Anderson Cancer Center, Houston, TX_.

Introduction: Blockade of immune checkpoints has improved clinical outcomes for patients with metastatic non-small cell lung cancer (NSCLC), but its role in the perioperative setting for early-stage disease is unclear. We generated preclinical models of resectable NSCLC expressing an antigen that permits quantitative assessment of the immune response and compared survival, tumor recurrence, and immune response after neoadjuvant or adjuvant immunotherapy.

Experimental Procedures: We transfected murine 344SQ NSCLC cells with an ovalbumin-expression plasmid to generate OVA+ cells that can be identified with an antibody against the peptide SIINFEKL bound to H-2Kb of MHC-I. We implanted 344SQ-OVA+ cells in the flank of syngeneic mice and then randomized mice with established tumors to either 3 doses of neoadjuvant IgG, anti-PD-1, anti-CTLA-4, anti-PD-1+anti-CTLA-4, or to observation. Primary tumors were resected in all mice 2 days after mice in the neoadjuvant group had received their last dose of therapy. Two days post-surgery, mice in the observation group were treated with 3 doses of adjuvant IgG, anti-PD-1, anti-CTLA-4, anti-PD-1+anti-CTLA-4. Mice were euthanized 4 weeks after injection or when moribund and their survival recorded and lung metastases counted. We determined the composition of CD3+CD8+ tumor-infiltrating lymphocytes (TILs) with flow cytometry using tetramers against SIINFEKL-specific T cell receptors and immunohistochemical staining of tumors.

Results: Single agent and combined immunotherapy significantly prolonged survival compared to controls in both neoadjuvant and adjuvant groups (p<0.05). Combined therapy was superior to either monotherapy in the neoadjuvant setting (combo vs. anti-PD-1 and vs. anti-CTLA-4 p<0.05), whereas there were no significant differences between combined treatment and monotherapies in the adjuvant setting. Moreover, combined therapy in the neoadjuvant setting significantly prolonged survival compared to adjuvant combined treatment (p<0.05). Neoadjuvant combined therapy produced the most profound reduction in lung metastases when compared to monotherapy (combo vs. IgG, vs. anti-PD-1 and vs. anti-CTLA-4 p<0.01), or to adjuvant combined treatment. Neoadjuvant combined therapy was associated with the greatest amounts of SIINFEKL-tetramer+ TILs in resected primary tumors and also with increased CD8+ TIL density in both resected primary tumors (combo vs. IgG and vs. anti-PD-1 p<0.05) and secondary lung metastases (combo vs. IgG, vs. anti-PD1 and vs. anti-CTLA-4 p<0.05). Functional studies investigating the immune response of tumors treated with perioperative immunotherapies are ongoing.

Conclusions: Neoadjuvant combined immune checkpoint blockade is superior to adjuvant immunotherapy at prolonging survival, reducing distal recurrence and inducing anti-tumor immunity in preclinical models of resectable NSCLC.

#1720

Analysis of multiplexed immunotherapy targets and secreted ligands using computational tumor microenvironment profiling.

Jenifer Caldara,1 Brooke Hirsch,1 Joseph Krueger,1 Chris Bunker,2 Jeff Kim,2 Annelies Laeremans,2 Mohammed Qutaish,2 Allison Harney1. 1 _Flagship Biosciences, Westminster, CO;_ 2 _Advanced Cell Diagnostics, Newark, CA_.

As the number and diversity of cellular and signaling pathway therapeutic targets in immuno-oncology increases, more therapy combinations are aimed at multiple cell types in the tumor microenvironment (TME). To develop rational combinations of therapies that will be effective in any individual patient, multivariate biomarker assays will be required to understand each patient's specific tumor and immune profile. Understanding the mechanistic effects of immunotherapy includes the tissue context of direct immunotherapy targets and downstream effector molecules such as secreted cytokines. Although the content of tumor-infiltrating immune cells and gene expression of the total tumor can be ascertained through several methods, spatial arrangements and proximity of cells and co-localization of biomarkers can only be achieved with tissue-based assays. Duplex chromogenic in situ hybridization assays using the RNAscope platform allows for the visualization of gene expression of mRNA targets directly in tissue for multiple cell types or biomarkers. Computational Tissue Analysis (cTA™) was used to quantify cellular spatial distributions including regional density maps, proximity measurements, and colocalization of biomarkers in both the tumor nests and the surrounding stroma that would be challenging by visual inspection of duplex RNAscope assays in non-small cell lung cancer (NSCLC) tissue microarrays (TMAs). Often, neither immune effector cells nor the therapeutic target is sufficient in isolation to understand blocked anti-tumor immune responses and therefore the potential efficacy of a single or combination therapeutic strategy. Multiplex RNAscope assays and cTA™ in combination allow the assessment of end points that measure spatial relationships between cell types expressing certain biomarkers. As these are performed in the context of the tissue microarchitecture within and across multiple tissue sections, they are a significant advancement over traditional tissue-based manual end points.

#1721

Diversity evenness of TCR repertoire in the tumor and periphery reflects antitumor activity and immune-related adverse events, respectively.

Akihiro Hosoi,1 Hirokazu Matsushita,1 Kazuyoshi Takeda,2 Ryuji Suzuki,3 Kazuhiro Kakimi1. 1 _The University of Tokyo Hospital, Tokyo, Japan;_ 2 _Graduate School of Medicine, Juntendo University, Tokyo, Japan;_ 3 _Repertoire Genesis Inc., Osaka, Japan_.

To develop more effective combination immunotherapies lacking immune-related adverse events, we investigated the nature of the intratumoral immune response and systemic immune response induced by immunotherapies.

B16 melanoma cells were subcutaneously inoculated into IFNγ venus mice that express venus fluorescent protein as a reporter of IFNγ production. On days 5 and 9 after tumor inoculation, mice received intraperitoneal injections of 200 μg anti-PD-1 (RMP1-14), anti-CTLA-4 (9H10), anti-4-1BB (3H3), anti-CD4 (GK1.5) mAbs or a combination of anti-PD-1 and anti-4-1BB mAb. Fourteen days after tumor inoculation, cells in the tumor, draining lymph node, spleen were analyzed by flow cytometry and CDR3 region of TCR α and β chains sequencing. We decomposed TCR diversity into richness and evenness. Diversity Evenness 50 (DE50) was calculated as the ratio of how many clonotypes amongst the most frequent were necessary to account for 50% of the total read counts divided by the total number of read counts present.

The tumor growth suppression was achieved only by anti-PD-1, anti-PD-1/4-1BB combined, or by anti-CD4 treatment, but not by anti-CTLA-4 or anti-4-1BB monotherapy in our model. IFNγ venus signals of intratumoral CD8+ T cells was higher than those of spleens, suggesting that CD8+ T cells in the tumor were activated and expanded to a greater or lesser extent by these immunotherapies. Anti-tumor effects were associated with increased CD8+ T cell effector function and TCR diversity with enrichment of certain TCR clonotypes in the tumor. In the tumor of mice where the tumor growth was well-controlled by the combination of anti-PD-1/4-1BB or anti-CD4 treatment, DE50 values were significantly lower than those of control. Activation of tissue-reactive T cells were also examined. IFNγ venus\+ T cells were increased in the spleens of all mice with the expansion of many clonotypes in mice receiving immunotherapies. Mononuclear cell infiltrations into liver, kidney, heart, lung and pancreas were observed particularly in mice that received anti-4-1BB and the anti-PD-1/4-1BB combination. DE50 values in the spleen were reduced in these mice.

In conclusion, effective immunotherapies increase TCR diversity with extended activation of selective CD8+ T-cells specifically in the tumor but not in the periphery, while polyclonal activation of T cells and emergence of dominant clones in the periphery was associated with tissue damage. We propose DE50 for the evaluation of diversity evenness of TCR repertoire that reflects anti-tumor activity and immune-related adverse events.

#1723

**CD4** + **and CD8a** \+ **PET imaging in syngeneic mouse models for prediction of immuno-therapeutic response.**

Lotte K. Kristensen,1 Camilla Fröhlich,2 Camilla Christensen,3 Maria C. Melander,2 Thomas T. Poulsen,2 Gunther R. Galler,2 Johan Lantto,2 Ivan D. Horak,2 Michael Kragh,2 Carsten H. Nielsen,1 Andreas Kjaer3. 1 _Minerva Imaging, Copenhagen, Denmark;_ 2 _Symphogen A/S, Ballerup, Denmark;_ 3 _Dept. of Clinical Physiology, Nuclear Medicine & PET and Cluster for Molecular Imaging, Dept. of Biomedical Sciences, Rigshospitalet and University of Copenhagen, Copenhagen, Denmark_.

Introduction: The clinical success of immune-checkpoint inhibitors in oncology has stimulated development of immune-based agents and revolutionized treatment for many types of cancer. With only a subpopulation of patients benefiting from immuno-therapeutic agents, the development of a predictive in vivo biomarker of response is critical to enhance the therapeutic efficacy of such agents. The level of immune infiltration in tumors seems to reflect the outcome of immuno-therapy, and treatment failure is usually attributed to the so-called "cold" tumors that do not attract T-cell infiltration. Thus, we sought to develop specific PET radiotracers for non-invasive in vivo detection, tracking and quantification of CD4\+ and CD8a+ tumor-infiltrating lymphocytes (TILs).

Experimental procedures: A panel of syngeneic mouse tumor models was analyzed for the level of TILs using flow cytometry and immunohistochemistry (IHC), and for response to Sym021, a humanized anti-PD1 antibody cross-reactive with mouse PD1. Radiotracers were generated from F(ab)'2 fragments of rat-anti-mouse CD4 and CD8a antibodies conjugated to the p-SCN-Bn-Deferoxamine (p-SCN-Bn-DFO) chelator and radiolabeled with 89Zr. Immuno-reactivity of 89Zr-DFO-CD4-F(ab)'2 and 89Zr-DFO-CD8a-F(ab)'2 was assessed with murine T-cells. Tracers were assessed in vivo in a panel of syngeneic mouse models following optimization of dose and imaging time-points.

Results: Based on flow cytometry and IHC analysis, the syngeneic tumor models were characterized as "hot" or "cold" by the level of TILs. The "hot" tumor phenotype clearly correlated with response to Sym021 treatment. 89Zr-labeled CD4- and CD8a-targeting radiotracers were successfully generated with a radiochemical purity >99% and an immuno-reactivity >85%. The optimal imaging parameters were 24 hours post-injection of 1 MBq of tracer. Co-injection with 30 μg unlabeled CD4/CD8a-F(ab)'2 significantly decreased spleen and lymph node uptake, whereas tumor uptake was increased. PET imaging in syngeneic mouse models revealed differential uptake of 89Zr-DFO-CD4-F(ab)'2 and 89Zr-DFO-CD8a-F(ab)'2, with an increased uptake primarily seen in "hot" tumors. In addition, an increase in CD8a+ TILs after fractionated external radiation therapy (XRT) was successfully detected by 89Zr-DFO-CD8a-F(ab)'2 PET imaging.

Conclusions: We have developed 89Zr-DFO-CD4-F(ab)'2 and 89Zr-DFO-CD8a-F(ab)'2 PET imaging radiotracers for whole-body detection and assessment of CD4\+ and CD8a\+ status. These radiotracers can be used as tools to predict efficacy of immuno-therapeutic treatment in preclinical drug development. In addition, they may be used to develop non-invasive in vivo biomarkers for identifying patients responding to immuno-therapeutic agents, such as Sym021.

#1724

Evaluation of niraparib in combination with anti-PD1/anti-PD-L1 in preclinical models.

Sarah Wang, Kaiming Sun, Yonghong Xiao, Bin Feng, Keith Mikule, Sridhar Ramaswamy, Jeffrey Hanke, Jing Wang. _TESARO Inc., Waltham, MA_.

Niraparib is an orally available and selective poly(ADP-ribose) polymerase (PARP)-1/-2 inhibitor approved for maintenance treatment of adult patients with recurrent epithelial ovarian, fallopian tube, or primary peritoneal cancer who are in complete or partial response to platinum-based chemotherapy. PARP inhibition may enhance the immune response in tumors treated with anti-PD-1 therapy via generation of cytosolic DNA that activates T cells through the stimulator of interferon gene (STING) pathway, rendering tumors immunologically "hot" with an increase in infiltrating lymphocytes.

In this study, we explored the responses and mechanism of action of niraparib and anti-PD-1/anti-PD-L1 combination therapy in preclinical models. Out of a cohort of 14 immune-competent mouse tumor models, the combination treatment demonstrated enhanced anti-tumor activity in eight tumor models derived from BRCA-proficient and BRCA-deficient genetic backgrounds. Substantial increases compared to monotherapies in anti-tumor activity was observed in 5 models, indicative of synergy between niraparib and anti-PD-1/anti-PD-L1 therapy. The combination triggered durable responses that were coincident with induction of immune memories in a BRCA-deficient ovarian syngeneic model. Mechanistically, niraparib treatment increased the number of infiltrating CD8+ and CD4+ cells within the intratumoral region. The enhanced immune cell infiltration was accompanied by elevated interferon-stimulated gene expression. Pathway analyses using transcriptome profiling identified interferon response gene signatures as the significantly differentially-upregulated gene sets following niraparib treatment. Consistently, niraparib treatment activated the STING pathway in vitro in BRCA-deficient MDA-MB-436 human triple negative breast cancer cells. STING pathway markers including p-STING(Ser366), p-TBK1(Ser172) and p-NFκB p65 were elevated following niraparib treatment and was accompanied by an increase in IFNB mRNA expression. In summary, our data suggested that niraparib treatment in combination with anti-PD-1/anti-PD-L1 therapy enhanced immune cell infiltration, interferon-stimulated gene expression and tumor responses.

#1725

A bi-functional mechanism of action: Activating the NLRP3 inflammasome and triggering apoptosis in cancer via a HK2-VDAC modulator.

Vered Behar, Reut Yosef Hamo, Eyal Dor-On, Oren M. Becker. _Vidac Pharma, Jerusalem, Israel_.

Introduction: Cancer cells undergo metabolic reprogramming to enable the efficient conversion of glucose needed for massive cell growth and proliferation, a well-documented phenomenon known as the Warburg effect. A key enzyme in this process is hexokinase 2 (HK2), which catalyzes the first step of glucose metabolism. Unlike HK1, which is ubiquitously expressed in normal cells, high levels of HK2 are found in cancer where it is required for cancer initiation and transformation. HK2 in cancer cells is attached to the outer mitochondrial membrane via the VDAC1 channel. VDAC1/HK2 association blocks pro-apoptotic signals as well as allows a continuous flux of mitochondrial ATP to HK, leading to apoptosis prevention and a high rate of glycolysis.

Temporal high HK2 expression, and binding to VDAC, is also found in a variety of activated immune cells to support their changing metabolic needs. It has been recently published that detachment of HK2 from VDAC is one of the first events leading to the NLRP3-inflammasome activation, resulting in IL-1β and IL-18 secretion from macrophages (Cell 166:624 (2016)).

Methods: A novel small molecule VDAC/HK2 modulator, VDA-1102, is being developed as a bi-functional drug for the treatment of solid tumors – triggering apoptosis in cancer cells while simultaneously activating the NLRP3-inflammasome in macrophages to induce an anti-tumoral immune response.

Results: In vitro studies established that VDA-1102 selectively detaches HK2, but not HK1, from VDAC leading to cancer cell apoptosis, glycolysis inhibition, and prevention of cancer cell proliferation. VDA-1102 treatment of mouse primary bone marrow-derived macrophages and of human macrophage cell line, THP-1 cells, established a dose-dependent NLRP3-inflammasome activation and cytokine secretion. In vivo efficacy studies demonstrated significant tumor growth delay in syngeneic solid tumor models.

Analysis of tumor-associated macrophages indicated a treatment-induced change in these macrophage phenotype from M2 to M1. This change was associated with a notable increase in spleen size, an increase ratio of naïve T cell in the spleen, and a significant increase in CD8+ and CD4+ tumor-infiltrating T cells.

Conclusions: This data supports the notion that VDA-1102 is a bi-functional drug that targets both cancer and the innate immune system. In cancer cells it induces apoptosis, whereas in macrophages it activates the NLRP3-inflammasome machinery and stimulates an anti-tumor immune response. Our findings support further development of VDA-1102 to evaluate its potential as an anti-cancer therapy, either as a monotherapy or in combination with checkpoint inhibitors in high HK2-expressing solid tumors.

#1726

A novel delivery platform containing up to 14 neoantigens can induce robust immune responses in a single formulation.

Valarmathy Kaliaperumal, Genevieve Weir, Rajkannan Rajagopalan, Arthvan Sharma, Heather Torrey, Alecia MacKay, Ava Vila-Leahey, Cynthia Tram, Andrea Penwell, Leeladhar Sammatur, Marianne Stanford. _Immunovacine, Halifax, Nova Scotia, Canada_.

Neoantigens are emerging targets for personalized cancer vaccines that provide patient specific cancer immunotherapies. Many algorithms have been developed to select the most immunogenic neoantigens to include, however not all neoantigens chosen will generate equivalent immune responses, nor may they induce effective anti-tumour activity. To maximize immunological activity, selected peptides should be delivered simultaneously in a formulation that can stimulate potent, sustained immune responses to many different peptides. The DepoVaxTM platform is an oil-based system that uses lipids to incorporate many different types of antigens and adjuvants into a single formulation. Using a set of neoantigens identified from murine B16-F10 melanoma, we optimized a DepoVax formulation method that allows us to incorporate up to 14 neoantigens with a polynucleotide based adjuvant in a single formulation. These selected neoantigens irrespective of their solubility and hydrophobicity were formulated in DepoVax with the contents completely soluble in oil.

C57BL/6 mice were vaccinated with 14 synthetic long peptide neoantigens (each 27 amino acids in length) prepared in DepoVax or in an aqueous formulation containing poly ICLC adjuvant. The immune responses were assessed eight days later by IFN-γ ELISPOT using splenocytes. Several of the peptides generated strong immune responses that were significantly higher in mice vaccinated with the DepoVax formulation compared to the aqueous formulation. To assess the contribution of CD8+ and CD4+ T cell responses, splenocytes from vaccinated mice were stimulated with an immunogenic peptide and intracellular IFN-γ/TNF-α producing CD8+ or CD4\+ T cells were detected by flow cytometry. The highest production of TNF-α was detected by CD8\+ T cells. Biological activity of the vaccines was assessed after one month storage at -20, 5 and 25 °C by IFN-γ ELISPOT assay; no significant difference was detected compared to the initial results. Analytical characterization of 14 peptides in DepoVax carried out using high-performance liquid chromatography (RP-HPLC), detected no significant chemical modifications or degradation of peptides after storage at -20 °C for up to 3 months compared to the initial results.

These results demonstrate that the DepoVax platform can incorporate at least 14 neoantigens in a single formulation. Neoantigens formulated in DepoVax are stable for at least 3 months and our manufacturing method can incorporate peptides with a wide range of physical and chemical characteristics. This formulation generates strong CD8+ T cell responses, in excess of those induced by an aqueous formulation. The DepoVax platform is a promising solution to inducing robust immune responses to multiple neoantigens in a single formulation.

#1727

Antitumor activity associated with dual targeting of CD38 and programmed death-1 (PD-1) pathways in preclinical models.

Natalie A. Bezman,1 Michelle Kinder,2 Amy D. Jhatakia,1 Bethany K. Mattson,2 Darlene Pizutti,2 Edward W. Thompson,2 Dorie A. Capaldi,2 Mark W. Mendonca,2 Aravind Anandam,3 Gopal Dhar,3 Lavanya Kovvuri,3 Archana Devi,3 Swagatam Ray,3 Vivek Surse,3 Elizabeth Saravia,1 Robert F. Graziano,1 Natalie A. Hutnick,2 Alan Korman1. 1 _Bristol-Myers Squibb, Princeton, NJ;_ 2 _Janssen Research and Development, Spring House, PA;_ 3 _Biocon Bristol-Myers Squibb R &D Center, Syngene International, Bangalore, India_.

Background: Blockade of the programmed death-1 (PD-1) pathway has shown efficacy across a range of tumor types. However, some tumors do not respond and a combination strategy with other treatments may be required. CD38, a leukocyte receptor and ectoenzyme, was recently shown to be upregulated following PD-1 pathway blockade in mouse tumor models (Chen et al. ASCO-SITC 2017). Daratumumab is a fully human monoclonal antibody (mAb) that binds to CD38. It has been shown to exhibit immunomodulatory properties in patients with multiple myeloma (Krejcik et al. Blood. 2016). Studies in syngeneic mouse models and in vitro assays were conducted to investigate the potential of targeting CD38 in combination with anti-PD-1 to increase antitumor response.

Methods: mAbs targeting mouse CD38 (mCD38-mg2a; mCD38) and mouse PD-1 (mPD1-mIgG1-D265A; mPD-1) were tested alone or in combination in several syngeneic models: colon adenocarcinoma (MC38), plasmacytoma (J558), and lung carcinoma (M109). The effect of these mAbs on the phenotype and functionality of tumor-infiltrating and circulating lymphocytes was evaluated using flow cytometry and immunohistochemistry. In addition, the effect of daratumumab on human regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs) was assessed.

Results: Combined anti-mCD38/anti-mPD-1 mAb treatment enhanced tumor regression and extended survival compared with single-mAb treatment in mouse tumor models. In the MC38 model (n=10/group), 55% of mice were tumor free (TF) following combined treatment, whereas 0% and 25% were TF following individual administration of anti-mCD38 or anti-mPD-1 mAbs, respectively. Similar effects were observed in J558- and M109-bearing mice (n=10/group for both models): with combined treatment, 85% of J558 and 20% of M109 mice were TF, compared with 50% (J558) and 0% (M109) for anti-mCD38 and 25% (J558) and 0% (M109) for anti-mPD-1 mAbs, respectively. Enhanced antitumor activity with combination treatment was associated with increased infiltration and activation of intratumoral CD4+ and CD8+ T cells, as well as increased levels of Ki67 on circulating T cells in MC38 and J558 models. The antitumor activity of anti-mCD38 mAb was dependent on active Fc function, suggesting that CD38+ cells may limit antitumor response. Consistent with this hypothesis, anti-mCD38 mAb led to a reduction in the frequency of CD38+ immunosuppressive Tregs and MDSC populations in mice. In vitro, daratumumab mediated antibody-dependent cellular cytotoxicity of human Tregs and led to the depletion of human monocytic MDSCs in blood of patients with lung cancer (n=17).

Conclusion: These results demonstrate the combined effect of targeting both CD38 and PD-1 pathways in regulating antitumor immunity, and suggest that dual targeting of CD38 with daratumumab and PD-1 with nivolumab may represent a promising combination strategy for treating cancer.

#1728

**Nonalcoholic steatohepatitis (NASH) impairs treatment of intrahepatic metastases with CD4** + **T cell dependent RNA vaccine.**

Bernd Heinrich,1 Zachary J. Brown,1 Vormehr Mathias,2 Qiong Fu,1 Chi Ma,1 Su Jong Yu,1 Ugur Sahin,2 Tim F. Greten1. 1 _National Institutes of Health (NIH), Bethesda, MD;_ 2 _Biopharmaceutical New Technologies (BioNTech) Corporation, Mainz, Germany_.

Nonalcoholic fatty liver disease (NAFLD) is the most common liver disorder in Western industrialized countries. NAFLD along with its advanced form non-alcoholic steatohepatitis (NASH) are becoming an increasing risk factor for the development of cirrhosis and subsequently for hepatocellular carcinoma (HCC). We showed a selective CD4+ T cell loss in mice fed with methionine-choline deficient (MCD) diet, a model to induce NASH (Ma et al. Nature 2016). This selective loss of CD4 T cells may not only lead to immune escape by tumor cells but also lessen the effectiveness of immunotherapy. Kreiter et al. created a RNA vaccine from B16 melanoma cells encoding the neo-epitope M30, which is presented by MHC class II and recognized by CD4\+ T cells. The vaccine showed therapeutic immune response in mouse models of subcutaneous B16-tumors and lung metastases (Kreiter et al. Nature 2015). Here we present an in vivo study of M30 RNA vaccine in a mouse model of B16 melanoma liver metastasis induced by intrahepatic injection. Mice were separated into groups and fed regular chow or MCD diet. B16F10-GFP-Luc melanoma cells were intrahepatically injected and mice were treated with M30 RNA or irrelevant RNA as control via tail vein injection. Intrahepatic tumor growth was monitored by bioluminescence imaging over the course of treatment. Mice were sacrificed and immunomonitoring of tumor surrounding liver tissue and tumor infiltrating lymphocytes (TIL) was performed. Mice treated with M30 RNA vaccine showed reduced tumor growth of intrahepatic metastasis compared to irrelevant RNA. Mice fed MCD diet could not control tumor growth either receiving M30 RNA vaccine treatment or irrelevant RNA. This impaired function of the treatment was related to a loss of CD4+ T cells in the NASH liver and a more immunosuppressive immune cell phenotype in the tumor environment.

This study indicates impaired efficacy of cancer immunotherapy due to fatty liver disease and subsequently changed immune response due to loss of CD4+ T cells as the main effector immune cell. As the M30 RNA vaccine reduced tumor growth in non-fatty liver, this data might be considered when planning future immunotherapy treatment strategies for comorbid patients with fatty liver disease as CD4+ T cell based treatment strategies might be impaired in patients with NASH.

#1729

Integrative immune profiling of syngeneic tumor models provides predictive immune signatures for treatment response with HMBD002, a novel anti-VISTA neutralizing antibody.

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

Myeloid-derived suppressor cells (MDSCs) are a group of immature myeloid cells with potent immune suppressive activity. Accumulation of pathologically activated MDSCs within the tumor microenvironment has emerged as an important mechanism of primary and acquired resistance to immunotherapy. MDSCs have high surface expression levels of V-domain immunoglobulin (Ig)-containing suppressor of T-cell activation (VISTA), a co-inhibitory negative checkpoint regulator (NCR). Immune suppression by VISTA+ MDSCs has been implicated in the failure of, and resistance to, anti-PD1/PDL1 therapy and represents a unique axis for NCR targeting in the non-responder population. Elucidating the dynamics of VISTA+ MDSCs within tumors is imperative for predicting response and patient stratification.

HMBD-002 is a novel anti-VISTA neutralizing monoclonal antibody, developed using Hummingbird Bioscience's proprietary Rational Antibody Discovery platform, against a specific functional epitope on VISTA predicted to be involved in ligand binding. HMBD002 binds human, cyno and murine VISTA protein with high affinity (kD <5nM) and demonstrated a strong, dose dependent inhibition for the interaction of VISTA with its putative ligand VSIG3. In ex vivo PBMC models of immune activation, HMBD002 treatment showed potent induction of cytokine release. Further, HMBD002 monotherapy in a CT26 syngeneic BALB/C model showed significant delay on tumor progression with no observable toxicity.

To investigate predictive immune signatures for HMBD002 antibody efficacy, as a monotherapy and in combination with anti- PD-L1 therapy, we established multiple mouse models of tumor immune subtypes using syngeneic cell lines. Tumor biopsies were analyzed before and during treatment, to determine the evolution of immune response and its effect on tumor burden within responders and non-responders. The combination of HMBD-002 and anti-PD-L1 was more effective than either antibody alone, especially in tumors that showed abundant MDSC infiltration. Immune phenotyping revealed that this was associated with a significant increase in activated CD8+ T effector cells and concomitant increase in IFN-gamma.

Profiling syngeneic models for deeper insights into the interplay between the tumor, immune system and therapy, will facilitate the clinical development of immune checkpoint and combination therapies. Hummingbird Bioscience anticipates commencing First-in-Human trials of HMBD002 in 2019.

#1730

A translational platform using primary human immune cells in vitro, syngeneic and humanized models in vivo to support and advance immuno-oncology drug discovery.

Martin O'Rourke,1 Shilina Roman,1 Gary Salmon,1 Ian Waddell,1 Julia Schueler,2 Edgar Wood3. 1 _Charles River Labs, Cambridge, United Kingdom;_ 2 _Charles River Labs, Freiburg, Germany;_ 3 _Charles River Labs, Morrisville, NC_.

Charles River Laboratories (CRL) has established a powerful translational immuno-oncology platform with the capability of progressing biologics or small-molecule modulators of immune response from in vitro to in vivo assays using human and mouse variants of current checkpoint inhibitors and small molecules. Utilizing a blood donor panel, our in vitro platform includes primary human immune cell assays that profile T-cell activation, T cell mediated-cancer cell kill, expansion of T-cell populations, mixed lymphocyte reactions (MLR), T-cell invasion, and antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). The platform is currently being expanded to determine the effect of activated immune cell populations on tumor cell spheroid cultures. It has been validated with standard-of-care chemotherapeutics, including anti-CTLA4, anti-PD1 and a selection of small-molecule inhibitors of targets known to modulate immune responses including IDO inhibitors. Ex vivo analysis of activated mouse splenocyte response to checkpoint inhibitors, measured as cytokine release, and modulation of immune cell populations, as determined by flow cytometry, supports the translation of important compounds from the bench to preclinical models. Syngeneic mouse tumor models have frequently been used to profile immune responses in tumors; CRL has optimized and profiled existing checkpoint inhibitors to support immuno-oncology drug discovery using mouse and rat antibody variants of anti-CTLA4 and anti-PD1. To confirm the translational development of our platform in human tissue, CRL has developed and optimized humanized mouse models using subcutaneous implanted patient-derived xenografts (PDX) with human engraftment via CD34+ haematopoeitic stem cells in NOG mice, which were treated with anti-CTLA4 and anti-PD1. Infiltration of human immune cells and PDL-1 expression was detected by flow cytometry (FC) and immunohistochemistry (IHC) in hematopoietic organs and tumor tissue, supporting the initial in vitro response in primary immune cells. Here we present a screening platform, validated with standard-of-care chemotherapeutics, which will support translation of compounds from in vitro primary immune cell assays to modulation of mouse immune cell population in spleen and tumors, resulting in efficacy and tumor immune cell activation in humanized mouse models.

#1731

Preclinical evaluation of the anti-tumor activity of Fc-fused interleukin-7 in both monotherapy and combination therapy.

Ji-Hae Kim,1 Donghoon Choi,2 Man Kyu Ji,2 Seat-byeul Jo,1 Han Wook Park,1 Yeon Kyung Oh,2 Youngmin Kim,1 Hyekang Kim,1 Byung Ha Lee,3 Se Hwan Yang,3 Young Chul Sung,2 Seung-Woo Lee1. 1 _POSTECH, Pohang-si, Republic of Korea;_ 2 _Genexine, Republic of Korea;_ 3 _NeoImmuneTech, MD_.

A remarkable progress of cancer immunotherapy in a recent decade, including immune checkpoint blockades (ICB), has shed a new light on the medical treatment of cancer patients. These successes of immunotherapies affirm the notion that modulation of immune-related environment, although not directly targeting a tumor cell, might lead to a better efficacy for cancer treatment. Interleukine-7 (IL-7), a member of the common γ chain family cytokine, plays important roles in the development and homeostasis of lymphocytes in both mouse and human, in particular T lymphocytes. Positive effects of recombinant IL-7 on anti-tumor activity in preclinical models have placed IL-7 as a strong candidate for a novel immunotherapeutic agent in clinics; however, a short half-life of recombinant protein has remained a challenge. Here, we investigated anti-tumor effects in mice of the long-acting form of recombinant human IL-7 fused with hybrid Fc (IL-7-hyFc) in syngeneic tumor models. A dramatic inhibition of tumor growth was achieved when IL-7-hyFc is given in a single subcutaneous injection with palpable tumor burdens. IL-7-hyFc administration significantly enhanced the expression level of CXCR3 on T cells and the frequency of CD8+ tumor-infiltrating lymphocytes (TILs). Of interest, the fraction of PD-1+CD8+ TILs was decreased by IL-7-hyFc, with the cell surface level of PD-1 being diminished. Therefore, IL-7-hyFc is able to expand tumor antigen specific CD8+ effector T cells, resulting in the enhanced infiltration and functional recuperation of TILs. Nonetheless, the tumor growth inhibition by IL-7-hyFc was not observed in mice with large tumor burdens. To increase the therapeutic efficacy of IL-7-hyFc in this model, we combined single injection of the conventional chemotherapeutics cyclophosphamide (CTX) with a moderate dose in which CTX confers immunogenic tumor cell death without severely depleting immune compartment. The combinatorial treatment with IL-7-hyFc and CTX augmented the infiltration of CD8+ TILs, leading to an increased survival in a large established tumor model. In sum, IL-7-hyFc confers the effective anti-tumor responses through reconstructing CD8+ T lymphocytes; this activity was limited when the tumor burden was high but restored along with combination with the chemotherapeutics. Thus, these results imply that IL-7-hyFc can be applied to various cancer immunotherapy regimens as monotherapy or a combination partner with conventional and other immunotherapy, like ICB.

#1732

Gp96-Ig/costimulator combination platform improves T cell priming and enhances immunity, memory, and tumor elimination.

Louise Giffin, Louis Gonzalez, Jason Rose, Jeff Hutchins. _Heat Biologics, Inc., Durham, NC_.

In this study, we report that addition of checkpoint inhibition and T cell costimulators into the Heat Biologics gp96-Ig-based vaccine platform elicits stronger antigen-specific CD8+ T cell activation and expansion, stimulates memory precursor cell activation, and enhances rejection of murine tumors. Despite the dramatic successes of checkpoint inhibitors in limited populations of cancer patients, 60-90% of patients are still failing to respond to these therapies. It is widely believed that approaches such as ours that target multiple facets of the immune system, including combinations with checkpoint inhibition and costimulator agonism, will be required to improve patient outcomes. Heat Biologics' first-generation ImPACT vaccine is a cell-based therapy that utilizes representative tumor cell lines expressing the antigen chaperone, gp96-Ig, to efficiently prime antigen-specific CD8+ T cells. Heat recently developed a next generation vaccine called ComPACT (COMbination Pan-Antigen Cytotoxic Therapy) that incorporates expression of a T cell costimulator (OX40L-Ig) and gp96-Ig within a single vaccine cell line. In multiple murine tumor and tumor-naive models, ComPACT elicits more potent activation of antigen-specific T cells as well as expansion of CD127+KLRG1- memory precursor cells, leading to a detectable memory response. Furthermore, ComPACT lacks the toxic systemic inflammatory cytokine production and proliferation of non-specific CD4+ T cells and Tregs observed with systemic administration of OX40 agonist antibodies. ComPACT also leads to high frequencies of IFNγ+, TNFα+, granzyme B+ and IL-2+ antigen-specific CD8+ T cells in mice at both priming and boosting, which increases rejection of established murine melanoma (B16.F10) and colon cancer (CT26) tumors and increased overall survival. Here, we have further assessed ImPACT and ComPACT-OX40L in combination with the anti-PD1 checkpoint inhibitor and with expression of an additional cell-secreted T cell costimulator, TL1A-Ig. In various murine models we show that these treatments synergize effectively to amplify antigen-specific T cells, program a functional memory response, and eliminate tumors. The combination of ComPACT-OX40L/TL1A with αPD1 or αPD-L1 may therefore translate into an efficacious approach to treat human cancers.

#1733

Wnt antagonists synergize with immune checkpoint inhibitors to enhance anti-tumor responses.

Jenny Ross, Timothy Hoey, Ann Kapoun, John Lewicki, Austin Gurney, Christopher L. Murriel. _OncoMed Pharmaceuticals, Inc., Redwood City, CA_.

Aberrant Wnt signaling through overexpression or activating mutations in Wnt pathway proteins is responsible for the initiation and progression of numerous cancers. While enhanced Wnt signaling has been shown to play a major role in cancer stem cell biology, more recent studies have implicated Wnt in the development of resistance to anti-tumor immune responses. Here, we show that in various syngeneic murine tumor models, targeting Wnt signaling using the Fzd receptor monoclonal antagonist antibody, OMP-18R5 (vantictumab) or the pan-Wnt decoy receptor Fc fusion protein OMP-Fzd8-Fc (ipafricept) in combination with immune checkpoint inhibitors anti-CTLA-4 or anti-PD1 induce synergistic anti-tumor responses leading to decreased tumor volume and increased infiltration of activated CD8+ T cells into the tumor microenvironment. In addition, we show that combined Wnt and immune checkpoint inhibition decrease regulatory T cells (Tregs), enhance cytotoxic T cell activity and increase antigen presentation by APCs. More importantly, we demonstrate that anti-PD1 and the Wnt antagonists decrease immune suppressive myeloid cell populations, which may enhance therapeutic efficacy and anti-tumor responses. Therefore, these results suggest that co-targeting Wnt and immune checkpoint proteins may provide valuable opportunities for novel combination strategies for immunotherapeutic clinical development.

#1734

In vivo **immunomodulatory effects by uPAR-retargeted oncolytic Measles virus.**

Natasha K. Khatwani, Yuqi Jing, Valery Chavez, Jaime Merchan. _University of Miami, Miami, FL_.

Background: Oncolytic viruses offer an advantage over other treatment options, as they both induce cytotoxicity as well as immune modulation. Oncolytic measles virus (MV) is a safe and promising oncolytic viral vector that is being tested in early clinical trials for treatment of cancer. While the oncolytic activity of MV has been clearly defined, the virus' in vivo immunomodulatory effects have not been well established, as CD46 (the endogenous MV receptor) is expressed in human, but not murine cells. We have developed MV-m-uPA, a fully retargeted MV that can selectively target mouse tissues in a m-uPAR dependent manner. The goals of this study are to characterize the in vivo immunomodulatory effects of MV-m-UPA.

Methods: Immunocompetent Balb/c mice were injected subcutaneously with CT-26 tumors. When tumor growth reached 4-5 mm, mice were treated with either vehicle control or MV-m-UPA via tail vein (IV) or intratumor (IT) every other day for two doses. Tumors were resected and changes in the immune microenvironment were analyzed by flow cytometry at days 5 and 10 after treatment.

Results: IV and IT administration of MV-m-UPA induced differential, time-dependent changes of components of innate and adaptive immunity. At early time points (day 5), intravenous virus treatment was associated with increases in MDSCs and NK cells, while the opposite effects were observed after IT administration. Analysis of CD8+ T cell subpopulations showed that IV treatments led to a decrease in TIM3+/PD1+ (exhausted) CD8+ T cells, while TIM3-/PD1- CD8+ cells increased after IV administration. The opposite effect was observed after IT administration. While both CD4+ T helper and FOXP3+ CD4+ cells increased after IV treatments, no major changes occurred after IT treatments. Interestingly, while a significant decrease in TAM 2 cells was observed at days 5 and 10 in both groups, TAM 1 cells increased only in the IV group at day 10.

Conclusions: In summary, IV MV-m-uPA administration induces overall decreases in TAM 2 and MDSCs, while NK and TAM 1 cells increase, changes that may be associated with tumor control. On the other hand, FOXP3+ cells increase, which may be a mechanism of immune suppression after IV administration. IT administration is associated with a decrease in MDSCs, TAM 2, as well as a decrease in FOXP3+ CD4+ cells, while no significant changes were observed in TAM 1 cells. The above results demonstrate for the first time the dynamic changes that occur within the immune microenvironment upon administration an oncolytic measles virus in a fully immunocompetent cancer model. These findings may help better understand positive and negative changes as a result of MV infection and may lead to identification of rational combinations of viruses with selected immunotherapeutic agents.

#1735

Expression of receptor tyrosine kinases on lymphocytes in patients with renal cell carcinoma and healthy donors.

Maria Volkova,1 Dmitry Khochenkov,1 Grigory Raskin,2 Anna Olshanskaia,1 Saida Aschuba,1 Yulia Khochenkova,1 Anastasia Bondarenko,3 Ilya Tsimafeyeu4. 1 _N.N. Blokhin Russian Cancer Research Center, Moscow, Russian Federation;_ 2 _A.M. Granov Russian Scientific Centre of Radiology and Surgical Technologies, Saint-Petersburg, Russian Federation;_ 3 _I.M. Sechenov First Moscow State Medical University, Moscow, Russian Federation;_ 4 _Kidney Cancer Research Bureau, Moscow, Russian Federation_.

Background: Very little is known about receptor tyrosine kinases (RTK) expression on peripheral blood mononuclear cells (PBMC) in humans including renal cell carcinoma (RCC) patients. The primary objective of study was to evaluate expression levels of major RTKs on PBMC and tumor infiltrating lymphocytes (TIL) isolated from RCC patients. The secondary aim was to compare levels of RTK expression in RCC patients before surgery and on the 180th day after surgery (lymphocyte lifetime) and to compare with expression in healthy donors (HD). In addition, we compared RTK and PD-L1 expression in TIL.

Methods: Tumor and blood samples were obtained from 20 patients with primary RCC immediately after surgical resection. Blood samples were collected from 10 HD. Tumors were harvested into RPMI1640 medium (Gibco) and processed within 4 h. TIL isolation was performed under modified protocol [Baldan 2015]. Isolated TIL and PBMC were prepared for flow cytometry. Cells were double stained with anti-CD45 FITC-conjugated mouse antibody and with PE-conjugated mouse antibodies to VEGFR1-2, PDGFRα-β, FGFR2 (all Sony Biotech) and were analyzed on NovoCyte 2000R flow cytometer (ACEA Biosciences). Expression of RTK was evaluated with NovoExpress Software. 20 tumors from same patients were stained with PD-L1 IHC assay (clone SP142 (Ventana).

Results: PBMC/TIL express RTKs (Table). In HD PBMC express all RTKs in 2-3 times higher than PBMC of RCC patients (all P<0.05). TIL also had lower expression of RTK (all P<0.05). There was no significant recovery of RTK expression on 180th day except of VEGFR2. Level of FGFR2 was lower on TIL (P=0.03). 50% of patients had PD-L1 expression (1-11% of positive TIL). We found negative correlation of PDGFRα, β and PD-L1 expression (P=0.04).

Expression of RTK, %, mean | PBMC, HD | PBMC, RCC, before surgery | PBMC, RCC, 180 days after surgery | TIL, RCC

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

VEGFR1 | 78.1 | 28.8 | 43.4 | 28.1

VEGFR2 | 79.6 | 27.1 | 57.8 | 44.3

PDGFRα | 80.1 | 44.9 | 49.1 | 52.3

PDGFRβ | 75.5 | 62.6 | 47.4 | 52.3

FGFR2 | 72.1 | 41.4 | 35.1 | 23.2

Conclusions: PBMC and TIL had similar low RTK expression levels in RCC patients. Lymphocytes of healthy humans had significantly higher expression of RTK. PD-L1 and PDGFRα-β expression could correlate.

### Modifiers of the Tumor Microenvironment 2

#1736

The combination of CXCR4 and checkpoint receptor inhibition improves survival in an orthotopic murine glioma model.

Adela Wu,1 Pina Cardarelli,2 Miho Oyasu,2 Daniel Menezes,3 Paul Ponath,2 John Cogswell,2 Russell Maxwell,1 Andrew Luksik,1 Alice Hung,1 Eileen Kim,1 Zineb Belcaid,1 Henry Brem,1 Drew Pardoll,1 Michael Lim1. 1 _Johns Hopkins University School of Medicine, Baltimore, MD;_ 2 _Bristol-Myers Squibb, Redwood City, CA;_ 3 _Celgene, San Francisco, CA_.

Introduction: Angiogenesis plays an important role in the malignancy of glioblastoma (GBM) as well as in cancer immunotherapy. In addition, PD-1 checkpoint receptors are upregulated in GBM. We investigate the treatment effects of combination immunotherapy with checkpoint inhibitor anti-PD-1 and anti-CXCR4, an antagonist of a chemokine receptor involved in immune cell homing as well as vasculature development, in a murine glioma model.

Methods: C57Bl/6 mice were implanted with GL261-Luc+ glioma cells and randomized into 4 treatment arms: 1) control, 2) anti-PD-1 monotherapy, 3) anti-CXCR4 monotherapy, and 4) combination anti-PD-1 and anti-CXCR4 therapy. Overall survival and median survival were assessed. Brain samples from untreated mice were stained for CXCR4 expression on plasma membrane, and immunohistochemistry studies were conducted on human glioblastoma specimens as well. Immune cell activation and cell population changes were assessed through flow cytometry analysis.

Results: Both murine and human glioma specimens demonstrated robust positive expression of CXCR4 on tumor-infiltrating immune cells and endothelial cells of vasculature specific to the tumor bed. Combination therapy with anti-PD-1 and anti-CXCR4 conferred survival benefit compared to control and both monotherapy arms. Long-term survivors that had received combination therapy demonstrate immune memory and decreased populations of immunosuppressive, tumor-promoting immune cells. For instance, the monocytic myeloid-derived suppressor cell population was decreased in the brain in mice treated with combination therapy. The pattern of change in microglia was similar in the brain compartment for the combination treatment group as well.

Conclusion: Anti-CXCR4 and anti-PD-1 synergistic immunotherapy not only modulates the immune cell make-up of the glioma microenvironment but also affects vasculature within the tumor region. Dual therapy targeting both checkpoint and chemokine receptors results in improved survival rates.

#1737

Efficacy of immunotherapy is attenuated with age in triple-negative breast cancer.

Sandra S. McAllister, Jaclyn Sceneay, Tyler Laszewski, Molly DeCristo, Jessalyn Ubellacker, Kristin Wilson, Yuanbo Qin, John Hutchinson. _Harvard Medical School, Boston, MA_.

Age is associated with increasing immune dysfunction that includes significant changes to both the innate and adaptive immune responses. These age-related changes could present limitations to the application of immunotherapy in breast cancer, as over 50% of patients are over 60 years old at diagnosis. Triple-negative breast cancer (TNBC) is one of the most aggressive subtypes of breast cancer, but remains the only subtype that lacks any type of targeted therapy. Early results from clinical trials of anti-PD-1 therapy in metastatic TNBC patients have shown promise, with approximately 20% of patients demonstrating a partial or complete response. While TNBC incidence is similar in young and old patients, differences in disease progression and outcome with age suggest that treatment needs to be tailored to both age as well as breast cancer subtype. It is not known whether age impacts response to immunotherapy, particularly checkpoint blockade, in breast cancer. Using multiple preclinical models of age-dependent TNBC, we found that aged mice had significantly reduced ability to respond to either anti-PD-L1 or anti-CTLA4. Old mice showed decreased numbers of circulating CD4-positive and CD8-positive T cells, as well as NK cells. Furthermore, PD-1 expression was increased on these populations relative to young mice, suggesting increased immune cell exhaustion with age. The old mice also demonstrated increased central and effector memory T cells, but decreased naive T cells compared to young mice, consistent with reports from clinical studies of aged individuals. These findings suggest decreased ability to respond to neoantigens with age. Importantly, using high-content flow cytometric analyses and RNA-seq, we defined cellular and molecular mechanisms that correlate with response in young mice that are attenuated or lacking in old mice. Many of the differentially expressed genes that defined response to anti-CTLA4 in young mice and resistance to treatment in the old mice were also predictors of response to immunotherapy in cancer patients. Our data suggested novel combination therapies that, when combined with checkpoint blockade, improved responses and survival in old mice with TNBC. Our findings should suggest age-stratified treatments to ensure that TNBC patients see the most benefit possible and prevent overtreatment with futile and harsh chemotherapies.

#1738

PD-1/PD-L1 expression and tumor-infiltrating immune cells in triple-negative breast cancer: Characterization of preclinical primary tumor and bone metastasis models in humanized mice.

Tiina E. Kähkönen,1 Mari I. Suominen,1 Jussi M. Halleen,1 Azusa Tanaka,2 Michael Seiler,2 Teppo Haapaniemi,3 Jenni Bernoulli1. 1 _Pharmatest Services, Turku, Finland;_ 2 _Taconic Biosciences, Hudson, NY;_ 3 _BioSiteHisto Ltd., Tampere, Finland_.

Expression of programmed cell death-1 receptor (PD-1), programmed death-ligand 1 (PD-L1) and tumor-infiltrating lymphocytes (TILs) in triple-negative breast cancer (TNBC) have been shown in many studies supporting development of immunotherapies against TNBC. In order to develop novel immunotherapies especially against bone metastatic TNBC, better understanding of PD-1/PD-L1 axis, TILs, and tumor associated macrophages (TAMs) in the bone microenvironment is warranted. Aim of this study was to characterize immune cell reaction between TNBC primary tumor and bone metastasis in humanized mice to support immunotherapy drug discovery against bone metastasis.

MDA-MB-231(SA) human breast carcinoma cells were inoculated into mammary fat pad or tibia of female CIEA NOG mice engrafted with human pluripotent CD34+ cells and tumor growth was followed for 3 weeks. Immunohistochemical stainings of primary tumors and intratibial tumors were performed against human specific antigens to characterize expression of PD-1, PD-L1, Granzyme B, CD4, CD8 and CD68/CD163. TILs and TAMs were assessed by 4-scale immunoscoring system and PD-L1 expression was determined by Tumor Proportion Scoring (TPS). PD-L1 positivity of the TAM's was omitted in TPS interpretation.

Carcinoma cells of orthotopical tumors exhibited mainly low or moderate heterogeneous expression of PD-L1 (TPS score 1-49%). Moderate or high numbers of CD4 and CD8 positive TILs (scoring 2-3) and low PD-1 expression were observed. Granzyme B expression correlated with CD8 positivity. In comparison, when MDA-MB-231(SA) carcinoma cells were inoculated into bone marrow, tumors had corresponding PD-L1 expression and tumor-infiltrating CD4+ and granzyme B+ human immune cells were observed. However, in the bone microenvironment less CD8+ immune cells were observed and PD-1 expression was negative. When analyzing all tumor area, intratumoral and peritumoral variation of the marker expression and cell location of TILs and TAMs was observed especially in the bone tumors but also in the primary tumors. TILs and TAMs occurred in scattered as well as aggregate form in tumor area.

Taken together, TNBC primary tumors and bone metastases had mainly low or moderate, but clear membranous expression of PD-L1. TILs consisted of CD4+, CD8+, and granzyme B+ cells in the primary tumors, but less CD8+ cells were observed in the bone metastases. PD-1 expression was negative in bone metastases. The obtained results comparing primary tumor and bone metastasis highlights the importance of understanding the influence of tumor microenvironment. Immune system is not only strong regulator of cancer progression, but it also regulates bone turnover. Therefore, it is crucial to use predictive preclinical models when assessing novel immunotherapies.

#1739

Tumor extracellular vesicles are required for tumor-associated macrophage programming.

Daniel C. Rabe, Jiyoung Lee, Felicia Rustandy, Marsha R. Rosner. _University of Chicago, Chicago, IL_.

Triple-negative breast cancers (TNBC) are highly infiltrated by tumor-associated macrophages (TAMs) that promote tumor growth, survival, metastasis and therapeutic resistance. Although cytokines such as CCL5 have been implicated in TAM recruitment to TNBC tumors, the mechanism by which tumor cells educate TAMs is not understood. Here we show that tumor extracellular vesicles (EVs) are both necessary and sufficient for programming TAMs toward a pro-metastatic phenotype. The mechanism involves CCL5 regulation of tumor EVs, which activate TLR2 and TLR3, leading to secretion of a common set of cytokines that further stimulate tumor cell invasion and metastasis as well as alter the tumor microenvironment. Cytokine expression is significantly correlated to CCL5 expression and up-regulated in TNBC patient tumors. These results demonstrate for the first time that tumor EVs are key mediators of TAM education, phenocopy the pro-metastatic and drug resistant state of the tumors to TAMs, and illustrate the potential clinical relevance of these findings to TNBC patients.

#1740

Epigenetic reprogramming of the tumor microenvironment by entinostat increases tumor sensitivity to multivalent immunotherapy combinations with an IL-15 superagonist plus vaccine or immune checkpoint blockade.

Kristin C. Hicks,1 Karin M. Knudson,1 Frank R. Jones,2 Peter Ordentlich,3 Shahrooz Rabizadeh,4 Hing C. Wong,5 James W. Hodge,1 Jeffrey Schlom,1 Sofia R. Gameiro1. 1 _NCI-CCR, Bethesda, MD;_ 2 _Etubics Corporation, Seattle, WA;_ 3 _Syndax Pharmaceuticals, Inc., Waltham, MA;_ 4 _NantCell, LLC., Culver City, CA;_ 5 _Altor BioScience Corporation, Miramar, FL_.

The clinical promise of cancer immunotherapy relies on the immune system recognizing and eliminating tumor cells identified as non-self. However, the tumor microenvironment (TME) can greatly impede a tumor targeted immune response via increasing immunosuppressive cells or hampering T and NK cell maturation, recruitment, and function through numerous pathways, including upregulation of immune checkpoints such as PD-L1. Hence, there is an unmet clinical need to develop effective therapeutic strategies that can reprogram the TME to restore tumor immune recognition and reverse immune evasion. We recently demonstrated that Entinostat, a class I histone deacetylase (HDAC) inhibitor, reverses tumor immune escape to T cell-mediated lysis. We hypothesize that the immune-mediated tumor elimination promoted by the IL-15/IL-15Rα superagonist ALT803 in combination with PD-L1 checkpoint blockade or a therapeutic adenoviral vaccine targeting CEA (Ad-CEA) will be augmented by the epigenetic reprograming of the TME induced by Entinostat. In preclinical studies, ALT803 has been shown to exhibit potent antitumor activity in multiple murine models of cancer through the expansion of NK and CD8+ T cells with high effector function. Here, we demonstrate that Entinostat increased extracellular expression of immune-relevant proteins on murine colon and breast carcinoma cells in vitro. Additionally, the frequency of T cells with an activated phenotype were increased in non-tumor bearing mice. In the MC38-CEA murine model of colon carcinoma, the addition of Entinostat augmented the antitumor activity promoted by ALT803 plus Ad-CEA resulting in increased survival. Furthermore, in the 4T1 murine model of triple-negative breast cancer, the combination of Entinostat with ALT803 and a monoclonal antibody targeting PD-L1 significantly reduced primary tumor weight relative to ALT803 plus anti-PD-L1 therapy and resulted in a significant reduction of the number of 4T1 tumor-forming cells in the lung. The immune mechanism associated with antitumor efficacy of these multivalent therapies was examined in both the periphery and TME. Overall, these studies provide a rationale for combining Entinostat with multivalent immunotherapy combinations, including cytokines, antibodies targeting PD-L1, and therapeutic cancer vaccines.

#1741

**Cancer-associated fibroblasts regulate intratumoral CD8** + **/FoxP3** + **T cells via interleukin 6 in the tumor immune microenvironment.**

Takuya Kato,1 Kazuhiro Noma,2 Yuki Katsura,2 Hiroaki Sato,2 Satoshi Kohmoto,2 Toshiaki Ohara,2 Hiroshi Tazawa,2 Yasuhiro Shirakawa,2 Masaru Inagaki,1 Toshiyoshi Fujiwara2. 1 _National Hospital Organization Fukuyama Medical Hospital, Fukuyama, Japan;_ 2 _Okayama University, Okayama, Japan_.

Cancer-associated fibroblasts (CAFs) in the tumor microenvironment (TME) are the most abundant cell population and play a central role in tumor progression. It is currently considered that the TME may strongly affect tumor immunosuppression. We investigated whether CAFs can regulate tumor-infiltrating lymphocytes (TILs) and their role in tumor immunosuppression. 140 cases of esophageal cancer were analyzed for CAFs, and CD8+ or forkhead box protein 3 (FoxP3)+ TILs by immunohistochemistry (IHC). We performed cytokine assays in a co-culture model using murine or human fibroblasts and cancer cells. Murine-derived fibroblasts and cancer cells were also inoculated into BALB/c or BALB/c-nu/nu mice, and the tumors treated with recombinant interleukin 6 (IL-6) or anti-IL-6 antibody. In clinical samples, patients with high CD8+ TIL numbers in intra-tumoral sites had significantly longer OS than those with low numbers (HR = 0.42, 95% CI = 0.25-0.70; P = 0.001). On the other hand, the high FoxP3+ TIL group for intra-tumoral tissues had significantly a shorter OS (HR = 2.82, 95% CI = 1.66-4.78; P < 0.001). However, in peri-tumoral tissues, no significant correlations between CD8+ or FoxP3+ TIL numbers and prognosis were identified. Furthermore, CD8+ TILs and CAFs were negatively correlated in intra-tumoral tissues (P < 0.001), while FoxP3+ TILs and CAFs, were positively correlated (P < 0.001). In vivo, Co-cultured Colon26 cancer cells and NIH/3T3 fibroblasts resulted in subcutaneous tumors with accelerated growth in BALB/c mice, along with decreased CD8+ and increased FoxP3+ TILs, compared with cancer cells alone. Furthermore, the tumor progression ratio of co-cultured group to cancer cells alone group was demonstrated more strongly in BALB/c rather than BALB/c-nu/nu mice, which are immunodeficient mice. In vitro, IL-6 was secreted at high levels in both murine and human cancer cell/fibroblast co-cultures rather than cancer cell/fibroblast alone. Treatment with IL-6 significantly increased growth of Colon26 subcutaneous tumors in immune-competent BALB/c mice (P < 0.001), whereas no difference was observed in BALB/c-nu/nu mice. IHC demonstrated fewer CD8+ TILs in Colon26+IL-6 subcutaneous tumors than in untreated tumors (P < 0.001). In contrast, FoxP3+ TILs increased in IL-6-treated tumors (P < 0.001). IL-6 antibody blockade of tumors co-cultured with fibroblasts resulted not only in regression of tumor growth but also in the accumulation of CD8+ TILs in intra-tumoral tissues. In conclusion, CAFs regulate immunosuppressive TIL populations in the TME via IL-6. IL-6 blockade, or targeting CAFs, may improve pre-existing tumor immunity and enhance the efficacy of conventional immunotherapies.

#1742

Dual inhibitor of immunokinase and pan-RAF for the treatment of KRAS-mutated cancers.

Sungpyo Hong,1 Soon Kil Ahn,1 Ho-Seok Kwon,2 Yongbin Park,3 Min-Hyo Ki,3 Hee Jong Shin3. 1 _University of Incheon, Incheon, Republic of Korea;_ 2 _Samjin Pharm. Co. Ltd, Incheon, Republic of Korea;_ 3 _Samjin Pharm. Co. Ltd., Seongnam, Republic of Korea_.

Tumor-associated macrophages (TAMs) represent most of the white blood cell population in solid tumors. TAMs usually display an M2-like phenotype due to signals from the tumor microenvironment, such as IL-10, VEGFA and CSF-1/M-CSF. These anti-inflammatory and pro-tumorigenic macrophages promote tumor growth and metastasis. The presence of TAMs is usually correlated with a poor clinical outcome in cancer patients. Modulation of the immunosuppressive factors within the tumor microenvironment is a key issue in tumor immunology. TAMs subvert the anti-cancer function of tumor-infiltrating T lymphocytes. TAMs also modify the immune cell population within the tumor microenvironment to decrease anti-tumor immune cells while simultaneously increase the immunosuppressive cell population to promote tumorigenesis. As a result, targeting of TAMs may be a promising new approach to cancer treatment.

CSF-1 is the crucial growth and differentiation factor for macrophage and CSF-1R is exclusively expressed by cells of the monocyte lineage, suggesting that CSF-1R is an attractive therapeutic target to enable interference with TAMs.

In preclinical models, macrophage depletion by CSF-1R inhibitor increased the antitumor effects of VEGF-targeted therapies. Furthermore, failure of antiangiogenic therapy is caused by the pro-angiogenic Tie2-expressing monocytes infiltrating tumor tissues. These data provide the rationale for the combination of antiangiogenic drugs with macrophage targeting strategies to increase the therapeutic efficacy and to prevent drug resistance.

Therefore, we tried to find inhibitors of immunokinase using structure-based molecular modeling and identified a selective inhibitor, SJP-1601. SJP-1601 potentially inhibited CSF-1R, Tie2, and VEGFR2 in vitro kinase assay. Interestingly, SJP-1601 also inhibited B-RAF, C-RAF and B-RAF V600E in kinase panel assay. We orally administered SJP-1601 in colorectal cancer xenografts with mutant K-RAS and observed the dose-dependent reduction of tumor volume. In addition, oral administration of SJP-1601 attenuated tumor growth correlated with enriched CD8+ T cells and decreased regulatory T cells in tumor stroma in BALB/c mice bearing 4T1 tumor cells or C57BL/6 mice bearing MC38 tumor cells. These data confirmed the preclinical merit of CSF1-CSF1R signaling blockade alone or as a part of combinatorial therapies to offer synergistic immunotherapeutic effects in treatments of human cancer.

In this study, we discovered an orally active dual inhibitor of immunokinase & pan-RAF. SJP-1601 not only improved the efficacy of adoptive T cell therapy through inhibition of immunosuppressive macrophage recruitment and activation in immunocompetent mice but also potentiated the response of xenograft with mutant KRAS by preventing pan-RAF. Our findings suggest that SJP-1601 could be an excellent preclinical candidate for the treatment of cancer with mutant K-RAS.

#1743

Rationale for evaluating PEGylated recombinant human hyaluronidase PH20 (pegvorhyaluronidase alfa; PEGPH20) in patients with hyaluronan (HA)-accumulating colorectal cancer.

Renee Clift, Benjamin J. Thompson, Darin Taverna, Barbara Blouw, Jisook Lee, Curtis B. Thompson, Daniel C. Maneval. _Halozyme Therapeutics, Inc., San Diego, CA_.

Colorectal cancer is the third most common cancer, and the third most common cause of cancer-related death, among both men and women in the United States. To address this unmet clinical need, ongoing efforts have focused on classifying different subtypes of colorectal cancer, with the goal of identifying therapies with the highest likelihood of success for a given tumor subtype. One marker that has been associated with poor clinical outcome in multiple solid tumor types, including colorectal cancer, is the accumulation of the glycosaminoglycan hyaluronan (HA), which, in preclinical tumor models, increases interstitial fluid pressure, compresses tumor vasculature, and restricts the access of therapeutics. PEGylated recombinant human hyaluronidase PH20 (pegvorhyaluronidase alfa; PEGPH20), which enzymatically degrades HA, is currently being evaluated in combination with existing therapies in clinical trials for HA-accumulating pancreatic, gastric, non-small cell lung, and bile duct cancers. Here, we review the available data for PEGPH20 as published by the authors to evaluate the potential utility of PEGPH20 in treating colorectal cancer. We first studied PEGPH20 in two HA-accumulating murine colon tumor models: CT26 transduced with hyaluronan synthase-3 (HAS3) and MC38 (Charles River Laboratories). In CT26-HAS3 tumors, PEGPH20 monotherapy induced 43% tumor growth inhibition and was associated with tumor blood vessel decompression. In combination with anti-CTLA4 treatment, PEGPH20 further enhanced tumor growth inhibition (79%, p≤0.002 vs anti-CTLA4 alone and PEGPH20 alone). In MC38 tumors, combination of PEGPH20 with anti-PD-L1 treatment significantly increased infiltration of CD8+ T cells (2.8-fold, p<0.05) and NK cells (3-fold, p<0.0001) compared with anti-PD-L1 alone. We then investigated the prevalence of HA accumulation in human colorectal cancer. To do this, we histologically analyzed 138 patient tumor specimens for stromal content and presence of HA. In 38 samples (28%), ≥50% of the tumor stromal area stained positive for HA. Additionally, Phase 1 studies with single agent PEGPH20 included several subjects with colorectal cancer. Pharmacodynamic measurements before and after treatment demonstrated increased tumor perfusion as measured by median volume transfer constant (Ktrans), decreased tumor metabolic activity as measured by 18F-FDG-PET/CT in one patient, and decreased tumor HA following PEGPH20 administration. Collectively, these data warrant further investigation of the potential for PEGPH20 to benefit patients with HA-high colorectal cancer.

#1744

Reversal of advanced colitis-associated colon cancer by OMX, a novel oxygen carrier that immunosensitizes the hypoxic tumor microenvironment.

Kevin G. Leong, Yuqiong Pan, Changan Guo, Padmini Narayanan, Jonathan A. Winger, Stephen P. Cary, Natacha Le Moan, Ana Krtolica. _Omniox Inc., San Carlos, CA_.

Chronic inflammation of the colon increases cancer development risk. Ulcerative colitis, characterized by excessive inflammation initiated by innate immune cells and exacerbated by a dysregulation in adaptive immunity, can give rise to colitis-associated colon cancer (CAC). Whereas overactivity of effector T cells and loss of immunosuppressive cells are hallmarks of ulcerative colitis, the opposite is true for CAC, with CAC tumors exhibiting a lack of effector T cell infiltration and a preponderence of immunosuppressive Treg cells and myeloid-derived suppressor cells (MDSCs). Recently, hypoxia has been identified as a potential driver in the pathogenesis of ulcerative colitis, with hypoxia persisting upon progression to CAC tumor formation. We have previously demonstrated that (i) hypoxia generates an immunosuppressive tumor microenvironment that limits effector T cell infiltration and activation, (ii) OMX, a first-in-class anti-cancer therapy designed to reverse tumor hypoxia to enhance immunotherapeutic efficacy, accumulates in preclinical rodent and spontaneous canine tumors and reduces tumor hypoxia, and (iii) OMX promotes effector T cell infiltration, reduces Treg cells, and enhances checkpoint inhibitor efficacy, resulting in greater tumor control. Given that CAC tumors are hypoxic and immunosuppressed, we hypothesized that hypoxia drives CAC tumor immunosuppression, and accordingly, that reversal of hypoxia with OMX may restore immunosensitivity and elicit an anti-tumor response. Here, using a chemically induced mouse model of CAC generated by administering azoxymethane (AOM) followed by repeated cycles of dextran sulfate sodium (DSS) exposure, we show that OMX treatment exhibits anti-tumor efficacy in advanced CAC tumors. We characterized CAC tumor progression from 8 to 12 weeks post-tumor induction, and confirmed previous reports that advanced CAC tumors are indeed hypoxic, and that immunosuppressive Treg cells and MDSCs are more abundant in CAC tumors relative to adjacent normal mucosa or control non-AOM/DSS-treated colons. Moreover, we observed a negative correlation between hypoxia and CD8+ T cell infiltration into CAC tumors. OMX single agent treatment reduced both CAC tumor number and total CAC tumor burden. Of note, OMX treatment reversed colon length shortening that was characteristic of tumor-bearing mice, indicative of a restoration of colon crypt regeneration and hence normal colon biology. Investigations into the immunological mechanism(s) responsible for OMX anti-tumor efficacy are currently underway. Taken together, our data suggest that OMX, by delivering oxygen to hypoxic CAC tumor regions, may be sufficient to induce an immunological change in the CAC tumor microenvironment from an immunosuppressive to an immunopermissive state, leading to tumor responses and a restoration of normal physiology.

#1745

Inhibition of chemokine receptor 2 (CCR2) with a small molecule antagonist enhances the effectiveness of checkpoint inhibition by altering the tumor microenvironment in mouse colorectal tumors: Reducing tumor size and increasing long term survival.

James J. Campbell, Christine Janson, Linda Ertl, Chris Li, Zhenhua Miao, Antoni Krasinski, Rebecca Lui, Venkat Mali, Jeffrey McMahon, Yibin Zheng, Yu Wang, Xiaoping Zang, Vicky Chhina, Marta Vilalta, Alice Kumamoto, Ton Dang, Shirley Liu, Simon Yao, Penglie Zhang, Thomas J. Schall, Rajinder Singh. _ChemoCentryx, Inc, Mountain View, CA_.

Mouse CT26 colorectal tumors are heavily infiltrated by tumor-specific CD8 T cells, but nevertheless grow rapidly in Balb/c mice. These tumors are partially responsive to anti-PD-1 (α-PD-1) monoclonal antibody therapy, which suggests that an active suppression of the tumor-specific cytotoxic T cells exists in the untreated tumor. As chemokine receptor 2 (CCR2) is expressed by a potentially suppressive leukocyte subset within these tumors (monocytic myeloid derived suppressor cells aka M-MDSCs), we aimed to test whether CCR2 blockade could enhance the anti-tumor effects of α-PD-1.

We have found that the therapeutic effects of α-PD-1 therapy are appreciably enhanced by specific blockade of CCR2 via a small molecule antagonist. This combined α-PD-1/CCR2i approach significantly decreases overall tumor size and increases the proportion of long-term survivors, with more than 50% of the mice (up to 73%) showing complete regression of a previously established tumor. The effects of this combined therapy are dependent on the presence of CD8+ T cells, as tumors do not respond to the therapy in CD8-depleted mice. The anti-CT26 tumor response is specific: long term survivors are resistant to re-inoculation with the CT26 tumor (even without further dosing of either drug) but are not resistant to the 4T1 breast tumor. CCR2 antagonism alters the tumor microenvironment by reducing the number of M-MDSC per gram of tumor (a CCR2hi population phenotypically defined as CD11b+/Ly6G-/Ly6Chi). Reduction in tumor size is inversely proportional to the ratio of CD8 T cells to M-MDSC.

These data are consistent with a hypothesis that CCR2 antagonism enhances α-PD-1 therapy by preventing M-MDSC from accumulating within the tumor, thus reducing their suppressive effects on cytotoxic T cells.

#1746

Epigenetic modulation of the tumor microenvironment enhances immune checkpoint efficacy in a murine model of pancreatic cancer.

Brian Christmas, Blake Scott, Todd Armstrong, Nilofer Azad, Elizabeth Jaffee. _Johns Hopkins University School of Medicine, Baltimore, MD_.

This project aims to test the hypothesis that the epigenetic modulatory drug (EMD), entinostat, is capable of altering the inflammatory environment of pancreatic ductal adenocarcinoma (PDAC) and sensitizing it to checkpoint blockade inhibition. Recently the use of antibody therapy targeting immune checkpoints, such as CTLA-4 and PD-1, has become a major focus of cancer immunotherapy. In responsive patients, these therapies have resulted in long-term control of chemotherapy-resistant disease. The most compelling activity has been seen in the minority of patients with immunogenic tumors where T cell infiltration naturally occurs. These benefits are not observed in nonimmunogenic tumors, such as PDAC, with low expression of tumor-associated antigens (TAA) and a lack of intrinsic T cell infiltrate. Therapies that can alter the tumor microenvironment (TME) and allow infiltration of effector T cells, decrease of immunosuppressive cells, and stimulate TAA expression may convert nonimmunogenic tumors into cancers sensitive to checkpoint inhibitors. Recent work with EMDs has shown that they are capable of altering the immunogenicity of the TME by inducing the expression of cancer testis antigens as well as increasing tumor cell expression of MHC class II. We aim to test the hypothesis that treatment with EMDs can sensitize the inflammatory environment of PDAC to checkpoint blockade inhibition by evaluating changes of immune cells within the TME via flow cytometry, immunohistochemistry, and gene expression array. We are using a murine model of hepatic metastases of pancreatic cancer, which involves injecting syngeneic pancreatic tumor cells into the spleen followed by a hemisplenectomy, resulting in the consistent formation of hepatic metastases. We have evaluated entinostat, a histone deacetylase inhibitor, in combination with anti-PD-1 and demonstrated a significant survival benefit compared to either agent alone. Flow cytometric analysis of the TME shows that the combination of entinostat and anti-PD-1 causes a significant shift from an M-MDSC dominated to a more G-MDSC population. Additionally, the M-MDSC population expressed less arginase-1 while the G-MDSC population expressed less PD-L1, indicating inhibited immunosuppressive function. By adding anti-CTLA-4 to entinostat and anti-PD-1 combination therapy, the infiltration of granzyme-B-producing CD8+ T effector cells is significantly increased, correlating with improved survival. Current studies aim to further elucidate the functionality of the MDSC populations by isolating them to perform ex vivo immunosuppression assays as well as colorimetric arginase activity assays. Additionally, future studies aim to identify changes in gene expression profiles of isolated MDSC populations as well as general immune populations using the Nanostring platform.

#1747

**Hyaluronan (HA) accumulation restricts CD8** + **T cell numbers and skews tumor-associated macrophage (TAM) phenotype in mouse syngeneic pancreatic tumors.**

Benjamin Thompson,1 Trevor Kimbler,1 Jisook Lee,1 Chunmei Zhao,1 Kelly Chen,1 Renee Clift,1 Curtis B. Thompson,1 Sanna Rosengren,2 Daniel C. Maneval1. 1 _Halozyme Therapeutics, Inc, San Diego, CA;_ 2 _Formerly of Halozyme Therapeutics, Inc, San Diego, CA_.

The glycosaminoglycan hyaluronan (HA) is abundant in the tumor microenvironment (TME) of diverse solid tumor types, and tumor accumulation of HA is associated with poor patient outcome. We previously demonstrated, in multiple HA-rich mouse syngeneic tumor models, that enzymatic degradation of HA using PEGylated recombinant human hyaluronidase PH20 (pegvorhyaluronidase alfa; PEGPH20) enhanced tumor growth inhibition and increased tumor accumulation of T and NK cells when combined with a checkpoint inhibitor. However, the ability of HA accumulation in the TME to alter the composition of tumor-infiltrating immune cells has not been well characterized. Here, we addressed this question by performing a detailed immune phenotypic analysis of HA-poor and HA-rich tumors using a mouse syngeneic pancreatic tumor model. To achieve this, we transduced Pan02 cells with a lentivirus encoding hyaluronan synthase 3 (HAS3, Pan02-HAS3) or empty vector (EV, Pan02-EV). After peritibial implantation, Pan02-HAS3 tumors contained 13.4-fold higher levels of HA than Pan02-EV tumors, as determined by HA ELISA. Both variants grew with similar kinetics to the Pan02 parental line. Flow cytometric analysis of tumor-infiltrating immune cells in four independent and identical studies revealed an average 6.2-fold reduction in the number of CD8+ T cells in Pan02-HAS3 tumors compared with Pan02-EV. Likewise, numbers of CD4+ T cells and NK cells were reduced by an average of 4.8- and 2.9-fold, respectively, in Pan02-HAS3 tumors compared with Pan02-EV. In contrast, we did not observe consistent differences in the numbers of tumor-associated macrophages (TAMs), neutrophils/granulocytic myeloid-derived suppressor cells (G-MDSC), monocytes/monocytic (M)-MDSC, or dendritic cells (DCs) between the two tumor variants. However, within the TAM population, cells displaying an "M2-like" phenotype (CD206high MHCIIlow) were present at an average 10.5-fold higher frequency in Pan02-HAS3 tumors compared with Pan02-EV. Similarly, the percentage of DCs that were CD103+ CD11b- was reduced by 1.4-fold in Pan02-HAS3 tumors compared with Pan02-EV. Together, our data indicate that HAS3-driven HA accumulation in Pan02 mouse syngeneic tumors induces several aspects of immunosuppression: it restricts the numbers of tumor-infiltrating lymphocytes, and skews TAMs and DCs toward immunosuppressive phenotypes. These findings are consistent with our previous work, which demonstrated that combining PEGPH20 with anti-PD-L1 increased tumor-infiltrating lymphocyte numbers and reduced the frequency of CD206high MHCIIlow TAMs in Pan02-HAS3 tumors. Our results support the ongoing clinical evaluation of PEGPH20 in combination with immunotherapy in HA-rich tumor indications (NCT02563548 and NCT03193190).

#1748

Head and neck cancer-derived exosomes facilitate carcinogenesis in the murine 4NQO chemically induced oropharyngeal cancer.

Beatrice M. Razzo,1 Chang-Sook Hong,1 Kellsye P. Fabian,2 Nils Ludwig,1 Priyanka Sharma,1 Walter J. Storkus,3 Theresa L. Whiteside1. 1 _UPMC Hillman Cancer Institute, Pittsburgh, PA;_ 2 _NIH, Bethesda, MD;_ 3 _University of Pittsburgh School of Medicine, Pittsburgh, PA_.

Purpose: An increasing body of evidence suggests that tumor-derived exosomes (TEX) promote tumor formation and progression. This study aims to show that exosomes produced by the SCCVII murine tumor cell line promote progression of pre-malignant oropharyngeal lesions to malignant tumors. The 4-Nitroquinoline 1-oxide (4NQO) tumor model is chemically induced in immunocompetent mice. It recapitulates all the histological stages and molecular changes associated with of oral carcinogenesis described for human head and neck squamous cell carcinoma (HNSCC). Experimental design: Exosomes were isolated from supernatant of a murine as well as human HNSCC cell-lines by mini size-exclusion chromatography (miniSEC). Morphology, number, protein and the molecular cargo of the recovered exosomes were determined. Exosome-mediated in vitro inhibition of lymphocyte proliferation and induction of CD8+ T-cell apoptosis were demonstrated. C57BL/6 mice were treated with 4NQO supplied in drinking water (100ug/ml) for 16 weeks and all developed oral and esophageal pre-malignant lesions. Exosomes were administered via a single tail-vein or retro-orbital injection at week 18. Tumor sizes and overall tumor burden per mouse were measured with a caliper. Proliferation indexes of individual tumors were measured by immunohistochemistry. Splenocytes were harvested and were analyzed for the presence and frequency of myeloid derived suppressor cells (MDSC) and Tregs, and for in vitro responsiveness of T cells to exogenous activators. Results: SCCVII-derived exosomes were found to carry an immunosuppressive cargo, including CD39, CD73, PD-L1, FasL and COX-2. One-time 90ug injections (IV) of exosomes to groups of >8 mice induced a statistically significant increase in the number of developing tumors and in the overall tumor burden per mouse, relative to control mice injected with PBS. The average number of tumors per mouse was 6.3 versus 3.6, respectively (p < 0.005). TEX derived from human HNSCC cell lines also induced a greater though not statistically significant number of tumors in 4NQO-treated mice. The MDSC frequency was increased in the exosome-injected mice relative to controls. The Treg frequency was not altered. Conclusions: The data demonstrate that the delivery of Head and Neck Cancer-derived exosomes enhanced carcinogenesis of the 4NQO-induced tumors in mice. Augmented carcinogenesis was associated with systemic immunosuppression and increases in the frequency of MDSCs in spleens of mice treated with exosomes.

#1749

CXCR4 inhibition modulates tumor microenvironment and robustly inhibits growth of B16-OVA melanoma.

Ruchi Saxena,1 Yan Wang,2 James W. Mier1. 1 _Beth Israel Deaconess Medical Ctr., Boston, MA;_ 2 _X4 Pharmaceticals, Cambridge, MA_.

Background: The chemokine receptor CXCR4 is expressed on a range of immune cells. Additionally, CXCR4 has been found to be overexpressed in a variety of cancers and promotes cancer cell proliferation and metastasis, possibly by activating prosurvival signals that render cancer cells resistant to immune attack. Blockade of immune inhibitory pathways is emerging as an important therapeutic approach for the treatment of cancer. In our previous studies, X4-136, a CXCR4 antagonist, alone and in combination with various immune checkpoint inhibitor,s exhibited potent antitumor activity in the B16-OVA murine melanoma model. We report here results from additional in vivo studies as well as in vitro mechanistic experiments to determine the impact of CXCR4 inhibition on tumor cell gene expression and on immune phenotypes within the tumor microenvironment.

Methods: B16-OVA cells were implanted into C57BL/6 mice. Seven days post implantation, mice were treated with X4-136, anti-PD-L1, or the combination for 16 days. At sacrifice, tumors were excised and flash frozen in liquid nitrogen for Western blot analysis or treated with collagenase for the analysis of subpopulations of T-cells by FACS. Gene expression was determined by PCR and protein expression was analyzed by Western blot.

Results: X4-136 alone demonstrated more robust activity than anti-PD-L1. The antitumor activity of the anti-PD-L1 was further enhanced in combination with X4-136. Analysis of infiltrating immune cells by flow cytometry showed that X4-136 alone or in combination led to an increase of CD8+ T cells as well as CD8 and perforin double-positive T cells. A trend of decrease in both immunosuppressive populations, Treg and MDSC, was observed. This combination also led to a decrease in the expression of HIF-2α and cyclin D1 in comparison to tumors from vehicle-treated mice by Western blot analysis, suggesting an antiproliferative effect. Additional analysis of impacted genes and pathways will also be presented.

Conclusion: X4-136 alone increased tumor-infiltrating CD8+ T-cells and exhibited potent antitumor activity in the B16-OVA murine melanoma model. The enhanced antitumor activity was observed when X4-136 was added to anti-PD-L1 treatment. The activities observed were associated with the improved CD8+ T cells to Treg cells ratio and with improved tumor microenvironment.

#1750

Inducing therapeutic lymphangiogenesis for potentiating cancer immunotherapy in melanoma.

Lambert Potin, Lea Maillat, Priscilla S. Briquez, Jeffrey A. Hubbell, Melody A. Swartz. _University of Chicago, Chicago, IL_.

Tumor-associated lymphangiogenesis is well known to promote metastasis and to correlate with poor prognosis in melanoma and other cancers, yet its functional roles in antitumor immunity had remained largely unexplored. In a recent study, we investigated the role of the vascular endothelial growth factor C (VEGF-C)—the main driver of lymphangiogenesis—in regulating antitumor immune response. We showed that in the B16 F10 injectable melanoma model, VEGF-C inhibition prevented T cells from infiltrating the tumor and reduced the efficacy of immunotherapies. Here, we sought to extend this concept to exploit the immunologic benefits of VEGF-C for therapeutic applications. We engineered a VEGF-C variant with an extracellular matrix-binding domain (MB-VEGF-C) allowing retention into the microenvironment after intratumoral injection. MB-VEGF-C induced increased lymphatic vasculature in tumors without reducing tumor growth rate. In an autochthonous melanoma model with reduced immune infiltrates, MB-VEGF-C intratumoral injections promoted the infiltration of cross-presenting DCs, as well as CD8+ and CD4+ T cells. Crucially, in these tumors that are normally unresponsive to immune checkpoint blockade (ICB), we could restore the responsiveness to ICB by delivering MB-VEGF-C together with immune adjuvants. Taken together, our findings suggest that VEGF-C and tumor-associated lymphangiogenesis, albeit promoting metastasis, can also be exploited to promote a T cell-inflamed microenvironment and to subsequently potentiate immunotherapies.

#1751

MSC-1 is a first-in-class humanized monoclonal antibody that modulates the tumor microenvironment by inhibiting a novel cancer immunotherapy target, LIF.

Angus Sinclair,1 Robin Hallett,1 Patricia Giblin,1 Isabel Huber-Ruano,2 Judit Anido,2 Naimish Pandya,1 Kimberly Hoffman,1 Ada Sala,3 Monica Pascual,3 Vanessa Chigancas,2 Swetha Raman,4 Johan Fransson,1 Jean-Philippe Julien,4 Robert Wasserman,1 Jeanne Magram,1 Joan Seoane3. 1 _Northern Biologics, Inc, Toronto, Ontario, Canada;_ 2 _Northern Biologics, Inc, Spain;_ 3 _Vall d'Hebron Institute of Oncology, Spain;_ 4 _The Hospital for Sick Children, Toronto, Ontario, Canada_.

Leukemia Inhibitory Factor (LIF) is a member of the IL-6 family of cytokines and is involved in many physiological and pathological processes including the promotion of an immunosuppressive environment to support embryo implantation, down-regulation of autoimmune processes and the regulation of stem cell homeostasis and differentiation. In cancer, LIF is hypothesized to have a complex role tumor development and progression, creating an immunosuppressive tumor microenvironment as well as promoting the activity of cancer initiating cells (CICs). LIF is highly expressed in a subset of tumors across multiple tumor types, e.g. glioblastoma multiforme (GBM), non-small cell lung cancer (NSCLC), colon, ovarian, prostate, and pancreatic cancer, and correlates with poor prognosis. Given the pleiotropic role LIF is hypothesized to play in cancer, inhibition of LIF represents an exciting new therapeutic concept sitting at the intersection of two key therapeutic approaches in oncology: blockade of tumor evasion of the immune system and blockade of tumor growth via inhibition of CICs. We have identified and developed MSC-1, a first-in-class humanized IgG1 monoclonal antibody that is a potent and selective inhibitor of LIF. MSC-1 cross reacts with mouse and non-human primate LIF and inhibits LIF signaling by blocking the recruitment of gp130 to the LIF-LIFR-gp130 signaling complex. The efficacy of MSC-1 was evaluated in multiple mouse tumor models and the mechanism of action investigated. LIF inhibition with MSC-1 or shRNAs reduced tumor growth in multiple syngeneic tumor models (NSCLC, ovarian and colon), and clear target engagement was shown for MSC-1. Investigations into the mechanism of action identified that inhibition of LIF with MSC-1 reprogrammed the tumor microenvironment by decreasing immunosuppressive M2 macrophages and increasing the number of intratumoral NK cells and total/activated T cells. MSC-1 also decreased immunosuppressive M2 macrophages in an orthotopic GBM xenograft model and human GBM organotypic tumor slices in an ex vivo model. Similarly, immunosuppressive macrophage genes were decreased when monocytes were co-cultured with supernatants from a GBM cell line in which LIF expression had been knocked-down. Given the effects of MSC-1 on intratumoral immune cells, we hypothesized that MSC-1 could be effectively combined with checkpoint inhibitors and we are currently evaluating MSC-1/checkpoint inhibitor combination therapy. Taken together, these findings form the basis of a robust therapeutic hypothesis, whereby MSC-1 treatment will lead to clinical activity in multiple cancer indications. A Phase I dose-escalation and expansion study of MSC-1 is planned to initiate early 2018 in patients with advanced solid tumors that will incorporate target engagement and PD biomarkers, as well as safety and efficacy endpoints.

#1752

Transient exposure of trilaciclib, a CDK4/6 inhibitor, modulates gene expression in tumor immune infiltrates to promote a pro-inflammatory tumor microenvironment.

Anne Y. Lai, Jessica A. Sorrentino, Jay C. Strum, Patrick J. Roberts. _G1 Therapeutics, Research Triangle Park, NC_.

While immune checkpoint inhibitors (ICIs) are efficacious and lead to durable responses in patients with various cancers, only a minority of patients respond. An approach to increase the response rate of ICIs is to combine them with chemotherapy to induce immunogenic cell death, leading to an enhanced antitumor response. However, chemotherapy can cause damage to cell types of the immune system, potentially diminishing the activity of the chemotherapy/ICI combination. Therefore, a targeted approach to preserve immune system function during chemotherapy could allow optimal efficacy of chemotherapy alone and chemotherapy/ICI combinations. Trilaciclib, a short-acting intravenous CDK4/6 inhibitor, preserves hematopoietic stem and progenitor cells and enhances immune system function during chemotherapy. While combining chemotherapeutic agents such as oxaliplatin or 5-FU with an ICI against PD-1 or PD-L1 delayed tumor growth in MC38 and CT26 mouse tumor models, the addition of intermittent dosing of trilaciclib to chemotherapy/ICI regimens led to further tumor regression and significantly enhanced the durability of response to chemotherapy/ICI. These preclinical results support the hypothesis that intermittent dosing of trilaciclib with chemotherapy/ICI can preserve and/or enhance an antitumor response. To gain insight into the effect of transient exposure of trilaciclib on the tumor microenvironment, we examined the proliferation status, cellular composition, and gene expression of tumor immune infiltrates from MC38 tumor-bearing mice post-treatment. All major intratumor immune cell types (T, NK, and MDSC subsets) are highly proliferative and sensitive to CDK4/6 inhibition. Cell proliferation was reduced by more than 75% after trilaciclib exposure, but was fully restored 48 hours post-treatment. While this brief proliferation arrest did not affect the frequency of each immune population, the overall expression of 59 genes functionally enriched for lymphocyte activation and upregulation of the pro-inflammatory cytokine interferon-γ was significantly enhanced. In parallel, several genes involved in immunosuppressive reactive oxygen species metabolic processes were downregulated. These findings suggest that transient cell-cycle arrest in tumor immune infiltrates can lead to modulation of gene expression, resulting in a pro-inflammatory tumor microenvironment that is favorable for increasing ICI activity. Additional roles of trilaciclib in preserving and enhancing antitumor response in combination with chemotherapy/ICIs are currently being investigated. A phase 2 study to assess the safety and efficacy of trilaciclib or placebo with carboplatin, etoposide, and atezolizumab in first-line extensive stage SCLC patients is ongoing (NCT03041311).

#1753

SIRPa inhibition monotherapy leads to dramatic change in solid tumor microenvironment and prevents metastasis development.

Justine Durand,1 Vanessa Gauttier,2 Aurore Morello,2 Sabrina Pengam,2 Bernard Vanhove,2 Nicolas Poirier2. 1 _INSERM UMR1064, Nantes, France;_ 2 _OSE Immunotherapeutics, Nantes, France_.

Targeting immune checkpoints of the adaptive immunity has shown great therapeutic efficacy to fight cancers, but in a limited proportion of patients. Myeloid cells represent a major immune cell type infiltrating many solid tumors, and are often associated with a poor outcome. While TAMs (mainly MDSCs and macrophages) are involved in the regulation of immune responses and in tissue repair in healthy individuals, they can be co-opted by cancer cells to exert suppressor functions, preventing action of other immune cells. In addition to their suppressor functions, TAMs can also participate in tumor growth and metastasis through several other mechanisms. SIRPalpha (SIRPa) is an immune checkpoint expressed by nearly all myeloid cells which interacts with the ubiquitous receptor CD47 and is now well described to regulate macrophage functions, including phagocytosis. Here we evaluated the impact of SIRPa signaling inhibition on tumor microenvironment and metastasis development in orthotopic tumor models in immunocompetent mice.

We first found that administration of an antagonist anti-SIRPa monoclonal antibody (mAb) in monotherapy significantly reduces tumor growth in an orthotopic mouse triple-negative mammary carcinoma (4T1) model (n=20, p<0.01) and significantly reduces lung metastasis development (p<0.05). Tumor microenvironment analysis by flow cytometry at early time-point (10 days post-treatment) revealed significant increased frequency of M1 inflammatory macrophages (including M1/M2 ratio), NK cells and both increased of intra-tumoral activated CD8+ T cells and memory CD4+ T lymphocytes. Similarly, using SIRPa mutant mice (deletion of the intracellular signaling domain) in an orthotopic mesothelioma (AK7) model, we found that absence of SIRPa signaling significantly reduces tumor growth as compared to wild-type mice (n= 8, p<0.01). Tumor microenvironment analysis by flow cytometry revealed also significant accumulation of intra-tumoral F4/80+ macrophages, CD11c+ dendritic cells, CD8+ T cells and increased CD8/MDSC ratio. Intra-tumoral CD8+ T lymphocytes significantly over-expressed activation markers (e.g. CD69) in SIRPa mutant mice. Finally, using the orthotopic B16F10 melanoma model, we found that curative treatment (initiated two weeks after tumor inoculation) with the antagonist anti-SIRPa monoclonal antibody significantly reduced tumor growth in this aggressive model (n=7, p<0.05), with two mice treated with the anti-SIRPa mAb in complete remission and two others in partial remission at the end of the study.

In conclusion, we found that inhibition of SIRPa signaling in monotherapy rewire innate and adaptive immune cells in cancer. Such straight modifications in the tumor microenvironment led to a reduction in tumor growth in different orthotopic solid tumor models and prevention of metastasis development.

#1754

Smac mimetics APG-1387 synergizes with immune checkpoint inhibitors in preclinical models.

Wentao Pan,1 Douglas D. Fang,2 Guangfeng Wang,2 Miaoyi Wu,2 Yingfeng Li,2 Keji Lian,2 Dingxiong Cheng,2 Yanhui Kong,2 Guoqin Zhai,2 Ming Guo,2 Shaomeng Wang,3 Dajun Yang,2 Yifan Zhai2. 1 _Sun Yat-Sen University Cancer Center, Guangzhou, China;_ 2 _Ascentage Pharma Group Corp., Limited, Suzhou, China;_ 3 _University of Michigan, Ann Arbor, MI_.

APG-1387 is a novel, SMAC-mimetic, small molecule inhibitor for inhibitor of apoptosis proteins (IAPs). IAPs are a class of proteins which negatively regulate caspases and apoptosis. The IAP proteins are frequently dysregulated in many cancers and have been suggested as contributing to apoptosis-resistance of cancer cells to a variety of anticancer drugs. DNA amplification of the cellular IAP-1 (cIAP-1) and cellular IAP-2 (cIAP-2) genes (BIRC2 and BIRC3, respectively) has been observed in various human cancer types, including lung cancer, pancreatic cancer, and liver carcinoma. Dysregulation of IAP proteins has also been frequently observed at the protein level in multiple cancer cell lines and tumor samples. IAPs contribute to tumor cell survival, chemo-resistance, disease progression, and poor prognosis. In addition, IAPs play an important role in immune regulation. For example, IAPs regulate ubiquitin (Ub)-dependent pathways that modulate innate immune signaling via activation of nuclear factor κB (NF-κB). Due to their significant biological functions in cell death and immune responses, IAPs have been emerging as drug targets in a wide range of malignancies. Several IAP inhibitors have been developed and their clinical benefits are currently in investigation in multiple clinical trials in both single agent and combination regimen with immune checkpoint inhibitors. APG-1387 has been developed as a novel drug candidate targeting IAPs. Mechanistically, in both cancer cells and xenograft tumors, APG-1387 induces degradation of cIAP-1 and XIAP proteins, as well as caspase-3 activation and PARP cleavage, which leads to apoptosis. Anti-proliferative and anti-tumor activities of APG-1387 have been well demonstrated in a panel of cancer cell lines and xenograft tumor models. In this study, we explored the synergistic effect of APG-1387 in combination therapy with immune checkpoint inhibitors. We found that APG-1387 enhanced human PBMC co-stimulation of anti-CD3 and anti-CD28, and T cell proliferation in vitro. In syngeneic tumor tissues, the combination treatment with APG-1387 and anti-PD-1 antibody led to an increase in CD3+ and CD8+ T cell populations and a decrease in Treg cell population. Synergistic effect of the combination was further demonstrated in syngeneic tumor models. Taken together, our results suggest that the treatment with APG-1387 may enhance the effect of anti-PD-1 antibody therapy. Consequently, APG-1387 has been approved by FDA for phase I clinical trials as single agent and in combination with anti-PD-1 antibody in solid tumors.

#1755

PEGylated adenosine deaminase (ADA2) prevents adenosine-mediated increase in tumor growth and improves antitumor immune responses.

Luz Marina Londoño-R, Jessica Cowell, Lin Wang, Qiping Zhao, Lei Huang, Chris Thanos, Michael J. LaBarre, Xiaoming Li, Caglar Cekic. _Halozyme Therapeutics, Inc., San Diego, CA_.

Adenosine deaminase 2 (ADA2) is a human serum protein that catalyzes the reaction of deamination of adenosine to inosine. Besides catalytic activity, ADA2 has a heparin-binding domain and a growth factor-like domain. Adenosine accumulates in the tumor microenvironment and suppresses anti-tumor immune responses, while promoting tumor cell metastasis. Previously, we showed that treatment of mice with PEGylated forms of ADA2 (PEG ADA2) can decrease the accumulation of tumor-associated adenosine. Here, we analyzed the TCGA RNA-seq database using OncoLnc tool and observed that high expression of the ADA2 gene (CECR1) in certain immunotherapy responsive tumors correlated with increased survival of patients. We then tested different engineered ADA2 variants, using the inactive enzyme variant as a control, for the reversal of adenosine-mediated responses in human T cells, mouse peritoneal macrophages, and mouse and human tumor cell lines. ADA2 variants with higher catalytic activity were able to reverse adenosine-mediated suppression of human T-cell proliferation, cAMP accumulation in tumors and human T cells, and polarization of macrophages into an immunosuppressive, pro-tumor phenotype, whereas the inactive ADA2 variant failed to cause any of these effects. In addition, the variant that lacks the ability to bind to heparin but has improved catalytic activity (PEG NME8058) showed similar responses to those with the catalytically improved variant with intact heparin-binding domain (PEG NME8062). These results suggest that the improved immune cell activity with ADA2 in the presence of adenosine requires the enzyme activity of this molecule. Next, we assessed PEG NME8062 in MC38 and CT26 colon carcinoma syngeneic tumors in C57BL/6 and Balb/c mice, and observed delayed growth in both tumors. Using the inactive enzyme variant, we have shown that anti-tumor effect of PEG NME8062 depends on enzyme activity. Delayed tumor growth was also associated with increased density of key anti-tumor immune cell populations in both tumor models. These data suggest that PEG ADA2 can reduce tumor growth in an enzyme-activity-dependent manner and promote anti-tumor immune responses in vitro and in vivo. These findings have important implications for targeting purinergic signaling for cancer therapy.

#1756

Discovery and characterization of AB680, a potent and selective small-molecule CD73 inhibitor for cancer immunotherapy.

Kenneth V. Lawson, Lixia Jin, Jenna L. Jeffrey, Jarek Kalisiak, Fangfang Yin, Kristen Zhang, Ada Chen, Debbie Swinarski, Matt J. Walters, Steve Young, Ulrike Schindler, Powers P. Jay. _Arcus Biosciences, Hayward, CA_.

Introduction. Extracellular adenosine (ADO) suppresses immune function via inhibition of T cell and NK cell activation and is present in high concentrations in the tumor micro-environment (TME). Intra-tumoral generation of ADO depends on the sequential catabolism of ATP by two ecto-nucleotidases, CD39 (ATP→AMP) and CD73 (AMP→ADO). Inhibition of CD73 eliminates a major, non-redundant, pathway of ADO production in the TME and can reverse ADO-mediated immune suppression. Here we present the characterization of AB680, a novel, highly potent, reversible and selective small molecule inhibitor of CD73, currently in preclinical development as a potential anti-tumor agent.

Methods. The potency of CD73 inhibitors was evaluated by measuring AMP hydrolysis by CHO-CD73 cells using a malachite green assay. Potency was also measured using human T-cells and soluble recombinant CD73. Selectivity against related ecto-nucleotidases was also assessed. In the presence of human serum, CD73 inhibition was measured by quantitation of AMP hydrolysis via luminescence. The ability of AB680 to reverse AMP-mediated immune suppression of human CD4+/CD8+ T cells was determined by adding exogenous AMP during T cell activation by anti-CD3/CD28/CD2 beads. The pharmacokinetic properties of AB680 were evaluated in multiple preclinical species. A projected human dosing schedule for AB680 was determined via allometric scaling.

Results. AB680 is a highly potent, reversible and selective inhibitor of CD73 (IC50 < 0.01 nM on human CD8+ T-cells), which retains high potency in the presence of human serum. AB680 is > 10,000-fold selective against related ecto-nucleotidases and a large panel of unrelated enzymes, receptors, and ion channels. AB680 does not show significant inhibition of the major CYP450 isoforms or the hERG potassium channel. AB680 potently reverses AMP and ADO-mediated suppression of immune function in vitro. In the presence of high concentrations of AMP, AB680 robustly restored CD25 expression, IFN-γ production and proliferation of human CD4+ and CD8+ T-cells. The pharmacokinetics (PK) of AB680 were assessed in rodent and non-rodent species. The PK properties of AB680 are characterized by very low clearance and long half-lives in preclinical species, resulting in projected human PK properties suitable for intravenous (i.v.) dosing on a schedule consistent with typical mAb dosing cycles. High-dose infusions of AB680 in preclinical species were well tolerated.

Conclusions. AB680 is a highly potent and selective small-molecule inhibitor of CD73 which can mitigate AMP and ADO-mediated tumor immunity by potently blocking the production of adenosine in the TME. AB680 exhibits a favorable projected human PK profile suitable for parental administration and is expected to enter clinical development in 2018.

#1757

Mobilization of tumor-primed, marrow-infiltrating lymphocytes into peripheral blood with inhibitors of E-selectin or E-selectin and CXCR4.

William E. Fogler, Theodore A. Smith, Rachel K. King, John L. Magnani. _GlycoMimetics, Inc., Rockville, MD_.

Marrow-infiltrating lymphocytes (MILs) primed to tumor antigens have been described in patients with hematologic malignancies and in metastatic disease arising from carcinomas. The presence of tumor-reactive MILs in these patients has suggested the possibility of their utilization in T-cell immunotherapeutic approaches. Inherent in this approach are considerations that mediate MIL interactions with the microenvironment and how these may be governed for adoptive or active immunotherapy. Both E-selectin and CXCR4 are known to regulate the homing and retention of T cells to the bone marrow. GMI-1271 and GMI-1359 are potent, small-molecule glycomimetic antagonists of E-selectin and E-selectin/CXCR4, respectively. GMI-1359 is a potent small-molecule glycomimetic dual antagonist targeting E-selectin and CXCR4. In the present studies tumor-specific MILs were established in BALB/c mice that had been induced to reject the syngeneic CT26 colon carcinoma via treatment with anti-CTLA-4 T cell checkpoint antibody, and the subsequent effects of antagonizing E-selectin and/or CXCR4 with GMI-1271 or GMI-1359 on the mobilization and distribution of these bone marrow-derived tumor-specific CD8+ T cells were determined. CT26-immune mice were treated for three days with saline, GMI-1271 (40 mg/kg), or GMI-1359 (40 mg/kg) and 12 hours following the last injection, the phenotype and functional activity of CD8+ T cells were determined in bone marrow and peripheral blood. Additional controls included CT26-immune mice treated with G-CSF (0.125 mg/kg) and tumor-naïve mice treated with saline. Treatment of mice with GMI-1271 and to a greater extent with GMI-1359 led to an approximate 3-4 fold increase in CD8+CD62L+CD44- naïve and CD8+CD62L+CD44+ central memory T cells in peripheral blood. This was not observed following treatment of tumor-immune mice with G-CSF. Treatment of mice with GMI-1271 or GMI-1359 did not affect distribution of CD8+CD62L-CD44+ effector memory T cells in peripheral blood. The increase in percentages of CD8+ naïve and central memory T cells in peripheral blood following treatment with GMI-1271 or GMI-1357 functionally correlated with increased production of IFN-γ ex vivo in response to irradiated CT26 tumor cells or the immunodominant CT26 peptide, AH-1. Collectively these results demonstrate the mobilization or redistribution of marrow-infiltrating tumor-specific CD8+ T cells into peripheral blood as a consequence of E-selectin and/or E-selectin and CXCR4 antagonism. Once in the periphery, these MILs could (1) be collected for adoptive immunotherapy approaches or (2) serve as a systemic augmentation of T cells for combination with immune stimulants as a foundation to boost active immunotherapy.

#1758

Intratumoral IL-12 mRNA promotes cytotoxic T cell-dependent anti-tumor immunity and enhances responses to PD-L1 blockade in pre-clinical models.

Susannah L. Hewitt,1 Nadia Luheshi,2 Dyane Bailey,1 John Zielinski,1 Ameya Apte,1 Faith Musenge,1 Russell Karp,1 Grace Adjei,2 Andrew Leinster,2 Sushma Gurumurthy,1 Ankita Mishra,1 Kristen Arnold,1 Darren Potz,1 Robert W. Wilkinson,2 Ronald Herbst,3 Joshua P. Frederick1. 1 _Moderna Therapeutics, Cambridge, MA;_ 2 _MedImmune Ltd., Cambridge, United Kingdom;_ 3 _MedImmune, Gaithersburg, MD_.

As a central mediator of TH1 immune responses, interleukin 12 (IL-12) in the tumor microenvironment (TME) plays a key role in driving anti-tumor immunity. Recombinant IL-12 has been reported to promote CD8+ cytotoxic T cell (CTL) dependent anti-tumor immunity in a wide variety of preclinical models. However, systemic recombinant IL-12 was poorly tolerated in early clinical trials. We are therefore investigating whether intra-tumoral (IT) delivery of a messenger RNA encoding IL-12 formulated in lipid nanoparticles can drive local IL-12 expression in solid tumors to induce TME transformation and anti-tumor immunity.

Here we report that IT IL-12 mRNA induces dose-dependent local IL-12 protein expression in mice bearing both subcutaneous syngeneic and patient-derived xenograft tumors. In multiple syngeneic tumor models (e.g. MC38, A20), mouse IL-12 mRNA (mIL-12 mRNA) treatment promoted dose-dependent tumor regression and significantly improved overall survival. Flow cytometry and luminex analysis revealed that anti-tumor activity of mIL-12 mRNA was accompanied by transformation of the TME characteristic of a TH1 immune response. IFNγ induction and natural killer cell activation was accompanied by antigen presenting cell maturation, increased CTL numbers and activation, and increased PD-L1 expression. Furthermore, rejection of established MC38 tumors in response to mIL-12 mRNA was CTL-dependent, and animals demonstrated immunity to subsequent re-challenge with MC38 tumor cells.

Since mIL-12 mRNA enhanced PD-L1 expression in the TME, we next investigated whether PD-L1 blockade would enhance responses to mIL-12 mRNA. We found that the combination of a single dose of mIL-12 mRNA and PD-L1 blockade led to significantly more complete responses compared to mIL-12 mRNA monotherapy in the PD-L1 resistant MC38 model. Furthermore, A single dose of mIL-12 mRNA induced rejection of un-injected MC38 tumors in a dual flank model, and this was further enhanced by combination with PD-L1 blockade.

These preclinical data demonstrate the potential for IT IL-12 mRNA to drive TME transformation into a proinflammatory state consistent with a TH1 immune response and to ignite CTL-dependent anti-tumor immunity, even in a PD-L1 resistant setting.

#1759

AVID200, a highly potent TGF-beta trap, exhibits optimal isoform selectivity for enhancing anti-tumor T-cell activity, without promoting metastasis or cardiotoxicity.

Maureen D. O'Connor-McCourt,1 Gilles Tremblay,1 Anne Lenferink,2 Traian Sulea,2 John Zwaagstra,2 James Koropatnick3. 1 _Formation Biologics, Montreal, Quebec, Canada;_ 2 _National Research Council Canada, Montreal, Quebec, Canada;_ 3 _Lawson Health Research Institute, London, Ontario, Canada_.

The purpose of the studies presented here is to examine the ability of the novel TGF-β-neutralizing agent, AVID200, to reverse immunosuppression in the tumor microenvironment, and to determine the safety profile of AVID200 in non-human primates. TGF-β is a secreted protein that is aberrantly produced by tumors, and which promotes cancer progression primarily by suppressing both the innate and adaptive immune systems. AVID200 is a computationally-designed, avidity-enhanced, receptor ectodomain-based trap that binds and neutralizes TGF-β1 and -β3. We demonstrate, in tumor cell-based assays, that AVID200 potently neutralizes TGF-β1 and -β3 with low pM potency. Using a syngeneic 4T1 triple negative breast cancer (TNBC) model, we report that AVID200, in a dose-dependent manner, enhances the capacity of T-cells isolated from draining lymph nodes to specifically recognize and kill 4T1 tumor cells. The anti-tumor T-cell-activating potency of AVID200 was observed to be higher than that of the pan-neutralizing TGF-β antibody, 1D11. AVID200 was designed to have minimal activity against TGF-β2. This isoform specificity was chosen since it has been reported that inhibition of the TGF-β2 isoform can promote metastasis. We report that AVID200 does not increase the number of foci in a metastasis assay, whereas pan TGF-β neutralizing agents do. This supports the concept that blockade of TGF-β2 is undesirable. Also, it has been reported that pan-TGF-β blockers can exhibit cardiac toxicity in animal models and in humans. It has been proposed that this is due to TGF-β2 neutralization since this is the main isoform implicated in normal cardiac function. To determine if AVID200 exhibits cardiotoxicity, we tested AVID200 in a pilot non-human primate study. No adverse cardiac events were observed at doses of up to 30 mg/kg. An update on GLP toxicology studies will be presented at the meeting. In conclusion, AVID200 is a novel TGF-β trap that potently blocks TGF-β-1 and -3 isoforms, which results in reversal of immunosuppression in the tumor microenvironment. It has minimal activity against TGF-β2 and accordingly does not detectably promote cardiac toxicity and metastasis. Thus, AVID200 is a promising new immunotherapy that selectively inhibits the TGF-β1 and -3 isoforms, thereby enhancing desirable anti-tumor immunity, while avoiding the tumor-promoting and cardiotoxic effects resulting from TGF-β2 neutralization.

#1760

The hexavalent CD40 agonist HERA-CD40L augments multi-level crosstalk between T cells and antigen-presenting cells.

Christian Merz, Jaromir Sykora, Viola Marschall, David M. Richards, Meinolf Thiemann, Harald Fricke, Oliver Hill, Christian Gieffers. _Apogenix AG, Heidelberg, Germany_.

Introduction: HERA-CD40L is a novel hexavalent CD40 agonist engineered with the HERA-Technology developed by Apogenix. We have previously shown that the natural binding mode via the receptor/ligand binding domains and the high clustering capacity for the cognate receptor clearly distinguish HERA-CD40L from other, e.g. antibody-based, CD40-targeting compounds. Here, we report on the effects of HERA-CD40L on crosstalk between T cells and antigen presenting cells (APC) and the functional consequences in vitro. Materials & Methods: Biological activity of CD40 agonists was analyzed using co-cultures of primary T cells with B cells or monocytes/macrophages. All primary cells were isolated by negative selection using magnetic sorting from healthy donor buffy coats. Expression of CD markers upon CD40 ligation on B cells and monocytes was analyzed by flow cytometry (FC). Monitoring of T cell-induced killing of tumor cells primed in direct co-cultures with APC was done on a real-time cell analysis system (xCELLigence). For analysis of phagocytosis, we developed an FC-based assay employing primary monocytes/macrophages and Jurkat A3 cells. Results: Treatment of primary B cells and monocytes with HERA-CD40L induced expression of co-stimulatory molecules, like CD86, and promotes M1 maturation of naïve (M0) monocytes. In vitro, treatment of alternatively activated M2 macrophages with HERA-CD40L induced an M2 to M1 phenotype switch (re-programming) which concurs with CD16 downregulation and a dose-dependent decrease of phagocytic activity of re-programmed macrophages compared to M0 or M2 macrophages. Primary B cells and M1 macrophages enhanced the proliferation and cytotoxic activity of naïve T cells in direct co-cultures in the presence of HERA-CD40L. The activating effect on T cells required direct cell-cell contact with APC and was not observed in indirect co-cultures. Functionally, neutralization of either MHC-I or CD80/CD86 in direct co-cultures inhibited full activation of the T cells in vitro as shown by kill assays with various tumor cell lines. Conclusion: The hexavalent CD40 agonist HERA-CD40L produced by the Apogenix HERA-Technology is a potent immune-regulator acting on B cells and myeloid cells. HERA-CD40L promotes activation of B cells, maturation of APC and induces an M2 to M1 phenotype switch which inhibits tolerance-inducing phagocytic activity of the repolarized macrophages in vitro. In response to CD40 ligation on APC, an efficient anti-tumor response is conferred to primary T cells through cell-cell interactions via MHC-I and CD80/CD86.

#1761

Combination of a T cell activating immunotherapy with immune modulators alters the tumor microenvironment and promotes more effective tumor control in preclinical models.

Alecia MacKay,1 Genevieve Weir,1 Holly Koblish,2 Ava Vila- Leahey,1 Valarmathy Kaliaperumal,1 Cynthia Tram,1 Peggy Scherle,2 Marianne Stanford1. 1 _Immunovaccine Inc, Halifax, Nova Scotia, Canada;_ 2 _Incyte Corporation, PA_.

Combinations of immune therapies for cancer treatment will likely improve clinical responses, and a treatment that stimulates a robust T cell response may be a key component of therapy in patients with poorly infiltrated tumors. DPX-Survivac is a T cell activating therapy targeting survivin, formulated in DepoVax™ (DPX), an oil based delivery platform. In clinical studies, the MHC class I peptide antigens in DPX-Survivac induced strong and sustained T cell responses when used in combination with metronomic cyclophosphamide (mCPA) in ovarian cancer patients. Epacadostat is an indoleamine-2,3-dioxygenase 1 (IDO1) inhibitor which has shown to reduce immune suppression in tumors and has demonstrated encouraging results in clinical trials. Using preclinical mouse tumor models, we evaluated the combination of these three immune therapies. C57Bl/6 mice were implanted subcutaneously with murine pancreatic adenocarcinoma (Panc02) cells. Groups of mice were vaccinated with DPX-Survivac vaccine (containing murine H2D peptides) by subcutaneous injection and treated with mCPA (20 mg/kg/day, PO) and epacadostat (6 mg/day, PO). The combination of the three treatments provided a significant delay in tumor progression, and improvement in survival over untreated animals. Similar findings were also observed in the HPV16 E7 expressing C3 tumor model, using an HPV16 minimal peptide epitope (HPV16 E749-57) formulated in DPX. In this model, mice were terminated at defined endpoints to evaluate systemic immune responses in the spleen by IFN-γ ELISPOT and profile tumor infiltration by flow cytometric analysis. Although antigen-specific immune responses in the spleen were not increased by the triple combination in comparison to the DPX-based vaccine, there was a significant impact on several immune subtypes found in the tumor. Notably, antigen-specific CD8+ T cells (as detected by dextramer analysis) were increased and regulatory CD4+CD25+FoxP3+ T cells (Tregs) were decreased. In comparison, the Treg population in the spleen was highest in the triple therapy group (p=0.0046 compared to DPX/mCPA alone). This may indicate a selective exclusion of Tregs from the tumor microenvironment is induced by epacadostat, which can facilitate the anti-tumor immune response mediated by CD8+ T cells induced by DPX. Other immune modulating therapies, such as anti-PD-L1, may further enhance the tumor control induced by this treatment. The combination of DPX-based, T cell activating therapy with epacadostat, a drug that reduced tumor immune suppression is a rational, synergistic combination that is currently being evaluated in advanced ovarian cancer patients in the DeCidE1 clinical trial (NCT0278520).

#1762

Shifting the tumor microenvironment with first-in-class semaphorin 4D mab for combination immunotherapy.

Elizabeth E. Evans,1 Holm Bussler,1 Crystal Mallow,1 Christine Reilly,1 Sebold Torno,1 Maria Scrivens,1 Alan Howell,1 Leslie Balch,1 John E. Leonard,1 Terrence Fisher,1 Clint Allen,2 Paul E. Clavijo,2 Gregory Lesinski,3 Christina Wu,3 Siwen Hu-Lieskovan,4 Antoni Ribas,4 Emily G. Greengard,5 Ernest S. Smith,1 Maurice Zauderer1. 1 _Vaccinex, Inc., Rochester, NY;_ 2 _National Institute of Health (NIH), Bethesda, MD;_ 3 _Winship Cancer Institute of Emory University, Atlanta, GA;_ 4 _UCLA, Los Angeles, CA;_ 5 _University of Minnesota Masonic Children's Hospital, Minneapolis, MN_.

Purpose: In preclinical models, tumor growth inhibition by anti-semaphorin 4D (SEMA4D, CD100) blocking antibody is enhanced when combined with various immunotherapies. Immune checkpoint combinations with humanized VX15/2503 anti-SEMA4D are currently being evaluated in several clinical trials. Methods: Expanded mechanistic studies in preclinical models investigate the effect of SEMA4D signaling through its Plexin receptors (PLXN) on MDSC function and chemokine secretion in the tumor microenvironment. Humanized VX15/2503 anti-SEMA4D is now also being evaluated as single agent or in combination with other immunotherapies in four clinical trials:: (i) a Phase 1b/2a combination trial of VX15/2503 with avelumab in NSCLC (CLASSICAL-Lung); (ii) a phase 1 combination trial with nivolumab or ipilimumab in melanoma patients who have progressed on any anti-PD-1/PD-L1; (iii) a neoadjuvant integrated biomarker trial in patients with metastatic colorectal and pancreatic cancers treated with VX15/2503 in combination with nivolumab or ipilimumab; and (iv) a Phase 1/2 trial of VX15/2503 in children with solid tumors and children and young adults with osteosarcoma. Results: SEMA4D plays a multi-faceted role within the tumor microenvironment by creating a barrier at the tumor-stroma margin to restrict immune cell infiltration and promoting immunosuppressive activity of myeloid-derived cells. SEMA4D directly enhanced ability of MDSC to suppress T cell proliferation and antibody blockade reversed these effects, both in vitro and in vivo. Furthermore, SEMA4D-PLXN signaling modulates expression of chemokines that recruit MDSC. Importantly, anti-SEMA4D MAb can enhance activity of co-administered immunotherapies in murine colon, head and neck (HNSCC), and melanoma models. For example, anti-SEMA4D plus anti-CTLA-4 results in 100% survival and 90% complete tumor rejection (CR) (p<0.0001) in an HNSCC model representative of a T cell inflamed tumor with high MDSC suppression. Entinostat has broad immunomodulatory effects, including reduction of MDSC, and combination treatment of established Colon26 tumors with anti-SEMA4D and entinostat results in maximal tumor growth delay and 90% CR (p<0.0001). Conclusions: SEMA4D blockade represents a novel approach to promote functional immune infiltration into the tumor, reduce mesenchymal suppression, and enhance immunotherapy. VX15/2503 treatment was well tolerated in a Phase I trial in patients with advanced refractory solid tumors. Several clinical trials are in progress to evaluate safety, tolerability, efficacy, and biological endpoints, including immunophenotyping tumors and blood of patients treated with VX15/2503 in combination with immune checkpoint antibodies. Finally, a pediatric phase 1/2 trial based on the previously described role of SEMA4D as an oncogene in osteosarcoma and its immunomodulatory effects is being conducted.

#1762A

CD200 and CD200R1 expressions and their prognostic roles in patients with non-small cell lung cancer.

Katsuhiro Yoshimura,1 Yuzo Suzuki,1 Yusuke Inoue,1 Masato Karayama,1 Yuji Iwashita,1 Tomoaki Kahyo,1 Akikazu Kawase,1 Masayuki Tanahashi,2 Hiroshi Ogawa,2 Naoki Inui,1 Kazuhito Funai,1 Kazuya Shinmura,1 Takafumi Suda,1 Haruhiko Sugimura1. 1 _Hamamatsu University School Of Medicine, Hamamatsu, Japan;_ 2 _Seirei Mikatahara General Hospital, Hamamatsu, Japan_.

Background: CD200 is a member of the immunoglobulin superfamily and is expressed in various cells. CD200 interacts its receptor CD200R, which is mainly expressed in myeloid lineage cells, and modulate cancer immune-microenvironments. High CD200 expressions in hematologic malignancies were associated with poor prognosis and a clinical trial of anti-CD200 antibody has already conducted. However the role of CD200/CD200R signaling in solid cancers including non-small-cell lung cancer (NSCLC) have yet to be elucidated.

Methods: We evaluated CD200 and CD200R1 protein expression using immunohistochemically stained tissue microarrays containing 653 resected NSCLC specimens. The tumor expression levels were assessed using the H-score method that ranged from 0 to 300. The stromal expression levels were also assessed semi-quantitatively. The cut-offs were determined using the minimum P-value method for overall survival (OS). The associations between their expression levels and clinicopathological data were retrospectively analyzed. In addition, we investigated CD200 and CD200R1 expressions among lung-cancer cell-lines. In vitro, endogenous immunorelated factor levels and cell proliferation changes were evaluated according to CD200 knockdown and CD200-Fc fusion protein administration.

Results: The median age of patients was 69.5 years old (23-88 years) and 444 (67.8%) patients were male. 444 (68.0%) patients were smoking history and 431 (66.0%) patients had adenocarcinoma. The median follow-up duration was 3.6 years. CD200 expression was mainly observed in tumoral area but not in stroma, whereas CD200R1 expression was observed in both tumoral and stromal area. CD200 and CD200R1 expressions were inversely correlated. Higher tumoral CD200 expression and lower stromal CD200R1 expression were significantly prominent in female, in never smokers, and in adenocarcinoma. CD200 expression was significantly associated with TTF-1 positivity and EGFR mutations. Higher tumoral CD200 expression was an independent favorable prognostic factor for NSCLC in multivariate Cox regression analyses for OS (hazard ratio (HR), 0.65; 95% confidence interval (CI), 0.45-0.94). In contrast, higher CD200R1 expressions in both tumor and stroma were poor prognostic indicators for OS (Log-rank, p=0.019 for tumor, and p<0.001 for stroma). In vitro, we observed that both CD200 and also CD200R1 were expressed in several lung-cancer cell-lines. Treatment with CD200-Fc on PC9 did not change any cytokine levels. Knockdown of CD200 in H1299 did not show any effects on cell proliferations, but significantly decreased endogenous TNFα levels.

Conclusion: CD200R1 expression had poor prognostic capability in resected NSCLC. Inversely, CD200 expression was an independent favorable prognostic factor. CD200/CD200R axis was involved in patients' prognoses and would be a candidate of potent therapeutic target for NSCLC.

#1763A

Single-cell RNA-sequencing reveals the immune contexture of triple-negative breast cancer tumors.

Si Qiu,1 Ruoxi Hong,2 Zhenkun Zhuang,1 Shusen Wang2. 1 _BGI-Shenzhen, Shenzhen, China;_ 2 _Sun Yat-Sen University, China_.

Triple-negative breast cancer (TNBC) is aggressive and associated with a higher risk of early relapse. The lack of estrogen receptor, progesterone receptor, and HER2 expression precludes the use of targeted therapies, and the high level of immune cell infiltration suggests that TNBC patients may benefit from immunotherapy. The design of immunomodulatory strategies for the TNBC treatment will tremendously benefit from a comprehensive understanding of the tumor infiltrated immune cell landscape of this cancer. To this end, we performed deep single cell RNA sequencing to 7,066 immune cells collected from 8 TNBC patients using the MIRALCS platform. We identified 13 immune cell subsets according to their transcriptome features. We found that the frequency of identified T cells and macrophages varied greatly across patients. We explored the diversity of tumor-infiltrating T lymphocytes and their functional states, and described the complicated and patient specific macrophage phenotypes. We also examined the single-cell expression level of immunotherapeutic target molecules in different immune cell subgroups. Our study gives an insight into the heterogeneity of the immune cell contexture of TNBC tumors, provides a valuable resource to understand the immune ecosystem of TNBC and may accelerate immunotherapies development on this breast cancer subtype. 

### Therapeutic Antibodies, Including Engineered Antibodies 1

#1763

BiTEs vs CAR-Ts: Preclinical targeting of CD133+ brain tumor initiating cells using immunotherapy-based treatment strategies.

Parvez Vora,1 Jarrett Adams,2 Mohini Singh,1 Chitra Venugopal,1 Nazanin Tatari,1 Chirayu Chokshi,1 Maleeha Qazi,1 Sabra Salim,1 Sujeivan Mahendram,1 David Bakhshinyan,1 Max London,2 Neil Savage,1 Minomi Subapanditha,1 Nicole McFarlane,1 James Pan,2 Jonathan Bramson,1 Sachdev Sidhu,2 Jason Moffat,2 Sheila Singh1. 1 _McMaster University, Hamilton, Ontario, Canada;_ 2 _University of Toronto, Toronto, Ontario, Canada_.

Glioblastoma (GBM) is a uniformly fatal primary brain tumor, characterized by extensive cellular heterogeneity. Numerous studies have implicated CD133+ brain tumor initiating cells (BTICs) as drivers of chemo- and radio-resistance in GBM. We have recently demonstrated that a CD133-driven gene signature is predictive of poor overall survival and targeting CD133+ treatment-refractory cells may be an effective strategy to block GBM recurrence.

Bispecific T-Cell engaging antibodies (BiTEs) and Chimeric antigen receptors (CARs) present promising immunotherapeutic approaches that have not yet been validated for recurrent GBM. Using CellectSeq, a novel methodology that combines the use of phage-displayed synthetic antibody libraries and DNA sequencing, we developed the CD133-specific monoclonal antibody 'RW03'. We constructed CD133-specific BiTEs that consist of two arms; one recognizes the tumor antigen (CD133) and the second is specific to the CD3 antigen that binds to human GBMs and PBMC-derived T cells, respectively. We observed BiTEs redirecting T cells to kill GBMs, with greater efficiency observed in CD133high GBMs, validating BiTE target specificity. Incubating T-cells with BiTEs and the CD133high GBMs resulted in increased expression of T cell activation markers. In parallel, we derived the single chain variable fragment (scFv) from RW03 we engineered a second-generation CAR T cell. CD133-specific CAR-T cells were cytotoxic to CD133+ GBMs. Co-culturing CD133 CAR-T cells with GBMs triggered T cell activation and proliferation. Treatment of GBM tumor-bearing mice with CD133-specific CAR-T cells yielded extended survival in mice and significant reductions in brain tumor burden.

Furthermore, we uniquely adapted the existing chemoradiotherapy protocol for GBM patients for treatment of immunocompromised mice engrafted with human GBMs. Within this model, we have initiated treatment of recurrent GBM directed against CD133+ BTICs, to allow for a direct prospective comparison of toxicity and efficacy of BiTEs and CAR-T cell strategies.

#1764

Neurovascular-targeting antibodies discovered using yeast biopanning enhance drug delivery and improve survival for glioblastoma.

Paul A. Clark, Benjamin J. Umlauf, Eric V. Shusta, John S. Kuo. _Univ. of Wisconsin-Madison, Madison, WI_.

INTRODUCTION: Median survival for newly diagnosed glioblastoma (GBM) patients remains less than two years despite aggressive clinical interventions. Antibody-based GBM-targeting could improve brain delivery and accumulation of therapies, but it has been challenging to identify universal tumor cell antigens due to intra-tumor and inter-patient GBM heterogeneity. We hypothesized that antigens of the neurovascular extracellular matrix (nvECM) within GBM-disrupted blood-brain barrier (BBB) may localize and enhance efficacy of therapeutic agents.

METHODS: Yeast surface display biopanning and high throughput screening identified Variable Lymphocyte Receptors (VLR, i.e. jawless vertebrate lamprey antibodies) that preferentially accumulate at nvECM. Lead VLR candidates were tested for tumor targeting via combination with chemotherapeutic doxorubicin (DOX) liposomes and assessed for anti-GBM effects in vitro and against orthotopic mouse xenografts in vivo.

RESULTS: VLR clones were enriched for binding to mouse brain endothelial cell (bEnd.3 line) ECM with two rounds of biopanning, then screened for preferential binding to brain-derived ECM. Lead VLR clone P1C10 was chosen for further testing for nvECM specificity vs. fibroblast (3T3 line) ECM in a high throughput screen, high affinity (Kd=48±6.1 nM), and immunolabeling of normal mouse and human brain, and GBM samples. Clone P1C10 demonstrated tumor-specific targeting in orthotopic U87 GBM xenografts, with 50% more DOX fluorescence detected at GBM after intravenous injection of P1C10-DOX liposomes compared to control, non-specific VLR-DOX. Finally, administration of P1C10-DOX liposomes (15 mg/kg, i.p., 1/week) significantly improved survival of mice bearing U87 xenografts, compared to non-targeted VLR-DOX (Kaplan-Meier, p<0.01).

CONCLUSIONS: We report combining yeast surface display biopanning and screening to identify VLRs that preferentially accumulate at disrupted nvECM of GBM-disrupted BBB, and demonstrate GBM-targeting specificity in vitro and in vivo with significant survival benefit. These results support the nvECM immune-targeting strategy to improve anti-GBM therapeutic efficacy, potentially improving survival and quality-of-life in brain cancer patients.

#1765

A humanized anti-CD47 monoclonal antibody that directly kills human tumor cells and has additional unique functional characteristics.

Robyn Puro, Katherine Liu, Benjamin Capoccia, Michael Donio, Ronald Hiebsch, Myriam Bouchlaka, Alun Carter, Pamela Manning, Kathleen Crowley, Robert Karr. _Arch Oncology, St. Louis, MO_.

CD47 is a cell surface glycoprotein that interacts with signal regulatory protein alpha (SIRPα) on macrophages and dendritic cells triggering a "don't eat me" signal that inhibits phagocytosis. Many tumors evade immune surveillance by overexpressing CD47, thereby preventing their recognition by phagocytes. Blocking the interaction of SIRPα/CD47 promotes phagocytosis and tumor cell destruction leading to a reduction in tumor burden.

We have developed a humanized anti-CD47 antibody, AO-176, that blocks the interaction between CD47 and SIRPα and exhibits several additional novel functional characteristics. These characteristics include the induction of cell death in multiple human tumor cell lines in a cell autonomous manner (not ADCC), assessed by an increase in phosphatidylserine/7AAD positive staining. A second novel characteristic is enhanced binding to tumor cells at acidic pH. AO-176 binds to human tumor cell lines in the high pM to low nM range at physiologic pH, however, binding is enhanced up to 20-fold at an acidic pH of 6.5. The acidic pH of the tumor microenvironment which ranges from 6.4-7.2 is characteristic of solid tumors and correlates with tumor progression and metastasis. As a result of this enhanced binding at acidic pH, AO-176 has the potential added advantage of tumor-specific targeting. A third novel characteristic exhibited by AO-176 is its selective binding to tumor cells while exhibiting reduced binding to normal cells including red blood cells (cynomolgus monkey and human), endothelial, epithelial and skeletal muscle cells. In addition to these novel characteristics, AO-176 also exhibits dose-dependent efficacy in multiple mouse tumor models.

Taken together, the unique combination of functional characteristics of AO-176, including induction of cell-autonomous killing, enhanced binding to tumor cells at acidic pH, significantly reduced binding to normal cells and potent in vivo efficacy provides the preclinical rationale for further development.

#1766

**Dinutuximab targets GD2** + **breast cancer stem cells and inhibits TNBC tumor growth.**

Venkata Lokesh Battula,1 Khoa Nguyen,1 Anitha Somanchi,1 Hong Mu,1 Naoto T. Ueno,1 Dean Lee,2 Michael Andreeff1. 1 _UT MD Anderson Cancer Ctr., Houston, TX;_ 2 _Nationwide Children's Hospital, Columbus, OH_.

Breast cancer stem-like cells (BCSCs), also known as breast cancer initiating cells, are a fraction of cells in the primary tumor that are highly tumorigenic, chemotherapy resistant, and are able to cause metastases. Therefore, targeting BCSCs is an important strategy that could complement standard chemotherapy for breast cancer. We have identified that ganglioside GD2 as an important maker for BCSCs and that ST8SIA1 (GD3-synthase) is an enzyme that regulates GD2 expression in BCSCs (Battula et al., JCI, 2012). GD2 is over expressed in triple negative breast cancer (TNBC) compared to non-TNBC. Inhibition of GD2 expression by knockdown of ST8SIA1 completely inhibited tumor growth and metastases in xenograft models. Therefore, we hypothesize that targeting GD2+ BCSCs will inhibit TNBC tumor growth. To test our hypothesis, we used the chimeric anti-GD2 monoclonal antibody (Dinutuximab) that has been FDA approved for the treatment of neuroblastoma in children. The main mode of action for dinutuximab is antibody dependent cell-mediated cytotoxicity (ADCC). To test the activity of dinutuximab against GD2+ BCSCs, we treated MDA-MB-231 and SUM159 breast cancer cells with dinutuximab alone at different concentrations (1, 12.5, 25, 50, and 100µg/ml) for 24, 48, and, 72hrs or in combination with IL-21 activated NK cells. As control, cells were treated with rituximab (anti-CD20 antibody) and NK cells. Although only a moderate effect was observed on GD2+ cells when treated with dinutuximab alone, we found a dramatic decrease in the number of live (annexin-vneg and DAPIneg) GD2+ cells when treated with dinutuximab in combination with NK-cells. This combination decreased the percentage of GD2+ cells from 20.1±0.3% to 5.8±0.2% (p<0.001). No significant effect on GD2+ cells was observed in the rituximab + NK cell group, indicating that the dinutuximab + NK-cell combination is most affective in inducing cytolysis of GD2+ cells. Next, to investigate the effect of dinutuximab on in-vivo tumor growth, we implanted firefly luciferase expressing MDA-MB-231 cells into nude mice (these mice were chosen because of the presence of NK cells). When palpable tumors were formed (tumor volume 25-30mm3), the mice were treated with dinutuximab or rituximab at 1.4mg/kg twice a week for 6 weeks. Tumor growth was measured weekly by bioluminescence imaging as well as by calipers. We found a >10 fold decrease in tumor growth in mice treated with dinutuximab compared to control. In contrast, tumor volumes reached up to 250mm3 within 10 weeks of tumor implantation in the rituximab treated group (p<0.001). Conclusion: Our data suggest that dinutuximab targets GD2+ BCSCs with the help of NK cells by ADCC and inhibits tumor growth. We are currently in the process of developing a phase I/II clinical trial using dinutuximab in patients with TNBC. In addition, preclinical testing of dinutuximab in combination with NK cells using TNBC PDX models is underway.

#1767

Synergy between intratumoral immunotoxin and systemic anti-CTLA-4 promotes massive inflammation and leads to complete regression of tumors in mice.

Yasmin Leshem,1 James O'Brien,2 Yoram Reiter,3 Ira Pastan1. 1 _NIH, Rockville, MD;_ 2 _Pittsburgh Medical School, Pittsburgh, PA;_ 3 _Technion, Haifa, Israel_.

Background: The immunotoxin SS1P consists of an anti-mesothelin Fv attached to Pseudomonas exotoxin A and kills tumor cells by inhibiting protein synthesis. Because of its unique mechanism of cell killing and unlike other anti-cancer drugs, SS1P does not suppress the immune system. Here we determined if SS1P can cooperate with anti-CTLA-4 to induce anti-tumor immunity and cause tumor regression.

Methods: A BALB/c breast cancer cell line was transfected with human mesothelin (66C14-M) and implanted in two different locations. SS1P was injected directly into one tumor and anti-CTLA-4 given IP. Twelve days after treatment initiation, the tumors were harvested and evaluated for immune related gene expression by Nanostring Technologies. A portion of each tumor was sent to pathology and stained for CD8.

Results: In mice treated with anti-CTLA-4 and SS1P, complete tumor regressions occurred in 25/30 (86%) SS1P injected tumors and in 16/30 (53%) tumors at the un-injected location. No complete regressions occurred with either drug given separately. This indicates that the combination therapy has a synergistic and systemic anti-tumor effect. Pathological evaluation of injected tumors from combination-treated mice revealed large central abscesses surrounded by a collar of inflammation, containing a mixture of lymphocytes and mononuclear cells. The collar of inflammation was unique to the combination treated mice. Only very small abscesses were observed in tumors from control mice. Immunohistochemistry of tumors from mice treated with SS1P and anti-CTLA-4 showed an increase in CD8+ cells located mainly in the inflammation collar. Analysis of gene expression in tumors from combination-treated mice, compared to PBS-treated tumors, showed that 341/747 (45%) immune transcripts were elevated by 2-fold or more including CD8a, PDL2 and CD56 (8, 15 and 24-fold, respectively). In mice treated with anti-CTLA-4 and PBS, the increase of transcripts was lower: 127/747 (17%) genes. Only 18 out of 747 transcripts (2%) were elevated in the SS1P-treated tumors. This suggests that SS1P by itself does not initiate an inflammatory process. However, SS1P greatly potentiates the inflammatory process initiated by anti-CTLA-4.

Conclusions: Combining local SS1P with anti-CTLA-4 promotes massive inflammation that cures injected and un-injected tumors and provides a strong rational to explore this combination therapy in patients.

#1768

An anti-TGF-β1/2 antibody that spares TGF-β3 retains full anti-tumor efficacy and generates an improved metabolic profile.

Yu-an Yang,1 Srividya Vasu,2 Howard Yang,1 Maxwell P. Lee,1 Sushil Rane,2 Amer M. Mirza,3 Lalage M. Wakefield1. 1 _National Cancer Institute, Bethesda, MD;_ 2 _National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD;_ 3 _XOMA Corp, Berkeley, CA_.

TGF-β family members are overexpressed in many advanced cancers and correlate with metastasis and poor prognosis. Based on encouraging preclinical data, therapeutics that target the TGF-β pathway are now in early phase clinical trials in oncology. While the three isoforms of TGF-β have essentially identical activities in vitro, there is relatively little known about how they might differ in vivo. Using a panel of twelve mouse allograft models of metastatic breast cancer, we showed that while TGF-β1 protein was consistently up-regulated in mammary tumors compared with normal mammary gland, the opposite was true for TGF-β3. Furthermore, in human breast cancer high TGF-β3 mRNA or protein expression was associated with better outcome, particularly in estrogen-receptor positive breast cancers. Collectively the data suggest that TGF-β1 and TGF-β3 may have opposing effects on breast cancer progression. Using an antibody that selectively neutralizes only TGF-β1 and TGF-β2, we explored the effect of sparing TGF-β3 on therapeutic outcome in the 4T1 and TSAE1 models of metastatic breast cancer. While the TGFβ1,2 antibody and two pan-TGF-β antibodies had similar efficacy against the metastasis endpoint in these very aggressive models, transcriptomic analysis of primary tumors after two weeks of antibody therapy suggested that sparing TGF-β3 might have positive effects on the metabolic profile of treated animals. To address this issue directly, we showed that mice without tumors had a significantly improved glucose tolerance following treatment with anti-TGF-β1/2 antibodies for 2-3 weeks when compared with mice treated with pan-TGF-β antibodies. Addressing potential human relevance, we showed that high expression of transcripts that were selectively upregulated in the primary tumors when TGF-β3 was spared correlated with good outcome in human breast cancer. The data suggest that use of isoform-selective TGF-β antagonists may offer advantages over the use of pan-TGF-β blocking agents for the treatment of breast cancer.

#1769

Antibody blockade of IL1RAP signaling reduces metastasis in a breast cancer model.

David Liberg,1 Per-Ola Önnervik,2 Matteo Riva,2 Liselotte Larsson,1 Göran Forsberg,1 Karin von Wachenfeldt2. 1 _Cantargia, Lund, Sweden;_ 2 _Truly Labs, Lund, Sweden_.

Blockade of tumor inflammation has potential for cancer therapy, both as a primary mechanism to counter tumor growth but also in combination with other therapeutics. IL-1 signaling has been shown preclinically to be involved in tumor development and chemoresistance of pancreatic cancer, and blockade of IL-1 was recently shown to have a significant clinical impact on development of lung cancer. IL-1 receptor associated protein (IL1RAP) is a coreceptor for the IL-1 receptor (IL1R1) and is required for IL-1 signaling. IL1RAP is expressed in a number of tumor tissues, including lung and pancreatic cancer, both on tumor cells and on infiltrating immune cells. We have, using antibodies directed against IL1RAP, shown the ability to target and kill IL1RAP-expressing tumor cells by ADCC, to inhibit IL-1 signaling in those cells and to reduce growth of transplanted human tumors in vivo. To study effects of IL1RAP targeting on the tumor microenvironment and in an immune competent setting, an antibody towards mouse IL1RAP was generated. This antibody potently blocks mouse IL-1 (IC50 = 13 nM), binds to IL1RAP protein with high affinity (Kd = 4,2 nM), labels IL1RAP-expressing cells and can be administered to mice with good pharmacokinetics. In vivo imaging shows that the antibody is not generally distributed in tissues but localizes to tumor sites after injection. Treatment of mice with orthotopically implanted 4T1 breast cancer cells did not reduce primary tumor growth significantly but reduced both the number of (47% reduction, p=0.02) and size of lung metastases. Interestingly, 4T1 tumor cells express low levels of IL1RAP and are not responsive to IL1RAP blockade, but the effects instead relate to effects on the tumor microenvironment. We conclude that targeting of IL1RAP can, in addition to induce ADCC of tumor cells and block their response to IL-1, also inhibit metastasis by affecting the tumor microenvironment.

#1770

Morab-202, a folate receptor alpha-targeted antibody-drug conjugate, shows a highly potent activity against a panel of FRA-expressing tumors.

Caroline Mignard,1 Coralie DURIX,1 Katherine Rybinski,2 Xin Cheng,2 Keiji Furuuchi,2 Earl Albone,2 Toshimitsu Uenaka,2 Marc Hillairet de Boisferon1. 1 _Oncodesign S.A., Dijon, France;_ 2 _Morphotek inc, Exton, PA_.

Folate receptor alpha (FRA) is a membrane protein with high affinity for binding and transporting folate into cells. Overexpression of FRA may confer a growth advantage to tumors by increasing folate uptake and affecting cell proliferation via alternative cell signaling pathways (1). FRA levels have been found to be elevated in tumors of epithelial origin compared to normal tissue as cancers of the breast (including TNBC (2)), colon, lungs and ovary (3).

In this study, we report the development of MORAb-202, an anti-FRA antibody-drug conjugate (ADC), consisting of a FRA-binding antibody (MORAb-003, farletuzumab) with a cathepsin-cleavable form of eribulin (eribulin mesylate, marketed as Halaven®), a highly potent anti-mitotic agent that induces cell-cycle arrest and cell death by targeting microtubules.

We first study expression of FRA on a large panel of tumors patient-derived xenograft (PDX) and Cancer Cell Line-derived Xenograft (CDX). Then, we performed in vitro and in vivo anti-proliferation assays and compare antitumor activity of MORAb-202 with free eribulin accordingly to the FRA expression level. FRA expression was found to be determinant in the sensitivity of tumor cells to the cytotoxic effect of the ADC. Moreover, in case of high expression of FRA, MORAb-202 showed a higher antitumor activity compared with free eribulin.

These results suggest that FRA expression could be used as a response-predictive biomarker for this targeted therapy. The ability to identify and treat patients with an effective therapy based on the known expression of the tumor marker is a key point in predictive medicine progress. These findings support the clinical development of MORAb-202 ADC as a novel targeted therapy for patients with FRA-expressing tumors.

The ADC described in this abstract is investigational, as efficacy and safety have not been established. There is no guarantee that this ADC will be available commercially.

1-Siu MK et al., PLoS One. 2012;7(11) 2-Nacela BM. et al., PLoS One. 2015, 10(3) 3-Cheung A. et al., Oncotarget. 2016 7(32), 52553-52574 4-Arrowsmith J. & Miller P. 2011-2012. Nat. Rev. Drug Discov. 12, 569 (2013). 5-Paul S.M. et al. Nat. Rev. Drug Discov. 9, 203-214 (2010). 6-Hidalgo, M. et al. Cancer Discov. 4, 998-1013 (2014).

#1771

BAY 1834942 is an immunotherapeutic antibody blocking the novel immune checkpoint regulator CEACAM6 (CD66c).

Joerg Willuda,1 Mark Trautwein,2 Jessica Pinkert,3 Wolf-Dietrich Doecke,1 Hans-Henning Boehm,3 Florian Wessel,3 Yingzi Ge,3 Eva Maria Gutierrez,3 Joerg Weiske,1 Christoph Freiberg,2 Uwe Gritzan,4 Julian Glueck,2 Dieter Zopf,1 Sven Golfier,1 Oliver von Ahsen,1 Ruprecht Zierz,1 Sabine Wittemer-Rump,1 Heiner Apeler,2 Ziegelbauer Karl,1 Rienk Offringa,3 Bertolt Kreft,1 Beckhove Philipp3. 1 _Bayer AG, Berlin, Germany;_ 2 _Bayer AG, Wuppertal, Germany;_ 3 _DKFZ/Bayer Joint Immunotherapy Lab, Heidelberg, Germany;_ 4 _Bayer AG, Cologne, Germany_.

CEACAM6 (CD66c) was previously shown to act as a novel immune checkpoint regulator suppressing the activity of effector T cells against tumors (Witzens-Harig et al., Blood 2013). CEACAM6 is a GPI-linked protein that is strongly expressed at the tumor cell surface in multiple cancer indications such as non-small cell lung adenocarcinoma (NSCLC), colorectal carcinoma (CRC), gastric adenocarcinoma and pancreatic cancer. In general, elevated CEACAM6 expression is associated with advanced tumor stages and poor prognosis. In vitro experiments showed that engagement of T-cells with CEACAM6, either expressed on tumor cells or presented on beads, resulted in suppression of TCR-mediated T-cell activation and ZAP70 phosphorylation. Based on these findings, we hypothesized that antibodies targeting CEACAM6 may be employed to enhance T-cell responses against CEACAM6-expressing cancers. Here we report the generation and characterization of BAY 1834942, a humanized monoclonal antibody selectively blocking the inhibitory impact of CEACAM6 on human T cells. There is no rodent ortholog of CEACAM6 precluding in vivo efficacy studies. In tumor cell / T cell co-culture systems, BAY 1834942 increased secretion of T-cell cytokines and effector molecules (e.g. IFNγ, TNFα, IL-2, granzyme B) and resulted in improved tumor cell killing. The effects of BAY 1834942 were dose-dependent, only observed in the context of CEACAM6-expressing tumor cells and could be reproduced in experiments using tumor cell lines and T-cell preparations from different sources, including T cells derived from tumor infiltrating lymphocytes from pancreatic cancer. BAY 1834942 is cross-reactive with the cynomolgus CEACAM6 ortholog and was well-tolerated in monkey toxicology studies. In summary, BAY 1834942 is a novel checkpoint inhibitor with potential for the treatment of patients with CEACAM6 expressing cancers, both as single agent and in combination with other checkpoint inhibitors. First-in-man trials are expected to commence in 2018.

#1772

Immunotherapy of colorectal cancer by the T-cell targeted DART® protein MGD007: Cellular mechanisms of action.

Paul A. Moore,1 Yinhua Yang,1 Kalpana Shah,1 Daorong Liu,1 Vatana Long,1 Douglas H. Smith,2 Zhuangyu Pan,2 Ralph Alderson,1 Liqin Liu,1 Syd Johnson,1 Ezio Bonvini1. 1 _MacroGenics, Inc., Rockville, MD;_ 2 _MacroGenics, Inc., South San Francisco, MD_.

Introduction

MGD007 (glycoprotein A33 x CD3), a DART protein designed to redirect T cells to target gpA33-expressing colon cancer, is presently undergoing clinical evaluation (NCT02238805). The gpA33 target was selected based on its universal expression profile across primary and metastatic CRC, including expression on putative cancer stem cell (CSC) populations. MGD007 activity in CRC cell cytolysis and its prolonged PK in non-human primates have previously been reported (Cancer Res 2014;74(19 Suppl): Abstract nr 669.1). Here we further characterize MGD007 cellular mechanisms associated with redirected T-cell killing, cytokine responses and modulation with steroids.

Method

Redirected killing assays were performed using luciferase labelled gpA33+ve Colo205 or RECA0201-GF colorectal cancer stem-like cells (CSLC) with freshly isolated PBMC or fractionated T-cell populations; Treg cells (CD4+, CD127lo, CD25+) were expanded for 14 days in presence of IL-2 and rapamycin and confirmed to be suppressive; steroids (budesonide and dexamethasone) were evaluated at pharmacologically relevant concentrations; multi-parameter FACS and ELISA were performed to determine cell surface marker expression and cytokine levels respectively.

Results

MGD007 displays potent redirected T-cell killing of gpA33+ve CRC cells, including complete lysis of CRC stem cell-like models. Importantly, MGD007 mediated cytolysis can be supported by various T-cell populations, including Treg cells. Following prolonged in vitro exposure to MGD007 and gpA33+ve tumor cells, expanded T cells acquire a memory phenotype and retain potent CTL activity when challenged with fresh gpA33+ve target cells; however, much decreased cytokine release was observed compared to that observed following initial T-cell exposure. The addition of dexamethasone or budesonide to freshly isolated effector cells and gpA33+ve target cells also reduces cytokine release levels to baseline in the presence of MGD007, with minimal impact observed on MGD007-mediated killing.

Discussion

MGD007 supports targeted lysis of CRC, including CSC subpopulations, and can leverage suppressive Tregs in addition to conventional T cells for cytolytic activity. Biological activity modulation is also feasible through induction of cytolytic Tmem cells with diminished cytokine release potential via sequential exposure to MGD007 or the simultaneous exposure to low-dose steroids. These data support further clinical development of MGD007 for the treatment of CRC patients.

#1773

HPN424, a half-life extended, PSMA/CD3-specific TriTAC for the treatment of metastatic prostate cancer.

Bryan Lemon,1 Wade Aaron,1 Richard Austin,1 Patrick Baeuerle,2 Manasi Barath,1 Adrie Jones,1 Susan D. Jones,1 Kathryn Kwant,1 Che-Leung Law,1 Anna Muchnik,1 Kenneth Sexton,1 Laurie Tatalick,1 Holger Wesche,1 Timothy Yu1. 1 _Harpoon Therapeutics, South San Francisco, CA;_ 2 _MPM Capital, Cambridge, MA_.

Metastatic, castrate-resistant prostate cancer (mCRPC) is diagnosed in up to 50,000 patients each year in the US alone, and roughly 27,000 patients will succumb to it every year. Once metastasized beyond regional lymph nodes, the 5-year survival rate is 30%. While novel therapeutics like abiraterone and enzalutamide have improved the treatment options for mCRPC, no curative treatment is available, and new therapies are urgently needed. HPN424 is a ~50-kDa antibody derivative called TriTAC (Tri-specific T cell Activating Construct) under development for the treatment of mCRPC. It is designed to simultaneously bind to CD3ε on T cells and to prostate specific membrane antigen (PSMA, FOLH1) on prostate cancer cells. A third domain of HPN424 binds non-covalently to serum albumin for extension of serum half-life. PSMA is expressed in >90% of malignant lesions of patients, and outside the central nervous system, its expression on normal tissue is largely restricted to the prostate. HPN424 binds human PSMA with sub-nanomolar affinity. When incubated in co-cultures with resting, human T cells and prostate cancer cells, it activates T cells and induces cytokine production, proliferation and redirected target cell killing with EC50 values in the single digit picomolar range. When administered to mice bearing human prostate cancer xenografts and human T cells, HPN424 eradicates subcutaneous tumors. The affinities of HPN424 for human and cynomolgus monkey CD3 and albumin are comparable, while HPN424 binds only marginally to cynomolgus PSMA. HPN424 is very well tolerated in non-human primates, even at high doses, indicating that CD3-binding has little if any pharmacological effect in the absence of target binding. Pharmacokinetic analysis supports weekly administration in humans. Our preclinical data suggest that HPN424 will be highly efficacious, safe and convenient for the treatment of patients with mCRPC.

#1774

External beam radiation therapy and androgen deprivation therapy increase PSMA expression in prostate cancer.

Marigdalia K. Ramirez-Fort,1 Sean S. Mahase,2 M. Junaid Niaz,2 He Liu,2 Vincent Navarro,2 Goran Rac,1 Harry S. Clarke,1 Scott T. Tagawa,2 Joseph M. Jenrette,1 Neil H. Bander,2 Christopher S. Lange3. 1 _Medical University of South Carolina, Charleston, NY;_ 2 _Weill Cornell Medicine, New York, NY;_ 3 _State Univ. of New York, Downstate Med Ctr, Brooklyn, NY_.

Purpose/Objective(s) Prostate-specific membrane antigen (PSMA), also known as folate hydrolase 1 (FOLH1), is a highly specific biomarker and therapeutic target for prostate cancer (PC). J591, a monoclonal antibody specific to PSMA, is the primary method used to detect PSMA expression. It is currently under investigation as a vehicle to deliver targeted therapies to PC cells. Androgen deprivation therapy (ADT) upregulates PSMA. Preclinical models of androgen resistant (AR) xenografts demonstrate PSMA upregulation by ADT increases the therapeutic index of J591-drug conjugates, suggestive of enhanced drug uptake. We sought to evaluate the effect of external beam radiation (EBRT) on PSMA expression in PC cells ± ADT. Methods Androgen sensitive LNCaP and AR CWR22Rv1 PC cell lines were cultured in standard and charcoal-stripped media for two weeks. Both cell lines were grown in both media conditions, and received either no EBRT, EBRT in 2 Gray (Gy) daily fractions to a maximum of 10 Gy, or 7.5 Gy in 1 fraction. Cell surface PSMA expression was measured by flow cytometry as mean fluorescence intensity 24 hours after each 2 Gy fraction, or on days 1, 3, 5, 7, and 9 after 7.5 Gy. Results LNCaP in standard media upregulated PSMA by 2.97-fold (SEM ± 0.72) with fractionated EBRT to a cumulative dose of 4 Gy, peaking on days 2 and 3 after 4 Gy and 6 Gy cumulative doses, respectively. CWR22Rv1 in standard medium, upregulated PSMA expression by 3.21-fold (SEM ± 0.44) after a cumulative dose of 8 Gy, peaking on day 4. Under ADT (charcoal-stripped media), EBRT did not further increase LNCaP cell PSMA expression (maximal fold-change of 1.01 (SEM ± 0.02)) relative to ADT alone. Compared to CWR22Rv1 with ADT alone, ADT plus EBRT upregulated PSMA by 3.73-fold (SEM ± 0.44) after a cumulative fractionated dose of 8 Gy, and peaking on days 4 and 5. However, 7.5 Gy in one fraction did not significantly increase PSMA expression of LNCaP or CWR22Rv1 in standard media. LNCaP showed a maximal fold-change of 1.14 (SEM ± 0.29) on day 9. CWR22Rv1 showed a maximal fold-change of 1.27 (SEM ± 0.02) on day 1. Conclusions We show PSMA expression is independently enhanced by ADT and by low-dose fractionated EBRT, but not with single-fraction ablative EBRT. These observations warrant additional studies using in vitro and in vivo PC models to validate our findings. PSMA upregulation may aid in optimizing J591 targeting for treatment of local and disseminated or micrometastatic disease. Validation of PSMA upregulation by ADT and EBRT supports use of J591-drug conjugates as an adjunct treatment to these standard therapies for PC. Furthermore, if fractionated EBRT upregulates PSMA, then targeted brachytherapy using appropriately selected J591-radionucleotide conjugates may increase its own therapeutic index in a time-dependent manner as a result of regional low-dose rate beta or alpha emission.

#1775

Development of fully humanized N-cadherin monoclonal antibodies for treatment of castration resistant prostate cancer.

Evelyn A. Kono, Naoko Kobayashi, Kirstin Zettlitz, Keyu Li, Joyce Yamashiro, Chun Wang, Anna Wu, Robert E. Reiter. _David Geffen School of Medicine at UCLA, Los Angeles, CA_.

Enzalutamide (ENZ) is an androgen receptor (AR) antagonist that targets various stages of the AR signaling pathway, and approved for the treatment of castration resistant prostate cancer (CRPC). Studies show increased survival in men with metastatic CRPC after chemotherapy when treated with ENZ. However, emergence of potential AR-independent mechanisms of castration resistance and resistance to these next generation AR inhibitors has proven to be a challenge. Previously, we reported an association of N-cadherin, a mesenchymal cadherin expressed on the cell surface, with tumor progression and castration resistance and resistance to AR inhibitors may lead to increase in N-cadherin. Targeting N-cadherin positive cells with specific monoclonal antibodies (mAb) affect tumor growth in both AR positive and negative prostate cancers. Co-targeting AR and N-cadherin with ENZ and mAb respectively show promise in addressing not only castration resistant tumor progression but also ENZ resistance. Ectopic expression of N-cadherin in N-cadherin negative cells enhanced cell growth and invasion in androgen-deprived conditions. Endogenously expressing N-cadherin prostate cancer cell lines (PC3, LAPC9) and androgen dependent prostate cancer cells ectopically expressing N-cadherin (LNCaP, MDA-PCa-2b, VCaP) were evaluated in vitro for invasion, growth, and self-renewal in the presence of monoclonal antibodies raised against various extracellular domains of N-cadherin and their effects in combination with ENZ. In vivo studies on promising candidates were performed using castration resistant N-cadherin and AR positive, as well as androgen-dependent tumors implanted subcutaneously in mice as xenografts. The tumors were analyzed for response to ENZ, mAb alone, or in combination. At end-point, tumors were harvested and further analyzed. Final candidate mAbs were then selected and re-formatted into fully humanized N-cadherin monoclonal antibodies and further evaluated. A molecular model of the mouse chimeric antibodies were built based on structure of similar antibodies and the CDR loops were grafted to the human framework region and back-translated into nucleotide sequences. Codon-optimized DNA encoding the humanized variable domains connected by a 15 glycine-rich amino acid linker was synthesized by GeneArt and sub-cloned into expression vectors containing light and heavy chain constant regions. Clones were then screened and selected. Candidates were transiently expressed in 293 cells and supernatant were collected and purified for further analysis. These newly generated humanized mAbs engineered from the promising human anti-mouse N-cadherin antibody candidates exhibited similar outcomes to their chimeric counterparts, revealing potential in providing methods for diagnosis and treatment of advanced diseases.

#1776

High-affinity anti-galectin-3 antibodies targeting oncogenic properties in serous ovarian cancer.

Dharmarao Thapi,1 Marina Stasenko,1 Thomas White,1 Sven J. Walderich,2 Noah Feit,1 Frances Weis-Garcia,1 David R. Spriggs1. 1 _Mem. Sloan Kettering Cancer Ctr., New York, NY;_ 2 _Weill Cornell Medical College, New York, NY_.

Objective: Galectin-3 (LGALS3) regulates the interaction of surface proteins with the extracellular membrane domain and mediates a signal cascade leading to invasion, oncogene activation and growth. We targeted LGALS3 cancer promotion by developing high-affinity anti-galectin-3 antibodies directed either at the carbohydrate recognition domain (CRD) of the galectin-3 carboxyl-terminus (to block sugar binding) or at the N-terminus (NT) (to inhibit N-terminal mediated oligomerization of galectin-3) in order to prevent formation of the cell surface galectin lattice and suppress the oncogenic effects of the glycoprotein.

Methods: Two distinct types of murine monoclonal antibodies (MAb) were generated by creating hybridomas from mice immunized either with a fusion protein derived from the 128-amino acid LGALS3 carboxyl-terminus fused to a human IgG1-Fc backbone followed by immunization with a recombinant human LGALS3 protein, or the mice were further immunized with a 25-amino acid peptide derived from the LGALS3-N-terminus conjugated to KLH. The resulting antibodies were confirmed to recognize either the LGALS3 CRD or NT by ELISA. Functional assays (Matrigel invasion, inhibition of laminin binding, and oncogene expression) and cell surface expression by FACS analysis were utilized to confirm MAb binding and activity. Mice bearing A2780 and SKOV3 ovarian cancer cell lines were treated with purified MAb to determine the effects of MAb on tumor growth in vivo. Data were analyzed for statistical significance using Student's t-test.

Results: Initial screening yielded two candidate antibodies, of which one (named 14D11.2D2) had superior binding to galectin-3 by ELISA and Surface Plasmon Resonance (SPR). The antibody dissociation constant was 14.6 nM by SPR. At a concentration as low as 150 nM, the 14D11.2D2 MAb decreased galectin-3 binding to laminin by 36.6% compared to untreated control. In a Matrigel invasion assay, the anti-CRD MAb significantly inhibited invasion by MUC16 expressing SKOV3 cells and by wild-type MUC16 positive OVCAR3 cells. MUC16 expressing A2780 cells showed a statistically nonsignificant decrease in invasion. In vivo experiments with MUC16 expressing A2780 or SKOV3 tumors showed a significant inhibition of tumor growth in mice treated with anti-CRD MAb. Six candidate antibodies directed against the LGALS3-N-terminus have been identified. Characterization of two NT antibodies (1F5.D4 and 7D4.A1) is under way via in in vitro and in vivo experiments.

Conclusion: High-affinity CRD and NT anti-galectin-3 antibodies inhibit MUC16-induced cellular invasion and growth. Anti-LGALS3 MAbs hold therapeutic potential for inhibition of glycosylation-dependent invasion.

#1777

REGN4018, a novel MUC16xCD3 bispecific T-cell engager for the treatment of ovarian cancer.

Alison Crawford, Lauric Haber, Kristin Vazzana, Lauren Canova, Priyanka Ram, Jennifer Principio, Arpita Pawashe, Curtis Colleton, Marcus Kelly, Sosina Makkonen, Carlos Hickey, Paurene Duramad, Stephen Godin, John Lin, Eric Smith, Gavin Thurston, Jessica R. Kirshner. _Regeneron Pharmaceuticals, Tarrytown, NY_.

Advanced ovarian cancer has a high rate of recurrence, thus there is a need for therapies that can produce durable responses and extend overall survival. Infiltrating CD3+ T cells are reported to correlate with improved clinical outcome in stage III/IV ovarian cancer. Bispecific antibodies that engage T cells via CD3 and a tumor antigen on ovarian tumor cells may be able to exploit these tumor-infiltrating T cells and have drawn interest as a novel strategy of antitumor immunotherapy.

Mucin16 (MUC16) is a well described antigen highly expressed in ovarian cancer and several other tumors. We generated a human bispecific antibody (REGN4018) that binds MUC16 on tumor cells and CD3, bridging MUC16-expressing cells with CD3+ T cells. REGN4018 demonstrates MUC16-directed T cell activation and polyclonal T cell killing of MUC16-expressing tumor cells in vitro and in vivo. Binding and cytotoxicity are minimally affected by high concentrations of CA-125, the shed form of MUC16 that is used as a biomarker for ovarian cancer.

Several murine tumor models were developed to determine the in vivo anti-tumor effects of REGN4018. As a xenogenic tumor model, human OVCAR-3 cells were grown as ascites in immunodeficient mice [NOD-SCID-IL2Rgamma deficient (NSG)] pre-implanted with human PBMC. Significant tumor inhibition by REGN4018 was observed at doses of ≥ 0.1 mg/kg. To enable investigation of clinical bispecific antibodies in tumor-bearing immune competent mice, mice were genetically engineered to humanize both CD3 and a part of MUC16 covering the antibody binding region. ImmunoPET imaging demonstrated localization of REGN4018 in both T cell-rich organs such as the spleen and lymph nodes as well as in MUC16-expressing tumors. REGN4018 inhibited growth of murine tumors transfected with human MUC16 in syngeneic tumor models at doses ≥ 0.05 mg/kg.

The safety and tolerability of REGN4018 were evaluated in cynomolgous monkey studies. REGN4018 administration resulted in a minimal and transient increase in serum cytokines and C-reactive protein with no overt toxicity. Collectively, these data show the potent anti-tumor activity and tolerability of REGN4018 and provide strong support for the clinical testing of REGN4018 in patients with MUC16-expressing ovarian cancers.

#1778

Preclinical characterization of the safety and antitumor activity of IMAB027-vcMMAE, an anticlaudin 6 antibody-drug conjugate.

Özlem Türeci,1 Maria Kreuzberg,2 Korden Walter,2 Stefan Wöll,2 Ramona Schmitt,2 Tomohiro Yamada,3 Ikumi Nakajo,3 Ugur Sahin4. 1 _CI3 – Cluster of Individualized Immune Intervention, Mainz, Germany;_ 2 _Formerly of Ganymed Pharmaceuticals GmbH, Mainz, Germany;_ 3 _Astellas Pharma, Inc, Tokyo, Japan;_ 4 _TRON – Translational Oncology at the University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany_.

Background Claudin 6 (CLDN6) is a tight junction membrane protein whose expression in normal tissue is confined to embryonic cells, but is aberrantly expressed in various human cancers. The anti-CLDN6 monoclonal antibody (mAb), IMAB027, has shown promising antitumor activity in preclinical human CLDN6-positive (CLDN6+) cancer models. Conjugation of IMAB027 with monomethyl auristatin E (MMAE) may utilize the precision tumor-targeting of the mAb to deliver a highly effective cytotoxic drug to the tumor. In this report we present the preclinical characterization of this antibody–drug conjugate, IMAB027–vcMMAE.

Methods Internalization of IMAB027 in various CLDN6+ human ovarian (OC) and testicular cancer (TC) cell lines was assessed by immunofluorescence, flow cytometry, and Fab-ZAP internalization assay. Binding characteristics of IMAB027–vcMMAE were examined by flow cytometry. Cell viability and IMAB027–vcMMAE-mediated cytotoxic effects (direct and indirect [bystander]) were assessed in cell cultures by the XTT metabolic assay. Apoptosis was evaluated by caspase 3/7, annexin V, and TUNEL assays. Xenograft mouse tumors were generated by injecting human OC cells into nude mice to assess the safety and antitumor activity of IMAB027–vcMMAE.

Results IMAB027–vcMMAE binds robustly to, and is internalized by, cell lines expressing CLDN6. IMAB027–vcMMAE reduced the viability of CLDN6+ OC and TC cells by up to 100% with EC50 values in the ng/mL order. IMAB027–vcMMAE induced apoptosis in CLDN6+ cells in a dose-dependent manner. Additionally, after conjugation, IMAB027–vcMMAE retained IMAB027's ability to induce CLDN6+ cell death via antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity. Cell lines that did not express CLDN6 were unaffected by IMAB027–vcMMAE in monocultures; however, in cocultures of CLDN6+ and CLDN6-negative cells, IMAB027–vcMMAE exerted bystander effect, resulting in the death of cocultured CLDN6-negative cells in addition to the target-bearing CLDN6\+ cells. In vivo, significant antitumor effects were observed after a single intravenous administration of 16 mg/kg IMAB027–vcMMAE in mouse OC xenografts. Further, xenograft tumors with low and/or heterogeneous CLDN6 expression treated with IMAB027–vcMMAE showed efficient tumor size reduction. Repeated dosing of IMAB027–vcMMAE was well tolerated in mice, with no physical abnormalities, changes in behavior, or alterations in appearance observed.

Conclusions IMAB027–vcMMAE is a specific antibody–drug conjugate against CLDN6 that induces potent antitumor activity in CLND6+ tumor cells in vitro and in vivo. Furthermore, IMAB027–vcMMAE was able to induce antitumor effects in tumors with low and/or heterogeneous target expression, which may be driven by bystander activity.

#1779

Anti-Müllerian hormone type II receptor (AMHRII), a cancer target for GM103, a novel antibody-drug conjugate (ADC).

Olivier Dubreuil,1 Jean-Marc M. Barret,1 Raghav Mohan,2 Martine Pugnière,3 David Pepin,2 Delphine Desigaud,1 William McDowell,4 Anaïs Manin,4 Nicolas Camper,4 André Nicolas,5 Patricia K. Donahoe,2 Jean-François F. Prost1. 1 _GamaMabs Pharma, Toulouse, France;_ 2 _Massachusetts General Hospital, Boston, MA;_ 3 _Cancer Research Institute Montpellier, Montpellier, France;_ 4 _PolyTherics, Cambridge, United Kingdom;_ 5 _Curie Institute, Paris, France_.

The anti-Müllerian hormone (AMH) belongs to the TGF-β family and plays a key role during fetal sexual development. Its receptor, AMHRII, is selectively expressed in normal sexual organs in healthy adults. RT-PCR and In Situ Hybridization studies have confirmed that AMHRII expression in normal tissues is restricted to ovary, testis and adrenal gland. AMHRII is re-expressed in gynecologic cancers including ovarian tumors especially in progenitor cancer cells, which makes it an outstanding antigen to be exploited as a target for a novel ADC.Recently, AMHRII expression was also demonstrated in major cancers such as colorectal, non-small cell lung and hepatocarcinoma cancers. GM103 is an ADC, composed of a humanized IgG1 antibody against AMHRII, linked via a cleavable linker to a potent auristatin derivative, using a site-directed conjugation methodology. In this study, we evaluated the in-vitro and in-vivo (xenograft and PDX) efficacy profile of GM103.In vitro experiments using GM103 showed high affinity for AMHRII (KD < 60 pM) and AMHRII-expressing tumor cells with no interference with AMH binding. Interestingly, Biacore experiments demonstrated that GM103 binding to CD16a was reduced whereas binding to FcRn was maintained, which was similar to that of the antibody alone. Using cultured in-vitro cancer cell lines, GM103 demonstrated a powerful, specific and target-dependent cytotoxic activity. Both immunofluorescence and flow cytometry studies demonstrated a rapid cellular internalization of GM103 within 60 minutes when cells were treated with GM103 at 10µg/mL. The presence of GM103 in the tumor cell lysosomes was confirmed by immunofluorescence using an anti-LAMP1 antibody.Twelve AMHRII expressing primary patient-derived ovarian cancer cell lines (previously verified by qPCR) were grown as spheroids and treated with GM103 at 20µg/mL for two weeks. The results of these experiments demonstrated cellular growth inhibition of at least 75% in 9 of the 12 cell lines tested. In an in-vivo study, tumor-free survivors were observed following a single IV dose of GM103, when an AMHRII+ COV434 xenograft model was used at concentrations as low as 5mg/kg. In addition, when GM103 was tested at 10 mg/kg against a hepatocarcinoma PDX model, a tumor growth inhibition similar to that of Sorafenib, the current standard of care, was observed.In conclusion, these results demonstrated that AMHRII is a very good target for treating AMHRII+ solid tumors with an ADC. GM103 showed effective in-vitro and in-vivo cytotoxicity that could be exploited with highly cytotoxic payloads.

#1780

Anetumab ravtansine has single-agent activity in mesothelin-expressing human ovarian cancer models and potentiates the activity of chemotherapeutics and targeted agents.

Christoph A. Schatz,1 Maria Quanz,1 Urs B. Hagemann,1 Sabine Zitzmann-Kolbe,1 Beatrix Stelte-Ludwig,1 Sven Golfier,1 Charlotte Kopitz,1 Cem Elbi,2 Karl Ziegelbauer,1 Dominik Mumberg1. 1 _Bayer AG, Berlin, Germany;_ 2 _Bayer AG, Whippany, NJ_.

Ovarian cancer remains an area of high unmet medical need, with 239,000 patients newly diagnosed per year. Here we describe the mesothelin-targeting antibody drug-conjugate anetumab ravtansine as a novel treatment option for ovarian cancer. Internalization of anetumab ravtansine and co-localization with lysosomal markers in ovarian cancer cells was accompanied by rapid resynthesis of mesothelin, which may allow consecutive anetumab ravtansine treatment cycles. The strong antitumor activity of anetumab ravtansine was preserved during repeated treatment cycles in the OVCAR-3 ovarian cancer in vivo model. Mechanistically, treatment with anetumab ravtansine caused mitotic arrest characterized by increased pHH3 signal and monopolar spindle structures. Interestingly, anetumab ravtansine monotherapy also induced DNA damage as indicated by focal γH2AX signals. Ultimately, the cells engaged in apoptotic cell death. Strong in vitro monotherapy activity was demonstrated in a set of 11 ovarian cancer cell lines with IC50s between 3 and 90nM. The in vivo efficacy of anetumab ravtansine dosed at 2.5 mg/kg Q3Dx3 i.v. anetumab ravtansine was evaluated in 9 human ovarian cancer models with varying mesothelin expression levels. Strong in vivo efficacy at this moderate dose with T/C <0.2 was seen in OVCAR-3 cell line and the PDX model OvCa6668. Efficacy was generally higher in tumors with strong mesothelin expression. No activity was seen in mesothelin-negative ovarian cancer models. Combination with the PI3K inhibitor copanlisib was additive in the OVCAR-3 and OVCAR-8 cell line in vitro, showing increased apoptosis in the combination treatment. In vivo, anetumab ravtansine + copanlisib in OVCAR-3 tumors consistently resulted in greater efficacy than with either treatment alone. Both single treatments and the combination were well tolerated. Combination of anetumab ravtansine with bevacizumab, the only antibody therapy approved for ovarian cancer to date, also showed increased in vivo efficacy in the combination while being well tolerated. Next combination with PEGylated doxorubicin (PLD) was investigated. The combination of anetumab ravtansine plus PLD in OVCAR-8 cells showed additive effects and in vivo in OVCAR-8 tumors, the combination consistently showed better therapeutic efficacy than either therapy alone. Overall, these data support the development of anetumab ravtansine in ovarian cancer and suggest combinations with copanlisib, bevacizumab or PLD.

#1781

HPN536, a T cell-engaging, mesothelin/CD3-specific TriTAC for the treatment of solid tumors.

Richard Austin,1 Wade Aaron,1 Patrick Baeuerle,2 Manasi Barath,1 Adrie Jones,1 Susan D. Jones,1 Che-Leung Law,1 Kathryn Kwant,1 Bryan Lemon,1 Anna Muchnik,1 Kenneth Sexton,1 Laurie Tatalick,1 Holger Wesche,1 Timothy Yu1. 1 _Harpoon Therapeutics, South San Francisco, CA;_ 2 _MPM Capital, Cambridge, MA_.

Mesothelin (MSLN) is a GPI-linked tumor antigen overexpressed in a variety of solid tumors, including ovarian, pancreatic, lung and triple-negative breast cancer. Normal tissue expression is restricted to single-cell, mesothelial layers lining the pleural, pericardial, and peritoneal cavities. Overexpression of MSLN is associated with poor prognosis in lung adenocarcinoma and triple-negative breast cancer. MSLN has been used as cancer target antigen for numerous modalities, including immunotoxins, vaccines, antibody drug conjugates and CAR-T cells. Early signs of clinical efficacy have validated MSLN as target, but therapies with improved efficacy are still needed to address the significant, unmet medical need posed by MSLN-expressing cancers. HPN536 is a ~50-kDa antibody derivative called TriTAC (Tri-specific T cell Activating Construct) designed to simultaneously bind to MSLN on tumor cells and to CD3ε on T cells with an affinity of 1 nM and 14 nM, respectively. Transient bispecific binding leads to the formation of an immunological cytolytic synapse, T cell activation and redirected tumor cell killing. A third domain of HPN536 binds non-covalently to serum albumin with an affinity of 8 nM to extend serum half-life life. Because TriTACs are built using single domain antibodies, TriTACs are much smaller than full size antibodies and are anticipated to demonstrate improved penetration of human tumors compared to full sized antibodies. HPN536 is produced by eukaryotic cell culture and secreted as a highly stable, single polypeptide. It binds with similar affinity to human and cynomolgus MSLN, albumin and CD3. When incubated in co-cultures with resting, human or cynomolgus T cells and human tumor cells, T cells are induced to release cytokines, to proliferate, and to specifically lyse MSLN-positive target cells with EC50 values at single-digit picomolar concentrations. In an exploratory toxicological study in non-human primates, HPN536 was well tolerated and showed pharmacokinetics in support of weekly dosing in humans. Preclinical characterization suggests that HPN536 is an efficacious and safe novel therapeutic candidate for the convenient treatment of patients with MSLN-expressing malignancies.

#1782

Discovery and activity of STRO-002, a novel ADC targeting folate receptor alpha for ovarian and endometrial cancer.

Xiaofan Li, Cristina Abrahams, Sihong Zhou, Stellanie Krimm, Robert Henningsen, Heather Stephenson, Jeffrey Hanson, Mary Rose Masikat, Krishna Bajjuri, Tyler Heibeck, Cuong Tran, Gang Yin, James Zawada, Ganapathy Sarma, Joy Chen, Maureen Bruhns, Willy Solis, Alexander Steiner, Adam Galan, Toni Kline, Ryan Stafford, Alice Yam, Venita I. De Almeida, Mark Lupher, Trevor Hallam. _Sutro Biopharma, Inc., South San Francisco, CA_.

Folate receptor alpha (FolRα) is a glycosylphosphatidylinositol linked cell-surface glycoprotein that is widely expressed in serous and epithelial ovarian cancer, endometrial adenocarcinoma, non-small cell lung cancer and triple negative breast cancer. In contrast, FolRα expression is highly restricted on normal tissues, making it a highly promising target for cancer therapy using antibody drug conjugates (ADCs). We have designed a novel, FolRα-targeting ADC, STRO-002, with potent cytotoxic activity on FolRα expressing tumors in vitro and in vivo, including in cells with low expression levels (~0.2 million copies/cell) of FolRα.

STRO-002 contains the anti-FolRa human IgG1 antibody (SP8166) conjugated to a proprietary cleavable drug-linker (SC239). SC239 contains a tubulin-targeting 3-aminophenyl hemiasterlin warhead, SC209, which has potent cytotoxic activity and is a weak substrate for efflux pumps. SP8166 was discovered and optimized using a Fab ribosome display selection and screening platform based on Sutro's Xpress CF+TM system. Four non-natural amino acid p-azidomethyl phenylalanine (pAMF) residues are incorporated into SP8166 at two defined sites on each heavy chain. These sites were selected based on optimal stability and activity in vitro and in vivo. The SC239 drug-linker is conjugated via a cleavable valine citrulline p-aminobenzyl carbamate linker functionalized with dibenzocyclooctyne (DBCO). The rapid and selective reaction of DBCO and pAMF results in a well-defined, homogeneous ADC with a drug-antibody ratio (DAR) of ~4.

STRO-002 has potent cytotoxic activity (0.1-3 nM) on multiple FolRα-positive ovarian cancer cell lines in vitro and demonstrates strong anti-tumor response in KB, Igrov1 and OvCAR3 xenograft models in vivo. On Igrov1 xenografts, STRO-002 exhibits dose-dependent tumor growth inhibition starting at a single dose as low as 2.5 mg/kg. Evaluation of in vivo activity of STRO-002 in additional xenograft and PDX models, as well as in combination studies with chemotherapeutic agents is ongoing. Data from exploratory safety studies of STRO-002 in cynomolgus monkey and SC209 (active catabolite) in rats show a favorable safety profile. Our data suggests that STRO-002 is a promising clinical candidate for ovarian cancer, including tumors with low expression levels of FolRα, and IND enabling studies are currently being conducted.

#1783

UMG1, a novel humanized monoclonal antibody against a glysosylated-CD43-related epitope, induces antibody-dependent cellular cytotoxicity (ADCC) on human T-cell acute lymphoblastic leukemia cells.

Cirino Botta,1 Maria E. Gallo Cantafio,1 Chiara Buracchi,2 Maria A. Siciliano,1 Maria Cucè,1 Caterina Riillo,1 Franca M. Tuccillo,3 Daniele Caracciolo,1 Emanuela Altomare,1 Mariamena Arbitrio,1 Maria T. Di Martino,1 Marco Rossi,1 Andrea Biondi,4 Giuseppe Gaipa,4 Pierosandro Tagliaferri,1 Pierfrancesco Tassone1. 1 _Magna Graecia University of Catanzaro, Catanzaro, Italy;_ 2 _Tettamanti Research Center,Centro Maria Letizia Verga, "San Gerardo" Hospital, Monza, Italy;_ 3 _Molecular Biology and Viral Oncogenesis Unit, Department of Experimental Oncology, Istituto Nazionale Tumori "Fondazione G. Pascale", Naples, Italy;_ 4 _Tettamanti Research Center, Centro Maria Letizia Verga, "San Gerardo" Hospital, Monza, Italy_.

T-cell acute lymphoblastic leukemia (T-ALL) is a hematologic malignancy accounting for about 25% of all acute leukemias. Due to the poor therapeutic scenario and severe prognosis of T-ALL, novel drugs are eagerly awaited. The targeting of tumor-associated antigens by monoclonal antibodies (mAb) for induction of immune-mediated cellular cytotoxicity is presently a promising immunotherapeutic strategy. We previously developed a novel mAb direct against a heavy glycosylated oncofetal epitope of CD43 (UN1) with known potential application as therapeutic and diagnostic tool. By screening different cancer cell lines, we observed that UN1 is highly and selectively expressed on malignant T-ALL cells. The expression of UN1 was then evaluated in 38 T-ALL patient-derived blasts and high correlation for a specific subset of patients (about 80%) belonging to the cortical T-ALL group (EGIL T3) was detected. Accordingly, we developed a humanized mAb, (UMG1) and an afucosylated enginereed version (a-UMG1). Therefore, to elucidate the mechanism of action of these mAbs, we investigated complement-mediated cytotoxicity (CDC), ADCC and antibody-dependent cellular phagocytosis (ADCP). To this aim, T-ALL cells (HPB-ALL and CCRF-CEM) have been cultured in the presence of complement, peripheral blood mononuclear cells (PBMCs), NK-92-CD16+ cells or macrophages, using increasing concentrations of both mAbs. Notably, we observed that mAbs treatment alone did not exert either cellular cytotoxicity or CDC on target cells. Conversely, both mAbs induced significant ADCC mediated by PBMCs and NK-92-CD16+ cell line in term of NK degranulation and cytotoxicity and macrophage-mediated ADCP. In vivo results demonstrated a powerful activity of UMG1 in 3 different models of T-ALL in NSG mice in the presence or absence of NK-92-CD16+ cells. Specifically, in two subcutaneous models, we observed a strong ability of both mAbs to delay tumor growth and increase survival of treated mice. As expected, the combinatory treatment with NK-92-CD16+ cell line strongly improved the activity of a-UMG1. Importantly, in the orthotopic disseminated model, which better reproduces the human T-ALL disease, we observed 5 out of 20 treated mice alive and free of disease 100 days after injection. Furthermore, to explore the possibility of combination therapy, the modulation of UN1 expression by chemotherapeutic agents in T-ALL was investigated. Interestingly, we observed that methotrexate and doxorubicin , alone or in combination, increased UN1 expression at low nanomolar concentrations, and improved ADCC in vitro. In conclusion, we demonstrated that UMG1 and a-UMG1 represent a novel promising immune-therapeutic tool for the treatment of T-ALL patients.

#1784

IL-7R targeting therapy for immunoregulation and overcoming steroid resistance in cancer and autoimmune disease.

Masahiro Yasunaga,1 Shino Manabe,2 Yasuhiro Matsumura1. 1 _National Cancer Ctr. EPOC, Kashiwa, Japan;_ 2 _RIKEN, Wako, Japan_.

The occurrence of autoimmune reactions caused by immune checkpoint blockade in the treatment of cancer indicates the importance of the cross-disciplinary study of malignancy and autoimmune disease. Meanwhile, although steroids have been commonly used for the treatment of malignancies and immune diseases, steroid resistance is a serious prognosis factor and remains an unsolved problem. IL-7R signaling, which physiologically regulates lymphocyte growth and survival, including antigen-responsive T lymphocyte selection, has been implicated in the development of malignancies and autoimmune diseases. However, the biological significance of IL-7R-signaling in steroid treatment is poorly understood. Here, we identified the relationship between unique IL-7R-signaling and steroid-resistance in lymphoid malignancy and demonstrated the presence of steroid-resistant IL-7R-positive lymphocytes in a mouse bone marrow and spleen or in a mouse model of autoimmune arthritis following steroid treatment. We further showed that an anti-IL-7R-antibody conjugated with SN-38 (A7R-ADC-SN-38) has strong anti-tumor effects against both parent and steroid-resistant malignant cells. Although A7R-ADC-SN-38 efficiently eliminated IL-7R-positive cells, IL-7R-negative mature lymphocytes were preserved. Furthermore, inflammation in the mouse autoimmune arthritis model was suppressed to a greater extent by A7R-ADC conjugated to MMAE than by A7R-ADC-SN-38. Strong and specific elimination of enhanced IL-7R-positive cells, a common pathogenesis of both lymphoid malignancy and autoimmune disease, might prevent the development of malignancy or autoimmune disease in high-risk patients. Thus, the use of A7R-ADC may be a promising strategy for the immunoregulation of both malignancy and autoimmune disease and may serve as a new option to steroid therapy. Thus, A7R-ADC may be a promising strategy to treat malignancies and autoimmune diseases and may serve as a novel alternative to steroid therapy. Thus, A7R-ADC may be a promising strategy to treat malignancies and autoimmune diseases and may serve as a novel alternative to steroid therapy. We have evaluated the effectiveness of A7R-ADC in the treatment of other autoimmune or inflammatory diseases involving IL-7R signaling in a preclinical setting for general use. In addition, IL-7R metastatic solid tumors which acquired Il-7R-dependent homing ability of lymphocytes to be spread into many organs may also be promising therapeutic targets of A7R-ADC. We are proceeding the study to advance A7R-ADC for clinical use in both cancer and autoimmune disease

#1785

Pre-clinical development of a novel CD3-CD123 bispecific T-cell engager using Cross-Over-Dual-Variable-Domain (CODV) format for the treatment of acute myeloid leukemia (AML).

Helene Bonnevaux,1 Stephane Guerif,1 Jana Albrecht,2 Erwan Jouannot,1 Laurent Bassinet,1 Agnès Vergezac,1 Christian Beil,2 Christian Lange,2 Wulf Dirk Leuschner,2 Anne Caron,1 Celine Amara,1 Cedric Barriere,1 Justine Siavellis,3 Valerie Bardet,3 Ernesto Luna,4 Donald Drake,4 Ercole Rao,2 Corina Oprea,1 Peter Wonerow,2 Chantal Carrez,1 Veronique Blanc,1 Karl Hsu,5 Dmitri Wiederschain,5 Paula G. Fraenkel5. 1 _Sanofi R &D, Vitry sur seine Cedex, France; _2 _Sanofi R &D, Germany; _3 _INSERM, Paris, France;_ 4 _Sanofi-Pasteur, Orlando, FL;_ 5 _Sanofi R &D, Cambridge, MA_.

Acute myeloid leukemia (AML) is characterized by the accumulation of abnormal blast cells in the bone marrow and blood. While high intensity chemotherapy and allogeneic stem cell transplantation cure a subset of patients with AML, many patients are ineligible or do not respond sufficiently to these therapies. One potential reason for treatment failure in a particular patient may be the inability to reach and eliminate residual leukemic stem cells (LSCs) located in the bone marrow. T-cell mediated cytotoxicity, targeting LSCs with high expression of certain leukemic antigens, represents an attractive therapeutic strategy for relapsed and refractory AML. In this study, the proprietary Cross-Over-Dual-Variable-Domain (CODV) format was applied to a fully humanized IgG1 backbone with reduced Fc functionality, resulting in a bispecific T-cell engager (TCE), CD123-CODV-TCE, that binds to both CD3 on T cells and CD123 (α-chain of the interleukin-3 receptor) on AML blasts and LSCs. CD123-CODV-TCE displayed high affinity for human CD123 and medium affinity for human CD3 proteins. As expected, CD123-CODV-TCE activated CD4-positive and CD8-positive T cells only in the presence of cells expressing the CD123 target, such as THP1 (an AML tumor cell line), and induced killing of these cells with an EC50 in a picomolar range. Potential cytotoxic activity of CD123-CODV-TCE was also evaluated on CD123-expressing normal blood cells such as plasmacytoid dendritic cells (pDC) and monocytes. CD123-CODV-TCE was shown to deplete pDC and monocyte from human Healthy Donor (HD) Peripheral Blood Mononuclear Cells (PBMC) with an EC50 in the picomolar range. This efficacy correlated with the release of numerous cytokines, thus highlighting the potential risk of cytokine release syndrome as described for other TCE's. In an in vivo disseminated AML model using CD123+ Molm13-luc human AML cell line, treatment of the mice with CD123-CODV-TCE suppressed AML tumor growth in the bone marrow compartment following co-injection of primary human T cells. In this murine pre-clinical model, CD123-CODV-TCE displayed favorable pharmacokinetic properties with a terminal half-life of 3 days. To investigate CD123-CODV-TCE activity on myeloid blast cells and LSC, in vivo efficacy studies were performed in NSG mice injected with primary human AML cells obtained from patients. In this model, CD123-CODV-TCE induced the killing of primary AML cells by activating human autologous T cells. Taken together, these results indicate that CD123-CODV-TCE can potently and specifically kill CD123+ leukemic cancer cells in vitro and in vivo. CD123-CODV-TCE therefore represents a potential candidate for future clinical development in relapsed and refractory AML.

#1786

APVO436, a bispecific anti-CD123 x anti-CD3 ADAPTIR™ molecule for redirected T-cell cytotoxicity, induces potent T-cell activation, proliferation and cytotoxicity with limited cytokine release.

Michael R. Comeau, Robert E. Miller, Robert Bader, Rebecca Gottschalk, Mollie Daugherty, Toddy Sewell, Lynda Misher, Lara Parr, Melissa DeFrancesco, David Bienvenue, Catherine J. McMahan, Gabriela H. Hoyos, Jane A. Gross. _Aptevo Therapeutics, Seattle, WA_.

Introduction: Depletion of CD123 over-expressing malignant cells provides a potential new treatment option which may improve patient outcomes in several hematological malignancies. CD123 is over-expressed in AML, MDS, ALL, CML, HCL and BPDCN and infrequently expressed by normal cells making it an attractive target which is being pursued using a number of different approaches including T-cell engaging immunotherapy. Cytokine release syndrome is a significant concern with T-cell activating therapeutics which has led to severe complications in clinical trials. We have developed APVO436, a bispecific anti-CD123 x anti-CD3 ADAPTIR molecule for redirecting T-cell cytotoxicity to CD123-expressing tumor cells. A potential advantage of the ADAPTIR platform is reduced cytokine release upon T-cell engagement compared to other formats (Mol Cancer Ther. 2016 Sep;15(9):2155-65). Here we present in vitro and in vivo activity of APVO436 and compare the activity of APVO436 to another anti-CD123 x anti-CD3 bispecific containing the amino acid sequence of MGD006.Methods: Binding, T-cell activation and proliferation were assessed using multi-color flow cytometry. Cytotoxic activity was determined using chromium release assays and flow cytometry. PBMC samples were obtained from normal donors and AML patients. In vivo studies were performed using NSG mice transplanted with human PBMC's. The CD123 and CD3 binding domain sequences for flotuzumab (MGD006) were obtained from patent W02015026892 engineered in Macrogenic's dual-affinity re-targeting format as reported in Sci Transl Med. 2015 May 27;7(289):289ra82.Results: Dose-dependent cytotoxicity of CD123 expressing tumor cell lines and primary AML cells was induced by APVO436 at low effector to target ratios, accompanied by T-cell activation and proliferation. APVO436 induced significantly lower levels of several T-cell cytokines including IFNγ, IL-2, and TNFα compared to the molecule in the dual-affinity re-targeting format. In vivo, APVO436 significantly reduced established tumor burden in xenograft murine models.Conclusions: APVO436 potently induces T-cell activation, proliferation and CD123+ cell depletion with AML and normal donor samples and CD123 expressing tumor cell lines with limited levels of T-cell cytokine release compared to another CD123 x CD3 targeting bispecific format suggesting a potential safety advantage. APVO436 inhibits tumor-growth in sub-cutaneous tumor models with IV-implanted human T cells, indicating migration and engagement of T cells at the tumor site. These data are supportive of further investigation of APVO436 as a potential treatment option for AML and other hematological malignancies. GLP toxicology studies have been completed in non-human primates and APVO436 is advancing to clinical testing.

#1787

COVA4231, a potent CD3/CD33 bispecific FynomAb with IgG-like pharmacokinetics for the treatment of acute myeloid leukemia.

Kristina Klupsch,1 Vanessa Baeriswyl,1 Roland Scholz,1 Joana Dannenberg,1 Roger Santimaria,1 David Senn,1 Elena Kage,1 Adrian Zumsteg,1 Isabella Attinger-Toller,1 Ulrike von der Bey,1 Susann König-Friedrich,1 Wibke Lembke,1 Clara Albani,1 Severin Wendelspiess,1 Lucijana Dinkel,1 Chelsea J. Gudgeon,2 Roland B. Walter,2 Julian Bertschinger,1 Simon Brack1. 1 _Covagen, one of the Janssen Pharmaceutical Companies of J &J, Schlieren, Switzerland; _2 _Fred Hutchinson Cancer Research Center, Seattle, WA_.

Acute myeloid leukemia (AML) is an aggressive blood cancer with neoplastic infiltration of leukemic blasts in blood, bone marrow and viscera. Current treatment options have limited efficacy, and the 5-year survival rate is only 27%. CD3 bispecific antibodies that re-direct T cells towards the tumor have shown promising efficacy in hematological malignancies. The CD3/CD19 bispecific antibody blinatumomab has recently been approved by the FDA for the treatment of B-cell acute lymphocytic leukemia (ALL) (Kantarjian H et al (2017) NEJM). We evaluated COVA4231, a CD3/CD33 bispecific FynomAb, preclinically as therapeutic candidate for AML. CD33 is a cell surface receptor expressed on blasts of the majority of AML patients (Walter RB (2014) Expert Opin Ther Targets). COVA4231 was constructed by fusing the CD33-specific Fynomer D5 - a small (7 kDa) globular protein derived from the human Fyn SH3 domain with engineered affinity for CD33 - to a CD3-specific antibody with a novel silent IgG1 Fc. COVA4231 induced T cell activation and cytokine release in a CD33-dependent manner, and elicited potent T cell mediated cytotoxicity of CD33-expressing target cells in vitro with EC50 in the range of 4 - 29 pM (E:T cell ratio of 2:1, KG-1 or MOLM-13 as target cells). COVA4231 showed potent activity against 13 out of 15 primary AML blast samples ex vivo at E:T cell ratio of 1:1. The in vivo efficacy of COVA4231 was investigated in a subcutaneous HL-60 leukemia xenograft model in NSG mice in the presence of human T cells. COVA4231 was highly active across a wide dose range (0.05 - 5 mg/kg) and prevented tumor outgrowth in all treated mice. COVA4231 demonstrated an IgG1-like pharmacokinetic profile in mice with a terminal half-life 15.8 days (total drug), providing the opportunity to avoid continuous intravenous infusion protocols required for established CD3 bispecific formats (e.g. (scFv)2). In conclusion, COVA4231 is a highly active therapeutic candidate in vitro, in vivo and ex vivo, has IgG-like pharmacokinetics and is a promising therapeutic candidate for further preclinical and clinical development.

#1788

Enhanced in vitro/in vivo cytotoxicity against Burkitt lymphoma/primary mediastinal large B cell lymphoma by polatuzumab vedotin (hu- anti-CD79b-vc-MMAE, PV) alone or in combination with obinutuzumab.

Aradhana Awasthi Tiwari,1 Dina Edani,1 Christeen Azmy,1 Janet Ayello,1 Christian Klein,2 Mitchell S. Cairo1. 1 _New York Medical College, Valhalla, NY;_ 2 _Roche Pharmaceutical Research & Early Development, Zurich, Switzerland_.

Background: Mature B-NHL, including Burkitt lymphoma (BL) and primary mediastinal large B cell lymphoma (PMBL) express CD20+/CD79b+ and have an excellent prognosis, however, subset of patients relapse secondary to chemoimmunotherapy resistant disease and have a dismal prognosis (≤ 20% 5 yr. EFS, Cairo et al. Blood. 2007; Gerrard/Cairo et al., Blood, 2013, Goldman/Cairo et al. Leukemia, 2013). PV has been demonstrated to possess significant preclinical activity against indolent CD79b+NHL (Polson et. al.Can. Res.2009). We previously observed that obinutuzumab (Anti-CD20 mAb) significantly enhanced cell death and increased overall survival against BL (Awasthi/Cairo et al., BJH 2015) in xenografted NSG mice. However, additive/synergistic effects of PV with obinutuzumab against mature PMBL/BL are unknown.

Objective: To determine the efficacy of the PV or obinutuzumab/RTX alone or in combination against PMBL and rituximab (RTX) sensitive/resistant BL cell lines.

Methods: Raji4RH (provided by M. Barth, MD, Roswell Park Cancer Institute) and Raji/ Karpas1106P (ATCC, USA) were cultured in RPMI. Tumor cells were incubated with PV, and/or anti-CD79b, MMAE (generously supplied by Genentech Inc.) with obinutuzumab /rituximab (100ug/ml) for 4 hr with NK cells at 10:1 E: T ratio and cytotoxicity was determined by DELFIA cytotoxicity assay. Six to 8 week old female NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ), were divided into 5 groups: PBS, isotype control, PV, antiCD79B mAb and MMAE (5mg/kg). Mice were xenografted with intravenous injections of Luc+ BL and PMBL cells and tumor burden was monitored by IVIS spectrum system.

Results: OS of mice receiving PV alone was significantly increased compared to antiCD79b or isotype control in Raji (35.5 vs.17 vs.19.5 days, p=0.0001, 0.0003), Raji4RH (50 vs.18 vs.18.5 days, p=0.0001, 0.0001) and Karpas1106P (150 vs 89 vs 64 days, p=0.03, 0.003), respectively. Obinutuzumab+NK, rituximab+NK compared to PV+NK cells significantly enhanced cell lysis in Raji, 65.9±2.4% vs. 38.9±5.4% vs. 44.24±8.1% (p=0.001 & p=0.001), Raji4RH, 52.8±9.4% vs. 16.04±7.2% vs.47.0±8.2% (p=0.03 & p=NS) and Karpas1106P, 66.10±5.3% vs.48.2±3.9% vs. 61.6±10.06% (p=0.004 & NS), respectively. PV+ obinutuzumab+NK, significantly improved cytotoxicity compared to PV+ rituximab+NK in Raji, 93.6±6.1% vs 79.9±5.3% (p=0.007), Raji4RH, 78.07±2.05% vs 63.5±0.16% (p=0.004) and Karpas1106P, 88.3±6.3 %vs.73.03±3.03% (p=0.003), respectively.

Conclusion: Our preliminary data indicates that PV significantly increased survival in BL and PMBL NSG xenografts compared to anti-CD79b Ab alone. Furthermore, PV in combination with obinutuzumab significantly enhances cytotoxicity in BL and PMBL compared to obinutuzumab or PV alone.

#1789

**CD30** + **T regulatory cells, but not CD30** + **CD8 T cells, are impaired following brentuximab vedotin treatment in vitro and in vivo.**

Ryan A. Heiser, Bryan M. Grogan, Luke S. Manlove, Shyra J. Gardai. _Seattle Genetics, Bothell, WA_.

Brentuximab vedotin (BV) is an antibody-drug conjugate (ADC) directed against CD30, a TNF receptor superfamily (TNFRSF) member highly expressed on Reed Sternberg cells in Hodgkin lymphoma (HL) and also commonly expressed in a number of other lymphoid malignancies such as ALCL and CTCL. BV consists of a monoclonal antibody conjugated to monomethyl auristatin E (MMAE), a highly potent microtubule-disrupting agent. MMAE-based ADC antitumor activity primarily results from intracellular payload release, leading to mitotic arrest and apoptotic cell death, although secondary MOAs may exist. CD30 is mostly absent on resting peripheral lymphocytes, but is known to be transiently upregulated on both CD4+ and CD8+ T cells following activation. Recently, CD30 was identified, by separate research groups, as a marker differentially upregulated by human intratumoral T regulatory cells (Tregs). This recent observation raises the possibility that BV could target and eliminate intratumoral CD30+ Tregs. Abundant evidence shows that tumor-specific CD8+ T cells are paramount to antitumor immunity. In contrast, tumor-resident T regulatory cells have been shown to counteract immunosurveillance and promote tumor escape. Given that CD30 may be expressed on both activated CD8+ T cells and intratumoral Tregs, we explored the outcome of BV treatment on each cell type. In this work, we show that BV directly depleted inducible and primary CD30+ Tregs in vitro, in a dose-dependent manner, while CD30+ CD8+ T cells were unaffected. Moreover, BV selectively depleted Tregs in a Treg:CD8 T cell co-culture suppression assay, resulting in expansion of proliferating CD8+ T cells. In vivo, using a humanized mouse model of graft-versus-host disease (xeno-GVHD), treatment of mice with BV significantly reduced splenic Treg numbers, while amplifying total xeno-reactive CD8+ cytotoxic T cells. In an effort to understand BV's selective impairment of Tregs, we evaluated CD30 expression following in vitro activation with CD3/CD28. Freshly isolated Tregs showed notably accelerated CD30 expression kinetics along with significantly higher peak receptor number compared to CD8+ T cells. Furthermore, assays confirmed that the heightened receptor expression on Tregs translated into increased BV internalization and drug-linker cleavage. Finally, CD8+ T cells were much more efficient at effluxing rhodamine than Tregs, suggesting lowered drug accumulation and exposure over time. Together, these data raise the novel possibility that brentuximab vedotin may be able to positively impact the Treg:CD8 T cell balance in the tumor microenvironment through selective depletion of CD30+ Tregs, but not activated CD8+ T cells.

#1790

TNFR2-targeted elimination of Tregs and tumor-residing T cells in advanced cutaneous T cell lymphoma.

Denise L. Faustman,1 Heather Torrey,1 Michael Khodadoust,2 Audrey Defusco,1 Danielle Baum,1 Ziba Rahbar,2 Youn H. Kim2. 1 _MGH/Harvard Medical School, Charlestown, MA;_ 2 _Stanford University School of Medicine/Cancer Institute, Palo Alto, CA_.

Background: Tumor necrosis factor receptor 2 (TNFR2, or TNFRSF1B) is a lymphoid marker of the most potent regulatory T cell (Treg) subtype and a commonly expressed oncogene in human tumors. TNFR2 Tregs are also enriched in the tumor microenvironment. TNFR2 antagonistic antibodies have been recently developed to inhibit NFkB-driven growth through the TNFR2 receptor, showing both Treg and tumor inhibition with specificity for the tumor microenvironment (Sci Signaling 2017). TNFR2 is a candidate oncogene in cutaneous T cell lymphoma (CTCL), with recurrent point mutations and gain of function alteration of TNFR2 resulting in abnormal expression of TNFR2 on CD4+CD26- tumor cells (Nat Genet 2015). Methods: We designed monoclonal antibodies to target the TNFR2 oncogene and directly kill human tumor cells in CTCL. TNFR2-directed monoclonal antibodies were screened for their ability to induce the death of leukemic cells in patients with Stage IV CTCL (Sézary syndrome) on a diversity of prior treatment regimens, as well as their ability to induce killing of tumor-associated Tregs and induce effector T cell (Teff) proliferation. Studies were performed in vitro on sorted CD4+CD26- Sézary cells or V-beta specific populations when a tumor was typed. Results: Baseline blood samples from patients with CTCL showed significant burdens of tumor cells within the CD26- subset of CD4 cells, in contrast to control blood cells. In CTCL subjects, numbers of Tregs (CD4+CD25hiFoxp3) were also elevated at baseline (CTCL vs Control, 11% vs 7%, p< 0.05), numbers of Teffs were depressed (CTCL vs Control, 3% vs 8%, p< 0.05), and Treg/Teff ratios were abnormally elevated (CTCL vs Control, 8% vs 1%, p< 0.05). Regardless of underlying therapy used in vivo, TNFR2 antagonism showed dose-responsive killing of the tumor cells within the CD26- fraction of peripheral CD4 T cells. TNFR2 antibody antagonism also showed specificity for tumor cells over CD26- cells from paired controls. In vivo treatment of CTCL subjects with an anti-proliferative agent such as methotrexate hindered TNFR2 antagonism driven killing, demonstrating the specificity of TNFR2 antagonism for rapidly proliferating cells. In dose response experiments in vitro, TNFR2 antagonism also had the desired dual effect of potent Treg killing combined with potent unleashing of Teff proliferation. Conclusions: TNFR2 can potentially be targeted in CTCL to directly stop the growth of tumor cells by antibody-induced cell death. TNFR2 antagonism may also provide the ability to eliminate the potent Tregs of the tumor microenvironment and unleash Teff proliferation.

#1791

Evolutionary dynamics of non-Hodgkin's lymphoma CAR T cell therapy.

Philipp M. Altrock, Gregory Kimmel, Frederick L. Locke. _Moffitt Cancer Center and Research Institute, Tampa, FL_.

Non-Hodgkin Lymphoma (NHL) is the most common hematologic malignancy in the United States with an estimated 72,000 new cases (4.3% of all cancer cases) and 20,000 deaths (3.4% of all cancer deaths) in 2017; the median 5-year survival rate is 71%. Despite a possible cure, with front-line chemotherapy, there exist patients that do not response or relapse and develop refractory disease. These patients have a median overall survival of less than seven months. Chimeric antigen receptor (CAR) T-cell therapy for refractory NHL relies on expansion of engineered T- cells that specifically target tumor cells expressing CD19. Here we combine mathematical modeling with statistical data-analysis based on recent results of clinical studies of CAR T-cell dynamics in individual patients. We use statistical and mathematical modeling to elucidate the key mechanisms that drive evolutionary dynamics of anti-CD19 CAR T-cell therapy. To this end, we integrate patient specific tumor burden profiles, inflammatory cytokine profiles, and CAR T cell population dynamics into our model. We find that the success of therapy may depend on dynamic regulation of inflammatory cytokines in the tumor microenvironment, as well as on specific properties of the heterogeneous CAR T-cell population. Relative abundances of juvenile and effector T cells are key factors that drive the duration of treatment response, and the tumor-killing rate of this CD19-specific immunotherapy. Our modeling framework elucidates disease and treatment specific eco-evolutionary dynamical properties, but can also quantify genetically engineered T-cell population properties related to neurological toxicity and long-term homeostatic mechanisms that determine patient survival.

## ENDOCRINOLOGY:

### Growth Factors and Nuclear Receptor Signaling

#1792

Upregulation of INSR/IGF1Rby C-myc promotes TSCC tumorigenesis and metastasis through the NF-κB pathway.

Anxun Wang, Jingjing Sun, Mei Yu, Zhiyuan Lu, Wei Wang. _Sun Yat-sen Univ., Guangzhou, China_.

INSR/IGF1R has been detected to be involved in tumorigenesis and metastasis in various malignancies. The aim of our study was to compare the function role and mechanism of INSR/IGF1R in the progression of tongue squamous cell carcinoma (TSCC). We found that INSR had the same up-regulated expression pattern as IGF1R in TSCC tissues. Up-regulation of INSR/IGF1R was correlated each other and associated with lymph node metastasis and poor prognosis. Functional studies established that INSR/IGF1R knockdown dramatically impeded TSCC in vitro cell growth, migration, invasion and in vivo tumorigenesis and tumor metastasis, whereas ectopic overexpression of INSR/IGF1R enhanced these parameters. INSR/IGF1R promoted TSCC tumorigenesis and metastasis by positively modulating the NF-κB pathway. Furthermore, the up-regulation of INSR and IGF1R in TSCC resulted directly from the binding of C-myc to their promoters. Collectively, our current data demonstrate that the INSR/IGF1R axis, directly targeted by C-myc, plays an important role in the development and progression of TSCC, through NF-κB pathway, and that INSR and IGF1R are potential prognostic factors for TSCC.

#1793

Gastrin-releasing peptide causes transactivation of the EGFR and HER2 in non-small cell lung cancer cells.

Terry W. Moody,1 Irene Ramos Alvarez,2 Robert T. Jensen2. 1 _NCI-CCR, Bethesda, MD;_ 2 _NIDDK, Bethesda, MD_.

Gastrin releasing peptide (GRP) is a 27 amino acid peptide which is present in many lung cancer cell lines (Moody et al., Science 1981; 214: 1246). High expression of the GRP receptor (R) is associated with advanced non-small cell lung cancer (NSCLC) (Mattei et al., Arch Pathol Lab Med 2014; 138: 98). GRP stimulates but the GRPR antagonist PD176252 inhibits the proliferation of NSCLC cells (Moody et al., Eur J Pharmacol 2003; 474: 21). Here the effects of GRP on the transactivation of the EGFR and HER2 were investigated in NSCLC cells. Six of 8 NSCLC tested had GRP-R mRNA whereas all the NSCLC cells had EGFR and HER2 mRNA. Addition of GRP to NCI-H1299 cells elevated cytosolic Ca2+, increased tyrosine phosphorylation of the ERK, EGFR and HER2 and increased colony number. The effects of GRP were blocked by PD176252. The transactivation of the EGFR and HER2 was inhibited by gefitinib (EGFR tyrosine kinase inhibitor (TKI)), lapatinib (EGFR and HER2 TKI), GM6001 (matrix metalloprotease inhibitor), PP2 (Src inhibitor), N-acetylcysteine (anti-oxidant) and transforming growth factor TGF) α antibody. By ELISA, GRP increased secretion of TGFα from lung cancer cells. PD176252, gefitinib, lapatinib and trastuzumab inhibited NSCLC growth. Activation of the GRPR in NSCLC cells may facilitate the formation of EGFR homodimers and EGFR/HER2 heterodimers.

#1794

The FES-related tyrosine kinase associates with and activates the insulin-like growth factor 1 receptor at sites of cell adhesion.

Joanna Stanicka,1 Leonie Rieger,1 Orla T. Cox,1 Sandra O'Shea,1 Michael Coleman,1 Ciara O'Flanagan,1 Barbara Addario,1 Nuala McCabe,2 Richard Kennedy,2 Rosemary O'Connor1. 1 _Univ. College Cork, Cork, Ireland;_ 2 _Queens University Belfast and Almac Diagnostics, Belfast, United Kingdom_.

Insulin-like Growth Factor-1 signaling has a well-described function in facilitating tumourigenesis and promoting tumour growth. Attempts to suppress IGF signals at the level of the IGF-1 Receptor have been disappointing, with kinase inhibitors and blocking antibodies generally showing poor efficacy in clinical trials. To address the mechanisms for this lack of efficacy we sought to identify proteins that modulate the cytotoxic response to IGF-1R tyrosine kinase inhibition using a functional siRNA screen. We identified the non-receptor tyrosine FES-related (FER) kinase as a mediator of sensitivity to the IGF-1R tyrosine kinase inhibitor in MCF-7 cells. We found that FER and the IGF-1R co-locate and can be co-immunoprecipitated from different cell types. Ectopic expression of FER strongly enhanced IGF-1R expression and phosphorylation on the atuophosphorylation sites at tyrosines 950 and 1131. FER phosphorylated these sites in an IGF-1R kinase-independent manner and also enhanced IGF-1-mediated phosphorylation of SHC, and activation of either the AKT or MAPK signaling pathways in breast cancer cell lines. The IGF-1R, β1 Integrin, FER and its substrate cortactin were all observed to be co-located in cell adhesion complexes, the disruption of which reduced IGF-1R expression and activity. High FER expression correlates with phosphorylation of SHC in breast cancer cell lines and with a poor prognosis in patient cohorts. FER and SHC phosphorylation and IGF-1R expression could be suppressed with a known ALK inhibitor that shows high specificity for FER kinase. Overall, we conclude that FER-enhances IGF-1R expression, phosphorylation and signaling to promote cooperative growth and adhesion signaling that may facilitate cancer progression and resistance to IGF-1R inhibition.

#1795

**Darolutamide demonstrates greater in vivo** **activity than enzalutamide in prostate cancer model with therapy-resistant androgen receptor mutation.**

Bernard Haendler, Tatsuo Sugawara, Simon J. Baumgart, Pascale Lejeune. _Bayer AG, Berlin, Germany_.

Resistance mechanisms of prostate cancer to anti-androgen treatment include the emergence of different androgen receptor (AR) mutations. Here we report that darolutamide, a novel investigational AR antagonist, exhibits in vitro antagonism for several AR mutants identified in the clinic. In vivo studies additionally show that darolutamide has a superior activity to enzalutamide in the KuCaP-1 prostate cancer model, which harbors the AR W742C mutation.

Methods: AR mutants leading to therapy resistance were tested in PC-3 cells transiently transfected with an androgen-controlled reporter and stimulated with 0.1 nM R1881. In vivo efficacy studies were performed on SCID CB17 male mice inoculated subcutaneously with KuCaP-1 tumor fragments. Oral treatment started on day 35 post tumor inoculation at a mean tumor size of about 150 mm3. Mouse body weight and tumors were measured at least twice weekly. The study was stopped on day 68 post tumor implantation, and efficacy evaluated by calculating the % ΔT/ΔC. Efficacy was declared according to NCI criteria with ΔT/ΔC ≤ 42%. For gene expression analysis, RNA was extracted from 20 mg of tumor.

Results: Several clinically relevant AR mutants including W742C, W742L, H875Y, F877L, T878A, F877L/T878A, T878G and M896V were tested. Enzalutamide and apalutamide had IC50 values approximately 200 nM and higher for the W742C and W742L mutants, and showed agonistic activity for the F877L and F877L/T878A mutants. For darolutamide the IC50 values were below 200 nM, except for the T878A and F877L/T878A mutants where the value was 1-1.5 µM, and no agonistic activity was observed. As the activity of darolutamide for the W742C mutant was about threefold better than that of enzalutamide in vitro, we performed in vivo studies with the KuCaP-1 xenograft model which harbors this mutation to further confirm these results. At day 68 post tumor implantation, the ΔT/ΔC values for enzalutamide were 70% and 99% for the 30 and 100 mg/kg daily treatments, respectively. The situation was different for darolutamide where a marked reduction of tumor growth was observed when mice were given oral doses of 40 or 100 mg/kg twice-daily. At day 68, the ΔT/ΔC was 45% for the 40 mg/kg twice-daily doses and 26% for the 100 mg/kg twice-daily doses. The treatments were well tolerated, with only limited body weight loss. Expression of androgen target genes was determined at the end of the study and a strong reduction was observed in the 100 mg/kg daily enzalutamide group. In conclusion, darolutamide showed in vitro antagonism for several AR mutants identified in the clinic. For the W742C mutation, a threefold better activity was measured compared to enzalutamide. This also translated in vivo where a strong efficacy was observed in the KuCaP-1 xenograft for darolutamide, but not for enzalutamide.

#1796

Repurposing CRISPR to study aberrant AR signaling in models of castration-resistant prostate cancer.

Evangelia E. Kounatidou, Craig Robson, Luke Gaughan. _Newcastle University, Newcastle, United Kingdom_.

Prostate cancer (PC) is currently the most commonly diagnosed noncutaneous cancer affecting men worldwide. Despite the initial response to androgen ablation, patients relapse with tumors becoming refractory to clinically approved anti-androgens, resulting invariably in castration-resistant PC (CRPC). In CRPC, androgen receptor (AR) signaling is inappropriately restored. Resistance to available hormone therapies in PC has been linked to aberrant splicing of nascent AR transcripts and subsequent expression of constitutively active AR splice variants. Although we have information on the expression of receptor variants in advanced disease, the mechanisms that regulate these aberrantly functioning proteins are markedly understudied. Therefore, a better understanding of how splice variants are generated and function in this disease setting is critical for the development of more refined targeted therapies. By modeling clinically relevant AR splice variants, we will identify and exploit mechanisms of receptor regulation for advanced prostate cancer therapy. The CRISPR-Cas9 technology has been exploited to generate a CWR22Rv1 derivative cell line that lacks AR-FL and hence only expresses AR-Vs. This has allowed us to study the function of AR-Vs in an AR-FL-free background and therefore attribute the observed outcomes exclusively to AR splice variants. In addition, a novel CRISPR-based approach, currently developed in our lab, allows selective targeting of a catalytically inactive form of Cas9 (dead Cas9, dCas9) fused to a FLAG tag, to native chromatin regions and subsequent isolation of protein/RNA complexes associated with these sites. We have taken advantage of spatial mRNA splicing and transcription coupling in order to isolate splicing factors responsible for the generation of the AR variant 7 (AR-V7) in the CWR22Rv1 cell line and its AR-FL CRISPR knockout derivative. Three gRNAs were designed against the AR cryptic exon 3 genomic region. Targeting of FLAG-tagged dCas9 to that site was exploited for in situ capture and isolation of endogenous splicing factors specific to AR-V7, upon rapid FLAG immunoprecipitation and characterization by mass spectrometry. Identification of the AR-V7 spliceosome will fill a critical knowledge gap in our understanding of how AR-Vs are generated and provide key targets for future therapeutic intervention in advanced PC patients.

#1797

Characterization of a novel androgen receptor coregulator RIPK1.

Cheng-Lung Hsu,1 Jai-Shin Liu,2 Ting-Wei Lin,3 Chun-Cheng Lin,2 Wen-Guey Wu2. 1 _Chang Gung Memorial Hospital, Taipei, Taiwan;_ 2 _National Tsing Hua University, Hsinchu, Taiwan;_ 3 _National Tsing-Hua University, Hsinchu, Taiwan_.

Using bicalutamide-androgen receptor (AR) DNA binding domain-ligand binding domain as bait, we observed enrichment of FxxFY motif-containing peptides. Protein database searches revealed the presence of receptor-interacting protein kinase 1 (RIPK1) harboring one FxxFY motif. RIPK1 interacted directly with AR and suppressed AR transactivation in a dose-dependent manner. Domain mapping experiments showed that the FxxFY motif in RIPK1 is critical for interactions with AR and the death domain of RIPK1 plays a crucial role in its inhibitory effect on transactivation. In terms of tissue expression, RIPK1 levels were markedly higher in benign prostate hyperplasia and non-cancerous tissue regions relative to the tumor area. With the aid of computer modeling for screening of chemicals targeting activation function 2 (AF-2) of AR, we identified oxadiazole derivatives as good candidates and subsequently generated a small library of these compounds. A number of candidates could effectively suppress AR transactivation and AR-related functions in vitro and in vivo with tolerable toxicity via inhibiting AR-peptide, AR-coregulator and AR N-C interactions. Combination of these chemicals with antiandrogen had an additive suppressive effect on AR transcriptional activity. Our collective findings may pave the way in creating new strategies for the development and design of anti-AR drugs.

#1798

A detailed comparison between second-generation AR antagonists reveals differences in the overall impact on gene regulation patterns in prostate cancer cells.

Simon J. Baumgart, Ekaterina Nevedomskaya, Ralf Lesche, Henrik Seidel, Bernard Haendler. _Bayer AG, Berlin, Germany_.

Androgen receptor (AR) signaling is essential at all prostate cancer stages and is clinically addressed by surgical or chemical castration, AR antagonists and androgen synthesis inhibitors. Second-generation AR antagonists include the approved drug enzalutamide and the late-stage clinical investigational oral compounds apalutamide and darolutamide. Comparing the gene expression patterns and the overarching epigenetic modifications elicited by AR antagonists will be essential for a detailed understanding of the molecular mode of action of these compounds.

Methods: The transcriptome and AR binding patterns were analyzed by RNA-seq and qPCR, respectively. Genome-wide changes of the active transcription histone modification H3K27 acetylation were determined by ChIP-seq.

Results: RNA-seq was performed in the androgen-dependent VCaP and LNCaP cell lines for a detailed analysis of the genome-wide transcriptional effects of the AR antagonists darolutamide, enzalutamide and apalutamide. Gene set enrichment analysis indicated that many genes involved in androgen response were up- or down-regulated by androgen treatment with R1881. Principal component analysis showed that treatment with pharmacologically relevant doses of AR antagonists had similar effects at the 8-hour time point, however some differences were visible at the 22-hour time point. A strong expression down-regulation of a number of genes from the Wnt and epithelial-mesenchymal transition (EMT) pathways was observed for darolutamide as compared to enzalutamide and apalutamide treatment in the VCaP cell line. In LNCaP cells the major differences between the AR antagonists were observed in the androgen-regulated genes, which is consistent with varying degrees of inhibition by the drugs in this cell line. Next we determined the global H3K27 acetylation profiles which characterize open chromatin and active transcription. ChIP-seq analysis performed in LNCaP cells revealed a strong impact of androgen treatment on the H3K27 acetylation profiles of numerous genes involved in hormonal regulation. Darolutamide significantly reversed genome-wide H3K27 acetylation patterns, almost to non-androgen stimulated levels. More specifically, a reduction of H3K27 peaks was observed at KLK3 and FKBP5 regulatory elements. In addition, we determined the impact of AR antagonists on AR binding at the KLK2 and KLK3 regulatory elements. Darolutamide and enzalutamide strongly reduced androgen-stimulated AR binding whereas the impact of apalutamide was slightly lower.

Conclusions: AR antagonists strongly reversed the effects of the androgen R1881, both at the level of gene expression, and of H3K27 acetylation and AR binding. A detailed comparison between second-generation AR antagonists showed interesting differences in the regulation of genes in the Wnt and EMT pathways.

#1799

ATRX, DAXX or MEN1 mutant pancreatic neuroendocrine tumors are a distinct "alpha-cell signature" subgroup.

Saurabh V. Laddha,1 Chang S. Chan,1 Peter Lewis,2 Matthew Koletsky,3 Kenneth Robzyk,4 Edaise Da Silva,4 Paula J. Torres,4 Brian Untch,4 Promita Bose,4 Timothy Chan,4 David S. Klimstra,4 David C. Allis,3 Laura H. Tang4. 1 _Rutgers University (RCINJ), New Brunswick, NJ;_ 2 _Wisconsin Institute for Discovery, Madison, WI;_ 3 _The Rockefeller University, New York, NY;_ 4 _Memorial Sloan-Kettering Cancer Center, New York, NY_.

Pancreatic neuroendocrine tumors (PanNETs) or islet cell tumors are a rare neuroendocrine malignancy with an annual incidence of less than 1 per 100,000 per year. Current classification scheme for PanNETs include grade and stage. While well-differentiated PanNETs can be successfully treated with surgery, there are few treatments for metastatic PanNETs. A greater understanding of the cells of origin of PanNETs, tumor progression and pathway pathogenesis may guide the development of novel therapeutic options. The most commonly mutated genes in PanNETs are ATRX, DAXX, and MEN1. Little is known about the cells-of-origin for non-functional neuroendocrine tumors. Here, we genotyped 64 PanNETs for mutations in ATRX, DAXX, and MEN1 and found 37 tumors (58%) carry mutations in these three genes (A-D-M mutant PanNETs) and this correlates with a worse clinical outcome than tumors carrying the wild-type alleles of all three genes (A-D-M WT PanNETs). We performed RNA sequencing (n=33) and Illumina 450K DNA methylation (n=32) analysis on randomly selected PanNETs to reveal two distinct subgroups with one group consisting exclusively of A-D-M mutant PanNETs. Pair-wise correlation of gene expression between all PanNETs, showed A-D-M mutant PanNETs are more homogeneous as a group than A-D-M WT PanNETs. Among A-D-M mutant PanNETs, mutants with the same genotype (mutations in ATRX/DAXX/MEN1) did not show greater gene expression correlation. Two biomarkers differentiating A-D-M mutant from A-D-M WT PanNETs were high ARX gene expression and low PDX1 gene expression with PDX1 promoter hyper-methylation in the A-D-M mutant PanNETs. Moreover, A-D-M mutant PanNETs had a gene expression signature similar to that of alpha cells (pval < 0.009) of pancreatic islets including increased expression of HNF1A and its transcriptional target genes. We validated our subtype classification and A-D-M mutant panNET alpha signature using two independent PanNETs expression dataset. This gene expression profile suggests that A-D-M mutant PanNETs originate from or transdifferentiate into a distinct cell type similar to alpha cells.

#1800

Progesterone receptor attenuates STAT1-mediated interferon signaling in breast cancer.

Merit Goodman,1 Gloria Trinca,1 Katherine Walter,1 Evangelia Papachristou,2 Clive D'Santos,2 Tianbao Li,3 Qi Liu,3 Zhao Lai,4 Prabhakar Chalise,1 Rashna Madan,1 Victor Jin,3 Jason Carroll,2 Christy Hagan1. 1 _University of Kansas Medical Center, Kansas City, KS;_ 2 _University of Cambridge, United Kingdom;_ 3 _University of Texas Health San Antonio, TX;_ 4 _Greehey Children's Cancer Research Institute, TX_.

Why some tumors remain indolent and others progress to clinical relevance remains a major unanswered question in cancer biology. Interferon signaling in nascent tumors, mediated by STAT1, is a critical step through which the surveilling immune system can recognize and destroy developing tumors, thereby blocking their progression. We used an unbiased approach aimed at identifying nuclear protein interactions, called RIME, to identify novel progesterone receptor (PR) protein-protein interactions in T47D breast cancer cells. Using this technique, we identified a novel interaction between PR and STAT1. This interaction led to decreased interferon-induced STAT1 phosphorylation, a phenotype that was further potentiated in the presence of PR ligand. In human breast cancer samples, PR-positive tumors exhibited lower levels of phospho-STAT1 as compared to their PR-negative counterparts, indicating that this phenotype translates to human tumors. Breast cancer cells lacking PR expression exhibited higher levels of interferon-stimulated gene (ISG) RNA, the transcriptional endpoint of interferon activation, indicating that unliganded PR alone could decrease transcription of ISGs. Moreover, the absence of PR led to increased recruitment of STAT1, STAT2 and IRF9 (key transcription factors necessary for ISG transcription) to ISG promoters. These data indicate that PR, both in the presence and absence of ligand, attenuates interferon-induced STAT1 signaling, culminating in significantly abrogated activation of genes transcribed in response to interferons. PR-positive tumors may use downregulation of STAT1-mediated interferon signaling to escape immune surveillance, leading to the development of clinically relevant PR-positive tumors. Selective immune evasion of PR-positive tumors may be one explanation as to why over 70% of breast cancers are PR-positive at the time of diagnosis.

#1802

Constitutively active androgen receptor splice variants AR-V3, AR-V7 and AR-V9 are co-expressed in castration-resistant prostate cancer metastases.

Heini M. Kallio,1 Reija Hieta,1 Anniina Brofeldt,1 Matti Annala,1 Kati Kivinummi,1 Teuvo L. Tammela,2 Matti Nykter,1 William B. Isaacs,3 Hans G. Lilja,4 G Steven Bova,1 Tapio Visakorpi1. 1 _University of Tampere, Tampere, Finland;_ 2 _Tampere University Hospital, Tampere, Finland;_ 3 _Johns Hopkins University School of Medicine, Baltimore, MD;_ 4 _Memorial Sloan-Kettering Cancer Center, New York, NY_.

Prostate cancer (PC) is the most commonly diagnosed male cancer both in the United States and in Europe. Approximately 20-25% of cases will develop metastatic disease, which eventually progresses to lethal castration-resistant form of the disease (CRPC). Even though the exact mechanism by which CRPC develops remains to be fully understood, several mechanisms of castration resistance have been identified. Importantly, androgen signaling remains active even in the CRPC stage. This has led to the clinical development of second-generation AR-targeting drugs enzalutamide and abiraterone that target the ligand-binding domain of AR directly and indirectly, respectively. Unfortunately, a significant subset of patients show primary resistance to these agents. One potential explanation for this is the presence of AR splice variants (AR-Vs). AR-Vs are alternatively spliced isoforms of the AR mRNA usually resulting in truncated AR protein product. Even though AR-Vs lack variable portions of the AR COOH-terminal domain including the ligand-binding domain (LBD), they are constitutively active as transcription factors. The aim of this work was to study AR-Vs and also AR rearrangements, mutations and copy number variations (CNVs) to better understand the emergence of CRPC. This was done by analyzing specimens from different stages of prostate cancer by next-generation sequencing methods. Our sample cohorts included hormone-naïve PCs and lymph node metastases as well as locally recurrent and metastatic CRPCs. AR mutations and CNVs were detected only in CRPC specimens. Genomic structural rearrangements of AR (AR-GSRs) were observed in 5/30 metastatic CRPC patients but they were not associated with the expression of previously known AR-Vs, and their variant allele fractions were low. The main AR-Vs detected were AR-V3, AR-V7 and AR-V9, whose expression levels were higher in CRPC cases in comparison to prostatectomy samples. The differences were statistically significant for either variant alone or when their expression fractions were combined (p=0.0006). In addition, metastatic CRPC cases expressed significantly more AR-V3, AR-V7 and AR-V9 when compared to non-androgen deprived pelvic lymph node metastases (p=0.0282). The expression of these AR-Vs was strongly associated with the levels of full-length AR. Out of 25 CRPC metastases that expressed any AR variant, 17 cases harbored expression of all three of these AR-Vs. In conclusion, AR-V3, AR-V7 and AR-V9 tend to be co-expressed in metastatic CRPC highlighting the fact that targeting of the AR ligand-binding domain might not be sufficient to achieve a treatment response in certain patients. Consequently, targeting of AR via regions common to all AR-Vs is likely to provide additional benefit to patients suffering from CRPC.

#1803

Dynamic pro-survival signaling mediates resistance to androgen receptor targeted therapy in AR-v7 splice variant expressing prostate cancer models.

Amanda Pilling, Clara Hwang. _Henry Ford Health System, Detroit, MI_.

Patient survival rates for metastatic prostate cancer are limited by therapeutic failure and disease progression. Treatment of castrate resistant prostate cancer (CRPC) remains a clinical challenge as current treatment options offer modest benefit and are not curative. Second generation androgen axis inhibitors such as enzalutamide and abiraterone acetate provide initial clinical benefit for patients. However, responses are not durable and disease progression is inevitable, resulting in limited patient survival. Several mechanisms of resistance have been identified that relate to persistent AR signaling including AR splice variants (ARVs) which encode an AR protein lacking the ligand binding domain (LBD). Since we have demonstrated that enzalutamide induces an apoptotic response in sensitive prostate tumor cells, we hypothesized that anti-apoptotic signaling is a critical mechanism of resistance to AR-targeted therapy in resistant tumor cell populations. Using 22Rv1 cells, a cell line model that expresses high levels of AR-v7, and cell lines that express low or no ARVs, we demonstrate differential activation of intrinsic/Bcl2-mediated apoptosis signaling in response to enzalutamide. Specifically, in 22Rv1 cells we demonstrate robust induction of anti-apoptotic signaling including increased Bcl-2 and Mcl-1 expression and inactivation of the apoptosis sensitizer Bad in response to enzalutamide, where this signaling is opposed in enzalutamide-sensitive cells. Analysis of potential upstream regulators revealed phosphorylation and activation of Akt through PI3K signaling in the ARV-expressing 22Rv1 cells in response to AR antagonism. Pharmacological inhibition of PI3K/Akt signaling in combination with enzalutamide prevented upregulation of Mcl-1 and inactivation of Bad, thus lowering the apoptotic threshold and sensitizing 22Rv1 cells to enzalutamide. In conclusion, this data demonstrates active engagement of pro-survival kinase signaling that mediates an increased apoptotic threshold in response to AR-directed therapy. Therefore, resistance to AR-directed therapy in ARV-expressing tumors may extend beyond the recognized mechanisms of treatment failure (i.e. lack of LBD for drug binding, constitutive AR transcriptional activity) and involve dynamic anti-apoptosis signaling. We demonstrate that strategies that lower the apoptotic threshold can increase sensitivity to AR antagonist in ARV-expressing cells and represents a novel therapeutic approach with potential to manage or overcome therapy resistance in this lethal form of prostate cancer.

#1804

Novel indole compound LX-039 as an oral selective estrogen receptor degrader (SERD) for treating estrogen receptor positive (ER+) breast cancer.

Jianyu Lu,1 Lihong Hu,1 Ping Wang,2 Yusong Zhu,2 Peng Zou,3 Dongdong Wu,2 Charles Z. Ding,2 Shuhui Chen2. 1 _WuXiAppTec, Wuhan, China;_ 2 _WuXiAppTec, Shanghai, China;_ 3 _WuXiAppTec, Suzhou, China_.

Objective: Endocrine therapy (ET) now is the standard of care (SOC) for ER+ breast cancer treatment. The representative drug of ET is tamoxifen, a selective estrogen receptor modulator (SERM) that inhibits ER+ breast cancer growth by antagonizing ER function. Tamoxifen displays promising efficacy but also brings problems such as increased endometrial cancer risk and acquired resistance. SERD fulvestrant controls ER+ breast cancer by degrading in-cell ER. It shows improved efficacy in advanced or tamoxifen-resistant (TamR) breast cancer patients and is recently approved as first-line treatment for ER+ breast cancer. However, due to its poor PK profile, this drug could only be administrated intramuscularly, which restricts its applicable patient population. Thus, developing an oral SERD is still an unmet medical need.

Method & Results: We have discovered an oral SERD, LX-039. LX-039 is a novel indole series compound. It robustly degraded ER in MCF-7 breast cancer cells (IC50 = 1.53 nM) and inhibited MCF-7 cell proliferation (IC50 = 2.56 nM). Besides, LX-039 exhibited good PK profile across species, F for mice, rat and dog were 32.2%, 44.5% and 48.1% respectively. Dose to p.o. AUC showed linear relationship, and very high p.o. AUCs could be obtained at high doses. For rat @500 mpk, p.o. AUC reached to 4.52M nM.h, and dog @300 mpk, p.o. AUC reached to 1.5M nM.h. LX-039 displayed promising in vivo antitumor efficacy. In naive MCF-7 breast cancer model, TGI was 87% @ 20 mpk; in TamR MCF-7 model, TGI was 70% @100 mpk. What makes LX-039 more advantageous is its safety profile. LX-039 showed purer ER antagonism. In rat uterine growth inhibition assay, the inhibition rate reached to 94% while inhibition rate of GDC-0810 and AZD-9496 were 65% and 89%, respectively. It had a clean CYP inhibition profile by various CYP assays, which indicates a low DDI risk. LX-039 selectively antagonizes ER without affecting other nuclear receptors such as AR, GR and PR. It showed a clean inhibition profile for a selected kinase/GPCR panel (IC50 >3 μM) and was negative in the GABA gated ion channel assay (IC50 > 10 μM) and the mini Ames test. In the 14 day rat non-GLP tox study, NOAEL of LX-039 was determined to be 300 mpk.

Conclusion: The antitumor efficacy of LX-039 as demonstrated in the preclinical tumor models is highly encouraging and warrants testing the compound in the clinic as an oral SERD for treating ER+ breast cancer. The IND enabling studies of LX-039 are ongoing, and IND filing scheduled in the mid of 2018.

#1805

Development of a selective androgen receptor degrader (SARD) for treatment of castration-resistant prostate cancer.

ZhongKe Yao,1 Suzanne Wardell,2 Ivan Spasojevic,2 John Norris,2 John Katzenellenbogen,1 Donald McDonnell,2 Jatinder S. Josan1. 1 _Virginia Polytechnic Inst. & State Univ., Blacksburg, VA; _2 _Duke University School of Medicine, NC_.

Current treatments for prostate cancer are centered on blocking androgen-signaling axis, which is the target of several clinical androgen receptor (AR) antagonists such as enzalutamide. Recent studies have shown that a single AR mutation (F876L) converts enzalutamide from an antagonist to an agonist—a fate shared by earlier antagonists as well such as bicalutamide and flutamide. While hormonal therapy is often successful in the initial stages of the disease, prostatic tumors inevitably become resistant to these therapeutics. Remarkably though, several mechanistic studies suggest that the androgen receptor signaling remains active even in these hormonal refractory cancers, which suggests that AR remains a viable target in the castration-resistant prostate cancers (CRPC). Thus, novel therapeutics that target and degrade AR could provide unique druggable opportunities for complete and more sustained AR inhibition in prostate cancers. Based on an AR-gelsolin interaction in a mammalian two-hybrid luciferase assay, we conducted a screen of an in-house library of 170,000 compounds, re-assayed and validated the top 2K hits. The top 500 hits were then characterized in a battery of assays such as AR-driven gene expression, inhibition of cell proliferation in WT and mutant cell lines, ChIP-seq, transcriptional and conformational profiling, and AR degradation studies. We have identified a selective androgen receptor degrader (SARD) series that shows efficacy against both WT and AR mutants. Further structure-activity relationship (SAR) studies revealed analogs with IC50 equivalent or superior to enzalutamide. In vivo studies are currently under way to evaluate the efficacy of the optimized lead in mouse tumor xenografts. These preclinical findings highlight the utility of SARDs as an effective therapeutic prostate tumor strategy in the context of AR mutations and overexpression that bestow resistance to the second-generation AR antagonists.

#1806

**Distinct** PMEPA1 **gene isoforms regulate androgen-responsive or TGF-β-responsive prostate cancers.**

Hua Li, Shashwat Sharad, Lakshmi Ravindranath, Gyorgy Petrovics, Yongmei Chen, Inger L. Rosner, Albert Dobi, Shiv Srivastava. _Ctr. for Prostate Disease Research, Rockville, MD_.

Introduction: PMEPA1 gene has been defined as androgen and TGF-β responsive gene to inhibit AR and TGF-β signaling via negative feedback loops. Our previous studies identified five PMEPA1 isoforms (PMEPA1-252, PMEPA1-287, PMEPA1-237, PMEPA1-259 and PMEPA1-344) in prostate cancer (CaP) cell lines and CaP RNA-Seq data. PMEPA1-252 decreased androgen receptor (AR) protein levels, suppressed AR signaling and inhibited CaP cell growth. In contrast, PMEPA1-287 and PMEPA1-259 inhibited the transcript levels of TGF-β responsive genes and promoted CaP cell growth. Previous studies showed that knockdown of PMEPA1 gene in PC-3 cells facilitated bone metastasis via activating TGF-β signaling and increasing bone metastasis associated genes. In this study, we further study the roles of PMEPA1 isoforms in the context androgen resistance and bone metastasis process in CaP.

Methods: The expression vectors harboring PMEPA1 isoforms were transfected into DU-145 and PC-3 cells. The protein levels of PMEPA1 isoforms, TGF-β receptor I, SMAD2 and 3 were detected by immunoblotting. The TGF-β signaling activity was measured by SMAD reported dual-luciferase assay in DU-145 and PC-3 cells. The transcript levels of PMEPA1 isoforms and AR or TGF-β responsive genes including KLK3 (PSA), THBS1, NEDD9, bone metastasis associated genes, such as SPARC, CTGF, IL11 and PTHRP were assessed by quantitative PCR (Q-PCR).

Results: PMEPA1-287 and PMEPA1-259 inhibited SMAD reporter activity in DU-145 and PC-3 cells. No PMEPA1 isoform was found to down-regulate the protein levels of TGF-β receptor I and SMAD2 and 3. Increased TGF-β signaling was detected in androgen independent LAPC4-AI and C4-2B cells. In contrast, AR signaling and PMEPA1-252 increased in C4 cells, but not in C4-2 and C4-2B cells. Decreased transcript level of PMEPA1-259 was detected in C4-2 and C4-2B cells, while PMEPA1-287 was found to decrease in C4-2 cells but increase in C4-2B and LAPC4-AI cells. No significant changes of transcript levels of PMEPA1-344 and PMEPA1-237 were detected. In 120 matched human benign and malignant frozen prostate tissues, only PMEPA1-252 associated with SPARC showing inverse correlation. The expression of SPARC was not responsive to either androgen or TGF-β in CaP cells, but increase significantly in C4-2B cells compared to LNCaP cells. These findings suggested PMEPA1 isoforms drive bone metastasis and androgen resistance development via interrupting AR/TGF-β signaling and bone metastasis associated genes.

Conclusions: Our results provide a plausible explanation for the dual function of PMEPA1 gene through its alternative isoforms in the process of prostate cancer progression from androgen dependent to TGF-β dependent/androgen resistant stages of the disease. These findings may lead to new therapeutic opportunities in standard hormone ablation and TGF-β targeted therapies of CaP.

#1807

Inter-individual variation in response to estrogen in the mammary gland.

Karen A. Dunphy,1 Amye L. Black,1 Sallie S. Schneider,2 D Joseph Jerry1. 1 _Univ. of Massachusetts Amherst, Amherst, MA;_ 2 _Pioneer Valley Life Sciences Institute, Springfield, MA_.

Background: Previous research has highlighted the paradoxical nature of estrogen in both contributing to and reducing the risk of breast cancer. Exposure to xenoestrogens may affect this delicate balance between the protective effects of estrogen and its contribution to breast cancer risk. The effects of xenoestrogens may differ among individual women, as genetic diversity has been shown to impact responses to chemicals and breast cancer susceptibility. We hypothesize that there are subsets of individuals uniquely sensitive to estrogen and xenoestrogens.

Methods: In order to identify high and low estrogen response groups, we utilized primary human breast tissues and primary human breast epithelial cells. Explant cultures of primary human breast tissues maintain the normal tissue architecture, while primary human breast epithelial cells are more efficiently used for mechanistic studies. Primary breast tissues were treated with 17β-estradiol (E2), an estrogen receptor alpha (ERα) specific agonist PPT, and an estrogen receptor beta (ERβ) specific agonist ERB041 in order to examine estrogen responsiveness. TUNEL assays were used to examine apoptotic responses. Because expression of estrogen receptor in primary human breast epithelial cells is lost in culture, we treat with TGFβ receptor inhibitors RepSox and SB431542 to restore estrogen receptor expression. We also take conditionally immortalized human breast epithelial cell (ciHMEC) lines and TERT immortalized normal breast epithelial cell lines and transfect in ESR1 to examine responses to E2 and xenoestrogens BP3 and PP.

Results: Our results show that, based on quantitative PCR analysis of estrogen receptor target genes, individuals vary in response to estrogen receptor agonist treatment. Our results from treatment of primary breast epithelial cells with TGFβ inhibitors show an increase in the levels of ESR1 expression by 2-3 fold compared to control. We are testing whether this is sufficient to restore estrogen-induced responses. Current results from the ciHMEC and normal TERT immortalized breast epithelial cell lines also illustrate the differences in response among individuals. Some individuals have estrogenic responses at physiological doses of BP3 and PP which are similar to responses seen from treating with pregnancy levels of E2.

Conclusions: These models demonstrate the variation in estrogenic responses between individual patient samples. Estrogenic compounds PPT, ERB041, PP, and BP3 can induce similar responses to those of E2 in certain individuals, as reflected in target gene expression and transactivation data. Responses to E2 and xenoestrogens vary among TERT immortalized lines and ciHMEC lines, just as responses to E2 and other estrogen receptor agonists varied among individual explant patient samples. The ciHMEC lines can be used to define the mechanistic differences in estrogen sensitivity among women.

#1808

The anti-estrogen endoxifen altered bone morphology and reduced muscle function in mice.

Laura Wright, Khalid Mohammad, Theresa Guise. _Indiana University, Indianapolis, IN_.

Endoxifen, the predominant CYP2D6 metabolite of the selective estrogen receptor modulator (SERM) tamoxifen, is currently being developed as a novel anti-estrogen therapy for the treatment of estrogen receptor (ER)+ breast cancer. Genetic polymorphism of CYP2D6 is a predictor of response to tamoxifen in patients, implicating endoxifen as one of the most active and relevant tamoxifen metabolites. Breast cancer patients treated with adjuvant endocrine therapies including aromatase inhibitors (AIs) and SERMs often report unmanageable musculoskeletal toxicities that can result in treatment discontinuation. While ER binding affinity and anti-tumor effects have been established in preclinical models, the effects of endoxifen on the musculoskeletal system are not fully known. Twenty-week female C57BL/6 mice underwent sham surgery or ovariectomy (OVX) and were treated daily with vehicle, the AI letrozole, or the SERM endoxifen. Body composition was assessed prospectively by DXA. Bone indices and marrow adipose tissue volume were measured by μCT and muscle contractility of the extensor digitorum longus (EDL) was measured ex vivo. After eight weeks, trabecular bone volume fraction (BV/TV) decreased by 50% in OVX-vehicle and OVX-letrozole mice, whereas BV/TV increased threefold in endoxifen mice relative to sham-vehicle. Despite the presence of significantly more trabecular bone following endoxifen treatment, cortical bone analyses revealed impaired periosteal and endosteal expansion of bone, resulting in reduced polar moment of inertia, which is a measure of resistance to fracture. Uterine weight increased significantly in endoxifen-treated mice relative to all OVX groups, similar to what is observed with tamoxifen treatment. Peripheral body fat, bone marrow adipose tissue, and circulating leptin were reduced by endoxifen, suggesting that the drug may elicit positive systemic metabolic effects. At the termination of the study, muscle-specific force was reduced in OVX-endoxifen mice relative to sham-vehicle, OVX-vehicle, and OVX-AI mice, despite no change in muscle mass. While endoxifen shows promise as a potent anti-estrogen therapy for the treatment of ER+ breast cancer, it may be important to monitor patients for endometrial proliferation, morphological changes in bone that increase fracture risk, and for musculoskeletal side effects that could reduce drug compliance.

#1809

Expression of genes in the nuclear receptor superfamily defines a set of prognostic biomarkers in endometrial cancer.

Erling A. Hoivik, Ingvild L. Tangen, Karen M. Mauland, Mari K. Halle, Henrica J. Werner, Jone Trovik, Camilla Krakstad. _University of Bergen, Bergen, Norway_.

The nuclear receptor (NR) superfamily in humans consists of 48 transcription factors that form a network to sense lipophilic molecules from the diet, metabolism and hormone production, which ultimately regulate development, circadian rhythm, metabolism, proliferation and differentiation in the normal state. In cancer, biomarker signatures that provide either prognostic or predictive information are useful when suggesting therapeutic strategies. However, when performing differential gene expression analysis on global datasets, there is little overlap among the gene sets identified, even among similar datasets of same cancers. The fourth-eight NRs in cancers could represent a unique predefined pan-cancer subset of potential biomarkers in gynecologic cancers. Of note, NRs, in particular, estrogen, progesterone and androgen receptors (ESR1, PGR, AR), are strongly involved in the progression of gynecologic cancers, as revealed by us and others exemplified by endometrial cancer. Further, NRs are appealing targets, as they are regulated by ligand-receptor interactions, and approximately 15% of all pharmacologic drugs target factors within this group. We investigate the association of NR expressions and clinical outcome, and reveal NR signatures that may provide useful information as a biomarker in gynecologic cancers, with emphasis on endometrial cancer.

#1810

Stapled peptide inhibitors of mutant estrogen receptor/coactivator interactions.

Thomas E. Speltz,1 Jeanne M. Danes,1 Sean W. Fanning,2 Christopher G. Mayne,3 Emad Tajkhorshid,3 Geoffrey L. Greene,2 Jonna M. Frasor,1 Terry W. Moore1. 1 _University of Illinois at Chicago, Chicago, IL;_ 2 _The University of Chicago, Chicago, IL;_ 3 _The University of Illinois at Urbana-Champaign, Urbana, IL_.

A current limitation in treating estrogen receptor-positive breast cancer is the development of resistance to endocrine therapy. Among the many possible mechanisms of resistance, mutations in the estrogen receptor ligand binding domain have been recently shown to be a contributing factor. Specifically, estrogen receptor mutations Y537S and D538G have been reported to contribute to resistance by decreasing the efficacy of the clinically used drug tamoxifen. A standing hypothesis in estrogen receptor pharmacology is that coactivator binding inhibitors can be used in place of traditional endocrine therapy to block cellular proliferation mediated by estrogen receptor. To validate the coactivator binding inhibitor hypothesis within a cellular context, we have prepared cell-permeable stapled peptides to function as inhibitors of the estrogen receptor/steroid receptor coactivator interaction in both wild-type and mutant estrogen receptors. Guided by x-ray crystallography, we have applied molecular dynamics and structure-based rational design to develop stapled peptides with high affinity for the estrogen receptor. In targeting the wild-type receptor, we have found that stapled peptides mimicking steroid receptor coactivators have even higher affinity against mutant receptors Y537S and D538G. In addition to inhibiting the estrogen receptor/coactivator interaction in biochemical assays, we find inhibition of estrogen receptor function within multiple breast cancer cell lines. The stapled peptides reported in this research support the coactivator binding inhibitor hypothesis and shed light on estrogen receptor coactivator interactions in cells expressing both wild-type and mutant receptors.

#1811

O-GlcNAc-dependent regulation of progesterone receptor function in breast cancer.

Gloria M. Trinca,1 Merit L. Goodman,1 Evangelia K. Papachristou,2 Clive S. D'Santos,2 Prabhakar Chalise,1 Rashna Madan,1 Chad Slawson,1 Christy R. Hagan1. 1 _University of Kansas Medical Center, Kansas City, KS;_ 2 _Cancer Research UK Cambridge Institute, University of Cambridge, Cambridge, United Kingdom_.

Emerging clinical trial data implicate progestins in the development of breast cancer. While the role for the progesterone receptor (PR) in this process remains controversial, it is clear that PR, a steroid-activated nuclear receptor, alters the transcriptional landscape of breast cancer. PR interacts with many different types of proteins, including transcriptional co-activators and co-repressors, transcription factors, nuclear receptors and proteins that post-translationally modify PR (i.e. kinases and phosphatases). Herein, we identify a novel interaction between PR and OGT, the enzyme that catalyzes the addition of a single N-acetylglucosamine sugar, referred to as O-GlcNAc, to acceptor serines and threonines in target proteins. This interaction between PR and OGT leads to the post-translational modification of PR by O-GlcNAc. Moreover, we show that O-GlcNAcylated PR is more transcriptionally active on PR-target genes, despite the observation that PR mRNA and protein levels are decreased when O-GlcNAc levels are high. O-GlcNAcylation in breast cancer is clinically relevant, as we show that O-GlcNAc levels are higher in breast cancer as compared to matched normal tissues, and PR-positive breast cancers have higher levels of OGT. These data predict that under conditions where O-GlcNAc levels are high (breast cancer), PR, through an interaction with the modifying enzyme OGT, will exhibit increased O-GlcNAcylation and potentiated transcriptional activity. Therapeutic strategies aimed at altering cellular O-GlcNAc levels may have profound effects on PR transcriptional activity in breast cancer.

#1812

Mechanism of telapristone acetate (CDB4124) on progesterone receptor action in breast cancer cells.

Batzaya Davaadelger, Seema A. Khan, J Julie Kim. _Northwestern University, Chicago, IL_.

Progesterone is a steroid hormone that plays an important role in the breast. Progesterone exerts its action through binding to Progesterone Receptor (PR), a transcription factor that belongs to the nuclear receptor family. Deregulation of the progesterone signaling pathway is implicated in the formation, development and progression of breast cancer. Next generation selective progesterone receptor modulators (SPRMs) have potent anti-progestin activity and are selective for PR, reducing the off-target effects on other nuclear receptors. To date, there is limited information on how the newer generation of SPRM's such as Telapristone acetate (TPA), affect PR function at the molecular level. In this study we investigated the molecular mechanisms by which TPA antagonizes PR action in T47D breast cancer cells. First, we performed ChiP sequencing to obtain a global analysis of the effect of TPA on the PR cistrome. We observed that TPA decreases PR genome wide recruitment to the chromatin. Approximately 19,865 and 19,892 PR binding peaks were identified in the progestin, R5020 or R5020 + TPA data sets, respectively, with 83% (18,113) of overlapping PR binding regions. HOMER motif analysis indicated similar enriched motifs between R5020 and R5020+TPA groups, identifying progesterone response elements as the most significant motif. We validated our ChiP-seq data with PR ChIP by examining the promoter regions of known PR target genes. PR occupancy was decreased at these sites with the addition of TPA. In addition, TPA decreased R5020- driven expression of these genes. To further investigate the mechanism by which TPA inhibits PR transcriptional activity we performed Rapid immunoprecipitation mass spectrometry of endogenous proteins (RIME). TPA significantly influenced co-regulator recruitment to PR. After applying a stringent cut-off to our data set, 3 unique proteins were identified in the R5020+TPA group: TRPS1, LASP1 and AP1G1. TRPS1 has been shown to be a transcriptional corepressor and is overexpressed in majority of human breast cancer. Follow-up studies revealed an increase in the levels of PR-TRPS1 complex with TPA treatment. In addition, silencing TRPS1 with siRNA increased PR occupancy to the known PR gene promoters, suggesting that TRPS1 inhibited PR binding to these regions upon TPA treatment. In addition, knockdown of TRPS1 attenuated the inhibition of ACSL1, NOTCH2, PACSIN1 and GREB1 gene expression after TPA treatment. Taken together our results show that TPA decreases recruitment of PR to chromatin, recruits co-repressors such as TRPS1 to the PR complex thereby regulates PR target gene expression. This is the first study showing TRPS1 complexes with PR and regulates its activity. Ongoing studies are focused on the potential role of TRPS1 as a predictive biomarker of SPRM response in breast cancer.

#1813

The role of androgen receptor variant AR-V9 in prostate cancer.

Yeung Ho,1 Jamie L. Van Etten,1 Christine Henzler,2 Yingming Li,1 Rendong Yang,3 Elizabeth Tseng,4 Ting Hon,4 Tyson Clark,4 Scott M. Dehm1. 1 _Univ. of Minnesota Mansonic Cancer Ctr., Minneapolis, MN;_ 2 _Univ. of Minnesota Supercomputing Institute, Minneapolis, MN;_ 3 _Univ. of Minnesota The Homel Institute, Minneapolis, MN;_ 4 _Pacifici Biosciences, Menlo Park, CA_.

The expression of androgen receptor (AR) variants is a frequent, yet poorly-understood mechanism of clinical resistance to AR-targeted therapy for castration-resistant prostate cancer (CRPC). Among the multiple AR variants expressed in CRPC, AR-V7 is considered the most clinically-relevant AR variant due to broad expression in CRPC, correlations of AR-V7 expression with clinical resistance, and growth inhibition when AR-V7 is knocked down in CRPC models. Therefore, efforts are under way to develop strategies for monitoring and inhibiting AR-V7 in castration-resistant prostate cancer (CRPC). The aim of this study was to understand whether other AR variants are co-expressed with AR-V7 and promote resistance to AR-targeted therapies. To test this, we utilized RNA-seq to characterize AR expression in CRPC models. RNA-seq revealed the frequent coexpression of AR-V9 and AR-V7 in multiple CRPC models and metastases. Furthermore, long-read single-molecule real-time (SMRT) sequencing of AR isoforms revealed that AR-V7 and AR-V9 shared a common 3'terminal cryptic exon. To test this, we knocked down AR-V7 in prostate cancer cell lines and confirmed that AR-V9 mRNA and protein expression were also impacted. In reporter assays with AR-responsive promoters, AR-V9 functioned as a constitutive activator of androgen/AR signaling. Similarly, infection of AR-V9 lentiviral construct in LNCaP cells induced androgen-independent cell proliferation. In conclusion, these data implicate co-expression of AR-V9 with AR-V7 as an important component of constitutive AR signaling and therapeutic resistance in CRPC.

#1814

NF1 as an estrogen receptor-α co-repressor in breast cancer.

Eric C. Chang,1 zeyi Zheng,1 Meenakshi Anurag,1 Jin Gao,1 Burcu Cakar,1 Xinhui Du,1 Jing Li,1 Philip Lavere,1 Jonathan T. Lei,1 Purba Singh,1 Sinem Seker,1 Wei Song,1 Jianheng Peng,1 Tiffany Nguyen,1 Doug Chan,1 Xi Chen,1 Kimberly C. Banks,2 Richarad B. Lanman,2 Maryam Shafaee,1 Susan Hilsenbeck,1 Charles Foulds,1 Matthew J. Ellis1. 1 _Baylor College of Medicine, Houston, TX;_ 2 _Guardant Health, Redwood City, CA_.

NF1 has been best known as a GAP (GTPase Activating Protein) that inactivates Ras. However, we are now finding evidence that it also functions as an ER co-repressor, whose loss leads to endocrine therapy resistance. Sequencing tumor DNA from >600 ER+ breast cancers treated by tamoxifen adjuvant monotherapy, we found that frameshift (FS) and nonsense (NS) NF1 mutations, which can create an NF1-null state, strongly correlate with relapse risk (HR=2.6, submitted). Surprisingly, no recurrent missense NF1 mutations inactivating GAP activity were found in our cohort, and such mutations are rare in primary cancers in general. We thus posulated that complete loss of NF1 protein (e.g., caused by NS/FS mutations), but not GAP inactivation alone, is required to drive endocrine therapy resistance.

Here we demonstrate that NF1 loss (by gene silencing) in ER+ breast cancer cells greatly enhances ligand-dependent ER transcriptional activity in vitro and in vivo, causing estradiol (E2) hypersensitivity and tamoxifen agonism. Mechanistically we show that NF1 can bind directly to ER, an interaction enhanced by tamoxifen but not by E2. Binding is mediated by leucine/isoleucine-rich motifs in NF1, analogous to other ER co-repressors. Mutations in these motifs (some of which are targeted by somatic mutation in cancer) inhibit ER binding and transcriptional activity without impacting GAP activity; conversely, inactivating GAP activity does not impact ER binding and repression. To validate NF1 as an ER co-repressor, we examined proteomic data from >100 breast cancer patients in the CPTAC data base and found that proteins whose levels are positively correlated with NF1 are highly enriched with factors known to bind nuclear receptors; by contrast, levels of another GAP, p120, which lacks ER binding sites, are negatively correlated with these molecules. Importantly, preclinical treatment studies indicate that while NF1-deficient ER+ breast cancer should not be treated by tamoxifen or aromatase inhibitors, fulvestrant, which degrades ER, remains effective. However, fulvestrant monotherapy can activate the Ras-MAP pathway, which may promote cell survival and acquired fulvestrant resistance unless combined with dabrafinib and trametinib to inhibit Raf and MEK —a clinical trial for this combination is in development.

Our data suggest that NF1 is a dual negative regulator at the intersection of two potent oncogenic signaling pathways, Ras and ER. Combination therapy targeting both the ER and the Ras-Raf pathways should be investigated for NF1-deficient cancers driven by ER. 

## EXPERIMENTAL AND MOLECULAR THERAPEUTICS:

### Acquired Resistance Against Molecularly Targeted Therapies

#1815

Massively parallel identification of conserved drug resistant mutations in kinases.

Nicole S. Persky,1 Desiree Hernandez,1 Jonathon Cordova,2 Amanda Walker,1 Lisa Brenan,1 Federica Piccioni,1 Sasha Pantel,1 Yenarae Lee,1 Amy Goodale,1 Xiaoping Yang,1 Yoichiro Mitsuishi,3 Mariana Do Carmo,1 Cong Zhu,1 Aleksandr Andreev,1 David E. Root,1 Cory M. Johannessen1. 1 _The Broad Institute of MIT and Harvard, Cambridge, MA;_ 2 _University of New Mexico, Albuquerque, NM;_ 3 _The Dana Farber Cancer Institute, Boston, MA_.

Drug resistant mutations that arise in therapeutic targets often limit clinical responses. However, the discovery of such mutations has historically been performed one gene or mutation at a time, often over decades of experimental and clinical testing, limiting our understanding of conserved mechanisms of drug resistance.

We hypothesized that deep mutational scanning of canonical kinases may expedite this process and identify novel conserved elements that cause drug resistance when mutated (similar to the well-studied "Gatekeeper" residue). To test this, we generated cDNA-expression libraries containing all possible amino acid substitutions in CDK6, CDK4, ERK2, and EGFR. We screened each library against clinically utilized, ATP-competitive small molecule inhibitors. We then mapped the phenotypic data for over 40,000 missense mutations onto the aligned crystal structures of each protein and searched for shared structural attributes associated with drug resistance.

This analysis revealed 4 equivalent amino acid sites whose mutation conferred drug resistance to ATP-competitive inhibitors in all of our screens: the Gatekeeper residue, as well as three uncharacterized residues. One of these sites, which we have termed the "Keymaster", was additionally found to cause resistance in published data sets of sub-saturation BRAF, HER2, BCR-ABL, and MEK1 mutagenesis screens against their respective inhibitors. We confirmed that drug resistant phenotypes are caused by these alterations utilizing growth assays and protein target phosphorylation detection assays. Mechanistically, we show preliminary evidence that Keymaster-mutant proteins are competent for drug binding, but may display elevated basal activity. Consistent with our findings, we additionally identified mutations at Keymaster residues in reported patient tumors in a number of oncogene kinases, suggesting that Keymaster mutations could be drivers of tumorigenesis, as well as drug resistance. These efforts may prove useful for characterizing somatic kinase mutations of unknown function, designing next-generation therapeutics and deepening our understanding of kinase regulation.

#1816

**Mechanism of resistance to single treatment with MET inhibitor in** EGFR **-mutant lung cancer cell secondarily acquiring** c-MET **amplification.**

Youngjoo Lee, Yu-Ra Choi. _National Cancer Ctr. Korea, Goyang-si, Republic of Korea_.

Amplification in c-MET is the second most common mechanism of resistance to EGFR tyrosine kinase inhibitors (TKI) in lung cancer harboring activated EGFR mutations. An acquired resistance mechanism to single treatment with MET kinase inhibitor remains unknown. In this study, we modeled acquired resistance to experimental type I MET kinase inhibitor PHA66572 in MET-amplified HCC827 lung cancer cell line, which was established under chronic exposure to gefitinib in our lab. We found that reactivation of the EGFR pathway emerges as a resistance mechanism in MET secondarily amplified EGFR-mutant lung cancer cells after prolonged exposure to PHA66572. Any gene alteration influencing sensitivity to the drugs was not observed. Long treatment with only MET inhibitor resulted in partial resensitization to EGFR-TKI in these resistant lung cancer cells. Thus, the combination of gefitinib and PHA66572 could completely suppress the cell growth. Our findings support an intimate relationship between the EGFR and MET signaling pathways in HCC827 lung cancer cell line.

#1817

Establishment and characterization of non-small cell lung cancer cell line variants selected for resistance to osimertinib.

Peter J. Ferguson,1 Mark D. Vincent,1 James Koropatnick2. 1 _London Regional Cancer Program, London, Ontario, Canada;_ 2 _University of Western Ontario, London, Ontario, Canada_.

Many non-small cell lung cancers (NSCLC) are oncogenically driven by mutant epidermal growth factor receptor (EGFR). EGFR tyrosine kinase inhibitors (TKIs) are commonly used as a first-line treatment against such tumors, but NSCLC tumors often recur with a mutant EGFR (often with a T790M mutation in exon 20) that confers resistance to these drugs [Ther Adv Respir Dis 10(6): 549-565, 2016]. Third generation EGFR TKIs (e.g., osimertinib) have been designed that are highly active aganst both T790M EGFR and the original non-T790M-mutated, oncogenic EGFR. Clinical resistance to third generation TKIs has been observed, but model systems in which to study the mechanisms of resistance are few. The human NSCLC cell line H1975, derived from an EGFR-TKI-resistant tumor, contains EGFR mutations T790M and L858R (exon 21). To explore mechanisms mediating resistance to third generation TKIs, we selected variants of the H1975 cell line for resistance to osimertinib to create models in which mechanisms of resistance can be characterized and tested for potential methods to overcome that resistance. Cells were exposed continuously to a single concentration of osimertinib, in some cases with the inclusion of verapamil to avoid possible selection of multidrug resistant cells. After 4 weeks, with weekly changes of drug-medium, clonal cell lines were selected in the presence of 6 and 10 μM osimertinib (one each) or in 5 μM osimertinib plus 10 μM verapamil (3 cell lines). These clonal lines were, respectively, 90-, 95-, 38-, 227-, and 244-fold resistant to osimertinib. The RAD51 inhibitor 2-(benzylsulfonyl)-1-(1H-indol-3-yl)-1,2-dihydroisoquinoline (IBR2), which enhances cytotoxicity of several EGFR inhibitors against numerous cell lines (Ferguson et al., JPET 2017, doi.org/10.1124/jpet.117.241661), decreased osimertinib-resistance by up to 80% in these cell lines. Sequence analysis indicates that cell line H1975/osi-6b retained the same EGFR sequence as that in the parent cell line. H1975/osi-5b/VPL appears to have undergone epithelial-to-mesenchymal transition. Further analyses of the resistant cell lines are being undertaken. These cell lines are a valuable resource in which to test methods to circumvent resistance to third generation EGFR TKIs.

#1818

Activation of IGF-1R pathway and NPM-ALK G1269A mutation confer resistance to crizotinib in NPM-ALK positive lymphoma.

Yanrong Li, Kai Wang, Jingdong Zhang. _Cancer Hospital of China Medical University, Liaoning Cancer Hospital & Institute, No. 44 Xiaoheyan Road, Shenyang, Liaoning, PR China, Shenyang, China_.

ALK-positive Anaplastic Large Cell Lymphoma (ALCL), harboring NPM-ALK fusion gene, is sensitive to dual ALK/MET inhibitor crizotinib. However, cancer cells eventually develop resistance to crizotinib after remarkable initial responses, the mechanisms of crizotinib resistance in NPM-ALK positive ALCL remained unclear.

To investigate the mechanisms of ALCL cells resistant to crizotinib, we established a model of acquired resistance to crizotinib by gradually exposing sensitive NPM-ALK-positive ALCL cell line to higher concentration of crizotinib. We found that NPM-ALK G1269A mutation were selected in 400nM resistant clones and activation of IGF-1R pathway. Then we generated a G1269A-mutated sub-lines by limiting dilution, which were sensitive to second-generation ALK inhibitor NVP-TAE684. IGF-1R inhibitor picropodophyllotoxin had no effect on these sub-clones and didn't change sensitivity to first and second generation ALK TKIs. But IGF-1R inhibitor increased sensitivity to crizotinib and NVP-TAE684 in 400nM crizotinib-resistant cells. Thus, we have shown that IGF-1R pathway and NPM-ALK G1269A mutation contribute to resistance to ALK TKIs and combined ALK/IGF1R inhibition improves therapeutic efficacy.

#1819

Acquired T790M mutation after first generation EGFR tyrosine kinase retreatment.

Sung Yong Lee, Jee Youn OH, Jae Jeong Shim, Kyung Ho Kang, Kyung Hoon Min. _Korea University Guro Hospital, Seoul, Republic of Korea_.

Introduction The first or second generation EGFR TKI drugs showed good overall response and progression free survival rates in EGFR mutation positive patients. However, most of them progress within 12 months from treatment start due to acquired resistance. In clinical practice, many physicians frequently offer these patients retreatment with EGFR TKIs after a chemotherapy break, because EGFR TKI retreatment can reduce the regrowing EGFR TKI sensitive clones. We evaluated induction of T790M mutation in previously sensitive EGFR mutation positive cancer patients that repeatedly treated EGFR TKIs. Method We reviewed the medical records of patients who were rechallenged with first generation EGFR TKIs. Those patients were previously responded to gefitinib or erlotinib and progressed after several chemotherapies. We analyzed the clincopathological characteristics, treatment modality and their outcome, and development of T790M mutation. EGFR mutations in exons 18, 19, 20, and 21 were evaluated with PNA mediated PCR clamping method. Results Three patients showed acquired T790M mutation after first generation EGFR TKIs retreatment. The acquired T790M mutation was detected in the tumor tissue in two patients and in the bronchoalveolar lavage fluid in the other patient. Three patients were taking osimertinib or olmutinib, two patients showed good clinical efficacy and low adverse events, however the other patient showed serious adverse event (interstitial lung disease). Conclusions Re-treatment with first generation EGFR TKIs after drug holiday in sensitizing EGFR mutant patients are considered to be an effective option, because EGFR TKI retreatment can reduce the regrowing EGFR TKI sensitive clones. Moreover, retreatment of first generation EGFR TKI may also induce or expand EGFR TKI resistance by T790M mutation and it could be treated with third generation EGFR TKI.

#1820

Overcoming drug resistance in NSCLC with SRC inhibitor.

Soumavo Mukherjee, Dhananjay Suresh, Ajit P. Zambre, Anandhi Upendran, Raghuraman Kannan. _Univ. of Missouri, Columbia, MO_.

Lung cancer accounts for about 27 percent of all cancer deaths in the United States. Approximately 85 percent of lung cancers are non-small cell type (NSCLC) and, when treated with both chemotherapy and targeted therapies, have a five-year overall survival rate of approximately 16 percent. These statistics clearly emphasize the need for developing treatment methodologies more effective than the current standard of care. Here, we evaluated the efficacy of combined inhibition of SRC and AXL in NSCLC.Dasatinib, a BCR/abl and src family tyrosine kinase inhibitor (TKI) is a drug that has a potential to be used against both EGFR and KRAS mutated lung cancer patients, alone (phase II clinical trial) and in combination with cetuximab (in phase III clinical trial), osimertinib and erlotinib (both in phase II clinical trials) by exploiting its SRC inhibition capabilities. However, NSCLC patients treated with dasatinib often develop resistance and is the major reason for the low median survival in stage IV patients. Recently, AXL pathway has been shown to be upregulated in Dasatinib resistant cells causing activation of EMT, AKT and survival pathways. It has been suggested that auto-phosphorylation of DDR2 and subsequent activation of SHP2/RAS/AKT pathway as a result of AXL upregulation is one of the major reasons for failure to induce apoptosis in cells during dasatinib resistance. Therefore, we examined whether inhibition of AXL resensitized TKI resistant NSCLC to dasatinib. Our results show that AXL knockdown can resensitize cells to dasatinib. IC50 values decreased significantly following AXL inhibition. We have also demonstrated that nanoparticle-mediated AXL knockdown also produces similar resensitization. The details of the study will be presented.

#1821

Genome-wide genetic screens define the drug resistance landscape of BRAF mutant colon cancer.

Jonathan Brammeld,1 Marco Ranzani,1 Elizabeth A. Coker,2 Stacey Price,1 Theodoros I. Roumeliotis,1 Barbara Lupo,3 Mi Petljak,1 Steven P. Williams,1 Francesco Iorio,1 Francesco Sassi,3 Nanne Aben,4 Kosuke Yusa,1 Livio Trusolino,3 Lodewyk Wessels,4 David J. Adams,1 Mathew Garnett,1 Bissan Al-Lazikani,2 Jyoti S. Choudhary,2 Andrea Bertotti,3 Ultan McDermott1. 1 _Welcome Trust Sanger Institute, Cambridge, United Kingdom;_ 2 _Institute of Cancer Research, London, United Kingdom;_ 3 _Candiolo Cancer Institute, FPO-IRCCS, University of Torino, Torino, Italy;_ 4 _The Netherlands Cancer Institute, Amsterdam, Netherlands_.

Background: Almost 10% of colon cancers harbor the canonical activating V600 BRAF mutation that in melanoma cancers has been shown to dramatically sensitize cells to targeted therapy and prolong survival. In the colon, such tumors are associated with worse survival. Recent clinical trials of combination therapies targeting the EGFR and MAPK pathways in these tumors have demonstrated impressive response rates. However, the clinical benefit has been limited by the rapid emergence of drug resistance. Identifying and targeting the genes complicit in this process and thus re-sensitize resistant cancer cells may be of clinical benefit. The landscape of drug resistance in cancer patients is composed of (a) loss-of-function (LoF), (b) gain-of-function (GoF) and (c) point mutation-mediated perturbations of genes, with often no single mechanism being predominant. Therefore, capturing the full breadth of resistance genes for any drug would require multiple genetic screens to be executed in parallel.

Methods: We performed 3 genome-wide genetic screens in parallel in BRAF mutant colon cancer cells treated with a BRAF/MEK/EGFR inhibitor combination, to capture all of the resistance mechanisms described above. To screen for LoF events we used a genome-wide CRISPR/Cas9 sgRNA library. For GoF resistance events, a lentiviral-based insertional mutagenesis vector was used to randomly integrate the SFFV enhancer/promoter sequence throughout the genome. Finally, to saturate the genome of each cell line with point mutations we used N-Ethyl-N-nitrosourea (ENU) to randomly mutagenize every base in the genome. Each genetic screen was analysed separately to identify the relevant genes and pathways that confer drug resistance before aggregating the outputs of all three screens for a more comprehensive view of the drug resistance landscape.

Results: The most recurrent and strongest hit from the CRISPR knockout screen was the inhibitor of Src family kinases, CSK. A Gaussian Kernal Convolution analysis for recurrent integrations detected in resistant clones following the insertional mutagenesis screen identified five genes as likely resistance candidates - BRAF, SOS1, MET, FRS2 and KRAS. Finally, Illumina exome sequencing of resistant clones following ENU mutagenesis revealed statistical enrichment for non-synonymous point mutations in three genes in the MAPK pathway, namely MAP2K1, NRAS and KRAS. Of note, loss of CSK was also shown to confer resistance to targeted therapies in other cancer types.

Conclusion: Genome-wide genetic screens identified genes in the MAPK pathway likely to cause resistance clinically, and many of which are amenable to therapeutic targeting. Of note, the resistance as a result of loss of CSK instead activates Src signalling. It can be overcome through the addition of a Src inhibitor, and may be relevant across a range of cancer types as a resistance mechanism.

#1822

Emergence of MET D1228N mutation as a resistance mechanism following an exceptional response to crizotinib in a MET-amplified triple-negative breast cancer patient.

Benjamin M. Parsons, David R. Meier, Grzegorz T. Gurda, Kristopher A. Lofgren, Paraic A. Kenny. _Gundersen Medical Foundation, La Crosse, WI_.

The MET proto-oncogene encodes a receptor tyrosine kinase which is activated by Hepatocyte Growth Factor binding. Recent reports have described single agent activity for crizotinib in MET-amplified or mutant lung adenocarcinoma, squamous cell carcinoma and papillary renal cell carcinoma. Here we describe a stage IV triple-negative breast cancer patient in her early forties, previously treated with doxorubicin/cyclophosphamide, carboplatin/paclitaxel and with capecitabine. PET/CT imaging after progression on capecitabine revealed extensive metastatic disease including a large left pleural effusion, bilateral hypermetabolic nodular pleural implants, innumerable bilateral lung nodules and widespread mildly hyper-metabolic skeletal metastases in the ribs, spine and pelvis. A next-generation sequencing assay detected a 30-fold amplification of MET. MET amplification, overexpression and hyperactivity was confirmed by FISH and by immunohistochemistry for total MET and phospho-MET, respectively. Single agent crizotinib (250 mg, twice daily) was well-tolerated. Immunostaining of a biopsy taken after 9 days of treatment showed elimination of active phosphorylated MET, demonstrating in-tumor crizotinib efficacy at the clinical dose. CT imaging at 10 weeks showed a resolution of her metastatic disease, meeting RECIST 1.1 criteria for a complete response. This response was sustained at 22 weeks. At 37 weeks, the patient progressed in the pleura. An ultrasound guided biopsy revealed a robust resurgence of MET phosphorylation while still taking crizotinib, suggesting an alteration rendering MET recalcitrant to crizotinib. Next generation sequencing revealed a MET D1228N mutation, previously implicated in crizotinib resistance in lung cancer. This mutation was not detected in the pre-crizotinib biopsy (locus sequenced at 6300x) suggesting de novo acquisition in response to crizotinib. Transition from this type I MET inhibitor to a type II inhibitor (cabozantinib, initially 60 mg/day, later 100 mg/day) resulted in clinically stable disease for a period of 7 weeks, at which point the patient again progressed. Although MET-amplification is rare in breast cancer (~0.6% of cases), these data indicate that substantial clinical benefit may be achieved using single agent MET inhibition however the optimal selection of second-line agents to use upon progression will be critical.

#1823

Identification of molecular determinants of vinorelbine resistance in BRAF(V600E) mutated chemorefractory metastatic colorectal cancer patients.

Loredana Vecchione,1 Ines Beumer,2 Antonio Mulero-Sanchez,3 Mireille Snel,2 Filippo Pietrantonio,4 Chiara Cremolini,5 Fotios Loupakis,6 Antonia Martinetti,4 Giovanni Fuca',4 Roberto Moretto,5 Gabriella Fontanini,7 Matteo Fassan,8 Sara Lonardi,6 Marta Schirripa,6 Ulrich Keilholz,1 Rene' Bernards9. 1 _Charité - Universitätsmedizin Berlin, Berlin, Germany;_ 2 _Agendia, Amsterdam, Netherlands;_ 3 _NKI/AvL, Amsterdam, Netherlands;_ 4 _Fondazione IRCCS, Istituto Nazionale dei Tumori, Milan, Milan, Italy;_ 5 _Azienda Ospedaliera-Universitaria Pisana, Pisa, Italy;_ 6 _IOV- IRCCS, Padova, Italy;_ 7 _University of Pisa, Pisa, Italy;_ 8 _University of Padua, Padua, Italy;_ 9 _Agendia and NKI/AvL, Amsterdam, Netherlands_.

Background BRAF(V600E) colon cancers (CCs) are characterized by a distinct gene expression profile when compared to KRAS mutant and KRAS-BRAF double wild type (WT2) CCs. Most importantly, 20% of WT2 CCs are BRAF-like by gene expression profile (1,2). By using a loss of function genetic approach, we previously found that Vinorelbine (VBN) might represent a new therapeutic option for BRAF-like metastatic colorectal cancer (mCRC) patients (3). Recently, in a phase II study, Cremolini et al (4) reported no activity of VBN in BRAFV600E mutated chemorefractory mCRC patients. We hypothesize that the lack of response could be driven by the loss of the BRAF-like signature after several lines of treatment and/or the acquisition of a multidrug resistant, an EMT and/or a hypoxia phenotype.

Material and methods We retrospectively collected formalin-fixed-paraffin-embedded (FFPE) tumor tissue of primary tumor or metastatic lesions of mCRC patients enrolled in the Cremolini et al study. In particular, both chemonaive tissue (before the start of any treatment) and chemorefractory tissue (before the start of VBN) were collected to perform gene expression analysis and whole genome sequencing (WGS). Agendia's full genome arrays were used for gene expression analysis and the TrueSeq Nano Dna protocol was used for WGS analysis. In parallel, two independent genome wide CRISPR screens for resistance to VBN were performed in VACO432 CRC cell line by using the Gecko half library A and the Brunello library.

Results and conclusions Matched paired samples were available for six out of twenty patients from the Cremolini et al cohort (Female: 0%, Male: 100%, median age at the start of VBN: 53 (26-71), Stage IV: 100%, chemorefractory: 84%). Samples were available for both gene expression and WGS. For WGS, genomic DNA was extracted from peripheral blood mononuclear cell (pbmc) for four patients and from normal colon FFPE tissue for two patients. All samples passed the quality control steps for both gene expression analysis and WGS. Six hits were identified from the CRISPR screens. Comparative genomic and transcriptomic analysis of the patients´ data will be integrated with CRISPR screens results and presented at the meeting.

References 1. Popovici V et al. Identification of a poor-prognosis BRAF-mutant-like population of patients with colon cancer. Journal of clinical oncology : official journal of the American Society of Clinical Oncology. 2012;30(12):1288-95 2. Tian S et al (2013). A combined oncogenic pathway signature of BRAF, KRAS and PI3KCA mutation improves colorectal cancer classification and cetuximab treatment prediction. Gut 62, 540-549 3. Vecchione L et al. A Vulnerability of a Subset of Colon Cancers with Potential Clinical Utility. Cell. 2016;165(2):317-30 4. Cremolini C et al: ESMO Open Aug 2017, 2 (3) e000241; DOI: 10.1136/esmoopen-2017-000241

#1824

Mechanisms of resistance to anti-EGFR/HER2 therapy in breast cancer.

Luis Daniel Borrero-García,1 Brian Vidal,2 Suranganie Dharmawardhane1. 1 _University of Puerto Rico Medical Sciences Campus, San Juan, PR;_ 2 _Rowan University, NJ_.

Therapy resistance to epidermal growth factor receptor family (EGFR and HER2) targeted therapies has been an obstacle for treatment success in breast cancer. Therefore, our goal is to investigate the mechanisms of resistance to these therapeutics and look for alternative ways to overcome such resistance. We previously created and characterized syngeneic gefitinib and lapatinib (EGFR and HER2 therapeutics) therapy resistant variants of the therapy sensitive SKBR3 human breast cancer cell line. We found that key mechanisms of resistance involved downregulation of miR-200a and upregulation of miR-221/222 families, which are known to regulate epithelial to mesenchymal transition (EMT). To validate the role of miR-221/222 in therapy resistance, western blots were performed for the miR-221/222 target Trichorhinophalangeal syndrome type I (TRPS1) protein. Downregulation of TRPS1 is known to promote EMT and metastasis. As expected, our results showed a downregulation of TRPS1 in the therapy resistant cells compare to the therapy sensitive cells. Since miR-200a was significantly downregulated in the therapy resistant cells, we ectopically expressed miR-200a in the EGFR therapy resistant SKBR3 cells using a lentiviral vector. MiR-200a expressing cells were selected by puromycin resistance. Preliminary data showed a decrease in the fold resistance of cells resistant to lapatinib transfected with lentiviral vector overexpressing miR-200a. These results suggest that overexpression of miR-200a in lapatinib resistant cells can be used to overcome therapy resistance in breast cancer.

#1825

PIK3CA E542K mutation in bladder cancer confers resistance to PI3K targeted therapy but synergy with BET inhibition.

May Faek Elbanna, Nur Damayanti, Sreenivasulu Chintala, Roberto Pili. _Indiana University School of Medicine, Indianapolis, IN_.

Background: We have previously reported that two bladder cancer PDX models that carry distinct PIK3CA helical domain (HD) mutations respond differently to dual PI3K/mTOR inhibitor (LY3023414), where one model is sensitive (RP-B-02, PIK3CA E545K MUT) while the other is resistant (PIK3CA, E542K MUT) (Wei L et al Oncotarget 2016). Thus, we transduced these mutations in isogeneic cells to determine whether each mutation is indeed associated with a distinct oncogenic potential, as well as differential therapeutic response. Subsequently, we tested rational drug combinations capable of overriding resistance to PI3K targeted therapy (i.e. bromodomain inhibitor). Methods: Isogeneic HEK cells and urothelial cells (SV-HUC) were transduced with plasmids carrying either wild type (WT) or mutant PIK3CA (E545K and E542K mutants). Proliferation and growth were compared both in 2D and 3D culture respectively. We tested the therapeutic response to LY3023414 in 2D and 3D culture. Western blotting was used to test target modulation in the PI3K/mTOR pathway in response to treatment. JQ1 and LY3023414 combination studies were done in vitro. Calcusyn™ was used to determine whether synergy between the two agents exists in the context of either mutation. Results: PIK3CA E542K mutation has a growth advantage compared to the E545K mutation. This observation was more significant in 3D culture, which more faithfully represents the complexity of the tumor microenvironment. Isogeneic HEK cells expressing the E542K mutation were significantly more resistant to LY3023414 treatment both in 3D and 2D. This resonates with the resistance we initially reported in the bladder cancer PDX models carrying the E542K mutation. Interestingly, LY3023414+JQ1 combination was synergistic in the RP-B-01 (E542K MUT, but not in the RP-B-02 cell line (E545K MUT). Conclusion: PIK3CA mutation status is predictive of response to PI3K targeted therapy in bladder cancer, where PIK3CA E542K mutation confers resistance to PI3K targeted therapy. The synergistic combination of LY3023414 and the bromodomain inhibitor JQ1 can override resistance to PI3K targeted therapy. This drug combination may be of clinical significance in the context of PIK3CA E542K, but not E545K mutation.

#1826

Oncogenic KRAS mediates resistance to MET targeted therapy in non-small cell lung cancer (NSCLC) with MET mutations that induce exon14 skipping.

Ken Suzawa,1 Michael D. Offin,1 Christopher Kurzatkowski,1 Daniel Liu,2 Morana Vojnic,1 Roger S. Smith,1 Marissa Mattar,1 Inna Khodos,1 Elisa de Stanchina,1 Joshua K. Sabari,1 William W. Lockwood,2 Alexander E. Drilon,1 Marc Ladanyi,1 Romel Somwar1. 1 _Memorial Sloan-Kettering Cancer Center, NY;_ 2 _British Columbia Cancer Center, British Columbia, Canada_.

Mutations in MET that induce skipping of exon 14 and lead to reduced ubiquitin ligase-mediated turnover of this receptor tyrosine kinase (RTK) are detected in 3-4% of non-small cell lung cancer (NSCLC), approaching the prevalence of ALK-rearranged lung cancers. Preclinical and clinical studies have revealed that MET exon14 alterations are actionable oncogenic drivers that are amenable to therapy with MET kinase inhibitors such as crizotinib. However, similar to most kinase-driven cancers, despite initial benefit, acquired resistance to therapy is inevitable. Next-generation sequencing (MSK-IMPACT 468 gene panel) was performed on samples from 81 NSCLC patients with MET exon14 alterations, including 7 with paired pre- and post-treatment tumor samples. A concurrent KRAS G12 mutation was identified in 5 patients. In 4 of these patients, the KRAS mutation was present prior to receiving crizotinib. The KRAS mutation was acquired post-crizotinib in the remaining patient. These findings implicate KRAS activation as a potential mechanism of acquired resistance. Using isogenic and patient-derived in vitro and in vivo models harboring MET exon14 skipping alteration, we confirmed that the KRAS mutation results in constitutive activation of RAS/ERK signaling and cells expressing both MET exon14 skipping and KRAS mutations are refractory to MET inhibition. Dual inhibition of MET and MEK with crizotinib and trametinib, respectively, has an additive effect in cell line and xenograft models. Whereas concurrent KRAS mutation is an extremely rare event in EGFR- and ALK-driven NSCLC, our findings confirm KRAS mutation as a recurrent mechanism of primary or secondary resistance to MET-directed therapies in lung cancers harboring MET exon14 alterations. We provide a new potential therapeutic strategy for NSCLC patients with both MET exon14 alterations and KRAS mutations.

#1827

Direct activation of the tumor suppressor protein phosphatase 2A as a therapeutic strategy for TKI-resistant lung adenocarcinoma.

Rita Tohme,1 Jaya Sangodkar,2 Neil Dhawan,2 Sudeh Izadmeher,2 Neelesh Sharma,3 Michael Ohlmeyer,2 Goutham Narla1. 1 _Case Western Reserve Univ., Cleveland, OH;_ 2 _Mount Sinai, New York, NY;_ 3 _University Hospitals, Cleveland, OH_.

Most lung adenocarcinoma (LUAD) patients develop acquired resistance to tyrosine kinase inhibitors (TKI) via mechanisms enabling the sustained activation of the MAPK and PI3K oncogenic pathways downstream of the tyrosine kinase EGFR. The tumor suppressor protein phosphatase 2A (PP2A) acts as a negative regulator of these pathways. We hypothesize that activation of PP2A simultaneously inhibits the MAPK and AKT pathways and is a promising therapeutic strategy for TKI-resistant LUAD. LUAD cell lines A549, H1975, and H1650 modeling intrinsic and acquired modes of TKI-resistance were treated with Small Molecule Activator of PP2A (SMAP) developed in our lab. RNAseq canonical pathway analysis revealed that PP2A activation resulted in significant upregulation in major apoptotic cell death pathways and downregulation in growth and proliferation pathways, namely the canonical PI3K and MAPK signaling pathways. Kinase enrichment analysis followed by principal component analysis indicated that SMAP treatment induces a gene signature similar to a combination of the selective AKT and MEK inhibitors MK2206 and AZD6244, respectively. Cell-cycle arrest, caspase-dependent apoptosis, and reduced colony formation ability were observed in TKI-resistant cells. SMAP treatment caused a dephosphorylation of AKT and ERK resulting in downregulation of the AKT and MAPK pathways in H1650. A549 and H1975, with lower baseline pAKT levels, experienced a significant dephosphorylation of ERK. These results suggest that the dephosphorylation effect mediated by PP2A activation is highly dependent on the availability of the phosphatase's target. Indeed, stably overexpressing myristoylated AKT in H1975 led to a dual-pathway inhibition upon SMAP treatment. The therapeutic potential of PP2A activation in vivo was first evaluated in a transgenic EGFRL858R mouse model harboring an activating transgene directing the expression of mutant EGFR to the Clara cells. The reticulonodular pattern observed with magnetic resonance imaging was recapitulated by hematoxylin and eosin staining of lung samples, where mice treated with SMAP showed less diffuse lung cancer and a significant decrease in total nodules. Immunohistochemistry revealed increased TUNEL staining, decreased PCNA staining, and dephosphorylation of both ERK and AKT. Single-agent SMAP demonstrated significant tumor growth inhibition, as well as ERK and AKT dephosphorylation, in a TKI-resistant patient-derived xenograft model, in a comparable fashion to a combination of AZD6244 and MK2206 kinase inhibitors. Combination of SMAP and the TKI Afatinib resulted in an enhanced effect on cell death and tumor growth inhibition in a H1975 xenograft model. SMAP treatment was well tolerated and had no notable toxicities in vivo. These collective data support the development of PP2A activators for the treatment of TKI-resistant LUAD.

#1828

Hyperactivation of mTORC1 drives acquired resistance to the pan HER tyrosine kinase inhibitor neratinib in HER2 mutant cancers.

Dhivya R. Sudhan,1 Ariella B. Hanker,1 Angel Guerrero-Zotano,1 Luigi Formisano,1 Yan Guo,1 Qi Liu,1 Francesca Avogadri-Connors,2 Richard E. Cutler,2 Alshad S. Lalani,2 Richard Bryce,2 Alan Auerbach,2 Carlos L. Arteaga1. 1 _Vanderbilt University Medical Center, Nashville, TN;_ 2 _Puma Biotechnology Inc, Los Angeles, CA_.

Background: Tumor genomic profiling has identified patients with cancers harboring activating ERBB2 (HER2) mutations that are sensitive to HER2 targeted therapies. In the SUMMIT phase II 'basket' trial, a subset of patients with ERBB2 mutant cancers have exhibited significant clinical benefit from treatment with the pan-HER irreversible tyrosine kinase inhibitor (TKI) neratinib. However, durable responses to neratinib are few, suggesting mechanisms of de novo and acquired drug resistance. Thus, we sought to identify druggable mechanisms of resistance to neratinib.

Methods: We utilized 5637 bladder cancer (with HER2S310F) and OVCAR8 ovarian cancer (with HER2G776V) cells. Drug resistant cells were developed by exposing cells to increasing concentrations of neratinib over 6 months (5637, 600 nM; OVCAR8, 2 µM). Neratinib resistant H1781 lung cancer cells (with HER2G776>VC) and MCF7 breast cancer cells (with L755S or V777L) knock-in mutations are currently being developed. For immunoblot and drug sensitivity assays, neratinib resistant cells were maintained drug-free for 96 hours and then retreated with neratinib and other inhibitors. Candidate pathways/genes driving neratinib resistance were identified by performing RNA sequencing and whole exome sequencing in drug-resistant and -sensitive cells.

Results: Neratinib-resistant 5637 and OVCAR8 cells were cross-resistant to the HER2 TKIs afatinib and lapatinib. Immunoblot analysis of both cells treated with neratinib showed effective suppression of HER2, EGFR and HER3 phosphorylation. However, they exhibited a striking increase in S6 kinase (S6K) activity and S6 phosphorylation compared to drug-sensitive parental cells, which was maintained in the presence of supra-pharmacological levels of neratinib (1 µM). S6 phosphorylation and viability of drug resistant cells was completely ablated by the combination of neratinib and the mTORC1 inhibitor everolimus, but not with the PI3Kα inhibitor alpelisib, the pan-PI3K inhibitor buparlisib, or the AKT inhibitor MK-2206, suggesting PI3K- and AKT-independent activation of mTORC1. Gene set enrichment analysis (GSEA) of RNA seq data from the drug-resistant cells revealed significant enrichment of K-Ras pathway components in addition to mTORC1 pathway. Consistent with these results, whole exome sequencing revealed activating alterations of the Ras pathway including a truncating mutation in RASA2 and a P200L mutation in PIK3CA Ras binding domain; thereby suggesting potential Ras mediated mTOR activation driving neratinib resistance. Studies are underway to confirm the contribution of Ras pathway in mTOR mediated neratinib resistance.

Conclusions: These data suggest that hyperactivation of mTORC1 promotes acquired resistance to neratinib across histologically distinct ERBB2-mutant cancers.

#1829

PRAS40 as a mediator of insulin-like growth factor-1 receptor-induced resistance to epidermal growth factor receptor inhibition in head and neck cancer.

Michael I. Dougherty,1 Christine E. Lehman,1 Rolando E. Mendez,1 Linnea E. Taniguchi,1 Julie Wulfkuhle,2 Emanuel F. Petricoin,2 Daniel G. Gioeli,1 Mark J. Jameson1. 1 _University of Virginia, Charlottesville, VA;_ 2 _George Mason University, Manassas, VA_.

BACKGROUND: Despite the known growth-promoting role of the epidermal growth factor receptor (EGFR) in head and neck squamous cell carcinoma (HNSCC), EGFR tyrosine kinase inhibitors (TKIs) have shown low efficacy in this disease. The insulin-like growth factor-1 receptor (IGF-1R) has been shown to induce resistance to EGFR TKIs in HNSCC predominantly through anti-apoptotic pathways. PRAS40 is an inhibitor of mTOR that ceases inhibition upon phosphorylation by Akt. Phosphorylated PRAS40 in turn inhibits FOXO3a, contributing to an overall pro-survival state. This study evaluates the role of PRAS40 in IGF-1R mediated resistance to EGFR TKIs.

METHODS: In vitro experiments using alamarBlue, CyQuant, reverse phase protein microarray (RPPA), and immunoblot techniques to evaluate protein expression/phosphorylation and correlate with cell physiologic behavior.

RESULTS: Proliferation assays were used to separate 6 HNSCC cell lines into 2 groups: those rescued from EGFR inhibition by IGF-1R activation and those not rescued. RPPA analysis identified a correlation between PRAS40 phosphorylation and rescue status. Immunoblot analysis confirmed the RPPA findings: in rescued cell lines, PRAS40 phosphorylation decreases with EGFR inhibition, but phosphorylation is restored by IGF-1R activation. However, these treatments have little effect on PRAS40 phosphorylation in non-rescued cell lines. In a representative cell line from each group, p70S6K phosphorylation was found to follow this pattern as well, suggesting possible involvement of mTOR in the rescue mechanism. However the addition of temsirolimus, an mTORC1 inhibitor, to treatment with an EGFR TKI was not sufficient to overcome IGF-induced rescue.

CONCLUSIONS: PRAS40 phosphorylation is tightly correlated with IGF1R activation in HNSCC cells that exhibit IGF1R-induced rescue from EGFR TKI treatment. This phenomenon is absent in non-rescued cells, suggesting a potential role for pPRAS40 in IGF1R-based therapeutic resistance. While PRAS40 phosphorylation results in mTOR activation, the inability of mTOR inhibition to overcome IGF-induced rescue from EGFR antagonism suggests an important alternative downstream pathway. One possible mechanism is through inhibition of FOXO3a, a function of pPRAS40 that has been previously reported in other cell types.

#1830

A clinical paired resistant model elucidated novel dual salvage signaling that confers alectinib resistance in ALK-rearranged lung cancer.

Takahiro Tsuji,1 Hiroaki Ozasa,1 Takashi Nomizo,1 Tomoko Funazo,1 Yuto Yasuda,1 Yuichi Sakamori,1 Hironori Yoshida,1 Kiyoshi Uemasu,1 Hitomi Ajimizu,1 Ryoko Okutani,1 Shunsuke Aburaya,2 Wataru Aoki,2 Mitsuyoshi Ueda,2 Koh Furugaki,3 Yasushi Yoshimura,3 Toyohiro Hirai,1 Young Hak Kim1. 1 _Kyoto Univ. Graduate School of Medicine, Kyoto, Japan;_ 2 _Kyoto Univ. Graduate School of Agriculture, Kyoto, Japan;_ 3 _Chugai Pharmaceutical Co., Ltd., Kamakura, Japan_.

Conventional drug-resistant cancer cell line models have contributed to the elucidation of drug-resistant mechanisms. However, whether or not these models reflect patients in clinical settings is often controversial. We herein report a patient-derived drug resistant model system named the "clinical paired resistant model". This model consists of 2 cell lines derived from a treatment-naïve patient (drug sensitive cell line model) and again after disease progression (drug resistant cell line model). The clinical paired resistant model of alectinib, the second generation ALK inhibitor, revealed the properties that cancer cells acquired during treatment using phosphoproteome and immunoblotting analyses; the proto-oncogene protein tyrosine kinase Src and hepatocyte growth factor receptor MET were activated after the development of alectinib resistance. No secondary mutations were detected in the coding region of ALK tyrosine kinase in the alectinib resistant model. In this alectinib-resistant model, the inhibition of Src and MET using saracatinib and PHA-665752 significantly restored alectinib sensitivity in vitro (17.2-fold change in IC50). Downstream signaling molecules for proliferation and survival, phosphorylation of Akt and ERK1/2, were inhibited and caspase 3/7 activity was significantly increased when the cells were treated with all three inhibitors (saracatinib, PHA-665752, and alectinib). Combined knockdown of SRC and MET restored alectinib sensitivity and inhibited downstream signaling in combination with ALK inhibition using alectinib, suggesting that the dual salvage signaling of MET and Src conferred alectinib resistance. A xenograft generated from our paired resistant model (N=5-6, in each group) indicated that combination therapy with a saracatinib and crizotinib, the first generation ALK inhibitor which also inhibits MET, significantly decreased tumor size in vivo as compared with saracatinib or crizotinib monotherapy. We also established a conventional alectinib resistant cell line model in vitro by exposing NCI-H2228 cells (EML4-ALK variant 3a/b) to 300 nM of alectinib for 3 months and found MET and Src were also activated in the model. Our clinical paired resistant model permits the detection of drug-resistant mechanisms without exploring the common characteristics of numerous drug-resistant patients. Our results demonstrate that MET and Src are potential therapeutic targets in patients with alectinib resistance and that the clinical paired resistant model may be a new strategy to elucidate drug-resistant mechanisms in relatively rare cancers.

#1831

PKCδ as a novel target for HER2-positive, trastuzumab resistant breast cancer.

Debra L. Wyatt, Andrei Zlobin, Emily Ma, Mitchell F. Denning, Clodia Osipo. _Loyola Univ. Cardinal Bernardin Cancer Ctr., Maywood, IL_.

Breast cancer is the most common cancer diagnosis and second leading cause of cancer related deaths among women in the United States. This poor prognosis is in part due to the high rates of resistance. In particular, resistance to anti-HER2-based therapy remains a clinical challenge for women with breast cancer suggesting that better treatment strategies are needed. Results demonstrate that resistance to trastuzumab correlates with lower PKC activity compared to trastuzumab sensitive breast cancer cells, suggesting that downregulation of PKC activity may contribute to trastuzumab resistance. The exact mechanism by which PKCs contribute to trastuzumab resistance is not well understood. Here, we hypothesized that activation of PKCs could prevent trastuzumab resistance and thus inhibit the survival and recurrence of HER2+ breast cancer. Our results show that in two distinct trastuzumab-resistant cell lines (HCC1954 and BT474), activation of PKC by the phorbol ester TPA inhibits both proliferation of bulk cells and survival of cancer stem cells (CSC) as assessed by mammosphere formation. Further, treatment using a pharmacological PKC inhibitor (GF109203X) almost completely rescued the anti-proliferative and anti-CSC survival effects of TPA in two distinct trastuzumab-resistant cell lines. Measurement of PKC isoforms at the transcript and protein levels showed that PKCδ was overexpressed in the resistant cell line compared to the sensitive cell line. PKCδ depletion using a siRNA prevented the anti-proliferative and anti-CSC survival effects of TPA, suggesting that PKCδ was the target of TPA activation in trastuzumab resistant cells. TPA-mediated activation of PKCδ was found to cause G2/M cell cycle arrest, almost complete inhibition of AKT phosphorylation at serine 473, and decreased expression of the CSC marker, ALDH1A1. These results suggest that phorbol ester-mediated activation of PKCδ could be a novel therapeutic strategy for HER2+ breast cancer that is resistant to trastuzumab-based therapy.

#1832

**Acquired resistance mechanisms to afatinib in lung cancer cells harboring** HER2 **alterations.**

Hidejiro Torigoe, Kazuhiko Shien, Tatsuaki Takeda, Takahiro Yoshioka, Kei Namba, Hiroki Sato, Ken Suzawa, Hiromasa Yamamoto, Junichi Soh, Masakiyo Sakaguchi, Shuta Tomida, Shinichiro Miyoshi, Shinichi Toyooka. _Okayama Univ. Hospital, Okayama, Japan_.

Human epidermal growth factor receptor 2 (HER2) plays an important role in the pathogenesis of various cancers. HER2 alterations have been suggested to be a therapeutic target in non-small cell lung cancer (NSCLC), just as in breast and gastric cancers. We previously reported that the pan-HER inhibitor afatinib could be a useful therapeutic agent as HER2-targeted therapy for patients with NSCLC harboring HER2 alterations. However, acquired resistance to afatinib was observed in the clinical setting, similar to the case for other HER inhibitors. Thus, elucidation of the mechanisms underlying the development of acquired drug resistance and exploring means to overcome acquired drug resistance are important issues in the treatment of NSCLC. In this study, we experimentally established afatinib-resistant cell lines from NSCLC cell lines harboring HER2 alterations (Calu3, H2170, and H1781), by stepwise-escalation or high-concentration exposure methods, and investigated the mechanisms underlying the acquisition of drug resistance. The established cell lines showed several unique afatinib-resistance mechanisms, including MET amplification, loss of HER2 amplification and gene expression, epithelial-to-mesenchymal transition (EMT) and acquisition of cancer stem cell (CSC)-like features. The afatinib-resistant cell lines showing MET amplification were sensitive to the combination of afatinib plus crizotinib (a MET inhibitor), both in vitro and in vivo. The resistant cell lines with the loss of HER2 amplification obtained MEK inhibitors sensitivity in vitro and in vivo. Other resistant cell lines that showed EMT or had acquired CSC-like features remained sensitive to docetaxel, like the parental cells. These findings may provide clues to countering the resistance to afatinib in NSCLC patients with HER2 alterations.

#1833

Identification of resistance mechanisms to EGFR treatment in the real world using a clinicogenomic database.

Gaurav Singal,1 Gerald Li,1 Vineeta Agarwala,2 Gaurav Kaushik,1 Claire O'Connell,2 Garrett A. Cobb,1 Thomas Caron,2 David Bourque,1 Ameet Guria,1 Shannon Frank,2 Garrett Frampton,1 Ken Carson,2 Amy Abernethy,2 Vincent A. Miller1. 1 _Foundation Medicine, Inc., Cambridge, MA;_ 2 _Flatiron Health, New York, NY_.

Introduction

Use of targeted therapies often results in resistance mediated by genomic evolution, but discovery of these mechanisms has historically been opportunistic. A more scalable approach is needed to uncover resistance mechanisms from real world patient experience.

Methods

A real-world clinicogenomic database (CGDB) was created of patients with NSCLC who received Foundation Medicine's FoundationOne next generation sequencing (NGS) assay as part of routine care, for whom electronic health record (EHR) data was available in the Flatiron Health Database. Data from NGS testing and the EHR were linked through a HIPAA compliant de-identification and linking process (Singal et al, ASCO 2017). Patients who received first and second generation EGFR inhibitors (afatinib, cetuximab, erlotinib, gefitinib, lapatinib) were segmented into those undergoing NGS testing before treatment ("pre-treatment cohort") and those biopsied at least 3 months after treatment start ("post-treatment cohort"). All tumor samples were from different patients; paired samples were not available. The prevalence of EGFR and non-EGFR alterations in pre- vs post-treatment cohorts were compared using Fisher's exact test.

Results

The NSCLC cohort included 2139 patients, of whom 370 were treated with EGFR inhibitors (NGS testing pre-treatment cohort n=237, post-treatment cohort n=133). Of 51 distinct EGFR short variants identified, only T790M was significantly enriched in the post-treatment cohort (3.4% pre-treatment vs 32.3% post-treatment, p < 0.0001). Analysis of non-EGFR genes demonstrated significant post-treatment enrichment of amplifications in both AKT2 (0.84% vs 7.5%, p=0.001) and FGF10 (1.3% vs 6.0%, p=0.02). These findings may represent resistance mechanisms, concordant with prior literature (Lin et al., AJCR 2014). Conversely, the frequency of ERBB2 short variants was significantly lower in the post-treatment cohort (7.2% vs 0%, p=0.0005).

Conclusion

Population-based analyses of a scalable, real-world clinicogenomic database, derived from data generated as part of routine patient care, can recapitulate known and previously hypothesized mechanisms of resistance to EGFR inhibitors. Extension of this approach with increasing sample size and longitudinal follow-up over time may elucidate novel mechanisms of resistance to a broad array of cancer therapies.

#1834

Src inhibition overcomes neratinib resistance in HER2-positive breast cancer.

Neil Conlon,1 Michelle Lowry,2 Susan Breslin,2 Lorraine O'Driscoll,2 Alexander J. Eustace,1 John Crown,3 Norma O'Donovan,1 Denis M. Collins1. 1 _Dublin City University, Dublin, Ireland;_ 2 _Trinity College Dublin, Dublin, Ireland;_ 3 _St Vincent's University Hospital, Dublin, Ireland_.

Introduction: Despite the recent successes of HER2-targeted therapies, drug resistance still represents a significant clinical problem. Neratinib, an irreversible pan-HER tyrosine kinase inhibitor, has been recently FDA approved for the extended adjuvant treatment of early-stage HER2-positive breast cancer and little is known of its mechanism of resistance in breast cancer. Using a neratinib-resistant cell line model of HER2-positive breast cancer, HCC1954-N, the aim of this study was to characterise cross-resistance to other HER-targeting tyrosine kinase inhibitors (TKIs) and investigate the role of Src in acquired neratinib resistance.

Methods: The effect of HER-family targeting TKIs neratinib, lapatinib, afatinib and, the Src inhibitor, dasatinib on proliferation of HCC1954-N and HCC1954-Par cells was assessed by acid phosphatase assays and three-dimensional Matrigel assays. Cell cycle progression was examined by flow cytometry using a propidium iodide assay. Caspase 3/7-Glo assay was used to assess apoptosis induction. To assess the prevention of the development of neratinib resistance, HCC1954 cells were treated twice weekly with neratinib, dasatinib, or the combination and stained with crystal violet when confluent.

Results: The HCC1954-N cell line was resistant to neratinib and to afatinib and lapatinib compared to parental HCC1954, but neratinib was still the most potent of the three anti-proliferative agents. The combination of neratinib and dasatinib was synergistic in the neratinib-resistant HCC1954-N cell line in both adherent (CI value = 0.1 ± 0.03) and three-dimensional (CI value = 0.36 ± 0.02) conditions. Neratinib plus dasatinib did not result in cell cycle arrest; however, the combination caused a significant increase in the sub-G1 cell cycle population (p value = 0.018), indicating induction of apoptosis. This was further validated by increased activation of caspase 3 and 7 (p value = 0.015) in HCC1954-N cells treated with neratinib plus dasatinib. The addition of dasatinib to neratinib treatment prevented the development of neratinib resistance in parental HCC1954 cells.

Conclusions: Further pre-clinical investigation of the combination of neratinib plus dasatinib is warranted as a potential therapeutic intervention for patients with neratinib-resistant HER2-positive breast cancer.

#1835

**PPARγ agonist in combination with** bcr/abl **tyrosine kinase inhibitors in patients of chronic myeloid leukemia in chronic phase with suboptimal molecular response.**

Hemant Malhotra,1 Ajay Yadav,2 Ashwin Mathur,2 Debashish Biswas,2 Bharti Malhotra2. 1 _RK Birla Cancer Ctr., Jaipur, India;_ 2 _SMS Medical College, Jaipur, India_.

Introduction & background:

Ten to 20 % of patients of Chronic Myeloid Leukemia in chronic phase (CML CP) have suboptimal molecular response (MR) to first line Imatinib maleate (IM). Treatment options include IM dose increase or switch to a 2nd generation tyrosine kinase inhibitor (TKI), Nilotinib or Dasatinib. Recently, synergy has been demonstrated when PPAR γ agonists are added to TKIs. We describe our initial results on 15 patients of CML CP treated with the combination of IM and pioglitazone, a PPAR γ agonist.

Patients and methods:

Fifteen patients of CML CP with suboptimal MR (bcr/abl:abl RQ-PCR between 1 to 10) after more than two years of IM were recruited in this pilot study. Because of economic considerations, these patients were not candidates for 2nd gen. TKIs. Patients given IM 600 mg/day and pioglitazone 30 mg/day. Blood counts and biochemistry monitored monthly, bcr/abl tested every 3 months using a cartridge based IS assay using the Cepheid GenXpert.

Results & conclusions:

Twelve of the 15 patients achieved significant MR, with 6/16 achieving a major MR at 9 months. Only 3/15 did not show a response (Table). Treatment with pioglitazone was well tolerated. One patient was not analysed due to intolerance to pioglitazone are first dose. Two patients had grade 1 elevation of hepatic enzymes which returned to normal after one week treatment cessation.

We conclude that the combination of imatinib with pioglitazone is effective and well tolerated in patients with a sub-optimal MR to TKI in patients with CML-CP. The combination could be a cost-effective strategy in treating imatinib non/sub-optimal responders in the developing world. The combination needs further and larger studies for confirmation and evaluation of the mechanisms of the synergy. | |  | |  | |

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

S. No. | Patient

ID/Sex | Diagnosis

date | BCR-ABL

(Before Pio) | BCR-ABL (After Pio)

|  | |  | At 3 month | At 6 month | At 9 month

1 | SCM/M | Oct 2012 | 5.35 | 2.74 | 1.83 | 0.005

2 | MD/F | Aug 2006 | 1.13 | 0.88 | 0.48 | 0.1

3 | GPJ/M | Jan 2014 | 2.86 | 0.43 | ND | 0.5

4 | RRC/M | Oct 2008 | 2.86 | 1.07 | 0.25 | ND

5 | RK/M | Mar2008 | 1.39 | 0.77 | 0.35 | 0.41

6 | RDS/F | Oct 2000 | 10.1 | 5.99 | 4.87 | 0.67

7 | RDR/F | April 2003 | 9.04 | 5.78 | 0.36 | 0.05

8 | MM/M | May 2011 | 4.48 | ND | ND | 0.14

9 | RD/F | July 2010 | 8.33 | 1.29 | 0.44 | 0.73

10 | RB/F | July 2015 | 3.25 | 3.02 | 2.89 | 1.39

11 | BRR/M | Jan 2010 | 7.13 | 5.61 | 3.82 | 1.77

12 | BK/F | Nov 2004 | 2.06 | 0.04 | 0.03 | 0.07

13 | DPS/M | Jan 2011 | 6.15 | 5.47 | 1.02 | 0.84

14 | JKM/M | April 2007 | 4.22 | 15.4 | 9.11 | 7.62

15 | CS/M | Oct 2012 | 1.72 | 1.17 | 0.84 | 0.08

#1836

Induction of secreted soluble decoy EGFR variants by splicing interference overcomes drug resistance in human non-small cell lung cancer.

Jeong Eun Park,1 Lee Spraggon,2 Elisa de Stanchina,2 Luca Cartegni1. 1 _Susan Lehman Cullman Laboratory for Cancer Research, Ernest Mario School of Pharmacy, Rutgers University, Piscataway, NJ;_ 2 _Memorial Sloan Kettering Cancer Center, New York, NY_.

Activation of the epidermal growth factor receptor (EGFR) is involved in the oncogenesis of multiple cancers, particularly lung cancer. Current treatments are limited by the appearance of TKI resistance from secondary somatic mutations in the EGFR gene, such as the T790M mutation (>50% of biopsies) in the EGFR kinase domain following erlotinib or gefitinib treatment. As these tumors are still EGFR-dependent, EGFR remains a prime therapeutic target in NSCLC. We have recently described multiple natural soluble decoy isoforms for multiple RTKs, including EGFR, which depend on the differential inclusion of functional domains by alternative splicing. We employ antisense oligo nucleotide (ASO) to reprogram EGFR pre-mRNA processing in order to increase expression of mRNA variants that encode for dominant-negative, soluble decoys EGFR (sdEGFR), at the expense of the oncogenic full-length EGFR receptor. These alternative splicing isoforms are generated via intronic polyadenylation (IPA) of pre-mRNA in a U1-snRNP (U1)-dependent manner. More specifically, we are able to induce the sdEGFR variants by using specific ASOs, designed to block the upstream U1 binding site and thus activate the appropriate IPA sites. Here, we show that our novel strategy effectively induces the expression of potent natural inhibitors of EGFR signaling in treatment-refractory lung cancer models in vitro and in vivo, leading to suppression of downstream pathways and tumor growth inhibition. The ASO-induced natural sdEGFR compounds function in a dominant-negative manner and induce dramatic cell death in NSCLC cells harboring multiple activating and resistance EGFR mutations, and thus provides a novel alternative strategy for treatment of refractory NSCLC, to overcome resistance mediated by the EGFR secondary mutations (including T790M, C797S and others).

#1837

Overcoming trastuzumab resistance in HER2-overexpressing breast cancer by utilizing PHB2, a tumor suppressor of multiple resistance pathways.

Tetsuro Yoshimaru,1 Yosuke Matsushita,1 Mitsunori Sasa,2 Yasuo Miyoshi,3 Toyomasa Katagiri1. 1 _Tokushima University, Tokushima, Japan;_ 2 _Tokushima Breast Care Clinic, Tokushima, Japan;_ 3 _Hyogo College of Medicine, Nishinomiya, Japan_.

Overexpression of the human epidermal growth factor receptor 2 (HER2) is associated with aggressive tumor behavior and poor prognosis in breast cancer. Although anti-HER2 monoclonal antibody, trastuzumab shows considerable efficacy and extends the overall survival of patients with HER2 overexpressing breast cancer, about half of individuals with HER2-overexpressing breast cancer do not respond to trastuzumab-based therapies due to multiple resistance mechanisms. Therefore, acquired and de novo resistance to trastuzumab remains an important issue in the clinical treatment of HER2-overexpressing breast cancer. Our previous studies demonstrated that Brefeldin A-Inhibited Guanine nucleotide-exchange protein 3 (BIG3), which is exclusively overexpressed in the majority of breast cancers, functions as an A-kinase anchoring protein that binds protein kinase A (PKA) and protein phosphatase 1 (PP1Cα), thereby inactivating prohibitin2 (PHB2) suppressive activity through its dephosphorylation on Ser39. We further developed the stapled dominant-negative peptide (stERAP; stapled ERAP), which is designed to specifically inhibit the BIG3-PHB2 interaction and improve duration of their anti-tumor effects. Here we newly report that stERAP has significant suppressive effects on the HER2 signaling network by activating the tumor suppressive ability of PHB2. Intrinsic PHB2 released from BIG3 by stERAP was rapidly serine- and threonine-phosphorylated via PKCα and/or TTK, which are essential for its tumor suppressive activity, and effectively disrupted the HER2-HER3 interaction associated with acquired resistance to trastuzumab, resulting in significant reduction of proliferation of HER2-overexpressing breast cancer cells (IC50 = 54 nM in SK-BR-3 cells). We also confirm that stERAP inhibited HER2 phosphorylation on Thr488, HER2-HER3 interaction, and NF-κB pathway, which are associated with trastuzumab-resistance, respectively, resulting in significant suppression of growth of trastuzumab-resistant breast cancer cells. More importantly, stERAP treatment led to the enhancement of trastuzumab-sensitivity, resulting in a synergistic growth suppression of HER2-overexpressing and trastuzumab-resistant breast cancer cells, respectively. Our findings strongly suggest that stERAP is a novel promising anti-cancer drug to suppress the growth of trastuzumab-resistant breast cancer in clinical use.

#1838

**A pre-existing rare PIK3CA** E545K **subpopulation confers clinical resistance to MEK plus CDK4/6 inhibition in NRAS melanoma and is dependent on S6K1 signaling.**

Gabriele Romano,1 Pei-Ling Chen,1 Ping Song,2 Jennifer McQuade,1 Roger Liang,1 Mingguang Liu,1 Whijae Roh,1 Dzifa Duose,1 Fernando Carapeto,1 Jun Li,1 Jessica Teh,3 Andrew Aplin,3 Merry Chen,1 Jianhua Zhang,1 Alexander Lazar,1 P Andrew Futreal,1 Rodabe Amaria,1 David Zhang,2 Jennifer Wargo,1 Lawrence N. Kwong1. 1 _UT MD Anderson Cancer Ctr., Houston, TX;_ 2 _Rice University, Houston, TX;_ 3 _Thomas Jefferson University, Philadelphia, PA_.

Combined MEK and CDK4/6 inhibition (MEKi+CDK4i) has shown promising clinical outcomes in NRAS mutant melanoma patients. Here, we interrogated longitudinal biopsies from a patient who initially responded to MEKi+CDK4i therapy but subsequently developed resistance. Whole exome sequencing and functional validation identified an acquired PIK3CAE545K mutation as conferring drug resistance. We demonstrate that PIK3CAE545K pre-existed in a rare subpopulation that was missed by both clinical and research testing, but was revealed upon multi-region sampling due to PIK3CAE545K being non-uniformly distributed. This resistant population rapidly expanded after the initiation of MEKi+CDK4i therapy and persisted in all successive samples even after immune checkpoint therapy. Functional studies identified activated S6K1 as both a key marker and specific therapeutic vulnerability downstream of PIK3CAE545K-induced resistance. These results demonstrate that comprehensive analysis of pre-treatment samples can reveal rare pre-existing resistant subpopulations and posit S6K1 as a common downstream therapeutic nexus for the MAPK, CDK4/6, and PI3K pathways.

#1839

Suppression of Spry1 sensitizes cutaneous melanoma to BRAF-targeted therapy.

Giorgio Giurato,1 Francesca Colizzi,2 Aurora Rizzo,2 Debora Martorelli,2 Barbara Montico,2 Katy Mastorci,2 Dania Benedetti,2 Alessandro Weisz,1 Riccardo Dolcetti,3 Sigalotti Luca,4 Elisabetta Fratta2. 1 _University of Salerno, Salerno, Italy;_ 2 _Centro di Riferimento Oncologico, Aviano, Italy;_ 3 _University of Queensland Diamantina Institute, Brisbane, Australia;_ 4 _University of Udine, Udine, Italy_.

Background: About 50% of cutaneous melanoma (CM) harbor the activating BRAFV600E mutation, which exerts most of the oncogenic effects through the activation of the MAPK signaling pathways. BRAF inhibitors (BRAFi) have showed important clinical activities in CM, but their effectiveness is impaired by the emergence of an early drug resistance. Accordingly, a better understanding of the molecular basis of BRAFi resistance may provide insights useful to develop new therapeutic strategies in CM. Spry gene family includes four members (Spry1-4), which differ in their tissue distribution, activity and interaction partners, thus suggesting nonredundant functions. Although interactions between Spry proteins and several MAPK pathway components have been reported, the specific role of Spry1 in CM has yet to be defined.

Methods: Bioinformatic analysis on data available on TCGA was performed using the pipeline suggested on the TCGA portal. Spry1 expression levels were measured in BRAFV600 mutant CM cell lines using quantitative real-time PCR (qRT-PCR) and Western blotting. CRISPR based strategy was used to knock out Spry1 gene in BRAFV600 mutant CM cell lines established in our Institution. Transfected cells were sorted into single cells and subsequently expanded. Spry1 knockout (Spry1KO) clones were validated by Sanger sequencing and Western blotting, respectively. Cell viabilities before and after BRAFi treatment were evaluated by MTT and clonogenic assays. Apoptosis was assessed by Annexin V/PI staining. Modulation of MAPK signaling pathways and apoptosis were examined by qRT-PCR and Western blotting. P53 nuclear translocation was evaluated through multispectral imaging flow cytometry analysis.

Results: Specific bioinformatic analysis, using TCGA, was performed to provide more information regarding pathways associated with MAPK signaling. Our analysis revealed that Spry proteins are differentially expressed in CM. In line with these findings, Spry1 was found high expressed in a panel of CM cell lines with BRAFV600 mutation both at mRNA and protein level. Preliminary data demonstrated that the expression and/or phosphorylation of key proteins involved in MAPK signaling were modulated in Spry1KO clones with respect to the parental cell line. Furthermore, Spry1 inactivation associated with: i) enhanced activation and nuclear translocation of the tumor suppressor p53, and ii) decreased mRNA and protein levels of several antiapoptotic proteins. Of note, the treatment of Spry1KO clones with BRAFi reduced CM cell survival in clonogenic assays, and induced apoptosis in a dose- and time-dependent manner. Further studies are ongoing in order to determine the molecular changes involved in Spry1KO clones in response to BRAFi treatment.

Conclusions: Our results suggest that Spry1 gene may exert oncogenic functions in CM and its upregulation can potentially contribute to BRAFi resistance.

#1840

Glycosylation of HER2+ human breast cancer cells control sensitivity to trastuzumab.

Ramiro M. Perrotta, Tomás Dalotto-Moreno, Alejandro Cagnoni, Karina Mariño, Gabriel Rabinovich, Mariana Salatino. _IBYME-CONICET, CABA, Argentina_.

Galectins decode glycan-containing information in a number of cell receptors adjusting signaling thresholds and modulating cellular functions. Upon specific binding to cell surface glycans these lectins control receptor clustering, endocytosis and signaling, influencing many physiological and pathological conditions. In particular, galectin-1 (Gal1), a 14kD prototype galectin, binds to terminal N-acetyllactosamine residues on glycosylated proteins in the absence of α2-6 sialic acid capping (Gal1 permissive glycophenotype). In early studies, our group demonstrated that tumors usurp the Gal1 pathway to evade immune surveillance and to promote aberrant angiogenesis, influencing sensitivity to anti-VEGF therapy. Here we aim to explore the glycosylation signature of HER2+ breast cancer cells and to investigate the effects of tumor-derived Gal1 on RTK activation and resistance to trastuzumab (TZ). We first selected three HER2+ human breast cancer cell lines with different response to TZ: JIMT-1 (resistant-TZR), BT-474 and SK-BR-3 (sensitive-TZS). To identify specific glycan structures, we used a panel of biotinylated plant lectins with different carbohydrate specificities. We found that TZS cell lines exhibited a Gal-1 restrictive glycophenotype characterized by high α2,6 sialic acid capping. Profiling of N-type glycans (WAX-HPLC) confirmed that TZSs cell lines exhibit higher frequency of α2,6 sialic acid residues compared to TZR JIMT-1. This effect was further substantiated by RT-PCR analysis of specific glycosyltransferases responsible of Gal1 ligands biosynthesis. These results were supported by analysis of raw data from public databases arrays (GSE62327) showing that patients who presented complete response to TZ exhibited higher levels of ST6GAL1, a glycosyltransferase responsible for α2-6 sialic acid capping. In accordance with the glycophenotype, TZR cell line bound and expressed higher levels of Gal1 when compared to TZS cell lines by Western blot, RT-PCR and ELISA of conditioned medium, suggesting a positive autocrine loop that could modulate cell behavior. Moreover, in silico analysis of raw data from the Long HER Study (GSE44272) revealed that patients with poor response to TZ expressed higher levels of Gal1 mRNA than long-term responders, reinforcing our hypothesis from a clinical standpoint. Finally, in order to elucidate Gal1 implications in TZ resistance, we knocked down Gal1 in JIMT-1 cell line using shRNA strategies. Remarkably, absence of Gal1 expression sensitized JIMT-1 cells to TZ-inhibition in vitro, and the resistant phenotype was further reestablished by addition of human recombinant Gal1. In summary, our study suggests that individual HER2+ human breast cancer cells display particular "glycosylation signatures" which, in association with Gal1 expression pattern, may control resistance to anti-HER2 targeted therapy and may predict breast cancer clinical outcome.

#1841

Integrative analysis of resistance to BRAF-targeted therapies in lung adenocarcinomas.

Sandra Ortiz-Cuaran,1 Julien Mazières,2 Aurélie Swalduz,3 Washington René Chumbi Flores,4 Yohan Loriot,5 Virginie Westeel,6 Anne Pradines,7 Claire Tissot,8 Christelle Clement Duchene,9 Christine Raynaud,10 Xavier Quantin,11 Radj Gervais,12 Etienne Brain,13 Isabelle Monnet,14 Etienne Giroux Leprieur,15 Séverine Neymarc,3 Virginie Avrillon,3 Solène Marteau,1 Séverine Martinez,3 Gilles Clapisson,3 Nathalie Girerd-Chambaz,3 Celine Mahier,16 Nathalie Hoog-Labouret,16 Frank de Kievit,17 Karen Howarth,17 Emma Green,17 Clive Morris,17 Maurice Pérol,3 Jean-Yves Blay,3 Pierre Saintingy1. 1 _Centre Léon Bérard / Cancer Research Center of Lyon, Lyon, France;_ 2 _Hôpital Larrey, Centre Hospitalier Universitaire, Toulouse, France;_ 3 _Centre Léon Bérard, Lyon, France;_ 4 _Hôpital de la Croix-Rousse, Lyon, France;_ 5 _Institut Gustave Roussy, Paris, France;_ 6 _Jean Minjoz University Hospital, Besançon, France;_ 7 _Centre de Recherche en Cancérologie de Toulouse, CRCT, Toulouse, France;_ 8 _Hôpital Nord, CHU de Saint-Étienne, Saint-Étienne, France;_ 9 _Institut de cancérologie de Lorraine Alexis-Vautrin, Vandœuvre-lès-Nancy, France;_ 10 _Centre Hospitalier Victor-Dupouy, Argenteuil, France;_ 11 _Centre Hospitalier Universitaire Montpellier, Montpellier, France;_ 12 _Hospital Center University (CHU) Caen, Caen, France;_ 13 _CLCC Rene Huguenin Institut Curie, Saint Cloud, France;_ 14 _Centre Hospitalier Intercommunal de Créteil (CHI), Créteil, France;_ 15 _APHP - Ambroise Paré Hospital, Boulogne-Billancourt, France;_ 16 _Unicancer, Paris, France;_ 17 _Inivata Ltd., Cambridge, United Kingdom_.

Background: BRAF mutations occur in 2 to 3% of patients (pts) with non-small cell lung cancer (NSCLC). In these pts vemurafenib, a selective oral BRAF inhibitor is associated with a response rate (RR) of 42%, rising to 64% for combination treatment with dabrafenib and trametinib. Despite initial responses, most pts ultimately develop resistance to therapy. Mechanisms of resistance to BRAF inhibitors in NSCLC have only been reported in 2 pts (acquired KRAS G12D and primary resistance due to BRAF G469L)

Objective: To assess the molecular mechanisms of resistance and to monitor disease response to treatment using liquid biopsies in NSCLC pts treated with BRAF inhibitors.

Strategy: We performed a longitudinal genomic analysis of circulating-tumor DNA (ctDNA) in BRAF-mutated NSCLC pts treated in the AcSé vemurafenib program (NCT02304809) (n=44), or with the combination of dabrafenib and trametinib (n=6). We have collected 24 samples at baseline, 45 during follow-up and 9 at progressive disease (PD). ctDNA genotyping of 36 genes was performed using the Inivata InVisionFirst™ assay. Functional analyses of potentially resistant mutations and in vitro strategies to revert the resistant phenotype are ongoing.

Results: Our preliminary analyses showed that BRAF mutations were detected at diagnosis in 16/24 pts, including 12 BRAF V600E mutations and 4 non-V600E mutations (i.e. G466V, G596R, G469A and K601E). 4/12 (34%) of BRAF V600E-mutated pts presented coexistent mutations, in FGFR2, CTNNB1, IDH1 or PI3KCA, whereas concomitant mutations in KRAS, NRAS or MYC were found in 3/4 (75%) of non-V600E cases. Analyses of response to treatment vs mutational profile will be presented. For the remaining 8/24 pts, TP53 mutations were found in 5 pts in absence of BRAF mutations, and no mutations were detected in 3 pts. Mechanisms of resistance were evaluated in 9 pts. One patient who progressed after 11 months on vemurafenib had MAP2K1 C121S and NFE2L2 p.31-32:GV/X mutations. In this patient, longitudinal ctDNA profiling revealed agreement between the %AF of BRAF and TP53 mutations and response to treatment, and detectable levels of the BRAF V600E and the MAP2K1 C121S mutations up to 6 months before the clinical confirmation of PD. Acquired PI3KCA H1047R and E545K mutations were seen in two pts, respectively, who progressed after 15 and 7 months of vemurafenib. Finally, a fourth patient who relapsed after 3 months on vemurafenib, presented a KRAS G12C mutation. All 4 cases also presented detectable levels of the BRAF V600E mutation at PD. In 3/8 pts, we detected the BRAF V600E mutation at PD but no other mutations; drivers of resistance may be present in genes outside this panel. ctDNA sequencing data on additional 7 pts at PD will be presented.

Conclusion: Our results suggest that ctDNA genotyping might be an informative tool for monitoring disease response and resistance in NSCLC pts treated with BRAF-targeted therapies.

#1842

**Novel** KIF5B-RET **\+ NSCLC cell lines demonstrate differential responses to RET inhibitors.**

Laura Schubert, Anh T. Le, Andrea E. Doak, Robert C. Doebele. _University of Colorado Anschutz Medical Campus, Aurora, CO_.

Chromosomal rearrangements involving the rearranged during transfection (RET) tyrosine kinase occur in 1-2% of NSCLCs. This results in the expression of a constitutively active fusion kinase that promotes cancer cell growth, proliferation and survival. In lung cancer, RET is most commonly fused to KIF5B, but can have many other 5' partners such as CCDC6, NCOA, TRIM33 and TRIM24. Clinical trials with several multi-kinase inhibitors, which potently inhibit RET, have had low response rates between 18-53%. Novel, more highly specific RET inhibitors are now in clinical development. Collectively, the clinical trial data suggest that patients with KIF5B-RET rearrangements have exceptionally low responses to RET inhibitors in relation to non-KIF5B-RET fusions. In order to understand the role of RET 5' partners in regulating response to RET inhibitors we have generated two novel KIF5B-RET+ patient-derived cell lines, CUTO22 (K23;R12) and CUTO32 (K15;R12). We have confirmed that each of these cell lines contain the KIF5B-RET fusion and express the RET fusion protein. MTS proliferation assays have revealed that the CUTO22 cell line is very sensitive to the highly specific RET inhibitor, CEP-32496/RXDX-105, and the multi-kinase inhibitor ponatinib. Conversely, the CUTO32 cell line is dramatically resistant to both RXDX-105 and ponatinib. Phospho-RET (pRET) is inhibited by RXDX-105 and ponatinib in a dose-dependent manner in both the CUTO22 and CUTO32 cell line. Interestingly, despite successful RET inhibition, downstream signaling in pERK and pAKT remains active. In a time-course experiment, pRET is inhibited for up to 24 hours while pERK and pAKT is sustained. Additionally, knockdown of RET with siRNA did not reduce downstream signaling activation in CUTO22 or CUTO32 cells. Combined inhibition of possible bypass pathways, EGFR or mTOR, did not significantly enhance sensitivity to RET inhibitors in CUTO22 and CUTO32 cell lines. To isolate the role of the 5' partners in response to RET inhibitors we have utilized CRISPR/Cas9 technology to generate Kif5b-Ret and Trim24-Ret rearrangements in Ba/F3 cells. We have confirmed with PCR or RT-PCR that these cell lines harbor the intended Ret fusions and express the RET protein. Both the Kif5b-Ret+ and Trim24-Ret+ Ba/F3 cell lines are highly sensitive to multiple RET inhibitors. In conclusion, these data suggest that RET inhibitors successfully inhibit the target protein, but not downstream signaling, suggesting that KIF5B-RET may have unique co-alterations or reliance on alternative pathways to promote growth and proliferation. 

### Cell Cycle, Drug Resistance, and Combinations

#1843

Combination therapy targeting Rb/Wee1 kinase pathways for rhabdomyosarcoma treatment.

Simin Kiany,1 Gangxiong Huang,2 Melanie Justice,3 Khandan Keyomarsi,1 Eugenie Kleinerman1. 1 _UT MD Anderson Cancer Center, Houston, TX;_ 2 _Fujian Medical University, Fuzhou, China;_ 3 _University of Notre Dame, South Bend, IN_.

Introduction

Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma. New therapeutic strategies are needed particularly for high-risk patients where the survival rate is 20-40%. The FDA approved CDK4/6 inhibitor palbociclib (PD), and the WEE1 kinase inhibitor MK-1775 have anticancer effects when used as single agents. PD causes cell arrest at G1 phase (requiring intact Rb), while MK-1775 promotes cell death by abrogating DNA damage during the G2/M checkpoint phase by forcing premature mitosis entry. As the two drugs target tumor cells at different phases of the cell cycle, cells that are not in the targeted phase may escape single therapy. We hypothesized that sequential combination therapy will increase therapeutic efficacy. The use of PD first, will induce the cells to go under G1 arrest. Upon recovery, more cells will synchronize in the S/G2 phase, increasing their susceptibility to MK1775, resulting in synergistic cell killing.

Methods

Rb+ RMS (Rh-30) and Rb\- osteosarcoma (OS) cell lines (CCH-D, LM7, and SASO2) were treated with increasing doses of PD for 6 days, then stained with propidium iodide (PI) and analyzed for cell cycle status to determine whether PD induced G1 arrest. To determine the optimal recovery time for Rb+ cells to be synchronized in S/G2 phase, cells were treated with PD (IC50-IC75) for 6 days and then were harvested either immediately or after 3-24 h recovery in drug free medium. Cells were stained with PI for cell cycle analysis. A 12-day long combination therapy assay was performed with or without recovery time between the two treatments. Combination index (CI) was calculated by using the statistical program calcusyn to determine whether the combinatorial drug treatment had acted in an antagonistic, additive or synergistic manner.

Results

Six days of treatment with PD resulted in reversible G1 arrest only in Rb+ cell lines, confirming that Rb, the downstream target of CDK4, must be intact to induce G1 arrest by palbociclib. Six days of treatment with PD followed by 9-12 h recovery in fresh medium (the optimal recovery time prior to treatment with MK-1775), led to re-entry of the Rb+ Rh-30 cells into the cell cycle and successful synchronization at S/G2 phase. By contrast, Rb- cells failed to arrest in G1 phase following treatment with PD and were not synchronized at S/G2 phase. Since cells synchronized in S/G2 phase are more vulnerable to MK1775, combination therapy resulted in a synergistic effect in the Rb+ cells but an antagonistic effect in the Rb- cells.

Conclusion:

Our results suggest that the combination therapy strategy of PD preceding MK1775 may be a novel therapeutic approach for patients with Rb\+ RMS. This strategy will be investigated in xenograft and PDX RMS model.

#1844

Centriole amplification sensitizes cells to paclitaxel.

Amber L. Lasek, Eric M. Britigan, Beth A. Weaver. _University of Wisconsin-Madison, Madison, WI_.

Paclitaxel (Taxol) is a widely utilized treatment for primary and metastatic breast cancer. However, only about 50% of patients respond and no predictive biomarker for paclitaxel efficacy is currently available. Identification of a clinically validated predictive biomarker would substantially improve patient outcomes. Aneuploidy, an aberrant chromosome number, was recognized in the late 1800s as a hallmark of tumor cells. This led to the hypothesis that aneuploidy drives tumorigenesis. However, experiments in animal models have shown that the effect of aneuploidy on tumors depends on the rate of ongoing chromosome missegregation, also known as chromosomal instability (CIN). Low rates of CIN are weakly tumor promoting, while high rates of CIN lead to cell death and tumor suppression due to loss of one or more essential chromosomes. Combining two independent insults that each cause low CIN results in high CIN, cell death, and tumor suppression. We have recently shown that, in primary breast cancer patients, paclitaxel does not reach sufficiently high concentrations to cause mitotic arrest, as it does at typically used concentrations in cell culture. Instead, low clinically relevant doses of paclitaxel cause CIN due to multipolar spindles. We hypothesize that a preexisting low rate of chromosomal instability (CIN), which occurs in about 50% of breast tumors, sensitizes cells to paclitaxel treatment, since paclitaxel increases their rate of CIN over a maximally tolerated threshold. Centrosome amplification is common in breast cancer and can cause CIN through induction of multipolar spindles. To test whether CIN induced by centrosome amplification sensitizes cells to low, clinically relevant doses of paclitaxel, we inducibly overexpressed Polo-like kinase 4 (Plk4), the master regulator of centriole duplication, to generate supernumerary centrioles. Plk4 overexpression induced centriole amplification and increased the incidence of multipolar divisions, even in subclinical levels of paclitaxel. Consistent with our hypothesis, CIN induced by supernumerary centrosomes potentiates the decrease in cell viability caused by paclitaxel treatment. Decreased cell viability is accompanied by an increase in mitotic errors, CIN, and cell death and a reduction in the percentage of cycling cells. This data suggests that the subset of patients whose tumors exhibit centrosome amplification prior to treatment may be more likely to benefit from paclitaxel therapy.

#1845

TP53 **status determines the fate of cancer cells exposed to ionizing radiation and DNA-PK inhibitor, M3814.**

Qing Sun,1 Yige Guo,1 Xiaohong Liu,1 Frank Czauderna,1 Frank Zenke,2 Andree Blaukat,2 Lyubomir T. Vassilev1. 1 _EMD Serono Research and Development Institute, Inc., Billerica, MA;_ 2 _Merck Biopharma, Merck KGaA, Darmstadt, Germany_.

Inhibition of DNA double strand break repair in cancer cells has been proposed as a new therapeutic approach for potentiation of radiotherapy. M3814 is a potent and selective inhibitor of DNA-PK, a key component of non-homologous end joining repair pathway and is currently under clinical investigation in radiation combination trials. We studied the mechanism of cancer cell response to combined treatment with ionizing radiation and M3814 using cancer cell lines with different p53 status. M3814 blocked the repair of radiation-induced DSBs and suppressed a negative regulatory loop between DNA-PK and ATM causing enhanced phosphorylation and activation of ATM. In the p53 wild-type cells, ATM and its targets, CHK2 and p53, were activated more strongly by the combination treatment than by radiation alone, leading to a complete p53/CHK2-dependent cell cycle block and premature cell senescence. The p53-null or mutant cancer cells were unable to fully arrest the cell cycle and a substantial fraction entered S phase with unrepaired DNA, leading to mutations, chromosomal aberrations, aneuploidy, micronucleation and ultimately cell death, primarily due to mitotic catastrophe. Two isogenic pairs of p53 null/wt cells (A549 and HT-1080) were generated and used to demonstrate the critical role of p53 in the choice of M3814-modified radiation response. Time-lapse imaging of cell death kinetics using a small panel of p53 wild-type and p53 null/mutant cancer lines revealed clear differences in outcome dependent on p53 status. However, standard growth/viability assays failed to show a role of p53 in the response type, due to inability to discriminate between cell cycle arrest and cell killing. Similarly, screening results obtained from large cancer cell panels (>100 lines) did not identify p53 as a marker of response. Our data revealed an important role of p53 in determining the mode of cancer cells' response to radiation in the presence of M3814 and suggest that p53 mutation status should be considered in the design of clinical trials. The failure of standard growth/viability screens with large cancer cell line panels to identify p53 status as a response indicator raises an alert for the utility of these screening platforms currently widely used in cancer drug discovery and development.

#1846

HPV oncoproteins cause specific types of chromosomal instability in head and neck cancer.

Laura C. Funk, Denis L. Lee, Randall J. Kimple, Paul F. Lambert, Beth A. Weaver. _UW-Madison, Madison, WI_.

Aneuploidy, a karyotype that differs from a multiple of the haploid, is a hallmark of cancer. Aneuploidy can be caused by an ongoing rate of chromosome missegregation during mitosis, known as whole chromosomal instability (CIN). Work from our lab and others has shown that while low rates of CIN can promote tumorigenesis, high levels of CIN are tumor suppressive. Combining two insults that each cause low CIN results in high CIN, cell death, and tumor suppression. We recently showed that the chemotherapy drug paclitaxel causes CIN due to multipolar divisions in breast cancer. Preliminary evidence suggests that tumors which exhibit CIN prior to therapy are sensitized to paclitaxel because it increases their level of CIN over a maximally tolerated threshold. Radiation also induces CIN, and rectal adenocarcinomas which exhibit CIN prior to treatment are sensitized to chemoradiation therapy. Human papillomavirus (HPV), specifically HPV subtype 16, is a growing cause of head and neck cancer worldwide. In cervical cancer, HPV is associated with CIN, which has been attributed largely to the expression of its E6 and E7 oncoproteins. HPV+ head and neck cancer patients show improved responses to radiation therapy than HPV- head and neck patients, potentially due to an increased basal rate of CIN. Here, we tested the ability of HPV E6, E7, or E6+E7 expression to induce CIN in normal oral keratinocytes (NOKs). Expression of E6 alone robustly induced misaligned chromosomes and multipolar spindles with supernumerary centrosomes, while E7 expression alone produced more moderate effects. Combined expression of E6 and E7 exacerbated the phenotypes observed in NOKs expressing E6 alone. Expression of E6, either alone or in combination with E7 caused reduced protein levels of the mitotic kinesin CENP-E, which is sufficient to induce misaligned chromosomes. Interestingly, HPV+ patient-derived xenograft (PDX) models of head and neck squamous cell carcinoma showed less evidence of mitotic defects than NOKs expressing E6 alone. A subset of HPV+ PDX tumors showed an increase in misaligned chromosomes when compared to HPV- tumors, but no increase in lagging chromosomes or multipolar spindles was observed. Together, these data suggest HPV infection causes an incompletely penetrant increase in specific types of CIN in head and neck cancer and that a basal rate of CIN may be predictive of treatment response independent of HPV status.

#1847

The ubiquitin proteasome system controls cell cycle gene expression through regulation of histone acetylation in multiple myeloma.

Laure Maneix,1 Polina Iakova,1 Shannon Moree,1 Luke Fletcher,1 Premal Lulla,1 Sarvari V. Yellapragada,2 Andre Catic1. 1 _Baylor College of Medicine, Houston, TX;_ 2 _Michael E. DeBakey Veterans Affairs Medical Center, Houston, TX_.

Transcription factors (TFs) are generally short-lived proteins that undergo active turnover. The dynamic interaction of TFs and co-regulators with promoters and enhancers allows cells to continuously adjust gene expression. Whereas the composition and binding of TFs at genomic sites is the focus of a widespread research effort, relatively little is known about how these complexes are removed by the ubiquitin-proteasome system (UPS).

Multiple myeloma (MM), the second most common hematopoietic malignancy, has become a model disease for drugs that interfere with the UPS through either blocking or facilitating protein elimination. The proteasome inhibitor Bortezomib, for instance, is used as first-line treatment in MM. Since TFs are prime targets of proteasomal degradation, our research is focused on defining how nuclear proteolysis regulates transcriptional dynamics in this disease. This is of particular relevance since the therapeutic mechanisms of action of proteasome inhibitors are still ill-defined.

Following proteasome inhibition in multiple myeloma cell lines, we performed chromatin-immunoprecipitation for histone H3 acetylation (K27) and multiple histone deacetylases (HDACs) and used next generation sequencing (ChIP-seq) to identify unique gene clusters that are actively regulated by the proteasome and quantify epigenetic changes in dependence of protein turnover. Our findings reveal that cell cycle and mitosis-related genes, particularly subsets of genes involved in centromere formation and sister chromatid segregation, are associated with nuclear protein turnover and transcriptionally repressed by proteasome inhibition. Among the main transcriptional co-repressor complexes, we found NCoR1 to play a key role in controlling MM growth by adjusting cell cycle gene expression. Following proteasome inhibition, stabilization of NCoR1 at the promoters of these genes favors the recruitment of histone deacetylases to further inhibit gene activity. Concordantly, analysis of a panel of MM patients shows that the expression levels of certain histone deacetylases correlate with patient survival, specifically when treated with proteasome inhibitors. We are currently examining how NCoR1 is degraded, with a particular emphasis on the impact on histone acetylation at cell cycle gene promoters. Exploring how histone modifications and proteasome activity crosstalk in a therapeutically relevant manner in MM will help us to better understand how this degradation pathway impacts myeloma proliferation.

This research project will contribute to our understanding of epigenetic and transcriptional dynamics in MM. With our focus on the continuously changing abundance of TFs and co-regulators at promoters of cell cycle genes, we seek to unlock new therapeutical pathways and more specific targets for MM treatment compared to blunt proteasome inhibition.

#1848

A comprehensive image cytometry method to identify and functionally characterize a rare subpopulation of platinum-induced aneuploid tumor cells in high-grade serous carcinoma.

Yu Wing Yeung,1 Oliver Fung,2 Mikhail Bashkurov,3 Robert Rottapel,4 Andras Kapus1. 1 _St. Michael's Hospital, Toronto, Ontario, Canada;_ 2 _McGill University, Montreal, Quebec, Canada;_ 3 _Network Biology Collaborative Centre, Toronto, Ontario, Canada;_ 4 _University Health Network, Toronto, Ontario, Canada_.

Background: Although platinum treatment effectively reduces tumor bulk for high grade serous carcinomas of tubal-ovarian origin, there are often scattered tumor cells exhibiting reparative and aneuploid features that remained within the peritoneal cavity. The biological importance of these aneuploid cells has not been well elucidated. In this study, we used HGSC cell line models and developed an improved image cytometry approach to identify these aneuploid cells for functional interrogation.

Methods: We have developed a comprehensive image cytometry approach to assess DNA ploidy and cell cycle phase distribution within a standard flow cytometry software platform and validated this methodology using phase-specific markers (EdU, pRb, Geminin, pH3). We further introduced a novel 'brightness vs. nuclear size' scatter plot to enhance the cell cycle assessment at the single-nucleus level. The method was applied to correlate the kinetics of the DNA damage and checkpoint response with the cell cycle status and cell viability on HGSC cell lines (OVCAR3, TOV3133G) after in-vitro carboplatin treatment.

Results: Using this improved image cytometry method, we identified the emergence of a rare subpopulation of large aneuploid tumor cells at 1 week after platinum treatment. These large aneuploid cells remained viable (PI- and cleaved-PARP negative) but were uniquely dependent on anti-apoptotic signaling for their continual survival. While the bulk population of treated tumor cells underwent mitotic catastrophe following premature G2-M checkpoint exit, a rare subpopulation escaped cell death likely via mitotic slippage as they exhibit characteristic features of aneuploidy. These large aneuploid cells remained KI-67 positive and clonal dilutional experiments are being performed to assess their regrowth potential.

Conclusions: This study has identified a rare subpopulation of tumor cells that avoided mitotic cell death after platinum treatment and then survived in an aneuploid state. The derivation of aneuploid cells via mitotic slippage subsequent to platinum treatment is an under-recognized phenomenon but may pose as an important drug-resistant mechanism due to enhanced survival strategies co-opted by these aneuploid cells.

Research funding: Ontario Institute for Cancer Research

#1849

Determining the molecular requirements for paclitaxel-induced spindle multipolarity.

Christina Scribano. _University of Wisconsin-Madison, Madison, WI_.

Paclitaxel (TaxolTM) and other microtubule poisons are standard chemotherapy agents used in the treatment of breast cancer. However, only a subset of patients experience positive therapeutic effects from these drugs, underscoring the importance of further mechanistic study. Recently we showed that paclitaxel does not accumulate in primary breast cancers at sufficient concentrations to cause mitotic arrest, as it does at typically used concentrations in cell culture. Instead, paclitaxel therapy induces multipolar spindles in breast cancers. Breast cancer cell lines treated with low nanomolar, clinically relevant doses of paclitaxel exhibit chromosome missegregation due to multipolar divisions. We have now extended this to demonstrate that low nanomolar concentrations of other clinically used microtubule poisons (including ixabepilone, vinorelbine, and eribulin) also induce multipolarity in breast cancer cells, suggesting that multipolar mitotic divisions may be a common mechanism of cytotoxicity for microtubule-targeted drugs. Importantly, supernumerary centrosomes are not required for microtubule poison-induced multipolar spindles. To gain mechanistic insight into the formation of paclitaxel-induced multipolar spindles, a variety of mitotic regulators were tested for their ability to affect spindle multipolarity in breast cancer cells treated with clinically relevant concentrations of paclitaxel. Our results demonstrate that the mitotic kinesin Eg5 and the kinase Plk1 promote both the formation and the maintenance of paclitaxel-induced spindle multipolarity, while the mitotic kinesin CENP-E and the kinase Mps1 do not. Interestingly, we found that decreasing the time to anaphase onset decreases the number of multipolar divisions in paclitaxel, resulting in reduced cytotoxicity. Previous studies have shown that Mps1 inhibition sensitizes cells to paclitaxel, and this combination therapy has entered clinical trials (NCT02138812 and NCT02366949). However, our evidence suggests that Mps1 inhibition reduces the efficacy of paclitaxel as a result of decreased time to anaphase onset and fewer multipolar divisions. Additionally, our results suggest that inhibitors of spindle pole clustering increase the number of paclitaxel-induced multipolar divisions and therefore have the potential to increase its efficacy. Identification of the cellular requirements for multipolarity induced by paclitaxel, and of agents that drive the metaphase-to-anaphase transition in the presence of multipolar spindles, may improve the clinical utility of paclitaxel and other microtubule poisons.

#1850

CDK inhibitor PHA-848125 preferentially inhibits proliferation of triple negative breast cancer and synergizes with cisplatin.

Douglas G. Cheung,1 Marta Buzzetti,2 Carlo M. Croce,1 Gianpiero di Leva2. 1 _The Ohio State University, Columbus, OH;_ 2 _University of Salford, Salford, United Kingdom_.

Triple-negative breast cancer (TNBC), defined by the lack of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2), is highly aggressive with a poor prognosis as it lacks targeted therapies. Several inhibitors of the cyclin-dependent kinase 4 and 6 (CDK4/6) have been approved by the FDA for the treatment of ER-positive breast cancer. However, the effectiveness of these inhibitors against TNBC tumors is less than expected. Here, we tested the efficacy of PHA-848125, a potent ATP-competitive CDK2 inhibitor, on a panel of breast cancer cell lines showing a selective impaired proliferation in TNBC cells (IC50 of ER-positive MCF7 cells: 25.9 μM vs. IC50 of TNBC MDA-MB-231 cells: 430.8 nM). TNBC cells treated with PHA-848125 exhibited effects consistent with CDK2 inhibition, including G1 cell cycle arrest and reduced phosphorylation levels of the retinoblastoma protein (pRB) and CDK2. Interestingly, PHA-848125 was also effective against pRB-null TNBC cell lines, unlike other approved CDK inhibitors. Oral administration of PHA-848125 to TNBC xenograft mice and MMTV-PyMT transgenic mice impaired both tumor growth and metastasis at a tolerable dose (40 mg/kg), but not in ER-positive xenograft mice. A combination treatment of PHA-848125 and cisplatin inhibited tumor growth and induced apoptosis synergistically in vitro and in vivo. Thus, our preclinical experiments set the rationale for the clinical evaluation of PHA-848125, either alone or in combination with cisplatin, for the treatment of TNBC.

#1851

Combined inhibition of PARP and the unfolded protein response synergistically reduces viability in BRCA1-mutant breast cancer cells.

Jennifer E. Endress, Isaac S. Harris, Jacob Stewart-Ornstein, Galit Lahav, Joan S. Brugge. _Harvard Medical School, Boston, MA_.

As single agents, small molecule inhibitors of poly-ADP-ribose polymerase (PARP) have shown remarkable efficacy in BRCA1/2-mutant tumors due to their synthetic lethality in DNA damage response-deficient cells. While PARP inhibitors, such as olaparib, have gained FDA-approval for gynecological malignancies, recurrent disease remains a challenge. To identify drug combinations with the potential to improve the initial response to PARP inhibition, we carried out a high-throughput drug screen in which we examined the ability of olaparib to synergize with more than 500 small molecules in BRCA1-mutant breast cancer cells. The compounds investigated included not only conventional "anti-cancer" agents but also lesser-studied metabolic and stress-inducing drugs. All compounds were tested across 10-point dose curves using an image-based readout to avoid false positives/negatives associated with more traditional metabolic readouts of cell viability (i.e. Cell Titer Glo). This screening approach revealed that inhibition of the unfolded protein response (UPR) sensitizes breast cancer cells to PARP inhibition. We have further characterized interaction of one class of inhibitors that target IRE1 (inositol-requiring enzyme 1), which initiates adaptive responses to ER stress through unconventional mRNA splicing of the transcription factor X-box binding protein 1 (XBP1). Combining olaparib with an inhibitor of IRE1-mediated XBP1 splicing (STF-0831010) synergistically reduced cancer cell viability in BRCA1-mutant SUM149 breast cancer cells. This combination led to increased DNA damage, indicated by increased γ-H2AX staining. This study provides evidence that UPR inhibition sensitizes cells to PARP inhibition, suggesting a previously un-recognized connection between PARP inhibition and protein homeostasis. Furthermore, it identifies a potential new drug combination to increase the efficacy of PARP inhibitors in the clinic.

#1852

Combination of the CDK4/6 inhibitor abemaciclib with xentuzumab, a humanized IGF-1 and IGF-2 ligand co-neutralizing monoclonal antibody, results in synergistic antineoplastic effects in human breast cancer cell lines.

Ulrike Weyer-Czernilofsky,1 Rosa Baumgartinger,1 Susanne Schmittner,1 Xueqian Gong,2 Sean Buchanan,2 Richard P. Beckmann,2 Carlos Marugan,3 Raquel Torres,3 Karsten Boehnke,3 Maria Jose Lallena,3 Flavio Solca,1 Norbert Kraut,1 Thomas Bogenrieder1. 1 _Boehringer Ingelheim RCV GmbH & Co KG, Wien, Austria; _2 _Eli Lilly and Company, Indianapolis, IN;_ 3 _Eli Lilly and Company, Madrid, Spain_.

Background: The proliferative and pro-survival signals driven by the insulin-like growth factor (IGF) ligands, IGF-1 and IGF-2, are transmitted through their binding to the IGF-1 receptor (IGF-1R). In addition, IGF-2 promotes proliferation through activation of the insulin receptor variant A (IR-A) that is expressed during embryonic development as well as in many cancers. IGF survival signaling has been implicated in driving resistance to cancer therapies with diverse mechanisms of action, due to cross-talk between cellular signaling networks. Recent studies[1][2] suggest that the combination of IGF-1R signaling inhibitors with CDK4/6 inhibitors can result in enhanced anti-tumor activity. The aim of this study was to explore the potential of the IGF-1/-2 ligand blocking antibody, xentuzumab (BI 836845[3]), to enhance the anti-tumor activity of the CDK4/6 inhibitor abemaciclib, alone or in combination with fulvestrant, in human breast cancer (BC) cell lines.

Methods: The anti-proliferative activity of the xentuzumab/abemaciclib combination was evaluated using CellTiter-Glo and propidium iodide staining in a panel of 51 and 20 BC cell lines, respectively. Detailed studies of abemaciclib (+/- fulvestrant), xentuzumab and combinations thereof were performed in MCF7 cells. Cell cycle analysis was done by FACS and BrdU ELISA, cellular signaling was assessed by Western blotting, proliferation was evaluated by Incucyte, CellTiter-Glo and alamarBlue assay. Apoptosis was measured by detection of cleaved PARP and caspase 3.

Results:

Among a panel of BC cell lines, enhanced anti-proliferative activity of xentuzumab+abemaciclib vs. abemaciclib alone was observed specifically in hormone receptor positive (HR+) cell lines. Combined treatment resulted in more pronounced cell cycle arrest in MCF7 cells, associated with synergistic blockade of IGF survival signaling and suppression of cell cycle genes downstream of CDK4/6. The triple combination with fulvestrant more effectively inhibited tumor cell proliferation than the doublet abemaciclib+fulvestrant, and led to induction of apoptosis.

Conclusion: The study results show that addition of the IGF-1/-2 neutralizing antibody xentuzumab to abemaciclib, in the absence or presence of fulvestrant, leads to improved anti-proliferative activity and, in the triple combination, results in cellular death in MCF7 HR+ breast cancer cells. A phase Ib trial evaluating the abemaciclib+xentuzumab combination, including triplets with endocrine therapy in HR+BC patients, is currently ongoing.

References:

[1] Miller ML et al. (2013). Sci Signal 6;ra85

[2] Heilmann AM et al. (2014). Cancer Res 74:3947-58

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

#1853

The Chk1 inhibitor, SRA737, synergizes with niraparib to kill cancer cells via multiple cell death pathways.

Laurence Booth,1 Jane Roberts,1 Andrew Poklepovic,1 Ryan J. Hansen,2 Bryan Strouse,2 Snezana Milutinovic,2 Christian Hassig,2 Paul Dent1. 1 _Virginia Commonwealth University, Richmond, VA;_ 2 _Sierra Oncology, Inc., Vancouver, British Columbia, Canada_.

Targeting the DNA damage response (DDR) network is a promising strategy for the development of new cancer therapies. Checkpoint kinase 1, Chk1, is a central mediator of the DDR network and the potent, selective oral Chk1 inhibitor, SRA737, is being investigated in clinical trials. A distinct class of DDR inhibitors targeting PARP (PARPi) are approved for the treatment of ovarian cancers; however, tumors with functional homologous recombination (HR) repair are less sensitive to their effects, thereby limiting the clinical potential of these agents. Several reports have described the synergistic combination of Chk1i and PARPi, although the mechanism of anti-tumor activity has not been well defined. We explored the efficacy and mechanism of cytotoxicity of SRA737 in combination with the PARPi, niraparib, in HR repair proficient tumor cell lines. In short-term cell viability assays, the combination of SRA737 and niraparib elicited greater tumor cell death than either agent alone, as early as 12 hours after exposure to drug. Combination indices determined from colony forming assays indicated synergistic activity (CI < 0.7) using clinically achievable concentrations of each agent. Quantitative immunofluorescence studies revealed activation of ATM and phosphorylation of H2AX within 4 hours of treatment, indicating induction of DNA double strand breaks and activation of DDR signaling. Concurrent changes in the phosphorylation of mTOR, AMPK and the downstream target ULK1 suggested an induction of autophagy. Consistent with this hypothesis, the single agents, as well as the combination, led to decreases in p62 and LAMP2 levels and simultaneous increases in ATG5 and Beclin1 expression, and ATG13 phosphorylation. Autophagic flux was confirmed in cells expressing an LC3-GFP-RFP reporter plasmid. Genetic knockdown of autophagy components resulted in partial rescue of cell viability, suggesting that autophagy-dependent cell death may represent a mechanism of cytotoxicity for this DDR combination. Given that rescue of cell death was incomplete following abrogation of autophagy, we additionally examined the involvement of apoptotic pathways. SRA737 and niraparib treatment resulted in reduced levels of anti-apoptotic proteins, BCL-XL and MCL-1, and increased levels of the pro-apoptotic protein, BIM. Moreover, knock down of pro-apoptotic proteins or over-expression of anti-apoptotic proteins partially rescued combination-induced lethality. Collectively these results argue that toxic autophagy, as well as the intrinsic and extrinsic apoptosis pathways, contribute to SRA737 and niraparib-induced tumor cell killing. The involvement of multiple mechanisms of cell death may decrease the likelihood of cancer cells to acquire resistance to these agents. These findings support further investigation of SRA737 in combination with PARPi, including niraparib, in HR repair proficient cancers.

#1854

Treatment of head and neck squamous cell carcinoma cells with BMS754807 and Dasatinib induce synergistic cytotoxicity through altered focal adhesion signaling.

Christine E. Lehman,1 Rolando MEndez,1 Mark Axelrod,2 Julia Wulfkuhle,3 Emanuel Petricoin,3 Daniel Gioeli,4 Mark Jameson1. 1 _University of Virginia Health System, Charlottesville, VA;_ 2 _University of Virginia School of Medicine, Charlottesville, VA;_ 3 _George Mason University, Manassas, VA;_ 4 _University of Virginia, Charlottesville, VA_.

Head and neck cancer (HNC) is the 6th most common cancer worldwide and the 9th most common cancer in the United States. Singular, targeted cancer therapies rarely produce a robust, complete and durable response and often do not substantially prolong patient survival. Adaptive survival responses often blunt the cytotoxic effects of drugs resulting in resistance. Therefore, targeting compensatory signaling pathways may provide improved therapeutic responses. Even with the current best treatment, 5-year survival for all patients with head and neck squamous cell carcinomas (HNSCC) is only approximately 65%, further highlighting the need for improved combinatorial treatments. Previous work in our laboratory has shown that inhibitors of Insulin Like Growth Factor 1 Receptor (IGF1R) and Src family kinases, when used in combination, synergistically inhibit HNSCC growth in vitro. Therefore, to explore the mechanisms which may contribute to this synergistic cytotoxicity, we utilized reverse phase protein arrays to assess the proteome of HNSCC lines treated with BMS754807 and Dasatinib to inhibit IGF1R and Src, respectively. We identified focal adhesion signaling as a potentially vital node in the cell signaling network of synergistic cytotoxicity upon treatment of HNSCC with BMS754807 and Dasatinib. Focal Adhesion Kinase (FAK) and Paxillin phosphorylation was decreased synergistically with the combination of BMS754807 and Dasatinib. Using western blot analysis, we confirmed a decrease in phosphorylation of FAK Y576/577 and Paxillin Y118 upon combined treatment with BMS754807 and Dasatinib. Interestingly, FAK is upregulated in radioresistant HNSCC patients and, metastases have increased phosphorylation of FAK compared to normal tissues. FAK is amplified in up to 26% of HNSCC and overexpression of Paxillin is associated with unfavorable prognosis, highlighting the importance of focal adhesion signaling in head and neck cancers. Our data suggests that interactions between integrins, receptor tyrosine kinase, and non-receptor kinase signaling regulate cell survival in HNSCC. Disruption of IGFR and Src activity leads to convergent inhibition of focal adhesion signaling and synergistic cytotoxicity in a variety of HNSCC cell lines.

#1855

NUC-1031 overcomes resistance associated with gemcitabine in cancer patients.

Jennifer Bré,1 Awa Sarr,1 Peter Mullen,1 In Hwa Um,1 Sarah P. Blagden,2 Paul A. Reynolds,1 David J. Harrison1. 1 _University of St Andrews, St Andrews, United Kingdom;_ 2 _University of Oxford, Oxford, United Kingdom_.

NUC-1031 (NuCana plc) is a phosphoramidate transformation of gemcitabine and the first anti-cancer compound based on ProTide technology (Slusarczyk et al., 2014). It overcomes the main resistance mechanisms associated with gemcitabine and is currently in Phase I, II and III trials for biliary, ovarian, and pancreatic cancers respectively. High expression of dCK is required for activation of gemcitabine, which competes directly with deoxycytidine (dC) as a substrate for dCK. We aimed to compare the mode of action of NUC-1031 with gemcitabine. A panel of pancreatic and ovarian cancer cell lines were treated for 2h to 24h with NUC-1031 or gemcitabine at the EC50 dose and were observed for 96h. Cell morphology and death were observed using time-lapse microscopy. Cell cycle analysis was performed using BrdU labeling and flow cytometry. Exogenous dC was added to the cells either when they were plated or at the time of the treatment, to observe the effect on the sensitivity of cells to NUC-1031 and gemcitabine. Variation of expression of dCK was determined using RT-qPCR and western blotting. Cytospin, followed by immunocytochemistry, was performed to determine the intracellular localization of dCK. Expression of dCK was assessed in tissue samples from patients in the clinical studies using a modified Allred scoring system. After 48h, most cells treated with NUC-1031 show a 24h period when they neither divide nor die, after which they then undergo apoptosis. By contrast, cells treated with gemcitabine start to die sooner, showing features of necrosis and apoptosis. A higher proportion of cells treated with NUC-1031 exhibited a G2/M arrest compared to those treated with gemcitabine or control samples. Protein expression of dCK was increased in MiaPaCa2 cells soon after treatment with gemcitabine compared with NUC-1031. The pool of endogenous nucleotides was altered by addition of dC, inducing significant resistance to gemcitabine, both when dC was added at the time of the treatment and when cells were plated (EC50 respectively 2.5 and 9 times higher than in control cells). NUC-1031 was associated with a moderate decrease in sensitivity when dC was added at the time of the treatment and when cells were plated (EC50 3 times higher than in control cells). Immunohistochemistry of 50 primary tumours from patients enrolled in a Phase 1 study for NUC-1031 showed low-level dCK expression in almost all tumours studied. Our findings indicate that NUC-1031 has a more targeted mode of action to induce cell death than gemcitabine. Also, although a small amount of dCK may be required for maximal sensitivity to NUC-1031, dCK levels are not a limiting step for NUC-1031 efficacy.

#1856

Combination of the novel ERK inhibitor AZD0364 with the MEK inhibitor selumetinib significantly enhances antitumor activity in KRAS mutant tumor models.

Vikki Flemington,1 Iain Simpson,1 Jason Breed,1 Emma Davies,1 Francis Gibbons,2 Phillip Hopcroft,1 Nicola Lyndsay,1 Christopher Jones,3 Clifford Jones,4 David Robinson,1 Claire Rooney,1 Karen Roberts,1 Linda Sandin,1 Richard Ward,1 Pei Zhang,1 Elizabeth Pease1. 1 _AstraZeneca, Cambridge, United Kingdom;_ 2 _AstraZeneca, Waltham, MA;_ 3 _Lundbeck, Denmark;_ 4 _RedX Pharma, United Kingdom_.

The RAS/MAPK pathway is a major driver in oncogenesis and is dysregulated in approximately 30% of human cancers, primarily by mutations in BRAF or RAS genes. While BRAF and MEK inhibitors improve BRAF mutant melanoma patient outcomes, single-agent pathway inhibitors have demonstrated limited clinical benefit. Therefore, combined inhibition of multiple nodes within the RAS/MAPK pathway may be necessary to effectively suppress pathway signaling and achieve meaningful clinical benefit, specifically in patients with KRAS mutant tumors. AZD0364 is a potent and highly selective inhibitor of ERK1 and ERK2. AZD0364 exhibits high cellular potency against a direct substrate (e.g., inhibition of phosphorylation of p90RSK in BRAF mutant A375 cells, IC50 = 6 nM) and is highly (10/329 kinases tested are inhibited at >50% at 1 µM). In an unbiased cell proliferation screen of 750 tumor cell lines, >50% of cell lines that are sensitive to AZD0364 have RAS/MAPK pathway genetic alterations such as BRAF, NRAS or KRAS mutations. In a subset of KRAS mutant NSCLC cell lines, combined treatment of AZD0364 and selumetinib (AZD6244, ARRY-142886) is highly synergistic. This combination results in deeper and more durable suppression of the RAS/MAPK pathway that is not achievable with single-agent treatment, as assessed by phospho-p90RSK, change in transcriptional signatures and induction of apoptotic biomarkers. The AZD0364 and selumetinib combination also significantly suppresses RAS/MAPK pathway output and tumor growth in vivo to a greater extent than achievable with either agent given as a monotherapy. This combination is well tolerated in vivo and delivers 65% tumor regression in the NCI H358 KRAS mutant NSCLC xenograft model. This combination also results in significant tumor regressions in both A549 and HCT116 KRAS mutant xenografts. These data demonstrate that combined AZD0364 and selumetinib is well tolerated, effectively suppresses RAS/MAPK pathway signalling and delivers durable regressions in preclinical models. The combination of ERK and MEK inhibition represents a viable clinical approach to target KRAS mutant tumors.

#1857

**Hydrophilic As** 4 **S** 4 **nanoparticles inhibit K562 cell activity through ROS modulation and autophagy induction.**

Tao Wang, Tao Wen, Jie Meng, Jian Liu, Haiyan Xu. _Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Beijing, China_.

Realgar is crystalized As4S4 that has been demonstrated to be effective in treating hematologic malignancies by oral administration [1]. Nevertheless, the low bioavailability due to its poor solubility in neutral or acidic aqueous solution not only leads to the over dose administration of arsenic in clinical practice but also hinders the study of pharmaceutical mechanism for realgar. We previously reported a solid dispersion of realgar (e-As4S4), in which the crystal realgar (r-As4S4) was fabricated into nanoparticles encapsulated by the hydrophilic polymer using the hot melting co-extrusion technique [2]. The e-As4S4 exhibited much higher bioavailability and improved treatment efficacy in the acute myeloid leukemia (AML) animal model compared with r-As4S4.

This study investigated the effect of e-As4S4 has inhibitory on K562 cells and the underlying mechanisms. We showed that e-As4S4 down regulated the cellular ROS and HIF1-alpha in a concentration dependent manner. The low concentration of e-As4S4 induced both erythroid and megakaryocytic differentiation in the cells, evidenced by the up-regulation of CD235a and CD41 respectively, and the higher concentration induced cell cycle arrested in G2/M phase and apoptosis. Consistently, the proliferation rate and oxygen-consuming rate of the cells were reduced. Meanwhile the autophagy was detected when the cells were treated with e-As4S4, as well as the variation phosphorylation for p38 and Erk. These effects together induced the significant elimination of Bcr-Abl. In conclusion, e-As4S4 induced the bi-direction differentiation by modulating cellular ROS and inducing autophagy of the cells. These results therefore suggest that e-As4S4 represents a promising orally administrated drug for the treatment of CML.

Reference

1. Zhu HH et al. Oral tetra-arsenic tetra-sulfide formula versus intravenous arsenic trioxide as first-line treatment of acute promyelocytic leukemia: a multicenter randomized controlled trial. J Clin Oncol. 2013; 31:4215-21.

2. Ma Q et al. Fabrication of water-soluble polymer-encapsulated As4S4 to increase oral bioavailability and chemotherapeutic efficacy in AML mice. Sci Rep. 2016; 6: 29348.

Acknowledgement This work was supported by National Key R&D Program of China (2017YFA0205504) and CAMS Innovation Fund for Medical Sciences (CIFMS, 2016-I2M-3-004).

#1858

**EDNRB isoform 3 confers temozolomide resistance in human melanoma cells by modulating membrane potential, reactive oxygen species and mitochondrial Ca** 2+ **.**

Long Cui,1 Bo Liang,1 Xiaoting Chen,2 Chi Chiu Wang1. 1 _The Chinese University of Hong Kong, Hong Kong, Hong Kong;_ 2 _Southern Medical University, Guangzhou, China_.

Background: The role of Endothelin receptor type B (EDNRB) isoform 3 involved in Temozolomide (TMZ) induced melanoma cell death has not yet been elucidated.

Methods: The subcellular localization of EDNRB isoform 3 was determined by confocal and immunoblotting assays. Silencing EDNRB isoform 3 was performed by CRISPR/Cas9. Apoptosis was assessed by Annexin V/PI staining and caspases 3/7/9 activity. Mitochondrial membrane potential, reactive oxygen species and mitochondrial Ca2+ were measured by flow cytometry. Apoptosis protein array was applied.

Results: Confocal and immunoblot analyses indicate mitochondrial localization of EDNRB isoform 3 and the first N-terminal (1-22) amino acids are sufficient for its mitochondrial targeting. EDNRB isoform 3 depleted A375 cells significantly inhibit apoptosis. EDNRB isoform 3 mediates chemoresistance appears to be acting via a mitochondrial apoptosis pathway as manifested by the prevention in mitochondrial depolarization, reduced reactive oxygen species, enhanced mitochondrial Ca2+ uptake and decreased caspase 9 activation. Additionally, apoptosis array shows that lack of EDNRB isoform 3 has relatively lower expression of phosphorylation of p53 at S392 and a slightly higher expression of Paraoxonase 2.

Conclusion: Our findings raise the possibility of targeting EDNRB isoform 3 as new therapeutic strategy in combination with TMZ for melanoma treatment.

#1859

Inhibition of proteasome deubiquitinase activity prevents cytoprotective aggresome formation in cancer cells.

Ellin-Kristina Hillert,1 Slavica Brjnic,1 Magdalena Mazurkiewicz,1 Rolf Larsson,2 Mårten Fryknäs,2 Lisa Swanton,3 Stephen High,3 Stig Linder,1 Padraig B. D'Arcy1. 1 _Karolinska Institute, Stockholm, Sweden;_ 2 _Uppsala University, Uppsala, Sweden;_ 3 _University of Manchester, Machester, United Kingdom_.

Proteasome inhibitors kill cancer cells by inducing an acute proteotoxic stress response characterized by the cytosolic accumulation of poly-ubiquitinated proteins, ER stress and the production of reactive oxygen species (ROS). The aggresome pathway has been described as an escape mechanism from proteasome inhibitor-induced cytotoxicity that functions by sequestering the accumulated protein. We show that b-AP15, an established small molecule inhibitor of proteasome deubiquitinase activity, induces the accumulation of cytosolic poly-ubiquitin in the absence of aggresome formation. In contrast to the clinically used proteasome inhibitor bortezomib, the accumulated poly-ubiquitin complexes induced by b-AP15 remain localized in the cytoplasm and fail to form typical aggresome structures. Inhibition of aggresome formation is dependent on ubiquitin but independent of tubulin dynamics, and may result in the accumulation of a range of substrates of the intracellular transport system on microtubules. Finally, b-AP15 induces hyper-ubiquitination of the aggresome chaperone HDAC6, as well as altering HDAC6 association with the proteasome. This suggests a potential mechanism for the present findings, which will require further investigation. Our data suggests that b-AP15 inhibition of aggresome formation may circumvent a potential resistance pathway following cytotoxicity due to proteasome inhibition via the blocking of microtubule transport.

#1860

Tumor-treating fields (TTFields) effects on glioblastoma cells are augmented by mitotic checkpoint inhibition.

Almuth F. Kessler, Greta E. Frömbling, Franziska Gross, Mirja Hahn, Wilfrid Dzokou, Ralf-Ingo Ernestus, Mario Löhr, Carsten Hagemann. _Univerity Hospital Würzburg, Würzburg, Germany_.

Introduction. Tumor Treating Fields (TTFields) supplementing the standard therapy led to a significant increase in progression free and overall survival of glioblastoma (GBM) patients. These alternating electric fields with low intensity (1-3 V/cm) and intermediate frequency (100-300 kHz) disrupt cell division through inhibition of spindle-fiber formation. The spindle assembly checkpoint (SAC) diminishes therapeutic effects of spindle damaging agents by monitoring correct sister chromatid attachment to functional spindle microtubules and ensures their equal segregation. Inhibition of its key regulator, Monopolar spindle 1 (MPS1), combined with anti-mitotic drugs, led to a synergistic effect on GBM growth in mice. Therefore, we hypothesized that MPS1 inhibition may increase the efficacy of TTFields.

Methods. TTFields were applied using the inovitro system. U87 and GaMG human GBM cells (30,000) were treated with TTFields (200 kHz, 1.7 V/cm) and/or with 4 µM of the MPS1 inhibitor MPS1-IN-3 (IN-3). Cell numbers were evaluated after 24 h, 48 h and 72 h of treatment and also at 24 h and 72 h after end of treatment (eot). Alterations of the cell cycle were detected by FACS analysis, aberrant mitotic figures and malformed nuclei by immunofluorescence microscopy and apoptosis by Annexin V staining and TUNEL-assay after 72 h of treatment.

Results. In all experiments U87 and GaMG yielded similar results. The combination of TTFields and IN-3 caused a more pronounced effect on cell proliferation (78.6% decrease of U87 cell number vs. TTFields (P = 0.0337), 52.6% vs. IN-3 (P = 0.0205) after 72 h). The number of viable cells was reduced (62% less GaMG cells than seeded) and the ratio of dead/alive cells increased. Significantly less cells re-entered the G1 phase (P < 0.0001), while the number of cells in G2/M- and sub-G1-phase was increased. Very distinct mitotic figures and aberrant nuclei were visible, leading to apoptosis in 44% of the U87 cells (TTFields 14%, P = 0.0002; IN-3 4%, P < 0.0001). After discontinuation of TTFields treatment alone, it took 24 h for the cells to start recovery. In contrast, the number of cells treated with IN-3 and TTFields, further decreased by 92% at 72 h after eot (P = 0.0288).

Conclusions. TTFields are an approved new treatment modality for GBM. A combination of physically damaging the spindle apparatus by TTFields and chemical inhibition of the SAC led to earlier and prolonged effects, which significantly augment TTFields efficacy and even may bridge TTFields treatment interruption, promising a new targeted multimodal treatment Option.

#1861

Combination of eribulin plus AKT inhibitor evokes synergistic cytotoxicity in soft tissue sarcoma cells.

Kohichi Takada, Naotaka Hayasaka, Yutaka Kawano, Hajime Nakamura, Yohei Arihara, Kazuyuki Murase, Shohei Kikuchi, Satoshi Iyama, Koji Miyanishi, Masayoshi Kobune, Junji Kato. _Sapporo Medical Univ Sch of Med, Sapporo, Japan_.

[Background]

Prognosis of unresectable or metastatic (UM)-soft tissue sarcoma (STS) is poor, with overall survival (OS) less than 1.2 years after diagnosis. Recommended therapy for most UM-STS patients is doxorubicin (Dox) as first line palliative chemotherapy. Recently, three new drugs, pazopanib, trabectedin and eribulin, were introduced for treatment of UM-STS after Dox therapy. Only eribulin could significantly improve OS. However, outcomes with eribulin for UM-STS did not satisfy an unmet clinical need. Therefore, despite recent advances in its treatment, prognosis of UM-STS remains dismal, underscoring the need to develop novel strategies for UM-STS. Resistance to anti-cancer drugs frequently arise that would hamper results of cancer therapy. Mechanisms of eribulin resistance have been largely unknown. Overexpressed p-AKT underlies the pathogenesis of a broad range of human malignancies including STS. The high expression of p-AKT has been identified as a negative surrogate marker for survival of STS patients. Therefore, AKT is an attractive target for STS therapeutics.

[Aims]

Our study aimed to investigate the molecular mechanism of eribulin-resistance and to evaluate the effect of combination therapy with eribulin and AKT-inhibitor on STS cells.

[Methods]

Eribulin-resistant STS cell lines were established by culturing HT1080 and SK-LMS-1 cells with stepwise increases in the eribulin concentration for more than 3 months. Alterations in the phosphorylation status of kinases in eribulin-resistant cells compared to parental cells were screened using a Human Phospho-Kinase Array Kit. The effect of eribulin and AKT-inhibitor MK-2206 on STS cell lines was examined using an MTT assay. To elucidate the cytotoxic mechanism of eribulin and MK-2206, immunoblotting and flow cytometry analyses were performed.

[Results]

In established eribulin-resistant cell lines, those IC50s were 2.40- to 27.14-fold greater than in the parental cell lines. Phospho-Kinase Array revealed increased p-AKT (S473) levels in eribulin-resistant cells compared to the parental cells. Eribulin treatment increased p-AKT levels in STS cells in a dose-dependent fashion. Combination treatment of eribulin and AKT-inhibitor MK-2206 inhibited the proliferation of STS cells accompanied with G1 and/or G2/M arrest by down-regulating CDKs, cdc2 and cyclins. Furthermore, eribulin combined with MK-2206 synergistically induced apoptosis in STS cell lines. The apoptosis was via Caspase 8 and 9 pathways.

[Conclusion]

This study demonstrated that activated-AKT was associated with eribulin-resistance in

STS cells and that eribulin combined with MK-2206 suppressed STS growth synergistically through G1 and G2/M arrest and caspase-dependent apoptosis. Our results uncover a potential clinical application of eribulin and MK-2206 combination therapy to improve clinical outcomes for UM-STS patients.

#1862

CX5461 in combination with radiotherapy I effective against gynecological tumors.

Mohammed Ismael, Roger Webb, Mazhur Ajaz, Helen Coley. _Univ. of Surrey, Guildford, Surrey, United Kingdom_.

An increased rate of cellular proliferation is a hallmark of cancer and may be accompanied by increase in ribosome biogenesis and dysregulation in rRNA synthesis. In this regard CX-5461 has been developed as a novel RNA POL1 inhibitor and is currently in Phase I/II clinical trials. We have set out to explore the use of this agent in combination with radiation therapy and to understand the associated molecular mechanisms using this approach. A panel of human tumour cell lines treated with CX-5461 indicated a broad spectrum of sensitivity (MTT assay) with IC50 values varying from 25nM - >2µM. No clear relationship between p53 status and sensitivity to CX-5461 was indicated and the mode of cell death was cell context dependent - either via autophagy (LC3I→ LC3II conversion, with P62 sequestosome decrease), sometimes via apoptosis (annexinV staining, caspase-3 activation), also in the presence of increased senescence (β-galactosidase activity). Combination treatment experiments involving CX-5461 alongside radiation therapy (0-16Gy) showed marked synergistic effects for some cancer cell lines, particularly the cervical cancer CASKI line (HPV16+ve). Highly significant effects were seen when low dose radiation and low dose CX-5461 were used together in CASKI cells: combination index CI=< 0.2 with 6nM CX-5461 and 2Gy (isobologram analysis). Cell cycle analysis of CASKI cells treated in combination indicated cell cycle disruption with prolonged S-phase, giving some insight into some of the associated mechanisms involved. In addition, we have found that BRCA-mutant PEO1 ovarian cancer cells were sensitive to CX-5461, IC50 approx. 100nM, whereas carboplatin-resistant PEO1CarbR (with reversed BRCA mutant status) cells gave an IC50 value >3µM In addition, proteomic studies have shown that PEO1CarbR cells overexpress a number of nucleolar proteins and we are investigating the relevance of this in relation to CX-5461 sensitivity. Our further studies aim to identify potential biomarkers of response to CX-5461 and may also inform the design of clinical trials of this novel agent.

#1863

Ursolic acid sensitizes rhTRAIL-resistant triple negative breast carcinoma to rhTRAIL-induced apoptosis.

Jasmine Manouchehri, Michael Kalafatis. _Cleveland State Univ., Cleveland, OH_.

Recombinant human tumor necrosis factor-related apoptosis-inducing ligand (rhTRAIL) possesses the ability to induce apoptosis in cancer cells independent of their p53 status while exhibiting minimal toxicity to normal, non-transformed cells, and thus, it is a promising anti-cancer therapeutic. However, rhTRAIL-induced apoptosis is not as effective in a majority of breast cancers due to the up-regulation of anti-apoptotic proteins, down-regulation of pro-apoptotic proteins, and/or down-regulation of death receptors (DRs) 4 and 5. A combinatorial approach of rhTRAIL with the "mother nature"-derived compound ursolic acid (UA) has been applied to sensitize rhTRAIL-resistant triple negative breast carcinoma. UA is derived from the leaves and berries of various plants and found in the coatings of fruits and does not exhibit toxicity to normal, non-transformed cells. UA has been revealed to possess the ability to up-regulate DR5 and diminish the expression of anti-apoptotic proteins survivin and FLIP in cancer cells and thereby, making UA an encouraging choice to be utilized as a sensitizing agent. The aim of this study was to determine the capacity of UA to sensitize rhTRAIL-resistant triple negative breast cancer (TNBC) BT-20 and HCC1937 cells to rhTRAIL-induced apoptosis and elucidate the underlying mechanisms for UA's sensitization. The combinatorial treatment of UA and rhTRAIL augmented the induction of apoptosis when compared to single agent UA and rhTRAIL treatments as detected by Annexin V/PI assays and through by the execution of the extrinsic pathway as marked by the activation of caspase 8, activation of the executioner caspases 3 and 7, and eventual PARP cleavage (a hallmark of apoptosis). The underlying mechanisms for UA's sensitization of rhTRAIL-resistant TNBCs were established to be through the down-regulation of the anti-apoptotic protein FLIP and through the up-regulation of DR4 and DR5. Overall, these findings reveal that UA is an efficacious sensitizing agent for rhTRAIL-resistant TNBCs.

#1864

Pharmacologic inhibition of NAMPT sensitizes pancreatic cancer cells to the antineoplastic effects of metformin.

Gerardo Ferbeyre, Maxime Parisotto, Marie-Camille Rowell, Véronique Bourdeau, Andreea R. Schmitzer. _Universite de Montreal, Montreal, Quebec, Canada_.

The use of the anti-diabetic drug metformin has been correlated with a reduced cancer incidence, suggesting an unexpected anti-neoplastic activity for this compound. Since metformin treatment is safe and economical, there is considerable interest in exploring its anticancer activity in patients. Pancreatic cancer (pancreatic ductal adenocarcinoma: PDAC) is one of the most aggressive neoplastic diseases, for which there is no treatment significantly increasing patient's survival. Metformin use was associated with reduced pancreatic cancer incidence or better survival in diabetics. In vitro, metformin decreases cell survival and growth of pancreatic cancer cells and appears to target tumor-initiating cells. In vivo, metformin decreases growth of human pancreatic cancer cell lines xenografts in mice. However, clinical trials using metformin failed to decrease pancreatic cancer progression in patients, raising important questions about molecular mechanisms that protect tumor cells from the antineoplastic activities of metformin. We discovered a new mechanism of resistance to the anti-oncogenic properties of metformin in PDAC cells through up-regulation of NAMPT and increase of NAD+ synthesis. Using an inhibitor specific to NAMPT, FK866, we sensitized PDAC cells to the effects of metformin in vitro. In vivo, FK866 increased the efficiency of metformin treatment on KP4 cells xenografts. As both metformin and FK866 clinical trials have failed to efficiently treat cancer, the combination of these two compounds may be a promising strategy to treat pancreatic cancer and maybe other malignancies.

#1865

The combined treatment of 150 kHz Tumor Treating Fields (TTFields) and sorafenib inhibits hepatocellular carcinoma in vitro.

Karnit Gotlib,1 Einav Zeevi,1 Rosa S. Schneiderman,1 Tali Voloshin,1 Moshe Giladi,1 Adrian Kinzel,2 Eilon D. Kison,1 Uri Weinberg,3 Yoram Palti1. 1 _Novocure, Haifa, Israel;_ 2 _Novocure, Munich, Germany;_ 3 _Novocure, Luzern, Switzerland_.

Hepatocellular carcinoma (HCC) remains a major health problem worldwide as the third cause of cancer related mortality and the primary cause of death. Currently, sorafenib, an oral multikinase inhibitor that blocks various signaling pathways, is the only drug that has been approved for patients with advanced hepatocellular carcinoma, yet its survival benefit is still limited. Tumor Treating Fields (TTFields) therapy is an effective anti-neoplastic treatment modality delivered via noninvasive application of low intensity, intermediate frequency, alternating electric fields. TTFields are employed as a local treatment with the intent to target dividing cells by disrupting microtubules leading to mitotic catastrophe, abnormal chromosome segregation and the induction of different forms of cancer cell death. The aim of this work is to explore the potential of the use of TTFields alone and in combination with Sorafenib as a treatment for HCC.

HepG2 and Huh-7D12 cells were treated with various TTFields frequencies for 72 hours using the inovitro system. Efficacy of the combined treatment of TTFields and Sorafenib was tested by applying TTFields at the optimal frequency together with various drug concentrations. Cell counts, induction of apoptosis, cell cycle and clonogenic potential were determined at the end of treatment.

The optimal TTFields frequency leading to the highest reduction in cell counts was found to be 150 kHz for both HepG2 and Huh-7D12 cells. TTFields application (1.0 - 1.7 V/cm, 72 hours) at 150 kHz led to 53-64% reduction in cell counts and to additional reduction of over 70% in the clonogenic potential. The combined treatment of TTFields and Sorafenib led to a significant reduction in the number of HepG2 and Huh-7D12 cells (2-way ANOVA, p<0.001 in both cell lines) as compared to each treatment alone.

The results presented in this work demonstrate that TTFields can be an effective treatment against HCC cells and that the combination with Sorafenib may further enhance treatment efficacy. Based on the above, there is a strong rational to continue exploring the potential of the use of TTFields together with standard of care for the treatment of HCC in the clinical settings.

#1866

Small molecule NSC59984 is a radio-sensitizer dependent on ERK2 and DDR but independent of wild-type p53.

Shengliang Zhang, Lanlan Zhou, Niklas Finnberg, Wafik El-Deiry. _Fox Chase Cancer Center, Philadelphia, PA_.

Radiotherapy is a common and effective therapeutic strategy applied to various types of tumors. Mutations that commonly occur in p53 as a tumor suppressor gene, are a hallmark of tumor, and confer cellular radio-resistance. Targeted therapy is considered as a promising approach to increase efficacy and specificity of radiotherapy. We explore utilization of a small molecule targeting mutant p53 to enhance efficacy of radiotherapy in colorectal cancer. Colorectal cancer cells carrying with mutant p53 were treated with NSC59984 followed by ionizing radiation treatment. Combined NSC59984 with radiation significantly reduced colony formation in mutant p53-expressing colorectal cancer cells in a dose-dependent manner, suggesting that NSC59984 enhances the efficacy of radiotherapy. NSC59984 induces mutant p53 degradation via the ERK2 pathway. We investigated the role of ERK2 in the efficacy of the combination of NSC59984 and radiation treatment. ERK2 blockade partially abrogated the effect of NSC59984 on the radio-sensitivity, and this correlated with the rescue of mutant p53. Our results suggest that NSC59984 increases the efficacy of radiotherapy via activation of ERK2 or ERK2-dependant mutant p53 degradation. Mutant p53 and ERK2 are important regulators involved in repair of DNA double-strand breaks. We examined the DNA damage response (DDR) system and found that reduced Rad51 protein in cells treated with NSC59984, and the Rad51 protein was increased by the blockade of the ERK2 pathway. On the basis of the above results, we conclude that small-molecule NSC59984 enhances cancer cell sensitivity to radiotherapy via the ERK2 pathway and effects on DDR.

#1867

Combined and sequential chemotherapy efficacy of trifluridine/tipiracil and regorafenib in colorectal cancer cell lines.

Kazuaki Matsuoka,1 Fumio Nakagawa,2 Junji Uchida,2 Teiji Takechi1. 1 _Taiho Pharmaceutical Co., Ltd., Tokyo, Japan;_ 2 _Taiho Pharmaceutical Co., Ltd., Tokushima, Japan_.

Background: Trifluridine/tipiracil (FTD/TPI or TFTD, also known as TAS-102) is recommended for treating metastatic colorectal cancers refractory to standard chemotherapies. Regorafenib is an oral multikinase inhibitor that blocks the activity of several protein kinases associated with angiogenesis, oncogenesis, and the tumor microenvironment. We evaluated the antitumor effects of FTD/TPI combined with regorafenib on a colorectal tumor xenograft mouse model. We also investigated whether cytotoxicity was enhanced when FTD was used sequentially with regorafenib in vitro.

Method: The colorectal cancer cell lines SW620 and COLO 205 were implanted into nude mice subcutaneously. FTD/TPI (150 mg/kg/day) was orally administered twice daily from days 1 to 14 and regorafenib (10 mg/kg/day) was administered once a day at approximately 2-4 h after FTD/TPI. For the in vitro cytotoxic assay, SW620 cells were treated with FTD and regorafenib as follows: (1) exposure to 0.1-4.0 µM FTD alone, (2) sequential exposure to 0.1-4.0 µM FTD for 24 h followed by 10 µM regorafenib for 24 h, or (3) sequential exposure to 10 µM regorafenib for 24 h followed by 0.1-4.0 µM FTD for 24 h. All treatments were evaluated by a colony formation assay. Molecules involved in mechanism of action of FTD and regorafenib and in apoptosis were evaluated by western blot.

Results: Tumor growth inhibition by combination therapy was 86.4% and 92.3% for SW620 and COLO 205 xenografts, respectively; this was significantly superior to the growth inhibition observed after either monotherapy for the two evaluated cancer xenografts (P < 0.01). Thus, FTD/TPI combined with regorafenib is significantly more effective than either monotherapy in colorectal cancer xenografts. This combination therapy was tolerated without 20% body-weight reduction and drug-related death. FTD followed by regorafenib treatment in SW620 cells showed stronger cell killing effect than FTD alone. In contrast, regorafenib followed by FTD treatment attenuated its cell killing effect rather than that to FTD alone. In comparison with FTD alone, FTD followed by regorafenib reduced thymidine synthase (TS) and inhibited phosphorylation of ERK1/2 (pERK1/2) with the induction of proapoptotic marker, cleavage of PARP. In contrast, regorafenib followed by FTD induced TS and pERK1/2. Thus, FTD followed by regorafenib treatment is more effective than regorafenib followed by FTD treatment in vitro.

Conclusion: FTD/TPI combined with regorafenib is significantly more effective than either monotherapy in preclinical models. Moreover, FTD followed by regorafenib is more effective than vice versa in vitro, suggesting that in patients sequential therapy with FTD/TPI prior to regorafenib may be helpful in treating colorectal cancer.

#1868

Upregulation of p62/Nrf2 and downregulation of p-eIF2a provide more susceptible to docosahexaenoic acid-induced cell death in LMP-1 expressing nasopnaryngeal carcinoma cells.

Kaipeng Jing,1 Soyeon Shin,1 Seung-Hyeon Han,2 Tong Wu,3 Kyu Lim4. 1 _Dept. of Biochemistry, School of Medicine, Chungnam National University, Daejeon, Republic of Korea;_ 2 _Dept. of Biochemistry, Department of Medical Science, School of Medicine, Chungnam National University, Daejeon, Republic of Korea;_ 3 _Department of Pathology and Laboratory medicine, Tulane University School of Medicine, New Orleans, LA;_ 4 _Dept. of Biochemistry, Department of Medical Science, School of Medicine, Cancer Research Institute, Chungnam National University, Daejeon, Republic of Korea_.

Docosahexaenoic acid (DHA) induces apoptotic cell death through several mechanisms in cancer cells. We have previously demonstrated that DHA triggers apoptosis by increasing reactive oxygen species (ROS) accumulation and the ROS-mediated apoptosis caused by DHA is associated with Nrf2 signaling activation. Here we report that DHA-induced cell death is more susceptible through upregulation of p62-Nrf2 and downregulation of p-eIF2α in LMP-1-expressing nasopharygeal carcinoma (NPC) cells. Treatment of four human NPC cells (CNE, CNE-LMP1, HONE, HONE-EBV) with DHA for 24 hr resulted in a dose-dependent inhibition of cell growth. The DHA effect was due to the induction of apoptosis, given that DHA increased the cleaved form of PARP as well as the number of TUNEL-positive cells. The inhibition of CNE-LMP1 and HONE-EBV cells after DHA treatment is more susceptible compared with CNE and HONE cells without LMP1 by MTT assay. The level of p62 and Nrf2 of LMP1-NPC cells were increased after DHA pretreatment compared to control NPC cells. On the other hand, the level of p-eIF2α produced reverse result. The activation of Nrf2 signal seems to result from decreased Nrf2 inhibitor, Keap1, because DHA remarkably attenuated Keap1 expression levels. Moreover, silencing Nrf2 by small interfering RNAs inhibited the cytotoxic effect of DHA, indicating that Nrf2 activation plays a positive role in the process of DHA-induced apoptosis. Increased staining for LMP1 and p62 was observed in NPC tissues when compared with the nonneplastic (chronic inflammation) tissues. These results suggest that differential regulation of p62/p-eIF2α/NRF2 contributes to susceptible cell death by DHA in LMP-1-expressing NPC cells. Thus, utilization of DHA may represent a promising therapeutic approach for chemoprevention and treatment of human NPC. [This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (NRF-2015R1D1A1A01056887) and by the framework of international cooperation program managed by National Research Foundation of Korea (2015K2A2A6002008)].

#1869

Role of Nef secretion modification region (SMR) peptides and the chaperone mortalin in breast cancer and leukemia cells.

Ming B. Huang, Vinvent C. Bond. _Morehouse School of Medicine, Atlanta, GA_.

BACKGROUND: Mortalin/GRP-75(mt-hsp70, HSPA9) is a mitochondrial chaperone protein, also

found in the cytoplasm, endoplasmic reticulum and cytoplasmic vesicles. It functions in many

cellular processes such as mitochondrial biogenesis, intracellular trafficking, cell proliferation,

signaling, immortalization and tumorigenesis, Thus, inhibition of mortalin is a promising avenue

for cancer therapy. Previous studies in our lab have suggested that mortalin contributes to breast

cancer development and progression. We showed tumor exosome secretion was decreased by

knockdown of mortalin expression using HIV-1 Nef peptides. Specifically, these peptides can

block exosome secretion and also mediate cell cycle arrest in MDA-MB-231 and MCF-7 breast

cancer cells.

AIMS: This study aims to investigate further the function and mechanism of interaction of PEG-

SMR-CLU peptide and SMR-CPP peptide, and chaperone mortalin and to explore the effect of

SMR-derived peptides and mortalin on human breast cancer and leukemia cell lines.

RESULTS: Our results demonstrate that the modified SMRwt peptides not only affect breast

cancer cell proliferation and arrest breast cancer cell growth, but they also inhibit migration of

breast cancer cells. SMRwt peptides also block exosome-mediated release of complement, and

allow the cells to die via complement induced cell death, and reduced mortalin protein expression.

We further determined that expression levels of the mesenchymal markers vimentin (VIM) was

decreased in presence of SMRwt peptides and MKT-077.

CONCLUSIONS: Mortalin may promote cell proliferation and invasion via induction of epithelial

mesenchymal transition (EMT) of cancer cells, which has a potential therapeutic target in cancer

immunotherapy. SMRwt peptides block tumor exosome release and reduce the expression of

mortalin. This may be a means to decreases breast cancer cell invasion and metastasis, which

may have important clinical applications for late stage breast cancer chemotherapy. Furthermore,

SMRwt peptides and inhibition of mortalin suppressed the migration and invasive capacity of

cancer cells and is associated with a diminished EMT. These findings support a role for SMR

peptide and mortalin in the induction of EMT, prompting a further investigation into their

therapeutic value in cancer metastasis.

### Experimental Agents and Combinations for Hematologic Malignancies 2

#1870

The anti-proliferative and pro-apoptotic effect of MDM2-p53 antagonists evaluated in human tumor cells lines and chronic lymphocytic leukemia patient samples.

Elaine Willmore,1 Yan Zhao,1 Carmela Ciardullo,1 Laura Woodhouse,1 Huw D. Thomas,1 Maria Ahn,2 Lynsey Fazal,2 Martin E. Noble,1 Ian Hardcastle,1 Steven Howard,2 Gianni Chessari,2 John Lunec,1 Steve Wedge1. 1 _Newcastle University, Newcastle upon Tyne, United Kingdom;_ 2 _Astex Pharmaceuticals, Cambridge, United Kingdom_.

We investigated the cellular response to two MDM2-p53 antagonists (RG7388 and Cpd 1) in a panel of 19 tumor cell lines and in patient-derived chronic lymphocytic leukemia (CLL) cells. RG7388 (Idasanutlin) is an MDM2-p53 antagonist developed by Roche and Cpd1 a novel potent and selective MDM2-p53 antagonist developed as part of an ongoing Alliance between Newcastle University, Cancer Research UK and Astex Pharmaceuticals. RG7388 and Cpd1 potently inhibited the proliferation of many cell lines, with MDM2-amplified SJSA-1 osteosarcoma cells and cells from haematological malignancies being the most sensitive (e.g. GI50 values in the acute myeloid leukemia cell line MOLM13 were 33 + 16 nM for RG7388 and 8 + 1 nM for Cpd 1). We also examined induction of apoptosis by measuring caspase 3/7 activation (24 h treatment). SJSA-1 cells showed a 20-fold increase in caspase activation and Molm13 cells a 6-fold increase (measured at 300nM with RG7388). However, cell lines from solid tumours such as the colorectal carcinoma HCT116 or the hepatocellular carcinoma HepG2 did not show any induction of apoptosis, even at concentrations up to 1µM RG3788. In primary CLL cells, Cpd 1 reduced cell viability (48 h) with an average LC50 of 131 + 46 nM (n = 4), compared to 400 + 55 nM for RG7388 (n =23). We also quantified mRNA levels in 25 primary CLL samples and found that RG7388 induced a predominantly pro-apoptotic gene signature: following 6h treatment, 1µM RG7388 resulted in an average 8-fold induction of PUMA and 3.5-fold increase in BAX compared to a 1.6-fold induction of CDKN1A (p21). Our data confirm that targeting the MDM2-p53 interaction is an effective strategy to inhibit growth and viability of tumor cells, and that some cells, including those expressing high levels of MDM2 or those derived from hematological malignancies, display a striking apoptotic response.

#1871

Development of pelorol analogues to activate the SHIP1 lipid phosphatase; a novel paradigm to suppress B-cell receptor signaling in human B-cell cancers.

Graham Packham,1 Elizabeth Lemm,1 Beatriz Valle-Argos,1 Lindsay Smith,1 Nichola Weston-Bell,1 Freda Stevenson,1 Matthew J. Carter,1 Mark S. Cragg,1 Francesco Forconi,1 Andrew J. Steele,1 Jennifer Cross,2 Curtis Harwig,2 Georg Lenz,3 Lloyd Mackenzie,2 Pavel Klener4. 1 _University of Southampton, Southampton, United Kingdom;_ 2 _Aquinox Pharmaceuticals Inc., Vancouver, British Columbia, Canada;_ 3 _University of Munster, Munster, Germany;_ 4 _Charles University General Hospital in Prague, Prague, Czech Republic_.

Signaling via the B-cell receptor (BCR) is a major driver of malignant B-cell proliferation and survival in B-cell malignancies including chronic lymphocytic leukemia (CLL). The role of kinases in BCR signalling is well understood and kinase inhibitors are effective therapies for B-cell cancers. However, resistance is increasingly common and new drugs are required. In this study we investigated an alternate strategy to block BCR signaling via small molecule activation of SHIP1, an inositol lipid phosphatase which suppresses PI3 kinase (PI3K)-mediated signaling by catalysing the conversion of the PI3K product PI(3,4,5)P3 (PIP3) to PI(3,4)P2. We focused on the exemplar compound, AQX-C5, which is structurally related to the natural product pelorol. AQX-C5 interfered with the ability of anti-IgM to activate PIP3-dependent signaling, including downstream phosphorylation of AKT, ERK1/2 and p70-S6K, and induction of MYC, in primary CLL cells. AQX-C5 also induced CLL cell apoptosis and overcame the survival-promoting effects of anti-IgM or CD40-ligand/interleukin-4. CLL cells were more sensitive to pro-apoptotic effects of AQX-C5 compared to non-malignant B cells. The AQX-C5-induced apoptosis in CLL cells was associated with down-modulation of the BCL2-related survival protein MCL1 in anti-IgM-stimulated cells, and induction of the pro-apoptotic protein NOXA. In addition to effects on PIP3-dependent signaling, AQX-C5 triggered down-modulation of CXCR4, a chemokine receptor important for homing of malignant cells to supportive tissue microenvironments. CXCR4 downmodulation was selective, since AQX-C5 did not alter surface expression of other receptors, including IgM and the transferrin receptor. In contrast to AQX-C5, the PI3Kδ inhibitor idelalisib failed to alter CXCR4 expression, indicating that these effects of AQX-C5 may be mediated via PI(3,4)P2 accumulation rather than PIP3 depletion. We evaluated the in vivo anti-lymphoma activity of AQX-C5 in a xenograft model using the TMD8 cell line, derived from a diffuse large B-cell lymphoma. Similar to the BTK inhibitor ibrutinib, AQX-C5 substantially reduced the rate of tumor growth and tumor mass at the end of the experiment, without evidence for gross toxicity. These findings suggest that SHIP1 activators, such as AQX-C5, may be interesting therapeutic agents for various B-cell cancers.

#1872

Pharmacological inhibition of WIP1 sensitizes AML cells to MDM2 inhibitors.

Maria Chiara Fontana,1 Jacopo Nanni,1 Giorgia Simonetti,1 Andrea Ghelli Luserna di Rorà,1 Antonella Padella,1 Samantha Bruno,1 Cristina Papayannidis,1 Giovanni Marconi,2 Mariachiara Abbenante,1 Michele Cavo,1 Giovanni Martinelli1. 1 _University of Bologna, DIMES, Istituto Seragnoli, Bologna, Italy;_ 2 _Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, Meldola, Italy_.

Introduction: The wild-type (wt) p53-inducible protein phosphatase WIP1 (gene PPM1D), is frequently mutated in solid tumors and in blood of healthy-individuals, but its role is still unknown in Acute Myeloid Leukemia (AML). WIP1 suppresses p53 after cell-stress. The aim of this study is to investigate whether the inhibition of WIP1 (GSK2830371) could increase the sensitivity to MDM2 inhibitors (Nutlin-3a) in order to obtain a novel therapeutic strategy for AML patients restoring p53 activity.

Methods: Whole Exome Sequencing (WES) and targeted Next Generation Sequencing (NGS) have been performed to investigate the mutational state of PPM1D in newly diagnosed AML patients. We performed in vitro WST-1 cell-viability and Annexin V-PI apoptosis assays in AML cell lines (MOLM-13, MV-4-11) and AML primary cells.

Results: WIP1i and Nut-3a reduced the cell viability as single agent in all the treated cells in a time and dosage-dependent manner. The IC50 values in TP53-wt cells showed that MOLM-13 (10 υM) are more sensitive than MV-4-11 (20 υM) to WIP1i. Combination index analyses confirmed that WIP1i and Nut-3a had a synergistic effect (C.I.<1) in both cell lines. Annexin V/PI staining performed after treatment with different concentration of Nut-3a (0.5 υM for MOLM-13, 15 υM for MV4-11) and WIP1 inhibitor (10 υM for MOLM-13, 20 υM for MV4-11) demonstrated that the combination significantly increased the number of apoptotic cells in comparison to single agents. In particular, after 48 hours of incubation with Nut-3a and WIP1i as single agents, the percentage of apoptotic cells was 34.4% and 21.4% for MOLM-13 and 17% and 18% for MV-4-11, respectively; while the combination induced 71.6% and 54.5% of apoptotic cells. The decrease of cells in G2/M phase induced by single agents, was enhanced by the drugs combination in both cell lines and, in particular, MV-4-11 showed an increasing percentage of S phase cells allowing us to hypothesize that the apoptotic death induced by the combination of drugs occurs when cells are blocked in S phase. AML primary cells were also tested, showing a similar pattern when the two drugs are combined. Finally, by WES and targeted-NGS, 7 mutations have been detected in 2/37 and 4/96 patients. One patient harbored a truncating mutation on exon 6, known to be gain of function, also confirmed by Western Blot (WB), increasing the inhibition of p53; while the other mutations affected the exon 1 and have not been described yet.

Conclusions: This pre-clinical study allows us to affirm for the first time that WIP1i potentiates the sensitivity to Nut-3a in AML. Further studies on TP53-mutated and wt cells will help to confirm these preliminary data, Gene Expression Profile and WB will identify the proteins playing a relevant role in the biological alterations induced by WIP1i. Supported by: ELN, AIL, AIRC, project Regione-Università 2010-12 (L. Bolondi), FP7 NGS-PTL project, HARMONY project, Fondazione del Monte BO e RA project.

#1873

Preclinical activity of tipifarnib in cutaneous T-cell lymphoma.

Rebecca Kohnken,1 Betina McNeil,1 Jing Wen,1 Kathleen McConnell,1 Francis Burrows,2 Pierluigi Porcu,1 Anjali Mishra1. 1 _The Ohio State University, Columbus, OH;_ 2 _Kura Oncology, San Diego, CA_.

Cutaneous T-cell lymphoma (CTCL) is a rare form of non-Hodgkin lymphoma characterized by malignant infiltration of skin-homing CD4+ T-cells, manifesting clinically as patches and plaques on the skin (Wong HK, et al. Br J Haematol 2011). The disease will often progress from an indolent skin-limited form, Mycosis Fungoides (MF), to an aggressive leukemic variant, Sezary Syndrome (SS) (Kohnken R, et al. Curr Hematol Malig Rep 2016). As there are few effective therapies available for advanced-stage CTCL, we sought to explore potential novel therapeutic modalities. Farnesyl transferase inhibitors (FTIs) are a relatively new class of anticancer drugs, the exact mechanism of which is unknown. Tipifarnib is one such FTI, which has demonstrated clinical activity as a single agent in patients with relapsed and refractory lymphomas (Witzig, TE, et al. Blood 2011). We utilized CTCL patient-derived cell lines to evaluate the potential efficacy of tipifarnib in vitro. EC50 doses ranged from 7.5nM for HuT78 to 50nM for SeAx. Importantly, there was no cytotoxic effect on normal donor CD4+ T-cells in vitro. Based on these promising findings, we evaluated tipifarnib in vivo using a recently described interleukin-15 (IL-15) transgenic mouse model of CTCL (Mishra A, et al. Can Discov 2016). Following two weeks of dosing in 5-week old mice, we found a significant decrease in gross lesion severity in tipifarnib-treated mice (0.5±1.22 out of a possible score of 5) compared to vehicle-treated mice (3.33±0.57, p=0.0076). Histologically, IL-15 transgenic mice develop characteristic malignant CD4+ T-cell infiltrates in the dermis and epidermis. Tipifarnib-treated mice, however, have minimal lesions in the skin. These dramatic changes are reflected in a significantly decreased histologic severity score in tipifarnib-treated mice (3.8±0.75 out of a possible score of 7 compared to vehicle-treated mice (7±0, p=0.0002). These findings demonstrate that tipifarnib is active in patient-derived cell lines and the mouse model of cutaneous T-cell lymphoma. Further studies to elucidate the mechanism of antitumor activity of farnesyl transferase inhibition in this cell type are in progress.

#1874

The PIM kinase inhibitor TP-3654 demonstrates efficacy in a murine model of myelofibrosis.

Avik Dutta, Dipmoy Nath, Yue Yang, Golam Mohi. _University of Virginia, Charlottesville, VA_.

Myelofibrosis (MF) is the deadliest form of myeloproliferative neoplasms (MPN). The JAK2V617F mutation has been found in ~50% patients with MF. Ruxolitinib, a JAK1/JAK2 inhibitor, has been approved for treatment of MF. However, Ruxolitinib treatment has failed to induce disease remission or cure fibrosis. Therefore, there is a need to develop novel targeted therapies for MF. We found that PIM1 expression is significantly increased in mouse and human MPN/MF hematopoietic progenitor cells. Moreover, PIM1 knockdown markedly inhibited proliferation in JAK2V617F-expressing cells but not in wild-type JAK2-expressing cells.

In this study, we investigated the efficacy of the second-generation pan-PIM kinase inhibitor, TP-3654, in hematopoietic cells expressing JAK2V617F and in a murine model of MF. We found that TP-3654 (0.5-1.0 μM) treatment significantly reduced proliferation of murine Ba/F3-EpoR cells expressing JAK2V617F or human JAK2V617F-positive HEL and UKE-1 cells. TP-3654 Treatment also resulted in marked apoptosis in Ba/F3-EpoR-JAK2V617F, HEL and UKE-1 cells but not in wild-type JAK2 expressing Ba/F3-EpoR cells. Additionally, TP-3654 and Ruxolitinib synergistically induced apoptosis in JAK2V617F-expressing hematopoietic cells. Furthermore, TP-3654 significantly inhibited MPN/MF CD34+ hematopoietic progenitor colony growth.

We previously reported the generation of JAK2V617F knock-in mice. Whereas heterozygous JAK2V617F knock-in mice exhibit a polycythemia vera phenotype, mice expressing homozygous JAK2V617F rapidly develop high-grade MF. We have utilized the homozygous JAK2V617F mice to test the in vivo efficacy of TP-3654 alone or in combination with Ruxolitinib against MF. Treatment of TP-3654 alone significantly reduced the increase in white blood cell (WBC) and neutrophil counts as well as spleen size in mice expressing homozygous JAK2V617F compared with vehicle treatment. Combined treatment of TP-3654 and Ruxolitinib almost completely normalized the WBC and neutrophil counts and the spleen size in homozygous JAK2V617F mice. Histopathologic analysis revealed marked reduction in fibrosis in the bone marrow and spleens of TP-3654-treated mice whereas Ruxolitinib treatment did not significantly reduce fibrosis. Combined TP-3654 and Ruxolitinib treatment almost completely eliminated fibrosis in the bone marrow and spleens of homozygous JAK2V617F mice. TP-3654 treatment was well tolerated and did not cause any significant toxicity in wild type mice. RNA-sequencing analysis on purified LSK (Lin-Sca-1+c-kit+) cells from these drug-treated mice revealed that the genes related to TNFα and WNT signaling pathways were significantly down-regulated by treatment of TP-3654 alone or TP-3654 and Ruxolitinib combination compared with vehicle. Overall, our results suggest that TP-3654 alone or in combination with Ruxolitinib may have therapeutic potential for treatment of MF.

#1875

Oxidative metabolism as a novel therapeutic target to eradicate T-ALL with mitochondrial complex I inhibitor IACS-010759.

Natalia Baran,1 Alessia Lodi,2 Shannon Sweeney,2 Vinitha Mary Kuruvilla,1 Antonio Cavazos,1 Anna Skwarska,3 Sriram Shanmuga Velandy,1 Karine Harutyunyan,1 Ningping Feng,1 Jason Gay,1 Marcin Kaminski,4 Elias J. Jabbour,1 Adolfo Ferrando,5 M. Emilia Di Francesco,1 Joseph R. Marszalek,1 Stefano Tiziani,2 Marina Konopleva1. 1 _MD Anderson Cancer Center, Houston, TX;_ 2 _UT at Austin, Austin, TX;_ 3 _Gdansk University of Technology, Gdansk, Poland;_ 4 _St. Jude Children's Research Hospital, Memphis, TN;_ 5 _The Columbia University, New York, NY_.

Adult T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy with limited treatment options, largely driven by the activating Notch1 mutations. Oncogenic Notch1 facilitates c-Myc signaling and glutamine oxidation, induces metabolic stress and increased reliance on oxidative metabolism maintained by AMPK and modulates metabolism under energy stress by mTOR (Kishton, Cell Metabolism 2016; Chan, Blood 2007).

In this study, we report pre-clinical activity of the novel OXPHOS inhibitor (OXPHOSi) IACS-010759 in NOTCH-mutated T-ALL, and characterize the cellular and metabolic responses to OxPhos inhibition. Exposure to IACS-010759 (0-370 nM) in vitro for 5 days drastically reduced T-ALL viability, with EC50 ranging from 0.001-10 nM for T-ALL cell lines and 13-45 nM for T-ALL PDX models (n=5). Oral administration of IACS-010759 at 7.5 mg/kg daily was tolerable in both, aggressive T-ALL PDX and in Notch-1 mutated murine T-ALL model, significantly reduced leukemia burden and extended survival. Functional metabolic characterization of T-ALL confirmed that IACS-010759 effectively inhibited mitochondrial respiration and caused striking dose-dependent decrease in basal and maximal OCR, ATP and NADH production. Pharmacological inhibition of Complex I with IACS-010759, similar to knockout of Complex I subunit NDUSF4 using CRISPR-CAS9, induced catastrophic changes in mitochondria, with induction of ROS, DNA damage and compensatory mTOR pathway activation. Further, OXPHOSi led to downregulation of mitochondrial Complex I, II, III and IV, decrease of wide range of TCA cycle enzymes and proteins involved in the mitochondrial transport. This translated into decrease of TCA cycle intermediates and reduction in ATP and NADH content by metabolomic analysis. Using stable isotope-resolved metabolomics (SIRM) flux analysis, IACS-010759 (30 nM at 24 hr) significantly decreased flux of glucose through the TCA cycle and redirected it towards glycolysis, additionally increased utilization of glutamine for fueling the TCA cycle, in particular through reductive metabolism, uncovering reliance on glutaminolysis as an additional therapeutic target. Consistent with this hypothesis, combined therapy of OXPHOSi with Glutaminase (GLS-i) or mTOR inhibitors caused additive or synergistic reduction of viability of T-ALL cells, and elicited anti-leukemia activity in T-ALL resistant to Complex I inhibitor alone. Ongoing in vivo studies will address the impact of Complex I Inhibition in the context of genetic GLS knockout utilizing Notch1-mutated GLS fl/fl murine model (Herranz, Nat Med 2016). Taken together, our findings indicate that OXPHOSi, alone and more so in combination with GLS inhibition, constitutes an novel therapeutic modality that targets unique metabolic vulnerability of Notch1- mutated T-ALL cells.

#1876

INCB052793, a JAK1 selective inhibitor, is highly efficacious in PDX and xenograft models of acute myeloid leukemia (AML) expressing elevated endogenous pSTAT3/pSTAT5.

Ashish Juvekar,1 Sindy Condon,1 Xiaoming Wen,1 Bruce Ruggeri,1 Peggy Scherle,1 Reid Huber,1 Yunlong Li,1 Wenqing Yao,1 Song Mei,1 Deepak Bhasin,2 Maria Mancini2. 1 _Incyte Corporation, Wilmington, DE;_ 2 _Champions Oncology, Baltimore, MD_.

Acute myeloid leukemia (AML) is characterized by infiltration of abnormally differentiated, clonal and highly proliferative cells of the hematopoietic system that acquire successive genomic alterations. AML is the most common acute leukemia in adults. Current therapies are of limited utility and involve a combination of cytarabine and anthracycline based regimens with allogeneic stem cell transplantation for eligible candidates, but there is an urgent need to improve therapies for AML. JAK/STAT pathway dysregulation plays a role in the pathogenesis of AML and the JAK2 V617F mutation is present in only a small percentage of these patients.

Studies were conducted to evaluate the in vitro and in vivo activities of INCB052793, a highly JAK1-selective inhibitor having 100-fold selectivity for JAK1 over JAK2 in cell lines, xenograft and PDX models of human AML having elevated endogenous pSTAT3 and or pSTAT5 activation. In vitro, INCB052793 effectively inhibited p-Stat3 and/or p-Stat5 phosphorylation MV411, Molm 16 and Molm 13 cell lines and caused marked reductions in p-Akt and c-Myc levels in MV411 and Molm16 cells. Given these observations, oral administration of INCB052793 was evaluated at doses of 3-30 mg/kg twice daily in MOLM-16 xenografts and FLT3-ITD AML xenograft models, MV-4-11 and Molm-13, in SCID mice. INCB052793 administration significantly inhibited tumor growth in MOLM-16 xenografts in a dose dependent manner and resulted in complete downregulation of p-Stat3 and p-Stat5 levels in MOLM-16 tumors. Similarly, INCB052793 administration was highly effective in inhibiting tumor growth in FLT3-ITD AML models, MV-4-11 and Molm-13. Administration of INCB052793 in a systemic PDX model of AML with elevated endogenous levels of p-Stat3 and p-Stat5 resulted in amelioration of disease severity and demonstrated a significant effect on median survival in leukemic SCID mice. All dosing regimens of INCB052793 in both xenograft and PDX models were well tolerated. Since azacitidine and cytarabine are standards of care for the treatment of AML, the efficacy of INCB052793 was benchmarked against optimal dosing regimens of these agents. In the AML xenograft models evaluated, INCB052793 was comparably or more efficacious in reducing tumor burden than azacitidine and cytarabine. The combination of INCB052793 with cytarabine showed superior efficacy in comparison to single agents in the MOLM-16 xenograft model, and combinatorial studies are in progress in additional AML models. These findings suggest the therapeutic potential of INCB052793 as a single agent and in combination with standard of care chemotherapeutic regimens for the management of AML.

#1877

Selective inhibitor of nuclear export (SINE) compound, eltanexor (KPT-8602), synergizes with venetoclax (ABT-199) to eliminate leukemia cells and extend survival in an in vivo model of acute myeloid leukemia.

Melissa A. Fischer,1 Pia M. Arrate,1 Hua Chang,2 Agnieszka E. Gorska,1 Londa S. Fuller,1 Haley E. Ramsey,1 Trinayan Kashyap,2 Christian Argueta,2 William Senapedis,2 Erkan Baloglu,2 Yosef Landesman,2 Sharon Shacham,2 Michael R. Savona1. 1 _Vanderbilt University Medical Center, Nashville, TN;_ 2 _Karyopharm Therapeutics Inc, Newton, MA_.

SINE compounds (KPT-330/selinexor and KPT-8602/eltanexor) are a novel class of small molecules with therapeutic potential across a broad range of cancers. SINE compounds target exportin 1 (XPO1), which is an essential nucleo-cytoplasmic transport protein, responsible for the nuclear export of >200 cargo proteins including many tumor-suppressor proteins (TSPs) and oncogenes, such as p53, IκB, BCL-2, eIF4E and MYC. XPO1 is overexpressed in numerous solid and hematologic malignancies, often correlating with poor prognosis. An important family of oncoproteins regulated by SINE compounds are the pro-survival BCL-2 family proteins, BCL-2 and MCL-1, as both are transported from the nucleus by forming a complex with the XPO1 cargo protein, elF4E. BCL-2 and MCL-1 are dysregulated and linked to maintenance and survival in numerous cancers. Early studies with venetoclax (VEN/ABT-199), a potent, selective inhibitor of BCL-2, have revealed response rates from 50-80% across a variety of hematologic malignancies. However, resistance often arises via upregulation of other BCL-2 family antiapoptotic proteins, most commonly MCL-1. Thus, we hypothesized that co-treatment of VEN and SINE compounds would show enhanced cell killing in AML due to the ability of SINE to further block the prosurvival signals of both BCL-2 and MCL-1. Co-treatment with eltanexor and VEN demonstrated synergistic loss of viability in multiple hematologic malignancy cell lines, particularly MV-4-11 cells. Further in vitro analysis showed this enhanced cell death was due to an increase in apoptosis that led to a loss of clonogenicity in methylcellulose assays. We used in vivo studies to test the effect of this combination in a systemic AML xenograft model. For this, MV-4-11 cells were transplanted into NSGS mice and allowed to engraft before treating the mice with vehicle, eltanexor, VEN, or a combination of the two compounds. When the vehicle mice became moribund at 4 weeks after transplant, all treatment groups were sacrificed to compare leukemic burden in the hematopoietic tissues. Flow cytometric analysis revealed a drastic elimination of leukemic cells in the bone marrow, spleen, and peripheral blood of the combination-treated mice compared to vehicle and single-treatment groups. Immunohistochemical analysis of the spleen and bone marrow confirmed this finding, and also revealed the reduction in leukemic cells was due, in part, to an induction of apoptosis (an increase in cleaved caspase 3 and decrease in survivin) in splenic tissue sections, in agreement with the in vitro results. Finally, the dramatic clearance of leukemia cells in the mouse caused by eltanexor and VEN treatment led to prolonged survival. Our results demonstrate the potential of this combination regimen in the treatment of AML.

#1878

Molecular mechanisms of the cardiotoxicity of the proteasomal-targeted anticancer drugs bortezomib, carfilzomib, ixazomib, oprozomib, and delanzomib.

Brian B. Hasinoff. _University of Manitoba, Winnipeg, Manitoba, Canada_.

Bortezomib, carfilzomib, ixazomib, oprozomib, and delanzomib are anticancer drugs that target the proteasomal system. Bortezomib, carfilzomib, and ixazomib, which are approved for clinical use, have shown some cardiotoxicity. Carfilzomib and oprozomib are epoxyketones that form an irreversible bond with the 20S proteasome, whereas bortezomib, ixazomib, and delanzomib are boronic acids that form slowly reversible adducts. A primary neonatal rat myocyte model was used to study these cardiotoxic mechanisms. Using LDH release as a measure of damage in the myocyte model, after drug treatments of either 1, 4 and 72 hr all these drugs induced myocyte damage at low- to sub-micromolar concentrations. Oprozomib induced significantly less myocyte damage than any of the other drugs. Utilizing a fluorogenic substrate the kinetics of the slow-binding inhibition of the chymotrypsin-like proteasomal activity of human 20S proteasome were also determined in order to see if differences in the binding kinetics could explain the degree of myocyte damage. Oprozomib inhibited the 20S proteasome with a second-order binding "on" rate constant that was 60-fold slower than for ixazomib, the fastest binding drug. The first-order dissociation "off" rate constants for the three boronic acids were also determined. Delanzomib dissociated from its complex with the 20S proteasome with a half-time that was more than 20-fold slower than for ixazomib, the fastest dissociating drug. In conclusion, while all these drugs were potently toxic to myocytes at submicromolar concentrations, oprozomib was considerably less toxic than any of the other drugs. As a group the boronic acid drugs were not significantly more toxic than the epoxyketone drugs. The dissociation half-times of the boronic acid complexes were also not correlated with myocyte damage. The fact that oprozomib bound to the 20S proteasome more slowly than any of the other drugs may be the reason, in part, that it is less toxic. Support: CIHR; a Canada Research Chair in Drug Development.

#1879

Pyrazolopyrimidines as novel selective allosteric MALT1 inhibitors with in vivo activity in ABC-DLBCL.

Andreas Weiss,1 Thomas Radimerski,1 Daniel Wyss,1 Rita Andraos,1 Alexandra Buhles,1 Dario Sterker,1 Jean Quancard,1 Carole Pissot,1 Oliver Simic,1 Marc Bigaud,1 Frederic Bornancin,1 Achim Schlapbach,1 Catherine Regnier,1 Paul McSheehy,1 Elisabeth Buchdunger,1 Markus Wartmann,1 Martin Renatus,1 Ralf Endres,1 William Sellers,2 Francesco Hofmann1. 1 _Novartis Insts. for BioMedical Research, Basel, Switzerland;_ 2 _Novartis Insts. for BioMedical Research, Cambridge, MA_.

Diffuse large B cell lymphoma (DLBCL) is the most common histological subtype of Non-Hodgkin's lymphoma, comprising 30% to 40% of all newly diagnosed cases. DLBCL is a biologically and clinically diverse disease with more than a dozen subtypes classified by the World Health Organization (WHO). Gene expression profiling groups DLBCL into three molecular subtypes, named according to their cell of origin, and which include germinal center B-cell-like (GCB) DLBCL, activated B-cell-like (ABC) DLBCL and primary mediastinal B cell lymphoma (PMBL). The current standard of care (SoC) is R-CHOP chemo-immunotherapy. Although the SoC is curative in a substantial proportion of patients, 40% of patients, especially in the ABC-DLBCL subtype, do not achieve durable remissions and suffer from progressive disease. In ABC-DLBCL, recurrent mutations in the B-cell receptor (BCR) and Toll-like receptor (TLR) pathways lead to constitutive NF-κB signaling. Mutations in the BCR pathway include gain-of-function mutations of CD79A/B (~20%) and CARD11 (~10%), and loss-of-function mutations of A20 (~25%). In the TLR pathway, MYD88 is commonly mutated (~37%). In recent years, knowledge of these aberrantly regulated pathways and their underlying mutations guided the development and investigation of newer molecular targeted agents, e.g. BTK, SYK, PI3K, PKC and MALT1 inhibitors. Among these strategies, inhibition of MALT1 provides the advantage that it is downstream of most of the reported BCR pathway mutations, including CARD11 mutations, and most of the targeted kinases with respect to potentially emerging resistance. However, no MALT1 inhibitor is in the clinic until now.

Here, we report the identification of pyrazolopyrimidines as a new class of allosteric MALT1 inhibitors and their optimization for in vivo. The lead compound shows nanomolar potency in biochemical and cellular MALT1 protease reporter assays. In addition, pyrazolopyrimidines are highly selective for MALT1 and demonstrate differential cell killing of CARD11 mutant ABC-DLBCL cells vs control cells in proliferation assays. Furthermore, we demonstrate in vivo activity of pyrazolopyrimidines in CD79 and CARD11 mutant ABC-DLBCL xenograft models, with the lead compound causing regression in a CARD11 mutant xenograft model.

#1880

Validation of protein arginine methyltransferase 5 (PRMT5) as a candidate therapeutic target in the canine model of non-Hodgkin lymphoma.

Kyle A. Renaldo,1 Lindsay E. Courtney,1 Konstantin Shilo,2 Robert A. Baiocchi,3 William C. Kisseberth1. 1 _Department of Veterinary Clinical Sciences, The Ohio State University, Columbus, OH;_ 2 _Department of Pathology, The Ohio State University, Columbus, OH;_ 3 _Division of Hematology, Department of Internal Medicine, The Ohio State University, Columbus, OH_.

Introduction: Lymphoma is a common, aggressive malignancy in dogs. While generally responsive to upfront combination chemotherapy with traditional cytotoxic drugs, remission times are short and cures rare. New treatment strategies are therefore needed. Protein arginine methyltransferase 5 (PRMT5) is a type II protein arginine methyltransferase (PRMT) enzyme whose relevance to cancer biology has become increasingly evident as more studies have shown this enzyme to be involved in the regulation of multiple major signaling pathways affecting cell death, proliferation, and malignant transformation. While the mechanism behind many of these cell-transforming capabilities of PRMT5 remains unknown, knockdown of PRMT5 expression by genetic or pharmacologic means has been shown to modulate the methylation status of target proteins and exert antitumor effects in vitro and in vivo. Consequently, PRMT5 small-molecule inhibitors are in early preclinical development.

Methods: In this study, we characterized patterns of PRMT5 expression and correlated these with histologic subtype using canine lymphoma tissue microarrays (TMAs). We characterized expression of PRMT5 in canine lymphoma tissues and methylation targets in canine B-cell lymphoma cell lines and treated them with different PRMT5 inhibitors to determine effects on PRMT5, target proteins and antitumor activity. We also assessed PRMT5 inhibition ex vivo in patient B-cell lymphomas via flow cytometry and assessment of apoptosis with annexin V/PI staining.

Results: Canine diffuse large B-cell lymphoma showed cytoplasmic staining for PRMT5 (48.8% strong, 50.0% weak, n = 165) compared to negative or weak staining in normal and hyperplastic lymph nodes (n = 40). Lymphoblastic T-cell lymphoma samples showed strong nuclear staining (40%, n= 10). No nuclear staining was appreciated in normal or hyperplastic lymph nodes. Similarly, canine B-cell lines showed high expression of PRMT5. PRMT5 small-molecule inhibitors inhibited growth of CLBL-1 and 17-71 canine lymphoma cell lines. Histone (H3R8 and H4R3) methylation status was suppressed at their respective IC50s. Ex vivo treatment of canine patient tumor cells showed suppression of growth at 24 hours.

Conclusion: We have demonstrated that PRMT5 is expressed in canine lymphomas and that PRMT5 inhibition can suppress the growth of canine lymphoma cells, supporting the continued use of the spontaneous canine lymphoma model for the preclinical development of PRMT5 inhibitors.

#1881

The microtubule-disrupting drug BNC105 is a potent inducer of apoptosis in AML patient samples.

Daniel J. Inglis,1 Debora A. Casolari,2 Tran Nguyen,2 Donna M. Beaumont,1 Nicole L. Wittwer,1 David Ross,3 Richard D'Andrea,2 Tina C. Lavranos1. 1 _Bionomics Ltd., Thebarton, Australia;_ 2 _University of South Australia, Adelaide, Australia;_ 3 _SA Pathology and Royal Adelaide Hospital, Adelaide, Australia_.

BNC105 is a phase II potent and highly selective disruptor of tumor microvasculature that causes the rapid onset of hypoxia and necrosis in solid tumors. BNC105 targets the colchicine-binding site on tubulin, causing chronic disruption of adhesion molecules, and was developed to be best-in-class with high specificity to actively dividing cells. It has one of the largest therapeutic windows of its class and has been shown to have direct cytotoxic activity on tumor cells. It is this highly tumor-specific mechanism of action that has positioned BNC105 as a therapeutic with high potential in the hematologic cancer setting. Previous studies of BNC105 have shown that treatment with BNC105 results in the activation of c-Jun N-terminal kinase (JNK), phosphorylation of ATF2, and the induction of ATF3 and Noxa, leading to acute apoptosis in chronic lymphocytic leukemia (CLL) cells. These findings led to the commencement of a phase 1/2 trial of BNC105 in patients with CLL. The present study was designed to investigate the effect of BNC105 treatment on acute myeloid leukemia (AML), a disease that currently has limited treatment options. To assess the utility of BNC105 therapy in this setting, six AML cell lines representing different subtypes, including the high-risk FLT3-ITD subtype, were initially used in proliferation and cytotoxicity assays. The production of reactive oxygen species (ROS), cell cycle distribution and cell signaling by Western blot were all assessed after treatment. All tested AML cell lines were highly sensitive to treatment with BNC105 with an IC50=0.2nM to 1.3nM after 48 hours treatment. Analysis of apoptosis induction revealed cell line-specific effects; however, a consistent dose-dependent increase in phosphorylation of JNK was observed across all cell lines. AML patient samples obtained from the South Australian Cancer Research Biobank (SACRB) were exposed to BNC105 at clinically relevant doses for up to 72 hours and cellular viability and apoptosis induction were assessed by Annexin V/ 7AAD staining and caspase 3 and 7 activation measured. BNC105 induced caspase activity and significantly decreased viability in a dose- and time-dependent manner, including the FLT3 mutant subtype patient samples. In comparison, bone marrow mononuclear cells from healthy controls were much less affected by BNC105. Effects of BNC105 on the leukemic stem cell (LSC) phenotype population were also investigated. The LSC-containing population, measured by CD34/CD38 and GPR56 or CD93 staining, was targeted by BNC105 in all AML patient samples tested. These results suggest that AML cells can be directly targeted by BNC105 at clinically relevant concentrations and hence further clinical investigation of BNC105 is warranted for AML treatment in a patient population with high unmet need.

#1882

Preclinical evaluation of the tyrosine kinase inhibitor ARQ 531 in AML.

Ola A. Elgamal,1 Bridget Carmichael,1 Amy Lehman,2 Shelley J. Orwick,1 Minh Tran,1 Virginia M. Goettl,1 Shaneice Mitchell,1 Rosa Lapalombella,1 Jae Yoon Jeon,3 Sharyn D. Baker,4 Sudharshan Eathiraj,5 Brian Schwartz,5 Erin Hertlein,1 John C. Byrd4. 1 _The Ohio State University, Division of Hematology, Department of Internal Medicine, Comprehensive Cancer Center, Columbus, OH;_ 2 _The Ohio State University, Center for Biostatistics, Columbus, OH;_ 3 _The Ohio State University, Division of Pharmaceutics and Pharmaceutical Chemistry, Columbus, OH;_ 4 _The Ohio State University, Division of Hematology, Department of Internal Medicine, Comprehensive Cancer Center and Division of Pharmaceutics and Pharmaceutical Chemistry, Columbus, OH;_ 5 _ArQule, Inc, Burlington, MA_.

Acute Myeloid Leukemia (AML) is a rapidly progressing hematopoietic malignancy arising from bone marrow myeloid progenitor cells. Treatment with cytotoxic chemotherapy has not changed for over four decades resulting in poor survival. The dismal prognosis could be attributed to the heterogeneity of this disease, where multiple genetically aberrant clones exist within the same patient. Mutations in the FMS-like tyrosine kinase (FLT3) occurs in 30% of AML patients, typically as an internal tandem duplication (ITD) resulting in a constitutively active FLT3 survival pathway. This has prompted the generation of selective FLT3 inhibitors such as Quizartinib and Gilteritinib which are currently being pursued in clinical trials. Still, acquired resistance to these selective FLT3 inhibitors due to the acquisition of tyrosine kinase domain mutations (TKD) can occur. This suggests that the use of an agent with a broader kinome inhibition profile (such as the recently granted FDA approved Midostaurin) could achieve more durable clinical benefit.

ARQ 531 is a novel potent BTK inhibitor currently being investigated in a Phase 1 trial in patients with relapsed/refractory hematological malignancies (ClinicalTrials.gov Identifier: NCT03162536). We have found that ARQ 531 also has inhibitory activity against members of the Src family of kinases (SFK; including downstream target SYK) as well as FLT3. SYK directly binds to and trans-activates FLT3 which is essential for FLT3-ITD tumorigenicity, suggesting that ARQ 531 has therapeutic potential in AML. Therefore we have investigated the in vitro and in vivo efficacy of ARQ 531 in AML.

Our preliminary studies demonstrate cytotoxicity for ARQ 531 in patient-derived primary AML cells harboring FLT3 wild type and FLT3-ITD, as well as multiple AML cell lines. Importantly, ARQ 531 is effective in a MOLM-13 tyrosine kinase inhibitor (TKI) resistant cell line harboring a FLT3-ITD-TKD-D835Y mutation. Furthermore, we show that ARQ 531 can reduce the level of phosphorylated FLT3, but unlike selective FLT3 inhibitors, it can also inhibit Src family phosphorylation and SYK phosphorylation. Additionally, ARQ 531 exhibited an anti-clonogenic effect on primary patient blasts using Methocult colony forming unit assay. Finally, to investigate the in vivo effect of ARQ 531, we used an aggressive AML MOLM-13 disseminated xenograft mouse model. NSG mice were randomized one-week post engraftment to either vehicle or daily oral gavage of 50 mg/kg ARQ 531. The estimated median survival for the ARQ 531 group was 23 days compared to 21 days for the vehicle group (p = 0.002) suggesting in vivo efficacy for ARQ 531 in AML.

Collectively, we provide for the first time promising preclinical efficacy for ARQ 531 in AML supporting further mechanistic investigation of this agent, and potentially, expansion of the ongoing clinical studies to include AML patients.

E. H. and J.C. B. contributed equally as co-senior authors to this work

#1883

A novel Axl and Mertk dual inhibitor ONO-7475: A new therapeutic agent for the treatment of FLT3-ITD and -wild-type acute myeloid leukemia (AML) overexpressing.

Kohei Tanaka, Cuifang Li, Tomomi Hirosaki, Hikaru Kato, Yoshinori Ishikawa, Miho Oka, Hiroshi Egawa, Ryohei Kozaki, Toshio Yoshizawa. _Ono Pharmaceutical Co., Ltd., Osaka, Japan_.

Introduction: The TAM (Tyro3, Axl, and Mertk) receptor tyrosine kinases (RTKs) together with their ligands Gas6 and Pros1 play diverse roles in survival signaling in cancers. Currently, the consensus indicates that therapeutic targeting of TAM RTKs will prove direct antitumor activity and may complement existing immunotherapy regimens to unleash the full power of the anticancer immune response. A number of small-molecule kinase inhibitors targeting TAM RTKs are being developed and tested in preclinical models. ONO-7475 is a highly potent and oral dual inhibitor of Axl and Mertk, currently in a phase 1 clinical study as a therapeutic agent for AML (NCT03176277). Our previous studies demonstrated that ONO-7475 induced complete tumor remission in the FLT3-internal tandem duplication (ITD)-positive AML cell line MV-4-11 mouse xenograft model (Yasuhiro et al., Blood 2014). However, the antitumor activity against FLT3-ITD AML cells was due to Axl inhibition (Ruvolo et al., Haematologica 2017) and the mechanism of Mertk regulation in AML has not been determined. To evaluate this question, we screened 28 AML-derived cell lines for FLT3-wild type (WT) and analyzed the influence of Mertk in proliferation.

Methods: Twenty-eight FLT3-WT AML cell lines were treated with ONO-7475 for 72 hours, and the cell viabilities (IC50 values) were determined. Phospho-Axl (P-Axl), Axl and Mertk were determined by Western blot analysis. P-Mertk was determined by immunoprecipitation. Gene knockdown was achieved in FLT3-WT AML by transfecting with Axl, Mertk, Gas6, Pros1, or scrambled control siRNA.

Results: Six out of 28 FLT3-WT cell lines responded to ONO-7475, and two cell lines, MOLM16 and MKPL1, were highly sensitive (IC50: 4.7nM and 83.5nM). Western blot analysis demonstrated that Axl, Mertk and P-Mertk were overexpressed in these ONO-7475-sensitive cells, and both are classified as megakaryoblastic leukemia (FAB-M7). SiRNA knockdown studies showed that the cell growth in MOLM16 was partially dependent on Mertk (63.5%) but totally dependent on double genes, Axl and Mertk. Furthermore, knockdown of the ligands Gas6 and Pros1 also partially inhibited the cell growth in MOLM16, respectively (46.0%, 49.2%).

Conclusion: Our data suggest that Mertk may play an important role in survival signaling in FLT3-WT AML. Therefore, targeting both Axl and Mertk by ONO-7475 could be a new strategy for the treatment of FLT3-WT patients with AML in addition to FLT3-ITD. Additional work to investigate and clarify for target populations in both FLT3-ITD and -WT AML is currently under way.

#1884

Dual targeting of cell cycle checkpoint and histone deacetylase overcomes bortezomib resistance in multiple myeloma.

Takayuki Tabayashi, Yuka Tanaka, Yasuyuki Takahashi, Yuta Kimura, Tatsuki Tomikawa, Tomoe Anan, Morihiko Sagawa, Reiko Watanabe, Michihide Tokuhira, Masahiro Kizaki. _Saitama Medical Center, Saitama Medical University, Saitama, Japan_.

Although the introduction of novel agents, such as proteasome inhibitors, immunomodulatory drugs, and histone deacetylase inhibitors (HDACi) has improved the clinical outcomes of the patients with multiple myeloma (MM), most patients eventually relapse and develop drug resistance. Proteasome inhibitor, bortezomib (BTZ), is a key drug for the treatment of MM, and the prognosis of the patients with BTZ resistance is very poor. Therefore, novel therapeutic approaches to overcome BTZ resistance are urgently needed. WEE1 is a cell cycle checkpoint kinase and a key regulator of DNA damage surveillance pathways. In response to extrinsically induced DNA damage, WEE1 induces cell cycle arrest, allowing for the damaged DNA to be repaired before the cell undergoes DNA replication, and prevents cells from mitotic lethality. WEE1 is overexpressed in various cancers, and relates to poor prognosis. In addition, the inhibition of WEE1 has been shown to induce cancer cells to premature mitotic entry leading to apoptotic cell death, suggesting that combining WEE1 inhibitor with DNA damaging agent is effective in cancer therapy. Resent research has shown that HDACi increase reactive oxygen species production resulting in increased DNA damage and impaired DNA repair. These data suggest that the combination of WEE1 inhibitor and HDACi might be an attractive therapeutic option against this incurable hematological malignancy. In the present study, we investigated the efficacy of WEE1 inhibitor MK-1775, a potent and highly-selective small molecule inhibitor of WEE1, in combination with HDACi in various MM cells. MTS assays showed that single agent MK-1775 inhibited the proliferation of various MM cell lines, including BTZ-resistant cell line, in a dose- (0 to 10 μM) and time- (0 to 72 h) dependent manner. In combination with vorinostat which is a pan-HDACi, MK-1775 inhibited the proliferation of BTZ-resistant MM cells more effectively than single agent. Furthermore, the combination of vorinostat and MK-1775 was able to re-sensitize BTZ-resistant MM cells to BTZ. Assays for apoptotic cell death demonstrated that MK-1775 induces both early and late apoptosis in MM cells. Western blotting analysis was performed to investigate the molecular mechanisms of MK-1775-induced cell death, showing that MK-1775 inhibited the expression of the target molecules of Bcl-2 and MCL1, and CDK1 phosphorylation. Moreover, the marked increase in PARP and Caspase 3 cleavage, and histone H2AX expression was observed following treatment with MK-1775 in combination with vorinostat. These findings suggest that the combination of MK-1775 and vorinostat induces synergistically DNA damage and promotes premature mitotic entry resulting in apoptotic cell death in MM cells. In conclusion, our data suggest that WEE1 in combination with HDACi might be a promising molecular target for the treatment of BTZ-resistant MM.

#1885

Selective Polo-like Kinase 1 (PLK1) inhibitor PCM-075 is highly active alone and shows synergy when combined with FLT3 inhibitors in models of acute myeloid leukemia (AML).

Karena Kosco, Maya Ridinger, Penn Whitley, Peter Croucher, Jeffrey N. Miner, Mark Erlander. _Trovagene, San Diego, CA_.

First generation FLT3 inhibitors have been useful in treating AML (sorafenib, midostaurin). Second generation inhibitors with more selectivity are currently in P3 trials in AML (quizartinib, gilteritinib). Despite the advances, resistance to FLT3 inhibitors and disease progression while on FLT3 inhibitors remains an important problem for patient care. We analyzed the activity of a highly-selective PLK1 inhibitor, PCM-075 (formerly NMS-1286937), in models of acute myeloid leukemia (AML) alone and in combination with various chemotherapies and targeted therapeutics including FLT3 inhibitors. PCM-075 is a potent, highly-selective adenosine triphosphate competitive inhibitor of PLK1, a serine/threonine kinase, which is over-expressed in hematologic malignancies and solid tumors such as AML, breast, prostate, ovarian, lung, gastric and colon. PCM-075 is highly active in blocking proliferation and inducing G2/M arrest in multiple AML cell lines. In a therapeutic model, PCM-075 was capable of inducing significant tumor growth inhibition (TGI)1, and increasing survival in an in vivo disseminated leukemia model (AML-NS8 Cells)2. Orally administered PCM-075 is currently in development for multiple tumor types and is in clinical trials for the treatment of AML (NCT03303339). Synergistic interactions between Quizartinib, other FLT3 inhibitors and PCM-075 were examined in cell culture models. In vivo, combination studies in an AML FLT3 mutant 21 day treatment xenograft model (MV4-11) revealed synergistic interactions between Quizartinib and PCM-075. PCM-075 (dosed orally 30 mg/kg, QD for days 1-10 and 12 -21) in combination with Quizartinib (1 mg/kg, QD for 21 days) resulted in 97.3% (96.2-98.4) TGI, compared to 77.9% (70.0-85.8) with Quizartinib and 80.2% (70.4-90.0) with PCM-075 as monotherapy. Body weight in treated mice remained within 20% of controls. To better understand the mechanism of the synergistic activity of PCM-075, we have conducted in vitro RNA-based profiling studies examining the combination of PCM-075 with FLT3 inhibitors for impact on specific gene pathways in AML cell lines and this will be presented. Combination therapy is the mainstay of current oncology treatment regimens and these results suggest that combining PCM-075 with FLT3 inhibitors could be useful in the treatment of AML and a potential next step for patients developing resistance to FLT3 inhibitors. 1 Mol Cancer Ther. 2012 Apr;11(4):1006-162 PLoS One. 2013;8(3):e58424

#1886

A novel inhibitor of eIF4F protein translation complex sensitizes DLBCL cells to BCL-2 targeted therapy.

Lee-or Herzog,1 Nancy Nguyen,1 Honyin Chiu,1 Sharmila Mallya,1 Ze'ev A. Ronai,2 David A. Fruman1. 1 _University of California, Irvine, Irvine, CA;_ 2 _Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA_.

The purpose of this project is to investigate the feasibility of using small molecules targeting cap-dependent translation to sensitize B cell lymphoma cells to killing by BCL-2 inhibitors (venetoclax/ ABT-199). We hypothesized that diffuse large B-cell lymphoma (DLBCL) cell survival requires cap-dependent translation, which is facilitated by eukaryotic translation initiation factor 4E (eIF4E)-eIF4G interaction and is promoted by mTOR complex 1 (mTORC1). We used the compound SBI-756, a novel inhibitor targeting the scaffolding protein eIF4G (1). SBI-756 is more potent and selective than previously reported compounds targeting the eIF4E cap-binding protein, or compounds targeting eIF4A helicase. By treating DLBCL cells (germinal center B-cell subtype) with SBI-756 (sub μM concentrations), we discovered profound synergistic induction of apoptosis when combined with venetoclax. Cell viability was reduced even more compared to mTOR kinase inhibitor (TOR-KI) treatment combination. Moreover, SBI-756 was found to reduce the viability (IC50 = 653nM) of VAL lymphoma cells lacking 4E-BP1, while VAL were insensitive to TOR-KI treatment. By using Proximity Ligation Assay (PLA) we showed that SBI-756 treatment prevents eIF4E-eIF4G association in intact lymphoma cells. Dual luciferase assays validated this and indicated a dose-dependent reduction in cap-dependent translation following SBI-756 treatment. Also, Western blot analyses confirmed that SBI-756 treatment did not change mTOR substrates' phosphorylation, indicating that the SBI-756 effect is specific to preventing the eIF4E-eIF4G interaction. Furthermore, SBI-756 treatment reduced polysome formation with a corresponding increase in abundance of monosomes. Lastly, induced expression of 4E-BP1 mutant, which irreversibly binds eIF4E, sensitized DLBCL cells to venetoclax treatment to a similar extent as TOR-KI treatment. Ongoing studies will investigate: (1) The molecular mechanism by which translation inhibition by SBI-756 synergizes with venetoclax to prime DLBCL cells toward apoptosis, and (2) pharmacokinetic and pharmacodynamic effects of venetoclax and SBI-756 treatment on DLBCL progression in vivo. Hence, this project highlights a novel combination for treatment of aggressive lymphomas, and establishes its efficacy using preclinical models.

Reference: 1. Feng Y., et al. SBI-0640756 attenuates the growth of clinically unresponsive melanomas by disrupting the eIF4F translation initiation complex. Cancer Res 2015;75(24):OF1-8.

#1887

Preclinical evaluation of a novel interleukin-1 receptor-associated kinase 4 (IRAK4) inhibitor in combination with PI3K inhibitor copanlisib or BTK inhibitors in ABC-DLBCL.

Martin Lange, Antje Margret Wengner, Ulrich Bothe, Ulf Boemer, Reinhard Nubbemeyer, Holger Siebeneicher, Holger Steuber, Judith Guenther, Lisette Potze, Nicole Schmidt, Oliver Politz, Wolf-Dietrich Doecke, Eleni Lagkadinou, Thomas M. Zollner, Franz von Nussbaum, Dominik Mumberg, Andreas Steinmeyer, Michael Brands, Karl Ziegelbauer. _Bayer AG, Berlin, Germany_.

Activated B-cell-like diffuse large B-cell lymphoma (ABC-DLBCL) is frequently characterized by aberrant activation of both B-Cell Receptor (BCR) & TLR/MYD88 signaling pathways. Constitutive BCR signaling via Bruton's tyrosine kinase (BTK) and PI3K pathways leads to downstream activation of NF-κB and AKT signaling. In addition, IRAK4 mediated activation of the TLR/MYD88 pathway further activates NF-κB signaling and pro-survival pathways. Simultaneous blockade of TLR/MYD88 signaling via IRAK4 inhibition in combination with pharmacological blockade of PI3K/BCR signaling pathways may therefore provide a novel treatment strategy in ABC-DLBCL.

BAY 1830839 is a novel small molecule inhibitor of IRAK4 identified by a medicinal chemistry optimization program. Key features of the compound are high potency (IC50 of 3 nM) in a biochemical assay, excellent kinase selectivity and a good overall PK profile making the compound a valuable tool for in vivo studies.

In vitro, treatment of IRAK4 inhibitor BAY 1830839 in combination with BTK inhibitors or copanlisib, a pan class I PI3K inhibitor with predominant activity towards PI3Kα and PI3Kδ, synergistically inhibited NF-κB activation and cell viability in human ABC-DLBCL cell lines. In vivo, IRAK4 inhibition alone did not exhibit anti-tumor effects but in combination treatment with ibrutinib, a covalent inhibitor of BTK, synergistic anti-tumor activity with significantly improved efficacy over ibrutinib monotherapy was observed in the human ABC-DLBCL xenograft models TMD-8 and OCI-LY10 (MYD88mut/CD79A/Bmut). Moreover, IRAK4 inhibitor BAY 1830839 showed synergistic anti-tumor activity in combination with copanlisib with significant improvement of copanlisib monotherapy efficacy in the ABC-DLBCL PDX models LY2988 and LY2266 (MYD88mut/CD79A/Bmut and MYD88wt/CD79A/Bwt, respectively). In addition, the combination of IRAK4 inhibition with pharmacological blockade of PI3K-/ BCR signaling led to reduced activity of the downstream pro-survival STAT3 pathway and IL-6/IL-10 production as detected in tumor xenografts, validating our biological rationale and the expected mechanism of action.

In summary, IRAK4 inhibition in combination with pharmacological blockade of PI3K or BCR signaling blocks pro-survival NF-κB & JAK-STAT pathway activation and subsequent IL-6/IL-10 production. Enhancing activity of clinically used PI3K or BTK inhibitors by combination with IRAK4 inhibition indicates a potential new treatment approach for ABC-DLBCL patients progressing on Standard of Care therapy.

#1888

The FAD-directed LSD1 specific inhibitor, INCB059872, is a promising epigenetic agent for AML therapy by inducing differentiation of leukemic stem/progenitor cells.

Antony Chadderton, Min Ye, Melody Diamond, Valerie Roman, Michael Weber, Chunhong He, Liangxing Wu, Swamy Yeleswaram, Alan Roberts, Wenqing Yao, Gregory Hollis, Reid Huber, Peggy Scherle, Bruce Ruggeri, Sang Hyun Lee. _Incyte Corporation, Wilmington, DE_.

Numerous studies have elucidated that the most pivotal functions of lysine specific demethylase-1 (LSD1) are associated with regulating normal or malignant hematopoiesis by maintaining stem cell self-renewal and regulating myeloid differentiation. In preclinical models, studies with either pharmacological inhibition or genetic knockdown of LSD1 demonstrated that LSD1 is essential for differentiation of progenitor cells during normal hematopoiesis. In the clinic, AML manifests itself via clonal expansion of abnormal differentiation and proliferation of myeloid cells and, therefore, the inhibition of LSD1 activity with small molecule inhibitors could be a promising therapeutic approach for AML. Previously, we reported upon the identification of a flavin adenine dinucleotide (FAD) directed LSD1 specific inhibitor, INCB059872, which is efficacious in preclinical mouse models utilizing human AML cell lines and primary AML cells by inducing cell differentiation as indicated by the induction of CD11b and CD86 markers. Using a larger panel of myeloid and HSC flow cytometry markers, our currents efforts expanded upon these observations to ascertain whether INCB059872 enhanced lineage commitment at hematopoietic stem cell (HSC) and/ or promoted monocytic/granulocytic differentiation of human primary AML cells ex vivo and in human systemic AML PDX models. In both human AML PDX models and human primary AML samples, INCB059872 increased myeloid differentiation with increasing populations of monocytes (CD14+) and granulocytes (CD15+). Furthermore, INCB059872 induced the differentiation of early hematopoietic progenitors, CD34+/CD38- to more committed CD34+/CD38+ multipotent/oligopotent progenitors, which in turn gave rise to lineage specific progenitors in the human AML PDX models. These studies support further exploration of INCB059872 as a promising novel epigenetic agent for AML therapy whose mechanism of action lies in part through the induction of differentiation of leukemic stem/progenitor cells to more committed hematopoietic lineages.

#1889

Synergistic effects of glutaminase and proteasome inhibition in multiple myeloma cell lines.

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

Background: Multiple Myeloma (MM) accounts for approximately 2.1% of all cancer deaths with a 5-year median survival rate of 49.6%. Besides chemotherapy, treatment options include monoclonal antibodies, immunomodulatory agents, and proteasome inhibitors. Carfilzomib is a selective proteasome inhibitor administered intravenously in patients with relapsed or refractory MM. With cancer cell metabolism emerging as a critical regulator of tumor progression, combining carfilzomib with an inhibitor of the metabolic machinery represents a novel therapeutic strategy to prevent MM progression. RP10107 is a potent, and selective glutaminase (GLS-1) inhibitor that demonstrated high potency against mouse (IC50=21.2 nM), rat (IC50=18.2 nM) and human (IC50=26.4 nM) enzymes with selectivity over GLS-2 (>380-fold). The objective of this study was to evaluate the effect of a combination of carfilzomib and RP10107 in MM cell lines.

Methods: Glutamate concentrations in MM cell lines (MM-1S and RPMI-8226) following treatment with RP10107 was estimated by LC-MS/MS. Synergism between RP10107 and carfilzomib was determined using different concentrations of the compounds in a 5 x 5 grid. Synergism, additivity, or antagonism was calculated based on the BLISS score. Apoptosis was determined by Annexin V/7AAD staining using a MUSE® Annexin V and dead cell assay kit (Millipore) while the effect on cell cycle was estimated using Guava Cell Cycle Reagent (Millipore). Expression of cleaved PARP and cleaved caspase-8 were determined by Western Blotting.

Results: Treatment of MM cell lines with RP10107 resulted in an increased ratio of glutamine to glutamate with a doubling noticed at concentrations as low as 30 nM. A high degree of synergism between RP10107 and carfilzomib was noticed in both the MM cell lines. Addition of 3 nM Carfilzomib to 30 or 500 nM RP10107 resulted in near complete inhibition of RPMI-8226 and MM-1S cell growth, respectively. Additionally, incubation with the combination (1 μM RP10107 + 15 nM Carfilzomib) for 72 h caused a G0/G1 arrest with a corresponding increase in the percent of apoptotic cells (2.5-fold) compared to the individual agents. Incubating RPMI-8226 cells with a combination of RP10107 and Carfilzomib for 48 h resulted in a 2.2- and 13.6-fold increase in expression of cleaved caspase-8 and cleaved PARP, respectively.

Conclusions: Addition of RP10107, a glutaminase inhibitor, potentiated Carfilzomib activity in MM cells. Findings provide a rationale for use of the combination in future clinical trials involving MM patients thereby providing a safer and more effective alternative to currently available therapy.

#1890

**Inecalcitol respectively induces or increases CD38 expression at the surface of CD38** - **or CD38** + **AML cell lines representative of all 9 FAB subtypes except M6.**

Enguerran Mouly, Cécile Planquette, Emilie Rousseau, Rémi Delansorne. _Hybrigenics Pharma, Paris, France_.

Acute myeloid leukemia (AML) is a heterogeneous disease classified into 9 FAB subtypes according to the origin and/or maturity of the malignant cells: M0 undifferentiated, M1 minimally mature myeloblastic, M2 mature myeloblastic, M3 promyelocytic, M4 myelomonocytic, M4eos eosinophilic, M5 monocytic, M6 erythrocytic and M7 megakaryocytic. Overcoming this heterogeneity by the induction of a common surface antigen which would already be the target of an existing immunotherapy would be of great potential therapeutic interest to standardize a common immunotherapy approach to AML. Inecalcitol, a vitamin D receptor agonist characterized by a high anti-proliferative effect and a low calcemic potential, has been shown to stimulate CD38 expression at the surface of the M3 subtype HL-60 cell line and of the M5 subtype U-937, MOLM-13 and THP-1 cell lines (ASH 2016, AACR 2017). We now report the effect of inecalcitol on AML cell lines representative of the 7 other FAB subtypes: KG-1 (M0), UOC-M1 (M1), Kasumi-1 (M2), OCI-AML2 (M4), EOL-1 (M4eos), HEL-92.1.7 (M6) and CMK (M7). Inecalcitol induced CD38 at the surface of CD38- cell lines (HL-60, U-937 and EOL-1) and strongly increased CD38 density at the surface of already CD38+ cell lines (KG-1, UOC-M1, Kasumi-1, OCI-AML2, MOLM-13, THP-1 and CMK) in a concentration-dependent manner, but remained without effect on CD38- HEL-92.1.7 cells. We have quantified the number of CD38 molecules at the surface of all cell lines (except HEL-92.1.7 which was unresponsive to inecalcitol) using the CellQuant calibrator (Biocytex) analysis by flow cytometry. In basal vehicle-treated condition, the number (in thousands, mean ± sem, n=2 or 3) of CD38 molecules per CD38+ cell were: Kasumi-1, 1.5 ± 1.0; MOLM-13, 2.5 ± 0.6; KG-1, 2.7 ± 0.9; THP-1, 7.0 ± 0.2; CMK, 12.1 ± 1.5; OCI-AML2, 16.1 ± 1.2. With 10nM inecalcitol, a maximal increase in CD38 density was usually obtained within 3 days, or after 7 days for KG-1 and CMK: Kasumi-1, 16.1 ± 3.3 (x11); MOLM-13, 43.3 ± 0.3 (x17); KG-1, 15.4 ± 3.7 (x5.7); THP-1, 17.9 ± 0.6 (x2.3); CMK, 15.4 ± 3.3 (x1.3); OCI-AML2, 73.5 ± 3.5 (x4.6). After 3 days of exposure to 10 nM inecalcitol, CD38 molecules appeared at the surface of initially CD38- cells and became quantifiable: U-937, 7.5 ± 0.4 (n=3); HL-60, 24.4 ± 4.9 (n=3) and EOL-1 ranged from 5.5 to 72.7 (n=2). In conclusion, after exposure to inecalcitol, CD38 appeared at the surface of CD38- AML cell lines and the density of CD38 increased at the surface of CD38+ AML cell lines, altogether representative of all 9 FAB classification subtypes M0 to M7, except M6 (erythrocytic) which represents 5% of all AML cases. These results suggest that treatment by inecalcitol may render 95% of AML patients sensitive to highly sensitive to anti-CD38 immunotherapy such as daratumumab.

#1891

Cross-resistance and synergy between idelalisib and bendamustine in chronic lymphocytic leukemia.

Sara E. F. Kost,1 Ali Saleh,1 Edgard M. Mejia,2 Marina Mostafizar,2 Eric D. J. Bouchard,3 Versha Banerji,1 Aaron J. Marshall,2 Spencer B. Gibson,1 Sachin Katyal,1 James B. Johnston1. 1 _CancerCare Manitoba, Winnipeg, Manitoba, Canada;_ 2 _University of Manitoba, Winnipeg, Manitoba, Canada;_ 3 _Université de Saint-Boniface, Winnipeg, Manitoba, Canada_.

Idelalisib (IDE) is an inhibitor of the δ isoform of PI3 kinase and has shown high activity in chronic lymphocytic leukemia (CLL) either when given alone or in combination with bendamustine (BEN) /rituximab (BR). In the present study, we have determined whether there is cross-resistance between BEN and IDE in primary CLL cells in vitro and whether synergy is observed upon combining these agents. In primary CLL cells in vitro, cross-resistance was not observed between BEN and IDE suggesting different modes of cytotoxicity. In contrast to BEN, sensitivity to IDE was not influenced by prior clinical treatment or the presence of a del 17p. Marked synergy in cytotoxicity was seen between BEN and IDE, which was paralleled by changes in γ-H2AX staining. These findings suggest that the synergistic antitumor effect was related to enhanced DNA damage by the drug combination. The degree of cytotoxic synergy varied and synergy was observed in some cases that were resistant to BEN or IDE, and those with del 17p. Synergy between BEN and IDE was also seen in the B cell lines, BJAB and I83, and in healthy donor peripheral blood mononuclear cells indicating that the phenomenon was not CLL-specific. To simulate the microenvironment, CLL cells were pre-stimulating with CD40L and IL4 prior to drug treatment. Interestingly this treatment caused the cells to become resistant to IDE, but BEN sensitivity was unaffected. However, combining BEN and IDE produced greater synergy than observed when cells were incubated without IL4/CD40. To examine the role of PI3Kδ for synergy, B cells from C57 BL/6 mice, both wild type and with non-functioning PI3Kδ protein, were exposed to BEN and/or IDE. Cells with the non-functioning PI3Kδ protein were more resistant to IDE than the wild-type cells and showed lesser synergy confirming the importance of this protein for the synergistic effect. In summary, these results confirm that IDE can be active against BEN-resistant CLL cells, while BEN may remain active against IDE-resistant samples, demonstrating different modes of anti-tumor activity. However, the unique yet inconsistent synergy seen between these two agents suggest an overlapping mechanism of action. Ongoing studies are evaluating the mechanism for this synergy.

#1892

**Discovery of targeted therapies for acute myeloid leukemia patients with** NUP98-NSD1 **and** FLT3-ITD **.**

Jarno Kivioja,1 Angeliki Thanasopoulou,2 Mika Kontro,3 Ashwini Kumar,1 Bhagwan Yadav,1 Muntasir Mamun Majumder,1 Kimmo Porkka,3 Juerg Schwaller,2 Caroline A. Heckman1. 1 _Institute for Molecular Medicine Finland-FIMM, Helsinki Institute of Life Science, University of Helsinki, Helsinki, Finland;_ 2 _University Hospital Basel, Department of Biomedicine, Basel, Switzerland;_ 3 _Hematology Research Unit Helsinki, University of Helsinki, and Helsinki University Hospital Comprehensive Cancer Centre, Department of hematology, Helsinki, Finland_.

NUP98-NSD1 fusion resulting from the t(5;11)(q35;p15.5) is a clinically significant indicator of adverse prognosis in AML. While the understanding of NUP98-NSD1-driven leukaemogenesis has increased over the years, these patients still suffer from highly progressive disease with no effective therapies. To address this challenge, we analyzed primary patient samples and experimental cell models by high-throughput drug screening and RNA sequencing.

Primary samples included bone marrow mononuclear cells (BM MNCs) from three NUP98-NSD1/FLT3-ITD AML patients and ten healthy donors. Experimental cells included lineage-depleted balb/c BM cells transduced with chimeric NUP98-NSD1 and FLT3-ITD retroviruses alone or together, and Ba/F3 cells transduced with human-cloned NUP98-NSD1 lentivirus. The cells were seeded on 384-well plates containing up to 306 approved and investigational drugs in five concentrations over a 10,000-fold range and incubated for 72h at +37°C. Cell viabilities were measured using CellTiter-Glo®. Drug sensitivity scores (DSS) were calculated from area under the dose response curve and select DSS by subtracting DSS of mock-transduced parental cells from the experimental model.

We discovered 14 drugs with highly significant mean DSS difference (p < 0.001) between patients and healthy controls. Further analysis revealed that only two of these drugs (dasatinib and navitoclax) had positive correlation between DSS and BM blast percentage (Pearson's R: 0.83-0.98) as well as higher mean DSS in the context of other AML patients with (n = 9) and without (n = 38) FLT3-ITD. Experimental balb/c cells expressing NUP98-NSD1 had significantly increased sensitivity to BCL2 inhibitors (n = 3) (p < 0.05), while cells co-expressing NUP98-NSD1 and FLT3-ITD had significantly increased sensitivity to FLT3 (n = 11) and MEK inhibitors (n = 6) in comparison to cells expressing FLT3-ITD alone. In pilot combination screen, select efficacy of FLT3 and MEK inhibitors was further increased in the dual positive cells by dasatinib (100 nM). In synergy screens, navitoclax-dasatinib and navitoclax-quizartinib combinations were highly synergistic in primary cells and balb/c cells co-expressing NUP98-NSD1 and FLT3-ITD. Gene expression analysis showed significant up-regulation of dasatinib target genes LCK and FGR, and navitoclax target gene BCL2A1 in the primary AML cells. Interestingly, BCL2A1 was also upregulated in the balb/c BM cells expressing NUP98-NSD1 alone or with FLT3-ITD, but not in cells expressing FLT3-ITD alone.

Taken together, we have identified potential candidate drugs and drug combinations for t(5;11) positive AML. Although the sample size was small, our data suggests that further research into BCL2 inhibitors in combination with TKIs may eventually translate to better survival outlook of AML patients with t(5;11).

#1893

The evaluation of INCB059872, an FAD-directed inhibitor of LSD1, in preclinical models of T-ALL.

Melody Diamond, Yvonne Lo, Antony Chadderton, Min Ye, Valerie Roman, Michael Weber, Chunhong He, Liangxing Wu, Swamy Yeleswaram, Alan Roberts, Wenqing Yao, Gregory Hollis, Reid Huber, Peggy Scherle, Bruce Ruggeri, Sang Hyun Lee. _Incyte Corporation, Wilmington, DE_.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological tumor that is derived from the clonal expansion of immature T-cell progenitors. Multiple genetic and epigenetic alterations are attributed to the development of malignant T cell transformation. Among these, there is supporting evidence for a role of lysine specific demethylase (LSD1) in T-ALL. Oncogenic transcription factors, such as TAL-1, Notch, and ZEB2, form a complex with LSD1 to alter gene expression in T-ALL cells. In addition, LSD1 is aberrantly expressed in ALL, including B-ALL and T-ALL. Furthermore, the overexpression of LSD1 under control of the Sca-1 promoter in transgenic mice triggered T leukemogenesis via acquisition of self-renewal activity and alteration in the differentiation program to T-cell lineages. Together with the known function of LSD1 in regulating the activity of self-renewal in hematological malignancies, these studies prompted evaluation of the efficacy of the potent, selective, and orally bioavailable FAD-directed LSD1 inhibitor, INCB059872, in preclinical models of T-ALL. Expression of LSD1 was abundant in human-T-ALL cell lines as detected by immunoblotting. In vitro, INCB059872 treatment significantly inhibited the proliferation of a subset of human T-ALL cell lines. In vivo, once daily oral administration of INCB059872 inhibited tumor growth significantly in multiple human T-ALL subcutaneous xenograft models including Molt-4, RPMI-8402, CCRF-HSB-2, and CCRF-CEM, but was ineffective against DND-41 xenografts. The anti-tumor efficacy observed with INCB059872 had no clear genetic correlation with Notch mutation status of T-ALL tumors. Combination efficacy studies of INCB059872 with standard care of agents or targeted therapeutic agents in T-ALL models are currently being evaluated. These data suggest exploring the potential clinical development of INCB059872 as a therapy for T-ALL patients.

#1894

Identification of novel OTX015-containing combinations for lymphoma treatment.

Eugenio Gaudio,1 Chiara Tarantelli,1 Filippo Spriano,1 Alberto J. Arribas,1 Luciano Cascione,1 Emanuele Zucca,2 Anastasios Stathis,2 Francesco Bertoni1. 1 _Università della Svizzera Italiana, Institute of Oncology Research, Bellinzona, Switzerland;_ 2 _Oncology Institute of Southern Switzerland, Bellinzona, Switzerland_.

Introduction. Epigenetics represents and intriguing target of therapy for lymphomas. The epigenetic mechanisms are regulated by several players (e.g. HDAC, HAT, BET) and among them the BET proteins became of interest a few years ago with demonstrated preclinical anti-cancer activity with the first compounds and early clinical responses first in class clinical-grade BET inhibitor OTX015 (MK-8628/birabresib). Despite a widespread anti-proliferative activity, the cytotoxicity effect of BET inhibitors is limited, and, here, we aimed to identify drugs that improve OTX015 activity performing a screening with the BET inhibitor with a library 348 compounds in two lymphoma cell lines.

Methods. Two cell lines derived from germinal center B cell (GCB) diffuse large B cell lymphoma (DLBCL) (OCI-LY-19 and WSU-DLCL2) were exposed to OTX015 (single dose, 100 nM) in combination with two different doses (20 and 1,000 nM) of 348 compounds. Compounds giving a 1.5-fold decreased proliferation with the combination than with the individual compounds were further evaluated in two additional lymphoma cell lines, exposed (72h) to increasing doses of OTX015 alone and in combination with increasing doses of other selected compounds. Synergy was assessed with Chou-Talalay combination index (CI): synergism (<0.9), additive (0.9-1.1), antagonism/no benefit (> 1.1).

Results. OCI-LY-19 and WSU-DLCL2 were treated with OTX015 (100 nM) and 348 compounds as single agents and in combination. The combinations of OTX015 with a series of compounds achieved improved anti-tumor activity than single agents. Besides HDAC and mTOR inhibitors, in accordance with what previously reported (Boi, Gaudio, Bonetti et al, Clinical Cancer Res 2015), the ABL/SRC inhibitor dasatinib, the AKT1/2/3 inhibitor MK-2206, the JAK2 inhibitor TG101209 and the LRRK2 inhibitor LRRK2-IN appeared interesting combination partners. These screening results were validated in the DLBCL cell lines with targeted experiments, as well as in chronic lymphocytic leukemia (CLL) (OTX015 plus MK-2206) and mantle cell lymphoma (MCL) (REC1) (Dasatinib, LRRK2-IN). Experiments in additional cell lines and assessing the effects on the apoptosis are on-going and will be presented.

Conclusion. A chemical screening has identified novel OTX015-containing combinations active in lymphoma cell lines that are worth of further investigations.

Work supported by a San Salvatore Foundation grant.

#1895

Landmark development of PI3K delta specific inhibitors for hematologic cancer therapeutics by data-driven drug design.

Suji Hong, Jong Woo Kim, Chi Woo Lee. _Bioway, Chuncheon-si, Republic of Korea_.

PI3K δ is hyper activated primarily in B cell malignancy and plays a central role in the malignant cell retention in proliferation, survival and homing at the lymphoid tissue and bone marrow in a variety of signaling pathways. Our BW compounds(BW-101, BW-108) are highly potent and selective PI3K δ inhibitors with marked selectivity over the other PI3K isoforms(α, β, γ). BW compounds are designed for the purpose of advancing the PI3K δ inhibition activity and overcoming the toxicities in which are hepatotoxicity and immunotoxicity due to small ratio of PI3K γ/PI3K δ. BW compounds are proven to be best-in-class in PI3K δ inhibition activity and to possess a bigger ratio of PI3K γ/PI3K δ related to the immunotoxicity than that of Idelalisib As small molecules, they inhibit PI3Kδ in the way of competing with ATP strongly at PI3Kδ binding site that issues in apoptosis of malignant B and T cell through blocking AKT down pathway. BW compounds have shown the good PK profiles in rat so that their in vivo efficacies were superior to those of Idelalisib in xenograft studies. Such a result demonstrated the therapeutic potential of these compounds in acute leukemia mediated via the PI3Kδpathway. In the side of toxicity, BW compounds also are showing desirable properties. 

### Identification of Molecular Drug Targets

#1896

Patient-derived HER3 mutations transform ER+ and HER2+ breast cancer cells via MAPK pathway activation.

Rosalin Mishra,1 Samar Alanazi,1 Long Yuan,1 Thomas Solomon,1 Tarjani M. Thaker,2 Natalia Jura,2 Joan T. Garrett1. 1 _Univ. of Cincinnati College of Pharmacy, Cincinnati, OH;_ 2 _University of California, San Francisco, CA_.

We sought to investigate the role of patient-derived HER3 mutations in HER2+ and ER+ breast cancer cells using ectopic expression of HER3 mutants. We aim to identify mechanism(s) by which HER3 mutants enhanced HER2-mediated or hormone-driven transformation and if HER3 mutations confer resistance to HER2 or ER+ inhibitors.

A series of HER3 mutations (F94L, G284R, D297Y, D313H, K329T, T355I, L792V and E1261A) were introduced and stable cell lines were generated in MCF10AHER2 and ER+ MCF-7 and T47D cells using lentiviral transduction. HER3T355I mutant was transforming in ER+ cells. HER3T355I mutant had increased p-HER3 and p-ERK1/2 expression compared to controls expressing wild-type HER3. Receptor tyrosine kinase array results indicated that T47D and MCF-7 overexpressing HER3T355I had increased p-HER4 and p-HER1 expression, respectively. HER3T355I counteracted the effect of the ER inhibitor, 4-hydroxytamoxifen but not fulvestrant. Cells expressing HER3T355I were subjected to lapatinib in presence or absence of ER inhibitor fulvestrant and specific ERK1/2 inhibitor, SCH772984. The data indicated that induced transforming activity observed in HER3T355I is via HER4/MAPK pathway in T47D and HER1/MAPK signaling in MCF-7 cells. Immunoblotting data indicated that cyclin D1 and p-ERK1/2 expressions were altered in response to combined treatment of lapatinib with or without fulvestrant and SCH772984 indicating that cyclinD1 mediated signaling downstream of the MAPK pathway. HER3T355I and HER3WT demonstrated reduced cell proliferation and matrigel colony formation in presence of lapatinib and fulvestrant/SCH772984. We noted crosstalk between ERα and HER3 in T47D cells. Based on structural modelling, T355I likely disrupted the interactions between domain III and the hinge region pocket that stabilized the tethered conformation of HER3T355I, potentially making the hinge more flexible, shifting the equilibrium from the tethered to the untethered state.

Laboratory studies indicated that several HER3 mutants acquired gain-of-function phenotype and were resistant to lapatinib and partially resistant to neratinib in MCF10AHER2 cells. These mutants increased HER2-HER3 hetero-dimerization. However, there was no significant alteration in AKT signalling in HER3 mutants.

We are currently using a proteomics approach to identify HER3-interacting proteins, and determine how HER2 inhibition affects the repertoire of HER3 binding partners in HER2+ breast cancers. We will also mine for HER3 binding partners in ER+ breast cancers by using ReCLIP, a

methodology in which reversible cross-linking precedes HER3 immunoprecipitation and mass spectrometry-based identification of HER3-interacting proteins.

We aim to identify kinases (other than HER2) that phosphorylate HER3 upon therapeutic inhibition of HER2 and PI3K or anti-endocrine therapy.

#1897

Determining the role of PRCP/PREP in triple negative breast cancer.

Ricardo E. Perez, Lei Duan, Carl G. Maki. _Rush University, Chicago, IL_.

Triple negative breast cancer (TNBC) accounts for 15-20% of all breast cancer cases. Currently, the only effective treatment for TNBC is chemotherapy agents. A well-established characteristic of TNBC is the ability of these cells to become metastatic following chemotherapy treatment resulting in increased mortality. New forms of treatment which target TNBC are urgently needed. Insulin receptor substrate 1 (IRS1) has been extensively studied in the regulation of the insulin and insulin-like growth factor receptor signaling cascades, specifically in the induction of the intracellular PI3K/AKT/mTORC1 and MAP kinase pathways. The roles of such pathways in cancer are currently being studied and considered for therapeutic targeting. We previously showed that the stability and levels of IRS1 are maintained by prolylcarboxypeptidase proteins PRCP/PREP in pancreatic cancer cells. Our hypothesis is that PRCP/PREP are potential therapeutic targets in TNBC. Specifically, we hypothesize that blocking the expression or reducing the activity of PRCP/PREP will inhibit the PI3K/AKT/mTORC1 pathway in TNBC cells and, in turn, reduce growth and survival in these cells. To test this, we will be treating a number of TNBC cell lines with a PRCP/PREP inhibitor and determining IRS1/2 protein levels, AKT/mTORC1 signaling, and growth and survival. TNBC cells were screened for sensitivity against the PRCP/PREP inhibitor, Y-ox. Sensitive cells treated with Y-ox showed earlier reduction in the IRS1/AKT/mTORC1 signaling, as well as earlier loss of cell viability compared to those cells with relative resistance to Y-ox. Similarly, cells treated with the IRS1/2 inhibitor NT-157 and the AKT inhibitor, MK-2206 showed loss of cell viability similarly seen with the PRCP/PREP inhibitor, Y-ox. In conclusion, the results shown in this study show a possible new therapeutic target against triple negative breast cancer in the inhibition of PRCP/PREP leading to loss of cell proliferation and viability.

#1898

Regulation of cysteine supply for survival in hepatocellular carcinoma an breast cancer cell lines.

Do Hyung Kim, Seung Jin Lee. _College of Pharmacy, Chungnam National University, Daejeon, Republic of Korea_.

Several types of tumor highly rely on extracellular cystine to supply cysteine for essential biological processes, therefore, cystine transporter, system xc-, has been suggested as a potential target for cancer therapy. However, it has not been known about cellular heterogeneity and regulatory mechanisms for cystine-dependency. Cystine dependency of 9 hepatocellular carcinoma cell lines and 6 breast cancer cell lines was measured using cystine-deprived medium or sulfasalazine, an inhibitor of system xc-. Cultivation with cystine-deficient media significantly reduced the growth in 9 cell lines, moderately in 3 cell lines, but in 2 cell lines including SNU398 cells. Sulfasalazine dose-dependently decreased the growth of cystine-dependent cell lines, which was prevented by the addition of 2-mercaptoethanol which reduces cystine to be shuttled via L-type transporter system. To identify regulatory mechanisms to render cells to be less dependent on extracellular cystine, siRNA kinase library was screened in SNU387 cells. The siRNAs to decrease viability of SNU387 cells in cystine-deficient media were identified. These research suggest cellular heterogeneity for the dependency on extracellular L-cystine and the kinase which may support cysteine supply in the absence of extracellular cystine could be a potential candidate for targeting cysteine metabolism.

#1899

Targeting interferon induced transmembrane protein-1 (IFITM1) attenuates the aggressive phenotype of inflammatory breast cancer.

Olivia Provance, Eric Geanes, Joshua Ogony, Asona Lui, Eric Young, Sumedha Gunewardena, Joan Lewis-Wambi. _University of Kansas Medical Center, Kansas City, KS_.

Inflammatory breast cancer (IBC) is the most aggressive and lethal subtype of breast cancer, with triple-negative IBC being the most difficult to treat IBC subgroup. Improved understanding of mechanisms that lead to aggressiveness of triple-negative IBC could provide a basis for therapeutic targets. Our laboratory has identified a novel interferon (IFN)-α signaling target called interferon induced transmembrane protein-1 (IFITM1), which we hypothesize to promote the aggressive phenotype of triple-negative IBC. CRISPR/CAS9 was utilized to silence IFITM1 in SUM149 TN-IBC cells. In vitro, we found that knockout of IFITM1 increased in colony and mammosphere formation, inhibited migration as observed by the wound healing assay, and enhanced sensitivity to mainstay chemotherapeutic agents (taxol and doxorubicin) analyzed by flow cytometry, MTT and Tunel staining. Western blot analysis shows a decrease in c-Src and p-Src, vimentin, and CD44v expression, which are important regulators of migration and invasion. RNA sequencing analysis further indicates that knocking down IFITM1 promotes overall downregulation of pathways including breast cancer invasion, supporting our in vitro findings. In vivo, IFITM1-CRISPR/KD SUM149 cells were injected into the mammary fat pads of immunosuppressed female NSG mice to assess tumor growth. We found that mice injected with IFITM1-CRISPR/KD SUM149 cells showed significantly reduced tumor growth compared to mice injected with CRISPR/control SUM149 cells, which was observed at all time points examined. We used the mammary intraductal (MIND) method to assess tumor invasion in vivo. IFITM1-CRISPR/KD SUM149 cells showed decreased invasion outside of the mammary duct and decreased vimentin expression, further corroborating our in vitro data. Lastly, through tail vein injections to model metastasis, 33% of mice injected with SUM149 wildtype cells developed liver metastasis while 0% of mice injected with IFITM1-CRISPR/KD SUM149 mice developed liver metastasis. We have ongoing studies to further understand the mechanism by which IFITM1 regulates cellular migration, as well as invasion and angiogenesis in triple-negative IBC, which are all shown to be downregulated through RNA sequencing analysis. Overall, these findings indicate that IFITM1 may be driving the aggressiveness of triple-negative IBC and they suggest that IFITM1 may serve as a potential therapeutic target.

#1900

Disruption of EGFR and integrin b1 signaling in triple-negative breast cancer through targeting SHC1.

Ya-Fang Chang, Elizabeth Cordell, Yuhao Wang, Geoffrey Greene. _University of Chicago, Chicago, IL_.

Triple-negative breast cancer (TNBC) accounts for 15-20% of breast cancer cases and is more prevalent among young women and African American women. SHC1 (Src Homology 2 Domain Containing Transforming Protein 1) is an adaptor protein that can transmit cell surface receptor signaling and activate the Ras-ERK pathway. SHC1 is found to be overexpressed in breast cancers and is important in the regulation of tumor progression and drug resistance in ER+ breast cancer. Elevated SHC1 correlates with poor prognosis in breast cancer patients. However, the biological significance of SHC1 in TNBC is not known. In this study, we performed siRNA-mediated knockdown to determine the importance of SHC1 in cell proliferation and drug response in TNBC. We found that although siSHC1 slightly inhibited the tumor growth of TNBC cells, siSHC1 + paclitaxel significantly decreased cell proliferation and increased caspase-3/7 activity in vitro. In invasion assays, siSHC1 significantly decreased the invasion of TNBC cells. To better understand the systemic regulation by SHC1, proteomic analysis was performed after siSHC1 or Scr transfection. Pathway analysis revealed the involvement of PI3K/AKT, FAK, and actin cytoskeleton pathways, by disruption of EGFR and integrin β1 signaling, in the control of TNBC progression and drug sensitivity. In tumor explant studies, siSHC1 significantly enhanced paclitaxel-induced tumor shrinkage and reduced lung metastasis. Our data suggest that SHC1 is a potential therapeutic target in TNBC.

#1901

Structural basis for selective therapeutic targeting of BFL-1 in cancer by a covalent stapled peptide inhibitor.

Edward P. Harvey, Hyuk-Soo Seo, Rachel M. Guerra, Gregory H. Bird, Sirano Dhe-Paganon, Loren D. Walensky. _Dana-Farber Cancer Institute, Boston, MA_.

BCL-2 family proteins are master regulators of the mitochondrial apoptotic pathway and dictate whether a cell will live or die in response to stress. Cancer cells hijack the anti-apoptotic arm of this signaling network, overexpressing BCL-2, MCL-1, and BFL-1, to enforce cellular immortality. Whereas BCL-2 has been drugged by the selective small-molecule inhibitor, venetoclax, and new MCL-1 inhibitor molecules have just entered phase I clinical testing, anti-apoptotic BFL-1 remains undrugged. Importantly, the expression of BFL-1 can cause resistance to these molecules and has been independently linked to the pathogenesis and chemoresistance of human cancers, including melanomas and hematologic malignancies. Given the emergence of BCL-2 family proteins as high-priority cancer targets, solving their structures, alone and in complex with their natural BH3 domain ligands, yields important blueprints for drug design. Whereas the structures of numerous "BH3-in-groove" complexes have been solved, the corresponding panel of apo structures remains incomplete. Here, we report the crystal structure of apo BFL-1 at 1.69 Å resolution, revealing similarities and key differences among unliganded anti-apoptotic targets. Unlike all other BCL-2 proteins, apo BFL-1 contains a surface-accessible cysteine within its BH3-binding groove, allowing for selective covalent targeting by BH3-based stapled peptide inhibitors. The crystal structure of a complex between BFL-1 and a cysteine-reactive and stapled NOXA BH3 α-helix demonstrated the sulfhydryl bond and fortuitous interactions between the acrylamide-bearing moiety and a newly formed hydrophobic cavity. Comparison of the apo and BH3-liganded structures further revealed an induced conformational change. The two BFL-1 structures expand our understanding of the surface landscapes available for selective targeting of BFL-1, and inform a new therapeutic strategy for overcoming BFL-1-dependent apoptotic blockades in human cancer.

#1902

Potential role of DYRK family kinases in harmine-induced apoptosis in neuroblastoma.

Katie L. Uhl,1 Chad R. Schultz,1 Dirk Geerts,2 Andre S. Bachmann1. 1 _Michigan State University, Grand Rapids, MI;_ 2 _University of Amsterdam, Amsterdam, Netherlands_.

The purpose of this study was to determine the effect of the natural product Harmine in human neuroblastoma (NB). NB is an early childhood malignancy arising from the developing peripheral nervous system. In up to 25% of cases MYCN gene amplification is correlated to high tumor stage and poor patient prognosis. High stage NB relapses frequently in spite of multimodal therapy and is then virtually untreatable. There is a clear need for specific, novel therapeutics. Harmine is a tricyclic β-carboline alkaloid from the harmal plant with cytostatic and cytotoxic effects on tumor cells. It is capable of blocking the activity of mitogen activate protein kinase (MAPK) and the dual specificity tyrosine-phosphorylation-regulated kinase (DYRK) family proteins. These kinases inhibit apoptosis and encourage proliferation. Four human NB cell lines were used to study the effects of Harmine treatment: SK-N-BE(2)-C and KELLY (MYCN amplified) as well as SK-N-AS and SK-N-FI (MYCN single copy). Molecular interaction models of Harmine bound to DYRK family kinases were generated by computational docking using x-ray structures. The anti-cancer properties of Harmine were analyzed by real-time cell viability assays, in a dose-dependent manner, over a 72 hour period. The IC50 values were 169.9, 170.8, and 791.7 μM for SK-N-BE(2)-C, KELLY, and SK-N-FI, respectively, after 72 hours. Apoptosis assays for caspase activation, PARP cleavage, and annexin V induction, were performed using Western blot and flow cytometry. NB cell line exposure to 100 μM Harmine resulted in caspase-3/7 and caspase 9 activation, PARP cleavage, and annexin V-positive stained cells as early as 24 hours after treatment, indicating apoptosis induction. These results led us to investigate the clinical correlations of DYRK family gene expression in NB tumors. The patient results support our hypothesis that Harmine induces NB cell death through a cellular mechanism that involves DYRK family kinases and triggers caspase-mediated apoptosis.

#1903

Antiproliferative effect of cannabinoid receptor agonists on rectal cancer lines.

Alette Ortega Gómez,1 Pedro Luna-Merlos,1 Victor Manuel García Hernández,1 Abelardo Meneses García,1 Horacio Astudillo- de la Vega,2 Eric Alejandro García López,3 Abel Santamaría Del Angel,4 Erika Betzabé Ruiz García1. 1 _Instituto Nacional de Cancerologia, Mexico City, Mexico;_ 2 _HOCMN SXXI IMSS, Mexico City, Mexico;_ 3 _Instituto Nacional de Ciencias Médicas y Nutrición, Mexico City, Mexico;_ 4 _Instituto Nacional de Neurología y Neurocirugía, Mexico City, Mexico_.

Rectal cancer is considered a major health issue in several countries that present advanced and metastatic stages and a limited response to standard treatments. Though the antineoplasic activity of cannabinoids has been documented in several studies, scarce information is available about the effect of these agents in cell lines of rectal cancer. In this work, we explored the antiproliferative effects of cannabinoid receptor agonists in rectal tumor cells. The effects of the three agonists anandamide, methanandamide and CP55940 were evaluated in the SW837 rectal tumor cell line. The three agents tested showed time and concentration dependent effects on cell viability, induction of cell death and morphological changes. The three agonists were effective to induce late apoptosis and necrosis and internucleosomal DNA fragmentation. In addition, we evaluated for the first time the patterns of CB1 and CB2 cannabinoid receptor expression in SW837. Though some differences in their effects were found, our data support the concept that these agents mostly share common antiproliferative/cell death mechanisms. Our results also confirm the antineoplasic potential of cannabinoids receptor agonists against rectal cancer, and suggest the design of cannabinoid based coadjutant therapies at the clinical level.

#1904

Polyprenoic acid, the first-discovered hepatocyte nuclear factor 4 alpha agonist, inhibits multistep liver carcinogenesis.

Taro Yamashita,1 Hikari Okada,2 Masao Honda,2 Shuichi Kaneko2. 1 _Kanazawa University Hospital, Kanazawa, Japan;_ 2 _Kanazawa University, Kanazawa, Japan_.

Hepatocyte nuclear factor 4 alpha (HNF4α) is one of the most ancient nuclear receptors known as a master regulator of hepatocytes in lipids, carbohydrates, and drug metabolism. It also works as a tumor suppressor to regulate cell cycle in hepatocellular carcinoma (HCC), but the ligand activating HNF4α has not yet been discovered thus far. Here we evaluated the effect of polyprenoic acid (PA), formerly known as an acyclic retinoid, on HNF4α activation in dysplastic nodule and HCC in vitro and in vivo. PDGF-C transgenic (Tg) mice was evaluated by Gd-EOB-DTPA-enhanced MRI to examine the effect of PA on HCC development in vivo. Whole exome sequence analysis was performed using Illumina Hiseq 2000 system. Contrast enhanced CT scan data of patients enrolled in previous phase II/III randomized placebo-controlled study were collected and analyzed (n = 124 for PA and 127 for placebo). PA treatment regressed the liver dysplastic nodule and a subset of HCCs in PDGF-C Tg mice evaluated by MRI. This tumor regression was accompanied with the reduction of accumulated gene mutations, inactivation of cell cycle, and transcriptional activation of HNF4α-target genes. The binding of PA on ligand binding domain of HNF4α was shown by docking simulation in silico and immunoprecipitation-mass spectrometry analysis in vitro. Transcriptional activation of HNF4α by PA was also verified using a luciferase reporter assay and binding of HNF4α on DNA binding elements. PA treatment in HCC cells immediately resulted in the degradation of HNF4α by ubiquitin-proteasome system with activation of DNA damage responses when cultured in lipid depleted medium. In vivo knockdown of HNF4A cancelled the effect of PA on regression of liver tumor growth in PDGF-C Tg mice. PA treatment (600 mg/day) suppressed the HCC development from dysplastic nodule compared with placebo in human with statistical significance (HR 0.41; 95% CI, 0.17~0.98, P = 0.046). Taken together, our data indicated that PA is the first-discovered HNF4α ligand to activate its function. PA activates the DNA damage responses and suppresses the accumulation of gene mutations especially in dysplastic nodule, warranting the future prospective study to evaluate the effect of PA on suppression of HCC development from dysplastic nodules in human.

#1905

New targeted therapeutics for gastric cancer.

Jean F. Ruiz Calderon,1 Marcia Cruz-Correa,1 George A. Calin,2 Linette Castillo-Pichardo,3 Suranganie Dharmawardhane1. 1 _UPR-Medical Science Campus, San Juan, PR;_ 2 _University of Texas MD Anderson Cancer Center, Houston, TX;_ 3 _Universidad Central del Caribe School of Medicine, Bayamon, PR_.

Gastric cancer is a health disparity with 2.3 times higher incidence rates in Hispanics than in Non-Hispanic Whites (NHWs). Very few studies have been conducted to characterize this health disparity in Hispanics; however, studies from East Asian patients, where gastric cancer is also a health disparity, have revealed that p21-activated kinases (PAKs 1,2 and 4) are biomarkers for gastric cancer. Even though inhibition of PAKs impedes gastric cancer progression, current PAK inhibitors have failed to successfully complete clinical trials. Thus, we hypothesize that the inhibition of upstream effectors of PAK, the Rho GTPases Rac and Cdc42 is a rational treatment for gastric cancers with high PAK expression. The purpose of this study is to correlate Rac/Cdc42 and PAK expression and activation with Hispanic gastric cancer malignancy, and to test the efficacy of our recently developed Rac/Cdc42 inhibitor MBQ-167 (Humphries-Bickley, et al., Mol Cancer Therap. 2017, 16:805-818). In a related Aim, to identify new targets and biomarkers for Hispanic gastric cancer, gastric cancer tissues and normal gastric mucosa from biopsies and/or surgery were collected from Puerto Rican patients for expression profiling with customized qRT-PCR arrays for mRNA and Agilent arrays for non-coding RNAs (ncRNAs), and western blotting. Western blotting of gastric cancer cell lines revealed that NCI-N87 metastatic gastric cancer cells express more PAK isoforms and phospho (active)-PAK compared to the AGS non-metastatic gastric cancer cells; accordingly, MBQ-167 reduces the viability of NCI-N87 cells without affecting AGS cells. Therefore, to test the efficacy of Rac/Cdc42 inhibition in gastric cancer, immunocompromised mice with subcutaneous tumors from GFP-NCI-N87 cells were treated 3X week with vehicle or 1mg/kg BW of MBQ-167. Tumor growth calculated by image J analysis of fluorescent digital images. A drastic reduction in NCI-N87 tumor growth was observed in the MBQ-167 treated mice with a 61% inhibition in tumor size compared to the controls, and a tumor growth delay of >90% after the second doubling time. Gene expression profiling revealed that 13 genes were overexpressed (>1.5 fold change) in Puerto Rican gastric cancer tissue in comparison to the normal gastric mucosa, including Rac1, PAK1, STAT3, KDR (VEGFR2), CXCR4, IGF1, VEGFA, and IL-6, all of which are associated with Rac/Cdc42/Pak signaling. Differential profiling using a custom-made Agilent array for ncRNAs demonstrated upregulation of a number of pro-cancer microRNAs (miRNAs) such as miR-106b and downregulation of tumor suppressors such as let-7a and miR-145, as well as upregulation of the long ncRNA CCAT2, which we have shown to be important for colon cancer malignancy. Taken together, our data show that Hispanic gastric cancers have known and novel biomarkers, and MBQ-167 is a viable targeted therapeutic option for gastric cancers with elevated Rac/Cdc42/PAK signaling.

#1906

PFKFB4 **, much more than just a glycolytic gene.**

Emma L. Phillips,1 Frederic Bethke,1 Jörg Balss,1 Stefan Pusch,1 Stefan Christen,2 Martina Schnölzer,1 Antje Habel,1 David Capper,1 Andreas Von Deimling,1 Sarah-Maria Fendt,2 Peter Lichter,1 Violaine Goidts1. 1 _DKFZ, Heidelberg, Germany;_ 2 _CCB-KU Leuven Center for Cancer Biology, Leuven, Belgium_.

Glioblastoma is one of the most aggressive primary brain tumors in adults, with a dismal median overall survival of only 14 months after diagnosis. Glioblastoma stem-like cells (GSCs) are particularly resistant to current therapies, capable of self-renewal and tumour initiation and are hence thought to be major contributors to patient relapse. A kinome and phosphatome wide screen revealed glycolysis gene 6-Phosphofructo-2-Kinase/Fructose-2,6-Biphosphatase 4 (PFKFB4) as an important candidate gene for GSC survival. Notably, silencing PFKFB4 in an orthotopic xenograft mouse model of glioblastoma completely rid the mice of the tumor. Here we show a brand new function of PFKFB4, independent of its role in glycolysis - namely in the regulation of HIF1α, which is upregulated in GSCs. Gene expression profiling revealed a downregulation in HIF1α target genes in PFKFB4-silenced GSCs, and HIF1α protein levels are also dramatically reduced upon silencing. Mass spectrometric analysis of immunoprecipitated PFKFB4 protein revealed a novel interaction partner, F-box only protein 28 (FBXO28), an E3 ubiqutin ligase. Co-immunoprecipation assays show that FBXO28 forms an SCF multi-protein ubiquitin ligase complex with CUL1 and SKP1. Ubiquitylation studies of HIF1α show that PFKFB4 stabilizes this crucial protein in GSCs by preventing its targeting by FBXO28 for proteasomal degradation. These new findings, coupled with its cancer specific expression in a variety of tumor entities, makes PFKFB4 a compelling target.

#1907

Claudin 6 is a carcinoembryonic antigen with cancer stem cell marker features.

Özlem Türeci,1 Meike Wagner,2 Claudia Paret,3 Maria M. Kreuzberg,4 Stefan Wöll,4 Korden Walter,4 Sabine C. Häcker,5 Ikumi Nakajo,6 Tomohiro Yamada,6 Ugur Sahin7. 1 _CI3 – Cluster of Individualized Immune Intervention, Mainz, Germany;_ 2 _Formerly of TRON – Translational Oncology at the University Medical Center of the Johannes Gutenberg University Mainz and University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany;_ 3 _University Medical Center of the Johannes Gutenberg University, Mainz, Germany;_ 4 _Formerly of Ganymed Pharmaceuticals GmbH, Mainz, Germany;_ 5 _Formerly of TRON – Translational Oncology at the University Medical Center of the Johannes Gutenberg University Mainz and Unimedizin, Mainz, Germany, Mainz, Germany;_ 6 _Astellas Pharma Inc., Tokyo, Japan;_ 7 _TRON – Translational Oncology at the University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany_.

Background Claudin 6 (CLDN6) is a tight junction membrane protein whose expression in normal tissue is confined to embryonic cells, but is aberrantly expressed in various human cancers, such as ovarian cancer (OC) and testicular cancer (TC). A monoclonal antibody against CLDN6, IMAB027, has shown promising antitumor activity in preclinical human CLDN6-positive (CLDN6+) cancer models. In this series of nonclinical studies, we investigated CLDN6 expression in normal and cancer tissues, as well as the localization and possible function of CLDN6 in cancer cells.

Methods Expression of CLDN6 was assessed in a wide range of human tissues (eg, lung, colon, skin, ovary) and cultured cells by quantitative RT-PCR, immunohistochemistry (IHC), flow cytometry, and western blotting. To investigate the effect of dedifferentiation on CLDN6 expression, human-induced pluripotent cells were generated by transfecting foreskin fibroblasts with a reprogramming cocktail, and then CLDN6 expression was evaluated. To characterize CLDN6 as a potential novel marker to identify cancer stem cells (CSCs) in OC, coexpression of CLDN6 with known CSC surface markers were analyzed by flow cytometry, and CLDN6+ and CLDN6-negative cells were tested in colony formation and sphere formation assay. Human OC cell lines were transplanted intraperitoneally into nude mice and assessed for metastasis to investigate tumorigenecity of CLDN6+ cells.

Results Except for low mRNA levels measured in placenta, testis, umbilical cord, cerebellum, and lung samples, no CLDN6 (mRNA or protein) was detected in the vast majority of normal tissues. Additionally, there was also a lack of CLDN6 protein expression in tissue zones where stem cells for tissue homeostasis would normally be found as determined by IHC with an anti-CLDN6 antibody. CLDN6 was expressed on the cell surface of several solid tumors, including ovarian, testicular, uterine, and lung cancer tissues; OC and TC samples had high level expression. CLDN6 expression was strongly activated in human-induced pluripotent stem cells generated from fibroblasts. CLDN6 showed selective coexpression with known CSC markers such as CD44, CD24, and CD90 in OC and TC cell lines. In addition, some CLDN6+ OC cells exhibited CSC-like behavior in vitro: CLDN6+ populations were clonogenic and formed well-defined spheres in low attachment conditions; these spheres had the ability to self-renew into secondary spheres. Analysis of OC metastases in mouse xenografts showed when xenografts were generated by OC cells that had <10% of CLDN6+ cells, the metastases were enriched in CLDN6+ cells, suggesting CLDN6\+ cells had selective growth advantage.

Conclusions CLDN6 is a cancer cell-specific surface molecule aberrantly expressed in several cancers, and its expression may be an identifier for cells with CSC-like traits. These characteristics make CLDN6 an attractive target candidate for tumor-specific therapeutic antibodies.

#1908

Search for mediators of the lethal effect of COPZ1 depletion in thyroid tumor cells.

Maria Chiara Anania,1 Katia Todoerti,2 Italia Bongarzone,1 Elena Cetti,1 Tiziana Di Marco,1 Antonino Neri,3 Angela Greco1. 1 _Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy;_ 2 _IRCCS-CROB, Referral Cancer Center of Basilicata, Rionero in Vulture, Italy;_ 3 _Fondazione IRCCS Ca' Granda, Ospedale Maggiore Policlinico and Department of Oncology and Hemato-oncology, University of Milan, Milan, Italy_.

We previously identified COPZ1 as an example of "non-oncogene" addiction for thyroid cancer cells, since its silencing drastically impairs the viability. Notably, the inhibition of COPZ1 has no effect on normal cells such as primary and normal immortalized thyrocytes. COPZ1 belongs to the coatomer protein complex I, involved in assembly of coated vesicles on Golgi membranes, the retrograde transport of proteins in the ER-Golgi secretory pathway, endosome maturation, autophagy and lipid homeostasis. We found that COPZ1 depletion leads to abortive autophagy, ER stress, unfolded protein response (UPR) and apoptosis. To better identify intracellular pathways that are activated upon COPZ1 silencing and that contribute to cell death, we performed a gene expression profiling of COPZ1-depleted 8505C cells (anaplastic thyroid tumor cells), 72h after siRNA transfection, by using Affymetrix Gene ST 2.0 array. Hierarchical clustering was applied on most variable genes in the entire dataset and specific gene expression patterns were identified in COPZ1-silenced cells compared to the control (non targeting siRNA-transfected cells), by means of SAM software (q-value 0). Functional annotation analyses by means of DAVID 6.8 bioinformatic tool and Gene Set Enrichment Analysis were performed. We found 321 genes specifically modulated in COPZ1-depleted 8505C cells; the main altered functional categories dealt with cell cycle regulation, chromosome organization and protein metabolism. Specifically, gene sets associated to the UPR, interferon signalling, vesicular transport and the translation machinery were found upregulated, whereas those related to DNA metabolism, telomere maintenance, cell cycle check points, cytoskeleton and cell adhesion, non-coding RNA and mRNA metabolism were found downregulated. Furthermore, we performed a proteomic profiling of 8505C cellular extracts (at 48h after COPZ1 depletion) by a nLC-ESI-MS/MS analysis. Bioinformatic analysis identified 271 down-regulated and 291 up-regulated proteins compared to the control. Overall, GeneTrail2 analysis revealed that the most changing pathways are related to RNA metabolism involved in splicesome and RNA processing, thus likely explaining the extensive transcriptional remodelling observed in the later time point. More interestingly, the gene expression profiling was also carried out in Nthy-ori 3-1 cells (immortalized normal human thyroid cells) that, unlike tumor cells, are resistant to cell death after COPZ1 depletion. We found no significant differentially expressed genes in COPZ1-depleted cells in comparison to the control, in keeping with the absence of a lethal effect, despite COPZ1 silencing. In conclusion, we identified novel functions of COPZ1 and further pathway interactions, in addition to that already known, that can be considered other critical nodes in the oncogenic network whose inhibition will result in cell collapse.

#1909

NR4A2 role in head and neck cancer: Mechanistic and functional analysis.

Sanjib Chaudhary, Ramesh Pothuraju, Pranita Atri, Satyanarayana Rachagani, Surinder K. Batra, Muzafar A. Macha. _University of Nebraska Medical Center, Omaha, NE_.

Background: Nuclear receptor related 1 protein (NURR1/NR4A2) is a transcription factor that mediates numerous functions in differentiation/development, maintenance and metabolism. In cancer cells, NR4A2 promotes proliferation, migration, transformation and chemo-resistance in many cancers. Here, we explore the role of NR4A2 in head and neck squamous cell cancers (HNC) tumorigenesis and define its target genes. Methods: NR4A2 expression was determined by immunohistochemistry (IHC) and in publically available databases (Oncomine, TCGA). NR4A2 was knockedout/knockdown by CRISPR or shRNA. Coimmunoprepitation and reverse chromatin immunoprecipitation were done to study the interaction between EGFR/STAT3. ChIPSeq analysis was done by using Illumina NextSeq 500 Genome Analyzer and integrated with microarray (HPV+/- patients) to identify the true target genes of NR4A2 using BETA Software and validated by PCR/qPCR. Results: IHC and database analysis demonstrated overexpression of NR4A2 in HNC patients. NR4A2 was significantly overexpressed in HPV+ HNC patients, SCC47 and SCC104 cell lines. Functionally, CRISPR/shRNA knockout/knockdown of NR4A2 in cell lines significantly decreased colonogenicity, proliferation and migration. Expression of NR4A2 was positively correlated with EGFR expression (R2=0.92), and EGF treatment (100ng/ml; 24-48h) induced NR4A2 (~2 fold) expression coupled with epithelial to mesenchymal transition in SCC1 and SCC10B cells (HPV-). We also observed EGF mediated NR4A2 induction was abrogated by panEGFR inhibitor (afatinib) with concomitant decrease in STAT3 expression. Physical interaction between EGFR and STAT3 with EGF treatment regulated NR4A2 expression. Whole genome integration of ChIPseq and microarray demonstrates 9% (2015) and 23% (3002) of the enriched up and down-regulated target genes, respectively were canonically regulated in HPV+ HNC. ChIP PCR validation indicates enrichment of NR4A2 in some of the target genes, wnt10, p53, cxcl13, cln10 and hoxb8 in HPV+ cells. Conclusions: Our data indicate overexpression of NR4A2 in HNC, and its positive correlation with EGFR expression. We also noted the physical interaction between EGFR and STAT3 which functionally attribute the NR4A2 expression. Integration and validation data showed the differential enrichment of NR4A2 target genes in HPV+/- cell lines which suggest its possible discriminatory role in HNC pathogenesis which needs to be confirmed.

#1910

Investigating AURKA and TPX2 as therapeutic targets in KRAS-induced pancreatic ductal adenocarcinoma (PDAC).

Sandro M. Gomes-Filho, Edmilson O. dos Santos, Bianca Dazzani, Eduardo M. Reis, Daniela S. Basseres. _University of São Paulo, São Paulo, Brazil_.

KRAS oncogenic mutations are found in over 90% of pancreatic ductal adenocarcinomas (PDACs), and no effective therapies are available for KRAS-induced malignancies. Therefore, identification of KRAS targets with therapeutic potential is warranted. Given that we have shown that Aurora kinase A (AURKA) is a KRAS target in lung cancer, our goal was to evaluate the potential of AURKA and its activator TPX2 as therapeutic targets in PDAC. Publicly available genomics data from 179 PDAC cases was used to investigate the association of AURKA or TPX2 expression with the presence of KRAS mutations (CbioPortal), as well as with patient survival (SurvExpress). AURKA and TPX2 expression was also analyzed by RNAseq and qPCR in 14 PDAC patient samples and matched normal pancreatic tissue. Finally, we targeted KRAS, AURKA or TPX2 in KRAS-mutant PDAC PANC-1, MIAPaCa-2 and AsPC-1 cells by RNA interference or with an Aurora inhibitor and analyzed clonogenic and anchorage-independent growth. Based on the PDAC genomic dataset, KRAS mutations were significantly associated with increased AURKA and TPX2 expression, which in turn correlated with decreased overall survival. Moreover, when compared to adjacent normal tissue, PDAC tumors displayed higher expression of AURKA and TPX2. Targeting KRAS by RNA interference in KRAS-mutant PDAC cells led to reduced levels of AURKA and TPX2, thus confirming that AURKA and TPX2 are KRAS targets in PDAC. Furthermore, pharmacological inhibition of AURKA led to decreased cell viability and proliferation. Finally, AURKA or TPX2 targeting by RNA interference reduced PDAC cells clonogenic and anchorage-independent growth. In conclusion, we have shown that AURKA and TPX2 are KRAS targets in PDAC, that their expression is associated with worse prognosis, and that AURKA or TPX2 targeting reduces PDAC cell oncogenicity. These results support our hypothesis that AURKA and TPX2 are therapeutic targets for KRAS-mutant PDAC.

#1911

Uncovering novel targets of recurrent glioblastoma using transcriptomic profiling in a patient-derived xenograft model.

Nicolas Yelle,1 Chirayu Chokshi,1 Parvez Vora,1 Kevin R. Brown,2 Maleeha A. Qazi,1 Mohini Singh,1 Jarrett J. Adams,2 Chitra Venugopal,1 Sachdev Sidhu,2 Jason Moffat,2 Sheila K. Singh1. 1 _McMaster University, Hamilton, Ontario, Canada;_ 2 _University of Toronto, Toronto, Ontario, Canada_.

Glioblastoma (GBM) is the most common and aggressive adult primary brain tumor feared for its near uniformly fatal prognosis despite advances in multimodal therapy including surgical resection, chemotherapy and radiation. Poor patient survival due to tumor relapse is thought to be linked to intratumoral heterogeneity (ITH), driven by various environmental cues including chemotherapy and radiation treatment. ITH can be explained at the cellular level by the existence of multiple populations of cancer cells, including cancer stem cells (CSCs), which have acquired stemness properties like self-renewal, proliferation, and multilineage differentiation. In brain tumors, CSCs or brain tumor initiating cells (BTICs), have been shown to be resistant to both chemotherapy and radiation treatment, allowing them to escape therapy and allowing for tumor recurrence. To profile ITH as it evolves through therapy delivery, we have developed a novel and dynamic BTIC patient-derived xenograft (PDX) model of treatment-refractory human GBM, allowing for multimodal profiling of GBM BTICs through tumor engraftment, remission, and recurrence. In this study, we present the transcriptomic profiling at each stage, and novel target selection and validation through CRISPR/Cas9 knockouts, well-established in vitro stem cell assays, and in vivo characterization of their tumor initiation, development, and maintenance properties. Despite the fact that the BTIC population is responsible for GBM recurrence and thus patient demise, it remains a largely unknown landscape. Consequently, therapies that focus on targeting the BTIC compartment within the bulk tumor would provide better treatment and prognosis for patients with brain tumors. The study we present provides a unique therapeutic window into the recurrence of GBM, which drives patient mortality, yet is profiled far less than primary treatment-naïve GBM.

#1912

A microbial glucuronidase target implicated in intestinal reactivation of tyrosine kinase inhibitors.

Bret David Wallace, Jeffrey Hymes, Matthew Redinbo, Ward Peterson. _Symberix, Durham, NC_.

Many drugs are inactivated by liver UGT enzymes and reactivated by enteric bacterial β-glucuronidases (GUS), an axis of drug metabolism that contributes to gut toxicities of anti-neoplastic agents, NSAIDs, and potentially other drug classes. For example, toxic reactivation of irinotecan by E. coli GUS is the causative event leading to intense, delayed diarrhea. Receptor tyrosine kinase (RTK)-targeted cancer drugs that undergo this UGT-GUS axis of metabolism are also associated with a high incidence of diarrhea. However, glucuronides of RTK inhibitors (RTKi) are not substrates of E. coli GUS. We recently reported that the human gut microbiome expresses nearly 300 distinct GUS enzymes (GUSome). The objective of this work is to identify the GUS enzyme(s) capable of metabolizing the glucuronide of the RTKi, sorafenib, that may contribute to sorafenib-induced diarrhea.

We used UGT1A9 supersomes to make sorafenib-glucuronide (S-G) from commercially purchased sorafenib. S-G was screened against a library of 15 purified GUSome enzymes to identify the specific GUS orthologs that can cleave S-G, which was determined by monitoring sorafenib absorbance at 280 nm. Similar approaches were used to identify GUS enzymes that can cleave other RTKi's.

Of the GUS enzymes evaluated for cleavage of S-G, only the GUS enzyme from an uncultured Eubacterium species was active. The structurally similar regorafenib-glucuronide and two glucuronides of other RTKi's (axitinib and imatinib) are also substrates of Eubacterium GUS. All four RTKi's are associated with diarrhea in at least 40-60% of patients. In contrast to E. coli GUS, very little is known about Eubacterium GUS.

We have identified a set of RTKi-glucuronides as the first substrates of Eubacterium GUS, which may contribute to diarrhea associated with targeted anti-cancer drugs.

#1913

Investigation of targeting SKP2 to improve the treatment response in glioma.

Jing Wang, Hong-Kai Su, Zhong-Ping Chen, Yue Qu, Hai-Ping Cai. _Sun Yat-sen University Cancer Center, Guangzhou, China_.

Glioma is the most frequent tumor in central nervous system (CNS), constituting about 30% of CNS tumors and 80% of malignant brain tumors. The current standard strategy for glioma treatment is surgery plus adjuvant chemotherapy and concurrent radiotherapy. However, the prognosis of glioma is still poor. It has been proved that the existence of glioma stem cells and the timely repair of DNA damages are the main causes of treatment failure. Skp2 (S-phase kinase associated protein-2), a member of F-box proteins, forms the Skp2 SCF complex with Skp1, Cullin-1, and Rbx1, serves as one of the important E3-ligases in the process of ubiquitination, and is responsible for substrate recognition. Skp2 is overexpressed in a variety of human cancers and promotes cancer progression by inducing p27 degradation. Importantly, Skp2 deficiency profoundly restricts cancer progression in multiple genetic mouse tumor models. Our group has demonstrated that Skp2 is involved in the regulation of pool size and self-renewal ability of hematopoietic stem cells, as well as breast cancer stem cells and nasopharyngeal carcinoma stem cell-like cells. However, the exact role of Skp2 in the regulation of glioma stem cells and DNA damage repair in glioma has not been elucidated yet. We proposed that targeting Skp2 could promote radio- and chemotherapy sensitivity through suppressing function of glioma stem cells. We first detected the expression of SKP2 in glioma tumor tissues and found that SKP2 was highly expressed in glioma and related to the prognosis of glioma patients. We also adopted gene knockdown methods and inhibitor pretreatment with small molecules in order to suppress the level of Skp2, and then checked the sphere formation and self-renewal ability of glioma stem cell upon radio/chemo treatments. In this way we evaluated the role of Skp2 in repressing the glioma cell proliferation and subsequently triggering apoptosis. Our project will clarify the novel mechanisms about how Skp2 is involved in the glioma radio- and chemotherapy resistance, which will provide a potential target to improve the radio/chemo therapeutic effect through targeting one single target.

#1914

Elongation factor 2: A novel target for cancer progression.

Beatrice Malacrida,1 Catríona M. Dowling,1 Rasmus K. Flygaard,2 Maeve Kiely,1 John C. Coffey,1 Lasse B. Jenner,2 Patrick A. Kiely1. 1 _Univ. of Limerick, Limerick, Ireland;_ 2 _Aarhus University, Aarhus, Denmark_.

Protein translation is one the most energy demanding and highly regulated processes in cells. For this reason, dysregulation of protein synthesis is linked with different types of disease, including cancer. The link between protein synthesis and cancer progression is becoming a central theme among cancer research. Although numerous studies have identified protein synthesis as a process central to cancer progression, further research is required to identify potential novel targets and/or biomarkers.

Recently, RACK1 (Receptor for Activated C Kinase 1), a scaffold protein involved in many cell regulatory pathways including cell proliferation and migration, has been found to be part of the ribosomal machinery. Here, RACK1 is able to regulate the synthesis of particular sets of proteins and the translation itself. In fact, RACK1 is located close the mRNA exit channel where is able to recruit kinases, phosphatases and specific mRNA binding proteins.

We have identified elongation factor 2 (eEF2) as novel RACK1 interacting protein in our two and three dimensional colon cancer models. Elongation factor 2 is one of the main proteins regulating the elongation phase of protein synthesis, responsible for the translocation of the mRNA along the ribosome. We have confirmed the binding between eEF2 and RACK1 by isolating and purifying a native human form of eEF2 and combining it with a purified human version of RACK1.

Interestingly, we have demonstrated that stress conditions and IGF-1 stimulation modulate eEF2 activity in colon cancer cells. Moreover, we have examined the expression of EEF2 and have found that EEF2 gene is dysregulated in our cohort of matched normal and cancer colonic patient tissues. Interestingly, EEF2 expression is upregulated in mucinous cancers (a rare and aggressive type of colorectal cancer) compared to non-mucinous cancers and the expression of EEF2 is as well upregulated in cancers that have recurred within 2 to 3 years after the first surgery.

We believe that eEF2 is playing an important role in cancer progression and migration especially by regulating and modulating protein synthesis in certain conditions. A better understanding of the role of this protein will help in the identification of novel targets that can be then translated into therapeutic strategies against colorectal cancer.

#1915

Validation of ero1l as a therapeutic target for the treatment of multiple myeloma.

Weichih Chen, Werner Geldenhuys, Lori Hazlehurst. _West Virginia University, Morgantown, WV_.

Validation of ERO1L as a therapeutic target for the treatment of multiple myeloma

Wei-Chih Chen1, Werner Geldenhuys1 and Lori Hazlehurst1,2,3

1. Department of Pharmaceutical Sciences, School of Pharmacy.

2. Osborn Hematopoietic Malignancy and Transplantation Program, West Virginia University.

3. West Virginia University Cancer Institute, Morgantown, WV, USA.

Multiple myeloma (MM) is a hematologic malignancy of plasma cells and remains an incurable disease. MM is exquisitely sensitive to agents which induce ER stress including inhibitors that target the proteasome such as Bortezomib. However, MM patients eventually relapse with drug resistant disease, indicating that additional therapies are required to improve patient outcomes. Endoplasmic reticulum oxidase 1-alpha (ERO1L) is an endoplasmic reticulum (ER) resident protein that plays a critical role in disulfide bonds formation and contributes to proper folding of secretory proteins. High expression of ERO1L has been reported to be associated with poor prognosis in several cancer indications. However, the role of ERO1L in MM remains unclear. Our data indicate the ERO1L expression is increased in MM cells compared to normal controls. Because MM cells are known to secrete high levels of paraprotein resulting in basal ER stress, we investigated the role of ERO1L in tumor growth. Our data indicate that overexpression of ERO1L in U266 MM cells enhanced cell growth using both in vitro and in vivo models. Moreover, inducible reduction of ERO1L using shRNA strategies inhibited colony formation in U266 cells. Using a pharmacological inhibitor of, ERO1L referred to as EN460, we show that inhibition of ERO1L induced markers of ER stress and triggered apoptosis. Together our findings indicate that ERO1L maybe a tractable strategy for the treatment of MM.

Keywords: multiple myeloma (MM), endoplasmic reticulum oxidase 1 alpha (ERO1L)

#1916

Targeting adhesion signaling network blocks tumor growth in gallbladder cancer.

Mahendra K. Singh,1 Kaustav Majumder,2 Bhuwan Giri,1 Nivedita Arora,2 Shrey Modi,1 Vrishketan Sethi,1 Claudia P. Garcia,1 Juan C. Roa,3 Vikas Dudeja,1 Sulagna Banerjee,1 Ashok K. Saluja1. 1 _Univ. of Miami Miller School of Medicine, Miami, FL;_ 2 _University of Minnesota, Minneapolis, MN;_ 3 _Pontificia Universidad Católica de Chile, Santiago, Chile_.

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#1917

CHD1 depletion leads to androgen-independent growth of prostate cancer cells lines and regulates the TGFβ system in an EZH2-dependent manner.

Simon J. Böcker, Simon J. Baumgart, Annabell Roth, Vijayalakshmi Kari, Steven A. Johnsen, Oliver Hahn. _University Medical Center Göttingen, Göttingen, Germany_.

Introduction:

Therapy of metastasized prostate cancer (PCA) largely depends on androgen deprivation therapy (ADT). Despite initial response, tumors inevitably reach castration resistance (CRPC), a stage at which chemotherapy and second-generation antiandrogens (e.g. Enzalutamide) represent the main therapeutic options. Acquired resistance to these drugs is an emerging problem, raising a need for new approaches. Deletion or mutation of the Chromodomain Helicase DNA binding protein 1 (CHD1) gene, encoding an ATP-dependent chromatin remodeler, is more prevalent in CRPC compared to earlier tumor stages. Therefore, we investigated the effects of CHD1 depletion in androgen-sensitive LNCaP cells.

Experimental procedures:

LNCaP cells were grown in media with either 10% fetal bovine serum (FBS), 10% charcoal-stripped serum (CSS) or 10% CSS plus 1nM testosterone. Growth was visualized by image-based cell cytometry. After 10 days of growth, RNA sequencing (RNA-seq) was performed to investigate changes in gene expression (between the different growth conditions with and without CHD1 depletion). Results were verified by qRT-PCR and Western blot. The identified treatment options were tested with small-molecule inhibitors in different concentrations. Finally, we performed chromatin immunoprecipitation followed by next-generation sequencing (ChIP-seq) in order to identify the epigenetic mechanisms underlying the differential regulation.

Results:

We identified a correlation between androgen-independent growth and CHD1 depletion which could be compensated by addition of 1 nM testosterone. Moreover, we identified CHD1-dependent regulation of Enhancer Of Zeste Homolog-2 (EZH2), which could be confirmed at the protein level. Treatment with 1 µM JQ-EZ (an EZH2 inhibitor) showed a stronger effect on the proliferation of CHD1-knockdown cells compared to controls. ChIP-seq and RNA-seq analysis helped to identify a subset of EZH2-bound genes depending on CHD1 knockdown and androgen deprivation. Among those genes, we identified the I-SMAD genes SMAD6 and SMAD7 which are regulators of TGFβ and BMP signaling to be upregulated after androgen-deprivation and downregulated with CHD1 knockdown. We also showed differential regulation of PMEPA1, a TGFβ-dependent inhibitor of PCA bone metastasis. Coherently, we found upregulation of mesenchymal markers such as CDH2, pointing towards a role in EMT.

Conclusion:

We identified a new regulatory mechanism for CRPC harboring a CHD1-deletion leading to a more mesenchymal state in an EZH2-dependent manner. First experiments with an EZH2 inhibitor in our system indicate a possible therapeutic use. CHD1 could hereby be used both as a biomarker for early metastasis as well as for therapeutic stratification.

#1918

**Patient-derived hormone-naive prostate cancer xenograft models reveal** GRB10 **as an AR-repressed gene driving the development of castration-resistant prostate cancer.**

Jun Hao, Xinpei Ci, Hui Xue, Rebecca Wu, Xin Dong, Fang Zhang, Sifeng Qu, Fan Zhang, Anne M. Haegert, Peter W. Gout, Colin Collins, Martin E. Gleave, Dong Lin, Yuzhuo Wang. _Univ. of British Columbia, Vancouver, British Columbia, Canada_.

Prostate cancer is the most commonly diagnosed noncutaneous cancer and the leading cause of cancer-related death in North American men. Although androgen-deprivation therapy is initially effective in controlling the growth of hormone-naive prostate cancers (HNPC) in patients, currently incurable castration-resistant prostate cancer (CRPC) inevitably develops. Thus, we aim to identify CRPC driver genes that may provide new targets to enhance CRPC therapy. In this study, patient-derived xenografts (PDXs) of HNPCs that develop CRPC following host castration were examined for changes in expression of genes at various time points after castration using transcriptome profiling analysis; particular attention was given to pre-CRPC changes in expression indicative of genes acting as potential CRPC drivers. Eighty genes were found to be significantly upregulated at the CRPC stage while 7 of them also showed elevated expression prior to CRPC development. Among the latter, Growth Factor Receptor Bound Protein 10 (GRB10) was the most significantly and consistently upregulated gene. Moreover, we found GRB10 expression was elevated in clinical CRPC compared to HNPC in several clinical cohorts. Further investigation suggests that GRB10 is transcriptionally regulated by androgen receptor through an androgen-responsive element located in GRB10's intron. Functionally, we found that GRB10 knockdown markedly reduced prostate cancer cell proliferation and activity of AKT, a well-established CRPC mediator. Also, a positive correlation between AKT activity and GRB10 expression was found in clinical cohorts. In conclusion, GRB10 acts as a driver of CRPC and sensitizes AR pathway inhibitors, and hence GRB10-targeting provides a novel therapeutic strategy for the disease.

#1919

Characterization of URST1 as a prognostic biomarker and therapeutic target for oral cancer.

Atsushii Takano,1 Yohei Miyagi,2 Yataro Daigo3. 1 _The University of Tokyo, Tokyo, Japan;_ 2 _Kanagawa Cancer Center Research Institute, Yokohama, Japan;_ 3 _Shiga University of Medical Science, Otsu, Japan_.

Oral cancer shows a high mortality rate, and its incidence is increasing gradually worldwide. Current therapies for oral cancer include surgery, radiotherapy, and chemotherapy, however, local and regional recurrences account for up to 90% of treatment failures after surgery and radiotherapy. Therefore, next generation biomarkers and therapeutic strategies for oral cancer are eagerly awaited. To identify biomarkers and therapeutic targets for oral cancer, we screened genes that overexpressed in the majority of oral cancers by using our original gene expression profile database and identified up-regulated in solid tumor 1 (URST1) as a candidate. Immunohistochemical staining showed that URST1 expression was observed in the majority of oral cancer in 64 of 96 (67%) that had received curative surgery. URST1 expression was associated with poor prognosis for oral cancer patients (P = 0.032 by log-rank test). Reduction of URST1 expression by siRNAs against URST1 (si-URST1) significantly suppressed growth of oral cancer cells. Flow cytometric analysis revealed that si-URST1 increased the population of cancer cells at G2/M. In addition, exposure of cancer cells to selective URST1 inhibitor suppressed the growth of oral cancer cells. These data indicate that URST1 is a possible prognostic biomarker and therapeutic target for oral cancer.

#1920

Targeting structural RET and MET kinase alterations in lung adenocarcinoma patients.

Dennis Plenker,1 Carina Lorenz,1 Miriam Bertrand,2 Richard Riedel,1 Joop de Langen,3 Johannes Brägelmann,1 Reinhard Büttner,1 Jürgen Wolf,1 Roman K. Thomas,1 Johannes Heuckmann,2 Martin L. Sos1. 1 _Univ. Hospital of Cologne, Cologne, Germany;_ 2 _NEO New Onfology, Cologne, Germany;_ 3 _VU University Medical Center, Amsterdam, Amsterdam, Netherlands_.

Structural rearrangements that activate receptor kinases account for an ever-increasing pool of druggable targets in lung adenocarcinoma patients. Among these tumors RET kinase fusions and MET alterations represent two genetically distinct groups that share a common lack of clinically effective strategies. While RET rearranged tumors show a limited susceptibility to currently available RET inhibitors, MET rearranged tumors have not been yet fully appreciated as a relevant group that may benefit from treatment with MET targeted drugs. Using systematic molecular profiling of genetically engineered RET rearranged in vitro and models and patient-derived in vivo models we identified the type II kinase inhibitors ponatinib and AD80 as the most potent drugs. While both inhibitors are effective against gatekeeper mutant RET we identified a novel resistance mutation RETI788N that triggers a selective resistance against AD80 and other RET inhibitors but retains susceptibility to ponatinib. Furthermore, we studied the clinical and preclinical activity of MET targeted drugs. We specifically characterized genomic rearrangements of KIF5B-MET and STARD3NL-MET in cellular models that were found in two distinct LADC. In parallel we identified and characterized a MET kinase domain duplication that developed in an EML4-ALK rearranged positive tumor as a resistance mechanism to ceritinib. All three patients showed a partial response to crizotinib that effectively inhibits MET and ALK in these tumors. Thus, our molecular characterization of drug-target engagement in genetically defined models may further enhance the clinical efficacy of kinase inhibitors in lung tumors driven by rare oncogenic kinase alterations.

#1921

**The sensitivity of ovarian cancer cells to the HDAC inhibitor belinostat is improved by inhibiting Bcl-x** L **or Mcl-1 anti-apoptotic proteins.**

Charlène Duboc,1 Edwige Abeilard,1 Anne Sophie Voisin-Chiret,2 Pascal Gauduchon,1 Laurent Poulain,1 Marie Villedieu1. 1 _Normandie University, UNICAEN, Inserm U1086 ANTICIPE, Comprehensive Cancer Center F. Baclesse, BioTICLA group, Caen, France;_ 2 _Normandie University, UNICAEN, CERMN CNRS INC3M, Caen, France_.

The identification of novel therapeutic strategies is an important urgent requirement for the clinical management of ovarian cancers, which remain the leading cause of death from gynecologic cancer. Our previous studies have shown that the anti-apoptotic proteins Bcl-xL and Mcl-1 cooperate to protect resistant ovarian cancer cells from apoptosis as their concomitant inhibition results in massive apoptotic cell death. Moreover, their BH3-only pro-apoptotic partners Bim and Puma are crucial actors in the induced cell death. This suggests that ovarian cancer cell apoptosis can be triggered by inhibiting Bcl-xL and Mcl-1 and/or by promoting their BH3-only partners, especially Bim and Puma. The expression of these Bcl-2 family proteins has been reported to be modulated by HDAC (histone deacetylases) inhibitors in various cancer models. The objective of the present study was to evaluate the efficacy of the FDA-approved pan-HDAC inhibitor belinostat (i) to reduce the [Bcl-xL and Mcl-1] / BH3-only proteins ratio and (ii) to trigger apoptosis in chemoresistant ovarian cancer cell lines, alone or in combination with other pharmacological molecules that modulate these Bcl-2 family proteins. HDAC inhibition by belinostat led to a G2/M blockade and consequently reduced cell proliferation. Moreover, belinostat used at high concentrations (from 1000 to 2000 nM) efficiently triggered apoptosis in chemoresistant ovarian cancer cells. This cytotoxic effect was associated with both an up-regulation of Bim and Puma protein expression and an inhibition of Bcl-xL protein expression. Used at sub-toxic concentrations, belinostat also up-regulated Bim and Puma proteins but failed to down-regulate Bcl-xL and Mcl-1 protein expression. We therefore investigated whether Bcl-xL or Mcl-1 inhibition could sensitize ovarian cancer cells to sub-toxic concentrations of belinostat. Interestingly, the combination of belinostat with the BH3-mimetic ABT-737, that inhibits Bcl-xL, induced massive apoptosis. Moreover, Mcl-1 direct or indirect inhibition was also very efficient to sensitize ovarian cancer cells to belinostat. These results suggest that belinostat, alone or in association with pharmacological molecules that inhibit Mcl-1 or Bcl-xL, represents a promising approach for the treatment of chemoresistant ovarian cancers.

#1922

Targeted silencing of SOX2 by an artificial transcription factor showed antitumor effect in lung and esophageal squamous cell carcinoma.

Etsuko Yokota,1 Tomoki Yamatsuji,1 Munenori Takaoka,1 Minoru Haisa,1 Nagio Takigawa,1 Noriko Miyake,1 Tomoko Ikeda,1 Tomoaki Mori,2 Ohno Serika,2 Takashi Sera,2 Takuya Fukazawa,1 Yoshio Naomoto1. 1 _Kawasaki Medical School, Okayama, Japan;_ 2 _Okayama University, Okayama, Japan_.

S0X2 is a transcription factor essential for early mammalian development and for the maintenance of stem cells. Recently, SOX2 was identified as a lineage specific oncogene, recurrently amplified and activated in lung and esophageal squamous cell carcinoma (SCC). In this study, we have developed a zinc finger-based artificial transcription factor (ATF) to selectively suppress SOX2 expression in cancer cells and termed the system ATF/SOX2. We engineered the ATF using six zinc finger arrays designed to target a 19 bp site in the SOX2 distal promoter and a KOX transcriptional repressor domain. A recombinant adenoviral vector Ad- ATF/SOX2 that expresses ATF/SOX2 suppressed SOX2 at the mRNA and protein levels in lung and esophageal SCC cells expressing SOX2. In these kinds of cells, Ad-ATF/SOX2 decreased cell proliferation and colony formation more effectively than the recombinant adenoviral vector Ad-shSOX2, which expresses SOX2 short hairpin RNA (shSOX2). Ad-ATF/SOX2 induced the cell cycle inhibitor CDKN1A more strongly than Ad-shSOX2. Importantly, the ATF did not suppress the cell viability of normal human cells. Moreover, Ad-ATF/SOX2 effectively inhibited tumor growth in a lung SCC xenograft mouse model. These results indicate that ATF/SOX2 would lead to the development of an effective molecular-targeted therapy for lung and esophageal SCC.

#1923

Identification and validation of Integrin-Linked Kinase (ILK) signaling as a therapeutic target for ovarian cancer.

Michael A. Ulm,1 Suriyan Ponnusamy,2 Adam C. ElNaggar,1 Ramesh Narayanan2. 1 _UTHSC - West Cancer Center, Memphis, TN;_ 2 _UTHSC, Memphis, TN_.

Ovarian cancer affects approximately 25,000 women and contributes to around 14,000 deaths in the United States each year. Although significant advances have been made in the discovery of molecular diagnostics and therapeutics for other cancers, ovarian cancer still lacks reliable diagnostic and prognostic markers as well as appropriate targeted therapeutics. A major reason for the lagging development of targeted therapeutics is the molecular heterogeneity of high grade serous ovarian cancer and the lack of better understanding of driver mutations and pathogenesis. Although gene expression data sets are available, most of them lack appropriate pair-matched controls to define the alterations that result in the transformation of normal ovarian cells to highly proliferative cancerous cells. We compared differential gene expression of high-grade treatment-naïve ovarian cancer tissue samples to matched-pair adjacent benign ovarian tissue specimens from 24 patients. A significant upregulation of the integrin-linked kinase (ILK) pathway was identified in 22 of the 24 cancer specimens, identifying a potential player that could contribute to the transformation of normal ovarian cells to cancerous cells. To determine the therapeutic potential of ILK inhibition, ILK expression was reduced using siRNA in SKOV3 cells, which resulted in decreased cell proliferation. This is consistent with prior studies demonstrating reduced adhesion to and invasion of collagen gels and organotypic meso-mimetic ovarian cancer cultures with down-regulating ILK using siRNA resulted in. We then assessed the ability of a targeted agent, compound 22, a small molecule ILK-selective inhibitor, to inhibit proliferation in various ovarian cancer cell lines with different phenotypic characteristics (SKOV3, OVCAR3, OVCAR8, OV90). Compound 22 reduced the phosphorylation of Akt, a downstream target of ILK, and also inhibited cellular proliferation. Our initial findings validate ILK as a potential therapeutic target for molecular inhibition in ovarian cancer and warrants further investigation.

#1924

Role of CXCL16 and ADAM10 in ovarian cancer pathogenesis.

Hina Mir, Neeraj Kapur, James W. Lillard, Shailesh Singh. _Morehouse School of Medicine, Atlanta, GA_.

Ovarian cancer is the leading cause of death from gynecological malignancies. The five-year survival rate of ovarian cancer ranges from 30 to 92%, depending on the severity of the disease. Our laboratory has shown the role of chemokines and chemokines receptors in cancer, including ovarian cancer. In this study, we have shown higher expression of CXCR6, which is the only receptor of chemokine CXCL16, in ovarian cancer cell lines and clinical samples. We have tested the biological significance of CXCR6 and soluble CXCL16 (sCXCL16) using ovarian cancer cell lines in vitro. We show higher migratory and invasive potential of high CXCR6 expressing ovarian cancer cell lines in the response of sCXCL16. Migratory and invasive potential in response to sCXCL16 was significantly inhibited when CXCR6 and CXCL16 interaction was blocked using anti-CXCR6 monoclonal antibody. ADAM10 basal mRNA level was higher in cells with high migratory and invasive potential. Similar to cell lines, higher expression of CXCR6 and CXCL16, as well as ADAM10, was observed in tissue, which showed a positive correlation with tumor stage. These results suggest the clinical and biological significance of CXCR6-CXCL16 axis in the pathogenesis of ovarian cancer. Therefore, therapeutics directed to the CXCR6-CXCL16 axis may provide better therapeutic outcome and overall survival.

#1925

High-throughput drug library screening identifies potent drugs and novel drug targets for high-risk acute leukemia in children.

Priscilla Wander,1 Sandra S. Pinhanços,1 Bianca Koopmans,1 M. Emmy M. Dolman,1 Pauline Schneider,1 Patricia Garrido Castro,1 Luke Jones,1 Susan T.C.J.M. Arentsen-Peters,2 Mark Kerstjens,2 Jan Molenaar,1 Huib N. Caron,3 Rob Pieters,1 Michel C. Zwaan,2 Ronald W. Stam1. 1 _Princess Maxima Center, Utrecht, Netherlands;_ 2 _Erasmus MC, Sophia Childrens Hospital, Rotterdam, Netherlands;_ 3 _Emma Children's Hospital/AMC, Amsterdam, Netherlands_.

BACKGROUND: Acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) represent the most common types of cancer among children. Although the survival rates of pediatric ALL and AML have improved substantially over the last decades, the prognosis for some subgroups of pediatric leukemia remain poor. MLL-rearranged infant ALL, MLL-rearranged AML, and NUP98-rearranged AML, for instance, still have 5-year event-free survival rates of only ~40%. Evidently, these high-risk subtypes of childhood leukemia urgently require new treatment strategies in order to improve clinical outcome.

AIM: Identification of effective therapeutic drugs for high-risk leukemia subtypes harboring a translocation in the MLL or NUP98 gene, by using a drug repurposing strategy involving high-throughput screening of > 4000 compounds.

METHODS: Primary leukemic cells derived from six patients with either MLL-rearranged infant ALL, pediatric MLL-rearranged AML, or pediatric NUP98-rearranged AML at diagnosis, were screened using commercially available drug libraries comprising a total of 4165 compounds. Drug sensitivity was assessed after 4 days treatment with 10 nM, 100 nM and 1000 nM of the library compounds using MTT assays. Potential hits were subsequently validated using cytotoxicity assays with a more extensive range of drug concentrations.

RESULTS: The results of our drug screens showed that the most effective drugs, by far, were found for MLL-rearranged infant ALL. For MLL-rearranged AML the number of identified potent drugs was considerably less; notably, all of these compounds identified overlapped with the effective agents also found for MLL-rearranged infant ALL. Remarkably, for NUP98-translocated AML, only a few potential drug hits were identified, suggesting that this type of leukemia might be extremely chemo-resistant.

In addition to novel candidate compounds, we also found several agents either recently described to have strong anti-leukemic effects against MLL-rearranged leukemia, such as the BCL2 inhibitors Venetoclax (ABT199) and Navitoclax (ABT263), or the MDM2 inhibitor Idasanutlin (RG7388), as well as finding drugs currently being used in the treatment of MLL-rearranged acute leukemias. Taken together, these observations indicate that high-throughput drug screening on primary patient samples is indeed able to identify effective compounds, validating our experimental approach.

Interestingly, while validating newly identified drug hits, we observed a remarkable pattern for compounds effective against MLL-rearranged acute leukemia: the top hits seem to either directly or indirectly associate with p53 activity, albeit from various angles.

CONCLUSION: Our data suggests a potential role for p53 in MLL-rearranged acute leukemia, and indicates that targeting p53 may well become an important therapeutic strategy for these types of childhood leukemia.

### New Targets 1

#1926

Npy1r as a prognostic marker and a novel drug target in estrogen receptor-positive breast cancer.

Raksha Bhat,1 Suhas Vasaikar,2 Leon Bae,1 De Angelis Carmine,2 Maria Letizia Cataldo,2 Sarmistha Nanda,2 Bing Zhang,2 Rachel Schiff,2 Meghana V. Trivedi1. 1 _Univ. of Houston College of Pharmacy, Houston, TX;_ 2 _Baylor College of Medicine, Houston, TX_.

Despite the crucial role of some G protein-coupled receptors (GPCRs) in cancer progression and metastases reported in recent studies, the function of majority of GPCRs, as a class, remains largely unexplored in breast cancer. In this study, we evaluated transcriptome, proteome and phosphoproteome data for 398 non-sensory GPCRs (IUPHAR v1.0) using the landmark TCGA breast cancer proteogenomics dataset and Illumina HiSeq dataset. Neuropeptide Y Receptor Y1 (NPY1R) was the top most hypo-phosphorylated (at S368) GPCR in 92 out of 99 evaluable samples. Furthermore, NPY1R gene expression was found to be significantly lower in tumor samples than tumor-matched normal (n=112). Among tumor samples, the LumA subtype had significantly higher NPY1R expression than LumB, Basal and Her2 subtype (p<0.05, One-way ANOVA, Tukey test). Interestingly, the trend of NPY1R gene expression, protein expression and phosphosite expression was decreasing in the order of LumA, LumB, Basal and Her2 subtype (p<0.01, Jonckheere trend test). Using publicly available dataset of 22,277 genes on survival in 2,422 patients, we found that higher NPY1R expression predicted better overall survival and recurrence-free survival in estrogen receptor-positive (ER+) breast cancer patients. In light of this, we interrogated NPY1R gene expression in endocrine sensitive and resistant cell line models and the impact of NPY1R antagonist in ER+ breast cancer cell lines. NPY1R expression was increased in response to estradiol in various ER+ breast cancer cell line models (MCF7, T47D, and BT474) in publicly available datasets. Conversely, we found that tamoxifen or estrogen deprivation (ED) treatment reduced NPY1R expression in MCF7 and T47D cells. In our endocrine-resistant (tamoxifen-resistant, estrogen deprivation-resistant, and fulvestrant-resistant) cell line models, NPY1R expression remained significantly lower compared to the parental cells in vitro and in vivo (p<0.05, One-way ANOVA, Tukey test). Treatment of MCF7 parental cells with 100 nM NPY significantly reduced estrogen-stimulated cell growth (p<0.05, One-way ANOVA, Tukey test). Chronic treatment of cells with 1 μM BIBP3226, an NPY1R antagonist, for 2 weeks reversed the effect of NPY on the estradiol-stimulated cell growth. Estradiol-stimulated cell growth was modest (~40%) in ED-resistant MCF7 cells compared to that (~250%) in parental MCF7 cells. While 100 nM NPY treatment did not affect the estrogen-stimulated growth in ED-resistant MCF7 cells, which can be explained by the undetectable expression of NPY1R, it caused significant inhibition of the estrogen-stimulated growth in the same cells treated chronically with BIBP3226. Molecular mechanisms to explain these effects of chronic NPY1R antagonism are underway. Our ongoing studies will further elucidate the role of NPY1R as a novel drug target in ER+ breast cancer.

#1927

Targeting IL-13RA2 in melanoma and pancreatic cancer.

Rafal Zielinski,1 Izabela Tworowska,2 Stanislaw Skora,3 Aleksandra Rusin,1 Radjendirane Venugopal,3 Arumugam Jayakumar,3 Izabela Fokt,1 Yaan Kang,1 Jason Fleming,1 Yue Huang,4 Denise Herpai,4 Waldemar Debinski,4 Waldemar Priebe1. 1 _UT MD Anderson Cancer Ctr., Houston, TX;_ 2 _RadioMedix, Inc, Houston, TX;_ 3 _Houston Pharmaceuticals, Inc, Houston, TX;_ 4 _Brain Tumor Center of Excellence, Comprehensive Cancer Center of Wake Baptist Medical Center, Winston-Salem, NC_.

Background. Interleukin receptor alpha 1 (IL-13RA1) is a component of a heterodimeric IL-13 receptor that is shared with IL-4 and it is ubiquitously expressed in normal tissues and also tumors. In contrast, IL-13RA2 is a monomeric receptor which is overexpressed in various solid tumors, but is virtually absent in normal tissues. Ligands specifically targeting IL-13RA2 but not IL-13RA1, such as Pep-1L and IL-13.E13K.D2.Cys, have been developed and validated [1,2].

Objective. To assess the expression of IL-13RA2 in pancreatic ductal adenocarcinoma and metastatic melanoma specimens and to develop tracers for selective recognition of the tumors.

Methods. The expression of IL-13RA2in patient specimens, patient-derived xenografts (PDX) and established cell lines was assessed using Western blot, immunochemistry or immunofluorescence. The ligands were conjugated to a chelator and labeled with Yttrium 86. Ligand specificity was confirmed using IL-13RA2 positive and negative cells and blocking with a "cold" ligand. Biodistribution studies and PET/CT imaging were conducted to assess distribution of the tracer in IL-13RA2 positive tumors models.

Results. IL-13RA2 receptor is overexpressed in melanoma and pancreatic cell lines and tumor tissues. Western blot analysis of 56 patients tumor tissue lysates obtained from pancreatic cancer PDX models showed the presence of IL-13RA2 in over 60% of patients. Similarly, tissue microarray analysis of metastatic melanoma samples identified IL-13RA2 receptor expression in 40% of tumor specimens. Uptake study performed in receptor positive and negative cells confirmed the specificity of radiotracer binding. In vivo experiments indicated fast clearance of 86Y- Pep1L ligand with glomerular filtration as a primary mechanism of the tracer removal. Mice bearing IL-13RA2 positive tumors had up taken the tracer with "tumor to muscle" ratio of 5.6 1 h after probe administration. Yttrium 86-labelled IL-13-E13K.D2.Cys was also taken up by IL-13 RA2 positive tumors.

Conclusion. IL-13RA2 is an attractive target for the development of comprehensive theranostic strategies for melanoma and pancreatic cancer.

References

1. Pandya, H., et al., An interleukin 13 receptor alpha 2-specific peptide homes to human Glioblastoma multiforme xenografts. Neuro Oncol, 2012. 14(1): p. 6-18.

2. Nguyen, V., et al., A novel ligand delivery system to non-invasively visualize and therapeutically exploit the IL13Ralpha2 tumor-restricted biomarker. Neuro Oncol, 2012. 14(10): p. 1239-53.

#1928

**Identification of interleukin 13 receptor alpha 2 (IL-13Ra2) overexpression in LSL-Kras** G12D/+ **, LSL-** Try53R172H/+ **,** **and PDx-1-Cre (KPC) genetically engineered mouse model of pancreatic ductal adenocarcinoma as a target for receptor directed anticancer therapy.**

Akiko Suzuki, Pamela Leland, Bharat H. Joshi, Raj K. Puri. _FDA-CBER, Silver Spring, MD_.

Pancreatic ductal adenocarcinoma (PDA) is a uniformly lethal disease because it escapes early diagnosis and at an advanced stage there are no effective therapies available. Previously, we have demonstrated that IL-13R

α2 is overexpressed in 71% of human PDA specimens. We have targeted IL-13Rα2 in an orthotopic mouse model of human PDA and shown effectiveness of the receptor targeting agent, IL-13-PE, a recombinant immunotoxin comprised of IL-13 and Pseudomonas exotoxin. To determine the effectiveness of IL-13-PE and develop receptor targeting approaches, we used a LSL-KrasG12D/+, LSL-Try53R172H/+ and PDx-1-Cre (KPC) genetically engineered mouse model that spontaneously develops PDA in C57BL/6J immunocompetent mice and simulates human PDA disease. These spontaneous PDA tumors showed significant overexpression of IL-13R

α2 mRNA compared to normal mouse pancreas and surrounding non-cancerous pancreatic tissues as determined by RT-qPCR. By in situ hybridization (ISH) and immunohistochemistry (IHC) analysis, we confirmed these results at mRNA and protein levels of tumor and non-tumor tissue specimens by hybridizing the tumor sections with biotinylated anti-sense riboprobe and immunostaining with anti-murine IL-13R

α2 antibody. We established two tumor cell lines from KPC mice PDA, which maintained abundant expression of IL-13R

α2 mRNA and protein levels. The overexpressed IL-13R

α2 on PDA tumor cells is found biologically active, as it binds to IL-13-PE in a concentration dependent manner killing tumors cells at IC50 of ~25ng/ml (concentration of IL-13-PE that killed 50% of tumor cells). In a clonogenic assay, PDA tumor cells formed colonies, which were again sensitive to IL-13-PE in a concentration dependent manner with IC50 of ~28ng/ml. Based on these data, we have initiated assessment of anti-cancer activity of IL-13-PE against spontaneously developed PDA tumors developed in mice between the ages of 14-16 weeks. These mice were randomized to either receive excipient control i.p. or 50 µg/Kg IL-13-PE every alternate day for two weeks and then evaluated for general health and tumor growth by ultrasound technique. Five week follow up data reveal that mice treated with excipient alone developed larger tumors compared to treated mice and the general health of IL-13-PE treated mice was better compared to excipient-treated mice. These animals are being followed for their overall survival. Our studies indicate that IL-13R

α2 is overexpressed in genetically engineered mouse model of PDA suggesting that IL-13R

α

2 is a useful drug target not only in orthotopic mouse model but also in spontaneously developed PDA in mice, which may mimic its biological function more closely to human PDA.

#1929

ST8SIA1 is a novel therapeutic target in TNBC: Regulates FAK-PI3K-AKT-mTOR signaling to promote tumor growth and metastasis.

Khoa Nguyen, Bin Yuan, Yuanqing Yan, Kim-Anh Do, Naoto T. Ueno, Michael Andreeff, Venkata Lokesh Battula. _UT MD Anderson Cancer Ctr., Houston, TX_.

We identified ganglioside GD2 as a breast cancer stem cell (BCSC) marker and that GD3 synthase (ST8SIA1) regulates GD2 expression and BCSC function. We have reported that GD2 is up-regulated in TNBC and inhibition of its expression by knockdown of ST8SIA1 inhibits tumor growth and metastasis. Here we hypothesize that ST8SIA1 is overexpressed in TNBC and regulates cell signaling downstream of GD2 to promote tumor growth and metastasis. RNA sequencing data analysis of TCGA dataset with over 1100 primary and metastatic breast tumors revealed that ST8SIA1 was overexpressed in around 10% of all breast cancer patients. Basal type tumors expressed the highest levels of ST8SIA1 compared to luminal-A, luminal-B, and HER2-enriched (p<0.01) tumors. In addition, TNBC, (n=115) had 4.63-fold higher expression of ST8SIA1 than did hormone receptor-positive tumors, including ER+, PR+, and HER2+ tumors (n=852, p<0.001). Further analysis of ST8SIA1 expression in different TNBC subtypes revealed that the mesenchymal-subtype of TNBC had the highest expression (p<0.001) among the 7 TNBC subtypes. A survival analysis by univariant analysis indicated that patients with ST8SIA1high tumors had significantly lower overall and disease-free survival rates than did patients with ST8SIA1low tumors (p=0.0148 and p=0.0109, respectively with 60months follow-up). To investigate the mechanism of the ST8ISA1-mediated regulation of tumor growth and metastasis, we performed a proteomic analysis of GD2+ and GD2- SUM159 and MDA-MB-231 cells using antibody micro-arrays (Kinexus). After systematic data analysis and functional validation by western blotting, we found that FAK, PI3K, AKT, ERK, mTOR, and 4EBP1 were highly phosphorylated in GD2+ compared to in GD2- SUM159 cells. As it is known that GD2 acts as a co-receptor on the cell surface and activates downstream signaling, we hypothesized that ST8SIA1 regulates PI3K-AKT-mTOR signaling in BCSCs through GD2. To test our hypothesis, we knocked out ST8SIA1 in SUM159 cells using CRISPR-Cas9 approach. As expected knockout (KO) of ST8SIA1 reduced percentage of GD2+ cells from 20±5% to <1% in SUM159 cells. In addition, ST8SIA1-KO cells did not induce tumor growth or cause metastases in-vivo. Interestingly, we found that lack of ST8SIA1 led to inhibition of FAK, AKT, ERK, 4EBP1, and mTOR phosphorylation, suggesting that this signaling axis is regulated by ST8SIA1 in TNBC cells. Conclusion: ST8SIA1 is up-regulated in basal-like and mesenchymal subsets of TNBC tumors and is associated with poor prognosis in TNBC patients. Furthermore, we found that ST8SIA1 tightly regulates PI3K-AKT-mTOR signaling pathway downstream of GD2. Targeting ST8SIA1 could be a novel therapeutic strategy in TNBC therapy.

#1930

Targeting the c-Cbl tumor suppressor protein as a novel therapeutic approach in overcoming acquired tamoxifen resistance in luminal breast cancer.

Xiaowen Wang,1 Hsing-Yu Chen,2 Jennifer Stripay,3 Mark Noble1. 1 _University of Rochester, Rochester, NY;_ 2 _Harvard University, Cambridge, MA;_ 3 _St. Jude Children's Research Hospital, Memphis, TN_.

Background and aims: Treatment of luminal breast cancer (LBC) with tamoxifen (TMX) is associated with the emergence of resistance in ~40% of patients. Such resistance is associated with emergence of tumor initiating cells (TICs, also known as cancer stem cells), resistance to other therapeutic agents and multiple other obstacles to successful treatment. This study aims to investigate the role of tumor suppressor gene c-Cbl in TMX resistance and identify novel therapeutic approaches in treatment of acquired TMX resistant LBC that address unmet clinical needs.

Methods: TMX resistant MCF7 and T47D cell lines were generated by chronic exposure to TMX in vitro. Unbiased drug screening using the NINDS-II library revealed previously approved drugs with unexpected activities to restore c-Cbl activity. Assays, including dose-response studies, western blot, co-immunoprecipitation analyses and tumorsphere assays, were used to determine the mechanisms of action and utility of our lead agent (CRA-1). Finally, response to treatment was verified in xenograft mouse models.

Findings: In TMX-resistant LBC cells, c-Cbl was inhibited due to complex formation with the Cool-1/ßpix (C1ßp) protein. Restoration of c-Cbl function by C1ßp knockdown or by treatment with our lead c-Cbl restorative agent (CRA-1, which is approved for other purposes and is suitable for prolonged use in patients) restored sensitivity to TMX and decreased TIC function. Treatment with CRA-1+TMX increased c-Cbl activation via the non-canonical redox/Fyn/c-Cbl (RFC) pathway by harnessing TMX's estrogen receptor-independent pro-oxidative activity. This treatment also decreased levels of the c-Cbl/C1ßp inhibitory complex, and of levels and/or activity of multiple direct and indirect c-Cbl targets important in TMX resistance (e.g. EGFR, HER2, Notch-1, sox2, ß-catenin). Treatment with CRA-1+TMX also increased sensitivity to cyclophosphamide (CPP), one of the standard chemotherapeutic agents to treat TMX resistant LBCs. In vivo, treatment with MPT+TMX + ultra-low-dose of CPP completely suppressed tumor growth in a xenograft animal model for 20 weeks without any obvious toxicity. Tumors thus far have not recurred (15 weeks) after treatment was stopped.

Conclusions: Our study offers a new approach to overcoming acquired TMX resistance that also: (i) has the potential for rapid clinical translation due to the use of drugs already approved for other purposes; (ii) eliminates TICs; (iii) suppresses tumor growth in vivo with only a small fraction of clinically relevant CPP dosages; and (iv) exhibits greatly reduced toxicity effects and applicability for extended treatment periods.

#1931

Targeting master neuronal transcription factor BRN2 in neuroendocrine prostate cancer.

Daksh Thaper, Ravi Munuganti, Shaghayegh Norouzi, Sahil Kumar, Soojin Kim, Kriti Singh, Sepideh Vahid, Jennifer Bishop, Amina Zoubeidi. _University of British Columbia, Vancouver, British Columbia, Canada_.

Introduction: Resistance to newly developed androgen receptor pathway inhibitors (ARPIs), such as Enzalutamide (ENZ), rapidly emerges and patients generally die within two years. In particular, a subset of patients who relapse following ARPI therapy exhibit lineage switching whereby tumours shed their dependence on AR signaling and emerge with neuroendocrine features. These tumours, termed treatment induced neuroendocrine prostate cancer (t-NEPC), carry an extremely poor prognosis and, to date, treatment remains decades old cytotoxic chemotherapy which carries a short-lived response at the cost of significant toxicity. Therefore, targeted therapies for this deadly disease are desperately needed. Thus, the need to develop targeted treatments for this devastating disease is of paramount importance. Recently our group identified the neural transcription factor BRN2 as a major clinically relevant driver of NEPC and aggressive tumor growth, both in vitro and in vivo, suggesting targeting BRN2 is a promising strategy to prevent neuroendocrine differentiation or treat NEPC. Methods: Study the effects of BRN2 inhibition using siRNA and CRISPR K/O models. Results: Inhibition of BRN2 by siRNA and by CRISPR/Cas9 knockout drastically reduced cell proliferation in 42DENZR (NEPC) cell lines. This data was re-capitulated in human NEPC NCI-H660 cells. Loss of BRN2 initiated drastic epigenetic changes in NEPC cell lines as well as in G1 arrest through up-regulation of CDKN1A/1B. This was confirmed using our first in field BRN2 inhibitors. Targeting BRN2 also lead to downregulation several known targets in NEPC like EZH2, AURKA, SOX2 and Peg10. Conclusion: No therapies exist for highly lethal NEPC. Hence, the described work aims to verify BRN2 as a central driver of NEPC, and lay the pre-clinical foundation for the integration of targeted therapies into the treatment landscape to improve survival and quality of life for patients suffering from deadly form of prostate cancer.

#1932

Discovery of ENPP1 inhibitors as agonists of STING pathway.

Sunil Sharma, Alexis Weston, Trason Thode, Eric Gomez, Mohan Kaadige, Hariprasad Vankayalapti. _Stingray Therapeutics, Houston, TX_.

The discovery of STING (Stimulator of interferon genes) pathway and the second messenger 2′3′-cGAMP that activate it opened up several new possibilities for the development of anti-cancer and anti-pathogen therapeutics. STING is a pattern-recognition receptor anchored on endoplasmic reticulum and has pivotal roles in surveillance and innate immune response. Binding of 2′3′-cGAMP with STING induces conformational changes, which subsequently leads to perinuclear migration of STING and recruitment of TANK-binding kinase 1 (TBK1) and IFN regulatory factor 3 (IRF3). TBK1 phosphorylates IRF3, which then translocates to the nucleus and drives the transcription of innate immune genes, including IFN-β. Ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) is a dominant hydrolyzing enzyme of 2′3′-cGAMP. In ENPP-/- cells, 2′3′-cGAMP is more active in stimulating STING activation, and this is predicted to be due to increased half-life. Similarly, hydrolysis resistant 2′3′-cGAMP analogs are reported to be potent STING agonists. We hypothesized that inhibition of ENPP1 activity with an orally available small molecule would be a novel approach to activate STING pathway when compared with the cyclic dinucleotides (CDNs) due to their parenteral route of administration. We have utilized computational methods successfully discovered small molecule candidates that showed a strong binding affinity towards ENPP1. With the help of in vitro enzymatic inhibition and direct binding assays we have identified few lead candidates with an IC50 of <100 nM. Cellular assays further showed target specific inhibition of ENPP1 activity and subsequent activation of STING pathway. Additionally, our lead series of compounds showed no effect on other members of the ENPP family, a kinome panel, and several epigenetic regulators, suggesting the specificity towards ENPP1. In summary, we have developed small molecule inhibitors of ENPP1 and one of our lead candidate's is currently under investigation for ADME-Tox, PK and efficacy. The results from these studies will be presented.

#1933

**A novel small molecule inhibitor of MRCK shows utility in blocking radiation induced invasion of glioblastoma cells** in vitro **and** in vivo **.**

Heather J. McKinnon,1 Joanna Birch,2 Laura McDonald,1 Mairi Sime,1 Daniel Croft,1 Diane Crighton,1 Martin Drysdale,1 Lesley Gilmore,2 Christopher Gray,1 Jennifer Konczal,1 Duncan McArthur,1 Patricia McConnell,1 Mokdad Mezna,1 Alexander Schuettelkopf,1 Karen Strathdee,2 Justin Bower,1 Michael F. Olson,1 Anthony J. Chalmers2. 1 _Beatson Inst. for Cancer Research, Glasgow, United Kingdom;_ 2 _Institute of Cancer Sciences, Glasgow, United Kingdom_.

Myotonic dystrophy kinase-related CDC42-binding kinases MRCKα and MRCKβ regulate actin-myosin contractility and are members of the AGC family of serine/threonine kinases. MRCKs are closely related to the Rho-regulated ROCK kinases, which is reflected in their shared abilities to phosphorylate a similar set of substrates, including myosin II light chain proteins (MLC) and myosin phosphatase target subunit 1 (MYPT1). However, MRCK and ROCK may phosphorylate these substrates at different subcellular localisations leading to diverging effects on cell invasion and migration.

Glioblastoma (GBM) is an aggressive and incurable primary brain tumor. Patients are treated with surgery, radiotherapy and chemotherapy but prognosis remains poor, with a median survival of 15 months. Complete surgical resection of GBM is difficult because these tumours are frequently invasive. Recent research has indicated that, while radiotherapy extends life expectancy of patients, it can provoke a more infiltrative phenotype in those GBM cells that survive treatment.

We have developed potent and selective small molecule MRCK inhibitors exemplified by BDP-9066, which inhibits pMLC in a selective cellular assay with an EC50 28.3 nM, whilst displaying high selectivity over ROCK1 where the EC50 10.6 µM. This compound, and others in the series, have been used in numerous cellular assay systems to demonstrate that MRCK inhibition suppresses the invasion and migration of cancer cells including, breast, skin SCC and glioblastoma.

We have used BDP-9066 to demonstrate that MRCK inhibition is effective at perturbing radiation-induced migration of GBM cells in culture at concentrations that block phosphorylation of MLC. Using a clinically relevant intracranial mouse model that recapitulates key histological features of the disease, we have shown that BDP-9066 inhibits the invasion of G7 GBM cells into the contralateral hemisphere of the brain. These experiments highlight the potential utility of MRCK inhibition in the treatment of GBM in patients.

#1934

Anti-Progranulin (GP88) antibody AG01 therapeutic effect in triple negative breast cancer invasiveness.

Rupa Guha,1 Jianping Dong,2 Binbin Yue,2 Ginette Serrero2. 1 _University of Maryland School of Medicine, Baltimore, MD;_ 2 _A &G Pharmaceutical, Inc., Columbia, MD_.

Breast cancer (BC) is the second leading cause of death in women worldwide. 15-20% of BC is triple negative breast cancer (TNBC).This subtype is very important clinically because of its high, invasiveness and short metastasis-free survival. Because of the lack of ER, PR and Her2 receptors, TNBC patients cannot be treated with anti-estrogen or Her2 targeted therapies. Standard of care (SOC) includes chemotherapies like anthracyclines, cyclophosphamide, taxanes and radiation therapy. There is an unmet need for novel targeted therapies for TNBC that can be used as single agent or in combination to potentiate SOC therapy to avoid or reduce toxicity, non-specific effects and unresponsiveness. Therefore it is very important to identify biomarkers overexpressed in TNBC that play a critical role in TNBC invasiveness and can be used as therapeutic targets. Progranulin (PGRN, GP88) is a secreted glycoprotein autocrine growth/survival factor functions as a biological driver of tumor cell proliferation, tumorigenesis, survival, invasiveness and drug resistance in several cancers, including breast cancer (BC). In ER\+ BC, GP88 stimulates proliferation and survival and its overexpression confers estrogen independence and anti-estrogen resistance. GP88 is overexpressed in ER+ BC and in TNBC. GP88 expression in ER+ tumor biopsies is an independent prognostic factor of recurrence. High GP88 is also found in 30% of TNBC. GP88 is secreted in the serum of BC patients at an increased level compared to healthy subjects. GP88 represents an important therapeutic and diagnostic target in breast cancer. We have developed a recombinant neutralizing anti-human progranulin monoclonal antibody AG01 that inhibits its biological effect in vitro and in vivo and has fulfilled IND-enabling activities including toxicology study in non-human primates. Since GP88 is associated with poor outcome in BC patients, we investigated AG01 effect on the proliferation, survival, migration and invasion of TNBC MDA-MB-231 cells that overexpress PGRN/GP88.. AG01 reduced cell proliferation, migration and invasion significantly in a dose and time-dependent fashion. Western blot analysis showed evidence of regulatory changes upon AG01 treatment such as decreased expression of Ki67, p-Src, p-AKT, p-FAK and p-MAPK in these cells. In addition, AG01 treatment significantly reduced MDA-MB-231 cell migration and cell invasion in transwell assays. These data will be presented here along with in vivo tumor inhibition study. PGRN/GP88 represents a potential therapeutic with companion diagnostic target to provide novel solutions in TNBC and address the issue of toxicity, non-specific therapy and unresponsiveness often associated with SOC. We plan to further our in vivo studies based on on-going anti-PGRN pharmacokinetic study in athymic nude mice to help in the selection of in vivo efficacious AG01 dose in upcoming human phase 1 clinical study.

#1935

Accessing the cancer DUBome with UbiPlex: A bespoke drug discovery platform for deubiquitinase enzymes.

Gerald Gavory, Colin O'Dowd, Oliver Barker, Christina Bell, Frank Burkamp, Stephanie Burton, Eamon Cassidy, Joana Costa, Anthony Dossang, Matt Helm, Ashling Henderson, Peter Hewitt, Caroline Hughes, Mary McFarland, Hugues Miel, Natalie Page, Lauren Proctor, Shane Rountree, Ewelina Rozycka, Steven Shepherd, Adam Treder, Mark Wappett, Steven Whitehead, Tim Harrison. _Almac Discovery, Belfast, United Kingdom_.

Over the past decade, protein ubiquitination has emerged as an important post-translational modification with regulatory functions in all important cellular processes. Deubiquitinating enzymes (DUBs) including ubiquitin specific proteases (USPs) are cysteine proteases that catalyse the de-ubiquitination of protein substrates.

As a result of their increasing implications in the etiology of numerous pathological conditions including cancer and immuno-oncology, DUBs have emerged as an attractive and promising target class for the development of first-in-class medicines with high therapeutic impact. However, despite 15 years of intense research DUBs have proven largely refractory to drug discovery efforts.

Herein, we further describe the application of UbiPlex™, our purpose-built drug discovery platform for the identification and development of DUB inhibitors. In particular, we will highlight the versatility and robustness of UbiPlex™ by reporting the outcome of our focussed library screening campaign on multiple DUBs in parallel and by describing the de novo hit ID, orthogonal validation and hit optimization activities on two USPs of relevance to cancer.

Multiple series of novel, highly potent (e.g. IC50 < 20 nM) and non-covalent inhibitors have been developed. Excellent selectivity profiles against a large panel of DUBs and other non-related enzymes (e.g. proteases) will be described. Further profiling indicated that these inhibitors are cell-permeable and exhibit potent target engagement in cells (e.g. EC50 < 30 nM). Finally, we will describe our progress towards the development of lead molecules with drug-like properties with the aim of rapidly establishing proof-of-concept studies in vivo.

In summary, this work further exemplifies the broad tractability and druggability of the DUBome and reports the discovery and profiling of novel highly potent and selective inhibitors beyond USP7. These molecules may provide opportunities for the development of new therapeutics for cancer and associated disorders.

#1936

The central role of NFAT signalling in the mechanism of action of the TRPV6 oncochannel inhibitor and clinical candidate SOR-C13.

Christopher Rice,1 Tyler Lutes,2 Michelle Davey,1 Vett Lloyd,2 Tyson J. MacCormack,2 John M. Stewart,1 Dominique Dugourd1. 1 _Soricimed Biopharma Inc, Moncton, New Brunswick, Canada;_ 2 _Mount Allison University, Sackville, New Brunswick, Canada_.

SOR-C13, a 13-mer peptide derived from soricidin, the paralytic peptide in saliva of the Northern Short-tailed shrew, has completed an open-label, all comers Phase I Clinical Trial for the treatment of epithelially-derived cancers. SOR-C13 is a first-in-class drug candidate for the treatment of solid tumors. SOR-C13 specifically targets and inhibits the TRPV6 calcium channel - a recognized oncochannel whose over-expression in a number of epithelial cancers (e.g. breast, ovarian, prostate) is associated with poor prognosis. An increase in TRPV6 facilitates calcium influx into the cell, which activates the calcium binding protein calmodulin. This complex in turn activates calcineurin that de-phosphorylates and activates Nuclear Factor of Activated T-cells (NFAT), a gene transcription factor involved in multiple oncogenic processes. To demonstrate the role of NFAT activation in the mechanism of action (MOA) of SOR-C13, breast cancer cells (T-47D) were transfected with a NFAT dual reporter plasmid, treated with the peptide, and NFAT activation monitored. Additionally, RT-qPCR TaqMan array profiling was performed on prostate (PC-3), breast (T-47D), ovarian (OVCAR-3 and SK-OV-3), and pancreatic (BxPC-3 and SU.86.86) cancer cell lines treated with SOR-C13 compared with untreated cells. The TaqMan array panel consisted of 187 genes involved in cancer calcium signalling associated with the MOA of TRPV6 and directly, or indirectly, involved in NFAT signalling. SOR-C13 treatment significantly inhibited NFAT activation (p < 0.05) in T-47D cells transfected with a NFAT reporter plasmid. Molecular profiling of the 6 cancer cell lines showed that many genes involved in NFAT signalling were modulated by SOR-C13 treatment. Affected genes included genes involved in cell proliferation (e.g. TGF-beta, TLR9), metastasis (e.g. ADAM12, CEACAM6), resistance to apoptosis (e.g. WISP1) and angiogenesis (e.g. FLT4). Cytokines (e.g. IL-6) and chemokines (e.g. CXCL12), involved in cancer progression and associated with NFAT signalling, as well as transcription factors (e.g. GATA4, NF-kB) were down regulated upon SOR-C13 treatment. These results highlight NFAT signalling in the mechanism of action of SOR-C13. The impact of SOR-C13 on the expression of genes involved in resistance to apoptosis, proliferation, metastasis and angiogenesis as well as on the expression of immune cytokines and transcription factors in multiple cancer cells, makes SOR-C13 an attractive, novel anti-cancer drug.

#1937

Inhibition of a novel cancer target, PDE10, suppresses Wnt/β-catenin signaling and colon tumorigenesis: Benefits from combining with ornithine decarboxylase inhibitors.

Gary A. Piazza,1 Ashleigh Neese,1 Kevin Lee,1 Adam Keeton,1 Yulia Maxuitenko,1 Veronica Ramirez Alcantara,1 Kristy Berry,1 Jacob Valiyaveettil,1 Antonio Ward,1 Luciana Madeira da Silva,1 Jennifer Scalici,1 Bing Zhu,1 Tyler Mattox,1 Xi Chen,1 Margie Clapper,2 Harry Cooper,2 Wen-Chi Chang2. 1 _Univ. of South Alabama Mitchell Cancer Inst., Mobile, AL;_ 2 _Fox Chase Cancer Center, Philadelphia, PA_.

Sulindac has been shown in clinical trials to reduce the number and size of precancerous colon adenomas, but is not recommended for the long-term management of polyposis syndromes because of potentially fatal toxicities resulting from cyclooxygenase (COX) inhibition and the suppression of prostaglandin synthesis. Here we characterize the activity of a non-COX inhibitory sulindac derivative, MCI-030, that inhibits a novel cancer target, phosphodiesterase 10A (PDE10), recently reported to be overexpressed in colon tumors and essential for tumor cell growth (Oncogene 34:1499-509, 2015). PDE10 is an attractive cancer target because of limited expression and no known function in normal peripheral tissues. MCI-030 potently and selectively inhibits the growth of multiple colon tumor cell lines expressing PDE10 with IC50 values of 0.3 μM, but does not affect the growth of normal colonocytes (NCM460) that do not express PDE10. Sulindac inhibits colon tumor cell growth with IC50 values approximately 200 fold higher and modest tumor cell selectivity. MCI-030 is PDE10 selective, but sulindac inhibits multiple PDE isozymes (PDE2, 3, 5, and 10). MCI-030 treatment of colon tumor cells increased intracellular cGMP levels and activated protein kinase G at concentrations that inhibit recombinant PDE10 and tumor cell growth. Inhibition of PDE10 by MCI-030 or siRNA increased the phosphorylation and degradation of β-catenin and inhibited Wnt-induced nuclear translocation to suppress TCF-mediated transcription of cyclin D and survivin, leading to cell cycle arrest and apoptosis. With attractive pharmacokinetics and ability to achieve high concentrations in colon mucosa following oral administration, MCI-030 was tested in the APC+/min-FCCC mouse model of colon tumorigenesis. At 7-8 weeks of age, mice endoscopically determined to be adenoma-free were assigned to receive either control or MCI-030 supplemented diet (1500ppm) for 14 weeks. MCI-030 reduced the multiplicity of colonic adenomas by 50% (control: 3.95 ± 0.81 vs. MCI-030: 1.95 ± 0.58, P < 0.05) and incidence by 36.7%. MCI-030 also reduced the multiplicity of polypoid adenomas (36%), microadenomas (69%), and flat adenomas (100%, P < 0.05). These data indicate that MCI-030 strongly inhibits tumorigenesis in a predictive preclinical mouse model of colorectal cancer without discernable toxicity and could be effective together with colonoscopy in humans to reduce polyp formation and prevent malignant progression. In addition, in vitro colony formation and transformation assays using human colon tumor cells showed benefits of combining MCI-030 with the ornithine decarboxylase inhibitor, DFMO, supporting testing of this novel combination in the APC+/min-FCCC mouse model as a strategy to further improve efficacy. Funding from NCI R01CA131378, R01CA148817, R01CA197147, and R01CA155638.

#1938

Store-operated calcium signaling is an effective therapeutic target in AML.

Bridget Carmichael,1 Amy Lehman,2 Ola A. Elgamal,1 Shelley J. Orwick,1 Jean Truxall,1 Larry Beaver,1 Kumar V. Penmetsa,3 Srikant Viswanadha,4 John C. Byrd,5 Erin Hertlein1. 1 _The Ohio State University, Department of Internal Medicine, Division of Hematology, Columbus, OH;_ 2 _The Ohio State University, Center for Biostatistics, Columbus, OH;_ 3 _Rhizen Pharmaceuticals SA, La Chaux-de-Fonds, Switzerland;_ 4 _Incozen Therapeutics Pvt. Ltd, Hyderabad, India;_ 5 _The Ohio State University, Department of Internal Medicine, Division of Hematology, and Division of Pharmaceutics, College of Pharmacy, Columbus, OH_.

Acute myeloid leukemia (AML) is the most prevalent adult leukemia characterized by genetic or mutational disruption of myeloid differentiation, growth arrest, and apoptosis. Standard "7 + 3" chemotherapy consisting of cytarabine in combination with an anthracycline such as daunorubicin has been used for more than four decades. However this course only results in 10 year disease free survival in 15% of patients age <60 years and 2% among those > 60 years. Several new successful therapies in AML are shown to work by promoting differentiation, and/or inducing apoptosis of the terminally differentiated cells. However these therapies, while promising, have the limitation of working in only a subset of AML with specific genetic profiles. The discovery of novel agents applicable to a broader patient population still represents a critical unmet need in this disease. Intracellular calcium is a common signaling molecule used in a variety of cellular processes including cell cycle progression. One of the major pathways which regulates calcium entry is store operated calcium entry (SOCE) mediated through calcium release-activated Ca2+ channels (CRAC), which respond to depleted calcium stores in ER lumen. This primarily occurs via interaction of ORAI1 (expressed on the plasma membrane) and STIM1 (expressed on the endoplasmic reticulum). Leukemic cells in particular often rely on the influx of calcium through CRAC for proliferation, therefore targeting these channels are a promising therapeutic option. In the current study, we explore the preclinical use of RP4010, a novel inhibitor of ORAI1 developed by Rhizen Pharmaceuticals, in AML. RP4010 is very effective at inhibiting proliferation of both AML cell lines and primary AML patient samples (IC50 values ranging from 0.2 - 1 μM using a tetrazolium-based colorimetric assay (MTS) assay). Importantly, RP4010 efficacy is not altered in the presence of stromal cell support. RP4010 also decreased colony growth in CFU assays. Finally, we evaluated the in vivo efficacy using an MV4-11 xenograft model. RP4010 treatment at 50 mg/kg once daily by oral gavage significantly prolonged survival compared to the vehicle control (p = 0.019). Furthermore, at the time of sacrifice, splenic tumor burden (percentage of human CD45 positive cells) was reduced in the mice treated with RP4010 compared to the vehicle group (p = 0.019). In conclusion, we provide here compelling evidence that inhibition of ORAI1 with RP4010 is a promising therapeutic strategy in AML. A Phase 1/1b study in R/R Non-Hodgkin Lymphoma (NHL) patients is currently underway in the US (ClinicalTrials.gov Identifier: NCT03119467), and our data would support expansion to other hematological malignancies as well, particularly AML. Senior authors J. C. B and E. H contributed equally to this work.

#1939

BR101801, a first-in-class dual target inhibitor of DNA protein kinase (DNA-PK) and phosphoinositide 3-kinase delta (PI3Kδ), triggers antitumor immunity.

Byeongwook Jeon, Jin Sang Wang, Baek kyung Kim, Bo Ram Lee, Mi kwon Son, Yeon seo Choi, Nam-Hoon Kim, Jayhyuk Myung, Dal-Hyun Kim. _Boryung Pharmaceutical, Central Research Institute, Republic of Korea_.

Introduction: Regulatory T cells (Tregs) and conventional T cells (Tconvs) are differentially regulated by PI3K isoforms. Tregs are dependent on the PI3Kδ isoform, whereas Tconvs are regulated by PI3Kα and PI3Kβ instead of PI3Kδ as a complementary pathway. Moreover, recent reports have indicated that DNA-PK inhibition reduced Tregs maintenance and function by blocking phosphorylation of Nr4a. Idelalisib, which is an approved PI3Kδ inhibitor, was shown to reduce the number of suppressive Tregs in the peripheral blood of chronic lymphocytic leukemia patients in duration- and patient age-dependent manners. As BR101801 is a first-in-class dual target inhibitor of DNA-PK and PI3Kδ, the efficacy of BR101801 as a candidate for cancer immunotherapy is presented in this study.

Methods: BALB/c mice were subcutaneously inoculated into the abdomen on day 0 with 5x104 4T1 cells. BR101801 (50 mg/kg, once daily) or vehicle was administered by oral gavage from day -1. On day 30, mice were sacrificed, after which tumor tissue and spleen were extracted for ex vivo analysis. Human CD3+ lymphocytes were isolated from PBMCs using positive selection on magnetic beads. CD3+ lymphocytes stimulation was achieved with an anti-CD3/CD28 T cell activator. FACS analysis was performed using antibodies (CD4, CD25, Foxp3, PD-1, CTLA-4).

Results: Administration of BR101801 led to significant suppression of 4T1 tumor growth (TGI : 50.48%) based on a similar pattern as the positive control PI3Kδ inhibitor (PI-3065). Interestingly, 4T1 cells did not express PI3Kδ at a detectable level. Indeed, BR101801 did not affect the growth of 4T1 cells up to 10 μM in vitro. These results mean that BR101801 induces immune-mediated tumor regression without a direct cytotoxic effect on 4T1 cells. Ex vivo analysis of the spleen showed that the population of Tregs decreased while that of CD8 lymphocytes increased upon BR101801 treatment, reciprocally. Immuno-oncological activity was confirmed in human PBMCs. BR101801 did not influence the viability of CD3+ lymphocytes and CD4+ lymphocytes but specifically decreased the population of Tregs. In addition, PD-1 and CTLA-4 expression in Tregs was reduced in a dose-dependent manner, and the results were better than those obtained using idelalisib and TGR-1202. Furthermore, the remaining effect of BR101801 was confirmed to be effective even up to 24 hours after drug removal.

Conclusions: BR101801 has immunogenic potential to alter the balance of immune tolerance to anti-tumor immunity by encouraging antigen exposure, disrupting Tregs, and boosting CD8 cells. Therefore, this study suggests the potential of BR101801 to be used as a first chemical immune checkpoint inhibitor.

#1940

Thymine DNA glycosylase (TDG) as a novel target for melanoma.

Rossella Tricarico,1 Pietro Mancuso,1 Vikram Bhattacharjee,1 Laura Cosentino,1 Emmanuelle Nicolas,1 Margret Einarson,1 Neil Beeharry,1 Karthik Devarajan,1 Rich Katz,1 Dorjbal G. Dorjsuren,2 Anton Simeonov,2 Yuwaraj Kadariya,1 Guillaume Davidson,3 Joseph R. Testa,1 Irwin Davidson,3 Lionel Larue,4 Robert W. Sobol,5 Timothy Yen,1 Alfonso Bellacosa1. 1 _Fox Chase Cancer Ctr., Philadelphia, PA;_ 2 _National Center for Advancing Translational Sciences, NIH, Rockville, MD;_ 3 _CNRS/INSERM/ULP, Illkirch, France;_ 4 _Institut Curie Centre de Recherche, Orsay, France;_ 5 _Mitchell Cancer Institute, University of South Alabama, Mobile, AL_.

Melanoma is an aggressive neoplasm with increasing incidence that bears the infamous distinction of being a recalcitrant cancer, i.e. a cancer with poor prognosis, lacking progress in diagnosis and treatment. In addition to conventional therapy, melanoma treatment is currently based on targeting the BRAF/MEK/ERK signal transduction pathway and immune checkpoints; however, advanced therapeutic approaches based on novel targets are urgently needed. We reasoned that the base excision repair enzyme Thymine DNA Glycosylase (TDG) could be such a target for its dual role in safeguarding genome stability and in effecting active DNA demethylation downstream the Ten-Eleven Translocation (TET) dioxygenases. TDG knockdown in melanoma cell lines causes cell cycle arrest, senescence and death by mitotic alterations, and impairs xenograft formation. Importantly, untransformed melanocytes are not affected by TDG knockdown, and adult mice with conditional knockout of TDG are viable. Candidate TDG inhibitors, identified through a high-throughput screen, reduced viability and clonogenic capacity of melanoma cell lines. Candidate TDG inhibitors increased cellular levels of 5-carboxylcytosine, the last intermediate in DNA demethylation, which is specifically removed by TDG, indicating successful targeting. These findings suggest that TDG may provide critical functions in cancer cells, but not in normal cells, that make it a highly suitable anti-melanoma drug target. By potentially disrupting both DNA repair and the epigenetic state, targeting TDG may represent a completely new approach to melanoma therapy.

#1941

Constitutive IRAK4 activation contributes to NF-κB activity and chemoresistance in colorectal cancer.

Qiong Li,1 Lin Li,2 Daoxiang Zhang,2 Hongmei Jiang,2 Andrea Wang-Gillam,2 Marianna B. Ruzinova,2 Kian-Huat Lim2. 1 _Renji Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai, China;_ 2 _Washington University School of Medicine, St. Louis, MO_.

Aberrant activation of the NF-κB transcription factors is a major molecular mechanism that contributes to the aggressive behavior and treatment resistance of colorectal cancer (CRC). Understanding how these factors are activated will provide novel therapeutic opportunities to improve treatment response and outcome of CRC patients. In the present study, we showed that the Interleukin-1 Receptor-Associated Kinases 1 and 4 (IRAK1 and IRAK4) are constitutively activated by phosphorylation in a majority of CRC cell lines and patients samples. Notably, we observe significant positive correlation between phospho-IRAK4 and phospho-NF-kb/p65 staining by immunohistochemistry in patient CRC samples. Moreover, we showed that pharmacologic IRAK4 inhibitors dose-dependently suppressed phosphorylated -IRAK1, -IRAK4 and -IKKa/b by western blots, as well as significantly reducing NF-κB-driven luciferase activity in CRC cells. Furthermore, IRAK4 inhibitor dose dependently inhibit colony formation and anchorage-independent (AI) growth of CRC cells in soft agar. In three CRC cell lines, we observed various degrees of synergism between IRAK4 inhibitors and each chemotherapeutic agents including 5-FU, oxaliplatin and irinotecan, mainly through induction of apoptosis. Lastly, we showed that IRAK4 inhibitor augments the therapeutic effect of 5-FU and oxaliplatin in nude mice bearing CRC xenografts. Together, our data established IRAK4 kinase inhibitors as a promising novel class of targeted agent in CRC.

#1942

Synthetic lethal interaction between SETD2 loss and inhibition of PI3Kβ in clear cell renal cell carcinoma (ccRCC).

Esteban Terzo,1 Yun C. Chiang,2 Anna Chytil,1 Valerie M. Jansen,1 Kimryn Rathmell1. 1 _Vanderbilt University Medical Center, Nashville, TN;_ 2 _University of North Carolina, Chapel Hill, NC_.

Renal cell carcinoma (RCC) is a high-risk and high-mortality cancer for which several new targeted therapies exist. However, responses of primary clear cell RCC (ccRCC) tumors and metastases to these targeted agents are variable, and complete responses are relatively uncommon underscoring the need to identify reliable biomarkers to predict tumor behavior. Bi-allelic inactivation of the tumor suppressor gene SETD2, encoding a lysine methyltransferase, occur in approximately 20% of ccRCC tumors while the SETD2 protein deficiency rate has been reported to be as high as 34%. The phosphatidylinositide 3-kinases (PI3Ks) have emerged as important therapeutic targets for the treatment of solid and hematological malignancies, including ccRCC. Here, we show that genetic and pharmacologic inhibition of the PI3Kβ isoform results in inhibition of SETD2 deficient (-/-) ccRCC-derived 786-O and A498 cells, which exhibit increased sensitivity to PI3Kβ-specific inhibitors TGX221 (IC50 = 2.3 - 4.5 µM, respectively) and AZD8186 (IC50 = 1.79 - 0.24 µM, respectively) as compared to SETD2 proficient (+/+) 786-O cells (IC50 = >10 µM). Inhibition of the p110δ isoform alone had a modest effect on ccRCC cell growth and migration. Importantly, genetic (with siRNA) and pharmacologic (with BYL719) inhibition of p110α had no effect on the ccRCC cell lines. Inhibition of AKT with the small molecule inhibitor MK2206 recapitulated the effects seen with AZD8186 in ccRCC cell lines with SETD2 loss, suggesting PI3Kβ mediates the synthetic lethality via AKT signaling in the context of SETD2 loss in ccRCC. Our work strongly suggests a synthetic lethal-type of interaction between PI3Kβ inhibition and SETD2 loss and offers a rationale for further translational and clinical investigation of small molecule inhibitors of PI3Kβ in RCC. Xenograft studies are ongoing to investigate the in vivo efficacy of AZD8186.

#1943

PARG inhibitors exhibit synthetic lethality with XRCC1 deficiency and a cellular mechanism of action that is distinct from PARP inhibition.

Leenus Martin,1 Tzuling Cheng,1 Dominic I. James,2 Habiba Begum,2 Kate M. Smith,2 Allan Jordan,2 Ian Waddell,2 Kedar Vaidya,1 Marcus Fischer,1 Bing Yao,1 Jason Drummond,1 Leah Cleary,1 Ruben Martinez,1 James Sutton,1 Nandini Ravindran,1 James Joseph,1 Eleni Venetsanakos,1 Michael Dillon,1 Jeffrey H. Hager,1 Lisa D. Belmont1. 1 _Ideaya Biosciences, Inc., South San Francisco, CA;_ 2 _Cancer Research UK Manchester Institute, Manchester, United Kingdom_.

Poly(ADP-ribose) glycohydrolase (PARG) hydrolyzes poly(ADP-ribose) (PAR) chains that are polymerized by PARP enzymes, completing the PAR cycle. Small molecule inhibitors of PARG result in a dose dependent increase in cellular PAR after DNA damage. Here we demonstrate that depletion of XRCC1, a scaffolding protein with an essential role in base-excision repair (BER), sensitizes cancer cells to PARG inhibition. XRCC1 deficient cells exhibit increased nuclear PAR foci in response to PARG inhibition even in the absence of DNA damaging agents. Inhibition of PARP1 with RNAi or small molecule inhibitors rescues cell growth inhibition and reduces the amount of cellular PAR accumulation in PARG inhibitor treated cells. This indicates that the cellular growth inhibition is dependent upon cellular PAR levels, demonstrating selectivity of the small molecule inhibitors for PARG. We hypothesized that inhibition of PAR hydrolysis could result in depletion of cellular NAD as this could prevent recycling of PAR to NAD. Consistent with this hypothesis, PARG inhibition enhanced NAD depletion after treatment of cells with the DNA damaging agent methyl methanesulfonate (MMS). Live cell imaging of XRCC1 depleted cells treated with a PARG inhibitor revealed that cells have large membrane protrusions, similar to the morphology of cells that have been treated with a NAMPT inhibitor, which results in depletion of cellular NAD. Furthermore, addition of the NAD precursor, nicotinamide mononucleotide (NMN) rescued proliferation of PARG inhibited cells. Taken together, these data support a hypothesis in which PARG inhibitors are cytotoxic to sensitive cancer cells via depletion of NAD, ultimately starving the cell of ATP. Thus, PARG inhibition is a novel strategy for exploiting synthetic lethality in cancer cells. The defects that sensitize cancer cells to PARG inhibition are distinct from those that sensitize to PARP inhibitors, namely defects in homology directed repair. Approximately 15% of breast cancer samples exhibit low or no XRCC1 by IHC. A subset (approximately 35%) of the XRCC1 low patient samples also have defects in BRCA1, suggesting that the majority of XRCC1 low tumors may not be responsive to PARP inhibitors. Small molecule PARG inhibitors are currently being evaluated for efficacy in XRCC1 low xenograft models.

#1944

High-throughput fluorescence-based assay for screening of Arginase I inhibitors for cancer immunotherapy.

Yvonne Grobben,1 Joost C. Uitdehaag,1 Nicole Willemsen-Seegers,1 Werner W. Tabak,1 Martine B. Prinsen,1 Suzanne J. van Gerwen,1 Jan van Groningen,2 Johan Friesen,2 Helma Rutjes,2 Jos de Man,1 Rogier C. Buijsman,1 Guido J. Zaman1. 1 _Netherlands Translational Research Center B.V., Oss, Netherlands;_ 2 _Pivot Park Screening Centre, Oss, Netherlands_.

Arginase I (ArgI) is an important small molecule drug target in cancer immunotherapy. Arg1 converts L-arginine into L-ornithine and urea. Recruitment of ArgI-expressing myeloid-derived suppressor cells (MSDCs) at a tumor site results in the depletion of L-arginine, which causes reduced proliferation of T-cells and natural killer cells and inhibition of the antitumor immune response. In patient material, MSDC-induced T-cell suppression can be reverted by arginase inhibitors. ArgI inhibitors work synergistically with checkpoint inhibitor therapy in syngeneic mouse models.

In order to find novel inhibitors for ArgI, we developed a set of tools to enable screening and hit validation. The first is an activity assay that enables the high-throughput screening (HTS) of compound libraries. Previously reported arginase assays are poorly compatible with HTS due to the requirement of multiple reactions steps, harsh assay conditions, or the use of low-turnover substrates other than L-arginine.

We developed a novel ArgI activity assay, which has a homogenous format and requires only two addition steps before readout. The assay makes use of a fluorescence readout and has a high robustness (Z'-factor > 0.7). Progression of the assay can be followed in real time, allowing for kinetic experiments. To investigate the false positive hit rate, the assay was screened at the Pivot Park Screening Centre with a library consisting of 233 compounds with known interference in other assay formats. Using a simple background signal control, the number of false positives in this library was minimized to below 0.2%. The assay was subsequently used to accurately determine the dissociation constants and binding kinetics of the reference inhibitors ABH, NOHA and CB1158.

In order to validate the binding of screening hits to ArgI, we developed a thermal shift assay. We observed a shift in the ArgI melting temperature of up to 3.8°C after addition of the most potent inhibitors. In addition, we developed a Surface Plasmon Resonance binding assay. This showed that ABH and CB1158 have long residence times on ArgI.

To allow the development of novel inhibitors through rational drug design, we successfully crystallized human ArgI in a novel space group with higher symmetry (P63) compared to those previously reported (space group P3), and without the presence of hemihedral twinning. We determined a series of high resolution (< 1.7 Å) crystal structures at various pH values and with several ligands. These demonstrate that a series of peptide flips lies at the basis of the pH-dependent symmetry of ArgI and its unusually high pH optimum of 9.0 to 9.5.

Finally, to assess the cellular activity of ArgI inhibitors, we examined 102 cancer cell lines for ArgI activity and correlated the results to public gene expression profiles. The novel assay portfolio will help to deliver a new generation of ArgI inhibitors.

#1945

Degradation of ERG transcription factor as a targeted therapy in B-cell and T-cell acute lymphoblastic leukemia.

Xiaoju Wang, Caleb Cheng, Yuanyuan Qiao, Lanbo Xiao, Cynthia Wang, Abhijit Parolia, Arul Chinnaiyan. _The University of Michigan, Ann Arbor, MI_.

Acute lymphoblastic leukemia (ALL) occurs in children and adults and is one of the most common forms of pediatric cancer. The ideal individualized therapy for B- and T-cell ALL heavily depends on the present molecular drivers. However, even with the success of ALL therapy, only a few strategies target disease-specific molecular lesions. Philadelphia chromosome positive (Ph+) ALL, which harbors the BCR:ABL1 rearrangement, are sensitive to tailored inhibitors. Unfortunately, most ALL cases in both children (97-98%) and adults (70-80%) are Ph-; therefore, agents designed to directly target other molecular lesions are urgently needed. ERG, an ETS-family transcription factor, plays a key role in hematopoietic differentiation, megakaryopoiesis, and megakaryoblastic leukemia associated with Down syndrome. ERG overexpression is predictive of a poor prognosis in acute myeloid leukemia and T-ALL. Transgenic expression of ERG causes T-ALL in mice and knockdown of ERG reduces human T-ALL cell proliferation. We have previously identified a consensus peptide (EIPs) that specifically binds to and leads to the proteosomal degradation of the ERG protein in prostate cancers having the TMPRSS2:ERG gene fusion. Here, we demonstrated that wild type ERG was overexpressed at the mRNA and protein levels in a subset of B- and T-cell ALLs, but not in precursor CD4+, CD8+, or CD19+ cells. Pull-down assay found that streptavidin-linked beads were enriched for ERG proteins. Treatment of EIPs induced proteolytic degradation of ERG protein in Hal-01 cells, and therefore selectively inhibited ERG-driven cell growth and proliferation. More importantly, structure-based optimization created the next generation of ERG degraders (EIPv2). EIPv2 treatment significantly decreased ERG protein in Hal-01 cells starting from 3 hrs and nearly depleted ERG proteins at 48 hrs, resulting in 10-fold more potency than EIPs (IC50=2.3µM vs 25.7µM for EIPv2 and EIPs, respectively). Together, our results validate the development of peptidomimetics for ERG depletion as a targeted therapy for ALL. Unlike small-molecule antagonists, which require continuous dosing that eventually induces drug resistance, most protein degraders have relatively higher and longer pharmacological effects without requiring continuous exposure to high doses. Such an approach can abolish the presence of an oncoprotein, which, in theory, can overcome resistance to target inhibitors.

#1946

SJP1604, a novel nucleolin-targeted anti-cancer drug for acute myeloid leukemia using aptamer-drug conjugation technology.

Jihyun Um,1 Dohyeong Lee,1 Yongbin Park,1 Sung Hwan Moon,2 Su Jin Lee,2 Min-Hyo Ki,1 Hee Jong Shin,1 Eui Hwan Jo1. 1 _Samjin Pharm. Co., Ltd., Seongnam, Republic of Korea;_ 2 _Aptabio Therapeutics Inc., Seongnam, Republic of Korea_.

Acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) are cancers characterized by the rapid growth of abnormal white blood cells. Generally, cytarabine (Ara-C), azacitidine, and decitabine are treated as the standard first-line chemotherapy for AML/MDS. Although the therapy leads to high rates of remission, approximately 30% of the treated patients are refractory and more than 50% of them face a relapse because of the occurrence of drug resistance and high toxicity. Therefore, there is a strong unmet need for the next-generation targeted therapy to overcome drug resistance and reduce toxicity in AML/MDS.

Aptamers mostly consist of oligonucleotides, which can specifically bind to their target molecules with high affinity. Here, we designed a synthetic DNA aptamer-nucleoside drug conjugate (SJP1604) specific for nucleolin, which is highly expressed only on the cell membrane of cancer cells. In particular, AML and ALL (acute lymphoblastic leukemia) cells are known to express the highest mRNA level of nucleolin among various cancer cells, relatively.

Owing to the unique conformational property of SJP1604, it can be delivered into cancer cells with its high targeting ability and plasma stability. In this study, we verified that SJP1604 selectively targeted cancer cells by nucleolin-binding on the cancer cell membrane in the drug uptake assay. SJP1604 also exhibited a long-lasting plasma stability in 50% human plasma up to 48 hours in sharp contrast to unstability of general aptamers.

Furthermore, SJP1604 inhibited not only the growth of human AML/MDS cell lines (MV-4-11, HL-60, MOLM-13, THP-1 and KG-1) but also drug-resistant cell lines (cytarabine-, azacitidine- and decitabine-resistant MOLM-13, cytarabine-resistant HL60 and cytarabine-resistant MV-4-11). Interestingly, SJP1604 showed the significantly reduced IC50 values with the decreased expression level of nucleolin in cytarabine-, azacitidine- and decitabine-resistant MOLM-13 while cytarabine caused no effect on the cell growth and the expression of nucleolin of the same resistant cell lines. Intravenous administration of SJP1604 (150 mg/kg) led to tumor regression and improved survival rate in MOLM-13 xenograft mouse model.

In conclusion, these findings suggest that SJP1604 could be developed as a first-in-class drug for novel targeted therapy of AML/MDS with less drug toxicity as well as an orphan drug for overcoming drug resistance of AML/MDS, using aptamer-drug conjugation technology.

#1947

Rovalpituzumab tesirine as a therapeutic agent for neuroendocrine prostate cancer.

Loredana Puca,1 Verena Sailor,1 Katie Gavyert,1 Etienne Dardenne,1 Kumiko Isse,2 Michael Sigouros,1 David M. Nanus,1 Scott T. Tagawa,1 Juan Miguel Mosquera,1 Laura Saunders,2 Himisha Beltran1. 1 _Weill Cornell Medical College, New York, NY;_ 2 _AbbVie Stemcentrx LLC, San Franciscisco, CA_.

Background: Delta-like protein 3 (DLL3) is expressed on the surface of small cell lung cancer (SCLC) tumor-initiating cells, and the DLL3 targeted antibody-drug conjugate, Rovalpituzumab tesirine (Rova-T™; SC16LD6.5), has shown promise for patients with SCLC. Neuroendocrine prostate cancer (NEPC) is an emerging late stage subtype of castration resistant prostate cancer with limited therapeutic options. Based on clinical and molecular similarities with SCLC, we investigated expression of DLL3 and the use of Rovalpituzumab tesirine in NEPC xenografts.

Methods: We evaluated mRNA and/or protein expression of DLL3 in a cohort of 361 patients (552 samples) ranging from benign prostate (BEN), localized prostate adenocarcinoma (PCA), castration resistant adenocarcinoma (CRPC), and castration resistant NEPC and correlated with pathologic and genomic features. mRNA was assessed by RNAseq. Protein was assessed by immunohistochemistry (DLL3 SP347 antibody). Prostate cancer cell lines were engrafted in mice and treated with SC16LD6.5 in vivo.

Results: DLL3 was expressed at the mRNA and/or protein level in 0/132 Benign (0%), 1/254 (0.4%) PCA, 10/90 (11.1%) CRPC and 47/76 (61.8%) NEPC samples. DLL3 IHC was of higher intensity in NEPC and co-localized with classical neuroendocrine (NE) markers. DLL3 was amongst the most differentially expressed genes by RNA-seq in NEPC versus CRPC (p=<0.0001, fold change=71), and positively correlated with ASCL1 expression (r= 0.88) and RB1 genomic loss (83%), and inversely with AR expression. siRNA knockdown of DLL3 did not alter AR signaling or NE score. In vivo treatment of the NEPC, NCI-H660, xenografts with vehicle, IgG1LD6.5, or SC16LD6.5 (0.3mg/Kg, intraperitoneal single dose) showed complete responses to SC16LD6.5 with no recurring tumors after 100 days; while the tumor developed from the CRPC line, DU145, which do not express DLL3, continued growing despite the treatment and were sacrified when tumors reached the maximum size allowed by our IACUC protocol.

Conclusions: DLL3 is expressed on the surface of the majority of NEPC cases evaluated and is not expressed in primary prostate cancer or benign tissues. Modulation of DLL3 expression does not appear to affect AR or NEPC signaling in cell lines. SC16LD6.5 (Rova-T) demonstrates preferential preclinical activity in NEPC that express DLL3 compared to CRPC-adenocarcinoma that are DLL3 negative, in vivo. A Phase 1 Basket trial investigating Rova-T is now open with a dedicated NEPC arm (NCT02709889).

#1948

Polo-like kinase 4 inhibition produces polyploidy and apoptotic death of lung cancers.

Masanori Kawakami,1 Lisa Maria Mustachio,1 Lin Zheng,1 Yulong Chen,1 Jaime Rodriguez-Canales,1 Barbara Mino,1 Jonathan M. Kurie,1 Jason Roszik,1 Pamela Andrea Villalobos,1 Kelsie L. Thu,2 David W. Cescon,2 Jennifer Silvester,2 Ignacio Wistuba,1 Tak W. Mak,2 Xi Liu,1 Ethan Dmitrovsky1. 1 _The University of Texas MD Anderson Cancer Center, Houston, TX;_ 2 _Princess Margaret Cancer Centre, Ontario, Canada_.

Despite current treatments, lung cancers remain a major public health problem. Innovative ways are needed to treat or prevent these cancers. Centrosomes are critical for fidelity of mitosis. Abnormal centrosome numbers can cause aberrant mitosis and cell death. Polo-like kinase 4 (PLK4) is a serine/threonine kinase regulating centriole duplication and its deregulation alters centrosome number and mitosis. CFI-400945 is a highly selective PLK4 inhibitor that deregulates centriole duplication, causing mitotic defects and death of aneuploid cancers. Here, we explored CFI-400945 activity against lung cancer using in vitro and in vivo models. CFI-400945 caused polyploidy, growth inhibition and apoptotic death of murine and human lung cancer cells, despite expression of mutated KRAS or p53. Analysis of DNA content by propidium iodide (PI) staining revealed cells with > 4N DNA content (polyploidy) markedly increased after CFI-400945 treatment. Centrosome numbers and mitotic spindles were scored by individually staining treated and control cells with γ-tubulin, α-tubulin, and DAPI. CFI-400945 treatment produced supernumerary centrosomes and mitotic defects in the examined lung cancer cell lines. In vivo antineoplastic activity of CFI-400945 was established in mice with syngeneic lung cancer xenografts. Lung tumor growth was statistically significantly reduced at dosages that were well tolerated. Phospho-histone H3 staining of resected lung cancers following CFI-400945 treatment confirmed the presence of aberrant mitosis. PLK4 expression profiles in human lung cancers were explored using The Cancer Genome Atlas (TCGA) and RNA in situ hybridization (RNA ISH) of microarrays containing normal and malignant lung tissues. PLK4 expression was significantly higher in the malignant versus normal lung and conferred an unfavorable survival (P < 0.05) indicating the clinical relevance of PLK4 expression in lung cancer. Intriguingly, cyclin dependent kinase 2 (CDK2) antagonism (that confers multipolar anaphase catastrophe by inhibiting centrosome clustering) showed synergistic effects (CI < 1) on lung cancer cell growth when combined with CDI-400945. This is an appealing cooperating regimen since each agent increases multipolar cell division and cancer cell death. CFI-400945 is undergoing phase I clinical trial testing (NCT01954316). Taken together, targeting PLK4 for inhibition holds promise for lung cancer therapy either as a single agent or when combined with another agent that deregulates mitosis.

#1949

A compound screen reveals potential novel therapeutic targets for chordoma: Metabolic stress response and epigenetic control of brachyury.

Lucia Cottone,1 Edward Hookway,1 Graham Wells,2 Lorena Ligammari,1 Patrick Lombard,1 Ralph Mazitschek,3 Josh Sommer,4 Udo Oppermann,2 Adrienne M. Flanagan1. 1 _University College London, London, United Kingdom;_ 2 _University of Oxford, Oxford, United Kingdom;_ 3 _Massachusetts General Hospital, Boston, MA;_ 4 _Chordoma Foundation, Durham, NC_.

Chordoma is a rare bone cancer, showing notochordal differentiation, that develops in axial skeleton in adults and children. Patients have a median survival of 7 years and radical surgical resection is the main treatment for this morbid disease, which does not respond to cytotoxic chemotherapy. We have previously demonstrated that EGFR inhibitors represent the almost unique family of kinase inhibitors to exert an effect on chordoma cell lines proliferation. However not all cell lines respond to these agents and drug resistance is likely to occur. Genomic studies have revealed that chordomas do not harbor recurrent alterations in kinases whereas chromatin-remodelling genes are altered in at least 20% of cases. The transcription factor brachyury (T) is the diagnostic hallmark of chordoma and is strongly implicated in its pathogenesis. T is regulated during embryonic development at the epigenetic level, suggesting that epigenetic inhibitors may represent a novel therapeutic approach for this disease. In this study, we have undertaken a medium throughput focused compound screen (n=91) using validated small molecule inhibitors of enzymes involved in chromatin biology and metabolic pathways. The alamar blue assay was employed to assess cell viability. Screening revealed activity in a number of compounds targeting the jumonji domain-containing lysine demethylases, including GSK-J4 and KDOBA67, two structurally closely related compounds that mainly target KDM6A (aka UTX) and KMD6B (JMJD3). These compounds were effective in all five chordoma cell lines (UCH1, UCH2, MUG-Chor, UM-Chor, UCH7) tested. In contrast to EGFR inhibitors, these compounds induced downregulation of T at the transcriptional and protein level. Preliminary results suggest this is achieved via the induction of a metabolic stress response as well as through the epigenetic regulation of T, the latter being brought by increased levels of H3K27me3. We also found that Halofuginone, a highly specific inhibitor of the enzyme glutamyl-prolyl tRNA synthetase already tested in phase I autoimmunity clinical trials, induced a metabolic stress response, similar to KDM6 inhibitors, in all chordoma cell lines. Moreover, Halofuginone treatment of a chordoma PDX model demonstrated 44% tumor growth inhibition (p=0.0052). In conclusion, we have identified epigenetic and metabolic pathways that represent potential novel targets for the treatment of chordoma.

#1950

**Targeted silencing of Na** V **1.5 channel suppresses cell proliferation and invasion in ovarian cancer cells through inhibition of PI3K/AKT and Integrinβ-1/FAK/Src axis.**

Mumin Alper Erdogan, Bulent Ozpolat. _UT MD Anderson Cancer Center, Houston, TX_.

Ovarian cancer (OCa) is the leading cause of gynecological cancer related deaths. OCa is one of the most aggressive cancer and associated with poor prognosis and survival rates (5-year survival rates about 50%). OCa cells have highly invasive metastatic phenotype due to mutations, altered signaling pathways and deregulated of control mechanisms and patients with metastatic disease have poorer prognosis (5-year survival is 20%). The major reasons for patient death include significant intratumoral heterogeneity, early metastasis and development of resistance to currently used chemotherapeutics. Therefore, identification of novel molecular targets and therapeutics strategies are urgently needed to enhance the efficacy of current therapies and prolong patient survival. Ion channels are important signaling molecules expressed in a wide range of tissues where they have significant involvement in determining a variety of cellular functions: solute transport, volume control, enzyme activity, secretion, invasion, gene expression, excitation-contraction coupling and intercellular communication. Studies indicated that there are significant differences in the regulation and the function of ion channels between normal and cancer cells. Voltage gated sodium channels (VGSC) is a group of ion channels that has been correlated with OCa because of their higher expression in highly metastatic ovarian cancer cells. Importantly, VGSC activity contributes to many cellular behaviors integral to metastasis in breast and other cancers including OCA. In this study, we investigated the NaV1.5 as integral component of the metastatic process in human OCa. The aim of the current study was to reveal molecular mechanisms underlying the effects of NaV1.5 down-regulation and investigate the effects on OCa in vitro. In this study, the ovarian cancer cell lines (HEYA8, SKOV3-IP1, SKOV3-TR (taxol resistant)) were used. NaV1.5 and Control non-silencing small interfering RNAs (siRNA) were employed for therapy. As in vitro experiments, cell proliferation, colony formation, invasion, western blot analysis were performed. Our results showed that specific NaV1.5 siRNA treatments caused a significant reduction in cell proliferation, colony formation, and invasion capacity in OCa cells (p<0.0001). To reveal molecular mechanisms underlying the effects of NaV1.5 down-regulation, we evaluated signaling pathways regulating cell proliferation and invasion/metastasis by western blot analysis and found that NaV1.5 expression promotes expression of PI3K/AKT, Integrinβ1/FAK/Src and P70S6K. In conclusion, our results suggest that Nav1.5 channels may contribute OCa tumorigenesis and metastasis through the upregulation of oncogenic pathways and serve as a potential therapeutic target in OCa.

#1951

Role of mitochondrial peptide Humanin in the response of experimental breast cancer to chemotherapy.

Camila Zuccato,1 Mariela A. Moreno Ayala,1 Maria F. Gottardo,1 Matias Luis Pidre,2 Antonela S. Asad,1 Alejandro J. Nicola Candia,1 Victor Romanowski,2 Adriana Seilicovich,1 Marianela Candolfi1. 1 _University of Buenos Aires School of Medicine, Buenos Aires, Argentina;_ 2 _Facultad de Ciencias Exactas, Universidad Nacional de La Plata, La Plata, Argentina_.

Humanin (HN) is a mitochondrial-derived peptide with potent cytoprotective effect in many cell types. Administration of HN has been proposed as a therapeutic approach for several chronic diseases, such as diabetes, neurodegenerative conditions and cardiovascular diseases. Although HN has been shown to protect normal tissues against the toxic effects of chemotherapy, its role in tumor chemoresistance is poorly understood. Here, we aimed to elucidate the role of HN in the progression and cytotoxic response of experimental triple negative breast cancer (TNBC). We detected HN expression in murine and human TNBC cells, which was upregulated in the presence of chemotherapeutic drugs, i.e. Doxorubicin (DOXO) and Cisplatin, as assessed by flow cytometry. Addition of HN protected TNBC cells from various cytotoxic insults, such as serum deprivation, proapoptotic cytokine TNF-α or chemotherapy with DOXO. We next evaluated the role of endogenous HN in the apoptotic response of TNBC cells, using a plasmid encoding a short hairpin RNA to inhibit HN expression (pUC.shHN). To readily assess transduction efficiency, the plasmid also encodes the red fluorescent protein dTomato as a reporter gene. Transfection of TNBC 4T1 tumor cells with pUC.shHN increased the percentage of apoptotic cells when compared to cells transfected with control plasmid, as assessed by flow cytometry after staining with propidium iodide. When transfected 4T1 cells were incubated with different concentrations of DOXO, pUC.shHN reduced their clonogenic capacity. We also evaluated the effect of HN in the progression of experimental mouse 4T1 TNBC treated with DOXO. HN was readily detected in tumor samples and lung metastases. Systemic administration of HN reduced tumor apoptotic rate, accelerated tumor progression and increased the development of lung metastases. In addition, administration of HN impaired the antitumor and antimetastatic effect of chemotherapy. In summary, our findings suggest that HN inhibits the response of TNBC cells to cytotoxic stimuli, facilitating tumor progression and chemoresistance. Thus, blockade of HN could constitute a therapeutic strategy to improve the efficacy of chemotherapy in breast cancer.

#1952

Design and characterization of an NQO1-activated spiroisoindolinone derivative for glioma treatment.

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

Primary brain tumors and cancers metastasizing to CNS are the most challenging and lethal malignancies to treat. Chemotherapy with radiation has been a mainstay in their treatment, however, the adverse effects such as the bone marrow suppression, inefficient drug entry through the BBB, intratumoral heterogeneity, and the overexpression of MGMT repair protein are all impediments for successful treatment. Therefore, there is a great need for innovative exploitation of targets selectively expressed in CNS cancers for effective therapy. Such an approach is also likely to spare the normal tissues from the therapeutic insult and prevent the adverse effects. A potential cancer-specific target is the antioxidant enzyme NAD(P)H quinone oxidoreductase 1 (NQO1). NQO1 is a 2-electron reductase responsible for the detoxification of quinones. Its expression is typically quite low in normal tissues but is highly overexpressed in many solid tumor types, including brain cancers. Based on these observations, we have designed a BBB permeable small molecule NQO1-directed drug, spiroisoindolinone GNQ-9 for glioma therapy. GNQ-9 was prepared through rhodium(III)-catalyzed redox-neutral coupling reaction of N-acyl ketimine generated in situ from 3-hydroxyisoindolinone with benzoquinone. GNQ-9 exhibited potent antiproliferative activities against a variety of cancer cell lines, particularly, the NQO1 overexpressing tumor cells and showed minimal cytotoxicity towards a panel of normal cell counterparts (astrocytes, HIVECs, and fibroblasts from lung, pancreatic, breast, and colon). Cytotoxicity studies revealed a strong correlation between NQO1 bioreduction and anticancer activity of GNQ-9. Further, cotreatment with dicoumarol (DIC) significantly reversed the cytotoxic effect of GNQ against U87MG cells demonstrating the NQO1 dependent-cytotoxicity. The ability of GNQ-9 to participate in a redox cycle with NQO1 allows for outstanding potency, as one molecule of GNQ-9 processed by NQO1 generates several ROS molecules and induce oxidative stress and DNA damage. This was confirmed by GNQ-9 generated ROS production using the DCF-DA and DHE. GNQ-9 was also an intercalator and a topo II inhibitor. Other studies revealed that GNQ-9 induces autophagy and apoptosis through ROS with an increased expression of LC3-II and beclin-1 proteins. A marked loss of mitochondrial membrane potential was also noted. Further, the efficacy of GNQ-9 was tested in intracranial xenograft model developed by injecting the luciferase-expressing and NQO1-proficient U87 glioblastoma cells; a significant reduction in the tumor growth was noted with GNQ-9 as a single agent. Serum ALT and AST levels in the tumor-bearing nude mice were not altered, suggesting a lack of toxicity. We conclude that GNQ-9, as an NQO1-targeted drug heightens the tumor redox and induces selective apoptosis in gliomas (supported by CPRIT grants RP130266 and RP170207 to KSS).

#1953

Expression and activity of MT1-MMP in endothelial cells is regulated by Semaphorin 4D.

Amr Bugshan, Zaid Khoury, Asma Buhamrah, John R. Basile. _University of Maryland, Baltimore, MD_.

The semaphorins and plexins, originally shown to control axon growth cone guidance, are now known to influence many aspects of cell adhesion and migration. Our group and others have demonstrated that carcinomas over express Semaphorin 4D (S4D), particularly under conditions of hypoxia, for the purpose of supporting tumor-induced angiogenesis. However, S4D is a transmembrane protein that must be proteolytically cleaved to attract endothelial cells into a malignancy, a process that in head and neck squamous cell carcinoma (HNSCC) is controlled by membrane type 1-matrix metalloproteinase (MT1-MMP). We wanted to test the possibility that S4D acts through its receptor, Plexin-B1 (PB1), to enhance activity of endothelial cell MT1-MMP, facilitating extracellular matrix degradation and further promoting angiogenesis. We observed upregulated transcription, expression and cell surface localization of MT1-MMP in endothelial cells treated with S4D, occurring in a PB1, RhoA and NF-κB-dependent manner. We also demonstrated that S4D-induced endothelial cell collagen degradation, migration and capillary tube formation are suppressed when PB1 is silenced or its downstream effectors RhoA and NF-κB are inhibited. Endothelial MT1-MMP contributes to the ability of these cells to digest their way through the basement membrane and extracellular matrix, a requirement for the creation of the vascular networks that supply a developing tumor with oxygen and nutrients and allow it access to the systemic circulation for metastasis. These results demonstrate that S4D promotes angiogenesis at multiple levels, and suggest that it would be a valid target for anti-angiogenic intervention in the treatment of solid malignancies.

#1954

**Functional analyses of** CLDN18-ARHGAP26 **fusion gene in gastric cancers.**

Izuma Nakayama,1 Eiji Shinozaki,1 Tetsuo Mashima,2 Tokuichi Kawaguchi,3 Seiji Sakata,3 Noriko Yamamoto,3 Satoko Baba,3 Akito Dobashi,3 Yuki Togashi,3 Minoru Sugawara,4 Shunji Takahashi,1 Kensei Yamaguchi,1 Kengo Takeuchi,3 Tetsuo Noda3. 1 _Japanese Foundation For Cancer Research Cancer INST. Hospital, Tokyo, Japan;_ 2 _Japanese Foundation For Cancer Research, Cancer Chemotherapy Center, Tokyo, Japan;_ 3 _Japanese Foundation For Cancer Research, Cancer Institute, Tokyo, Japan;_ 4 _Japanese Foundation For Cancer Research, Cancer Precision Medicine Center, Tokyo, Japan_.

[Introduction]Comprehensive molecular analyses by TCGA have classified Gastric Cancers (GC) into 4 groups: EB virus -associated (EBV), Microsatellite instability (MSI), Genomically stable (GS) and Chromosomal instability (CIN). Among these 4 subtypes, patients with GS subtype have few actionable genetic alterations and thus a limited number of molecular-targeted treatments are available. A fusion gene between CLDN18 and ARHGAP26 was first reported by TCGA to be enriched in GS subtype (13.8%), but the prospective functions of this gene product remain unknown.

[Methods]We have previously established 124 GC-Patient derived xenografts (PDXs) from surgically resected specimens in our institute. There are various types of GCs in the PDX panel, including intestinal and diffuse type tumors or tumors of primary and metastatic site. In an attempt to assess roles of CLDN18-ARHGAP26 fusion gene in gastric cancers, we used the cultured cells obtained from GC-PDXs. We first prepared tissue micro-array of formalin-fixed and paraffin-embedded (FFPE) specimen of PDXs and screened for the patients harboring a CLDN18-ARHGAP26 fusion genes by FISH and RT-PCR. In addition, fusion positive cases were sequenced to determine the breakpoints by direct sequencing. Then, we conducted several functional analyses, including assays for cellular proliferation and mobility, by RNAi-knockdown experiments.

[Results]Among 124 GC-PDXs, we detected two CLDN18-ARHGAP26 fusion positive cases. Both of these 2 cases were derived from tumors with diffuse-type GC patient and had identical breakpoint between CLDN18 (exon 5) and ARHGAP26 (exon 10). We successfully established cell lines of these 2 cases. Furthermore, we would verify that PDX derived cells preserved the original patient properties, as transplantation of the PDX-derived cells histologically recapitulated the original tumors. To address its function, we introduced siRNA into cultured cells to reduce their protein levels. Our data so far indicate that the CLDN18-ARHGAP26 fusion might promote anchorage independent cell growth.

[Conclusion] The CLDN18-ARHGAP26 fusion gene potentially contributes to malignant phenotypes of GC. Further analyses in vivo are awaited.

#1955

Suppression of hormone receptor-positive and triple-negative breast cancers by new inhibitors of the transcription factor FOXM1.

Mary J. Laws,1 Sung Hoon Kim,1 Yvonne Ziegler,1 Noah Bindman,1 Ping Gong,1 Valeria Sanabria Guillen,1 Mayuri Yasuda,1 Divya Singh,1 Dorraya El-Ashry,2 John A. Katzenellenbogen,1 Benita S. Katzenellenbogen1. 1 _Univ. of Illinois at Urbana-Champaign, Urbana, IL;_ 2 _University of Minnesota, Minneapolis, MN_.

The transcription factor FOXM1 is up-regulated and overexpressed in aggressive, therapy-resistant forms of hormone receptor-positive and triple negative breast cancers, and is associated with less good patient survival. FOXM1 pathway signaling is also a key driver in other aggressive cancers including those in prostate, lung, ovary, and gastrointestinal tract, and in glioblastoma. Therefore, our goal has been to identify FOXM1 inhibitors effective in suppressing breast cancer proliferation both in vitro and in vivo and displaying potency, selectivity and good pharmacokinetic properties for in vivo efficacy. We screened large chemical libraries of over 130,000 compounds from which several were identified and characterized as potential FOXM1 inhibitors. These were then chemically modified and optimized for potency and good pharmacokinetic properties. The best were shown to effectively suppress the proliferation of FOXM1 expressing breast cancer cells, decrease the expression of FOXM1 and FOXM1- regulated genes, and inhibit the chromatin binding of FOXM1 to FOXM1 binding sites in these genes. Inhibitors that had long half-lives (t1/2 greater than 24H) were selected for studies in experimental preclinical mouse models. One inhibitor had good oral efficacy in suppressing the growth of FOXM1-containing breast tumors in NOD-SCID-gamma (NSG) mice, and several others had good efficacy in tumor suppression by subcutaneous but not oral administration. Our findings identify and characterize several compounds that effectively antagonize FOXM1 actions and tumor growth that may be suitable for further clinical evaluation in targeting aggressive breast cancers driven by FOXM1. 

### Therapeutic Resistance: New Targets and New Inhibitors

#1956

miR-181d degradation mediated genetic heterogeneity and acquired resistance.

Valya Ramakrishnan,1 Johnny Akers,2 Thien Nguyen,2 Aaron Wang,2 Bandita Adhikari,2 Brian Hirshman,2 Jie Li,1 Jann Sarkaria,3 Wei Hua,4 Mao Ying,4 Masayuki Nitta,5 Tao Jiang,6 Bob Carter,2 Clark C. Chen1. 1 _University of Minnesota, Minneapolis, MN;_ 2 _University of California San Diego, San Diego, CA;_ 3 _Mayo Clinic, Rochester, MN;_ 4 _Fudan University, Shanghai, China;_ 5 _Tokyo Womens University, Tokyo, Japan;_ 6 _Capital Medical University, Beijing, China_.

When unrepaired, alkylated DNA can induce cell death or trigger mutagenesis. Cellular capacity for repair of these lesions by O6-methylguanine methyltransferase (MGMT) dictates the equilibrium between cell viability and genetic diversity. Treatment of glioblastoma cells with temozolomide (TMZ) induced ATM- and Rad3-related (ATR) kinase dependent polyribonucleotide nucleotidyltransferase 1 (PNPT1) degradation of miR-181d. miR-181d suppresses MGMT expression; its degradation increases the mean MGMT expression of the cell population. miR-181d degradation also magnifies the cell-to-cell variability in MGMT expression, expanding the genetic heterogeneity of the population. This expanded heterogeneity enhances the "fitness" of the population and constitutes a novel form of chemotherapeutic resistance. These effects can be suppressed by overexpression of miR-181d, suggesting miRNA delivery as a strategy for glioblastoma therapy. To characterize the mechanism of acquired resistance, we profiled the expression of 2400 miRNAs before and after TMZ treatment. In independent patient-derived neurosphere lines, the majority of miRNAs remained unchanged after treatment. However, miR-181d was consistently suppressed after TMZ treatment. Our previous work demonstrated miR-181d as the master regulator of MGMT. We confirmed TMZ-induced suppression of miR-181d using independent in vitro and in vivo models as well as matched pre- and post-TMZ treated clinical specimens. TMZ-induced miR-181d suppression persisted after transcriptional inhibition, suggesting degradation as the primary mechanism. We performed an siRNA screen and identified polyribonucleotide nucleotidyltransferase 1 (PNPT1) as the gene responsible for miR-181d degradation. CRISPR inactivation of PNPT1 eliminated TMZ-induced suppression of miR-181d; this was rescued by wild-type PNPT1 but not by PNPT1 harboring RNAse-inactivating mutations. TMZ-induced degradation of miR-181d requires ATR kinase. Silencing or inhibition of ATR eliminated binding of PNPT1 to miR-181d and prevented degradation of miR-181d. TMZ-sensitizing effects of ATR inhibition were reversed by anti-miR-181d, suggesting miR-181d is essential in this process. In addition to elevating the mean MGMT expression of the population, single-cell analysis revealed that miR-181d degradation broadened the cell-to-cell variability in MGMT expression in vitro. In matched clinical pre- and post-TMZ treated specimens, variability in MGMT expression was significantly elevated in post-TMZ samples. This was recapitulated using The Cancer Genome Atlas (TCGA) database. We propose that miR-181d degradation-mediated expansion of genetic heterogeneity enhances the "fitness" of the population, constituting a novel form of chemotherapeutic resistance. These effects are suppressed by miR-181d overexpression, suggesting miRNA delivery as a strategy for glioblastoma therapy.

#1957

Aurora kinase A drives the evolution of resistance to third-generation EGFR inhibitors in lung cancer.

Khyati N. Shah,1 Roma Bhatt,1 Julia Rotow,1 Julia Rohrberg,1 Victor Olivas,1 Golzar Golzar Hemmati,1 Gregor Krings,1 Henry J. Haringsma,2 Andrew D. Simmons,2 Thomas C. Harding,2 Andrei Goga,1 Collin Blakely,1 Trever Bivona,1 Sourav Bandyopadhyay1. 1 _UCSF, San Francisco, CA;_ 2 _Clovis, San Francisco, CA_.

With the advent of precision medicine, EGFR inhibitors are a major breakthrough in the treatment of EGFR-mutant non-small cell lung cancer (NSCLC). Although these EGFR-TKI therapies often elicit profound initial therapeutic responses, their effects are transient due to residual disease. This residual disease and subsequent disease progression occurs through tumor evolution and molecular drivers behind the formation, maintenance and evolution of residual disease and acquired resistance have remained elusive. Although in many cases, pre-existing clones with bona fide genetic resistance have been identified, majority of patients have undetectable resistance causing genetic alterations, suggesting that nongenetic alterations may drive altered cell state and signaling associated with EGFR inhibitor resistance. Using EGFR mutant lung cancer cells, we developed several in vitro models of acquired resistance to third-generation EGFR-TKI inhibitors, osimertinib and rociletinib, and a chemical screen revealed that Aurora kinase inhibitors are highly synergistic when combined with third-generation EGFR inhibitors. Resistant cells harbored high activation of AURKA mediated by upregulation of its co-activator protein TPX2. In in vitro and in vivo models of acquired resistance, the combination induced potent cell death by reactivating BIM-mediated apoptosis. We found that tumors from patients progressing on first- and third-generation EGFR TKIs often harbored high levels of TPX2, indicating that AURKA is likely activated and driving resistance in a significant fraction of EGFR-mutant lung cancers. By tracking the kinetics of AURKA activation, we tested if AURKA activity is required for the formation and maintenance of residual cells leading to acquired resistance. In our sensitive in vitro models, either single-agent EGFR-TKI, MLN8237 or the combination enhanced the magnitude of response and forestalled the emergence of resistance as compared to monotherapies. We tested the combination in an EGFR L858R patient-derived xenograft (PDX) tumor model generated from a residual disease surgical specimen. In this PDX the combination robustly impaired tumor growth compared to both single agents alone and in most cases induced tumor regression This novel synthetic lethal interaction between EGFR TKIs and Aurora kinase inhibitors has important implications for the development of new treatment strategies for third-generation EGFR-TKI. These results invite a paradigm aimed at preventing the emergence of resistance.

#1958

Benztropine enhances temozolomide sensitivity by restricting the growth of drug-resistant glioma stem cells.

Damian A. Almiron Bonnin,1 Myung Chang Lee,2 Joseph M. Howard,1 Alison L. Young,1 Matthew C. Havrda1. 1 _Dartmouth Geisel School of Medicine, Lebanon, NH;_ 2 _Dartmouth College, Lebanon, NH_.

Glioblastomas (GBMs) are the most aggressive type of primary brain tumor in adults, the second leading cause of cancer-related death in children, and are incurable using currently available treatment modalities. GBMs are characterized by the presence of primitive glioma stem-like cell subpopulations that resist chemotherapy and sustain disease progression. In this study, we found that Benztropine, a muscarinic acetylcholine receptor (mAChR) inhibitor, suppresses glioma cell proliferation and sensitizes these cells to treatment with Temozolomide. Modeling the development of Temozolomide resistance in vitro, we identified a subpopulation of glioma cells, defined by the co-expression of CD44, CD15, and PDGFRa that was resistant to Temozolomide. This chemo-resistant subpopulation was phenotypically similar to oligodendroglial progenitor cells (OPC-like); a stem-like cell population important in the initiation and progression of glioblastoma known to be modulated by mAChR signaling. The addition of the mAChR inhibitor, benztropine, dramatically reduced the emergence of this Temozolomide-resistant sub-population. To begin understanding the mechanism of action of benztropine on GBM cells, we analyzed the effect of this drug on a panel of mitotic drivers that have been implicated in glioma initiation and progression. Sub-lethal doses of Benztropine alone or in combination with temozolomide repressed the PDGF-signaling axis as characterized by a significant reduction of PDGFA secretion and PDGFRA activation. We also developed an in vivo model of Temozolomide resistance to determine whether Benztropine could improve the survival of animals harboring glioma xenografts that had recurred following treatment with Temozolomide. These in vivo studies demonstrated that Benztropine alone improves animal survival as

compared with vehicle controls and that Benztropine combined with Temozolomide reduces glioma growth following initial Temozolomide treatment and improves survival in mice xenografted with human GBM cells. Taken together, our results suggest that Benztropine might be targeting glioma stem cells by altering the glioma stem cell microenvironment. These findings support a potential role for the use of adjuvant Benztropine for the clinical management of GBM.

#1959

Therapeutic rationale for SHP2 phosphatase inhibitors: Targeting drug-resistant breast cancer via blockade of FGFR1 signaling.

Hao Chen, Sheng Zhang, Zhong-Yin Zhang, Michael Keith Wendt. _Purdue University, West Lafayette, IN_.

Human epidermal growth factor receptor-2 (Her2) amplified breast cancer represents ~25% of breast cancer. Clinically used tyrosine kinase inhibitors (TKIs), such as lapatinib and neratinib, are capable of blocking Her2 signaling and provide therapeutic benefits. Unfortunately, resistance to TKIs is reducing survival of patients. Hence, there is a critical need for novel therapies targeting drug-resistant breast cancer. Expression of fibroblast growth factor receptor-1 (FGFR1) can be induced by acquired resistance to lapatinib. Also, FGFR1 signaling is a critical growth factor receptor signaling in drug resistance network. Src Homology Phosphatase-2 (SHP2) is a protein tyrosine phosphatase that facilitates growth factor receptor signaling and is strongly linked with cancer progression. Currently, there are two types of inhibitors targeting SHP2, one with allosteric binding mechanism (SHP099) and one with active-site mechanism (11a-1). However, there is still a mechanistic gap in knowledge concerning the roles of SHP2 in drug-resistant breast cancer. Our findings support the core hypothesis that SHP2 phosphatase is required for FGFR1 signaling, contributing to drug resistance. First, we stably overexpressed SHP2 and FGFR1 in normal murine-mammary-gland (NMuMG) cells. SHP2 could facilitate FGFR1 signaling driving cell growth. Conversely, pharmacologic inhibition of SHP2 could partially reduce phosphorylation of FRS2, AKT and ERK, downstream of FGFR1. Next, we identified the growth-inhibitory effects of SHP2 inhibitors in Her2 transformed cells that are sensitive (HMLE-HER2) and resistant (HMLE-HER2-LapR) to TKIs. Both SHP2-targerting compounds had significantly improved growth-inhibition effects in 2D growth assays on HMLE-HER2-LapR cells, which overexpress FGFR1. Both SHP2 inhibitors also inhibited the growth of SK-BR-3 and ZR-75-1 cells. While in 3D growth assays with the same cell lines, the growth inhibition effects were much better compared with 2D growth assays. To better identify whether TKIs-resistant cells could be targeted by SHP2 inhibitors, we assessed whether SHP2 inhibition could block signaling recovery after neratinib treatments. The results showed that both of inhibitors could significantly delay p-AKT and p-ERK after neratinib treatments. To further identify potential therapeutic application of SHP2 inhibitors, we assessed the efficacy of 11a-1 alone, or in combination with neratinib. To this end, we found that 11a-1 could decrease growth and migration of TKIs-resistant cells when used in combination with neratinib. In summary, continued understanding of the detailed mechanisms of SHP2 in FGFR1 signaling and continued development of compounds capable of inhibition SHP2 function may provide a therapeutic strategy for targeting SHP2 in combination with TKIs to prolong patient response rates in this subtype of breast cancer.

#1960

T790M-independent EGFR TKI resistance is associated with a broad multi-drug resistant phenotype but selective vulnerabilities to spindle assembly complex (SAC) and CDK inhibitors.

Monique B. Nilsson, Jacqueline Robichaux, Huiying Sun, Pan Tong, Lixia Diao, Mia Hofstad, YouHong Fan, Jing Wang, John Heymach. _UT MD Anderson Cancer Ctr., Houston, TX_.

While EGFR mutant NSCLC patients are initially responsive to EGFR targeted therapies, resistant disease inevitably emerges and in nearly half of resistance cases, tumors lack secondary EGFR mutations such as T790M and are refractory to 2nd and 3rd generation EGFR tyrosine kinase inhibitors (TKI). The identification of treatment regimens with efficacy against T790M-negative resistance remains a major clinical challenge. To address this unmet need, we derived a panel of NSCLC cell lines with acquired resistance to the EGFR TKI, erlotinib. A subset of EGFR TKI resistant variants were negative for secondary EGFR mutations, were resistant to 2nd and 3rd generation EGFR TKIs including osimertinb, afatinib, and dacomitinib, and had undergone epithelial to mesenchymal transition (EMT) as demonstrated by loss of E-cadherin, enhanced expression of N-cadherin and Axl and an increased invasive phenotype as determined by Boyden chamber assay. Proteomic profiling revealed that although EGFR TKI resistant cells displayed similar mesenchymal and invasive phenotypes, there was significant heterogeneity in protein expression and pathway activation among resistant variants derived from the same parental cell line. To identify therapeutic agents with activity against EMT-associated EGFR TKI resistance, we performed high-throughput drug screening to test the efficacy of 1,321 compounds. EMT-associated EGFR TKI resistance was accompanied by the acquisition of broad spectrum drug resistance. Compared to parental cells, mesenchymal EGFR TKI resistant cells were significantly more resistant to chemotherapeutic agents used to treat NSCLC including pemetrexed, irinotecan, vinblastine, and gemcitabine. EGFR TKI resistant cells displayed acquired resistance to 147 other tyrosine kinase and serine/threonine kinase inhibitors. In contrast, both parental cells and mesenchymal EGFR TKI resistant variants were highly sensitive to CDK inhibitors and agents targeting spindle assembly checkpoint (SAC) components including PLK1, Aurora, KSP, and survivin. These finding were validated by MTS and clonogenic assays. Treatment with SAC inhibitors induced the accumulation of cells in G2/M phase, enlarged nuclear size, and polyploidy. To clinically validate these findings, we established a cell line (MDA-011) from the pleural effusion of an EGFR mutant NSCLC patient with T790M-negative resistance to erlotinib. In vitro, MDA-011 cells were resistant to erlotinib and osimertinib. MDA-011 cells were highly sensitive to CDK inhibitors and SAC inhibitors as determined by MTS and clonogenic assays. These data indicate that EMT-associated resistance to EGFR TKIs is associated with broad spectrum drug resistance but vulnerabilities to CDK and SAC inhibition which can potentially be exploited to overcome resistant disease in NSCLC patients.

#1961

Gaps in the armor: Targeting HuR to sensitize pancreatic cancer.

Christopher W. Schultz,1 Kevin O'Hayer,1 Teena Dhir,1 Kathryn M. Bormes,1 Samantha Z. Brown,1 Henry Thomsett,1 Saswati Chand,1 Aditi Jain,1 Wei Jiang,1 Grace McCarthy,1 Charles J. Yeo,1 Austin Goetz,1 Avinoam Nevlar,1 Jonathan R. Brody,1 Jordan M. Winter,1 Ranjan Preet,2 Dan Dixon,2 Jessica Bowers,3 Kelly Rhodes,3 Robert Getts3. 1 _Thomas Jefferson, Philadelphia, PA;_ 2 _University of Kansas Medical Center, Kansas City, KS;_ 3 _Genisphere, Hatfield, PA_.

HuR is an RNA binding protein involved in a coordinated cellular response to stressors. Upon insults such as chemotherapy or radiation treatment, HuR translocates from the nucleus to the cytoplasm where it binds the 3'UTR of target mRNAs. HuR's interaction with target mRNAs leads to the upregulation of target genes and ultimately treatment resistance. This is particularly relevant in the case of pancreatic ductal adenocarcinoma (PDA). PDA is highly resistant to standard chemotherapy such as FOLFIRINOX or gemcitabine/nab-paclitaxel. Using a tumor microarray (TMA), we found 79% of patient tumor samples (n=70) were positive for active cytoplasmic HuR, while little to no cytoplasmic localization was detected in normal tissue. In addition, HuR CRISPR knockout cell lines have a xenograft lethal phenotype. Previously published data also demonstrated that reduction of HuR in xenografts with a DOX-inducible shRNA system caused a 3.6 fold decrease in tumor size. HuR reduction was also shown to potentiate a PARP-inhibitor (olaparib) treatment from a 5.6-fold reduction alone to 9.3-fold reduction in tumor size when combined with shHuR, demonstrating the role of HuR in drug resistance. The aim of our current study was: A) to target HuR directly using nanoparticle delivery of siRNA against HuR; and B) to use the FDA approved small molecule pyrvinium pamoate (PP) to inhibit HuR's translocation and sensitize PDA cells to concurrent therapies. Using 3DNA, a 60nm nanoparticle composed of a sphere of crosslinked DNA, we have successfully delivered siRNA against HuR in vivo utilizing targeting moieties against receptors known to be overexpressed on the surface of PDA cells: EGFR, folic acid receptor, and transferrin receptor. Bi-weekly IP treatment of siHuR bound to 3DNA was safe and effective at extending life in a xenograft model as indicated by Kaplan Meier analysis (p=0.01). We are currently testing siHuR-3DNA dendrimer therapy's ability to sensitize PDA cells to oxaliplatin or olaparib in vivo. We are also investigating the use of PP to target HuR's localization. PP has previously been shown in bladder cancer to inhibit the translocation of HuR in vitro and in vivo. We have reproduced this finding in PDA cells, and have shown impressive drug potency with IC50s as low as 38nM in 2D cultures and 16nM in a 3D mouse PDA organoid model. Combination index (CI) values determine drugs interactions where 1 is additive, <1 is synergistic and >1 is antagonistic. We have determined that PP can enhance therapies such as gemcitabine (CI of 0.55), olaparib (CI of 0.40) and palbociclib (CI of 0.37) in vitro, and are currently validating these findings in vivo. Taken together, our data demonstrate that HuR inhibition via 3DNA delivery of siHuR and/or PP treatment can sensitize PDA cells to chemotherapy and targeted therapies. By inhibiting a resistance driver (HuR) in pancreatic cancer we aim to improve current therapies for this devastating disease.

#1962

Dianhydrogalactitol (VAL-083) has a distinct mechanism of action that suggests combination with PARP inhibitors as an effective therapeutic strategy.

Jeffrey A. Bacha,1 Guangan He,2 Anne Steino,1 Dennis M. Brown,3 Zahid H. Siddik2. 1 _Delmar Pharmaceuticals Inc., Vancouver, British Columbia, Canada;_ 2 _The University of Texas MD Anderson Cancer Center, Houston, TX;_ 3 _Delmar Pharmaceuticals Inc., Menlo Park, CA_.

Ovarian cancer is usually treated with platinum (Pt)-based chemotherapy, but patients frequently develop Pt-resistance. Dysfunctional p53 is implicated in Pt-resistance, comprising a therapeutic challenge in high grade serous ovarian cancer (HGSOC), where p53 is 96% mutated. Attempts to overcome Pt-resistance in HGSOC include agents blocking the DNA repair pathways, most notably the PARP inhibitors (PARPi), leading to accumulation of DNA double strand breaks (DSBs) and cancer cell death. However, PARPi resistance often arises, leading to a 5-year survival rate of 40% in HGSOC. VAL-083 is a first-in-class DNA damaging agent with demonstrated clinical activity against a range of tumors, including ovarian. VAL-083 rapidly induces interstrand cross-links at guanine-N7 leading to DSBs, activation of the homologous recombination (HR) DNA repair pathway, S/G2 cell cycle arrest and cancer cell death. Notably, VAL-083 induces cell death through two parallel pathways; one p53-independent and one p53-dependent. We have shown that VAL-083 is able to overcome cisplatin resistance in a panel of ovarian cancer cells, independent of p53 status. We have also shown that VAL-083 maintains activity independent of DNA repair mechanisms such as O6-methylguanine DNA methyltransferase (MGMT), non-homologous end-joining (NHEJ) and mismatch repair (MMR), implicated in resistance to chemotherapeutics, including cisplatin and PARPi. Cancer cells thus rely heavily on functional HR for repair of VAL-083-induced DSBs, proposing combination therapy with agents further inducing DSBs or blocking their repair, including PARPi. Taken together, these data propose VAL-083 for treatment of Pt-resistant HGSOC and for combination therapy with PARPi. Here, we study the cytotoxicity of VAL-083 in combination with PARPi (olaparib, niraparib, rucaparib, veliparib or talazoparib) against HR-proficient and HR-impaired ovarian cancer cells, the impact of p53 status and Pt-resistance. VAL-083 cytotoxicity alone and in combination with PARPi was investigated using the MTT assay in HR-proficient and HR-impaired A2780 ovarian cancer cells. The impact of p53 status was studied by CRISPR/cas 9 knockout of p53 in wildtype A2780 cells and in a panel of p53-mutated ovarian cancer cells. We report increased VAL-083 cytotoxicity against HR-impaired A2780 cells. We further report superadditivity in both HR-proficient and HR-deficient A2780 cells between VAL-083 and olaparib, niraparib, talazoparib or rucaparib. In conclusion, our results demonstrated a distinct DNA damaging mechanism for VAL-083, resulting in the ability to overcome Pt-resistance, target HR-impaired tumors and overcome MGMT, MMR and NHEJ-related chemoresistance. In addition, VAL-083 activity was independent of p53 status and superadditive with PARPi in HR-proficient and HR-deficient A2780 tumor cells.

#1963

ARQ 531, a potent reversible BTK inhibitor, exhibits potent antitumor activity in ibrutinib-resistant diffuse large B-cell lymphoma.

Sudharshan Eathiraj,1 Yi Yu,1 Ron Savage,1 Jennifer A. Woyach,2 Sean D. Reiff,2 Amy J. Johnson,2 Brian Schwartz1. 1 _Arqule Inc., Burlington, MA;_ 2 _The Ohio State University, Columbus, OH_.

Background: B-cell receptor (BCR)-mediated signaling plays an important role in the pathogenesis of a subset of diffuse large B-cell lymphoma (DLBCL). Despite major advances in the treatment, ~40% of the relapsed/refractory DLBCL patients still experience early treatment failure after initial response to chemotherapy. ARQ 531, a reversible inhibitor of BTK and BTK-C481S mutant, also potently suppresses BCR signaling. Here we demonstrate that ARQ 531 targets additional kinases and suppresses multiple oncogenic pathways, this inhibitory potency is coupled to broad anti-tumor activity in DLBCL subtypes including tumors resistant to BCR targeted therapy.

Methods: Biochemical inhibition and broad kinase profiling were assessed using recombinant proteins. Binding kinetics and the residence time with BTK and BTK-C481S were measured by Surface Plasmon Resonance (SPR) assay. Binding mode of ARQ 531 with BTK was determined by protein crystallography. Pathway inhibition assessments, in vivo efficacy and in vivo target inhibition were performed in DLBCL tumor models derived from TMD8, SUDHL-4 and DOHH-2 cell lines.

Results: ARQ 531 potently inhibited BTK (IC50 = 0.85 nM), the binding potency was accompanied by long residence time (51 min). Crystal structure of BTK/ARQ 531 complex showed that ARQ 531 occupies the ATP-binding pocket. Kinase selectivity profile suggested that ARQ 531 inhibits sub-families of Tec, Src, Trk kinases. Significant anti-proliferative activity (GI50 = < 1µM) was observed in hematological malignant cell lines characterized by addiction to BTK signaling and primarily resistant to ibrutinib. Pathway inhibition analysis suggested that ARQ 531 targets multiple oncogenic signaling pathways in both ABC- and GCB-DLBCL cell lines. Unlike ibrutinib, ARQ 531 suppressed both the upstream activating signals (via inhibition of a select member of Src kinase family) and the downstream signaling pathways (via pAKT and pERK kinases). In GCB-DLBCL cell lines (SUDHL-4 and DOHH-2), ARQ 531 potently suppressed expression of anti-apoptotic c-Myc and BCL6 oncoproteins in a dose dependent fashion, and concomitantly induced apoptotic cleavage of PARP protein. In the ibrutinib-resistant SUDHL-4 mouse xenograft model, ARQ 531 potently suppressed tumor growth (>80% inhibition) compared to the control group when dosed orally at 75 mg/kg. Additionally, preliminary data suggest that ARQ 531 crosses the blood, brain-barrier.

Conclusion: ARQ 531 is a potent reversible inhibitor of BTK, its distinct kinase selectivity profile offers significant advantage for simultaneous inhibition of multiple therapeutically relevant targets. ARQ 531 attenuated the expression of key oncogenic drivers via inhibition of downstream BCR activating kinases. These results highlight the therapeutic potential of inhibition of BCR signaling inhibition by ARQ 531 in the treatment of DLBCL.

#1964

Preclinical efficacy of the HSP90 inhibitor SNX-5422 in targeting C481S mutant BTK and ibrutinib resistant CLL.

Timothy L. Chen,1 Bonnie Harrington,1 Jean Truxall,1 Ronni Wasmuth,1 Amy Lehman,1 Eric Orlemans,2 John C. Byrd,1 Jennifer A. Woyach,1 Erin Hertlein1. 1 _The Ohio State University, Columbus, OH;_ 2 _Esanex Inc., Indianapolis, IN_.

While chronic lymphocytic leukemia (CLL) is effectively treated by the Bruton's tyrosine kinase (BTK) inhibitor ibrutinib, patients that do relapse on ibrutinib (approximately 19%) have an extremely poor clinical outcome without further intervention. Before clinical diagnosis of relapse, signs of molecular relapse can be identified through deep sequencing for mutations in BTK (C481S) and the immediate downstream BTK target, PLCγ2. The BTK C481S mutation (which reduces ibrutinib binding) can be identified at frequencies as low as 5%, and expansion of this mutant close inevitably leads to clinical relapse. Therefore, identifying methods to target this mutant clone before clinical relapse represents a novel and potentially effective therapeutic strategy.

HSP90 is a chaperone protein responsible for maintaining the stability of a large number of client proteins involved in cancer development. Cancer cells, particularly those reliant on a mutated oncogene, rely heavily on HSP90 for survival, making this an attractive clinical target. Here we have explored the preclinical efficacy of SNX-5422, a chemically unique, orally bioavailable HSP90 inhibitor, in ibrutinib resistant CLL. We determined that SNX-5422 can degrade C481S mutated BTK by utilizing BTK-null B-cell lines over-expressing wildtype or C481S BTK. Whereas ibrutinib reduces BTK activity in only the wildtype BTK transfected cell line, SNX-5422 degrades both wildtype BTK and C481S mutant BTK and impairs downstream signaling. In fact, C481S mutant BTK shows increased degradation compared to wildtype BTK suggesting that the C481S mutant BTK may rely on HSP90 more for stabilization.

We next examined the in vivo efficacy of SNX-5422 in a mouse model of ibrutinib-resistant CLL. Leukemic splenocytes isolated at removal criteria from TCL1 mice continuously treated with ibrutinib were adoptively transferred into syngeneic WT B6 mice. Upon reaching 10% leukemia burden in the peripheral blood, the mice were randomized into 1 of 4 treatment groups: vehicle, SNX-5422 (50 mg/kg MWF), ibrutinib (continuous in drinking water), or SNX-5422 plus ibrutinib (concurrent). Mice treated with the combination of SNX-5422 and ibrutinib exhibit prolonged survival and decreased disease burden in the peripheral blood compared to the vehicle or single agent groups.

In summary, we show that SNX-5422 degrades C481S BTK in vitro and prolongs survival when combined with ibrutinib in vivo. We therefore propose that upon molecular relapse, the use of SNX-5422 may prevent clinical relapse and deepen the response to ibrutinib. A phase 1 study is currently open at Ohio State to investigate whether the addition of SNX-5422 to an established dose of ibrutinib can eliminate the C481S mutant clone and prevent or delay disease progression in subjects with CLL (NCT02914327).

EH and JAW contributed equally as senior authors.

#1965

Biologic evaluation of chalcone derivatives as ABCG2- and ABCB1-mediated drug resistance reversal agents.

Chaoyun Cai. _St. John's Univ. College of Pharmacy and Health Sciences, Fresh Meadows, NY_.

The overexpression of ATP-binding (ABC) transporters, including ABCG2 and ABCB1, has been shown to be related to multidrug resistance (MDR). In this work, we investigated a series of chalcones and bis-chalcones as ABCG2- and ABCB1-mediated resistance reversal agents. The results revealed that methoxyl chalcones have better reversal activities than hydroxyl chalcones toward ABCG2-mediated resistance. One methoxyl chalcone has good reversal activities in both ABCG2 and ABCB1 overexpressing cell lines. The compound can increase the accumulation of anticancer drugs in ABCG2 or ABCB1 overexpressing cell lines while combined with anticancer drugs, due to its inhibition of ABCG2 and ABCB1 (IC50 (ABCB1) =1.54 μM, IC50 (ABCG2) = 1.15 μM). In addition, this compound did not significantly alter the expression and localization of ABCG2 or ABCB1 in ABCG2 or ABCB1 overexpressing cell lines.

#1966

A novel small-molecule compound targeting PBX1-DNA interaction impedes cancer cell survival and carboplatin resistance.

Yao-An Shen,1 Jin Jung,1 Yohan Suryo Rahmanto,1 Licia Selleri,2 Ie-Ming Shih,1 Chi-Mu Chuang,3 Tian-Li Wang1. 1 _The Johns Hopkins Medical Institutions, Baltimore, MD;_ 2 _University of California, San Francisco, San Francisco, CA;_ 3 _National Yang-Ming University, Taipei, Taiwan_.

Pre-B-cell leukemia homeobox-1 (PBX1), a transcriptional factor and downstream effector of Notch signaling pathway, plays pivotal roles in a wide spectrum of tumors, as well as developmental, inflammatory, autoimmune, and neurodegenerative disorders. Exploiting the crystal structure of the Pbx1-DNA complex, we developed a novel small-molecular inhibitor T417 that can directly block Pbx1-binding to DNA, unlike other existing compounds that interfere with protein-protein interactions. When T417 docks into the hydrophobic pocket of Pbx1 protein, the small molecule can dampen PBX1 transcription activity by hindering its binding to the promoter regions of PBX1 downstream target genes. Intriguingly, the amount of PBX1 expression in cells can dictate its response to T417. Increment of PBX1 expression is found in the high-grade serous carcinoma (HGSC) and carboplatin-resistant (CR) cells, and its expression is generally low in normal tissues as compared to the transformed tissues, making it such an arguably unique therapeutic target in ovarian cancer cells. This expression pattern illustrates the very minimal toxicity of T417 on normal tissues and organs in animal models, while it imposed in vitro and in vivo detrimental effects toward HGSC and CR cells. Besides, T417 holds synergistic cytotoxic effects with DNA damage-related drugs including PARP inhibitor and platinum-based drug. As PBX1 was shown to participate in maintaining cancer stem cell (CSC)-like phenotypes and promoting resistance to antitumor drugs, T417 is able to hammer out the stemness traits of CR cells to revert to a differentiated status through tacking PBX1 signaling cascade. The novel PBX1-targeting compound selectively interferes with PBX1-binding to DNA, which potentially points to powerful therapeutics and broad applications for the treatment of different human malignancies and stem cell therapy.

#1967

Synthesis, in vitro evaluation in ovarian cancer cells and molecular modeling analysis of novel 2-aminothiazole and 2-aminopyridine derivatives.

Shagufta Naz,1 Humaira Nadeem,1 sadia sarwar,1 Rehan Z. Paracha,2 Jun Q. Yu,3 Fazlul Huq3. 1 _Riphah International University, Islamabad, Pakistan;_ 2 _National University of Science and Technology, Islamabad, Pakistan;_ 3 _Discipline of Biomedical Science, School of Medicine, The University of Sydney, Sydney, Australia_.

Background: Resistance to chemotherapy remains a major hurdle in complete cure against ovarian cancer. Among 75 percent of respondents to chemotherapy, the majority would relapse within two years. Cancer cells can overcome therapy-induced damage by activation of back-up signaling pathways and flexible modulation of affected networks resulting in chemo-resistance. Platinum-based drugs are the most active agents in use against the ovarian cancer. Non platinum-based chemotherapy regimens are usually given in the 'platinum-resistant' setting.

Methods: A series of N-protected and N-deprotected amino acid derivatives of ethyl-2-aminothiazole-4-carboxylate (4a-d & 6a-d) and 2-aminopyridine (5a-d & 7a-d) have been synthesized and characterized by FT-IR, 1H NMR, 13C NMR, mass spectrometry and elemental analysis. The compounds were evaluated in silico for selected target inhibition. AutoDockVina virtual screening program was used to target multiple markers in cell signaling pathway (including p110a/PI3K, EGFR, VEGFR, PDGFR, BCR-ABL, RET, c-KIT, c-Raf, ALK, ,B-Raf, , CTLA-4). Anti-tumor activity was determined in parent (A2780) and cisplatin resistant (A2780CISR) ovarian cancer cell lines by MTT reduction assay.Binding affinity data of standard drugs and synthesized compounds 4c, 6b and 7c were compared. Binding mode and 2D interactions of the compounds with p110a isoform of PI3K, EGFR, VEGFR and PDGFR were determined.

Results: Among selected targets, p110a isoform of PI3K is the main target involved in development of ovarian cancer; all three ligands were found to be active against this target. Compound 4c&6b exhibited excellent inhibition of p110a isoform of PI3K with binding energy -8.3 Kcal/mol and -8 Kcal/mol that is comparable to the reference compound i.e. -9.8 Kcal/mol. Overall, compound 4c&6b exhibited greatest inhibition with lowest binding energies against most of the targets.All the tested compounds exhibited dose dependent anti-proliferative effect against both cell lines. IC50 and RF values of cisplatin and tested compounds were determined. Compounds 4c, 6b and 7c showed interesting results as they were more active in cisplatin resistant cell line than in parent cells. The RF values of these compounds were 0.83, 0.46 and 0.53 respectively.

Discussion: RF value of a compound provides an idea if the compound can induce more cell death than corresponding drug in resistant cell lines. RF value less than 1 for a compound/drug is an indication of ability of that compound to bring about more cell death than standard drug in resistant cells. These data warrant further investigations into the mechanism of reversal of resistance induced by potential compounds.

#1968

A novel glucocorticoid receptor (GR) antagonist overcomes GR-mediated chemoresistance in triple-negative breast cancer.

Nadine Jahchan, Haiying Zhou, Wayne Kong, Dan Mc Weeney, Ted Tracy, James Stice, Dena Sutimantanapi, Chelsea Chen, Tom Huang, Yosup Rew, Xiauhui Du, Tatiana Zavorotinskaya, Daqing Sun, Qiuping Ye, Erica Jackson, Valeria Fantin. _ORIC Pharmaceuticals, South San Francisco, CA_.

The glucocorticoid receptor (GR) is a member of the nuclear receptor superfamily, which is activated by its endogenous steroid hormone ligand cortisol, and by synthetic glucocorticoids such as dexamethasone. Several preclinical studies have shown that GR mediates resistance to both targeted therapies and conventional chemotherapies in a variety of epithelial cancers including prostate, lung, bladder, renal, ovarian and triple-negative breast cancers (TNBC) (Gassler et al., 2005; Li et al., 2017; Zhang et al., 2007). In TNBC, both GR activation and a disrupted cortisol secretion cycle are associated with chemotherapy resistance, increased disease recurrence and poor prognosis (Pan et al., 2011; Skor et al., 2013). Therefore a molecule that inhibits GR activation could attenuate the development of therapy resistance and improve patient outcomes. We have developed novel GR inhibitors that effectively block GR transcriptional activity in cells by competing for ligand binding and by blocking GR-coactivator interactions. In vitro, treatment of TNBC cells with the GR antagonist OP-3713 blocks GR transcriptional activity and enhances the efficacy of chemotherapeutic agents. In rodents the predominant glucocorticoid is corticosterone (Siswanto et al., 2008), which is a weak agonist of human GR. Therefore, to fully activate GR in human xenograft cancer models it is necessary to provide exogenous cortisol. Using xenograft models of TNBC, we find that tumors grown in mice with physiologically relevant circulating cortisol levels to activate GR are significantly less sensitive to chemotherapy than those grown in the absence of cortisol. Furthermore, inhibition of GR by OP-3713 prevents tumor relapse following chemotherapy treatment. We have begun to elucidate the mechanisms by which GR mediates chemoresistance in TNBC, and the basis for the reversal by OP-3713. Our findings underscore the important role of GR as a mediator of resistance in TNBC and highlight the therapeutic potential of GR inhibitors in combination with clinically relevant chemotherapeutic agents.

#1969

Cancer exosomes as a new mediator to spread drug resistance to EGFR tyrosine kinase inhibitors.

Kenneth KW To, Christy WS Tong, Mia MX Wu. _Chinese Univ. of Hong Kong, Hong Kong, Hong Kong_.

Aim: The study aims to identify new therapeutic targets from cancer exosomes, which may spread drug resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) in the treatment of non-small-cell lung cancer (NSCLC). Background: EGFR TKIs have exhibited extraordinary clinical efficacy in advanced lung cancer. However, their usefulness is compromised by drug resistance mediated by various mechanisms, including the emergence of a secondary EGFR T790M mutation. Exosomes are small membrane vesicles derived from the cellular phenomenon of membrane blebbing under both normal and pathological conditions. They mediate intercellular cross-talk by transferring receptors, antigens and cytokines from donor to recipient cells. It has been reported that EGFR TKI resistance mutation exists before drug exposure. Selective pressure from EGFR TKI therapy may thus cause the expansion of the latent resistant subclones, resulting in the emergence of a resistant phenotype in the entire tumor population. We hypothesize that exosomes may mediate the transfer of the secondary EGFR mutant or other mediators from the resistant subclones to neighboring sensitive cells. Method: The possible exosome-mediated transfer of secondary EGFR mutant (or other protein mediators) from EGFR T790M-bearing NSCLC (H1975) cells to a sensitive NSCLC cell line (HCC827) was evaluated. Exosomes were isolated from H1975 cells-conditioned medium by the standard differential centrifugation method. The secretion of protein mediators into exosomes by resistant H1975 cells was studied by protein array. The EGFR kinase activity of the exosomes was evaluated by biochemical assay. The uptake of fluorescence-labeled exosome from the resistant cells by the sensitive cells was visualized by fluorescence microscopy. The activation of the EGFR signaling pathway in the recipient sensitive cells after incubation with exosomes derived from the resistant cells was examined by Western blot analysis. Result: Exosomes were successfully isolated from H1975-conditioned medium and confirmed by the expression of exosomal markers CD63 and HSP70. Compared with exosomes derived from sensitive HCC827 cells, exosomes secreted from H1975 cells were enriched with apoptosis-regulating proteins including Src. H1975-derived exosomes increased the viability of HCC827 cells in the presence of gefitinib and inhibited the apoptosis induced by gefitinib. Western blot analysis showed that the exosomes can block the reduction of BCL-2 and full-length PARP, while abolishing the increase of BAX and cleaved PARP induced by gefitinib in HCC827 cells. Conclusion: H9175-derived exosomes may be involved in the spreading of drug resistance to EGFR TKIs by regulating cell apoptosis. New treatment strategies may be designed to target and inhibit this exosome-mediated transfer of mediators implicated in the acquired resistance to EGFR TKIs.

#1970

**Novel Mcl-1 inhibitor effectively restores** in vivo **regorafenib sensitivity in resistant colorectal cancer.**

Karson J. Kump,1 Jingshan Tong,2 Ahmed S. Mady,1 Lei Miao,1 Siwei Li,1 Duxin Sun,1 Jian Yu,2 Lin Zhang,2 Zaneta Nikolovska-Coleska1. 1 _University of Michigan, Ann Arbor, MI;_ 2 _University of Pittsburgh, Pittsburgh, PA_.

Colorectal cancer (CRC) is one of deadliest malignancies and approximately 25% of patients present with metastatic disease at the time of diagnosis, necessitating efficacious therapeutic options. Treatment of CRC has been greatly improved by targeted therapies such as the FDA-approved multi-kinase inhibitor, regorafenib, which elicits its anti-cancer effects by inducing apoptosis. Ours and other studies identified the anti-apoptotic Mcl-1 protein as a significant resistance factor to regorafenib treatment in CRC, associated with inactivating mutations in the tumor suppressor F-box and WD repeat domain-containing 7 (FBW7). These mutations result in abrogated Mcl-1 degradation, which contributes to survival of cancer cells and ineffective regorafenib induced apoptosis. Therefore, there is sound rationale for Mcl-1 inhibition to circumvent FBW7 inactivity and restore the efficacy of regorafenib. Our group has discovered and developed several classes of selective small molecule Mcl-1 inhibitors that bind in the conserved BH3 groove. In HCT116 colon cancer cells, the lead compound, 483LM, engages endogenous Mcl-1 as demonstrated by a CETSA assay and leads to a rapid accumulation of Mcl-1 protein levels, indicative of protein stabilization. 483LM induces BAX/BAK dependent apoptosis in cancer cells, upregulates the pro-apoptotic protein, Noxa, and displays favorable in vivo DMPK properties. Using a genetic Mcl-1 knock-in (KI) CRC cell line to mimic the effect of FBW7 loss, we confirmed the in vitro resistance of these KI cells to single agent regorafenib treatment and restored apoptosis when treated in combination with 483LM. Importantly, the in vivo combination treatment of 483LM and regorafenib completely overcome Mcl-1-mediated regorafenib resistance in HCT116 KI CRC xenograft tumors. Pharmacodynamic evaluation of tumor samples revealed significant caspase-3 activation in 483LM treated tumors. The proximity ligation assay revealed the ability of 483LM to induce significant disruption of PUMA:Mcl-1 complexes in treated tumor samples. These data confirm the previous reports of the involvement of Mcl-1 in regorafenib resistance in CRC and provides rationale for further investigation into the clinical application of pharmacological Mcl-1 inhibition in combination therapies to combat resistance. In addition, these results provide evaluation and characterization of a novel Mcl-1 selective small molecule inhibitor with potent in vivo activity and potential to treat various malignancies.

#1971

A novel approach to overcome resistance to HER2 targeted therapy in a new syngeneic model of breast cancer brain metastasis.

Normand Pouliot,1 Aadya Nagpal,1 Miriam Fuentes,1 Effie Mouhtouris,1 Francesca Avogadri-Connors,2 Alshad S. Lalani,2 Heloisa Sobreiro Selistre de Araujo3. 1 _Olivia Newton John Cancer Research Institute (ONJCRI) and School of Cancer Medicine, La Trobe University, Heidelberg, Australia;_ 2 _Puma Biotechnology, Inc, Los Angeles, CA;_ 3 _Federal University of São Carlos, SP, Brazil_.

The human epidermal growth factor receptor-2 (HER2) subtype of breast cancer is aggressive and associated with a high propensity to metastasize to the brain. Whilst the introduction of HER2-targeted therapies has significantly improved survival outcomes in affected patients, effective treatment of brain metastasis remains a challenge due to acquired resistance and limited drug permeability across the blood brain barrier. Moreover, progress in identifying more effective treatments, particularly against therapy-resistant brain-metastatic HER2 breast cancer, has been hampered by the lack of robust and clinically relevant preclinical models. Here, we introduce a new Balb/c syngeneic model of spontaneous breast cancer brain metastasis that naturally expresses HER2 (RCSI-Her2). In vivo metastasis assays confirmed that this model gives rise to high incidence of spontaneous brain metastases from mammary tumors or experimental brain metastases following intra-cardiac inoculation. RT-PCR, western blotting and immunohistochemical analyses of RCSI-Her2 cells, tumors or brain metastases confirmed the lack of hormone receptor expression but high levels of HER2. Accordingly, RCSI-Her2 cell proliferation is inhibited by HER2 inhibitors, lapatinib or neratinib, but not by anti-estrogens, indicating phenotypic and functional similarities to human HER2 breast cancer. Importantly, short term in vitro proliferation assays show that the combination of neratinib with αvβ3 integrin inhibitors (Cilengitide or DisBa-01) potently and synergistically inhibits the proliferation of RCSI-HER2 or brain-metastatic triple negative breast cancer cells (4T1Br4). Moreover, treatment of neratinib-resistant RCSI-Her2 variants with Cilengitide or DisBa-01 restores sensitivity to neratinib. Preliminary in vivo experiments indicate that neratinib is well-tolerated in immune-competent mice, inhibits RCSI-HER2 primary tumour growth and extends survival. Transcriptomic analysis (RNAseq) of neratinib-sensitive and resistant variants treated or not with neratinib to identify signaling pathways implicated in resistance is underway. The results from this analysis will be presented. To our knowledge, RCSI-Her2 is the only model that fully recapitulates the spontaneous spread of HER2 breast cancer to the brain in immune-competent hosts. This model provides a unique tool to validate the efficacy of neratinib/αvβ3 integrin inhibitors in vivo against advanced brain-metastatic and therapy-resistant HER2 breast cancer.

#1972

Targeting the IL-6 signaling pathway overcomes cetuximab resistance in head and neck squamous cell carcinoma.

Rachel A. O'Keefe, Neil E. Bhola, David S. Lee, Daniel E. Johnson, Jennifer R. Grandis. _University of California, San Francisco, San Francisco, CA_.

Head and neck squamous cell carcinoma (HNSCC), the sixth most common cancer worldwide, has a five-year survival rate of 50%. The last few decades have seen only marginal improvements to the survival rate due to high rates of resistance to currently available treatments. The only oncogene-targeted agent that has been FDA approved for the treatment of HNSCC is cetuximab, a monoclonal antibody directed against the epidermal growth factor receptor (EGFR). The response rate for single-agent cetuximab is below 20%, and both intrinsic and acquired resistance to cetuximab remain major obstacles in the treatment of HNSCC. Identification and targeting of mediators that confer cetuximab resistance are needed to improve patient outcomes in HNSCC and in other cancers in which cetuximab is employed. To address this unmet need, we generated cetuximab-resistant variants of the human HNSCC cell lines PE/CA-PJ49 and FaDu. Because secretion of interleukin 6 (IL-6) has been proposed as a mechanism of cetuximab resistance in HNSCC, we assessed IL-6 levels in these models of acquired resistance. IL6 mRNA expression was increased in PE/CA-PJ49 and FaDu cells that have acquired resistance to cetuximab compared to the parental cells from which they were derived. In addition, cetuximab-resistant PE/CA-PJ49 variants exhibited increased levels of glycoprotein 130 (gp130), a molecule that oligomerizes with IL-6R alpha (IL-6Rα) to form the IL-6 receptor. Since IL-6 is known to promote tumor cell proliferation and survival, we hypothesized that cetuximab-resistant PE/CA-PJ49 and FaDu variants would be sensitive to inhibition of components of the IL-6 signaling pathway. To test this hypothesis, we treated PE/CA-PJ49 parental cells and cetuximab-resistant variants with cetuximab and/or inhibitors of the IL-6 pathway. Colony formation assays revealed that cetuximab-treated parental cells formed fewer colonies than vehicle-treated cells, while cetuximab treatment did not affect the ability of cetuximab-resistant PE/CA-PJ49 variants to form colonies. In contrast, siRNA-mediated knockdown of IL6 or IL6R (encoding IL-6Rα) markedly reduced the colony-forming ability of the cetuximab-resistant variants, but not parental cells. These results suggest that targeted inhibition of IL-6 signaling inhibits proliferation and/or survival in cetuximab-resistant PE/CA-PJ49 cells. Ongoing studies are testing our hypothesis using small-molecule and antibody-based inhibitors of the IL-6 pathway in our cell line models and in cetuximab-resistant patient-derived xenografts. By conducting these experiments, we aim to assess the therapeutic potential of targeting the IL-6 pathway in cetuximab-resistant HNSCC.

#1973

Targeting PARG in pancreatic cancer: Implications for synthetic lethal therapeutic strategies.

Saswati N. Chand,1 AnnJosette Ramirez,1 Aditi Jain,1 Avinoam Nevler,1 Cinthya Yabar-Lowder,1 Joseph A. Cozzitorto,1 Dominic I. James,2 Allan Jordan,2 Kate M. Smith,2 Ian Waddell,2 Charles J. Yeo,1 Jordan M. Winter,1 Jonathan R. Brody1. 1 _Thomas Jefferson University, Philadelphia, PA;_ 2 _Cancer Research UK Manchester Institute, University of Manchester, Manchester, United Kingdom_.

Metastatic pancreatic ductal adenocarcinoma (PDA) has an average survival of less than one year. There is a pressing need to identify patient subgroups for treatment with novel targeted agents and the necessity to combat increasing incidence of therapeutic resistance. In a previous study, we identified that poly(ADP) ribose glycohydrolase (PARG) is a critical player in mediating resistance to PARP inhibitor (PARPi); therefore targeting PARG is a strategy to enhance PARPi therapy in PDA and can be optimized to benefit patients with or without homologous repair (HR) deficiencies. We developed and characterized multiple PARG inhibition models in both DNA- repair proficient (MIA PaCa-2) and deficient (Hs766t) PDA lines; doxycycline-inducible shPARG knockdown, CRISPR- mediated PARG knockout and small molecule inhibition via a series of potent first-in-class, cell-active PARG inhibitors (PARGi). Our data show that PARG inhibition is synthetic lethal with DNA damage repair deficiency in PDA cells. This was further validated in isogenic colorectal cell lines with varying DNA repair functionality: DLD1 lines with BRCA2 (+/+, +/-, -/-) and RKO lines with FANCC (+/+, +/-, -/-). We have also shown that PARG inhibition enhances PARPi sensitivity through increased accumulation of DNA damage, apoptosis and persistence of detrimental PARylation. Moreover, PARP1 was trapped on the chromatin in response to both PARPi treatment as well as DNA damaging agents such as oxaliplatin. Complementary xenograft experiments were performed wherein MIA.shPARG cells were injected in nude female athymic mice. At an average tumor volume of 50mm3, respective groups were fed DOX- chow to induce PARG knockdown and treated with olaparib intraperitoneally at 100mg/kg five times a week. PARG inhibition by doxycycline induction significantly decreased tumor volumes (50% decrease, p-value 0.0165), which was further enhanced with olaparib treatment (70% decrease, p- value 0.0004), when compared with control arms. Similar results were obtained when DOX-fed mice with MIA.shPARG cells were treated with olaparib at 50mg/kg. Furthermore, in an attempt to mimic and break long-term in vivo PARPi resistance, doxycycline-mediated PARG inhibition was induced in the olaparib treatment arm on day 56 (with established tumors, and exposed to olaparib for 3weeks i.e. 15 injections). This resulted in a significant decrease in tumor volume when compared to control untreated arm (46% decrease, p-value 0.0025) and the olaparib only treatment arm (25% decrease, p-value 0.0124). We are currently validating these results in a DDR-deficient HST.shPARG cell line, as well as with CRISPR knockouts of PARG. Together these studies validate PARG as a therapeutically relevant and "druggable" target in both HR-proficient and deficient PDA cells, and lays the groundwork to optimize PARPi-based as well as other DNA targeted therapies in the treatment of PDA.

#1974

Inhibition of the P-glycoprotein with a novel agent in overexpressing P-glycoprotein chemotherapy resistant colorectal cancer cells.

Christopher L. Farrell, Rebecca Mahan, Catherine Blauvelt, Taylor Kaye Servais, Jessica Lane. _Presbyterian College, Clinton, SC_.

Multidrug resistance (MDR) plays a key role in chemotherapy failure and poor patient outcome. One of the ways multidrug resistance occurs is through the efflux of chemotherapeutic agents using the P-glycoprotein transporter protein on the surface of the tumor cell. There are several medications that work through P-glycoprotein (Pgp), which can either induce or inhibit the activity of the transporter. The purpose of this study was to evaluate known P-glycoprotein inhibitors and their effects on chemotherapy resistance in Caco2 cells, a colorectal cancer cell line. The experiments consisted of feeding the colorectal cancer cells with commonly known drugs that are Pgp substrates such as vinblastine for 4-8 months to establish an overexpressing Pgp transporter. An untreated negative control was used to show that Pgp was not overly expressed. Once the treated cells were overexpressing Pgp, the cells were seeded into a 96 well plate with a matrix of increasing chemotherapy and bergamottin, a known P-glycoprotein inhibitor. A cell proliferation assay was performed according to manufacturer protocol, with a Presto-Blue Cell Proliferation Assay kit to assess the effects of the chemotherapy agent, vinblastine and the bergamottin. Absorbance was recorded at 595 nanometers. This assay was tested on several multidrug resistant colorectal cell with different substrates and normal P-glycoprotein expressing cancer cells. The overexpressing Pgp cell lines required higher doses of vinblastine to exhibit cell death compared to the negative cells. The positive cells treated with bergamottin only showed change in cell death with increasing bergamottin concentration. From these results, it was determined that the use of the novel P-glycoprotein inhibitor was able to reverse the chemotherapy induced multidrug resistance in the cells with high Pgp expression. Patients whose tumor cells develop MDR have a poor prognosis compared to patients whose cells are responsive to chemotherapy. Because the chemotherapy agents are ineffective in killing the all of the tumor cells including cells which have become metastatic, the overall survival rate for patients with MDR-positive tumors is markedly decreased. Identify these patients as potential candidates for the Pgp inhibitor could significant increase their chances of survival with this aggressive form of cancer.

#1975

Disulfiram targets hypoxia-induced cancer stem cells and reverses chemoresistance in non-small cell lung cancer.

Kate Butcher,1 Vinodh Kannappan,1 Karim Azar,1 Rajagopal Kilari,1 Ogechi Nkeonye,1 Mark R. Morris,1 Christopher McConville,2 Yaohe Wang,3 Weiguang Wang1. 1 _University of Wolverhampton, Wolverhampton, United Kingdom;_ 2 _University of Birmingham, Birmingham, United Kingdom;_ 3 _Queen Mary University of London, London, United Kingdom_.

Background: This study aims to repurpose disulfiram (DS), a drug used to treat alcohol dependence, into an effective treatment for non-small cell lung cancer (NSCLC). NSCLC is the leading cause of cancer related death worldwide because of early metastasis and chemoresistance. Cancer stem cells (CSCs) play a key role in resistance and metastasis. Our previous studies indicate that tumor hypoxia induced activation of the nuclear factor-κB (NF-κB) pathway, a pivotal regulator of CSCs. Therefore, development of an NF-κB and CSCs targeting drug will improve NSCLC therapeutic outcomes. New drug development is an expensive and time-consuming procedure. Cytotoxicity of DS is copper (II) (Cu)-dependent. DS/Cu induces reactive oxygen species, inhibits NF-κB activity and demonstrates excellent in vitro anti-CSCs activity in a wide range of cancers. The clinical application of DS as an anticancer drug is impeded by its very short half-life in the bloodstream. To improve the drug delivery efficiency, we developed a poly lactic-co-glycolic-DS (PLGA-DS), which demonstrates strong anti-cancer efficacy in mouse NSCLC models. Methods: NSCLC cell line A549 and H23 cultured as spheroids, hypoxia and PCDNA-3.1 NF-κB p65 transfect A659 cell lines. The cytotoxic effect of DS/Cu analyzed using sphere reformation, clonogenic and MTT assays. Flow cytometric analysis to detect CSC markers ALDH and CD44, and analysis of apoptosis by Annexin V. Western blot analysis to determine expression of embryonic stem cell markers Nanog and OCT4, and identify the importance of the NF-κB pathway together with HIF and including expression of associated apoptotic proteins Bax and Bcl2. Results: CSC and hypoxic cultured cells expressed high levels of CSC markers and were resistant to first and second line NSCLC anticancer drugs (doxorubicin, oxaliplatin, paclitaxel and gemcitabine). High NF-κB expression was detected in CSCs and hypoxia-cultured NSCLC cell lines. After transfection with p65 subunit of NF-κB, A549 cells expressed CSC markers and became resistant to a wide range of anticancer drugs. DS (5-10 nM) supplemented with Cu (10 μM) induced cytotoxicity to hypoxic cultured NSCLC cells, DS (1 μM) in combination with Cu inhibited sphere reformation. DS/Cu effectively inhibited NF-κB activity, abolished the CSC population and synergistically enhanced the cytotoxicity of the above conventional anticancer drugs. Our study also shows that PLGA-DS extends the half-life of free DS in the blood stream from 30 seconds to 7 hours. Intravenous injection of PLG-DS in combination with oral Cu can effectively inhibit the growth of subcutaneous and lung NSCLC xenografts. Conclusion: DS/Cu specifically inhibits NF-κB pathway and targets CSC in NSCLC cell lines. PLGA encapsulation improves delivery of DS which demonstrated very strong anticancer activity in NSCLC xenografts in vivo.

#1976

Identification of 4 novel markers in the chemotherapy resistance of esophageal adenocarcinoma.

Jonathan P. Fleegel, Joeseph Abdo, Devendra Agrawal. _Creighton University, Omaha, NE_.

Esophageal adenocarcinoma (EAC) has seen a steady and steep rise in prevalence since the 1970's. This increase in incidence is correlated with the increase in gastro-esophageal reflux disease and Barrett's esophagus (BE). Unfortunately, where many other cancers have seen progress with advancements in treatments, EAC has not. This cancer is very resistant to almost all first line chemotherapy treatments including the most common platinum based treatments. Although few targeted therapies have been developed, they only show a modest survival advantage for eligible individuals and are not commonly integrated into patient treatment regimens. In our study, we have identified several markers associated with resistance to platinum based chemotherapies that have not previously been documented in EAC. Our study utilized a large-scale discovery mass spectrometric experiment such that the proteome of the 3 different tissue samples could be analyzed, (EAC, BE, and Normal). Part of the novelty of our experiment was that all the tissues collected were from esophagetomies of patients before they had received any chemotherapy. From the data generated we have identified 4 novel markers that are overexpressed in the BE and EAC tissue compared to the normal tissue. In BE tissue DAD1 was over expressed in 9/10 samples, ISG15 (10/10), S100P (10/10) and UBE2N (10/10). In the EAC samples, DAD1 was significantly overexpressed in 20 /20 samples, ISG15 (20/20), S100P (20/20) and UBE2N (19/20). DAD1 inhibits apoptosis and is upregulated by cisplatin and then contributes to cisplatin resistance. S100P has been shown to increase drug resistance in gastric cancer and decrease chemosensitivity 5-FU in vitro in colorectal cancer cells while facilitating proliferation and inhibiting apoptosis. UBE2N plays a role in glycolytic control and in various cellular damage responses. And ISG15 is important in development and metastatic progression of adenocarcinomas through the enhancement of cancer stem cells. We are confident that expression of these four markers can provide clinical utility in assessment of at risk patients with Barrett's esophagus while simultaneously informing treatment regimens. Our goal is to investigate the specific mechanisms by which these markers contribute to cancerous development and chemotherapy resistance in order to elucidate targets for future treatment of EAC.

#1977

KIFC1 is a potential target to prevent treatment resistance in prostate cancer.

Gonzalo Torga,1 Ke-Chih Lin,2 Bernat Navarro Serer,1 Cathleen Nguyen,1 Robert H. Austin,2 Kenneth J. Pienta1. 1 _Johns Hopkins University, Baltimore, MD;_ 2 _Princeton University, Princeton, NJ_.

Metastatic prostate cancer remains incurable. Taxane-based chemotherapy used to be restricted to the hormone-resistant metastatic setting. Unfortunately, most patients with castration-resistant prostate cancer (CRPC) rapidly develop resistance to chemotherapy. Recently, two trials demonstrated increased survival in men with high-risk metastatic hormone-sensitive prostate cancer treated with adjuvant chemohormonal therapy. Polyploidy has been demonstrated in most types of tumors to some extent and proposed to drive tumor progression by increasing the potential for cellular transformation. However, whether this is a cause or a consequence of cancer remains unclear. Different molecular mechanism can lead to polyploidy, such as mitotic slippage, endo-replication or cell fusion. In cancer biology, it has been associated with epithelial-mesenchymal transition (EMT), as well as with gain of stem-like potential. Experiments with drug gradients in our engineered microfluidic device have shown docetaxel therapy leads to polyploidy with a stiff distribution pattern of heterogeneous cell populations across the gradient. Moreover, in these experiments we evidenced polyploidization events occurring concurrently with the development of drug resistance. To confirm these findings, we conducted experiments in conventional cell culture using different chemotherapeutic agents in several prostate cancer cell lines and confirmed the development of polyploidy was a common feature among them. Interestingly, this feature occurred at some extent for all drugs, independently of the mechanism of action of the drug tested. Recent publications demonstrated the association of multinucleation with chemotherapy, androgen-deprivation therapy and radiotherapy. Although the data supporting the association of polyploidy with resistance is extensive, the effects of these therapies on tumor heterogeneity remains unknown. Most polyploid cells undergo cell death due to a mitotic catastrophe subsequent to multipolar cell division, however a small percentage of them survive and produce viable progeny of chemotherapy resistant clones via asymmetric cell division by undergoing centrosome clustering mediated by KIFC1. In this work, we focus on the characterization of the subset of cells within the tumor population that leads to the polyploid phenotype, particularly the subpopulation with potential to reverse polyploidy and repopulate tumor heterogeneity after therapy. We hypothesize a subset of polyploid cells with stem-like features may be the reservoir of therapeutic resistance in cancer. Given KIFC1 is required for centrosome clustering and clinical data suggests its association with polyploidy, poor prognosis and previous taxane-therapy in prostate cancer, we studied KIFC1 as a potential target to prevent relapse after therapy.

#1978

Antibody-drug conjugates with DNA inhibitor linker-payloads can overcome resistance in cultured tumor cells made resistant to conjugates with cleavable-linked auristatins.

Xingzhi Tan, Matthew Sung, Frank Loganzo. _Pfizer, Inc., Pearl River, NY_.

Antibody drug conjugates (ADCs) are targeted biotherapeutics delivering cytotoxic payloads to tumor-expressed antigens. The most common class of ADC payloads are microtubule inhibitors (MTI) and DNA inhibitors, exemplified by clinically approved ado-trastuzumab emtansine (TDM1; MTI DM1), brentuximab vedotin (MTI auristatin), gemtuzumab ozogamicin and inotuzumab ozogamicin (DNA double strand breaker calicheamicin). Auristatin payloads are in active development, primarily employing proteolytically cleavable linker mcValCitPABC. There are many benefits of cleavable-linked permeable payloads, including bystander activity in a heterogeneous tumor environment. However, due to the complexity of human cancer, tumors become refractory to most drug therapies. We hypothesized that cultured tumor cells chronically treated with mcValCitPABC-auristatin ADCs would acquire varied resistance mechanisms. As model ADCs, we used trastuzumab-mcValCitPABC-mono methyl auristatin E (TvcMMAE) and trastuzumab-mcValCitPABC-Aur0101 (Tvc0101). Her2 expressing gastric carcinoma line, N87, was chronically exposed to the respective ADCs at cytotoxic doses in a cyclical fashion (3 days on; ~1 - 3 weeks off). Resistance was acquired very slowly (over ~1 year), and cells were characterized for their resistance profile. N87-TvcMMAE cells had >1000X relative resistance (RR) to T-auristatin and TDM1 ADCs compared with parental N87, and showed moderate to high resistance (~50 – 300X) to unconjugated MTIs including auristatins and DM1. The cells were cross-resistant (>100 - 1000X) to T-ADCs delivering several payload classes, including NAc-calicheamicin and cyclopropylpyrroloindolone (CPI). Her2 and MDR1 levels were unchanged in N87-TvcMMAE cells, however, a slight increase of MRP1 was observed on immunoblots. Cells made resistant to Tvc0101 ADC showed a different profile. N87-Tvc0101 cells had >1000X RR to T-auristatin ADCs and >400X to TDM1, but were sensitive (<4X) to unconjugated auristatins and DM1. Interestingly, these cells retained sensitivity to T-ADCs delivering DNA inhibitor payloads, including NAc-calicheamicin (<5X) and CPI (<14X). These data are striking since N87-Tvc0101 cells had significantly reduced Her2 levels, yet responded to other Her2 ADCs. Immunoblots indicated no observed changes in MDR1 or MRP1 protein. The cross-resistance profile of each cell model is distinct, despite using the same parental cell background and ADCs differing in the auristatin. These data suggest that different resistance mechanisms develop upon treatment with structurally similar mcValCitPABC-auristatin ADCs, and that resistant tumors may respond to ADCs delivering a different linker-payload yet targeting the same antigen, even in cases where antigen levels have decreased.

#1979

HQP1351, a novel multikinase inhibitor in clinical development, overcomes drug resistance for the treatment of gastrointestinal stromal tumors in preclinical models.

Guangfeng Wang,1 Haibo Qiu,2 Ping Min,1 Miaoyi Wu,1 Shuo Dang,1 Chunyang Yang,1 Fei Zhang,1 Wei Zhuang,2 Zhiwei Zhou,2 Douglas D. Fang,1 Dajun Yang,1 Yifan Zhai1. 1 _Ascentage Pharma Group Corp., Limited, Suzhou, China;_ 2 _Sun Yat-Sen University Cancer Center, Guangzhou, China_.

Gastrointestinal stromal tumors (GIST) harbor driver mutations of signal transduction kinases such as KIT. Besides surgical resection for primary localized tumors, imatinib remains the first-line treatment for advanced and metastatic GISTs. Imatinib targets a few kinases including KIT that often carries the primary driver mutations commonly located on exon 11 and exon 9. Unfortunately, a large proportion of the patients ultimately develop progressive disease owing to the secondary resistance mutations in KIT gene. We have developed an orally bioavailable multikinase inhibitor HQP1351, which is currently in clinical trials for the treatment of T315I mutant CML. Here, we demonstrated that HQP1351 inhibited both wild type and mutant (i.e., KITL576P, KITV559D, and KITV559D/T670I) KIT in biochemical assays. Using a panel of imatinib-resistant and sensitive GIST cancer cell lines derived from patient samples, we showed that HQP1351 exhibited more potent anti-proliferative activities than ponatinib (range of IC50 values: 0.027-0.133 µM vs. 0.021-0.730 µM), the latter of which is currently in clinical development in GIST to overcome the resistance to imatinib (NCT03171389). In addition, the treatment of GIST cancer cells with HQP1351 in vitro led to more profound inhibition of pharmacodynamic markers, including p-c-KIT, p-AKT, p-ERK1/2, and p-AKT, in comparison with ponatinib. Correspondingly, in multiple xenograft tumor models derived from these GIST cancer cell lines carrying the secondary mutations of KIT, HQP1351 exhibited superior antitumor activities to ponatinib. Collectively, considering that HQP1351 has already been in clinical trials, the above results suggest that therapeutic application of HQP1351 in imatinib-resistant GIST patients deserves further investigation in human.

#1980

S116836 overcomes BCR-ABL T315I mutation mediated imatinib resistance in chronic myeloid leukemia.

Pranav Gupta,1 Guannan Zhang,1 Anna Maria Barbuti,1 Ke Ding,2 Jingxuan Pan,3 Brian J. Druker,4 Zhe-Sheng Chen1. 1 _St. John's Univ. College of Pharmacy, Fresh Meadows, NY;_ 2 _Key Laboratory of Regenerative Biology and Institute of Chemical Biology; Guangzhou Institute of Biomedicine and Health; Chinese Academy of Sciences; Guangzhou, China;_ 3 _State Key Laboratory of Ophthalmology; Zhongshan Ophthalmic Center; Sun Yat-sen University; Guangzhou, China;_ 4 _Division of Hematology and Medical Oncology, The Knight Cancer Institute, Oregon Health and Science University, Portland, OR_.

Chronic Myeloid Leukemia (CML) is largely caused by the Philadelphia (Ph) chromosome carrying the Break point Cluster Region-Abelson (BCR-ABL) oncogene. Imatinib is a BCR-ABL-targeted therapy and considered the standard of care in CML management. Resistance to imatinib therapy often develops because of mutations in the BCR-ABL kinase domain. In this study, we evaluated S116836, a novel BCR-ABL inhibitor, for its anti-cancer activity against BCR-ABL expressing BaF3 cells. S116836 shows potent activity against wild-type and T315I mutated BaF3 cells as compared with imatinib. S116836 inhibited the phosphorylation of BCR-ABL and its downstream signaling in BaF3/WT and BaF3/T315I cells. S116836 inhibited the mRNA expression of BCR-ABL in BaF3/WT and BaF3/T315I cells. Mechanistically, S116836 increased the cell population in the G0/G1 phase of the cell cycle, induced apoptosis and elevated ROS production in both BaF3/WT and BaF3/T315I cells. Moreover, S116836 inhibited tumor growth in BaF3/WT and BaF3/T315I tumor xenografts. Taken together, our results indicate that S116836 exhibits anti-proliferative effects and inhibits the imatinib-resistant T315I BCR-ABL mutation. S116836 may be a novel drug candidate for overcoming the resistance to imatinib in CML patients.

Keywords: S116836, Chronic myeloid leukemia, BCR-ABL, T315I, Resistance

#1981

Pan-cancer pathways gene expression profiling in mantle cell lymphoma reveals upregulation of DNA damage repair genes in ibrutinib-resistant tumor.

Makhdum Ahmed, Elizabeth Lorence, Hui Guo, Shengjian Huang, Victoria Zhang, Hui Zhang, Liang Zhang, Krystle Nomie, Michael Wang. _MD Anderson Cancer Center, Houston, TX_.

Introduction Mantle cell lymphoma (MCL) accounts for 7% of all non-Hodgkin lymphoma cases and is an incurable subtype of B-cell lymphoma. Ibrutinib, a first-in-class, once-daily, oral covalent inhibitor of Bruton's tyrosine kinase (BTK), was approved by the FDA in 2013 for relapsed-refractory MCL with an overall response rate of 68%. Unfortunately, resistance to ibrutinib inevitably develops and once patients relapse after ibrutinib treatment, the 1-year survival rate is only 22%, and median survival is only 8 months. If the response to ibrutinib can be predicted by genetic biomarkers, the ability to personalize therapy in the clinic would greatly benefit; therefore, we aimed to identify the gene expression profile associated with ibrutinib resistance in clinical samples.

Methods We used clinical samples (N=16) collected from relapsed MCL patients to extract RNA, and the RNA samples were used to conduct gene expression profiling of 770 genes using Nanostring® Pan Cancer Pathways Panel in the nCounter® system (Seattle, WA). Gene expression profiling analysis was adjusted for technical and biological covariates such as binding density and sample type. We adjusted the analysis for multiples statistical comparisons using the Benjamini-Yekutieli method.

Results Of the 16 clinical samples, 5 (31%) were resistant to ibrutinib as identified in clinical annotations. In univariate analysis, binding density was significantly higher in ibrutinib-resistant samples compared with the sensitive samples (P-value <0.001). After adjusting for binding density and sample type, we reported top 20 genes differentially expressed in the resistant samples compared with the ibrutinib-sensitive samples. SMC1B, FGFR1, CDK2, PCNA, RFC4 are among the top 20 differentially expressed genes and, belong to cell cycle control, DNA damage repair, MAPK and PI3K pathways. The pathway scores revealed that the ibrutnib-resistant samples had higher expression of DNA damage repair genes but lower expression of apoptotic genes, which is in contrast with the ibrutinib sensitive samples. Additionally, ibrutinib-resistant samples had significantly higher expressions of PI3K and NOTCH pathway genes and lower expression of JAK-STAT pathway genes as compared with the sensitive samples. These findings of differential expression of apoptosis and DNA repair genes in ibrutinib-resistant and sensitive samples were independently validated by RNA-seq experiments, presented elsewhere.

Conclusions In ibrutinib-resistant MCL samples, the ability to increase DNA damage repair and relative lower expression of apoptotic genes may explain the resistance mechanism. MCL cells may be vulnerable to therapeutic intervention targeting DNA damage repair pathways.

## TUMOR BIOLOGY:

### Epithelial Cancer Stem Cell Biology

#1982

Obesity reversibly enhances mammary epithelial estrogen receptor alpha expression and progenitor activity.

Tamara Chamberlin, Joseph V. D'Amato, Lisa M. Arendt. _Univ. of Wisconsin School of Veterinary Med., Madison, WI_.

Obesity has been identified as an important risk factor for breast cancer in post-menopausal women and is significantly correlated with poor prognosis in breast cancer patients. Women with an increasing body mass index (BMI) are more likely to develop estrogen receptor alpha (ERα) positive tumors that are larger at the time of diagnosis and demonstrate an increased risk for metastasis. Given that obesity is becoming a global epidemic, understanding how obesity alters the biology of the normal breast is of significant clinical relevance. To examine the effects of obesity on mammary tissue, mice were fed a high fat diet to induce obesity and compared with mice fed a control diet. We also examined reduction mammoplasty tissue from women with a known BMI. In both obese female mice and women, mammary epithelial cell populations demonstrated significant decreases in basal/myoepithelial cells, when examined using either flow cytometry or cell type-specific markers (SMA and p63) in tissue. ERα expression was significantly increased in luminal cells in obese mammary tissue, compared with control mice or breast tissue from lean women. Functional assays demonstrated significantly enhanced mammary epithelial progenitor activity in obese mammary epithelial cells as well as elevated numbers of ERα positive epithelial cells that were co-labeled with markers of proliferation. Weight loss in a group of obese mice reversed increases in progenitor activity and ERα expression observed in obese mammary tissue. Obesity enhances the percentage of ERα positive epithelial cells and reduces the number of basal/myoepithelial cells within normal mammary tissue in both women and female mice. Both aspects of this phenotype have been linked to breast tumor susceptibility. These changes may contribute to lifetime risk for the development of ERα positive tumors in postmenopausal women. However, the changes in epithelial cell populations induced by obesity are reversible with weight loss, suggesting that weight loss interventions in obese women may effectively reduce breast cancer risk by decreasing the number of potential tumor progenitor cells in the breast epithelium.

#1983

**Enriching EpCAM** + **/Sca-1** \+ **mouse bronchioalveolar stem cells to generate lung adenocarcinoma.**

Takashi Semba,1 Ryo Sato,1 Akiyoshi Kasuga,1 Makoto Suzuki,2 Hideyuki Saya,1 Yoshimi Arima1. 1 _Keio University, Tokyo, Japan;_ 2 _Kumamoto University, Kumamoto, Japan_.

Background: Clarifying the cells of origin in human lung adenocarcinoma will contribute to a better understanding of oncogene-induced lung adenocarcinomas and to the development of new treatments. Alveolar type II cells and club cells (Clara cells) have been shown to be the cells of origin by lineage-tracing approaches and conditional transgenic mice. Additionally, bronchioalveolar stem cells (BASCs), which are adult murine distal lung epithelial stem cells, are considered to be the cells of origin in lung adenocarcinomas because BASCs have both self-renewal abilities and differentiation properties. We attempted to enrich BASCs from mouse lungs and determine whether or not BASCs have the potential to form tumors driven by the oncogene.

Methods: The epithelial cell adhesion molecule (EpCAM)+/CD45-/CD31- lung cells were isolated from the Ink4a/Arf-/- C57BL/6 mice by using a fluorescence activated cell sorting (FACS). The cells were then subjected to 3D culture on Matrigel with B27, EGF, KGF, ROCK inhibitor in serum-free DMEM/F-12. For detection of BASCs, we investigated the expressions of EpCAM and Sca-1 by flow cytometric analyses. We also investigated the expressions of SP-C and CC-10 by immunofluorescence analyses. The cells were then infected with bicistronic retroviruses having green fluorescent protein (GFP) and either KRASG12V or EML4-ALK genes. The GFP-positive oncogene-expressing cells were sorted by FACS and expanded under 3D culture conditions. Cells were then transplanted into the recipient C57BL/6 mice so that we could investigate their tumorigenic activities.

Results: Flow cytometric analyses revealed that the EpCAM+/Sca-1\+ cells, which are putative BASCs, were enriched in the 3D culture of the isolated EpCAM+/CD45-/CD31- lung cells from Ink4a/Arf-/- mice. The percentage of EpCAM+/Sca-1\+ cell fraction was 36.97%, 92.63%, 99.98%, at primary EpCAM+/CD45-/CD31- lung cells and after the first and second passage, respectively. We also detected a small population of SP-C+/CC-10+ cells in colonies formed under our 3D culture conditions. The oncogene-expressing cells maintained the population of EpCAM+/Sca-1\+ cells; 88.64-99.89% and 72.72-99.12%, KRASG12V and EML4-ALK, respectively. These cells formed differentiated tumors which were similar to human lung adenocarcinomas, having papillary, acinar, and solid components.

Conclusions: The EpCAM+/Sca-1+ lung cells, putative BASCs, from Ink4a/Arf-/- mice were enriched in 3D culture. These cells formed histologically different types of lung adenocarcinomas after the transduction of KRASG12V or EML4-ALK, suggesting that we had established the source of the lung adenocarcinoma cells.

#1984

Cell fate reprogramming of liver tumor-initiating stem-like cells via phosphorylated NUMB and TBC1D15.

Keigo Machida, Hifzur R. Siddique, Mengmei Zheng, Peleg Winer, Dinesh Babu Uthaya Kumar, Ahmed Rokan, Linda Sher, Stanley M. Tahara, Michael Elowitz, Chengyu Liang, Hidekazu Tsukamoto. _University of Southern California Keck School of Medicine, Los Angeles, CA_.

Tumor-initiating stem-like cells (TICs) are defective in their control of asymmetric division. Stem cells are maintained through asymmetric self-renewing divisions in which one daughter cell commits to a specific fate while the other retains the multipotent characteristics of its parent. The p53 interacting protein NUMB preserves this intrinsic cellular asymmetry and functions as a vital barrier against unchecked expansion as seen in TICs. How TICs overcome this control of asymmetric division to cause cancer is unknown. The hypothesis is that accentuated TLR4-NANOG-mediated NUMB phosphorylation and TBC1D15 upregulation promote self-renewal of TICs in liver oncogenesis caused by HCV and alcohol. Here, we determined the causal roles of NUMB phosphorylation and TBC1D15 oncoprotein in alcohol-induced liver tumorigenesis in HCV-Tg mice. Liver-specific TBC1D15 deficiency or non-phosphorylatable mutations of NUMB, reduce liver tumor incidence and tumor-associated NANOG+ TICs in HCV NS5A Tg mice fed alcohol Western diet. TICs lost asymmetric division capacity and overexpressed TBC1D15, which associated with NUMB to promote aPKCζ activity and NUMB phosphorylation. Thus, TIC self-renewal is dependent on TBC1D15 and NUMB phosphorylation. Similarly, aPKCζ-mediated NUMB phosphorylation and induced TBC1D15 are mutually required for self-renewal. Furthermore, TBC1D15 activated NOTCH via binding to NOTCH intracellular domain to trigger its proteolytic. TBC1D15 interacts with all NOTCH isoforms, activates NOTCH pathway, and induces Nanog and TIC self-renewal in a NOTCH-dependent manner. TBC1D15 cooperates with the NOTCH pathway to support TIC tumorigenic activity and an inhibitor of this interaction is potentially therapeutic. Candidate small molecule antagonists have been tested for their ability to block the interaction of TBC1D15-NOTCH/NICD. The originality of this findings lies within the discovery of the novel oncoprotein TBC1D15 and its unique oncogenic activities involving p53 degradation and cooperation with the NOTCH pathway in TICs and CD133+ Huh7 cells. These mechanistic findings have laid down the foundation for a new translational path for discovery of new therapeutic targets.

#1985

Upregulation of miR-328 contributes to ovarian cancer stem cell maintenance by downregulating DDB2.

Amit K. Srivastava, Tiantian Cui, Chunhua Han, Ananya Banerjee, Shuri Cai, Lu Liu, Xiaoli Zhang, Zaibo Li, Selvendiran Karuppalyah, Altaf A. Wani, Qi-En Wang. _Ohio State Univ., Columbus, OH_.

Cancer stem cells (CSCs) are considered to play a central role in the cancer progression, metastasis and the development of drug resistance. MicroRNAs (miRNAs) have important roles in regulating CSC properties and are considered to be potential therapeutic targets. Diverse aberrantly expressed miRNAs have been reported in ovarian cancer cells. However, there have been few reports about miRNAs that were associated with stemness and progression of ovarian cancer. In this study, miRNA Nanostring profiling analysis was performed to screen crucial miRNAs associated with characteristics and maintenance of CSCs in ovarian cancer. We found that miR-328-3p was remarkably upregulated in ovarian CSCs isolated from both ovarian cancer cell lines and primary ovarian tumors compared to their corresponding bulk cancer cells. We further demonstrated that enforced expression of miR-328-3p in ovarian cancer cell lines expanded the population of ALDH+ cells, enhanced their sphere formation ability, as well as increased their tumorigenicity. While inhibition of miR-328-3p limited the ALDH+ cell population, reduced their sphere formation capacity, and decreased their tumorigenicity. The orthotopic ovarian xenograft assay also demonstrated that inhibition of miR-328-3p impedes tumor growth and metastasis. The mechanistic investigation revealed that repressed ERK1/2 phosphorylation in ovarian CSCs, mainly due to reduced level of reactive oxygen species (ROS), contributes to the enhanced expression of miR-328-3p, and the maintenance of CSCs. Finally, we identified DDB2 as a direct target of miR-328-3p. Given our previous finding that DDB2 is capable of limiting the CSC population in ovarian cancers, we conclude that highly expressed miR-328-3p in ovarian CSCs, probably due to repressed ERK1/2 activity, inhibits DDB2 expression, resulting in the expansion of these CSCs. Thus, targeting miR-328 could be exploited to a novel strategy to eradicate CSCs in ovarian cancer.

#1986

IRAK1 augments cancer stemness and drug resistance via the AP-1/AKR1B10 signaling cascade in hepatocellular carcinoma.

Bowie Yik Ling Cheng,1 Doris Hoi Wing Leung,2 Eunice Yuen Ting Lau,3 Irene Oi Lin Ng,1 Kin Wah Lee2. 1 _University of Hong Kong, Hong Kong, Hong Kong;_ 2 _Hong Kong Polytechnic University, Hong Kong, Hong Kong;_ 3 _Queen Elizabeth Hospital, Hong Kong, Hong Kong_.

Frequent relapse and drug resistance can be attributed to the existence of tumor-initiating cells (T-ICs) within the tumor bulk. From transcriptome sequencing of 16 pairs of clinical HCC samples, we found that Interleukin-1 receptor-associated kinase 1 (IRAK1) in the TLR/IRAK pathway was significantly upregulated in hepatocellular carcinoma (HCC). IRAK1 overexpression in HCC was further confirmed at the mRNA and protein levels and correlated with advanced tumor stages and poor patents survival. Interestingly, IRAK4, an upstream regulator of IRAK1, was also found to be consistently upregulated. We demonstrated that IRAK1 regulates liver T-IC properties, including self-renewal, tumorigenicity and liver T-IC marker expression. IRAK1 inhibition sensitized the HCC cells to doxorubicin and sorafenib treatment in vitro through the suppression of the apoptotic cascade. Pharmacological inhibition of IRAK1 with a specific IRAK1/4 kinase inhibitor consistently suppressed liver T-IC populations. Through RNA sequencing analysis by comparing gene expression profiles between IRAK1-knockdown and control cells, we identified Aldo-Keto Reductase Family 1 Member 10 (AKR1B10) as a novel downstream target of IRAK1. Clinically, AKR1B10 was found to be overexpressed in HCC, which was significantly correlated with IRAK1 expression. Functional analysis demonstrated that knockdown of AKR1B10 negated the IRAK1-induced T-IC functions via modulation of the AP-1 complex. Using an HCC xenograft model, we found that an IRAK1/4 inhibitor in combination with sorafenib synergistically suppressed the tumor growth. In conclusion, targeting the IRAK4/IRAK1/AP-1/AKR1B10 signaling pathway may be a potential therapeutic strategy against HCC.

#1987

Expansion of pancreatic cancer stem-like cells through PGE2 accumulation in inflammatory environment.

Takatsugu Ishimoto, Kota Arima, Luke Bu, Tomoyuki Uchihara, Keisuke Miyake, Tsugio Eto, Rumi Itoyama, Hideo Baba. _Kumamoto Univ. Graduate School of Medicine, Kumamoto, Japan_.

Chronic inflammation has a crucial role in cancer development and the progression of various tumors, including pancreatic ductal adenocarcinoma (PDAC). The arachidonate cascade is a major inflammatory pathway that produces several metabolites, such as prostaglandin E2 (PGE2). 15-hydroxyprostaglandin dehydrogenase (15-PGDH) participates in the degradation of PGE2 and has attracted attention in recent years. Although inhibition of 15-PGDH is reported to lead to PGE2 accumulation and expand tissue stem cell fraction, the role for pancreatic tumor progression is still unknown. The aim of this study is to elucidate the regulatory mechanism and functional role of 15-PGDH for cancer stem-like cell expansion during tumor progression in pancreas. We found that 15-PGDH expression is frequently down-regulated in PDAC cells and significantly correlated with the number of infiltrating macrophages in human PDAC tissues. Moreover, direct co-culture assay revealed that macrophage derived interleukin-1 beta down-regulates 15-PGDH expression in PDAC cells. Furthermore, pharmacological blockade of 15-PGDH led to PGE2 accumulation, and promoted growth and sphere formation through the expansion of ALDH1-positive cells. We also elucidated the molecular mechanism that PGE2 accumulation by 15-PGDH inhibition increases CYP26A1 expression and subsequently depletes all-trans retinoic acid, and results in ALDH1 up-regulation. Finally, genetic ablation of 15-Pgdh promoted tumorigenesis in KrasLSL-G12D; Ptf1aCre/+ mice through the expansion of ALDH1-positive cells. Our findings highlight the role and significance of PGE2 degradation pathway for PDAC tumor progression.

#1988

The pivotal roles of adipocytes on regulation of cancer stemness and drug resistance in hepatocellular carcinoma.

Shilpa Gurung, Terence Kin-Wah Lee. _The Hong Kong Polytechnic University, Hong Kong, Hong Kong_.

Hepatocellular carcinoma (HCC) is one of the deadliest cancers in the world with less than 10% 5-year survival rate. Liver cancer stem cells (CSCs) are sub-populations of the cancer cells that have been found to play a crucial role in tumor relapse and therapeutic resistance. Increasing reports showed the significant correlation between non-alcoholic fatty liver disease (NAFLD) and HCC development. Based on this evidence, we hypothesize adipocytes, one of the key cellular factors within the tumor microenvironment, play a crucial role in HCC development and progression via regulation of liver CSCs. To verify this, we have employed a co-culture system to dissect the potential cross-talk between fully differentiated adipocytes and HCC cells. Our pilot data showed that adipocytes enhanced the self-renewal ability of HCC cells through indirect paracrine secretion. Based on this finding, we examined the effect of the conditioned medium of adipocytes on regulation of liver CSC. Consistently, we found that HCC cells pre-incubated with conditioned medium of adipocytes showed enhanced in vivo tumorigenicity and chemo-resistance to doxorubicin and sorafenib. Furthermore, conditioned medium of adipocytes promoted the migration and invasion abilities of HCC cells. To delineate the molecular mechanism of how adipocytes, exert its CSC enhancing effect on HCC cells, we have collected the conditioned medium derived from adipocytes and analyzed their secretome profiles using Orbitrap Liquid Chromatography-Mass spectrometry. Upon analysis, we identified 209 proteins, among which we have focused on two proteins (CSF1 and FABP4) for further analysis as these are preferentially secreted from adipocytes. Respective inhibitors of the proteins are used to study the specificity of the protein in the medium and the downstream studied with the help of RNA sequencing which has been performed on conditioned medium treated HCC cells. Collectively, we demonstrated the pivotal role of adipocytes on regulation of liver CSCs through paracrine secretion. Thus, targeting adipocytes-derived signaling cascade may be a novel therapeutic strategy for treatment of fatty liver induced HCC.

#1989

Mechanism of tumor suppressor miRNA let-7 downregulation in ovarian cancer: The epithelial-mesenchymal transition factor Snail is associated with stemness and represses let-7.

Nozomi Hojo,1 Alyse Huisken,1 Hanmin Wang,1 Evgeny Chirshev,1 Sang Nguyen,2 Carlotta Glackin,3 Yevgeniya Ioffe,1 Juli Unternaehrer1. 1 _Loma Linda University, Loma Linda, CA;_ 2 _University of California Riverside, Riverside, CA;_ 3 _Beckman Research Institute, City of Hope, Duarte, CA_.

Purpose:We aimed to understand how stemness is regulated by the Snail/let-7 axis in ovarian cancer cells, toward the goal of developing novel treatments that target stem-like cells to prevent recurrence.

Experimental Procedures:We determined the correlation between mesenchymal and stem cell phenotype in a panel of high grade serous ovarian cancer (HGSOC) cells, including patient-derived cells. Cell surface expression of cancer stem cell (CSC) markers was analyzed by flow cytometry. Expression at the protein level was detected by Western blot, at RNA level by q-RT-PCR. Epithelial-mesenchymal transition (EMT) was induced with epidermal growth factor (EGF). Snail knockdown (KD) was achieved by lentiviral small hairpin (sh)RNA expression and puromycin selection. Migration was determined by wound healing assay, chemoresistance by MTT assay. Chromatin immunoprecipitation of Snail was followed by qPCR. Let-7i promoter luciferase with and without Snail were co-transfected into 293T cell lines and followed by luciferase assays for detection of bioluminescence. Let-7 overexpression was by transfection of miRNA mimics. Patient-derived xenografts (PDX) were established subcutaneously in NOD-SCID-Gamma (NSG) mice. Six week old nude (J:NU) mice, 5-8 per experiment, underwent ovarian bursa injections of luciferized HGSOC cells (OVCAR8 vs PDX) with control vs. Snail KD. Bioluminescence was quantified by IVIS Lumina III.

Results:We characterized several cell lines that accurately model HGSOC for their epithelial (E) vs. mesenchymal (M) and stem cell properties using a novel index. OVSAHO, Kuramochi, COV318, and OVCAR8 were selected for further study in increasing order of M characteristics. M cell status correlated with morphology, stemness, and invasiveness. Increasing the tumor suppressor miRNA let-7 levels or decreasing Snail levels disrupted CSC phenotype, reduced cells' migratory ability, and sensitized cells to cisplatin. Snail bound promotors of let-7 , and luciferase assays demonstrated direct repression of let-7 transcription by Snail. Metastatic tumor burden was significantly reduced in orthotopic xenografts using OVCAR8 cells expressing Snail shRNA. Similarly, in PDX derived from ascites or ovarian tumors, there was reduced tumor burden in Snail KD animals.

Conclusions:The EMT factor Snail represses let-7, placing Snail at the nexus of dedifferentiation via loss of let-7, and invasiveness via EMT. Orthotopic PDX demonstrate that Snail targeting reduces tumor burden. Cell line and patient-derived data supports the relationship between Snail expression, let-7 downregulation, and the induction of CSC. We hypothesize that Snail expression destabilizes differentiation and introduces the CSC state in part via let-7 repression. We propose that Snail is a potential target for recurrent, metastatic EOC.

#1990

**Aberrant cell surface expression of GRP78 in breast cancer cells marks a stem-like population that has increased metastatic potential** in vivo **.**

Tyson W. Lager,1 Henry C. Conner,1 Ian H. Guldner,1 Michael Z. Wu,2 Yuriko Hishida,2 Tomoaki Hishida,2 Sergio Ruiz,3 Amanda E. Yamasaki,1 Juan Carlos Izpisua Belmonte,2 Peter C. Gray,2 Jonathan A. Kelber,4 Siyuan Zhang,1 Athanasia D. Panopoulos1. 1 _University of Notre Dame, Notre Dame, IN;_ 2 _The Salk Institute for Biological Studies, La Jolla, CA;_ 3 _Spanish National Cancer Research Centre, Madrid, Spain;_ 4 _California State University-Northridge, Northridge, CA_.

Reliable approaches to identify and target stem-cell mechanisms that mediate aggressive cancer could have great therapeutic value, based on the growing evidence of embryonic signatures in metastatic cancers. However, how to best identify and target stem-like mechanisms aberrantly utilized by cancer cells has been challenging. We harnessed the power of induced pluripotent stem cells (iPSCs) to identify embryonic mechanisms exploited by cancer. A screen comparing the cell surface proteome of iPSCs and breast cancer cells identified GRP78, a heat shock protein that is normally ER-restricted, but has been shown to be aberrantly expressed on the cell surface of several cancers, where it can act as a signaling molecule by poorly understood mechanisms. Although cell surface GRP78 (sGRP78) has emerged as an attractive chemotherapeutic target, understanding how sGRP78 is functioning in cancer has been complicated by the fact that GRP78 can function to regulate a variety of cellular responses, using a diverse array of reported binding partners, which can vary by cell type. Therefore, without insight into the specific GRP78-dependent mechanisms that are responsible for mediating aggressive cancer, it will be difficult to determine how to best target GRP78. We have discovered that (1) sGRP78 is expressed on iPSCs (but not their somatic parental populations) and plays an important role in reprogramming, (2) sGRP78 promotes cellular functions such as proliferation/survival and migration in both stem cells and breast cancer cells (3) overexpression of GRP78 in breast cancer cells leads to an induction of a previously established CD24-/CD44+ 'cancer stem cell' (CSC) population (4) sGRP78+ breast cancer cell populations are enriched for genes involved in stemness and appear to be a subset of previously established CSCs (5) sGRP78+ breast cancer cell populations show a significantly enhanced ability to seed metastatic organ sites in vivo (6) GRP78 interacts with Dermcidin (DCD) at the cell surface of cancer cells and iPSCs, where it is important in regulating stem cell and cancer cell migration and survival/proliferation. These collective findings suggest that sGRP78 marks a stem-like population in breast cancer cells that has increased metastatic potential in vivo, and that sGRP78 and DCD cooperate to regulate key cellular functions important in mediating tumorigenesis. Overall, this work has implications for understanding how cancer cells exploit embryonic-like mechanisms, which could provide novel strategies for chemotherapeutic targeting of aggressive breast cancer cell populations.

#1991

Vulnerability of platinum-resistant ovarian cancer to FAK inhibition.

Carlos J. Diaz Osterman,1 Lisa M. Bean,1 Florian J. Sulzmaier,1 Kristin N. Taylor,1 Shulin A. Jiang,1 Isabelle Tancioni,1 Kristen Anderson,1 Christine Jean,1 Xiao Lei Chen,1 Elizabeth G. Kleinschmidt,1 Vihren N. Kolev,2 David T. Weaver,2 Jonathan A. Pachter,2 Denise C. Connolly,3 Alfredo Molinolo,1 David D. Schlaepfer1. 1 _UCSD, La Jolla, CA;_ 2 _Verastem Incorporated, Needham, MA;_ 3 _Fox Chase Cancer Center, Philadelphia, PA_.

Platinum (CP)-resistant ovarian cancer (OC) has few effective treatment options. Adaptive chemotherapy resistance occurs in part through cancer stem cell (CSC) generation. Wnt/beta-catenin signaling is a driver of CSC survival via induction of gene expression, including aldehyde dehydrogenase (ALDH) enzymes. The gene for focal adhesion kinase (FAK) is commonly amplified in advance-stage OC and this is associated with decreased patient survival. How FAK is activated in OC and whether this is connected to CSC survival is unknown. Here, we find that FAK Y397 phosphorylation (a marker of FAK activation) is increased in non-necrotic Pax8-positive OC tumor tissue after neo-adjuvant chemotherapy compared to paired pre-treatment tumor biopsy samples. FAK activation occurs after CP plus paclitaxel treatment of xenograft tumors, within 60 min of CP treatment of OC cells, and FAK Y397 levels are constitutively-elevated in OC cells pre-adapted to exhibit elevated CP resistance. Pharmacological FAK inhibition (VS-4718, 0.1 µM) selectively prevented CP-resistant tumorsphere formation in vitro and combined with CP to promote cell apoptosis. Combinatorial VS-4718, CP, and paclitaxel chemotherapy exhibited additive inhibitory effects in preventing CP-resistant tumor growth in mice. VS-4718 monotherapy of tumor-bearing mice reduced tumor-associated ALDH activity and resulted in an 85-fold reduction in secondary tumors formed in limiting dilution assays. CRISPR-mediated FAK knockout in OVCAR3 cells combined with stable re-expression of FAK wildtype or a kinase-inactive FAK (K454R) mutant revealed that intrinsic FAK activity was essential for beta-catenin activation, ALDH-1A1 expression, and OVCAR3 tumorsphere growth. As activated constructs of beta-catenin but not YAP1 rescued FAK KO OVCAR3 phenotypes, these studies provide important insights into a FAK signaling linkage to beta-catenin in promoting CSC survival and adaptive resistance to CP chemotherapy. These studies provide the foundational support for a Phase I-II clinical trial for treatment of recurrent platinum-resistant ovarian cancer (NCT03287271) termed ROCKIF: Re-sensitization of carboplatin-resistant Ovarian Cancer by Kinase Inhibition of FAK. As FAK is activated in tumor cells surviving carboplatin-paclitaxel chemotherapy, co-targeting of this FAK/beta-catenin adaptive resistance pathway may expose a vulnerability of CP-resistant tumors.

#1992

Epigenetic and transcriptomic profiling of mammary gland development and tumor models disclose regulators of cell state plasticity.

Christopher Dravis, Chi-Yeh Chung, Geoff Wahl. _Salk Institute for Biological Studies, La Jolla, CA_.

Cell state reprogramming during tumor progression significantly contributes to the genesis of intra-tumoral heterogeneity. Discovering how cell state plasticity is specified during normal mammary development, and how this relates to breast cancer, could provide new treatment approaches. We used chromatin accessibility assays and transcriptional profiling to infer lineage potential within distinct normal mammary cell populations. We show that fetal and adult basal cells exhibit features consistent with multi-lineage differentiation potential. SOX family transcription factor DNA-binding motifs were enriched in the open chromatin of stem/progenitor cells, implicating SOX factors in mammary cell plasticity. We provide evidence that SOX10 expression correlates with multiple indicators of cell state plasticity, and that it directly contributes to tumor development in multiple animal models. Strikingly, we show that SOX10-positive tumor cells uniquely exhibit neural crest cell features, and that SOX10 binds to genes that regulate neural crest cell identity. Collectively, these data help to resolve conflicts concerning cell fate and cell potential in the mammary cell hierarchy, reveal that SOX10 is a plasticity factor driving mammary tumor progression, and demonstrate the remarkable scope of cell state reprogramming that can occur during tumorigenesis.

#1993

Rapid clonal evolution of dormant micrometastatic disease in head and neck cancer.

Angelica M. Lagunas, Jianchun Wu, David L. Crowe. _Univ. of Illinois Cancer Ctr., Chicago, IL_.

Head and neck cancer (HNC) is prone to locoregional recurrence which results in poor clinical outcome. Recent HNC clinical studies demonstrated that cervical lymph node dissection results in improved patient outcome even in early disease. These results suggest that dormant micrometastatic cancer occurs early in head and neck carcinogenesis. We used our established in vivo chemical carcinogenesis model of head and neck cancer to characterize dormant micrometastatic disease using genetic lineage tracing and null mutant mice. Histopathology was analyzed by hematoxylin and eosin staining. Telomere DNA damage response was determined by immunofluorescent 53BP1 localization at telomeres, and by ATM/ATR western blotting. Keratin expression and proliferation index were determined by immunohistochemistry. Apoptotic cells were determined by TUNEL analysis. Telomerase activity was determined by telomere repeat amplification protocol. Alternative lengthening of telomeres was determined by PML localization at telomeres, telomere circular DNA analysis, and chromosome orientation fluorescence in situ hybridization. Genomic instability was determined by high throughput sequencing. Cancer stem cells were sorted by flow cytometry and transplanted subcutaneously in Matrigel. Advanced HNC exhibited significant metastatic disease in the majority of lymph nodes. Early HNC and even premalignant lesions exhibited micrometastatic disease in most lymph nodes. These micrometastatic lesions contained multiple HNC clones that were dependent for proliferation on paracrine chemokine signaling from a tumorigenic keratin 15+ cancer stem cell population. In situ, the micrometastatic K15+ cancer stem cell population exhibited increased telomere DNA damage response including activated ATM and ATR which correlated with low proliferation index. Clonal evolution of K15+ micrometastatic cancer stem cells was dependent on extremely short telomeres. These cells expressed both telomerase and recombination-based alternative lengthening of telomeres (ALT) activities. K15+ micrometastatic cancer stem cells exhibited high levels of genomic instability resulting from telomere fusions and expressed metastasis-specific gene expression programs. These patterns of micrometastatic gene expression revealed targeted therapeutic vulnerabilities. These results indicate that dormant micrometastatic disease develops rapidly during head and neck carcinogenesis from normal epithelial cells with short telomeres and is sensitive to targeted therapies.

#1994

Pre-existence of poly-resistant cancer stem cells in high-grade ovarian cancer.

Wa Xian,1 Frank McKeon,2 Christopher Crum,3 Jingzhong Xie,2 Bailiang Wang,1 Yusuke Yamamoto,4 Suzy Torti,5 Molly Brewer,5 Matthew Anderson,6 Amir Jazaeri,7 Giulio Draetta,7 Shan Wang,2 Rajasekaran Mahalingam,2 Hong Chen,2 Marcin Duleba,2 Wei Rao1. 1 _University of Texas, Houston, TX;_ 2 _University of Houston, Houston, TX;_ 3 _Harvard Medical School, Boston, MA;_ 4 _National Cancer Institute, Tokyo, Japan;_ 5 _University of Connecticut, Farmington, CT;_ 6 _Baylor College of Medicine, Houston, TX;_ 7 _University of Texas/MD Anderson Cancer Center, Houston, TX_.

Introduction: High-grade ovarian cancer (HGOC) shows excellent responses to standard-of-care surgery and paclitaxel/carboplatin therapy only to relapse 6-24 months later with typically resistant disease. While the origin of this recurrent, resistant disease is unclear, most believe it is acquired by the action of chemotherapeutics. Using novel stem cell technology that enables the cloning of cancer stem cells (CSCs) from epithelial cancers, we have generated large libraries of CSCs from multiple cases of HGOC. And while the vast majority of these CSC clones are killed by standard-of-care chemotherapeutic drugs, a minor fraction shows profound resistance not only to paclitaxel/carboplatin but to a wide range of structurally unrelated chemotherapeutic drugs to which these cells had no prior exposure. We describe screens for drugs that selectively target this resistant CSC population.

Methods: Libraries of 10- to 100,000 CSC clones were generated from individual, therapy naïve, HGOC resections using technology we developed for cloning so-called "adult" stem cells from normal columnar epithelia (Wang et al., 2015).

Results: Paclitaxel/carboplatin resistant CSCs were identified in CSC libraries derived from therapy naïve tumors at ratios of 1:50 to 1:300. By copy number variation, these resistant variant clones proved distinct from the bulk of CSCs, and by gene expression analysis varied from sensitive clones by more than 700 differentially expressed genes. Independent resistant clones from the same library clustered with other resistant clones by both copy number variation and gene expression profiles, suggesting the possibility that resistance within a single tumor is dominated by a single type of resistant CSCs. Clones resistant to paclitaxel/carboplatin were screened in a 384-well format against a wide range of experimental drug-like molecules. These pre-existing resistant clones also proved to be profoundly resistant to a large number of structurally unrelated chemotherapeutic drugs. This same screening program identified drugs that act alone or in combination with paclitaxel to eliminate these resistant clones, suggesting a route to personalized medicine for addressing the problem of recurrent disease in HGOC.

Conclusions: Tumors from patients with HGOC possess clonogenic CSCs including variants that are resistant to a broad spectrum of chemotherapeutics to which they have not been exposed. It is likely that such CSCs would survive standard-of-care chemotherapy and contribute to the recurrent disease seen in HGOC. We have identified known and experimental drugs that specifically eliminate these resistant variants and the overall platform represents a potential strategy to addressing the problem of recurrent disease in these patients.

#1995

PTHrP drives tumor initiation signaling pathways in the PyMT model of breast cancer progression.

Rui Zhang,1 Jiarong Li,1 Dunarel Badescu,2 Andrew Karaplis,3 Jiannis Ragoussis,2 Richard Kremer1. 1 _McGill Univ. Health Ctr., Montreal, Quebec, Canada;_ 2 _McGill Univ., Montreal, Quebec, Canada;_ 3 _Lady Davis Institute for Medical Research, Montreal, Quebec, Canada_.

Parathyroid hormone-related peptide (PTHrP) was first discovered in cancer patients as the primary cause of malignancy associated hypercalcemia (MAH) & is overexpressed in most human breast tumors. We previously showed that PTHrP ablation, in the MMTV-PyMT murine model of breast cancer progression can dramatically prolong tumor latency, slows tumor growth & metastases. Here we examined the signaling pathways under the control of PTHrP in the pre-neoplastic stage of tumor development (hyperplasia) by generating MMTV-PyMT model in which the mammary epithelium, expressing membrane-targeted GFP, is distinguished from the membrane-targeted red fluorescent backlight of stromal & nonepithelial-derived mammary tissues. We constructed Pthrpflox/flox; Cre\+ mT/mG tumor mice (PTHrP conditional KO) & Pthrpwt/wt; Cre+ mT/mG tumor mice (WT control). Next, we applied FACS to enrich the GFP+ mammary epithelial cells isolated from PTHrP KO & control tissues for subsequent RNAseq analyses. We then examined differentially expressed genes (DEGs) by comparing purified cell populations from PTHrP KO & control tissues. We identified 939 DEGs (p value <0.01) & used DAVID bioinformatics resources to systematically analyze the KEGG pathways. Among the most significant pathways, extracellular matrix (ECM), focal adhesion,PI3K-Akt, RAS and WNT pathways were up-regulated in WT control cells compared to KO cells. In summary, PTHrP controls critical signaling pathways involved in breast cancer initiation at the pre-neoplastic stage & suggests that PTHrP ablation is a promising therapeutic strategy in breast cancer.

#1996

Nuclear factor (erythroid-derived 2)-like 2 promotes tumor-initiating cell lineage and drug resistance in hepatocellular carcinoma.

Hoi Wing Leung, Terence Kin Wah Lee. _The Hong Kong Polytechnic University, Hong Kong_.

Hepatocellular carcinoma (HCC) is the fifth most common malignancy worldwide. The long-term prognosis of HCC remains unsatisfactory due to high recurrence rates and chemoresistance. The efficacy of sorafenib, the only FDA-approved molecularly targeted drug in advanced hepatocellular carcinoma (HCC) is limited by acquired resistance. Increasing evidences showed that liver tumor-initiating cells (T-ICs) is the source of acquired resistance and metastasis. We have previously established sorafenib-resistant HCC cells both in vitro and PDTX models and demonstrated that these cells are endowed with enhanced T-IC properties. Interestingly, nuclear factor (erythroid-derived 2)-like 2 (NRF2) expression was elevated in these resistant cells. The transcription factor NRF2 has long been considered as a tumour suppressor, as it can regulate cellular antioxidant response to protect cells from oxidative stress, chemotherapeutic agents and radiotherapy. In HCC, NRF2 level was up-regulated, and its expression was correlated with tumor differentiation, metastasis, and tumor size, revealing its oncogenic role. By lentiviral based knockdown approach, we found that NRF2 repression suppressed liver T-IC properties including self-renewal, in vivo tumorigenicity, HCC invasiveness and expression of liver T-IC markers. In addition, we found that NRF2 repression not only sensitized HCC cells to sorafenib but also to other chemotherapeutic drugs including doxorubicin and fluorouracil. Mechanistically, the signaling cascade related to sonic hedgehog pathway was greatly suppressed in NRF2 knockdown HCC cells, when compared with control counterparts. Collectively, NRF2 was involved in drug resistance, invasion and migration, tumorigenicity and self-renewal via regulating T-IC properties at least in part through direct regulation of sonic hedgehog pathway in HCC. Targeting NRF2 mediated signaling cascade alone or in combination with other treatment modalities may be a new a potential therapeutic approach for treatment of HCC.

#1997

Pro-inflammatory cytokine IL-6 induces the upregulation and deacetylation of FRA-1, thus promoting colon cancer stemness and aggressiveness.

Tingyang Wang, Jimin Shao. _Zhejiang University School of Medicine, Hangzhou, China_.

Colon cancer is an aggressive epithelial tumor type encompassing cancer stem cells (CSCs) that not only contribute to its grow at the primary site but also to its malignant progression, resistance to chemotherapy, and distant metastases thus underlying poor patient outcomes. We have previously reported that significant upregulation of the gene encoding for the Fos-related antigen-1 (FOSL1) upon pro-inflammatory cytokine interleukin-6 (IL-6)-driven epithelial-to-mesenchymal transition (EMT) in colon cancer. However, the underlying molecular and cellular mechanism through which inflammatory cytokines like IL6 regulate CSCs plasticity during colon cancer onset and progression development remain unclear. Here, we show that IL-6 enhanced the stemness, drug resistance and metastasis of colon cancer cells by upregulation and deacetylation of the transcription factor FRA1. Ectopic expression of FRA1 in colon cancer cells induced stem-like properties and metastasis, while its knockdown suppressed these IL-6 induced effects. IL-6 enhanced FRA1 transcription through the STAT3 pathway. Also, we show that FRA1 directly interacts with the promoter of the CSC transcriptional factor Nanog and transactivates its expression to modulate colon cancer stemness. Furthermore, deacetylation of lysine 116 (K116) in FRA1 was essential for its activity on Nanog transcription. Preliminary analyses with Trichostatin A (TSA), an inhibitor of histone deacetylases (HDACs) I and II, but not nicotinamide (NAM), an inhibitor of the sirtuin (SIRT) family of deacetylases, showed that some member of the HDACs family might be responsible for FRA1 deacetylation. Finally, we examined the expression of IL6/STAT3-pY(705)/FRA1/k116ac/Nanog/HDACs/β-catenin in 120 paraffin-embedded human colon cancer tissues by immunohistochemistry (IHC). We found that while FRA1 was mainly localized in the nucleus of cancer cells, K116ac of FRA1 was present both in the nucleus and cytoplasm and showed a low level of Lys116 acetylation in the FRA1 nuclear fraction. Moreover, analysis of patient-derived colon cancer samples supported the clinical relevance of the IL-6/STAT3/FRA1/Nanog axis in contributing to the increased colon cancer stemness and metastasis. We propose that a transcriptional regulatory cross-talk between pro-inflammatory and cancer stemness signaling pathways drives the reprogramming of colon cancer cells to CSCs thus promoting progression towards malignancy and distant metastasis. Combinations of anti-IL6 therapies with HDACs inhibitors may offer a potential novel approach for colon cancer treatment. Keywords: Inflammation, cancer cell stemness, transcription, deacetylation, colon cancer

#1998

**Novel interplay between TANK-binding kinase (TBK1) and Yes-associated protein (YAP1) in** KRAS **mutant NSCLC.**

Biswarup Saha, Neha Jaiswal, Namrata Bora-Singhal, Srikumar Chellappan. _H Lee Moffitt Cancer Center and Research Institute, Tampa, FL_.

Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related mortality in the United States. Lung adenocarcinomas are highly correlated with smoking and are characterized by mutations in KRAS, EGFR, BRAF and other oncogenes. Among them, KRAS mutations are widespread in adenocarcinomas in smokers and known to be a key player in various downstream signaling pathways contributing to genesis and progression of these tumors. More recently, the non-canonical IκB kinase, Tank Binding Kinase 1 (TBK1), has been found to facilitate tumorigenesis in KRAS mutant cancers. TBK1 has well documented functions in immune response, cell survival as well as in mitosis. While it has been suggested that TBK1-mediated regulation of Akt signaling might facilitate oncogenesis, the molecular mechanisms underlying TBK1 function downstream of KRAS is not fully elucidated. In a similar vein, Yes associated protein 1 (YAP1), the oncogenic component of the Hippo signaling cascade has been found to promote KRAS mediated oncogenesis and could substitute for the loss of Kras in mouse models of pancreatic cancer. In the present study, we have demonstrated a unique and novel molecular interplay between TBK1 and the oncogenic components of Hippo effector molecules, YAP1/TAZ. YAP1 and its ortholog, TAZ are known to be transcriptional co-activators, function to maintain organ size but often get activated in various types of cancer.

We find that TBK1 physically interacts with YAP1, and knock-down (KD) or knocking-out (KO) of TBK1 resulted in a significant elevation of YAP1/TAZ expressions at protein level. We have tested four different KRAS and EGFR mutant NSCLC cell-lines; interestingly, the upregulation of YAP1 upon depletion of TBK1 was only restricted to the KRAS mutant cell-lines. Further, depletion of TBK1 led to the enrichment of YAP1 in the nucleus; notably, there were only minimal changes in the levels of MST1/2 and LATS, raising the possibility that these changes occur independent of the classic hippo signaling pathway. Depletion of TBK1 also resulted in the induction of EMT-like features in these cells, and increased the proportion of stem-like side-population. Mechanistically, TBK1 physically interacts with YAP1 in the cultured cells, and could phosphorylate it in vitro, on T110, T114, S128 and S131 residues. The underlying molecular mechanism(s) by which TBK1 regulates YAP1 expression are under investigation, and we hypothesize that this regulatory event is exclusive to KRAS mediated oncogenesis, especially in the context of NSCLC.

#1999

Study of CD44 variant 6 (CD44v6) in prostate cancer chemo-/radio resistance in vivo.

Jie Ni,1 Paul Cozzi,2 Julia Beretov,1 Joseph Bucci,1 Peter Graham,1 Yong Li1. 1 _St George Hospital, Kogarah, Australia;_ 2 _University of New South Wales Sydney, Australia_.

Background: Chemo-/radio-resistance is an important reason for prostate cancer (CaP) progression and metastasis. CD44 is a well-documented cancer stem cell (CSC) biomarker, and one of its variants, CD44 variant 6 (CD44v6) is closely associated with aggressive behaviour and correlates with poor prognosis in a variety of human cancers. Our previous study has demonstrated increased expressions of CD44v6 in metastatic CaP cell lines and human CaP tissues, which was associated with CaP progression and chemo-/radio-resistance in vitro. However, the role of CD44v6 in CaP progression and therapeutic resistance in vivo is still uncertain.

Aim: The aim of this study was to investigate the role of CD44v6 in CaP development and chemo-/radioresistance as well as underlying pathways in vivo, and find whether it is a suitable therapeutic target for CaP therapy.

Methods: CD44v6 gene was knocked down (KD) in PC-3M CaP cell line using short hairpin RNA (shRNA). Subcutaneous (s.c.) and orthotopic CaP animal models were established using PC-3M-CD44v6-KD and PC-3M-CD44v6-scramble (scr) control cells, to assess the effect of CD44v6 on CaP tumourigenesis, chemotherapy (docetaxel) response and radiation response. Signal transduction proteins in PI3K/Akt/mTOR pathway (Akt, p-Akt, mTOR and p-mTOR) as well as proliferation and apoptosis makers were assessed by immunohistochemistry in xenografts from both animal models.

Results: Both s.c. and orthotopic xenografts from PC-3M-CD44v6-KD cells displayed supressed tumour growth and increased responsiveness to docetaxel (40mg/kg) and radiation (2Gy/day for 4 consecutive days) compared to control xenografts from PC-3M-CD44v6-scr cells in NOD/SCID male mice. Down-regulation of the PI3K/Akt/mTOR pathway proteins (p-Akt and p-mTOR), proliferation marker (Ki-67) and angiogenesis marker (CD31), as well as up-regulation of apoptotic responses (cleaved Caspase-3) to chemotherapy, DNA damage responses (ɣ-H2AX) to radiation were found in PC-3M-CD44v6-KD s.c. xenografts, respectively.

Conclusions: CD44v6 is actively involved in CaP tumorigenesis and treatment responses to chemo-/radio-therapy via PI3K/Akt/mTOR signalling pathway in in vivo mouse models and holds promise as a therapeutic target for the treatment of CaP.

Key words: CD44v6, prostate cancer, chemo-/radio-resistance, animal model, metastasis

#2000

The regulatory role of endothelial nitric oxide synthase signaling in the growth of prostate cancer stem-like cells.

Weijie Gao, Dinglan Wu, Yuliang Wang, Zhu Wang, Taiyang Ma, Franky Leung Chan. _The Chinese University of Hong Kong, Hong Kong_.

Cumulative studies indicate that there is a small subpopulation of cancer cells present in cancers or solid tumors referred as cancer stem-like cells (CSCs) as they share some common characteristics with stem cells, particularly self-renewal and multipotency, and these CSCs may contribute to the tumor initiation, treatment resistance and relapse. Recent advances also indicate that prostate cancer stem-like cells (PCSCs) may play a critical role in the development of castration-resistant prostate cancer (CRPC) and also its metastasis. Here, we demonstrated using an established CRPC xenograft model (VCaP-CRPC) that both the xenograft tumors and a xenograft-derived cell line contained more PCSC population, which showed higher expression levels of endothelial nitric oxide synthase (eNOS) and elevated intracellular NO production. PCSCs, isolated from prostaspheres derived from prostate cancer cell lines (DU145, LNCaP and VCaP) using a newly developed agar-based non-attachment 3D-culture method, exhibited up-regulation of eNOS and increased intracellular NO levels. Interruption of eNOS signaling by NOS inhibitors and shRNA-knockdown could significantly suppress the sphere formation capability of prostate cancer cells. Exogenous overexpression of eNOS could promote the tumor growth capacity of prostate cancer cells and their metastasis in vivo, while knockdown of eNOS could prevent the tumor growth and metastasis. Combined transcriptome-sequencing analysis and qPCR validation of prostaspheres upon eNOS-knockdown identified some potential targets of eNOS/NO signaling in the PCSC-enriched prostaspheres. In summary, our results suggest that eNOS/NO signaling may play a positive role in the growth regulation of PCSCs and eNOS might be a potential target for advanced prostate cancer. This study is supported by a General Research Fund from the Research Grants Council of Hong Kong (project code 14107617) and an Innovation and Technology Fund (GHP/003/16GD) from the Innovation and Technology Commission of Hong Kong.

#2001

Transcriptional integrator p300 in human prostate tumor-initiating cells.

Martina Gruber, Florian Handle, Zoran Culig. _Innsbruck Medical Univ., Innsbruck, Austria_.

Introduction

p300 is a well-known coactivator of the androgen receptor (AR) and has been shown to play an important role in prostate cancer (PCa). The expression of p300 has been correlated to tumor volume and PCa progression. Therefore, we hypothesize that p300 may play an important role in tumor-initiating cells and therapy resistance. Due to its multifunctional role p300 may serve as a promising new therapeutic target for treatment of PCa. Ultimately, it is our aim to find new innovative therapeutic targets for castration resistant PCa.

Material & Methods

The tumor-initiating capacity of the AR-negative PCa cell lines PC3 and Du145, as well as primary prostate epithelial cells was characterized by seeding a limiting number of cells per flask. Colony formation efficiency, colony type and detailed morphological information about each colony were determined by an automated pipeline that we have developed. Stable cell lines with doxycycline inducible p300 sh RNAs were generated by lentiviral transduction. Knockdown of p300 was verified by Western Blot and reduced p300 activity was confirmed by measuring histone h3 acetylation.

Results

In concordance with the literature we identified three colony types (holo-, mero- and paraclones) in PC3 and Du145 cell lines as well as primary PCa cells. Serial clonogenic assays confirmed in both cell lines that holoclones show the highest colony formation potential and maintain their tumor-initiating capacity over numerous rounds. In PC3 we detected 10% and in Du145 30% holoclones. Under optimal growth conditions p300 was detected in all colony types, however no differences in protein expression of p300 as well as on mRNA level were observed. Knockdown of p300 did not alter colony formation efficiency or the relative distribution of colony types.

Conclusion

Taken together, we confirmed the presence of tumor-initiating holoclones in PCa cell lines and primary prostate epithelial cells and established stable cell lines with inducible knockdown of p300. In contrast to several other types of malignancies, p300 has no specific role in regulation of stemness in our PCa models at the tested conditions. As a next step we will determine the effects of p300 knockdown during chemotherapy and radiation therapy to evaluate if p300 depletion may enhance the treatment efficacy and which role p300 plays in therapy resistance.

#2002

Ganoderma lucidum **extract (GLE) reduces stem cell markers in triple-negative breast cancer models via STAT3 regulation.**

Tiffany J. Rios, Alicia Marti, Gabriela Ortiz, Michelle M. Martínez-Montemayor. _Universidad Central del Caribe - School of Medicine, Bayamon, PR_.

Triple-negative breast cancer (TNBC) is a subtype of cancer described by the lack of estrogen receptor, progestogen receptor, and HER2 receptor. It accounts for approximately 15-20% of all breast cancer (BC) cases worldwide. TNBC aggressiveness might be explained by the presence of BC stem cells (BCSCs), which are a subpopulation of cancer cells involved in tumor initiation, metastasis, and therapy resistance. Interestingly, the signal transducer and activator of transcription 3 (STAT3) participates in the development and progression of BCSCs, but its role in TNBC progression remains unclear. Moreover, BCSCs can be identified by the CD44+/CD24- and ALDH+ phenotype, which correlates with a worse prognosis in TNBC patients. Furthermore, CSCs can be characterized based on the expression of transcription factors involved in stemness (Nanog, Sox2, and Oct4A), where activation of STAT3 leads to enhanced Nanog, Oct4 and Sox2 expression. Importantly, a major challenge arises to develop nontoxic targeted therapies to improve the outcome of TNBC patients. In this regard, our laboratory investigates the role of Ganoderma lucidum extract (GLE), a medicinal mushroom commonly used in complementary and alternative medicine. We hypothesize that GLE will decrease stem cell properties via STAT3 regulation in TNBC cells. Our in vitro results demonstrate that GLE significantly downregulates STAT3 and JAK2 in SUM-149 and MDA-MB-231, TNBC cell lines. In addition, GLE significantly decreases the secretion of IL-6 at 24hr in MDAMB231 cells. Further flow cytometry results show that GLE significantly decreases the CD44+/CD24- population at 24hr and 72hr in MDA-MB-231 cells. In this regard, GLE significantly decreases the expression of ALDH at 24hr and 72hr in MDAMB231 cells. Moreover, GLE decreases the expression of Nanog at 24hr, and all three transcription factors at 72hr. Additionally, our in vivo studies demonstrate that GLE significantly decreases tumor volume of mice injected with CD44+/CD24- sorted cells of the MDA-MB-231 cell line. Finally, tumor weight of GLE treated mice was marginally significantly lower (p=0.053) compared to vehicle. We can conclude that GLE decreases stemness of TNBC via STAT3 regulation.

#2003

P38α suppresses tumor initiating cell phenotypes through inhibition of NFATc4 in non-small cell lung cancer.

Zhi-Jie Xiao, Jing Liu, Si-Qi Wang, Xu-Yuan Gao, Vicky Pui-Chi Tin, Maria Pik Wong. _Univ.. of Hong Kong, Hong Kong, Hong Kong_.

Lung cancer development is often mediated through oxidative and inflammatory stresses caused by tobacco and environmental carcinogens, suggesting dysregulation of stress mediators might play a role during carcinogenesis. The p38 MAPK pathway is activated by multiple stress stimuli and inflammatory cytokines, and mediates a wide range of cellular processes in a context-specific manner. P38α, a member of the p38 family, is known to regulate the self-renewal and differentiation of adult lung stem cells. While p38α has been reported to mediate tumor suppressive or supportive roles in different cancers, inactivation of p38α in the lung epithelium is involved in KRASG12V induced tumorigenesis. However, whether p38α could be involved in regulating tumor initiating cells (TIC) of lung cancer remains unclear. To investigate this possibility, immunohistochemistry was first used to analyse the nuclear expression of phosphorylation-activated p38α (p-p38α) in 90 human primary lung adenocarcinomas. Results showed higher expression of p-p38α was significantly correlated with longer progression free and overall survivals, as well as a more differentiated tumor histological phenotype, indicating p38α might play a tumor suppressive role and drive tumor differentiation in lung cancer. To further address its functional role on lung TIC, p38α was suppressed by siRNA or pharmacologically inhibited by SB203580. This led to increase of the TIC subset that coexpresses ALDH and CD44 (ALDH+/CD44+-TIC), as well as up-regulated expression of the pluripotency genes SOX2, OCT4, and NANOG in lung cancer cell lines. Functionally, p38α inhibition by SB203580 promoted tumor spheres formation for two consecutive generations. NFAT is a family of transcription factors reported to regulate TIC in various cancers which are activated via de-phosphorylation leading to nuclear translocation. We hypothesize the TIC supportive function of p38α inhibition might be mediated through loss of phosphorylation and subsequent activation of NFATc4. In line with this hypothesis, we showed, using Western blot, p38α inhibition increased the expression of nuclear NFATc4. Moreover, NFATc4 knockdown led to suppressed sphere formation and pluripotency genes expression in lung cancer cell lines. Together, our results indicated p38α mediates a tumor suppressor pathway through downstream NFATc4 inactivation and TIC inhibition. The p38α -NFATc4 stress pathway might serve as a potential target for long term lung cancer treatment through TIC regulation.

#2004

Identification of MLK4 as a novel regulator of cancer stem cells in triple-negative breast cancer.

Chang-Ching Lin, Miao-Chia Lo, Rebecca Moody, Nicholas Stevers, Samantha Tinsley, Mari Gasparyan, Max Wicha, Duxin Sun. _Univ. of Michigan, Ann Arbor, MI_.

Cancer stem cells (CSCs) are a small population of cancer cells that are capable of self-renewal and tumor initiation. As a result, targeting CSCs is a potential therapeutic strategy for preventing cancer relapse and metastasis. Mixed lineage kinase 4 (MLK4), a serine/threonine kinase, is known to regulate mesenchymal glioma stem cells and to drive tumorigenesis in colorectal cancer. We found that MLK4 was highly expressed in triple-negative breast cancer (TNBC) compared to other breast cancer subtypes according to the database from The Cancer Genome Atlas. Furthermore, multiple datasets indicated that higher expression of MLK4 was associated with poor prognosis in breast cancer. Despite the correlation of MLK4 and clinical outcome, the function of MLK4 in breast cancer is still not known. In this study, we found that knockdown of MLK4 significantly decreased secondary mammosphere formation, the CD44+/CD24- CSC population, invasion and migration in TNBC cell lines. In addition to suppressing CSC phenotypes in vitro, knockdown of MLK4 also inhibited tumor growth in NOD/SCID mice. Secondary transplantation of tumor cells demonstrated that silencing of MLK4 significantly decreased tumor-initiating cell frequency. As accessed by qPCR, knockdown of MLK4 led to decreased expression of mesenchymal genes (CD44 and VIM) and concomitant increased expression of epithelial genes (CDH1 and claudin genes). Immunohistochemical staining revealed that knockdown of MLK4 in xenografts resulted in increased expression of CK19, a luminal-epithelial marker. These studies indicate that MLK4 plays an important role in the function of mesenchymal-like CSCs in TNBC. Silencing of MLK4 in TNBC cell lines can transform CSC in these tumors to a more differentiated epithelial cell state. Together, these studies identify that MLK4 is a potential therapeutic target for CSCs in TNBC.

#2005

CSCs induced from iPSCs indicate the trace of cancer cell culture conditioned media.

Akimasa Seno,1 Tomonari Kasai,1 Heizo Tokutaka,2 Masaharu Seno1. 1 _Okayama University, Okayama, Japan;_ 2 _SOM Japan, Inc., Tokyo, Japan_.

Cancer stem cells (CSCs) show drug resistance and could be a source of cancer cells. Although these cells are recently considered more important than ever in cancer studies, the extremely small number of CSCs in actual cancer tissues makes the analysis difficult. Previously, we demonstrated that CSCs could be differentiated from induced pluripotent stem cells (iPSCs) in the presence of cancer cell conditioned media. We have confirmed the conditioned media from various types of cancer cell lines were successful to convert iPSCs into CSCs showing self-renewal property, differentiation potential, and tumorigenicity. To identify their differences between the generated CSCs, gene expression was compared by spherical self-organizing map (sSOM). Comparing with more than 1000 cells including stem cells, induced CSCs, CSCs obtained from cancer tissue or cancer cell line, and cancer cells or cancer tissues, known cancer stem cell markers were found not enough to classify CSCs. In the sSOM analysis, "ideal probe" (IP), which was supposed to characteristically be expressed in each group, was set to nomiate the marker gene. The distance close to the IP depicted some candidate marker genes in each group. The gene expression commonly shared among different types of CSCs was also searched. This IP revealed known cancer related gene Endthelin 1 as a commonly expressed gene. Moreover, when the CSCs were compared with the parental hiPSC and cancer cells, of which conditioned media was used for the conversion, sSOM showed that CSCs were not always differentiated towards their origin, but which genes should be responsible for generating CSCs. These results indicate that the induced CSCs should be the initial state of cancer cells and imply some important role of Endothelin 1 in CSC development.

#2006

Tobacco smoke-induced Marcks phosphorylation promotes inflammatory and stemness properties in lung cancer.

David C. Yang, Yichin Wu, Mu-Jen Huang, Kent E. Pinkerton, Reen Wu, Ching-Hsien Chen. _University of California Davis, Davis, CA_.

Exposure to tobacco smoke is a major risk factor associated with lung cancer development and progression; however, the mechanisms of how tobacco smoke activates pathways promoting cancer malignancy remain to be established. Previously, phosphorylation of MARCKS at Ser159/163 (phospho-MARCKS) was shown to promote malignancy of neoplastic disease, but it is still unclear how phospho-MARCKS is upregulated in lung cancer. Herein, we investigated if phospho-MARCKS is elevated in response to smoke exposure and plays a key role in smoke-related lung cancer. In a screen of 200 patients with lung cancer, we confirmed that strong phospho-MARCKS staining was observed in lung cancer specimens from smokers and was positively correlated, as compared to non-smokers. In addition, we demonstrated that increased smoking pack-year was noted in lung cancer patients with high phospho-MARCKS. Surprisingly, there is an apparent trend that patients with both tobacco use and high phospho-MARCKS levels had the worst overall survival. MARCKS was found to become activated in airway epithelium and lung cancer in response to smoke both in vitro and in vivo. MARCKS inhibition significantly attenuated smoke-induced oncosphere formation and migratory capability. Mechanistically, upregulation of phospho-MARCKS by smoke occurred in parallel with increased inflammatory cytokine expression (e.g. TNF-α, IL-8) and stemness marker expression (e.g. Oct4, Nanog, CD133, Sox2). Conversely, targeting of MARCKS phosphorylation with the MPS peptide resulted in down-regulation of smoke-induced stemness and cytokine genes. Our data suggests that phospho-MARCKS plays a role in modulating smoke-mediated lung cancer progression and is a potential prognostic biomarker in smoke-related lung cancer.

#2007

A novel CRISPR/Cas9 reporter system to monitor TNBC cancer stem cells in real time.

Nathan A. Truchan,1 Johanna Buschhaus,1 Michael Brooks,1 Andre Halabu,1 Hongwei Guo,2 Hebao Yuan,1 Kathryn Luker,1 Max Wicha,1 Duxin Sun,1 Joseph P. Burnett1. 1 _University of Michigan, Ann Arbor, MI;_ 2 _College of Pharmacy, Guangxi Medical University, Nanning, China_.

Increasing evidence suggests that breast cancers arise from so-called cancer stem cells (CSC), which, consistent with their normal adult counterpart, are uniquely capable of long term self-renewal and differentiation into all bulk cancer cell types that exhibit unique epigenetic states within a tumor. While FACS has been instrumental to isolating these cells and quantifying efficacy of CSC targeted drugs in vitro and in vivo, having to remove cells from their native environment is an inherent limitation, thereby only providing a snapshot of information with each experiment. In order to directly visualize CSC dynamics in real time the CRISPR/Cas9 system was used to precisely knockin mCherry directly downstream of the final exon of ALDH1A3 in SUM149 triple-negative breast cancer cells. With regard to CSC properties, ALDH1A3 high cells were shown to be dramatically correlated with the ALDEFLUOR assay and functionally, increased ammosphere and tumor formation in vitro and in vivo. Strikingly, a single ALDH1A3 high cell was capable of reconstituting the entire heterogeneity of the parental cell line. With the ability to directly visualize CSCs we sought to utilize live cell imaging to observe population changes in response to varying therapeutics. As evident by mCherry fluorescence intensity we confirm previous findings that suggest conventional chemotherapeutics such as docetaxel enrich for CSCs while the natural product sulforpahane reduces the population. Taken together, we believe our mCherry reporter cells could be adapted to rapidly screen for novel inhibitors of CSCs in triple-negative breast cancers.

#2008

Stem cell-associated proteins P63 and Bmi-1 may cooperate in maintaining cancer-initiating cell populations in squamous cell carcinomas.

Linan Ha, Devin Reilly, Roshini Ponnamperuma, Andrea George, Wendy C. Weinberg. _FDA, Silver Spring, MD_.

Overexpression of p53-related protein p63, particularly ΔNp63α, is seen in squamous cell carcinomas of the head and neck, lung, cervix and skin. We use primary murine epidermal keratinocytes as a model of human squamous epithelial cells and mimic these elevated levels using adeno- and lentiviral vectors. In previous studies, we established that ΔNp63α inhibits induction of p16ink4a and p19arf, blocks oncogene-induced senescence, and cooperates with activated v-rasHa to enhance conversion of papilloma to carcinoma in a nude mouse grafting model. We evaluated the relationship between ΔNp63α and Bmi-1, a member of the polycomb complex known to repress the INK4a/ARF gene locus, and modulated Bmi-1 expression in the presence or absence of elevated ∆Np63α to assess its impact on p16ink4a and p19arf expression. Bmi-1 protein levels correlate with those of ΔNp63α; however, higher levels of Bmi-1 block p16ink4a but not p19arf. Consistent with this, Bmi-1 is required for the binding of ΔNp63α to the p16ink4a but not the p19arf promoter. These findings may have further important implications in HNSCC as other previously published models have demonstrated that Bmi-1 is important for prevention of senescence in stem cells. Using FACS analysis and cell sorting, we are defining cell populations of keratinocytes with respect to the expression of ΔNp63α, Bmi-1, and other markers of stem cells and senescence and extending these approaches from murine to human HNSCC models with the aim of correlating these distinct patterns with cancer-initiating potential and tumor phenotype.

#2009

**Breast cancer stem cells are associated with HER2-mediated radioresistance and** **the molecular subtype conversion of HER2-negative breast cancer.**

Jae-Sung Kim. _Korea Institute of Radiological and Medical Sciences, Seoul, Republic of Korea_.

Although it has been proposed that the benefits of HER2-targeted therapy in HER2-negative breast cancer are associated with molecular subtype conversion, the underlying mechanism and the clinical biomarkers are unclear. Herein, we showed that breast cancer stem cells (BCSCs) mediate HER2 subtype conversion and radioresistance in HER2-negative breast cancer cells and evaluated serum HER2 as a clinical biomarker for HER2 subtype conversion. We found that the CD44+/CD24-/low BCSCs from HER2-negative breast cancer cells overexpressed HER2/EGFR and showed the radioresistant phenotype. In addition, we showed that trastuzumab treatment sensitized the radioresistant phenotype of the CD44+/CD24-/low cells with decreased levels of HER2/EGFR, which suggested that HER2-targeted therapy in HER2-negative breast cancer may be effective by targeting BCSCs that overexpress HER2/EGFR. Importantly, our clinical data showed that serum HER2 could be a clinical biomarker for the evaluation of HER2 subtype conversion in recurrent HER2-negative breast cancer patients. Therefore, our data provide in vitro and in vivo evidence for the molecular subtype conversion of HER2-negative breast cancer.

#2010

Aggressive features of gastrointestinal melanoma as compared to skin melanoma.

Michiko Akiyama,1 Yoko Matsuda,2 Tomio Arai,2 Hidehisa Saeki1. 1 _Nippon Medical School, Tokyo, Japan;_ 2 _Tokyo Metropolitan Geriatric Hospital & Inst. of Gerontology, Tokyo, Japan_.

The incidence of malignant melanoma is increasing worldwide. Melanomas are most commonly localized in the skin but can arise anywhere in the body where melanocytes exist. Gastrointestinal melanoma (GM) is a rare form of this disease. The prognosis of GM is poor compared to that of skin melanoma (SM), however GM has not been well characterized. GM is difficult to diagnose at an early stage because of the anatomical location. Furthermore, it is usually challenging for pathologists to diagnose GM, especially given the small amount of biopsy samples usually collected. In the present study, we examined the differences between GM and SM. Design: The clinicopathological characteristics, morphological feature, mitosis rate, expression of melanoma stem cell markers (nestin, SOX2, and ABCB5), and presence of the BRAFV600E mutation were evaluated for 10 cases of GM and 31 of SM. Results: Patients with GM tended to be older than those with SM. Moreover, GMs were more likely to be amelanotic (50% vs. 7%, P=0.001) and to display round cells (70% vs. 23%, P=0.02) than were SMs, and the mitosis rate was higher in GMs than in SMs (P<0.05). The incidence of lymph-node metastasis (60% vs. 32%, P<0.05) and distant metastasis (10% vs. 6.5%, P=0.02) was higher in GMs than in SMs at the point of resection. Expression of stem cell markers did not differ between groups; however, in SM, advanced-stage disease was associated with higher expression of nestin than was early-stage disease (P<0.05). Immunohistochemically, the expression of BRAFV600E was lower in GMs than in SMs (1.0 vs 3.3, P=0.01). Conclusion: Our investigation revealed that, compared to SM patients, GMs were generally older, more likely to display round cells, and be amelanotic. Moreover, they had a lower frequency of BRAFV600E mutations than did SM patients. We believe that identification of these features may aid in the diagnosis of GM and SM and contribute to new targeted therapies for GM.

Clinicopathological characteristics of melanomas of the skin and gastrointestinal tract

---

|

Skin | Gastrointestinal tract

N | 31 | 10

Age | 66.7 ± 16.8 | 75.7 ± 14.9

Cell morphology

Spidle | 24 (77.4%) | 3 (30.0%)

Round | 7 (22.6%) | 7 (70.0%)

Melanin

Negative | 2 (6.5%) | 5 (50.0%)

Positive | 29 (93.5%) | 5 (50.0%)

Cell proliferation markers

MIB-1 index | 25.6 ± 19.1 | 35.0 ± 18.1

Stage I,II | 22.2 ±17.2 | 21.7 ±10.4

Stage III,IV | 30.8 ±21.5 | 40.7 ±18.1

Total mitosis | 14.5 ± 22.7 | 35.8 ± 30.7 *

Stage I,II | 8.6 ± 12.8 | 3.8 ± 3.5

Stage III,IV | 23.8 ± 31.3 | 49.4 ± 26.2**

Immunohistochemical score

Nestin | 7.6±3.9 | 6.7±2.8

Stage I,II | 6.3±3.9 | 5.7±2.5

Stage III,IV | 9.6±2.9 ** | 7.1±3.0

BRAF | 3.3±3.3 | 1.0±1.5*

Stage I,II | 2.9±3.2 | 1.0±1.0

Stage III,IV | 4.1±3.3 | 1.0±1.2

*P<0.05 vs skin melanoma. **P<0.05 vs stage I, II by student-t test.

#2011

Expression analysis of transcribed-ultraconserved regions in cancer stem cell population using colorectal cancer organoids.

Ririno Honma,1 Naoya Sakamoto,1 Akira Ishikawa,1 Daiki Taniyama,1 Kaho Fukada,1 Yohei Sekino,1 Shoichiro Mukai,1 Kazuhiro Sentani,1 Naohide Oue,1 Takao Hinoi,2 Hiroyuki Egi,1 Hideki Ohdan,1 Wataru Yasui1. 1 _Hiroshima University, Hiroshima, Japan;_ 2 _NHO, Kure Medical Center, Chugoku Cancer Center, Kure, Japan_.

Background: The transcribed-ultraconserved regions (T-UCRs) are a novel class of long noncoding RNAs transcribed from ultraconserved regions (UCRs) that are located in both intra- and intergenic regions and absolutely conserved among the orthologous regions in most of the vertebrates. There are at least 481 UCRs, and these regions can produce 962 T-UCRs. T-UCRs have distinct signatures in human cancers. However, little has been known about the correlation between T-UCRs and tissue/cancer stem cells so far. Organoid is a novel 3D in vitro stem cell culture technology, which sustains stem cell-driven formation of near-physiologic, self-renewing tissues through using the specific niche factors in a dish. As it can be established from patient-derived tissue samples and recapitulates the genetic diversity of the original tissues, patient-derived tumor organoids could be a reliable model of disease. Because of its biologic features, organoid is assumed to harbor many stem and progenitor cells, so that it is suitable for stem cell analysis. In this study, we aimed to identify T-UCRs that are specifically expressed in cancer stem cell of colorectal cancer (CRC) and validate their functional role in CRC stem cells.

Methods: We focused on 61 T-UCRs that were reported as the dysregulated T-UCRs in CRC by microarray analysis. We examined them using RNA samples isolated from 3 pairs of CRC and non-neoplastic organoids by qRT-PCR. Biologic roles of the T-UCRs upregulated in CRC organoids were analyzed using a vector containing the whole sequences of the T-UCRs.

Results: Through the validation, we found that Uc.91+, Uc.182+, Uc.249+A and Uc.266+A were specifically upregulated in CRC organoids compared to those in normal organoids. We also validated the T-UCRs expression in several CRC cases using both primary tumors and organoids, which further supported the validity of CRC stem cell specific signature. Each of the expression of the 4 T-UCRs was highly upregulated in 2 CRC cell lines, HCT-116 and SW480, while we did not see any similarities in representative genetic abnormalities between the 2 CRC cell lines. Further in-depth in vitro studies are ongoing to clarify the solid evidences, especially the direct effect of T-UCRs onto the stem cells features such as dormancy and sphere-forming ability.

Conclusion: We identified that Uc.91+, Uc.182+, Uc.249+A and Uc.266+A were specifically upregulated in CRC organoids, and the 4 T-UCRs showed similar trend of expression in several CRC cell lines. These 4 T-UCRs are promising candidates that could potentially have an important role in cancer stem cell biology.

### Epithelial-to-Mesenchymal Transition and EMT and TGFbeta in Metastasis

#2012

The generation of a NSCLC EMT reporter cell line for metastatic lung cancer drug discovery and development.

Metewo S. Enuameh,1 Sangeeta Kumari,1 Chauzhong Zou,1 John Foulke,2 Elizabeth Turner,2 Weiguo Shu,1 Robert Newman1. 1 _ATCC, Gaithersburg, MD;_ 2 _ATCC, Manassas, VA_.

Cancer metastasis continues to be the leading cause of mortality in cancer patients around the world. One of the prominent biological processes implicated in cancer metastasis is epithelial-to-mesenchymal transition (EMT). EMT has also been shown to have roles in many aspects of cancer initiation and progression, including tumorigenesis and drug resistance. Despite the accumulation of a large body of data on the association of EMT with cancer, to date, EMT has not been an active target for therapeutic development partly due to the lack of appropriate in vitro models. Utilizing some of the basic biology of EMT, we have created a novel advanced in vitro model for use in both basic research and discovery of new anti-EMT drugs.

In lung cancer, vimentin (VIM) intermediate filament (IF) proteins are associated with EMT, and the metastatic spread of cancer. Vimentin expression is generally upregulated when epithelial cells transition to the mesenchymal phenotype. We capitalized on this biological phenomenon, using CRISPR/Cas9 gene editing to generate a VIM RFP (red fluorescent protein) reporter cell line in the A549 non-small cell lung cancer (NSCLC) cell line; one of the most widely used and recognized lung cancer cell lines. The A549 VIM RFP cell line harbors a C-terminal red fluorescent protein (RFP) tag on the vimentin gene which enables end-point or real-time tracking of the EMT status as cells transition from epithelial to mesenchymal phenotype under defined conditions. The EMT reporter cell line was verified at the nucleic acid (genomic and mRNA) and protein levels as well as in cell-based assays. Bio-functional evaluation of the A549 VIM RFP cell line shows sensitivity to metastatic NSCLC drugs PP1 (SRC inhibitor) and A83-01 (ALK5 inhibitor). These results provide the foundation for the use of this cell line in high throughput screening (HTS) applications including the identification of new anti-EMT drugs for metastatic NSCLC. Furthermore, the A549 Vim RFP reporter cell line is also a convenient and sensitive model for basic science research on the mechanisms of metastasis.

#2013

microRNAs targeting EMT transcription factors in breast cancer and their relation to lymph node metastasis.

Elisa Pérez-Moreno,1 Valentina Zavala,1 Gabriela Valarezo,1 Wanda Fernández,2 Pilar Carvallo1. 1 _Pontificia Universidad Católica de Chile, Santiago, Chile;_ 2 _Hospital San Borja Arriarán, Santiago, Chile_.

Metastases derived from primary tumors are responsible for the high cancer-associated death rates. Lymph nodes near to the primary breast tumor have a high chance to develop a secondary tumor, representing one of the first signs of metastasis. For this reason, inhibition of metastasis should be a major goal for breast cancer treatment. Metastasis is a non-random process that initiates with a phenotypic change of the tumor cells, named Epithelial-Mesenchymal Transition (EMT), and is leaded by the transcription factors SNAIL, SLUG, ZEB and TWIST. Because these changes in tumor cells must be plastic and reversible, epigenetic alterations are necessary for EMT. In this sense, microRNAs have emerged as candidate molecular biomarkers and novel therapeutic targets associated to metastasis. The aim of this study is to identify microRNAs that regulate the expression of EMT-transcription factors in breast cancer tumors, and that are involved in lymph node metastasis. For this purpose, we used microRNA microarray data from 50 fresh frozen breast tumors, 28 from patients with lymph node metastasis. Transcription factor expression was assayed by immunohistochemistry. Microarray data analysis using RankProd (R package) revealed approximately 40 microRNAs down-regulated in breast tumors expressing EMT-transcription factors (p<0.05). Using in silico analysis, we predicted 28 microRNAs to be regulators of SNAIL, SLUG and/or TWIST. Four not previously validated microRNAs were selected: miR-202, miR-210, miR-331 and miR-34b. We used luciferase reporter assays to assess the regulation of the 3´UTR of selected transcription factors by each microRNA, at two different concentrations: 10nM and 50nM. We found that miR-210 caused a 50% decrease in luciferase activity through SNAIL and SLUG 3'UTR when tested at 50nM. miR-331, predicted as regulator of SLUG 3'UTR, decreased luciferase activity at both evaluated concentrations in a dose-dependent manner, in a 20% and a 37%. In the case of TWIST, miR-34b at 50nM, decreased luciferase activity in a 65%. No inhibition of luciferase activity was found with mir202. In addition to transcription factors, two of these microRNAs have been predicted as regulators of chemokine receptors CXCR4 and CCR7. Both receptors direct lymphocyte migration to lymphoid organs, and their role has been also implicated in cancer. Our results suggest that down regulation of specific microRNAs, and up regulation of their targets, may induce a metastatic behavior of primary breast tumor cells, promoting invasion and colonization of lymph nodes. In this sense, changes in the expression of microRNAs may serve as biomarkers of breast cancer prognosis in patients.

#2014

CAMSAP3, a tubulin minus-end binding protein, attenuates epithelial-to-mesenchymal transition and alters microtubule dynamic in non-small cell lung cancer.

Varisa Pongrakhananon,1 Onsurang Wattanathamsan,1 Masatoshi Takeichi2. 1 _Chulalongkorn University, Bangkok, Thailand;_ 2 _RIKEN Center for Developmental Biology, Kobe, Japan_.

Cancer metastasis become the major cause of mortality in cancer patients, which the cancer migration exerts this aggressiveness. Epithelial to mesenchymal transition (EMT) plays an important role on cell movement and survival of the cancer cells during metastasizes. The establishment of front-rare polarization is involved with the alteration of cytoskeleton and cell-cell interaction, contributing to the notable morphological transformation from cobble stone-like epithelial to fibroblast-like mesenchymal feature. Microtubule dynamic has gained attention as a modulator behind these phenotypic changes and the EMT-related signal trafficking. Tubulin behavior has been shown to be governed by microtubule-binding proteins including a family of Calmodulin-regulated spectrin-associated proteins (CAMSAPs), a tubulin minus-end binding proteins. To illustrate the role of CAMSAPs on mesenchymal transition, CAMSAP2 and 3 genes were knockout using CRISPR-Cas9 system. Wound scratching and transwell migration assays demonstrate that non-small cell lung cancer becomes more motile with an elevation of actin stress fiber and focal adhesion protein paxillin following CAMSAP3 knockout, whereas CAMSAP2 depletion shows no significant effect. The mesenchymal markers including Slug, N-Cadherin and ZEB1 were substantial up-regulated, in opposite to the epithelial marker E-cadherin. Furthermore, CAMSAP3 removal causes a decrease of tubulin dynamic and consequently sustains an active state of EMT-regulatory proteins. This study reveals the negative

regulatory effect of CAMSAP3, a non-centrosomal microtubule minus-end binding protein, on mesenchymal transition and cell motility.

#2015

Exosomes secreted by highly migratory premalignant lung epithelial cells promote epithelial mesenchymal transition and migration.

Manash K. Paul,1 Suman Dutta,1 Bharti Bisht,1 Salehi-Rad Ramin,1 Paul Pagano,1 Gal Bitan,1 John D. Minna,2 Steven M. Dubinett1. 1 _University of California Los Angeles, Los Angeles, CA;_ 2 _University of Texas Southwestern Medical Center,, Dallas, TX_.

Lung cancer is a highly metastatic disease. Contrary to the common belief that metastasis is a late event, recent findings have suggested that micrometastasis may also occur as an early phenomenon. An increasing body of evidence supports a role for epithelial-mesenchymal transition (EMT) in inducing field cancerization. Reports suggest that cancer-derived exosomes can act via intra-cellular communication to induce EMT and migration. However, the role of premalignant cell-derived exosomes in EMT induction, cellular migration and establishment of field cancerization in unknown. Herein, we describe the role of premalignant cell-derived exosomes on early metastatic behavior.

Our laboratory has recently discovered a unique highly motile (HM) subpopulation of human bronchial epithelial cells (HBECs), using a novel "constricted migration" selection strategy, with enhanced metastatic potential in vivo. The HBECs used for selection were modified with changes reciprocating premalignancy (p53null, activated Kras-G12D). Comparative RNA-seq datasets illustrate increased expression of key EMT genes in HM-HBECs. This unique subpopulation of HM-HBECs offer a unique model to investigate premalignant cell migration. We used exosomes derived from HM, SM (slow motile) and unselected (UN) HBECs to evaluate EMT induction, migration, and invasion in vitro and in vivo.

Exosomes were isolated by ultracentrifugation and characterized for size distribution using dynamic light scattering (DLS), nanoparticle tracking analysis (NTA), electron microscopy, and characterized by western blotting with antibodies specific for Alix, Flotillin, CD9 and CD63. Exosome labeling for cell uptake studies was performed using Dil dye entrapment and by transient transfection with CD63-GFP, releasing GFP+ exosomes. 6X108 HBEC cells produced approximately 6 x 108 exosomes with size ranging from 50 to 130 nm. The exosome signature of HM-, SM-, and UN-HBECs were characterized by mass spectroscopy and micro RNA sequencing. Exosomes from HM-HBECs induce UN-HBECs to obtain a HM phenotype with enhanced EMT, faster migration and increased invasive capacity. Western blot and qPCR data suggest a time-dependent activation of an EMT signature. Further in vivo experiments are in progress to evaluate the role of HM-HBEC derived exosomes on metastatic homing, tumor formation and spread of premalignant HBEC cells and tumor cells.

Our data suggest that premalignant cell-derived exosomes possess distinct molecular signatures and could potentially mediate early migration, resulting in the creation of a premalignant field. As such, exosomes afford novel targets in premalignancy for early intervention and cancer interception.

#2016

Potential role of polo-like kinase 1 in epithelial-mesenchymal transition in melanoma.

Gagan Chhabra, Chandra K. Singh, Mary A. Ndiaye, Nihal Ahmad. _Univ. of Wisconsin, Madison, WI_.

Mammalian polo-like kinase 1 (Plk1), an important serine/threonine kinase, has been shown to be a critical regulator of mitosis and cytokinesis. A number of studies, including those from our laboratory, have shown that Plk1 is significantly overexpressed in several cancers, including melanoma. Plk1 has also been linked with poor disease prognosis in cancer patients. A limited number of recent studies have suggested that Plk1 may be involved in epithelial-mesenchymal transition (EMT) in different cancers. However, the role of Plk1 in EMT process in melanoma has not been studied. Indeed, EMT plays a driving role in the acquisition of cancer metastasis and an important hallmark of EMT is downregulation of epithelial marker E-cadherin, and upregulation of mesenchymal marker, N-cadherin. In this study, we investigated whether the kinase activity of Plk1 plays a role in EMT process by modulating EMT markers in melanoma cells. To determine this, we performed western blots, RT-qPCR, cell migration and cell invasion assays using A375 melanoma cells stably transfected with plasmids for wild-type (WT) Plk1 overexpression, constitutively active Plk1 (T210D) and kinase-inactive (K82R) Plk1, as well as shRNA-mediated Plk1 knockdown. We observed that overexpression of WT Plk1 upregulated the mesenchymal markers N-cadherin, Vimentin, and Fibronectin, and downregulated the epithelial marker E-cadherin. We also found that the modulation in EMT markers by Plk1 in melanoma cells was associated with the upregulation of transcription factors Snail and Zeb1. Interestingly, downregulation of endogenous Plk1 by specific shRNA resulted in a downregulation of N-cadherin, Vimentin, Fibronectin, Snail and Zeb1. Further, when we assessed the EMT-related molecular changes in A375 melanoma cells containing constitutively active and kinase-inactive Plk1, the cells containing constitutively active Plk1 showed a significant decrease in epithelial marker as well as marked increases in mesenchymal markers. However, the cells containing kinase-inactive Plk1 showed the opposite trend. Moreover, A375 melanoma cells containing constitutively active Plk1 showed higher migration and invasion potential, whereas cells containing kinase-inactive Plk1 showed limited cell migration and invasion. Overall, these results suggest that Plk1 is involved in the EMT process and its kinase activity is important for EMT-related changes in melanoma cells. Further studies are in progress to determine the cause-and-effect molecular mechanisms of Plk1 mediated EMT regulation.

#2017

Phenotypic plasticity and class switching in ovarian cancer.

Sharmila A. Bapat, Sagar Varankar, Swapnil Kamble. _National Centre for Cell Science, Pune, India_.

The multifaceted evolution of cancer involves differential responses of tumor cell populations to intrinsic and extrinsic stimuli. Numerous reports have examined the role of epithelial-mesenchymal transition (EMT) in the progression of high-grade serous ovarian carcinoma (HGSC). An earlier study from our group successfully resolved molecular subclasses in HGSC wherein metastasis was mediated by cooperative cell migration in the epithelial subclass and EMT in the mesenchymal subclass. We now explore the transcriptional circuitry in expression datasets and cell lines regulating these states. This led to the identification that Tcf21 and Slug expression correlated with the epithelial and mesenchymal phenotypes, respectively, and inversely correlated with each other. Probing the differential altered localization and expression of TCF21 and Slug further resolved a spectrum of intermediary phenotypic states along the epithelial to mesenchymal spectrum, each of which is associated with the expression of specific markers. Further, functional assays reveal the inherent plasticity of each phenotype with migratory mechanisms emerging as a differential parameter. Rigidity and high degree of differentiation of the epithelial and mesenchymal state is evident from a lack of responsiveness to specific microenvironmental conditions. Importantly, such cellular plasticity induced shifts from a steady-state phenotype following modulation of the microenvironment only in the intermediate phenotypes to exhibit flexibility to transit towards either the two ends of the spectrum. Specifically, a tendency to acquire an epithelial phenotype on growth factor deprivation was noted. The effects of BMP7 and TGFβ to induce epithelial or mesenchymal phenotypes, respectively, were also noted exclusively within the intermediary states. The stress generated by chemotherapeutic agents like paclitaxel led to enrichment of and dominant effects of the epithelial state. We further queried if this plasticity is also a feature of HGSC tumors. Marker expression (Tcf21, E-cadherin, Parp1, Slug, Anxa2 and Hyaluronan) was scored based on their frequency, intensity and location in tumors to derive a definitive scoring system comprising biomarker and tumor class indices. Application of such scoring could effectively stratifiy 95 human HGSC cases comprising primary and metastatic tumors into epithelial and mesenchymal classes, and further resolve a third class likely to represent the intermediate states. A salient feature noted was that metastases and chemotherapy could lead to class switching. Our study thus provides a comprehensive view of the intrinsic and extrinsic molecular events governing cellular plasticity in HGSC.

#2018

Rescue of ΔNp63α inhibits urothelial cell invasion by attenuating epithelial-to-mesenchymal transition (EMT).

Kirtiman Srivastava, Victoria Smith, Eric A. Fernandez, Conor Breen, Karen D. McCloskey. _Queen's University Belfast, Belfast, United Kingdom_.

Background: Loss of the transcription factor, p63 is associated with poor prognosis in muscle-invasive bladder cancers. The current study aimed to investigate the tumor-suppressive role of p63 in urothelial cancers.

Methods: A 3-dimensional (3D) organotypic raft model of urothelial cancer was established by culturing human urothelial cancer cell lines (HT1376, T24) on human bladder fibroblast-embedded collagen-I. Control rafts contained normal primary human urothelial cells (HUC). Cells were also cultured as monolayers on collagen-I coated dishes before harvesting total RNA and protein for qPCR and Western blot analyses respectively.

Results: Non-invasive HUC and HT1376 had an epithelial phenotype, characterized by compact and cuboidal morphology whereas invasive T24 cells were elongated with a spindle morphology, typical of mesenchymal cells and indicative of epithelial-to-mesenchymal transition (EMT), which is necessary for invasion. The number of invasive incidents in 3D rafts established with T24 cells was higher compared to those with HUC or HT1376 cells (N=3, p<0.05). Consistent with this, the invasive cell line had reduced mRNA and protein expression of total p63 (N=3), a differentiation marker, and the epithelial marker E-cadherin (N=3), in addition to upregulated expression of the mesenchymal markers N-cadherin (N=3) and fibronectin (N=3) and pERK-Y204 (N=2) compared to the non-invasive cells.

Treatment of invasive T24 cells with histone deacetylase (HDAC) inhibitors (vorinostat (pan) or entinostat (HDAC1,3)) attenuated the number of invasive incidents (N=3, p<0.05), and restored protein expression of total p63 (N=3) and E-cadherin (N=2) while normalizing pERK-Y204 (N=2 for T24). The tumor-suppressive properties of the ΔNp63α isoform were studied by adenoviral-mediated overexpression of ΔNp63α in T24 cells, which mimicked the effects of HDAC inhibitors and diminished EMT by rescuing E-cadherin expression while depleting fibronectin (N=2). Overexpression of ΔNp63α also reduced the number of invasive incidents in 3D models established with T24 cells (N=2).

Conclusion: Invasive T24 cells exhibited an EMT phenotype, which coincided with depleted p63 expression. Rescue of ΔNp63α expression by treatment with HDAC inhibitors or through experimental overexpression attenuated the EMT phenotype and cell invasion.

#2019

Extracellular ATP induces EMT and metastatic activities in non-small cell lung cancer cells.

Yanyang Cao, Xuan Wang, Xiaozhuo Chen. _Ohio Univ., Athens, OH_.

Cancer is a leading cause of death worldwide. Metastasis, the spread of cancer cells from the primary tumors to distant organs, is responsible for approximately 90% of all cancer-related deaths. Metastasis is a multistep process in which loss of cell-cell adhesion, increased proteolysis, and cell motility have been shown to be critical steps. Recent studies have indicated that intratumoral ATP concentration is ~1,000 times higher than normal tissues of the same cell origins in the range of at least several hundred μM. Extracellular ATP (eATP), a tumor microenvironment molecule, potentially has a crucial role in supporting tumor invasion and metastasis. Our previous studies showed that eATP is internalized by cancer cells through macropinocytosis and other endocytosis and promotes cancer cell growth, survival, and drug resistance (1-4). Based on all these, we hypothesize that eATP functions as both a signaling molecule and an energy source to stimulate epithelial mesenchymal transition (EMT) and metastasis. Various bioassays were used in human non-small cell lung cancer (NSCLC) A549 and HOP-92 cells to test our hypothesis. Our results reveal that eATP treatment led to a substantially increased number of floating cancer cells, and these cells were viable and formed clones in clonogenic assays. Fence assay indicated that eATP not only induces cancer cell detachment but also increases the number of secondary colonies formed. Moreover, treatment of eATP induces a dose- and time-dependent increase in the migration and invasive capacities of the A549 and HOP-92 cells. Confocal images demonstrated that eATP induces the formation of F-actin-dependent filopodia necessary for cell migration. Using Western blot analysis, we found that the expression of epithelial markers was significantly reduced, while the mesenchymal markers were increased after eATP treatment. Purinergic receptor (PR) inhibitors and P2X7 receptor knockdown only slightly attenuated these effects, suggesting PR signaling is only partially responsible for eATP-induced migration and invasion. These results indicate eATP is an EMT and metastasis stimulator and imply novel targets for inhibiting/preventing metastasis.

References:

1. Qian Y et al. Cancer Lett 2014;351:242-51.

2. Chen X, Qian Y, Wu S. Free Radical Biol Med 2015;79;253-63.

3. Qian Y et al. Mol Cancer Res 2016;14(11):1087-96.

4. Wang X et al. Oncotarget 2017;8:87860-77.

#2020

SAHA and EGCG affect metastatic potential to varying degrees in three triple-negative breast cancer cell lines.

Kayla A. Lewis. _University of Alabama at Birmingham, Birmingham, AL_.

Although triple-negative breast cancers (TNBC) have distinct molecular aberrations, tumors of the same histological grade may have dramatically different responses to the same therapy. TNBC tumors are basal-like, aggressive, and harbor a proclivity to metastasize to the brain and lungs. By modifying the cancer epigenome we have shown it is possible to reduce the metastatic potential of triple-negative breast cancer. Suberoylanilide hydroxamic acid (SAHA, clinically Vorinostat) is a histone deacetylase (HDAC) inhibitor, and epigallocatechin-3-gallate (EGCG) is a green tea polyphenol that acts as a DNA methyltransferase (DNMT) inhibitor. In combination, we found that 3 μM SAHA and 5 μM EGCG alter mammosphere formation, and the expression of E-cadherin and N-cadherin, which are genes involved in the epithelial-to-mesenchymal transition (EMT). Epigenetic enzyme activity was also assessed to determine if SAHA and EGCG were capable of modulating the functionality of DNMTs and HDACs. Because our previous work has identified a decrease in cellular viability with the combination of SAHA and EGCG, we aimed to investigate the effect on cell cycle arrest and genes involved in cell cycle progression. Our prior findings have indicated that the combination of SAHA and EGCG decreases the expression level of miR-221/222, which is known to directly inhibit translation of p27 and PTEN. p27 is involved in regulating the transition from G1 to S phase, and with the combination of SAHA and EGCG the expression of p27 was significantly increased, suggesting potential cell cycle arrest between G1 and S phase. Because the PTEN/Akt pathway plays a role in progression from G1 to S phase through p-Akt, we also investigated changes in the expression of PTEN with SAHA and EGCG. Together our findings are reflective of tumor heterogeneity; despite the same breast cancer subtype they respond differently to chemotherapeutics. Further studies will include research into specific epigenetic modifications associated with the epithelial-like reversion seen in this project.

#2021

The CXCL12/CXCR4 axis drives epithelial-mesenchymal transition in renal cell carcinoma.

Alisa Zhilin-Roth, Jill A. Macoska. _University of Massachusetts Boston, Boston, MA_.

Introduction. Renal cell carcinoma (RCC) is the one of the most common cancers diagnosed in adult men with more than 40000 new cases annually. Recurrence or metastasis of the disease occurs in more than 30% of patients and death from disease occurs in most of these patients due to limited therapeutic options. Clearly, a better understanding of factors contributing to RCC metastasis might improve therapeutic development and patient outcomes. One of the steps required for tumor cell progression from localized disease to metastatic disease is the acquisition of invasive abilities through epithelial to mesenchymal transition (EMT). EMT promotes loss of epithelial markers by downregulation of E-cadherin, gain of mesenchymal markers like Vimentin, and N-cadherin, and cytoskeletal rearrangement that enables cells to leave the primary site and intravasate into the blood or lymphatic systems and thereby colonize distant sites. Metastasis and colonization are highly directed mechanisms in which cancer cells migrate toward specific preferred organs that express specific protein receptors. One of these receptors is CXCR4, which is upregulated in advanced cancer including RCC and is correlated with poor survival. In this study we explored the hypothesis that CXCR4/CXCL12 signaling promotes migratory ability by inducing EMT in vitro in the RCC cell lines Caki-2 and 786-0.

Methods. To pursue these studies we utilized in vitro mammalian cell culture, cell proliferation (WST) assays, cell migration (scratch) assays, and protein analysis methods including immunoblot and immunofluorescence.

Results. These studies showed that RCC cell lines express CXCR4 that is activated by CXCL12 to induce cellular proliferation and promote a wound healing migratory response dependent on EMT cytoskeletal rearrangement by Rho GTPases. Moreover, CXCR4/CXCL12 induces down-regulation of E-cadherin, up-regulation of vimentin, N-cadherin and the upregulation of EMT transcription factors SLUG/SNAIL, consistent with EMT signaling activation. Lastly, CXCR4/CXCL12 activation promotes phosphorylation of AKT and ERK, suggesting that these signaling pathways are coupled to CXCR4/CXCL12-mediated RCC progression.

Conclusions. Our findings suggest that CXCR4/CXCL12 activation in RCC has a pivotal role in EMT induction by the acquisition of a migratory and invasive phenotype. Understanding the underlying signaling pathways employed by the CXCL12/CXCR4 axis may shed new light on the migration/ invasion mechanisms that allow RCC cells to spread to distant organs and assist with the design and testing of new therapeutic strategies to treat advanced metastatic RCC.

#2022

Oncogenic protein kinase Cι drives melanoma cell epithelial-mesenchymal transition by activating vimentin through Par6/RhoA signaling.

Wishrawana S. Ratnayake, André H. Apostolatos, Christopher A. Apostolatos, Avijit Dey, Rekha Patel, Mildred Acevedo-Duncan. _Univ. of South Florida, Tampa, FL_.

Melanoma is one of the fastest growing cancers in the United States, predicting a 14% increase in 2017 compared to 2016. The five year survival rate drops to 4% when melanoma is metastasized and responsible for ˃90% melanoma related deaths. Most available drugs target BRAF (V600E) mutation which occurs in ~60% melanoma cases, yet there is a poor prognosis and tumors acquire resistance to BRAF mutation inhibition. PKC-ι (iota) is an oncogene involved in cell cycle progression, tumorigenesis and cell survival in many cancers. We believe PKC-ι is an effective therapeutic target for invasive melanoma. We reported that PKC-ι is overexpressed in melanoma cells [Int. J. Oncol. 51(5), 1370-1382, (2017)]. In the current study, we have investigated the effects of novel PKC-ι inhibitor [4-(5-amino-4-carbamoylimidazol-1-yl)-2, 3-dihydroxycyclopentyl] methyl dihydrogen phosphate (1 µM) along with its nucleoside analog, 5-amino-1-((1R,2S,3S,4R)-2,3-dihydroxy-4-methylcyclopentyl)-1H-imidazole-4-carboxamide (2.5 μM) on cell migration and invasion of two malignant melanoma cell lines (SK-MEL-2 and MeWo) compared to a normal melanocyte cell line (MEL-NEO-F). Cell viability and WST-1 assays showed that both inhibitors show lesser cytotoxicity to MEL-F-NEO cell line at higher concentrations (˃7.5 μM) compare to significant toxicity on melanoma (˃1 μM). Both inhibitors decreased the levels of total and phosphorylated levels of PKC-ι. Furthermore, both inhibitors increased the levels of E-cadherin and RhoA while decreasing the levels of Vimentin; a mesenchymal marker associated with EMT. Treatments with inhibitors significantly decreased the phosphorylated Vimentin (S39) and also altered the phosphorylation at S33 and S56, thereby preventing the Vimentin intermediate assembly. Immunoprecipitation and reversed immunoprecipitation showed a strong interaction of PKC-ι and Vimentin and immunofluorescence staining proved the observation. mRNA expression levels of PKC-ι, Vimentin decreased upon PKC-ι and the expression levels transcription factors such as SNAIL1, ZNF703, PRX1 will be tested using qPCR, Western blots and ELISA techniques. PKC-ι inhibition downregulate Par6 thereby stabilize RhoA even upon TGFβ1 stimulation. We have also showed that PKC-ι inhibition limits the translocation of activated NF-κB p65/p52 complex and β-catenin thereby regulates NF-κB and WNT/β-catenin signaling. Overall, results show that PKC-ι is essential for melanoma progression and metastasis through activation of Vimentin via TGFβ/Par6/RhoA pathway. Therefore PKC-ι could be used as effective therapeutic targets for malignant melanoma.

#2023

RhebL1 is involved in epithelial mesenchymal transition of breast cancer cells.

Hyunji KIM, Changhoon LEE. _Dongguk University, Goyang, Republic of Korea_.

RhebL1 belongs to the ras superfamily of G proteins and is highly conserved across many species. However, the cellular function of RhebL1 have not yet been reported especially in breast cancer. In this study, we found that RhebL1 is highly expressed in MDA-MB-231 and SK-BR-3 breast cancer cells. By using the public microarray data sets, we found that overall and relapse free survival were decreased in breast cancer patients with high expression of RhebL1. The involvement of RhebL1 in epithelial mesenchymal transition of breast cancer cells was examined. Gene silencing of RhebL1 decreased the expression of mesenchymal markers such as N-cadherin, Vimentin, snail and slug and increased the expression of epithelial marker E-cadherin in MDA-MB-231 and SK-BR-3 breast cancer cells. In contrast, overexpression of RhebL1 increased the expression of mesenchymal markers and decreased the expression of E-cadherin in MCF7 breast cancer cells. Next, we examined the effects of RhebL1 expression on migration and invasion since migratory and invasive capabilities were strongly related to EMT. RhebL1 overexpression significantly increased the migration and invasion of MCF7 cells and gene silencing of RhebL1 reduced the migration and invasion of MDA-MB-231 and SK-BR-3 cells. Furthermore, we identify a novel RhebL1-binding protein, RBP1, which mediates the segregation of protein phosphatase 2A from Akt, leading to induced Akt phosphorylation under conditions by binding to RhebL1. The collective results indicate that RhebL1 is involved in EMT of breast cancer cells via interaction with RBP1-Akt-PP2A complex. These results suggest that RhebL1 may be a key regulator modulating metastatic breast cancer cells.

#2024

Hyaluronic acid conjugated nanoparticle delivery of siTWIST reduces tumor burden and enhances chemosensitivity in ovarian cancer.

Carlotta A. Glackin,1 Sophia A. Shahin,1 Shirleen Simargi,1 Ruining Wang,2 Altagracia Contreras,1 Liliana Parra,1 Loiuse Qu,1 Wei Wen,1 Thanh Dellinger,1 Julia Unternaehrer,3 Fuyujiko Tamanoi,2 Jeffrey Zink2. 1 _City of Hope Beckman Research Inst., Duarte, CA;_ 2 _University of California, Los Angeles, CA;_ 3 _Loma Linda University, CA_.

Purpose: TWIST is a transcription factor critical to embryonic development, and aberrantly activated in many cancers. It regulates epithelial-to-mesenchymal transition (EMT), the process underlying metastatic spread and drug resistance. The majority of epithelial ovarian cancer (EOC) patients respond well to chemotherapy, but relapse with metastatic and drug-resistant disease. We are investigating the role of TWIST-mediated relapse, with the goal of developing treatments that sensitize chemoresistant tumors and improve survival.

Experimental Procedures: We employ siRNA to target TWIST mRNA and have created a mesoporous silica nanoparticle with hyaluronic acid (siTWIST-MSN-HA) as a delivery vehicle. Microscopy was conducted to ensure cancer stem cell (CSC) targeting via CD44, and efficient uptake of MSN-HAs. We tested TWIST knockdown combined with cisplatin in vitro and in vivo. At necropsy, total tumor, metastasis, and ascites were evaluated to determine effects of siTWIST-MSN-HA compared to cisplatin alone. qPCR was conducted on tumors to examine effect of siRNA against TWIST, and its target genes. Mice were imaged via bioluminescence to observe CSC localization of siTWIST-MSN-HA compared to siTWIST-MSN (No HA). Tissue sections were stained via IHC to determine MSN-HA tumor targeting. CD44 staining was conducted to reveal HA co-localization in tumor.

Results: siTWIST-MSN-HAs significantly knocked down TWIST, and sensitized cells to cisplatin compared to control in vitro. Following necropsy, tumor quantification revealed mice treated with siTWIST-MSN-HAs plus cisplatin exhibited a startling 75% loss of overall tumor burden (p=0.0012), 88% loss of total metastasis (p=0.001), and further 86% loss of total ascites (p=0.002) compared to cisplatin-alone treatment groups.

EMT target expression by qPCR analysis revealed loss of TWIST1, vimentin, N-cadherin, and gain of E-cadherin in tumors treated with siTWIST-MSN-HA compared to controls. Bioluminescent images of mice at necropsy revealed highly specific tumor localization of MSN-HA Dylight (680nm) compared to MSN without HA. MSN-HA exhibited CSC targeting at metastatic sites, where most of the signal was emitted from the primary tumor; and negligible quantities of MSN-HAs were detected elsewhere. MSN-HA RITC (576nm) reveals highly specific tumor targeting via IHC; MSN-HA nanoparticles localized primarily in tumor cells and not in control organs. CD44 staining of these tissues reveals MSN-HAs co-localized with CD44 in CSCs.

Conclusions: These studies demonstrate TWIST as a promising target for metastasis and acquired drug resistance in EOC. MSN-HAs provide CSC specific CD44 targeting with high efficacy and reduce tumor burden via siTWIST therapy. Thus, siTWIST-MSN-HAs provide a promising platform to improve survival from metastatic, drug-resistant ovarian cancer.

#2025

CD147/EMMPRIN mediates epithelial-mesenchymal transition of head and neck squamous cell carcinoma.

Shinsuke Suzuki. _Akita Univ., Akita, Japan_.

Epithelial-mesenchymal transition (EMT) is a key mechanism in the progression of solid tumors. This process is characterized by the development of more migratory, invasive cell behavior. Transforming growth factor-β (TGF-β) has been well characterized as a potent inducer of EMT. CD147, also known as extracellular matrix metalloproteinase inducer (EMMPRIN), a member of the immunoglobulin superfamily, is highly expressed in cancer cells and induces malignant characteristics in various tumors, including head and neck squamous cell carcinoma (HNSCC). Accumulating evidence indicates a contribution of CD147/EMMPRIN to tumorigenesis of solid tumors. However, the function of CD147/EMMPRIN in EMT and TGF-β-induced tumorigenicity in HNSCC has not yet been explored. To determine the role of CD147/EMMPRIN in EMT and TGF-β-induced tumorigenic behavior, SAS, a tongue squamous cell carcinoma cell line, was stimulated with TGF-β. After stimulation with TGF-β, we found that SAS cells acquired a spindle-shaped morphology. Immunoblotting analysis showed that the expression of vimentin was upregulated and E-cadherin was downregulated after TGF-β treatment. This result suggests that EMT was induced by TGF-β in SAS cells. In addition, expression of CD147/EMMPRIN was upregulated in SAS cells using TGF-β treatment. TGF-β stimulation enhanced the migratory ability of the cells. Furthermore, the TGF-β-induced migratory ability, and the change in cellular morphology was diminished by CD147/EMMPRIN knockdown. These results suggest that CD147 is involved in TGF-β-induced EMT and the migratory capacity of HNSCC.

#2026

Quantifying the mesenchymal and cancer stem cell properties of C6 glioma cells and their morphological changes after epithelial-to-mesenchymal transition.

Arpan De, Dilshan Harshajith Beligala, Christian Andres Burgos, Sanjhi Dhaval Gandhi, Michael Eric Geusz. _Bowling Green State Univ., Bowling Green, OH_.

Epithelial-to-mesenchymal transition (EMT) is a major event converting tumor cells into more migratory or metastatic cancer cells. Previous results suggest that EMT is controlled by a circadian clock in C6 rat glioma cells, favoring EMT and a larger mesenchymal cell population at a particular time of day along with expression of EMT protein markers ZEB1 and vimentin. To determine how well the distinct morphology of these post-EMT cells correlates with protein expression indicative of mesenchymal cancer cells (TWIST) and cancer stem cells (OCT4), a standard method was used to induce EMT in C6 cells resembling changes occurring in the tumor cell microenvironment. The serum-based cell culture medium was exchanged with a serum-free stem cell medium (SCM) containing growth factors EGF, FGF2, and PDGF-AB. After 48 hours, nearly all cells were of the mesenchymal phenotypic shape. Confocal microscopy of immunofluorescence and Hoechst nuclear staining indicated that the average percentage of OCT4-positive cells increased about 10-fold after 48 hours in SCM, from 7.32 to 75.3, and the average percentage of E-cadherin-positive/OCT4-negative (epithelial-like) cells declined about 3-fold, from 10.4 to 3.62. However, the average percentage of all E-cadherin-positive cells increased from 13 to 31.8. In a second experiment, the average percentage of OCT4-positive cells increased from 19.9 to 96, and the average percentage of TWIST-positive cells increased from 39.3 to 74.5. In SCM, 99.8% of the TWIST-positive cells were also OCT4-positive. Of the cells in SCM identified as isolated mesenchymal cells by their distinctive elongated bipolar morphology, 93% were positive for OCT4 and all were positive for TWIST, indicating an increased population of mesenchymal cancer stem cells in the favorable microenvironment. When considering all co-labeled cells, TWIST and OCT4 were correlated significantly by the intensity of their corresponding fluorescence. In a third experiment, 85% of cells were OCT4-positive after 48 hours in SCM, and 5% of the cells were OCT4-positive and negative for cell proliferation marker PCNA, suggesting that these may be quiescent cancer stem cells. Mesenchymal-to-epithelial transition (MET) was then induced by returning cells in SCM to serum-containing medium, which produced cells with a flattened cell morphology, indicating an epithelial or pseudoepithelial differentiation. MET events are being examined for any evidence of circadian clock regulation of MET timing. This study shows that mesenchymal cells with cancer stem cell properties generated by EMT can be reliably identified by their morphology. It also characterizes isolated mesenchymal cancer cells that are suitable for single-cell molecular analyses to understand their important role in gliomas.

#2027

P62/IMP2 promotes metastasis in hepatocellular carcinoma (HCC) by regulating EMT in different signaling pathway.

Mengtao Xing,1 Jianxiang Shi,2 Jitian Li,1 Beibei Chen,1 Giulio Francia,1 Jianying Zhang2. 1 _UT El Paso, El Paso, TX;_ 2 _Zhengzhou University, Zhengzhou, China_.

Insulin- like growth factor 2 mRNA binding proteins (IMPs) Family contains three members: IMP1, IMP2 and IMP3. IMPs are oncofetal proteins, expressed during embryogenesis and lost in most tissues in adults. Overexpressed IMPs have been reported in various types of cancers. They share similar protein structure in mammals and have similar functions: to bind specific mRNAs, stabilize and extend their half-life. IMP2 was first reported as a tumor-associated antigen in HCC. However, only a few research was done on what a role of p62/IMP2 plays on HCC progression.

Previous studies show that IMP2 is overexpressed in HCC tissues and in several human HCC cell lines. Western blotting was performed for different HCC cells has a relatively low expression in SNU449 and higher expression in HepG2 and Hep3b. Stable IMP2 overexpression SNU449 cell line was made by IMP2 cDNA plasmid transfection; stable IMP2 knockout Hep3b and HepG2 cell lines was made by IMP2 Crispr-Cas9 KO plasmid transfection. Wound healing assay has shown that overexpression of p62/IMP2 can significantly enhance cell migration ability in SNU449 cells. On the contrary, the depletion of p62/IMP2 can reduce the migration ability in HepG2 and Hep3b cells (p<0.05). Furthermore, we analyzed HCC gene expression data from TCGA. Median count of IMP2 was used to separate HCC expression data into high and low IMP2 expression group to conduct differential gene expression analysis. Gene Ontology over-representation analysis shows differentially expressed genes (adjusted p<0.05) mainly enriched in membrane part and plasma membrane. The expression of several genes involved in EMT regulation such as CTNNB1, SMAD2, SNAI2, AKT2 shows significant difference (adjust p<0.05). Western blotting assay was performed to show whether IMP2 is involved in EMT regulation. Wnt/beta-catenin pathway and TGF-beta/SMAD pathway were both activated and further upregulated SNAIL, which is an E-cadherin inhibitor. Western blotting results showed that E-cadherin was inhibited in IMP2 overexpressed SNU449 cells, which is supportive to our hypothesis.

#2028

ZEB2 promotes cancer cell survival and angiogenesis by cooperating with transcription factor Sp1.

Dongjoon Ko. _Korea Research Institute of Bioscience and Biotechnology(KRIBB), Daejon, Republic of Korea_.

The metastatic cascade essential for tumor progression consists of local invasion, intravasation, extravasation, micrometastasis, cancer cell survival in the circulation, and metastatic colonization. In particular, epithelial-mesenchymal transition (EMT) has been demonstrated a critical process whereby epithelial cells undergo molecular changes to acquire mesenchymal phenotypes through upregulation of EMT-inducing transcription factors. We previously showed that ZEB2, through cooperation with the transcription factor Sp1, plays an important role in the transcriptional upregulation of mesenchymal genes such as integrin α5 and vimentin during cancer cell invasion. Here, we show that ZEB2 cooperates with Sp1 for promoting not only EMT and invasion but also diverse cellular functions during metastasis. ZEB2 induces expression of Sp1-regulated genes such as VEGF, survivin, bcl-2 and cyclin D1 in an Sp1-dependent manner. Collaboration between ZEB2 and Sp1 induces cancer cell survival and proliferation, endothelial cell activation, and tumor angiogenesis. Furthermore, Sp1 stabilizes ZEB2 protein, supporting the presence of a positive feedback loop between ZEB2 and Sp1. Clinical data show that ZEB2 expression was correlated to Sp1 expression, and that those factors were a crucial for predicting survival in cancer patients. Our findings demonstrate cooperative roles of ZEB2 and Sp1 in regulating cancer cell survival and angiogenesis beyond invasion during cancer progression.

#2029

Notch signaling inhibitor increases E-selectin/ligand interactions and alters cell migration of mesenchymal-like breast cancer cells.

Christian A. Showalter, Monica M. Burdick. _Ohio University, Athens, OH_.

Activation of Notch signaling has been shown to promote cell migration properties and hematogenous metastasis by inducing a dramatic phenotypic change in breast cancer cells via the epithelial-to-mesenchymal transition (EMT). In response to these findings, Notch signaling inhibitors are being explored as therapeutics to prevent EMT in cancers. However, research examining the effects of Notch inhibitors on the activity of sialofucosylated ligands used by circulating tumor cells to adhere to vascular endothelial E-selectin during metastasis is lacking. To advance knowledge in this area, our study investigated the use of an indirect Notch signaling inhibitor (DAPT) on modifying E-selectin ligand activity of breast cancer cell lines by shear flow adhesion assays performed at physiological bone marrow shear stress. DAPT exposure to mesenchymal-like MDA-MB-231 and MDA-MB-468 (induced to the mesenchymal-like state by exogenous epidermal growth factor) breast cancer cells increased E-selectin ligand activity. Furthermore, mesenchymal-like MDA-MB-468 cells after DAPT exposure demonstrated E-selectin ligand activity similar to that of epithelial-like MDA-MB-468 cells. This supports the hypothesis by our group that epithelial-like and mesenchymal-like phenotypes determine activity of functional E-selectin ligands in breast cancer cells, as we have previously shown that epithelial cells display high E-selectin ligand activity while mesenchymal cells display little or no ligand activity. Cell migration ability was assessed using wound closure assays. Mesenchymal-like MDA-MB-468 cells exposed to DAPT exhibited decreased migration compared to mesenchymal-like MDA-MB-468 cells exposed to the DMSO diluent control, whereas mesenchymal-like MDA-MB-231 cells demonstrated no difference in migration compared to DMSO exposed cells. Altogether, our results suggest Notch inhibitors may both increase fluid-shear resistant adhesion and alter migration ability of mesenchymal-like breast cancer cells, and that factors present in the blood might impact the metastatic potential of cancer cells via regulating cellular phenotypes.

#2030

Eribulin disrupts TGF-β-mediated Smad2/3-dependent transcription of EMT promoting proteins in triple negative breast cancer cells.

Roma Kaul, April L. Risinger, Susan L. Mooberry. _University of Texas Health Science Center at San Antonio, San Antonio, TX_.

Microtubule targeting agents (MTAs) are of significant utility in the treatment of breast cancer. While decades of research have shown that these drugs cause mitotic arrest in cells by suppressing the dynamic instability of microtubules, recent evidence demonstrates that the ability of MTAs to disrupt the microtubule-dependent transport of key signaling proteins in interphase likely contributes to their anticancer actions. Ligand-mediated activation of TGF-β receptors leads to the activation of downstream pathways that induce the expression of Snail and Slug, key transcriptional-repressors that promote EMT through Smad-dependent and independent pathways. Eribulin was shown by Yoshida and colleagues to reverse epithelial-to-mesenchymal transition (EMT) in preclinical models of triple negative breast cancer (TNBC) within 7 days. We tested the hypothesis that the initial disruption of microtubule dynamics and structure by eribulin could rapidly inhibit TGF-β-dependent signaling related to EMT as compared to other clinically relevant MTAs. A panel of four TGF-β responsive triple negative breast cancer (TNBC) cells, BT-549, MDA-MB-231, HCC1937 and Hs578T, were pre-treated with clinically relevant concentrations of the MTAs eribulin, vinorelbine, paclitaxel, or ixabepilone, which caused maximum disruption of the interphase microtubule network within 2 h. These cells were then stimulated with 2 ng/mL of TGF-β ligand for 30 min - 3 h, followed by evaluation of downstream TGF-β targets. Immunofluorescence experiments showed that the microtubule destabilizers eribulin and vinorelbine inhibited the nuclear accumulation of phosphorylated-Smad 2/3 to an extent greater than the microtubule stabilizers paclitaxel and ixabepilone or the vehicle control. Further, eribulin and vinorelbine caused significant inhibition of TGF-β-induced Snail expression in all four cell lines when compared to vehicle or microtubule stabilizer-treated cells. Knock-down studies confirmed that the effects of MTAs on Snail expression are mediated by Smad 2/3. Consistent with current literature, a 7-day eribulin treatment began to reverse their mesenchymal characteristics as measured by loss of N-cadherin in two TNBC cell lines. These data support the premise that rapid, eribulin-mediated, inhibition of TGF-β signaling contributes to its ability to reverse EMT, through inhibition of Smad-2/3 dependent transcription. Ongoing studies are investigating whether eribulin differentially inhibits Smad 2/3 protein interactions with its upstream scaffold SARA that is integral for optimal signaling as compared to other MTAs. Dissecting how MTAs differentially disrupt the TGF-β-Smad-2/3 signaling pathway might facilitate a better understanding of the molecular contexts of breast cancers that might benefit from treatment with specific MTAs. These studies are supported by Eisai Inc.

#2031

The noncanonical pathways of transforming growth factor β in ovarian carcinoma.

Liora Jacobs Catane,1 Claes G. Trope,2 Ben Davidson,2 Reuven Reich1. 1 _Hebrew Univ. of Jerusalem, Jerusalem, Israel;_ 2 _Oslo University Hospital, Oslo,, Norway_.

Introduction: Of all the gynecological malignancies, ovarian cancer (OC) has the highest mortality rate. This can be attributed to the late detection and acquired drug resistance. It metastasizes mainly in the serosal cavities, and produces cancerous cells dispersed in the peritoneal and pleural fluids called effusions. Transforming Growth Factor β (TGFβ) is a family of growth factors that play a significant role in cellular processes. There are three homologous isoforms of TGFβ expressed in humans: TGFβ1-3. These 3 ligands activate 3 transmembrane receptors, TβRI-III. The 3 receptors and the ligand TGF-β2 have different splice variants. TGFβ signaling induces canonical and non-canonical signaling. The Non-canonical pathways consist of a variety of different pathways, including MAP kinase, Rho-like GTPase, and phosphatidylinositol-3-kinase (PI3K)/ AKT pathways.

Aims and methods: Our first aim was to investigate the metastatic role of TGFβ receptor variants. This was accomplished through the creation of specific receptor variant knockouts (KO's) using the CRISPR/Cas9 genome editing system and subsequent metastatic tests. Next we characterized chosen non-canonical pathways and downstream effectors in the CRISPR KO's through Western Blot analysis and qPCR respectively. The Second aim of this study was to characterize the non-canonical pathways In-vivo. Using human tumor tissue samples, we examined the expression and activation of the different pathways via Western Blot analysis and checked for correlation with clinical parameters.

Results: KO cells showed significantly lower migration and invasion capabilities compared to Wild Type (WT) cells. Our results show a decrease in the activation of ERK1/2, JNK1 and JNK2 in the TβRI variant 1 KO in ES-2 cells (p=0.058, p=0.002 and p<0.0001 respectively). The downstream effectors SNAIL and TWIST show significantly higher levels in the KO compared to WT cells. Of the pathways examined, p-AKT was found to be higher in post chemotherapy samples (p=0.029) and JNK1 was higher in effusions from patients with poor chemotherapy response (p=0.045). Site-related findings show that p85 (PI3K), AKT and p-AKT were highest in metastasis, medium in solid ovarian carcinoma and lowest in effusions (p<0.0001, p=0.0024 and p=0.0021 respectively).

Closing statement: These findings provide new insight on the importance of TGFβ's role in the progression of OC, and is, as far as we know, is the first study done on the metastatic roles of the receptor variants.

#2032

CXCR7 promotes the migration and invasion of head and neck squamous cell carcinoma through Smad2/Akt signaling.

Nayoung Kim, Solbi Kim, Hyewon Ryu, Hyo Jin Lee. _Chungnam National University College of Medicine, Daejeon, Republic of Korea_.

Background: The chemokine receptor, CXCR7, has been shown to play an important role in the progression of several types of cancer. However, there have been few reports on the biological role of CXCR7 in head and neck squamous cell carcinoma (HNSCC). In this study, we investigated the functional role of CXCR7 and the underlying molecular mechanism of disease progression in HNSCC.

Methods: We characterized the expression of CXCR7 in tumor specimens from patients with HNSCC. The biological functions of CXCR7- and CXCR7-mediated signaling pathways were investigated in HNSCC cell lines with CXCR7 overexpression and treatment of SDF-1α, a major ligand of CXCR7, as well as knockdown of CXCR7.

Results: CXCR7 was differentially expressed in human HNSCC patients. High expression of CXCR7 was associated with an aggressive tumor behavior. Overexpression of CXCR7 dramatically promoted cell migration and invasion in HNSCC cells, although it was not involved in cell proliferation and colony formation. CXCR7 knockdown using siRNA in HNSCC cells recovered the cell migratory and invasive behavior of HNSCC cells. CXCR7 overexpression also induced the epithelial-mesenchymal transition. Vimentin, Slug, and Twist were increased but E-cadherin and Ep-CAM were decreased by CXCR7 expression. Akt phosphorylation and Smad2 signaling activation were induced in HNSCC cells with CXCR7 overexpression. Treatment with a PI3K inhibitor reduced Slug and Twist levels while suppression of Smad2 signaling by siRNA reduced Akt phosphorylation, as well as Slug and Twist. Furthermore, inhibition of Smad2 decreased tumor cell migration and invasion in HNSCC.

Conclusions: CXCR7 contributed to cell migration and invasion of HNSCC through the Smad2/Akt signaling axis, suggesting that CXCR7 might be a therapeutic target for the treatment of HNSCC.

#2033

Transforming growth factor-b-induced epithelial-mesenchymal transition is attenuated by telomerase-specific oncolytic virotherapy in human esophageal cancer.

Tomoya Masuda,1 Hiroshi Tazawa,1 Takeshi Ieda,1 Yuuri Hashimoto,1 Shunsuke Tanabe,1 Kazuhiro Noma,1 Yasuo Urata,2 Shunsuke Kagawa,1 Yasuhiro Shirakawa,1 Toshiyoshi Fujiwara1. 1 _Okayama University Graduate School of Medicine, Okayama, Japan;_ 2 _Oncolys BioPharma, Inc, Tokyo, Japan_.

Background: Epithelial-mesenchymal transition (EMT) is known to be one of the poor prognostic factors. EMT is a biological process, by which epithelial cancer cells lose their epithelial characteristics and gain mesenchymal properties, including invasion, metastasis and drug resistance, resulting in tumor recurrence and poor prognosis. Esophageal cancer is one of the most common cause of cancer-related death worldwide, especially in East Asia. Despite the recent advances in the treatment of esophageal cancer, antitumor therapy to inhibit EMT program remains to be developed. We developed a telomerase-specific replication-selective oncolytic adenovirus (OBP-301; Telomelysin), in which the human telomerase reverse transcriptase promoter drives the expression of E1A and E1B for virus replication. In this study, we explored the biological effect of OBP-301 on EMT in human esophageal cancer cells.

Methods: Transforming growth factor-β (TGF-β) was used to induce EMT in human esophageal cancer TE-4 cells. To investigate whether OBP-301 infection affects TGF-β-induced EMT, we performed western blot and real-time PCR analysis for EMT-related markers. We also examined the migration capability using transwell migration assay. In vitro antitumor effect of OBP-301 and chemotherapeutic agents on TGF-β-treated TE-4 cells were assessed by a XTT assay.

Results: Administration of TGF-β induced mesenchymal characteristics, including spindle-shaped morphology, increased migration capability, and upregulation of mesenchymal markers N-cadherin and vimentin, in TE-4 cells. When TGF-β-treated TE-4 cells were infected with OBP-301, upregulation of N-cadherin and vimentin expression was attenuated. Upregulation of EMT-transcription factors, Snail, Slug, and ZEB1 by TGF-β was reduced by OBP-301 infection. OBP-301 also inhibited TGF-β-induced enhancement of migration capability. Moreover, interestingly, EMT-induced TE-4 cells acquired chemoresistance, whereas they were sensitive to OBP-301-mediated lytic effect.

Conclusion: Our data suggest that telomerase-specific oncolytic virotherapy is a promising antitumor strategy to inhibit TGF-β-induced EMT and warrants clinical trials in human esophageal cancer. Currently, we are conducting a phase I/II clinical study of OBP-301 in combination with radiotherapy for esophageal cancer.

#2034

Traf2-and Nck-interacting kinase (TNIK) inhibitor, NCB-0846, suppresses TGF-β1-induced epithelial-mesenchymal transition in lung cancer.

Teppei Sugano,1 Mari Masuda,1 Yuko Uno,2 Naoko Goto,1 Masahiro Seike,3 Masaaki Sawa,2 Akihiko Gemma,3 Tesshi Yamada1. 1 _Division of Chemotherapy and Clinical Research, National Cancer Center Research Institute, Tokyo, Japan;_ 2 _Carna Biosciences, Inc, Kobe, Japan;_ 3 _Department of Pulmonary Medicine and Oncology, Graduate School of Medicine, Nippon Medical School, Tokyo, Japan_.

Epithelial mesenchymal transition (EMT) has been implicated in cancer cell migration, invasion, and metastasis. Transforming growth factor-β (TGF-β) signal was known to be an essential EMT promotor. Development of therapeutics for targeting EMT through the TGF-β signal may be beneficial for the management of cancer metastasis. Recently, we found that a Traf2- and Nck-interacting kinase (TNIK) inhibitor, named NCB-0846, was capable of attenuating tumor-initiating cells among human colorectal cancer (1, 2). The cross link between EMT and cancer stemness has been revealed in several studies. Therefore we evaluated whether this small-molecule compound could have efficacy to inhibit TGF-β-induced EMT. NCB-0846 reduced the expression of mesenchymal markers (Vimentin and N-cadherin) and upregulated the expression of epithelial marker E-cadherin in A549 and H2228 cells. NCB-0846 suppressed the phosphorylation and nuclear translocation of Smad proteins and also inhibited migration, invasion, and metastasis. NCB-0846 inhibited TGF-β1-induced EMT through the down-regulation of TGFBR1 in mRNA levels. MiR-186-5p and miR-320 family were identified as candidate miRNAs that could target TGFBR1 and inhibited TGFBR1 protein expression.NCB-0846 might be a novel therapeutics drugs that targets the invasion and metastasis through inhibiting TGF-β-induced EMT in lung cancer.

1. Masuda M, Sawa M, Yamada T. Therapeutic targets in the Wnt signaling pathway: Feasibility of targeting TNIK in colorectal cancer. Pharmacology & therapeutics. 2015;156:1-9. Epub 2015/11/07.2. Masuda M, Uno Y, Ohbayashi N, Ohata H, Mimata A, Kukimoto-Niino M, et al. TNIK inhibition abrogates colorectal cancer stemness. Nature communications. 2016;7:12586. Epub 2016/08/27.

#2035

Knockdown of TGFβ-activated ATF4 inhibits triple negative breast cancer metastases independently of cellular stress.

Jenny C. Chang,1 Alberto Ramirez,1 Maria P. Molina,2 Francisca E. Cara,2 Wei Qian,1 Wen Chen,1 Anthony J. Kozielski,1 Roberto R. Rosato,1 Juan A. Marchal,3 Jose A. Lorente,4 Pedro Sanchez-Rovira,2 Sergio Granados-Principal2. 1 _Houston Methodist Research Institute, Houston, TX;_ 2 _Complejo Hospitalario de Jaen, Jaen, Spain;_ 3 _Biosanitary Research Institute of Granada, Granada, Spain;_ 4 _GENYO, Centre for Genomics and Oncological Research (Pfizer / University of Granada / Andalusian Regional Government), Granada, Spain_.

Background. Triple negative breast cancer (TNBC) is a very aggressive form of breast cancer which is characterized by a poor survival rate and high incidence of metastases. The integrated stress response (ISR) is activated under stress conditions (hypoxia, nutrient deprivation, or endoplasmic reticulum stress) and reduces the protein synthesis through phospho-eIF2α/ATF4 (activating transcription factor 4) to regulate cell fate. ATF4, which is overexpressed in breast cancer including TNBC, regulates tumor growth, autophagy, drug resistance, and metastasis during ISR through PERK and GCN2 pathways. We have reported enhanced ATF4 expression in unstressed MCF10A cells treated with TGFβ1. Here, we investigate the potential TGFβ-mediated stress-independent control of ATF4 activity and its impact on the TNBC-associated metastasis.

Methods. SUM159PT and BT549 TNBC cell lines were treated with human recombinant TGFβ1 (10 ng/ml) and the TGFBRI kinase inhibitor LY2157299 (5µM) for 72h. Effects of TGFβ on ATF4 expression were assessed by silencing SMAD2/3 and SMAD4 with TGFβ1 for 24h in SUM159PT and BT549 cells. Changes in ATF4 expression by IRS were assessed after PERK, GCN2, PKR, HRI, and eIF2α inhibition with TGFβ1 for 72h in SUM159PT, BT549 and MDA-MB-231 cell lines. ATF4 expression levels were determined by RT-PCR and/or western blot. Effects on TGFβ-induced metastasis were analyzed by ATF4 knockdown for 48h by using two different siRNA sequences (#1 and #2) following treatment with TGFβ1 for 24h. Migration and invasion were performed by wound healing and transwell assays, respectively. Results were compared to a scrambled siRNA as negative control (SCR).

Results. Our results show that ATF4 expression (at RNA and protein levels) was abrogated by LY2157299 upon TGFβ activation, suggesting that ATF4 is active downstream of TGFBRI. This result was supported by further SMAD2/3 and SMAD4 knockdown following treatment with TGFβ1, which was correlated with decreased ATF4 expression. Additionally, we assessed whether ATF4 inhibition can reduce the TGFβ-induced metastatic properties of TNCB cell lines. We found that ATF4 depletion inhibited migration and invasiveness in the three cell lines tested. ATF4 is known to exert a pro-metastatic role downstream of ISR through PERK and GCN2 pathways. Our results demonstrate that, upon treatment with TGFβ1, knockdown of the IRS mediators PERK, PKR, GCN2, HRI, and eIF2α did not correlate with a consistent decrease of ATF4 levels in the three cell lines.

Conclusion. In conclusion, our results show for the first time that ATF4 is a downstream target of the canonical TGFβ/SMAD pathway in a ISR-independent fashion, and its depletion correlates with an inhibition of the TGFβ1-mediated migration and invasion of TNBC cell lines. Therefore, ATF4 may represent a therapeutic target in TNBC patients with active TGFβ signaling pathway.

#2036

Alternative activation of TGFβ signaling pathways in breast cancer tumors with different subtype is related to a differential expression of transcription factors that induce EMT.

Victoria Ortega-Hernández,1 Wanda Fernandez,2 Pilar Carvallo1. 1 _Pontificia Universidad Catolica de Chile, Santiago, Chile;_ 2 _Hospital San Borja Arriarán, Santiago, Chile_.

Breast cancer is the leading cause of cancer death among women worldwide being distant metastases the main cause of disease. Patients with triple negative breast cancer tumors develop distant metastases earlier compared to luminal tumors. Epithelial-mesenchymal transition (EMT) induced by TGFβ signaling pathways, is the main mechanism to promote metastasis through the expression of the transcription factors TWIST, SNAIL, SLUG and ZEB1. Expression of EMT transcription factors has been studied in cancer cell lines however studies in breast cancer tumors with different subtype have not been reported. In addition, the molecular mechanism that induce EMT program is not known yet. In this study we evaluated the expression of the four transcription factors as well as the state of TGFβ/SMAD, ERK/MAPK and PI3K/AKT pathways in breast cancer cell lines HCC1937 (triple negative) and T47D (luminal) after TGFβ treatment. The same analysis was done by immunohistochemistry in 100 breast cancer tumors with different subtype. After TGFβ treatment T47D showed activation of ERK/MAPK and PI3K/AKT pathways and expression of only two transcription factors: SNAIL and SLUG. HCC1937 cells showed activation of TGFβ/SMAD and ERK/MAPK pathways and expression of TWIST, SLUG and ZEB1 but not SNAIL. Similar results were observed in breast cancer tumors, being TGFβ/SMAD pathway active in the majority of triple negative tumors, whereas an active state of PI3K/AKT was observed in luminal tumors. Active ERK/MAPK pathway was observed in both triple negative and luminal tumors. In relation to expression of transcription factors triple negative tumors with an active state of the TGFβ/SMAD pathway showed expression of transcription factors TWIST, SNAIL and SLUG, whereas those tumors with an active state of ERK/MAPK pathway showed only ZEB1 expression. Luminal tumors with an active PI3K/AKT pathway frequently showed ZEB1 expression. Our results showed that expression of a specific EMT transcription factor induced by TGFβ is related to the tumor subtype and depends on the activated signaling pathway. These results suggest different mechanisms of EMT induction by TGFβ in relation to breast cancer tumor subtype.

#2037

E-cadherin enhances immune control of metastatic melanoma.

Bradley D. Shields, Brian Koss, Fade Mahmoud, Stephanie D. Byrum, Alan J. Tackett. _Univ. Of Arkansas for Medical Sciences, Little Rock, AR_.

The advent of checkpoint blockade therapies has transformed the clinical management of metastatic melanoma. These monoclonal antibodies, targeted against CTLA-4 and PD-1, have shown durable and long-lasting responses in a subset of melanoma patients. However, response rates remain incomplete, even when using combination therapy (CTLA-4 plus PD-1). Identifying predictive or controlling features of responsiveness has been urgent goal in oncology research. Our group and others have previously shown responding tumors display high levels of E-cadherin, a cell-cell adhesion molecule which defines an epithelial-like phenotype. The varying clinical responses observed to checkpoint blockade in metastatic melanoma, paired with the knowledge that E-cadherin expression correlates with such responses, provides impetus to study the significance of E-cadherin in the context of checkpoint blockade. Here, we present a novel B16F10 melanoma mouse model in which E-cadherin is exogenously expressed (B16.E-cad). The parental B16F10 line does not express E-cadherin and is poorly immunogenic and notoriously resistant to treatment with immune checkpoint inhibitors. Thus, B16.E-cad and the vector control model epithelial-like (responsive) and mesenchymal-like (non-responsive) phenotypes respectively. We find, compared to vector control, B16.E-cad exhibits delayed tumor growth, reduced metastatic potential, and increased overall survival in vivo, despite similar proliferation rates in vitro. Analysis of immune cell infiltration (FACS) performed on tumors grown in C57/BL6 mice revealed no differences between vector, B16.Ecad and B16.SIY. Conversely, transplantation of B16.E-cad into Rag1-/-mice abrogated tumor growth delay indicating an immune-mediated tumor response. Additionally, B16.Ead tumors displayed greater responses to combination checkpoint inhibitor therapy. Further, imunohistochemistry for E-cadherin performed on patient pretreatment tumor biopsies revealed negative staining was predictive of a non-responsive status. These data indicate an epithelial-like phenotype in melanoma tumors facilitates tumor immunosurveillance and responsiveness to immune checkpoint blockade.

#2038

The role of epithelial-mesenchymal transition in ovarian cancer.

Hasanthi Assalaarachchi, Helen M. Coley. _University of Surrey, Guildford, Surrey, United Kingdom_.

Epithelial ovarian cancer (EOC) is one of the leading causes of death due to malignant disease in women. Due to its asymptomatic nature, 80% of patients are diagnosed at late stages, when the disease has metastasized and 5 year survival rates are then around 30%. Typically late stage EOC rapidly develops resistance to chemotherapy.

Recently, attention has focused on a possible involvement of EMT: epithelial-mesenchymal-transition- in the development of resistance to chemotherapy drugs. EMT refers to a change in cell morphology with the loss of the apical-basal polarity of a cell, the loss of cell-cell junctions and the gain of a spindle-like shape with migratory and invasive properties. Our work sets out to understand the role of EMT in EOC focusing on its contribution to the development of resistance towards carboplatin and paclitaxel.

Firstly, an in-vitro model of EMT was developed with OVCAR-3 EOC cells, using a cocktail of 20ng/ml EGF, 50ng/ml IL-6 and 10ng/ml TGF-β. Western blot results showed significantly increased expression of slug and vimentin with decreased E-cadherin expression in the in-vitro derived EMT model compared to the control untreated cells. In addition, using established drug resistant cell lines of OVCAR-3, PEO1, CaOV3 and SKOV-3 the EMT signature was compared with that of the corresponding parental line. Western blot examination indicated decreased E-cadherin expression (P>0.05) and significantly increased slug and vimentin expression (P<0.05) in the resistant cell lines, similar to the cytokine-treated model of EMT induction. In order to observe the effect of drug resistance on migratory properties, OVCAR-3 (paclitaxel-sensitive and resistant) and PEO1 (carboplatin-sensitive and resistant) were used in scratch/wound healing assays. Drug treatment was carried out and wound closure was measured with CellProfiler after 72 hours. A significantly increased migratory capacity (wound closure) was observed in drug resistant cells in the absence and presence of chemotherapy drugs, when compared with parental cells (P<0.05). In order to clarify any possible role for slug in the induction of drug resistance, we carried out SiRNA silencing of OVCAR-3 cells. Silenced cells (alongside scramble control) were subjected to drug treatment for 48 hours and were then assessed for apoptosis using the annexin assay (FITC and PI). On silencing the slug gene, a significant resensitization to carboplatin could be observed compared to control cells (P<0.05).

Further work includes analyses of the panel of drug sensitive and drug resistant (carboplatin and paclitaxel) EOC cell lines to identify other gene changes associated with EMT, using PCR array approaches. This work will help with the identity of candidate biomarkers of clinical drug resistance for EOC patients.

#2039

Cordycepin inhibits breast cancer migration and invasion by targeting epithelial to mesenchymal transition-inducing transcription factors (EMT-TFs).

Junjiang Fu,1 Chunli Wei,1 Jingliang Cheng,1 Md. Asaduzzaman Khan,1 Lisha Yang,1 Hui Zou2. 1 _Southwest Medical University, Luzhou, China;_ 2 _Southwest Medical University/Hunan Normal University, Changsha, China_.

Cancer metastasis is one of the major causes of human death. The developmental stage epithelial to mesenchymal transition (EMT) is associated with cancer metastasis. Cordycepin (CD) is a natural compound, found in traditionally used medicinal mushroom Cordyceps. In this study, we have investigated the anticancer and antimetastatic activity of CD, and whether this compound regulates epithelial to mesenchymal transition-inducing transcription factors (EMT-TFs). By employing cell viability assay and real-time cell analysis in BT549, 4T1 and MDA-MB-435 cell lines, we found that CD can potentially inhibit cancer cell growth, migration and invasion. By using quantitative PCR (qPCR) and Western blot analysis, we also studied the expression of EMT associated proteins N-Cadherin and E-Cadherin, and their transcription factors TWIST1, SNAIL1, SLUG and ZEB1 in BT549 and 4T1 cell lines. By dual-luciferase reporter assay, we confirmed that CD is able to inactivate EMT signaling pathway with mediatory of TWIST1 and SLUG. CD treatment also inhibited the growth and metastasis of cancer cell-derived xenograft tumors in mouse model. Taken together, CD inhibits breast cancer cell migration and invasion by targets EMT-TFs, which might be a possible CD mechanism of antimetastatic action. In addition, CD derivatives by conjugating thymoquinone, a promising anti-cancer compound from nature, were designed to investigate whether there are dramatically improvements on their antimetastatic activities. In conclusion, this study suggests CD as a potential anticancer and antimetastatic molecule, which might be a promising therapeutic agent against breast cancer. Funding: This work was supported by the NSFC (81172049, 81672887).

#2040

Withaferin A inhibits epithelial-to-mesenchymal transition in non-small lung cell cancer cells via regulation of SMAD and NFkB signaling.

Al Hassan Kyakulaga, Farrukh Aqil, Radha Munagala, Ramesh Gupta. _University of Louisville, Louisville, KY_.

Background: Metastasis remains the primary cause in lung cancer. Epithelial-to-mesenchyme transition (EMT) has been identified as the hallmark for metastasis in NSCLC, and is being explored for drug targeting. Aberrant signaling mechanisms for cytokines such as TGF-β1 and TNFα are known to induce EMT. In this study we demonstrate the effect and mechanisms of WFA inhibition of EMT in NSCLC cell lines.

Objective: The present study was done to determine the effect of WFA on TGF-β1 and TNFα induced EMT, migration and invasion in human NSCLC cells. Furthermore, to determine underlying the mechanisms of WFA inhibition of EMT.

Methods: Two human NSCLC cell lines (H1299 and A549) were cultured in DMEM media. To induce the EMT, cells in serum free media were challenged with TGF-β1 (5 ng/mL) or TNFα (5 ng/mL) alone or in combination. To determine the effects of WFA, cells were either pre- or co-treated with different concentrations of WFA for various time points. Western blot analysis, electrophoretic mobility shift assay (EMSA), RT-PCR and immunofluorescence staining were used to determine effects of WFA on Smad2/3 and NFkB activation. Cell adhesion, migration and invasion assays were used to assess in vitro anti-metastatic activity.

Results: Our findings indicate that the cytokines TGF-β1 and TNFα cooperate to promote the acquisition of EMT phenotype, migration and invasion of NSCLC cells in vitro. There was a marked increase in the expression of EMT transcription factors Snail, Slug, ZEB and EMT markers such as vimentin and N-cadherin. Cells treated with TGF-β1/TNFα had significantly increased migratory and invasive capacities. WFA treatment significantly inhibited the TGF-β1 induced Smad2/3 activation and TNFα-induced translocation of NFkB into the nucleus. We observed a dose and time dependent inhibition of the phosphorylation of Smad2/3 and NFkB in both cell lines. The cell adhesion, migratory and invasive capacities of both H1299 and A549 cells were greatly diminished by WFA in the presence or absence of cytokines. Conclusion: Based on the current finding, it can be concluded that WFA inhibits EMT via negative regulation of Smad2/3 and NFkB signaling in NSCLC cells.

#2041

Suppression of mutant p53-induced EMT by statins have double-edged effects on the patients with lung adenocarcinoma according to p53 mutation status.

Shigeto Nishikawa, Toshi Menju, Terumasa Sowa, Koji Takahashi, Ryo Miyata, Hiroyuki Ishikawa, Daisuke Nakajima, Masatsugu Hamaji, Hideki Motoyama, Akihiro Aoyama, Toshihiko Sato, Fengshi Chen-Yoshikawa, Makoto Sonobe, Hiroshi Date. _Kyoto Univ. Graduate School of Medicine, Kyoto, Japan_.

Background: Epithelial-mesenchymal transition (EMT) is known to be pivotal for driving metastasis and recurrence in lung cancer. Some reports have shown statins suppressed EMT by inactivating mutant p53 functions in vitro. Although several clinical trials regarding conventional treatments with statins have been performed, the effect of statins on the prognosis is still controversial. The purpose of the present study is to clarify the impact of statins on EMT and the prognosis of patients with lung adenocarcinoma.

Methods: First, we transfected wild-type p53 or mutant p53 (R175H, R273H) to H1650 lung adenocarcinoma cells and administrated simvastatin. We evaluated morphologic changes by microscopic examination and analyzed EMT markers (E-cadherin, vimentin) through Western blotting of whole cell lysate. We also analyzed their invasive ability by Matrigel invasion assay. Second, a total of 282 lung adenocarcinoma specimens were collected from patients who underwent surgery in our institute from January 2001 to December 2007. We analyzed EMT markers through immunostaining of tumor specimens and we determined p53 mutation by direct sequencing. The association between EMT, p53 mutation status, and statin use as well as the patients' clinical information was statistically analyzed after propensity score matching. Correlations were analyzed using Pearson's chi-square test and overall survival was compared using the log-rank test.

Results: Mutant p53 (R175H, R273H) induction to H1650 cells showed EMT-like morphologic changes. E-cadherin expression was decreased and vimentin expression was increased in H1650 harboring mutant p53 (H1650mut.p53). Additionally, H1650mut.p53 obtained more aggressive invasiveness compared to H1650 expressing wild-type p53 (H1650wt.p53). Simvastatin-treated H1650mut.p53 lost EMT character and aggressive invasiveness; on the other hand, simvastatin exacerbated invasive abilities of H1650wt.p53. Approximately 20% of the patients were prescribed statins as a treatment of hyperlipidemia or coronary artery disease. Statin administration was correlated to less EMT only in the patients with mutant p53, which was consistent with the results of our in vitro experiments. Moreover, the statin-administrated group showed significantly better survival compared to the non-statin group, which was observed only in the patients with mutant p53. On the other hand, statins significantly impaired the prognosis of patients with wild-type p53, especially in EGFR mutants.

Conclusion: Statins suppressed EMT and improved the prognosis of patients with lung adenocarcinoma in a p53 mutation-dependent manner, whereas they impaired the prognosis of patients with wild-type p53 harboring EGFR mutations. 

### Novel Mechanisms Regulating Angiogenesis

#2042

Hypoxia induces miR-153 through the IRE1α-XBP1 pathway to fine-tune the HIF1α/VEGFA axis in breast cancer angiogenesis.

Huichun Liang, Zhongmei Zhou, Ceshi Chen. _Kunming Inst. of Zoology, Kunming, China_.

It is well documented that hypoxia activates the hypoxia-inducible factor (HIF1α)/vascular endothelial growth factor A (VEGFA) axis to promote angiogenesis in breast cancer. However, it is unclear how this axis is negatively regulated. A number of studies suggest that miR-153 inhibits tumor growth and metastasis by targeting SNAI1, ZEB2, MTDH, ADAM19, AKT1, HECTD3, and MCL1. We recently reported that miR-153 was induced by mifepristone and inhibited the breast cancer stem cells by down-regulating KLF5. In this study, we demonstrated that miR-153 directly inhibits expression of HIF1α by binding to the 3'UTR of HIF1A mRNA, as well as suppresses tube formation of primary human umbilical vein endothelial cells (HUVECs) and breast cancer angiogenesis by decreasing the secretion of VEGFA. Importantly, expression of miR-153 was induced by hypoxia-stimulated ER stress, which activates IRE1α and its downstream transcription factor XBP1. XBP1 directly binds to the promoter of the miR-153 host gene PTPRN and activates transcription. These results indicate that hypoxia induces miR-153 to fine tune the HIF1α/VEGFA axis in breast cancer angiogenesis and miR-153 could be used for breast cancer anti-angiogenesis therapy.

#2043

PlGF/VEGFR-1-dependent activation of Dll4/Notch4 pathway contributes to liver vessel abnormalities and tumor growth in hepatocellular carcinoma.

Annemilaï Tijeras-Raballand,1 Christian Hobeika,2 Jean-Olivier Contreres,3 Patricia Hainaud,3 Matthieu Martinet,1 Philippe Bonnin,2 Clarisse Eveno,2 Evelyne Dupuy,3 Marc Pocard,2 Valerie Paradis,4 Eric Raymond,5 Sandrine Faivre,4 Armand de Gramont1. 1 _AFR Oncology, Paris, France;_ 2 _Inserm U965- Lariboisière University Hospital, Paris, France;_ 3 _Institut des Vaisseaux et du Sang, Paris, France;_ 4 _Beaujon University Hospital, Clichy, France;_ 5 _Paris Saint-Joseph Hospital, Paris, France_.

Background: Hepatocellular carcinoma (HCC) is characterized by strong abnormalities of liver vasculature linked to proangiogenic pathways in the tumor and its microenvironment. Upregulation of Notch4 and its ligand Dll4 in endothelial cells (ECs), as well as PlGF is involved in liver tumor growth, arterialization and sinusoid remodeling. However, the specific role of PlGF and its receptor VEGFR-1 toward Dll4/Notch4 in ECs and tumor vascular remodeling remains to be characterized.

Material and Methods: In vitro, we used primary culture of HUVECs, isolated in our laboratory from human umbilical cords. Active forms of Notch4 and Dll4 in HUVECs were assessed by Western blot. In vivo, differential expression of VEGF-A, PlGF and their receptors were evaluated by qRT-PCR and immunostaining in ASV-B transgenic mice developing stage-defined HCC. Finally, we assessed in vivo PlGF and Notch inhibition, using siRNA PlGF, DAPT and a Notch inhibitor, LY3039478, alone or in combination with sorafenib.

Results: In vitro, we first investigated the relative contribution of VEGF-A, VEGF-E and PlGF on Notch4 pathway, showing that all 3 growth factors increased Dll4 and Notch4 expression in HUVECs, the effect being more pronounced with PlGF. Downregulation of VEGFR-1 by si RNA abrogated VEGF-A and PlGF stimulation on Dll4 expression and Notch4 activation, whereas VEGFR-2 siRNA had no effect. PlGF silencing significantly reduced Dll4 dependent expression and Notch4 activation in response to VEGF-E and VEGF-A. To confirm in vivo the prevalence of PlGF/VEGFR-1 pathway on vessel abnormalities, we evaluated the differential expression of PlGF, VEGF-A, VEGFR-1 and VEGFR-2 in our ASV-B HCC model. VEGFR-1 expression increased with HCC progression and was restricted to macrophages and sinusoidal endothelial cells. PlGF levels were maximal at the stage that coincides with the beginning of liver vessels abnormalities in HCC. Silencing in vivo PlGF or blocking Notch4 using DAPT delayed tumor growth, reduced arterial vessel length, and sinusoids abnormalities while decreasing Dll4 and active Notch4 expression. Using a clinically relevant Notch inhibitor, LY3039478, alone or in combination with sorafenib, liver size and tumor macronodules number were significantly lower in all treatment arms vs placebo, Y3039478/sorafenib combination showing increased tumor control. Angiogenesis measured by mean blood flow in the coeliac trunk (TCm) decreased in all treatment arms vs placebo, confirmed by CD31 staining.

Conclusions: These data suggest that PlGF/VEGFR-1-Dll4/Notch4 pathways are involved in vessel remodeling and tumor growth. In vivo, silencing PlGF or inhibiting Notch4 prevent tumor vessels remodeling/arterialization and delay tumor growth, further warranting new antiangiogenic strategies for HCC treatment.

#2044

Lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1) modulates VEGF-C and VEGF-D secretion in the triple negative breast cancer cell line MDA-MB-231.

Sara Caceres, Andrew C. Little, Joel A. Yates, Rabia A. Gilani, Sofia D. Merajver. _University of Michigan, Ann Arbor, MI_.

Breast cancer commonly gives rise to lymph node metastases, and it has been suggested that tumor cell migration is facilitated by the generation of new lymphatic vessels, or lymphangiogenesis. Lymphatic endothelial cell hyaluronic acid receptor 1 (LYVE-1) is a hyaluronic acid (HA) receptor commonly used as a lymphatic vessel endothelial marker. LYVE-1 has been observed in a variety of breast cancer epithelial cells, and its lymphangiogenic function remains unclear. LYVE-1 has been casually linked to the vascular endothelial growth factors VEGF-C and VEGF-D, which are well appreciated for their roles in lymphangiogenic responses. Therefore, we hypothesize that LYVE-1 may impact lymphangiogensis via a VEGF-C/VEGF-D mediated pathway. We utilized LYVE-1-targeted RNAi in MDA-231 cells followed by HA stimulation and measured production and secretion of both VEGF-C and VEGF-D by western blot and ELISA, respectively. Surprisingly, LYVE-1 knockdown resulted in elevated levels of basal VEGF-C secretion, while LYVE-1 knockdown followed by HA stimulation decreased secreted levels of VEGF-C, with no observed changes in intracellular levels when compared to their controls. Conversely, LYVE-1 knockdown displayed no significant changes in basal VEGF-D levels, although, secreted VEGF-D levels were elevated following HA stimulation. Collectively, our preliminary findings suggest that LYVE-1 modulates VEGF-C/D secretion, potentially defining a novel function for LYVE-1 in MDA-231 triple negative breast cancer epithelial cells.

#2045

Mechanistic insights into the role of cAMP-regulated EPAC1 in tumor-induced angiogenesis and metastatic potential in triple-negative breast cancer.

Muthu Dhandapani,1 Aaron Goldman,1 Naveen Kumar,2 Peeyush Prasad,2 Eshna Jash,2 Itender Singh,3 Kodaganur S. Gopinath,4 Krishnamurthy S,5 Prabhusankar P,6 Seema Sehrawat7. 1 _Mitra Biotech, Woburn, MA;_ 2 _Shiv Nadar University, India;_ 3 _Washington University School of Medicine, St.Louis, MO;_ 4 _Bangalore Institute of Oncology, India;_ 5 _Kidwai Memorial Institute of Oncology, India;_ 6 _Salem Cancer Institute, India;_ 7 _Harvard Medical School, Boston, MA_.

Background: Breast cancer is the leading cause of cancer-related mortality in women. Triple negative breast cancer (TNBC) is one of most aggressive subtypes, which is often accompanied by metastasis, a feature that requires development of new blood vessels (neovasculature) to disseminate tumor cells throughout the body. We recently reported that down-regulation of exchange factor directly activated by Cyclic AMP (cAMP), also known as EPAC1, leads to a reduction in proliferation and inhibition of cell migration.

Methods: Here, using gene knockdown by siRNA and a 3-dimensional in-vitro TNBC co-culture model with Human Vascular Endothelial Cells (HUVECs), we studied microvascular density and the role of EPAC1. Angiogenesis proteome profiling was employed to examine protein networks that were regulated by EPAC1 in the TNBC cell line, MDA-MB-231. Immunofluorescence along with Electric cell-substrate impedance sensing (ECIS) was used to study vascular permeability. To confirm these findings in a translational context, we employed a human tumor explant assay that predicts clinical response to therapy (CANscriptTM) and studied CD34+ nodes to determine how neovasculature associates to drug response.

Results: We determined that downregulation of EPAC1 in the TNBC cell line, MDA-MB-231, leads to reduction of proteins involved in cell migration, metastasis, angiogenesis and adhesion. Interestingly, we determined that loss of EPAC1 leads to increased expression of negative regulators in these same pathways. Immunofluorescence imaging showed that EPAC1 downregulation in MDA-MB-231 cells leads to diminished expression of proteins such as Paxillin, MENA, MMP-9 and tubulin. indicated that EPAC1 role in vascular permeability. Finally, CANscriptTM demonstrated a link between response to therapy and microvascular density, which was reduced under drug pressure in patients that were predicted to respond to treatment.

Conclusion: Our results suggest that EPAC1 is a driver of microvascular density in the tumor microenvironment, a feature that may play a key role in distant metastasis and therapy failure. Ex-vivo modeling of neovasculature may be a novel strategy to predict clinical response and distant metastasis.

#2046

Structural analysis of VEGFR2 function blocking antibodies.

Anita Wary. _York Community High School, Elmhurst, IL_.

The vascular endothelial growth factor receptor-2 (VEGFR2) is a receptor tyrosine kinase that is expressed highly in endothelial cells (ECs). By binding to VEGF165 (VEGF-A) VEGFR2 signals in ECs mediate the formation of new blood vessels, a process known as angiogenesis. Growth of tumor and metastasis depends on angiogenic activities of ECs. Thus, an agent that blocks VEGFR2 signaling specifically in tumor ECs could inhibit tumor growth and metastasis. The IMC-1121B is an antiangiogenic agent that inhibits the VEGFR2 signaling. Here we modeled the structure of the 1121B Fab fragment in complex with domain-3 of VEGFR2, and the structure of a different neutralizing anti-VEGFR2 antibody, 6.64, also in complex with VEGFR2 domain-3. The two Fab fragments interact at opposite ends of VEGFR2 domain-3; 1121B blocks VEGF binding, whereas 6.64 appears to prevent receptor dimerization by perturbing the domain-3:domain-4 interface. Blocking of VEGFR2 signaling activities by two different antibodies could be a powerful approach to block angiogenesis, thereby inhibiting growth of tumor and metastasis.

Key reference: Franklin MC et al., Structure 2011;19:1097-1107.

#2047

N **-glycosylation modulates endothelial cell receptor tyrosine kinase VEGFR-2 ligand-dependent activation and signaling.**

Kevin B. Chandler, Deborah R. Leon, Jenevieve Kuang, Rosana D. Meyer, Nader Rahimi, Catherine E. Costello. _Boston Univ., Boston, MA_.

Activation of vascular endothelial growth factor receptor-2 (VEGFR-2), an endothelial receptor tyrosine kinase (RTK), is essential for tumor angiogenesis in tumors of diverse origin. The extracellular domain of VEGFR-2 contains seven immunoglobulin-like domains, each with multiple potential N-glycosylation sites. Changes in glycosylation influenced by hypoxia, the shunting of glycolytic intermediates via the hexosamine biosynthesis pathway into glycan synthesis, and pro-inflammatory cytokines have the potential to alter VEGFR-2 signaling in tumors. However, the role of glycosylation in VEGFR-2 signaling and function remain largely unknown. The objective of this study is to determine the functional importance of N-glycosylation in VEGFR-2 activation and angiogenic signaling.

Porcine aortic endothelial (PAE) cells with ectopic expression of either wild type VEGFR-2, or one in a series of VEGFR-2 constructs with single glycosylation site mutations in the ligand binding domain, were treated with VEGF-A for 0, 5, 10 or 30 minutes. VEGFR-2 tyrosine phosphorylation was measured via Western blot with an anti-pTyr-1054-VEGFR-2 antibody to assess activation, and with anti-pTyr-1175-VEGFR-2 and anti-pTyr-1212-VEGFR-2 antibodies. Activation of key molecules involved in downstream signaling of VEGFR-2 was also monitored. To establish the glycosylation status of VEGFR-2 available for ligand-mediated signaling, we isolated plasma membrane localized VEGFR-2, performed enzymatic proteolysis, enriched glycopeptides via hydrophilic interaction liquid chromatography (HILIC), and analyzed glycopeptides with an Agilent 6550 Quadrupole Time-of-Flight (Q-TOF) MS using collision-induced dissociation.

Mutation of one N-glycosylation site near the VEGF binding site increased ligand-dependent phosphorylation (at Tyr-1054, Tyr-1175 and Tyr-1212) within 5 min of ligand addition, and initial observations suggest that this resulted in higher levels of activated c-Src compared to wild type VEGFR-2, which may have implications for endothelial cell proliferation and migration. However, phosphorylation of other downstream signaling targets including Akt and PLCγ1 was not affected. Mutations at other N-glycosylation sites near the VEGF ligand binding site did not appear to impact ligand-mediated activation of VEGFR-2, suggesting that glycans located at certain N-linked sites may influence ligand-mediated activation while others have minimal impact. Targeting VEGFR-2 N-glycosylation may be a valuable strategy to inhibit tumor angiogenesis.

This research is supported by NIH grants P41 GM104603, R01 CA191970, and F32 CA196157.

Novel aspect: These results demonstrate that site-specific N-glycosylation of VEGFR-2 modulates ligand-mediated phosphorylation of key VEGFR-2 tyrosine residues involved in downstream signaling.

#2048

Interferon-γ triggers an anti-tumorigenic chain reaction in the tumor vessels of colorectal carcinoma.

Michael Stürzl,1 Victoria Langer,1 Daniela Regensburger,1 Clara Tenkerian,1 Annika Klingler,1 Maximilian Waldner,1 Christoph Becker,1 Valerie Meniel,2 Robert Grützmann,1 Carol-Immanuel Geppert,1 Nathalie Britzen-Laurent,1 Elisabeth Naschberger1. 1 _Univ. of Erlangen-Nuremberg, Erlangen, Germany;_ 2 _Cardiff University, Cardiff, United Kingdom_.

In colorectal carcinoma (CRC), a Th1-tumor microenvironment (TME) is associated with improved prognosis of the patients. The lead cytokine of the Th1-response is interferon (IFN)-γ. IFN-γ is predominantly regarded as an immunomodulatory cytokine. At present, its putative tumor blood vessel-directed anti-tumorigenic effects have not been investigated comprehensively. We demonstrate that the vascular effects of IFN-γ in CRC trigger a two-step anti-tumorigenic chain reaction. First, IFN-γ exerts direct angiostatic activity on endothelial cells. This was detected in vitro using primary endothelial cell cultures and in vivo in Th1-dominated inflammatory reactions of the colon using mouse models with specific deletion of the IFN-γ receptor-2 in endothelial cells and treatment with neutralizing anti-IFN-γ antibodies. Angiostatic IFN-γ effects could be validated in human CRC tissues using the cellular IFN-γ activation marker guanylate binding protein-1 which confirmed reduced angiogenic activity of vessels exposed to IFN-γ at that single cell level. IFN-γ-induced angiostasis was associated with a highly significantly improved cancer-related 5-year survival of the CRC patients (n=388). In a second step, angiostatic activity of IFN-γ resulted in the maintenance of mature tumor vessels in CRC tissues which expressed and secreted the anti-tumorigenic protein SPARCL1 in human CRC tissues (n = 42). This was significantly repressed in CRC tissues lacking a Th1 response. Functional analyses showed that SPARCL1 inhibited angiogenic activity of cultivated endothelial cells in different in vitro tests of angiogenesis (endothelial cell proliferation, migration, spreading, 3D-sprouting and capillary formation in matrigel), both after retrovirally triggered recombinant expression in endothelial cells or after adding the recombinantly purified human SPARCL1 protein. Interestingly, secreted SPARCL1 has also been shown to inhibit proliferation and migration of human CRC tumor cell lines. We could confirm the anti-tumor cell activity of soluble purified SPARCL1 using different tumor cell lines derived from mouse colon tumors (MC38) or mouse melanomas (B16F10), respectively. SPARCL1 inhibited proliferation and migration of both cell lines significantly. Using a mouse model system with a general knock out of the SPARCL1 gene, we obtained first evidence that the growth of metastatically spread MC38 cells in the lungs is repressed by SPARCL1 in vivo. Altogether our study demonstrates that the tumor vessel-directed effects of an IFN-γ-dominated tumor microenvironment in CRC trigger a two-step anti-tumorigenic chain reaction. This provides further insight into the potent anti-tumorigenic activity of a Th1-TME in CRC that may be of relevance for selection of patients' for anti-angiogenic therapy regiments. Moreover, our findings indicate novel pathways which may be prone to tumor immune evasion.

#2049

HNSCC-derived exosomes promote angiogenesis and tumor progression through reprogramming of the tumor microenvironment in vitro and in vivo.

Nils Ludwig,1 Saigopalakrishna S. Yerneni,2 Beatrice M. Razzo,1 Theresa L. Whiteside1. 1 _UPMC Hillman Cancer Center, Pittsburgh, PA;_ 2 _Carnegie Mellon University, Pittsburgh, PA_.

Head and neck squamous cell carcinoma (HNSCC) is the seventh most common malignant disease. For solid tumors, such as HNSCC, an adequate blood supply is of critical importance for tumor development, growth and metastasis. Tumor-derived exosomes (TEX) accumulate in the tumor microenvironment (TME) and serve as a communication system between the tumor and normal stromal cells. TEX may be one of the mechanisms responsible for induction of tumor angiogenesis. This study evaluates in vitro and in vivo effects mediated by TEX that result in promotion of tumor angiogenesis.

Biologically-active exosomes produced by SCC47 and PCI-13 tumor cell lines were isolated by mini size exclusion chromatography (mini-SEC). Exosomes were also isolated from plasma specimens of HNSCC patients or healthy donors. Exosome morphology, size and numbers were characterized using TEM and q-Nano. Molecular profiles were evaluated by western blots with the exosome-specific marker TSG101 blotted as control. The angiogenesis-inducing potential of TEX was measured in arrays with human endothelial cells (HUVEC). Uptake of labeled TEX by HUVEC cells was demonstrated by confocal microscopy. Proliferation, migration, chemotaxis and the tube formation by HUVECs in response to TEX were investigated. The matrigel plug model and the 4-nitroquinoline-1-oxide (4-NQO) oral carcinogenesis were used to confirm that exosomes induce the same results in vivo. In vitro and in vivo experiments were repeated with plasma-derived exosomes obtained from HNSCC patients and healthy donors.

TEX were found to be potent inducers of angiogenesis in vitro and in vivo through functional re-programming and phenotypic modulation of endothelial cells. HNSCC-derived exosomes carried angiogenesis markers (coagulation factor III, IGFBP-3, thrombospondin 1 and uPA) and were internalized by HUVECs within 4 h. Cell line derived exosomes stimulated proliferation (PCI-13 p < 0.05; SCC47 p < 0.01), migration (p < 0.05) and tube formation (p < 0.001) by endothelial cells and promoted formation of defined vascular structures in vivo. Proliferation, migration and tube formation of plasma-derived exosomes obtained from HNSCC patients were significantly enhanced compared to healthy donors' exosomes (p < 0.05).

Our data suggest that HNSCC-derived exosomes promoting angiogenesis, are an adverse factor in carcinogenesis and a potential biomarker of angiogenesis. Moreover, future efforts should focus on eliminating or silencing TEX and thereby adding new options for improving existing anti-angiogenic therapies.

#2050

Tumor stage-dependent transcriptomic signatures of endothelial cells reveal an intense paracrine crosstalk in liver capillarization.

Stefan Thomann,1 Sofia Weiler,1 Martin Dittmer,1 Daniel Kazdal,1 Simone Marquard,1 Stephanie Roessler,1 Carsten Sticht,2 Norbert Gretz,2 Carolin Mogler,3 Claudia Ball,4 Hanno Glimm,4 Eduard Ryschich,1 Peter Schirmacher,1 Kai Breuhahn1. 1 _University of Heidelberg, Heidelberg, Germany;_ 2 _University of Heidelberg Medical Faculty Mannheim, Heidelberg, Germany;_ 3 _Technical University of Munich, Heidelberg, Germany;_ 4 _German Cancer Research Center, Heidelberg, Germany_.

Introduction:

The liver blood vessel network, consisting of liver sinusoidal endothelial cells (LSECs) and capillary endothelial cells (CECs), undergoes distinct modifications in the process of liver cancer development. The selective expansion of the CEC compartment is thereby the most prominent modification leading to a change of the predominant endothelial phenotype. An in depth molecular analysis of the dynamic vascular modifications in hepatocellular carcinoma (HCC) is however still missing.

Methods:

An inducible mouse model with hepatocyte-specific expression of constitutively active yes-associated protein (YAPS127A) was used for stage dependent purification of endothelial cells. Cell sorting allowed the subtyping of liver endothelial cells into LSECs (CD31+, CD146+, Lyve-1+) and CECs (CD31+, CD146+, Lyve-1-) followed by their consecutive transcriptomic analysis. Predicted paracrine interactions were functionally tested in the endothelial cell line SVEC4-10. Gene expression data of 242 HCC patients were used to confirm the findings in human HCC (Roessler et al. 2010).

Results

Immunofluorescence revealed a progressive replacement of LSECs by CECs in livers, hyperplastic lesions, and HCC derived from YAPS127A mice. FACS analysis confirmed the selective expansion of CECs from 4±2% in healthy livers to 37±16% in hepatomegaly to 61±30% in tumor-bearing livers. Expression profiling of CECs and LSECs revealed dynamic CEC-selective changes in early and late phases of hepatocarcinogenesis including an enrichment of migration specific pathways. Phylogenetic tree analysis of the gene set "cytokine –cytokine receptor interaction" displayed endothelial cell (EC)- and stage-specific secretion patterns suggesting a dynamic crosstalk between the EC populations. The promigratory role of the identified HGF/c-Met axis was confirmed in vitro by single cell tracking and spheroid sprouting. In vivo the inhibition through the c-Met inhibitor Cabozantinib led to a 67% reduction of CEC sprouting, in line with with decreased tumor cell proliferation. Human HCC gene expression data from 242 HCC patients confirmed the prognostic relevance of an identified 19 gene capillarization signature.

Conclusions

Our results illustrate a dynamic crosstalk between the two major EC populations in the process of liver tumor development, already detectable in early phases of capillarization. The identified HGF/c-Met axis represents an essential signal pathway in the process of arterial branch elongation and its inhibition through clinically relevant c-Met inhibitors may represent a promising approach to perturbate disease-associated capillarization.

References

Weiler SME, et al., Gastroenterology, 2017. 152(8): p. 2037-2051 e22

Roessler S, et al., Cancer Res. 2010 Dec 15;70(24):10202-12

#2051

Perfusable 3D angiogenesis in a high-throughput microfluidic culture platform.

K M. Bircsak,1 V van Duinen,2 S J. Trietsch,3 A J. van Zonneveld,4 T Hankemeier,5 A Saleh,1 P Vulto3. 1 _Mimetas US, Gaithersburg, MD;_ 2 _Leiden University, Leiden, Netherlands;_ 3 _Mimetas BV, Leiden, Netherlands;_ 4 _Leiden University Medical Center, Leiden, Netherlands;_ 5 _Mimetas US, Leiden, Netherlands_.

The transition from 2D to 3D cell culture is a first step towards more physiologically relevant in vitro cancer models. To adequately capture the complex tissue architectures observed in vivo, 3D microfluidic techniques incorporate and achieve long-term gradient stability, continuous perfusion and patterning of cancer cell layers as stratified co-cultures. We used a standardized high-throughput (n=40) microfluidic 3D tissue culture platform called the OrganoPlate® to generate precisely controlled gradients, without pumps, ideal for growing blood vessels and inducing controlled 3D angiogenic sprouting. The blood vessel is grown against an extracellular matrix (ECM) gel with cancer cells and is subsequently exposed to pro- and antiangiogenic compounds to direct sprouting towards 3D cancer cell clusters. Utilizing high-content confocal time-lapse imaging and analysis, angiogenic potential was measured in various cancer models. The exposed vasculature shows many of the important hallmarks of cancer angiogenesis found in vivo, including tip cells induction and migration and stalk cells formation. Importantly, the stalk cells develop a perfusable lumen that is connected to the parental vessel as demonstrated with perfusion of high-molecular-weight (150KD) fitc-dextran through microvascular structures. This model will be used as an in vitro cancer screening platform to unravel the important drivers in angiogenesis and vasculogenesis and the mechanism of action of antiangiogenic compounds. By combining this culture platform with mural cells, cell-cell interactions can be studied. In parallel, we will combine this 3D cancer angiogenesis platform with our current Tumor-on-a-Chip models to create tissue models with integrated vasculature.

#2052

A novel PHD2/VHL- mediated regulation of YAP1 and its role in vascular mimicry and tumor angiogenesis.

Namrata Bora Singhal, Biswarup Saha, Srikumar Chellappan. _Moffitt Cancer Center, Tampa, FL_.

Non-small cell lung cancer (NSCLC) is the leading cause of cancer related death worldwide. In this study, we show that transcriptional co-activator YAP1, the oncogenic component of the Hippo pathway, may contribute to the progression of NSCLC by supporting tumor angiogenesis and vascular mimicry of cancer stem-like cells (CSCs). NSCLC CSCs were found to have higher mRNA expression of VEGF receptor II (KDR) and Angiopoietin-2 (AngPT-2); depletion of YAP1 inhibited the promoter activity as well as the mRNA expression of VEGF, KDR and AngPT-2, with a corresponding reduction in vascular mimicry as well as tumor growth in a mouse xenograft studies. These results suggest a possible mechanism by which YAP1 regulates tumor angiogenesis.

A role for YAP1 in tumor angiogenesis was further supported by the finding that NSCLC cells grown in hypoxic conditions showed higher levels of YAP1. There was little to no changes in the canonical Hippo pathway proteins like LATS1/2, MST1/2, SAV and MOB as well as in the expression of TAZ, an orthologue of YAP1. Elevated YAP1 was found to associate with HIF1α under hypoxic conditions and enhance its transcriptional activity; YAP1 could increase HIF1α-mediated induction of the VEGF promoter confirmed by chromatin immunoprecipitations and transient transfection assays. Elevated levels of YAP1 and HIF1α interaction was detected in lung tumor tissues compared to normal lung tissue, as detected by proximity ligation assay (PLA), suggesting that the higher association of YAP1 with HIF1α and resulting transcriptional activity might have contributed to tumor growth.

An examination of the underlying mechanism by which YAP1 levels are elevated under hypoxic conditions revealed a novel regulation of YAP1 protein by prolyl hydroxylase PHD2 and E3 ubiquitin ligase VHL, which are mainly known to regulate HIF1α under normoxia. PHD2 was found to hydroxylate proline residue(s) of YAP1 between aa 284 to aa 289 as seen by mutational studies. YAP1 was found to directly associate with PHD2 as well as with VHL. Depletion of PHD2 or treatment with DMOG, an inhibitor of prolyl hydroxylases, reduced YAP1 association with VHL ligase. This further elevated YAP1 levels in the nucleus. Interestingly, disruption of the YAP1-PHD2 interaction using a domain specific peptide enhanced the angiogenic tubule formation by endothelial cells. Our data therefore identifies a novel non-canonical pathway of regulation of YAP1 that supports angiogenesis and tumor growth.

#2053

**Exosomes from AC133** + **/CD34** \+ **stem cells mediate a paracrine effect in breast cancer cells.**

Ghada Ben Rahoma, Neha Tuli, Rachana Maniyar, Sanjukta Chakraborty, Sarnath Singh, Abraham Mittelman, Jan Gelibter, Raj K. Tiwari. _New York Medical Collge, Valhalla, NY_.

Exciting advances in the treatment of breast cancer have occurred over the past decade. However, the efficacy of the current therapeutic modalities is still limited by drug toxicity, resistance, and lack of predictive and prognostic biomarkers. Breast cancer remains the second leading cause of cancer death among women in the U.S. Thus, the development of new therapeutic targets and further understanding of the tumor microenvironment is extremely crucial for accelerating the progress against breast cancer. Human AC133+/CD34\+ stem cells serve as a highly promising and novel therapeutic option for targeting tumor angiogenesis. We and others have demonstrated the incorporation of bone marrow-derived AC133+/CD34+/KDR\+ endothelial progenitor cells in the neovasculature around implanted tumors supporting their growth and spread. Many mediators have been implicated in the crosstalk between AC133+/CD34+/KDR\+ endothelial progenitor cells, endothelial cells, and the tumor cells, but most of these have limited clinical benefits as reported by several trials. In this study, we analyzed the secretome of the AC133+/CD34\+ stem cells that were isolated by positive selection from human umbilical cord blood and evaluated their role in breast cancer progression. Our results show that AC133+/CD34\+ stem cells exhibited significant growth potential that was manifested as seventy-five fold increase in cell number after 10 days in culture. Flow cytometry demonstrated that AC133+/CD34\+ stem cells preserve their capacity to differentiate into AC133+/CD34+/KDR\+ endothelial progenitor cells even after long term in vitro expansion. In order to evaluate the effect of AC133+/CD34\+ stem cells on breast cancer cells, we performed a simple proliferation (XTT) assay using conditioned medium (CM) from AC133+/CD34\+ stem cells and tested on MCF-7 and MDA-MB-231 proliferation. As expected, CM significantly induced proliferation of breast cancer cells. This effect was in part due to the high expression of a repertoire of proinflammatory and proangiogenic cytokines in the CM of the AC133+/CD34\+ stem cells. In particular, angiogenin, GRO, IL-8, MCP, PDGF.BB, TIMP2. Further, we examined if exosomes, a component of paracrine secretion are involved in the paracrine effect of the AC133+/CD34\+ stem cells. Interestingly, exosomes from AC133+/CD34\+ stem cells significantly enhanced MCF-7 and MDA-MB-231 proliferation at a comparable level as the CM. Further analysis of the exosomes reveals that the pro-proliferative miR-141-3p, miR-182-5p, miR-200b-3p, and miR-203a are highly expressed in AC133+/CD34\+ exosomes. The analysis of the paracrine interactive mediators between breast cancer cells, AC133+/CD34\+ stem cells, and AC133+/CD34+/KDR\+ endothelial progenitor cells is likely to yield viable novel clinically translatable therapeutic targets.

#2054

Tumor endothelial cells survive under lactic acidosis.

Dorcas A. Annan,1 Nako Maishi,1 Tomoyoshi Soga,2 Randa Dawood,1 Yasuhiro Hida,1 Kyoko Hida1. 1 _Hokkaido University, Sapporo, Japan;_ 2 _Keio University, Tsuruoka, Japan_.

Tumor endothelial cells (TECs) which line tumor blood vessels differ functionally, structurally and genetically from normal endothelial cells (NECs). Recent findings have furthermore shown that TECs are hyper-glycolytic, making them a significant source of lactic acid within the tumor. Similar to TECs, tumor cells are also preferably glycolytic and release large amounts of lactic acid. Consequently, TECs may be influenced by both intrinsic and tumor-derived lactic acid. However, the effects of lactic acidic on TECs have not been elucidated. Moreover, with the quest to find novel therapeutic antiangiogenic targets, it is of much essence to identify adaptive mechanisms supporting TECs under conditions of lactic acidosis. In this study, metabolomic analysis was performed in TECs and NECs. TECs had significantly higher intracellular levels of glycolytic metabolites, lactate, ATP and amino acids than NECs. Additionally, unlike NECs, TECs proliferated when exposed to conditions of lactic acidosis (20mM lactate, pH 6.9) and not under lactosis (20mM lactate, pH 7.3). This observation implied that TECs survive and sustain their growth under low pH conditions but in the presence of lactate. Furthermore, qRT -PCR analysis revealed an elevated expression of proton-coupled transporters in TECs than in NECs. The TECs were sensitive to drugs targeting some of the transporters. This indicated that proton-coupled transporters may be involved in the survival and proliferation of TECs in lactic acidosis. Next, the effects of lactic acidosis on tumor blood vessels, particularly endothelial cellular junctions was investigated. VE-cadherin expression was analysed by immunocytochemistry in endothelial cells exposed to lactosis and lactic acidosis. The VE-cadherin was more disorganized under lactic acidosis than in lactosis. Lactic acidosis may therefore contribute to the poor integrity of tumor blood vessels. More studies are on-going to elucidate the molecular effects of lactic acidosis on TECs and the role played by proton-coupled transporters in TECs survival in the tumor microenvironment. These will lead to the identification of a proton-coupled transporter as an antiangiogenic therapeutic target.

#2055

Staphylococcus aureus **alpha toxin activates Notch in endothelial cells.**

Sonia L. Hernandez,1 Mildred Nelson,1 Georgia Sampedro,1 Bianca Lec,1 Jared Emolo,1 Naina Bagrodia,1 Ann M. Defnet,1 Lydia Wu,1 Juliane Bubeck-Wardenburg,2 Jessica Kandel1. 1 _The University of Chicago, Chicago, IL;_ 2 _Washington University, Chicago, MO_.

Background. Staphylococcus aureus infection is one of the leading causes of morbidity in hospitalized patients in the United States. The secreted agent hemolysin alpha toxin (Hla) requires the receptor A Disintegrin And Metalloproteinase domain-containing protein 10 (ADAM10) to mediate its toxic effects; ADAM10 in turn activates the Notch pathway. Notch proteins function in developmental and pathological angiogenesis via the modulation of key pathways in endothelial (EC) and perivascular cells. Thus, we hypothesized that Hla would activate Notch in EC in vitro and in vivo.

Methods. Human umbilical vein endothelial cells (HUVEC) were treated with recombinant Hla (rHla), Hla-H35L (genetically inactivated Hla), 5mM EDTA (a known Notch activator), or HBSS, and probed with a Luciferase reporter regulated by a Notch promoter. Mice engineered to express yellow fluorescent protein (YFP) upon Notch activation received a non-lethal daily subcutaneous injection of rHla (0.025 µg/5µL) or vehicle (PBS) and their retinal endothelial YFP interrogated at p6. 6 week old male Notch reporter mice received a 1-4 × 107 50 µl subcutaneous inoculation of S. aureus strains USA300/LAC (WT), or its isogenic Δhla mutant lacking Hla, and their skin biopsies were analyzed by histology after 36 hours, 8 and 16 days. Notch activation in endothelial cells of human liver sections from patients whose blood cultures were positive or negative for S. aureus was evaluated by immunohistochemistry.

Results. Luciferase assays demonstrated that Hla (0.01 µg/mL) increased Notch activation by 1.75±0.5-fold as compared to HBSS controls (p<0.05) and EDTA (5.4±1.4-fold activation relative to HBSS, p<0.01), whereas Hla-H35L had no effect. Retinal EC in YFP Notch reporter mice revealed significantly greater YFP intensity in EC after Hla injection than controls in two independent litters. Subcutaneous infection of S. aureus in the Notch reporter mice revealed a significant upregulation of Notch activation in response to Hla 36 hours after infection, both in intensity and area (4.2X10-5 ±1.8X10-5 vs. 7.9X10-4 ±6X10-4 mean intensity relative to DAPI area, p=0.008, and 6.7x106

±3.1x106 vs. 7.7x10-5 ±6x105 YFP area/ DAPI area, p=0.002). Increased EC Notch activation in response to Hla was maintained 8 and 16 days after inoculation. IHC showed EC in human liver had higher Notch expression than controls.

In sum, our results demonstrate that the S. aureus toxin Hla can potently activate Notch in endothelial cells, an effect which could have effects in development and pathological angiogenesis such as cancer.

#2056

CPAP promotes angiogenesis via interacting with and enhancing the activity of STAT3 in HCC.

Ruo Yu Chen. _National Cheng Kung University, Tainan City, Taiwan_.

Hepatocellular carcinoma (HCC) is one of the malignant hypervascular tumors in which angiogenesis plays a vital role in tumor growth and metastasis. Our previous studies indicated that CPAP is overexpressed in HCC, and is positively correlated with recurrence and vascular invasion. In this study, we demonstrated that overexpressed CPAP increases tumor growth, angiogenesis, as well as metastasis ex vivo and in vivo. The expression level of CPAP has a positive correlation with activated STAT3 in HCC. Mechanistically, CPAP can act as a transcriptional co-activator of STAT3 to enhance IL-6-induced STAT3 activation by directly binding with STAT3. Overexpressed CPAP up-regulates several target gene expressions of STAT3 that are involved in angiogenesis, such as IL-8, CD44 and MCAM. The expression of CPAP mRNA is positively correlated with the levels of IL-8, CD44 and MCAM mRNAs in HCC. Importantly, IL-6/STAT3-mediated angiogenesis is significantly increased by overexpressed CPAP, and can be blocked by decreased expression of IL-8. Taken together, our results suggest that overexpressed CPAP interacts with STAT3 to enhance its activity and therefore leads to angiogenesis. Our findings shed light on the importance of CPAP to act as a potential therapeutic target in inhibiting the angiogenesis pathway.

#2058

Lymph circulating tumor cells are phenotypically different from tumor cells circulating in the blood.

Odalys J. Torres-Luquis, Sulma Mohammed. _Purdue University, West Lafayette, IN_.

Breast cancer is the most common cancer in women. However, metastasis is the leading cause of death. When the tumor cell migrates from the primary tumor it can disseminate into the blood circulation or lymphatic system. It has been proven that 80% of malignant carcinoma chooses the lymphatic system over the vascular system. Despite the importance of sentinel lymph node (SLN) metastasis as a staging marker, lymphatically disseminated tumor cells (LCTCs) in transit from the primary tumor to the SLN have never been captured, characterized and compared to blood borne tumor cells (BCTCs) in the same host. Here we use a microsurgical technique to collect draining lymph in situ from a growing tumor prior to its entry in SLN in syngeneic animals and patients with breast cancer. Unlike BCTC, LCTCs are found in clusters that exhibit a hybrid Epithelial-Mesenchymal phenotype, display a Cancer Stem Cell CD44hi,CD24hi,ALDHA1hi signature, grow as mammospheres and are highly tumorigenic. We also showed that the lymph is enriched in tumor-derived exosome proteins and EGF growth factor. Characterization of LCTCs vs. BCTCs and the compilation of tumor-derived factors en route to the SLN can provide insights into the metastatic process and has relevance to cancer immunotherapy.

#2059

Alcohol enhances estrogen-responsive breast cancer neovascularization.

Natalie C. Cruz,1 Rachana Maniyar,2 Robert Suriano1. 1 _College of Mount Saint Vincent, Bronx, NY;_ 2 _New York Medical College, Valhalla, NY_.

The pleiotropic effects of estrogen in breast cancer development have been observed at both the initiation and progression phases. Interestingly, alcohol, as a dietary component, has been observed to possess estrogenic activity, but the synergistic interaction between alcohol and estrogen and its cellular targets remains undefined. Our previous study identified neovascularization as a cellular target of estrogen that was mediated by the mobilization of bone marrow-derived endothelial progenitor cells (BM-EPCs) and their subsequent incorporation within the tumor mass. The present study was undertaken to define the synergistic interaction between estrogen and alcohol using a cell culture model. To examine the effects of both estrogen and alcohol on breast cancer progression, we developed an in vitro model using the murine mammary tumor cell line, TG1-1, and the murine endothelial cell line, SVEC4-10. Neovascularization was assayed by the tubulogenesis and scratch wound assays as well as quantitating the expression of various provasculogenic markers. Conditioned media, generated by culturing the murine mammary cancer cell line, TG1-1, in estrogen and ethanol, was observed to enhance tubule formation and migration of the murine endothelial cell line, SVEC4-10. In addition, the TG1-1 conditioned media enhanced the prosurvival MAP kinase-signaling pathway in SVEC4-10 cells. Presumably, the observed enhancement of both endothelial cell tubulogenesis and migration is in part attributed to provasculogenic proteins secreted by TG1-1 cells. In order to address this, Western blot analysis using TG1-1 cell lysates indicated an increase in VEGF, MMP-9, and eNOS expression in presence of alcohol and estrogen. As a whole, our data suggest that both estrogen and alcohol function to enhance expression of key provasculogenic molecules in breast cancer cells, which in turn modulates the morphologic, migratory, and survival properties of endothelial cells.

#2060

**The pseudogene** FLT1P1 **functions as a tumor metastasis suppressor in a preclinical model of colorectal cancer.**

Xiangcang Ye, Fan Fan, Rajat Bhattacharya, Delphine R. Boulbes, Rui Wang, Ling Xia, Lee M. Ellis. _UT MD Anderson Cancer Ctr., Houston, TX_.

Chromosome 3p21.3 is one of the unstable genomic loci that are frequently altered by epigenetic modifications or interstitial deletions in human solid tumors. Several genes in 3p21.3 are known to be tumor suppressors. This locus includes a non-coding gene, fms-related tyrosine kinase 1 pseudogene 1 (FLT1P1), of which the role is largely unknown. Our previous published study (Ye et al. MCR, 2015) found that FLT1P1 was transcribed primarily as an antisense that inhibits VEGFR1 protein expression through interaction with miR-520a in human colorectal cancer (CRC) cells. In this study, we showed that the expression of FLT1P1 antisense is silenced by promoter hyper-methylation in xenograft tumors. Our data further showed that the loss of the FLT1P1 antisense not only up-regulated the expression of VEGFR1 and other genes relating to tumor angiogenesis, but also induced the EMT phenotype in tumor cells. Moreover, the FLT1P1 silenced cells demonstrated an increase in the number of metastasis to the liver following spleen injection in a mouse model. These observations suggest that the transcribed FLT1P1 antisense is a tumor metastasis suppressor. Our finding warrants further investigation of the genetic and epigenetic alterations of FLT1P1 and their impact on metastasis of CRC cells.

#2061

Genipin inhibits invasion and migration of colon cancer cells by suppression of HIF1α accumulation and VEGF expression.

Dae-Hee Lee, Sang-Choul Oh, Suk-young Lee, Hong Jun Kim. _Korea Univ. Medical Center, Seoul, Republic of Korea_.

Expression of hypoxia-inducible factor-1 (HIF-1) and vascular endothelial growth factor (VEGF) is known to play an important role in cancer progression in various cancer cell lines. Although genipin, a constituent of Gardenia fruit, has shown to have anti-tumor activity, its role in suppression of HIF-1 protein and downstream genes has yet to be clarified. We examined the effect of genipin on intracellular level of HIF-1α and extracellular level of VEGF proteins using colon cancer cell line HCT116. We observed genipin suppressed accumulation of HIF-1α under hypoxia in various cancer cell lines including HCT116. We found out suppressed accumulation of HIF-1α occurred through modulation of protein degradation. Genipin was also observed to suppress expression of VEGF and invasion of colon cancer cells through blocking of the ERK signaling pathway. Taken together, our results provide a new insight into the potential role of genipin in suppressing cancer progression of colon cancer cells.

### Pediatrics 1: Genomics, Epigenetics, and Biomarkers

#2062

**Selective gene dependencies in** MYCN **-amplified neuroblastoma include the core transcriptional regulatory circuitry.**

Adam D. Durbin,1 Mark W. Zimmerman,1 Neekesh V. Dharia,1 Brian J. Abraham,2 Brian J. Abraham,2 Amanda Balboni-Iniguez,1 Nina Weichert-Leahey,1 Shuning He,1 John M. Krill-Burger,3 David E. Root,3 Francisca Vazquez,3 Aviad Tsherniak,3 William C. Hahn,1 Todd R. Golub,3 Richard A. Young,2 A. Thomas Look,1 Kimberly Stegmaier1. 1 _Dana-Farber Cancer Institute, Boston, MA;_ 2 _Whitehead Institute for Biomedical Research, Cambridge, MA;_ 3 _Broad Institute of MIT and Harvard, Cambridge, MA_.

Childhood neuroblastomas with MYCN gene amplification form a particularly high-risk subset of this disease and are difficult to treat effectively. This has focused attention on tumor-specific gene dependencies that reflect important pathways in tumorigenesis, and thus could provide valuable targets for the development of novel therapeutics. Using genome-scale CRISPR-Cas9 approaches that allow unbiased detection of genes critically involved in tumor cell growth and survival, we identified 147 candidate genes associated with selective vulnerabilities in nine MYCN-amplified neuroblastoma cell lines, compared to findings in over 300 other human cancer cell lines representing multiple tumor cell types. We then used genome-wide ChIP-seq analysis to test the hypothesis that a small number of transcription factors - MYCN, HAND2, ISL1, PHOX2B, GATA3, and TBX2, all represented in the selective dependency group - are members of the transcriptional core regulatory circuitry (CRC) that underlies cell state in MYCN-amplified neuroblastoma. We show that these transcription factors bind as dense clusters at defined epicentres within the enhancers of their own genes, as well as those of the other CRC transcription factor genes, creating a positive feed-forward autoregulatory loop that establishes and maintains high levels of gene expression. To disable the CRC, we tested a combination of BRD4 and CDK7 inhibitors, which we postulated would act synergistically by targeting both transcriptional initiation and elongation required to synthesize regulatory transcription factors. MYCN-amplified neuroblastoma cells treated with both drugs were killed synergistically, in vitro and in vivo, and accompanied by rapid downregulation of CRC transcription factor gene expression. This study defines a set of critical dependency genes in MYCN-amplified neuroblastoma, a subset of which comprises the oncogenic transcriptional regulatory circuitry that underlies cell state and survival in this tumor.

#2063

SAMD9/SAMD9L **mutations in familial monosomy 7.**

Victoria Bryant,1 Jasmine Wong,2 Jason Schwartz,1 Tamara Lamprecht,1 Jing Ma,1 Charles Mullighan,1 Mignon Loh,2 Kevin Shannon,2 Jeffery Klco1. 1 _St. Jude Children's Research Hospital, Memphis, TN;_ 2 _University of California, San Francisco, San Francisco, CA_.

The purpose of this study was to define the mutations causing familial monosomy 7 with myelodysplastic syndrome (MDS) and/or acute myeloid leukemia (AML). We have previously reported frequent germline mutations in the genes SAMD9 and SAMD9L in pediatric MDS. We obtained samples on 32 individuals in 7 families with multiple siblings affected with MDS/AML and monosomy 7. We conducted targeted sequencing of genes frequently mutated in MDS/AML and amplicon sequencing of SAMD9 and SAMD9L. We performed cell cycle analysis assays on SAMD9/9L mutations to study their effects on cell proliferation. Our data show that 5 of the 7 families have germline pathologic mutations in SAMD9 (n=1) and SAMD9L (n=4), with significant clinical variability in the phenotypes displayed, ranging from transient monosomy 7 to overt AML with monosomy 7. Consistent with previous data, the mutant SAMD9 or SAMD9L allele was underrepresented in the bone marrow. This decrease in the mutant allele paralleled the extent of monosomy 7, suggesting that only the wild-type allele was retained in cells with monosomy 7. Functional analysis revealed that all germline SAMD9/9L mutations displayed a gain-of-function ability to suppress cell cycle progression. The patients with more aggressive disease were found to harbor somatic mutations in SETBP1, ETV6 or RUNX1. In many individuals we found additional somatic revertant mutations in cis with the germline mutation, which overcame the growth-suppressive effects of the germline mutation. Our data show that germline mutations in SAMD9/9L represent a new class of predisposition genes for familial monosomy 7 with MDS/AML and should be screened for in all children with MDS.

#2064

Differences in methylation patterns by pediatric germ cell tumor histologic subtype.

Lindsay A. Williams,1 Lauren Mills,1 Anthony J. Hooten,1 Erica Langer,1 Michelle Roesler,1 A Lindsay Frazier,2 James Amatruda,3 Jenny N. Poynter,1 Jenny N. Poynter1. 1 _University of Minnesota, Minneapolis, MN;_ 2 _Dana-Farber/Boston Children's Cancer and Blood Disorders Center, Boston, MA;_ 3 _University of Texas Southwestern Medical Center, TX_.

Anomalous DNA methylation contributes to carcinogenesis by regulating gene expression and may be important in germ cell tumor (GCT) development as germ cells undergo tremendous epigenetic reprogramming during embryogenesis. Histologic subtypes of GCTs show differences in global methylation patterns with less differentiated tumors (e.g. embryonal carcinoma) experiencing lower levels of global methylation relative to more differentiated tumors (e.g. teratoma). Differences in promoter methylation have also been reported in GCTs. In particular, developmental signaling pathways and the BMP/TGF beta pathway have been consistently found to be differentially methylated. In this analysis, we conducted an epigenome wide association study to identify gene-specific methylation differences by GCT histology. Using the Illumina HumanMethylation450K array we determined methylation profiles for 154 pediatric GCTs (21 germinomas, 54 mixed, 9 teratomas, 70 yolk sac tumors [YST]) from fresh frozen and formalin fixed paraffin embedded tumor tissue samples. Fisher's exact tests were used to assess differences in the distributions of age group, sex, and tumor location by histologic subtype (alpha=0.05 for a two-sided test of significance [SAS v9.4]). Bioconductor package minfi was used to identify differential methylation between GCT histologic subtypes by comparing the methylation beta values. To identify differential methylation of genes predictive of tumor histology, a generalized linear model (glm) with LASSO was fit (R v3.4.1) to the quantile normalized beta values for all probes from all samples. There were significant differences in age, sex, and tumor location by histologic subtype (all p<0.01). We observed unique clusters for both germinoma and YST, but a single cluster for mixed tumors and teratoma. The methylation clusters did not differ by age, sex or FFPE status after accounting for histologic subtype. When stratifying by age group (0-10 vs. 11-19 years), we observed 2,043 differentially methylated regions (DMRs) (FWER <0.05); however, these differences did not persist once we adjusted for tumor histology. Differential methylation tests across histologic subtypes resulted in 8,049 DMRs (FWER < 0.05). Germinoma clustered most consistently and displayed a unique methylation pattern relative to other tumor types, which were more similar to one another. Fitting a glm with LASSO to the methylation data identified 21 probes that were predictive of histologic subtype with misclassification error < 20%: ANKRD24, ASL, ATG13, CCDC53, CKB, FAM224B, FZD10, KCNMB3, LINC00939, LOC101927248, MIR378C, MMP14, MUC1, OGFOD3, PLP2, PLPP3, PRR5, SERPINA6¸ SGSM2, VARS, and YWHAZ. In our analyses, we identified a large number of genes with differential methylation by tumor histology. These data may be informative in understanding GCT etiology and the timing of GCT initiation.

#2066

The functional noncoding genome in childhood sarcomas plays important regulatory roles in tumor pathogenesis.

Sheetal A. Mitra,1 Anirban P. Mitra,2 Yang Liu,1 Jonathan D. Buckley,3 Timothy J. Triche3. 1 _Children's Hospital Los Angeles, Los Angeles, CA;_ 2 _University of Southern California, Los Angeles, CA;_ 3 _USC-Children's Hospital Los Angeles, Los Angeles, CA_.

Pediatric soft tissue sarcomas such as rhabdomyosarcomas (RMS) and Ewing sarcomas (ES) occur in children and adolescents. These tumors typically have a very primitive stem cell-like appearance with few mutations, thus underscoring the importance of other genetic and epigenetic mechanisms responsible for initiating and maintaining tumorigenesis. Noncoding RNAs (ncRNA) are known to play an important role in development and cancer and hence we hypothesized that they would regulate the genomes of these developmental sarcomas. Whole-transcriptome profiling was performed on primary tumors from 200 ES, and 80 RMS patients. Cox regression models were used to generate prognostic metafeatures (MF) for the RMS cohort. A 30-protein-coding MF (pcMF, p= 0.001) and a 34-noncoding MF (ncMF, p<0.001) both predicted survival, but the accuracy of the ncMF was higher than the pcMF (96% vs. 71%, p<0.001). Pathway analyses showed association of pcMF with musculoskeletal development and signaling pathways while ncMF enriched for cellular assembly, cell cycle, apoptosis, and cancer-associated functions. Further, the ncRNA profile was driven by expression of a 600kb very long noncoding RNA (vlncRNA), expressed at high levels only in surviving patients. For ES, we identified two highly expressed tumor-specific ncRNAs, a long ncRNA (lncRNA, 2.6kb) FEZF1-AS1, and another, 400kb vlncRNA, C9orf92. While both the ncRNAs were regulated by EWS-FLI1, only FEZF1-AS1 was directly regulated by the fusion protein binding to GGAA repeats in its promoter. In vitro Matrigel assays and tail vein mouse models of tumor metastasis showed that FEZF1-AS1 promoted tumor invasion while the C9orf22 vlncRNA inhibited tumor growth and metastasis. FEZF1-AS1 is present in the human fetal brain and a subset of genes induced by both EWS-FLI1 and FEZF1-AS1 allow for cell organization and neuronal development. Chromatin immunoprecipitation assays show that FEZF1-AS1 modifies histone activating marks across the ES genome while RNA-IP assays indicate binding of the lncRNA to chromatin modifying proteins such as EZH2 and HDACs. RNA-ISH and protein IF showed co-localization of vlncRNA with AP1. vlncRNA overexpression decreased protein levels of the MET oncogene in ES metastatic cell line. Matrigel assays showed that ES cells with decreased MET expression had reduced invasiveness, a feature associated with metastasis and poor outcome. Outcome data for 140 ES patients showed high expression of the 400kb vlncRNA correlated with increased survival rates (p<0.01). In conclusion, our studies demonstrate that noncoding RNAs of diverse type are highly functional in childhood sarcomas and predict outcome better than any coding RNA. Functional studies indicate that they regulate necessary pathways of cell growth and metastasis in ES pathogenesis. These findings highlight the importance of the regulatory noncoding genome in childhood tumors.

#2068

Single-cell RNA-seq profiling of the transcriptional response to Rb loss in the retinoblastoma cell of origin.

Sunhye Lee,1 Hardeep P. Singh,1 Martin Triska,1 Kevin T. Stachelek,1 Maxwell M. Bay,2 Brendan H. Grubbs,2 Matthew E. Thronton,2 Haoze Hu,2 Neil I. Segil,2 Michael A. Bonaguidi,2 David Cobrinik1. 1 _Children's Hospital Los Angeles, Los Angeles, CA;_ 2 _University of Southern California, Los Angeles, CA_.

Retinoblastoma is a rare childhood tumor initiated by biallelic inactivation of the RB1 gene and loss of functional retinoblastoma (Rb) protein. The tumors are thought to originate in cone photoreceptor precursors since Rb knockdown induces cone precursor proliferation and elicits cone-precursor-derived retinoblastoma-like tumors in orthotopic grafts. The identification of the retinoblastoma cell of origin and the authentic initiating mutation presents an opportunity to define the detailed molecular mechanisms controlling malignant transformation of a human cancer. In this project, we seek to define the transcriptional and epigenetic cell state transitions that follow Rb depletion as post-mitotic human cone precursors are induced to re-enter the cell cycle. Prospectively isolated cone precursors were transduced with lentivirus carrying either of three RB1 shRNAs or a SCR control shRNA and then re-isolated by FACS. Single cell RNA-sequencing (scRNA-seq) was performed at multiple time points over 15 days by generating full-length cDNAs corresponding to polyadenylated RNAs followed by sequencing. Among the 572 individual cells that passed quality control, an average of 6.6 million mapped reads and ~21,000 genes (~93,000 transcript isoforms) were detected per transcriptome. In addition, 300 cells per shRNA group were evaluated for genome-wide open chromatin using uATAC-seq at day 0 and 15. Cell transcriptomes were pseudotemporally ordered using "Waterfall" in order to define cellular trajectories and corresponding transcriptomic responses to Rb loss. Using transcriptomic, epigenetic, and network analyses, we define cell state transitions and identify potential mediators of such transitions, that are associated with the initiation of retinoblastoma development.

#2069

Therapeutic targeting of ependymoma as informed by oncogenic enhancers.

Stephen C. Mack, Global Ependymoma Network of Excellence. _Baylor College of Medicine, Houston, TX_.

Genomic sequencing has driven precision-based oncology therapy; however, genetic drivers remain unknown or non-targetable for many malignancies, demanding alternative approaches to identify therapeutic leads. Ependymomas comprise histologically similar tumor entities driven by distinct molecular mechanisms, such as fusion oncoproteins, genome-wide chromosomal instability, or disruption of DNA methylation patterns. Despite these differences, ependymomas commonly resist chemotherapy and lack available targeted agents for clinical trial development. In the case of genomically balanced ependymomas, and those driven by fusion oncoproteins, we hypothesized that the chromatin landscapes could uncover oncogenes that would inform actionable targets for therapy and reveal specific transcriptional circuitries to identify molecular origins of the disease. We therefore interrogated the oncogenic drivers of ependymoma by characterizing the active regulatory landscapes of 42 primary tumors through an integrative analysis of H3K27ac ChIP-seq, RNA-seq, whole-exome sequencing (WES), whole genome sequencing (WGS), copy number profiling, and DNA methylation profiling. Across the landscape of ependymal tumors, enhancer mapping revealed putative oncogenes, including NFIX, MIRLET7BHG, FGFR1, WEE1, and MTSS1L, and delineated subgroup specific enhancer lesions. Reconstruction of enhancer networks permitted the identification of core transcription factors (TFs) that establish ependymoma cell state, and lineage-specifying TFs that dictate molecular subgroup identity. Lineage-associated TFs point to distinct spatio-temporal origins of ependymoma subgroups such as FOXJ1 TF activity and expression observed preferentially in subsets of hindbrain ependymomas. To translate our results into a potential clinical paradigm, we demonstrate cancer dependencies on super enhancer associated genes and lineage TFs, and establish the utility of chromatin landscape analysis to predict novel targets for cancer therapy.

#2070

**The prevalence of** DICER1 **pathogenic variation in cancer population databases.**

Jung Kim,1 Kris Ann P. Schultz,2 D. Ashley A. Hill,3 Douglas R. Stewart1. 1 _National Cancer Institute, Rockville, MD;_ 2 _Children's Hospitals and Clinics of Minnesota, Minneapolis, MN;_ 3 _George Washington University School of Medicine and Health Sciences, DC_.

DICER1 is an RNaseIII endonuclease that is crucial for processing pre-microRNA into mature microRNA. The DICER1 syndrome is a rare, autosomal dominant tumor-predisposition disorder associated with pleuropulmonary blastoma, a rare pediatric lung cancer. Somatic missense variation in "hotspot" codons in the RNaseIIIb domain (E1705, D1709, G1809, D1810, E1813) is observed in DICER1-associated tumors. Previously, we found in the general population (non-TCGA ExAC) that the prevalence of pathogenic DICER1 variation is approximately 1:10,000. In this study, we investigated the prevalence of pathogenic DICER1 variation in common adult and pediatric cancers. We examined two publicly-available cancer datasets: TCGA (adult; 32 cancer types; 9,173 exomes) and TARGET (pediatric; 3 cancer types; 236 exomes). TCGA bam files were downloaded and germline variants were called. Germline VCF files were obtained from TARGET investigators. All datasets were annotated with snpEff and ANNOVAR. Variants were binned into 4 categories: likely benign (LB), variant of unknown significance (VUS), likely pathogenic (LP), or pathogenic (P). We reviewed publicly-available pathology images for the TCGA tumors. The prevalence of DICER1 P/LP variants (in all cancers combined) was 1:834 and 1:118 in TCGA and TARGET, respectively. To reduce potential prevalence inflation, we conducted a more stringent prevalence calculation by only considering loss-of-function and hotspot variants. The prevalence of P variants was approximately 1:4586 in TCGA and 1:236 in TARGET. With these more stringent criteria, a single participant with a uterine corpus endometrial carcinoma (UCEC in TCGA) harbored a two pathogenic germline DICER1 (hotspot and splice donor) variants, and a second UCEC harbored a somatic DICER1 (hotspot) variant. In TARGET, a single participant with a neuroblastoma harbored a germline DICER1 canonical splice variant. There were no atypical or unusual features noted on pathology review of publicly-available slides from TCGA tumors. In TCGA, across many types of cancer, the prevalence of pathogenic DICER1 variation was higher compared with the general population (~1:4,600 vs. ~1:10,000), a finding driven by one cancer type (UCEC). In the smaller TARGET dataset, we observed a single pathogenic germline variant in neuroblastoma. This is the first comprehensive analysis of DICER1 pathogenic variation in large adult and pediatric cancer populations. The novel association of DICER1 variation with UCEC merits additional investigation.

#2072

Genomic studies of Brazilian patients with hepatoblastoma: Insight into somatic mutations using whole-exome sequencing.

Talita F. Aguiar,1 Tatiane Rodrigues,2 Maria Prates,2 Fernanda Aparecida dos Santos,1 Gustavo Fernandes,2 Cecília Maria Lima da Costa,1 Isabela Werneck da Cunha,1 Monica Cypriano,3 Silvia Regina Caminada de Toledo,3 Jorge Estefano S. de Souza,1 Eugênia Valadares,4 Raquel Borges,5 Vicente Odone,6 Israel Tojal,1 Dirce Carraro,1 Carla Rosenberg,2 Ana C.V. Krepischi2. 1 _AC Camargo Cancer Center, São Paulo, Brazil;_ 2 _University of São Paulo, São Paulo, Brazil;_ 3 _Pediatric Oncology Institute (GRAACC), São Paulo, Brazil;_ 4 _Baleia Hospital, Belo Horizonte, Brazil;_ 5 _Children's Hospital, Porto Alegre, Brazil;_ 6 _ITACI, São Paulo, Brazil_.

Hepatoblastomas (HB) are embryonal tumors of the liver with histological features that resemble different stages of liver differentiation. The identification of molecular pathways involved in HB development can expand the understanding of the connections between disruption of normal differentiation and cancer. Exome sequencing (244K Agilent SureSelect Target Enrichment) analysis was performed for 6 HBs matched with their non tumoral liver tissues (fresh frozen tissues). Bioinformatic analysis of exome data identified somatic variants in 69 genes which were chosen for validation using a target sequencing panel (SureSelectXT Target Enrichment System for Illumina Paired-End Sequencing Library). The gene panel was composed of the detected 69 genes and other 48 genes related with HB or cancer, and it was used to investigated additional 13 HB samples as a validation group. 60% of the patients were male, and the mean age at diagnosis was 36 months. 13% of this cohort presented pulmonary metastasis. All patients received pre-surgery chemotherapy (SIOPEL and COG protocol).

Results: A total of 71 somatic rare coding mutations (missense and loss-of-function) were validated in 53 genes considering the entire HB group. The somatic analysis reveals pathogenic mutations in the CTNNB1 gene and a recurrent missense mutation in the CX3CL1 gene; the role of these mutations was explored by IHQ studies of their proteins as well as by gene expression analysis by RT-PCR. We also used results from Illumina 450k to evaluated the methylation levels of CX3CL1 and CX3CR1 genes. Methylation values for CpG sites in each sample were measured as β-and CpG sites were grouped into categories, promoter (1stExon; 5'UTR; TSS1500; TSS200) or gene body, information provided by Illumina, based on UCSC data (GRhC 37). The methylation level of each category was obtained by averaging the β-values of all CpGs mapped in the category for each gene, followed by Wilcoxon test correction by calculating the false discovery rate (FDR).

Conclusion: Most investigated HBs carry few potentially pathogenic genetic mutations (≤ 5 mutations). This observed low frequency of somatic mutations is a result similar to previous studies. The proposed explanation is based on the fact that pediatric tumors would originate from precursor cells with pluripotent characteristics; therefore, such tumors may require fewer mutations than adult solid tumors to develop. The congenital HB case of our cohort is discrepant from this scenario since a relatively high number of somatic mutations were found compared to the HB group. To our knowledge, this is the first comprehensive genomic characterization of Brazilian HBs. Next steps include expanding the casuistry of exome sequenced tumors, including two cases of HB associated with Hirschprung disease.

Grants: FAPESP (2016/04785-0; 2017/11212-0), FAPESP (2013/08028-1), CNPq (141625/2016-3).

#2073

Transcriptome profiles of cancer stem-like cells in patient-derived diffuse intrinsic pontine glioma (DIPG).

Stefanie Galbán,1 Carlos Espinoza,1 Karan Bedi,1 Uday B. Maachani,2 Mark M. Souweidane,2 Mats Ljungman,1 Marcian Van Dort,1 Brian D. Ross1. 1 _University of Michigan, Ann Arbor, MI;_ 2 _Weill Cornell Medicine, New York, NY_.

Diffuse intrinsic pontine glioma (DIPG) is a rare, but lethal childhood cancer with a 5-year survival less than 1 %. Genetic profiling of DIPG biopsies and post-mortem tissue have recently identified mutations in PI3KCA, PTEN, TP53, ATM/MPL, histones and PDGF receptor overexpression. PI3KCA and PTEN mutations as well as PDGF receptor overexpression indicate upregulation of the PI3K/AKT/mTOR signaling axis, representing druggable targets. We recently developed a multifunctional kinase inhibitor (ST-182), which targets the PI3K/AKT/mTOR and MAPK pathways which is often upregulated in various malignancies as a compensatory mechanism when PI3K is inhibited. We evaluated ST-182's efficacy for targeting these pathways in patient derived DIPG by western blotting and reverse phase protein array analysis (RPPA). Phosphorylation changes of ERK and AKT, downstream signaling inhibition as well as diminished proliferation was shown in two DIPG cell lines (SU-DIPGIV and XIII) when treated with ST-182, indicating efficacy of co-targeting these pathways as a new therapeutic advance for DIPG.

FACS analysis of DIPG cells (SU-DIPGXIII) identified a large percentage (>10%) of DIPG cells as ALDH positive indicating aggressive stem like features. Characterization of these distinct DIPG populations (ALDH+,-) at the transcriptome level was performed to understand differences in pathway signaling and to identify potential drug resistance mechanisms to ST-182. Utilizing an innovative transcriptome analysis approach, we identified elevated levels of MYC, E2F and DNA repair genes in ALDH+ cells, supporting stem like phenotype of ALDH+ DIPG cells. MYC has long been identified as a crucial player in maintaining embryonic stem cell pluripotency and self-renewal, whereas E2F provide transcriptional control of stem cell fate and DNA repair mechanisms maintain and regulate cancer stem cells. Pharmacological targeting of MAPK/PI3K/mTOR by ST-182 demonstrated up regulation of NFkB, apoptosis, hypoxia, p53 and inflammatory response in ALDH+ and ALDH- cells and down-regulation of MYC, E2F and DNA replication indicating efficacy of targeting these pathways in preventing/reversing stem-like phenotypes in the ALDH+ cell population.

Our findings indicate efficacy of ST-182 for the treatment of ALDH+ cancer stem cells providing impetus for evaluation of molecularly targeted MAPK/PI3K/mTOR therapy. Our comprehensive transcriptome studies provide a new direction for the treatment of DIPG through novel insights into the underlying transcriptomic basis of drug resistant cancer stem cells. Development of new compounds such as ST-182 provides opportunities to implement precision medicine to improve treatment outcomes for DIPG patients.

#2074

Single-cell RNA-seq reveals heterogeneity for stem cell markers, LRG5 and CD271 to predict a subpopulation in Ewing sarcoma cells with lower levels of BRAF.

Roxane Khoogar, Peter Houghton. _UT Health Science Ctr. at San Antonio, San Antonio, TX_.

Introduction: Ewing Sarcoma (ES) is an aggressive bone and soft tissue tumor, which has a high rate of recurrence among all childhood cancers. This suggests the need for a better understanding of heterogeneous cancer populations. In many cancers, recurrence and therapy resistance has been suggested to be due to the relative state of quiescent cells. Our previous work has shown that low EWSR1-FLI1 expressing cells have a significantly lower proliferative activity, which is similar to a quiescent state. Therefore, we used cells with low EWSR1-FLI1 expression to model and predict cells with a quiescent state.Methods: Using siEWSR1 to suppress EWSR1-FLI1, we studied proliferative quiescence. The Fluidigm C1 system was used to perform SMART-Seq whole transcriptome amplification using EW8 cells. We sequenced single- cell RNA-Seq libraries prepared from 700 ES cells. We developed a computational analysis to profile single cells. Then, we used gene expression signatures derived from the quiescent population to classify parental cells with ensemble classifiers. Results: Results revealed that EW8 cells show considerable heterogeneity at the transcriptome level in control (scrambled RNAi) as well as in the quiescent populations. We identified a subpopulation of EW8 cells with a negative correlation between the expression of LGR5 and Nerve Growth Factor Receptor (NGFR/CD271). Our preliminary data from 255 single cells indicate that LGR5 (0-43.57 TPM, median 0) and CD271 (0-80.05 unique transcripts, median 6.33) were correlated (r= -0.12, P<0.0001) in EW8 cells. CD271 is a surface membrane protein, and a mesenchymal stem cell marker, that regulates stemness, and heterogeneity. A subpopulation of cells with low CD271 was distinguished and studied for its co-expression pattern with LRG5 and BRAF. Conclusions: Our results suggest that there are multiple subpopulations within EW8 parental (Control) cells. We can characterize them with proliferation, stemness, motility and dormancy-associated markers. Ongoing studies are aimed to determine, if EW8 cells having elevated LGR5 exhibit different sensitivity to cytotoxic agents. Data derived from this work are expected to provide insights into the extent to which CD271 contributes to dormancy, and ES heterogeneity. Whether CD271 regulates proliferation to promote a dormant state in this subpopulation is being studied.

#2075

Whole genome sequence analysis informs precision medicine of pediatric cancers.

Bogdan Tanasa,1 Alex Lee,1 Marcus Breese,1 Avanthi Shah,1 Stan Leung,1 Heng-Yi Liu,1 Aviv Spillinger,1 Kimberly Hazard,2 Arun Rangaswami,2 Sheri Spunt,2 Norm Lacayo,2 Tabitha Cooney,1 Eric Alejandro Sweet-Cordero1. 1 _UCSF, San Francisco, CA;_ 2 _Stanford University, Stanford, CA_.

Several recent studies (including BASIC3, iCAT, INFORM, PEDS-MIONCOSEQ) have used whole exome sequencing (WES) and RNA-seq in order to identify targetable chromosomal alterations in a large variety of pediatric cancers. Few studies have attempted to evaluate whether a more comprehensive approach including WGS and RNA-seq could be used to identify novel events relevant to the pathogenesis of advanced pediatric cancer.

We analyzed a total of 59 patients (37 solid tumors, 11 brain tumors, and 10 leukemia/lymphomas) to determine the feasibility of using whole-genome sequencing (WGS) technology in conjunction with RNA-seq in order to identify actionable/druggable alterations in the pediatric cancer genomes. WGS analysis has been performed on 75 samples, that were collected from 45 patients, either at diagnosis or at relapse.

For WGS analysis of germline-tumor pairs, after performing the sequence alignment with BWA-MEM to gender-specific hg38 genomes, we have used and verified a set of computational methods: 1) MuTect2 for SNV calling, 2) cn.mops for CNV calling, 3) DELLY and LUMPY for SV calling.

Here we present our findings on the sets of SNV, CNV, SV, and gene fusions that we have identified by WGSA and RNA-seq, respectively, with a particular emphasis on the druggable alterations, and on the tumor response in the murine PDX models of the pediatric cancers. Consistent with previous observations, the mutational burden across pediatric cancers was low. While common mutations were identified, there was a long-tail of mutations that occurred at a low frequency. As anticipated, samples obtained post-chemotherapy had a higher mutational burden than treatment naive samples. TP53 was the most commonly mutated gene, but we also identified SNVs in other genes commonly mutated in cancer, such as ASXL1, NOTCH2, and RB1.

Other novel recurring variants were discovered, further analysis of which is ongoing. Our results indicate that integrated WGS and RNA-seq analysis is feasible in the clinical setting and can reliably identify variants reported on commercially available gene panel testing. However, this approach also resulted in additional clinically relevant findings and allows for novel discovery that will further advance our understanding of these rare and highly aggressive pediatric malignancies.

#2076

Identification of novel fusion genes and expression variants in primary and patient-derived xenograft samples of pediatric leukemia using error-corrected RNA sequencing.

Sonali P. Barwe,1 Anilkumar Gopalakrishnapillai,1 Nitin Mahajan,2 Todd Druley,2 E. Anders Kolb,1 Erin L. Crowgey1. 1 _Nemours/A. I. duPont Hospital for Children, Wilmington, DE;_ 2 _Washington University, St. Louis, MO_.

Introduction

Chromosomal rearrangements generating gene fusions are more common in pediatric malignancies compared to adults, and possess diagnostic, prognostic and therapeutic value. Traditionally chromosomal rearrangements including structural variants (SVs) are identified using karyotyping and fluorescence in situ hybridization (FISH); however, these methods are not sensitive for small rearrangements and single nucleotide variants (SNVs). The need to detect these cryptic SV and SNVs in pediatric cancers demands the development of assays to analyze complex RNA molecules.

Methods

Primary bone marrow samples obtained from Nemours Biobank were used for RNA isolation. Targeted error-corrected RNA sequencing using ArcherDX HemeV2 kit was conducted on 30 primary pediatric leukemia samples and their corresponding mouse passaged xenograft samples. RNA data (fastq) was analyzed via a custom cloud environment leveraging ArcherDx Version 5.1.3 software. The gene fusion data produced by the Archer panel was initially correlated with diagnostic FISH data available for each primary sample.

Results

Ten out of 30 primary bone marrow samples possessed gene fusions detected by routinely tested FISH probes for diagnostic purposes, and including ETV6-RUNX1, BCR-ABL1, TCF3-PBX1, and KMT2A. In addition, this approach detected cryptic gene fusions in 10 samples that were negative for chromosomal rearrangements via FISH, including SPTAN1-ABL1, RUNX1-MKL1, NUP98-NSD1, P2RY8-CRLF2 and TCF3-HLF. The remaining 10 samples, which did not possess detectable gene fusions, showed abnormal exon usage and domain duplications for several, key oncogenes along with novel mutations.

A comparison of the primary sample and mouse passaged xenograft sample revealed that majority of gene fusions representing the abundant clone remained consistent between the primary and xenograft sample in secondary and tertiary passages. Certain gene fusions representing minor clones appeared and disappeared in xenograft samples and subsequent passages in mice in comparison to the primary patient sample, highlighting the heterogeneity of the disease. Thus, the presence of major driver mutations at similar allelic frequencies in xenografts compared to primary samples and over multiple passages confirms the utility of xenograft models for preclinical drug testing.

Discussion

Using a novel approach that utilizes targeted error-corrected sequencing, all the aberrations detected by clinical diagnostic testing were verified, plus several novel fusion events were identified with high confidence. This method also validated the concordance between primary and xenograft samples. Characterization of these novel cryptic fusions and exonal variants in leukemogenesis will enable identification of new drug targets and prognostic factors for pediatric leukemia.

#2077

**Genome-wide association study identifies the** GLDC/IL33 **locus associated with survival of osteosarcoma patients.**

Roelof Koster,1 Orestis A. Panagiotou,1 William A. Wheeler,2 Eric Karlins,1 Julie M. Gastier-Foster,3 Silvia Regina Caminada de Toledo,4 Antonio S. Petrilli,4 Adrienne M. Flanagan,5 Roberto Tirabosco,5 Irene L. Andrulis,6 Jay S. Wunder,6 Nalan Gokgoz,6 Ana Patiño-Garcia,7 Fernando Lecanda,7 Massimo Serra,8 Claudia Hattinger,8 Piero Picci,8 Katia Scotlandi,8 David M. Thomas,9 Mandy L. Ballinger,9 Richard Gorlick,10 Donald A. Barkauskas,11 Logan G. Spector,12 Margaret Tucker,1 Belynda Hicks,1 Meredith Yeager,1 Robert Hoover,1 Stephen J. Chanock,1 Sharon A. Savage,1 Lisa J. Mirabello1. 1 _DCEG, National Cancer Institute, NIH, Bethesda, MD;_ 2 _Information Management Services (IMS), Inc., Rockville, MD;_ 3 _Nationwide Children's Hospital, and The Ohio State University Department of Pathology and Pediatrics, Columbus, OH;_ 4 _Laboratorio de Genética, Pediatric Oncology Institute, GRAACC/UNIFESP, São Paulo, Brazil;_ 5 _Royal National Orthopaedic Hospital NHS Trust, Middlesex, United Kingdom;_ 6 _Litwin Centre for Cancer Genetics, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, University of Toronto, Ontario, Canada;_ 7 _Department of Pediatrics, University Clinic of Navarra, Universidad de Navarra, Pamplona, Spain;_ 8 _Laboratory of Experimental Oncology, Orthopaedic Rizzoli Institute, Bologna, Italy;_ 9 _The Kinghorn Cancer Centre, Garvan Institute of Medical Research, Australia;_ 10 _Division of Pediatrics, The University of Texas MD Anderson Cancer Center, Houston, TX;_ 11 _Keck School of Medicine, University of Southern California, Los Angeles, CA;_ 12 _Department of Pediatrics, University of Minnesota, Minneapolis, MN_.

Survival rates for osteosarcoma, the most common primary bone cancer, have changed little over the past three decades and are particularly low for patients with metastatic disease. To date, few prognostic factors have been identified to be associated with survival in patients with osteosarcoma. We conducted an international, multi-institutional genome-wide association study (GWAS) to identify germline genetic variants associated with overall survival in 632 patients with osteosarcoma. Subjects were genotyped as part of our previously published osteosarcoma susceptibility GWAS, including 523 patients of >80% European ancestry and 109 patients from Brazil. We performed a time-to-event analysis and estimated hazard ratios (HR) and 95% confidence intervals (CI) using Cox proportional hazards models, with and without adjustment for metastatic disease. Cox models were adjusted for age at diagnosis, gender, significant principal components, and study/center. SNPs that reached P<10-4 in patients of European ancestry were followed up in patients from Brazil. The results were combined across case sets using a random-effects meta-analysis, and between-sets heterogeneity was evaluated using Cochran's Q chi-squared statistic and the I2 metric. Age (P<0.001) and presence of metastases at diagnosis (P<0.001) were associated with patient survival. In the combined analysis of 632 patients, the strongest association was SNP rs55933544, which was inversely associated with overall survival at genome-wide significance (HR=1.89 per copy of the T allele; 95% CI 1.5-2.4; P=1.3×10-8; I2=0%). Rs5593354 is located in intron 23 of the GLDC gene on chromosome 9p24.1, adjacent to the IL33 gene. This association was consistent across both the European (HR=1.9; P=1.6×10-6) and Brazilian (HR=2.1; P=8.4×10-3) case sets, and after adjustment for metastatic disease at diagnosis (combined HR=1.92; P=1.3×10-8), suggesting that this locus affects overall survival independent of metastatic disease. Kaplan-Meier curve analyses indicated a statistically significant difference between survival rates over time (log-rank test P<0.001) for both the dominant and multiplicative models for rs55933544. Using publicly available data, the risk allele was associated with lower expression of IL33, and lower expression of IL33 in osteosarcoma tumors was associated with worse survival in an independent set of 112 osteosarcomas (P<0.05). In conclusion, we have identified the GLDC/IL33 locus on chromosome 9p24.1 as associated with overall survival in patients with osteosarcoma. Further studies are needed to confirm this association and shed light on the biologic underpinnings of this susceptibility locus.

#2078

Analysis of DNA structural variations in context of the gene regulatory landscape of pediatric ALL.

Laura Oksa,1 Miikka Vouttilainen,1 Tapio Vuorenmaa,2 Saara Laukkanen,1 Mari Lahnalampi,2 Olli Lohi,1 Merja Heinäniemi2. 1 _University of Tampere, Tampere, Finland;_ 2 _University of Eastern Finland, Kuopio, Finland_.

Acute lymphoblastic leukemia (ALL) is the most common pediatric leukemia. It comprises of multiple subtypes with combinations of chromosomal rearrangements, indels and copy number variations. Furthermore, there is a significant number of somatic mutations located outside of protein coding regions of the genome. These DNA alterations can be classified based on whether they 1) introduce novel transcription factor binding sites, 2) create aberrant (super-)enhancers, or 3) lead to the loss of regulatory boundaries at oncogene loci upon disruption of CTCF binding sites.

We performed whole genome sequencing (WGS) on two T-ALL and 16 pre-B ALL patients from diagnostic and remission samples, and measured transcriptional activity using Global Nuclear Run-on sequencing (GRO-seq). These results were integrated with chromatin topology information and transcript annotations to yield comprehensive characterization of the gene regulatory landscape. The effect on gene expression was further analyzed based on monoallelic expression.

Altogether, we found 49 translocations, 153 deletions, 9 duplications and 22 inversions across the pre-B ALL samples. Majority of the changes were located within the annotated gene regions. At 25 of these 233 sites, we found evidence that active enhancers resulted in transcription-coupled genetic instability at DNA breakpoints. In a pre-B ALL case, our analysis successfully captured complex translocation event involving ETV6 (ETV6-MUTYH, ETV6-chr7, ETV6-chr21, ETV6-TXNRD2), and deletion at chr22 that hit to active promoters of PATZ1 and DRG1 genes leading to monoallelic expression of these genes.

In T-ALLs, we found 13 translocations (8 intergenic) and 9 deletions. In a T-ALL case, we found that translocation between TCR-beta locus and chr9 brought an active promoter to the proximity of TMEM38B resulting in increased gene expression. We also found a deletion in active promoter at chr9 leading to decreased expression of MTAP and CDKN2A genes. Moreover, a large deletion in chr11 brought oncogenic transcription factor LMO2 and RAG1 close to each other.

In summary, we present a genome-wide approach to link structural variation events with gene regulatory features in ALL cells. This extends previous approaches that have mainly focused on genetic changes at coding regions.

#2079

Collection of patient-derived xenografts (PDX) to study the biology and therapy of bone sarcomas.

Maria Cristina Manara,1 Giordano Nicoletti,1 Manuela Ferracin,2 Camilla Cristalli,1 Alberto Righi,1 Mauro Magnani,3 Michela Pasello,1 Piero Picci,1 Patrizia Nanni,2 Pier-Luigi Lollini,2 Katia Scotlandi1. 1 _Rizzoli Orthopaedic Inst., Bologna, Italy;_ 2 _University of Bologna, Bologna, Italy;_ 3 _University of Urbino, Urbino, Italy_.

Osteosarcoma (OS) and Ewing's sarcoma (EWS) are the two most common pediatric solid tumors, after brain tumors. Multimodal treatments have significantly improved prognosis in localized disease but outcome is still poor in metastatic patients, for whom therapeutic options are often inadequate. Preclinical drug testing to identify promising treatment options that match the molecular make-up of these tumors is hampered by the lack of appropriate and molecularly well-characterized patient-derived models. To address this need, a panel of patient-derived- xenografts (PDXs) was established by subcutaneous implantation of fresh, surgically resected OS and EWS tumors in NSG mice. Tumors were re-transplanted to next mice generations and fragments were collected for histopathological and molecular characterization. A model was considered established after observing stable histological and molecular features for at least three passages. To evaluate the similarity of the model with primary tumor, we performed a global gene expression profiling and tissue microarrays (TMA), to assess tumor specific biomarkers on tissues from OS/EWS tumors and their PDXs (1st and 3rd passage). Moreover, we verified the feasibility of these models for preclinical drug testing. We implanted 61 OS and 29 EWS samples: 14/38 (37%) primary OS and 9/23 (39%) OS lung metastases successfully engrafted; while among EWS, 5/26 (19%) primary samples and 1/3 (33%) metastases were established. Comparison between patient samples and PDXs, highlighted that histology and genetic characteristics of PDXs were stable and maintained over passages. In particular, correlative analysis between OS and EWS samples and their PDXs was extremely high (Pearson's r range r=0.94-0.96), while patient-derived primary cultures displayed reduced correlation with human samples (r=0.90-0.93), indicating that in vitro adaptation superimpose molecular alterations that create genetic diversion from original tumors. No significant differentially expressed gene profile was observed from the comparison between EWS samples and PDXs (fold change > 2, adjusted p <0.05 at paired t-test). In OS, the comparison between OS patient-derived tumors and PDX indicated differences in 397 genes, mostly belonging to immune system functional category. This is in line with the idea that human immune cells are gradually replaced by murine counterparts upon engraftment in the mouse. As proof-of concept, two EWS PDX and one OS PDX have been treated with conventional and innovated drugs to test their value in terms of drug-sensitivity prediction. Overall, our study indicated that PDX models maintained the histological and genetic markers of the tumor samples and represent reliable models to test sensitivity to novel drug associations. Grants AIRC (IG 18451 to KS; IG15324 to P-LL), Italian Ministry of Health (ER2017 2364984-ERANET. to KS; RF-2013-02357552 to P-LL)

#2080

Role of differentially expressed micro RNAs in non-WNT, non-SHH medulloblastoma.

Raikamal Paul,1 Rakesh Jalali,2 Epari Sridhar,2 Aliasagar Moiyadi,3 Neelam Shirsat1. 1 _Advanced Center for Treatment Research and Education in Cancer, Tata Memorial Center, Homi Bhabha National Institute, Training School Complex, Anushakti Nagar, Kharghar, India;_ 2 _Tata Memorial Hospital, Tata Memorial Centre, Kharghar, India;_ 3 _Advanced Centre for Treatment, Research and Education in Cancer (ACTREC), Tata Memorial Centre (TMC), Kharghar, India_.

Medulloblastoma is a common malignant brain tumor in children. It has four distinct molecular subgroups namely WNT, SHH, Group 3 and Group 4. The molecular subgroups differ in their clinical characteristics including age, incidence of metastasis and survival. Group 3 and Group 4 medulloblastomas have overlapping gene expression profile but are distinct in their clinical behavior. Group 3 medulloblastomas have the worst overall survival rates whereas Group 4 medulloblastomas have intermediate survival. Group 3 and Group 4 tumors are not associated with any known signaling pathway and due to their overlapping expression profiles it is often difficult to accurately identify the subgroup, which is very crucial for the appropriate treatment design. MiR-592, located in the intronic region of GRM8 gene, is over expressed in Group 4 medulloblastoma along with its host gene, thus acting as a good marker for Group 4 tumors. MiR-204, on the other hand is expressed from the 6th intron of TRPM3 gene and expressed in some Group 3 and majority of Group 4 medulloblastomas. In order to delineate the role of miR-592 and miR-204 in medulloblastoma biology, effect of expression of these microRNAs on medulloblastoma cell growth and behavior were investigated. The micro RNAs were expressed in a lentiviral mediated inducible manner in Group 3 medulloblastoma cell lines D283, D425 and D341 and their effects on cell proliferation, anchorage-independent growth and invasion potential were studied. MiR-592 expression had marginal effects on the anchorage independent growth potential and the tumorigenic potential of the Group 3 cell line where as miR-204 expression led to significant decrease in the anchorage independent growth, tumorigenic potential, as well as invasion potential of the medulloblastoma cell lines. Further in order to decipher the molecular mechanisms underlying these micro RNA mediated effects, RNA-sequencing analysis of miR-592 and miR-204 expressing medulloblastoma cells were performed to identify genes that are differentially expressed upon micro RNA expression. The results helped us to identify the molecular pathways that are being affected by the micro RNA expression and how they affect the malignant behavior of medulloblastoma cells.

#2081

Empowering rare disease cohort biomarker discovery via comparative assessments of gene expression analysis platforms for FFPE pediatric brain tumor specimens.

Mateusz P. Koptyra, Namrata Choudhari, Zhang Zhe, Mariarita Santi, Angela Waanders, Adam Resnick. _Children's Hospital of Philadelphia, Philadelphia, PA_.

The gene expression landscape for biomarker discovery is still limited for many pediatric brain tumors due to insufficient numbers of biorepository collected fresh frozen specimens. The majority of available tissue are formalin-fixed paraffin embedded (FFPE) pathology diagnostic specimens. These specimens, are often of limited quantity and contain compromised RNA material. A number of emerging commercial platforms are described as supporting quantitative expression analysis for low quantity and poor quality materials. Utilizing available platforms, we designed a study to evaluate RNA and miRNA levels in pediatric brain tumor FFPE specimens with limited/compromised access.Experiments were performed with specimens and/or data obtained from Brain Tumor Tissue Consortium (CBTTC) at Children's Hospital of Philadelphia (CHOP). The mRNA gene expression and miRNA target analysis were performed with FFPE material utilizing two commercial platforms: HTG EdgeSeq and Nanostring. The analysis included 5 specimens of low grade glioma or primitive neuroectodermal tumors for mRNA gene expression and 4 specimens of medulloblastoma for miRNA target analysis. FFPE specimens were processed according to manufacturer's protocols. The results were compared with the RNAseq or miRNA sequencing data derived from corresponding tumors' flash frozen specimens' RNA material.

We evaluated requirements of each platform for data generation and established between platform analysis correlations. In performed tests, the HTG platform required lowest amounts of specimen's material. For the majority of analyzed genes (>700 genes), the gene expression profile was relatively similar between all three approaches, however each of the platform presented distinctive distribution profile for normalized data. The RNAseq mean read counts values were correlated highly with NanoString (0.81) and of lesser value with HTG (0.7) platform. The RNAseq presented significantly higher variance distribution than other platforms. The miRNA target analysis (>600 genes) distribution of normalized data revealed significantly lower dynamics for NanoString when compared with miRNAseq or HTG panel data. The miRNA mean read counts were highly correlated with HTG (0.77) and little with NanoString (0.22), while miRNASeq presented the highest variance distribution. In summary, we found a significant level of agreement between all three platforms tested for gene expression data generation. As for miRNA target analysis, the HTG platform presented significantly higher agreement with miRNAseq data. We conclude that tested technologies can support data generation from archived FFPE specimens, however, the platform selection process should involve pre-selection data quality analysis, as well as sample size requirements, gene cohort selection, and pricing evaluations.

#2082

High-throughput droplet digital PCR for MYCN amplification detection in FFPE neuroblastoma samples: Cost-effective, rapid, feasible, and reliable alternative to FISH.

Dinesh Babu Somasundaram, Sheeja Aravindan, Zhongxin Yu, Muralidharan Jayaraman, Ngoc TB Tran, Shibo Li, Terence Herman, Natarajan Aravindan. _University of Oklahoma Health Sciences, Oklahoma City, OK_.

MYCN amplification directly correlates with the clinical course of neuroblastoma (NB) and poor patient survival, and serves as the most critical negative prognostic marker. Although FISH remains the gold standard for clinical diagnosis of MYCN status in NB, its limitations warrant the identification of rapid, reliable, less technically challenging, and inexpensive alternate approaches. In the present study, we examined the concordance of droplet digital PCR (ddPCR, in combination with IHC) with FISH for MYCN detection in FFPE NB samples. In 112 NB cases, ddPCR analysis demonstrated a 96-100% concordance with FISH. Consistently, IHC grading revealed 92-100% concordance with FISH. Comparing ddPCR with IHC, we observed a concordance of 95-98%. The results demonstrate that MYCN amplification status in NB cases can be assessed with ddPCR, and suggest that ddPCR could be a technically less challenging method of detecting MYCN status in FFPE specimens. More importantly, these findings illustrate the concordance between FISH and ddPCR in the detection of MYCN status. Together, the results suggest that rapid, less technically demanding, and inexpensive ddPCR in conjunction with IHC could serve as an alternate approach to detect MYCN status in NB cases, with near-identical sensitivity to that of FISH.

Funding: NIH 1P20GM103639-01 from the COBRE Program of NIH; OUHSC Department of Radiation Oncology Research Development funds

#2083

Innate immunity polymorphisms and risk of pediatric cancer.

Gabriela E. Mercado,1 Gabriela A. Rosales-Bravo,1 David Velazquez-Fernandez,2 Marta Zapata-Tarres,3 Luis E. Juarez-Villegas4. 1 _Universidad Nacional Autonoma de Mexico, Mexico City, Mexico;_ 2 _Instituto Nacional de la Nutricion y Ciencias Medicas Salvador Zubiran, Mexico City, Mexico;_ 3 _Insituto Nacional de Pediatria, Mexico City, Mexico;_ 4 _Hospital Infantil de Mexico Federico Gomez, Mexico City, Mexico_.

In Mexico, cancer represents the second leading cause of death in the pediatric population. The identification of susceptibility genes associated with cancer has been one of the main challenges of human genomics. Several studies have proposed that genetic variation in immune-related genes could be a determinant of susceptibility in different types of cancer. In the present study we evaluated the possible association between 10 polymorphisms in genes that participate in innate immunity and the risk of pediatric cancer. We analyzed genotyping data from 101 patients from Hospital Infantil de México "Federico Gómez" (HIM) with confirmed diagnosis of pediatric cancer. The genotypes were compared with 274 healthy adults without a medical history of pediatric cancer. For each polymorphism we calculated allelic and genotypic frequencies and Hardy-Weinberg equilibrium (HWE). The association between cancer and pediatric cancer was analyzed using Pearson chi-square and odds ratio (OR); survival analyses were performed by the log rank test and Kaplan-Meier curves. Our results show that risk of pediatric cancer in our sample is significantly associated with the genetic variants IL8-251 (p= 0.04327, OR= 0.701, IC 95% = 0.496-0.990), IL6-174 (p= 0.0037, OR= 2.413, IC 95% = 1.310-4.445), and IL10-592 (p= 0.0062, OR= 0.670, IC 95% = 0.502-0.894). However, we did not find any significant association between selected polymorphisms and overall or disease-free survival.

#2084

Reprogramming the epigenome as an effective treatment option for neurofibromatosis1 associated malignant peripheral nerve sheath tumors.

Chandra S. Mayanil,1 Mariam M. Khan,1 Vineet Mohanty,1 Raj Kumar,2 Guifa Xi,1 Shunsuke Ichi,3 Bal Ram Singh,2 Barbara Mania-Farnell,4 Tadanori Tomita1. 1 _Lurie Children's Hospital of Chicago & Northwestern University Feinberg School of Medicine, Chicago, IL; _2 _Institute of Advanced Sciences, Dartmouth, MA;_ 3 _Japanese Red Cross Medical Center, Shibuya-ku, Tokyo, Japan;_ 4 _Purdue University at Calumet, Hammond, IN_.

Neurofibromatosis type 1 (NF1) is common, occurring in 1 in 3000 live births, and results in skin pigmentation and the growth of tumors along nerves in the skin, brain, and other parts of the body. It arises from a mutated and non-functional tumor suppressor gene NF1. Tumors of the peripheral nerves, called neurofibromas and of the main eye nerve, called optic glioma, are debilitating and deadly. Epigenetic mechanisms have been shown to play an important role in the progression of neurofibromas to malignant peripheral nerve sheath tumors (MPNSTs). MPNSTs with polycomb repressive complex 2 (PRC2) loss showed complete loss of trimethylation at lysine 27 of histone H3 (H3K27me3). We hypothesized that owing to the loss of this repressive complex PRC2, there is an overexpression of genes related to tumorigenesis in MPNSTs because of reciprocal gain of the activation marks such as trimethylation at lysine 4 of histone H3 (H3K4me3) and acetylation of lysine 27 of histone H3 (H3K27Ac) at their promoters. Using RT-PCR, we observed an increase in the expression of candidate oncogenes genes PAX2, FOXN4, IGF2 and TLX1 in NF1 associated MPNST cell line ST88-14 versus the normal Schwann cells and normal human fibroblasts. Western blots using purified histones revealed that there was an increase in the expression of H3K4me3, H3K9me2, H3K18Ac and H3K27Ac. Recently, aqueous extracts of Ocimum Sanctum has been used as investigational cancer preventive drug. We wanted to know the effects of extracts of O. Sanctum on MPNST cell line ST88-14. Treatment of ST88-14 cells with the hydrophilic fraction of methanolic extract of Ocimum Sanctum (OSHP-1) not only showed a dose dependent decrease in the above histone marks but also showed an increase in the expression of N-CAM 140KD, characteristic of mature Schwann cells, in addition to a significant decline in proliferation. Further evaluation of the nuclear extracts showed that OSHP-1 treatment increased the expression of HDAC2, a component of NuRD complex and decreased that of lysine acetyl transferase 7 (KAT7) in a dose dependent manner suggesting that both HDAC2 and KAT7 may be responsible for the decrease in the acetylation of H3K27Ac and therefore reversal of the tumorigenic MPNSTs to a differentiated Schwann cell phenotype. Chromatin immunoprecipitation (ChIP) data revealed H3K27Ac occupancy on candidate genes (PAX2, FOXN4, IGF2 and TLX1) promoters was reduced by OSHP-1 treatment. In conclusion, OSHP-1 reprograms the epigenome and may be an effective treatment option for NF1 associated MPNSTs.

#2085

Exploring mutation signatures in pediatric cancers.

Francis A. San Lucas,1 Philip Lupo,2 Austin Brown,2 Michael Scheurer,2 Paul Scheet1. 1 _University of Texas MD Anderson Cancer Center, Houston, TX;_ 2 _Baylor College of Medicine, Houston, TX_.

Background: Mutation signatures have been catalogued across most common cancers, providing insights into their underlying mutational processes and suggesting strategies for categorization and subphenotyping. However, these analyses have been limited to mostly adult cancers, likely due to the relative rarity of pediatric analogs and a corresponding lower mutation burden of these tumors. Here we attempted to compare mutation signatures across several pediatric cancers, given these constraints, with their corresponding adult versions.

Data and Methods: From the ICGC data portal, we downloaded clinical and mutation data for pediatric brain (medulloblastoma, glioblastoma, neuroblastoma) and blood-based (acute lymphoblastic leukemia, acute myeloid leukemia) cancers. We also obtained data for related adult cancers, including glioblastoma and acute myeloid leukemia. For each cancer, we characterized mutation patterns using trinucleotides centered at somatic SNVs and then estimated the composition of underlying putative COSMIC mutation signatures. For cancers with low mutation rates (impeding estimates of mutation signatures), we grouped mutations from similar samples based on clinical and molecular statuses to infer mutation signatures.

Results: Each of the pediatric cancer types strongly exhibited the common cancer mutation Signature 1 (spontaneous deamination of 5-methylcytosine), as might be expected since it has been described across all adult cancer types. Further, pediatric acute myeloid leukemia and glioblastoma both harbored mutation signatures seen in their adult counterparts in addition to unexpected patterns of defective DNA mismatch repair and DNA double-strand break repair. Interestingly, for pediatric glioblastoma we identified Signatures 3, 8, 15 and 16, which were previously unidentified in adult glioblastoma and have been recently implicated in pediatric medulloblastoma.

Conclusion: Our findings may be influenced by the sparsity of mutations in pediatric cancers, highlighting the need for quantifying precision in measures of mutation signatures. This notwithstanding, from our initial observations, pediatric cancers appear to exhibit a more diverse set of mutation signatures than their adult counterparts. Further, pediatric cancers of different histologies, particularly those of brain cancers, may share molecular mechanisms more than currently believed. 

### Surveying the Tumorigenic Microenvironment

#2086

Comprehensive microbial and immunological screening of gastroesophageal biopsies: A multi-parametric analysis to study carcinogenesis.

Chao Zhang, Prashant V. Thakkar, Felice Schnoll-Sussman, Bridget McClure, Greg Sonnenberg, Doron Betel, Manish A. Shah. _Weill Cornell Medical College, New York, NY_.

The development of gastric cancer in the context of chronic H. pylori infection is multifactorial, encompassing both bacterial factors and the altered immune microenvironment. We proposed an unbiased study to evaluate the relationships among microbiome composition, host immune response and genomic characterization from next generation sequencing of gastric biopsy samples. Endoscopic biopsies collected from patients with active H. pylori infection (n=21), prior infection (n=22) and no prior infection (n=26), and were sequenced at 10X to 30X coverage. In total, 77 gastric biopsies from 69 patients were freshly frozen for whole genome sequencing and RNASeq analysis.

Detecting the microbiome from human biopsy sequencing data directly is challenging due to the low microbial content. A novel computational pipeline was developed to address this problem specifically. In our analysis population, in addition to the robust H. pylori signal, several bacteria associated with other cancers were also detected in several biopsy samples, such as Prevotella melaninogenica, Veillonella parvula and Fusobacterium nucleatum. H. pylori infection was associated with reduced microbial biodiversity compared to prior infection or control tissue (p=0.02). H. pylori active infection samples have a distinct non-H. pylori microbiome compared to prior infection and control samples. We also identified 5 patients with prior infection and 1 control patient with occult H. pylori infection.

To characterize the immune infiltration in the mucosal biopsy samples, we define a 176-gene immune panel. The result of unsupervised clustering of gene expression revealed a much higher immune infiltration in H. pylori positive samples compared to uninfected samples, especially for CD8+, Th2 and Th17 cell populations. Two orthogonal experimental essays (ELISA and Flow Cytometry) confirmed the RNAseq-based expression profiling of inflammatory cytokines such as GRO, IL8, TNFa and SCD40L. Importantly, in 2 patients with prior H. pylori infection, the pro-inflammatory immune signature characteristic of H. pylori persisted. Finally, amongst H. pylori active infection patients, the biodiversity of the other bacteria present was inversely correlated with local immune infiltration (eg. greater the bacterial diversity, the less robust the pro-inflammatory immune signature).

In summary, this study established a methodology for microbiome and immune profiling of gastric biopsy samples. We have identified a pro-inflammatory immune response signature associated with H. pylori infection, confirmed and validated by orthogonal assays. Notably, in several samples from previously eradicated H. pylori infection, the pro-inflammatory immune signature persisted, suggesting a potential long-term impact of H. pylori infection on mucosal immunity, that might contribute to gastric tumorigenesis.

#2087

Fibroblast-kinome siRNA screening identifies PIK3Cδ as a mediator of Triple-negative breast cancer (TNBC) invasion.

Teresa Gagliano, Viviana Vella, Kamila Bienkowska, Angeliki Ditsiou, Georgios Giamas. _University of Sussex, Brighton, United Kingdom_.

Background: Kinases represent one of the main therapeutic targets in cancer treatment as their impairing relates to tumor growth and progression. Despite increasing evidence that the tumor microenvironment (TME) signaling influences the behavior of surrounding cancer epithelial cells, still relatively little is known about what changes in stromal cells influence tumor cells' behavior and how they affect their response to target therapy. TNBC patients are still lacking an effective therapy, as not much is known about the biology of this BC tumor subtype. Clinical trials focus mainly on targeting epithelial cancer cells by using a combination of kinase inhibitors and standard chemotherapy however these therapeutic regimens are not considering the action of stromal cells that may influence response to treatment.

Aims: • Identify fibroblasts-expressed kinases that modulate tumor cells' invasion • Characterize the mechanism by which these kinases promote/reduce tumor cell invasion.

Methods: Human Mammary Fibroblast (HMF) and normal lung fibroblast (MRC5) cells were used in a high-throughput siRNA kinome screening targeting 710 Kinases. 24 hours after transfection, fibroblasts were co-cultured with MDA-MB-231 in 3D for spheroid formation. Matrigel and chemoattractants were then added to promote invasion. Spheroids pictures taken at different time-points were analyzed and results were expressed as changes in spheroid surface formation (ΔRatio= ΔCtrl/ΔKinase).

Results: PIK3Cδ was among the most prominent targets, whose silencing decreased TNBC invasion. PIK3Cδ is essential for regulating chemokine production in leukocytes and promotes migration during inflammation, while PIK3Cδ inhibitors (CAL -101) interfere in tumour-stroma interactions without directly killing cancer cells. Despite PIK3Cδ being expressed mainly in leucocytes, we detected high PIK3Cδ protein expression in fibroblast cell lines and primary fibroblasts derived from TNBC patients; still, PIK3Cδ was hardly detectable in breast cancer cell lines. CAL-101 treatment affected fibroblasts viability, while it had limited/no effects on breast cancer cells. Moreover, CAL-101 resulted in reduced phosphorylation of AKT, a PIK3Cδ target. Pretreatment of fibroblasts with CAL-101 significantly decreased TNBC cells' invasion in 2D & 3D co-culture experiments. Interestingly, using transwell systems we saw that TNBC co-culturing increased PIK3Cδ expression in fibroblasts, suggesting a feedback loop that 'fuels' tumor progression. Additional proteomic/genomic studies, provided us with evidence about the PIK3Cδ-mediated paracrine signaling leading to TNBC invasion.

Conclusion: Using a novel 3D co-culture invasion assay, we identified stromal PIK3Cδ as a key mediator of TNBC invasion. Our results suggest that targeting PIK3Cδ in the TME may represent a novel transformative strategy for TNBC therapy.

#2088

Characterization of an inducible alcoholic liver fibrosis model for hepatocellular carcinoma investigation in a transgenic porcine platform.

Kyle M. Schachtschneider,1 Nasya Mendoza-Elias,1 Daniel P. Regan,2 Kelly D. Garcia,1 Lauretta A. Rund,3 Regina M. Schwind,1 Lawrence B. Schook,3 Ron C. Gaba1. 1 _Univ. of Illinois at Chicago, Chicago, IL;_ 2 _Colorado State University, CO;_ 3 _Univ. of Illinois at Urbana-Champaign, Urbana, IL_.

Hepatocellular carcinoma (HCC) is a deadly tumor that spans more than 780,000 new diagnoses and causes 750,000 annual deaths worldwide. While global cancer incidence and mortality are generally decreasing, HCC incidence is rising and projected to continually increase given the growing prevalence of chronic liver diseases. Alcoholic liver disease represents a common progressive chronic liver ailment that incites liver cirrhosis—a precancerous state of liver scarring—that increases the risk for HCC development. The health status of the liver can also affect HCC tumor biology and treatment responses. Therefore, a large animal model capable of exhibiting both HCC and alcohol induced liver cirrhosis would be a valuable resource for preclinical research investigating HCC in its native comorbid cirrhotic microenvironment. This study utilized the innovative Oncopig Cancer Model (OCM)—a transgenic swine model that recapitulates human cancer through development of site and cell specific tumors via induced expression of heterozygous KRASG12D and TP53R167H transgenes—to develop alcohol induced fibrosis in a porcine model capable of developing HCC tumors.

Liver injury was induced in Oncopigs (n=5) via transcatheter infusion of absolute ethanol and ethiodized oil (1:3 v/v dosed at 0.75 mL/kg) into the hepatic arterial circulation. Histological assessment of resected liver specimens collected 8 weeks post-induction revealed METAVIR stage F2-F4 fibrosis, stage A2-A3 inflammation, and a median percent fibrosis of 15.3% (range 5.0-22.9%). All METAVIR and inflammation scores were elevated compared to age matched controls (F0-F1, A0-A1, median percent fibrosis 8.7%, range 5.8-12.1%). Liver injury was then induced via the same protocol in a second OCM cohort (n=5) for a time course study in which post-induction disease surveillance via general health assessment, lab analysis, and serial liver biopsies was performed every 2 weeks for 20 weeks post-induction. Histology revealed METAVIR stage F1-F3 fibrosis as early as 2 weeks post-induction. However, as clinically observed for patients presenting with pre-cirrhotic alcohol induced liver damage, a lack of persistent alcohol exposure resulted in liver recovery as evidenced by METAVIR F1-F2 fibrosis levels at 20 weeks post-induction. No behavioral or biochemical abnormalities were observed during the monitoring period to suggest liver decompensation.

In conclusion, this study successfully validated a protocol to develop a pre-cirrhotic METAVIR F2-F3 model of alcoholic liver disease within 8 weeks using transarterial ethanol-ethiodized oil infusion. While these results support the OCM's capability to serve as a model for HCC in a cirrhotic liver background, the data indicate prolonged exposure to alcohol may be required to develop an irreversible METAVIR stage F4 porcine model of cirrhosis.

#2089

Highly multiplexed analysis of immune cell subsets in non-small cell lung cancer: validation of protein and RNA analysis by the Nanostring Digital Spatial Profiling (DSP) platform.

James Ziai,1 Patrick Caplazi,1 Jérémie Decalf,1 Yan Liang,2 Patricia de Almeida,1 Daniel Zollinger,2 Alison Van Schoiack,2 Joseph Beechem,2 Jane Grogan,1 Matthew Albert1. 1 _Genentech, Inc, South San Francisco, CA;_ 2 _Nanostring Technologies, Seattle, WA_.

Background: Highly multiplexed spatial analysis of tumor tissue is necessary to improve understanding of anti-tumor immunity. Moreover, spatial interrogation of protein and nucleic acid targets simultaneously can better characterize immune cell subsets and targets such as cytokines that are difficult to detect by immunohistochemistry (IHC). NanoString's Digital Spatial Profıling (DSP) technology can detect and quantify proteins and RNA at highly multiplexed levels. We compared DSP to flow cytometry (FCM), IHC and in-situ hybridization (ISH) to assess its sensitivity and specificity. Ongoing studies will assess the prevalence and distribution of immune cell subsets, cytokines and tumor cells in 10 non-small cell lung cancer (NSCLC) cases.

Methods: 20 FFPE NSCLC cases and 1 peripheral blood mononuclear cell (PBMC) pellet were assayed on the DSP platform. Regions of interest (300 um x 400 um) or single cell masks were selected from H&E or fluorescence-stained sections. Serial sections were then probed with a cocktail of primary antibodies conjugated to DNA oligos via a UV-cleavable linker for 29 protein and probes for 39 RNA targets. Oligos from the ROIs were then released via UV-mediated linker cleavage and quantitated on the nCounter® Analysis System. Repeat analysis was performed on 10 cases to assess reproducibility between serial sections. A subset of cases was stained for CD3, CD4, CD8, CD45, PD-1, and PD-L1 by IHC and counted by image analysis. FCM was performed on the PBMC sample. Counts from both methods were compared to nCounter counts.

Results: Comparison of DSP to FCM in PBMC showed good correlation (R2=0.65). Correlation between DSP counts and IHC was robust for CD3 (R2=0.72), CD4 (R2=0.80), and CD45 (R2=0.90). 21 of 29 protein markers showed good/excellent reproducibility (R2 >0.70) on separate DSP runs. 7 of 29 showed fair/good reproducibility across tissue sections (R2=0.50-0.70). RNA analysis of NSCLC samples by both DSP and ISH is ongoing. Analysis of both PBMC and NSCLC samples suggests that target abundance and section-to-section variability may explain differences in performance and reproducibility between sections. For example, low abundance in NSCLC tissue and section-to-section variability was observed for CD19 and CD56 with corresponding lower correlation between replicates (R2=0.68 and R2=0.52, respectively).

Conclusion: DSP shows high section to section reproducibility and fair/good correlation with FCM and IHC. We find performance can be affected by differences in antibody clones or lots and/or section to section variability, particularly for low abundance cell types. Multiplexed RNA analysis of NSCLC samples is ongoing and will document the sensitivity and specificity of DSP compared to traditional ISH and the potential utility of DSP in assessing cytokines in the tumor microenvironment.

#2090

Designer microenvironments to control oncogenic phenotype.

Simone C. Rizzi, Mathieu Heulot, Charlotte Mermier, Katia Antoniello, Sophie Crettaz, Jeremy Touati. _QGel SA, Lausanne, Switzerland_.

"Half of the secret of the cell lies outside the cell (Mina Bissell)". This key aspect, over the genetic influence on cancer, has been largely neglected in the context of oncology drug discovery. In fact, the impressive reciprocity between extracellular matrix (ECM) components and cells, is responsible for numerous biological processes from homeostasis to disease development and progression. For example, alterations of cell-ECM interactions were shown to phenotypically revert cancer to healthy cells and vice versa, regardless of their genetic features. Furthermore, changes in cell adhesion to the ECM were reported to alter cancer cell sensitivity to chemo-therapies, and stiffening of the ECM was observed to favor cancerous cell phenotypes. Here, we present a case to address the question: how do specific modifications of ECM features generate different cell phenotypes. The answer will enable the investigation of ECM-induced drug resistance, disease progression and identification of new drug targets. Today, research with cancer cells and their interactions with the ECM employs natural matrices that provide biologically relevant yet undefined environments that cannot be systematically altered. However, recent advances in biomaterial engineering applied to cell biology have paved the road to overcome these limitations. Here, we use industrially-available QGel matrices, namely synthetic ECM analogues, whose biological, biochemical and structural features can be precisely tuned to systematically test their impact on cell behavior. We have investigated the impact of 60 different ECM combinations on A549 cells, a well differentiated human lung adenocarcinoma cell line with residual characteristic of alveolar type II epithelial cells. The contribution of matrix degradability, stiffness and cell attachment sites derived from several ECM proteins were individually depicted. When A549 were seeded as single cells in the different ECMs, two distinct phenotypes were identified within 4 weeks of culture: (i) full cell spheroid morphology (mass), which is the major phenotype observed in Matrigel, was obtained within MMP-degradable hydrogels; and (ii) acinar morphology (acini) that matured over time from full cell spheroids within non-degradable hydrogels containing specific integrin binding sites. We speculate that the acini phenotype reflects the healthy nature of the alveolar epithelial cells, while the mass morphology has cancerous characteristics. Interestingly, our data also showed that both phenotypes could be reverted when, during cell passaging, the ECMs were switched accordingly. This suggests that ECM-cell interaction solely reversibly influences the cell morphology and no selective pressure of specific clones occurs. Encouraged by these findings, we are now characterizing the gene expression profiles of the different phenotypes and exploiting this QGel platform to study drug responses.

#2091

Quantitative compositional and spatial analysis of tumor microenvironment (TME) in primary melanoma.

Robyn D. Gartrell,1 Andrew Chen,1 Douglas K. Marks,1 Thomas D. Hart,2 Gen Li,1 Alan Wu,1 Yan Lu,1 Camden Esancy,1 Zoe Blake,1 Bret Taback,1 Raul Rabadan,1 Howard L. Kaufman,3 Charles G. Drake,1 Basil Horst,1 Anthea Monod,1 Yvonne Saenger1. 1 _Columbia University Medical Center, New York, NY;_ 2 _Columbia University, New York, NY;_ 3 _Rutgers Robert Wood Johnson Medical School, New York, NY_.

Background: Stratifying melanoma patients for adjuvant immunotherapy trials has quickly become an urgent need. Tumor-infiltrating lymphocyte (TIL) analysis is predictive but insufficiently precise for clinical application. A novel pathology method, quantitative multiplex immunofluorescence (qmIF), allows for complex analysis of the tumor microenvironment (TME) for development of new biomarkers. Given that CD3+CD8+ cytotoxic lymphocytes (CTLs) promote antitumor immunity while CD68+ macrophages (Mφ) impair immunity, we hypothesized that precise quantification and spatial analysis of Mφ and CTLs would correlate with survival in melanoma.

Methods: We applied qmIF to 104 stage II/III primary melanomas from Columbia University Medical Center (CUMC); known cause of death is available for 64 patients. 5-µm slides were stained using qmIF for DAPI, CD3, CD8, CD68, SOX10, HLA-DR and Ki67. Tumor areas were preselected by a dermatopathologist and visualized using Mantra. Tissue and cell segmentation, multiparameter immune phenotyping, and quantitative spatial analysis (qSA) were performed using inForm. Local spatial analysis was implemented by partitioning images into 100-µm square windows. The interquartile range of cellular ratios among windows was used as the measure of dispersion.

Results: We find high CTL and low Mφ density in stroma (p=0.0038, p=0.0006) correlate with disease-specific survival (DSS). Correlation was strongest for stromal HLA-DR+ CTLs (p=0.0005). CTL distance to HLA-DR- Mφ associates with poor DSS (p=0.0016), while distance to Ki67- tumor cells associates inversely with DSS (p=0.0006). Low CTL/ Mφ ratio in stroma confers a hazard ratio (HR) of 3.719 for death from melanoma and correlates with shortened overall survival (OS) in the complete 104-patient cohort by Cox analysis (p=0.009). HLA-DR- Mφ are more evenly dispersed throughout the stroma of survivors (n=42) than patients known to have died of melanoma (n=22) (p=0.0036), while conversely, HLA-DR+ CTLs are less evenly dispersed in survivors (p=0.023).

Conclusions: CTL/ Mφ ratio is prognostically superior to conventional clinical biomarkers in nonmetastatic melanoma. Local spatial features such as leukocyte dispersion and intercellular distances also associate with survival, highlighting the importance of microenvironmental structure. Future efforts will further parse the large output of data from qmIF to generate biomarkers that are increasingly precise, spatially informed, and quantitatively robust.

#2092

Tumor-infiltrating myeloid cells: Using MultiOmyx to distinguish between TAMs, TANs, and MDSCs in the pancreatic tumor microenvironment.

Anna Juncker-Jensen, Nicholas Hoe, Judy Kuo, Qingyan Au, Shijun Zhu, Eric Leones, Flora Sahafi. _NeoGenomics, Aliso Viejo, CA_.

Myeloid cells are the primary recruited effector cells during inflammation. A subset of these, consisting primarily of tumor-associated M1/M2 macrophages (TAMs), tumor-associated neutrophils (TANs), and myeloid-derived suppressor cells (MDSCs), accumulate in tumors where they establish an inflammatory tumor microenvironment (TME) that is favorable for tumor progression (1-3). While these tumor-infiltrating myeloid cells (TIMs) are known to be major regulators of tumor-associated immune-suppression, angiogenesis, metastasis, and resistance to anti-cancer therapy much still remains to be understood about how they function within the TME and how they co-operate with tumor-resident lymphocytes to regulate antitumor immunity. Targeting TIMs to either eliminate them or to convert them from their immune-suppressive to an immune-stimulatory state has emerged as a new strategy complementing current cancer immunotherapy strategies. However, a major impediment to understanding the complexity of the distinct functions of subsets of TIMs and their spatial distribution within the TME is the ability to phenotypically characterize TIMs in FFPE tissues by standard immunohistochemistry, as most IHC studies do not utilize a panel of antibodies broad enough to characterize multiple myeloid cell subsets in the same sample.

We will be using MultiOmyx, a proprietary, multiplexing assay with similar staining characteristics as standard IHC stains but with the significant advantage that up to 60 protein biomarkers can be interrogated from a single FFPE section (4). MultiOmyx protein immunofluorescence (IF) assays utilize a pair of directly conjugated Cyanine dye-labeled (Cy3, Cy5) antibodies per round of staining. Each round of staining is imaged and followed by novel dye inactivation chemistry, enabling repeated rounds of staining and deactivation. Using this MultiOmyx multiplexing assay in combination with a proprietary algorithm that takes into account the staining pattern for each specific biomarker, we will seek to identify different subsets of TIMs and their spatial relationship in tumor tissue from patients with pancreatic ductal adenocarcinoma (PDAC), characterized by an excessive amount of desmoplastic stroma seeded with inflammatory cells.

TIM Panel: CD11b, CD14, CD15, CD16, CD33, CD68, CD163, HLA-DR, Arginase, PANCK.

Human Myeloid Cell Subset Markers: M1 TAMs (CD68+HLA-DR+CD163-), M2 TAMs (CD68+CD163+HLA-DR+), TANs (CD11b+CD15+CD16+HLA-DR+Arginase+), M-MDSCs (CD11b+CD14+CD33+HLA-DRLoCD15-), G-MDSC (CD11b+CD15+CD33+Arginase+HLA-DRLoCD14-).

References

1.De Palma M, Lewis CE. Cancer Cell. 2013;23(3):277-286.

2. Rivera LB et al. Cell Rep. 2015;11(4):577-591.

3. Klemm F, Joyce JA. Trends Cell Biol. 2015;25(4):198-213.

4. Gerdes MJ et al. PNAS. 2013;110:11982-11987.

#2094

Pathology beyond 2D: Imaging and analysis of cells and the tumor microenvironment in three dimensions.

James R. Mansfield,1 James Wainright,1 Christopher Bagnall,2 Geraint Wilde,1 Meredith Price,3 Benjamin E. Willcox,2 Mark Browne1. 1 _Andor Technology, Belfast, United Kingdom;_ 2 _University of Birmingham, Birmingham, United Kingdom;_ 3 _Bitplane, Belfast, United Kingdom_.

The success of cancer immunotherapy treatments and the development of immuno-oncology into the "fifth pillar" of cancer treatment has fueled a rapid growth in interest into methodologies that investigate immune cells in the context of the tumor microenvironment. However, nearly all technologies that investigate the immune contexture of tumors either rely on homogenization or disaggregation of the sample and the concomitant loss of spatial relationships, or rely on a thin section (5 um or less), two-dimensional sample of a biopsy, which is a poor representation of an inherently three-dimensional structure. An image of a thin section will tend to over-estimate the numbers of proximal cells and underestimate the numbers of more distant cells due to sampling error. To address this, we have implemented an imaging system capable of 3D confocal and super-resolution imaging, tiling of large sample areas and imaging samples over time along with software for the visualization and analysis of the datasets.

We present here a series of examples showing the utility of 3D fluorescence imaging of labeled cells in thick (ca. 50 micron) FFPE tissue sections. By using a multimarker immunostaining scheme, this enables the phenotyping of cells within the tumor microenvironment and the calculation of distance metrics between cells in three dimensions. This methodology is an inherently better means of assessing cellular distributions in the tumor microenvironment and can give a much better representation of the complicated 3D structure of a tumor than imaging of thin sections.

#2095

Automated mapping and analysis of stromal cells in tumor microenvironment in pancreatic adenocarcinoma andcholangiocarcinoma using deep learning.

Sunyoung S. Lee,1 Jin Cheon Kim,2 Seongwon Lee,3 Jilliam Dolan,4 Andrew Baird4. 1 _Roswell Park Cancer Institute, Williamsville, NY;_ 2 _ImmunoAI, Republic of Korea;_ 3 _National Institute for Mathematical Sciences, Republic of Korea;_ 4 _University at Buffalo, School of Medicine, Buffalo, NY_.

Objective: A characteristic histologic feature of pancreatic adenocarcinoma (PAD) and cholangiocarcinoma (COL) is extensive desmoplasia (DP) alongside leukocytes and stromal cells (SCs). Advances in mRNA-sequencing have enhanced our understanding of cancer biology in relation to selective changes in expressivity amongst SCs. DP changes secondary to aberrant expression in SCs creates a barrier to absorption and penetration of therapeutic drugs, but few models exist to analyze the spatial and architectural elements composing the complex tumor microenvironment (TME) in association with mRNA levels.

Methods: The histopathology images (H&E stain) and mRNA-seq of 178 PAD and 36 COL patients (pts) were obtained from the Cancer Genome Atlas (TCGA) and analyzed with the deep learning (DL) algorithm, which characterizes histological features in comparison to the corresponding mRNA-seq, allowing for rapid automated analysis of large quantities of data. Ninety genes enriched in leukocytes (CD8+ T cells, B cells, CD4+ regulatory T cells, macrophages, neutrophils, NK cells, and plasmacytoid dendritic cells), 7 genes for cytolytic activities (GZMA, PRF1, GZMH, GZMK, NKG7, CD3E, and CD247), and 5 genes involved with fibroblastic and DP changes (PDFGRA, ACTA2, COL1A1, COL1A2, and PDPN) were analyzed. For each pt, mRNA levels of select genes were analyzed against histologic features, including degree of DP reaction, number of leukocytes, and degree of leukocyte clustering and isolation from tumor cells.

Results: DL analysis demonstrates that the number of fibroblasts and degree of DP correlates with and predicts the mRNA expression of genes associated with fibroblastic and DP changes. The mRNA level of CXCL12 correlates with the degree of leukocyte clustering and spatial isolation in PAD and COL. The 5 genes associated with DP and fibrosis do not have a linear relationship with CXCL12 mRNA levels (R2 <0.1) in COL and (R2=0.2196 to 0.6279) in PAD. Cytolytic activity, measured by the mRNA levels from 7 genes, does not correlate with CXCL12 expression (R2 <0.1) in COL, and (R2=0.3530 to 0.6060) in PAD.

Conclusion: A DL model enables automated analysis and mapping of DP changes within stromal and malignant cells, revealing the spatial and architectural relationship in the TME with varying gene expression. This demonstrates that the degree of leukocyte clustering and isolation from tumor cells correlates with CXCL12 mRNA levels in PAD and COL. CXCL12 expressivity appears to be a contributing factor, limiting access of leukocytes to tumor cells and diminishing an important mechanism combating tumor progression. Varying degrees of DP and cytolytic activities of immune cells within the TME were also observed in association with CXCL12 expression in PAD and COL. Further biomarker-driven prospective studies in the context of immunotherapy and anti-fibrosis are warranted.

#2096

Multicellular modeling and identification of protein ligand-mediated and exosome-mediated crosstalk signaling cascades in the heterogeneous ovarian tumor microenvironment.

Tsz-Lun Yeung,1 Jianting Sheng,2 Samuel C. Mok,1 Stephen T. C. Wong2. 1 _University of Texas MD Anderson Cancer Center, Houston, TX;_ 2 _Houston Methodist Research Institute, Houston, TX_.

Ovarian cancer is the most lethal gynecologic malignancy in the US. While research efforts have been dedicated to identify disease related genes and mutations in cancer cells, the stroma-tumor interactions in the tumor microenvironment and their roles in disease progression is relatively unexplored. The identification of stroma-tumor crosstalk networks with prognostic value presents a unique opportunity for developing new treatment strategies. To capture such an opportunity, a multicellular computational modeling platform, the Cell-Cell Communication Explorer (CCCExplorer), has been developed to identify novel ligand- and exosome-mediated crosstalk networks among different cell types within tumor microenvironment.

With transcriptome profiling data generated from laser microdissected ovarian cancer cells and cancer associated fibroblasts (CAFs) in high-grade serous ovarian caner tissue as inputs, CCCExplorer identified TGF-β–dependent and TGF-β–independent Smad signaling networks as protein ligand-mediated crosstalk signaling cascades activated in CAFs associated with poor patient survival rates. Validation studies by co-culturing ovarian cancer cells with CAFs indicated that activation of Smad signaling in CAFs promoted aggressive phenotypes of ovarian cancer cells while inhibition of Smad signaling in CAFs suppressed ovarian cancer progression both in vitro and in vivo. On the other hand, CCCExplorer used transcriptome data generated from exosomes isolated from CAFs and normal fibroblasts (NFs) as well as from CAF- and NF-derived exosomes treated ovarian cancer cells to compute and predict crosstalk cascades mediated by fibroblast-derived exosomes. Our results showed that CAF-derived exosomal microRNAs and LncRNAs promoted signaling cascade activation and the subsequent increase of chemoresistance, motility and proliferation in ovarian cancer cells. After functional validation of predicted crosstalk cascades, CCCExplorer can further be used to query underlying drug information databases to identify and rank candidate drug agents that could target cancer progression-associated crosstalk between ovarian cancer cells and CAFs.

Our findings demonstrate a powerful tool at the multi-cellular level of heterogeneous tumor microenvironment in which a computational platform can be used to understand, model, visualize, predict, and target crosstalk signaling cascades and coarse-grained behavior of heterogeneous tumor microenvironment. We are applying CCCExplorer to uncover activated crosstalk networks among ovarian cancer cells and stromal cells and to discover drug compounds targeting these pathways, potentially leading to faster cures for ovarian cancer after the validation and confirmation using in vitro and in vivo models.

#2097

Desmoplasia-mimetic gels with modular and temporal properties for modeling pancreatic cancer progression.

Hung-Yi Liu,1 Chien-Chi Lin2. 1 _Purdue University, West Lafayette, IN;_ 2 _Indiana University-Purdue University Indianapolis, Indianapolis, IN_.

Tumor microenvironment (TME) governs all aspects of cancer progression, and in vitro 3D culture platforms are increasingly developed to emulate the interactions between stromal tissues and cancer cells. To mimic the complex compositions and dynamically changing matrix mechanics in TME, we developed a dynamic hydrogel via integrating modular thiol-ene photopolymerization and enzyme-triggered matrix stiffening. Gelatin was dually modified with norbornene (NB) and 4-hydroxyphenylacetic acid (4-HPA). The former renders gelatin photo-crosslinkable and the later affords responsiveness to tyrosinase (TYR)-triggered stiffening. In addition, hyaluronic acid was covalently incorporated into gels. We characterized macromer modification, as well as enzyme-triggered stiffening and degradation. We also evaluated the influence of matrix compositions and dynamic stiffening on pancreatic ductal adenocarcinoma (PDAC) cell in 3D. Methods: Gelatin-norbornene (GelNB) was modified with 4-HPA via carbodiimide chemistry to yield GelNB-HPA. Gels were prepared by reacting NB moieties (GelNB, GelNB-HPA) with thiol motifs on PEG4SH or THA via photopolymerization (1mM LAP, 365nm light 5 mW/cm2, 2 min). Next, gels were swollen for a day, followed by incubating in TYR solution (1 kU/mL) for 6 hours. To study the influences of matrix compositions on PDAC cells, COLO-357 cells were encapsulated in gels, 1) GelNB with PEG4SH (Gel/PEG), 2) GelNB with THA (Gel/HA), 3) GelNB-HPA with PEG4SH (GelHPA/PEG), and 4) GelNB-HPA with THA (GelHPA/HA). Live/dead staining was used to evaluate cell viability and morphology. At day 14, total RNA was extracted from cell-laden gels for mRNA expression analyses using Taqman® array. Results: After TYR treatment, gels with HPA moieties formed additional crosslinks and exhibited higher moduli. Live/dead staining showed high cell viability after cell encapsulation process and throughout 14 days culture. Cells grew into spheroids but were visibly larger in Gel/PEG gels. However, cluster sizes were significantly smaller when the gels were soft and contained HA (i.e., Gel/HA) or stiffened but contained no HA (i.e., GelHPA/PEG). Interestingly, the morphology of the spheroids in GelHPA/HA gels became highly irregular. Results in TaqMan® array revealed changes unique to the presence of HA, to a stiffened microenvironment, or to the combination of both. We characterized mRNA expression from cells in GelHPA/HA gels. Results showed that these cells underwent epithelial-mesenchymal transition (EMT), as evidenced by downregulation of E-cadherin and upregulation of mesenchymal markers as N-cadherin, SNAIL-1 and vimentin. In sum, we designed a biomimetic hydrogel capable of mimicking the diverse biochemical compositions and dynamic biophysical environment of pancreatic desmoplasia, providing a diverse platform for studying pancreatic cancer.

#2098

Quantitative modeling as a systematic approach for drug combination evaluation in immuno-oncology (IO).

Gabriel Helmlinger,1 Yuri Kosinsky,2 Lulu Chu,1 Kirill Peskov,2 Veronika Voronova,2 Alexandra Borodovsky,1 Richard Woessner,1 Kris Sachsenmeier,1 Nidal Al-huniti1. 1 _AstraZeneca R &D Boston, Waltham, MA; _2 _M &S Decisions, Moscow, Russian Federation_.

Objectives: Multiple strategies for eliciting and enhancing antitumor immunity are currently being evaluated. However, a more systematic approach is needed, to analyze and translate such results into clinic practice, while rationally designing combination therapies based on mechanistic understanding of potential synergistic effects (1). The objective of this study was to provide predictive simulations, via a quantitative systems pharmacology (QSP) model, capable of categorizing the types of synergistic effects that may arise from IO agent combinations, across realistic baseline conditions prevailing in the tumor microenvironment (TME).

Methods: The QSP model was developed and qualified using in vivo mouse data published in the literature and from internal research. The following pharmacologic modalities were calibrated: PD-L1/PD-1, CTLA-4, CXCR2, A2AR inhibition, and OX40 agonism. Various combination scenarios were simulated for these modalities, at four baseline conditions prevailing in different syngeneic murine models.

Results: Simulated efficacy results were highly dependent on the baseline conditions. Several combinations and monotherapies were effective only within a specific baseline TME phenotype. These findings were in agreement with experimental data (2). At baselines with higher levels of MDSC, best results were obtained for a PD-L1 mAb combined with either an OX40 agonist or a CXCR2 inhibitor, with 90% of complete responders. Anti (PD-L1 + CTLA-4) combinations showed high efficacy in Treg prevalence, but only moderate efficacy (22% complete responders), under baseline conditions of a dual (Treg + MDSC) immunosuppressive TME.

Conclusion: This work provides a quantitative modeling framework to comparatively predict responses to IO combinations, based on realistic baseline conditions prevailing in the TME, while revealing mechanistic interactions underlying such responses in IO combinations.

References:

1. Melero I et al. Nat Rev Cancer 2015;15:457-72.

2. Mosely S et al. Cancer Immunol Res 2016;5:29-41.

#2099

Multi-spatial whole-lesion molecular heterogeneity analyses of an immunotherapy-resistant metastatic melanoma.

Akash Mitra,1 Whijae Roh,2 Alexandre Reuben,1 Jennifer Wargo,1 Alexandar Lazar,1 Philip A. Futural1. 1 _The University of Texas MD Anderson Cancer Center, Houston, TX;_ 2 _The Broad Institute, Cambridge, MA_.

Genomic and immune intratumor heterogeneity (ITH) can be a significant obstacle to treating patients with checkpoint blockade immunotherapy. The extent of ITH within a single lesion has begun to be addressed but there have been no comprehensive molecular analyses of an entire lesion, multiply sampled in three dimensions with reconstruction of the tumor in its host microenvironment reported thus far. This approach can allow a portrait of the entire tumor ecosystem to emerge and provide a richer, more comprehensive view of the interplay of microenvironment and tumor not bound by a single planar view of a complex entity. Here, we report on our analyses of an anti-CTLA4/anti-PD1 resistant metastatic lesion in toto with an attendant comparative longitudinal view of pre-immunotherapy lesion.

We performed 3-D reconstruction of metastasis obtained at palliative surgery from a patient progressing on anti-PD1 in the context of prior anti-CTLA4 therapy utilizing combined genomic (gene expression profiling, methylation, whole exome sequencing and neoantigen prediction) and immune (immunohistochemistry, RNA- and DNA- based T cell receptor sequencing) profiling of 67 distinct sampled regions from the entire lesion.

Limited point-mutation heterogeneity was found in melanoma driver genes across regions of metastasis. However, copy number alteration analysis revealed changes over time and space with gain of chromosome 7, loss of chromosome 10, and gain of chromosome 14 in certain regions. Differences in immune signatures were observed across regions of a tumor with pockets of immune activation and suppression throughout the metastasis. TCR profiling revealed the dominance of two distinct T cell clonotypes across different regions of the same met, suggesting spatial differences in tumor immunogenicity and T cell activation status. Neoantigen prediction and expression analyses were performed, revealing persistence of a predicted neoantigen detectable prior to CTLA-4 therapy, enriched in different regions of the tumor post-PD-1 blockade. Gene expression of this potential neo-antigen correlated with the distribution and dominance of a specific T cell subclone, suggesting a potential path of infiltration and migration of T cells through the lesion.

Our findings shed light on the three-dimensional genomic and immune landscape in metastatic melanoma in the context of treatment and resistance to immune checkpoint blockade. In particular, even when essentially "gated" on a relatively monotonous mutation landscape, immune intra-tumor heterogeneity can be substantial. This suggests that as immune markers move into the mainstream for use as biomarkers, the use of single biopsies to inform treatment choice may be confounded. These data further impress the need for comprehensive, integrated molecular phenotyping approaches to unravel immunotherapy response and resistance in metastatic melanoma.

#2100

**Using** in vitro **models to identify newly synthesised proteins involved in the progression of breast cancer.**

Amira F. Mahdi,1 Beatrice Malacrida,1 Kieran McGourty,1 Aoife J. Lowery,2 Patrick A. Kiely1. 1 _University of Limerick, Limerick, Ireland;_ 2 _National University of Ireland, Galway, Galway, Ireland_.

Triple negative breast cancer (TNBC) is a distinct subtype of breast cancer defined by tumours deficient in the expression of the three main immuno-histochemical markers: the estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth receptor 2 (HER2). Although accounting for only 15-20% of total breast cancer, TNBC is characterized by an aggressive phenotype and poor prognosis. As a result, it is the cause of a disproportionate amount of breast cancer deaths, with a higher incidence amongst younger women and ethnic minorities. In addition, lack of ER, PR and HER2 expression means there are no specific targeted therapies available. Significant efforts are being made to improve detection and survival rates for women affected by this disease. In order to achieve this, there is an urgent need to identify novel biomarkers and molecular targets so that we can more quickly and accurately diagnose breast cancer and develop a better understanding of disease progression in patients diagnosed with TNBC.

Genomic studies of patients with TNBC have shown that there is strong heterogeneity within the sub-type and a need for more precise classification at a molecular level. One target that has emerged from these studies is the epidermal growth factor receptor (EGFR). The EGFR is found to be over expressed in approximately 50% of TNBC cases and amplification of the EGFR gene is correlated with poor prognosis. Our study aims to investigate exactly how the progression of TNBC is influenced by specific micro-environmental cues and identify novel targets involved in the growth and migration of tumour cells.

We are investigating this target by developing cell culture based, in vitro models of EGFR driven cancer metastasis. Using Click-iT chemistry and mass spectrometry analysis, and by culturing the cells in both 2D and 3D in vitro models, we have identified proteins that are newly synthesised by the cancer cells as they are stimulated to migrate towards epidermal growth factor (EGF). Our approach has generated a list of proteins that span a myriad of different functions known to be important in cancer progression such as cell metabolism, intracellular calcium sensing, anti-oxidation and regulation of cell structure. We hypothesise that these newly synthesised proteins play a vital role in cancer cell migration and metastasis. Interaction and molecular function analysis of this list using STRING has shown an enrichment in calcium ion binding proteins. We are particularly interested the role of these calcium-regulated proteins in influencing the structure and behaviour of migrating cells. Currently, we are investigating the role of several of these proteins in regulating the cells response to the tumour microenvironment.

Directing pharmacological interventions towards these newly synthesised proteins has the potential to target invasive cancer cells and provide a highly specific and effective therapy for TNBC.

#2101

The growth of a xenograft breast cancer tumor model with engineered hyaluronan-accumulating stroma is dependent on hyaluronan and independent of CD44.

Chunmei Zhao,1 Benjamin J. Thompson,1 Kelly Chen,1 Mathieu Marella,2 Susan Zimmerman,1 Trevor Kimbler,1 Barbara Blouw,1 Sheryl Garrovillo,1 Lei Huang,1 Adrian Radi,1 Zhongdong Huang,2 H. Michael Shepard,2 Sanna Rosengren,2 Christopher D. Thanos,2 Daniel C. Maneval1. 1 _Halozyme Therapeutics, San Diego, CA;_ 2 _Formerly of Halozyme Therapeutics, San Diego, CA_.

The abnormal accumulation of hyaluronan in the solid tumor microenvironment is associated with poor prognosis in several human cancers. Hyaluronan often accumulates in the tumor stroma. To understand the role of hyaluronan-accumulating stroma in tumor progression, we engineered a hyaluronan-producing fibroblast cell line 3T3HAS3 by lentiviral transduction of the human hyaluronan synthase 3 gene into Balb/c 3T3 cells. The 3T3HAS3 cells produced significant amounts of hyaluronan in vitro, which bind to the cell surface of the human breast cancer cell line MDA-MB-468. When co-grafted with MDA-MB-468 cells in nude mice, 3T3HAS3 significantly enhanced tumor growth. Immunohistochemical analysis of tumor xenografts showed that MDA-MB-468 cells were surrounded by hyaluronan-accumulating stroma, closely resembling the morphology observed in human breast cancer specimens. Tumor growth of this co-graft model required hyaluronan production from 3T3HAS3 cells, as demonstrated by the delayed tumor growth upon hyaluronan removal by expression of the human PH20 gene in 3T3HAS3 cells, or by systemic administration of PEGylated recombinant human PH20 (PEGPH20). In contrast, neither the expression of CD44, a well-characterized hyaluronan receptor, in tumor and stromal fibroblasts, nor the binding of hyaluronan to CD44 in MDA-MB-468 tumor cells was essential for tumor growth. Small scale screening of signaling changes in xenograft tumors suggested that the AMPK/mTOR pathway may respond to hyaluronan removal by PEGPH20. Collectively, these data demonstrate that the growth of an engineered breast cancer xenograft model with hyaluronan-accumulating stroma is dependent on hyaluronan, and that hyaluronan-CD44 interaction may not be the main mechanism through which hyaluronan promotes tumor progression in certain tumors.

#2102

UFH-001: A novel triple-negative, CAIX-positive cell line for small-molecule targeting of human breast cancer.

Mam Y. Mboge,1 Zhijuan Chen,1 Lingbao Ai,1 Chingkuang Tu,1 Fabrizio Carta,2 Claudiu Supuran,2 Christopher J. Frost,3 Zaihui Zhang,4 Robert McKenna,1 Coy Heldermon,1 Susan C. Frost1. 1 _Univ. of Florida, Gainesville, FL;_ 2 _University of Florence, Florence, Italy;_ 3 _University of Louisville, Louisville, KY;_ 4 _Signalchem Lifesciences Corp, Richmond, British Columbia, Canada_.

Specific treatment options for patients with metastatic breast cancers, especially of the triple negative subtype (TNBC), are limited and most patients develop resistance to radiation and/or chemotherapy. Because of this, the mortality rate in women with TNBC remains high. Carbonic anhydrases (CAs), most specifically membrane-bound CAIX, has been shown to play an important role in tumor progression and as a result is validated as a therapeutic target in several aggressive cancers. In breast cancer, CAIX expression has been shown to be ubiquitous in hypoxic TNBC tissues and is considered a prognostic marker and a potential therapeutic target. Therefore targeting CAIX activity, using small molecule inhibitors, in these highly aggressive and metastatic TNBCs may serve to improve overall disease free survival and therapeutic outcome in clinics. Our goal for this study was to 1) Identify and/or develop a cell line that expresses CAIX in a hypoxia independent manner and is representative of TNBCs and 2) To target CAIX activity in this cell line and observe the effects of CAIX inhibition on its growth and metastasis. Our hypothesis is that CAIX inhibition, in the context of a hypoxic and/or acidic microenvironment, will dysregulate its ability to maintain the acidic pH preferred by cancer cells which favors their growth and migration. To achieve our aims, we newly developed and characterized an endogenous CAIX-expressing cell line named UFH-001. Our results show that CAIX expression and activity in this cell line, although endogenous under normoxic conditions, is increased more than 2 fold under hypoxic conditions. This cell line is also representative of TNBCs (ER/PR and HER2 negative) with a more epithelial-like morphology and phenotype. UFH-001 cells grow aggressively and have the ability to migrate/invade and form tumors in vivo. In these studies we have also shown how inhibition of CAIX activity, using sulfonamide-based inhibitors, affects UFH-001 growth in an apoptosis independent manner and decrease the migratory/inhibitory capacity of these cells. Taken together, our observations indicate that CAIX is a viable small molecular drug target for the treatment of patients with TNBCs.

#2103

Acoustic activation of the glioma-brain microenvironment.

Pavlos Anastasiadis,1 Kenisha Younger,1 Nathan B. Roberts,1 Joseph A. Frank,2 Victor Frenkel,1 Eduardo Davila,1 Graeme Woodworth1. 1 _University of Maryland School of Medicine, Baltimore, MD;_ 2 _National Institutes of Health, Baltimore, MD_.

Introduction

Glioblastoma (GBM) is a malignant brain tumor characterized by diffuse brain invasion, molecular and cellular heterogeneity, and immune evasion, making current chemo-radiation treatments and emerging immunotherapies minimally effective. Transcranial MRI-guided focused ultrasound (MRgFUS) is a novel therapeutic modality, enabling the safe and targeted application of ultrasound energy in the brain. Recently, we demonstrated how MRgFUS can be used to transiently disrupt the blood-brain barrier (BBB) for enhanced delivery of stem cells and locally administered nanoparticles. In addition to creating structural effects, MRgFUS exposures are also capable of generating effects at the molecular level by modulating the levels of cytokines, chemokines and trophic factors in a tempo-spatially controlled manner. In the current study, we investigated the effects of MRgFUS on the GBM immune microenvironment.

Materials and Methods

C57BL/6 mice were implanted with luciferase expressing mouse GL261 glioma cells in the right striatum. MRgFUS exposures were applied using an MRI-guided system. Tumor, spleen, and superficial lymph nodes were collected seven days after MRgFUS treatment. Flow cytometry and immunohistochemistry were performed for the following antibodies: CD45-PE, CD3-FITC, CD8-PErCp/Cy5.5, TIM3-BV605, PD-1-PacBlue, CD4-APC, Nk1.1-FITC, Cd11b-Percp/Cy5.5, CD11c-B650, CD86-APC, GR1-PacBlue, FoxP3-PE, and CD25-APC. Statistical analysis of the data was performed using unpaired T-tests.

Results

MRgFUS treatment of tumors resulted in a statistically significant increase in the percentage of Foxp3+ CD25+ T-regulatory cells (Tregs) in the spleen compared to untreated controls. Analysis of superficial cervical lymph nodes following MRgFUS treatment revealed that myeloid-derived suppressor cells (MDSCs) were significantly increased while CD8+ T cells were significantly decreased in MRgFUS-treated tumors compared to untreated controls.

Conclusions

These findings suggest that MRgFUS treatment of intracranial GBM tumors results in the redistribution of key immune cell types within the tumor microenvironment as well as the body lymphoid tissues. Specifically, we found that Tregs and MDSCs moved away from the tumor, and CD8+ T cells moved into the tumor at 1 week post-treatment. This suggests that MRgFUS has the potential to awaken the suppressed GBM microenvironment and augment immunotherapy in brain cancer. Additional studies to help elucidate the underlying mechanisms and further evaluate the potential of this approach are ongoing.

#2104

Near-infrared fluorescent tumor histology for multiplexed phenotypic identification of immunosuppressive cells.

Amy Flor,1 Annalise Vaccarello,1 Matt Levin,2 Helen Snyder,2 David Schwartz,2 Stephen Kron1. 1 _University of Chicago, Chicago, IL;_ 2 _Cell IDx, San Diego, CA_.

The purpose of our study was to develop near-infrared fluorophore-labeled antibodies to overcome autofluorescence interference in the visible spectrum, thereby enabling enhanced multiplexed identification of immune cells in human tumor tissue sections. Towards multiplexing, we conjugated each monoclonal primary antibody to a unique hapten tag. Detection of hapten-tagged primary antibodies was accomplished using high-affinity anti-hapten rabbit monoclonal secondary antibodies, which were each conjugated to a near-infrared fluorophore with Emax = 594, 650, 731, or 800 nm. For identification of immunosuppressive cells in tumor tissues, we used formalin-fixed, paraffin-embedded human tissue microarrays comprising malignant breast or prostate tumor core sections. Microarrays contained 12-60 patient biopsy cores with duplicate sections per case mounted on standard glass slides. Two antibody panels to detect immunosuppressive cell phenotypes were used in our study. The T regulatory panel targeted the antigens CD4, CD8, FOXP3, and LAG-3, allowing for phenotypic identification of CD4+ T helper cells, CD8+ cytotoxic T cells, CD4+ FOXP3+ Tregs, and highly immunosuppressive CD4+ FOXP3+ LAG-3+ Tregs. The macrophage panel targeted the antigens CD3, CD20, CD68, and CD163 for identification of CD3+ T cells, CD20+ B cells, CD68+ M1 macrophages, and highly immunosuppressive CD163+ M2 macrophages. Widefield fluorescence microscopy was used to image the stained tumor microarrays, utilizing a high-radiance mercury arc lamp, high-sensitivity digital CMOS camera, and single-band optical filter sets to detect near-infrared fluorescence. Triplicate images were taken per tissue core. Prior to image analysis, a spectral unmixing algorithm was created using a reference set of single-stained tissue slides in order to deconvolute minor spectral overlap of near-infrared fluorophores. The algorithm was then applied to the multiplexed digital raw images, and immunophenotyping analysis was conducted to enumerate cells of interest. Image analysis results indicated unique patterns of distribution of immune cells in tumor tissues correlated with tumor grade and stage. In breast cancer tissues, higher levels of LAG-3+ immunosuppressive cells were found in tumors of the aggressive, treatment-resistant "triple-negative" tumor subtype. In prostate cancer tissues, an increase in CD68+ macrophages and loss of immunostimulatory CD3+ T cells and CD20+ B cells were found to be associated with advanced tumor grade and Gleason score. The use of near-infrared fluorophores and hapten-tagged detection antibodies facilitated enhanced, rapid multiplexed phenotyping of immunosuppressive cells in tumor tissue. These findings demonstrate our approach to be a potentially paradigm-shifting technology for detection of immune infiltrates in patient samples.

#2106

Focal gene expression profiling of counterstained FFPE with correlation to morphology using TempO-Seq.

Elliot Imler,1 Milos Babic,2 Deanna Adams,1 Peter Shepard,2 Joanne Yeakley,2 Raymond B. Nagle,3 Bruce Seligmann1. 1 _BioSpyder Technologies, Inc., Tucson, AZ;_ 2 _BioSpyder Technologies, Inc., Carlsbad, CA;_ 3 _Univ. of Arizona Cancer Ctr., Tucson, AZ_.

We previously reported on use of the whole transcriptome TempO-Seq® assay to profile gene expression from 1 mm2 focal areas of 5 µm thick FFPE sections of normal and cancerous tissue to identify disease biomarkers and mechanistic pathways. We are now presenting data demonstrating that the TempO-Seq assay can be performed as an "in situ" assay on slides by an automated slide stainer, followed by counterstaining. Then, using an automated digital imaging platform, areas as small as 30 μm in diameter within the FFPE section can be profiled, permitting the gene expression data to be correlated directly to the specific morphology of that focal area. The pathologist uses a computer interface to select areas for profiling during the course of the histologic examination of the section, and then the TempO-Seq probes are automatically recovered from those region(s) of interest. After depositing them into PCR tubes, the assay is completed with PCR amplification and sample barcoding before pooling the samples into a library for sequencing. Analysis of the sequencing data is carried out automatically to report results. There is no use of laser capture or destruction of the tissue. The TempO-Seq processed slides can be archived and additional areas may be sampled at a later date. In this study, we sample replicate areas of matched normal vs. cancerous tissue, measuring gene biomarkers of clinical utility, gene fusions, and SNPs. We present gene expression profiles for replicate areas of pure stroma, single normal glands, single high-grade PIN glands, and single cancerous glands from prostate FFPE, demonstrating the statistical power of the TempO-Seq in situ assay as well as the correlation to focal histology and the use of focal differential expression to elucidate molecular pathways involved in the transition from normal tissue to cancer. We report measurements of clinically relevant biomarkers and signatures from clinical FFPE samples, plus therapeutic targets and drug resistance genes. This new approach demonstrates that complex molecular tests can be carried out by any pathologist in their own lab, and makes moot the issues of "% cancer" and amount of tissue required for testing. The approach we present brings extraction-free complex molecular testing of FFPE into the pathology lab and takes molecular pathology to a new level of simplicity, focal precision and correlation to morphology.

#2107

A novel real-time cell imaging assay to quantify macrophage efferocytosis.

Daniel Bravo, Jianyong Wang, Yongchang Shi. _Genentech, Redwood City, CA_.

Tumor-associated macrophages (TAMs) are found in the microenvironment of solid tumors, representing one of the most abundant immune cell types within tumor stroma. Studies have demonstrated that the presence of TAMs correlates with tumor progression and poor prognosis. The engulfment of apoptotic tumor cells by TAMs (a phagocytic process known as efferocytosis) results in clearance of neoplastic antigens, which prevents antigen presentation and activation of effector T cells. Additionally, it has been shown that TAMs produce cytokines that create an immunosuppressive environment that facilitates tumor evasion of immune surveillance and promotes tumor growth. Furthermore, TAMs have been reported to be key players in the promotion of tumor angiogenesis and metastasis. Since TAMs play such an important role in cancer progression, inhibition of their function in the tumor microenvironment becomes an attractive approach of cancer immunotherapy. Traditionally, quantifying efferocytosis in vitro has been a technically challenging task. Our lab has established a novel high-throughput assay that can be used to screen drug candidates that inhibit TAM efferocytosis. Our new assay uses the IncuCyte ZOOM real-time imaging platform to visualize macrophage efferocytosis of apoptotic cells that are labeled with a pH-sensitive probe (pHrodo) that only emits fluorescence in acidic enviroments. This technology enables us to quantify phagocytic events of apoptotic cells that have been processed into the phagolysosome. By using this method, we quantitatively characterized the efferocytosis activity of TAM-like macrophages that were differentiated from primary human monocytes. We tested several agents (including small-molecule compounds and antibodies) and quantified their activities in blocking efferocytosis of TAMs via IC50 concentrations and potency. This new high-throughput assay platform yields highly robust and reproducible data (Z'=0.69), is fully automated, noninvasive (no washing, fixing or lifting of cells), and works with low cell numbers. Our results show that our efferocytosis assay facilitates high-throughput functional screening and can be utilized to identify and characterize new cancer drug candidates, as well as enables research of efferocytosis mechanisms and functions of phagocytic cells.

#2108

Detection and molecular profiling of leptomeningeal disease in melanoma.

Inna Smalley, Brittany Evernden, Vincent Law, Rajappa Kenchappa, John Puskas, Elena Ryzhova, Nam Tran, Arnold Etame, Solmaz Sahebjam, Anthony Magliocco, Peter Forsyth, Keiran S. Smalley. _Moffitt Cancer Center, Tampa, FL_.

Approximately 5% of melanoma patients develop leptomeningeal disease (LMDz), a highly symptomatic complication with a dismal survival of 8-10 weeks. The aim of this study was to determine whether diagnosis and personalized treatment for melanoma-LMDz could be improved by assessing patient-derived cerebrospinal fluid (CSF) specimens. Circulating tumor cells in CSF (CSF-CTCs) were detected by Veridex CellSearch® System and the circulating melanoma cell kit, based on anti-CD146 and anti-high molecular weight melanoma associated antigen (HMW-MAA-PE (MEL-PE)). Of the 12 patients with definitive LMDz diagnosis, all but 1 patient (92%) had CSF-CTCs (range 23-3055 CTCs/ml). In contrast, only 3/8 (37%) melanoma patients without LMDz diagnosis had CSF-CTCs detected, with significantly lower CTC counts per ml CSF (range 0.13-0.6 CTCs/ml). Ex vivo studies of CSF uncovered that although patient-derived CSF does not appear to act as a chemo-attractant or a stimulant of invasion to initiate melanoma migration to the leptomeninges, it significantly reduced the ability of BRAF inhibitors to induce apoptosis in established melanoma cell lines (p<0.005). Patient-derived CSF composition is complex but includes many growth factors and mediators of adhesion and TGFβ signaling. In melanoma cells, the CSF stimulated signaling through mTOR/AKT and STAT. BRAF inhibition amplified these signals further. We show that ex vivo expansion of isolated CSF-CTCs is possible for ~25% of samples, in the presence of FBS, FGF and EGF. At this time, there is a very limited understanding of the mechanisms underlying melanoma metastasis to the leptomeninges, a truly devastating and rapidly terminal complication of melanoma. This is the first report of patient-derived CSF eliciting a protective effect in melanoma cells, suggesting melanoma cells find a protective niche in the central nervous system. Furthermore, we demonstrate that patient-derived CSF biopsies serve as a critical tool for improving diagnosis and personalized treatment for melanoma-LMDz patients.

#2109

Prognostic and functional significance of interleukin 22 in colorectal cancer.

Eleonora Cremonesi, Nadia Tosti, Francesca Amicarella, Benjamin Weixler, Silvio Däster, Valeria Governa, Giulio C. Spagnoli, Luigi Terracciano, Luigi Tornillo, Serenella Eppenberger-Castori, Giandomenica Iezzi, Raoul Droeser. _University Hospital of Basel, Basel, Switzerland_.

T cell infiltration has been recognized to significantly impact clinical outcome in human colorectal cancer (CRC). Interleukin 22 (IL-22), a cytokine secreted by IL-17-producing CD4+ T cells (Th17) is known to play a crucial role in inflammatory bowel disease and has been shown to have protumorigenic activity in mouse tumor models. However, its role in human CRC is still unclear. In a previous study we could demonstrate the dual role of Th17 in CRC. As consequence we evaluated the prognostic and functional impact of IL-22 producing cells in human CRC in this follow-up study. Upon staining of a tissue microarray (TMA), including 423 CRC cases, IL-22 positive cells were detected both within tumor cells and tumor infiltrating immune cells. Whereas, IL-22 expression by tumor cells did not impact on prognosis, densities of IL-22 positive immune cells were found to be significantly associated with early T stage and MMR-deficient microsatellite stability. Importantly, IL-22 expression by CRC infiltrating immune cells was predictive of improved overall survival independent of known prognostic factors such as T stage, N stage, tumor grade, vascular invasion, tumor border configuration and MMR status. Phenotypic characterization of IL-22 positive cells performed by flow cytometry revealed that they consist mainly of Th17 cells, also producing IL-17. In vitro experiments showed no direct effect of IL-22 on CRC cell proliferation. On-going studies are validating the prognostic effect on a TMA cohort with corresponding transcriptomic data and evaluating potential effects mediated by IL-22 on other cell types of CRC microenvironment.

#2110

Therapeutic intervention for pancreatic cancer using autologous exosomes.

Saini Setua, Sheema Khan, Murali Yallapu, Sonam Kumari, Meena Jaggi, Subhash C. Chauhan. _Univ. of Tennessee Health Science Center, Memphis, TN_.

Objective: Pancreatic cancer (PanCa) is the third deadliest cancer in United States with a poor survival rate. Despite extensive research efforts, there is not any substantial progress in cancer therapeutics due to impediments against intracellular drug delivery. Thus, novel delivery vehicles are required that are biocompatible and non-immunogenic. This is possible by utilizing an autologous biological material as delivery vehicles that can be applied as a personalized medicine. Towards this, our lab has optimized an exosomes based therapeutic approach, which utilizes exosomes isolated from the cultured tumor adjacent normal (NAT) fibroblast cells. We utilized this scaffold for safe and effective delivery of therapeutic payload. Methods: Following surgical resection, freshly collected, matched tumor adjacent normal tissues were utilized to isolate fibroblasts. Exosomes were purified from the isolated fibroblasts in presence of exosome-depleted FBS media. Physico-chemical characterization (DLS, TEM), presence of exosomal marker (CD63; immunoblotting), protein concentration (Bradford assay), cellular internalization (confocal microscopy) and miR-145 expression (qRT-PCR) in the exosomes were performed. Efficacy of exosomes to deliver therapeutics was investigated utilizing our recently identified drug, ormeloxifene (ORM). Drug encapsulation and loading efficiency was performed using UPLC. Functional assays, such as, proliferation were performed using clinically relevant PanCa cell lines (HPAF-II/AsPC-1). Results: NAT derived exosomes show effective size and zeta potential (size: 44.12 ± 0.89; Zeta potential: -14.9 mV), which is ideal for drug delivery purposes. The purification of exosomes was confirmed by analyzing the expression of exosomal markers, such as CD63. Immunofluorescence for CD63 expression confirmed the efficient delivery of exosomes in PanCa cells. Our results indicated high drug loading capacity of NAT derived exosomes as demonstrated though UPLC. ORM loaded exosome treatment efficiently delivered ORM into the cancer cells and inhibited tumorigenic features such as, proliferation of PanCa cells. Additionally, NAT derived exosomes showed enhanced expression of tumor suppressor microRNA, miR-145, suggestive of their therapeutic importance. We observed restoration of lost miR-145 levels in PanCa cells on incubation with NAT derived exosomes. This further indicates relevance for their utilization in the development of novel therapeutic modalities. Conclusion: Our observations offer importance of the utilization of NAT derived exosomes for personalized medicine as a therapeutic strategy in PanCa treatment.

#2111

The tumor microenvironment of preclinical tumor models may have an impact on the therapeutic index of nanotherapeutics.

Stephane Ferretti, Nicolau Beckmann, laura Holzer, Michael Obrecht, Marjorie Berger, Michael Rugaard Jensen. _Novartis Institutes for BioMedical Research, Basel, Switzerland_.

Rapid development in nanotechnology allows the incorporation of multiple therapeutic agents (e.g. liposomes) into nanoparticle with a size range from 1 to 1000 nm. These nanocarrier systems provide new approaches to treat aggressive malignancy. Nano-based delivery systems hold an advantage over traditional small molecule therapy in that they can deliver drugs preferentially to tumors due to the enhanced permeability and retention (EPR) effect. On one hand, high permeability of the tumor vessels and a lack of functional lymphatic vessels results in the EPR effect, driving nanoparticle extravasation. On the other hand, these phenomena lead to high interstitial fluid pressure (IFP), limiting nanoparticle extravasation. In this study, we have used dynamic-contrast enhanced (DCE) MRI with small (Dotarem, 5 nm) and large (Vistarem, 25 nm) contrast agents (CAs) to examine tumor vessel permeability in SJSA-1 osteosarcoma tumor-bearing rats. An inversion-recovery true fast imaging with steady state precession (TrueFISP) was employed to determine the transfer constant Ktrans in subcutaneous and intra-tibia tumor models. When the tumor cells were injected in both niches within the same animal, Ktrans was significantly lower in intra-tibia tumors with both CAs, and blood vessels significantly less and larger compared to subcutaneous tumors. Moreover, Ktrans negatively correlated with tumor bioluminescence for the intra-tibia model, suggesting that EPR decreased inversely with tumor size, while the opposite was noticed for the subcutaneous tumors. These data demonstrate for the first time that the EPR of tumors with a similar size within the same animal can be influenced by the microenvironment. Moreover, they show that the therapeutic index of a nanotherapeutics may be different if it's determined with either an orthotopic or ectopic model.

#2112

Cell by cell immuno- and cancer marker profiling for non-small cell lung cancer (NSCLC) tissue sample using non-enzymatic tissue dissociation and high-parameter flow cytometry.

Xiaoyang Wang,1 Pin-I Chen,1 Maria Jaimes,2 Huimin Gu,2 Keith Shults,1 Fariba Fazeli,1 Janine Fernandez,1 Vishal Sharma,3 Kalyan Handique,3 Bruce K. Patterson1. 1 _Incell Dx Inc, Menlo Park, CA;_ 2 _Cytek Biosciences Inc., Fremont, CA;_ 3 _Celsee Inc, Plymouth, MI_.

Non-small cell lung cancer (NSCLC) is the most common type of lung cancer, accounting for 80-85% cases of lung cancer. It has poor prognosis as most NSCLC patients are at advanced stage when diagnosed. The development of immunotherapy blocking the PD-1/PD-L1 ligand pathway, in recent years, has shed light on new treatment for lung cancer. An in-depth understanding of the interaction between immune system and tumor will help to identify novel markers for lung cancer treatment.

In this study, we performed a comprehensive study on 10 NSCLC patient tissue samples with paired blood samples for circulating tumor cells (CTCs). The solid tissue biopsy samples were dissociated into single cells by non-enzymatic tissue homogenization (IncellDx IncellPREPTM). The single cell suspensions were stained simultaneously with multiple (>20) immune check point markers (including PD-1*, PD-L1*, TIM-3*, LAG-3*, and CTLA-4*), cancer markers (such as EGFR* and ALK* fusion protein), and a cell cycle dye. The samples were interrogated on a novel, innovative, high parameter, spectral flow cytometer Cytek AuroraTM. Markers on subsets of immune cell and cancer cell populations were investigated.

Our results showed the association of high levels of immune check point marker and cancer marker expressions with the high level of aneuploidy (indicated by DNA index >1.05), and the aggressiveness of the cancer (indicated by the number of CTCs). High numbers of CTCs were associated with aneuploidy, increased Post G0-G1%, and high expression of LAG-3, TIM-3, and PD-1.

Multi-parametric flow cytometry allows simultaneous profiling of multiple immune and cancer markers on cancer samples at the single cell level. The knowledge acquired from these studies will enhance our understanding of cancer immune system, cancer cells, and the interaction between immune system and the cancer. It will potentially transform patient diagnosis, disease monitoring, and drug discovery. Our study demonstrated a powerful method to study solid tumors that may provide important information for successful precision cancer immunotherapy.

*PD-1: programmed death-1; *PD-L1: programmed death-ligand 1; *TIM-3: mucin domain-3-containing molecule-3; *LAG-3: lymphocyte-activation gene-3; *CTLA-4: cytotoxic T-lymphocyte antigen-4; *EGFR: epidermal growth factor receptor; *ALK: anaplastic lymphoma kinase

#2113

The effects of enzymatic digestion on epitope detection by flow cytometry.

Aaron J. Middlebrook,1 Caitlin Austin,1 Daniel Santos,1 Eileen Snowden,2 Warren Porter,2 Friedrich Hahn,2 Mitchell Ferguson,2 Rainer Blaesius,2 Smita Ghanekar1. 1 _BD Biosciences, San Jose, CA;_ 2 _BD Technologies, Raleigh-Durham, CA_.

The heterogeneous nature of solid tumors, coupled with the relatively small sample size of available biopsies, has led to an emerging need to glean as much information as possible from these valuable specimens. Current approaches to solid tumor analysis fail to completely reveal the diverse range of cellular compartments that comprise the tumor microenvironment. A comprehensive approach to tumor interrogation requires efficient tissue dissociation to facilitate analysis at the single-cell level. In contrast to current methods, single-cell analysis of tumor derived cell suspensions by flow cytometry has the potential to provide a more complete understanding of the many subpopulations within the tumor microenvironment and the cell to cell interactions that govern this space. In order to prepare a cell suspension from solid tumor tissue, most investigators subject the tumor sample to a combination of mechanical and enzymatic dissociation modalities. Regardless of the combination of enzymes used for dissociation, there exists a degree of non-specific proteolytic cleavage. In the work presented here, we set out to quantify the non-specific cleavage associated with a proprietary enzyme mixture developed for tumor dissociation. Using a mixed sample that included peripheral blood mononuclear cells and several cell lines, we were able to generate a sample that expressed approximately 90% of our current antibody catalog. By staining our sample with a rudimentary 3 color panel, we were able to identify 10 individual cell populations based on marker expression and/or scatter. This composite sample was left untreated or exposed to our propriety mixture of dissociation enzymes and then subsequently screened for all 251 antigens in our catalog. The impact of non-specific proteolytic cleavage on surface marker detection was gauged in terms of median fluorescence intensity of positive population and stain index. Our results indicate that of the 251 surface markers tested in our experimental series, less than 10 percent were significantly affected by enzymatic digestion. Of those markers that were affected only a fraction of them were affected to such an extent that it was impossible to detect positive expression. These results underscore the importance of understanding the factors that can influence surface marker detection and bolster our confidence that this particular mixture of enzymes is imparting minimal impact on surface marker detection.

#2114

3D co-culture of pancreatic tumor sphroids and stellate cells using minipillar array as a microtumor model of pancreatic ductal adenocarcinoma.

Hyun Ju Hwang, Min-Suk Oh, Seul-Ki Kim, Hyo-Jeong Kuh. _Collage of Medicine, The Catholic Univ. of Korea, Seoul, Republic of Korea_.

Purpose: Tumor microenvironmental (TME) factors play important roles in cancer progression and chemoresistance by inducing epithelial-mesenchymal transition (EMT) and related mechanisms. Pancreatic stellate cell (PSC) is a highly-enriched cell type in TME of pancreatic ductal adenocarcinoma (PDAC). Three dimensional (3D) tumor spheroids (TS) are considered as an in vivo-like tumor model demonstrating its clinical relevancy. We previously developed a minipillar array histochip for 3D culture and immunohistological analysis of TS in an increased throughput manner. Here, we present a minipillar-based co-culture of TS and PSC as a novel in vitro microtumor model of pancreatic ductal adenocarcinoma. Methods: Minipillar chips were obtained from MBD Co. (Cellvitro™ 55Cryo). 3D TS of Panc-1, human PDAC cells and PSCs (Sciencell) were cultured on minipillars and at the bottom of 96 well plates, respectively, as embedded in type I collagen. Changes in expression of extracellular matrix (ECM) and EMT-related factors in TS were assessed by confocal microscopy following cyrosection preparation. Results: Co-culture condition was optimized for the growth of TS, activation of PSC, and their interaction. Expression of EMT markers such as TGF-β1 and vimentin increased in co-cultured TS than mono-cultured TS. ECM remodeling as indicated by increased matrix deposition (collagen I, fibronectin) and fiber organization was observed in TS co-cultured with PSC compared to mono-cultured TS. Upon drug exposure (gemcitabine and paclitaxel), survival advantage was prominent in TS co-cultured with activated PSC. Conclusions: We established 3D model of PDAC by co-culturing TS and PSC using minipillar histochips. EMT-related changes including ECM remodeling and drug resistance were recapitulated in the model. Overall our results indicate usefulness of the present model not only in studying the role of TME factors and their interactions towards tumor progression but also in the evaluation of therapeutic efficacy of drug candidates and combinations.

#2115

An authenticated in vitro model for prostate microenvironment studies utilizing prostate epithelial cells and stromal-derived cells.

Luis G. Rodriguez, Russell E. McDaniel, Xiangshan Zhao, Chaozhong Zou. _ATCC, Gaithersburg, MD_.

Prostate cancer remains one of the most common cancers diagnosed in men and one of the leading causes of cancer death in men. Tumor development and progression have been shown to be highly influenced not simply by the genetic makeup of a cell, but by its surrounding stroma, particularly fibroblasts. It has been demonstrated that prostate cancer-associated fibroblasts (CAFs, which are located marginal to the prostate tumor), differ from prostate normal-associated fibroblast (NAFs, which are located distal to the prostate tumor), on their contribution to tumor progression. However, human prostate cancer in-vitro model systems have focused largely on prostate cancer epithelial cells exclusively. A need exists for a more physiologically relevant human cell model system to study prostate cancer progression within the context of its tumor microenvironment. In this study, we utilized prostate cancer-associated fibroblasts (CAFs), prostate normal-associated fibroblasts (NAFs) and normal prostate epithelial (PrE) cells; all three lines were immortalized by hTERT (human telomerase reverse transcriptase) alone and they were continuously passaged for at least 15 passages without any indications of a decrease in growth rate. All cell lines express appropriate specific cell lineage markers for either fibroblasts or epithelial cells. Fibroblasts expressed TE7 and alpha smooth muscle actin (a-SMA), while prostate epithelial cells expressed cytokeratin 5, low levels of prostate specific antigen (PSA) and high levels of p63 throughout their continuous passage; all characteristics in accord with their primary cell counterparts. Next, cell proliferation was measured for various prostate-derived epithelial cells under the influence of CAFs and NAFs cells. Normal prostate epithelial cell proliferation was inhibited and produced a visible morphological change in the cells in the presence of CAFs or CAF-conditioned medium. Meanwhile, the effects of stromal cells on prostate cancer cells was cell line dependent, demonstrating both promotion and inhibition of growth of selected cancer cells. Surprisingly, both CAFs and NAFs promoted cancer cell growth, but CAFs promoted a greater increase in cell proliferation than NAFs in some cancer cell lines. This study demonstrates that these three hTERT immortalized cells from human prostate are a valuable model system for the study of prostate cancer cell progression and tumor micro environment studies.

### The Metastatic Microenvironment

#2116

Tumor-specific effects of estrogen drive a murine model of human estrogen receptor-positive breast cancer bone metastasis independent of bone microenvironment changes.

Julia N. Cheng, Jennifer B. Frye, Susan A. Whitman, Andrew G. Kunihiro, Madison M. Egan, Julia A. Brickey, Janet L. Funk. _University of Arizona, Tucson, AZ_.

Estrogen receptor α-positive (ER+) breast cancers (BrCAs) have the greatest predilection for forming bone metastases (BMETs). To begin to query ERα's role in osteolytic BrCA BMET progression, tumoral vs bone microenvironment effects of 17β-estradiol (E2) were determined in a human xenograft murine model of ER+ BrCA BMET. For these studies, naïve or 17β-estradiol (E2)-treated female athymic mice aged 4 weeks (young) or 15 weeks (mature) were inoculated with 1x105 human ER+ MCF-7 BrCA cells via the left cardiac ventricle 2 days post-placement of 60-day release E2 pellets (0.05, 0.10, 0.18, 0.36, & 0.72 mg E2). For studies examining E2 effects on bone, MCF-7 cells were not inoculated. Osteolytic lesion formation was assessed by hind limb radiographs, and E2 effects on bone were assessed by DXA (Faxitron), μCT (Scanco), and biochemical markers of bone formation or resorption (P1NP and TRAcP, respectively; Immunodiagnostic Systems). BMET incidence and lesion size in young mice were E2 dose-dependent, achieving 100% incidence at the highest dose tested, with smaller lesions and lower incidence occurring in response to lower doses, and no lesions forming in the absence of E2 even after 8 months. In contrast, E2 effects on the bone microenvironment were not dose dependent, and resulted in identical increases in bone mineral density (BMD) and bone volume (BV/TV), primarily via an increase in bone formation. To further query the importance of tumoral vs bone effects of E2 in driving ER+ BrCA progression, the tumoral effects of an identical E2 dose (0.72 mg) were compared in the skeletally immature young mice, which accrued increased bone (BV/TV) due to increases in bone formation in response to E2, vs skeletally mature mice, where E2-induced increases in bone were less marked and primarily driven by decreases in bone resorption. In these two very different bone milieus, progression of E2-dependent BMET was identical. While the relative roles of E2-induced tumor cell proliferation vs osteolytic factor secretion cannot be distinguished here, the development of osteolytic lesions similar in size to those occurring in ER- models driven by tumoral secretion of osteolytic factors, despite a marked increase in bone volume, suggests that tumoral effects of E2 may drive osteolytic ER+ BrCA BMET progression in a bone-specific fashion.

#2117

Role of Neuropilin 2 in orchestrating the functions of osteoclasts in promoting prostate cancer bone metastasis.

Navatha Shree Polavaram, Arup Bag, Samikshan Dutta, Kaustubh Datta. _Univ. of Nebraska Medical Center, Omaha, NE_.

Background: Bone metastasis is one of the major clinical concerns that causes skeletal-related malignancies and increased mortality. Bone is one of the preferred sites for metastatic prostate cancer. The metastatic prostate cancer cells interact with bone cells (osteoblasts and osteoclasts), resulting in an imbalance in the bone homeostasis leading to increased activation of osteoblasts over osteoclasts. Our preliminary data indicated a non-tyrosine kinase receptor Neuropilin 2 (NRP2) is expressed in osteoclasts (OC) induced by PCa cells and acts as a negative regulator of osteoclasts' function. We hypothesize that PCa-induced NRP2 expression in OC is necessary for low osteolytic activity and thus favors an osteoblastic lesion in PCa bone metastasis.

Methods: Mouse OC precursors were isolated from bone marrow of C57BL/6 mice as well as transgenic CSF1R-cre; NRP2 Flox/Flox inducible mice where addition of 4-hydroxytamoxifen depletes NRP2 specifically from the myeloid cells. Differentiation of OCs was conducted under the conditions of RANKL and M-CSF and in conditioned medium (CM) collected from PCa cell line LNCaP C4-2B (promotes high osteoblastic and low osteoclastic activity) and PC3 (predominantly osteoclastic activity) to mimic the conditions in normal bone and PCa bone metastasis. NRP2 expression at protein and mRNA was evaluated. TRAP staining and pit formation were conducted to confirm the differentiation and function of OCs.

Results and Discussion: We observed an increase in NRP2 expression in OCs induced by RANKL and M-CSF and in PC3 and LNCaP C4-2B CM simultaneously. TRAP staining and activity confirmed the differentiation of OCs under these conditions. Interestingly, depletion of NRP2 and treatment either in RANKL and M-CSF or LNCaP C4-2B CM exhibited a drastic increase in osteoclast differentiation and function. mRNA analysis revealed an increase in expression of osteoclastic genes following NRP2 depletion in RANKL and M-CSF and LNCaP C4-2B CM. However, NRP2-depleted OC precursors when treated with PC3 CM showed no change in osteoclastogenesis. These findings advocate a role of NRP2 in inhibiting osteoclastic activity in PCa bone metastasis and that osteolytic PCa evades NRP2 inhibition. Using in vitro and transgenic mice, we will elucidate the molecular mechanisms through which NRP2 regulates osteoclast differentiation and function in normal bone and in LNCaP C4-2B CM. We will also address how PC3 CM-induced OCs escapes the inhibition of NRP2. Together, this approach will elucidate the role of NRP2 axis on OCs in promoting PCa-induced bone metastasis and will aid in determining whether NRP2 axis can be a therapeutic target.

#2118

Single-cell analysis reveals dynamic interaction between myeloma and bone marrow microenvironment.

Jaewoong Min, Daeun Ryu, Hae-Ock Lee, Areum Jo, Woong-Yang Park, Seok Jin Kim, Kihyun Kim. _Samsung Genome Institute, Seoul, Republic of Korea_.

Multiple myeloma (MM) is a monoclonal plasma cell (MPC) malignancy primarily propagating in the bone marrow. To understand molecular signatures of MM in association with tumor microenvironment, we performed single-cell RNA sequencing for bone marrow biopsies from seven newly diagnosed myeloma patients. Clinical parameters of 7 MM patients differed, which were recapitulated in the single-cell transcriptome analysis. In comparison to the normal bone marrow from healthy donors, patient-specific monoclonal immunoglobulin gene expression as well as high levels of plasma cell markers distinguished the MPC clusters in myeloma patients. For other cell type identification, reference transcriptome for bone marrow immune cells were utilized. Major cell populations were determined to be monocytes, T cells, B cells, and erythroid cells. T cells manifested a mature phenotype encompassing from naïve to cytotoxic effector gene expression characteristics. B cells and erythroid cell populations were in various developmental stages. Monocytes demonstrated relatively constant gene expression throughout healthy donors to myeloma patients. In more advanced tumor stages, the composition and gene expression characteristics of MPCs and bone marrow immune cells were altered. Taken together, single cell RNA sequencing reveals gene expression characteristics of both myeloma and bone marrow immune cells which provide potential therapeutic strategies targeting tumor or immune compartment.

#2119

Bone Morphogenetic Protein Receptor 1a in myeloid cells regulates mouse prostate cancer growth.

Desiree M. Straign,1 Sergey V. Novitskiy,2 Philip Owens1. 1 _Univ. of Colorado, Aurora, CO;_ 2 _Vanderbilt University, Nashville, TN_.

The Bone Morphogenetic Protein (BMP) pathway is a member of the TGFβ signaling family and has mirrored the complex roles in diverse contexts during cancer development. BMP signaling is rarely mutated and can be frequently overexpressed in many human cancers. The dichotomous role of BMPs as both tumor promoters and suppressors appears to be largely context based in both the cancer cell and the surrounding microenvironment. Myeloid cells including macrophages and neutrophils have been shown to be tumor promoting when stimulated from BMP signaling. We discovered that conditional deletion of BMPR1a in myeloid cells (LysM-Cre) would affect tumor progression and found smaller tumors in a syngeneic FVBn prostate cancer model. Specific changes occurred in myeloid cells both in tumor-bearing mice and non-tumor naïve mice throughout multiple tissues. We profiled myeloid subsets both in the bone marrow, spleen and primary tumor. We found that BMPR1a loss in myeloid cells altered the differentiation and lineage capability of distinct populations by histologic, flow cytometry and high-dimensional mass cytometry analysis. These results indicate a distinct genetic requirement for BMP signaling in myeloid cells during tumor progression.

#2120

Inhibition of multiple myeloma exosomes prevents bone loss and reduces tumor growth.

Sylvia T. Faict,1 Kim De Veirman,1 Ken Maes,1 Elke De Bruyne,1 Roy Heusschen,2 Jo Caers,2 Karin Vanderkerken,3 Rik Schots,1 Eline Menu1. 1 _Vrije Universiteit Brussel, Brussels, Belgium;_ 2 _Université de Liège, Liège, Belgium;_ 3 _Vrije Universiteit Brussel, Belgium, Belgium_.

Multiple myeloma, one of the most common hematological malignancies, will often manifest osteolytic lesions during disease development, throughout the whole body. MM is still considered an incurable malignancy, with a supportive micro-environment in the bone marrow. Extracellular vesicles such as exosomes are known to play an important tumor-promoting role in this micro-environment, by inducing angiogenesis and immune suppression. The aim of our study was to examine the effects of MM exosomes on osteolysis in vitro and in vivo, and to determine whether inhibition of exosome secretion can lead to a delay in tumor growth and prevent bone loss. We used the 5TGM1 model which is a syngeneic murine MM model, presenting typical MM characteristics such as osteolysis, angiogenesis and a serum M-spike. We examined the effects of conditioned medium from these MM cells and MM-derived exosomes on cell viability and proliferation of the MC3T3 pre-osteoblast cell line and on their ability to produce alkaline phosphatase (ALP). We saw a decrease in viability and ALP activity when adding exosomes or conditioned medium of the 5TGM1 myeloma cells to the osteoblasts. Moreover, RT-PCR analysis showed a decrease of various osteogenic differentiation genes when osteoblasts were exposed to 5TGM1 exosomes, demonstrating a diminished capacity of osteoblastic differentiation. We next added the exosome secretion inhibitor GW4869 to the 5TGM1 cells, thereby reducing the number of exosomes in the conditioned medium. This was confirmed through western blot of the typical exosome markers. The negative effects of the 5TGM1 conditioned medium on osteoblasts could be overturned by reducing the exosome content therein. In vivo, we injected 5TGM1 exosomes intravenously in healthy mice for three weeks and examined the effects on osteolysis by microCT and TRAP staining. We saw an increase in osteolysis, comparable to the typical osteolytic lesions in tumor-bearing mice. Furthermore, when inhibiting the exosome secretion in vivo in 5TGM1 mice, we observed a significant reduction in tumor load. Through this study we can conclude that exosomes from multiple myeloma cells can induce osteolytic lesions by inhibiting osteoblast proliferation and differentiation. These effects can be reversed by inhibiting the secretion of exosomes.

#2121

MET/VEGFR/FMS signaling contributes prostate cancer-induced osteoclast differentiation and bone resorption.

Kenta Watanabe,1 Michiko Hirata,1 Tsukasa Tomomi,1 Chiho Matsumoto,1 Hidenori Fujita,2 Yukari Yamada,2 Kenichi Matsuo,2 Kazuhiko Yonekura,3 Chisato Miyaura,1 Masaki Inada1. 1 _Tokyo University of Agriculture and Technology, Tokyo, Japan;_ 2 _Taiho Pharmaceutical Co., Ltd., Ibaraki, Japan;_ 3 _Taiho Pharmaceutical Co., Ltd., Tokyo, Japan_.

Blockage of both vascular endothelial growth factor (VEGF) receptor and hepatocyte growth factor (HGF) receptor MET signaling pathways has been reported to suppress tumor growth and angiogenesis synergistically, suggesting the possibility that the dual inhibition of VEGFR and MET signals may have potential effects on the prevention of tumor growth. Recently, we developed a novel VEGFR/MET-targeted tyrosine kinase inhibitor, TAS-115, and showed its antitumor properties in xenografts of human gastric carcinoma (Mol Cancer Ther 2013). Although bone metastases frequently occur in prostate cancer patients, the role of VEGF receptor and MET in cancer-induced bone resorption is not known. Patients with advanced prostate cancer show sclerotic bone metastases, which cause chronic pain and pathologic fractures; however, the invasion of prostate cancer cells into bone tissues first induces bone destruction by increased osteoclast-mediated bone resorption. In this study, we used TAS-115, which inhibits both MET and VEGFR, and examined its effects on human prostate cancer cell line (PC3)-induced bone resorption by directly injecting PC3 cells into the proximal medulla of tibiae in nude mouse in vivo, and by the co-culturing of calvarial bone with PC3 cells in vitro. When PC3 cells were injected into proximal tibiae in nude mouse, severe trabecular and cortical bone destruction was detected with subsequent tumor growth. Oral administration of TAS-115 almost completely inhibited both PC3-induced bone loss and PC3 cell proliferation. In an ex vivo bone organ culture, PC3 cells induced osteoclastic bone resorption effectively suppressed by the treatment of TAS-115. In the culture of the bone marrow cells, M-CSF dependent macrophage differentiation and following RANKL-induced osteoclast formation were suppressed by adding TAS-115. FMS-related receptor kinases such as ERK and Akt were also suppressed by the presence of TAS-115. FMS expression was only detected in macrophage and in osteoclast cell lineage. These results indicated that administration of TAS-115 restored bone destruction induced by PC3, mainly by inhibiting the FMS-dependent and RANKL-induced differentiation of preosteoclasts into mature osteoclasts. The additional inhibition of the tyrosine kinase FMS by TAS-115 has profound effects on prostate cancer-driven osteoclastogenesis, and its proliferation extends the capability of this agent to act as a powerful antidote to the devastating effects of metastatic spread to bone.

#2122

Induction of structural and functional effects of myeloma cells after daratumumab treatment.

Barbara Castella,1 Angelo C. Faini,1 Yuliya Yakymiv,1 Fabio Morandi,2 Alessandra Larocca,3 Stefania Oliva,3 Alberto L. Horenstein,1 Massimo Massaia,1 Fabio Malavasi1. 1 _University of Turin, Turin, Italy;_ 2 _Stem Cell Laboratory and Cell Therapy Center, Istituto Giannina Gaslini, Genova, Italy, Genova, Italy;_ 3 _AOU Città della Salute e della Scienza di Torino, Italy, Turin, Italy_.

Background CD38 is a pleiotropic cell surface glycoprotein with receptorial and enzymatic functions. The molecule is generally expressed at low levels by different hematological and solid tissues: plasma cells score the highest surface levels of CD38 among mature lymphoid cells. CD38 has become the target of therapeutic antibodies in multiple myeloma (MM). Daratumumab (Dara) has been approved as efficient monotherapy or in combination with other anti-myeloma agents. The results obtained are good in patients refractory to standard myeloma therapies. Dara mediates clinical effects through multiple mechanisms. These include complement- and antibody-dependent cell cytotoxicity, antibody-dependent phagocytosis, programmed cell death and modulation of enzymatic activities. Promising are Dara immunotherapeutic functions in virtue of its ability to induce cytotoxicity exploiting both arms of innate and adaptive immune responses. Recent studies suggest that Dara plays immunomodulatory roles in addition to its direct effects on cytotoxicity.

Results CD38 engagement by Dara on MM cells is followed by a selective polar aggregation of the target molecule in myeloma membranes, with subsequent release of microvesicles (MV) of 100-1,000 nm into the extracellular space. We validated the hypothesis that MV released by MM in the bone marrow (BM) niche may express functional ectoenzymes (CD38, CD39, CD73, and CD203a), potentially capable of metabolizing both ATP and NAD+ and to produce adenosine (ADO). Results indicate that MV obtained after Dara treatment tend to cluster around (and to be internalized in) NK cells, monocytes and MDSC, cells all expressing IgG Fc Receptors (FcR). NK cells, which apparently disappear in patients during Dara treatment: because of this, they were selected for testing MV-mediated effects. Comparative analysis of the genes modulated after exposing NK cells to the MV/Dara complex were followed by functional in vitro experiments. Both sets of results confirmed reduced proliferative ability and enhanced NK cell-mediated killing of MM cells. A further support to the immune modulatory roles exerted by Dara comes from the observation that MV surface represent a clustering of the expected CD38/Dara complex, flanked by a set of ectoenzymes involved in the generation of ADO. Moreover, MV express high amount of CD55 and CD59 molecules, receptors which impair the ability of the complement to exert in situ a cytolytic activity. Another set of observations indicate that PD-L1 tend to accumulate in the surface of MV after Dara treatment, anticipating a role in the modulation of immune checkpoint pathways (PD-1/PD-L1).

Conclusions Results of our observations indicate that MV obtained from MM cells treated with Dara may be a particulate system to influence the BM niche and the successive immune responses elicited by different mechanisms and cell effectors.

#2123

Myeloma derived macrophage inhibitory factor regulates BMSC IL6/8 via cMYC.

Rachel E. Piddock,1 Christopher R. Marlein,1 Amina Abdul-Aziz,1 Martin J. Auger,2 Kristian M. Bowles,1 Stuart A. Rushworth1. 1 _University of East Anglia, Norwich, United Kingdom;_ 2 _NNUH, Norwich, United Kingdom_.

Background

Multiple Myeloma (MM) is a plasma cell malignancy dependent on the bone marrow microenvironment. Macrophage inhibitory factor (MIF) acts directly on malignant plasma cells affecting homing and chemotherapy resistance, however the adaptive effect that MM derived MIF has on the tumor microenvironment is not yet defined. Here we investigate the function of MM derived MIF in the MM microenvironment by examining its effects on bone marrow mesenchymal stromal cells (BMSC).

Methods

Primary MM and BMSC were obtained from patient bone marrow. Proteome Profiler Human XL Cytokine Array was used. Inhibitors including JQ1 were

purchased from Merck. In-vivo experiments were performed using 8-10 week old NSG mice.

Results

First we injected 1 x 106 MM.1S MIF-knock down (KD) cells or MM.1S control-KD cells (containing the pCDH-luciferase-T2A-mCherry construct) into the tail vein of 6-8 week old NSG mice. MIF-KD in MM cells resulted in animals with significantly reduced tumor burden and improved overall survival. To further investigate the role of MIF in regulating the BM microenvironment we stimulated primary human BMSC with biologically appropriate doses of recombinant MIF and then used Human XL Cytokine Array and cytokine specific ELISA to assay the supernatant from these MIF stimulated BMSC. Results show that MIF induces IL-6 and IL-8 protein secretion from primary BMSC. To examine the mechanism of action of these observations we used a panel of inhibitors to screen for potential pathways responsible for MIF induced BMSC derived IL-6 and IL-8 expression. The c-Myc inhibitor JQ1 inhibited MIF induced IL-6 and IL-8 expression. Next, we looked at whether c-Myc regulates BMSC pro-tumoral interleukin production in-vivo. We injected NSG mice via the tail vein with human myeloma cell line U266 and then randomized animals to two groups; treating with either JQ1 (50mg/kg, IP) or alternatively vehicle control for 5 days, after which bloods were taken

to assess murine IL-6 levels (IL-8 is deleted from the mouse genome). Results show that murine IL-6 was significantly reduced in the JQ1 treated animals (carrying human MM) compared to vehicle control treated animals.

Summary

MM derived MIF is pro-tumoral through induction of c-Myc and downstream IL-6 and IL-8 in the BMSC of the tumor micro-environment. Identification of this novel pro-tumoral crosstalk mechanism which exists between MM and the bone marrow stroma provides scientific rationale for the clinical evaluation of new therapeutic targets in MM.

#2124

Therapeutic targeting of cancer-associated fibroblasts by a novel protein, ProAgio, effectively decreases TNBC growth and metastasis to the lungs.

Malvika Sharma, Ravi Chakra Turaga, Yi Yuan, Falguni Mishra, Zhi-Ren Liu. _Georgia State University, Atlanta, GA_.

Background: TNBC metastasis is responsible for a majority of the mortalities associated with breast cancer across the globe. Cancers metastasize to various distant organs such as lungs, liver, and bones, which accounts for a low 5-year survival rate. Lung offers the most favorable environment and is, therefore, the most common metastatic site in the majority of patients. Currently, there are no effective targeted therapies for breast cancer metastasis and hence there is a dire clinical need to establish treatment for this disease. It is noteworthy that tumor growth and metastasis is supported by the surrounding stroma, also known as the tumor microenvironment (TME) that promotes the proliferation of cancer cells by secreting various growth factors and cytokines. Despite growing awareness of the role of the TME in tumor progression, targeting cells that comprise the majority of the TME is still uncharted territory. TME is composed of a variety of cell types, with cancer-associated fibroblasts (CAFs) - cells that express high levels of integrin αVβ3 - being the predominant cell type. Here we report the development of a novel, rationally designed protein, ProAgio, which induces apoptosis of integrin αVβ3 expressing cells via a novel mechanism.

Objectives: To test the efficacy of ProAgio in targeting the TME in TNBC. To elucidate the mechanism through which ProAgio exerts an anti-metastatic effect.

Methods: We implanted 4T1 cells orthotopically in Balb/c mice and treated them with 10 doses of ProAgio (10mg/kg; i.p.) when the tumor volume reached around 250mm3. Another in vivo experiment was performed with the 4T1 model to see the effect of ProAgio in combination with a chemotherapeutic agent, doxorubicin (DOX) (3mg weekly i.p.). The tissues isolated were stained for collagen and α-SMA (CAF marker). Breast and lung fibroblasts were differentiated with TGF-β (5ng/mL) for two weeks and annexin V staining was used to measure their apoptosis.

Results: Our data demonstrate that ProAgio inhibits breast tumor growth and metastasis to lungs by decreasing collagen and CAFs in 4T1 tumor-bearing mice. Histological analyses of the tissues exhibit decreased collagen and α-SMA in both breast tumor and lungs upon ProAgio treatment. Mechanistically, ProAgio induces integrin αVβ3-mediated apoptosis in breast and lung CAFs. In addition, ProAgio shows no effect on 4T1 breast cancer cells as they lack αVβ3 expression. Our study demonstrates that a combination of ProAgio and DOX exhibits improved anti-tumor efficacy and enhances survival in the 4T1 murine model.

In conclusion, our study reports the first evidence of inducing apoptosis in CAFs and shows antitumor synergy for combined therapy, supporting the significant therapeutic potential of ProAgio as a stand-alone therapy and more effectively when combined with chemotherapy.

#2125

Cancer-associated fibroblasts-stimulated IL-11 promotes metastasis of gastric cancer cells mediated by upregulation of MUC1.

Xiaoxun Wang, Xiaofang Che, Ming Bai, Yibo Fan, Yunpeng Liu, xiujuan qu. _China Medical Univ., Shenyang, China_.

Background: Cancer-associated fibroblasts (CAFs) are major components of the tumor stroma and regulators of tumor progression. However, the molecular mechanism by which CAFs promote gastric cancer progression should be further explored.

Methods: Mouse model was used to certify that CAFs could promote peritoneal metastasis of gastric cancer cells. The expression of interleukin-11 (IL-11) was analyzed in two gastric cancer cell lines by real-time RT-PCR and ELISA. The role of IL-11 in gastric cancer cell migration and invasion explored by Transwell. Western blotting was conducted to detect the activation of STAT3 and ERK signaling pathway.

Results: In our study, we reveal that when CAFs co-cultured with gastric cancer cells secreted significant amounts of IL-11. CAFs promote the ability of migration and invasion through the production of IL-11 by stimulating JAK/STAT3 and MAPK/ERK pathways to upregulate MUC1 in gastric cancer cells. On the other hand, when IL-11 was deprived using a neutralizing antibody or inhibition of JAK/STAT3 and MAPK/ERK pathways with specific inhibitors, Stattic and PD-98059, markedly attenuated these CAFs-induced phenotypes in gastric cancer cells.

Conclusions: Taken together, these results revealed that CAFs play a significant role in the gastric cancer progression in the tumor microenvironment through IL-11-STAT3/ERK signaling by upregulating MUC1. Also, IL-11 targeted therapy can achieve an effective treatment against gastric cancer indirectly by exerting their action on stromal fibroblasts.

#2126

Characterization of colorectal liver metastasis at single-cell resolution reveals dynamic interplay in the tumor microenvironment.

Anuja Sathe, Jiamin Chen, Christina Wood-Bouwens, Alison Almeda, Billy Lau, Sue M. Grimes, George A. Poultsides, Hanlee Ji. _Stanford University, Stanford, CA_.

Colorectal cancer (CRC) metastasizes to the liver in over half of patients. Compared to primary tumors, the biology of CRC liver metastasis (CLM) is poorly characterized. We performed high-throughput microfluidics-based single-cell RNA sequencing (scRNA-Seq) to define the epithelial and stromal components of CLMs. Using the 10X Genomics platform, scRNA-Seq was performed on single-cell suspensions generated from dissociation of fresh surgical excisions. We detected 1,551 cells from 2 CLMs and 5,407 cells from paired normal liver. Cells were sequenced at an average depth of 1 million reads/cell using Illumina sequencing. Graph-based clustering and differential expression analysis was performed using the Seurat algorithm. Tumor epithelial cells (EPCAM+, CDH1+, TFF3+) consisted of heterogeneous subpopulations with diverse transcriptomes and activation of varying signaling pathways. The stroma consisted of myofibroblasts, endothelial cells and immune cells. Myofibroblasts consisted of multiple subtypes indicative of a range of differentiation between smooth muscle cells and fibroblasts (ACTA2+, THY1+, COLA1+). These cells were absent in normal liver and could be confirmed as a desmoplastic stroma on histology. The immune infiltrate was rich in monocyte-derived macrophages, with few dendritic cells, CD4 and CD8 T cells. We identified 2 macrophage subpopulations in both CLMs. These cells expressed markers belonging to both the M1 and M2 classes (IL1A+, IL1B+, TNF+, MARCO+, MSR1+, CD68+) suggestive of a spectrum of polarization states. Macrophages showed increased expression of the immune checkpoint inhibitor TIM3, while CD4 and CD8 cells showed upregulation of TIGIT. Immune cell populations also expressed a variety of cytokines such as chemokines and interleukins capable of regulating functional effector states. Tumor epithelial cells expressed receptors for various growth factors of the EGF, VEGF, PDGF, FGF and TGFGB families. Stromal cells expressed several heterologous growth factors that can act as receptors ligands and influence tumor cell growth. This included BMPs and INHA expressed in myofibroblasts that can modulate TGFB signaling, as well as FGFs, HGF and VEGF. Endothelial cells secreted PDGF and macrophages expressed AREG and EREG that can activate EGF signaling. These features of the cellular landscape of CLMs can be used for devising novel therapeutic strategies for patients unfit for surgery or for those with unresectable tumors. We will test inhibition of the identified heterologous growth factors and improving the antitumor potential of macrophages in an ex vivo air liquid interphase organoid system from a cohort of CLMs.

#2127

RSK2 phosphorylates T-bet to attenuate colon cancer metastasis and growth.

Ke Yao,1 Cong Peng,1 Yuwen Zhang,1 Tatyana A. Zykova,1 Mee-Hyun Lee,2 Hanyong Chen,1 Joohyun Ryu,1 Enyu Rao,1 Wei-Ya Ma,1 kangdong liu,2 Ann M. Bode,1 Bing Li,1 zigang dong1. 1 _Univ. of Minnesota Hormel Inst., Austin, MN;_ 2 _China-US Hormel Institute, Zhengzhou, China_.

Metastasis is the major cause of death for patients with solid malignancies. Approximately 80 and 20% of colorectal cancer (CRC) patients develop liver metastasis and lung metastasis, respectively. We found that at different stages of colon cancer, the patients' interferon (IFN) γ secretion from peripheral blood mononuclear cells (PBMCs), was decreased compared with healthy people. The ribosomal S6 kinase (RSK) family of kinases has multiple cellular functions, and we examined their role in this observed IFNγ decrease. Flow cytometry analysis of wild-type (WT) and RSK2 knockout (KO) mice, revealed that the level of IFNγ was significantly lower in RSK2 KO mice as compared to WT mice. Since IFNγ is a component of immunity, which contributes to protection against metastatic carcinomas, a colon cancer liver metastasis experiment was conducted. We found that metastasis was significantly increased in RSK2 KO mice, compared to WT mice. Transcription factor T-bet can directly activate Ifnγ gene transcription. In vitro kinase assay results showed that RSK2 phosphorylated T-bet at serines 498 and 502. We show that phosphorylation of T-bet by RSK2 is required for IFNγ expression, because knocking down RSK2 expression or over-expressing T-bet S498A/S502A mutants reduces IFNγ mRNA expression. To verify the function of the phosphorylation sites, a

constitutively active mutant T-bet (S498E/S502E) was over-expressed in bone marrow. Mutant T-bet restored the IFNγ mRNA levels and dramatically reduced the metastasis rate in these mice. Overall, these results indicate that phosphorylation of T-bet is required for inhibiting colon cancer metastasis and growth through a positive regulation of RSK2/T-bet/IFNγ signaling.

#2128

Autocrine fibronectin inhibits breast cancer metastasis.

Aparna Shinde, Ammara Abdullah, Luis Solorio, Michael Wendt. _Purdue University, West Lafayette, IN_.

The processes of the epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET) are strongly linked to breast cancer metastasis via their ability to generate cells of increased invasive capacity and enhanced tumor-initiating phenotypes. Numerous growth factors, cytokines and chemotherapies present in the tumor microenvironment are capable of inducing EMT, but the role of extracellular matrix (ECM) components in this process remains less well understood. Analysis of the METABRIC dataset strongly linked high-level FN expression to decreased patient survival. However, in vitro analysis of the MCF-10A progression series indicated that autocrine FN expression was associated with nonmetastatic cells. Therefore, we utilized differential bioluminescent imaging to track the metastasis of these isogenic epithelial and mesenchymal cells within heterogeneous primary tumors. Using this approach, we demonstrate that tumor cells expressing autocrine FN display a constitutively mesenchymal phenotype and are incapable of completing the metastatic process, even when grown within a tumor containing epithelial tumor cells. Importantly, depletion of FN allows tumor cells to exit their mesenchymal state, regain epithelial characteristics and initiate tumor growth in a metastatic microenvironment. Using a tessellated three-dimensional polymer scaffolding, we show that depletion of FN or pharmacologic inhibition of focal adhesion kinase prevents mesenchymal tumor cells from producing structural fibrils and thus supporting the growth and migration of metastatic-competent epithelial tumor cells. Finally, dynamic FN coating of this 3D growth scaffold resulted in formation of a functionalized fibrillar FN network that was sufficient to induce transient EMT events as metastatic cells migrated along FN fibrils. Taken together, our data indicate that autocrine expression of FN by tumor cells supports a stable mesenchymal phenotype limiting their metastatic potential. Instead, this tumor cell-derived production of fibrillar FN acts in a paracrine fashion to support transient EMT events in metastatic competent cells within a heterogeneous tumor microenvironment.

#2129

Breast tumor microenvironment & metastasis is controlled by activity of a2 isoform of V-ATPase in mammary epithelium.

Gajendra K. Katara, Arpita Kulshrestha, Manoranjan Sahoo, Kenneth Beaman. _Rosalind Franklin Univ. of Med. Science, North Chicago, IL_.

Extracellular matrix (ECM) critically impacts tumor progression and is influenced by both cancer and host tissue cells. While our understanding of cancer cell ECM remodeling is widespread, the importance of host tissue ECM, which provides an initial congenial environment for primary tumor formation, is only partly understood. Previously, we have shown that epithelial cell associated vacuolar (V)-ATPase 'a2' isoform (a2V) regulates stiffness of mammary gland ECM. Here, we investigated how a2V mediated alterations in ECM of breast tissues affects the tumor microenvironment and metastasis of breast cancer. We also established the correlation of a2V expression in the normal breast tissue with metastatic occurrence in cancer patients. In this study, using the MMTV-Cre-Lox system, we deleted the a2V gene in epithelial cells of mouse mammary glands. Tumors were transplanted in mammary fat pads of a2V conditional knockout mice (a2VcKO) using E0771/Py230 mouse mammary cancer cells. These tumors were evaluated for the histopathology, ECM stiffness, immune cell populations, cytokines, angiogenic factors and metastasis. We show that in a2VcKO, breast tumors exhibit less tumor rigidity, increased inflammation and a more necrotic tumor microenvironment. These mice showed an increased metastasis of tumors compared to control evident by cytokeratin (Ck)14 staining of cancer cells in lung tissues (3.3 fold increase, P=0.004). Immuno-phenotyping of breast tumor microenvironment revealed increased infiltration of CD45 cells with higher number of anti-tumor M1 macrophages (2.1 fold increase, P=0.007) and elevated expression of inflammatory cytokines TNF-α and iNOS in a2VcKO mice compared to control (P=0.01). The tumors in a2VcKO mice were much more highly vascularized and angiogenic compared to control (P=0.01) evident by increased number of Meca32+SMA+ cells. Clinically, in cancer patients, low a2V expression levels in normal breast tissue correlated directly with lymph-node metastasis. Thus, this study has identified a2V expression in epithelial cells as a key modulator of tumor microenvironment and its expression levels can significantly modulate breast tumor dissemination.

#2130

Novel ADAM9 variant induces prostate cancer metastasis.

Chia-Ling Hsieh, Shian-Ying Sung. _Taipei Medical Univ., Taipei, Taiwan_.

Prostate cancer often progresses from an androgen to castration-refractory prostate cancer and metastasizes to the bone and lymph nodes. However, there is no biomarker to predict the initiation of cancer metastasis. Therefore, it is an unmet need to understand molecules which induce metastasis to replace the prediction by PSA. Previous studies by us demonstrate the elevation of ADAM9 in PCa malignant progression. However, the role of ADAM9 in the tumor microenvironment is still not fully understood. Using laser capture microdissection of prostate tumor and stromal cells, indicating the increased of ADAM9 and reactive oxygen species in tumor-associated stromal cells. These inductions correlated with the increased expression of SOD-2 in tumor-associated stromal cells. Both electron microscope analysis and conditioned medium of stromal cells demonstrated the increased of soluble ADAM9 level in the medium. ELISA analysis of patient serum confirmed the increased of ADAM9 serum levels in prostate cancer patients. PCR and sequencing analysis confirmed a novel soluble ADAM9 (sADAM9v2) with deletion of membrane domain that induces cell migration through the activation of FAK and AKT phosphorylation. Our studies demonstrated the release of sADAM9 in tumor microenvironment correlated with reactive oxygen stress and regulate prostate cancer migration and metastasis. In conclusion, our studies showed that sADAM9 release by cancer-associated stromal cells could be a potential biomarker for prostate cancer progression.

#2131

Paracrine cytokine pathways mediate metastasis of breast cancer to lymphatics.

Kyungmin Ji,1 Zhiguo Zhao,1 Kamiar Moin,1 Yong Xu,1 Robert J. Gillies,2 Raymond R. Mattingly,1 Bonnie F. Sloane1. 1 _Wayne State University School of Medicine, Detroit, MI;_ 2 _Moffitt Cancer Center, Tampa, FL_.

Lymphatics, rather than blood vessels, are the primary route for breast cancer metastasis. The presence of breast cancer cells in regional lymphatics, i.e., lymphatic metastasis, is an important prognostic factor for patients. Delineating molecular mechanisms by which the breast cancer cells migrate toward and infiltrate into lymphatics is crucial to designing new therapies to prevent metastatic dissemination. To study tumor:lymphatic interactions, we are using a three-dimensional (3D) heterotypic co-culture model of human breast cancer cells (hBCCs) grown with human microvascular lymphatic endothelial cells (hLECs) in novel chambers that we designed and fabricated. These chambers support growth of the 3D co-cultures, live-cell confocal imaging in real time and noninvasive collection of conditioned media for secretomic analyses. We use live-cell assays developed in our laboratory for quantitative analysis of temporal and dynamic changes in BCC:LEC interactions in correspondence with changes in their malignant and proteolytic phenotypes. We cultured hLECs in the presence and absence of human MDA-MB-231 (231) triple-negative BCCs in 3D cultures for 4 days. In mono-cultures, the 231 cells grow in clusters that exhibit a stellate morphology and hLECs form branching networks with central nodes. We observed that the volumes of 3D structures formed by LECs and 231 cells were significantly greater in co-cultures than in mono-cultures of either cell type. In addition, 231 cells infiltrate into the LEC networks with the infiltration increasing over the 4-day period as assessed by the degree of overlap between 231 cells and hLECs in 3D reconstructions of the co-cultures. Moreover, soluble factors from LECs increase invasive outgrowths of 231 structures. This was demonstrated in 231 cells grown in media conditioned by LECs and in parallel co-cultures of 231 cells and LECs. The induction of invasiveness by LEC conditioned media is reduced by boiling and repeated freeze/thawing, suggesting that the active factor(s) is a protein. Our preliminary results suggest that LECs secrete soluble factors that may be therapeutic targets for reducing invasion of BCCs into lymphatic networks.

#2132

Anti-vascular inflammatory nanoparticles inhibit tumor progression and metastasis through vascular normalization.

Young Sun Choi,1 Soo Hyun Kang,1 Jong-Sup Bae,1 In-San Kim,2 You Mie Lee1. 1 _Kyungpook National Univ., Daegu, Republic of Korea;_ 2 _KIST, Seoul, Republic of Korea_.

Vascular networks in cancer are structurally and functionally abnormal. Abnormal blood vessel development in cancer plays an important role in cancer growth and metastasis, and blocks delivery of chemotherapeutic drugs. Recent studies have revealed that the normalization of abnormal tumor vasculature is one of the promising approaches to treat cancer. Inflammatory microenvironment is one of the newly focused hallmarks of cancer, and tumor vasculature has similar features to that of inflammatory vessels. Thus, vascular inflammation is emerged as an attractive target for cancer treatment. We hypothesized that amelioration of vascular inflammation in tumor may induce vascular normalization leading to the inhibition of tumor progression and metastasis. Here, we investigated the function of ant-vascular nanoparticles in tumor growth and metastasis in LLC allograft tumor models and MMTV-PyMT, spontaneous breast tumor models using TFG and TFMG which are engineered a protein-cage nano-particle (ferritin) with both EPCR-targeting peptides (γ-carboxyglutamic acid (Gla) domain of protein C) and PAR-1-activating peptides (TRAP) of thrombin on its surface. As a result, tumor growth was significantly inhibited in the nanoparticle-injected group, and the lymph node metastasis was also decreased in both animal models. It has been found that the survival rate of the nanoparticle-injected group was prolonged. TFG and TFMG normalized abnormal tumor vasculature, that is, pericytes coverage in vasculatures was significantly increased but the hypoxic regions was decreased in the nanoparticle injected group. Furthermore, when the experiment was conducted by administering cisplatin and nanoparticles together in the LLC allograft tumor model, the tumor size was synergistically increased in the case of the nanoparticle and cisplatin combination administration compared to the cisplatin alone group. Taken together, anti-vascular inflammation nanoparticles with anti-vascular inflammatory properties normalized the abnormal blood vessels and inhibited tumor formation and metastasis. It is suggested that inducing normalization of cancer blood vessels by improving the inflammatory state of blood vessels may be a promising therapeutic method for effective cancer treatment.

#2133

Mechanisms by which 27-hydroxycholesterol promotes breast cancer metastasis.

Liqian Ma, Amy E. Baek, Erik R. Nelson. _University of Illinois at Urbana Champaign, Urbana, IL_.

The purpose of this study is to investigate the mechanism by which the cholesterol metabolite, 27-hydroxycholesterol (27HC) promotes breast cancer metastasis. Breast cancer is the most commonly diagnosed cancer and among the leading causes of death in women in the United States. Elevated cholesterol is identified as a major risk factor of breast cancer onset and recurrence, while cholesterol-lowering drugs are associated with a good prognosis. Previous work has found that many of the effects of cholesterol on breast cancer progression are due to the actions of its primary metabolite, 27HC. 27HC is a ligand of both the estrogen receptor (ER) and liver X receptor (LXR). Intriguingly, the pro-metastatic effects of 27HC require the presence of myeloid-immune cells. This cell type has been implicated in suppressing acquired immunity, allowing cancer cells to escape immune-surveillance. Therefore, we hypothesize that 27HC suppresses the immune system to promote metastasis. To elucidate the immunomodulatory capacity of 27HC, we co-cultured vehicle- or 27HC-treated bone marrow-derived macrophages (BMDMs) with activated, CFSE-prelabeled T cells. As a readout of immune activation, we measured the resulting proliferation of T cells by flow cytometry. In support of our hypothesis, we found that 27HC-treated BMDMs inhibited T cell expansion in a dose dependent manner. In order to determine the mechanisms by which 27HC-treated BMDMs suppress T cell proliferation, we first evaluated the relative contributions of two receptors known to bind 27HC: the ERs and LXRs. To this end, BMDMs were treated with various combinations of pharmacologic agonists or antagonists of the ERs or LXRs. These treated BMDMs were subsequently co-cultured with activated T cells to study their impact on T cell proliferation. Interestingly, a combination treatment of suboptimal doses of an ER antagonist and LXR agonist moderately reduced T cell expansion, similar to 27HC. Therefore, our results suggest that 27HC affects the microenvironment of breast cancer by altering the activity of antigen-presenting cells through the modulation of the ER and LXR. This in turn reduces the expansion of T cell function, ultimately resulting in tumor progression. Our ongoing work is aimed at further elucidating the crosstalk between the ERs and LXRs and identifying the downstream targets of 27HC involved in mediating this immune-suppressive phenotype. Our work is of considerable importance given the prevalence of metastatic breast cancer and that the cholesterol axis is highly amenable to therapeutic targeting.

This work was supported by the National Cancer Institute of the National Institutes of Health (R00CA172357, E.R.N.).

#2134

IL6 and CXCL8 mediate redundant, targetable tumor-host interactions that drive osteosarcoma lung metastasis.

Ryan D. Roberts,1 Hakan Cam,1 Laura Brandolini,2 John M. Hinckley,1 Amy C. Gross1. 1 _Nationwide Children's Hospital, Columbus, OH;_ 2 _Dompe Farmaceutici, L'Aquila, Italy_.

Osteosarcoma (OS) kills patients through aggressive metastatic spread, primarily to lung tissues. Our lab has sought to understand the tumor-host interactions that facilitate growth of OS cells specifically within the lung. We previously identified two intercellular signaling molecules, IL6 and CXCL8, which exhibit enhanced expression when primary tumors become lung metastases. Here, we show that epigenetic changes drive aberrant expression of ΔNp63, endowing OS cells with the capacity to colonize lung tissue. IL6 and CXCL8 serve as downstream mediators induced directly by ΔNp63 and necessary for survival and proliferation within the lung. Inhibition of IL6 and CXCL8 signaling has no direct effect on the proliferation of OS cells, but IL6 inhibition has a profound effect on their ability to form colonies in soft agar. In preclinical mouse models of OS, inhibitors of IL6 and CXCL8 signaling profoundly impair the ability of OS cells to colonize lung tissue, completely preventing metastasis in some models. Co-culture studies suggest that strong paracrine interactions with bronchial epithelial and smooth muscle cells, but not macrophages or lung fibroblasts, drive increased expression of IL6 and CXCL8. These data suggest a mechanism where aberrant ΔNp63 expression within developing OS tumors generates subsets of tumor cells endowed with increased IL6 and CXCL8 expression. This triggers feed-forward paracrine loops with bronchial epithelial and smooth muscle cells that facilitate growth and survival within the lung, leading to metastasis.

#2135

Complex roles of discoidin domain receptors (DDRs) in tumor growth and experimental metastasis: role of collagen I in DDR-mediated tumor growth.

Benjamin D. Wasinski,1 R. Daniel Bonfil,2 Anjum Sohail,1 Seong Ho Kim,3 Lisa Polin,3 Allen-Dexter Saliganan,1 Mohamad Bouhamdan,1 Sayed Nabi,1 Hyeong-Reh C. Kim,3 Marco Prunotto,4 Rafael A. Fridman3. 1 _Wayne State University, Detroit, MI;_ 2 _Nova Southeastern University, Fort Lauderdale, FL;_ 3 _Wayne State University and Karmanos Cancer Institute, Detroit, MI;_ 4 _Hoffmann-La Roche, Basel, Switzerland_.

DDR1 and DDR2 constitute a unique set of receptor tyrosine kinases that signal in response to collagen. Evidence indicates that DDRs play key roles in cancer progression. However, a comparison of the effects of DDR1 and DDR2 on tumor growth and metastasis within the same cellular context has not been done. To accomplish this goal, we developed a doxycycline (DOX) regulated system (Tet-Off) to express human DDR1b or DDR2 in the human HT1080 fibrosarcoma cell line. The cells were then examined for their ability to form subcutaneous (s.c.) tumors, or experimental lung metastases after intravenous (i.v.) injection in SCID mice. To induce or repress DDR expression in vivo, mice were fed a regular diet or a diet with DOX, respectively. Because collagen I (COL I) is a ligand for both DDR1 and DDR2, a set of mice were inoculated s.c. with HT1080 cells suspended in COL I with or without DDR induction in –DOX and +DOX mice, respectively. Growth of s.c. tumors was evaluated by measuring tumor volumes and lung metastases were quantitated by Alu qPCR and histological examination. In the absence of COL I, expression of either DDR1b or DDR2 had no impact on s.c. tumor growth rate, when compared to cells without DDR induction (+DOX). However, in the presence of COL I, tumor growth rates were significantly higher only in HT1080 cells with induced expression of DDR1b or DDR2 (p<0.001 for both), when compared to control cells inoculated with COL I into +DOX mice (no DDR induction). This suggests that a pro-oncogenic effect of DDR1b and DDR2 can be readily uncovered only in tumors growing within a COL I-rich environment. Because the Hippo tumor suppressor pathway has been shown to be regulated by extracellular matrix, homogenates of COL I tumors generated by cells with or without DDR induction were examined for levels and activation of Hippo pathway components. These analyses were consistent with Hippo pathway inactivation in tumors generated by DDR1b- and DDR2-expressing cells in a COL I matrix, consistent with their higher rate of tumor growth. Experimental metastasis assays revealed that induction of DDR2 expression had no impact on lung metastatic burden. In contrast, induction of DDR1b expression dramatically suppressed HT1080 lung metastasis formation, compared to control cells (no DDR1b induction). Collectively, these results suggest that: 1. Ectopic expression of DDRs in HT1080 cells accelerates tumor growth only when co-inoculated with COL I, consistent with a pro-tumorigenic effect of activated receptors within a collagen matrix. 2. A DDR/COL I axis supports HT1080 tumor growth, in part, by inactivation of the Hippo tumor suppressor pathway, suggesting that COL I-initiated DDR signaling regulates the Hippo pathway in vivo. 3. DDR1b, but not DDR2, is a potent suppressor of lung metastasis in the HT1080 model. Taken together, these studies provide novel insights into the complex roles of DDRs in cancer.

#2136

Transcriptome analysis of osteoblasts fused with cancer-derived exosomes.

Nicholas H. Hum, Kelly A. Martin, Gabriela G. Loots. _Lawrence Livermore National Laboratory, Livermore, CA_.

Breast cancer tumors have a high rate of metastasis and certain aggressive subtypes favor the bone environment. Clinically, it is estimated that metastatic breast cancer has a prevalence of up to 70% of bone tumors. Exosomes are important in cell-cell communication, and are increasingly being recognized as key contributors for priming targeted tissues for metastasis. Despite numerous advances in exosome detection and cargo characterization, transcriptional effects of cancer exosomes on targeted cells remain difficult to characterize at physiologically relevant levels. This is largely due to a combination of limited exosome secretion/uptake and an inability to segregate affected cells from the population. Conventional approaches to circumvent these limitations include spiking cell environments with exosome quantities far in excess of physiological concentrations.

We sought to analyze the transcriptional effect of cancer exosomes on cells that have uptaken cancer-derived exosomes in a dynamic co-culture environment. In order to accomplish this, we first engineered a human breast cancer cell line (MDA-MB-231) to express GFP labeled exosomes. This novel cell line expresses three GFP fusion proteins for tetraspanin exosome markers (CD9, CD63, and CD81) known to be found on exosomes derived from MDA-MB-231 cells. Stable, intrinsic, fluorescent labeling of three exosome markers allows for an improved, more sensitive method for the analysis of exosome transfer. We can now examine transfer representing a more diverse pool of exosomes requiring no manipulation post exosome isolation prior to visualization.

To investigate breast cancer exosome-mediated alterations on gene expression in bone cells to mimic breast cancer bone metastasis, this GFP exosome expressing line (MDA-MB-231 exo-GFP) was co-cultured with a mouse osteoblast cell line (MC3T3) then analyzed via flow cytometry to quantify the uptake of cancer exosomes in target cell population over time. MC3T3 cells that become GFP positive, indicative of exosome mediated transfer, increased from 0.91% of the population on day 3 post co-culture to 17.3% on day 14. Subsequent cell sorting and RNA-seq of GFP positive MC3T3 populations yielded novel insights into the progressive transcriptional effect of cancer exosomes on target cells. Future research will expand upon this approach to examine the effects on bone cells non-metastatic and metastatic cancer cell lines have on the metastatic niche through exosome-mediated signaling. This approach introduces a novel method for prolonged transcriptional analysis of cancer and target cell co-culture allowing the segregation of exosome affected and unaffected subpopulations of target cells.

This study was funded by DOD grant BC151687. This work was conducted under the auspices of the USDOE by LLNL (DE-AC52-07NA27344).

#2137

Osteoblast-lineage cells protect AML cells from cytarabine-induced apoptosis via a mechanism sensitive to HDACi and reduced cell-cell contact.

Rosalie M. Sterner, Kimberly N. Kremer, Meagan R. Rollins, Amel Dudakovic, Jennifer J. Westendorf, Andre J. van Wijnen, Karen E. Hedin. _Mayo Clinic, Rochester, MN_.

The endosteal niche is a component of the bone marrow microenvironment that can serve to protect hematological malignancies such as acute myeloid leukemia (AML) from standard chemotherapies such as cytarabine (Ara-C). Surviving AML cells harbored by this niche can eventually lead to relapse. The endosteal niche is rich in osteoblast lineage cells. U937 or KG1a AML cell lines were cultured with or without osteoblast lineage cells (MC3T3 or W-20-17 cell lines), challenged with doses of 0µM, 0.1µM, 0.5µM, 1µM, 5µM, or 10µM of Ara-C, and assayed for apoptosis via annexin-V staining and flow cytometry. Osteoblast lineage cells (MC3T3 or W-20-17 cell lines) were able to protect AML cells (U937 or KG1a cell lines) from Ara-C-induced apoptosis. Histone deacetylase inhibitors (HDACi) globally alter gene expression within cells. When we pre-treated osteoblast lineage cells (MC3T3) with the HDACi vorinostat (suberoylanilide hydroxamic acid, SAHA), it reduced the ability of the osteoblast lineage cells (MC3T3) to protect AML cells (U937) from Ara-C-induced apoptosis, which we have previously confirmed in the KG1a AML cell line as well. This indicates that osteoblast lineage cell-mediated protection of AML from Ara-C occurs via an HDACi sensitive mechanism. To begin to further explore the mechanism of action, we co-cultured AML cells (KG1a or U937) with and without osteoblast lineage cells (MC3T3) in the presence or absence of a transwell. We found that the presence of the transwell reduced the osteoblast lineage cell-mediated protection, indicating that osteoblast lineage cell-mediated protection of AML from Ara-C is cell contact dependent. Thus, osteoblast lineage cells can protect AML cells from Ara-C induced apoptosis, this protection can be reduced by pre-treatment of the osteoblast lineage cells with the HDACi vorinostat, and osteoblast lineage cell-mediated protection from Ara-C is cell contact dependent. These studies begin to characterize the mechanisms of osteoblast lineage cell-mediated protection of AML from Ara-C. Manipulating the protective properties of osteoblast lineage cells of the endosteal niche may help make AML cells more susceptible to chemotherapeutics. Therefore, developing combination therapies that target the protective mechanisms of osteoblast-lineage cells may help to further deplete the bone marrow microenvironment of AML cells and prevent relapse of disease.

#2138

**Understanding the microenvironment contribution to ductal carcinoma** in situ **transition to invasiveness in breast cancer.**

Núria Moragas, Leire Recalde-Percaz, Aleix Noguera-Castells, Patricia Fernandez-Nogueira, Mario Mancino, Pedro Gascón, Gemma Fuster. _IDIBAPS, Barcelona, Spain_.

Breast cancer (BC) is thought to arise from the linear evolution of begins ductal carcinoma in situ (DCIS) to the invasive ductal carcinoma (IDC). DCIS is a poorly understood disease. Diagnosis of DCIS is increasing in the last few years. All DCIS are usually treated, however only 40% of them will progress to IDC if left untreated. Therefore, the challenge in the clinical control of the DCIS is to identify the individual patient risk to develop IDC and apply an adequate treatment and/or follow up.

Moreover, microenvironmental factors including neuronal factors have been related to the initiation and progression of breast cancer. For this reason, we are studying how factors related to the nervous system regulate the transition from DCIS to IDC. Our objective is to increase the molecular understanding of the DCIS and its surrounding microenvironment. For this purpose we used a BC progression to invasiveness in vitro model using MCF10A as healthy cell line, MCF10DCIS as mild-aggressive/ DCIS cell line and MCF10A-T as invasive/IDC cell line. First of all, we analyzed the expression of several neural related genes that we have previously described to be associated with breast cancer progression and invasion in our DCIS model. We found that netrin 1 (NTN1) and its receptor UNC5A are upregulated in MCF10DCIS and inhibited in MCF10AT both at the genetic and protein level. In addition, treatment with NTN1 in in vitro 2D and 3D assays reduced epithelial - mesenchymal transition (EMT) markers in MCF10AT. Hence, our results suggest that NTN1 via its receptor UNC5A could be negative regulators of the progression to invasiveness in DCIS.

Moreover, in our model, semaphorin 3F (SEMA3F) as well as its receptors, neuropilin 1 (NRP1) and neuropilin 2 (NRP2), are downregulated in MCF10DCIS at the genetic and protein level. Furthermore, we detected that treatment of MCF10DCIS with SEMA3F resulted in an up-regulation of EMT genes. Therefore, SEMA3F and its receptors NRP1 and NRP2 could be pro-invasive elements in the transition of DCIS to IDC. Our results suggest that NTN1 and UNC5A could be considered as good prognosis indicators meanwhile SEMA3F and its receptors expression in DCIS would be indicators of poor prognosis. All these results will contribute to a better knowledge of the DCIS phase in BC, to describe new therapeutical approaches and also to properly classify the risk of progression of each DCIS lesion.

#2139

Evaluation of a novel hypoxia-activated prodrug strategy in colorectal cancer cells.

Omar Hussain,1 Henrik Johansson,2 Simon J. Allison,1 Daniel S. Pedersen,2 Roger M. Phillips1. 1 _Univ. of Huddersfield, Huddersfield, United Kingdom;_ 2 _University of Copenhagen, Copenhagen, Denmark_.

Introduction: Current chemotherapeutic drugs are often ineffective against cancer cells that reside within the tumor microenvironment due to contributing factors such as hypoxia (low O2 tension) and acidic extracellular pH (pHe). Hypoxia is known to drive the aggressive cancer phenotype in many solid tumors, making it a key component of drug development. The use of hypoxia-activated prodrugs (HAPs) has been extensively studied over 40 years but to date no HAP has been approved for use in humans, largely because of poor activity and toxicity profiles. The design of prodrugs that are activated by both hypoxia and acidic pHe may provide a greater degree of selectivity and activity than HAPs alone, and this study was designed to evaluate a series of novel bromosugar-based HAPs under hypoxic and acidic pHe conditions.

Method: A library of 13 HAPs was evaluated against HCT116 colorectal cancer cells and ARPE-19 human retinal epithelial noncancerous cell line. Cytotoxicity was determined under (i) aerobic condition (21% oxygen) at pHe 7.4 or mild acidic conditions (pHe 6.5) and (ii) hypoxic condition (0.1% oxygen) at pHe 7.4 or mild acidic conditions (pHe 6.5). ARPE-19 cells were only tested under aerobic conditions and pHe 7.4 to reflect normal physiologic conditions. Compounds were tested at different concentrations ranging from 0.1-100µM from which an IC50 value was determined. Cell survival was measured after a 96-hour continuous drug exposure using the MTT assay.

Results: Among the compounds screened, DS10 demonstrated the most enhanced potency against HCT116 cells under hypoxic conditions with an IC50 value of 28.18 ± 7.01 µM as compared to an IC50 value of 66.39 ± 8.10 µM under normoxic conditions at pHe 7.4. Potency was further increased when DS10 was evaluated under a combination of hypoxic and acidic pHe conditions with an IC50 value of 16.30 µM ± 4.11. ARPE-19 cells showed an IC50 value of 42.95 µM ± 5.03 under aerobic conditions.

Discussion: The results of this study demonstrated that bromosugar derivatives may have the potential to selectively target hypoxic cancer cells in an acidic extracellular environment. Specifically, DS10 exhibited potential HAP properties with the ability to target cells that reside within the hypoxic and acidic microenvironment of tumors. Selectivity to tumor cells as opposed to noncancer cells was observed under these conditions, suggesting that DS10 is a promising compound to take forward for further evaluation.

#2140

Mesenchymal stromal cells expressing a PEAK1/Cripto axis sustain pro-survival NF-κB signaling in adjacent tumor cells to promote disease progression and therapy resistance.

Sarkis Hamalian,1 Robert Güth,1 Ioannis Zervantonakis,2 Erika Duell,1 Jia-Ren Lin,2 Preston Shisgal,1 Justin Molnar,1 Cameron Geller,1 Megan Agajanian,1 Julia Tchou,3 Peter K. Sorger,2 Joan S. Brugge,2 Jonathan A. Kelber1. 1 _California State University, Northridge, Northridge, CA;_ 2 _Harvard Medical School, Boston, MA;_ 3 _University of Pennsylvania Perelman School of Medicine and Abramson Cancer Center, Philadelphia, PA_.

The cellular and molecular heterogeneity of solid tumors, like breast cancer, is a significant hurdle in the effort to develop effective therapies. The complex breast cancer microenvironment (BCME) includes mesenchymal stromal cells (MSCs) that engage in paracrine/juxtacrine signaling with adjacent tumor cells to support disease progression. Since PEAK1 (Pseudopodium-Enriched Atypical Kinase One) is a cytoskeleton-associated kinase that regulates growth factor-integrin signaling crosstalk in mesenchymal cell types and recent evidence demonstrates that stromal expression of PEAK1 correlates with breast cancer recurrence, we hypothesized that PEAK1 expression in the mesenchymal compartment of the BCME promotes tumorigenesis. We report that PEAK1 is expressed in patient-derived cancer-associated fibroblasts (CAFs) from all breast cancer subtypes. Additionally, PEAK1hi CAF conditioned media promoted breast cancer cell (BCC) proliferation, migration and therapy resistance in vitro. We established in vitro co-culture and in vivo co-xenografting models for evaluating the effects of defined MSC populations on BCC proliferation/survival, metastasis and therapy response. Co-xenografting PEAK1hi CAFs or MSCs with BCCs increased the mass of HER2-positive and ER-positive primary tumors. In agreement with these data, co-culturing MSCs with BCCs promoted cancer cell proliferation and resistance to lapatinib or tamoxifen treatment, while MSC conditioned media supported BCC expansion under serum-free conditions in vitro. Notably, PEAK1 knockdown in MSCs abrogated their ability to promote tumor growth in vivo and therapy/stress resistance in vitro. Using the highly multiplexed cyclic immunofluorescence (CycIF) platform, we analyzed tumor cell states in these MSC-BCC co-cultures at different time points following lapatinib treatment. We demonstrate that MSCs sustain NF-κB (p65) signaling and anti-apoptotic gene expression within BCCs in the presence of lapatinib. Finally, we discovered that PEAK1 is required for MSC expression of Cripto, a GPI-anchored glycoprotein previously reported to activate NF-κB signaling in trans. Taken together, this work identifies new PEAK1-dependent stroma-tumor signaling vulnerabilities that may be exploited for improving patient responses to current therapeutic interventions.

#2141

Stromal SPARC deficiency skews prostate cancer toward neuroendocrine differentiation.

Claudia Enriquez,1 Valeria Cancila,2 Ivano Arioli,1 Claudio Tripodo,2 Mario Paolo Colombo,1 Elena Jachetti1. 1 _Istituto Nazionale dei Tumori, Milan, Italy;_ 2 _University of Palermo, School of Medicine, Palermo, Italy_.

Tumor progression is a multifaceted process in which, complex interactions between tumor and different types of stromal cells and extracellular matrix components, actively contribute to its phenotypic heterogeneity. Among extracellular matrix proteins, secreted protein acidic and rich in cysteine (SPARC) has been deeply studied since conflicting reports have described its expression to be either increased or decreased in different cancer settings, also depending on whether it is produced by the neoplasm or by the neighboring stroma. Nevertheless, the different contribution of tumor- or stromal-derived SPARC in prostate tumor microenvironment has not been addressed at least for tumor tissue histotype. Given this evidence, we aimed at providing new insights into the mechanism by which SPARC modulation influences prostate cancer development and progression.

We modeled human disease using TRAMP mice, which spontaneously develop autochthonous prostate tumors following the onset of puberty. Crossing TRAMP mice with Sparc-/- mice, we found the appearance of focal areas of neuroendocrine differentiation within adenocarcinoma. In patients this phenomenon commonly results after androgen ablation therapy and correlates with poor prognosis.

Interestingly, areas of neuroendocrine differentiation in Sparc-/- TRAMP mice were both positive for cytokeratin 8 and synaptophysin (usually expressed by luminal proliferating cells within adenocarcinoma or neuroendocrine cells, respectively), further suggesting a differentiation of adenocarcinoma cells to a neuroendocrine-like phenotype. Moreover, immunohistochemistry showed SPARC positivity not only in scattered tumor cells but also in fibroblasts and myeloid cells infiltrating TRAMP prostate. Accordingly, in vitro experiments suggested that stromal-derived SPARC limits neuroendocrine differentiation of prostate cancer cells, while they excluded a role of endogenous SPARC in this phenomenon. Indeed, prostate cancer cell lines co-cultured in presence of Sparc-deficient fibroblasts increased or acquired neuroendocrine features. This likely occurs through the effect of IL-6, a cytokine recently discovered to induce neuroendocrine differentiation, and that we found to be released by Sparc-deficient, but not sufficient, fibroblasts.

Data collected so far indicate that stromal SPARC deficiency skews prostate carcinogenesis toward neuroendocrine differentiation. A deeper understanding of the molecular mechanisms governing the balance between prostate adenocarcinoma and neuroendocrine tumors according to extracellular matrix composition will provide important insights for the development of new prognostic and therapeutic strategies.

#2142

High variability of TIGIT expression in Hodgkin's lymphoma.

Ronald Simon,1 Niclas C. Blessin,1 Martina Kluth,1 Kristine Fischer,2 Claudia Hube-Magg,1 Wenchao Li,1 Georgia Makrypidi-Fraune,1 Björn Wellge,1 Tim Mandelkow,1 Nicolaus F. Debatin,1 Guido Sauter,1 Waldemar Wilczak,1 Andrea Hinsch1. 1 _Univ. Medical Ctr. Hamburg-Eppendorf, Hamburg, Germany;_ 2 _dianova GmbH, Hamburg, Germany_.

Hodgkin's lymphoma (HL) is characterized by a high background of inflammatory cells which play an important role for the pathogenesis of the disease. T cell immunoreceptor with Ig and ITIM domains (TIGIT) is an inhibitory immune checkpoint receptor and a putative target for novel immune therapies. To study patterns of TIGIT expression in the T cell background surrounding malignant cells including Hodgkin cells, Reed-Sternberg cells and histiocytic cells, a microenvironment (ME) tissue microarray (TMA) was constructed from tissue punches measuring 2 mm in diameter obtained from formalin-fixed tissue samples of Hodgkin lymphoma lymph nodes (n=40) and normal human tonsil (n=2) as a reference. The ME-TMA was stained with brightfield and multiplex fluorescence immunohistochemistry (IHC) in order to evaluate expression levels of TIGIT and PD-1 as well as standard lymphocyte markers (CD8, CD4, FOXP3) in the lymphocytic background. TIGIT and PD-1 expression was found in all (100%) analyzed HL samples. In general, TIGIT localized to the same cells as PD-1. IHC based identification of T cell subtypes revealed TIGIT and PD-1 expression on CD8+ cytotoxic T cells, CD4+ helper T cells and FOXP3+ regulatory T cells. Strikingly, expression levels of TIGIT and PD-1 were highly variable among the analyzed samples irrespective of the histological subtype of HL. Interestingly, highest levels of both proteins were found in one sample of nodular lymphocytic-predominant HL (NLPHL). The high variability of TIGIT and PD-1 expression was also independent from the T cell subtype. HL with high TIGIT/PD-1 expression on CD8 positive cells sometimes showed low TIGIT/PD-1 expression on CD4 or FOXP3 positive T cells and vice versa. However, the T cell subtypes differed with respect to the TIGIT:PD-1 ratio. In the majority of analyzed HL, FOXP3+ regulatory T cells expressed higher levels of TIGIT than of PD-1. This was different for CD8+ and CD4+ cells, where similar fractions of HL showed either more TIGIT as PD-1, or more PD-1 as TIGIT, or comparable levels of both receptors. In conclusion, TIGIT (and PD-1) expression is highly variable between patients with Hodgkin's lymphoma. TIGIT may play a particularly important role in FOXP3 regulatory T cells in the lymphocytic background.

#2143

IL-6 mediated signaling regulates cytotoxic effect of Burkitt lymphoma cells by combined treatment of Danusertib and BKM120.

Jun Liu,1 Kwang-Sung Ahn,2 Jun-Shik Hong,3 Sung-Soo Yoon3. 1 _Seoul National University, Seoul, Republic of Korea;_ 2 _PDXen Biosystems Co., Daejeon, Republic of Korea;_ 3 _Seoul National University Hospital, Seoul, Republic of Korea_.

Even though Burkitt Lymphoma is relative has a good response to combined treatment, some part of outcome of chemotherapy is very poor. One of the obstacles is the chemoresistance due to several cytokines which are released from stromal cells. The effects of either Danusertib or BKM120 on Namalwa and BJAB, respectively, were different. On Namalwa the IC 50 of Danusertib was 24.64 µM (72 h) and BKM120 was 12.44 µM (72h). On BJAB the IC 50 of Danusertib was 6.63 µM (72h) and BKM120 was 1.42 µM (72h). But in the combined treatment, we found that BJAB showed less sensitive than Namalwa, the IC 50 on Namalwa was Danusertib 8.33 µM+ BKM120 2.08 µM (72h) and on BJAB was Danusertib 20.36 µM+ BKM120 5.09 µM (72h). To figure out the resistant pattern of these two cell lines we performed 3 steps treatment with 16 µM Danusertib and 4 µM BKM120, every step we culture 1.5×105/mL cells in 24hours first, and then treated with combined inhibitors 48 hours. We found that although Namalwa was sensitive to combined treatment at the first step, it was more easily to become resistant to combined treatment. To confirm the resistant pattern, co-culture system was used to identify what kinds of cytokines are involving in the chemoresistance of Burkitt lymphoma. We found that IL-6 was induced with stromal cell including inflammatory cytokines when cells were co-cultured with stromal cells. Also, IL-6 mediated signaling pathway such as JAK/STATIL-6 and AKT pathway, was activated by IL-6 and sIL-6. We suggest that IL-6 mediated signaling could mediate signaling regulates cytotoxic effect of Burkitt lymphoma cells by combined treatment of Danusertib and BKM120 as well as inflammatory effects may regulate the response of chemotherapeutic treatment.

#2144

Enhanced expression of β-catenin and Axl receptor tyrosine kinase in chronic lymphocytic leukemia (CLL) B-cells with co-culture on marrow stromal cells: Implications for drug resistance.

Sutapa Sinha,1 Charla Secreto,1 Justin Boysen,1 Steven L. Warner,2 David J. Bearss,2 Asish Ghosh,3 Neil E. Kay1. 1 _Mayo Clinic, Rochester, MN;_ 2 _Tolero Pharmaceuticals, Inc., USA, Lehi, UT;_ 3 _Stephenson Cancer Center/The University of Oklahoma Health Sciences Center, Oklahoma City, OK_.

Our study in CLL delineated a unique mechanism of how bone marrow stromal cells (BMSCs), may influence CLL B-cell signaling and drug resistance via modifying β-catenin and Axl status in leukemic cells. Purified primary CLL B-cells (n= 31) from previously untreated CLL patients were cultured alone or co-cultured with primary BMSCs from either normal individuals (n=23) or CLL patients (n=12) at a 50:1 ratio in AIMV medium. After 48 hours, separated CLL B-cells or BMSCs were examined by immunoprecipitation/Western blot analyses and real-time PCRs. We observed significant increases in expression of Axl at both mRNA and protein levels in CLL B-cells co-cultured with BMSCs compared to CLL B-cells cultured alone. We also detected significantly increased expression of β-catenin at the protein level in co-cultured CLL B-cells. But, we did not see any significant change in β-catenin or Axl expression in BMSCs co-cultured with CLL B-cells. The CLL B-cells from co-culture had an upregulation in downstream P-ERK-1/2 but no change in P-AKT(Ser473). High nuclear active-β-catenin and P-ERK-1/2 levels were also detected in co-cultured CLL B-cells. ERK associates with and inactivates GSK-3β resulting in the up-regulation of β-catenin. We next checked for P-GSK-3β(Ser9) in co-cultured CLL B-cells. Upregulation in P-GSK-3β(Ser9) suggests inactivation of GSK-3β and β-catenin accumulation in co-cultured CLL B-cells. Additionally, we determined the phosphorylation status of Axl in CLL B-cells in co-culture compared with cultured alone. There was no change either at Y702 or total tyrosine phosphorylation in CLL B cells. Thus, we assume that the role of Axl in co-cultured leukemic B-cells is independent of its kinase activity. Next we determined the effect of Axl inhibitor TP-0903 on CLL B-cells in co-culture vs cultured alone. Interestingly, both Axl and β-catenin expression levels were further upregulated in CLL B-cells exposed to TP-0903 compared to CLL B-cells without TP-0903 in co-culture, indicating possible roles for both Axl and β-catenin in stromal mediated CLL B-cell drug resistance. However, TP-0903 decreased P-Axl(Y702) as well as an Axl downstream mediator, P-Akt(S473) and reduced Mcl-1 expression in CLL B-cells even in presence of BMSCs. This was associated with induction of cell death and overrode BMSC mediated protection. Treatments of fludarabine or chlorambucil also led to increase in expressions of both β-catenin and Axl in co-cultured CLL B-cells. Moreover, inhibition of P-ERK-1/2 with inhibitor PD98059 in CLL B-cells cultured with BMSCs inhibited β-catenin as well as Axl expression levels. Here we show that stroma mediated increased expression in both β-catenin and Axl likely contribute to the survival and drug resistance of B-cells from patients with CLL. Understanding their role in drug metabolism is underway.

#2145

The role of MEN1611, a class I PI3-kinases (PI3Ks) inhibitor, in reprogramming the pro-tumoral inflammatory environment.

Stefania Capano,1 Alessio Fiascarelli,1 Andrea M Tomirotti,1 Mario Bigioni,1 Alessandro Bressan,1 Daniela Bellarosa,1 Andrea Pellacani,2 Monica Binaschi1. 1 _Menarini Ricerche S.p.A., Pomezia, Italy;_ 2 _Menarini Ricerche S.p.A., Firenze, Italy_.

MEN1611 (previously PA799) is a specific PI3Ks inhibitor with peculiar biochemical profile: it targets at the highest potency the mutated PI3Kα isoforms and the PI3Kγ isoform. PI3Kγ isoform is abundantly expressed in myeloid cells but not in cancer cells and promotes myeloid cell trafficking during inflammation and cancer. Previous studies have shown that selective inactivation of macrophage PI3Kγ is able to promote an immunostimulatory transcriptional program that restores CD8+ T cell activation and cytotoxicity. Based on these evidences, we sought to investigate whether the anti-tumoral activity of MEN1611, that has been demonstrated especially in tumors harboring PI3Kα mutations, should be mediated also by inflammatory cells. To this aim we have set up an in vitro model in order to study macrophages polarization: in the presence of colony stimulating factor, mouse bone marrow-derived monocytes were directed into M0 macrophages differentiation, followed by M1 (by lipopolysaccharides and interferon γ) or M2 stimulation (by interleukin 4). Potential changes in macrophage phenotype induced by MEN1611 were measured by quantitative RT-qPCR and mouse cytokine antibody array. Moreover in order to evaluate in vivo the immune modulatory capacity of MEN1611 two syngeneic tumor models were established: 4T1 breast tumor model and CT26.wt colorectal tumor model. M1 or M2 polarized macrophages displayed different cell morphologies: M2 cells exhibited an higher degree of elongation compared with M1 or unstimulated M0 cells. Their activation status was tracked and confirmed by gene expression analysis, enzymatic assays (iNOS and arginase activity) and cytokine array. MEN1611 treatment was able to revert M2 cell morphology towards an M1-like round cell shape. Analysis of gene expression in MEN1611-treated macrophages showed a significant increase of immunostimulating factors mRNAs, such as IL-1b, IL-12b and iNOS, in M2 macrophages. MEN1611 also enhanced the secretion of pro-inflammatory cytokines both in M1 and M2 macrophages. In syngeneic mouse models, immune cell subsets were characterized via flow cytometry: both 4T1 and CT26.wt tumors had a similar baseline relative amount of M1 and M2 macrophages and were characterized by CD4+ and CD8+ T cells infiltrates. The impact of MEN1611 treatment on macrophages and T lymphocytes subsets infiltrating tumors is currently studied. In conclusion, we have demonstrated the ability of MEN1611 to modulate macrophage polarization towards an immune-activating phenotype at clinically relevant doses. These findings might suggest the involvement of PI3Kγ as part of MEN1611 mechanism of action. 

### Translational Therapeutics in Cancer Models 2

#2146

Activity of entinostat alone and in combination with cisplatin in a panel of low passage adenoid cystic carcinoma patient-derived xenograft (PDX) models.

Amanda Mangold,1 Melissa Rundle,1 Nicole Spardy Burr,2 Alyssa Moriarty,1 Priscila Goncalves,3 Rogerio Castilho,4 Peter Ordentlich,5 Jeffrey Kaufman,2 Kyriakos Papadopoulos,1 Michael J. Wick1. 1 _START, San Antonio, TX;_ 2 _ACCRF, Needham, MA;_ 3 _Monter Cancer Center, Northwell Health, Lake Success, NY;_ 4 _University of Michigan School of Dentistry, Ann Arbor, MI;_ 5 _Syndax Pharmaceuticals, Waltham, MA_.

Background: Adenoid Cystic Carcinoma (ACC) is a rare cancer of secretory glands, most typically occurring in the salivary glands. No approved standard of care exists for systemic therapy. To identify potentially useful therapies, we screened Food and Drug Administration (FDA)-approved and investigational therapies using a well characterized panel of low passage ACC PDX models. Based on these results, combination treatments were evaluated for additive or super-additive effects. In line with the clinical chemo resistance observed in ACC patients, we previously reported that ACC tumor models are insensitive to platinum therapies. Interestingly, histone deacetylase (HDAC) inhibitors have been shown to sensitize cancer cells to anti-cancer agents in vitro. Entinostat is an oral class 1 selective HDAC inhibitor in Phase 3 testing for ER+ breast cancer in combination with hormone therapy. To better understand the potential effects of entinostat in ACC, we evaluated this agent alone and in combination with cisplatin in ACC PDX tumor models.

Methods: Low passage ACC models were established in immune-deficient mice from primary or metastatic patient tissue and confirmed by histologic comparative analysis. Drug sensitivity studies evaluating entinostat, cisplatin and the combination were performed in three models, including ACCx5M1, ACCx6 and ACCx9. Study endpoints included tumor volume and time from treatment initiation with tumor growth inhibition, delay and regression reported at study completion.

Results: Entinostat, cisplatin and the combination were all well tolerated with minimum cycle-based weight loss. Cisplatin tested alone (3 mg/kg weekly) was similar to control in all models, while single agent entinostat (5 mg/kg daily) showed significant anti-tumor activity in the ACCx5M1 (p<0.05) and ACCx9 (p<0.0001) models. Combination of entinostat and cisplatin exerted significant anti-tumor activity versus control in ACCx5M1 (p<0.01) and in ACCx9 versus control (p<0.0001) or entinostat alone (p<0.05). Sequence based and other characterization analysis is currently underway to correlate sensitivity and resistance between models.

Conclusion: Our studies identify entinostat as an agent of potential benefit in treating ACC and demonstrate that entinostat can induce low-dose cisplatin activity in the ACCx9 model, which harbors a mutation in the NOTCH1 gene that has been linked with chemo resistance and worse prognosis. Genomic and other characterization methods are currently underway to correlate sensitivity and resistance between models.

#2147

Development of preclinical models to accelerate the identification of next generation treatments for patients with acquired resistance to targeted therapies.

Olivier Déas,1 Ludovic Bigot,2 Guillaume Lang,1 Yohann Loriot,2 Fabrice Andre,2 Jean Charles Soria,2 Benjamin Besse,2 Stefano Cairo,1 Marie Tavernier,1 Katell Mevel,1 Enora Le Ven,1 Jean-Gabriel Judde,1 Luc Friboulet2. 1 _XenTech, Evry, France;_ 2 _Gustave Roussy, Villejuif, France_.

The last 20 years have witnessed the identification of an increasing number of druggable oncogenic drivers and the development and clinical use of specific inhibitors against these targets. Unfortunately, patients treated with targeted therapies consistently develop resistance and progression under treatment. Hence, important scientific, pharmaceutical and medical research efforts are directed towards understanding the mechanisms of acquired resistance to explore new therapeutic pathways.

The MATCH-R clinical trial enrolls patients with oncogene-driven cancer who have had previous clinical response to targeted therapy and subsequently experienced disease progression. In the framework of this project, Gustave Roussy and XenTech are joining forces to develop a panel of patient-derived xenografts (PDXs) derived from biopsies collected from these patients at the stage of acquired resistance. These PDX models will be used to improve knowledge on the mechanisms underlying resistance to treatment and to evaluate response to new treatments.

In this perspective, the development of 75 PDX-AR (Active Resistance) models is planned over 3 years. All the models are maintained under the same therapeutic pressure the parental tumor was submitted to at the time of biopsy, and will be subjected to extensive phenotypic and genotypic characterization.

The following models have been established so far:

• ENDx-MR-004-AR (endometrial): resistant to the combination of MEK and MDM2 inhibitors;

• LCx-MR-007-AR: (NSCLC): resistant to third generation EGFR inhibitor (osimertinib);

• UREx-MR-015A-AR (ureter) and VEx-MR-086A-AR (bladder): resistant to a FGFR inhibitor (erdafitinib);

• PARx-MR-010-AR (parotid): resistant to a NOTCH Inhibitor;

• TCx-MR-122-AR (colon): resistant to an ATR inhibitor.

To favor successful xenograft establishment, the first two passages were performed without drug treatment, which was applied from the third passage on. When doing so, some models showed resistance from the first passage under treatment, whereas others showed stabilization under treatment at the first passages and rapidly acquired resistance over passages. These different behaviors might underlie different mechanisms of resistance, irreversible (monoclonal) for the former, reversible (polyclonal) for the latter.

Parallel to the development of UREx-MR-015A-AR, we developed the UREx-MR-015B-SD (stable disease) model from a biopsy collected from a different metastasis in the same patient, but stabilized by the therapy. Comparative analysis of these two models will provide important insights into the mechanisms of resistance to FGFR inhibitors. The MATCH-R PDX project will provide a unique preclinical platform for identifying resistance mechanisms to current targeted therapies and developing next generation therapeutic strategies.

#2148

Image-guided catheter-based ultrasound thermal ablation of intramuscular and retroperitoneal sarcomas in the transgenic Oncopig cancer model.

Laurie A. Rund,1 Goutam Ghoshal,2 Emery Williams,2 Paul Neubauer,2 Lance Frith,2 R Tamas Heffter,2 Patrick Roady,1 Lawrence B. Schook,1 E Clif Burdette2. 1 _Univ. of Illinos at Urbana-Champaign, Urbana, IL;_ 2 _Acoustic MedSystems, Savoy, IL_.

Soft-tissue sarcomas (STS) are aggressive, often lethal mesenchymal tumors that represent roughly 1% of adult cancers. The 5-year survival rate of STS has remained unchanged for decades at 50%, highlighting the need for more effective therapies. We are developing protocols and assessing the treatment efficacy of 3D spatially-registered real-time image-guided needle/catheter based ultrasound (CBUS) ablative thermal therapy in STS induced in the transgenic Oncopig cancer model (OCM). The transgenic Oncopig carries a Cre recombinase inducible transgene encoding KRASG12D and TP53R167H; both mutations commonly in human cancers. Cre recombinase induces expression of the transgenes and transformation of cells in culture and rapid and reproducible development of leiomyosarcomas when introduced into the muscle of oncopigs. The flexibility of the model allowed induction of sarcomas in two clinically relevant intramuscular (IM) sites in both the hind limb and retroperitoneal (RP) regions, to closely simulate human disease. We have previously shown that these Oncopig tumors present the same transcriptional hallmarks as human STS including altered TP53 signaling, Wnt signaling activation and evidence of epigenetic reprogramming. Initial acute studies revealed that the tumors could be successfully grown in the muscle reaching mean tumor volume of 19.76cm3 (RP, n= 8) and 25.12cm3 (IM, n=14) cm3 within two weeks following AdCre (5.0x108 to 1.0x109 PFU) injections. Tumors were then treated utilizing ultrasound image guidance combined with 3D EM tracking to place the applicator in the target region. Sectored tubular transducers were introduced through a small skin incision and used to precisely deliver thermal energy to the treatment region. Tumors were treated for 6-9 minutes at 7 watts acoustic power (10.2W/cm2). Thermocouples were inserted into the tumors monitored temperature throughout the treatment. Post-treatment analysis revealed that the mean induced lesion volume exceeded the actual tumor volume for both IM (43.41 cm3) and RP (22.87 cm3) tumors with mean peak temperatures reaching 64.56 ͦ C (IM) and 58.75 ͦ C (RP). The mean peak thermal dose (thermal equivalent minutes at 43 ͦ C - TEM43) accumulated for IM tumors was 3.70E+08, and 1.46E+06 for RP tumors. Histopathology analysis verified that treatment areas reached past tumor margins using single applicator configuration. This study clearly demonstrates that the Oncopig is a suitable model in size and clinically relevant tumor type for evaluation of ultrasound ablation instrumentation. In addition, these STS treatment protocols can reliably and accurately target and treat tumor volumes plus surrounding margins. Future work will include monitoring of treatment lesions for up to 3 months post ablation. Funded by NIH 5R21CA195433

#2149

Use of PDXs and patient-derived cell lines to uncover unconventional drug therapies and combinations for the treatment of drug-resistant cancers.

Marguerite Buchanan,1 Catherine Chabot,1 Josiane Lafleur,1 Urszula Krzemien,1 Cathy Lan,1 Olga Savichtcheva,1 Jean-François Boileau,1 Cristiano Ferrario,1 Paul Savage,2 Morag Park,2 Gerald Batist,1 Adriana Aguilar-Mahecha,1 Mark Basik1. 1 _Jewish General Hospital, Montréal, Quebec, Canada;_ 2 _Goodman Cancer Center, McGill University, Montréal, Quebec, Canada_.

The use of patient derived models for pre-clinical drug testing provides a unique opportunity for testing non-conventional therapies and novel drug combinations to overcome drug resistance in cancer. We have established 22 models from breast cancer, 20 PDXs were engrafted in house and two were generated from live tissue obtained from PDXs established in a partner lab. The overall engraftment rate in house was of 24%. Engraftment varied according to BC subtype: 38% (3/8) for ER-HER2+ and 33% (17/51) for TNBC. No PDXs could be generated from ER+PR+Her2- tumors (0/13) or ER+HER2+ breast cancers (0/11). Post-chemotherapy drug resistant tumors engrafted with better success (18/55) compared to non-treated tumors (2/28). In 3 patients, we established PDX models with tissues collected at different anatomical sites, enabling the study of tumor heterogeneity. For one patient, 2 PDX models were established from serial samples after different lines of chemotherapy. Molecular profiling at the copy number and mutation level has confirmed the molecular stability of our models over engraftment passages. Drug studies were performed to validate clinical drug response and models were also treated with unconventional drugs and drug combinations to overcome drug resistance. Drugs that resulted in significant tumor regression in drug resistant TNBC models included irinotecan as well as olaparib in combination with an angiogenesis inhibitor. We successfully established one PDX model from a recurrent Her2+ gastric tumor resistant to trastuzumab. In this model, treatment with T-DM1 alone as well as with pertuzumab/trastuzumab resulted in complete tumor regression, while everolimus significantly inhibited tumor growth. This PDX study provided an alternative clinical management for the patient who subsequently received T-DM1 and showed clinical response, validating the results obtained in the PDX model. To develop a more agile model, we also established conditionally reprogrammed cell lines directly from patient tumors or from PDX models. Drug resistant conditionally reprogrammed cells from Her2+ breast cancers harboring a FGFR1 amplification were found to be sensitive to FGFR inhibitors, chloroquine, and a CDK4/6 inhibitor. Correlation of drug responses between conditionally reprogrammed cells and matched PDX models will be presented. These live tumor models enable timely and clinically pertinent drug testing, which has the potential to expand the known therapeutic arsenal as well as provide truly personalized therapy to cancer patients.

#2150

Rapid and efficient evaluation of drug sensitivity in a diverse panel of chordoma xenograft models.

Patricia Cogswell,1 Michael Wick,2 Melissa Rundle,2 Amanda Mangold,2 Josh Sommer,1 Joan Levy1. 1 _Chordoma Foundation, Durham, NC;_ 2 _START, San Antonio, TX_.

Background: Chordoma is a rare cancer found in the base of the skull and the spine that develops from residual embryonic notochord cells. Currently, no systemic therapies are approved for treatment of the disease, but emerging understanding of chordoma biology and tumor dependencies has revealed a number of potential therapeutic targets. To date, validation of novel targets and preclinical evaluation of corresponding therapies has been hampered by scarcity of preclinical models as well the high cost of starting in-vivo experiments. To facilitate more rapid and efficient evaluation of new therapeutic approaches, the Chordoma Foundation has developed a centralized Drug Screening Program (DSP) operated through a partnership with South Texas Accelerated Research Therapeutics (START).

Methods: Patients undergoing surgical resection are consented through the Chordoma Foundation's IRB approved biobanking protocol. Tumor tissue is shipped to START, engrafted in immunodeficient mice, and expanded. Resulting patient-derived xenograft (PDX) models are validated by confirmation of histology and expression of brachyury, a protein highly expressed in chordoma tumors. Additional PDX models and cell line derived xenograft (CDX) models are acquired from academic institutions and established at START for use in screening experiments. Drugs are nominated by academic and industry investigators and prioritized by the Chordoma Foundation on the basis of molecular rationale and existing in vitro data. Selected drugs are evaluated for efficacy in the panel of PDX and CDX models. Data is provided back to the nominating investigator, and, when permitted, is made public through an online data repository.

Results: A diverse collection of seven validated PDX and CDX models has been developed and is available for in-vivo drug screening experiments. These models represent different clinical subtypes of chordoma, including five derived from conventional chordomas and two derived from poorly differentiated tumors, the latter being an aggressive subtype of chordoma typically found in pediatric patients and young adults. To date, twenty-four drugs have been tested as single agents or in combination in various models. A range of activities have been observed, which may be dependent on molecular alterations present in the individual models. Inhibitors of EGFR and CDK4/6 demonstrated the greatest activity across models. This data has supported the initiation of two clinical trials specific for chordoma patients testing the targeted agents afatinib and palbociclib.

Conclusion: The Chordoma Foundation's DSP serves as a core facility for the chordoma research community, providing investigators with a rapid and low-cost means of evaluating novel targets and therapies. It overcomes a key barrier to chordoma drug development and may provide a template that can be employed to other cancer types.

#2151

Development of small-molecule mTORC1 radiotracers for evaluation of targeted therapy resistance using PET.

Adam C. Parslow,1 Christian W. Wichmann,2 Angela Rigopoulos,1 Uwe Ackermann,1 Jonathan M. White,2 Andrew M. Scott1. 1 _Olivia Newton-John Cancer Research Institute, Melbourne, Australia;_ 2 _Bio21 Institute, Melbourne, Australia_.

Overcoming resistance is an area of unmet clinical need for patients undergoing trastuzumab-based HER2-directed therapy. PTEN loss or PI3K mutation has been shown to induce mTOR activation and mediate trastuzumab resistance. During the BOLERO-3 phase III clinical trial, the addition of everolimus to trastuzumab plus vinorelbine significantly prolonged progression-free survival in patients with trastuzumab-resistant and taxane-pretreated, HER2-positive advanced breast cancer. It is critically important to identify HER2-positive breast cancer patients who are sensitive to mTOR inhibition. Our aim is to develop noninvasive mTORC1 binding small-molecule imaging probes to predict breast cancer patient resistance to treatment utilizing positron emission tomography (PET). Utilizing the benzofuran-based compound ChemBridge 5219657, we have developed a panel of potent inhibitors specific for mTOR complex 1. Alkyne derivatives suitable for conjugation with 2-[18F]fluoroethylazide were developed via Cu(I)-catalyzed click chemistry. Radiolabeling was performed on the automated iPhase FlexLab module providing reliable amounts of radiotracer. Synthesis of click precursor was achieved over 5 linear steps with a 24% isolated yield. Quality control was characterized using NMR, MS, UV- and radio-HPLC analysis. 18F-labeling conjugation with precursor resulted in a 40 ± 5% isolated radiochemical yield with >99% radiochemical purity and a molar activity of 12.5 ± 6.0 GBq/µmol.

Initial in vitro sensitivity to everolimus was performed via SRB proliferation analysis following cold-compound treatment on HER2-positive human breast cancer cell lines (BT-474 and HCC1419) and HER2 low expressing control cell lines (MDA-MB-468 and MDA-MB-231). In vitro 18F-labeled binding experiments demonstrated an increased uptake in BT-474 and HCC-1419 cells, 1.5 and 2.5-fold compared to the negative controls, respectively. NSG mice bearing BT-474, MDA-MB-231 and MDA-MB-468 xenografts were explored for in vivo dynamic PET imaging experiments confirming this selective uptake of radiotracer in everolimus-sensitive cells. Our synthesis strategies identified a reliable radiolabeling approach capable of producing sufficient amounts of radiochemically pure radiotracer for in vivo investigation. The increased tracer uptake in mTOR treatment-sensitive cell lines in vitro as well as in vivo provides an encouraging outcome warranting further preclinical studies for the investigation of HER2-positive therapy resistance in mTOR-sensitive models.

#2152

Optimizing therapies for drug-resistant ovarian cancer stem cells using aCAM-PDX model.

Altagracia Contreras,1 Sophia Allaf Shahin,1 Laura E. Ratliff,1 Shirleen I. Simargi,1 Ruining Wang,2 Binh Vu,2 Thanh H. Dellinger,1 Jeffrey I. Zink,2 Fuyuhiko Tamanoi,2 Julia Unternaehrer-Hamm,3 Carlotta A. Glackin1. 1 _City of Hope Beckman Research Inst., Duarte, CA;_ 2 _University of California, Los Angeles, Los Angeles, CA;_ 3 _Loma Linda University, Loma Linda, CA_.

Purpose: To develop rapidly operational CAM-PDX (chorioallantoic membrane-patient derived xenograft) tumor models that morphologically resemble human tumors in order to perform therapeutic/efficacy studies targeting TWIST, a transcription factor expressed in cancer stem cells critical to embryonic development, and aberrantly activated in many cancers. This CAM-PDX model was used to optimize mesoporous silica nanoparticle with hyaluronic acid (MSN-HA) and siTWIST in combination with cisplatin, as well as screen other drug therapies (e.g., doxorubicin) that will benefit ovarian cancer patients.

Experimental Procedures: Fertilized eggs were obtained from AA Lab Eggs, Inc. The eggs were incubated for 10 days at 100°F and 60% humidity. At day 10, a small window was opened on the surface of the egg and the CAM was dropped. A teflon ring was inserted, 50µg/µL of cancer cells (1 million cells) were deposited, and the window was covered with transparent film dressing and replaced in the incubator. 120µL of siTWIST (with MSN-HA) and/or 100µL of cisplatin were administered intravenously (day 13 and 14, respectively). On day 17, the eggs were imaged using Leica microscope. The CAM-PDX models were developed first with standard EOC cell lines and second using primary cells from patient tissues transfected with eGFP/ffluc for imaging growth, histology, and tumor biodistribution studies. Toxicity studies were performed by administering doxorubicin at varying doses and with different nanoparticle delivery methods.

Results: The study showed that we were able to grow patient-derived tumors in the egg CAM model. Moreove,r a histologic comparison of CAM tumors and ovarian patient tumors reveals highly similar morphology. Furthermore, when treated with chemotherapy, the tumor regressed, as it does in patients. Our toxicity study demonstrates that surrounding organs, as well as the embryo, were spared from detrimental off-target effects of doxorubicin when the drug was delivered inside nanoparticles, as opposed to free-drug, as demonstrated by final egg survivability count. Images show free-drug administration of doxorubicin damaged organs in the peritoneal cavity while nanoparticle administration of doxorubicin resulted in a clean cavity and sound organs. Our study also shows that our siTWIST MSN-HA sensitized drug-resistant cancer stem cells to cisplatin. Finally, our results also show that the optimal treatment for tumor dissemination in the PDX-CAM system is the combination of siTWIST MSN-HA and cisplatin compared to cisplatin alone.

Conclusions: This study provides a much-needed, cost-effective means to grow PDX lines from patient tissues. The CAM-PDX system provides a faster means of collecting information on the efficacy of novel MSNs for the delivery of siRNA and drugs than expensive mouse models. Thus, the CAM-PDX model is beneficial toward moving us closer to translation and application in other cancers.

#2153

Development of a novel MEK mutation driven drug resistant melanoma model by CRISPR/Cas9 technology.

Lysa-Anne Volpe, Metewo Selase Enuameh, Luping Chen, Elizabeth Turner, John Foulke, Fang Tian. _ATCC, Manassas, VA_.

Melanoma remains the most lethal form of skin cancer exhibiting high mortality rates due to a high likelihood of developing metastases and acquiring drug resistance. Approximately 40-50% of melanomas contain oncogenic BRAF mutations of which 75-90% harbor the BRAFV600E mutation. This mutation constitutively activates the mitogen-activating protein kinase (MAPK) signaling pathway leading to uncontrolled cell growth and oncogenesis. Recent combination therapies of BRAF and MEK specific inhibitors have shown improved progression-free patient response in phase II clinical trials. However, in the majority of patients, acquired resistance to MAPK pathway inhibitor therapies develops after approximately 12 months of treatment. Preclinical studies have suggested that MEK1 mutations confer resistance to BRAF and MEK inhibitors. In this study, we used the CRISPR genome editing technology to generate a drug resistant MEK1Q56P knock-in mutation within the A375 melanoma cell line which naturally harbors the BRAFV600E mutation. We validated this new isogenic cell model using both molecular and biofunctional approaches. Method and results: Single guide RNAs (sgRNAs) were designed and built into Cas9 plasmids to bind and cut desired regions in the MEK1 target region. The melanoma cell line A375 was co-transfected with single guide-containing Cas9 plasmids and donor plasmids. Single cells were cloned and expanded for subsequent screening of the desired gene mutation events. The introduction of the MEK1Q56P mutation in the cells was then confirmed via Sanger sequencing and NGS at the genetic level. Validation of the mutation in mRNA transcripts was carried out by RT-PCR followed by sequencing. The cell line maintains permanent and genetically stable resistance characteristics during cell culture expansion without the use of selective pressure. Drug responses to BRAF and MEK1 specific inhibitors and non-specific chemotherapy drugs were compared between the A375 MEK1Q56P isogenic cell line and the parental cell line in 2D and 3D culture environments. Results demonstrated that the isogenic MEK1Q56P cell line showed significant and specific resistance to BRAF inhibitors in comparison to the parental A375 line. Using gene editing we have targeted an endogenous loci within a melanoma cell line creating a novel in-vitro model to aid in basic and translational melanoma research as well as drug screening efforts. This new approach to cell line development provides direct in vitro bio-functional evidence of a drug resistant gene driving tumor cells survival under targeted anti-cancer treatments. Furthermore, this A375 MEK1Q56P isogenic cell line represents a new type of drug resistance model that contains a defined genetic resistance mechanism. This model provides an invaluable tool for developing next generation therapeutics that can overcome drug resistance in melanoma.

#2154

A potent and orally available tubulin inhibitor ABI-231 suppresses triple negative breast cancer tumor growth and metastasis.

Shanshan Deng, Raya Krutilina, Qinghui Wang, Tiffany N. Seagroves, Duane D. Miller, Wei Li. _University of Tennessee Health Science Center, Memphis, TN_.

Triple-negative breast cancer (TNBC) is the most aggressive type and accounts for at least 15% of all types of breast cancer in the USA. TNBC is defined as tumors that lack the expression of estrogen receptor, progesterone receptor and human epithermal growth factor 2, and it is always characterized by poor prognosis due to the lack of molecular targets and the rapid development of resistance to many chemotherapeutic drugs. Interfering with microtubule dynamics is a validated drug target. Currently, one of the standard cares for TNBC treatment is using taxanes based chemotherapy, such as paclitaxel, which targets microtubules. However, drug resistance and neurotoxicities often limit their clinical efficacy, thus there are continuous needs to develop more effective therapies that could overcome these clinical limitations. In this study, we evaluated the preclinical efficacy of our potent tubulin inhibitor, ABI-231, in treating TNBC in mouse xenograft models. ABI-231 inhibited tubulin polymerization and effectively overcame P-gp mediated multidrug resistance, and it is orally available. ABI-231 showed remarkable anticancer activities against two TNBC cell lines at low nanomolar concentrations, and it could inhibit the cancer cell colony formation. Treatment with ABI-231 suppressed invasion and migration of TNBC cells, and significantly induced apoptosis of TNBC cells in vitro. ABI-231 inhibited TNBC tumor growth in a dose-dependent manner without acute toxicity in an orthotopic TNBC xenograft model. ABI-231 had similar efficacy to that of paclitaxel as tested in this mouse model. In addition, ABI-231 significantly reduced metastasis of TNBC in a tail vein mouse model. Collectively, these preliminary studies strongly suggest that ABI-231 is potent to inhibit the growth and metastasis of TNBC both in vitro and in vivo, and this potent tubulin inhibitor is a promising drug candidate for the more effective treatment of TNBC. Supported by NIH grant R01CA148706.

#2155

Modeling therapeutic response in BRAF V600-mutant melanoma using patient-derived xenografts.

Tianshu Feng, Javad Golji, Ailing Li, Xiamei Zhang, David Ruddy, Darrin Stuart, Matthew Meyer. _Novartis Institutes for BioMedical Research, Cambridge, MA_.

Approximately 50% of metastatic melanoma harbor oncogenic mutations of BRAF, resulting in constitutive activation of the MAPK signaling pathway. Treatment using BRAF and MEK inhibitor combination significantly improved survival in patients with BRAF V600-mutant melanoma. However, the duration of response remains limited as most patients develop resistance. Previously, our group established a collection of patient derived xenografts (PDXs), and conducted a PDX clinical trial using a 1x1x1 experimental design. The PDX clinical trial showed that BRAF V600-mutant melanoma models varied in their response to BRAF and MEK inhibitors, similar to observations in the clinical setting. Therefore, we sought to elucidate the mechanisms governing differential responses to MAPK inhibition in vivo. The PDX models were classified into complete and partial responders based on their sensitivity to BRAF and MEK inhibitors. Using this classification, we characterized the downstream signaling associated with therapeutic response. Whereas complete responders showed almost full suppression of MAPK signaling, partial responders showed heterogeneous levels of MAPK signaling upon BRAF and MEK inhibition. Ongoing work seeks to identify determinants of therapeutic response using biased and unbiased approaches, in an effort to inform combination strategies that improve survival in patients with BRAF V600-mutant melanoma.

#2156

Lung niche factors for progenitor cell self-renewal sustain lung cancer stem cells and contribute to drug resistance to kinase inhibitors in non-small cell lung cancer.

Akinori Mitsui,1 Sachiko Yokokawa,1 Osamu Nagano,2 Hideyuki Saya2. 1 _Kyowa Hakko Kirin co., Ltd., Machida-shi, Tokyo, Japan;_ 2 _Keio university school of medicine, Shinjuku-ku, Tokyo, Japan_.

A mechanism underlying anti-cancer drug resistance relies on the existence of cancer stem cells (CSCs). However, CSCs are either absent or are few in number in xenograft tumors and syngeneic mouse models. We developed new lung cancer mouse models with CSCs to elucidate the role of CSCs in resistance to targeted therapeutics in non-small cell lung cancer (NSCLC). Mouse lung organoids containing progenitor cells were cultured in complete medium containing lung niche factors, FGF-10 and HGF, and were infected with a retrovirus encoding a Kras mutation (G12D), an Egfr deletion, and the EML4-ALK fusion protein. These induced CSCs were transplanted into mice, and the established tumors were cultured in vitro to test for drug sensitivity to trametinib, erlotinib, and crizotinib in the presence or absence of niche factors. The tumors that were poorly differentiated contained EpCAM-, Cd49f-, and Sca-1-positive CSCs. These mouse models allowed the prediction of clinical responses to targeted therapeutics, i.e., the tumor cells with the Egfr deletion and those with EML4-ALK fusion proteins showed strong responses to erlotinib and crizotinib, respectively. In addition, we found that Kras mutant cancer cells were resistant to trametinib due to activation of the HGF/Met pathway since HGF is an essential niche factor for lung progenitor cell proliferation. Combination treatment with trametinib and a Met inhibitor showed a synergistic effect in Kras mutant tumors. Based on these results, we propose a treatment approach that focuses on the use of niche factors for lung progenitor cells in NSCLC.

#2157

**Chemotherapeutic tolerability and estrogen dose response in the B6;129-** Rag2tm1FwaIL2rgtm1Rsky **/DwlHsd (R2G2) mouse model.**

Jamie L. Naden,1 Michele Melton,2 Athena Bast,2 Jermaine Gardner2. 1 _Envigo, Indianapolis, IN;_ 2 _Horizon Discovery, St. Louis, MO_.

The B6;129-Rag2tm1FwaIL2rgtm1Rsky/DwlHsd (R2G2) knockout mouse was developed by backcrossing an IL2rg (common gamma) knockout model to a RAG2 (recombinase activating gene) knockout model. The resulting mouse lacks various cytokines including IL-2, IL-4, IL-7, IL-9 and IL-15. In addition, this model lacks B cells, T cells, NK cells and has a deficit in lymphocyte development. This model was developed to provide another immunodeficient option for the oncology and immunology fields. The literature supports better tolerability of DNA-damaging oncology treatments for models that do not carry the SCID mutation. We have already reported in a white paper that the R2G2 mouse model is more tolerant of whole-body radiation than a model with the SCID mutation. Herein we describe a study examining chemotherapeutic tolerability of common DNA-damaging oncology drugs including 5-fluorouracil (5-FU), doxorubicin (Doxo), and cyclophosphamide (CTX) (n = 10 per group). 5-FU was given at 30, 60 or 100 mg/kg, intraperitoneally, twice weekly for five weeks. Doxo was given at 2 or 5 mg/kg, intraperitoneally, once weekly for three weeks. CTX was given at 100 or 140 mg/kg intraperitoneally, once weekly for three weeks. The low dose was chosen based on the average dose used in immunodeficient mouse models and the high dose was chosen based on the average dose used in immunocompetent mouse models. This was done to determine if the R2G2 mouse model can tolerate higher doses of these chemotherapeutic agents compared to other immunodeficient mouse models. Results show that the R2G2 mouse model tolerates higher doses of these chemotherapeutic drugs than doses found in the literature for SCID models. Exogenous estrogen tolerance is another common concern in oncology research as some immunodeficient mouse models cannot tolerate the subcutaneous estrogen pellets, developing negative secondary effects resulting in removal from study. We performed an estrogen pellet dose-response study using four doses of 60-day release 17-β estradiol pellets at 0.18, 0.36, 0.72, and 1.7 mg/pellet (n = 10 per group). R2G2 mice show dose-dependent effects of estrogen on morbidity. These data will allow researchers to determine the optimal dose for use in the R2G2 model. In conclusion, these data support that the R2G2 mouse model may be a good alternative to SCID models when administering DNA-damaging chemotherapies or when estrogen supplementation is required for xenograft growth.

#2158

Inhibition of NFkB restores tamoxifen responsiveness in resistant estrogen receptor positive breast cancer in a syngeneic mouse model.

Emily Smart, Irida Kastrati, Jonna Frasor. _University of Illinois, Chicago, IL_.

Breast cancer is responsible for 23% of new cancer cases per year worldwide and is the most frequently diagnosed cancer in women. Approximately 75% of breast cancers express the estrogen receptor (ER), and are subsequently treated with endocrine therapies. Despite initial therapeutic response, around 50% of ER positive tumors will recur and are often more aggressive and resistant to therapy. Consequently, overcoming resistance is paramount in moving towards more effective treatments for breast cancer. Studies are hindered by the lack of models that effectively recapitulate a physiologically relevant in-vivo environment with an intact microenvironment and functional immune system, both of which are important in affecting tumor response. The use of syngeneic mouse models overcomes this and allows us to more accurately replicate therapy response in-vivo. There are multiple mouse cell lines that are described as ER positive, however many do not respond to tamoxifen. These may represent a suitable archetype to study endocrine resistance and the underlying mechanism associated with this phenotype, both in-vitro and in a syngeneic in-vivo mouse model. J110 and Py2T cells are both described as ER positive and recognized as being tamoxifen resistant by our group and others. We have confirmed mRNA and protein expression of ER in these cells, however expression of ER target genes is unaffected by estradiol (E2) or tamoxifen treatment. Using a proliferation assay we demonstrated that cells are unresponsive to E2, tamoxifen and fulvestrant (ICI), suggesting resistance to ER ligands. A screen of pathways inhibitors found that IKK inhibitor VII and dimethyl fumarate, both directed at the NF-kB pathway, were able to sensitize cells to the anti-proliferative effects of tamoxifen. This effect translates in-vivo, where mice treated with tamoxifen and DMF, showed significantly reduced tumor growth compared to control groups. In agreement with our data, in-vitro studies have shown that repression of NF-kB can restore sensitivity to endocrine agents. There is considerable evidence that resistance and therapeutic failure in ER positive tumors may be partly attributed to the activation of NF-kB signaling, therefore NF-kB presents as an attractive target to overcome resistance. We have confirmed that NF-kB is active in mouse cell lines in-vitro, and that both cell lines are migratory and invasive in-vitro, suggesting a more aggressive phenotype than is typically observed in ER positive luminal cells. The data indicates that convergence between ER and NF-kB pathways may be responsible for therapy resistance and promoting a more aggressive phenotype, however the mechanism by which this occurs is unclear. Ultimately, we hope to provide evidence to support the effectiveness of therapy targeted to both ER and NF-kB in overcoming resistance in a subset of breast tumors.

#2159

**Use of in vitro** **cell sheet engineering as a chemotherapeutic model for epithelial cancer.**

Eun Hye Kim,1 Jaewang Lee,1 Daiha Shin,1 Sunwoo Park,2 Minsu Kwon,2 Jong-Lyel Roh1. 1 _Asan Medical Center, Univ. of Ulsan College of Medicine, Seoul, Republic of Korea;_ 2 _Eulji University School of Medicine, Seoul, Republic of Korea_.

Epithelial cancer is grown in the in vivo microenvironment mixed of keratinocytes, fibroblasts and matrix, vessels, and immune cells. The current in vitro 2-dimensional (2D) models for anti-cancer drug testing are commonly performed on a physical basis, resulting in significant difference from elucidating the real in vivo effects. To resolve the experimental gap, the 3D models have been developed to mimic the in vivo cancer microenvironment, while there is a lack of specific models for epithelial origin tumors. Therefore, we developed a new 3D model of using the in vitro cell sheet engineering for epithelial cancers and compared the results of several chemotherapeutic drug testing among in the 3D model, spheroid culture, and 2D-dish cell culture. The cell sheet included epithelial and subepithelial layers consisting of keratinocytes overlying the mixture of fibroblasts and endothelial progenitors. The spheroids of green fluorescent protein-tagged epithelial cancers were placed at the interface of epithelial and subepithelial layers. The spheroids with mixture of cancers and fibroblasts or cancer-associated fibroblasts (CAF) were made by the conventional hanging-drop method, and the 2D was simply seeded in the culture dish. The cell growth, viability, and hypoxia were calculated by cell counting kit (CCK), live/dead assay, propidium iodide and LOX-1 staining. The morphology, invasion, and mRNA and protein expressions were also compared among the different in vitro models. The results of live/dead assay and CCK showed higher viabilities in the cell sheet-based models before and after treatment with chemotherapeutic drugs. TGF-β and epithelial-mesenchymal transition-related mRNA and proteins were highly expressed in tumors growing in the cell sheet. Further, the incorporation of CAF into the cancer spheroids appeared to less decrease viable cancer cells by chemotherapeutic agents compared to the use of no or normal fibroblasts. Tumor angiogenesis and hypoxia were also apparently observed in the cell sheet model, which partly contributed to relative resistance to chemotherapeutic drugs. Taken together, the cell sheet-based cancer model might be applied to the in vitro observation of epithelial cancer growth and invasion, and anti-cancer drug testing.

#2160

MEN1611, a novel α-selective PI3K inhibitor in solid tumors.

Giuseppe Merlino,1 Alessio Fiascarelli,1 Mario Bigioni,1 Alessandro Bressan,1 Clelia Irrissuto,1 Andrea Pellacani,2 Maurizio Scaltriti,3 Monica Binaschi1. 1 _Menarini Ricerche S.p.A., Pomezia, Italy;_ 2 _Menarini Ricerche S.p.A., Firenze, Italy;_ 3 _Memorial Sloan Kettering Cancer Center, NY_.

The PI3K pathway regulates various cellular processes such as proliferation, growth, apoptosis and cytoskeletal rearrangement. This pathway is frequently de-regulated and considered an oncogenic driver in human cancer. In most solid tumors, the activation of the PI3K pathway is induced by mutations of the catalytic subunit PI3Kα, present in approximately 20% to 50% of breast cancer, 14% of colorectal cancer and in 5-10% of NSCLC. Besides being an actionable target, the PI3K pathway is implicated in de novo and acquired treatment resistant in various tumor types treated with targeted therapy. As a matter of fact, the most interesting antitumor activity with PI3K inhibitors has been observed in combination. Here, we present MEN1611 (previously PA799), a PI3-kinase inhibitor with a peculiar biochemical profile targeting with high inhibition potency the mutated forms of PI3Kα and PI3Kγ. In the current study we demonstrate the anticancer activity and the pharmacodynamic effects of MEN1611 as monotherapy or in combination with targeted therapies in both xenograft and PDX models of breast cancer and in colorectal cancer and in NSCLC models, all with defined genetic profiles. MEN1611, at a clinically relevant dose, showed good antitumor activity in combination with targeted therapy agents against breast cancer, colorectal cancer and NSCLC models bearing PI3Kα mutations. A reduced activity was instead observed when the tumor models were wild type for PI3Kα or had low levels of PTEN. The antitumor activity of MEN611 was supported by its pharmacodynamic activity. In all the models, both AKT and S6 phosphorylation, markers of PI3K inhibition, were significantly inhibited upon treatment. In agreement with the biochemical profile of the drug, in PTEN null xenograft models where the tumor driver is the PI3K β-isoform, we observed lower effects in term of antitumor activity and of molecular inhibition of the pathway. Last, MEN1611 induction of hyperglycemia and insulin release was studied in the tumor models at clinical relevant dose as a marker of class-effect toxicity. MEN1611 showed significant antitumor activity in combination with other targeted therapies in breast, colon and NSCL cancer PI3Kα mutant models. Our results support the evidence that MEN611 is a PI3Kα specific inhibitor and provided the basis for the planned phase IB/II.

#2161

Characterization of a panel of orthotopic glioblastoma multiforme (GBM) patient-derived xenograft (PDX) mouse models and efficacy evaluation of temozolomide and external radiation therapy.

Mette Munk Jensen,1 Michael J. Wick,2 Lotte K. Kristensen,1 Maria Z. Alfsen,1 Laura A. Paludan,1 Mark Juul,1 Melissa Rundle,2 Kyriakos P. Papadopoulos,2 Andreas Kjær,3 Carsten H. Nielsen1. 1 _Minerva Imaging, Copenhagen, Denmark;_ 2 _START, San Antonio, TX;_ 3 _Dept. of Clinical Physiology, Nuclear Medicine & PET and Cluster for Molecular Imaging, Dept. of Biomedical Sciences, Rigshospitalet and University of Copenhagen, Copenhagen, Denmark_.

Background Glioblastoma multiforme (GBM) is an aggressive cancer type with poor prognosis. The lack of effective treatment may be due to the complex molecular composition and the heterogeneity of the tumors. Standard of care treatment is surgery, external radiation therapy (XRT) and chemotherapy with temozolomide (TMZ); however, the response rate varies widely. Subcutaneous patient-derived xenograft (PDX) models are increasingly used during drug development. Implantation of GBM PDX tumors orthotopically in the brain displays an advantage as the tumors develop in a microenvironment that resembles that of the original patient tumor. In addition, the impact of the blood brain barrier (BBB) in terms of reduced drug bioavailability is also represented in the orthotopic GBM models. The aim of this study was to develop and characterize a panel of orthotopic GBM PDX models. The models were then used to study the efficacy of TMZ and XRT.

Methods A panel of 14 PDX GBM models was characterized for EGFR, EGFRvIII, MGMT and mGluR3 expression. Selected PDX GBM models were furthermore characterized for GBM stem cell markers by flow cytometry. Low passage subcutaneous tumors from seven PDX GBM models (ST108, ST112, ST146, ST545, ST610, ST1388 and ST2473) were used for assessment of XRT sensitivity. Mice were randomized to receive XRT (2 Gy/day for 5 days) or sham. Furthermore, subcutaneous tumors were enzymatically digested and used for intracranial stereotactic injection in nude mice. Tumor take and growth rate was determined by T2-weighted magnetic resonance imaging (MRI) and 18F-fluoro-ethyl-L-tyrosine (18F-FET) PET imaging. At confirmed tumor take, mice were treated with TMZ (20 mg/mg 5 days on/2 days off x3 or 100mg/kg/day for 5 days) or whole brain XRT (2 Gy/day for 5 days). Control groups received vehicle or sham XRT. Final endpoint was survival by humane endpoints and tumors were fixed in formalin for histologic evaluation.

Results A wide variation in expression levels of EGFR, EGFRvIII, MGMT and mGluR3 was observed in the PDX GBM panel. Three models had EGFRvIII mutation and all models had variable expression levels of EGFR. XRT showed a variable efficacy outcome on both tumor volume and survival in the PDX GBM models in the subcutaneous and orthotopic setting. TMZ showed efficacy in ST610 and ST2473 whereas ST146 and ST112 were resistant towards TMZ. Histologic evaluation showed typical characteristics of GBM tumors.

Conclusion A panel of GBM PDX models was characterized for several molecular markers and the panel reflected the heterogeneity of human GBM tumors. The orthotopic GBM PDX models showed a wide range of sensitivity towards both TMZ and XRT. The established panel of orthotopic GBM PDX models can be used as a platform for testing of new drugs in a setting that more closely mimic the GBM tumor microenvironment and impact of the BBB.

#2162

Comparing volatile and intravenous anesthetics in a mouse model of breast cancer metastasis.

Ru Li, Yujie Huang, Hengrui Liu, James P. Dilger, Jun Lin. _Stony Brook University, Stony Brook, NY_.

Introduction: Retrospective studies have suggested that anesthesia maintained with intravenous propofol during oncological surgery is associated with better outcome than anesthesia with volatile anesthetics. Here, we determine the effects of anesthetics on breast cancer lung metastasis in an animal model.

Methods: A primary tumor is generated in 6-8 week old Balb/c female mice by orthotopic implantation of 4T1 mouse breast cancer cells expressing firefly luciferase, into the mammary gland. When the primary tumor volume reaches 500 mm3, it is resected under three-hour anesthesia either with 2.7% inhaled sevoflurane or a 150 mg/kg intraperitoneal injection of propofol. A control group does not receive surgery or anesthesia. After surgery, lung metastasis is tracked and quantified with noninvasive bioluminescent imaging (IVIS). Two weeks later, the mice are euthanized, and the lungs are harvested for macroscopic and histological analyses, which include counting metastatic nodules, and CD11b/CD31 overlay staining.

As a step towards understanding the mechanism, we examine the effect of propofol and sevoflurane on angiogenesis. An in vivo matrigel plug assay is used to evaluate the effect of anesthetics on blood vessel formation. Balb/c mice are administered 2.7% sevoflurane or 150 mg/kg propofol 1 hour each week for three weeks. A control group receives no anesthetic. Blood vessels are visualized by perfusion of TRITC labeled dextran. No surgery is done in this experiment.

Results: In the 4T1 syngeneic model, surgical resection of the primary tumor under propofol anesthesia significantly reduced spontaneous lung metastasis in comparison with sevoflurane. Using IVIS imaging, similar levels of bioluminescence in the lung were observed in control and propofol treated mice two weeks after surgery. In contrast, mice anesthetized with sevoflurane had 1.5-fold higher levels (p<0.0001). This was consistent with manual counts of tumor nodules on the lungs: the number of tumor nodules increased by more than 300% in sevoflurane group versus control or propofol group (p<0.0001). The in vivo matrigel plug assay showed that exposure of Balb/c mice to sevoflurane led to a 2-fold increase in blood vessel density over control (no anesthesia). Propofol decreased blood vessel density.

Conclusion: We used a mouse model to recapitulate the human clinical situation in which the choice of anesthestic given during cancer surgery may be a risk factor for cancer metastasis. Our results show that the volatile anesthetic sevoflurane preferentially promotes metastasis of breast cancer cells in the lung. This may be related to the ability of sevoflurane to promote angiogenesis. The 4T1 mouse model successfully recapitulated the differential effects of anesthetic agents on breast cancer development seen in clinic, and further deepen our understanding of the mechanism behind that phenomenon.

#2163

**Establishment and characterization of an endocrine resistant ESR1** D538G **-mutant breast cancer PDX model.**

Megan Groves, Lizette Gamez, Alyssa Moriarty, Amanda Mangold, Melissa Rundle, Kyriakos P. Papadopoulos, Drew W. Rasco, Anthony W. Tolcher, Amita Patnaik, Michael J. Wick. _START, San Antonio, TX_.

Background: Chronic endocrine therapy (ET) to treat luminal type breast cancer can result in acquired mutations in the ligand binding domain (LBD) of ERα (ESR1), primarily at residues Y537 and D538, leading to constitutive ER activation and hormone-independent progressive disease. While ESR1-mutated breast cancers are insensitive to endocrine therapy, agents including selective estrogen receptor degrader (SERD) or modulator (SERM) therapies may be effective. However, few preclinical models of ESR1-mutant breast cancer are available for preclinical evaluation. Previously we reported the establishment and characterization of several ER+ breast PDX models and a cell-derived xenograft representing ESR1Y537S-mutated breast cancer.

Methods: We report here the establishment and characterization of ST2535 and ST2535/HI, ER+, ESR1D538G-mutated breast PDX models which are maintained in the presence (ST2535) or absence (ST2535/HI) of exogenous estradiol. We characterized these models using genomic analysis and evaluated both in vivo and compared efficacy results with those from ESR1Y537S-mutated models. Studies were performed evaluating model sensitivity to various test agents at standard dose and schedule regimens. Study endpoints included tumor volume and time from treatment initiation with %T/C values and tumor regression reported at study completion.

Results: Genomic analysis confirmed the identified ESR1-D538G mutation which was also reported in the donor patient. In addition, estrogen receptor expression (2+-3+) was confirmed in both models. Growth characteristics for both models were comparable with a doubling time of approximately 15 days after model stabilization in the F3 passage. Slight or no activity was reported in the ST2535/HI model with tamoxifen (T/C=90%) or fulvestrant (T/C>100%). Treatment with everolimus (T/C=92%) or palbociclib (T/C=90%) was ineffective. However, combination of fulvestrant and palbociclib reported a super additive effect in the study with a T/C=46%. Interestingly, all tested agents were notably less responsive in the ST2535/HI versus similar ESR1Y537S/HI models.

Conclusion: Overall, we have established and characterized two models of ESR1D538G-mutant breast cancer and compared in vivo activity to previously developed ESR1Y537S-mutant hormone independent models, all of which can be utilized for development of targeted therapies.

#2164

Conditional tumor cell reprogramming: From in vitro sensitivity to in vivo PDX modeling.

Yuan Long,1 Loc Van,2 Song Xi,1 Le Li,1 Yuhui Zheng,1 Ying Ling,1 Feifei Zhang,1 Jijun Cheng,1 Danyi Wen1. 1 _LIDE Biotech, Shanghai, China;_ 2 _Primordial Biotech, Philadelphia, PA_.

Tumor cell immortalization through conditional reprogramming, particularly from limited biopsy specimen, is an invaluable tool to generate propagating tumor cells for cell-based diagnostics, drug sensitivity assay and bio-banking in vitro. We have successfully reprogrammed primary tumor cells from a number of unreported tumor types, including lung carcinoma and adenoid cystic carcinoma, towards immortalization. The propagating cells exhibited typical colonized growth, which is well maintained upon cryo-frozen. In some cases, the cells can be passaged for multiple times and likely become useful cell lines. Like primary tumor cells, conditional reprogrammed tumor cells are reliable to test drug sensitivity in vitro. We attempt to extend conditional reprogrammed tumor cells to reconstitute and grow PDX models in vivo for testing drug efficacy. Preliminary data indicate that, in a 7-day in vivo assay utilizing hollow-fiber tubes grown subcutaneously in mouse, which we call OncoVee Mini-PDX, conditional reprogrammed tumor cells, like primary tumor cells and PDX tumor cells, can be successfully used to test drug efficacy in vivo. In summary, this study explores and evaluates applications of conditional reprogrammed tumor cells in drug efficacy tests both in vitro and in vivo.

#2165

Mini-patient-derived xenograft sensitive assay: A rapid systemic detection method for assessment of cancer therapeutics.

Xiaoqing Pan, Feifei Zhang, Yuan Long, Hui Liu, Lin Guo, Jijun Cheng, Danyi Wen. _LIDE Biotech, Shanghai, China_.

Patient-derived xenografts (PDX) have a high predictive power for efficacy of anticancer therapeutics. Limitations such as engraftment failure and time consuming for establishment and expansion of PDX models for drug efficacy testing need to be overcome for immediate application in patient decision-making for therapeutics. We developed a rapid in vivo drug sensitivity assay system so-called OncoVee® Mini-PDX, in which patient-derived tumor cells were arrayed within hollow fiber capsules in vivo over 7 days and screened against clinical relevant regimens. For validation study, the Mini-PDX was assessed by comparing the drug response in PDX tumor-bearing mice to response in Mini-PDX (capsule-containing) mice. The correlation between PDX tumorgrafts assay and Mini-PDX assay was evaluated using PDX models generated from 21 cancer patients. In the 21 cases, the overall predictive accuracy of the PDX assay for sensitivity was 69% and resistance was 88%, respectively. We also extended the approach to the patients with advanced malignancies. We report clinical cases in which Mini-PDX guided treatment improved the outcomes of treated cancer patients. In sum, Mini-PDX assay based on the capsule implantation was developed and optimized for quick assessment of the chemosensitivity of patients' cancers and found to be compatible with that of PDX tumorgrafts assay. This method hold promise for application of personalized chemotherapy for cancer patients.

#2166

Dasatinib displays antitumor efficacy in an orthotopic xenograft mouse model of histiocytic sarcoma.

Marilia Takada, Lauren Smyth, Vilma Yuzbasiyan-Gurkan. _Michigan State University, East Lansing, MI_.

Histiocytic sarcoma (HS) is an orphan hematological malignancy with an incidence of less than 1% of all non-Hodgkin's lymphoma, affecting both children and adults. It is a highly metastatic disease, and can affect any organ in the body including spleen, lungs, bone marrow, liver and lymph nodes. It is associated with a poor prognosis as treatment options are scarce and responses are short lived. Dogs spontaneously develop a similar form of HS with an appreciable frequency, and therefore present a unique translational model to investigate better treatments. Preliminary data from in vitro studies identified dasatinib, a multi tyrosine kinase inhibitor, as a highly potent drug against HS cell lines derived from tumors in dogs, but non-toxic to normal normal tissue surrogate fibroblasts. The current study was undertaken to evaluate if the efficacy of dasatinib could be re-capitulated in an in vivo orthotopic xenograft model of HS. The mouse model was generated in NOD-SCID mice by surgical injection of HS cells, transfected with a luciferase reporter construct, into their spleen. Tumor growth was monitored using In Vivo Imaging System. Mice were treated with 30 mg/kg of dasatinib intraperitoneally once a day or vehicle control, with five mice in each group. Dasatinib treated mice had a significantly decreased tumor growth rate in comparison to untreated mice. Most importantly, treatment with dasatinib significantly increased their survival time. All mice from the vehicle control group reached humane endpoints for euthanasia by day 27, while treated mice were clinically stable at day 53, with the difference in survival being highly significant (p<0.0001). In conclusion, here we report the first orthotopic xenograft mouse model of HS, that offers the possibility to test therapeutic response to novel agents, such as dasatinib. Our findings indicate that dasatinib is a promising treatment option for HS, and provide a rational for the initiation of clinical trials.

#2167

Efficacy assessment of BTK inhibitor ibrutinib in de novo and viral-induced B cell lymphoma.

Jessie Jingjing Wang, Meiling Zheng, Xuesong Huang, Yanrui Song, Wubin Qian, Likun Zhang, Jie Cai, Sheng Guo, Henry Qixiang Li, Davy Xuesong Ouyang. _Crown Bioscience, Inc., Taicang, Jiangsu, China_.

Bruton's tyrosine kinase (BTK) is crucial for B cell maturation. Its activation has also been considered as a major oncogenic driver for various B cell-derived lymphoid cancers. A BTK inhibitor, Ibrutinib, has been approved for mantle cell lymphoma, chronic lymphocytic leukemia, and Waldenström's macroglobulinemia, a form of non-Hodgkin's lymphoma (NHL). Ibrutinib has also shown encouraging efficacies in defuse large B cell lymphoma (DLBCL), specifically in activated B cell-like (ABC) subtype, but not in germinal center B cell-like (GCB) subtype. We have established a series of patient derived xenograft (PDX) models, recapitulating diverse genotypes of de novo DLBCL patients, including two MYD88L265P/CD79BY197N double mutants (LY2298, LY2264), one MYD88L265P single mutant (LY0257), and a variety of wild-types (LY2214, LY3604, and LY6934, etc.). This cohort of PDX models serve as perfect preclinical tools to investigate Ibrutinib responses in different genotypes. Our studies demonstrated a moderate to robust efficacies of Ibrutinib in the MYD88/CD79B double mutants, suggesting that CD79B mutation is the predictive biomarker for chronic activation of B cell receptor (BCR) signaling, as well as generally good responses to BTK inhibitor. Interestingly, the MYD88 single mutant model LY0257, where the disease is likely driven by constitutive activation of MYD88 signaling, also showed response to Ibrutinib when dosed in drinking water. In the wild type PDXs, two of seven models tested so far are either partially responsive or sensitive to Ibrutinib, while the others are resistant to the treatment. It seems that Ibrutinib response does not always require CD79B mutation, and BCR pathway addiction may occur by other means in DLBCL, even when MYD88 signaling is constitutively active. Notably, all 5 wild type DLBCL PDXs that are non-responsive to Ibrutinib are EBV positive verified by RNAseq. These EBV-transformed B lymphoma PDX models, which share the similar histopathology as de novo DLBCL, but have distinct molecular pathology signatures and different pathogenesis. In efficacy studies, none of the transformed B lymphoma PDXs showed response to Ibrutinib. Taken together, our DLBCL and EBV-induced lymphoma PDX models provide a valuable preclinical platform for evaluating BTK inhibitors, as well as future drug discovery efforts on other targets in the BCR and MYD88 pathways, such as PI3K, SYK, and IRAK4.

#2168

Anti-cancer drug sensitivity assay using in vitro primary culture cells.

Shiki Fujino,1 Norikatsu Miyoshi,1 Masayuki Ohue,2 Kazuhiro Saso,1 Tsunekazu Mizushima,1 Yuichiro Doki,1 Masaki Mori1. 1 _Osaka Univ. Graduate School of Medicine, Osaka, Japan;_ 2 _Osaka International Cancer Institute, Osaka, Japan_.

Primary culture of cancer cells derived from an individual patient's tumor provides important information regarding the tumor character. We established a novel culture method for primary colorectal cancer (CRC) with phenotypic heterogeneity as a human tumor model, named isolated tumor-derived cancer cells (iCCs). The success rate of iCC growth in vitro was 100%, passage was 90%, and establishment of a xenograft model from iCC was 80%. The gene expression of the cultured cells was similar to the clinical tumors, and exhibited a ductal structure of the tumor in the xenograft model even after several passages. The present in vitro analyses allow us to predict the clinical efficacy of chemotherapeutic drugs. For this purpose, we examined the sensitivities of iCCs isolated from patients. In this study four patients with distant metastasis received FOLFOX (folinic acid, 5-fluorouracil, and oxaliplatin) chemotherapy according to the treatment guidelines, and in vitro drug sensitivity was evaluated using the cultured cells, compared with each parental tumor in the individual clinical course. It is a model to predict the effect of chemotherapy to the individual tumor. Our results showed that the effect of chemotherapy was poor in three patients and good in one patient. In their clinical courses, the tumor size increased in two patients and decreased in two patients. In vitro drug sensitivity results reflected the clinical course in three of the four patients. In conclusion, our primary culture model is a novel tool for the prediction of anti-cancer drugs in the clinical courses.

#2169

Metastases of a temozolimide-sensitive patient-derived melanoma xenograft model show distinct biologic features and developed resistance against temozolomide.

Kerstin Klingner, Dorothee Lenhard, Elke Simon, Anne-Lise Peille, Julia Schüler. _Charles River Research Services Germany GmbH, Freiburg, Germany_.

Patient-derived tumor xenografts (PDX) play a major role in the development of new cancer therapies and their strengths and weaknesses have gradually been elucidated. In the current study we established a melanoma PDX from donor patient tissue. In addition, we were able to create two sublines from spontaneous metastases occurring in the murine host during the establishment phase of the original model. All three lines were characterized by tumor growth kinetics, antitumoral activity against standard of care Temozolomide and patho-histological examination. Furthermore, whole exome sequencing and RNAseq data of primary PDX and its metastases are available. Two out of the three sublines have corresponding cell lines for 2D and 3D testing. The PDX model was developed from a biopsy of a 68 year old woman undergoing surgery due to a non-pretreated melanoma. After seven subcutaneous passages in immune-compromised mice, individual animals showed tumor growth in the liver as well as the spleen. We were able to passage and characterize those metastases in parallel to the original model. The human origin of the lines as well as cell lines established in 2D was confirmed by str-analysis. All three in vivo lines depicted distinct growth kinetics: The doubling times varied significantly (Kruskal-Wallis, p< 0.0018) between 12.34 days (primary tumor, MEXF 2090P) and 30.78 day (spleen metastasis, MEXF 2090S) and 19.01 days doubling time for the liver metastasis model (MEXF 2090L). MEXF 2090S thereby depicted the slowest growth rate in our melanoma PDX panel (24 models, 10.09 days mean doubling time). The patho-histological examination revealed a well differentiated melanoma with low stroma content (3-5%) in all three lines. The molecular analysis (whole exome sequencing) identified distinct differences. Nevertheless, the potential driver mutations, Tp53 (R213Q) and Nras (Q61K), were recognized in all investigated samples. Temozolomide was applied to all three lines in vivo. 6 mice per group and line were treated either with Temozolomide (40 mg/kg/d, iv, twice a week for three weeks) or the control vehicle (10% DMSO, 90% NaCl). The primary tumor depicted statistically significant antitumoral activity with a T/C (test vs control) value of 36% (p< 0.05, t test, two-tailed) two weeks after the last treatment (experiment day 32). The two lines derived of metastases were resistant against the alkylating agent depicting T/C values of 76% (2090L) and 79% (2090S), respectively. Spontaneous metastases are a rare event in PDX models growing subcutaneously in NMRI nude mice. Thus, shedding some light into the biology of the metastatic event in the current model will help to understand and influence the metastatic process in general. In any case, those lines can serve as indispensable tools in the oncology drug development pipeline.

#2170

Single mouse preclinical trial: A tool for translational research.

Cyril Berthet. _Oncodesign S.A., Dijon Cedex, France_.

Roughly, 85% of preclinical agents entering oncology clinical trials fail to demonstrate sufficient safety or efficacy to gain regulatory approval. Hence, there is a need for experimental systems which better mimic the inter-patient response heterogeneity observed in the clinic. Patient-derived tumor xenograft (PDX) mouse models have emerged as a relevant oncology research tool to study tumor evolution, drug response, biomarkers, resistance phenomenon and adjusting chemotherapeutic approaches for individual patients. We will here expose the effectiveness of the single mouse preclinical trial paradigm for evaluating drug response, as mono or combo therapy using our well-characterized PDX collection. Based on the "1 PDX tumor/1 mouse/1 treatment" experimental design, a cohort of colorectal, breast, pancreas, lungs PDX models (minimum 20 of each) was used to explore response to Standard Of Care (SOC) and combo therapy used in clinic. We compared single-animal response data with treatment group data taken from historical experiments. We demonstrated that about >80% of the individual response matched the treatment group data, supporting the idea of using one single animal to predict the drug response. The response of each PDX to treatment was correlated to their histological and genetic profiles. This approach represents a new experimental design to address the tumor biology of cancer patients, and investigate targeted therapies in relevant mouse models, which could improve our ability to predict clinical trial responses, stratify patients for clinical trial, identify drug resistance mechanism, responsive sub-populations as well as biomarker. By combining genomic profiling data and drug screening data, and clinical data from patients, SMPT process is a helpful tool for precision cancer medicine and individualized drug selection.

#2171

Efficacy of chemotherapeutic agents in brain metastases of lung cancer model.

Rachel M. Tallman, Neal Shah, Sam Sprowls, Mark Pinti, Afroz Mohammad, Paul Lockman, Pushkar Saralkar. _West Virginia University, Morgantown, WV_.

Background: Lung cancer is not only the second most common cancer in the United States, but it is also the highest cause of cancer-related death worldwide. In 2017, it is estimated that there will be over 200,000 new cases and 150,000 deaths as a result of lung cancer. The associated high mortality can in part be attributed to the high rate of primary lung cancer metastasis to the brain. Median survival for untreated patients with metastatic brain lesions is approximately 4 to 9 months, while treated patients still typically die within two years. Treatment options are limited for patients with brain metastases due in part to chemotherapeutic ineffectiveness, as many agents used to treat the primary tumor are not able to penetrate the blood-brain barrier (BBB). This study aims to investigate the efficacy of various chemotherapies on brain metastases of lung cancer via bioluminescence imaging data as a surrogate for tumor burden and overall survival data.

Methods: Female athymic nude mice underwent intracardiac injection with brain-seeking PC-9 lung cancer cells transfected with Luc-2 and TdTomato. Tumor burden was monitored using bioluminescent imaging biweekly. Upon tumor burden establishment, mice were administered either Doxil (peglylated liposomal doxorubicin) (6 mg/kg), irinotecan (50 mg/kg), gemcitabine (60 mg/kg), docetaxel (10 mg/kg), eribulin (1.5 mg/kg), vinorelbine (10 mg/kg), cisplatin (5 mg/kg) + etoposide (10 mg/kg), cisplatin (5 mg/kg) + pemetrexed (100 mg/kg), osimertinib (25 mg/kg), or gefitinib (6.25 mg/kg). Upon sacrifice, brains were perfused to visualize tumors and permeability using fluorescent microscopy. Survival data were collected and plotted on a Kaplan-Meier curve for statistical analyses.

Results: Bioluminescence for nontargeted chemotherapy was nearly equal to that of vehicle. Osimertinib (25 mg/kg) demonstrated immediate decreased tumor burden and increased survival. Vehicle and all nontargeted agents had a survival of 42 +/- 3 days. Images taken of metastatic lesions indicate permeability has increased compared to normal brain, but is not as drastic as expected (1.5 to 1.8 fold increase) and displays heterogeneity between tumors.

Conclusion: The efficacy of chemotherapies in brain metastases of lung cancer is not necessarily associated with increased permeability. No smaller nontargeted agent showed improved survival or superior tumor control compared to vehicle, while the third-generation EGFR inhibitor osimertinib showed superior tumor burden control and survival in vivo. Future directions aim to investigate methods to increase drug permeation into tumors so that they may overcome the BBB and accumulate to cytotoxic levels in tumors.

#2172

Dying tumor cells (tumor debris) stimulate the growth of 4T1 breast cancer tumor in female athymic nude mice.

M Waheed Roomi, Aleksandra Niedzwiecki, Matthias Rath. _Dr. Rath Research Inst., Santa Clara, CA_.

Current cancer therapies are based on chemotherapy and radiation. These therapeutic approaches can initially reduce the tumor mass or tumor burden by killing tumor cells. It has been postulated that the resulting dying tumor cells, or tumor debris, can act as sources of tumor stimulation on surviving cells leading to reappearance of the cancer, and thereby drastically reducing the survival rate. In our study we investigated whether 4T1 debris induced by chemotherapeutic agent docetaxel would affect breast tumor growth in female nude mice. Female athymic nude mice were divided into three groups: Group 1 was inoculated with 4T1 cells; Group 2 with 4T1 cells together with their debris and Group 3 with debris alone. All mice were fed a regular diet. After four weeks, the mice were sacrificed; tumors were excised, weighed and processed for histology and immunohistochemistry for inflammation parameters. Tumor weight significantly increased in Group 2 mice compared to the control Group 1. Group 3 mice developed no tumors. Although the tumor histology for both Groups 1 and 2 were similar, there were significant differences in inflammatory parameters in the two groups. Tumors developed in Group 2 mice had intense staining patterns for TNF-alpha, IL-6, Ki-67, i-Nos and VEGF compared to tumors in the control Group 1. Our results show that dying cells from docetaxel treatment, or tumor debris, had stimulatory effect on growth of breast cancer 4T1 cells in nude mice which was associated with increased inflammation. This study represents a novel method to evaluate the effects of chemotherapy or radiation on surviving tumor cells and to investigate the effect of various inhibitors on the growth recurrence.

#2173

Testing neratinib-containing drug combination regimens on HER2 mutated non-amplified, ER+ breast cancer patient-derived xenografts.

Shunqiang Li,1 Tina M. Primeau,1 Stephanie L. Pratt,1 Katherine R. Harrill,1 Francesca Avogadri-Connors,2 Alshad S. Lalani,2 Cynthia X. Ma,1 Ron Bose1. 1 _Washington University School of Medicine, Saint Louis, MO;_ 2 _Puma Biotechnology, Inc., Los Angeles, CA_.

Introduction: HER2 activating mutations are a novel, druggable genomic alteration in metastatic breast cancer (MBC). These HER2 mutations are predominantly found in HER2 gene amplification negative, hormone receptor positive breast cancers. We have previously demonstrated that HER2 mutations can be potently inhibited by the second generation, irreversible pan-HER tyrosine kinase inhibitor, neratinib (Bose et al., Cancer Discovery 2013). Further, we performed a phase II clinical trial to treat HER2 mutated MBC and we found that neratinib monotherapy produced a clinical benefit rate of 31% and progression free survival of 16 weeks in a heavily pre-treated patient population (Ma et al., Clinical Cancer Research 2017). The objective of the current pre-clinical study is to develop combination strategies to improve the anti-tumor activity of neratinib in HER2 mutated breast cancer. As ER and CDK4/6 signaling activation are known resistance mechanisms to HER2 targeted agents in HER2 positive breast cancer, we hypothesized that neratinib in combination with agents that target ER (fulvestrant) or CDK4/6 (palbociclib) will induce synergistic anti-tumor effect in HER2 mutated MBC. In addition, we hypothesized that dual HER2 targeting (with trastuzumab) or in combination with chemotherapy agents (vinorelbine), may be more effective than neratinib alone. Since these partnering agents are well established in the treatment of breast cancer and are without overlapping toxicities with neratinib, rapid clinical translation is possible for the treatment of patients with HER2 mutated, ER+ MBC.

Description of our Human-in-Mouse Patient Derived Xenografts (PDX): We developed two HER2 mutated breast cancer PDX lines. Both lines come from HER2 mutation positive, HER2 gene amplification negative, ER+ MBC patients. PDX-51 has a HER2 exon 20 insertion mutation (ERBB2 G776insYVMA), a PIK3CA H1047R mutation and is wild-type for TP53. PDX-64 has a HER2 kinase domain missense mutation (ERBB2 L869R) and is wild-type for both PIK3CA and TP53. Both PDX lines are grown in female NSG strain mice without any exogenous estrogen supplementation.

Results: We tested combinations of neratinib with fulvestrant, palbociclib, trastuzumab, or vinorelbine for their anti-tumor activity and effects on cell proliferation and survival pathway signaling activities, including ER, CDK4/6 and HER2 pathways, in vivo. We will present data on the efficacy of these drug combinations. Tumor size measurements and reverse phase protein array (RPPA) data on the treated PDX lines will be shown.

#2174

Intracranial PDX models of breast cancer brain metastases and PET imaging for drug efficacy studies.

Carsten H. Nielsen,1 Michael J. Wick,2 Mette M. Jensen,1 Lotte K. Kristensen,1 Alyssa Moriarty,2 Melissa Rundle,2 Kyriakos P. Papadopoulos,2 Andreas Kjaer3. 1 _Minerva Imaging, Copenhagen, Denmark;_ 2 _START, TX;_ 3 _Department of Clinical Physiology, Nuclear Medicine & PET and Cluster for Molecular Imaging, Rigshospitalet and University of Copenhagen, Copenhagen, Denmark_.

Background: Overexpression of the human epidermal growth factor receptor 2 (HER2) is an independent risk factor for development of brain metastases. Between 37-55% of patients with HER2-positive metastatic breast cancer develop brain metastases and the incidence is increasing. A reason for the increase of brain metastases is the improved control of systemic disease by novel therapeutics such as ado-trastuzumab emtansine (T-DM1). However, the major impact of the blood-brain-barrier (BBB), BBB efflux pumps and the local microenvironment in the brain represent a challenge for treatment of brain metastases. The aim of this study was to develop a model system of HER2 positive breast cancer brain metastases for evaluation of drugs directed at brain metastases.

Methods: Subcutaneous tumors from a panel of HER2-positive PDX breast cancer models designated ST340, ST1339, ST1616B, ST1360B and ST3338 were enzymatically digested and used for intracranial stereotactic injection or intra carotid injection in nude mice. Contrast-enhanced T1- and T2-weighted Magnetic Resonance Imaging (MRI) were used to determine tumor take and growth.

Drug sensitivity studies to single agent T-DM1 administered intravenously were performed at confirmed tumor take in a subset of the models. Mice were treated with either saline or T-DM1, 10 mg/kg/week x4 or 2 mg/kg/week x4. Delivery of T-DM1 to the tumor site was evaluated by PET/CT imaging with 64Cu-trastuzumab.

Results: Intracranial tumor growth was detected by MR imaging in all models. T-DM1 treatment of animals with intracranial ST1339 or ST3338 tumors inhibited tumor growth and prolonged survival compared to non-treated animals. A variable response within the treatment groups was observed. Delivery of T-DM1 across the BBB to the intracranial tumors was visualized by 64Cu-trastuzumab PET/CT and high tumor uptake was associated with a response to T-DM1 therapy in the ST1339 model.

Conclusion: We have established a model system for evaluating drug sensitivity in HER2 positive breast cancer brain metastases. Treatment response to T-DM1 was observed in the models. 64Cu-trastuzumab PET imaging confirmed delivery of trastuzumab to the tumors, and high uptake was associated with an increased response to T-DM1 therapy.

Together, the established PDX models of breast cancer and brain metastases can be used as a relevant translational platform in combination with advanced non-invasive imaging for evaluation of new drugs.

#2175

Considerations in PDX mouse trial design and their relevance to human clinical trial outcomes.

Jingjing Jiang,1 Ying Yan,2 Tingting Tan,2 Wei Du,2 Jiali Gu,2 Ling Qiu,2 Katherine Ye,2 Zhenyu Gu1. 1 _GenenDesign Co, Ltd., Menlo Park, CA;_ 2 _GenenDesign Co, Ltd., Shanghai, China_.

Using cancer models to validate drug targets, evaluate drug candidates, and support clinical trial design has been important parts of preclinical studies in cancer drug research. To translate cancer model studies into clinical studies, great efforts have been made to generate a large number of patient derived xenograft (PDX) tumor models in certain cancer types and to demonstrate their similarities to cancer patients in tumor growth, histopathology, tumor complexity, molecular features and drug responses. Recently, focus has been shifted to use cancer model populations to mimic clinical trial design and predict drug responses in clinical trials.

We have developed over 1200 PDX models in multiple cancer types from naive or relapse tumor samples. Genomic profile and hotspot mutation analyses were performed to characterize drug targets and biomarkers used in clinical settings. Chemotherapies such as taxane and platinum, and targeted drugs such as cabozantinib, olaparib or sorafenib were tested at different doses and durations in PDX models such as lung cancer, gastric cancer or liver cancer. Drug response results from different regimens in PDX studies were analyzed by mRECIST method and compared with the corresponding results from clinical trials.

Our results demonstrated that selection of PDX models with histopathology and genetic features matched to the corresponding patient population in clinical trials is important for treatment result prediction. Some widely used doses for chemos in preclinical studies need to be reduced to achieve consistency with clinical results. Longer treatment time and more models than those normally used in preclinical efficacy studies also improve prediction value especially in cancer types with higher heterogeneity. Overall benefits of a targeted drug combined with one chemo over its combination with another chemo can be more accurately reflected in a large PDX population. In contrast PDX models derived from naive patient samples showed not much difference from models derived from chemo resistant tumors in their responses to new targeted treatments. Drugs targeting RAS/RAF signaling, PI3K/AKT signaling or cell cycle showed more uncertainty in PDX models if single biomarkers were used for drug response prediction.

In summary, a sufficient number of PDX models with pathological and molecular features similar to compositions of human cancer patients in clinical trials are necessary for using PDX mouse trial in predicting clinical outcome. Considerations should be given to mouse trial design similar to clinical trial design rather than traditional preclinical studies for targeting validation or proof-of-concept efficacy tests. 

### Tumor Heterogeneity 3

#2176

Whole-genome analysis of CNAs identifies four main evolution trajectories in multiple myeloma (MM) patients front-line treated with PI-based regimens.

Carolina Terragna, Andrea Poletti, Vincenza Solli, Marina Martello, Barbara Santacroce, Rosalinda Termini, Enrica Borsi, Chiara Benni, Lucia Pantani, Elena Zamagni, Paola Tacchetti, Giovanni Martinelli, Michele Cavo. _Seragnoli Inst. of Hematology & Medical Onc., Bologna, Italy_.

The inter- and intra-patients (pts) genetic heterogeneity of MM is the result of a competition between subclones harboring different genomic background, under the selective pressure of both microenvironment constrains and therapeutic treatment. Aim: to describe the clonal architecture changes driven by PI-based regimens in a cohort of homogeneously treated MM pts, by means of a genome-wide approach The study included 50 MM pts up-front treated with PI-based regimens. SNPs array data, derived from the BM CD138+ enriched cell fractions, as collected both at diagnosis (D) and at relapse (R), were analyzed with TAPS and Raw Copy R packages. In addition novel and personalized R-based scripts were designed, aimed at the detailed description of the whole genome CN changes. To be able to analyze the whole genome's changes between D and R, we first translated the signal derived from each of the 2,7x106 markers of the array (Cytoscan HD, Affymetrix) into gene-specific signals, therefore being able to describe CN changes of 20,172 genes. To highlight specific chromosomal regions affected by PI-based selective pressure, we than categorised the overall genomic CN changes, assuming that a limited number (9) of alternative genes' transitions might be observed between D and R. We therefore obtained dynamic profiles describing in details each genes' CNAs transitions, thus being able to highlight the following chromosomal regions particularly involved: chr 1p, 2p, 5, 15p, 19q (negative selection/extinction against CNs gains); chr 2q, 4 and 1p (positive selection/emersion of CNs losses and gains); chr13 and chr19p (mostly stable CN losses and gains). Each gene's CNAs transitions were also evaluated per pts, in order to describe the pts-specific evolutionary trajectories, thus highlighting an extremely high heterogeneity among pts, which might be roughly resumed in four main clonal evolution patterns: 32% of pts showed a stable, whereas 68% a branching evolution pattern. Among the branching patterns, three different behaviours were shown: 8% of pts showed a prevalent positive selection of CNAs, 24% showed a prevalent extinction of CNAs, whereas the remaining showed a blend of both behaviours. Of note, any correlation was observed between the presence of particular CNAs and the propensity to particular evolution patterns. Nevertheless, pts with stable evolution patterns had a slightly shorter median TTP, as compared to pts with branching patterns (22 vs 30 months, range 4-122 and 5-65, respectively). We concluded that the selective pressure of PI-based regimens mostly causes branching trajectories, even if any peculiar CNAs seem to guide at baseline this process. Overall, the clonal evolution patterns in MM remain still unpredictable, being probably driven by a combination of different factors, not yet fully depicted. Thanks to AIRC (MC), FB and FM (CT), AILBologna.

#2177

Sensitive single cell copy number profiling using a novel microfluidic droplet based platform.

Rui Li, Charles Couturier, Paul Savage, Jean Monlong, Guillaume Bourque, Kevin Petrecca, Morag Park, Jiannis Ragoussis. _McGill University, Montreal, Quebec, Canada_.

Intratumoral genetic heterogeneity may relate to aggressiveness and treatment-resistance. However, the genomic abnormalities underlie this heterogeneity remain poorly characterized. To better describe the picture of tumour heterogeneity, single cell approaches to accommodate larger number of cells are highly in demand. Here, we present results of single cell DNA sequencing by using a novel microfluidic droplet system. We applied this approach to discover copy number profiles in two common and therapeutically challenging cancers, isocitrate dehydrogenase wild-type glioblastoma (GBM) and breast cancer. The GBM cells were extracted from patients operated on site and cultured for 2 months in restricted media to isolate glioma stem cells. The breast cancer cells were extracted from patient-derived xenografts (PDXs). A novel microfluidic droplet based platform (10x GenomicsTM) was used to perform sensitive copy number profiling of thousands of single cells simultaneously. Briefly, the platform integrates single cell encapsulation, cell lysis, genomic amplification, and barcoding with oligonucleotides. The barcoded genomic DNA fragments from individual cells were pooled and converted into libraries that are compatible with Illumina sequencers. Thus far, we sequenced one glioma stem cell sample and two breast cancer PDXs. Uniquely mapped reads were counted in variable-bins with GC bias corrected by lowess fit. The Circular Binary Segmentation was performed and the integer copy number was estimated by least square fitting. The single cell copy number profiles were clustered by hierarchical clustering. We achieved approximately 2% and 6% coverage of the genome per cell for the glioblastoma and the breast tumors, respectively. The uniformity of coverage was comparable to other whole genome amplification and single cell multiplex approaches, while requiring 700k-2.1mi reads/single cell with the median absolute deviation of pair-wise differences in read counts between neighboring bins between 0.17-0.21 vs 0.16)1. We identified two major clonal subpopulations in the glioblastoma sample. One subclone had chr1q amplification and the other subclone had a 9.8Mb deletion on chr4. The clonal CNVs observed were validated using single-cell RNA-seq-based CNV analysis, and the frequent CNVs using fluorescent in situ hybridization. We also identified the HER2 copy number profile within a basal like triple-negative breast cancer as 40% of 2.5N and 25% of 3N. This was previously validated by fluorescence in situ hybridization2. By applying this new microfluidic system, our work will provide more insights into genomic heterogeneity for malignant properties of cancers. 1. Navin, N. et al. Tumour evolution inferred by single-cell sequencing. Nature 472, 90-94 (2011). 2. Savage P. et al. A Targetable EGFR-Dependent Tumor-Initiating Program in Breast Cancer. Cell Rep. 21, 1140-1149 (2017).

#2178

Single cell dynamics maintain an intrinsic drug resistant state in T cell leukemia and lymphoma.

Hamidullah Khan,1 Sushmita Roy,1 Ashish Anshu,1 Wasakorn Kittipongdaja,2 Stefan M. Schieke1. 1 _University of Wisconsin–Madison, Madison, WI;_ 2 _Medical College of Wisconsin, Milwaukee, WI_.

Cancer therapies commonly slow rather than cure disease and most patients ultimately relapse. Phenotypic variability and dynamics are a cause for relapsing disease and pose significant challenges to a successful cure. Here we show at the single-cell level that T lymphoma and leukemia cells are programmed to occupy a rare transient, drug-resistant state. Our work defines a panel of drug-resistance markers allowing early identification of the cells programmed to resist treatment and cause relapsing disease. Single-cell analyses in T cell lymphoma and leukemia lines reveal a small fraction of cells resisting cytotoxic drugs such as doxorubicin, methotrexate, and vorinostat. Repeat treatment cycles in recovered colonies indicate the transient and intrinsic nature of this drug resistant state. In cell fate analyses of bulk populations and xenograft tumors treated we observed that drug treatment leads to selective survival of label-retaining cells. Those cells then lead to population recovery and re-growth of tumors consistent with a relapse-inducing phenotype of the slow-cycling state. This was further supported by directly exposing isolated single cells to anticancer drugs with selective recovery and clonal expansion of cells in a pre-existing slow-cycling state at the time of drug exposure. Repeated exposure does not decrease the chemosensitivity of the surviving clones indicating that the drug-resistant state is transient and non-heritable. Label-retaining cells from different T cell lymphoma and leukemia lines display a transcriptional profile distinct from the parental population which is maintained in treatment-surviving cells. Cell line-independent similarities suggest T lineage-specific rather than disease-specific drug-resistance expression program. In particular, early growth response proteins 1-3 (Egr1-3) show increased expression levels in label-retaining cells serving as a biomarker for the drug-resistant subset of malignant T cells. Since Egr2 is critical for the induction of T cell anergy and activation, its overexpression in drug-resistant cells suggests a dual role of lineage-specific anergy mechanisms programming drug-response variability in malignant T cells. Tracing analyses demonstrated that single cells readily revert their phenotype to establish a clonal population with original heterogeneity indicating the dynamic nature of the slow-cycling state. This indicates that every single cell has a strong inherent drive to program daughter cells to occupy a rare transient slow-cycling state providing a high degree of stress- and, ultimately, treatment-resistance. The described resistance marker expression profile allows early identification of the drug-resistant state and helps to identify therapeutic targets in the rare-cell programming to prevent cells entering the relapse-inducing state in T cell leukemia and lymphoma.

#2179

Population remodeling in the acquisition of endocrine resistance in breast cancer.

Rong Hu,1 Surojeet Sengupta,1 Catherine Sevigny,1 Zhen Zhang,2 Yue Wang,3 Robert Clarke1. 1 _Georgetown Lombardi Comp. Cancer Center, Washington, DC;_ 2 _Johns Hopkins Medical Institutions, Baltimore, MD;_ 3 _Virginia Polytechnic Institute and State University, Arlington, VA_.

The cell-cell interactions that occur within the breast tumor microenvironment are critical determinants of cancer cell fate. In the face of treatment, the theory of clonal evolution dominates current thinking. Thus, individual cells acquire a mutation(s) that provides a selection advantage where Darwinian selection acts at the single-cell level. We studied the effect of antiestrogen treatment on the population remodeling of admixtures of sensitive LCC1 (S) cells and resistant LCC9 (R) human breast cancer cells. R cells were derived from S cells by selection against antiestrogens. We labeled S cells with GFP and R cells with mCherry, mixed the cells in different ratios, and treated with antiestrogen or vehicle. Unlike other resistance phenotypes (e.g., MDR1-driven cytotoxic drug models), R cells do not outcompete S cells, nor do S cells restrain R cell growth. Rather, R cells protect S cells from treatment. Full communication of the resistance phenotype occurs at a 1:1 mixture of S:R; ~75% of the R phenotype transfer is evident at 10:1. These results reflect improved fitness conferred by population interactions, not clonal evolution. SILAC analysis of S and R cells (pure populations) implicate differential regulation of estrogen (E2) response-early; E2 response-late; glycolysis; fatty acid metabolism; mTORC1 signaling; peroxisome; unfolded protein response (UPR); oxidative phosphorylation in S vs. R cells. Clustering of iTRAQ-TAT data from pure and admixed S:R populations shows that the 1:1 and 5:1 treated, and the 1:1 untreated admixtures, cluster with pure R cells. Untreated 5:1 admixed cells cluster with untreated S cells. Thus, R and S cells alter each other's molecular signatures. Studies using scratch labeling and small-molecule inhibitors show that R cells communicate with S cells via both juxtacrine and paracrine interactions mediated by gap junctional intracellular communication and secreted microvesicles, respectively. The molecular features modified reflect our unifying hypothesis of signaling and cell function control in endocrine resistance.

#2180

Investigating lung adenocarcinoma tumor heterogeneity with single-cell mass cytometry.

Maria-Fernanda Senosain-Ortega, Yong Zou, Deon B. Doxie, Caroline E. Roe, Jonathan M. Lehman, Jonathan M. Irish, Pierre P. Massion. _Vanderbilt University, Nashville, TN_.

The lack of accuracy in predicting behavior of early detected lung adenocarcinoma (ADC) presents a major challenge to patients and their providers. Indolent tumors may be overdiagnosed and overtreated. While imaging tools may contribute to better prediction of tumor behavior, unveiling tumor heterogeneity of ADCs at the single-cell level will elucidate mechanisms of tumor progression. Mass cytometry allows us to profile tumor heterogeneity and identify cell populations driven by the activation of specific signaling pathways. We report the development of an antibody panel for mass cytometry and preliminary experiments in cancer cell lines and in ADC. ADCs were collected at the time of surgery, dissociated into suspension and cryopreserved within 24 hours. A mass cytometry antibody panel was developed, including commercially available and in-house metal conjugated antibodies. Biaxial gating or unsupervised analysis approaches SPADE and viSNE were used to compare major populations of cells. Our comprehensive antibody panel includes markers for cellular lineage (14) (immune, epithelial, mesenchymal, fibroblasts), cancer cells (3), signaling pathways (15) and quality control markers (2). The in-house conjugated antibodies (9/34) were titrated in concentrations going from 0 to 1 ug/mL using cell lines with known expression of the marker of interest. A panel of previously validated antibodies was used to gate cell populations. Signal medians of positive and negative populations and standard deviations of negative population were used to determine the stain index and the signal/noise ratio. Based on titration curves, optimal concentrations of antibody were selected for use. In the tumor studied, four cell populations were identified: endothelial cells, fibroblasts, epithelial tumor cells and leukocytes. Basal kinase activity was detected in cancer cells and infiltrating leukocytes, both exhibiting p-STAT5 activation, and cancer cells showing high p-AKT activation. Using our Marker Enrichment Modeling algorithm, we identified 3 differentially enriched subpopulations of tumor cells, as well as different leukocyte populations. These preliminary data proved mass cytometry and the panel developed are suitable tools to characterize tumor heterogeneity in lung ADC. The work in progress studying indolent and aggressive ADCs promises to identify detailed phenotypes and respective signaling pathway activation that may ultimately allow us to better predict tumor behavior and integrate this knowledge in the clinical workflow.

Supported by CA196415.

#2181

Functionalizing intratumoral signaling heterogeneity in triple negative breast cancer.

Amanda L. Rinkenbaugh, Vidya C. Sinha, Xiaomei Zhang, Helen Piwnica-Worms. _MD Anderson Cancer Center, Houston, TX_.

Tumors are highly heterogeneous populations of cells, and measures of intratumoral heterogeneity (ITH) and diversity correlate with worse prognosis in many cancers, including breast cancer. Emerging studies are highlighting functional interactions between subclones, as well as among subclones and components of the tumor microenvironment. However, these studies have largely focused on soluble factors without interrogating the spatial distribution of subclones defined by activated signaling pathways. Previous work in this area has been severely limited by technical restrictions – existing techniques allowing measurement of many biomarkers simultaneously lose all information about the tissue architecture, while those that do retain spatial information can only assay a handful of markers at once. We will circumvent these limitations by undertaking imaging mass cytometry (IMC), which allows for simultaneous measurement of 30-40 antigens while retaining the spatial organization of the sample. Our objective is to dissect the signaling heterogeneity in tumors from patients and patient-derived xenograft (PDX) models of triple negative breast cancer (TNBC), through two main approaches: (1) characterization of signaling heterogeneity in human tumors and PDX models of TNBC through the use of imaging mass cytometry (IMC) and (2) modeling cell signaling heterogeneity in cell line-based models to determine mechanisms of cell-cell interaction and communication. We have constructed an IMC panel of antibodies that combines markers for tissue architecture, cell phenotyping, and signaling pathway activation. Profiling of a panel of TNBC PDX models, including matched sets of pre- and post-chemotherapy treatment, indicates emerging patterns of pathway activation. We will utilize these results to inform functional studies in our isogenic cell line models. As it is becoming more clear that tumors are composed of heterogeneous subclones, it is crucial that we understand the communication between subclones and with components of the microenvironment. Ultimately, we plan to use these results to identify novel vulnerabilities in subclonal interactions that can be targeted therapeutically in TNBC.

#2182

DigiPico: A whole-genome sequencing approach to investigate microscopic residual chemotherapy resistance disease in ovarian cancer.

Mohammad KaramiNejadRanjbar, Donatien Chedom Fotso, Yuhao Zheng, Christopher Yau, Ahmed Ahmed. _University of Oxford, Oxford, United Kingdom_.

Microscopic residual chemotherapy resistance disease (MRCD) following cancer therapy predisposes to recurrence. Genome analysis of MRCDs could, therefore, give insights into evolutionary mechanisms that might govern this process. However, to date, this has largely been restricted to studies utilizing targeted sequencing of known somatic mutations due to the challenges of whole-genome sequencing of subnanogram quantities of DNA. A previous report showed that obtaining accurate whole-genome sequencing is possible from as little as 10 to 20 cells with the added advantage of obtaining reconstructed long fragment reads. Adopting this technology, we were able to optimize a procedure to accurately obtain high-quality whole-genome sequencing data from minimally available clinical samples such as MRCDs, termed DigiPico. Using DigiPico, we were able to perform sequencing on several post-chemotherapy MRCDs and single tumor islets from a high-grade serous ovarian cancer (HGSOC) tumor. The data were found to be highly accurate, with over 96% of the single-nucleotide polymorphisms (SNPs) being detected in DigiPico sequencing results despite the extremely low amount of material available. Moreover, we found that DigiPico can be used for calculation of allele fractions with a consistency rate of up to 84% as opposed to a consistency rate of only 47% for when a standard multiple displacement amplification of similar amount of DNA is used during library preparation. The high-quality allele fraction information from DigiPico was then used to predict the copy number variation (CNV) in these clinical samples. Here, we showed the power of DigiPico for accurate SNP calling and CNV prediction from post-chemotherapy MRCDs and single tumur islets of a HGSOC tumor, the latter of which is extremely important for studying HGSOC as these tumors often show a great degree of structural alterations. This information can now be used for better understanding of how MRCDs might be able to remain unharmed during the course of chemotherapy and initiate recurrence after completion of treatment, which in turn can allow us to design better treatment strategies for HGSOC patients.

#2183

Clonal cooperation in cancer progression: A new paradigm in cancer.

Ana Martín-Pardillos,1 Angeles Valls-Chiva,1 Eva Bejar Serrano,1 Roberto Piñeiro Cid,2 Pablo Hurtado Blanco,2 Angel Días-Lagares,2 María Magdalena Castro,2 Juan Antonio Juan Antonio Cámara Serrano,1 Santiago Ramon y Cajal1. 1 _Vall d´Hebron Institut de Recerca, Barcelona, Spain;_ 2 _Health Research Institute in Santiago de Compostela – IDIS, Santiago de Compostela, Spain_.

Introduction: Cancer is a rapidly evolving, multifactorial disease that accumulates numerous genetic and epigenetic alterations. This results in phenotypic and molecular heterogeneity within the tumor, the complexity of which is further amplified through specific interactions between cancer cells and the tumor microenvironment. In this context, cancer may be perceived as an "ecomolecular" disease that involves cooperation between several neoplastic clones and their interactions with other cell types and secreted factors present in the microenvironment. Cancer is therefore analogous to complex ecosystems such as microbial consortia. We are currently aiming at dissecting the molecular mechanisms underlying the cooperation between different clones.

Methods: We produced clonal cell lines derived from the MDA-MB-231 breast cancer cell line, using UbC-StarTrack system, which allows tracking of multiple clones by color. The characteristics of each clone were determined by measuring migration, proliferation and metabolic activity. In vivo analysis by orthotopic breast injection and intravascular tail vein injection was performed to assess the tumorigenic capacity of these clones. We studied potential collaborations between clones by determining the influence of secreted factors on growth rate of different clones by medium complementation with supernatant or exosomes from different clones. In vivo, we used zebrafish as a model system to study the migration of individually or co-injected clones.

Results: Characterization of these clones in vitro revealed clear differences in proliferation, cell metabolic activity and morphology among them. In vivo, all the individually injected clones were able to form tumors but the growth rates differed among them. Injection of an equal mix of clones led to the formation of tumors where some clones displayed a growth or survival advantage. In vitro the complementation of growth medium with medium from other clone increased the proliferation rate of the other clones. Co-injection of clones in zebrafish increased the efficiency of migration.

Conclusions: These results confirm that even in stable cell lines heterogeneity is present. Malignant properties were enhanced when some clones were combined or treated with exosomes or medium from other clones. These results clearly support our hypothesis that tumor clones cooperate in cancer progression and that this cooperation is mediated by secreted factors. Finally, we are performing expression arrays of mRNA, microRNas and lncRNAs and determining epigenetic state by methylation arrays, in order to identify potential factors that are differentially expressed among clones and are therefore bona fide candidates for clonal cooperation promoting factors. It is anticipated that this knowledge will facilitate the design of new and more effective therapeutic approaches that are directed to the tumor ecosystem as a sum of different clones.

#2184

Enrichment and isolation of tumor cells using a unique flow-sorting approach to decrypt intratumoral heterogeneity in matched samples of squamous cell carcinoma of the lung.

Arthur Krause,1 Thomas Lorber,1 Valeria Perrina,1 Michael T. Barrett,2 Christian Ruiz,1 Lukas Bubendorf1. 1 _University Hospital of Basel, University of Basel, Basel, Switzerland;_ 2 _Mayo Clinic, Scottsdalce, AZ_.

Background:

Genomic intratumoral heterogeneity in cancers is a driving force for progression or recurrence. Availability of pure tumor cell material is critical for studying clonal evolution. In general, tumors are highly mixed with benign cells with a mean tumor cell purity of only 49%1 shown in lung squamous cell carcinoma (LSCC). Using a unique flow-sorting based approach enables us to overcome this limitation by highly enriching for tumor cells. Here, we aimed at precisely identifying different tumor populations in primary LSCC and matched distant metastases from 18 patients.

Methods:

Nuclei from snap-frozen and FFPE tumor specimens were extracted and subjected to flow-sorting using DNA content/ploidy as a parameter. Pan-cytokeratin (pCK) was used to differentiate diploid tumor cell populations from benign diploid stromal cells. DNA has been extracted from these individual tumor cell populations and the mutational profile and copy number status are currently being analyzed using whole exome seqeuencing (WES).

Results:

We successfully enriched for tumor cells with a purity of higher than 95% using the multi parameter flow-sorting approach. Besides the aneuploid tumors with higher ploidies, we discovered near-diploid tumor populations after enrichment with the pCK antibody. The pCK negative fraction consisting of stromal cells can serve as a germ line control in further downstream analysis as WES.

Conclusion:

The present study illustrates a unique method that allows high enrichment for tumor cells. Avoiding the dilution of DNA by benign stromal cells, together with the purity of aneuploid tumor cells, the precision of genomic analysis highly increases. At the same time, the diploid and pCK negative stromal cells can serve as a germ line control. Comprehensive genomic profiling of flow-sorted aneuploid and pCK positive diploid tumor populations represent a powerful technique to study intratumoral heterogeneity.

References:

1. Zack, T. I. Pan-cancer patterns of somatic copy number alteration. Nature Genetics. 45, 10 (2013).

#2185

Multiple region whole-genome sequencing reveals intratumor heterogeneity and branching clonal architecture of non-clear-cell renal cell carcinoma.

Bin Zhu*,1 Luana Poeta*,2 Manuela Costantini*,3 Tongwu Zhang*,1 Steno Sentinelli,3 Jianxin Shi,1 Kevin Brown,1 Xing Hua,1 Meredith Yeager,1 Mingyi Wang,1 Belynda Hicks,1 Stephen Chanock*,1 Michele Gallucci*,3 Vito Fazio*,4 Maria Teresa Landi*1. 1 _NCI, Bethesda, MD;_ 2 _University of Bari, Bari, Italy;_ 3 _Regina Elena Cancer Institute, Rome, Italy;_ 4 _University Campus Biomedico, Rome, Italy_.

Limited knowledge about the intratumor heterogeneity (ITH) of non-clear-cell renal cell carcinoma (nccRCC) hinders therapeutic efficacy. We performed multi-region whole-genome sequencing, genome-wide methylation profiling and deep targeted sequencing of both primary and paired metastatic tumors in 124 samples from 29 subjects with papillary renal cell carcinoma type 1, papillary renal cell carcinoma type 2 and collecting duct kidney tumors. Samples were taken from the tumors' center to the periphery at ~1.5cm from each other. We conducted integrative ITH analysis of single nucleotide variants, somatic copy number alterations, structural variants, transposon element insertions, telomere length and DNA methylation, and inferred their respective evolutionary history. We also analyzed the concordance between physical and lineage phylogenetic evolution, and inferred the regions from which the metastases likely originated.

ITH varied significantly across tumors and histological subtypes. For example, the average number of structural variants per tumor was 1.2 in papillary RCC type 1 vs. 23.6 in papillary RCC type 2. In papillary tumors, ITH was lowest for copy number alterations (~5% subclonal changes across all tumor types), intermediate for single nucleotide variants (~32%), and highest for structural variants (~60%), suggesting a tumor molecular evolution along these steps. Lineage trees commonly showed branching evolutions with a dominant subclone. Focal deletion of the CDKN2A gene characterized the collecting duct tumors. Methylation ITH was highest in the enhancer regions. Metastatic and primary tumors shared similar mutation signatures. Telomere length was generally longer in normal vs. tumor samples and was similar in physically adjacent samples.

In summary, our study characterized ITH of nccRCC with unprecedented details for multiple types of somatic alterations, and delineated the branching architecture of clonal evolution, including the metastatic phase.

#2186

Drug treatment induces phenotypic switch and stemness during acquisition of drug resistance.

Santhalakshmi Ranganathan, Annapoorni Rangarajan. _Indian Institute of Science, Bengaluru, India_.

The breast tumor heterogeneity and development of drug resistance has become a challenge for anti-cancer therapy. Tumor cells acquire resistance due to intra-tumor heterogeneity imposed by epithelial to mesenchymal transition. The drugs which target epithelial cells may fail to target mesenchymal and the intermediate phenotypes due to which the cancer cells acquire drug resistance. This concept provoked us to understand the phenotypic diversity among the tumor heterogeneity which might lead to drug resistance. Hence the aim of our study is to identify the mechanisms of anti-cancer drug resistance which would ultimately help in identification of new clinical targets.

Treatment of breast cancer cell lines MCF7 and MDA MB 231 with Doxorubicin showed a loss of epithelial markers and gain of mesenchymal markers in MCF7 cells, and vice versa in MDA MB 231 cells, thereby shifting them into an intermediate phenotype. Moreover, Doxorubicin treatment of MDA MB 231-Nanog stable cells led to an increase in Nanog positive cells confirming that the treatment-induced increase in stemness property of cancer cells. We predict that phosphoproteomic dataset of the anti-cancer drug-resistant cells let us analyze the possible molecular players involved in the phenotypic switch and hence drug resistance (work in progress). Thus, our current data suggest that

the anti-cancer therapy induces cancer cells to undergo a phenotypic switch and increases the stemness property which might be responsible to gain resistance. Identification of the critical regulators of the phenotypic switch by phosphoproteomics analysis is likely to help us investigate the possible mechanisms underlying anti-cancer drug resistance. Previous reports also suggest that conventional therapies fail to eliminate cancer stem cells thereby allowing tumor relapse.

These results suggest that understanding the protein abundance and phosphorylation state of key signaling molecules would elucidate specific signaling interactions or/and pathways differentially active in drug-resistant tumors would help us predict novel kinases, and hence identify drug targets.

#2187

A rapid autopsy program for cancer research.

Thomas G. Beach, Lucia I. Sue, Geidy E. Serrano, Anthony Intorcia, Jessica Walker, Michael Glass, Michael Callan. _Banner Sun Health Research Institute, Sun City, AZ_.

Many new technologies, such as genomics, transcriptomics, proteomics and metabolomics, are directed at molecular-level analyses of high-quality human normal and diseased tissues. At present, however, the relative lack of suitable tissue for this work is a critical roadblock to the full utilization of these new methods. Tissue is traditionally collected at the time of therapeutic surgical interventions, such as biopsy, or at the time of death, by autopsy. Biopsy is only done on individuals with disease and therefore normal control tissues are only obtained by chance. Biopsy tissue is often completely used for diagnostic purposes or is insufficient in quantity to constitute a shared resource, or for studies of intra-tumoral heterogeneity. Metastatic tissue is often not biopsied, precluding analysis of its evolving molecular changes. Both diseased and normal control tissue could potentially be obtained at autopsy, but autopsy tissue is generally only suitable when it is rapidly obtained after death. The Brain and Body Donation Program (BBDP) in Sun City, Arizona, is a not-for-profit longitudinal clinicopathological study of aging and is the world's only consistently-rapid autopsy program (3 h median). Board-certified pathologists diagnose all tissue microscopically. Many cases have both primary and metastatic tumor tissue. Both fresh-frozen & formalin-fixed samples are saved. All subjects are research volunteers with comprehensive clinical documentation. Our informed consent allows wide sharing of tissue and data, including with for-profit companies. More than 100 cancer autopsies have been done; fixed and frozen tissue is available from more than 25 cancer types, many of which have both primary and metastatic tumor tissue. Normal control tissue is available from more than 40 tissue types and RNA quality is high (Walker DG et al, Cell Tissue Bank 2016 17(3):361-75). The BBDP is listed on the NCI Specimen Resource Locator, and inquiries may be made directly to our website, www.brainandbodydonationprogram.org.

#2189

Genomic dynamics in breast cancer progression analyzed by miFISH analysis.

Irianna Marie Torres,1 Leanora Hernandez,2 Jausheng Tzeng,3 Russell Schwartz,4 Alejandro Schaffer,5 Edward Michael Gertz,5 Daniela Hirsch,6 Steven Brower,3 Miguel Sanchez,3 Gert Auer,7 Kerstin Heselmeyer-Haddad,1 Thomas Ried1. 1 _National Cancer Institute, Bethesda, MD;_ 2 _Sanford Burnham Prebys Medical Discovery Institute, CA;_ 3 _Englewood Hospital and Medical Center, Englewood, NJ;_ 4 _Carnegie Mellon University, Pittsburgh, PA;_ 5 _NCBI, Bethesda, MD;_ 6 _University of Mannheim, Mannheim, Germany;_ 7 _Karolinska Hospital, Stockholm, Sweden_.

Our previous studies of synchronous Ductal Carcinomas in Situ (DCIS) and Invasive Ductal Carcinomas (IDC) often showed a high degree of chromosomal instability already in DCIS, and frequently a direct clonal evolution from DCIS to IDC. We now asked the question whether this degree of instability is also present in DCIS that did not progress to IDC. In collaboration with Englewood Hospital and Medical Center, we are therefore conducting a retrospective study analyzing FFPE material from 20 DCIS patients with low-grade or high-grade DCIS who did not present with invasive breast carcinoma during their follow-up (5-10 years) and compared the findings to 10 DCIS patients who presented with synchronous IDC. Using our novel multiplex interphase FISH (miFISH) assay, we are able to determine copy number changes of five oncogenes (COX2, MYC, CCND1, HER2, ZNF217) and three tumor suppressor genes (DBC2, CDH1, TP53) and assessed intratumor heterogeneity (ITH) and clonal evolution during disease progression.

The miFISH analysis showed that low-grade DCIS displayed mostly stable patterns of gene copy numbers in a diploid genome, while high-grade DCIS frequently had increased nuclear DNA content (ploidy) associated with a higher degree of ITH. However, some low-grade DCIS exhibited complex and unstable patterns with higher ploidy. Also, a few high-grade DCIS were defined by few copy number changes, e.g., the gain of a whole chromosome 8 (DBC and MYC), resulting in a clonal population with a low instability index. DCIS and their synchronous IDC often displayed very similar miFISH patterns indicating direct expansion of dominant clones. Of note, the DCIS cells of one case were defined by the well described 1q gain/16q loss pattern and acquired subsequently a chromosome 8 gain in the IDC, confirming our previous observation that the gain of MYC is a pivotal step in breast cancer progression. Our data also show that DCIS with or without synchronous IDC exhibited similar patterns. This could indicate that DCIS in general, regardless of grading, should be treated as lesions with progressive potential. Phylogenetic tree modeling with our custom FISHtrees software will further elucidate the dynamics of DCIS lesions.

In summary, miFISH analysis and phylogenetic tree modeling allow an in-depth characterization of precursor lesions and their evolutionary trajectory. We anticipate that this approach will ultimately help to stratify progression risk in patients with DCIS.

#2190

A method for differentiating clonal driver mutations from subclonal emerging resistance mutations in circulating cell-free DNA.

Oliver Zill, Stephen Fairclough, Catalin Barbacioru, Justin Odegaard, Richard B. Lanman, AmirAli Talasaz, Darya Chudova. _Guardant Health, Inc, Redwood City, CA_.

Background: Cell-free circulating tumor DNA analysis provides a non-invasive method for obtaining actionable genomic information to guide personalized cancer treatment. Deep sequencing of cell-free DNA (cfDNA) can potentially provide insights into tumor heterogeneity across multiple tumor sites in a patient, including emerging treatment-resistant subclones. However, the increased informational complexity of polyclonal cfDNA in circulation poses analysis challenges, particularly in tumors with abundant copy number alterations. To facilitate interpretation of this added complexity, we developed methods to identify cfDNA copy-number driver alterations and cfDNA clonality,

Methods: We analyzed a large clinical sequencing database of somatic point mutations and copy number alterations from targeted cfDNA sequencing of 21,807 consecutive patients across >50 cancer types (Guardant Health, CA). We evaluated a minimal cfDNA clonality model that relies on the relationship between variant allele frequency (VAF) in cfDNA and the level of tumor DNA in circulation (ctDNA level), while accounting for copy number alterations.

Results: We found that the initial simple model of cfDNA clonality performed well on >90% of samples, given a relatively small targeted genomic region (70 genes, 150 kb). However, normalizing VAF by copy number is subject to error in some samples due to the effect of ctDNA level on variant detection, variable unique molecule coverage across samples, and non-linearity of VAF at high copy number. Therefore, we developed an improved cfDNA clonality model that incorporated these analytical and biological features, which was then trained on a portion of the large cfDNA data set. Our cfDNA clonality model accurately distinguished subclonal resistance from driver alterations in a test set of over 5,000 lung, colorectal, and breast cancer patients. Although numerous subclonal tumor-derived alterations were apparent in the initial test data set, leading to an apparent departure from mutual exclusivity in treatment-naïve tumors, robust mutual exclusivity was observed among cfDNA clonal driver alterations when our cfDNA clonality analysis method was applied. These results suggest our analytical approach can be used to identify treatment-associated emerging resistance alterations in patients from a single blood draw, including parallel evolution of distinct subclonal alterations.

Conclusion: Managing cancer will likely depend on identifying emerging treatment-resistant subclones at or in anticipation of progression. Highly accurate deep sequencing of cfDNA, along with comprehensive models of cfDNA clonality, can elucidate subclonal structure of the tumor and identify emerging treatment resistance.

#2191

Quantitative cell-level spatial analysis of tumor metabolism.

Tiffany M. Heaster,1 Bennett A. Landman,2 Melissa C. Skala1. 1 _University of Wisconsin-Madison, Madison, WI;_ 2 _Vanderbilt University, Nashville, TN_.

Sub-populations of tumor cells exhibit distinct responses to treatment. This cell-level heterogeneity can lead to growth of resistant populations, and tumor recurrence. Many cancer therapies alter tumor cell metabolism, and the metabolic microenvironment can further contribute to tumor heterogeneity and drug response. However, the relationship between the spatial diversity of tumor cell metabolism and treatment response requires further characterization. Two-photon fluorescence lifetime microscopy (FLIM) monitors cellular metabolism by measuring NAD(P)H and FAD autofluorescence, and enables single-cell resolution of metabolic heterogeneity within 3D samples. Previous studies have shown that FLIM predicts treatment response of 2D and 3D cultures in vitro, and in vivo tumors. Additionally, these studies demonstrated responsive cells have lower post-treatment NAD(P)H mean lifetime (NAD(P)H τm) than non-responsive cells. Thus, these published FLIM data were used to develop an algorithm to quantify the spatial distribution of cellular metabolism in response to treatment. Density-based clustering analysis defined sub-populations in organoids based on NAD(P)H τm. Accuracy of cluster analysis classification was first evaluated on 2D co-culture images of two cell lines plated at varying proportions. Following this validation, metabolic sub-populations and their spatial distributions were characterized in control and drug-treated 3D tumor organoids of human squamous cell carcinoma (FaDu). Descriptive spatial metrics were developed using graph-based network analysis integrated with standard image analysis to establish local (sub-population level) and global (organoid level) spatial relationships. High classification accuracy (94.7%) of cell lines was achieved in 2D breast carcinoma co-cultures. In all FaDu 3D organoids, metabolic sub-populations were largely segregated into clusters of identical metabolic phenotype, with ≥78% of cell neighbors belonging to the same sub-population. Clustering density was greater in populations with low NAD(P)H τm (responsive), exhibiting shorter intercellular distances than high NAD(P)H τm (non-responsive) populations (p <0.05). Additionally, responsive and non-responsive cell sub-populations displayed similar organization relative to both adjacent sub-populations and organoid centers. Geographically weighted principal component analysis demonstrates variability of each metabolic and spatial parameter as a function of cell location. This analysis is under refinement for use with in vivo FaDu xenograft images. This algorithm to quantify spatial distributions of metabolic sub-populations within tumors could enhance our understanding of tumor progression and therapeutic resistance.

#2192

Single-molecule single-cell DNA sequencing identifies ongoing copy number evolution in BRCA breast cancers.

Haowei Du,1 Alexander Davis,2 Ruli Gao,2 Medina Colic,2 Emi Sei,2 Min Hu,2 Angelica Gutierrez Barrera,2 Anna Casasent,2 Constance Albarracin,2 Banu Arun,2 Nicholas E. Navin2. 1 _Baylor College of Medicine, Houston, TX;_ 2 _The University of Texas MD Anderson Cancer Center, Houston, TX_.

BRCA2 germline mutation carriers have a lifetime risk of 72% for developing invasive breast cancer. Although BRCA2 has been implicated in double-stranded break repair, the role of this mutation in genome evolution and maintaining genome instability during the expansion of the primary tumor mass is not well understood. To investigate these questions, we developed a novel method for single-cell, single-molecule (SCSM) sequencing. SCSM uses a Tn5 transposase to directly fragment and ligate adapters for PCR amplification, thereby omitting the whole-genome-amplification (WGA) step, which introduces technical noise into single-cell sequencing datasets. This approach provides single-molecule data by positional barcoding, reducing WGA artifacts that are typically associated with amplification bias. We further utilize a dual-barcoding approach and acoustic liquid transfer technology to increase throughput (N=384) and reduce cost to $1 per cell using nanoliter volumes. We applied SMSC to sequence copy number profiles from ~1000 single cells from two patients with BRCA2-positive breast tumors. In both cases we identified several major clonal subpopulations that shared a set of truncal CNAs, suggesting that they evolved from a single normal epithelial cell in the breast, not multiple initiating cells. We also identified many small subclonal CNAs that further diversified the major clones, which suggests that copy number evolution was ongoing during the expansion of the primary tumor mass. These data demonstrate the technical feasibility of SMSC sequencing, and further show that minor copy number evolution is ongoing in BRCA-positive breast cancer patients.

#2193

Biopsy and analytical testing methods used to identify PIK3CA mutations in hormone receptor-positive, human epidermal growth factor receptor-negative metastatic breast cancer: A systematic literature review.

Elizabeth Anderson,1 Lea Mollon,1 Alejandra Aguilar,1 Joni Dean,1 Lisa Davis,1 Terri Warholak,1 Ayal Aizer,2 Emma Platt,3 Aditya Bardiya,2 Derek Tang3. 1 _University of Arizona, Tucson, AZ;_ 2 _Harvard Medical School, MA;_ 3 _Novartis, NJ_.

Introduction: This study aimed to identify biopsy and analytical testing methods for PIK3CA mutation in hormone receptor-positive (HR+), human epidermal growth factor receptor 2-negative (HER2-) metastatic breast cancer (mBC) via a systematic literature review. Understanding the available biopsy and analytical methods for PIK3CA mutation and its publication trends over time may support understanding of emerging technology trends.

Methods: A comprehensive search of PubMed/MEDLINE, EMBASE, Cochrane Central, and select conference abstracts was performed by two independent researchers that included, but was not limited to, keywords: "breast neoplasm," "PIK3CA protein," "hormone receptor positive," and "metastases." English-language studies in HR+, HER2- mBC reporting either biopsy or analytical testing methods for PIK3CA mutation and published between January 1993 through August 2017 were included. Content analysis was employed to quantify collected data elements.

Results: Of 558 studies included for full-text review, 36 met inclusion criteria. Total number of samples tested among this subgroup were 4,247 (tumor = 3,597; liquid = 650). Sample size ranged from 9 to 618 (median = 47). The most common methods used for identifying PIK3CA mutations were tumor biopsies (n = 33) while liquid biopsies only occurred in four studies. One study reported both liquid and tumor biopsy data. Formalin-fixed, paraffin-embedded (FFPE) tissue samples were used in most studies examining tumor samples (n = 28). Among liquid biopsies, circulating tumor cells (CTC) (n = 2), cell-free DNA (cfDNA) (n = 1), and circulating tumor DNA (ctDNA) (n = 1) were used. Techniques for DNA analyses included PCR (n = 14), NGS (n = 14), Sanger (n = 7), mass spectrometry (n = 5) and liquid chip technology (n = 1). Some studies used multiple methods (n = 5). Most studies using NGS were cross-sectional (n = 9); other studies using NGS (n=4) were clinical trials. Among clinical trials (n = 9), four used PCR only (sample size range 30 to 550), two used NGS only (sample size range 16 to 28), one used Sanger sequencing only (sample size = 21) and two used both NGS and PCR methods (sample size range 26 to 55). Of fourteen studies using NGS, thirteen were published from 2015 to 2017, which may be indicative of a transition to newer methodology. While PCR remained a commonly used analytical method regardless of sample size, NGS has become more common than PCR in recent publication years.

Conclusions: The majority of studies utilized PCR for PIK3CA analytical testing despite newer technologies available. NGS methodology was used more frequently in more recent publications. However, the relative accuracy between PCR and NGS testing is yet to be fully understood.

#2194

Subclonal variation and evolutionary dynamics of BRAF mutations in cancer.

Priyanka Gopal, Elif I. Sarihan, Mohamed E. Abazeed. _The Cleveland Clinic Taussig Cancer Inst., Cleveland, OH_.

Tumors have genetically distinct subclones that compete for space and resources and differentially resist efforts to make them extinct. We studied the intratumoral heterogeneity of BRAF mutations across several cancer types. We identified BRAF driver mutations as predominately clonal in some cancer types (e.g. melanoma) and subclonal in others (e.g. lung adenocarcinoma). Clonality corresponded to the amplification of BRAF and prevalence of V600 mutations in each cancer type. We mathematically and experimentally modeled the propagation and selection of tumors containing BRAF mutations and determined that the speed of clonal sweeps were associated with the extent of activation of MAPK signaling pathway and BRAF copy number. Consistent with these findings, tumors with "hard" sweeps were more likely to respond to BRAF and/or MEK inhibitors. Furthermore, some PDX models treated with cytotoxic therapy underwent BRAF mutant subclone expansion over time and this effect is mitigated by inhibitors of BRAF and/or MEK. Treatment strategies based on subclone composition resulted in significantly improved tumor control. Our study uncovers patterns of distinct BRAF clonal evolutionary trajectories and advances therapeutic strategies on the basis of BRAF mutation identity and subclone composition that merit testing in patients.

#2195

Temporal and spatial dynamics of metastatic colonization revealed by 26 rapid-autopsy tumor biopsies from a TNBC patient.

Xiaomeng Huang,1 Yi Qiao,1 Samuel W. Brady,1 Adam Cohen,1 Andrea H. Bild,2 Gabor T. Marth1. 1 _University of Utah, Salt Lake City, UT;_ 2 _City of Hope, Duarte, CA_.

Metastatic breast cancer is an advanced-stage disease in which the cancer cells have spread to distant organs. To understand the patterns of metastatic colonization in a patient who presented with aggressive disease, we have collected tumor biopsies at initial diagnosis, at mastectomy necessitated by the patient's relapse, twenty-six metastatic tumors across seven organs and two normal tissue control skin biopsies via a rapid autopsy procedure within hours after the patient's death.

All biopsy samples were subjected to 60X Illumina whole genome sequencing. Our analysis revealed extensive chromosomal changes including amplifications, deletions, LOH and translocations, as well as known driver mutations in RB1, TP53, and PTEN in all tumor samples. We used the CNV and LOH data to reconstruct the phylogenetic relationships among the tumor samples. We used the somatic SNV allele frequencies in copy number-normal regions to refine these phylogenetic relationships, and to establish the evolution of the tumor at the subclonal level across different metastatic sites, with an extension of our SubcloneSeeker algorithm.

Our analysis reveals the lung as the first metastatic site. In fact, this invasion occurred before relapse at the primary site in the breast, indicating early metastatic escape. This initial invasion was followed by distinct waves of massive metastatic expansion, beginning with the abdominal organs, then the lymph nodes, brain and bones. As the initial invasion, each metastatic wave involved other distinct sites in the lungs, these sites serving as incubators where subclones further evolved prior to seeding new metastatic sites. Finally, we see clear evidence for metastatic "recolonization" where such further evolved subclones invaded already established, earlier metastatic sites.

The high number of biopsied sites in this study, 30 in all, allowed us to reconstruct the evolution of the aggressive disease in our patient with unprecedented resolution, and to identify characteristic patterns of metastatic colonization. If confirmed in additional, similarly high resolution datasets, these patterns will lead to better understanding of the metastatic process, and guide effective clinical intervention.

#2196

Noninvasive description of natural tumor evolution.

Jerome Griffon, Delphine Le Guillou-Buffello, Oumeima Laifa, Lori Bridal, Michele Lamuraglia. _Laboratoire d'Imagerie Biomedicale (UPMC, CNRS, INSERM), Paris, France_.

Background: Our goal is to investigate how quantitative parameters of Contrast Enhanced Ultrasound (CEUS) and Shear Wave Elastography (SWE) change during tumor growth and link them with morphological and histological indexes.

Methods: An ectopic model of murine colorectal carcinoma (CT26) was implanted in 46 mice (BALB/c ) at Day 0. On 5 subsequent days, data were acquired from subsets of these mice (D7, n=8 ; D10, n=9 ; D14, n=10 ; D16, n=9, D17, n=10) by scanning each tumor along its major, longitudinal axis in vivo to obtain conventional B-mode, SWE (SSI, Aixplorer, SL15-4 probe) and dynamic CEUS (Sequoia 512, 7-14 MHz probe, cadence contrast pulse sequencing) data. The ellipsoidal volume of each tumor was estimated from measurements of the width and thickness of the largest plane along the longitudinal and transversal plane using B-mode display on the Aixplorer device. Quantitative maps of tissue elasticity distribution in the tumor were obtained from SWE data. Average values and standard deviation of SWE data were calculated within regions selected to outline the tumor cross section on B-mode images. CEUS was performed after a syringe-pump-controlled bolus injection in the caudal vein of 40 μL of SonoVue (Bracco Suisse, Geneva, Switzerland) in a weight-adjusted volume of 0.5 mL/kg. A lognormal model was fit to the time intensity curve inside the tumor to estimate parameters related to microvascular volume and flow. All tumors imaged on a given day were excised and marked to conserve orientation and approximate position relative to the US imaging plane. Tumors were frozen with liquid nitrogen in an Optimal Cutting Temperature compound cube and stored at -80°C.

Results: Significance of correlations were evaluated using the non-parametric Spearman correlation coefficient (RS). Average SWE parameters were not significantly correlated with the CEUS parameters (p > 0.03). Standard deviation of SWE in the tumor was well correlated with tumor volume (RS = 0.65 [p<6x10^-7]). For CEUS only the Time To Peak and Latency Time correlated with tumor volume (RS = 0.62 [p<4x10^-6] and RS = -0.72 [p<2x10^-8], respectively). Correlation with histological assessment of tumor microstructure is still in the process of analysis.

Conclusions: These preliminary results probe the relationship between tumor volume, functional-flow and elastic properties in tumors. Both CEUS and SWE parameters were found that correlate with tumor growth and thus, appear to be sensitive to changes occurring during tumor development. Histological analysis of tumor microstructure is underway to more fully probe this point. Lack of correlation between SWE and CEUS parameters suggests that they provide complementary information. Better understanding of the sensitivity of these non invasive parameters to tumor growth changes will contribute to their eventual integration in procedures of tumor response criteria in clinical practice.

#2197

Surface marker and gene expression profiling of tumors from PDX models of small cell lung cancer with varying sensitivity to growth inhibition by the p70S6K/AKT1/3 inhibitor M2698.

Friedrich Hahn,1 Warren Porter,1 Eileen Snowden,1 Mitchell Ferguson,1 Frances Tong,1 William S. Dillmore,1 Anderson Clark,2 Hong Zhang,2 Rainer Blaesius1. 1 _BD Technologies, Research Triangle Park, NC;_ 2 _EMD Serono, Billerica, MA_.

Small Cell Lung Cancer (SCLC) is characterized by rapid tumor growth and currently, there are few therapeutic options. As part of the development of M2698, a potent, selective inhibitor of p70S6K and AKT 1/3, the compound was tested in a preclinical screen of 45 patient derived xenograft (PDX) models of SCLC. In this screen, two mice were implanted subcutaneously with tumors for each model; one mouse was treated with vehicle while the other was treated with M2698 25 mg/kg QD po and tumors were measured 2x/week until the tumor in the vehicle-treated mouse reached ~1200 mm3. Tumor control (tumor stasis or regression) was seen in 12 (27%) of the models. From these data, a subset of models that were the most and the least sensitive to M2698 (n=7) were selected for implantation into a new set of mice. Tumors were profiled once they reached ~800 mm3 for tumor cell surface markers potentially correlating with sensitivity to M2698 seen in the previous study.

PDX model systems are capable of recapitulating the intra-tumor heterogeneity (ITH) observed in the original patient tumor. The relationship between ITH and drug response/acquired drug resistance has been clearly demonstrated. Thus, a more detailed examination of this complexity in PDX tissue enables one to elucidate the underlying mechanisms in a reproducible, biologically relevant system.

We have previously shown that cell surface marker profiles of PDX derived tumor tissue demonstrate high intra-model reproducibility whereas each model has a unique profile. In addition, some markers have a distinct heterogeneity. Gene expression profiling of tumor cell subpopulations identified by some of these specific markers suggest different biological roles for these subpopulations with relevance to their drug responses.

Here we show surface marker and gene expression profiles of multiple PDX models for SCLC. We have evaluated 50+ markers commonly used to identify tumor initiating cells (e.g. CD44, CD90, CD133, CD166, CD184), EMT or aggressiveness (e.g. CD166, EphB2, CD324, CD325) or drug targets (e.g. CD184, EGFR, Her2) to establish extensive marker profiles.

Our data reveal that surface marker profiles in SCLC PDX models are consistent among tumors from mice bearing the same model, show major differences from other PDX (e.g. breast) cancer types and heterogeneity exists for several markers of interest. By correlating gene expression and surface marker profiles in the current preclinical study, we aim to illuminate the interactions of major cell subpopulations within SCLC PDX tumor tissue and also investigate potential markers of sensitivity to growth inhibition by treatment with M2698. Overall, our workflow of deep phenotyping may provide tools for sample characterization, quality control and possibly study of cellular response to varying selective pressures such as drug challenges.

#2198

An integrated workflow for liquid biopsy of circulating multiple myeloma cells (CMMCs) with single cell resolution reveals tumor heterogeneity.

Mario Terracciano,1 Claudio Forcato,1 Edoardo Petrini,1 Alberto Ferrarini,1 Valentina del Monaco,1 Andrea Raspadori,1 Carrie Morano,2 Steven Gross,2 Chiara Bolognesi,1 Genny Buson,1 Thai Bui,2 Francesca Fontana,1 Gianni Medoro,1 Mark Connelly,2 Nicolò Manaresi1. 1 _Menarini Silicon Biosystems S.p.A, Italy;_ 2 _Menarini Silicon Biosystems Inc., PA_.

Background: Multiple myeloma (MM) is a bone marrow derived cancer of plasma cells, which remains an incurable disease. Because of the invasive and painful nature of bone biopsy, an alternate tumor monitoring strategy is needed. We have previously shown that Circulating Multiple Myeloma Cells (CMMCs) isolated by CellSearch® (CS) are prognostic and indicative of disease burden through remission and relapse. Here we report, for the first time, the molecular characterization of pure single CMMCs isolated from a multiple myeloma patient, by integrating CS and DEPArray™ (DA) NxT systems, providing access to copy-number alteration (CNA) profiling. Methods: 4.0ml of peripheral blood from a patient with multiple myeloma was tested. On CS, target CMMCs were enriched using anti-CD138 for cell capture, and stained immunofluorescently with CD38-PE, CD19 and CD45-APC. Nuclei were stained with DAPI and detected target cells counted. The enriched sample was then analyzed using the DA NxT system: single CMMCs (CD38+/CD19- and CD45-/DAPI+), along with some single White Blood Cells (WBCs: CD38-/CD19+ or CD45+/DAPI+), were isolated. The DNA of each single cell was amplified using the Ampli1™ WGA kit and used for highly-multiplexed, genome-wide single-cell CNA analysis using a Ampli1™ LowPass kit (LPCNA) on Illumina® MiSeq. Results: CS identified 128 CMMCs. From DA NxT we harvested 20 individual CMMCs for LPCNA analysis. Copy-number profiles of CMMCs confirmed their tumor origin, showing a high-level of genome instability with several gains and few losses of chromosomal segments. Moreover, an unsupervised hierarchical cluster analysis highlighted a conserved pattern of alterations, enabling the separation between CMMCs and WBCs groups. A pattern of copy-number gains shared by all CMMCs, coupled with gains and losses shared by a subset of CMMCs, suggests a branched evolution of different tumor subclones. WBC profiles were flat as expected. Conclusions: Cell enrichment by CS followed by individual cell sorting using DA NxT, enabled the isolation of single CMMCs with 100% purity. Ampli1™ single-cell analysis demonstrated CMMC molecular heterogeneity suggestive of tumor subclones presence. This platform combination provides a reliable and non-invasive method for MM characterization enabling translational research and future clinical application.

#2199

Establishment and high-throughput drug testing of multiple patient-derived cells from each renal cancer; intratumor heterogeneity of drug response and implications for precision medicine.

Khalid Saeed,1 Poojitha Ojamies,1 Teijo Pellinen,1 Samuli Eldfors,1 Riku Turkki,1 Johan Lundin,1 Harry Nisen,2 Petrus Järvinen,2 Kimmo Taari,2 Taija af Hällström,1 Antti Rannikko,2 Tuomas Mirtti,1 Olli Kallioniemi,3 Päivi Östling3. 1 _Institute for Molecular Medicine Finland, FIMM, University of Helsinki, Helsinki, Finland;_ 2 _Department of Urology, Helsinki University Hospital, Helsinki, Finland;_ 3 _Science for Life Laboratory, Dept of Oncology and Pathology, Karolinska Institutet, Stockholm, Sweden_.

Renal cell cancer (RCC) has become a prototype example of the extensive intra-tumor genetic heterogeneity and clonal evolution of human cancers. There is, however, little information on how the genetic heterogeneity will impact on drug responsiveness of the cancer cells. Establishment of multiple patient-derived cells (PDCs) from individual patients may help us to understand genomic clonal evolution pathways in cancer, explore intratumor heterogeneity of drug response across tumors, as well as help to define combinatorial treatments needed to target multiple subclones in a cancer patient. Here, we established several PDCs from different tumor regions of four RCC patients, verified clonal relationships of the PDCs with each others and with the uncultured tumor tissue by genome sequencing. PDCs retained cancer-specific copy number alterations and mutations in driver genes such as VHL, PBRM1, PIK3C2A, KMD5C and TSC2 genes, but also showed differences indicating genetic heterogeneity and clonal evolution, and thus generating a set of natural isogenic variants. Comprehensive drug-sensitivity testing of the PDC clones with 460 oncology drugs was performed. The drug testing analysis identified shared vulnerability of the PDCs towards several approved RCC drugs, such as mTOR-inhibitor (temsirolimus) and multi-kinase-inhibitor (pazopanib). The individual RCC PDC clones from different tumor regions in one patient showed distinct drug response profiles, confirming that genomic heterogeneity contributes to the variability in drug responses. We developed a capability to map drug response evolution on top of genomic evolution in cancer. In conclusion, our study suggested that comparison of drug response profiles among multiple (isogenic) PDC variants from a cancer patient may be informative for i) assessing intratumor heterogeneity in drug response ii) elucidating pharmacogenomic biomarkers among natural isogenic cell variants and iii) identifying drugs and drug combinations that may kill the multiple cancer subclones in a patient. Funding: EU-FP7-Systems Microscopy Network of Excellence, Sigrid Juselius Foundation, Cancer Society of Finland, Academy of Finland, the Magnus Ehrnrooth Foundation, TEKES FiDiPro Fellow Grant and Knut and Alice Wallenberg Foundation.

#2200

Intratumoral heterogeneity of copy number variation in lung cancer harboring L858R via immunohistochemical heterogeneous staining.

Takafumi Hashimoto. _Oita University, Oita, Japan, Japan_.

Background: Acquired resistance to EGFR-TKI is an inevitable event for advanced lung cancer patients harboring the EGFR-activating mutation. Although intratumoral heterogeneity is commonly observed in several cancers, such as renal cell carcinoma, few studies have shown its presence in EGFR-mutated lung cancer. We analyzed the intratumoral heterogeneity in EGFR-mutated (L858R) lung cancer through immunohistochemistry (IHC) for the primary site and targeted sequencing for specific cases in order to clarify the mechanism of acquired resistance.

Methods: Twenty resected primary lung cancers known to harbor EGFR L858R were analyzed. IHC was performed using an L858R mutant-specific rabbit monoclonal antibody (clone43B2; Japan) and the samples were scored by staining intensity (0-3) and proportion. For cases with heterogeneous L858R protein expression, the nucleic acids were extracted from each differently stained lesion, and targeted sequencing (Oncomine Cancer Research Panel) was performed. Single-nucleotide variations (SNVs) and copy number variations (CNVs) from each site were then analyzed. The cell proliferation and apoptosis were also evaluated by the ki-67 labeling index and TUNEL staining.

Results: Among 20 cases, 3 (15%) showed heterogeneous staining, defined as having both an L858R IHC-positive part and an IHC-negative one. Genetic analyses for cases with heterogeneous staining revealed an increase in the copy number of EGFR in the IHC-positive part (CNV 19.54) compared to the negative part (CNV 3.03), and an increase in the copy number of CCNE1 was observed in the IHC-positive part (CNV 18.59) compared to the negative part (CNV 2.59) in one case (case 1). In another case (case 2), an increase in the copy number of EGFR was observed in the IHC-positive part (CNV 8.21) compared to the negative part (CNV 2.90), and an increase in the copy number of MDM2 was observed in the IHC-positive part (CNV 6.65) compared to the negative part (CNV 1.37). Case 3 did not show any CNV. In three cases, no SNV changes were observed. An increase in the ki-67 labeling index in the L858R-positive part in case 1 and increased apoptosis in the L858R-positive part in case 2 were observed, suggesting the functional significance of CNV changes.

Conclusion: These cases exhibiting L858R IHC intratumoral heterogeneity suggest a heterogeneous effect on the cell activity due to CNV heterogeneity.

## EPIDEMIOLOGY:

### Screening, Early Detection, and Risk Prediction

#2201

Prostate-specific antigen velocity is associated with future prostate cancer mortality among men in the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial.

Erik Barr,1 Sonja I. Berndt,2 John D. Sorkin,3 Wen-Yi Huang,2 Kathryn Hughes Barry1. 1 _University of Maryland School of Medicine, Baltimore, MD;_ 2 _National Cancer Institute, Rockville, MD;_ 3 _Baltimore VA Medical Center Geriatric Research, Education, and Clinical Center, Baltimore, MD_.

Higher prostate-specific antigen (PSA) velocity (change in PSA over time) measured after diagnosis or treatment is associated with poorer prognosis for prostate cancer patients. However, less is known about the association between PSA velocity measured prior to a diagnosis of cancer and future prostate cancer mortality. We evaluated whether PSA velocity is associated with the risk of prostate cancer mortality among men without a history of prostate cancer in the screening arm of the Prostate, Lung, Colorectal, and Ovarian (PLCO) Cancer Screening Trial. As part of the PLCO trial, men in the screening arm were offered PSA screening at baseline in the trial and annually thereafter for five years. From the screening arm, we included 33,961 men who had at least two prediagnostic trial PSA measurements (to allow for PSA velocity calculation), among whom 167 later died of prostate cancer. We estimated PSA velocity and initial PSA concentration for each participant from the random slope and random intercept terms in a mixed effects model based on up to the first three trial PSA measures per person since participants varied in the number of available measures. We defined our study baseline for survival analysis as the time of the last PSA measurement used to calculate PSA velocity. Participants were 55 to 79 years of age at our study baseline, and we followed them for up to 18 years (through Dec. 31, 2012) for prostate cancer mortality, with a median follow-up time of 12.7 years (interquartile range: 10.7-14.4 years). We dichotomized PSA velocity as <0.4 vs. ≥0.4 ng/mL/year for comparison with previous studies. We analyzed the association between PSA velocity and prostate cancer mortality using Cox proportional hazards regression, adjusting for initial PSA concentration, age, race, body mass index, our study baseline year, and past finasteride use. Higher PSA velocity was associated with an increased hazard of prostate cancer mortality compared to lower PSA velocity [Hazard Ratio (HR)=2.5, 95% Confidence Interval (CI): 1.7-3.7]. Additional adjustment for family history of prostate cancer or comorbidities did not appreciably alter findings. When we stratified by age at our study baseline, we observed a stronger association among men who were less than 70 years of age (HR=3.1, 95% CI: 1.9-4.9) than among those who were 70 years of age or older (p-interaction=0.04). In conclusion, in this large prospective study, higher prediagnostic PSA velocity was associated with an increased risk of prostate cancer mortality. Our findings suggest a potential benefit of using longitudinal PSA measures in predicting future prostate cancer mortality.

#2202

Long term risk prediction of p16/Ki-67 dual stain in triage of HPV-positive women.

Megan A. Clarke,1 Barbara Fetterman,2 Mark Schiffman,1 Philip E. Castle,3 Eric Stiemerling,2 Diane Tokugawa,2 Nancy Poitras,2 Walter Kinney,4 Thomas Lorey,2 Nicolas Wentzensen1. 1 _National Cancer Institute, Rockville, MD;_ 2 _Kaiser Permanente, Berkeley, CA;_ 3 _Albert Einstein College of Medicine, Bronx, NY;_ 4 _Kaiser Permanente Medical Care Program, Oakland, CA_.

Background: Human papillomavirus (HPV) testing has been approved as a primary strategy for cervical cancer screening, either alone or in combination with cytology (co-testing), based on its high sensitivity and long-term reassurance against cervical precancer following a negative test result. However, nearly twice as many women will screen positive for HPV compared with cytology-based screening. Thus, effective management of HPV-positive women requires triage markers to distinguish those at high-risk who should be referred to colposcopy from those with benign infections who can safely return to routine screening. p16/Ki-67 dual stain cytology has previously shown good risk stratification for triage of HPV-positive women; however, studies with follow-up extending beyond 3 years are lacking. We evaluated the long-term risk prediction of p16/Ki-67 for detection of cervical precancer (cervical intraepithelial neoplasia grade 3 or worse, CIN3+) in a large population of HPV-positive women. Methods: 1,588 HPV-positive women screened with HPV/cytology co-testing were enrolled in 2012 at Kaiser Permanente Northern California. p16/Ki-67 cytology was performed on residual Surepath material and slides were evaluated for p16/Ki-67 positivity. Cervical histology endpoints were ascertained from the clinical database with follow-up through 2017. We conducted a Kaplan Meier analysis to estimate risk of CIN3+ by p16/Ki-67 and cytology (atypical squamous cells of undetermined significance or worse, ASC-US+, versus normal cytology). Risks were compared to internal benchmarks for colposcopy referral and for a one year return interval. Results: In women testing p16/Ki-67 positive at baseline, the 2-year risk of CIN3+ was 14.3%, compared with 2.2% in p16/Ki-67-negative women. For ASC-US+, the risk was 12.6% and 2.9% for normal cytology. The 5-year risk of CIN3+ in p16/Ki-67-positive women was 21.6% and 5.0% in p16/Ki-67-negatives. The 5-year risk of ASC-US+ was 17.1% compared to 8.2% for normal cytology. Among p16/Ki-67-negatives, the risk remained below the colposcopy referral threshold for 5 years while in women with normal cytology, the colposcopy referral threshold was crossed after year 3. Conclusion: In the first study evaluating long-term risk stratification of p16/Ki-67 dual staining, p16/Ki-67- negativity provided strong reassurance against CIN3+ for at least five years. In contrast, the risk in women with normal cytology crossed the colposcopy referral threshold after three years. These data support use of p16/Ki-67 for triage of HPV-positive women with the possibility of extending surveillance intervals in p16/Ki-67-negative women.

#2203

A prospective study of endometrial cancer detection in women presenting for evaluation of abnormal peri- and postmenopausal bleeding.

Nicolas A. Wentzensen,1 Megan Clarke,1 Viji Shridhar,2 Maureen Lemens,2 Matthew Hopkins,2 Lisa Ahlberg,2 Shannon Laughlin-Tommaso,2 Karl Podratz,2 Mark Sherman,3 Jamie Bakkum-Gamez2. 1 _NCI-DCEG, Bethesda, MD;_ 2 _Mayo Clinic, MN;_ 3 _Mayo Clinic, FL_.

Background: Endometrial cancer (EC) is the most common gynecologic malignancy in developed countries and the incidence and mortality of EC continue to rise in the US. When detected early, EC is highly curable, suggesting that early detection strategies could reduce EC mortality. Women who develop EC often have a prolonged history of abnormal bleeding, offering a window for identifying many at-risk women prior to the development of advanced cancer. To evaluate risk prediction, novel sampling and early detection strategies in women at increased risk of EC, we established a prospective cohort of women with abnormal bleeding that is followed for endometrial hyperplasia and cancer endpoints. Methods: We consecutively enrolled women ≥45 years old presenting to the Mayo Clinic for evaluation of abnormal uterine bleeding (AUB), postmenopausal bleeding (PMB), thickened endometrial stripe (ES) on imaging, or Lynch Syndrome (LS). From all women, an intravaginal tampon and an endometrial brushing via Tao brush were collected before endometrial biopsy. Epidemiological EC risk factor data, clinical data, and endometrial biopsy histology results were abstracted from medical records. Absolute risk of endometrial hyperplasia and EC were calculated overall and in strata based on clinical symptoms and risk factors. Results: Among the first 1,000 women enrolled into the study, 712 (71%) were overweight or obese, 534 (53%) were postmenopausal, and 214 (22%) were current hormone replacement therapy (HRT) users. Endometrial biopsy and, if indicated, subsequent hysterectomy, detected 39 ECs, 16 atypical hyperplasias, and 28 hyperplasias without atypia at baseline evaluation. Among 534 postmenopausal women, 453 (85%) reported any type of bleeding, with spotting (39%) and light bleeding (27%) being more common than heavy bleeding (14%). Among perimenopausal women with AUB (n=452), the absolute risk of EC at baseline was 0.9% and among women with PMB, the absolute risk of EC at baseline was 7.5%. Among women with PMB, EC risk was modified by HRT use (risk of 2.6% in women on HRT and 8.2% in women not on HRT, respectively), obesity (risk of 5.3%, 8.1% and 9.5% for women with normal, overweight, and obese BMI, respectively), and type of bleeding (risk of 9.7%, 4.9%, and 8.2% in women with spotting, light or heavy bleeding, respectively). Discussion: While risk of endometrial cancer in perimenopausal women with AUB is low, approximately 8% of women presenting for clinical work up of PMB have underlying EC. Clinical presentation and risk factors modify the absolute risk of EC and may inform clinical management. Additional recruitment and prospective follow-up is underway to improve estimates of short- and long-term EC risk in women with PMB. With its collection of tampon and Tao brush samples, the study will provide a unique resource to evaluate molecular markers of EC and EC risk in elevated-risk women.

#2204

Influence of chronic comorbidities on colorectal cancer screening adherence in a population-based Canadian cohort.

Dominika Bhatia, Rinku Sutradhar, Jill Tinmouth, Simron Singh, Lorraine Lipscombe. _University of Toronto, Toronto, Ontario, Canada_.

Background: Regular screening for colorectal cancer (CRC) is associated with a lower stage at diagnosis and improved survival. National guidelines recommend periodic screening for adults aged 50-74 years. Chronic diseases may distract from regular screening and increase CRC burden. We sought to determine whether having certain chronic conditions is associated with a lower rate of CRC screening adherence.

Methods: Using linked healthcare databases from Ontario, Canada, we assembled a population-based cohort of adults aged 50-74 years, who were eligible and overdue for routine CRC screening (fecal occult blood testing > 2 years, sigmoidoscopy > 5 years, or colonoscopy > 10 years) at any point between April 1, 2004 and March 31, 2016. We implemented Andersen-Gill extended Cox models for recurrent events using an age time scale to examine the impact of chronic conditions on the relative rate (RR, 95% confidence interval, CI) of becoming adherent with periodic CRC tests, adjusted for the relevant time-varying covariates.

Results: During the 12-year observation window, included individuals contributed a total of 19,818,050 person-years spent overdue for CRC tests. Over 70% of all CRC tests performed during the study period were fecal occult blood tests. At baseline, of the 4,642,422 individuals that met the inclusion criteria, 31% had hypertension, 12% had diabetes, 9% had chronic obstructive pulmonary disease (COPD), 7% had mental illness, 5% had cardiovascular disease (CVD), 1.6% had congestive heart failure (CHF), and 0.1% had end-stage renal disease (ESRD). After adjustment for sociodemographic and health system factors, the highest rates of nonadherence were observed for ESRD on dialysis (RR 0.66, CI 0.63-0.68), CHF (RR 0.75, CI 0.75-0.76), COPD (RR 0.84, CI 0.83-0.84), CVD (RR 0.85, CI 0.84-0.85), diabetes (RR 0.86, CI 0.86-0.87), and mental illness (RR 0.88, CI 0.87-0.88). Hypertension (RR 0.98, CI 0.97-0.98) and ESRD with kidney transplant (RR 1.00, CI 0.84-1.19) were associated with modest to no risk.

Conclusion: The presence of a major chronic condition is associated with lower adherence to CRC screening. Future studies should explore reasons for lower CRC screening adherence in individuals with chronic conditions and the appropriateness of secondary cancer prevention and early diagnosis in this population.

#2205

Prediction model of CA125 among premenopausal women.

Naoko Sasamoto,1 Ana Babic,2 Bernard A. Rosner,1 Allison F. Vitonis,1 Daniel W. Cramer,1 Shelley S. Tworoger,3 Kathryn L. Terry1. 1 _Brigham and Women's Hospital, Boston, MA;_ 2 _Dana-Farber Cancer Institute, Boston, MA;_ 3 _Moffitt Cancer Center, Tampa, FL_.

Background: Cancer antigen 125 (CA125) is a membrane-bound glycosylated mucin that has been reported to be the most promising biomarker for ovarian cancer screening, although results from two large randomized trials comparing screening using CA125 and transvaginal ultrasound to usual care have shown no clinically significant difference in ovarian cancer mortality. A major limitation of CA125 as an ovarian cancer screening biomarker has been low specificity and variation between individuals by personal characteristics. Identifying personal characteristics that influence CA125 levels could be used to create personalized thresholds for CA125 for each individual, thereby improving its performance as an ovarian cancer screening biomarker. However, reports on factors that influence CA125 among premenopausal women are limited.

Methods: We evaluated the association between reproductive and lifestyle factors and CA125 among 828 premenopausal population-based controls enrolled in the New England Case-Control study between 1992 and 2008. CA125 was measured using the CA125II assay at the CERLab at Boston Children's Hospital. We developed a prediction model of log-transformed CA125 using stepwise linear regression with <0.15 as significance level for entry and stay considering factors associated with CA125 in postmenopausal women and ovarian cancer risk factors, such as age, body mass index (BMI), race, smoking status, age at menarche, oral contraceptive use and its duration, parity, endometriosis, tubal ligation, unilateral oophorectomy, hysterectomy, family history of ovarian cancer, previous history of cancer, genital powder use, and caffeine intake as candidate predictors.

Results: In our univariate analyses, older age and endometriosis were associated with significantly higher CA125 while tubal ligation, hysterectomy, and time since last menstrual period were associated with significantly lower CA125 (p<0.05). Age, endometriosis, tubal ligation, hysterectomy, smoking status, caffeine intake, and interval days since last menstrual period were selected in our final prediction model, which explained 6% of the total variability of log-transformed CA125 levels.

Conclusion: Efforts to validate this CA125 prediction model in an independent cohort of premenopausal women and evaluate whether using the personalized CA125 cutoff based on this prediction model discriminates cases and controls better than the single 35 cutoff are ongoing.

#2206

A new sensitive, accurate liquid chromatography-tandem mass spectrometry (LC-MS-MS) method to measure circulating estrogens, androgens, and progesterone in postmenopausal and premenopausal women.

Lumi Duke,1 Paul H. Kim,2 Julianne Cook Botelho,1 Regina G. Ziegler,3 Hubert Vesper1. 1 _CDC Atlanta, Atlanta, GA;_ 2 _Battelle Memorial Institute, Atlanta, GA;_ 3 _National Cancer Institute, Bethesda, MD_.

Endogenous steroid hormones play a critical role in the etiology of breast cancer. However, the interrelationships among estrogens, androgens, and progesterone in determining risk are not well understood. Relative strengths of associations, independent and joint effects, differential effects for tumor subtypes, and effect modification by lifestyle and genetic profile remain unclear. A major reason is the limited accuracy, specificity, and sensitivity of many steroid hormone assays, especially at the low circulating hormone concentrations characteristic of postmenopausal women.

CDC developed a new sensitive, accurate isotope dilution LC-MS/MS method capable of measuring eight important steroid hormones in 200 µL of female serum. The method uses sequential liquid-liquid extractions for isolation of estradiol (E2), estrone (E1), estrone sulfate (E1S), testosterone (TT), androstenedione (A), dehydroepiandrosterone sulfate (DHEAS), progesterone (P), and 17-hydroxyprogesterone (17-OHP) without derivatization or hydrolysis. Chromatographic separation of the steroids is carried out using a phenyl-hexyl HPLC column and a gradient of methanol and methanol:water. Scheduled selected reaction monitoring by electrospray ionization in the positive and negative ion modes is applied to quantify the analytes. Two mass transitions, each specific to the fragmentation of the analyte, are monitored for each analyte: quantitation ion (QI) and confirmation ion (CI). The QI to CI ratios are used as criteria to identify possible interferences.

The method is able to measure each steroid in nearly all postmenopausal women as well as premenopausal women. Limits of detection in serum-based materials are: E2 1.72 pg/mL, E1 0.13 ng/dL, E1S 2.04 pg/mL, TT 0.57 ng/dL, A 0.82 ng/dL, DHEAS 0.22 µg/dL, P 0.86 ng/dL, and 17-OHP 0.41 ng/dL. Precision, expressed as coefficients of variation and evaluated at three levels for each analyte, ranged from 1.2 to 9.9%. Average bias, a measure of accuracy, ranged from -0.3 to 8.8%. The method is certified through the CDC Hormone Standardization Program for estradiol and testosterone and meets the requirements of the College of American Pathologists Proficiency Testing Program for androgens and progesterone.

This new, highly specific and sensitive method demonstrates high accuracy and precision while requiring small amounts of specimens. It can assay up to 140 samples at once. Thus, the method is suitable for large epidemiologic studies investigating the hormonal etiology of breast, endometrial, and ovarian cancer.

#2207

High-risk human papillomavirus detection in head and neck cancers using saliva.

Kai D Tang,1 Chris Perry,2 Ian Frazer,3 Chamindie Punyadeera4. 1 _The School of Biomedical Sciences, Queensland University of Technology, Brisbane, Australia;_ 2 _Princess Alexandra Hospital, Brisbane, Australia;_ 3 _The University of Queensland, Brisbane, Australia;_ 4 _Queensland University of Technology, Brisbane, Australia_.

Human papillomavirus (HPV), a virus vital to the etiology of cervical cancer, is strongly associated with the development of head and neck cancer. Therefore, it is pivotal to develop a noninvasive and cost-effective approach for detecting oral HPV. Accumulating evidence suggested the usefulness of salivary oral rinse as a diagnostic fluid to detect oral HPV DNA within a clinical setting. However, there is little known about the HPV DNA prevalence and type distribution in unstimulated whole mouth saliva (UWMS). In the current study, we show a positive correlation of HPV-16 E2 (r = 0.95, p < 0.0001) and E6/7 (r = 0.93, p < 0.0001) relative copy number as well as high-risk HPV genotypes (HPV 16, 18 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68) and fold change difference between the relative copy numbers of E2 and E6/7 (r = 0.46, p < 0.0098) in both salivary oral rinse and UWMS samples collected from OPC patients. More importantly, in line with the previous study, among HPV-16 positive head and neck cancer cases, most often a mixed HPV-16 form (episomal and integrated) was found in both saliva sampling methods. In conclusion, regardless of the sampling method, detection of HPV in saliva is an attractive, noninvasive method that could change the need for invasive biopsies in head and neck cancer patients.

#2208

Clinicopathological and genetic study of ovarian cancer in Algerian women: First report.

Farid Cherbal,1 Chiraz Mehemmai,1 Lamia Boumehdi,1 Feriel Khider,1 Amina Boucheffa,1 Hadjer Gaceb,1 Hassen Mahfouf,2 Rabah Bakour1. 1 _Unit of Genetics, LMCB, Faculty of Biological Sciences, USTHB, Algiers, Algeria;_ 2 _Public Hospital Academic Medical Oncology Services, School of Medicine, University of Algiers, Rouiba, Algeria_.

Background: Ovarian cancer represents the fourth most common cause of mortality among Algerian women. Of all gynecological malignancies, ovarian cancer causes the highest number of deaths in women in Algeria. In the present study, we sought to determine for the first time, clinical, tumor and genetic characteristics associated with ovarian cancer in Algerian women. We also screened for specific mutations in BRCA1 and BRCA2 genes in patients with positive family history of ovarian cancer.

Materials and Methods: The study population included 244 patients diagnosed with ovarian cancer. Ovarian carcinomas were diagnosed between 2011 and 2016. Data were collected from cancer registries of public hospitals that covered 10 provinces of Algeria. Patient and tumor information included: age at diagnosis, histological type, histological grade, TNM stage, preoperative tumors markers CEA and CA-125 serum levels, family history with ovarian cancer or any cancer, age at menarche, oral contraception and breast feeding. BRCA1 exon 11 and BRCA2 exon 22 were screened by PCR-direct sequencing in 11 patients with a family history of ovarian cancer for two common mutations that were previously found in Algerian population, respectively.

Results: The median age at diagnosis cancer was 51.02 years. The mean age at menarche was 13.04 years. We noticed that 43.85 % of women have been diagnosed with ovarian cancer at younger age (< 50 years). The proportion of ovarian cancer patients with premenopausal status was 39.75%. The commonest histological subtypes was serous adenocarcinoma (52.45%) followed by endometrioid (10.65%), mucinous (9.43%) and clear cell carcinoma (4.09%). We found that the proportion of tumors with histological grade II (41.8%) and grade III (27.86%) was commonest in 70% of the patients. Our results showed that 61.46% of the patients were diagnosed at stage II (39.34%), stage III (8.6%) and stage IV (13.52%), respectively. 73.36% and 18.08% of the patients were positive for CA-125 and CEA, respectively. We noticed that 29 patients (11.88%) had a positive family history of ovarian cancer. 141 patients (57.78%) had breastfed and 107 patients (43.85%) were using contraception.The BRCA1 mutation c.2125_2126insA and BRCA2 mutation c.8940delA have not been detected in our patients.

Conclusions: For the first time, we report here some clinical, biological, tumor and genetic characteristics of ovarian cancer in Algerian women. Our study showed that ovarian cancer in Algerian women has some similar clinicopathological and biological features with women of European descent. Interestingly, the median age of diagnosis in ovarian cancer patients was younger than average age in Europe and America. High frequency of patients with positive family history of ovarian cancer could be linked to genetic background of Algerian population. Further studies are needed to reaffirm our findings. BRCA1 and BRCA2 analysis are ongoing in our patients.

#2209

Lung cancer risk prediction using DNA methylation markers.

Florence Guida,1 Therese H. Nøst,2 Caroline Relton,3 Paul Brennan,1 Torkjel M. Sandanger,2 Marc Chadeau-Hyam,4 Mattias Johansson1. 1 _International Agency for Research on Cancer, Lyon, France;_ 2 _UiT-The Arctic University of Norway, Tromsø, Norway;_ 3 _University of Bristol, Bristol, United Kingdom;_ 4 _Imperial College London,, London, United Kingdom_.

There is an urgent need to improve lung cancer risk assessment as current screening criteria miss a large proportion of cases and result in a high rate of false positives on low-dose CT screening. A fixed effect meta-analyses of 4 epigenome-wide association studies of lung cancer revealed differential DNA methylation at 16 CpG sites (FDR<0.05). The current study aimed to evaluate the extent to which such methylation markers can improve upon smoking-based risk-discrimination among ever smokers. We used data on 662 ever smoking lung cancer case-control pairs from 4 individual prospective cohorts that measured DNA methylation using the Illumina Infinium HumanMethylation450 BeadChip in peripheral blood samples before diagnosis: The Italian part of the European Prospective Investigation into Cancer (EPIC) cohort, the Melbourne Collaborative Cohort (MCCS), the Norwegian Women and Cancer cohort (NOWAC) and the Northern Sweden Health and Disease Study (NSHDS). We adopted a training-testing design where the training was performed on MCCS and NSHDS (N=511 case-control pairs), and the testing on EPIC-Italy and NOWAC (N=151 case-control pairs). Logistic regressions with lasso penalties were performed in the training set to select the best set of CpGs jointly predicting lung cancer. A methylation score was trained by fitting a logistic regression model including the selected CpGs in the training set. A baseline score based on self-reported smoking information (duration, cigarettes/day for current smokers and time since smoking cessation for former smokers) was also developed. The discriminative performances of both scores, as well as integrated model where both smoking and methylation info was incorporated, were assessed by the AUC under the ROC curves in the validation set. The methylation score based on the 9 selected CpG sites yielded an AUC of 0.78 [0.73-0.84] compared to 0.73 [0.68-0.79] for the baseline-smoking score. The model integrating both scores yielded an AUC of 0.79 [0.73-0.84], a notable 0.06-increase in AUC from using the smoking score alone (P=0.008 for difference in AUC). In conclusion, specific methylation biomarkers have a strong potential to improve lung cancer risk assessment and current USPSTF criteria for CT-screening. During the conference, we will present absolute risk estimates based on the integrated risk prediction model.

#2211

Patient level polygenic risk scores and continuous estrogen receptor expression in breast cancer.

Anamay H. Shetty,1 Raza Ali,1 Fiona Blows,1 Carlos Caldas,1 Nicholas Inard,1 Javier Benítez,2 Jenny Chang-Claude,3 Georgia Chenevix-Trench,4 Fergus Couch,5 Angela Cox,6 Peter Devilee,7 Douglas Easton,1 Montserrat Garcia-Closas,8 Arto Mannermaa,9 Heli Nevanlinna,10 Marjanka Schmidt,11 Paul Pharoah1. 1 _University of Cambridge, Cambridge, United Kingdom;_ 2 _CNIO, Madrid, Spain;_ 3 _DKFZ, Heidelberg, Germany;_ 4 _QIMR Berghofer, Brisbane, Australia;_ 5 _Mayo Clinic, Rochester, MN;_ 6 _University of Sheffield, Sheffield, United Kingdom;_ 7 _Leiden University Medical Centre, Leiden, Netherlands;_ 8 _National Cancer Institue, Bethesda, MD;_ 9 _University of Eastern Finland, Finland;_ 10 _University of Helsinki, Helsinki, Finland;_ 11 _Netherlands Cancer Institute, Amsterdam, Netherlands_.

Introduction: Genome-wide association studies over the past 10 years have identified over 100 common variants associated with breast cancer risk. The association of these polymorphisms varies according to estrogen receptor status of the tumor, with some associated primarily with ER+ or ER- disease, or both subtypes. However, ER expression in breast cancer cells is continuous. The aim of this study was to establish whether the association between polygenic risk scores and ER expression in breast cancer is continuous in nature.

Methods: Data were taken from the international Breast Cancer Association Consortium (BCAC). Germline genotype data for 75 SNPs previously reported to be associated with breast cancer were available for 4,999 breast cancer cases for which ER expression data were also available. Automated ER expression on a continuous scale was measured using two image analysis algorithms: Astrogrid and Ariol. A third form of ER expression score, the manual Allred ordinal, was used. Genotype data were used to calculate polygenic risk scores using the equation:

PRS = ∑ βx

where β is the log-odds of the odds ratio (OR) of breast cancer associated with that single nucleotide polymorphism (SNP), and x is the number of variant alleles at a SNP locus. Five different PRSs were calculated:

i) SNPs associated with both ER+ and ER- disease (PRS-A, n= 74)

ii) SNPs associated with ER+ disease at P<0.01 (PRS-B, n= 65)

iii) SNPs associated with ER- disease at P<0.01 (PRS-C, n= 43)

iv) SNPs associated with ER+ but not ER- disease (PRS-D, n= 30)

v) SNPs associated with ER- but not ER+ disease (PRS-E, n = 8)

The ER-specific weights for the PRS were taken from previous OR estimates. Linear regression models adjusted for study were used to test for a relationship between the five PRSs and ER expression measured on the Ariol, Astrogrid and Allred scales.

Results: There was a strong positive correlation found between PRS-B against Astrogrid (β=0.0176, P=4.06x10-4), a weak negative correlation found with PRS-C (β=-0.012, P=0.019) and a weak positive correlation for PRS-A (β=0.012, P=0.0144). PRS-D and E showed far stronger association with Astrogrid than PRS-B and C, respectively (β=0.0186 and -0.017, P=1.97x10-4 and 8.1x10-4). There was a qualitatively similar association when the PRSs were correlated with Ariol and Allred, apart from the stronger correlations with PRS-B (β=91.2 and 0.0853, P=5.52x10-4 and 1.33x10-3).

Conclusion: We have shown a robust association between PRS and ER expression in the directions we predicted. This suggests that the continuous variation in ER expression is biologically plausible and linked to genetic polymorphism. The weakening of association with PRS-A and the stronger associations with PRS-D and E show that having SNPs that are associated with both ER subtypes in the same PRS weakens significance, and suggests that subtype-specific PRSs may be improved on by using SNPs exclusive to the respective subtype.

#2212

Risk prediction for Barrett's esophagus and esophageal adenocarcinoma: Incorporation of epidemiologic risk factors and 23 confirmed genetic loci.

Jing Dong, Aaron Thrift. _Baylor College of Medicine, Houston, TX_.

Background & Aims: We developed comprehensive risk prediction models for Barrett's esophagus (BE) and esophageal adenocarcinoma (EAC) that incorporate a polygenic risk score (PRS) and non-genetic factors.

Methods: We used pooled data from 3,288 BE, 2,511 EAC, and 2,177 controls from BEACON, the United Kingdom Barrett's Esophagus Gene Study, and United Kingdom Stomach and Oesophageal Cancer Study. A PRS was created from 23 BE/EAC risk loci. We developed and compared risk models with various combinations of non-genetics factors and the PRS. We assessed their predictive accuracy using the area under the receiver operating characteristic curve (AUC).

Results: Individuals in the highest quartile of the PRS had 2-fold higher risks of BE (odds ratio [OR], 2.22; 95% confidence interval [CI], 1.89-2.60) and EAC (OR, 2.46; 95% CI, 2.07-2.92) compared to those in the lowest quartile of the PRS. Risk models including only demographic/lifestyle factors (age, sex, smoking, body mass index, and nonsteroidal anti-inflammatory drugs) or only gastroesophageal reflux disease (GERD) symptoms had AUCs ranging from 0.637 to 0.667. The AUCs for models adding demographic/lifestyle factors to GERD symptoms were 0.793 and 0.745 for BE and EAC, respectively. Small absolute improvement in AUCs for each model was observed when including the PRS in the model (AUCs range, 0.656-0.799). Including the PRS in the model of non-genetic factors provided 3.0% and 5.6% improvement in the net reclassification index for BE and EAC, respectively.

Conclusions: Although adding the PRS improved discrimination and net reclassification, the absolute magnitude of improvement is not sufficient to justify its clinical use.

#2213

A simple risk prediction model for high-risk adenomatous polyps at the time of colonoscopy.

Devon J. Boyne,1 Lisa M. Lix,2 Susanna Town,1 Steven J. Heitman,1 Robert J. Hilsden,1 Darren R. Brenner1. 1 _University of Calgary, Calgary, Alberta, Canada;_ 2 _University of Manitoba, Winnipeg, Manitoba, Canada_.

Introduction: The prediction of high risk adenoma polyp (HRAP) may help to prioritize the urgency and guide the performance of colonoscopy procedures. Our objective was to develop and internally validate a simple, scalable clinical prediction model.

Methods: The study population consisted of 2,364 individuals aged 50 to 74 with no prior history of cancer who had a screening colonoscopy at the Forzani and MacPhail Colon Cancer Screening Centre in Calgary, Canada. A total of 190 HRAPs were identified (8.0%). A multivariable logistic regression model was created using colorectal cancer risk factors identified from prior research. Predictor variables were collected from a baseline health questionnaire and included patient demographic (age, sex, and ethnicity), lifestyle (body mass index, alcohol (daily vs. no), smoking (never vs. ever), physical activity (high vs. moderate to low), and non-steroidal anti-inflammatory drug (NSAID) use (yes vs. no)), medical (family history of colorectal cancer, personal history of diabetes, or fecal occult blood test within the past two years), and female-specific characteristics (menopausal status and hormone replacement therapy (yes vs. no)). The demographic variables were first used to create a baseline model and the benefits of adding the groups of lifestyle, medical, and female-specific variables into the model in various combinations was assessed. Five-fold internal cross validation was conducted. Performance was assessed using the C-statistic and Hosmer-Lemeshow goodness-of-fit test.

Results: The average age of the participants was 58 years of whom 54.9% were male and 85.24% were Caucasian. The clinical prediction model included demographic and lifestyle variables. On average, the predicted probability of having a HRAP was 8.0% (IQR: 4.1% to 10.6%). The bias-adjusted C-statistic was 0.66 (95% CI: 0.62 to 0.70) and there was no evidence of a lack of calibration according to the Hosmer-Lemeshow goodness-of-fit test. The addition of the medical history variables (∆ AUC = +0.0001; p = 0.98) or female-specific variables (∆ AUC = + 0.006; p = 0.32) or both medical and female-specific groups of variables (∆ AUC = +0.004; p = 0.45) did not significantly improve predictive performance.

Conclusions: A model based on demographic and lifestyle variables showed a modest predictive ability for having an HRAP at the time of colonoscopy in a population undergoing screening-related colonoscopies. Consideration of these factors may assist in guiding prioritization of limited screening resources. Next steps include external validation and testing the incremental predictive ability of circulating biomarkers.

#2214

Impact of comorbidities at diagnosis on prostate cancer treatment and survival.

Katarina L. Matthes,1 Manuela Limam,1 Giulia Pestoni,1 Leonhard Held,1 Dimitri Korol,2 Sabine Rohrmann1. 1 _Institute for Epidemiology, Biostatistics and Prevention, University of Zurich, Zurich, Switzerland;_ 2 _Cancer Registry Zurich and Zug, University Hospital Zurich, Zurich, Switzerland_.

Background: For Switzerland, it is currently unclear in which way comorbidities influence the choice of primary treatment in prostate cancer (PCa) patients and how comorbidities affect long-term survival of PCa patients. Thus, the aim of this study was to assess the associations of comorbidities with primary treatment of PCa patients and of comorbidities with PCa-specific mortality (PCSM) compared to other-cause mortality (OCM) in Switzerland.

Patients and Methods: We included 1527 men diagnosed with PCa in 2000 and 2001 in the canton of Zurich. Comorbidities at time of diagnosis were based on the Charlson Comorbidity Index (CCI). For each patient, the scores of the respective comorbidities were summed up and classified into three groups: CCI 0 = no comorbidities, CCI 1 = sum of scores equals to 1, CCI 2+ = sum of scores equals to 2 or larger. Multiple imputation methods were applied to missing data for stage, grade and comorbidities. Multinomial logistic regression analyses were used to explore the associations of comorbidities with treatment. Cox regression models were used to estimate all-cause mortality, and Fine and Gray competing risk regression models to estimate sub-distribution hazard ratios for the outcomes PCSM and OCM.

Results: Increasing age was associated with a decreasing probability of receiving curative treatment, whereas an increasing CCI did not influence the treatment decision as strongly as age. The probability of OCM was significantly higher for patients with comorbidities compared to those without comorbidities (CCI 1: 2.07 [95% CI 1.51 - 2.85], CCI 2+: 2.34 [1.59 - 3.44]); this was not observed for PCSM (CCI 1: 0.79 [0.50 - 1.23], CCI 2+: 0.97 [0.59 - 1.59]). In addition, comorbidities had a greater impact on the patients' mortality than age.

Conclusions: The results of the current study suggest that the chronological age is a stronger predictor of treatment choices than comorbidities. This study supports the inclusion of comorbidities in treatment choices in order to offer more appropriate treatment for PCa patients to counteract over- or undertreatment.

#2215

The role of primary lymph node sites in survival and mortality prediction in DLBCL: A SEER population-based retrospective study.

Mohamed Gomaa Kamel,1 Fatma A. Abd-Elhay,1 Sara Morsy,2 Amr E. El-Qushayri,1 Khaled M. Elhusseiny,3 Muhammed K. Elfaituri,4 Ahmed M. Kamel,1 Sherief Ghozy,5 Nourin A. Sherif,6 Nguyen Tien Huy7. 1 _Minia University, Minia, Egypt;_ 2 _Tanta University, Tanta, Egypt;_ 3 _Al-Azhar University, Cairo, Egypt;_ 4 _Tripoli University, Tripoli, Libyan Arab Jamahiriya;_ 5 _El Sheikh Zayed Specialized Hospital, Giza, Egypt;_ 6 _Mansoura University, Mansoura, Egypt;_ 7 _Institute of Tropical Medicine (NEKKEN), Nagasaki University, Nagasaki, Japan_.

Background Diffuse large B cell lymphoma (DLBCL) is the most common histological subtype of non-Hodgkin lymphoma (NHL), accounting for 25% of NHL subtypes. Usually associated with classic B-symptoms, such as fever, weight loss and night sweats in about 30% of patients, accompanied with elevated lactate dehydrogenase. Approximately in 25-40% of DLBCL cases, the disease arises from extra-nodal sites, such as stomach/gastrointestinal tract. Differences in clinical outcomes between extra-nodal and nodal site involvement suggest that both types should be considered as different entities. Our study aims to identify the role of primary nodal site as predictive tool for clinical and survival outcomes in patients with DLBCL.

Methods Our study was based on data from the Surveillance, Epidemiology, and End Results program database. We used the Kaplan-Meier curve, log-rank test, and Cox proportional hazard regression models to examine predictors affecting the DLBCL survival. We used the multi-variable analysis to get overall survival (OS) as well as the disease-specific survival (DSS).

Results From a total of 34,231 DLBCL patients were identified. The mean age was 61.3 years. The majority had multiple regions (M) lymph nodes (LNs) (18,044, 52.7%) followed by head, face and neck (HFN), intra-abdominal, intra-thoracic, axilla or arm (AA), inguinal or leg (IL) then pelvic LNs. Moreover, male, unmarried, black or Asian/Pacific Islander, older patients, advanced stages, and patients did not receive radiation and/or surgery were associated with a worse survival. We found a mean survival time of 61.07 months with OS and DSS of 39.1/54.1% only. The multiple regions, intra-abdominal, intrathoracic LNs were significantly associated with a worse survival.

Conclusions DLBCL incidence has increased in the recent years. The multiple regions, intra-abdominal, intrathoracic LNs were significantly associated with a worse survival, suggesting that DLBCL nodal primary site can serve as a simple yet prognostic tool. Hence, we strongly encourage future studies to compare DLBCL patients' survival affected by these aggressive primary sites with other unfavorable predictors in IPI. Moreover, biomarkers, within these aggressive sites, may demonstrate their aggressive behaviors. Furthermore, more studies need to shed the light on the biological theory beyond these aggressive primary sites and to validate its reliability.

#2216

A novel prognostic model based on the tumor and its microenvironment in tongue squamous cell carcinoma.

Pei Yu, Weiwang Wang, Zehang Zhuang, Jieyun Xu, Xiqiang Liu. _Guangdong Provincial Key Laboratory of Stomatology, Guangzhou, China_.

Objective: Pathological factors of tumor including tumor budding, invasive type and invasive depth has been reported to predict survival in tongue squamous cell carcinoma (TSCC). However, their prognostic value remains inconclusive and the role of tumor microenvironment is unknown. In the current study, both parameters of tumor and its microenvironment were examined and a new grading system associated with high impact on patient outcome has been proposed. Methods: A total of 246 patients with TSCC were included in this retrospective study. Using H&E stained sections, factors including pathological differentiation, T classification, lymph node metastasis (LNM), intensity of tumor budding, invasive pattern, depth, inflammation response (KM grade), tumor stroma percentage (TSP) were evaluated; the prognostic models including BD model (combination of tumor budding and invasive depth) and GMS grade (combination of KM grade and TSP) were also scored. The associations between variables and the survival was examined. Results: High intensity of tumor budding was associated with increasing T classification and positive LNM. Deep invasive depth was correlated with poorly differentiation, high degree of T classification, positive LNM, late clinical stage, and recurrence. BD score had strong correlation with T classification, LNM and clinical stage. Low KM grade showed significant correlation with late clinical stage and high tumor budding. TSP is positively related to tumor budding. GMS grade had significant association with clinical stage. Cox's regression models indicated that both tumor budding and KM grade were independent variables for predicting overall survival (OS) and disease-free survival (DFS). BD score and GMS grade had significant prognostic impact on survival but showed inferior separation between score 0 and 1, score 1 and 2, respectively. The novel designed model based on the combination of the inflammatory infiltration (KM grade) and BD score, iBD model, was proved to be strongly correlated with T classification, LNM, clinical stage, and recurrence. Patients with high iBD score showed reduced OS and DFS with excellent separation of score 0, 1 and 2. Conclusion: iBD scoring model was a promising predictor of survival and strongly associated with lymph node metastasis and recurrence in TSCC.

#2217

Concordance of PIK3CA mutations in hormone receptor-positive, human epidermal growth factor receptor 2-negative breast cancer: A systematic literature review.

Alejandra Aguilar,1 Joni Dean,1 Elizabeth Anderson,1 Lea Mollon,1 Lisa Davis,1 Terri Warholak,1 Ayal Aizer,2 Emma Platt,3 Aditya Bardiya,2 Derek Tang3. 1 _University of Arizona, Tucson, AZ;_ 2 _Harvard Medical School, MA;_ 3 _Novartis, NJ_.

Introduction: This systematic literature review sought to determine the concordance of PIK3CA mutations in hormone receptor-positive (HR+), human epidermal growth factor receptor 2-negative (HER2-) metastatic breast cancer (mBC) patients.

Methods: A comprehensive search of PubMed/MEDLINE, EMBASE, Cochrane Central, and select conference abstracts was performed by two independent researchers that included, but was not limited to, keywords: "breast neoplasm," "PIK3CA protein," "hormone receptor positive," and "metastases." Databases were searched for English-language publications in HR+, HER2- mBC published between January 1993 through August 2017. Studies were included if results reported were specific to patients with HR+, HER2- mBC and reported data comparing PIK3CA mutations from liquid biopsies versus tumor biopsies or primary tumor samples versus metastatic lesion samples. Concordance was assessed between tissue samples (mutations found in primary tumors versus those from metastatic lesions) and between biopsy methods (i.e., tumor biopsy versus liquid biopsy) when the assessment was done at a variety of time points including time of initial diagnosis and time of diagnosis at disease progression. Content analysis was employed to quantify collected data elements.

Results: Of 558 studies included for full-text review, four met inclusion criteria (number of patient samples [n]=358). One study (n = 9) found the concordance between the mutation status of primary breast tumors to the mutation status of metastatic lesions to be 100%. Three studies (n = 47; n = 55; n = 247) determined the concordance of PIK3CA mutations between tumor biopsies and liquid biopsies with concordance ranging from 70.4% to 94%. One study found that the concordance of circulating tumor DNA (ctDNA) against tumor biopsy was higher among metastatic lesions only (81.6%) relative to using all tumor samples (either primary or metastatic lesions) (70.4%).

Conclusions: Available evidence suggested high level of concordance across sampling methods as well as across time. However, additional evidence is needed to increase study generalizability as well as to assess other possible confounding factors, such as differential timing between tumor and liquid biopsies.

#2218

Trim29 regulates hemangiomas neovascularization through targeting HMOX1.

Qiang Huang, Wei Li, Xin Ge, Peng Li, Ya Gao. _Second Affiliated Hospital of Xi'an Jiaotang Univ., Xi'an, China_.

Infantile hemangiomas is the most common benign vascular tumors of childhood, characterized by rapid proliferation after birth and slow involution thereafter. Although several signaling pathways have been implicated in hemangiomas neovascularization, the mechanism is still unrevealed especially the initiative factor. Here we used gene microarray to profile proliferating and involuting phases of infantile hemangiomas and bioinformatic methods to find the distinguished genes. Log2FC>0.585 or <-0.585 and a nominal P value of 0.05 were used as the filtering criteria. A total of 354 significant genes were identified including 262 upregulated genes. 20 genes were selected for screening according to the microarray results. Trim29 was certified to be responsible for the proliferation of IH using Celigo cytometer method, which were reinforced in patient lesions. By overexpression of Trim29, proteomics was used to detect the downstream proteins.After comparing with the microarray results, HMOX1 was identified and selected for further study after reidentification in patient lesions. Through overexpression and knockdown assay in vitro, we found Trim29 could regulate the expression of HMOX1 and affect hemangiomas neovascularization. Propranolol could block this potential pathway to inhibit proliferation of IH in vivo. These results indicated Trim29 could regulate hemangiomas neovascularization through targeting HMOX1. This innovative pathway could be blocked by propranolol and gave a potential target gene for further investigation to find new drug treatment for IH.

## PREVENTION RESEARCH:

### Biomarkers, Intervention, and Early Detection for Cancer Prevention

#2219

Expression of PCNA and MCM2 in ameloblastoma.

Bhavana Mallipudi. _Sri Sai College of Dental Surgery, Hyderabad, India_.

Background:-Ameloblastoma is an odontogenic neoplasm with the most frequent osseous destructive lesions of the jaws. Ameloblastoma deserves special attention, because of its biological behaviour exhibiting great infiltrative potential, high recurrence rate and capacity to metastasize. The increased proliferation rate in the odontogenic epithelium can be a result of perturbations in the cell cycle regulators and dictates the aggressive behavior of the tumors. Investigating proliferative rates in the epithelial lining of these lesions helps in assessing their biological behaviour. Various cell proliferation markers are used as diagnostic and prognostic tools in oral lesions. Assessing the expression of these markers helps in estimating the proliferative index of the tissues.

Aim:- The aim of this study is to analyze the presence of, Proliferating cell nuclear antigen (PCNA) and the minichromosome maintenance-2 (MCM2) proteins, two well established markers of cell proliferation, in clinically diagnosed cases of ameloblastoma and correlate with aggressiveness and its types (Solid/multicystc{Follicular, plexiform} unicystic).

Materials and method:-14 clinically diagnosed cases of ameloblastomas (10 Solid/multicystic cases{6 cases of Follicular, 4 cases of plexiform} and 4 cases of unicystic) are obtained from the archieves of the Department of Oral and Maxillofacial Pathology to study the expression of MCM2 and PCNA. The data was analysed by Kruskals ANOVA, Mann-Whitney U test and Post hoc analysis.

Results and conclusion:- A Kruskal-Wallis ANOVA was used to detect if there was a significant difference in the expression of MCM2 and PCNA between the three variants of ameloblastoma. There was a significant difference between the three groups for mean percentage expression of MCM2 but there was uniformly higher expression of PCNA in all the three variants. Interestingly, further post hoc analysis revealed that the expression of MCM2 was significantly higher in the follicular variantas compared to the unicystic variant, suggesting that dual staining of MCM2 and PCNA may be a better marker for follicular variant.

Keywords:-Ameloblastoma, MCM2, PCNA.

#2220

Autoantibody detection of high-grade serous ovarian cancer with paraneoplastic antigens.

Laura C. Hurley,1 Nancy K. Levin,2 Madhumita Chatterjee,2 Michael A. Tainsky1. 1 _Wayne State University, Detroit, MI;_ 2 _Karmanos Cancer Institute, Detroit, MI_.

Improved early detection methods for ovarian cancer are necessary as the majority of cases present at late stage metastatic disease with a poor prognosis. Early stages of tumor development initiate an immune response, eliciting antibodies that target tumor associated antigens. Paraneoplastic neurological syndromes are rare autoimmune disorders that develop when the unregulated immune response against a tumor also targets healthy cells, due to shared tumor antigen expression with nervous system and muscle tissue. As these syndromes generally precede detection of a tumor, paraneoplastic antibodies are previously unexplored candidate biomarkers for early diagnosis of ovarian cancer. We have detected paraneoplastic antibodies in serum samples from patients with high-grade serous ovarian cancer (HGSOC) that did not present with paraneoplastic neurological syndrome symptoms using paraneoplastic diagnostic line blots. Ovarian cancer specific epitopes discovered previously in our laboratory by phage-display biopanning were evaluated for both homology to paraneoplastic antigens and for reactivity with paraneoplastic neurological syndrome patient sera. Western blot screening revealed a panel of paraneoplastic antigens that distinguished HGSOC patient sera autoantibody reactivity from healthy and benign sera. An indirect ELISA for measuring patient serum autoantibody reactivity was developed and correlated with western blot results. A panel consisting of 8 antigens: 5 paraneoplastic antigens, 2 well-characterized tumor associated antigens, and a non-reactive control was selected for screening on ELISA against patient serum samples representing each group of HGSOC early stage, HGSOC late stage, healthy, and benign. Biomarkers identified can contribute to an autoantibody diagnostic panel for early clinical diagnosis of ovarian cancer, which would allow for timely therapeutic intervention with potential to improve patient outcome.

#2221

Necroptosis and apoptosis in human colorectal tissue.

Timothy Su, Yong Li, Chang Yu, Martha J. Shrubsole, Xiangzhu Zhu, Xinqing Deng, Eugene Shubin, Wei Zheng, Harvey J. Murff, Douglas L. Seidner, Reid M. Ness, Qi Dai. _Vanderbilt Univ. Medical Ctr., Nashville, TN_.

Background: The molecular mechanisms for colorectal carcinogenesis still remain elusive. In recent years, necroptosis has emerged as a programmed cell death regulated by RIP kinases /mixed lineage kinase domain-like (MLKL) pathway, involving the release of intracellular "danger signals" which results in inflammation. On the other hand, classic apoptosis is regulated by Bax-induced caspase pathway and does not lead to inflammation. Transient receptor potential melastatin related 7 (TRPM7), a MLKL downstream target for the mediation of Ca2+ influx, was found to be involved in TNF-induced necroptosis (Nat Cell Bio 2014;16:55). Both necroptosis and apoptosis are important barriers to prevent normal cells from developing cancer. No study has examined how necroptosis biomarkers (MLKL, TRPM7) and pro-apoptosis biomarker (Bax) are related to inflammation (COX-2) and cell proliferation (Ki-67) and how MLKL relates to TRPM7 in human colorectal tissue. Methods: In our randomized trial (R01CA149633, NCT01105169) "Personalized Prevention of Colorectal Cancer Trial" conducted among colorectal polyp patients or those at high risk of colorectal cancer, colorectal biopsies from 250 participants were collected prior to intervention in the trial. Formalin-fixed paraffin-embedded tissues were serially sectioned, and three levels of serial sections spaced 50 µm apart were mounted on one slide for each tissue block. The protein expressions of COX-2, Bax, pMLKL, TRPM7, and Ki-67 were detected immunohistochemically following the EnVision+ System-HRP kit. Positive cells in the upper and bottom zones were evaluated separately by a zonal quantitative analysis procedure using BioQuant imaging software. Correlation analysis was used to compute Pearson product-moment correlation coefficient between necroptosis, apoptosis and inflammation biomarkers. Results: The expression of selected biomarkers showed stronger positive signals in the upper zone than in the bottom zone, with the exception of Ki-67 which showed stronger staining in the bottom zone of crypts. Epithelial Bax had strong positive correlation with COX-2 in the same zones (r=0.25 - 0.36, p< 0.001), and overall Bax expression showed weaker association with stromal COX-2 (r=0.13, p=0.04). pMLKL and TRPM7 were not significantly correlated with COX-2 except a weak correlation between stromal COX-2 and overall TRPM7 (r=-0.174, p=0.008). pMLKL expression was positively correlated with TRPM7 in the upper zone of crypts (r=0.19, p=0.004), and both pMLKL and TRPM7 were not significantly correlated with Bax expression. Conclusions: Our study confirmed a positive correlation of pMLKL and TRPM7, as suggested by previous cell studies, in human non-tumoral colorectal tissue. The positive correlation between Bax and COX-2 suggests that elevated inflammation may result in increased apoptosis. On the other hand, pMLKL is not related to COX-2, although stromal COX-2 is inversely associated with overall TRPM7.

#2222

Using rare variants to characterize lung cancer risk.

Daniel J. Craig,1 Mazzin Elsamaloty,2 Thomas M. Blomquist,1 Erin L. Crawford,1 James C. Willey1. 1 _University of Toledo College of Medicine, Toledo, OH;_ 2 _University of Toledo Main Campus, Toledo, OH_.

The human genome is exposed to a variety of exogenous and endogenous assaults capable of inducing DNA damage and contributing to diseases like cancer. Multiple DNA repair mechanisms are tasked with identifying and repairing these aberrant nucleotides, but damage is occasionally missed giving rise to somatic mutations. As we age, the relative abundance of somatic mutations increases, which may increase our risk for developing cancer. In addition, inter-individual variation in somatic mutation prevalence suggests an inter-individual variation in (a) inherited DNA repair capacity and/or (b) exposure to environmental mutagens. Therefore, somatic mutation prevalence may serve as an end-point biomarker for cancer risk, as it provides critical information regarding both.

While Next Generation Sequencing (NGS) has provided unparalleled access to the human genome and revolutionized the field of cancer genomics, its implementation into the clinical setting has been limited by technical errors introduced during library preparation and on the sequencing platform. This makes identification of rare variants below 10-4 mutations per nucleotide through NGS a challenge, and the use of somatic mutations to characterize cancer risk unfeasible. To address this issue, unique molecular indexes (UMI) were assigned to each genomic copy, enabling reliable identification of rare somatic mutations versus technical errors. We hypothesize that measurement of somatic mutation prevalence in normal bronchial epithelial cells (NBEC) will serve as an end-point biomarker for lung cancer risk by identifying both (a) inherited sub-optimal DNA repair/protection and (b) exposure to inhaled environmental mutagens including components of cigarette smoke, radon, and/or occupational hazards.

In our initial pilot study, we used genomic DNA derived from an A549 cell line to optimize an ERCC5 gene-specific, dual-indexed PCR method for UMI assignment, followed by sequencing on the Illumina MiSeq platform. In parallel analyses, UMI correction using 25,000 genomic copies provided over 10-fold greater sensitivity in identifying somatic mutations compared to no UMI correction. Based on this pilot, we obtained three clinical NBEC specimens via cytology brush biopsy of grossly normal (non-cancerous) airway from (a) heavy smoker with lung cancer, (b) heavy smoker without lung cancer, and (c) non-smoking control. UMIs were assigned to 50,000 genomic copies at 16 loci, increasing our theoretical limit of detection to approximately 1 in 4x107 nucleotides. Results thus far support further application of this approach in studies to assess NBEC somatic mutation prevalence as a biomarker for cancer risk.

#2223

Analytical validation of a novel circulating tumor cell platform (CMx) for early detection of cancer.

Hung-Jen Shao,1 Huangpin B. Hsieh,1 Wen-Sy Tsai,2 Jen-chia Wu,1 Jr-Ming Lai,1 Shih-En Chang,1 Mana Javey,1 Oscar Segurado,3 Mahul B. Amin,4 Ashish Nimgaonkar,5 Rui Mei1. 1 _CellMax Life, Sunnyvale, CA;_ 2 _Chang Gung Memorial Hospital, Taoyuan, Taiwan;_ 3 _MedicAffairs Consulting, San Jose, CA;_ 4 _University of Tennessee Health Science Center, Memphis, TN;_ 5 _Johns Hopkins Hospital, Baltimore, MD_.

Introduction Circulating Tumor Cells (CTCs) are shed into the blood early by in situ neoplasm and are the mechanism by which cancer metastasizes. CTCs are generally precursors to ctDNA and can be used for early cancer detection. However, to the best of our knowledge, no platform has been analytically validated to detect CTCs in early stage/pre-cancer when these cells are present at a very low concentration in blood.

Methods The CellMax CTC (CMx) biomimetic platform uses a proprietary surface modified microfluidic biochip that sensitively captures CTCs. The analytical performance of the test was established by spiking low EpCAM-expressing colorectal cancer cell line HCT116 into 2mLs of healthy donor blood to achieve three different concentrations, with ~3 cells as "low", ~12 cells as "medium" and ~96 cells as "high" concentrations. Cytokeratin 20, a more specific epithelial cell marker than pan-cytokeratin, and CD45, a WBC marker were used for staining.

Results Overall efficiency (O/E, intact cells recovered vs. input) for 40 spiked donor samples was 34% (4.6% standard deviation (SD) and 13.6% CV) and met the requirements for College of American Pathologists accreditation. No CTCs were detected in 5 unspiked controls, confirming 100% analytical specificity. Assay limit of detection (sensitivity) was a low 3-cells-per-2mL-blood and shown to be linear across all concentrations. The linear regression equation slope=0.23, intercept=0.82 and R2=0.97, demonstrating that the detection of CTCs was linear over the reportable range (3 to 129 CTCs per 2 mL of blood). The precision (reproducibility) of the assay as assessed by using triplicate samples at 3 levels of concentrations over 7 days, for 63 samples, was 36.9% O/E (Low, SD=9.2%), 34.2% (Medium, SD=12.1%), and 31% (High, SD=6%). The highest SD of 12.1% compares favorably to the SD for the CellSearch Assay (18%) in their publicly available 510K filing. In 398 routine quality control samples with cell lines spiked into growth medium, which better reflects process variability than blood variability, the mean O/E was 66.7%, SD was 9.9% and CV was 14.9%. Clinical proof for early detection was established under IRB by testing 170 patients with colorectal diseases ranging from pre-cancerous lesions to stage I-IV cancer: 28% (N=48) were colonoscopy-negative, 72% (N=142) had diagnostically confirmed colorectal neoplasms or cancer. Detection sensitivity for precancerous lesions, including adenomatous and dysplastic polyps, was 47% (with 94% specificity), which is higher than the sensitivity for the existing stool-based standard FIT (24%), and Cologuard (42%) per the latter's publicly available FDA submission.

Conclusion The analytical validation of CMx CTC platform showed better precision than the FDA-approved CellSearch and the clinical study confirmed higher sensitivity for the detection of pre-malignant colorectal cancer than existing stool-based tests.

#2224

Panel of autoantibodies to seven early tumor-associated overexpressed proteins can identify women with DCIS from women with benign breast tumors detected by mammography.

Sasha Elizabeth Stanton, Erik Ramos, Mary L. Disis. _Univ. of Washington, Seattle, WA_.

While mammography is essential to identify early breast cancer, the sensitivity is dependent on breast characteristics. Currently all breast masses identified by mammography have to be biopsied to determine if malignant. A serum-based biomarker used along with mammography to identify pre-malignant lesions to biopsy would both improve the sensitivity of mammography and reduce the number of biopsies needed. We have identified a panel of autoantibodies found in early breast cancer. An autoantibody biomarker is ideal because antibody immunity can be detected with very low levels of antigen with direct antigen recognition by B cells resulting in clonal amplification of antigen specific plasma cells. Work in our laboratory has identified tumor-associated proteins present in pre-malignant tumors that are necessary for survival of human breast cancer cells across breast cancer subtypes. We have found that increased autoantibodies to seven of these early tumor-associated proteins (PDIA6, KRT8, SERBP1, ARPC2, RRM2, AURKA, and NDC80) were present in the sera of women with pre-malignant breast atypia but not women with no breast atypia or benign breast atypia and may be a panel to increase sensitivity of mammography.

The presence of autoantibodies was evaluated in 191 individuals, 36 women with no breast atypia, 12 women with benign breast atypia, 36 patients with fibroadenoma, 12 patients with hyperplasia, 59 patients with ductal carcinoma in situ (DCIS), and 36 patients with invasive breast cancer (IBC). We found more women with pre-malignant breast atypia had increased autoantibodies to at least one of the seven early tumor associated proteins as compared to women with either no breast atypia or benign breast atypia. For example, a positive autoantibody response is over 2 standard deviations above the mean found in women with no breast atypia. There were 2.9% of women with positive autoantibody response to RRM2 in women without breast atypia. However, in women with breast atypia, there was a positive antibody response in 41.7% of individuals with hyperplasia, 48.6% of individuals with fibroadenoma, 34.5% of individuals with DCIS, and 28.6% of individuals with IBC. All seven autoantibodies could predict patients with hyperplasia, fibroadenoma, DCIS, and IBC from both women with no atypia and women with benign breast atypia. For example, the seven-antibody panel could identify DCIS from women with no breast atypia with AUC of 0.95 (95% CI 0.912 to 0.995) and from women with benign breast atypia with AUC of 0.71 (95% CI 0.519 to 0.898). This is a better AUC than seen with previous published autoantibody profiles in DCIS. Future studies will validate these findings with the goal of developing a serum biomarker that will improve sensitivity of mammography.

#2225

Limitations on mutation detection for early detection of cancer.

Imran S. Haque,1 Gabriel Otte,1 Olivier Elemento2. 1 _Freenome, South San Francisco, CA;_ 2 _Weill Cornell Medical College, New York City, NY_.

Introduction: Cell-free DNA (cfDNA) has potential utility for early non-invasive detection of cancer. We assess the feasibility of cfDNA mutational assays for early detection based on their physiological and economic requirements. We further review alternative biological signals of early cancer and the potential of machine learning to integrate these signals into reliable diagnostics.

Methods: A binomial model was used to assess depth and input requirements, with parameters derived from published data on cfDNA sequencing. Alternative strategies for early detection were assessed by literature review.

Results: 30,000x unique coverage is required for 95% sensitivity for 1 mutant read at 0.01% variant allele frequency, requiring 180ng cfDNA input at 50% process efficiency; current sequencing costs and reimbursement levels may make such tests economically infeasible (Table). 5th percentile plasma cfDNA concentration in the screening population is ~2.3 ng/mL, requiring >140mL blood collection for test failure rate <5%. Somatic mutational heterogeneity creates significant specificity challenges. Proteins, RNAs, and compartmentalized macromolecules are more abundant and may overcome cfDNA's issues of quantity, but irrelevant biological processes may confound the data. New techniques in machine learning, including latent variable modeling, may be used to integrate heterogeneous data from multiple sources to overcome these challenges.

Conclusions: Mutation-detection assays may not be feasible for early cancer detection due to limited sensitivity arising from low concentrations of cfDNA, heterogeneity leading to specificity challenges, and prohibitive cost. Integrating markers beyond mutations with modern machine learning may provide a potential route to statistically robust biomarker development for early cancer detection.

Table. Assay requirements for tumor liquid biopsy and mutation-based early cancer detection. | |  | |  | |

|

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

|  | |  | |

Sequencing cost by panel size

|

VAF 95% sensitivity | Corrected depth | Raw depth | Input volume (blood) | TEC-Seq1

58 genes

81 kb | Razavi et al2

508 genes

2000 Kb | WES

~20k genes

50,000 Kb

Tumor liquid biopsy | 0.1% | 3000x | 15,000x | 15 mL | $14 | $340 | $8300

Early cancer detection | 0.01% | 30,000x | 150,000x | 150 mL | $140 | $3400 | $83,000

1Phallen J, et al. Sci Transl Med. 2017 Aug 16;9(403). 2Razavi P, et al. J Clin Oncol. 2017;35(suppl):abstr 11526.

Model parameters: 1) No more than 5% of samples may fail because of insufficient cfDNA quantity; 2) 95% sensitivity to detect one read from any cancer-derived allele, assuming that one is present in the sample; 3) 50% process efficiency: half of the cfDNA molecules in the input blood sample are represented in the sequencer output; 4) 5x oversampling in sequencing for error correction; 5) 100% on-target rate in target enrichment; 6) "$1000 genome" sequencing costs: US $1000/(30 x 3 Gbp) of sequencing bandwidth; 7) only sequencing costs computed; all other costs (labor, equipment, facilities, depreciation, etc.) accounted at $0; 8) panel expansion neither reduces input requirements nor increases sequencing requirements.

VAF = variant allele frequency; TEC-Seq = targeted error correction sequencing; WES = whole exome sequencing.

#2226

TGF-β and CEACAMs regulated biomarkers detect early colorectal cancer.

Sobia Zaidi,1 Anil Korkut,2 Wilma Jogunoori,3 Jian Chen,2 Shoujun Gu,1 Shuyun Rao,1 Kazufumi Ohshiro,1 Rehan Akbani,2 Chuxia Deng,1 Bibhuti Mishra,1 Lopa Mishra1. 1 _George Washington Univ., Washington, DC;_ 2 _The University of Texas MD Anderson Cancer Center, Houston, TX;_ 3 _Veterans Affairs Medical Center, Washington, DC_.

Introduction: Development of colorectal cancer (CRC) is associated with alterations in key driving pathways, which include Wnt (APC-βcatenin), TGF-β members, p53 and pathways that regulate Ras activity. Members of the TGF-β superfamily regulate colon inflammation, have both tumor-suppressing and tumor-promoting activities, while colon cancer formation has been observed in TGF-β deficient mouse models. Through earlier studies, using mouse models followed by functional studies in human cell lines and tissues, we identified candidate set of TGF-β regulated biomarkers for early detection of CRC, that were altered in tissues from patients with adenomas, and could represent signs of early cancer stem cell development (J Clin Invest 2016;126(2); PLoS One 2016;11(4)). These markers are CEACAMs 1, 5 and 6; TGFBR2, SMAD4, Smad adaptor, SPTBN1. Here, we took an integrated approach to extend and validate these potential markers for early detection of CRC.

Methods and Results: 1) Analyzing the TCGA cohort of 9,125 samples and 33 cancer types, including CRC, revealed alterations in TGF-β members in ~40% of samples. 2) cBioportal cancer genomics data reveal reduced overall survival in CRC patients with decreased TGFBR1 and TGFBR2, together with increased CEA (CEACAM5). 3) TCGA analyses also reveal significant tendency of co-occurrence of genomic alterations in TGFBR1 and CEA. 4) mRNA stemness index score in 33 cancers types in TCGA, reveals increased transcriptome levels of a cancer stem cell signature in specific cancers, that concomitantly have decreased levels of TGF-β pathway members, supporting the mouse models revealing that TGF-β suppresses cancer stem cells. 5) Cluster analysis for miRNAs in the 33 cancers suggest a role for these in suppression of TGF-β pathway, depending on the cancer type. miRNA 92a-3p that targets 3 core genes, BMPR2, TGFBR2, and SMAD7, is overexpressed in many cancers. Colon cancer with high frequencies of hotspot mutations in BMPR2 and TGFBR2 did not have high expression of 92a-3p, perhaps indicating that there is little selective pressure for a second mechanism of inactivation. 6) We further identified hotspot mutations in the B3 domain of the CEA that interacts with TGFBR1, supporting a mechanism for previously observed CEA inactivation of TGF-β tumor suppressor function.

Conclusions: CEACAMs with TGF-β signaling members as a group could represent strong prognostic indicators of high-risk adenoma-carcinoma progression and invasive disease.

#2227

Multi-analyte profiling reveals relationships among circulating biomarkers in colorectal cancer.

Daniel Delubac,1 Eric Ariazi,2 Jonathan Berliner,2 Adam Drake,2 John Dulin,2 Riley Ennis,2 Erik Gafni,2 Kate Niehaus,2 Gabriel Otte,2 Jennifer Pecson,2 Girish Putcha,2 Corey Schaninger,2 Aarushi Sharma,2 Mike Singer,2 Abraham Tzou,2 Jill Waters,2 David Weinberg,2 Brandon White,2 Imran S. Haque3. 1 _Freenome Inc., South San Francisco, CA;_ 2 _Freenome, Inc., South San Francisco, CA;_ 3 _Freenome, South San Francisco, CA_.

Introduction: Blood-based tests hold great promise as cancer diagnostics but until now have largely been restricted to the analysis of a single class of molecules (eg, circulating tumor DNA, platelet mRNA, circulating proteins). The ability to analyze multiple analytes simultaneously from the same biological sample may increase the sensitivity and specificity of such tests by exploiting independent information between signals. Here, we describe an experimental and analytical system that we developed and implemented for the integrated analysis of multiple analytes from a single blood sample, which revealed examples of both correlations and orthogonality among individual analytes. Methods: De-identified blood samples were obtained from healthy individuals, as well as individuals with pre-malignant conditions and stage I-IV colorectal cancer (CRC). After plasma separation, multiple types of analytes were assayed: cell-free DNA (cfDNA) content was assessed by low-coverage whole-genome sequencing (lcWGS) and whole-genome bisulfite sequencing (WGBS), cell-free microRNA (cf-miRNA) was assessed by small-RNA sequencing, and levels of circulating proteins were measured by quantitative immunoassay. Results: lcWGS of plasma cfDNA was able to identify CRC samples with high tumor fraction (>20%) on the basis of copy number variation (CNV) across the genome. High tumor fractions, while more frequent in late-stage cancer samples, were observed in some stage I and II patients. Aberrant signals in each of the three other analytes—cf-miRNA profiles discordant with those in healthy controls, genome-wide hypomethylation at LINE1 (long interspersed nuclear element 1) CpG loci, and elevated levels of circulating carcinoembryonic antigen (CEA) and cytokeratin fragment 21-1 (CYFRA 21-1) proteins—were also observed in cancer patients. Strikingly, aberrant profiles across analytes were indicative of high tumor fraction (as estimated from cfDNA CNV), rather than cancer stage. Conclusion: Our data suggest that tumor fraction is correlated with cancer stage but has a large potential range, even in early stage samples. Previous literature on blood-based screens for detection of cancer has displayed discordance in the claimed ability of different single analytes to detect early stage cancer. Tumor fraction may be able to explain the historical disagreement, as we found that aberrant profiles among cf-miRNA, cfDNA methylation, and circulating protein levels were more strongly associated with high tumor fraction than with late stage. These findings suggest that some positive "early stage" detection results may in fact be "high tumor fraction" detection results. Our results further demonstrate that assaying multiple analytes from a single sample may enable the development of classifiers that are reliable at low tumor fraction and for detecting pre-malignant or early-stage disease.

#2228

Differential expression of MT-ATPase and COXIV genes in colorectal adenopolyps.

Lashanale Wallace, Anju Cherian, Felix Aikhionbare. _Morehouse School of Medicine, Atlanta, GA_.

Therapeutic decisions in colorectal cancer (CRC) will be enhanced if guided by more accurate prognostic and predictive biomarkers during the progression of adenomas to CRC. Given that most CRCs develop from adenopolyps via the adenoma-carcinoma sequence, a mechanism for the inhibition of this sequence in patients with a high risk of developing CRC is a pressing need. Variants in mitochondrial (mt) protein expressions have been correlated with several clinico-pathological features of cancers as the majority of energy for tumor transformation are of mitochondrial origin. Differences in mitochondrial efficiency may be reflected as in adenoma-carcinoma sequence. Reports have shown that cytochrome c oxidase (COX) is a key player in oxidative phosphorylation and reactive oxygen species (ROS) formation. In addition ATPase subunits are also associated with ROS formation and mtDNA maintenance. Here, we specifically searched for differentially expressed ATPase and COX subunits in early adenomas of CRC tissues as compared to late stages of CRC tissues. In addition mitochondria variants were analyzed in mitochondrial encoded subunits of complexes IV and V of the electron transport chain. Direct sequencing, high resolution restriction digestion, RT-qPCR and western blot techniques were used to assess differences in colorectal tumors. Tissue samples used included early adenomas classified as tubular adenoma (TA), tubulovillous adenoma (TV), and villous adenomas (v); cancer tissues, and normal surrounding tissues. Results suggest that most variants of complex IV were found in mitochondrial encoded cytochrome c oxidase subunit III (9207-9990). Of these variants 9414delC found in 60% TA and 20% CA samples was predicted to be disease causing. Furthermore, ATPase6 variant G9055A found abundantly in TA and V samples was confirmed using high resolution restriction digestion. Expression levels of ATPase6 progressively increased from early adenomas to late stage adenomas and cytochrome c oxidase mitochondrial subunits also varied within the adenoma carcinoma sequence. Interestingly, COX subunit 4 isoform 1 protein expression decreased by five-fold in cancer samples when compared to normal tissue and by three-fold when compared to TA. Therefore, this study suggest an important role of ATPase and COX IV-1 in tumor CRC progression in respect to the impact on mitochondrial ROS production and oxidative phosphorylation regulation.

#2229

Mammography assessment of microcalcifications using a pixel detector system.

Carlos Ávila Bernal, Gerardo Roque, Roberto Rueda, Jorge Racedo. _Universidad de los Andes, Bogotá, Colombia_.

Breast cancer is a major public health problem being the most frequently diagnosed malignancy and the second leading cause of dead from cancer among women in the United States. In 2015, there were expected around 231,840 new cases of invasive breast cancer, as well as 60,290 cases of in situ breast cancer, and 40,290 deaths. Worldwide, it represents 23% of the total cases diagnosed of cancer and 14% of the cancer deaths in female. Diagnosing breast cancer before it has the potential to invade surrounding tissue and metastasize can increase the chance of survival, reduce poor prognosis and the need of aggressive treatment. This is one of the main goals of early diagnosis that advocates mammography, the clinical examination and the breast self-examination as screening modalities for breast cancer. Recently, other technologies have been proposed like ultrasound and magnetic resonance imaging (MRI). Although these screening methods have reduced the relative risk of death, they have some limitations in some population groups. The aim of this work is adapting and testing a semiconductor pixel detector, Timepix in a custom-swine breast phantom to evaluate its technical feasibility to introduce this technology in a clinical setting for mammography assessment based on microcalcification detection. Swine model is commonly used in medical research based on anatomical and histopathological characteristics closely resemble those of humans, and this is an advantage over the wax-based phantoms that contains aluminum oxide (Al2O3) specks, nylon fibers and other inclusions to mimic microcalcifications and histopathological structures. The Timepix detector was used together with a Tungsten microfocus X-ray to detect hydroxyapatite crystals mimicking malignant microcalcifications with sizes ranged between 153-827μm, embedded into the swine phantom. To compare the detection performance in the swine phantom, a commercial phantom has been used (Model 015 CIRS Inc., Norfolk, VA, US). Both phantoms were tested on a clinical mammography system (Selenia Dimensions AWS 5000 ET19) and by using the experimental setup with the Timepix. This approach has potential to be used for mammography screening in different population groups based on its performance in microcalcifications detection. The results are highly relevant for further studies of the feasibility of pixel detectors for risk assessment and early diagnosis of breast cancer.

#2230

Low frequency variant detection in cell free DNA by applying molecular identifiers to targeted NGS.

Ashley Wood, Sukhinder Sandhu, Mida Pezeshkian, Vanessa Kelchner, Jordan RoseFigura, Justin Lenhart, Laurie Kurihara, Vladimir Makarov. _Swift Biosciences, Ann Arbor, MI_.

The growing use of liquid biopsy for early detection and monitoring of disease necessitates accurate variant detection at <1% allele frequencies due to a low population of disease DNA within circulating, cell-free DNA (cfDNA). Reliable, low-frequency variant detection by next-generation sequencing (NGS) is challenging due to background noise from PCR and sequencing errors. We employed molecular identifiers (MIDs) to uniquely label individual DNA molecules prior to amplification, facilitating the distinction of true variants from PCR and sequencing errors. We incorporated MIDs in both our amplicon library prep that uses multiplex PCR for targeted NGS and our whole genome library prep followed by targeting with hybridization capture using an 800kb pan-cancer panel. We performed low frequency spike-in experiments at <1% allele frequencies. We prepared MID libraries with various amplicon panels including a 17 amplicon EGFR pathway panel and a 104 amplicon SNP panel. Deep sequencing to >30,000x was done to maximize MID family size (number of PCR duplicates) and optimize generation of a consensus sequence. This analysis identified all known variants present at 1%, 0.5%, and 0.25% allele frequencies. Next, the hybridization capture libraries were prepared with low-frequency spike-in samples, sequenced to >8000x, and all known variants at 1% and 0.5% allele frequencies were maintained in the consensus data. In both cases, the number of false positives was reduced, resulting in improved specificity. Further, EGFR amplicon libraries and hybridization capture libraries were prepared using cfDNA samples from lung, ovarian, liver, stomach, and colon cancers. Variant calling based on MID generated consensus sequences identified mutations in cfDNA samples as well as corresponding tumor and normal samples when available. This study highlights the ability of MID technology to enable low frequency variant detection, critical to track known variants and identify novel pathogenic mutations in cfDNA samples.

#2231

Insulin and multi-system alterations in non-diabetic American adults: NHANES 1999-2016.

Neil E. Caporaso,1 Rena R. Jones,2 Lisa L. Kahle,3 Barry I. Graubard1. 1 _NCI, Rockville, MD;_ 2 _NCI, Silver Spring, MD;_ 3 _Information Management Services Corp, Rockville, MD_.

Epidemiological evidence for a relationship between insulin resistance and cancer is emerging, and insulins' complex actions suggest that its dysregulation results in multisystem alterations. Using cross-sectional demographic and laboratory data from the continuous NHANES conducted from 1999-2016. we surveyed the range of multisystem alterations observed in normal (not diabetic or prediabetic) adult subjects in relation to variation in insulin and glucose levels. The homeostatic model of insulin resistance (HOMA-IR) was used as the key outcome measure of insulin resistance. We examined cardiovascular (BP), respiratory (FEV1, FVC), renal (BUN, creatinine), inflammatory (C-reactive protein, fibrinogen), lipid (triglycerides, LDL, HLD), hematologic (WBC, hemoglobin, cell counts), and nutritional (vitamin levels) factors in relation to HOMA-IR, C-peptide, Hgb1Ac and fasting glucose levels after adjustment for demographic (age, gender, smoking, alcohol, race, SES) and anthropometric (BMI) factors. Every system exhibited highly significant associations with insulin/glucose measures. Strong correlations (p < 0.0001, Pearson, accounting for sample weights) of HOMA-IR were observed with waist size, systolic and diastolic blood pressure, triglycerides, HDL (inverse), LDL and inflammatory markers: fibrinogen, C-reactive protein, ferritin. Measured serum levels of vitamin C, vitamin D, and folate (RBC), were inversely associated with HOMA-IR. The relationship for selected variables, to HOMA-IR quartiles is shown below.

In a broad, representative sample of up to 9000 non-diabetic US subjects, we relate insulin resistance to adverse changes in anthropometric, cardiovascular, renal, metabolic, inflammatory and nutritional markers. Analyses accounting for NHANES sample design, confounding and potential interactions will be reported. Our preliminary results suggest that insulin resistance is linked to perturbations in normal function across numerous systems.

HOMA-IR Quartiles, adjusted (95% CI)

---

|

1st | 2nd | 3rd | 4th

Ferritin | 79.1(71-87) | 75.9 (70-82) | 82.3 (76-89) | 96.2 (82-110)

BP (d) | 69.7 (69-71) | 71.8 (70-73) | 71.4 (70-73) | 73.2 (72-75)

HDL | 60.8 (60-62) | 56.3 (56-57) | 54.1 (53-55) | 51.3 (50-52)

WBC | 6.37 (6.3-6.5) | 6.44 (6.3-6.6) | 6.68 (6.5-6.8) | 6.96 (6.8-7.1)

#2232

A high-sucrose diet does not enhance spontaneous metastases of Lewis lung carcinoma in mice.

Lin Yan, Sneha Sundaram. _USDA-ARS, Grand Forks, ND_.

Obesity is a risk factor for cancer, and high caloric intake contributes to obesity. We previously reported that a high-fat diet enhances metastasis in mice (Clin Exp Metastasis 2010). The purpose of this study was to determine the effects of a high-sucrose diet in comparison to a high-fat diet, on an isocaloric basis, on the spontaneous metastasis of Lewis lung carcinoma (LLC) in C57BL/6 mice. Mice (male) were fed a low-fat (AIN93G-based), a high-fat, or a high-sucrose diet for eight weeks before receiving a subcutaneous injection of 2.5x105 viable LLC cells. These diets contained 16% of energy from corn oil, 45% of energy from corn oil, or sucrose. The resulting primary tumor was resected 10 days later. The experiment was terminated 10 days after resection. There were no differences in caloric intake among the groups. The body fat mass of the high-sucrose group was lower than the high-fat group but higher than the low-fat group. The number and size of lung metastases were significantly higher in the high-fat group than in the low-fat group; however, they were similar between the high-sucrose and the low-fat groups. Compared to the low-fat diet, the high-fat diet, but not the high-sucrose diet, significantly increased plasma concentrations of insulin, inflammatory cytokines (leptin, plasminogen activator inhibitor-1, and monocyte chemotactic protein-1) and angiogenic factors (vascular endothelial growth factor and tissue inhibitor of metalloproteinase-1). These findings showed that the high-sucrose diet does not enhance metastasis in this LLC model. This null effect suggests that the high-sucrose diet, compared to the high-fat diet, is less capable of promoting adipogenesis and production of related inflammatory cytokines and angiogenic factors.

#2233

Breaking the obesity-cancer link: Nutritional modulation of inflammation and epigenetic reprogramming.

Laura W. Bowers,1 Shannon B. McDonell,1 Emily Rossi,1 Stephanie Montgomery,2 Joel Parker,1 Stephen D. Hursting1. 1 _Univ. of North Carolina, Chapel Hill, NC;_ 2 _University of North Carolina, Chapel Hill, NC_.

Purpose: Obesity is associated with poorer breast cancer-specific survival, but there is limited and conflicting evidence to support recommendations of post-treatment weight loss for outcome improvement. Consequently, we have examined the reversibility of the procancer effects of obesity via weight loss using preclinical studies combined with transcriptomic and epigenetic analyses.

Methods: C57BL/6 mice were randomized to a low-fat diet (LFD, 10% kcal from fat) or diet-induced obesity (DIO, 60% kcal from fat) regimen. After 15 weeks, DIO mice were randomized to remain on DIO (Obese) or begin 1 of 4 weight loss diets: LFD (formerly obese, FOb), high-carb 30% calorie-restricted (HCCR), low-carb 30% CR (LCCR), or intermittent calorie restriction (ICR). ICR mice received a 70% CR diet 2 days/week. Control mice received the LFD throughout study. At week 25, mice were orthotopically injected with E0771 mouse mammary tumor cells, a model of basal-like breast cancer. All mice were euthanized when a tumor in 1 mouse reached 1.5 cm in diameter. Paired RNA sequencing and reduced representation bisulfite sequencing (RRBS) were performed on normal mammary tissue.

Results: At study endpoint, Obese mice had a higher average body weight and body fat percent versus all other groups (P<0.05). These measures did not differ between Control and FOb mice, but were significantly lower in all 3 CR groups versus the other groups (P<0.05). Tumor volume and weight at sacrifice were significantly greater in the Obese mice relative to all groups (P<0.05) except the FOb mice, despite their weight loss to Control levels. In contrast, tumors in all 3 CR groups were smaller than FOb (P<0.05). Lung micrometastases were present in 5 of 6 mice in both the Obese and FOb groups and in 1 ICR mouse; none were detected in the Control, HCCR, or LCCR mice. Comparisons of RNA sequencing and RRBS data from Obese vs FOb mice and FOb mice vs all 3 CR groups indicated that there were concordant significant differences in the expression and CpG methylation of 169 and 119 genes, respectively, that were also found in significant KEGG pathways (q<0.05). For both comparisons, pathways with the highest number of differentially expressed and methylated genes included MAPK Signaling, Pathways in Cancer, and Leukocyte Transendothelial Migration.

Conclusions: This study demonstrates that weight normalization does not reverse the effects of chronic obesity in a preclinical model of basal-like breast cancer, except when more severe weight loss is achieved via calorie restriction. Multiomic profiling of normal mammary tissue indicated a sustained dysregulation in the transcription and methylation of pro-growth and pro-inflammatory genes, relative to CR mice. Greater understanding of the mechanisms mediating obesity-induced epigenetic reprogramming will enable the development of specific treatment strategies to improve obese breast cancer patient outcomes.

#2234

Dietary-AGE ingestion during puberty modifies the breast microenvironment to alter mammary gland development: Linking diet, development and breast cancer risk.

Jaime F. Randise,1 Bradley A. Krisanits,1 Lourdes M. Nogueira,1 Kristi L. Helke,1 Taaliah Campbell,2 Victoria J. Findlay,1 David P. Turner1. 1 _Medical University of South Carolina, Charleston, SC;_ 2 _Claflin University, Orangeburg, SC_.

Evidence supports the notion that critical events during mammary development permanently alter developmentally regulated programs which influence the breast microenvironment to increase breast cancer risk. This is analogous to metabolic memory in diabetic patients where early metabolic events have been found to be remembered and affect disease severity later in life.

Advanced glycation end products (AGEs) are highly reactive metabolites that irreversibly accumulate in tissues as we age. AGE accumulation can contribute to pro-inflammatory and -oxidant phenotypes when signaling through the receptor for advanced glycation end products (RAGE). The pathogenic effects of AGE-RAGE signaling include tissue degeneration, protein dysfunction, aberrant cell signaling, and reduced genetic fidelity. AGEs form during normal metabolism but critically, lifestyle factors such as poor diet, a sedentary lifestyle and being obese also contribute to the AGE accumulation pool. The permanence of AGE adducts and their ability to mediate chronic and persistent inflammatory and oxidative stresses is particularly compatible to the concept of metabolic memory.

Our dietary studies in pubertal FVB/n mice fed a high AGE diet show a significant dysregulation of mammary gland development and hyperplastic lesion formation. We observe delayed mammary ductal extension, increased ductal branching and aberrant terminal end-bud (TEB) morphology. The basal myoepithelial cell layer surrounding mammary ducts and TEBs was irregular and epithelial cell proliferation was increased. Molecular characterization of these hyperplastic lesions were defined using DCIS progression markers by histopathological staining and qRT-PCR. Elevated AGE levels accompanied increased RAGE expression and increased macrophage and fibroblast infiltration around the TEBs. In attempt to reverse the effects caused by a high AGE diet, mice were fed a control diet after a pubertal high AGE diet. Hyperplastic lesions persisted despite diet intervention. Importantly, hyperplastic lesions were not observed in mice fed a control diet during puberty, then switched to a high AGE diet. These data indicate that exposure to AGE induced changes during puberty may leave a long-lasting imprint analogous to metabolic memory.

In conclusion, increased AGE consumption during pubertal growth results in significant disruption of normal mammary development and the appearance of hyperplastic lesions by adulthood. Consumption of a high AGE diet despite a control diet intervention, reveals hyperplastic lesions indicative of metabolic memory. We hypothesize that the high AGE diet may leave a metabolic imprint on the mammary gland microenvironment, increasing the risk of future breast cancer development.

#2235

Prostatic fatty acids correlate with the progression and racial disparity of prostate cancer.

Xinchun Zhou,1 Jinghe Mao,2 Hao Mei,1 Timera Brown,2 Joshua Agee,1 Steven Bigler,3 Ruth Welti4. 1 _Univ. of Mississippi Medical Ctr., Jackson, MS;_ 2 _Tougaloo College, Tougaloo, MS;_ 3 _Baptist Health Systems in Jackson, Jackson, MS;_ 4 _Kansas State University, Manhattan, KS_.

Objectives: This study is aimed to correlate the levels of prostatic fatty acids with the pathogenesis, progression and racial disparity of prostate cancer (PCa).

Methods: Total fatty acids (TFA) and free fatty acids (FFA) were measured in 26 fresh frozen PCa tissues from 13 African American (AA) and 13 Caucasian American (CA) patients, and 21 benign prostatic tissues (BPT) from 12 AA and 9 CA men by gas chromatography with flame ionization detection and electrospray ionization mass spectrometry, respectively.

Results: In all populations, TFA in total was significantly higher in PCa than in ABP (1.8-fold, p=0.023). Saturated TFA (STFA) was 1.6-fold higher (p=0.024), mono-unsaturated TFA (MUTFA) was 1.9-fold higher (p=0.027), and poly-unsaturated TFA (PUTFA) was 2.1-fold higher (p=0.045) in PCa than in BPT. Six out of 16 individual TFA species were significantly higher in PCa than in BPT. Three out of 24 TFA parameters were significantly higher in high than in low grade PCa; and six out of 24 TFA parameters were significantly higher in high than low stage PCa. FFA in total was significantly higher in PCa than in BPT (1.3-fold, p=0.038). Saturated FFA (SFFA) in total was 1.2-fold higher (p=0.24), mono-unsaturated FFA (MUFFA) in total was 1.7-fold higher (p=0.027), and poly-unsaturated FFA (PUFFA) in total was 1.7-fold higher (p=0.045) in PCa than in BPT. Four out of 10 FFA species were significantly higher in PCa than in BPT. In stratified populations, the difference of TFA in total was not significant between AA PCa and AA BPT (p=0.71), but it was significantly higher in CA PCa than in CA BPT (2.7-fold, p=0.003). There were only 2 out of 16 TFA species in AA PCa significantly higher than in AA BPT, whereas 11 out of 16 TFA species in CA PCa significantly higher than in CA BPT. The differences in all groups of TFAs, including STFA, MSTFA, PUTFA, n-3, n-6 and n-9 TFA were not significant between AA PCa and AA BPT, however they were all significantly higher in CA PCa than in CA BPT. Out of 24 fatty acid parameters, two in AA population but 14 in CA population were significantly higher in high than low grade PCa, and one in AA population but 15 in CA population were significantly higher in high than low stage PCa. None of 14 FFA parameters were significantly different between AA PCa and AA BPT, but 10 of those were significantly higher in CA PCa than in CA BPT. Intriguingly, TFA in total, STFA, MUTFA, PUTFA, n-3TFA, n-6 TFA, n-9 TFA and ratio of n-6 to n-3 were all higher in AA BPT than CA BPT, but all lower in AA PCa than in CA PCa.

Conclusion: This study suggests that increasing in majority of prostatic fatty acids (both TFA and FFA) is risk for pathogenesis and progression of PCa. Higher levels of prostatic fatty acids in BPT are greater risk for PCa pathogenesis in AA men, whereas higher levels of prostatic fatty acids contribute more to the progression of PCa in CA men.

#2236

Metformin and pancreatic cancer risk in Taiwanese patients with type 2 diabetes.

Chin-Hsiao Tseng. _National Taiwan Univ. College of Medicine, Taipei, Taiwan_.

Background: Whether metformin may reduce pancreatic cancer risk is controversial and has rarely been studied in Asian populations. This study evaluated pancreatic cancer risk in type 2 diabetes patients with and without metformin treatment. Methods: An original cohort of 16232 never users and 153408 ever users of metformin who were newly diagnosed of type 2 diabetes between 1999 and 2005 were enrolled from Taiwan's National Health Insurance. A propensity score matched cohort of 16232 ever users and 16232 never users were derived from this original cohort. The patients were followed up until December 31, 2011 and analyses were conducted in both the original cohort and the matched cohort. Hazard ratios were estimated by Cox regression incorporated with the inverse probability of treatment weighting using propensity score. Results: In the original cohort, the incidence rate of pancreatic cancer in never users and ever users were 43.59 and 28.85 per 100000 person-years, respectively, with an overall hazard ratio of 0.658 (95% confidence interval: 0.438-0.988, P=0.0436). While compared to never users, the hazard ratio (95% confidence interval) for cumulative duration of metformin use in the first (<22.10 months), second (22.10-46.70 months) and third tertile (>46.70 months) was 1.445 (0.950-2.196), 0.595 (0.384-0.922) and 0.157 (0.094-0.264), respectively. Results in the matched cohort were very similar, with an overall hazard ratio of 0.492 (0.252-0.961) and the hazard ratio in the respective tertile of cumulative duration was 0.900 (0.368-2.198), 0.648 (0.266-1.581) and 0.094 (0.013-0.692). Conclusions: Metformin significantly reduces pancreatic cancer risk in a dose-response pattern.

#2237

**Diclofenac as a dual inhibitor of cyclooxygenase and aldo-keto-reductase 1B10 inhibits pancreatitis-enhanced carcinogenesis in** LSL-KrasG12D-Pdx-1-Cre **mice.**

Jie Liao, Leyu Sun, Haonan Li, Rong Xia, Xiaoming You, Dandan Xu, Guang-Yu Yang. _Northwestern Univ. Feinberg School of Medicine, Chicago, IL_.

Diclofenac is one of non-steroid anti-inflammatory drugs and is recently identified as a competitive inhibitor of aldo-keto reductase family 1B10 (AKR1B10). AKR1B10 is an NADPH-dependent enzyme that exhibits lipid substrate specificity, especially for farnesyl and geranylgeranyl involved in protein prenylation (such as for Kras) and plays an oncogenic role in pancreatic carcinogenesis. In the present study, we determined the chemopreventive effects and mechanism of diclofenac on inhibiting pancreatitis-carcinogenesis in LSL-KrasG12D/Pdx1-Cre mice (called Pankras mice), particularly on Kras prenylation. Pancreatitis was induced by single i.p. injection of Cearulein (250ug/Kg body weight) for 5-week old mice, and one week after caerulein treatment, Pankras mice were fed an AIN-93M diet or a diet with 5 or 20ppm diclofenac (n=12 mice/group) for 7 months. The results showed that Diclofenac significantly reduced the severity of chronic pancreatitis, as measured by the extent of acini loss, inflammatory cell infiltration and stromal fibrosis. The progression of low-grade mPanIN I to high-grade mPanIN II/III and to invasive carcinoma was significantly suppressed and the tumor incidence significantly reduced to 50% and 17% in the mice treated with either 5ppm or 20 diclofenac compared to 86% in Pankras mice. Inhibition of membrane-bound mutant Kras (prenylated) and its transmitted phosphorylation of mitogen-activated protein kinase's kinase/extracellular signal-regulated kinases were demonstrated in pancreatic tissues by western blot assay. Analysis of the eicosanoid profile revealed a significant reduction of prostaglandin E2. These results indicate that diclofenac is a highly potential agent for preventing chronic pancreatitis and carcinogenesis, mainly via targeting mutant Kras and inflammation (Supported by NIH R01 CA164041).

#2238

Self-microemulsifying drug delivery systems (SMEDDS) containing novel compounds for the chemoprevention of pancreatic cancer.

Preshita Desai,1 Arvind Thakkar,2 Jeffrey Wang,1 Sunil Prabhu1. 1 _Western Univ. of Health Sciences, Pomona, CA;_ 2 _University of New England, Portland, ME_.

Pancreatic cancer (PC) is the fourth leading cause of deaths in the United States, claiming approximately 43,000 lives every year. Existing trends suggest that it will be the second cause of death after lung cancer within the next 20 years. With mortality rates remaining high even after decades of research, scientists are now turning to develop chemopreventive strategies that may stop or delay the initiation and progression of the disease. Our study reports the chemoprevention potential of (1) a free drug combination of H1-receptor antagonist drug with Sulforaphane (SFN), a neutraceutical compound derived from broccoli and (2) lipid nanocarriers of H1-receptor antagonist drug (SP1 SMEDDS; formulation patent pending) with free SFN when administered at low doses in human pancreatic cancer cell lines (MIA PaCa-2 and Panc-1). IC50 concentrations were calculated for various treatment groups using MTS viability assay in accordance with published protocol, using 96-well microplates and incubation time of 72 h. The free drug combination revealed almost 70-fold and 20-fold enhancement in chemoprevention efficacy (based decrease in cancer cell viability) as compared to controls. Interestingly, the free drug combination (at 5µM) showed ~55 % (p<0.001) inhibition in cell lines whereas no measurable response was observed with individual free drug treatments at the same concentration. This confirmed the synergistic chemoprevention potential of the free drug combination. SP1 SMEDDS were optimized for particle size, encapsulation efficiency and chemoprevention efficacy. SP1 SMEDDS (10µM) by itself exhibited 54% (p<0.001) cell line inhibition whereas no measurable response was observed with free drug at that same concentration, confirming the potential of lipid nanocarriers in enhancing the chemoprevention activity. Moreover, the SP1 SMEDDS in combination with free SFN demonstrated synergistic activity with 500-fold, 140-fold and 7-fold enhancement in chemoprevention potential as compared to the free HI-receptor antagonist, SFN and the free drug combination, respectively. In conclusion, a novel lipid formulation of an H1-receptor antagonist in combination with free SFN was invented (provisional patent applied) to successfully elicit PC chemoprevention. Future studies will look into the efficacy of this novel formulation in in vivo PC models.

#2239

Is folic acid safe for non-muscle-invasive bladder cancer patients? An evidence-based cohort study.

Huakang Tu,1 Colin P. Dinney,1 Yuanqing Ye,1 Barton H. Grossman,1 Seth P. Lerner,2 Xifeng Wu1. 1 _University of Texas MD Anderson Cancer Center, Houston, TX;_ 2 _Baylor College of Medicine, Houston, TX_.

Background: Cancer patients are highly concerned about food choices and dietary supplements that may affect their treatment outcomes. Excess folic acid (synthetic folate) from supplements or fortification can lead to accumulation of unmetabolized folic acid in the systemic circulation and urine, and may promote cancer growth, especially among those with neoplastic alterations.

Objective: We investigated the prospective association between synthetic versus natural folate intake and clinical outcomes in non-muscle-invasive bladder cancer (NMIBC), which is a highly recurrent disease.

Design: In a cohort of 619 NMIBC patients, folate intake at diagnosis was assessed with a previously validated food frequency questionnaire and categorized according to tertiles. After a median follow-up of 5.2 years, 303 tumor recurrence and 108 progression events were documented from medical record review. Multivariable Cox proportional hazards and logistic models were used to estimate adjusted hazard ratios (HR) and odds ratios (OR) with 95% confidence intervals (95% CI).

Results: Synthetic folic acid intake was positively associated with risk of recurrence among NMIBC patients (medium vs. low intake: HR=1.72; 95% CI=1.20, 2.48; P=0.003; high vs. low intake: HR=1.80; 95% CI=1.14, 2.84; P=0.01). Patients with higher folic acid intake were more likely to have multifocal tumors at diagnosis (medium/high vs. low: OR=2.08; 95% CI=1.08, 4.02; P=0.03). In contrast, natural folate intake tended to be inversely associated with risk of progression (medium/high vs. low: HR=0.68; 95% CI=0.44, 1.04; P=0.08).

Conclusions: High intake of synthetic folic acid, in contrast to the natural forms, is associated with increased risk of recurrence in NMIBC and multifocal tumors at diagnosis, suggesting that folic acid may be unsafe for NMIBC patients. These findings provide some evidence for nutritional consultation regarding folate intake among NMIBC patients.

#2240

Diet and exercise-induced weight maintenance, alone and in combination with a whole tumor cell vaccine, delays mammary tumor growth and reduces tumor-infiltrating MDSCs expressing PD-L1 and IDO.

Yitong Xu, William J. Turbitt, Andrea M. Mastro, Connie J. Rogers. _Pennsylvania State University, University Park, PA_.

Obesity and physical inactivity increase breast cancer risk, while the prevention of weight gain by diet and exercise can be protective. Numerous biological mechanisms are proposed to explain the beneficial effects of weight maintenance, including changes in metabolic, inflammatory and immune mediators. We have previously shown that diet and exercise-induced weight maintenance (WM), in combination with a whole 4T1.2luc tumor cell vaccine (VAX), significantly reduced mammary tumor growth and metastases in the 4T1.2luc murine mammary tumor model. This reduction in growth and metastases occurred concomitantly with an elevation in tumor antigen-induced splenic IFN-γ production and a reduction in the accumulation of splenic myeloid-derived suppressor cells (MDSCs) at day 35 post-tumor implantation. The goal of the current study was to determine if WM+VAX alters the tumor microenvironment at an early stage of tumor growth (day 24 post-tumor implantation) which may contribute to reduced tumor growth and enhanced immune outcomes at day 35 post-tumor implantation. Female BALB/c mice were randomized into sedentary, weight gain (WG) or exercising (access to voluntary running wheel), weight maintenance (WM) groups (n=22-27/group). After 8 weeks on the intervention, all mice were orthotopically injected with 5x104 4T1.2luc cells into the fourth mammary fat pad and continued on their intervention. Once injected, both WG and WM mice were further randomized into vaccination (VAX) or vehicle control (VEH) groups (n=10-15/group) and administered 1x106 irradiated 4T1.2luc cells (VAX) or HBSS (VEH) at day 7, 14 and 21 post-tumor implantation. Mice were sacrificed at day 24 post-tumor implantation and tumor-infiltrating immune cells were isolated for analyses. Both WM+VEH and WM+VAX groups showed a significant reduction in primary tumor growth and splenomegaly compared to both WG groups (p<0.001). Flow cytometric analysis demonstrated that the combination of WM+VAX significantly reduced total tumor-infiltrating MDSCs and MDSCs expressing PD-L1 and indoleamine 2,3-dioxygenase (IDO) compared to WG+VEH (p<0.05). This finding was consistent with a 1.5-4.5 fold decrease in the gene expression level of Pdcd1, Ido1 and Ifng in the total tumor infiltrates. These results suggest that the combination of weight maintenance and the whole tumor cell vaccine may be altering both the number and immunosuppressive capacity of tumor-infiltrating MDSCs. Thus, preventing weight gain through diet and exercise may be an important recommendation in combination with immune-based therapies to enhance efficacy and improve clinical outcomes. This work is supported by R21 CA209144; T32GM108563. W.J.T is currently funded by the NIH T32DK062710 grant; NORC, UAB, Birmngham, AL.

#2241

Comparison of rare pathogenic germline variants associated with cancer predisposition syndrome in Taiwan and USA populations.

Zulfiqar Gulzar,1 Julian Lucas,1 Alex Atkins,1 stephen Su,1 Oscar Segurado,2 Rui Mei1. 1 _CellMax Life, Sunnyvale, CA;_ 2 _MedicAffairs Consulting, San Jose, CA_.

Objective To evaluate the analytical performance of a 98-gene NGS panel designed to detect highly- penetrant, rare pathogenic germline variants, strongly linked to predisposition of cancers in Taiwan and USA.

Methods This 98-gene panel was developed following an extensive review of genes with a strong clinical and genetic linkage evidence for cancer predisposition. Probes covering the exon sequences from these 98 genes were validated using 19 samples obtained from the Platinum Genomes Project and the Genome In A Bottle Consortium. Analytical sensitivity and specificity for the detection of clinically important variants within multiple cancers types were demonstrated in 30 reference samples derived from patients with familial cancers. Additionally, two clinical cohorts of 1885 and 374 subjects from Taiwan and USA, respectively, were screened for detection of rare pathogenic germline variants in our CAP accredited laboratory in Taiwan, and CLIA certified laboratory in the USA.

Results The analytical sensitivity and specificity of the assay panel were 99.95% and 100%, respectively. Similarly, the reportable variants for all 30 DNA samples containing clinically important SNVs in BRCA1/2, RET, APC, MEN1 and PTEN were called with no false positives. The concordance for the use of either blood (reference lab) or saliva (internal lab) as a sample source was found to be 99.9%. The mean age of the Taiwan cohort was 43, with 58% males and 42% females, while the US cohort had a mean age of 58 with 40% males and 60% females. Germline pathogenic variants were detected in 47/1885 (2.5%) and 25/374 (6.7%) individuals respectively. Outside of BRCA1 and BRCA2 positive cases in Taiwan (8.5% and 17%) and US (4% and 0%), the remaining germline positive cohort had pathogenic variants detected in MUTYH, MLH1, RET, ATM, APC, CHEK2, ERCC2, PALB2, PTCH1, WRN, BARD1, BRIP1, PMS2, PTEN, RAD51D, SDHD, RUNX1, BLM, HOXB13, and MSH6. Stratification of pathogenic variants among high risk and average risk individuals suggests these variants are distributed in both risk groups. Using a multigene approach, we identified 35/47 (87%) and 24/25 (96%) additional cases with rare pathogenic mutations respectively which would not have been identified if we were only looking for BRCA1/2 mutations.

Conclusion We have demonstrated in 2 clinical cohorts the analytical and clinical validity of a 98-gene panel for the detection of pathogenic germline variants predisposing to cancer. High sensitivity and specificity of this panel was demonstrated irrespective of the use of blood or saliva as samples.

#2242

Breastfeeding protects against pro-tumorigenic changes in the mammary gland by limiting epithelial luminal progenitor cell expansion.

Mustafa M. Basree,1 Neelam Shinde,2 Christopher Koivisto,3 Maria Cuitino,3 Raleigh Kladney,4 Allen Zhang,2 Hee Kyung Kim,2 Anthony Trimboli,3 Jianying Zhang,5 Gustavo W. Leone,3 Gina M. Sizemore,4 Sarmila Majumder,2 Bhuvaneswari Ramaswamy2. 1 _Kentucky College of Osteopathic Medicine, Pikeville, KY;_ 2 _The Ohio State University Wexner Medical Center, Columbus, OH;_ 3 _Medical University of South Carolina Hollings Cancer Center, Charleston, SC;_ 4 _The Ohio State University Comprehensive Cancer Center, Columbus, OH;_ 5 _The Ohio State University Center of Biostatistics, Columbus, OH_.

Introduction: Multiple epidemiological studies have shown that prolonged breastfeeding is associated with a reduced risk of developing triple-negative/basal-like breast cancers (TN/BLBC). However, no preclinical models that delineate the mechanism of this link exist. This understanding is critical not only to prevent TN/BLBC, but also to address disparity in breast cancer outcomes, as African-American women have a higher incidence of TN/BLBC and lower prevalence of breastfeeding. We present our data using mouse models that provides new insights into this link.

Experimental procedure: We modeled gradual involution (GI) and abrupt involution (AI) of mammary glands (MGs) in wild type FVB/N mice. Uniparous mice were assigned to AI or GI cohorts either by removal of all pups on day7 postpartum (AI) or allowing the pups to naturally wean (GI). MGs were harvested for analysis on postpartum day28, day56 and day120. We assessed MG morphology/histology using whole mounts and H&E stained sections, collagen deposition using Trichrome and PicroSirus red staining, inflammatory markers and immune cell infiltration using immunohistochemistry. Mammary epithelial cell hierarchy was analyzed by Fluorescence-Activated Cell Sorting of a single cell suspension prepared from the MGs. Gene expression was analyzed in mouse mammary epithelial cells using Affymatrix Gene ChIP Mouse Transcriptome array 1.0 and Gene Set Enrichment Analysis (GSEA) was used to analyze gene expression data.

Summary: Abruptly involuted MGs exhibited altered morphology including denser stroma, increased collagen deposition with higher levels of Type I collagen, increased inflammation (pStat3-Y705), increased immune cell infiltration and proliferation compared to GI glands. The mammary epithelial cell hierarchy was disrupted with marked expansion of the luminal progenitor (LP) population in the AI glands but not in the GI glands, a trend frequently observed in women heterozygous for BRCA1 mutation at higher risk of developing BLBC. Enrichment of an LP gene signature and Notch pathway was observed in mouse LP cells isolated from the AI glands. Most strikingly, the AI glands developed alveolar hyperplasia, and squamous metaplasia within 4 months of removal of the pups.

Conclusion: Involution leading to a pro-inflammatory milieu in the MG is well established. Whether involution that happens gradually after prolonged breastfeeding differs in its effect on MGs has not been well studied. Using novel animal modeling to study the differences in MGs following abrupt vs gradual involution, we report a distinct morphology in the mammary tissue favoring pro-carcinogenic changes following AI. Furthermore, we show for the first time, expansion of the LP population in AI glands, which is known to be cell of origin for TN/BLBC potentially linking the risk of TN/BLBC with lack of breastfeeding. Further studies are ongoing.

#2243

Cell-phone radiofrequency enhances angiogenesis and stimulates cell invasion of human head and neck cancer cells.

Ala-Eddin Al Moustafa. _MEACR, Montréal, Quebec, Canada_.

Today, cell-phone is the most widespread technology globally. It has been suggested that cell-phone radiofrequency (RF) use may increase the risk of human brain and probably head cancers. However, the outcome of cell phone RF on head and neck (HN) cancer progression has not been explored yet. Thus, first we examined the outcome of cell-phone RF on angiogenesis using the chorioallantoic membrane (CAM) of the chicken embryo as a model. Then we investigated the effect of cell-phone RF on cell invasion and colony formation in soft agar of two human HN cancer cell lines. Our data revealed that cell-phone RF promotes angiogenesis of the CAM. In addition, cell-phone RF enhances cell invasion and colony formation of human HN cancer cells; this is accompanied by a down-regulation of E-cadherin expression. Regarding the mechanism of cell-phone RF on angiogenesis, cell invasion and colony formation, we found that cell-phone RF activates Erk1 and Erk2 in our experimental models which could be the main pathway behind these events. These data suggest that cell-phone RF enhances HN cancer progression by stimulating angiogenesis, cell invasion and tumor formation via Erk1 and Erk2.

## BIOINFORMATICS AND SYSTEMS BIOLOGY:

### Application of Bioinformatics to Cancer Biology 2

#2244

Differential DNA methylation analysis of TCGA cholangiocarcinoma data.

Nitish K. Mishra, Niu Meng, Chittibabu Guda. _UNMC, Omaha, NE_.

Cholangiocarcinoma (CCA), also known as bile duct cancer, is a rare malignancy that develops in bile duct, arising from the epithelial cells (cholangiocytes) lining the bile duct. CCA is the second most common type of primary liver cancer, and its incidence has been rising in the Unites States. Most of the patients with CCA are diagnosed at an advanced stage with a median survival of less than 1 year despite administering chemotherapy. In the current study, we analyze the global patterns of DNA methylation in CCA data from TCGA. Pathway and gene ontology (GO) enrichment analysis of differentially methylated genes (DMGs) enabled us to understand how changes in methylation and gene expression affect the biologic pathways involved in the progression of CCA. TCGA level-3 DNA methylation data were downloaded for 485,578 CpG sites with annotations. Further, we normalized β value by using Beta Mixed Integer-quantile normalization (BMIQ) to adjust type I and type II probes. For a CpG site to be considered differentially methylated, the difference in the median β value in primary tumor and normal samples should be at least 0.2 and the BH adjusted p-value should be less than 0.005 and AUC ≥ 0.7. A total of 12,259 differentially methylated (Δβ >) CpG were observed; out of these 9,534 are hypermethylated and 2,725 are hypomethylated. About 4,714 differentially methylated CpGs (dm-CpGs) were observed within 1.5Kb from the TSS of corresponding genes. Chromosome 1 has maximum number of dm-CpGs, while chromosome 18 has lowest number of dm-CpGs. Differential DNA methylation frequency per Mb was also calculated and it was observed that chromosome 17 has the highest differential methylation frequency (9.61 dm-CpGs/Mb) while chromosome 18 has the lowest (1.47 dm-CpG/Mb). KEGG pathway analysis of genes with differentially methylated CpGs (Δβ ≥ 0.2) observed the enrichment of cancer, cell division and differentiation, amino acid metabolism, xenobiotics degradation, and immune response pathways. Hippo signaling pathway also got affected by DNA methylation (FDR 3.70e-18), which also got affected by differential gene expression. Change in DNA methylation and gene expression level of hippo signaling pathway genes plays a vital role in CCA.

#2245

Deconvolution of diverse immune cell populations within tumors using ACE Transcriptome.

Eric Levy, Sean M. Boyle, Pamela Milani, Shujun Luo, John West, Richard Chen. _Personalis, Inc., Menlo Park, CA_.

Comprehensive tumor immuno-genomic characterization is becoming an important tool for identifying new biomarkers correlated with patient response to immunotherapy. Both the abundance and composition of tumor-infiltrating lymphocytes have been associated with tumor progression and patient outcome. Prior studies have shown that an enrichment of cytotoxic immune cells, and depletion of immunosuppressive cells, can indicate better clinical outcome.

While common lab approaches are used to profile tumor samples for the presence and enumeration of immune cell types, these approaches can be limited by the number of markers and throughput. Thus, there is interest in using an accurate computational method leveraging NGS data to more comprehensively profile the distribution of immune cells in the tumor microenvironment. Through the use of marker genes that are expressed in a cell type specific manner, it is possible to computationally predict the fractions of multiple cell types in a mixed sample. This approach relies on an input of reference gene expression signatures, which are used to estimate the proportions of the cell types represented in the reference. The generation and validation of these reference signatures are critical for ensuring the accuracy of the results on a given platform.

We have used the ACE Cancer Transcriptome, from the ACE ImmunoID platform, to produce high-quality gene expression profiles of purified immune cells representing many lineages, including B cells, monocytes, NK cells, and T cells. These profiles were used to create reference signatures of immune cell type specific genes, enabling quantification of their cellular abundances. In addition, we test the accuracy of the deconvolution approach on mixtures of immune cells. Finally, we validate the approach on tumor samples with immune cells quantified by widely accepted orthogonal methods. Through these results, we show that deconvolution of TILs through our ACE Cancer Transcriptome can be a robust platform for elucidating the composition of immune cells in a tumor sample.

#2246

Identification of mutational signatures in high-risk neuroblastoma patients.

Susanne E. Reinsbach,1 Malin Larsson,2 Angela Martinez-Monleon,1 Rose-Marie Sjöberg,1 Niloufar Javanmardi,1 Anna Djos,1 Per Kogner,3 Susanne Fransson,1 Erik Larsson Lekholm,1 Tommy Martinsson1. 1 _University of Gothenburg, Gothenburg, Sweden;_ 2 _Linköping University, Linköping, Sweden;_ 3 _Karolinska Institutet, Stockholm, Sweden_.

Background: Mutational processes generate unique patterns of base substitutions generally referred to as mutational signatures. Mutational signatures can give insights into fundamental mutational processes underlying somatic mutations in tumors and have been shown to provide a starting point from which to evaluate therapeutic options. Neuroblastoma is clinically heterogeneous ranging from tendency of spontaneous regression to frequent formation of metastases. Earlier studies have identified several recurrent genetic alterations, including mutations in genes such as ALK and ATRX, as well as chromosomal rearrangements such as MYCN amplification, 11q deletion and 17 gain. Despite recent progress in the treatment of high-risk neuroblastoma, patients in this subgroup still have a poor prognosis, underscoring the need to further stratify patients in order to help inform clinical decision making.

Methods: Here, we sought to characterize high-risk patients and their underlying pattern of aberrations by conducting whole genome sequencing of 30 neuroblastoma patients. For each tumor/normal pair, the mutational landscape comprising single nucleotide and structural variants including copy number alterations was investigated. Non-negative matrix factorization was used to decompose single nucleotide variants to discover mutational signatures.

Results: De novo mutational signature analysis identified two mutational profiles (signature A and signature B) that were compared to known signatures of the pan-cancer study by Alexandrov et al. (2013). The landscape of signature A is closest to their signature 18 (0.97 cosine similarity) and revealed a strong bias towards C>A substitutions mainly found in neuroblastoma, and signature B is closest to signature 5 (0.82 cosine similarity) identified in most of the cancers. Out of 13 patients with a clear dominance of signature A, 12 patients were classified with MYCN amplification or 11q deletion. Furthermore, 5 out of 6 patients with a main contribution of signature B were metastasis or relapse cases.

Conclusions: Our results suggest that what is currently considered to be the typical neuroblastoma signature is mainly present in primary high-risk patients whereas the mutational landscape is altered in more advanced stages of the disease. To better understand the connection between the identified signatures and the high-risk subgroup, we are currently investigating the genome wide landscape of the observed C>A substitutions.

#2247

Accelerating cancer research using big data with BioKDE platform.

Xiaodong Pang,1 Mayassa J. Bou-Dargham,2 Yuhang Liu,2 Zihan Cui,2 Linlin Sha,2 Tingting Zhao,2 Jinfeng Zhang2. 1 _Insilicom LLC, Tallahassee, FL;_ 2 _Florida State University, Tallahassee, FL_.

Large volumes of data have been generated in biomedical field every day and made publicly accessible. A significant portion of the data are highly under-analyzed. On the other hands, biomedical researchers having problems that can be solved by these data do not have the expertise to access and analyze the data. The BioKDE (Biomedical Knowledge Discovery Engine) platform aims to bridge this gap by accelerating biomedical discovery through integration and mining of large volumes of public genomic data. We have integrated a large volume of genomics data from TCGA (the Cancer Genome Atlas) and GEO (Gene Expression Omnibus) database, which are linked with patient clinical information. Powerful and versatile query functions were built for the integrated data. Basic and advanced analysis tools have been implemented with user-friendly interfaces. Pipelines can be built by chaining the query tools with analysis tools to perform sophisticated data acquisition and analysis tasks very conveniently. Pipelines can be saved and shared among researchers to quickly replicate certain analyses using the same dataset or different datasets. A large number of novel discoveries have been made, which can be used directly to write scientific research papers using these discoveries alone or by combining with additional experimental validation studies. We will present a few case studies published recently to illustrate the power of the BioKDE. We are looking for collaborators to write papers together for the discoveries we made. The platform can be accessed at http://www.insilicom.com.

#2248

Dynamic changes in transposon expression and epigenetic regulators detected during early breast cancer transition to malignancy.

Isaac D. Raplee, Alexei Evsikov, Caralina Marin de Evsikova. _Univ. of South Florida, Tampa, FL_.

Introduction: Breast cancer is the second leading cause of cancer-related deaths in women. In 2017, about 315,000 women were diagnosed with breast cancer, with approximately 63,000 new cases of ductal carcinoma in situ (DCIS), the earliest form of breast cancer. Little is known as to why some patients diagnosed with atypia and DCIS remain cancer-free while others progresses to invasive ductal carcinoma (IDC). Hypothesis: While mutagenic role of transposonable elements (TEs) in cancer is known, we test the novel role of long terminal repeat (LTR) TEs as potential drivers promoting cell de-differentiation during early stages of breast cancer. Objective: To identify molecular signatures driving cell fate decisions at atypia and DCIS to transformation, we investigated expression of TEs in atypia, DCIS and IDC. Methods: We created a TE Enrichment Set Analysis (TESA) to identify TEs in RNA sequencing datasets across four stages of breast cancer, normal atypia, DCIS, IDC (n= 8-23 samples per stage). Results: After quality control steps to remove outliers, TEs, compared to genes, exhibit substantially less variation in their expression because the first principal component accounted for over 80% expression variation compared to 20% in transcript expression variation (p<0.05). Eighty-eight TEs were detected as significant across stages by ANOVA (α=0.05, FDR 5%). The majority of differentially expressed TEs were LTRs (67%, p<0.05) with the remaining split into DNA TEs (18%), SINEs (11%) and unclassified (4%). Despite being the largest TE portion in the human genome, no statistically significant differences were detected in LINEs (p>0.10). Six general patterns of TE expression were identified using k-means clustering algorithm. In general, the majority of TEs were upregulated during atypia or DCIS stages or both stages. To identify potential molecular mechanisms of TE de-repression, which induce LTR expression during atypia and DCIS, we measured the correlation between specific LTRs, including HERV-H, HERV-K, LTR7Y, and expression of epigenetic regulators, TETs, DNMTs, and TRIM28/KAP1 across all four stages. As expected, normal tissue has positive and negative correlations between LTRs and epigenetic regulators, however, at atypia there was a loss of some negative correlations and at DCIS all negative correlations between LTRs and DNMTS were lost (p<0.0001). Unexpectedly, at IDC stage, the negative correlations re-emerged between LTRs and DNMTs (p<0.05). Together, these data imply the epigenetic remodeling occurs prior to malignancy, which associates, and may potentially drive, LTR expression. Conclusion: Our TESA data provides experimental support that early genomic changes are a mechanism underlying malignancy. Translational bioinformatics is a technique to identify prognostic molecules for impending invasive breast cancer from biopsies from pre-malignant stages.

#2249

PWS: An integrated web service platform enabling precision medicine.

Zhenyu Duan. _Predicine, Inc, CA_.

Aiming at providing an integrated platform to accelerate global drug development with personalized medicine, Predicine Web Service (PWS) is built to enable real time project status report, interpretation of clinical impact for genomic aberrations, and novel biomarker discovery with data mining algorithms and analytics.

A large-scale Predicine database is built with KnowledgeBase layer and dynamically growing genomics data layer. The KnowledBase layer integrates genome annotations, ontologies, computed variant impacts, and curated clinical implications. The dynamic genomic data layer is designed to be scalable so that both Predicine patient genomic profiles and public domain genomics data can be imported with standardized data models. A wide range of curated databases and public domain genomics profiles are integrated, including COSMIC, ClinVar, dbSNP, 1000 Genomes, ExAC, CIViC, GeneDrug KnowledgeBase, MSK IMPACT, and TCGA. Data mining algorithm and analytics are developed to enable data driven interpretation and biomarker discovery.

An intuitive web interface is developed for users to select patient space with clinical and genomics criteria, to generate patient reports with ability to drill down to multi-level variant data, and to discover biomarkers leading to hypothesis of novel targets, drug response association, and combination therapy. The project management module securely communicates the status of projects and samples for pharmaceutical companies and other clients. On PWS, private domain patient data can be combined with comprehensive public domain data for comparative and comprehensive global analysis to enable precision medicine.

#2250

Tumor mutational burden: Guidelines for derivation and robustness of measurement.

Natalie Mola, Matthew Schu, Suzy Stiegelmeyer, Wendell Jones, Victor J. Weigman. _EA Genomics, A Q2 Solutions Company, Morrisville, NC_.

Tumor mutational burden (TMB) is defined as the number of somatic mutations per Mb of the genome and is used to represent accumulation of somatic mutations over the life of the tumor. Recent evidence has shown that high TMB scores are associated with improved response to immune therapies across multiple indications. However, the derivation of this score is not well characterized, nor is this score universally performed across laboratories.

Here we describe our methodology for calculating TMB, which considers an estimation of true germline mutations, the efficacy of different somatic callers, and the identification of regions within a capture panel that most reliably contribute to robust TMB scoring. To accomplish this, we only include positions that fall within high confidence regions of the genome as defined by NIST GIAB datasets HG001-HG005 v3.3.2. The high confidence regions encompass 88% of the exome.

Somatic variant calling is performed utilizing the tumor and normal samples using only positions where there is sufficient depth of coverage in both samples. Any remaining germline variants are excluded, leaving only somatic variants in the targeted, high-confidence regions for the final TMB calculation. TMB calculations utilizing high-confidence regions reduce the contribution of false positive variants in the mutational burden estimate.

We have compared TMB scores using this approach to those provided from evaluating somatic calls from TCGA, where matched tumor/normal pairs exist. In addition, we introduce a method to determine the effect on TMB scores when in silico determination of germline variants is used to derive somatic calls when matching normal samples are not available.

Defining the robustness of this score across and criteria for its derivation will help better position defining analytical range and future utility across indications and NGS panels

#2251

PathologyMap: The world's largest online pathology database for collaborative cancer research.

Ke Cheng,1 Agedi Boto,2 Harini Babu1. 1 _HistoWiz, Inc., Brooklyn, NY;_ 2 _Yale School of Medicine, New Haven, CT_.

Introduction: Currently there is no large centralized online whole slide imaging (WSI) repository for histopathology data, making collaboration difficult and bioinformatics-driven discovery impossible. We wanted to know if it was possible to build a self-sustaining, crowd-sourced digital pathology database for cancer research discovery. If such a database existed and was searchable, it would allow researchers to compare the histology of their mouse models with that of human tissues to determine which mouse model was closest to the true disease. Furthermore, researchers could get tissue corresponding to their mouse model for subsequent DNA, RNA, or protein analysis to ensure that the model has the qualities they are most interested in.

Methods: HistoWiz automates histology for cancer researchers guaranteeing a 48-hour turnaround from tissue specimen to digital slides in the cloud. This system feeds digital data into an intelligent tissue platform, PathologyMapTM. which employs a novel image-tagging technology to capture metadata from users and is searchable on the annotation fields. The search becomes more accurate with more historic data, and has the potential for breakthroughs.

Results: Currently there are 48,983 scanned slides and the database is growing at 220% per year. It allows researchers to find similarities in cancers extremely quickly by searching for slides from a specific species, strain, genotype, organ, lesion, experimental treatment condition, sex, age and biomarker. A search of the database reveals 58 slides that are stained for CD8. Perhaps its greatest utility for researchers not familiar with normal histology is that abundant normal controls are searchable in the database, allowing easy comparisons between the pathology studied and normal tissue. A search for liver returns at least 30 slides depicting normal liver histology from mice, monkeys, and humans. A search for KRAS yields at least 100 unique slides. A search for p53 reveals over 150 slides. A search for GFP-infected mouse embryo yields 120 slides. PathologyMapTM also reflects the current focus of cancer therapy. For instance, there are 60 slides stained for EGFR, most likely related to the focus on precision medicine in the treatment of tumors.

Conclusions: PathologyMapTM is the world's largest, most comprehensive WSI database. It not only allows for online viewing, sharing, archival, annotation, search and meta-analysis of cancer tissue images, but also access to the corresponding cancer tissue specimens. By using machine learning (ML) algorithms and allowing histology service users to contribute, annotate and compare cancer tissue data across different laboratories and hospitals around the world, PathologyMapTM will be vital for improving cancer diagnosis, discovering insights to advance cancer research, saving money by reducing repetitive research, and accelerating drug development.

#2252

Biomarkers for staging melanoma, a search at transcriptome level.

Daniel Ortega-Bernal,1 Claudia Rangel-Escareño,2 Elena Arechaga-Ocampo,1 Claudia H. Gonzalez-De la Rosa1. 1 _Universidad Autónoma Metropolitana, México, Mexico;_ 2 _Instituto Nacional de Medicina Genómica, México, Mexico_.

Background: Melanoma represents one of the most aggressive malignancies and has a high tendency to metastasize. Metastatic potential may not be predicted from the morphologic features alone, and the use of routine histologic prognostic factors is insufficient for melanoma staging. Because metastatic melanoma remains extremely difficult to cure, there has been an urgent need to find new markers to identify tumors capable of metastasizing. Here, we used a computational strategy based on transcriptome meta-analysis for melanoma staging. We aimed to identify biomarkers that differentiate between non-metastatic and metastatic melanomas.

Methods: Affymetrix HU 133A Plus 2.0 microarray data were obtained from the GEO database for the meta-analysis, representing benign nevi (N), stages I, II and III melanoma (EI-III), stage IV melanoma from primary site (EIV) and stage IV melanoma from metastatic site (Mm). Differential expression was analyzed using linear models (118 files) and time course (64 files). Transcripts with a log fold change of <-1/>1 and B-statistic >3 using linear models and evaluate by MB-statistic for Time Course were included in the study. The analysis focused on genes that encode secreted proteins, whose expression was upregulated throughout the progression of melanomas, starting at N and increasing gradually from EI-III to EIV and finally to Mm.

Results: We obtained 6137 differentially expressed genes with linear models (between EI-III vs. N, EIV vs. EI-III, Mm vs. EIV, EIV vs. N and Mm vs. N contrasts) and took the top 6137 genes ranked with Time Course. Our results show 17 possible biomarkers that grouped into three types: 1) genes capable of differentiating between EIV/Mm and EI-III/N (SPP1); 2) genes capable of differentiating between Mm and EIV/EI-III/N (CXCR4, FCGR1B, NGRN, PHTF1, RAB8B, ARHGEF2, ARMC9, BCL2A1, CALU, DLGAP5, DMXL2, FKBP11 and STARD3NL); and 3) genes capable of differentiating between Mm/EIV/EI-III and N (ADAM10, CLIC4 and STK36).

Conclusions: Our analyses, in agreement with previously reported results, identify SPP1 and CXCR4 as biomarkers of metastatic disease and poor clinical outcome, supporting the quality of our analysis. We also identified new biomarkers that may be used to predict undetectable metastasis. In order to provide a more precise evaluation, experimental validation or functional exploration of the reported gene expression differences is warranted.

#2253

Identifying pathogenic germline variants in 1,172 cancer patients utilizing a novel variant phasing tool and strict public database curation.

Amanda Polley,1 Charles Vaske,1 Steve Benz,1 Patrick Soon-Shiong,2 Shahrooz Rabizadeh,2 J Zachary Sanborn1. 1 _NantOmics, LLC, Santa Cruz, CA;_ 2 _NantOmics, LLC, Culver City, CA_.

The identification of pathogenic or likely pathogenic (P/LP) germline variants in cancer patients is vital in assessing potential genetic causes of cancer risk, as well as incidental rare disease risk. However, sequencing limitations, lack of pedigree data, and unreliable phenotypic data can hinder the discovery of these putative causal germline variants. In order to improve the identification of such variants, we have developed a germline analysis pipeline that phases variants and identifies rare P/LP variants utilizing data from the GnomAD and ClinVar databases.

In order to ensure our pipeline is annotating variants with accurate phenotypic data, we rate the quality of submitters to ClinVar based on their submission history. This history includes review status, agreement between population frequencies and stated clinical significance, and the quality of supporting evidence provided. The variant-phenotype relationship data extracted from ClinVar is valuable in directing us towards cancer-contributing variants, as well as incidental disease risk. However, there are many discrepancies between various institutions' assertion criteria submitted to ClinVar. Many variant entries in ClinVar are submitted as P/LP, yet a large number of these submissions lack evidence or criteria supporting the variant's clinical significance. Our pipeline identifies low-quality submissions, allowing for the inclusion of only high-quality P/LP variant annotations.

In addition to recognizing known P/LP variants from the ClinVar archive, our pipeline provides additional identification of potentially pathogenic novel germline variants via haplotype phasing. Haplotype phasing of germline variants is vital when determining the impact of multiple heterozygous variants within the same gene, but it is difficult to perform such phasing outside of family studies. Our pipeline utilizes normal DNA, tumor DNA, and tumor RNA to predict the phase of variants without the need for full pedigree information.

The pipeline was tested on 1,172 patient samples, with the goal of phasing clinically-associated cancer genes and discovering rare P/LP germline variants. We were able to phase variants in over 50% of the patients, allowing us to identify the nature of compound heterozygosity as it relates to disease risk in these patients. Additionally, our pipeline identified at least 1 high-quality ClinVar P/LP variant per patient in over 25% of the patients. In the vast majority of patients, we were able to identify rare homozygous germline variants. Gene panels specific to cancer type can be used to further investigate which rare variants most likely factor in the patient's disease. Based on these results, as well as our analysis of ClinVar, we assert the need for deep introspection of ClinVar submissions and highlight the utility of RNA data in variant phasing.

#2254

Dynamic network topologies analysis in proteomics data of NSCLC cell lines using a new pipeline based on machine learning tools.

Chiara Antonni,1 Lorenzo Tomassoni,1 Elisa Baldelli,2 Vienna Ludovini,3 Sara Baglivo,3 Mariaelena Pierobon,2 Emanuel F Petricoin,2 Lucio Crinò,4 Paolo Valigi,1 Fortunato Bianconi5. 1 _University of Perugia, Perugia, Italy;_ 2 _George Mason University, Manassas, VA;_ 3 _Azienda Ospedaliera di Perugia, Perugia, Italy;_ 4 _Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST), Meldola, Italy;_ 5 _Independent researcher, Montefalco, Italy_.

Background: Non-small cell lung cancer (NSCLC) is the most common subtype of lung cancer, characterizing ~80% of lung cancer diagnoses. Thus, the development of novel therapies for NSCLC is of primary importance in oncology. The aim of this study was to develop a novel cancer systems biology approach to understand the complex interactions in NSCLC and predict the response to target treatments. We combined proteomic experimental data with a machine learning tool to discover the diverse signaling networks of both KRAS mutant (MUT) and wild-type (WT) NSCLC cell lines (CL).

Material and Methods: Eight NSCLC CL (5 KRAS MUT and 3 KRAS WT) were subjected to reverse phase protein microarray (RPPA) to explore the activation level of 183 proteins treated with selumetinib (SE) and its combination with everolimus (EV) or tamoxifen (TA). Measures were taken at 6 time points: 5 minutes (min), 1, 2 , 6 and 24 hours (h). RPPA was performed also on the baseline and on CL with only dimethyl sulfoxide. Recursive Feature Elimination with Support Vector Machine (RFE-SVM) was used to identify the subset of proteins/features that optimally separated samples in 2 groups. In order to build the interaction network from this subset and browse the main pathways involved, we used Reactome FI and the Pathway Enrichment tool, two Cytoscape plugins for network-based data analysis.

Results: We applied RFE-SVM to discover the most divergent proteins between control and treated samples in the whole CL panel. For all the 3 drug combinations, the network in output contained not only the target of the corresponding drug, but also showed the activation of other parallel pathways. The analysis revealed also that all drugs were mainly effective in a short time, since the number of significant proteins was considerably higher at 5 min and 1 h. For instance, SE treatment activated NGFR, mTOR, PI3K-Akt and proteoglycans pathways in addition to the MAPK pathway. Network at 1 h contained 25 proteins linked by 76 interactions, while at 6 and 24 h the distinctive subsets of features included only 2 and 4 proteins. Then, we applied RFE-SVM to analyze KRAS MUT vs WT CLs. The algorithm showed that treated KRAS MUT CL had a more complex signaling network than the WT ones. Indeed, in SE WT samples MER and ERK were the main relevant proteins to distinguish from the control. In treated KRAS MUT samples, a complex network of 70 proteins and 278 interactions was active during the first hour.

Conclusion: The proposed framework processes high-throughput data and identifies new possible molecular targets in cancer research. This approach is highly flexible for a multitude of data and purposes. We applied it to a RPPA dataset of NSCLC CL and it revealed that all drug combinations had fast dynamics and that KRAS MUT CL had a more complicated response due to a higher number of active pathways.

#2255

Using tumor sample gene expression data to infer tumor purity levels with stochastic gradient boosting machines.

YuanYuan Li, Adrienna Bingham, David M. Umbach, Leping Li. _National Institute of Environmental Health Sciences/National Institutes of Health, Research Triangle Park, NC_.

Tumor purity is the percent of cancer cells present in a sample of tumor tissue. The noncancerous cells (stromal cells) in a tumor are thought to have an important role in tumor growth, metastatic progression, and drug resistance. They also strongly influence genomic analyses of tumor samples. The Cancer Genome Atlas (TCGA) has extensive RNA-seq data from tumor tissue samples as well as assessments of tumor purity for the samples. Our goal is to select a subset of genes whose expression levels are predictive of tumor purity for each tumor type as well as a subset of genes whose expression levels are predictive of all tumor type samples when pooled together. We hope that the genes selected may provide insight about the cell-type composition of tumor samples and about the similarities and differences in tumor microenvironments. We use data from the TCGA, which covers 11 different tumor types and includes genome-wide assessments on over 3,148 samples for gene expression. To identify predictive genes, we used XGBoost, a supervised machine learning algorithm based on the idea of a boosted regression tree ensemble. We carried out 100 repeated runs of 10-fold cross-validations (total of 1,000 train-test partitions) for each tumor type and, also, for all tumor types combined. Using the training-set samples, XGBoost selects a set of genes to predict tumor purity levels; the selected genes are subsequently used to predict the purity levels of the test-set samples. Across the 1,000 train-test partitions for all 11 tumor types, the average root-mean-squared error ranged from 0.09 to 0.16 for the test sets. For each tumor type, we selected the top 250 genes based on their aggregated feature importance scores, a measure of each gene's contribution to tumor purity estimation. No single gene was among the top 250 in all 11 tumor types; however, ACAP1, AMICA1, CSF2RB, CYTIP, GGT5, GLIPR1, IRF4, and PECAM1 were not only among the top 250 in more than 6 tumor types but also in the top 250 when all tumors were combined, suggesting those genes might serve as biomarkers for tumor purity. The most common pathways from gene ontology analysis of these top genes include various immune and signaling pathways. We used XGBoost to identify genes whose expression levels were associated with tumor purity levels in each tumor type. Our results suggest that assessed tumor purity levels in tumor samples can be faithfully recapitulated using certain subsets of genes. We believe that those genes selected for each tumor type by our unbiased approach might provide insight into the biology of the tumor microenvironment, e.g., the presence of cell type-specific marker genes would indicate the presence of specific cell types.

#2256

**Efficient large-scale cell classification and analysis for MultiOmyx** TM **assays: A deep learning approach.**

Mate L. Nagy, Arezoo Hanifi, Ahalya Tirupsur, Geoffrey Wong, Jun Fang, Nicholas Hoe, Qingyan Au, Raghav K. Padmanabhan. _Neogenomics Laboratories, Inc., Aliso Viejo, CA_.

Traditional immunohistochemistry (IHC) techniques utilize one slide per biomarker. When clinical samples are precious and the number of serial sections is limited, comprehensive biomarker profiling becomes difficult with IHC. MultiOmyx is a proprietary, multiplexing methodology capable of staining up to 60 biomarkers on the same slide. The output of the assay enables quantitative profiling of tissues at a single cell level. The assay generates data for millions of cells with billions of queryable data points. To detect and classify cells efficiently at this large scale, we developed an image analysis framework using Deep Learning. Our framework consists of seven major steps: (1) manual annotation of a small subset of the nuclear staining channel (DAPI); (2) training of a fully convolutional neural network [1] on this annotation-set to generate a feature map identifying cell centers; (3) application of the trained network in (2) on the nuclear stain (DAPI) of the entire dataset to delineate individual cells; (4) manual annotation of a small subset of each of the other biomarker channels; (5) training of a convolutional neural network on these annotation-sets for binary classification of each of the biomarkers; (6) application of these classifiers to the entire dataset; (7) combination of the binary classification results to identify phenotypes of interest. For our output, we provide both visual label maps and classification summary tables for individual and co-localized biomarkers at the region of interest level and the entire slide level. In addition, combining the phenotype and location information allows us to visualize complex spatial relationships in the tissue.

The benefits of using this Deep Learning framework are greatly felt through increased time efficiency without a loss in accuracy, when compared to more traditional computer vision methods requiring high levels of parameter fine-tuning. For future work, we plan on fully automating the approach as more manual annotation-sets are generated.

#2257

**AGIS** TM **-OncoGxKB: A simple tool for actionable genomic knowledgebase curation and clinical interpretation.**

Pengfei Yu, Cheng Yan. _Admera Health, South Plainfield, NJ_.

Nowadays, targeted therapy and immunotherapy have become the mainstream of cancer therapy because of their overall merit over traditional chemotherapy. With the increasing number of drug targets being discovered, cancer patients are more likely to benefit from these advanced therapies. However, the interpretation of actionable variants from patients is still a challenge in cancer clinical sequencing workflows. Here we present OncoGxKB, a crowdsource-based knowledgebase system tailored to provide clinical interpretation of variants in cancer. Based on genetic makeup of patient tumor, OncoGxKB provides actionable therapeutic strategies that maximize treatment efficacy and minimize side effects. The clinical recommendations provided cover targeted therapies, immunotherapies and clinical trials associated with actionable variants. Interpretations are rendered in tiers based on clinical evidence and stratified cancer types, following the newest AMP guideline. The crowdsource-based implementation keeps OncoGxKB comprehensive, updated, transparent and accessible to scientific community. The application programming interface (API) availability can also make the interpretation system be easily adopted to different existing pipelines. OncoGxKB is publicly available at: https://agis.admerahealth.com/OncoGxKB.

#2258

Chromothripsis and survival in human malignancies.

Aaron S. Mansfield,1 Enrique Garcia-Rivera2. 1 _Mayo Clinic, Rochester, MN;_ 2 _nference, Cambridge, MA_.

Chromothripsis is a complex genomic event characterized by a high number of chromosomal rearrangements and copy number changes that typically localize to a specific region on a single chromosome. Next generation sequencing has led to the initial discovery and description of these events which have since been observed in multiple human cancers. Whereas most mutagenic processes occur gradually, the multiple structural variations of each chromothriptic event likely occur at once. The clinical significance of chromothripsis remains uncertain, so we sought to characterize the relationship between chromothripsis and survival across human malignancies.

We analyzed 13,318 tumors of 36 types using publicly available data from The Cancer Genome Atlas with CTLPScanner, which uses a sliding window scan statistic to identify events consistent with a chromothripsis-like pattern (CTLP). Our cutoffs for CTLP of copy number variation ≥ 20 and a log10 likelihood ratio ≥ 8 were derived from a clinical training set previously described by CTLPScanner.

We detected one or more events consistent with a CTLP in 8,276 tumors (62.1%) for a total 24,879 events. There were no events in 5,042 cases (37.86%), a significant decrease from established rates of CTLPs. CTLPs were more common in ovarian serous cystadenocarcinomas (89.8%), esophageal carcinomas (86.5%) and sarcomas (81.2%) than in thymomas (26.6%) and thyroid carcinomas (15.2%). Chromosomes 8, 1 and 6 were most frequently involved with CTLPs. CTLPs in chromosome 6 were associated with uveal melanoma, chromophobic renal cell carcinoma and cholangiocarcinomas whereas CTLPs involving chromosome 12 were associated with testicular germ cell tumors. Multiple CTLPs were found in 5,262 tumors (39.5%, range of events 2-18+) and were most common in ovarian serous cystadenocarcinomas and sarcomas. The presence of CTLPs was associated with diminished overall survival across all tumor types (HR 1.5, p<2e-16). Given the potential for multiple CTLPs to have a greater effect on outcomes, we compared the survival of patients whose tumors contained 3 or more CTLPs (n=3,255, 24.4%) to those with 0-1 or 2 CTLPs. The presence of 3 or more CTLPs was associated with the worst survival amongst these groups (HR 1.67, p<2e-16).

We identified that CTLPs are common in human cancers despite their prior descriptions as rare events. This discrepancy is likely due to our detection parameters. In addition, we identified that having multiple co-occurring CTLPs is associated with a worse prognosis, suggesting this type of event is more clinically relevant than a single CTLP and can be used as a negative prognostic marker in human malignancies.

#2259

Prostate cancer molecular subtype impacts interpretation of clinical parameters.

Deli Liu. _Weill Cornell Medicine, New York, NY_.

Background: Molecular characterization of prostate cancer has revealed distinct subtypes with underlying genomic alterations. One of these core subtypes (SPOP mutant prostate cancer) has previously only been identifiable through DNA sequencing, making the impact on prognosis and routinely utilized risk stratification parameters unclear. We aimed to identify prostate cancer molecular subtypes from transcriptional data alone and determine associations with clinicopathologic outcomes and long-term prognosis.

Methods: We developed a novel gene expression signature, classifier (SCaPT), and decision tree to predict SPOP mutant subclass from RNA gene expression data and classify common prostate cancer molecular subtypes. We then validated and further interrogated the association of prostate cancer molecular subtypes with pathologic and clinical outcomes in retrospective and prospective cohorts of 8,158 patients. Associations with clinicopathologic variables, metastasis-free and prostate cancer-specific survival were the main outcomes.

Findings: The SCaPT model prediction showed high sensitivity and specificity in multiple cohorts across both RNA-seq and microarray gene expression platforms. We predicted 8~9% of cases to be SPOP mutant from both retrospective and prospective cohorts. Surprisingly, we found routine clinical risk factors were disconjugate when molecular subtype was considered. The SPOP mutant subclass was associated with lower frequency of positive margins, extraprostatic extension, and seminal vesicle invasion at prostatectomy. However, SPOP mutant cancers were associated with higher pretreatment serum prostate-specific antigen (PSA). The association between SPOP mutant status and higher PSA level was validated in three independent cohorts. Despite high pre-treatment PSA, the SPOP mutant subtype was associated with a favorable prognosis with improved metastasis-free survival, particularly in patients with "high-risk" preoperative PSA levels.

Interpretation: Using a novel gene expression signature to assign the SPOP mutant subclass and a decision tree algorithm to define prostate cancer molecular subclasses, we found the SPOP mutant subclass associated with higher preoperative PSA, less adverse pathological features, and favorable prognosis. These findings suggest a paradigm where interpretation of common risk stratification parameters, particularly PSA, must be modified to account for the molecular subtype of prostate cancer.

#2260

Exploring drug response metrics for biomarker discovery using the OncoPanel™ cell-based profiling service.

Charles R. Wageman, Jesse J. Parry, Lee R. Cavedine, Alyssa M. Croff, Steven M. Garner, Brogan A. Epkins, Natiya E. Robinson, Kristin C. Dempsey, Vanessa L. Norman, Usha Warrior, Alastair J. King. _Eurofins Panlabs, Inc., St. Charles, MO_.

Using drug response data, generated by the OncoPanelTM cell-based profiling service, we investigated the differences between traditional and normalized growth rate inhibition (GR) metrics in drug sensitivity classification and predictive genomic biomarker discovery. Traditional cellular response parameters can be confounded by numerous growth conditions, especially when the mechanism of action of a drug is coupled to cell cycle processes. Metrics such as IC50 or Emax can be influenced by cellular growth rate, adding bias to the apparent sensitivity to or efficacy of the drug. Use of GR metrics aims to eliminate this bias by normalizing the response data to a per-cell division rate. In this study, we analyzed various parameters for a selection of standard of care compounds when tested against a large panel of cancer cell lines. Compounds were tested against between 213 and 297 different human cancer cell lines. Cells were seeded in 384-well plates and allowed to incubate for 24 hours before acoustic energy-based compound addition was made over 10 half-log concentrations, in triplicate. Initial cell density was captured in a parallel plate to enable cell doubling and end-point GR calculations. After 3 or 10 days of incubation, the cells were fixed, stained with DAPI, and imaged using a high-content imaging platform. Cell proliferation dose response curves were fitted using a 4-parameter log-logistic model within a custom curve-fitting software platform. These same data were then re-analyzed using the GR metrics package developed for the statistical programming language R, to determine GR metrics (GR50 and GRmax). Exploratory analysis was conducted on four parameters: IC50, GR50, GRmax, and number of doublings. IC50 and GR50 were used to classify compounds into drug-resistant or sensitive groups, and GRmax was used to classify each as cytostatic, partially cytostatic, or cytotoxic. Each parameter was used in a predictive genomic biomarker search, using baseline mRNA expression data and mutation data from the COSMIC/Sanger and Broad/CCLE databases. Correlation analysis for doublings and IC50, or GR50, suggests that cell lines with faster doubling rates are associated with drug sensitivity, especially with compounds that target cell cycle processes, such as doxorubicin, carboplatin, cisplatin, and oxaliplatin. Genomic analyses revealed different predictive biomarkers for each choice of parameter. The relationships between significant biomarkers were explored across compounds and cancer cell types. In summary, the methods employed in the OncopanelTM cell-based profiling service enable various dose response parameter estimations, including GR metrics. Through careful use of growth-normalized data, we have expanded our capacity to discover predictive genomic biomarkers that are less influenced by cellular growth conditions.

#2261

Identification of MEIS-associated networks and gene sets involved in prostate cancer progression.

Calvin VanOpstall, Raj Bhanvadia, Hannah Brechka, Marc Gillard, Wen-Ching Chan, Jorge Andrade, Donald J. Vander Griend. _University of Chicago, Chicago, IL_.

Introduction: MEIS1/2 transcription factors, known for their role as HOX cofactors in development, have previously been shown to have prognostic value in patients with intermediate Gleason grade prostate cancer (PrCa). Patients with low expression of MEIS1/2 were shown to have decreased overall survival and time to biochemical recurrence. However, an understanding of the MEIS-associated transcriptional networks driving this tumor suppression has remained unknown.

Methods: Using an RNA-seq dataset composed of 25 primary prostate tumors and 51 annotated metastases obtained from dbGAP, we normalized transcript expression across samples before stratifying by MEIS1/2 expression levels. Primary tumor (PT) samples with MEIS1/2 expression levels in the lowest tertile were classified as the MEISLOW group, while PT samples with MEIS1/2 expression in the highest tertile were classified as the MEISHIGH group. Differentially expressed genes (DEGs) were detected using Cuffquant-Cuffnorm-Cuffdiff suite featureCounts, DESeq2, edgeR, or limma. Transcripts were further filtered by fold change ≥ 1.5, and genes detected by at least more than one method were collected into a list of high-confidence differentially expressed genes (DEGs). Gene set enrichment analysis (GSEA), and Ingenuity Pathways Analysis (IPA) (Ingenuity Systems) were then used to identify functional categories or pathways that were significantly enriched or different.

Results: Pair-wise analyses identified 1804 DEGs between the MEISHIGH and MEISLOW prostate primary tumor groups, which also demonstrated similar and non-significant expression between the MEISLOW and metastatic samples. These genes thus have similar expression between more aggressive MEISLOW tumors and metastasis, yet significantly distinct expression in the more indolent MEISLOW tumors. IPA analysis of this DEG list identified several networks involved in cellular proliferation and survival, as well as inflammatory response and cellular movement. GSEA results also demonstrate a positive enrichment in MEISHIGH tumors for gene sets involved in cell fate commitment, cell-cell adhesion, negative regulation of proliferation, and cell morphogenesis and differentiation, and a negative enrichment in expression of cMYC targets and genes involved in G1-S transition.

Conclusions: Together these data provide a list of key MEIS-associated target genes in PrCa progression, and support a model whereby primary prostate tumors expressing high levels of MEIS1/2 are less proliferative, more differentiated, and have increased cell-cell adhesion, thereby promoting a more indolent tumor phenotype. These data are in direct support of previous studies revealing improved prognosis in tumors with higher expression of MEIS1/2, and decreased expression of MEIS2 in castration-resistant prostate cancer cells.

#2262

Inference of kinase activity for cancer phosphoproteomics using substrate prediction scores.

Peter Liao, Jennifer Yori, Ruth Keri, Mehmet Koyuturk, Jill Barnholtz-Sloan. _Case Western Reserve University, Cleveland, OH_.

Background: Quantification of global protein phosphorylation abundance levels with residue-level resolution is feasible with mass spectrometry and phosphopeptide enrichment methods. Researchers have inferred kinase activity from phosphoproteomics data using a variety of methods to extract interpretable and biologically relevant information, but analysis of phosphoproteomics data is limited by the fact that detected phosphorylation sites often have unknown function and relevance.

Methods: We developed a novel method for aggregating and interpreting phosphoproteomics data that combines kinase prediction scores from NetworKIN with experimental summary statistics to estimate abundance changes in predicted kinase substrates. By weighting student t-statistic scores by kinase prediction scores and summing cumulative scores across all phosphopeptides detected in a given screen, we generated kinase activity scores that reflect the overall direction, magnitude and significance of observed changes in predicted kinase substrates. These kinase scores were assessed using permutation testing to determine their significance versus changes that would be expected from random chance. To evaluate our method, we applied it to a shotgun phosphoproteomic dataset generated to study phosphorylation responses to rapamycin, dasatinib and combination treatment in MDA-MB-231 breast cancer cells.

Results: We were able to confirm phosphoproteomic perturbations in expected "hallmark" kinase activities using prediction-based kinase activity inference scores, including decreased phosphorylation of Src and Abl substrates in response to dasatinib treatment, as well as decreases in p70S6K substrate phosphorylation in response to rapamycin. Compared to KSEA, we identified changes in substrate abundance for a variety of potential downstream kinases including DNAPK, PAK2, CDK3, and multiple members of the MAPK family that were observed to be downregulated in single-agent and in combination treatment.

Conclusions: Our novel method provides a representation of phosphoproteomic changes based on abundance of predicted kinase substrates. Using predicted substrates allows for a global view of phosphoproteomic changes that extends beyond the relatively small number of known kinase-substrate relationships. Using a drug combination with potential application in breast cancer as an example, we demonstrate that this method can be used to identify phosphoproteomic changes and can potentially be used to rationally design and investigate novel drug combinations.

#2263

The dysregulation of circadian genes across cancers.

Zexian Liu,1 Huan Lin,2 Zekun Liu,1 Kai Yu,1 Qi Zhao,1 Xiaolong Zhang,1 Yan Wang,1 Zhixiang Zuo1. 1 _Sun Yat-sen University Cancer Center, Guangzhou, China;_ 2 _Guangdong Provincial Hospital of Chinese Medicine, Guangzhou, China_.

As the daily oscillation in physiological processes, circadian is critical for health while disruption of the circadian rhythm has been associated with cancer in humans. Generally, circadian rhythm was proposed to play important roles in tumor suppression, while there were also evidences that disruption of canonical circadian pathway have anti-cancer effects. In this study, we systematically analyzed the aberrances including mutation, somatic copy number alternation, differential DNA methylation and differential expression of circadian genes across cancers based on TCGA data. It is observed that core circadian network genes (CCNGs) such as PER1/2/3, CLOCK, CRY1/2, ARNTL were highly mutated in a number of cancers such as uterine corpus endometrial carcinoma, skin cutaneous melanoma, stomach adenocarcinoma and colon adenocarcinoma. Generally, the expression for the majority of CCNGs was down regulated in a variety of the cancers such as bladder urothelial carcinoma, head and neck squamous cell carcinoma, breast invasive carcinoma, cervical and endocervical cancers, prostate adenocarcinoma, glioblastoma multiforme and stomach adenocarcinoma, while the expression of a number of CCNGs were related with overall survival in various cancers. The alternations of somatic copy number and DNA methylation were observed to be consistent with the differential expression of these CCNGs. Furthermore, the PCA based circadian gene expression signature was found to be related with the overall survivals and immunophenotypes among cancers, which indicated that circadian rhythm might play critical roles in regulation of immune signaling state in tumor environment. Taken together, our study provided comprehensive analyses of the aberrances and functional roles of circadian genes in cancer, while the results should be helpful for further investigation of the molecular mechanisms and therapy development for cancer.

#2264

RNA sequencing based analysis of transposon-induced tumors reveals novel insights into cancer pathogenesis and progression.

Emily T. Camilleri,1 Pauli J. Beckmann,1 Jon D. Larson,2 Morito Kurata,3 Jingmin Shu,1 Emily Pope,1 Wendy A. Hudson,1 Nuri A. Temiz,1 Susan K. Rathe,1 Rebecca S. LaRue,1 Anne E. Sarver,1 Milcah C. Scott,1 Jyotika Varshney,4 Jaime F. Modiano,1 Branden S. Moriarity,1 Aaron L. Sarver,1 Somasekar Seshagiri,4 David A. Largaespada1. 1 _Univ. of Minnesota, Minneapolis, MN;_ 2 _St Jude Children's Research Hospital, Memphis, TN;_ 3 _Tokyo Medical and Dental University, Tokyo, Japan;_ 4 _Genentech, Inc, San Francisco, CA_.

Current challenges in oncology include the discovery of drivers suitable for targeting using small molecules or antibodies and predictive animal models. We're addressing these challenges in several ways. We have developed mouse models using the Sleeping Beauty (SB) transposon system to perform unbiased, forward genetic screens to define strong candidate cancer genes. Further, our studies and others have utilized transposon mutagenesis to derive cancers from all three germ layers including carcinomas, sarcomas, neuroectodermal tumors, and hematopoietic malignancies. We hypothesize that data from these screens will help to identify genetic drivers of human cancer that are altered at the gene copy number or epigenetic levels. Our T2/Onc SB transposons create fusions with endogenous gene transcripts and RNA sequencing (RNA-seq) reveals the genes targeted in the tumor. From these results, correlations can be drawn between the alteration of specific genes and changes in tumor gene expression patterns. Thus, the goals of our recent research have been to discover novel associations between SB-induced tumor phenotypes and specific driver gene alterations (i.e. the tumor genotype). Moreover, RNA-seq has also revealed tumor molecular subtypes, in many cases with correlating transposon insertion mutations. Several examples will be described: mammary tumors, osteosarcoma (OS), medulloblastoma and central nervous system primitive neuro-ectodermal tumors. In each case, RNA-seq has revealed novel genotype-phenotype correlations including drivers of high cell cycle activity, metastasis, white blood cell exclusion from the tumor, SHH pathway activation, and hormone receptor signaling. These models provide a source of genetically heterogenous tumors with the same initiating mutation useful for identifying cooperating pathways and drivers of specific tumor phenotypes. Secondly, we are using SB transposon mutagenesis to understand the genetic basis of chemotherapy resistance in cancer including OS. In our OS model, SB mutagenesis is an ongoing process due to the constitutive expression of SB transposase, which permits the continued mobilization of transposons. We hypothesize that in vivo treatment with chemotherapy agents will allow tumor cells that contain transposon mutations in chemoresistance genes to persist. Preliminary studies with primary tumor cells derived from SB-enhanced OS tumors demonstrate resistance to combination chemotherapy treatment in vivo. Further analysis to identify candidate chemotherapy resistance genes are currently underway.

#2265

viSNE in Cytobank enables rapid exploratory data analysis for RNA-seq biomarker discovery.

Ashu Sethi, Hannah Polikowsky, Katherine A. Drake. _Cytobank, Inc, Santa Clara, CA_.

Biomarkers provide great potential for understanding and predicting patient outcomes. However, the large numbers of putative biomarkers assayed by many discovery technologies can be difficult to parse quickly to understand their potential as predictors of an outcome of interest. viSNE is a non-linear dimensionality reduction algorithm available on the Cytobank informatics platform that can rapidly assess whether a set of biomarkers can meaningfully group samples. We establish a method for applying viSNE to RNA-seq data. We demonstrate this method using data from The Cancer Genome Atlas for 1,011 samples from 5 different types of kidney cancer and matched normal tissue samples for 139 of the subjects. The tumors ranged from stage I to stage IV, and subjects varied by age, gender, and whether they were living when the sample was taken. Using FPKM data from the 806 most variable transcripts in these samples, viSNE was able to group the samples into six distinct "islands" that separated cancer samples from each of the five disease types and the matched normal tissue. We demonstrate that viSNE is robust to noise and is able to group the samples even when variables that do not contribute to the signal are present in the data, and illustrate how this can be leveraged as part of a larger biomarker discovery workflow with RNA-seq and other data types. viSNE allows rapid exploratory data analysis that helps to build understanding of correlations between biomarkers and patient outcomes and improve overall time-to-results in biomarker discovery pipelines.

#2266

Development and validation of a cancer variant capture panel using the open-sourced CIViC database.

Erica K. Barnell,1 Malachi Griffith,2 Katie M. Campbell,2 Kilannin Krysiak,2 Joshua F. McMichael,2 Adam C. Coffman,2 Susanna Kiwala,2 Obi L. Griffith2. 1 _Washington University School of Medicine, Saint Louis, MO;_ 2 _McDonnell Genome Institute, Saint Louis, MO_.

Background: The Clinical Interpretations of Variants in Cancer database (CIViC) mitigates the variant interpretation bottleneck currently hindering clinical adoption of customized treatment plans for oncology patients. The knowledgebase stores evidence items derived from peer-reviewed publications detailing the therapeutic, prognostic, predisposing, and diagnostic implications of genetic variants in cancer. The knowledgebase is freely accessible without login requirements, fees, or exclusive access, making it uniquely able to attract contributions from volunteer domain experts. Here we present a method to create a dynamic capture panel derived from a community consensus of clinically-relevant variants using the CIViC knowledgebase.

Methods: Using curated Sequence Ontology IDs that specify the variant type (eg., missense mutation, fusion), we elucidated variants that could be evaluated by DNA-sequencing. A CIViC score was developed for each DNA-based variant to identify high-quality variants. This score takes into account the evidence level (eg. case-study, clinical data) and the curated trust rating of variant-associated evidence statements. Variants that attained a CIViC score > 20 were eligible for the CIViC capture panel. CIViC-curated variant coordinates were used to design single molecule molecular inversion probes (smMIPs) for all eligible variants. Sequencing data from 27 samples across five tumor subtypes were compared using both the smMIPs assay and whole exome sequencing to assess panel performance.

Results: Of the 497 DNA-based variants in CIViC, 150 attained a CIViC score > 20 points and were targetable via smMIPs assay. Of these 150 variants, 134 had at least one evidence item that was derived from a clinical study, thereby supporting the CIViC score's ability to identify high quality variants. We designed 2,124 smMIPs to assess the 150 CIViC variants. This required tiling protein-coding exons for 41 genes and developing targeted smMIPs for 95 single nucleotide variants, insertions, and deletions. The CIViC panel design showed extensive overlap with variants identified in the 27 samples using whole exome sequencing. Predicted overlap ranged from 2-12 variants per sample with an average of 4.1 variants per sample.

Discussion: Using expert crowd-sourced curation to drive production of the CIViC capture panel creates an impartial method for panel development. Future validation of the initial capture probe design will allow for automated development of probes for additional variants in the database as curated evidence increases.

#2267

A novel method for classification of microsatellite instability (MSI) using the Oncomine Tumor Mutation Load assay.

Aleksandr Pankov,1 Sameh El-Difrawy,1 Warren Tom,1 Jeffrey Conroy,2 Sean Glenn,2 Sarabjot Pabla,2 Carl Morrison,2 Fiona Hyland,1 Simon Cawley1. 1 _Thermo Fisher Scientific Inc., South San Francisco, CA;_ 2 _OmniSeq Inc., Buffalo, NY_.

Cancer-associated changes in short tandem repeats (STRs) throughout the genome have been shown to be predictive of response to immunotherapy treatment. Traditional assays efficiently rely on a small number of genomic loci to assign an MSI status, but are only effective in a small number of cancer types. Genome-wide profiling of aberrations in STRs has the potential to reveal a comprehensive signature of MSI and could allow for the association of response with samples that are labeled as microsatellite stable (MSS) by traditional assays.To facilitate the discovery of informative repeat sites across the genome, we have extended the Oncomine Tumor Mutation Load assay* to allow for the detection of variants in STRs. Our method addresses challenges of semiconductor sequencing of long STR elements including indel and partial incorporation signal errors. We developed an algorithm to avoid and correct these errors in flowspace, rather than base space, to ensure accurate signal assessment. Our pipeline is able to determine the optimal anchor locations used to identify the exact position of a repeat sequence within a read and then quantify the size, sequence, and number of reads associated with each allele.

An additional complication of accurately quantifying the length of an STR is the increased error rates of the polymerase during DNA amplification. By evaluating the similarity of every loci between tumor and normal pairs of an individual using a metric derived from information theory, we are able to account for both the sequencing and PCR associated noise to robustly identify the genome-wide changes. Furthermore by evaluating a set of 24 MSI-High and 24 MSS samples along with their paired normal samples, we created a novel method to identify a highly informative subset of STR loci. Creating a classifier using only these highly informative loci, we are able to recapitulate the traditional MSI assay with 95% accuracy on these initial samples. This general methodology can be further extended to a model where MSI status is predicted using only the tumor sample, without the need for a control from the same individual. Due to the flexibility of this method, we are able to examine an increased number of potentially informative loci and use the total mutation load along with mutation calls in the MMR genes to provide comprehensive, multi-dimensional genomic perspective of samples.

While this novel method has been developed and tested on the Oncomine Tumor Mutation Load assay, we have created a novel general approach that can be used on the output of any semiconductor sequencing assay. The extended information derived from individual mutations and the total mutation load, along with STR aberration information allows for a comprehensive evaluation of every sample and promotes the discovery of novel genomic components important for understanding the mechanisms of immune-oncology. *For research use only

#2268

Differential splicing analysis of doxorubicin treatment response in cancer cells.

Edward Simpson,1 Yunlong Liu2. 1 _Indiana University-Purdue University Indianapolis, Indianapolis, IN;_ 2 _Indiana University School of Medicine, Indianapolis, IN_.

Background:Splicing of transcripts is an important epigenetic control mechanism that significantly influences expression of protein isoforms in a cell. Alternative splicing is known to contribute to development and progression of cancer, however the impact of cellular splicing profiles on cancer drug response has been largely unexplored. In this study, we integrated two large public data sets and report findings on the basal splicing profiles between doxorubicin treated sensitive and resistant cancer cell lines. We introduced a new method to analyze of splicing data using the quasi-binomial distribution that is incredibly simple, powerful and well suited for comparison of large groups of samples. We also identified exons whose inclusion or exclusion level is likely to be damaging to intended gene products. Finally, we performed RNA-binding protein motif enrichment at these identified exons to characterize proteins that may play a role in their regulation.

Methods:Drug response data from the Cancer Therapeutic Response Portal (CTRP) was integrated with RNAseq data from the Cancer Cell Line Encyclopedia (CCLE). AUC values from drug-response time curves in CTRP were used to classify cells as either doxorubicin sensitive, intermediate or resistant. The upper and lower tertiles provided a total of 253 sensitive and 258 resistant cell lines respectively. Splice junction read counts for skipped-exon events were calculated using the matched cell line data from CCLE. Response groups were then used to train a generalized linear model with quasi-binomial family. Results were filtered by group weight significance at an FDR of <=0.01. The remaining events were again filtered for >=10% difference in mean exon inclusion ratio.

Results:In total we identified 277 differentially spliced events between doxorubicin sensitive and resistant groups. When performing GO term over-representation our results demonstrated significantly enriched biological processes supporting the role of alternative splicing in EMT and cellular differentiation. This further confirms previous studies linking alternative splicing to these cancer hallmarks, suggesting that splicing may facilitate acquired drug resistance through survival mechanisms similar to those seen in cancer stem cells.

#2269

Transcription factor relationships associated with androgen-deprivation therapy response and metastatic progression in prostate cancer.

Nitya V. Sharma,1 Kathryn L. Pellegrini,1 Veronique Ouellet,2 Felipe O. Giuste,1 Selvi Ramalingam,1 Kenneth Watanabe,1 Eloise Adam-Granger,2 Lucresse Fossouo,2 Sungyong You,3 Michael R. Freeman,3 Paula Vertino,1 Karen Conneely,1 Adeboye O. Osunkoya,1 Dominique Trudel,2 Anne-Marie Mes-Masson,2 John A. Petros,1 Fred Saad,2 Carlos S. Moreno1. 1 _Emory University, Atlanta, GA;_ 2 _Université de Montréal, Montréal, Quebec, Canada;_ 3 _Cedars-Sinai Medical Center, CA_.

Patients with recurrent, aggressive prostate cancer typically undergo androgen-deprivation therapy (ADT), but the benefits are often short-lived, and responses are variable. Failure to respond to ADT invariably leads to metastatic disease, and ultimately death. To investigate differential responses to ADT, we performed whole-transcriptome analysis of 20 patient-matched pre-ADT biopsies and post-ADT prostatectomy specimens, and observed that all patients lost transcriptional signatures indicative of the androgen receptor (AR)-dependent subtype, after treatment. We also identified two subgroups of patients with either a strong or weak transcriptional response to ADT. The strong responders maintained the more aggressive subtype signal, while the weak responders lost expression of these genes and more resembled an AR-suppressed, basal subtype. We generated a strong responder transcriptional network using the PANDA program and integrated expression data from our cohort, protein-protein interaction, and DNA binding motif data. We also leveraged the expression data from a large public dataset of over 800 metastatic and primary samples to construct a metastatic lesion transcriptional network. We identified 20 common transcription factor coordinated groups (TFCGs) associated with both the strong responders and metastatic lesions, including GLI3/GLI2, SOX4/FOXA2/GATA4, ERF/ETV5/ETV3/ELF4, and a TFCG containing JUN, JUNB, JUND, FOS, FOSB, and FOSL1. Many TFCGs in the metastatic network were subsets of larger groups in the strong responders network, implicating these transcription factor associations as potentially critical for both the differential ADT response and metastatic disease progression.

#2270

Integrating 5hmC and gene expression data infers regulatory mechanisms linked to alternative mRNA splicing in breast cancer.

Cristina Mitrea, Priyanga Wijesinghe, Greg Dyson, Emily Girsch, Bin Bao, Adele Kruger, Aliccia Bollig-Fischer. _Wayne State University, Detroit, MI_.

Epigenetic mechanisms, such as 5-methylcytosine (5mC), are known to play a major role in breast cancer. However, the role of 5-hydroxymethylcytosine (5hmC) DNA modification remains understudied. We hypothesize that 5hmC mediates redox regulation of gene expression in the triple negative breast cancer (TNBC) subtype. To address this, our objective was to highlight genes that may be the target of this process by identifying redox-regulated, antioxidant-sensitive, gene-localized 5hmC changes associated with mRNA changes in TNBC cells. We proceeded to develop an approach to integrate data sets generated by a methodology called Pvu-sequencing, which measures 5hmC in the genome, and RNA-sequencing. The result of our approach to merge genome-wide, high-throughput TNBC cell line data to identify significant, concordant 5hmC and mRNA changes in response to antioxidant treatment, that perturbs redox signaling, produced a gene set with relevance to alternative mRNA splicing and cancer stem cell function. Key genes identified included TAR DNA binding protein (TARDBP), vimentin (VIM), and splicing factor proline and glutamine rich (SFPQ). Moreover, we have established a method that will be useful for continued research of 5hmC in TNBC cells and tissue samples.

#2271

Pathway analysis suggests biological processes driven by germline genetic associations with breast cancer prognosis.

Maria Escala Garcia,1 Qi Guo,2 Lodewyk Wessels,1 Gary Bader,3 Paul Pharoah,2 Georgia Chenevix-Trench,4 Douglas Easton,2 Sander Canisius,1 Marjanka Schmidt1. 1 _Netherlands Cancer Institute, Amsterdam, Netherlands;_ 2 _Centre for Cancer Genetic Epidemiology, University of Cambridge, Cambridge, United Kingdom;_ 3 _The Donnelly Centre, University of Toronto, Toronto, Ontario, Canada;_ 4 _QIMR Berghofer Medical Research Institute, Brisbane, Australia_.

Finding the impact of germline variants on breast cancer survival could provide novel insights in its etiology and help identify new therapeutic targets. While genome-wide association studies (GWAS) have made considerable progress in identifying germline variants associated with diverse risk phenotypes, survival analysis has been hampered by a lack of power. To overcome this limitation, we aimed to assess whether biological networks operating in breast cancer prognosis can be inferred by integrating patient genotypes and the associated survival data in a pathway analysis.

We analyzed ~7.3 million genotyped and imputed variants from 84,457 breast cancer patients in all, estrogen receptor (ER) positive and negative breast cancers and validated the results in 12,381 independent samples. First, a Cox model was fitted to obtain summary statistics for each variant. We then integrated the summary statistics into gene scores using the Pascal algorithm. Finally, we constructed pathways based on the gene scores and a protein-protein interaction network with the HotNet2 tool. To assess the validity of the significant networks (P<0.01) found by Hotnet2, we tested the association of the germline variants forming the subnetworks with prognosis. We selected genetic variants that best represented the genetic association of each subnetwork with survival by using a Lasso-penalized Cox model. Then we computed a polygenic hazard score (PHS) on the independent validation set and used it to run a univariate Cox model that allowed selecting the networks significantly associated with prognosis (P<0.05).

We found significant subnetworks in the ER-specific analyses, but not in those including all breast cancers. We then clustered the networks into functional pathways to identify significant (Padj<0.05) functional groups. The enriched processes included growth factor signalling, DNA repair and cell cycle functions. These pathways overlapped with similar biological processes obtained in a downstream characterization analysis based on the genotypes and mRNA expression data of the TCGA breast cancer study and were complemented by key enriched transcription factors (Padj<0.01).

The approach developed is novel in studying germline variants and breast cancer-specific mortality and shows an alternative method to handle underpowered datasets. The networks and the posterior functional characterization suggest that there is some genetic regulation of biological processes associated with breast cancer prognosis specifically for each ER-status subgroup.

#2272

Identification of predictive biomarkers for immunotherapy of brain and solid tumors by studying inter-cellular immune networks.

Ilan Volovitz, Gil Diamant, Marina Roitman, Nati Shapira, Roni Hagai, Barak Bensimhon, Merav Lustgarten, Rachel Grossman, Zvi Ram. _Tel-Aviv Medical Center, Israel_.

Immune checkpoint molecules (ICMs) make up the immune suppressive network (ISN) that blocks spontaneous or therapy-induced anti-tumoral immune responses. A decade of research into checkpoint inhibitors (CPIs) has identified only few predictive biomarkers for single-agent CPI therapy. Those discovered show mediocre predictive capability. A considerable fraction of predicted non-responders demonstrate response and vice versa. It is unlikely that current biomarker discovery tools based on single antigen immunohistochemistry or whole-tumor genomics/transcriptomics will uncover predictive biomarkers for therapy with CPI combinations. Brain tumors, specifically glioblastoma, are almost uniformly lethal. Preclinical studies have shown that more than one agent is needed to relieve the strong immunosuppression in these tumors. No rational approach to select the right combination of agents is currently available.

We have developed several unique methodologies that delineate the ISN formed between all immune and tumor cell subsets within individual human brain tumors. Our current ISN models incorporate cell-level data, frequency of all intratumoral immune subsets (up to 13 immune subsets), and gene-expression data with each subset's deep-sequenced transcriptome. We produce transcriptomes from all intratumoral immune subsets (as few as 100 cells) and tumor cells that are sorted using elaborate multicolor flow cytometric panels. These transcriptomes reveal each subset's full immune state: expression of all ICMs, all inter-cellular functional and communication molecules (e.g. cytokines, chemokines, receptors, ligands), and its active pathways.

The collected data is integrated using systems-biology tools to generate models of the ISN which delineate the interactions that each cell subset has with all other immune or tumor cell subsets within a tumor. Individual ISN data can be evaluated in respect to each patient's response to CPI treatment to determine which ISN patterns correlate with response, or lack of response, to CPI treatment. Emerging results may retrospectively explain the failure of some CPI clinical trials conducted on glioblastoma, and reveal new targets.

Studying the relationship between ISN patterns and clinical responses to checkpoint inhibition may enable identification of much needed predictive biomarkers. A systematic evaluation of the suppressive components found to be frequently active in many patients' ISNs may guide rational decisions in planning of clinical trials with improved chances to succeed. This methodology may, in the future, guide physicians in selecting reagents to treat an individual patient's tumor, bringing about the age of personalized medicine to the field of immuno-oncology.

#2273

Meta-analysis of cancer cell lines based on responses to Tumor Treating Fields (TTFields).

Gitit Lavy Shahaf,1 Moshe Giladi,1 Rosa S. Schneiderman,1 Noa Urman,1 Karnit Gotlieb,1 Einav Zeevi,1 Yaara Porat,1 Mijal Munster,1 Adrian Kinzel,2 Uri Weinberg,3 Eilon D. Kirson,1 Yoram Palti1. 1 _Novocure, Israel;_ 2 _Novocure, Germany;_ 3 _Novocure, Switzerland_.

Objective: Tumor Treating Fields (TTFields) therapy is an approved modality for the treatment of glioblastoma. These alternating electric fields were shown to exert an inhibitory effect in numerous cancer cell lines with some variability in the responses of different cell lines. The goal of the present study is to compare characteristics of cell lines based on their response pattern to TTFields.

Methods: Forty different human cancerous cell lines were treated for 72 hours with TTFields at respective cell-specific optimal frequency with the same nominal intensity (1.7 V/cm). Cell survival and clonogenicity were determined. Functional analysis of differentially expressed genes and mutations associated with response to TTFields was performed based on the Cancer Cell Line Encyclopedia (CCLE) database. Sensitivity to TTFields was compared with pharmacologic profiling (CCLE).

Results: TTFields application demonstrated varying degrees of cytotoxic effects in all cell lines tested. The inhibitory response to TTFields was found to be distributed around an average of 50% with cytotoxic effects ranging between 14% and 86% reductions in cell counts, and a clonogenic effect ranging between no effect and 88% reduction in the number of colonies. In line with TTFields' anti-mitotic properties, a correlation between treatment efficacy and cell doubling time was demonstrated. However, few cell lines demonstrated enhanced treatment efficacy despite long doubling time, suggesting other factors may also be involved in the response to treatment. Pharmacologic profiling based on IC50 values revealed increased sensitivity to lapatinib, PHA-665752 and PLX-4720 within the group of cell lines that were less sensitive to TTFields. Functional analyses of cell line gene expression and mutation data revealed enriched pathways related to DNA damage repair response such as the BRCA1 repair pathway, which validate previous data obtained using different methodologies. Other pathways that were found to be associated with sensitivity to TTFields include cell migration, hypoxia signaling, and oxidative stress.

Conclusion: This multiparameter, large-scale comparison of cancer cell line responses demonstrates the broad effectiveness of TTFields in various cell lines and defines the optimal frequency to be applied for each cell line. The data presented in this work suggest that beside anti-mitotic properties, TTFields may have effects on other cellular pathways. The pharmacologic profiling may offer a rational for combining specific agents with TTFields in cells that are less sensitive to the electric fields.

### New Software for Data Analysis

#2274

Cancer genomics visualization and interpretation using UCSC Xena.

Mary Goldman, Brian Craft, Jingchun Zhu, David Haussler. _UC Santa Cruz, Santa Cruz, CA_.

The UCSC Xena platform (http://xena.ucsc.edu/) allows biologists and bioinformaticians to securely analyze and visualize functional genomics data. Our unique Visual Spreadsheet shows multiple data types side by side enabling discovery of correlations across and within genes and genomic regions. Dynamic Kaplan-Meier survival analysis assesses survival stratification in addition to scatter plots, bar graphs, and boxplots all shown with statistical tests.

In addition to the commonly available SNPs, INDELs, CNV, and gene expression datasets, we support DNA methylation, exon-, transcript-, miRNA-, lncRNA-expression and structural variants. We also support clinical data such as phenotypes, subtype classifications and biomarkers. Our new whole genome views allow users to easily visualize non-coding regions for both copy number variation and somatic mutations. All of our data is available for download via our python API or through AWS S3 buckets.

Our expanding public Xena Data Hubs currently host 1500+ datasets from more than 35 cancer types, as well as Pan-Cancer datasets. In addition to serving seminal cancer genomic datasets to the scientific community, including the latest from the GDC, TCGA, TARGET, and ICGC, we also host 'normal tissue' datasets from GTEx. A recompute of TCGA, TARGET and GTEx datasets through the same bioinformatics pipeline allows users to compare expression between tumor and normal tissues.

In addition to exploring these public datasets, the UCSC Xena Browser can easily display an investigator's genomic and clinical data on their own Xena Hub. By empowering users to install and load data into their own hub, our architecture ensures that the investigator's data remains private. The lightweight Xena Data Hubs are straightforward to install on Windows, Mac and Linux operating systems and loading data is easy using either our application or command line interface.

#2275

Individualized drug combination based on single-cell drug perturbations.

Benedict Anchang, Kara Davis, Harris Fienberg, Sean Bendall, Loukia Karacosta, Garry Nolan, Sylvia K. Plevritis. _Stanford Univ., Santa Clara, CA_.

Currently, cancer drug combinations primarily focus on mutational heterogeneity of the primary tumor and do not account for single-cell variations that can give rise to drug resistance. Moreover, even with the increasing number of potential FDA-approved targeted drugs including immunotherapies, methods are needed to identify better combination therapy that leverages intratumor heterogeneity, thereby potentially mitigating the need for trials with large numbers of patients. Despite advances in single-cell technologies that capture intratumor heterogeneity, there are no drug combination strategies that utilize single-cell platforms. One idea that has been proposed is to use Mass Cytometry Time-of-Flight (CyTOF) for drug screening by producing drug perturbation effects at the level of the single cell, but the required analytics for the resulting complex data were not addressed. To address this unmet need, we have developed a novel algorithm to optimize combination therapy for an individual patient by analyzing distinct single-cell drug perturbation responses on a tumor sample. This model framework, called "DRUGNEM," can be applied to CyTOF data, single-cell RNA-seq, or any single-cell imaging data currently available. DRUGNEM is composed of three steps: (1) identify the subpopulations that make up the tumor and may respond differently to treatment; (2) reconstruct a drug-nested-effects model that integrates the drug effects across all subpopulations to capture sub-setting relationships among individual drug effects; and (3) systematically score potential drug combinations to identify or prioritize strategies that will be clinically (and economically) sustainable. Currently, DRUGNEM is optimized to select the minimum number of drugs that produces the maximal desired intracellular effect, predicated on the premise that fewer drugs lower treatment-related toxicities and costs, but the final selection criterion can be easily modified. As proof of concept, we applied the DRUGNEM framework to individualize drug combinations based on CyTOF data generated on de-identified malignant research samples from 30 ALL pediatric patients before and after exposure to 3 targeted FDA -pproved single drugs (dasatinib, tofacitinib and BEZ235). We found that the most common combination treatment strategy (dasatinib and BEZ235) might not be optimal for all 30 ALL patients, with 2 of the 30 likely responding best to tofacitinib alone. Using in vitro survival assays, we validated the DRUGNEM prediction of BEZ235 and dasatinib as a potential synergistic combination on ALL cell lines. In summary, DRUGNEM is a novel framework using single-cell technologies to guide drug-combination strategies and can be adapted to incorporate complementary molecular data and computational methods to ultimately achieve more effective therapy for the individual cancer patient.

#2276

Efficient curation and ontology mapping of clinical and phenotypic data.

Henrik Edgren, Beatriz Mano, Maria Laaksonen. _MediSapiens Ltd, Helsinki, Finland_.

Heterogeneity within and between data sets is one of the primary impediments to sound and efficient data analysis. This heterogeneity can arise from many sources: data collection practices can change over time, even within the same study; rigorous standards for data encoding may be missing, leading to inter-individual heterogeneity in data encoding; or the data may originally have been collected for a different purpose (e.g. EHR data). Additionally, narrative text, such as medical statements, are inherently unstructured and require curation before analysis.

No matter which source(s) heterogeneity derives from, curation of the data is necessary for efficient, accurate and reproducible data analysis. This not only includes correcting errors and standardizing encoding, but also extends to enriching the data through mapping it to relevant medical ontologies, such as SNOMED CT, or other standardized terminologies, such as Research Resource Identifiers.

To solve the most common issues in clinical and phenotypic data curation, we have developed the AccurateTM data curation and ontology mapping solution. It combines an intuitive web-based user interface for data cleaning with efficient solutions for semi-automated ontology mapping of both structured data and narrative text. For structured data, tokenized and stemmed data items are mapped against ontologies indexed in Elasticsearch. Term names, their synonyms and the local ontology structure are then used to query the target ontology, with a list of best matches returned along with a quality score for the mapping. Ontology tagging of narrative text is based on a sentence-based deep learning approach, analyzing sentences to classify and ontology map identified text units. In practice, combining a bidirectional long short term memory network with a conditional random field model into a named entity recognition system (bio-NER). Preliminary benchmarking of the bio-NER system on the MIMIC III data set suggests good specificity and sensitivity for identification of biomedically relevant concepts.

In summary, we here present an intuitive and highly efficient solution for curating clinical and phenotypic data, as well as enriching it using ontology mapping of both structured and narrative data.

#2277

Machine learning to identify prostate cancer mutations for screening cell-free DNA (cfDNA).

Clinton L. Cario, Lancelote Leong, Man-Tzu Wang, John Witte. _University of California, San Francisco, San Francisco, CA_.

Cell free DNA (cfDNA), a form of 'liquid biopsy', has recently emerged as a promising technology to screen, diagnose, and monitor many types of disease. However, in the context of cancer, one unfortunate complication of cfDNA's use as a biomarker is the weak signal-to-noise ratio that arises primarily due to DNA contamination from healthy tissue DNA. To circumvent this, many researchers have focused on a limited set of frequently observed coding variants within a single gene or a small panel of hand selected genes, trading breadth for depth to improve signal. This approach, while sensible, can lead to detection issues in highly heterogeneous diseases— such as prostate cancer— in which variants may not be universally present at high proportion across all patients. Furthermore, many of these panels ignore regions that are non-coding but functional, and possibly even crucial in tumorigenesis.

This project proposes a different, agnostic, and machine-learning based approach for panel generation. Using public whole genome sequence (WGS) datasets containing single nucleotide polymorphisms (SNPs) and copy variant information from the International Cancer Genome Consortium (ICGC) as training data and a large number of annotation features collected from a dozen biological databases, we've developed an open source push-button tool called orchid to build cancer variant prediction models. After training a Support Vector Machine (SVM) on known prostate cancer mutations in patients with few mutations (putatively driver enriched) and those in patients with many mutations (putatively passenger enriched), the model was used to suggest a set of mutations most "prostate cancer like". Mutations furthest from the classifying hyperplane are then selected to form the SVM panel.

Compared to manually curated panels, the SVM panel demonstrates superior in silico patient detection in both untrained ICGC data and in tumor/normal sequence data from a pilot study of 13 prostate cancer patients with multiple heterogeneous tumor foci. Moving forward, the SVM generated panel will be used to screen cfDNA from patients in the pilot study and 100 additional UCSF prostate cancer patients.

This research has clinical implications in creating diagnostic, prognostic, and predictive tools for prostate cancer. In particular, the SVM panel may be used to screen cfDNA for cancer mutations and to assess tumor heterogeneity and residual disease. Additionally, the published software tool orchid can be used for other tumor mutation classification tasks, such as determining tissue-of-origin from cfDNA fragments using only the locations of mutations in the fragments.

#2278

Scalability and reliability improvements to the Iso-Seq analysis pipeline enables higher throughput sequencing of full-length cancer transcripts.

Elizabeth Tseng, Tyson Clark, Meredith H. Ashby. _Pacific Biosciences, Menlo Park, CA_.

The characterization of gene expression profiles via transcriptome sequencing has proven to be an important tool for characterizing how genomic rearrangements in cancer affect the biological pathways involved in cancer progression and treatment response. More recently, better resolution of transcript isoforms has shown that this additional level of information can be useful in stratifying patients into cancer subtypes with different outcomes and responses to treatment. The Iso-Seq protocol developed at PacBio is uniquely able to deliver full-length, single molecule cDNA sequences, allowing for the unambiguous, direct determination of splice variants. This has led to the identification of novel cancer biomarker candidates and has yielded new insights into gene fusion events that contribute to cancer progression.1,2,3

Recent improvements to the Iso-Seq bioinformatics pipeline increases the speed and scalability of data analysis while boosting the reliability of isoform detection and cross-platform usability. Here we report evaluation of Iso-Seq runs of human UHRR samples with spiked-in synthetic RNA controls on the Sequel System and show that the new pipeline recovers both more human and synthetic isoforms while reducing the number of false positives. We also share the results of sequencing the well-characterized HCC-1954 breast cancer and matched normal cell lines, which will be made publicly available. Combined with the recent simplification of the Iso-Seq sample preparation protocol, the new analysis pipeline completes a streamlined workflow for revealing the most comprehensive picture of transcriptomes at the throughput needed to characterize cancer samples.

1. Kohli M, et. al. (2017) Androgen Receptor Variant AR-V9 Is Coexpressed with AR-V7 in Prostate Cancer Metastases and Predicts Abiraterone Resistance. Clin Cancer Res. ePub ahead of print. doi: 10.1158/1078-0432.CCR-17-0017

2. Komor MA, et. al. (2017) Identification of differentially expressed splice variants by the proteogenomic pipeline Splicify. Mol Cell Prot. ePub ahead of print. doi: 10.1074/mcp.TIR117.000056

3. Weirather JL, et. al. (2015) Characterization of fusion genes and the significantly expressed fusion isoforms in breast cancer by hybrid sequencing. Nucleic Acids Res, 43 (18), e116.

#2279

The GenePattern Notebook environment for reproducible cancer research.

Michael M. Reich,1 Thorin T. Tabor,1 Ted Liefeld,1 Barbara Hill,2 Helga Thorvaldsdottir,2 Jill P. Mesirov1. 1 _UCSD, La Jolla, CA;_ 2 _Broad Institute of MIT and Harvard, Cambridge, MA_.

As the availability of genetic and genomic data and analysis tools from large-scale cancer initiatives continues to increase, the need has become more urgent for a software environment that supports the entire "idea to dissemination" cycle of an integrative cancer genomics analysis. Such a system would need to provide access to a large number of analysis tools without the need for programming, be sufficiently flexible to accommodate the practices of non-programming biologists as well as experienced bioinformaticians, and would provide a way for researchers to encapsulate their work into a single "executable document" including not only the analytical workflow but also the associated descriptive text, graphics, and supporting research. To address these needs, we have developed GenePattern Notebook, based on the GenePattern environment for integrative genomics and the Jupyter Notebook system. GenePattern Notebook unites the phases of in silico research - experiment design, analysis, and publication - into a single interface.

GenePattern Notebook presents a familiar lab notebook format that allows researchers to build a record of their work by creating "cells" containing text, graphics, and executable analyses. Researchers add, delete, and modify cells as the research evolves, supporting the initial research phases of prototyping and collaborative analysis. When an analysis is ready for publication, the same document that was used in the design and analysis phases becomes a research narrative that interleaves text, graphics, data, and executable analyses. The online notebook format allows researchers to explain the analytical and scientific considerations of each step in any level of detail, promoting reproducibility and adoption. Notebooks can also be shared between researchers for collaborative development.

GenePattern Notebook features are designed to help nonprogramming users create and adapt notebooks. We have developed additional cell types allowing users to choose analyses, specify inputs, navigate results, send result files to new analyses, and create richly formatted text, all without the need for programming.

A free online GenePattern Notebook workspace is available at http://www.genepattern-notebook.org, where researchers can develop, share, and publish notebook documents. We have provided a collection of template notebooks that walk users through various genomic and machine learning analyses, and are collaborating with cancer research laboratories to create integrative cancer genomics notebooks.

#2280

Systematic analysis of causal noncoding mutations in pediatric B-cell acute lymphoblastic leukemia.

Bing He, Peng Gao, Yang Ding, Chia-Hui Chen, Hannah Kim, Stephen P. Hunger, Sarah K. Tasian, Kai Tan. _Children's Hospital of Philadelphia, Philadelphia, PA_.

B cell acute lymphoblastic leukemia (B-ALL) is the most prevalent childhood hematological malignancy and the leading cause of childhood cancer-related mortality. To date, the molecular pathogenesis of B-ALL has not been completely defined, limiting our ability to develop targeted therapies. Despite great advances in identifying mutations in cancer patients, the functional consequences of noncoding mutations are still difficult to interpret. Transcriptional enhancers are key determinants of tissue-specific gene expression. Several recent studies have reported novel mutations in cancer genome that disrupt enhance function and thus expression of their target genes. However, systematic detection of noncoding mutations that perturb either enhancer sequence or enhancer-promoter (EP) interaction is still challenging. Such analysis can help to identify novel driver mutations and improve our understanding of the disease.

Here we introduce a novel algorithm for identifying causal mutations that disrupt enhancer function and target gene expression in B-ALL. The algorithm takes as input

whole genome sequencing (WGS) and RNA-Seq data to predict causal noncoding mutations. We apply the algorithm to analyze data from 163 B-ALL patients and identify 332 noncoding mutations that disrupt EP interactions and gene expression. Our analysis recapitulate previously known translocations such as t(14;X) that leads to CRLF2 overexpression, as well as novel causal translocations that leads to deregulation of TMEM106A and MIR663B. Systematic analysis of the identified mutations indicated that SNVs in enhancer region have a higher variant allele frequency (VAF), suggesting enhancer mutations likely occur earlier than coding mutations during oncogenesis. In addition, pathway analysis show that the majority (57%) of mutations perturbing metabolic pathways are noncoding mutations. Further analysis and experimental follow-up of the predicted noncoding mutations may reveal novel insights into the pathogenesis of B-cell ALL.

#2281

Flexible discovery of recurrent coding and non-coding mutations in tumor whole genomes.

Anthony R. Soltis,1 Coralie Viollet,1 Harvey B. Pollard,1 Christopher A. Moskaluk,2 Robert F. Browning,3 Clifton L. Dalgard,4 Craig D. Shriver,5 Matthew D. Wilkerson4. 1 _The American Genome Center at the Uniformed Services University, Bethesda, MD;_ 2 _University of Virginia, Charlottesville, VA;_ 3 _Walter Reed National Military Medical Center, Bethesda, MD;_ 4 _The American Genome Center at the Uniformed Services University and John P. Murtha Cancer Center, Bethesda, MD;_ 5 _John P. Murtha Cancer Center, Walter Reed National Military Medical Center, Bethesda, MD_.

Analysis of somatic protein-coding mutations, particularly via tumor exome sequencing, has driven discovery of novel cancer genes in recent years. However, ~97% of the human genome is non-coding and not addressable by exome-targeting methods. Tumor whole genome sequencing, by contrast, allows for genome-wide discovery of somatic events and may identify novel non-coding driver mutations. In conjunction, flexible tools employing principled statistical methods are needed for discovery of recurrent somatic driver mutations from tumor whole genomes.

Here, we developed a computational toolset that addresses these needs and describe its application to a novel cohort of lung adenocarcinoma tumors generated by the APOLLO (Applied Proteogenomics OrganizationaL Learning and Outcomes) consortium. Our toolset flexibly computes feature-wise and hotspot somatic mutation enrichment statistics in both protein-coding and non-coding (e.g. promoters, enhancers) genomic regions. Additionally, we incorporated an affinity propagation-based clustering procedure that groups test regions by user-specified genomic covariates that can influence local mutation properties.

We applied our toolset to several TCGA datasets of tumor exome sequencing. We found strong agreement between our results and those of other tools: among the top 15 genes called at FDR < 0.01 by MutSigCV, we observed a median overlap of 75.7% with genes ranked in the top 15 by our methods. We next applied our toolset to tumor whole genome sequencing data from our APOLLO lung adenocarcinoma cohort. During analysis, we simultaneously controlled for several genomic covariates, including GC/CpG content, replication timing, and proximal mRNA expression. Gene-centric analysis identified several significantly mutated genes, including KRAS, STK11, and TP53 (FDR < 0.005). We examined matched mRNA-seq data from this cohort and found significant over-expression of KRAS mRNA in samples possessing a non-silent coding mutation in this gene (p < 0.02). We additionally assessed somatic mutation enrichment in promoters, 3' UTRs, non-coding genes, and lung-specific enhancers. From these results, we found significant enrichment of somatic mutations in the body of the long non-coding RNA NEAT1. Among the tumors with somatic NEAT1 mutations, a single sample was hypermutated at this locus and corresponded with the lowest observed RNA expression for this lncRNA in our cohort (TPM of 12.2 vs. overall mean TPM of 84.2).

In conclusion, we developed a flexible toolset for interrogating both coding and non-coding landscapes from tumor whole genomes. We applied our methods to a novel cohort of lung adenocarcinoma tumors and identified recurrently mutated genes and non-coding regions, including the NEAT1 lncRNA.

#2282

VIP: A system biology platform for cell line centric integrative analysis of molecular and functional genomics data.

Julio Fernandez, Ying Ding, Zhengyan Kan. _Pfizer, Inc., San Diego, CA_.

Cancer is a heterogeneous disease that is classified by its tissue of origin or molecular characteristics such as genetic alterations that drive tumorigenesis. Large-scale efforts using loss-of-function genetic screens to systematically identify genes essential for the proliferation and survival of cancer cells provide a powerful approach for studying cancer biology and discovering drug targets. However, cancer cell lines from different lineages or molecular background vary in their dependencies on specific genes or pathways. Functional ablation of a gene may be only lethal in one segment of cancer cell lines harboring certain molecular deficiencies or activation of a specific oncogenic signaling pathway, a phenomenon called synthetic lethality. A key challenge here would be to identify the true synthetic lethal interactions from a combinatorially large number of possible interactions between molecular aberrations and gene-level sensitivity to functional perturbation. With recent advances in large-scale studies such as CCLE and Achilles, genome-wide functional genomics screens using CRISPR-Cas9 or shRNA technologies as well as comprehensive molecular profiles have become available for a large number of cancer cell lines. Here we present the Variant Interpretation Portal (VIP), a system biology tool designed to enable integrative analysis of molecular and functional genomics data in cell lines for identifying synthetic lethal interactions. VIP consists of a command-line pipeline and a web-based user interface. The VIP pipeline rigorously filters out germline events and classifies genomic variants into multiple functional tiers. The VIP web portal provides users with a graphic interface to perform exploratory visual analyses of associations between two variables or multiple variables, where a variable could be a genetic alteration or a gene-level sensitivity profile. VIP also enables systematic search for significant association between one variable, such as the sensitivity profile, vs. a database of variables such as histology or molecular attributes. VIP is optimized to perform real-time analytics and enables the incorporation and analysis of user-provided data. The code can be accessed from https://github.com/juferban/vip.

#2283

PCR-based validation of novel cancer-predisposing mutations identified upon exome sequencing: An emphasis on primer design.

Ekaterina Sh Kuligina,1 Anna P. Sokolenko,1 Ilya V. Bizin2. 1 _N.N. Petrov Research Inst. of Oncology, St. Petersburg, Russian Federation;_ 2 _Peter the Great St. Petersburg Polytechnic University, St. Petersburg, Russian Federation_.

Whole exome sequencing (WES) is a robust instrument for the identification of novel genes for hereditary diseases. WES experiments usually produce hundreds or thousands of candidates, which require validation in primary DNA samples and subsequent case-control studies. The analysis of each candidate includes individual primer design for allele-specific polymerase chain reaction (PCR) assay or high-resolution melting (HRM) coupled with Sanger sequencing. For the time being, the selection of primers for PCR remains a time-consuming procedure. None of the available software instruments allows to generate a pool of primers for the entire coding region of the gene, therefore each coding fragment has to be selected and analyzed manually. Furthermore, the existing tools have limited capability to ensure that primers will not amplify a non-target fragment of the genome. The consideration of single nucleotide polymorphisms located within primer sequence is usually done without adjustment for their frequency. We developed a pipeline, which facilitates the process of large-scale primer design in an automatic mode. We named the tool PrimerWay and made it available at http://github.com/zoldrax/primer-way under the GNU General Public License. The developed pipeline is provided as a standalone python-based script which requires installed Primer3, tntBLAST and HTSlib. We created automatic upload of the sequence of interest with flanking regions using protein_id or genome coordinates from the reference genome. We utilized the ThermonucleotideBLAST (tntBLAST) instrument in order to ensure PCR specificity. While other computer tools focus mainly on the heuristic definition of sequence similarity, this software runs in-silico PCR based on the thermodynamic similarity and minimizes the risk of the amplification form a non-target template. Our pipeline detects polymorphic sites within the potential primer (5'-end or 3'-end) using a common subset of the dbSNP public database. If the SNP is rare and located at the 5'-end of the primer, it is highly unlikely to compromise further experiments. PrimerWay is capable to suggest the design for the analysis of long DNA sequences. It breaks the sequence for short consecutive fragments and generates a pool of candidate primers for each fragment. It suggests, which of the chosen fragments and primer pairs are most reliable for analysis, by considering the risks of forming dimers, recognizing the non-target sequences, or being compromised by SNPs; these risks are measured by the comprehensive penalty scores. Subsequently, it considers several variants of breaking the entire sequence for overlapping DNA fragments. The utility of PrimerWay tool was validated in a WES study for hereditary breast cancer, which utilized, inter alia, 23 candidate mutations and entire coding regions of two candidate genes. The research is supported by RSF grant 16-45-02011.

#2284

Semiautomated MRI segmentation workflow for glioblastoma treated by Tumor Treating Fields.

Joshua J. Timmons, Ed Lok, Pyay San, Kevin Bui, Eric T. Wong. _Beth Israel Deaconess Medical Center, Boston, MA_.

Background: Tumor Treating Fields (TTFields) therapy is an approved modality of treatment for glioblastoma. Patient anatomy-based finite element analysis (FEA) has the potential to reveal not only how these fields affect tumor control but also how they can be optimized to improve efficacy. Historically, this has been achieved with manual segmentation by an expert familiar with intracranial neuroanatomic structures, but this method is time-consuming, financially costly, and highly variable between individuals. While the availability of tools for segmenting MRIs obviates some of these obstacles, creating FEA models requires manual adjustments, including removal of disconnected voxels, incorporation of unsegmented structures that may alter the electric field, and addition of 36 electrodes matching those of the transducers on the scalp. Existing approaches are also not scalable to a large number of patient MRI scans.

Methods: Patient anatomy-based FEA models were created by segmenting MRI images—using the NewSegment algorithm from Statistical Paramedic Mapping 8 (SPM8)—into tissue "masks" using ScanIP from Simpleware (Exeter, UK). The electric properties of each tissue were applied to these masks in silico using COMSOL Multiphysics (Burlington, MA). The fidelity of the segmentations was compared using the Dice coefficient, and COMSOL generated electric field maps were compared for models with and without post-processing in ScanIP.

Results: A semiautomated workflow was developed to prepare FEA models for TTFields with fully automated electrode placement. The workflow encompassed image preprocessing, co-registration, segmentation, post-processing and mesh generation in ScanIP. Modified extended tissue probability maps were used to segment cerebellum and orbits in addition to white matter, gray matter, cerebrospinal fluid, skull, and scalp. Further automated post-processing in ScanIP cleaned up disconnected voxels, smoothed surfaces, and layered the masks to prevent erroneous boundary conditions. The Dice coefficient was greater for the model with ScanIP post-processing, as compared to the segmentations resulting from SPM8 NewSegment alone. COMSOL-generated electric field maps also displayed differences when models were generated with post-processing in ScanIP compared to those without.

Conclusions: These software and workflow may be used to accelerate the investigation of TTFields in patients by facilitating the creation of FEA models, like electric field maps, derived from patient MRI.

#2285

Regtools: Integrated analysis of genomic and transcriptomic data for discovery of mutations associated with aberrant splicing in cancer.

Yang-Yang Feng, Avinash Ramu, Zachary L. Skidmore, Jason Kunisaki, Kelsy C. Cotto, Obi L. Griffith, Malachi Griffith. _Washington University in St. Louis School of Medicine, St. Louis, MO_.

The interpretation of variants in cancer is frequently focused on direct protein coding alterations. However, most somatic mutations are in noncoding regions of the genome, and even exonic mutations may have unidentified noncoding consequences. Here we present Regtools, a software package designed to efficiently identify variants that may cause aberrant splicing in tumors. Our tool integrates variant calls from genomic data with junctions extracted from transcriptomic data in order to examine potential cis alterations to splicing near a somatic variant. Based on user-defined parameters and position relative to known exons, variants are first annotated as splicing relevant or not. Splicing junctions are inferred from transcriptomic sequencing data, and comparison of junctions to a reference transcriptome allows for identification and annotation of novel junctions and nearby regulatory or splicing motifs. From there, mutations are associated with junctions that overlap with a flanking region. In order to evaluate Regtools, we used it to analyze the transcriptional output of tumor-sequencing data from two cohorts of cancer patients, one of hepatocellular carcinoma and one of small cell lung cancer with 28 and 21 samples, respectively. We performed whole-exome and RNA sequencing on each sample. Somatic variants were called on whole-exome alignment data. For each cohort, we compared the junctional profiles between tumors and identified numerous examples of variants for which there are elevated levels of proximal novel or known junctions. Moreover, out of 754 (153 in HCC; 601 in SCLC) variants identified as splicing relevant by our approach, only 165 (20 in HCC; 145 in SCLC) were annotated as splicing relevant by Ensembl's Variant Effect Predictor, using the recommended "per_gene" option. This preliminary analysis illustrates the importance of an efficient, user-friendly computational tool for identifying important noncoding variants that would otherwise be undervalued or perhaps even completely ignored by traditional methods and annotators. Regtools is freely available and open source (https://github.com/griffithlab/regtools).

#2286

Feature-based clinical interpretation of whole exome and transcriptome data for precision cancer medicine.

Brendan Reardon,1 Nathanael Moore,2 Eric Kofman,1 Eliezer Mendell Van Allen1. 1 _Dana-Farber Cancer Inst., Boston, MA;_ 2 _Indianapolis University School of Medicine, Indianapolis, IN_.

Background: Clinical interpretation algorithms and knowledge bases (e.g. PHIAL, OncoKB) are being used for clinical decision making. These approaches are typically based generally limited to "first order" genomic relationships (e.g. BRAFV600E & RAF/MEK inhibition). The increasing complexity of molecular data generated at the point of care, including whole exome and transcriptome results, along with the expanded therapeutic landscape in cancer, necessitate novel algorithms to enable robust and modern clinical interpretation of a cancer patient's molecular data to accelerate precision cancer medicine. We introduce a paired feature-based clinical interpretation algorithm and knowledge system for cancer genomic data to inform treatment decisions at the point of care and provide researchers with rapid assessment of tumor actionability.

Methods: We expand upon PHIAL to predict actionability based on first-order genomics using SNVs (from both whole-exome sequencing and bulk RNA-seq), InDels, SCNAs, and fusions to further to infer global features of an individual tumor such as mutational burden, mutational signature profile, MSI-status, somatic-germline interaction, and connections between events. Predictive implication values were assigned to reflect the validities of the database's drug sensitivity, resistance, and prognostic claims.

Results: We benchmarked the feature-based approach against PHIAL & TARGET across two cohorts that include both whole exome and transcriptomic data - 150 castrate resistant prostate cancers and 110 metastatic melanomas. PHIAL identified 1281 putatively actionable or biologically relevant alterations, with a median of 3 events per patient and 94% of patients having at least 1 event. The feature-based approach identified 1767 putatively actionable or biologically relevant variants or features, with a median of 5 events per patient and 97% of patients having at least 1 event. Of the these patients, 27% had at least 1 variant associated with an FDA-approved therapy and 18% had events associated with a clinical trial. It also identified 29% of samples as having a putatively actionable global feature.

Conclusion: A DNA and RNA based interpretation method was able to identify and rank more putatively actionable first-order genomic alterations than PHIAL & TARGET, while also providing insight to global features of individual tumors. Increased accessibility of clinical interpretation through our cloud-based web portals and genomic reports may aid in sample contextualization, especially at the point of care.

Source code and an interactive web portal for this project are available at chips.broadinstitute.org.

#2287

Precise common and rare germline CNV calling with GATK.

Mehrtash Babadi, Samuel K. Lee, Andrey Smirnov, Lee Lichtenstein, Laura D. Gauthier, Daniel P. Howrigan, Timothy Poterba. _Broad Institute, Cambridge, MA_.

We propose, implement, and evaluate a novel method (GATK gCNV) for accurate discovery of rare and common copy-number variations (CNVs) from read-depth data obtained from whole genome sequencing (WGS), whole exome sequencing (WES), or custom gene panels. GATK gCNV utilizes a sophisticated Bayesian model to learn bias factors arising from sequencing and library preparation. This model accounts for ploidy of sex chromosomes and autosomal aneuploidies, treats GC bias probabilistically, and automatically determines the necessary level of model complexity in a data-driven manner. Unlike most existing read-depth methods, GATK gCNV maintains a high level of sensitivity in common CNV regions, due to a hierarchical hidden Markov model used for accurate genotyping of multi-allelic loci. Furthermore, GATK gCNV performs bias modeling and CNV discovery simultaneously and self-consistently, resulting in significantly improved sensitivity and precision. Our implementation utilizes the PyMC3/Theano framework for performing automatic differentiation variational inference (ADVI). In addition, GATK gCNV automatically scatters large tasks across multiple machines using the Cromwell/WDL framework, enabling the scalable processing of large cohorts.

We use GATK gCNV to compute copy-number transmission and de novo rates in a cohort of WES trios and observe consistency with observed population metrics. Furthermore, we benchmark GATK gCNV for sensitivity, precision, and reproducibility of both rare and common CNV calls. Using a cohort of WES blood normal samples from The Cancer Genome Atlas (TCGA), we show that GATK gCNV calls are in remarkable concordance with Genome STRiP calls on matched WGS data and gCNV substantially outperforms XHMM and CODEX. We also validate GATK gCNV calls on WGS data against calls made using PacBio long reads.

#2288

Assessing patient trial readiness for precision cancer medicine clinical trials through computational trial matching and rule-based logic applied to genomic and clinical data.

Catherine Del Vecchio Fitz, Khanh Do, James Lindsay, Suzanne Hector-Barry, Zachary Zwiesler, Priti Kumari, Tali Mazor, Tamba Monrose, Adem Albayrak, John Methot, Michael Hassett, Geoffrey Shapiro, Ethan Cerami. _Dana-Farber Cancer Institute, Boston, MA_.

More than 1,100 phase I treatment trials are registered with clinicaltrials.cancer.gov. Patient identification and recruitment to these trials is particularly challenging due to the vast molecular data now available for patients and the increasingly complex nature of the molecular eligibility criteria. Additionally, Phase I trial staff often manage a portfolio of dozens of trials, making patient identification, recruitment, screening, and enrollment an arduous task. To address the need for better and more efficient patient identification, we have developed MatchMiner, a novel open source computational platform for matching patient-specific genomic profiles to precision cancer medicine clinical trials, and a pre-screening tool that includes options for filtering and sorting patients based on clinical data elements. MatchMiner excels at identifying patients who are eligible for clinical trials based on genomic, cancer type, age, and gender criteria, but the great majority of patients are not currently ready for a trial. The pre-screening tool allows for the integration of additional clinical data, including genomic testing date, appointments, treatment plan history, and radiology scan impression text, in order to provide an assessment of 'trial readiness'. To integrate these tools into the clinical workflow, we have implemented a weekly MatchMiner Tumor Board Review process, where together with the DFCI Early-Phase Drug Development Center (EDDC) physicians and staff, a thorough assessment of potential patient-trial matches is performed. Currently we have scaled this initiative to include 18 genomically-driven trials. Each week, ~9000 patient-trial matches are computed across all open trials, which are then computationally filtered to a list of ~300 patient-trial matches utilizing rules-based logic applied to our clinical data sources. The MatchMiner team then performs a manual review of pre-filtered matches based on additional requirements, such as evidence of progression in the radiology scan text impressions. The final list of ~10-20 patients is reviewed with the EDDC, and patients deemed to be 'trial ready' are flagged. E-mail notifications are then sent out to the patient's oncologist, alerting them to a potential genomically-driven clinical trial opportunity. The combination of computational patient-trial match identification and rules-based computational pre-filtering based on additional clinical data has allowed us to screen thousands of patients per week, which would not be possible otherwise. To evaluate the success of this initiative, we are tracking patient consults and trial enrollments, and capturing feedback on workflow integration and utilization. Additionally, efforts to further automate trial readiness measurements to minimize the need for manual review are ongoing.

#2289

Precision-weighted RNA-seq analyses of molecular abundance (RoMA) for detecting differential gene expression.

Guoshuai Cai,1 Jennifer M. Franks,2 Michael L. Whitfield2. 1 _University of South Carolina, Columbia, SC;_ 2 _Geisel School of Medicine at Dartmouth, Hanover, NH_.

In this study, we propose a novel method, RoMA, to accurately detect differential expression and unlock the integration with numerous upstream and downstream analyses on mRNA abundance in RNA-seq studies. Various methods have been proposed, each with its own limitations. Some naive normal-based tests have low testing power with the invalid normal distribution assumptions for RNA-seq read counts, whereas count-based methods lack a biologically meaningful interpretation and have limited capability for integration with other analysis packages for mRNA abundance. RoMA incorporates information from both mRNA abundance and raw counts by modeling RPKM (reads per kilobase per million), which represents the relative abundance of mRNA transcripts, and borrowing mean-variance dependency from CPM (counts per million) as a precision weight accounting for the variability in sequencing depth. Studies on simulated data and two real datasets showed that RoMA provides an accurate quantification of mRNA abundance and a value adjustment-tolerant DE analysis with high AUC, low FDR and a desirable type I error rate. This study provides a valid strategy for mRNA abundance modeling and data analysis integration for RNA-seq studies, which will greatly facilitate the identification and interpretation of DE genes. The methods is implemented in a user-friendly R package (RoMA).

#2290

CellMiner and CellMiner Cross-Database (CDB) as a foundation for the exploration of pharmacogenomics through the use of cancerous cell-lines.

William C. Reinhold, Margot Sunshine, Sudir Varma, Fathi Elloumi, Yves Pommier, M.D., Ph.D.. _National Cancer Institute, Bethesda, MD_.

Determining the influence of molecular alterations on pharmacological responses in cancer cell lines from the omic perspective is the logical first step towards making oncology treatment for patients more effective, specific, and logical. Our established CellMiner (http://discover.nci.nih.gov/cellminer) and in development CellMinerCDB (https://discover.nci.nih.gov/cellminercdb/) web-applications provide high-quality, clean, and numerically extensive molecular and pharmacological data, providing a basis for making these types of assessments. CellMiner provides extensive data on the NCI-60 cancerous cell lines, Including are the most extensive set of both drug activity, and molecular data found for any of the databases. The substantial activity profiles are those generated by the high-quality efforts of the Developmental Therapeutics Program (https://dtp.cancer.gov). Included currently are activity data for 21,766 compounds, including 130 Food and Drug Administration (FDA)-approved, 75 clinical trial drugs, as well as molecular data including i) transcript expression for 25,683 genes, ii) genetic variants for 16,568 genes, iii) transcript expression for 360 microRNAs, iv) protein levels for 94 genes, v) DNA copy number (from aCGH) for 23,413 genes, and vi) DNA methylation levels for 17,559 genes. CellMinerCDB, while currently containing fewer output types of data and tools, does provides an interactive web-application that allows the exploration of the larger (~1,000) cell line databases. Included are i) Cancer Cell Line Encyclopedia (CCLE, http://www.broadinstitute.org/software/cprg/?q=node/11) from the Broad Institute of MIT and Harvard, ii) Cancer Therapeutics Response Portal (CTRP, https://portals.broadinstitute.org/ctrp/) from the Broad Institute of MIT, and iii) Genomics of Drug Sensitivity in Cancer (GDSC, http://www.cancerrxgene.org) from the Wellcome Trust Sanger and Massachusetts General Hospital. These databases each contain ~1000 cell lines, and so provide a broader spectrum of cancer types as well as greater numbers within the cancer types that overlap with the NCI-60 (cell lines). Descriptions of the data availability, retrieval, and web-application functionalities will be presented, including informative examples of data integration, and translational results. <!--EndFragment-->

#2291

RBP, a community for reproducible bioinformatics.

Luca Alessandrì,1 Neha Kulkarni,1 Riccardo Panero,1 Martina Olivero,2 Maddalena Arigoni,1 Marco Beccuti,1 Francesca Cordero,1 Raffaele A. Calogero2. 1 _University of Torino, Torino, Italy;_ 2 _Candiolo Cancer Institute FPO IRCCS, Torino, Italy_.

Background Reproducibility of a research is a key element in the modern science and it is mandatory for any industrial application. It represents the ability of replicating an experiment independently by the location and the operator. Therefore, a study can be considered reproducible only if all used data are available and the exploited computational analysis workflow is clearly described. However, today for reproducing a complex bioinformatics analysis, the raw data and a list of tools used in the workflow could be not enough to guarantee the reproducibility of the results obtained. Indeed, different releases of the same tools and/or of the system libraries (exploited by such tools) might lead to sneaky reproducibility issues.

Results To address this challenge, we established the Reproducible Bioinformatics Project (RBP, http://reproducible-bioinformatics.org/), which is an open-source project, whose aim is to provide an infrastructure, based on docker images and R package, to provide reproducible results in Bioinformatics. One or more Docker images are then defined for a workflow (typically one for each task), while the workflow implementation is handled via R-functions embedded in a package available at github repository (https://github.com/kendomaniac/docker4seq). Thus, a bioinformatician participating to the project has firstly to integrate her/his workflow modules into Docker image(s) exploiting an Ubuntu docker image developed ad hoc by RPB to make easier this task. Secondly, the workflow implementation must be realized in R according to an R-skeleton function made available by RPB to guarantee homogeneity and reusability among different RPB functions. Moreover she/he has to provide the R vignette explaining the package functionality together with an example dataset which can be used to improve the user confidence in the workflow utilization. Available workflows: (i) RNAseq/miRNAseq workflows (from fastq to differential expression analysis). (iii) ChIPseq (transcription factors and histones-marks peaks calling). (iv) DNA/RNA SNVs calling (based on GATK-best-practice), (v) Xenome-seq (removing mouse reads contaminating RNA/DNA in patient-derived-xenografts). (vi) HashClone (clonality markers detection tool to quantify minimal residual disease during patient follow-up)

Conclusions Reproducible Bioinformatics Project provides a general schema and an infrastructure to distribute robust and reproducible workflows. Thus, it guarantees to final users the ability to repeat consistently any analysis independently by the used UNIX-like architecture.

#2292

Comparison of Q Path and Aperio Image Analysis System for quantification of ER and PR by IHC in breast carcinoma TMAs.

Max Henry Jacobsen, Constance Temm, George Sandusky. _Indiana Univ. School of Medicine, Indianapolis, IN_.

According to the Centers for Disease Control and Prevention, breast cancer is still one of the most common cancer in women, and top one or two in cancer mortality rates for all races of women. Novel drug treatments are needed to develop new treatments and clinical validation with IHC are common in research. Some common methods of validations are using Tissue MicroArrays (TMAs). In this study 5 TMAs containing approximately 100 cores each were stained with Estrogen Receptor (ER) and Progesterone Receptor (PR). Paraffin-embedded tissue blocks were obtained from the Indiana University Heath Pathology Laboratory under IRB approved protocols. Immunostainings with ER and PR antibodies (DAKO) were preformed using the DAKO-flex platform system at the IU Health Pathology Laboratory. The slides were digitally imaged using the Aperio Digital Imaging System. The SVS digital images were quantified using two different image analysis systems. One imaging system was the Aperio TMALab software, the other was Q Path image analysis software. Both systems were compared not only on the basis of performance, but among other parameters such as user friendliness and the ability to use a SVS image file type. When comparing the systems, the algorithm of Q Path was altered to closely match the algorithm of the standard Aperio positive pixel algorithm. Two measurements of antibody quantification were compared, one being positive pixel percentage, the other being an H Score. A regression line was used to show correlation between systems. Standard deviations were used to compare data against a standard pathology hand count of 0, 1, 2, and 3. The results show a strong correlation (R-squared value at or greater than 0.85) between the two imaging systems for both the positive pixel algorithm and H Score. The tightness may be due to consistent and rigorously explored in the past 20 years with both of these nuclear antibodies. Further studies should help distinguish any other possible errors using novel biomarkers and also delineate any discrepancies between a nuclear expressions against both a cytoplasmic and/or cell membranous expressions. In conclusion, Aperio Imaging analysis systems and Q path image analysis systems were in strong correlation, and therefore interchangeable in scientific research.

#2293

Outstanding chromosomal expression patterning in human cancers.

Spencer Barnes, Mingzhou Song. _New Mexico State University, Las Cruces, NM_.

Cancer transcriptome is an effect of abnormal genetic and/or epigenetic regulation. In the past, transcriptome was studied at the level of individual transcripts for their differential expression. Spatial gene expression patterns along chromosomes were not yet characterized for cancer. We hypothesized that gene expression patterning is not randomly distributed along the chromosomes in cancer versus normal tissues. We analyzed cancer transcriptome from the National Cancer Institute Genomic Data Commons, and identified genomic regions with significant shifts in expression from normal tissues to tumors. We used a novel highly efficient method in the Ckmeans.1d.dp package for clustering genes by chromosomal location and expression level. We identified two chromosomal zones of note that exhibit similar expression patterns across several cancer types. In breast, lung, liver, and esophageal cancers, a zone near 6p22.2 contains genes that are similarly highly upregulated in tumor tissues compared to matched normal tissues. This zone contains a group of histone cluster 1 genes, among which are 14 known cancer genes. In colorectal, lung, liver, and esophageal cancers, a zone on 14q32.33 exhibits either heavy up- or downregulation, dependent on the specific cancer. This zone is rich in immunoglobulin heavy variable genes. Table 1 lists genomic zones of note specific to six cancer types. Our study provides an atlas of outstanding chromosomal expression patterning that can be used as markers for cancer in general and also for specific cancer types.

Table 1. Outstanding genomic zones specific to six human cancer types.

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Cancer type  | Chr:start-end (Mb)  | Cytogenetic band  | # Matched tumor/normal pairs

Breast | chr2:88-92 | p11.2 | 112

Colorectal | chr7:141-146 | q35 | 41

Esophageal | chr11:7-12 | p15.4 | 8

Kidney | chr12:85-91 | q21.33 | 23

Liver | chr8:15-22 | p22 | 50

Lung | chr8:121-126 | q24.13 | 57

#2294

Customizable weighted DNA copy-number networks inform oncogenic pathway alterations and chemoresistance risk.

Joe Ryan Delaney,1 Dwayne G. Stupack2. 1 _Duke University, Durham, NC;_ 2 _UCSD Moores Cancer Center, La Jolla, CA_.

Cancer is driven by mutations of many types: single nucleotide variants, insertion-deletion events within coding exons, and prolific DNA copy-number alterations (CNAs). CNAs have been often viewed as a passenger event due to seemingly inconsistent patterns between tumors and a large fraction of the genome altered, preventing traditional statistical analysis. Here, we present a tool which incorporates phenotypic data including haploinsufficiency and triploproficiency of genes and protein-protein interaction studies, which enables the identification of consistently altered pathways in cancer. This tool, HAPTRIG (for HAPloinsufficient and TRIplosensitive Gene) can be used with RNA, log2 CNA, absolute CNA data, or any other normalized gene-level data. As part of the pathway scoring process, control CNA networks are generated to provide a permuted-genome control, enabling detection of alterations in very heterogeneous cancer backgrounds. The customizable gene-scores which build these networks allow prioritization of altered network hubs: single genes which have exceptional influence within an altered pathway, facilitating hypothesis-testing in cell and animal models. A beta-version was validated in a recent publication from our lab to better predict tumor suppressor genes and oncogenes than Gene Set Enrichment Analysis, and additionally identified targetable pathway hubs which improved murine treatment in ovarian cancer chemo-resistant models. However, its applicability was limited in the beta. Now HAPTRIG can work with any user-supplied human dataset, not just TCGA samples, is an order of magnitude faster, able to run in parallel processors for unlimited speed improvement, and can produce pan-pathway outputs. The tool can use expected background rates from permuted data or compare to a second control dataset, which can be used to compare altered CNA pathways in tumor subtypes or unusual alterations of RNA compared to CNAs, among many other possibilities. We provide this tool for free community use online and in App form, so molecular biologists without bioinformatics training may produce well-informed hypotheses. We find that altered CNA pathways can predict chemoresistance risk groups within tumor types. A tumor suppressor gene hub was found to accelerate tumorigenesis upon deletion in a spontaneous mouse model. Overall, HAPTRIG represents a user-friendly tool to better understand a whole new landscape of tumor mutations: that of copy number alterations.

#2295

LinkedOmics: Analyzing multi-omics data within and across 32 cancer types.

Suhas Vasaikar,1 Pater Straub,2 Jing Wang,1 Bing Zhang1. 1 _Baylor College of Medicine, Houston, TX;_ 2 _Vanderbilt University School of Medicine, Nashville, TN_.

Background: The Cancer Genome Atlas (TCGA) project has performed molecular profiling of human tumors using genomic, epigenomic, transcriptomic, and proteomic platforms, and each tumor is comprehensively characterized by around 100,000 molecular attributes in addition to typical clinical attributes. To make these data directly available to the entire cancer research community, several data portals have been developed. However, none of the existing data portals allow systematic exploration and interpretation of the complex relationships between the vast amount of clinical and molecular attributes.

Methods: We developed LinkedOmics (http://www.linkedomics.org), a web platform that focuses on the discovery and interpretation of associations between clinical and molecular attributes. LinkedOmics includes three data analysis modules. The LinkFinder module allows flexible exploration of associations between a molecular or clinical attribute of interest and all other attributes, providing the opportunity to analyze and visualize associations between billions of attribute pairs for each cancer cohort. The LinkCompare module enables easy comparison of the associations identified by LinkFinder, which is particularly useful in multi-omics and pan-cancer analyses. The LinkInterpreter module transforms identified associations into biological understanding through pathway and network analysis. All modules provide user-friendly data visualization.

Results: The current version of LinkedOmics contains multi-omics data and clinical data for 32 cancer types and a total of 11,158 patients from the TCGA project. It is also the first multi-omics database that integrates mass spectrometry (MS)-based global proteomics data generated by the Clinical Proteomic Tumor Analysis Consortium (CPTAC) on selected TCGA tumor samples. In total, LinkedOmics has more than a billion data points. We used several case studies to demonstrate the utility of LinkedOmics in revealing functional impact of somatic mutation or copy number alteration on mRNA or protein expression, in deriving multi-omics based protein signature for poor prognosis, in performing pan-cancer analysis to identify survival-associated gene expression signature, and in connecting novel pan-cancer poor prognosis markers to tumor invasiveness and aggressiveness.

Conclusions: LinkedOmics provides a unique platform for biologists and clinicians to access, analyze and compare cancer multi-omics data within and across tumor types. With 5 case studies we demonstrated the power of LinkedOmics in cancer research. Although the current version of LinkedOmics includes only TCGA and CPTAC data, it can be easily extended to support other cohort-based or user provided multi-omics studies.

#2296

STAR-SEQR: Accurate fusion detection and support for fusion neoantigen applications.

Jeff Jasper, Jason G. Powers, Victor J. Weigman. _Q2 Solutions, Morrisville, NC_.

Genomic structural variation and associated RNA fusions are a common clinical feature known to be involved in the initiation and pathogenesis of cancer. This complex class of variants also has significant implications on therapeutic decisions and has emerging roles in evidence-based clinical applications. Consequently, fusion detection is an emerging aspect of precision medicine that enables clinical utility of fusions as a therapeutically-relevant target which includes small molecules, biologics, and neoantigen-directed approaches.Here we present STAR-SEQR, an algorithm that uses multiple levels of evidence to detect and quantify fusions from RNA-Seq data. STAR-SEQR is a fast and accurate tool that goes beyond fusion detection and also provides rich annotation and useful reporting features to aid in the adoption of fusions in clinical diagnostics. Notably the software produces fusion expression values, PCR primers, fusion RNA sequence, and predicted peptide sequence to facilitate downstream applications.To address the analytical performance of STAR-SEQR to existing tools, we utilized previously characterized synthetic datasets and real-world sample data with matching DNA sequencing. Of note, in a dataset of 50 synthetic fusions, STAR-SEQR had the highest accuracy of all tools tested and called 49/50 events with no false positives. STAR-SEQR has also been run on several large clinical datasets and the fusion events identified have displayed strong overlap with identified DNA structural variants. STAR-SEQR also performed well in the ongoing ICGC-TCGA DREAM SMC-RNA Challenge and achieved the highest F1 score in the simulated data round with an overall average of 0.96. Analytical lab testing has also been performed using the Seracare Fusion RNA Mix V2 sample containing 15 clinically significant fusions with known concentrations in the low range of detectability. STAR-SEQR accurately called 13/15 events with the other events having no identifiable read alignments. We have also benchmarked methods comparing FFPE material, range of read lengths, and library methods where STAR-SEQR demonstrated excellent tradeoff between sensitivity and precision and generally outperformed other tools.In summary, STAR-SEQR has excellent analytical performance that surpasses existing fusion detection tools. It is broadly applicable to RNA-Seq methods, computationally efficient, and robust across sample characteristics. Lastly, STAR-SEQR is explicitly designed to enable fusion-directed clinical diagnostics and support cancer immunotherapy approaches that utilize fusion neoantigens.

#2297

Differential analysis of gene expression across the human genome using recount2 and FANTOM-CAT.

Eddie Luidy-Imada,1 Tejasvi Matam,2 Leonardo Collado-Torres,3 Wikum Dinalankara,2 Aleksei Stupnikov,4 Christopher Wilks,5 Andrew E. Jaffe,6 Ben Langmead,5 Jeffrey T. Leek,6 Alexander Favorov,1 Luigi Marchionni1. 1 _Johns Hopkins University School of Medicine, Baltimore, MD;_ 2 _Johns HopkinsUniversity School of Medicine, Baltimore, MD;_ 3 _Lieber Institute for Brain Development, Baltimore, MD;_ 4 _Johns Hopkins Univ. School of Medicine, Baltimore, MD;_ 5 _Johns Hopkins University, Baltimore, MD;_ 6 _Johns Hopkins Bloomberg School of Public Health, Baltimore, MD_.

In recent years, in depth exploration of genomes structure and function has revealed a central role for non-coding RNAs (ncRNAs) in orchestrating key biological and cellular processes through the fine tuning of gene expression regulation. Most importantly a role for ncRNAs has also started to emerge in human disease pathogenesis. This further speaks to the importance of an in-depth characterization of ncRNA involvement in human diseases, including cancer.

In this work, we have built a comprehensive atlas of gene expression across the human transcriptome containing over 100,000 genes by leveraging two publicly available resources: the FANTOM CAGE Associated Transcriptome (FANTOM-CAT), and recount2. The FANTOM-CAT is a comprehensive meta-assembly of the human transcriptome encompassing coding and non-coding genes, including promoters, enhancers, and lncRNAs. recount2 is the largest, available collection of human RNA-seq data processed and quantified using a unified pipeline, containing over 4.4 trillion reads from over 70,000 human samples from the SRA, GTEx and TCGA projects.

Using FANTOM-CAT/recount2 gene expression summaries across human tissue samples from the GTEx project, we validated our approach by reproducing key findings recently described by the FANTOM consortium. In particular, we could identify and confirm the reported tissue specific transcription patterns for the distinct classes of coding and non-coding genes, such as messenger RNAs, intergenic long ncRNAs, and expressed divergent promoters and enhancers. Most importantly, we have leveraged the FANTOM-CAT/recount2 atlas to perform differential gene expression analysis across 13 cancer types in TCGA, identifying a comprehensive list of mRNAs, promoters, enhancers, and other ncRNAs differentially expressed between cancer and normal samples. Overall, this analysis revealed a set of molecular signatures encompassing coding and non-coding genes that are shared across cancer types or restricted to specific tissues of origin.

In summary, our findings establish the FANTOM-CAT/recount2 gene expression atlas as a reliable resource to study the role ncRNAs play in cancer and other human diseases, providing important cues to understand their biological function. Finally, we make the FANTOM-CAT/recount2 atlas available as a public tool to empower other researchers to study important biological and clinical phenotypes and identify new candidate ncRNAs for further investigation.

#2298

Understanding tumor biology complexity in the advanced cancer setting: PreciGENE® platform predictions correlate with exceptional responses to cancer treatment.

Amelie Boichard,1 Timothy V. Pham,2 Razelle Kurzrock1. 1 _University of California, San Diego, La Jolla, CA;_ 2 _CureMatch Inc., San Diego, CA_.

Cancer is a disease caused by an elaborate network of genetic and environmental factors, and its biologic complexity renders difficult the selection of optimized treatment options. New tools are needed to allow oncologists to quickly distinguish, among the plethora of possible combinations, those regimens that best fit the patient's specificities and increase the chances of survival. The PreciGENE analytical platform integrates genomic, transcriptomic and proteomic data to provide fully personalized therapeutic options. Here we present an analysis of 70 exceptional responders to cancer treatment for whom a molecular profile was published in the literature between February 2010 and June 2017. We aimed to evaluate the performance of the PreciGENE tool in the determination of optimized regimens for cancer therapy.

Methods: A PubMed search was conducted using the keywords "exceptional response" and "cancer." All cases published in the period defined and presenting tumor molecular data were kept for analysis. Molecular and drug descriptions were entered into the PreciGENE system that computed a score (%), with a high score representing a better fit between treatment and tumor profile. Correlation between the predictive score and the clinical response observed was evaluated using a Mann-Whitney test: complete or partial responses (CR/PR) were compared to stable or progressive diseases (SD/PD).

Results: A total of 202 treatment lines were retrieved, including 70 successful and 132 unsuccessful lines of therapy. Patients received an average of 1.6 ([CI95%] =1.5-1.8) treatment lines and molecular profiles retrieved an average of 3.7 [2.7-4.7] alterations per tumor. 84/202 (42%) regimens included at least two therapeutic agents. Among the regimens that led to an exceptional response (CR/PR), 31/70 (44%) were combination therapies. The predictive scores obtained for regimens that led to a positive outcome were significantly greater than those of regimens that had failed (mean [CI95%] = 60 [52-68]% for CR/PR vs 14 [10-19]% for SD/PD; p=.0001). Using a score threshold of 25% (corresponding to a therapeutic regimen matching 25% of the pathogenic alterations detected in the tumor), the PreciGENE platform results presented a sensitivity of 84% and a specificity of 77% for the prediction of the clinical response. The negative predictive value was 90%; the positive predictive value was 66%.

Conclusion: The algorithm used by the PreciGENE decision-support platform correctly ranked the treatment response in 70 published cancer patients having received 202 different regimens and presented an exceptional response to at least one therapeutic regimen. In this retrospective study, we show that such systems may empower oncologists in their choice of treatment. Prospective studies of the use of decision-support platforms in advanced cancer are warranted.

#2298A

Enhanced consideration of context dependence reveals the scope of somatic missense drivers in human cancers.

Collin J. Tokheim, Neha Nanda, Nicholas J. Roberts, Rachel Karchin. _Johns Hopkins Univ., Baltimore, MD_.

Large-scale sequencing studies of patient cohorts have enabled identification of many driver genes whose somatic alterations confer a selective advantage to cancer cells. However, not every mutation in a driver gene is necessarily a driver of cancer. As mutation-based biomarkers are increasingly used to decide drug regimens and placement in clinical trials, it is important to distinguish specific driver mutations. Improved computational methods are necessary for identifying the particular mutations in a given cancer that are drivers, especially for rare mutations (<1% of patients). We have previously developed the CHASM algorithm to identify cancer-specific driver missense mutations. Here, we present a completely re-worked version that leverages the context-dependence of driver mutations. We show that the new CHASM2 consistently outperforms comparable methods across a wide variety of benchmarks, including in vivo and in vitro experiments. Applied to 8,657 TCGA cancer samples in 32 cancer types, CHASM2 identifies 3,527 distinct driver somatic missense mutations. Our results suggest substantial heterogeneity of the driver mutation landscape across cancer types and a strikingly large prevalence of rare driver mutations. For particular cancer types driven by common mutations, current sample sizes are already saturating driver discovery. We performed validation experiments on missense mutations of rare and intermediate frequency in the tumor suppressor gene ATM and show CHASM2 distinguishes a radiosensitive phenotype in cultured cells. To our knowledge CHASM2 is the first sufficiently powered method to predict individual driver somatic missense mutations at multiple scales -- whole exome, targeted gene panel, and within a single gene. 

## MOLECULAR AND CELLULAR BIOLOGY / GENETICS:

### CDK and CDK Inhibitors: Mechanism and Therapy

#2300

**CDK4/6 inhibition triggers dissociation of MEIS2 from CRL4** CRBN **E3 ligase to induce interferon response and reprogram myeloma for IMiD vulnerability.**

Xiangao Huang,1 David Jayabalan,1 Maurizio Di Liberto,1 Zhengming Chen,1 Anna Schinzel,2 Scott A. Ely,1 Adriana Rossi,1 Morton Coleman,1 Joseph M. Lane,3 William C. Hahn,2 Tomer M. Mark,1 Ruben Niesvizky,1 Selina Chen-Kiang1. 1 _Weill Cornell Medicine, New York, NY;_ 2 _Dana-Farber Cancer Institute, Boston, MA;_ 3 _Hospital for Special Surgery, New York, NY_.

Inhibition of CDK4/6 has emerged as an effective therapy in human cancer. Palbociclib, the first selective CDK4/6 inhibitor (CDK4/6i), is FDA-approved for treatment of metastatic breast cancer, and abemaciclib has been fast tracked for approval as a front line breast cancer treatment. Recent evidence in mouse models of breast cancer further indicates that CDK4/6 inhibition triggers anti-tumor immunity, but the precise tumor intrinsic mechanism that mediates CDK4/6 inhibition for a clinical response remains to be defined. To address this, we inhibited CDK4/6 with palbociclib in sequential combination with immunomodulary drugs (IMiD)s in primary bone marrow myeloma cells (BMMCs), using normal bone marrow plasma cells as a control. IMiDs, lenalidomide (Len) and pomalidomide (Pom), are standard of care for multiple myeloma (MM) and lymphoma. Each rarely achieves complete remission despite a high overall response rate and durability. However, each markedly potentiates the clinical efficacy of diverse partners by unknown mechanisms. Cereblon (CRBN), a substrate receptor of the CUL4-ROC1-DDB1-CRBN (CRL4CRBN) E3 ubiquitin ligase, is requited for IMiD's anti-tumor activity. Binding of Len or Pom to CRBN accelerates the recruitment of transcription factors IKZF1/3 to CRL4CRBN for ubiquitination and degradation in MM cells. One of the targets of IKZF1/3 is IRF4, which is required for survival of MM cells and reduced after exposure to Len or Pom. Moreover, MEIS2, a homeobox transcription factor was identified to be an substrate of CRBN in crystal structure and by biochemical screen, implicating a role for MEIS2 in modulates IMiD's anti-tumor activity. However, evidence for MEIS2 expression in myeloma cells is lacking. Previously, we have shown that prolonged early G1 arrest (pG1) induced by sustained CDK4/6 inhibition reprogrammed cancer cells for vulnerability to diverse clinically relevant agents ex vivo and in animal models. To address if CDK4 inhibition reprograms MM cells for IMiD vulnerability, first we show that the intrinsic IMiD sensitivity in primary BMMCs ex vivo is a major determinant for the clinical response to IMiD therapy in myeloma patients. Second, we discovered that MEIS2 is aberrantly expressed and associated with CRBN in BMMCs. Third, MEIS2 is regulated by the cell cycle in primary MM cells and required for the survival of MM cells in part by maintaining IRF4 expression. Finally, CDK4/6 inhibition liberates CRBN from association with MEIS2 in pG1. This rapidly accelerates Len-mediated ubiquitination and degradation of IKZF1/3, loss of IRF4, de-repression of IRF7 and induction of interferon (IFN) responsive genes that culminated in TRAIL-mediated apoptosis. Dissociation of MEIS2 from CRBN thus represents a noel mechanism by which CDK4/6 inhibition reprograms myeloma for vulnerability to IMiDs through induction anti-tumor IFN response.

#2301

Dual targeting of CDK4/6 and MET in metastatic uveal melanoma.

Masahiro Ohara,1 Kengo Saito,2 Ken Kageyama,3 Mizue Terai,4 Hanyin Cheng,4 Andrew E. Aplin,4 Takami Sato4. 1 _Hiroshima General Hospital, Hatsukaichi, Japan;_ 2 _Graduate School of Medicine, Japan;_ 3 _Osaka City University, Osaka, Japan;_ 4 _Thomas Jefferson University, Philadelphia, PA_.

Uveal melanoma (UM) is the most common cancer of the eye in adults. Up to 50% of UM patients subsequently develop metastases, especially in the liver, for which no effective treatment has been identified. It has been reported that the retinoblastoma (RB) pathway is deregulated in more than 90% of UM despite the rarity of mutations in the RB1 gene itself. Cyclin D1 over-expression and inactivation of p16INK4a in UM are frequently present and make the RB protein constitutively hyperphosphorylated and functionally inactivated; therefore, CDK4/6 inhibition (CDK4/6i) is a rational strategy for treatment of UM. In this report, we investigated the antiproliferative activity of a selective CDK4/6 inhibitor on metastatic UM. A CDK4/6 inhibitor induced G0-G1 arrest in all three metastatic UM cell lines and suppressed their growth in in vitro and in vivo experiments. Interestingly, hepatocyte growth factor (HGF) decreased the effect of CDK4/6 inhibitor on metastatic UM cell lines and protected the metastatic UM cells from CDK4/6 inhibitor-induced cellular senescence. When CDK4/6i was combined with MET inhibitor, enhanced growth suppression was observed in metastatic UM tumors grown in human-HGF knock-in xenograft mouse models. HGF is enriched in the liver and the majority of liver metastases from UM express activated forms of MET; therefore, signaling through MET could contribute to the resistance mechanisms against CDK4/6i, especially in UM patients with hepatic metastasis. Together, these results provide a rationale for the use of MET inhibitor in combination with a CDK4/6 inhibitor for the treatment of metastatic UM.

#2302

Preclinical evaluation of CDK4/6 inhibitor and biomarker exploration in gastric cancer.

Hyun Joo Bae, Woo Sun Kwon, Hyun Jeong Kim, Sun Kyoung Kang, Inhye Jeong, Tae Soo Kim, Hyun Cheol Chung, Sun Young Rha. _Yonsei University, Seoul, Republic of Korea_.

Dysregulation of cell cycle is a hallmark of cancer and the p16INK4a-CDK4/6-Rb pathway is often disrupted in many types of cancer including gastric cancer. Inhibition of CDK4/6 in cancer cells has become a promising therapeutic strategy, but not tested in gastric cancer. In this study, we investigated the effect of abemaciclib (LY2835219), a dual inhibitor of CDK4 and CDK6, and identified potential biomarkers in 49 gastric cancer cell lines. The cytotoxicity of abemaciclib was assessed by CCK-8 assay. For exploration of predictive biomarkers, status of single nucleotide variants (SNVs) and copy number variations (CNVs) were analyzed by whole exome sequencing (WES). Also, RNA and protein expression levels of cell cycle related molecules were determined by RNA sequencing and Western Blot, respectively. In TCGA study (The Cancer Genome Atlas, Nature 2017) approximately 37% of stomach adenocarcinoma patients have defects in the p16INK4a-CDK4/6-Rb pathway (143/388patients); notable signatures include alterations of p16INK4a (14%), Rb1 (7%), CDK4 (4%), CDK6 (8%), cyclinD1 (6%), and cyclinE1 (12%). In our 49 gastric cancer cell lines, we confirmed 25 cell lines have defects in this pathway (51%) including alterations of p16INK4a (38%), Rb1 (10%), CDK6 (8%), cyclinD1 (4%), and cyclinE1 (2%). Based on the sensitivity of abemaciclib, we could divide 49 gastric cancer cell lines into: sensitive (IC50<0.5uM, 28/49 cell lines) and resistant (IC50≥0.5uM, 21/49cell lines) groups with the cut off 0.5uM which was determined by clinically achievable concentration from phase I trial. As a result of relation analysis between protein expression and sensitivity of abemaciclib, p16INK4a deletion did not correlate with sensitivity. Then, correlation analysis between sensitivity and RNA expressions of p16INK4a-CDK4/6-Rb pathway related genes were determined. Significant correlations were found in p16INK4a (r=0.4783, p=0.0016), Rb1 (r=-0.3206, p=0.0410), and cyclinE1 (r=0.5378, p=0.0003) whereas not in cyclinD1, CDK4, and CDK6. Protein expression levels of p16INK4a and cyclinD1 were not related to sensitivity of abemaciclib. In addition, with reproduction of the GDSC (Genomics of Drug Sensitivity in Cancer) information, focal recurrent copy number alterations of regions of chromosome 9p24.3-p21.1 (cnaPANCAN144) were correlated with CDK4/6 inhibitor sensitivity. Among ninety-one genes within these regions, 16 genes from cnaPANCAN144 were correlated with sensitivity of abemaciclib (p≤0.05). Our results indicate that abemaciclib is a potential therapeutic agent for gastric cancer and demonstrate that RNA expressions of p16INK4a, Rb1, and cyclinE1 would be predictive biomarkers based on molecular genetic profiling.

#2303

CDK4/6 inhibition blocks effects of IGFs and insulin in estrogen receptor positive and triple negative breast cancers.

Katelyn Hoff, Deepali Sachdev. _Masonic Cancer Center, University of Minnesota, Minneapolis, MN_.

Insulin-like growth factors (IGFs) and insulin signaling via the type I IGF receptor (IGF1R) and insulin receptor (IR) respectively, are potent activators of PI3K/Akt/mTOR. Drugs targeting IGF1R and the related IR were tested clinically including in combination with mTOR inhibitors. Inhibition of mTOR was not effective as inhibition of mTOR relieved the negative feedback loop regulating levels of the adaptor protein, insulin receptor substrate 1 (IRS-1), that mediates proliferative effects of IGFs and insulin and rapamycin enhanced phosphorylation of Akt. The ribosomal protein S6 kinase (S6K) phosphorylates IRS-1 on serine residues and targets it for proteasomal degradation, and this negative feedback regulation is important in attenuating IGF and insulin signaling. Cyclin dependent kinases (CDKs) 4 and 6 are required for cell cycle progression. CDK4/6 inhibitors have recently been approved for treatment of estrogen receptor positive (ER+), Her2- advanced breast cancers and these CDK4/6 inhibitors such as palbociclib block phosphorylation of retinoblastoma (Rb). IGFs and insulin stimulate cell cycle progression and increase cyclin D1 levels in breast cancers. Therefore, we hypothesized that CDK4/6 inhibition can be combined with IGF1R/IR targeting to block mitogenic functions of IGF/insulin signaling in breast cancer as this would not relieve the negative feedback regulation of IGF and insulin signaling. Palbociclib blocked growth of ER+ parental MCF-7 and T47D breast cancer cells that respond to hormonal therapy including tamoxifen, a selective estrogen receptor modulator. Parental MCF-7 and T47D were more sensitive to palbociclib compared to matched cells with acquired resistance to tamoxifen (MCF-7/TamR and T47D/TamR). Palbociclib also blocked IGF-I and insulin stimulated entry into cell cycle leading to G0/G1 arrest in ER+ breast cancer cells. Combination of palbociclib with an IGF1R inhibitory antibody, but not IR antibody, was more effective in inhibiting growth of tamoxifen sensitive parental ER+ breast cancer cells. In contrast combination of palbociclib with an IR antibody was effective in inhibition of MCF-7/TamR cells. Unlike mTOR inhibitors that upregulated IRS-1 levels leading to increased phosphorylation of Akt through IGF1R/IR, palbociclib did not affect IRS-1 levels and did not enhance phosphorylation of Akt in ER+ and TNBC cells. Further, palbociclib also blocked growth and cell cycle progression of triple negative breast cancer (TNBC) cells. Our data show that combining IGF1R/IR inhibitors with palbociclib may be superior to combining them with mTOR inhibitors for ER+ breast cancer and palbociclib can be a potential therapeutic strategy for TNBC. These data implicate that cotargeting CDK4/6 and IR could be a therapeutic option for patients with endocrine resistant disease.

#2304

In vivo E2F reporting reveals efficacious schedules of MEK1/2-CDK4/6 targeting and mTOR-S6 resistance mechanisms.

Jessica Teh,1 Phil F. Cheng,2 Timothy J. Purwin,1 Neda Nikbakht,1 Prem Patel,1 Inna Chervoneva,1 Ines Keibler,3 HooKim Kim,1 Michael A. Davies,4 Lawrence N. Kwong,4 Mitch P. Levesque,3 Reinhard Dummer,3 Andrew E. Aplin1. 1 _Thomas Jefferson Univ., Philadelphia, PA;_ 2 _University of Zurich, Philadelphia, Switzerland;_ 3 _University of Zurich, Zurich, Switzerland;_ 4 _MD Anderson University of Texas Houston, Houston, TX_.

Targeting cyclin dependent kinases 4 and 6 (CDK4/6) represents a viable therapeutic option in combination with BRAF inhibitor and/or MEK inhibitor in different genetic subsets of melanoma. However, continuous dosing of MEK inhibitor (MEKi) plus CDK4/6 inhibitor (CDK4/6i) elicits toxicities in patients. Utilizing an in vivo E2F reporter system, we analyzed the efficacy of MEKi plus CDK4/6i schedules in a quantitative and temporal manner. Continuous MEKi with intermittent CDK4/6i led to more complete responses as compared to either continuous CDK4/6i with intermittent MEKi or intermittent dosing of both drugs. Nevertheless, some tumors acquired resistance. Phospho-proteomic analysis of resistant tumors revealed increased phosphorylation of ribosomal S6 protein (RPS6). These data were supported by staining of patient biopsies from clinical trials, which also indicated that high phospho S6 levels may serve as a predictor of response to CDK4/6i plus targeted inhibitors. Enhanced phospho S6 provided a therapeutic window for the mTORC1/2 inhibitor, AZD2014, in MEKi plus CDK4/6i-resistant tumors. Mechanistically, aberrant regulation of NRAS was associated with resistance and sufficient to mediate resistance. This study highlights the use of an in vivo reporter model for optimization of schedules and supports targeting the mTORC1/2-S6 pathway as a salvage option to overcome MEKi plus CDK4/6i resistance.

#2305

Targeting 6-phosphofructo-2-kinase to increase the efficacy of ER and CDK4/6 inhibitors against breast cancer.

Yoannis Imbert-Fernandez, Lilibeth Lanceta, Sucheta Telang, Jason Chesney. _University of Louisville, Louisville, KY_.

We demonstrated that estradiol stimulates the expression and activity of an enzyme, 6-phosphofructo-2-kinase (PFKFB3), that regulates glucose metabolism via allosteric stimulation of 6-phosphofructo-1-kinase. PFKFB3 expression is increased in breast cancer cells that have metastasized to lymph nodes and the combination of the anti-estrogen, fulvestrant, with a PFKFB3 inhibitor, PFK158, synergistically increases apoptosis in ER+ breast cancer cells in vitro and in vivo. Given that estradiol simultaneously increases the expression of both the regulatory subunit of CDK4/6, cyclin D1, and PFKFB3, we postulated that the combination of palbociclib with PFK158 would yield synergy against breast cancer cells. In new preliminary studies, co-treatment with palbociclib and PFK158 resulted in a marked decrease in cell proliferation in two ER+ breast cancer cell lines that was accompanied by a synergistic decrease in phospho-Retinoblastoma (Rb). Importantly, exposure of ER+ breast cancer cells to palbociclib and PFK158 causes an increase in cell cycle arrest and apoptotic cell death in vitro. Notably, this combination has no effect on cell cycle arrest or apoptosis in normal mammary cells suggesting that this therapeutic strategy may be well tolerated and selective for breast cancer.

#2306

Transcriptional and post-transcriptional upregulation of p27 mediates growth inhibition of isorhapontigenin/ISO on human bladder cancer cells.

Jiheng Xu,1 Jingxia Li,1 Chuanshu Huang,1 Haishan Huang,2 Guosong Jiang1. 1 _New York Univ., Tuxedo Park, NY;_ 2 _Zhejiang Provincial Key Laboratory for Technology & Application of Model Organisms, School of Life Sciences, Wenzhou Medical University, Wenzhou, China_.

Our recent studies have demonstrated that Isorhapontigenin (ISO), a new derivative isolated from the Chinese herb Gnetum Cleistostachyum, effectively induces cell-cycle arrest at the G0/G1 phase and inhibits anchorage-independent cell growth through the miR-137/Sp1/cyclin D1 axis in human muscle invasive bladder cancer cells both in vitro and in vivo. Herein, we found that treatment of muscle invasive bladder cancer cells with ISO resulted in a significant upregulation of p27, a key cyclin-dependent kinase inhibitor. Importantly, knockdown of p27 caused a decline in the ISO-induced G0-G1 growth arrest and reversed ISO suppression of anchorage-independent growth in bladder cancer cells. Mechanistic studies revealed that ISO promoted p27 expression at mRNA transcription level through increasing direct binding of FOXO1 to its promoter, while knockdown of FOXO1 attenuated ISO inhibition of cell growth. On the other hand, ISO upregulated the 3'UTR activity of p27, which was accompanied by a reduction of miR-182 expression. In line with these observations, ectopic expression of miR-182 significantly blocked p27 3'UTR activity, whereas mutation of the miR-182 binding site at p27 mRNA 3'UTR effectively reversed this inhibition. Accordingly, ectopic expression of miR-182 also attenuated ISO upregulation of p27 expression and impaired ISO inhibition of cancer cell growth. Our results not only provide novel insight into understanding of the underlying mechanism related to regulation of muscle invasive bladder cancer cell growth, but also identify new roles and mechanisms underlying ISO inhibition of the growth of human bladder cancer cells.

#2307

**Differential effects of N-terminal vs C-terminal truncating** CDKN1A **mutations on cisplatin resistance in bladder cancer.**

Rahmat K. Sikder, Wafik S. El-Deiry, Philip H. Abbosh. _Fox Chase Cancer Center, Philadelphia, PA_.

Introduction

Approximately 15% of muscle invasive bladder cancers harbor mutations in the CDKN1A gene, which encodes the p53 target gene p21, with the majority of alterations truncating the peptide. We previously found that proximal truncation of p21 sensitized cells to cisplatin, while distal truncations made them more resistant. We hypothesized that truncations preserving the CDKi domain but ablating the PCNA binding domain would abrogate p21's ability to sequester PCNA but maintain cell cycle arrest capacities, enhancing the interaction between mono-ubiquitiniated-PCNA (ub-PCNA) and DNA polymerase eta (Pol η), resulting in increased TLS and DNA damage tolerance.

Methods

We used SW780 cell line for this study based on its resistance to cisplatin and p53 and p21 WT status. CRISPR sgRNAs designed to target amino acids 12 and 109 (SW780-sg12 and SW780-sg109) were used to introduce frameshifts and disrupt the majority of the peptide or only the PCNA the binding domain, respectively. sgRNA designed to target GFP was used as a control (sgGFP). We treated the cells with GI50 dose of cisplatin and analyzed post-cisplatin cell-cycle profile over 96 hours using PI-FACS and immunoblot. We treated all three cell types with increasing doses (0-40 μM) of cisplatin for 24 hours and measured the levels of ub-PCNA (K164) and Pol η using immunoblot.

Results

SW780-sgGFP arrested in S-phase at 48 and 72 hours but overcame this arrest at 96 hours and began to cycle again. SW780-sg12 were unable to arrest in S-phase, whereas SW780-sg109 were able to arrest in S-phase at 48 to 96 hours. This pattern of S-phase arrest was further confirmed by prolonged upregulation of cyclin D1 in SW780-sg109 and brief upregulation in SW780-sgGFP. ub-PCNA and Pol η accumulated in a dose responsive manner in all three cell types at 24 hours after cisplatin treatment. ub-PCNA increased abruptly at higher cisplatin doses in SW780-sgGFP, whereas the increase was gradual in CDKN1A-edited cells. Interestingly, in SW780-sg109, Pol η induction was strongest at all doses.

Conclusion

Cisplatin causes S-phase arrest in SW780-sgGFP cells, possibly by inducing replicative stress. However, this arrest is eventually bypassed and the cells progress through the cell cycle. S-phase arrest is not possible in the absence of a functioning p21, indicating that p21 plays a vital role in bringing about post-cisplatin cell cycle arrest. Interestingly, truncated p21 with intact CDKi domain is capable of causing stronger and more prolonged S-phase arrest compared to the full-length peptide. TLS may play a role in DNA damage tolerance in SW780 cells as indicated by elevated ub-PCNA and pol η. Stronger accumulation of Pol η in SW780-sg109, possibly enhances TLS and thus cisplatin resistance. Moving forward, we would like to confirm the anticipated competition between p21 and Pol η for PCNA and further verify this phenomenon in other bladder cancer cell lines.

#2308

Salicylic acid metabolites and derivatives inhibit CDK activity: Novel insights into aspirin's chemopreventive effects against colorectal cancer.

Ranjini Sankaranarayanan,1 Rakesh Dachineni,1 D. Ramesh Kumar,2 Eduardo Callegari,3 Siddharth S. Kesharwani,1 Teresa Seefeldt,1 Hemachand Tummala,1 Jayarama B. Gunaje1. 1 _South Dakota State Univ. College of Pharmacy, Brookings, SD;_ 2 _University of Kentucky, Lexington, KY;_ 3 _University of South Dakota, Vermillion, SD_.

Aspirin's potential as a drug continues to be evaluated for the prevention of colorectal cancer (CRC). Although multiple targets for aspirin and its metabolite, salicylic acid, have been identified, no unifying mechanism has been proposed to clearly explain its chemopreventive effects. Our goal here was to investigate the ability of salicylic acid metabolites, known to be generated through cytochrome P450 (CYP450) enzymes, and also its derivatives as cyclin dependent kinase (CDK) inhibitors to gain new insights into aspirin's chemopreventive actions. Using in vitro kinase assays, for the first time, we demonstrate that salicylic acid metabolites, 2,3-dihydroxybenzoic acid (2,3-DHBA) and 2,5-dihydroxybenzoic acid (2,5-DHBA), as well as derivatives 2,4-dihydroxybenzoic acid (2,4-DHBA), 2,6-dihydroxybenzoic acid (2,6-DHBA), inhibited CDK1 enzyme activity. 2,3-DHBA and 2,6-DHBA did not inhibit CDK2 and 4; however, both inhibited CDK-6 activity. Interestingly, another derivative, 2,4,6-trihydroxybenzoic acid (2,4,6-THBA) was highly effective in inhibiting CDK1, 2, 4 and 6 activity. Molecular docking studies showed that these compounds potentially interact with CDK1. Immunoblotting experiments showed that aspirin acetylated CDK1, and preincubation with salicylic acid and its derivatives prevented aspirin-mediated CDK1 acetylation, which supported the data obtained from molecular docking studies. We suggest that intracellularly generated salicylic acid metabolites through CYP450 enzymes within the colonic epithelial cells, or the salicylic acid metabolites generated by gut microflora may significantly contribute to the preferential chemopreventive effect of aspirin against CRC through inhibition of CDKs. This novel hypothesis and mechanism of action in aspirin's chemopreventive effects opens a new area for future research. In addition, structural modification to salicylic acid derivatives may prove useful in the development of novel CDK inhibitors in cancer prevention and treatment.

#2309

Increase in Cdk4 activity during progression of small intestinal neuroedocrine tumors.

Tanupriya Contractor, Chris R. Harris. _Raymond and Beverly Sackler Foundation, New Brunswick, NJ_.

In the USA, there are approximately 5000 new cases of small intestinal neuroendocrine tumors (SINETs) per year, many of which already show liver metastasis upon initial diagnosis. The genetic causes of SINETs have long been a mystery. The only known recurrent mutations are in the Cdkn1b gene, which is mutated in 8% of tumors. Cdkn1b encodes p27kip1, an inhibitor of Cdk4 and Cdk2. We analyzed proteins and genes linked to the function of Cdk4 in a set of early and late SINETs (pathological stages M0 or M1). Amplification of Cdk4 activators (CcnD1, CcnD2, CcnD3), or loss of copy of Cdk4 inhibitors (Cdkn1b as well as Cdkn2a), or mutation of Cdkn1b were uncommon events, but notably occurred almost exclusively in patients with late stage disease. Patients with late stage tumors also had genetic abnormalities in Phlda3, including loss of copy, loss of heterozygosity, or presence of the rs35383942 SNP. Altogether, more than 50 percent of the advanced tumors had one or more of these genetic events, compared to less than 15 percent of the early tumors. Phlda3 is known to affect Akt activity which in turn is known to repress p27kip1 by phosphorylation, and indeed we detected more p27kip1 phosphorylation in late stage tumors. Finally, advanced tumors showed higher expression of Cdk4 and increased phosphorylation of serine-807 or serine-811 of the Rb1 protein. Together, these data argue that there is genetic pressure to increase Cdk4 activity during progression of SINETs, and suggest that patients with advanced SINETs may respond to Cdk4 inhibitors.

#2310

Targeting transcription-associated CDKs is an effective way to combat glioblastoma and medulloblastoma with minimal effect on primary neurons.

Isha Bhutada, Srikumar Chellappan, Jaya Padmanabhan. _Moffitt Cancer Center and Research Institute, Tampa, FL_.

Glioblastoma multiforme (GBM) is the most prevalent and malignant brain tumor in adults. Currently there are no effective therapies to manage the disease efficiently. Medulloblastoma is the most common type of pediatric tumor and accounts for approximately 15% of all pediatric brain tumors. We aimed to determine if targeting the transcription-associated cyclin-dependent kinases, cdk7 and cdk9, using specific inhibitors, could interfere with the growth and metastatic properties of glioblastoma and medulloblastoma cell lines. We tested the effectiveness of Flavopiridol, a known inhibitor of cdk4, cdk6, pTEFb and cdk9; THZ1, a cdk7 inhibitor; and SNS032, which is known to inhibit cdk2, cdk7 and cdk9, on U87 and SNB19 glioblastoma, H4 neuroglioma and Daoy medullobastoma cells. Treatment with these pharmacological agents showed very strong cytotoxic effects on these cancer cells as measured by MTT (3-(4, 5-dimethylthiazol-2yl)-2, 5-diphenyl tetrazolium bromide) assay. Among these agents the cdk7 inhibitor was most effective and showed efficacy at nanomolar concentrations. Additionally, the inhibitors interfered with the cancer cell migration, as measured by wound healing assay, and anchorage independent growth, as measured by soft agar colony formation. In stark contrast, the inhibitors had no cytotoxic effects on primary neurons. Mechanistically, western blot analysis showed that the tumor cells treated with the inhibitors had reduced levels of Pol II C-terminal domain phosphorylation, indicative of its inhibition and suppression of transcription. Furthermore, inhibitor treatment resulted in a significant reduction in p70S6 kinase phosphorylation, suggesting that in addition to the transcriptional machinery, the translational machinery is also affected upon treatment with the aforementioned cdk inhibitors. Immunostaining analysis of the cells showed a marked reduction in the levels of P-Pol II and P-p70S6 kinase upon inhibitor treatment, confirming that the cdk inhibitors indeed interfere with both transcription and translation. These inhibitors appear to be working in a p53-dependent fashion. These novel findings shed light on potential mechanisms that can be targeted to combat both glioblastoma and medulloblastoma effectively. More specifically, our studies imply that cdk7 and cdk9 inhibitors may serve as potential therapies for effective management of brain tumor.

#2311

Determinants of CDK4 target engagement by palbociclib, ribociclib and abemaciclib.

Jennifer L. Green, Tyzoon Nomanbhoy. _ActivX Biosciences, La Jolla, CA_.

Palbociclib, ribociclib and abemaciclib compose a class of ATP-competitive CDK4 and CDK6 inhibitors that have demonstrated preclinical and clinical efficacy against various tumor types. While these inhibitors have favorably altered the landscape of cancer therapeutics, identification of patients likely to respond to CDK4/6 inhibition remains a challenge. Previously, we used our chemoproteomics platform (KiNativ), which measures competition of an ATP- or ADP-acyl-phosphate probe, to profile lysates generated from cells that were treated with palbociclib. We showed that palbociclib-CDK4 target engagement is cell line specific and that engagement correlates with sensitivity of the cell line to palbociclib-mediated growth inhibition. However, the circumstances that determine whether target engagement is achieved were not understood. In an effort to understand the molecular mechanisms that influence CDK4 target engagement, we used the same proteomics platform to interrogate the contribution of various known CDK regulators. We identified a family of proteins that prevents palbociclib target engagement upon overexpression and whose lack of expression correlates with palbociclib sensitivity. This interaction is specific for CDK4/6 as modulation of the drug's interaction with off-target kinases was not affected by overexpression of these proteins. Furthermore, we found that these proteins similarly prevent CDK4 target engagement by ribociclib and abemaciclib. Incorporation of the proteins identified here into a biomarker strategy should help to enrich for patients likely to respond to CDK4/6 inhibition therapy and expand the utility of these inhibitors.

#2312

Phospho-proteomics analysis to determine the signaling pathways affected by a novel CDC7-selective inhibitor TAK-931.

Kenichi Iwai,1 Masamitsu Gotou,1 Kazunori Yamanaka,1 Akihiro Ohashi2. 1 _Takeda Pharmaceutical Company Ltd., Fujisawa, Japan;_ 2 _Takeda Pharmaceuticals International Co., Cambridge, MA_.

Background: Cell division cycle 7 (CDC7) is a serine/threonine kinase, which plays important roles in initiation of DNA replication by phosphorylating MCM2. We developed the CDC7-selective small molecule inhibitor TAK-931. Treatment with TAK-931 results in delayed S phase progression, DNA replication stress (RS), centrosome disorganization, mitotic aberrations, and potent growth suppression in cancer cells. In this study, we conducted the time-dependent phospho-proteomics analysis to determine the signaling pathways affected by TAK-931.

Materials and Methods: TAK-931 was synthesized by Takeda Pharmaceutical Company, Ltd. The SILAC-labeled COLO205 cells were treated with TAK-931 at 100 nM for 0, 4, 24 hours, applying for the phospho-proteomics analysis in the PhosphoScout® Service at Evotec AG (Munich, Germany).

Results: The SILAC-labeled COLO205 cells with TAK-931 treatment were subjected to quantitative mass spectrometry, detecting 15,128 phosphorylation sites of the 4,426 phosphoproteins. At 4 h of treatment, only 2 phosphorylation sites were detected as significantly downregulated: MAX gene-associated protein (MGA) and alpha-enolase (ENO1). At 24 h of treatment, however, 51 phosphorylation sites were identified as being significantly altered, including 44 upregulation and 7 downregulation sites. The gene ontology (GO) enrichment analysis in the categories of biological process (BP) and cellular component (CC) revealed that 10 GO terms from BP and 2 GO terms from CC were significantly enriched in the treated cells after 24 h: cell cycle, cell cycle phase, cell cycle process, mitosis, nuclear division, organelle fusion, organelle organization, mitotic cell cycle, M phase of mitotic cell cycle, M phase, chromosomal part, and spindles. Network analysis on the changed phosphorylation prolife also revealed that significant network modules existed at 24 h of treatment, which included DNA replication/damage, mitotic progression/spindle assembly, nucleus assembly, GTPase activity, cell migration, cell adhesion, TP53, and protein translation.

Conclusion: The time-dependent phospho-proteomics analysis revealed the dynamics of phospho-signal transduction affected by TAK-931 in the cancer cell, which may help to provide a deeper understanding of the molecular mechanisms of TAK-931.

#2313

Effect of dinaciclib on drug-naive and letrozole- and palbociclib-resistant breast cancer.

Armina A. Kazi,1 Emily Shukdinas,1 Adam Schneider,1 Antony Sare,1 Saranya Chumsri2. 1 _Loyola University MD, Baltimore, MD;_ 2 _Mayo Clinic, Jacksonville, FL_.

Estrogen receptor-positive (ER+) breast cancer is the most common form of breast cancer. Therapeutic strategies to treat this breast cancer subtype have included aromatase inhibitors, such as letrozole and, more recently, letrozole in combination with cyclin-dependent kinase 4/6 inhibitor palbociclib. Despite the proven effectiveness of both these treatments, it has been shown in the lab and/or clinic that breast cancer cells can acquire resistance to them. Thus, there is a need to 1) further characterize breast cancer cells that have developed resistance to letrozole alone or in combination with palbociclib and 2) explore new treatment strategies that might be effective in treating both drug-sensitive and drug-resistant breast cancer. In this current study, drug-naïve (MCF7Ca), letrozole-resistant (LTLTCa), and letrozole and palbociclib-resistant (MCF7Calet+palb) breast cancer cells were investigated by Western blot analysis, MTT assays, mammosphere assays, and migration/invasion assay. Western blot analyses demonstrated that all cell lines expressed phosphorylated and total cyclin-dependent kinase 1 (CDK1) and -2 (CDK2), although LTLTCa cells and MCF7Calet+palb cells expressed higher levels of phosphorylated CDK2 (0.9-fold and 2.5-fold, respectively) and/or CDK1 (1.6-fold and 0.4-fold, respectively) compared to MCF7Ca cells. It was further observed that expression of breast cancer resistance protein (BCRP) was significantly increased in LTLTCa and MCF7Calet+palb cells (1.2-fold and 2.11-fold), while cell cycle arrest protein p21 (1.2-fold and 0.4-fold) and/or apoptotic cleaved PARP (0.09-fold and 0.12-fold) were significantly decreased in LTLTCa and MCF7Calet+palb cells. Based on Western blot results, the effect of dinaciclib, a broader-range CDK inhibitor that can target CDK1 and CDK2, was studied. In MTT assays, dinaciclib was shown to be more effective in inhibiting cell viability than palbociclib in all three cell lines. Dinaclicib's IC50s were 1.4 nM in MC7Ca, 9.5 nM in LTLTCa, 12.4 nM in MCF7Calet+palb cells, while palbociclib IC50s were 1.5 uM, 11.2 uM, and 118 uM, respectively. Dinaciclib also significantly reduced mammosphere formation 0.01-fold vs. untreated in both MCF7Ca and MCF7Calet+palb cells. Lastly, preliminary results indicate migration of MCF7Ca cells was significantly reduced by dinaciclib (27.6±4.9 vs. 733.7±33.8 migrated cells in untreated). Overall, these results suggest that acquired resistance to letrozole alone or in combination with palbociclib induces changes in breast cancer cells, and that dinaciclib may be an effective alternative treatment for drug-naïve ER+ breast cancer, letrozole- and letrozole- and palbociclib-resistant breast cancer.

#2314

Preclinical development of a highly potent and selective dual inhibitor of CDK4/6 for the treatment of cancer.

Laychiluh Bantie, Solomon Tadesse, Jimma Likisa, Mingfeng Yu, Benjamin Noll, Hugo Albrecht, Robert Milne, Shudong Wang. _University of South Australia, Adelaide, Australia_.

Cyclin-dependent kinases 4 and 6 (CDK4/6) in concert with D-type cyclins, are vital drivers of the cell cycle. Activated CDK4/6-cyclin D complexes regulate cell proliferation, senescence and apoptosis via phosphorylation of proteins including Rb, FOXM1 and SMAD3. Deregulation of the CDK4/6-cyclin D-Rb-E2F pathway has been found in over 90% of human cancers, and amplification of genes coding for cyclin D1 (CCND1) and CDK4/6, or deletions of the locus encoding p16INK4a are the major mechanisms. For instance, CCDN1 amplification and cyclin D protein overexpression are seen in ~ 35% and ≥ 50% of breast cancer cases, respectively. Similarly, ≥ 40% of prostate cancers overexpress CDK6 while loss of p16INK4a occurs in ~ 80% of head and neck squamous cell carcinoma. Specific mutations or translocations in the genes encoding CDK4/6-cyclin D-Rb-E2F pathway are aberrantly activated in melanoma and mantle cell lymphoma. Therefore, CDK4/6 inhibition can be an effective treatment strategy for these cancers.

With our extensive medicinal chemistry, biochemical and cellular assays, in vivo pharmacokinetic, safety and efficacy studies, we have identified a highly potent dual inhibitor of CDK4/6, 3-14, that showed excellent selectivity over a panel of >360 kinases. 3-14 is more potent anti-proliferative agent than palbociclib against a range of cancer cell lines including breast, ovarian, colon, prostate, pancreatic, lung and melanoma cancer cells. 3-14 arrested cells in G1 phase of the cell cycle, prevented colony formation and induced senescence in MCF7 breast cancer cells. 3-14 caused dose dependent inhibition of Rb phosphorylation at S780, S795 and S807/11 residues, and this was translated to the reduction in the levels of E2F regulated proteins such as cyclin E2, A2. Moreover, 3-14 possessed excellent pharmaceutical properties, and pharmacokinetics with high oral bioavailability in rodents. 3-14 (MTD = 250 mg/kg) is better tolerated than palbociclib (MTD = 150 mg/kg) in mice. In A2780 ovarian cancer xenograft, 3-14 (100 mg/kg p.o. daily) delayed tumor growth (i.e. T/C = 24.4 %) and increased animal life span (90%), and was more efficacious

compared to palbociclib (i.e. T/C = 38% and ILS = 73%). Both 3-14 and palbociclib didn't cause histopathological changes in the bone marrow, intestine, liver, heart and kidney except for the non-significant reduction in the neutrophils. Given its excellent efficacy and safety as well as pharmacokinetic profiles, 3-14 is warranted as a development candidate for further preclinical and clinical studies.

#2315

Inhibition of cyclin-dependent kinase 4 as a potential therapeutic strategy for treatment of synovial sarcoma.

Xiaoyang Li,1 Cassandra Garbutt,2 Francis John Hornicek,1 Zhenfeng Duan1. 1 _David Geffen School of Medicine at UCLA, Los Angeles, CA;_ 2 _Massachusetts General Hospital and Harvard Medical School, Boston, MA_.

Synovial sarcoma is a rare and aggressive form of soft tissue cancer that affects the extremities of the arms or legs, for which current chemotherapeutic agents have not been proven to be very effective. The cyclin-dependent kinase 4/6-retinoblastoma protein (CDK4/6-Rb) pathway is aberrant in a large proportion of cancer. Recent evidences on pre-clinical application of CDK4 inhibitors have been implicated in many types of human cancers, and the FDA has approved the CDK4 selective inhibitor for the treatment of breast cancer. However, the expression and therapeutic potential of CDK4 in synovial sarcoma remain unclear. In the present study, we examined the expression of CDK4 in synovial sarcoma cell lines by western blot and immunofluorescence assay, and in synovial sarcoma tissue microassays by immunohistochemical analysis. Cell viabilities were determined by MTT assay after exposure to different dosages of the selective CDK4 inhibitor. Flow cytometry analysis and wound healing assay were conducted to determine the mechanisms underlying the cytotoxic effects of the selective CDK4 inhibitor. CDK4 specific small interference RNA was used to validate the effect of targeting CDK4 by the selective CDK4 inhibitor in synovial sarcoma cells. We found that CDK4 was highly expressed in human synovial sarcoma, and was related to clinical stage and TNM grade in synovial sarcoma patients and poor prognosis in sarcoma patients. Cell viabilities determined by MTT assay after exposure to different dosages of the selective CDK4 inhibitor showed that this selective CDK4 inhibitor repressed synovial sarcoma cell proliferation and growth in a dose- and time- dependent manner. The selective CDK4 inhibitor inhibited the CDK4/6-Rb signaling pathway and promoted cell apoptosis without influence on the expression of CDK4/6, suggesting that the selective CDK4 inhibitor only repressed the hyperactivation, not the production of CDK4/6. The inhibition effect of the selective CDK4 inhibitor was confirmed by knockdown of CDK4 with specific small interference RNA. Flow cytometry analysis revealed that the selective CDK4 inhibitor induced G1 cell-cycle arrest by targeting CDK4/6-Rb pathway in synovial sarcoma cells. Furthermore, the wound healing assay exhibited that inhibition of CDK4/6-Rb pathway with the use of the selective CDK4 inhibitor significantly decreased synovial sarcoma cell migration in vitro. Our data highlight the role of dysregulated CDK4/6-Rb pathway and current selective CDK4/6 inhibitor may be a potential promising therapeutic agent in the targeted treatment of human synovial sarcoma.

#2316

In vitro and in vivo anticancer potential of adapalene, a third-generation retinoid against bladder cancer.

Jeevan Ghosalkar, Srikanth Iyer, Geena Malhotra, Kalpana Joshi. _Cipla Ltd, Mumbai, India_.

Bladder cancer is the ninth most common cause of cancer, with the highest recurrence rate of any malignancy. By 2025, the seven major markets for bladder cancer are expected to reach $1.17B. The disease is highly prevalent in developed nations. While smoking and industrialization have been closely linked to the development of the disease, the chances of new cases are expected to rise in the coming years. The current chemotherapy strategy involves myriad standard of care (SOC) drugs, including the promising regimen of Programmed Cell Death-1 (PD-1) modulators. However, they exhibit several limitations like adverse effects and drug resistance followed by relapse. Hence, there remains an unmet medical need in bladder cancer treatment including patients who do not respond following PD-1 therapy. In this scenario, alternate drug therapy, with a novel mechanism of action would be the path forward. This study attempts to establish the role of adapalene in the treatment of bladder cancer and to elucidate the underlying mechanism of action. Adapalene is a FDA approved, third generation synthetic retinoid drug, mainly used as a topical application in the treatment of acne vulgaris. It has been reported to inhibit cyclin-dependent kinase 2, which is an important regulator in progressing the cell cycle from G1 to S phase. This brings about a cell cycle arrest, consequently leading to apoptosis of the tumor cells. Few reports implicate its efficacy in colorectal and hepatoma carcinoma cell lines. However, its anti-cancer efficacy against bladder cancer as a stand-alone and/or in combination with the SOC drug has not yet been reported. Adapalene anti-proliferative potential was tested against a panel of five bladder cancer cell lines, including patient derived cells (PDX). The results indicated a dose dependent cytotoxicity with a mean IC50 of 6.84 µM and 14.01 µM in a 2D and 3D setup respectively. Further, we studied the effect of adapalene alone or in combination with cisplatin in colony formation assay using bladder cancer cell lines. Interestingly, it showed a synergistic effect in combination with cisplatin in 1218L (PDX) and T24; while the effect was additive in case of 1036L (PDX). Similarly, adapalene by itself and in combination showed significant apoptosis in HTB5 and HTB9 cells. Likewise, differential gene and protein expression studies using drug alone and combination showed promising results. Additionally, in vivo efficacy study was performed in 1218L model using adapalene alone and in combination with cisplatin. Adapalene by itself showed a significant antitumor efficacy. Notably, the combination group shows tumor growth inhibition by 78% as compared to cisplatin and adapalene alone, where the growth inhibition was 64% and 59% respectively. Thus, the promising anti-tumor efficacy of adapalene clearly demonstrates that it could be a potential candidate alone and in combination against bladder cancer.

#2317

Palbociclib monotherapy exhibits potent activity in cervical cancer cell lines.

Peter Van Dam,1 An Wouters,2 Christian Rolfo1. 1 _Antwerp University Hospital, Edegem, Belgium;_ 2 _Antwerp University, Edegem, Belgium_.

Background: Over the last decades, little progress has been made in the systemic treatment of patients with advanced or recurrent cervical cancer. We recently performed an silico analysis to identify potential driver pathways of cervical carcinogenesis and candidate targets for treatment.1 Expression2Kinases (E2K) analysis revealed a protein-protein interaction (PPI) network of 162 nodes (including 20 druggable kinases) consisting of 5 signaling modules associated with MYC signaling (module 1), cell cycle deregulation and cyclin signaling (module 2), TGFβ-signaling (module 3), a PI3K - MAPK signaling (module 4) and chromatine modeling (module 5).

Methods: The cervical cancer cell lines included in the study were CaSki, SiHa and HeLa. Cells were incubated for 24 or 72 hours with the PI3K pathway inhibitor BYL719 (Novartis), the multiple pathway inhibitor INC280 (inhibits cMET-dependent PI3K and RAS signaling, Novartis), or the CDK4/6 inhibitor PD-0332991 (palbociclib, Selleck Chemicals) in monotherapy. Sensitivity to drug treatment (0 - 10 μM) was investigated using the colorimetric sulforhodamine B (SRB) assay. IC50 values were calculated using WinNonlin Software.

Results: Incubation with BYL719 or INC280 for 24 or 72 hours in concentrations up to 10 μM was not able to establish a distinct cytotoxic effect in the three cervical cancer cell lines included in the study. Incubation with 10 μM BYL719 for 72h resulted in only 20% cell kill, while 10 μM INC280 for 72h caused around 30% cell kill in all three cell lines. However, incubation with palbociclib for 72 hours clearly induced a concentration-dependent cytotoxic effect, with IC50 values of 5.32 ± 0.03 μM, 7.69 ± 0.02 μM and 5.68 ± 0.12 μM for HeLa, SiHa and CaSki cells, respectively.

Conclusion: CDK4/6 inhibition seems effective in cervical cancer cell lines and should be further evaluated alone and in combination with chemotherapy for the treatment of advanced and recurrent cervical cancer.

References:

  1. Van Dam P, van Dam PJ, Rolfo C, Giallombardo M, Van Berckelaer C, Trin XB, Altintas S, Huizing M, Papadimitriou K, Tjalma W, Van Laere S. In silico pathway analysis in cervical carcinoma reveals potential new targets for treatment. Oncotarget 2016;7(3):2780-2795

#2318

Palbociclib, a CDK4/6 inhibitor, suppresses proliferation of triple negative breast cancer.

Ahrum Min,1 Yu Jin Kim,1 Hyemin Hang,1 Jee Min Lim,2 Seongyeong Kim,2 So Hyeon Kim,2 Koung Jin Suh,3 Kyung-Hun Lee,3 Tae-Yong Kim,3 Seock-Ah Im3. 1 _Seoul National University, Biomedical Research Institute, Seoul, Republic of Korea;_ 2 _Seoul National University, Cancer Research Institute, Seoul, Republic of Korea;_ 3 _Seoul National University Hospital, Seoul, Republic of Korea_.

Background: Abnormal cell cycle progression is a characteristic of cancer and targeting the cell cycle is a strategy for cancer treatment. TCGA reported that Cyclin D1 is overexpressed in 25% to 60% of invasive breast carcinomas, and CDK4/6 and CDKN2A/B aberrations are observed in 7.6% of breast cancer cases. Palbociclib is a specific inhibitor of CDK4/6, a key regulator of the G1 checkpoint. In hormone receptor positive breast cancer, the addition of palbociclib to endocrine treatment either aromatase inhibitor or fulvestrant significantly prolonged PFS in phase III trials. However, the effects of palbociclib have not been fully examined in triple negative breast cancer (TNBC) yet, although CCND amplification and CDKN2A loss are frequently observed in TNBC. Therefore, we investigated the effects of palbociclib on TNBC cells and attempted to identify the underlying mechanisms of palbociclib.

Methods: The cytotoxicity assay, cell cycle analysis and western blotting were conducted to determine anti-tumor effect and action mechanisms of palbociclib on breast cancer cell lines. These in vitro data were validated in vivo model as well.

Results: Human breast cancer cell lines showed heterogeneous response to palbociclib. Palbociclib induced G1 cell cycle arrest by blocking Rb phosphorylation, and inhibited cell proliferative signaling. In sensitive TNBC cells, palbociclib promoted senescence rather than apoptosis. In addition, palbociclib enhanced the antitumor effect of 5-FU by modulating thymidine synthase (TS) and E2F1 expression. The anti-tumor effects of palbociclib on TNBC cells were validated in TNBC-xenograft model as well.

Conclusions: CDK4/6 inhibitor palbociclib showed anti-tumor effect in vitro and in vivo xenograft model of TNBC and acts synergistically with 5-FU by downregulating TS. Our results suggest that palbociclib has therapeutic potential for the treatment of TNBC, not limited in hormone positive breast cancer type. Our results provide a rationale for the future clinical trials of palbociclib in the treatment of breast cancers.

#2319

The role of CDKN1A and CDKN2B in the reprogramming of AML cells toward cell cycle arrest.

Michael Roberts, Grace Crossland, Jeffrey Forrester. _Dickinson College, Carlisle, PA_.

AML is the most common acute leukemia in adults, showing rapid progression if left untreated. With current treatments, AML patients frequently achieve remission, but often experience relapse leading to the overall poor prognosis and high mortality of this cancer. We have employed human AML cell lines to examine the response of leukemia cells to phorbol esters. Phorbol esters mimic the action of diacyl glycerol (DAG), an activator of protein kinase C, which regulates several signal transduction pathways and cellular metabolic activities. In response to the phorbol ester PMA, AML cells undergo cell cycle arrest, differentiate into "normal" macrophage-like cells, and ultimately undergo programmed cell death. The transcriptome variations that accompany these changes have been explored by DNA microarray analysis and quantitative real-time PCR, revealing ~1250 genes that are differentially expressed as the cells transition from the cancer state to non-dividing, myeloid cells that initiate apoptosis. Two genes of great interest regarding cell cycle arrest are the cyclin-dependent kinase inhibitors CDKN1A and CDKN2B. We have shown that both of these genes are expressed at extremely low levels in AML cells, but are rapidly up-regulated during the PMA response, in keeping with their proposed function as tumor suppressor genes. We present here results from the transfection of expression plasmids containing the CDKN1A or CDKN2B genes into HL-60 cells, showing ~100 and ~1,000-fold overexpression, respectively. In addition, we have engineered a plasmid containing both genes and demonstrate their co-overexpression. RNA samples from cells transfected with CDKN1A, CDKN2B, and CDKN1A-CDKN2B have been subjected to RNA sequencing. RNA-seq analysis reveals changes in CDKN1A and CDKN2B-mediated gene expression, altering the AML transcriptome in ways that facilitate cell cycle arrest. Finally, we present results on the phenotypic and transcriptome changes of a stable HL-60 cell line containing an inducible CDKN2B gene after dox-induced overexpression, implicating CDKN2B as an important regulator of cell cycle progression in AML cells.

### Ferroptosis, Metabolism, and Cancer Cell Death

#2320

Heme oxygenase-1 mediates ferroptosis.

Shih-Kai Chiang,1 Shuen-Ei Chen,1 Ling-Chu Chang2. 1 _National Chung Hsing University, Taichung, Taiwan;_ 2 _China Medical University Hospital, Taichung, Taiwan_.

Ferroptosis is a form of oxidative cell death and has become a chemotherapeutic target for cancer treatment. BAY 11-7085 (BAY), which is a well-known IκBα inhibitor, suppressed viability in cancer cells via induction of ferroptotic death in an NF-κB-independent manner. This study was aimed to characterize the underlying mechanism by BAY and demonstrate its potential chemotherapeutic targets for cancer treatment. Reactive oxygen species scavenging, relief of lipid peroxidation, replenishment of glutathione and thiol-containing agents, as well as iron chelation, rescued BAY-induced cell death in human breast cancer cells (MDA-MB-231 and MDA-MB-468) and glioblastoma cells (DBTRG-05MG). The results from quantitative real-time PCR and western blotting analysis showed that BAY upregulated a variety of nuclear factor-E2-related factor 2 (Nrf2) target genes related to redox regulation, particularly heme oxygenase-1 (HO-1) and solute carrier family 7 membrane 1 (SLC7A11). Studies with pharmacological inhibitors and shRNA interventions suggested that the hierarchy of induction is Nrf2-SLC7A11-HO-1. The ferroptotic process induced by hHO-1 overexpression further indicated that HO-1 is a key mediator of BAY-induced ferroptosis that operates through cellular redox regulation and iron accumulation. Our study discovered that BAY induced ferroptosis via Nrf2-SLC7A11-HO-1 pathway and provided new insights to understand the mechanism of HO-1-induced ferroptosis, suggesting HO-1 as a new chemotherapeutic target for cancer treatment.

#2321

Molecular crosstalk between ferroptosis and apoptosis: Emerging role of ER stress-induced p53-independent PUMA expression.

Young-Sun Lee,1 Se Hoon Hong,1 Dae-Hee Lee,2 Min J. Jo,2 Yoo A. Jeong,2 William T. Kwon,1 Haroon A. Choudry,1 David L. Bartlett,1 Yong J. Lee1. 1 _Univ. of Pittsburgh Cancer Inst., Pittsburgh, PA;_ 2 _Korea University, Seoul, Republic of Korea_.

Ferroptosis is a type of programmed cell death that depends on iron and is characterized by the accumulation of lipid peroxides. In the present study, we investigated the nature of the interplay between ferroptosis and other forms of cell death such as apoptosis. Human pancreatic cancer PANC-1 and BxPC-3 and human colorectal cancer HCT116 cells were treated with ferroptotic agents such as erastin and artesunate (ART) in combination with the apoptotic agent tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). We observed synergistic interaction of erastin or ART with TRAIL as determined by cell death assay, caspase activation, poly [ADP-ribose] polymerase 1 (PARP-1) cleavage, flow cytometry analysis, and lipid peroxidation assay. Moreover, erastin and ART induced endoplasmic reticulum (ER) stress and promoted p53 upregulated modulator of apoptosis (PUMA) expression via C/EBP-homologous protein (CHOP). Synergy of erastin/ART and TRAIL was abolished in PUMA-deficient HCT116 cells and CHOP-deficient mouse embryonic fibroblasts, but not in p53-deficient HCT116 cells. The results suggest the involvement of the p53-independent CHOP/PUMA axis in response to ferroptosis inducers, which may play a key role in ferroptotic agent-mediated sensitization to TRAIL-induced apoptosis.

#2322

Ferroptosis induced by erastin in RAS mutant ovarian cancer cells.

Motoki Takenaka,1 Noriko Suzuki,1 Minako Mori,1 Hitomi Aoki,1 Tasuku Hirayama,2 Hideko Nagasawa,2 Ken-ichirou Morishige1. 1 _Gifu University School of Medicine, Gifu, Japan;_ 2 _Gifu Pharmaceutical University, Gifu, Japan_.

Although apoptosis has been thought as the principal mechanism of programed cell death in chemotherapy, other forms of non-apoptotic death also has been noted. The oncogenic RAS-selective lethal small molecule erastin triggers a unique iron-dependent form of cell death termed ferroptosis. This mechanism is expected to induce eradication of chemotherapeutic (e.g. platinum) resistant cancer cells. In this study we demonstrate this new form of cell death in ovarian cancer cells. The clear cell epithelial ovarian cancer cell lines, TOV-21G (KRAS_G13C) and KOC-7C (RAS wild type) were used in this study. Erastin dose-dependently inhibited growth in TOV-21G cells, but not KOC-7C cells. Treatment of TOV-21G cells with erastin resulted in increase of ROS. Iron chelator deferoxamine (DFO) rescued growth inhibition induced by erastin in TOV-21G cells. Although cisplatin inhibited growth in TOV-21G cells, this inhibition was not rescued by DFO. The GSH level was reduced by erastin, but this reduction was not rescued by DFO. In addition, the basal GSH level was higher and the basal ROS level was lower in KOC-7C cells than those in TOV-21G cells. Erastin induced ferroptosis by reducing the GSH level in RAS mutant ovarian cancer cells. Although DFO rescued growth inhibition induced by erastin, the pathway was not on System Xc-. The sensitivity to erastin might depends on the basal level of GSH in cells. Ferroptosis had great potential to become a new approach in anti-tumor therapies in ovarian cancer.

#2323

Involvement of glutamine in tolerance of human pancreatic cancer cell to inhibition of fatty acid synthesis.

Koji Nishi, Mina Suzuki, Noriko Yamamoto, Yumiko Iwase, Nagahiko Yumita. _Yokohama University of Pharmacy, Yokohama, Japan_.

Pancreatic cancer (PC) is the fifth leading cause of cancer-related mortality in Japan. Although the standard chemotherapy regimens, FOLFIRINOX (fluorouracil, leucovorin, irinotecan and oxaliplatin), gemcitabine and nab-paclitaxel, are widely used to treat patients with advanced pancreatic cancer, their antitumor effects (progression and survival rates) are less potent against this cancer type than other solid tumors. Previously, we found that inhibition of acetyl-CoA carboxylase (ACC), a major rate-controlling enzyme of fatty acid synthetic pathway, suppressed the proliferation of PC cells. However, in this study, we found the tolerance to the inhibition in a part of PC cells. Therefore, we investigated the mechanism of the tolerance to ACC inhibition in PC cells. The human PC cell lines, AsPC-1, BxPC-3 and PANC-1 were obtained from the American Type Culture Collection. Live and apoptotic cell numbers were determined using the MUSE Annexin V and Dead Cell Kit according to the manufacturer's instructions. PANC-1 cells showed the tolerance to inhibition of fatty acid synthesis by ACC inhibitor, 5-(tetradecyloxy)-2-furoic acid (TOFA), whereas TOFA induced apoptosis in AsPC-1 and BxPC-3 cells. In addition, PANC-1 did not show autophagy even under the condition of fatty acid starvation. Next, we investigated whether PANC-1 has the tolerance to nutrient starvation in nutrient-depletion medium, Earle's Balanced Salt Solution (EBSS) and serum-free medium respectively. PANC-1 showed the survival even in such mediums, whereas AsPC-1 and BxPC-3 almost dead. This result suggests that the tolerance of fatty acid starvation of PANC-1 are related to that of nutrient starvation. Interestingly, proliferation of PANC-1 cultured in glutamine-depletion medium dramatically was suppressed, even though low glucose medium did not affect. BPTES, a selective inhibitor of glutaminase, also suppressed the proliferation and induced apoptosis in PANC-1, whereas there are little effects of 2-dexy-glucose, inhibitor of glycolysis, on them. These results suggest that the TCA cycle plays an important role on the tolerance to inhibition of fatty acid synthesis and nutrient starvation of PANC-1, and can be novel therapeutic target of PC cells which have the tolerance to nutrient starvation like PANC-1.

#2324

Docosahexaenoic acid-induced Nrf2 may be correlated with cell death by amplification of oxidative stress via induction of klf9 in cervical cancer cells.

Kaipeng Jing,1 Soyeon Shin,1 Seung-Hyeon Han,2 Young-Joo Jeon,2 Jun-Young Heo,2 Gi-Ryang Kweon,2 Seung-Kiel Park,1 Jong-Il Park,1 Kyu Lim3. 1 _Dept. of Biochemistry, School of Medicine, Chungnam National University., Daejeon, Republic of Korea;_ 2 _Dept. of Biochemistry, Department of Medical Science, School of Medicine, Chungnam National University, Daejeon, Republic of Korea;_ 3 _Dept. of Biochemistry, Department of Medical Science, School of Medicine, Cancer Research Institute, Chungnam National University, Daejeon, Republic of Korea_.

NF-E2-related transcription factor-2 (Nrf2) is a key transcriptional factor regulating antioxidant gene expression and protection from oxidant damage. Docosahexaenoic acid (DHA) as one of ω3-polyunsaturated fatty acids has anticancer action through several mechanisms. Here we report that DHA-induced Nrf2 is related to cell death, not survival in HeLa cell. We confirmed DHA-induced apoptotic cell death using PARP cleavage, Anexin V staing, and TUNEL positive staining assay. DHA-triggering ROS production increased the level Nrf2 and decreased keap1 in dose- and time-dependent manner. Increased Nrf2 by DHA was partially Keap1 dependent. DHA activated Nrf2 signaling by increasing its translation and the Nrf2 was translocated from cytosol to nucleus. Upregulation of DHA-mediated Nrf2 was also confirmed by increase of heme oxygenase-1 (HO-1) expression. DHA-induced Nrf2 increased the level of Kruppel-like factor 9 (klf9) and then decreased thioredoxin reductase 2 (Txnrd2) in dose- and time-dependent manner. These results suggest that DHA-induced ROS increases Nrf2 and the Nrf2 is related to cell death by increase of klf9 and decrease of Txnrd2 in HeLa cells. Thus, utilization of ω3-PUFAs may represent a promising therapeutic approach for chemoprevention and treatment of human cervical cancer [This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (NRF-2015R1D1A1A01056887) and by the framework of international cooperation program managed by National Research Foundation of Korea (2015K2A2A6002008)]

#2325

Protein homeostasis adaptation to Hsp70 inhibition in cancer.

Sara Sannino,1 Christopher J. Guerriero,1 Amit J. Sabnis,2 Trever G. Bivona,2 Jeffrey L. Brodsky1. 1 _Univ. of Pittsburgh, Pittsburgh, PA;_ 2 _University of California, San Francisco, CA_.

Cancer cells experience acute stress conditions due to their increased proliferation rate and synthesis of misfolded proteins. However, by modulating the mechanisms that control protein-folding, cancer cells avoid cell death. Thus, it is perhaps not surprising that molecular chaperones, like Hsp70, are upregulated in cancer cells compared to their normal counterparts. These data suggest that these chaperones are potential targets for cancer therapy. Previous work in our lab demonstrated the dependence of patient-driven rhabdomyosarcoma cell survival on cytoplasmic Hsp70 activity, thanks to the use of a specific Hsp70 inhibitor, MAL3-101. In particular, we discovered that treatment of on RMS13 cell line with an Hsp70 inhibitor, known as MAL3-101, activates the PERK driven unfolded protein response that results in CHOP-dependent cell death (Sabinis et al., 2016). Nevertheless, the mechanism underlying how Hsp70 inhibition triggered apoptosis was poorly understood. By taking advantage of a MAL3-101-resistant cell line (RMS13-R), we have now determined which compensatory mechanism alters MAL3-101-driven cell death. We found that both endoplasmic reticulum-associated degradation (ERAD) and autophagy are upregulated in RMS13-R cells, underlying the increased demand on two protein degradation pathways upon inhibition of Hsp70. Specifically, autophagy related genes were upregulated, and increased conversion of LC3BI to LC3BII and accumulation of LC3BII was detected in RMS13-R cells. Further experiments demonstrated that autophagy was further induced by MAL3-101 treatment in RMS13-R cells, as evidenced by an increase in the messages and proteins corresponding to key autophagy components. Finally, we discovered that only autophagy inhibition—but not inhibition of ERAD—re-sensitized RMS13-R cells to Hsp70 inhibition, which was apparent from an induction of apoptotic markers and cell death. These data highlight a pro-survival role for autophagy induction upon exposure to an Hsp70 inhibitor in cancer, and provide a link between Hsp70, proteasomal degradation, the unfolded protein response, and autophagy in rhabdomyosarcoma.

#2326

Time-resolved studies of necroptosis reveal distinct regulatory functions for NFκB.

Marie Oliver Metzig,1 Simon Mitchell,1 Brooks Taylor,2 Alexander Hoffmann1. 1 _University of California, Los Angeles, CA;_ 2 _Stanford University, Stanford, CA_.

Resistance to regulated cell death (RCD) is an important hallmark of cancer and reason why chemotherapy fails. Efficient reprogramming of cancer cells requires a molecular and dynamic understanding of regulatory circuits. Research has uncovered several resistance mechanisms for apoptosis with nuclear factor κB (NFκB) as a key inhibitory transcription factor. Targeted induction of necroptosis, which was recently discovered as an alternative form of RCD, could be a promising strategy to overcome apoptosis resistance. However, whether NFκB has a regulatory role in necroptosis is less clear. Here, we develop a molecular and time-resolved understanding of how NFκB regulates necroptosis. We present a real-time experimental system to quantify necroptosis in single cancer cells to study the cytotoxic effect of substances while avoiding the bias of proliferation in endpoint protocols. Using live-cell microscopy, computational cell tracking and automated image analysis we quantitate necroptotic death rates in L929 cells, a murine fibrosarcoma cell line, treated with tumor necrosis factor (TNF). While it is known that L929 cells predominantly respond with necroptosis despite rapid activation of NFκB, our experiments reveal an unsuspected temporal phasing of necroptosis over a time course of 24 hours. By perturbing NFκB activation dynamics using targeted CRISPR/Cas9 of IκB and/or Rel proteins, we find that NFκB has at least two distinct roles regulating early and late phases of TNF-induced necroptosis. Further, we will present our molecular analysis that identified NFκB-responsive candidate genes and mapped them into a functional temporo-spatial context within the NFκB-necroptosis signaling network. Our combined approach may inform novel personalized anticancer strategies to exploit targeted necroptosis while avoiding NFκB-induced drug resistance.

#2327

Azole antifungal drugs induce cell death by suppressing mTOR through PI3K/Akt inhibition in human breast cancer.

Ju Ho Park,1 Hyesook Kim,2 So Hee Kim1. 1 _Ajou Univ., Suwon, Republic of Korea;_ 2 _Detroit R &D, Detroit, MI_.

Breast cancer is a major cancer in women and its incidence rate increases every year. So it needs a perfect treatment. Azole drugs structurally have an imidazole [clotrimazole (CTZ) and ketoconazole (KCZ)] or a triazole [fluconazole (FCZ) and itraconazole (ICZ)]. Recently, azole antifungal drugs have shown anticancer efficacy in clinical trial. However, their antitumor mechanism are vague. Here, we evaluated the effects, as apoptosis, cell arrest, and autophagy and tumor growth using human breast cancer cells. Apoptosis assay showed imidazole-induced cell death, pro-apoptosis maker, as cleaved PARP, cleaved caspase-3, Bax, increased and anti-apoptosis maker, as Bcl-2, decreased by CTZ and KCZ in both MCF-7 and MDA-MB-231 cells. We also checked imidazole of azole drugs blocked the cell cycle in G1 phage by downregulating the protein levels of cyclin D1, CDK2, and CDK6 and increased p21 and p27 in breast cancer. Further experiments showed that azole drugs also induced autophagy through the increased accumulation of LC 3B inside the cells by immunoblot analysis and immunofluorescence staining, increased beclin-1 and decreased p62 protein. With azole decreased that though 3-MA. Azole drugs inhibited PI3K/Akt/mTOR signaling pathway. Tumor growth was also inhibited in nude mice bearing MDA-MB-231 xenograft by CTZ, KCZ and ICZ through the accumulation of LC 3B in tumor xenograft. In conclusion, imidazole antifungal drugs induced apoptosis, cell cycle and autophagy by suppressing mTOR through the regulation of PI3K/Akt pathway in human breast cancer cells, suggesting azole antifungal drugs could be new potential therapeutics for breast cancer.

#2328

ADCs that induce necrosis and thwart apoptotic resistance.

James R. Prudent, David Marshall. _Centrose LLC, Madison, WI_.

Purpose: We have previously shown that targeting the Na,K-ATPase (NKA) with antibody drug conjugates is a powerful method for reducing tumors at levels found to be safe in cynomolgus monkeys. Currently no approved cancer drugs induce a potent necrotic type cell death even though experimental evidence shows that necrotic cell death has many notable and untapped benefits. Therefore, we wanted to better understand the driving mechanism by which ADCs targeting NKA inhibition induce necrosis.

Experimental Design: Various cancer cell types were subjected to increasing and decreasing concentrations of Na+, K+ and Ca++, as well as various ADCs using bufalin as the warhead/payload/toxin. The cells were also subjected to siRNAs specific to the NKA and bufalin alone. The cells were then analyzed for the appearance and/or presence of necrosis, apoptosis, and markers indicating various stress pathways.

Results: Conditions found to induce necrosis included: ADCs targeting specific NKA-protein complexes, cells subjected to media containing increased concentrations of K+, the specific Na,K-ATPase siRNAs, and bufalin alone.

Conditions not found to induce necrosis included: media containing increased Na+, decreased Na+ or K+, control siRNAs, or ADCs that targeted proteins not complexed to the NKA or expressed on the cell surface.

All cells undergoing necrosis lost their actin filaments, rounded up, and adherent cells detached from the surface and lost their motility. Markers indicated initial activation of the integrated stress responses (ISR). Minimal to no annexin staining indicated that apoptosis was not the dominate pathway in any experiment.

Conclusion: Proper ion flux is important for maintaining cellular homeostasis. Cell swelling is a key process that is used to correct for changes in ion flux, induce inflammation and kill unwanted cells. The NKA is a key regulatory protein in this process and when blocked, leads to uncontrolled cells swelling and necrotic death. Precise inhibition of ion flux focused on cancer cells should be further investigated as a potential method for treating patients.

#2329

Noncanonical mitophagy induced by Mieap and its role in tumor suppression via ferroptosis-like cell death.

Yasuyuki Nakamura, Makoto Yamamoto, Hirofumi Arakawa. _National Cancer Ctr. Research Inst., Tokyo, Japan_.

Parkin/Pink1-mediated mitophagy plays a critical role in mitochondrial quality control, in which the damaged mitochondria are sequestered by autophagosomes, and degraded by fusion between autophagosomes and lysosomes. Here, we report Mieap-mediated noncanonical mitophagy as a new function of tumor suppressor p53. Mieap was originally identified as a p53-target gene. Overexpression of exogenous Mieap induces large vacuoles in cancer cells. Mieap-induced vacuoles are generated from the mitochondria, and directly eat and degrade the cancer mitochondria. UVRAG regulates the Mieap-mediated noncanonical mitophagy. Surprisingly, Mieap-mediated noncanonical mitophagy induces cell death via the iron-dependent production of reactive oxygen species (ROS). Introduction of exogenous Mieap strikingly suppresses in vivo tumor growth of Mieap-deficient colorectal cancer cells. Deficiency of endogenous Mieap dramatically promotes cancer development, malignancy, and aggressiveness in colorectal and gastric cancer model mice. The cell death induced by Mieap-mediated noncanonical mitophagy occurs via the ROS production in in vivo hypoxic tumor microenvironment. These results suggest that Mieap-mediated noncanonical mitophagy plays a critical role in tumor suppression via ferroptosis-like cell death.

#2330

CaV1.3 calcium channel expression in prostate cancer cells during androgen-deprivation conditions.

Niamh McKerr, Conor Breen, Kirtiman Srivastava, Ian G. Mills, Karen D. McCloskey. _Queens University Belfast, Belfast, United Kingdom_.

Background Prostate cancer (PCa) remains one of the most frequently diagnosed cancers in males in the western world. Despite efforts to reduce tumour burden through treatments including androgen-deprivation therapy (ADT), patients often relapse 2 years post-therapy due to emergence of castration-resistant disease. The importance of ion channels including calcium channels in cancer biology is increasingly recognized. CACNA1D encodes the calcium channel, CaV1.3, which is overexpressed in PCa biopsies. In addition to canonical ion transport, the c-terminal region of CaV1.3 may modulate gene expression. The purpose of this study was to elucidate the expression of CaV1.3 during PCa progression and explore the effects of calcium channel blockers. Methods A cell line panel comprising normal prostate epithelium (RWPE-1), androgen-sensitive PCa (LNCaP, VCaP) and castration-resistant PCa (C4-2B, DU145 and PC3) were used. LNCaP were cultured in charcoal-stripped serum-containing media to mimic ADT or with the anti-androgen enzalutamide (10μM) for 4-14 days. qPCR, Western blotting and immunofluorescence imaging were performed. Cell viability assays were performed in the absence/presence of ADT, enzalutamide and the CaV1.3 blocker nifedipine (1μM). Apoptosis was measured with flow cytometry in cells stained with Annexin V/Propidium Iodide (AV/PI). Results CACNA1D mRNA had higher expression in LNCaP, VCaP and C4-2B vs. RWPE-1 (p≤0.05; N=3). Western blotting/densitometry showed higher protein expression of CaV1.3 in LNCaP, VCaP and C4-2B relative to RWPE-1 (P≤0.05; N=3). Immunofluorescence indicated cytoplasmic and perinuclear localisation of CaV1.3 in the majority of cell lines and diffuse cytoplasmic staining in VCaP (N=2). ADT and enzalutamide-treated LNCaP displayed time-dependent increased CACNA1D expression over 14 days however, CaV1.3 protein expression peaked at 4 days (N=3). Cell viability assays of LNCaP treated with ADT or enzalutamide (4 days) showed significant reduction in viability when nifedipine was combined with the treatment (N=3; P≤0.05). Similarly, increased % of apoptotic LNCaP cells were observed when nifedipine was added to treated LNCaP vs. ADT or enzalutamide alone (N=2). Conclusion CaV1.3 expression is differentially expressed in cells lines modelling PCa progression and enhanced during ADT. Blockade of CaV1.3 in combination with ADT treatments further reduced cell viability and increased the apoptotic population.

#2331

Antioxidant enzymes mediate the survival of ECM-detached ovarian cancer cells.

Calli A. Versagli, Cassandra Libbing. _St. Mary's College, South Bend, IN_.

Epithelial ovarian cancer (EOC) is the deadliest gynecological cancer and is one of the leading causes of cancer death in women. It has been speculated that the high death rate associated with EOC is due to the fact that approximately 70% of all cases are diagnosed after the disease has already metastasized, thus making the illness more difficult to treat. In order to successfully survive the process of metastasis, EOC cells must be able to survive without anchorage to the extracellular matrix (ECM); however, the molecular mechanisms behind how EOC cells accomplish this feat has yet to be unveiled. Here, we present data that suggest antioxidant enzymes, namely Catalase, Peroxiredoxin 1, and Peroxiredoxin 2 all play a unique role in the survival and proliferation of ECM-detached SKOV3 cells. SKOV3 cells were genetically engineered to be deficient in Catalase, Peroxiredoxin 1, or Peroxiredoxin 2, and all three engineered cell types show abrogated anchorage-independent survival in soft agar. Furthermore, Catalase elimination in ECM-detached SKOV3 cells results in increased cell death at 48 hours, whereas elimination of Peroxiredoxin 1 or Peroxiredoxin 2 in ECM-detached SKOV3 cells results in decreased proliferation at 72 hours. Our current research is working to understand the molecular mechanisms behind the changes in survival and proliferation seen in antioxidant enzyme deficient SKOV3 cells. In aggregate, our data reveal that antioxidant enzymes may serve as attractive targets for the development of chemotherapeutics to eliminate metastatic ovarian cancer.

#2332

Necroptosis induction in triple negative breast cancer therapy.

Angelique Nyinawabera,1 Smiti Gupta,1 Karthikeyan Chandrabose,2 Amit K. Tiwari1. 1 _Univ. of Toledo, Toledo, OH;_ 2 _Indira Gandhi National Tribal University, Madhya, India_.

"Triple-negative" breast cancer (TNBC), is the most aggressive form of malignant mammary tumors that do not express three cell-surface receptors (estrogen receptor-α, progesterone receptor, and HER2/ERBB3). TNBC accounts for about 15 percent of breast cancer cases, with >42,000 new cases each year and approximately 21,000 deaths. Currently there are no targeted treatments available for TNBC. We found an interesting target associated with TNBC, the mitochondrial fission protein GTPase, dynamin-related protein-1 (Drp1), for which one inhibitor (mdivi-1) is under active investigation. However, mdivi-1 has shown inconsistent antineoplastic activity, low potency and selectivity. This led us to use a 200,000-compound library of commercially available small molecules (Specs), in conjunction with the likely structure of the Drp1-mdivi-1 inhibitory complex, resulting in the identification of twelve thieno-pyrimidin-4-yl-hydrazinylidene class of compounds that were highly potent and selective Drp1 inhibitors. In our preliminary work we found one compound ANT4 with IC50 value of 180-220 nM that has up to 100-fold selectivity in different TNBC cells compared to normal breast cells and up to 30-fold compared to other normal cells. We discovered that ANT4 is a selective inhibitor of Drp1 and has a 100-fold greater affinity for the Drp1 complex. ANT4 inhibited TNBC colony formation, invasion and metastasis,150-fold more potently than Mdivi-1. Interestingly, we discovered that ANT4, in nanomolar range, selectively induced a unique non-apoptotic, necroptotic inducing (NANI) cell death in TNBC cells by increasing the expression of necroptotic cell death markers (RIP, p-RIP, MLKL, p-MLKL). Further, ANT4, inhibited the expression of pro-apoptotic proteins and enhanced the expression of anti-apoptotic proteins. In addition, ANT4 arrested the TNBC cells in S-phase without producing any significant nuclear condensation or release of reactive oxygen species (ROS). Further studies are underway to understand the Drp1 mediated NANI induction process in TNBC cells and to identify the structural requirements that are necessary for targeting cancers refractory to apoptosis. The findings of these studies will allow us to understand the biology of necroptosis-induced Drp1 inhibition and to develop this new class of small molecules for the treatment of TNBC patients.

#2333

Genetic characterization of programmed cell death in aneuploid yeast cells.

Megan Onyundo, Matthew Sanborn, Victoria Timmel, Nicanor Austriaco. _Providence College, Providence, RI_.

Aneuploidy is the genetic state of a cell that has a chromosomal number that is not an exact multiple of the haploid complement. It is a leading cause of spontaneous abortions and of mental retardation in humans, and is a characteristic defect in cancer. Yeast cells that are aneuploid manifest a diversity of phenotypes including cell cycle defects, genomic instability, protein imbalance, chaperone stress and proteotoxicity. We are investigating the links between aneuploidy on the budding yeast, Saccharomyces cerevisiae and programmed cell death. We have shown that aneuploid yeast cells are more sensitive to ethanol-induced cell death. In addition, we have also shown that caloric restriction protects aneuploid yeast from program cell death. [In addition to funding from the RI-INBRE Program, our laboratory is supported by grant NIGMS R15 GM110578, awarded to N. Austriaco.]

#2334

Sialylation of the TNFR1 death receptor promotes cancer cell survival.

Andrew T. Holdbrooks,1 Matthew J. Schultz,2 Zhongyu Liu,1 Daniel C. Bullard,1 Susan L. Bellis1. 1 _Univ. of Alabama at Birmingham, Birmingham, AL;_ 2 _Mayo Clinic, Rochester, MN_.

Studies have well-established that activation of TNFR1, a ubiquitously expressed cell surface receptor, by TNF can induce either cell survival or cell death signaling cascades, however, the mechanisms regulating TNFR1 to initiate these disparate outcomes are poorly understood. TNF-induced apoptosis is initiated by clustering of activated TNFR1 at the cell surface, followed by internalization of the receptor complexes and subsequent caspase activation. Conversely, surface retention of activated TNFR1 promotes cell survival signaling mediated by NFκB and MAPKs pathways. Our group has identified a glycosylation-dependent mechanism that controls this TNFR1 signaling switch (i.e. apoptosis vs. survival). Specifically, we have found that TNFR1 activity is modified by the addition of a distinct sugar, an α2-6 linked sialic acid, by the Golgi sialyltransferase, ST6Gal-I. Importantly, ST6Gal-I is highly upregulated in numerous cancer types, and through its sialylation of a select cohort of cell surface receptors, various studies have indicated ST6Gal-I as a potent tumor cell survival factor. The effect of sialylation on TNFR1 was examined in multiple cancer cell models, including leukemia and epithelial cancer cell lines with forced overexpression or knockdown of ST6Gal-I. These models show that TNFR1 sialylation blocks TNF-induced apoptosis (indicated by the inhibition of caspase activation), and data from our group suggests that the underlying mechanism of this inhibition of apoptosis involves the sialylation-driven interference of TNFR1 oligomerization and internalization. Considering sialylated TNFR1 is retained on the cell surface following activation, we hypothesize that cells with abundant ST6Gal-I not only block TNFR1-mediated apoptosis but also divert signaling towards survival, indicated by increased activity and expression of many pro-survival factors (NFkB, AKT, etc.). Based on these collective findings, we conclude that the ST6Gal-I-mediated sialylation of TNFR1 controls the cellular response to TNF by blocking TNFR1 internalization, resulting in apoptosis inhibition and the promotion of survival signaling. Considering the tumor microenvironment is rich in immune cell-derived TNF, we posit that ST6Gal-I protects tumor cells within this inflammatory milieu by providing a mechanism by which tumor cells can evade immune cell killing.

#2335

Disulfiram overcomes trastuzumab resistance by targeting cancer stem-like properties and HER2/Akt signaling in HER2-positive breast cancer.

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

Although trastuzumab has shown a significant clinical benefit for HER2-positive breast cancers, responses are limited by the frequent emergence of resistance. Trastuzumab resistance is a multi-factorial phenomenon thought to arise from the presence of cancer stem cells and interactions between truncated p95HER2 and HER family members. Disulfiram (DSF), an anti-alcoholism drug, is known to elicit cytotoxicity in numerous cancer cell types in the presence of copper (Cu). We sought to investigate the effect of DSF on apoptosis, HER2/Akt signaling, cancer stem-like properties and trastuzumab resistance in HER2-positive breast cancer cells in vitro and in vivo. DSF/Cu treatment induced apoptosis, associated with a significant downregulation of HER2, truncated p95HER2, phospho-HER2, HER3, phospho-HER3 and phospho-Akt levels in both trastuzumab-sensitive and -resistant cell lines. This was accompanied by the eradication of cancer stem-like populations, concomitant with the suppression of aldehyde dehydrogenase 1 (ALDH1) activity and mammosphere-forming ability. DSF administration also caused significant tumor suppression in trastuzumab-resistant xenografts, coinciding with the downregulation of BCSC-related markers and intracellular HER2 in vivo. These findings highlight the mechanisms of action of DSF in overcoming trastuzumab resistance in HER2-positive breast cancer.

#2336

**Targeting CDK1 and MEK/ERK overcomes apoptosis resistance in** BRAFV600E **human colorectal cancer cells.**

Peng Zhang,1 Hisato Kawakami,1 Weizhen Liu,2 Xiangyu Zeng,2 Klaus Stebhardt,3 Kaixiong Tao,2 Shengbing Huang,1 Frank A. Sinicrope1. 1 _Mayo Clinic College of Medicine, Rochester, MN;_ 2 _Huazhong University of Science and Technology, Wuhan, China;_ 3 _Johann Wolfgang Goethe-University, Frankfurt, Germany_.

Background. Mutation of the BRAF(V600E) oncogene occurs in ~8% of human colorectal cancers (CRC) and its inhibition is associated with treatment resistance that is due, in part, to paradoxical re-activation of MEK/ERK signaling. Recently, pathway analysis identified cyclin-dependent kinase 1 (CDK1) upregulation in a subset of human BRAFV600E CRCs. We determined whether CDK1 antagonism can enhance the efficacy of MEK inhibition in BRAFV600E CRC cell lines.

Methods. Manipulation of gene expression was achieved by siRNA and/or ectopic expression, or using isogenic cell pairs. Cells were treated with the CDK1 selective inhibitor RO-3306 or dinaciclib (CDK1,2,5,9 inhibitor) alone or combined with the MEK inhibitor cobimetinib. Apoptosis was quantified by Annexin V staining with flow cytometry; apoptotic signaling was analyzed by immunoblotting of caspase-8 phosphorylation and caspase cleavage. Long-term cell survival was evaluated by clonogenic assay. In vivo efficacy of the drug combination was determined in a murine flank xenograft model.

Results. BRAFV600E CRC cell lines expressed CDK1 whose siRNA knockdown or pharmacological inhibition by RO-3306 or dinaciclib was shown to sensitize them to apoptosis (annexin V labeling). The combination of RO-3306 or dinaciclib with cobimetinib cooperatively enhanced apoptosis and reduced clonogenic survival compared to drug monotherapy. Cells isogenic or ectopic for BRAFV600E displayed apoptosis resistance to CDK inhibitors, as did cells with ectopic expression of constitutively active MEK. CDK1 inhibition by RO-3306 or dinaciclib induced a caspase-8-dependent apoptosis shown by caspase-8 restoration in deficient NB7 cells that enhanced dinaciclib-induced caspase-3 cleavage. Furthermore, CDK inhibitors suppressed procaspase-8 phosphorylation at S387 shown by drug withdrawal that restored phospho-S387 and increased cell mitosis. In a CRC xenograft model, dinaciclib plus cobimetinib produced significantly greater tumor growth inhibition in association with a caspase-dependent apoptosis than did either drug alone. Analysis of TCGA datasets revealed overexpression of CDK1 in human CRCs compared to normal colonic tissue. An inverse relationship existed between expression of CDK1 and the cell cycle inhibitor p16. In CRC cells, ectopic p16 was shown to downregulate CDK1 expression.

In conclusion, our data establish CDK1 as a novel mediator of apoptosis resistance in BRAFV600E CRC cells lines whose combined targeting with CDK and MEK/ERK inhibitors may represent an effective therapeutic strategy.

#2337

Effect of Honokiol and Indol-3-carbinol in viability and apoptosis of colorectal cancer cell lines treated with Orexin-A.

Ana Maria Wielandt, Cyntia Villarroel, Elea Gros, Mauricio Moreno, Claudia Hurtado, Udo Kronberg, Francisco Lopez-Kostner. _Clinica Las Condes, Santiago, Chile_.

Introduction: Colorectal cancer (CRC) is the third leading cause of cancer deaths worldwide. Recently, much attention has been given to nutraceuticals. They are being investigated for prevention and treatment of cancer. It has been shown that they have antitumoral properties without secondary effects, and could be used as a complement to conventional therapy for CRC. Orexin-A (OXA) is a hypothalamic neuropeptide with various functions in central nervous system and peripheral tissues. Orexin receptor 1 (OX1R) is expressed in CRC tissues. Activation of OX1R by OXA in CRC cell lines triggers apoptosis and was proposed as a new alternative for therapy. The aim of this study was to evaluate different nutraceuticals that could decrease survival and increase apoptosis of CRC cells lines in synergism with OXA.

Material and Methods: Two CRC cell lines were obtained from ATCC: Caco2 and SW480. Cell lines were treated with Honokiol (HK) or Indol-3-carbinol (I3C) for 24h at different doses with or without 100nM OXA. Cell viability was determined using CellTiter Kit (Promega). Caspase 3/7 mediated apoptosis activity was assessed using ApoOne Kit (Promega). Real-time qPCR was performed to determine the effect of nutraceuticals on OX1R expression.

Results: In this study, HK decreases cellular viability on average of 47% in Caco2 and 80% SW480 cells lines, at 25uM, 50uM or 100uM. In Caco2 cell line an increase in caspase 3/7 mediated apoptosis of 60% and 76% was observed at 25uM and 50uM, respectively, unlike what was observed in SW480 in which there was an inhibition of 80% at the different doses used. The combined treatment of 50uM HK with 100nM OXA does not produce a synergistic effect in cell viability or apoptosis compared to use of HK alone in both cell lines. HK produces an inhibition in the OX1R expression that explains the lack of synergism with OXA. I3C produces a decrease of 25% to 95% of cell viability according to doses used with an increase in apoptosis of 6 % to 38% in Caco2 cell lines. The combined effect of 500uM I3C with 100nM OxA produces a synergistic effect of 95% decrease on cell viability, with an increase of OX1R expression, which is not explained by an increase in apoptosis mediated by caspase 3/7.

Conclusions: We found that HK and I3C can inhibit viability and induce apoptosis in Caco2 and SW480 cells. In Caco2 cell line this can be explained by an increase in 3/7 caspase-mediated apoptosis, unlike that observed for SW480 treated with HK. Only 13C has a synergistic effect with OXA on cell viability in Caco2 cell lines but was independent of caspase 3/7 mediated apoptosis at doses and times used. Treatments using new doses and times are necessary to improve response. In our results, I3C might be a better therapeutic agent than HK for CRC. In vivo studies and clinical trials are further required to evaluate physiologic efficacies of the combination treatment.

Fondecyt 1140012.

#2338

XAV-939, a Wnt/beta-catenin pathway inhibitor, sensitizes liver cancer cells to sorafenib: Implications in sorafenib resistance.

Kanchan Vishnoi, Rong Ke, Navin Vishwakarma, Ajay Rana, Basabi Rana. _University of Illinois, Chicago, IL_.

Sorafenib is the only FDA-approved drug currently available for the treatment of advanced hepatocellular carcinoma (HCC). However, development of sorafenib-resistance in patients poses a major challenge to make this treatment successful in long-term. There is an urgent need to explore the mechanisms underlying sorafenib-resistance and to develop strategies towards overcoming sorafenib-resistance. Aberrant activation of Wnt/β-catenin signaling in HCC is suggested to maintain tumor initiating cells, drug-resistance, tumor progression, and metastasis. Combining β-catenin inhibitors with sorafenib might thus be an effective therapeutic strategy to target drug-resistant HCC. To explore this, we used wnt/β-catenin pathway inhibitor XAV-939 in combination with sorafenib to study their effect on the apoptotic potential of HCC cell lines Huh7, Hep3B and HepG2. XAV-939 was demonstrated to effectively reduce the expression of β-catenin in wild-type expressing Huh7 and Hep3B cells. Flow cytometric analysis of these cells pre-treated with XAV-939 followed by sorafenib treatment showed a significant increase in apoptosis in a time dependent manner. Furthermore, these cells revealed increased mitochondrial depolarization which was accompanied by a parallel increase in cytosolic cytochrome c release and downregulation of pro-survival protein Mcl-1. These effects of XAV-939 were further validated following knockdown of endogenous β-catenin, which showed a potentiation of sorafenib effects with β-catenin knockdown. Further, to understand the mechanism associated with sorafenib-resistance, we developed sorafenib-resistant HCC cells by repeated exposure to increasing concentration of sorafenib. To our surprise the combination of sorafenib and XAV-939 was ineffective in increasing the apoptotic potential of these resistant cells, suggesting a differential role of wnt/β-catenin axis in sorafenib-resistance. Taken together, these results suggested that combination of wnt/β-catenin pathway inhibitor and sorafenib could be effective in sensitizing HCC cells to apoptosis initially, but might be ineffective once these cells acquire sorafenib-resistance.

#2339

Targeting ICMT in pancreatic cancer.

Kanjoormana A. Manu, Tin F. Chai, Jing T. Teh, Wan Zhu, Patrick J. Casey, Mei Wang. _Duke-NUS Medical School, Singapore, Singapore_.

Introduction: Pancreatic Cancer remains one of the most difficult to treat human cancers with the 5-year survival as low as 5%. No targeted therapy yet developed against Pancreatic cancer. Here we want to target Isoprenylcysteine carboxylmethyltransferase (ICMT), an enzyme that post translationally modifies a group of proteins including several small GTPases against Pancreatic Cancer. Method: ICMT inhibition was achieved using lentivirus expressing ICMT shRNA and a pharmacological inhibitor named Cysmethynil. We evaluated a panel of human pancreatic cancer cell lines and identified those that are sensitive to ICMT inhibition by cell viability assay. This responsiveness to ICMT inhibition was confirmed in in vivo xenograft tumor mouse models using both Cysmethynil and shRNA-targeting ICMT. Flow cytometry analysis after DAPI or PI staining is used to detect cell cycle arrest and apoptosis. We used western blot analysis to study autophagy, apoptosis and cell cycle markers. ShRNA for P21 is used for p21 knock down studies. SiRNA for p53 is used to study whether p21 induction is p53 dependent. BNIP3, ULK1 and ATG5 knock down was done using specific shRNAs to study their role in ICMT inhibition induced autophagy and apoptosis. BCL-xL is over expressed in the cells using retroviral vectors to study the role of apoptosis in ICMT inhibition induced cell death. Cell and mitochondrial respiration was determined by oxygen consumption rate analysis using Seahorse XF24 analyzer. Summary: In sensitive pancreatic cancer cells, ICMT inhibition induces mitochondrial respiratory deficiency and cellular energy depletion, leading to significant upregulation of p21 and p21 dependent induction of BNIP3 leading to autophagy and apoptosis. But unlike in prostate and liver cancer, ICMT inhibition induced apoptosis not the autophagy responsible for inhibition of cell viability in pancreatic cancer. Conclusion: These findings suggest ICMT is a potential therapeutic target against pancreatic cancers and atleast in pancreatic cancer, ICMT inhibition induced apoptosis but not autophagy responsible for its therapeutic efficacy.

### Gene Discovery

#2340

Integrin subunit alpha 5: Potential prognostic biomarker in lung adenocarcinoma.

Ji Hyung Hong,1 Jeong-Hun Lee,2 Na Ra Yoon,1 Suk Hee Hong,3 Yoon Ho Ko4. 1 _Incheon St.Mary's Hospital, The Catholic University of Korea, Incheon, Republic of Korea;_ 2 _Seoul National University College of Medicine, Seoul, Republic of Korea, Seoul, Republic of Korea;_ 3 _Seoul St.Mary's Hospital, The Catholic University of Korea, Seoul, Republic of Korea;_ 4 _Ujungbu St.Mary's Hospital, The Catholic University of Korea, Republic of Korea_.

Identifying patients at high risk for recurrence in resected non-small cell lung cancer (NSCLC) is the key to choosing patients who can benefit from postoperative adjuvant therapy. However, the criteria to stratify patients to receive postoperative adjuvant therapy are still clinicopathologic factor-oriented, such as differentiation, tumor size, the presence of pleural involvement and vascular invasion and so on. Even using adjuvant treatment under these criteria, 5-year survival rate is around 60% even in early-stage disease. Here, we extracted potential candidate genes for stratifying resected NSCLC patients according to correlation with their survival using public big data and validated the prognostic role of the extracted candidate gene in tumor samples of patients' cohort with resected NSCLC. Under hypothesis "X affects Y" (X: regulatory gene, Y: events related gene), we extracted X and Y gene pairs in the RNA expression data of stage I-IIIA NSCLC in The Cancer Genomic Atlas using correlation analysis, Cox regression modeling, and Bonferroni correction. Extracted 20 Y genes related death events among 20,501 genes 576 lung adenocarcinoma include: BEST3, SLC25A42, C1orf210, TLE1, CD109, VEGFC, CXCL17, LASS4, DKK1, LDLRAD3, EPGN, NKX2-1, FJX1, PLEKHB1, FLNC, RGS20, FUT1, LASS4, TTGB1, KRT6C. Then, here are the 38 X genes significantly related to each Y genes: C11orf52, PPFIBP1, GGTLC1, ST3GAL5, CRB3, TGFBI, HOPX, STK32A, ERBB3, C16orf89, IRX5, TMPRSS2, PRR15L, PIGR, LMO3, ITGA5, RAB17, TMEM125, LPCAT1, SNAI2, RAB25, ABCA3, MBIP, TMEM125, B3GNT8, NAPSA ARNTL2, C16orf89, SCNN1B, ELK3, CDKL2, SFTA2, GPR115, CISH, SFTA3, LAMC2, ESYT3, SFTPB. Among these extracted X and Y gene pairs, we choose only ITGA5 (X) and VEGFC (Y) pair according to their known function of lymphangiogenesis and vasculogenesis during tumorigenesis process. We hypothesized that IGTA5, which is related to VEGFC, could be potential biomarker in resected NSCLC. In TCGA data, patients with low expression of ITGA5 showed better recurrence-free survival (RFS) than those with high ITGA5 (54.4 vs. 32.8 months; P-value <0.001). Then, using tissue microarray of 100 patients with surgically resected NSCLC, the expression level of ITGA5 and VEGFC was assessed by immunohistochemistry and analyzed correlated with survival outcome for validation. The expression level of ITGA5 was significantly correlated with that of VEGFC (P-value=0.004). RFS was longer in patients with low ITGA5 expression than in those with high ITGA5 expression (55.6 vs. 21.6 months; P= 0.020). According to our data, ITGA5 that affects VEGFC might one of the potential biomarkers of the resected lung adenocarcinoma relevant survival outcome. Furthermore, the immunohistochemistry of the larger number of cohort and functional experiment of the regulation of ITGA5 toward VEGFC in cell levels of NSCLC are ongoing.

#2341

Sleeping Beauty transposon mutagenesis screen identifies cancer genes of uterine leiomyosarcoma driving sarcomagenesis and lung metastasis.

Michiko Kodama,1 Takahiro Kodama,1 Justin Y. Newberg,2 Jean C. Tien,3 Roberto Rangel,4 Aya Nakae,1 Kae Hashimoto,1 Seiji Mabuchi,1 Kenjiro Sawada,1 Tadashi Kimura,1 Nancy A. Jenkins,4 Neal G. Copeland4. 1 _Osaka Univ. Graduate School of Medicine, Suita, Japan;_ 2 _Moffitt Cancer Center, Tampa, FL;_ 3 _University of Michigan, Ann Arbor, MI;_ 4 _University of Texas MD Anderson Cancer Center, Houston, TX_.

Introduction

Uterine leiomyosarcoma (ut-LMS) is one of the most aggressive malignancies with extremely poor prognosis. The 5-year survival rate is 0-20% for patients with tumors beyond uterus and 50% for even early stage patients due to high hematological metastatic potential. Because of its rarity, large-scale genetic profiling could not be done and thus its molecular mechanisms remain largely unknown. To solve this problem, we conducted in vivo sleeping beauty (SB) transposon mutagenesis screen and identified the key genetic drivers of ut-LMS.

Methods

We crossed Amhr2-Cre knock-in mice, transposon transgenic mice (T2Onc2), SB transposase knock-in mice (Rosa-LSL-SBase), Pten floxed mice, and Kras G12D knock-in mice, generating experimental mice (Amhr2-Cre Tg/+;T2Onc2 Tg/+;SBase KI/+;Pten fl/fl;Kras LSL-G12D/+) and control mice (Amhr2-Cre Tg/+; Pten fl/fl;Kras LSL-G12D/+). The experimental mice have homozygous deletion of Pten, constitutive activation of Kras and SB transposon mobilization in uterine smooth muscle cells. Fifty-nine experimental mice and 14 control mice were aged until moribund.

Results

All of the experimental mice died of multiple uterine tumors by 2 months of age, while none of the control mice developed uterine tumors. These uterine tumors were histologically diagnosed as ut-LMS by their morphology and immunohistochemical positivity of desmin and alpha-SMA. These results clearly indicated that transposon mutagenesis was required for sarcomagenesis of ut-LMS. To identify genes mutagenized by SB transposon in these tumors, we performed splink HiSeq sequence of gDNA from 80 primary uterine tumors and 24 normal uterus of mice with uterine tumors. Gene-centric common insertion site analysis (gCIS) identified 19 CIS genes in primary uterine tumors but no CIS genes

in normal uterus, indicating the selective enrichment of transposon insertions in cancer genes in tumors. By further focusing on the transposon insertions with high sequence read counts, we identified trunk mutations driving sarcomagenesis that include inactivating mutations of NF1 and activating mutations of one of zinc fingers gene family. Its inhibition via siRNA in human ut-LMS cell lines, SK-LMS1, SK-UT1, and SKN, impaired cell proliferation and sphere formation, suggesting the importance of this gene for the survival of ut-LMS.

Next, to identify the metastatic drivers of ut-LMS, we induced the lung metastasis by tail vein injection of 9 cell lines established from SB uterine tumors. After the sequential in vivo passage of metastatic SB tumors, we have obtained 50 lung metastatic tumors in immunocompetent mice. Sequencing and gCIS analysis of 50 lung metastases identified 3 potential driver genes of lung metastases.

Conclusion

SB mutagenesis screen discovers multiple cancer genes of ut-LMS driving sarcomagenesis and lung metastasis.

#2342

Digital gene expression of up to 96 targets in 96 samples for cell line screening with nCounter® PlexSet™.

Giang T. Ong, Chris Merritt, Anisha Kharkia, Philippa Webster. _NanoString Technologies, Inc., Seattle, WA_.

Cell line screening studies require highly efficient protocols for studying many samples in parallel. We have developed a lyse-and-go protocol for digital gene expression profiling of 96-samples by 96-genes in parallel. This protocol is based on nCounter® PlexSet™ Reagents, which enable multiplexing of samples and gene targets. The NanoString® nCounter Analysis platform uses a novel molecular barcoding technology to measure multiplexed gene expression. The assay counts fluorescent barcodes hybridized to targets to provide precise digital data. This platform is used in a wide variety of research applications. The standard nCounter gene expression assay can be used to process 12 samples per run. We recognize an additional need for a higher sample-throughput assay that would enable researchers to quickly evaluate up to 96 multiplexed targets in samples processed in a 96-well plate-based format. The PlexSet Reagent protocol is simple and fast and can be divided into 3 steps: 1) lyse cells, 2) set-up hybridization with crude lysate, 3) initiate automated post-hybridization processing and digital counting. The data presented are crude cells lysates from various PBMC cell types, two suspension cell lines, and two adherent cell lines using the IO Lymphocyte Activity Panel to measure the gene expression of 5 housekeeper genes and 91 lymphocyte activity genes (96 genes in total). The data demonstrate that up to 96 high-quality, digital data points for gene expression in each of 96 samples can be generated from crude cell lysates in an efficient, high-throughput protocol and that the cell lysate data correlate well with purified total RNA.

#2343

Comprehensive evaluation of antibody-drug conjugate targets for drug development across tumor types: Analysis from TCGA.

Roman Groisberg, Jason Rosznik, Funda Meric-Bernstam, Vivek Subbiah. _UT MD Anderson Cancer Center, Houston, TX_.

Introduction: Cancer cells over-express proteins on their cell surface that can be exploited as drug targets and has led to development of two successful Anti-body Drug Conjugates (ADCs), Brentuximab and Trastuzumab Emtansine. Because a strong correlation exists between mRNA levels and protein expression we analyzed RNAseq data from TCGA to look at known ADC targets as a proof-of-concept to discovering new therapeutic targets.

Methods: We used mRNA expressions (transcripts per million, TPM) from publicly available RNA sequencing data from the Cancer Genome Atlas (TCGA) and the Genotype-Tissue Expression (GTEx) project to analyze the expression of ADC targets in tumor and normal tissues. We chose thirty-seven targets with known ADCs in clinical development. Graphs were generated to show distribution of each gene's expression across all tumor and normal tissue types in TCGA and GTEx. Potential targets were determined based on tumor expression of at least two times the highest expression in any normal tissue. Proteins known to be secreted in the blood stream at high levels were excluded as they are unlikely to make a good ADC target (eg. AFP, FOLH1).

Results: Using ERBB2 as the prototype gene, we were able to detect over-expression in all tumors that have HER-2 ove-rexpression in the literature including breast, bladder, NSCLC, Gastroesophageal, endometrial, HNSCC, ovarian, and colon cancer. Other genes with overexpression in select tumors including BSG (RCC, Melanoma, ACC), CD70 (RCC, DLBCL, mesothelioma, cervical), ENPP3 (RCC, endometrial, breast, lung adeno), FGFR2 (breast, gastric), FOLR1 (ovarian, lung adeno), GPNMB (lung, melanoma, HNSCC, renal), MSLN (lung adeno, mesothelioma, ovarian), SLC39A6 (breast), TDGF1 (thymoma, testicular, adrenocortical) were discovered as well. Tumors exhibited a significantly higher expression compared to normal tissue for ERBB2: breast cancer vs normal breast: p < 0.001; bladder cancer vs normal bladder: p < 0.001; lung adenocarcinoma vs normal lung: p < 0.001; ovarian cancer vs normal ovary: p < 0.001.

Conclusion: Overall, this large-scale analysis from TCGA reveals that RNA-seq can be used as an initial screen to identify novel targets for ADCs. Further analysis will identify previously unappreciated targets that can be exploited for drug development across diverse cancers. Further confirmation with protein expression is warranted.

#2344

Identification and validation of synthetic lethal cancer pairs from genome-wide CRISPR screens.

Benjamin Schwartz, Junping Jing, Euan Stronach, David Cooper, Boris Wilson, Sarah Middleton, Mugdha Khaladkar, Coco Dong, Usman Shabon, Yanhua Rao, Stefanie Riesenberg. _GlaxoSmithKline, West Chester, PA_.

Although most canonical somatic cancer mutations have now been cataloged, only a fraction are amenable to drug discovery efforts. These are limited to targets with gain of function mutations and druggable protein domains. Examples include kinases such as FLT3 and metabolic enzymes such as IDH1/2. However, excluded from this list are undruggable protein targets with gain of function mutations, such as KRAS, or tumor suppressors which acquire loss of function mutations, such as APC.

The concept of synthetic lethality is being used to address this gap in cancer discovery. Cells which carry these directly undruggable somatic mutations become susceptible to inhibition of secondary targets. This susceptibility is specific in context to cells carrying the cancer associated mutation, offering both a novel approach for establishing efficacy as well as a potential therapeutic window for normal cells. The successful development of PARP inhibitors for BRCA mutant ovarian and breast cancers has provided the first clinical validation of this idea.

Pooled, genome-wide knock out experiments are being pursued to systematically discover more of these types of synthetic lethalities. Typically, a sgRNA library is used to transfect a CAS9 expressing cancer cell line, and sequencing of the guides over time identifies lethalities specific to the cell line. Once this experiment is repeated over hundreds of cell lines, one can use the genomic information of the cell lines, such as mutations or copy number aberrations, to identify potential new combinations of molecular lethality.

We have analyzed pooled CRISPR knockout data in a large panel of cancer cell lines generated from two independent sources (Broad Institute and Open Targets) to identify these types of lethality. Besides identifying targets for prevalent cancer mutations, we have also identified lethal combinations based on genes aberrantly deleted/silenced in cancers, termed "collateral lethality." These include pairs of paralogs, many of which code enzymes with redundant functions.

In this presentation we will outline our analysis strategies and methods, and describe the development of a downstream experimental validation pipeline, using a specific target/predictor pair to exemplify our approach.

#2345

The genomic landscape of small cell carcinoma of the oesophagus.

Feng Wang,1 Dongbing Liu,2 Qi Zhao,1 Gang Chen,3 Xinming Liu,2 Yingnan Wang,1 Hong Su,2 Yanru Qin,4 Yifu He,5 Qingfeng Zou,6 Yanhui Liu,7 Youen Lin,8 Hui Sheng,1 Jiajia Hu,1 Zexian Liu,1 Zhixiang Zuo,1 Jinxin Bei,1 Ruihua Xu1. 1 _Sun Yat-sen University Cancer Center, Guangzhou, China;_ 2 _BGI-Shenzhen, Shenzhen, China;_ 3 _Fujian Provincial Cancer Hospital, Fouzhou, China;_ 4 _The First Affiliated Hospital of Zhengzhou University, Guangzhou, China;_ 5 _Anhui Provincial Hospital Affiliated to Anhui Medical University, China;_ 6 _Affiliated Cancer Hospital & Institute of Guangzhou Medical University, Guangzhou, China; _7 _Guangdong General Hospital, Guangzhou, China;_ 8 _Jieyang People's Hospital, Jieyang, Guangdong, China_.

Background & Aims—Small cell carcinoma of the oesophagus (SCCE) is a deadly malignancy but its genetic characteristics remain unknown and treatment is commonly adopted from therapies for its histologically identical counterpart, small cell lung cancer (SCLC). We conducted a large-scale genomic analysis of SCCE to determine the mutational landscape of this deadly disease.

Methods—We performed whole-exome sequence of tumour and para normal esophageal tissues from 55 patients with SCCE who underwent surgery at seven institutions in China. Comprehensive analyses were carried out to identify key genetic events in SCCE, including significant mutations, copy number variation, pathway disruption. We also performed ultra-deep targeted sequencing on 20 specimens and microarray assays on 24 specimens to validate somatic mutations and copy number variations respectively. Using an unsupervised hierarchical clustering approach, the genomic characteristics of SCCE were compared with other types of malignancies including oesophageal squamous cell carcinoma, oesophageal adenocarcinoma and et al. In vitro proliferation and migration assays were conducted to assess the function of PDE3A.

Results—The mutational spectrum of SCCE was dominated by C>T/G>A transitions and the mutational signatures classified the patients into 3 groups. We identified three novel significantly mutated genes (PDE3A, PTPRM and CBLN2) and mutations in five other well-known tumour-associated genes (TP53, RB1, NOTCH1, FAT1 and FBXW7). Non-silent alterations of chromatin remodeling genes were detected in 96.4%. Moreover, pathway enrichment analysis revealed that genes involved in the cell cycle, p53, Notch and Wnt signaling were frequently altered in SCCE. Mutations in NOTCH family correlated with shorter overall survival. Furthermore, comparison analysis of mutation signatures revealed that SCCE tumours were more closely related to oesophageal squamous cell carcinoma, rather than oesophageal adenocarcinoma and SCLC, suggesting that current therapies for SCCE may be inadequate. Functional characterizations suggested that PDE3A acts as a novel oncogene.

Conclusions—Our findings reveal key somatic alterations in SCCE, and provide insights to better understand the pathogenesis of SCCE and to develop better treatment strategies for patients with the condition.

#2346

ARDE: Detecting selectively expressed cancer driver mutations through integration of exome and transcriptome sequencing data.

Fang Wang, Shaojun Zhang, Yiwen Chen, Traver Hart, Kenna Shaw, Funda Meric-Bernstam, Gordon Mills, Ken Chen. _UT MD Anderson Cancer Ctr., Houston, TX_.

Integrating multi-omics data from cancer patient samples can potentially advance the discovery of cancer driver mutations (CDM) towards establishing a knowledgebase for cancer precision medicine. However, it remains unclear whether CDMs are selectively expressed at RNA levels unexpected from DNA copy numbers and whether identifying such selectivity will enable more accurate discovery of CDMs. To answer this question, we developed a novel statistical algorithm, ARDE (Allelic RNA DNA expression), which estimates tumor purity, allele-specific copy number and intratumor heterogeneity (ITH) from matched whole-exome and whole-transcriptome sequencing data, deconvolutes tumor allele-specific expression levels (ASEL) and uses a two-component mixture probability model to identify selectively expressed alleles (SEAs) in the tumor compartment of heterogeneous tumor tissues. We applied ARDE to analyze melanoma and breast samples from The Cancer Genome Atlas (TCGA). Tumor purities estimated by ARDE were highly correlated with estimation from pathology review, DNA methylation array, gene expression microarray and SNP array data. ITH scores can significantly stratify breast cancer subtypes and survival time of melanoma patients. The deconvoluted tumor ASEL showed consistently higher correlation with the averaged gene expression levels in tissue-matched single tumor cells than in normal cells. Higher proportions of somatic mutations are selectively expressed than germline. Top SEAs are highly enriched in deleterious and gain-of-function (GOF) mutations. Stratifying melanoma patients by the expression selectivity of BRAF V600E mutation resulted in a 10-fold improvement of significance in survival analysis. These results indicate that ARDE will likely be an effective, widely applicable approach for accurate discovery of CDMs from tumor tissue sequencing data. (See Table 1.)

Table 1. Top 10 significant positive-selectively expressed somatic mutations | |  | |  | |

|

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

Melanoma | Breast cancer

Gene | pSNV | Function | SIFT | Gene | pSNV | Function | SIFT

BRAF | p. V600E | GOF | D | AKT1 | p.E17K | GOF | D

NRAS | p. Q61R | GOF | D | PIK3CA | p.H1047R | GOF | T

CDKN2A | p. R114L | GOF | D | ERBB2 | p.L755S | GOF | D

IDH1 | p. R132C | Switch of function | D | TP53 | p.R273H | GOF | D

EEF1A1 | p. H349Y | |

D | GATA3 | p.M293K | |

D

RAC1 | p. P29S | GOF | D | TP53 | p.P278T | |

D

NRAS | p. Q61K | GOF | D | FOXA1 | p.S250F | |

D

SF3B1 | p. R625H | GOF | D | TP53 | p. R175H | GOF | D

NRAS | p. Q61L | GOF | D | PIK3CA | p.H1047L | GOF | T

MAP2K1 | p. P124S | GOF | D | ERBB2 | p.V777L | GOF | T

#2347

High-throughput scRNA-seq analysis using Celsee Nano-prep 250K sequencing chip on Celsee C-PREP GENESIS.

Vishal Premdev Sharma, William Chiro Chow, Brian Boniface, Kyle Gleason, Austin Payne, Priya Gogoi, Kalyan Handique. _Celsee,Inc., Plymouth, MI_.

For effective targeted therapeutics, it is critical to identify cancer mutations in cells that are often regional and heterogenic. Recent advances in single-cell mRNA sequencing (scRNA-seq) have made it possible to study intratumor heterogeneity and its role in cancer progression, metastasis, and treatment resistance. Next-generation sequencing (NGS)-based studies have identified critical genetic alterations in a variety of malignancies. However, the current workflow of existing technologies leads to significant loss of cells and thus loss of critical information. In this study we present a technology that decreases cell loss and increases efficiency of scRNA-seq. C-PREP GENESIS is a fully automated, random-access, multiple-sample processor for single-cell sequencing preparation. C-PREP GENESIS allows automated fluid dispensing and controlled pumping through Nanochips. The instrument utilizes unitized reagent cartridge for dispensing reagents for wash, lysis, cDNA synthesis and oil (for partitioning). With its automated thermocycling (4oC- 100oC), it can achieve the single cell to cDNA synthesis on beads with unique cell barcode and UMI tags in under 2 hours with an efficiency of ~80% of the 50,000 single cells captured on the nano-PREP 250K sequencing chip. The cDNA can then be retrieved from the chip for downstream library preparation. Alternatively, users can perform a targeted library prep PCR on the same instrument. Here we present scRNA-seq analysis of single cells obtained from patient tumor biopsy samples and cancer tissue culture single cells to discuss the critical mutations observed in single cells of the heterogenic tumor populations.

#2348

Low-cost and accurate human leukocyte antigen (HLA) class I typing of The Cancer Genome Atlas on the Seven Bridges Cancer Genomics Cloud.

Raunaq Malhotra,1 Alexandar Krasnitz,2 Anurag Sethi,1 Erik Lehnert,1 Elizabeth H. Williams,1 Davis-Dusenbery N. Brandi1. 1 _Seven Bridges Inc, Cambridge, MA;_ 2 _Cold Spring Harbor Laboratory, New York, NY_.

The Cancer Genome Atlas (TCGA)network has generated more than2.5petabytes of genomic dataover the last decade, with

petabyte-scale additions of dataexpected inthe coming years. Access and analysis of this informationonalocal compute

environment is challenging due tothe volume of dataand lack of sufficient computing resources at many research organizations.

The Cancer Genomics Cloud Pilot project from the National Cancer Institute (NCI)has helped indemocratizing access toTCGA

by co-localizing datawith computational resources onthe cloud. Funded as part of this project, the SevenBridges Cancer

Genomics Cloud (CGC)hosts nearly 5petabytes of public datafrom TCGA, the Simons Genome Diversity Project, the

Therapeutically Applicable Research toGenerate Effective Treatments (TARGET)initiative, The Cancer Imaging Archive (TCIA),

and the Clinical Proteomic Tumor Analysis Consortium (CPTAC). The CGCprovides academic researchers with asecure,

scalable, cloud-based cancer research platform that includes collaborative tools for accessing, uploading, analyzing, and

visualizing data. The platform uses resource descriptionframeworks, dataharmonization, and metadatacurationtofacilitate

effective querying, and bioinformatics tools are implemented onthe CGCusing the CommonWorkflow Language (CWL), an

emerging standard for describing computational workflows, tosupport computational reproducibility. Since its launch in2016, the

CGChas enabled researchers from around the world tounderstand humangenetics and cancer biology through the analysis of

large public datasets and private datainacloud computing environment. Inthis poster, we present anexample of analysis of

TCGAdataonthe CGC. The OptiType tool for HumanLeukocyte Antigenclass I typing was used toprofile 8,872RNA-Seq

samples present inthe TCGAdataset. All samples were accurately processed within2days using arobust, fault-tolerant, and

cost-efficient CWL descriptionof OptiType that enabled analysis for less than50cents per sample onaverage. This case study

demonstrates how cloud computing resources canfacilitate the successful analysis of large cohorts of datausing custom

pipelines in a robust, scalable, and reproducible manner.

#2349

Identifying genes and microRNAs that when lost, can drive neoplastic transformation of non-cancerous lung cells.

Chennan Li, Sagar M. Utturkar, Andrea L. Kasinski. _Purdue University, West Lafayette, IN_.

Despite the fact that the death rate continues to drop over the last decades, lung cancer is still by far the leading cause of cancer mortalities due to lack of highly accurate prediction method and effective targeted therapeutics. Thus, this calls for identification of novel biomarkers and therapeutic targets, particularly those targeting critical genes that drive lung cancer development and malignancy. KRAS and TP53 are two of the most commonly mutated genes in non-small cell lung cancer (NSCLC) which represents 85% of all cases in lung cancer. However, aberrant expressions of many other genes that act as drivers of lung cancer are yet unidentified. Particularly, microRNAs which are genome-encoded small RNA molecules are globally downregulated in many cancers, and disrupting microRNA biogenesis has been shown to promote tumor formation. We utilize the power of the CRISPR-Cas9 gene knockout system to screen for critical tumor suppressor genes and microRNAs in the human and mouse genomes that when lost, can drive neoplastic transformation of lung cells. Two non-cancerous mammalian lung model systems are used for this study. (1) The human bronchial epithelial cells that stably express KRAS G12V and TP53-targeting shRNA (HBEC-KP) are used as the baseline in the first approach. Importantly, the HBEC-KP cells are anchorage dependent and incapable of forming tumor in in vivo. We have transiently transfected HBEC-KP tdTomato-expressing cells with Cas9 and transduced the cells with lenti-sgRNA human library (A). Cells were either passaged in two-dimensions for over two-months or were selected for growth in soft agar assays. Resulting cells were harvested to identify sgRNAs enriched in each of the individual condition. Several known tumor suppressor genes and microRNAs (such as BRCA2, let-7a-3, miR-34a) are present among top hits, while MYC and TP53 are highly depleted, which suggests the validity of this data. Top hits will be selected and validated. (2) The second model being used is the KrasLSL-G12D mouse model. Genetic recombination induces lung hyperplasia in these mice, which will serve as the baseline to identify gene and microRNA knockouts that drive advanced tumor progression. The KrasLSL-G12D; RosaLSL-Cas9/LSL-Cas9 (KC) mice have been generated, and will be validated for Cre-induced Cas9 stable expression. KC mice will be intratracheally injected with the mixture of adeno-Cre and lenti-sgRNAs, and eventually sgRNAs that are highly enriched in individual tumors that develop will be identified through deep sequencing and bioinformatic analysis, and targeted genes and microRNAs downregulated in the tumors will be validated in functional assays and for loss in human NSCLC tumor samples.

#2350

Efficient survival multifactor dimensionality reduction method for genome-wide association study.

Jiang Gui,1 Xuemei Ji,1 Christopher I. Amos2. 1 _Dartmouth Geisel School of Medicine, Hanover, NH;_ 2 _Baylor College of Medicine, Houston, TX_.

The problem of identifying associations between SNPs and case-control outcome has been studied extensively and a number of new techniques have been developed. Little progress has been made, however in the analysis of SNP effect in relation to censored survival data. We present an extension of the two class multifactor dimensionality reduction (MDR) algorithm that enables detection and characterization of epistatic SNP-SNP interactions in the context of survival outcome. The proposed an Efficient Survival MDR (ES-MDR) method handles censored data by modifying MDR's constructive induction algorithm to use logrank Test. We applied ES-MDR to genetic data of over 470,000 SNPs from the OncoArray Consortium. We use onset age of lung cancer and case-control (n=27,312) status as the survival outcome and divided data into training and testing sets. We also adjust for subject's age, gender and smoking status. From training set, we identified strong association from SNPs in BRCA1 and IL17RC genes with lung cancer onset age. This result is validated in the testing set.

#2351

Transcriptome-wide association study reveals candidate causal genes for lung cancer.

Alisson Clemenceau,1 Maxime Lamontagne,1 Robert Carreras Torres,2 Ma'en Obeidat,3 Wim Timens,4 Philippe Joubert,1 Christopher I. Amos,5 James D. McKay,2 Yohan Bossé1. 1 _Institut universitaire de cardiologie et de pneumologie de Québec, Quebec, Quebec, Canada;_ 2 _International Agency for Research on Cancer, World Health Organization, Lyon, France;_ 3 _The University of British Columbia Centre for Heart Lung Innovation, St Paul's Hospital, Vancouver, British Columbia, Canada;_ 4 _University of Groningen, University Medical Center Groningen, GRIAC Research Institute, Groningen, Netherlands;_ 5 _Baylor College of Medicine, Houston, TX_.

Genome-wide association studies have identified robust susceptibility loci associated with lung cancer. As part of the OncoArray-TRICL consortium, we have recently completed the largest GWAS on lung cancer including 29,266 cases and 56,450 controls of European descent. The goal of this study is to integrate the complete GWAS results with a large-scale expression quantitative trait loci (eQTL) mapping study in human lung tissues (n=1,038) to identify candidate causal genes for lung cancer. Transcriptome-wide association study (TWAS) was used to integrate GWAS and lung eQTL signals and identify genes whose levels of expression in lung tissue are causally related to lung cancer. TWAS was performed on six histologic and smoking subgroups, namely overall lung cancer, adenocarcinoma, squamous cell carcinoma, small cell carcinoma, never-smokers, and ever-smokers. As expected, the main TWAS signal for all histologic subtypes and ever-smokers was on chromosome 15q25. The genes most strongly associated with lung cancer at this locus were IREB2 (PTWAS=4.97E-104), and to a lower extent, CHRNA5 (PTWAS=5.26E20) and HYKK (PTWAS=2.04E-17). TWAS identified causal genes were different from those reported in GWAS, including JAML on 11q23.3 in overall lung cancer (PTWAS=1.39E-6) and adenocarcinoma (PTWAS=2.09E-8), NOTCH4 on 6p21.32 in squamous cell carcinoma (PTWAS=1.24E-12), ZNRD1 on 6p22.1 in overall lung cancer (PTWAS=3.41E-14) and ever-smokers (PTWAS=1.29E-9), HIST1H2BD on 6p22.2 for small cell carcinoma (PTWAS=1.54E-6), and NEXN on 1p31.1 in never-smokers (PTWAS=2.64E-5). In addition, a new small cell carcinoma susceptibility locus was identified on 4q32.2 and associated with the expression levels of TMA16 (PTWAS=4.2E-6). In conclusion, lung tissue TWAS on lung cancer, histologic subtypes and smoking subgroups revealed novel causal genes in GWAS-nominated loci. A new locus for small cell carcinoma (4q32.2-TMA16) was also identified and will require further validation.

#2352

Defining a pediatric cancer dependency map.

Neekesh V. Dharia,1 Clare Malone,1 Amanda Balboni Iniguez,1 Lillian Guenther,1 Liying Chen,1 Gabriela Alexe,1 Adam D. Durbin,1 Mark W. Zimmerman,1 Andrew Hong,1 Pratiti Bandopadhayay,1 Mariella G. Filbin,1 Thomas Howard,1 Brenton Paolella,2 Iris Fung,2 Josephine Lee,2 Phil Montgomery,2 John M. Krill-Burger,2 Brian J. Abraham,3 Jennifer Roth,2 David E. Root,2 Richard A. Young,3 A. Thomas Look,1 Rameen Beroukhim,1 Jesse S. Boehm,2 William C. Hahn,1 Todd R. Golub,2 Aviad Tsherniak,2 Francisca Vazquez,2 Kimberly Stegmaier1. 1 _Dana-Farber Cancer Inst., Boston, MA;_ 2 _Broad Institute, Cambridge, MA;_ 3 _Whitehead Institute for Biomedical Research, Cambridge, MA_.

Many children with metastatic or recurrent pediatric solid tumors continue to have poor survival, and there is an immense need to identify novel therapeutic approaches. Moreover, these cancers typically have simple genomes with limited known druggable molecular events. In order to discover new vulnerabilities in pediatric solid tumors, we have performed genome-scale CRISPR-Cas9 loss-of-function screening and deep "omic" characterization in over 60 pediatric cancer cell lines to date, including neuroblastoma, medulloblastoma, Ewing sarcoma, malignant rhabdoid tumor and rhabdomyosarcoma lines, to begin defining a pediatric cancer dependency map. Global analyses of the pediatric dependency landscape have identified emerging classes of pediatric cancers, including epigenetic-driven, aberrant transcription factor-driven and receptor tyrosine kinase-driven malignancies. For example, the preferential dependencies identified in a subset of neuroblastoma, which has aberrantly high expression of the transcription factor MYCN, are highly enriched for an interconnected network of genes annotated to have transcription factor activity. In addition to the global evaluation, we have developed methods and tools for prioritizing targets for further validation within a cancer type. These tools computationally integrate the pediatric dependency data across multiple datasets to identify categories of genetic dependencies that are especially strong hits or enriched hits in a specific pediatric malignancy. As an example, the intersection of MYCN-amplified neuroblastoma specific dependencies and H3-lysine 27 acetylation (H3K27ac) profiling across MYCN-amplified neuroblastoma allowed us to identify a transcriptional core regulatory circuit (CRC) that may drive the malignant state. Furthermore, targeting transcription with the BRD4 inhibitor JQ1 and CDK7 inhibitor THZ1 caused synergistic killing of neuroblastoma cells suggesting a novel therapeutic approach to treating this disease. Thus, defining a comprehensive pediatric cancer dependency map and developing the methods and tools to prioritize vulnerabilities in different cancer types will allow us to discover both novel biology and new therapeutic opportunities in childhood malignancies.

#2353

Analysis of noncoding mutations in cancer revealed novel potentially pathogenic recurrent mutations.

Takahiko Koyama, Laxmi Parida. _IBM T.J. Watson Research Center, Yorktown Heights, NY_.

Introduction:

98% of mutations are non-coding mutations. Thus far, most non-coding mutations are ignored or merely classified as variant of unknown significance with a few exceptions, despite the fact that non-coding region plays significant role in regulations and other important biological processes. Although they would not be directly druggable, these non-coding mutations might be important for cancer etiology, response to treatments and prognosis assessments.

Method:

Mutations in non-coding genes, intron, upstream and downstream, and intergenic regions are collected from COSMIC and TCGA as defined in Ensemble GRCh37.75 gene definition and hg19 RefSeq. Over 15 million non-coding mutations are analyzed for recurrence over 10 samples. Mutations in neighborhood of IG genes and dbSNP150 polymorphisms are removed from further investigation. We analyzed remaining mutations for possible super enhancer and transcription factor binding.

Result:

911 distinct recurrent mutations are identified. Promoter mutations in protein coding genes like MPRS31, NUDFB9, SZT2, WASF3 in addition to TERT are discovered. Intronic mutations of well-known cancer genes such as HIF1A, NF1, KMT2C, and ATR are discovered with their associated cancer type specificity. As for non-coding RNA's, lincRNA CROCCP2, LINC00969 and miRNA MIR4477 appear frequently. Only 182 mutations are reluctantly classified as intergenic; intergenic loci RP11-96F8.1-KSR1P1, MLLT10P1-DEFB115, and bP-21201H5.1-IGHV1OR21-1 contain unusually high recurrent mutations.

Conclusion:

Many non-coding mutations worth further investigation are found. Still, it is quite possible that many of these mutations might be artifacts resulting from paralogous alignments, gene definition compatibility and other technical issues. Importantly, we discovered novel intronic mutations in genes already associated with treatments. Mutations in intergenic regions might be involved in regulatory mechanisms and chromosomal structures. Understanding these non-coding mutations will further refine our understanding of biology as well as cancer.

#2354

Candidate non-coding driver mutations in super-enhancers and long-range chromatin interaction networks across 1,800 whole cancer genomes.

Juri Reimand. _Ontario Inst. for Cancer Research, Toronto, Ontario, Canada_.

A comprehensive understanding of the mutations that drive tumorigenesis and disease progression is essential to understanding tumor biology and designing precision therapies. The landscape of driver mutations in the protein-coding genome has been well-characterized by large exome-sequencing studies. Surprisingly, many tumors do not have mutations in any known protein-coding driver. Non-coding driver mutations are hypothesized to explain many of these cases, but aside from a few regions, like the TERT promoter, our understanding of drivers in the complex regulatory genome remains limited. To fill this gap, we analyzed 150,000 cis-regulatory modules with clustered transcription factor binding sites in 1,844 whole cancer genomes from the ICGC-TCGA PCAWG project. Using a new statistical method, ActiveDriverWGS, we identified dozens of frequently mutated regulatory elements (FMREs) enriched in non-coding SNVs and indels (FDR<0.05) with many structural rearrangements and focal copy number alterations in additional samples. The FMREs were enriched in super-enhancers, long-range chromatin interactions and H3K27ac marks derived from primary tumors, suggesting a gene regulatory role of these mutations through three-dimensional genome organization. The interaction network of chromatin loops and FMREs revealed putative target genes located dozens to hundreds of kilobases away from the mutated regulatory elements. We found known and putative oncogenes and tumor suppressors whose expression significantly correlated with mutations in FMREs, suggesting novel oncogenic mechanisms. Most of the FMREs were also confirmed by additional driver discovery methods, lending confidence to our statistical approach. We also validated ActiveDriverWGS on protein-coding sequence and accurately recovered known driver genes. The non-coding regulatory genome is characterized by diverse mutational processes, regional hypermutations and technically challenging areas with suboptimal sequencing coverage. Thus our findings, most of which are reported for the first time, should be carefully vetted and experimentally validated in future studies. Our integrative analysis of somatic mutations, cis-regulatory regions and long-range chromatin interaction networks is a novel framework for cancer discovery and reveals the currently largest set of potential non-coding drivers in a pan-cancer cohort.

#2355

Penile cancer in vitro models useful for the identification of targeted therapies.

Hellen Kuasne,1 Mateus Barros-Filho,1 Juan Muñoz,1 Fabio Marchi,1 Cristovam Scapulatempo-Neto,2 Eliney Faria,2 Gustavo Guimarães,1 Ademar Lopes,1 Julia Mello,1 Maria Domingues,3 Sebastien Carréno,4 Silvia Rogatto5. 1 _AC Camargo Cancer Center, Sao Paulo, Brazil;_ 2 _Barrretos Cancer Hospital, Barretos, Brazil;_ 3 _São Paulo State University - UNESP, Botucatu, Brazil;_ 4 _Université de Montréal, Montreal, Quebec, Canada;_ 5 _University of Southern Denmark, Vejle, Denmark_.

Background: Penile carcinoma (PeCa) is a frequent disease in poor and developing countries showing high morbidity. Despite the recent progress in the molecular research of PeCa, the lack of well-characterized in vitro models precludes new advances in the knowledge of cellular processes and in preclinical tests for anticancer drugs' efficacy.

Material and Methods: Twenty PeCa were surgically removed, dissected and cultured in vitro (2D and 3D environment) using 3 KSFM: 1 DMEM/F12. We established five penile cancer cell cultures in vitro (passage 20), which were characterized by genomic (CytoScan HD - Affymetrix), translatomic (Clariom D Array - Affymetrix), epigenomic (Infinium Methylation EPIC BeadChip - Illumina) and proteomic profiles (Reverse phase protein array - RPPA). Chemosensitivity assay using cisplatin and EGFR inhibitors (Erbitux and Tarceva) was performed by MTT test.

Results: Two cell cultures presented typical epithelial cell morphology and epithelial markers expression such as cytokeratin and EpCAM and three were cancer-associated fibroblasts (CAFs). These cell cultures presented cancer behavior features including anchorage-independent growth, invasive potential, and capability to form tumor in nude mice. The genomic analysis revealed high similarity between the primary tumor and its derived cell culture. Transcriptomic and proteomic analysis revealed that the most relevant differentially expressed genes and proteins are components of key oncogenic pathways (MMPs, EGFR, STAT3, and PI3K/AKT/mTOR), supporting our previous data using PeCa fresh tissues. We also identified a set of genes potentially regulated by epigenetic modifications. A chemosensitivity investigation suggested that these cells are sensitive to cisplatin, a chemotherapy agent commonly used in advanced PeCa. One cell culture with EGFR overexpression was sensitive to anti-EGFR drugs (Tarceva and Erbitux).

Conclusion: In general, PeCa cell cultures recapitulated cancer behavior in vitro and showed the same dysregulated pathways than their corresponding primary tumors. These cells proved to be reliable in vitro models to dissect mechanisms promoting penile carcinogenesis and to test new targeted therapies. Our results support recent studies in which patients with advanced PeCa expressing EGFR can benefit from the treatment with EGFR-targeted therapies.

#2356

Prioritizing cancer gene drivers defined in large-scale cancer genome analyses using the SBCDDB.

Justin Y. Newberg, Michael B. Mann, Karen M. Mann. _Moffitt Cancer Center, Tampa, FL_.

Next-generation sequencing approaches to identify molecular signatures of cancer have uncovered few frequently altered genes, many infrequently altered genes, and various transcriptional changes associated with cancer initiation/progression. Prioritization of genetic and epigentic alterations in various contexts is therefore important for biological discovery and therapy development. Comparative oncogenomics is a powerful approach to identify drivers that impact cancer development. Importantly, data integration across different experimental systems and data types is imperative to the success of these oncogenomics approaches. Sleeping Beauty (SB) insertional mutagenesis coupled with next generation sequencing forms a powerful cancer gene discovery framework widely used to identify and prioritize candidate drivers in mouse models of human cancers. SB has been used to model over 20 different human cancers and identify cooperating drivers underlying tumor development. Integration of SB data with human cancer genomic and transcriptomic data from The Cancer Genome Atlas (TCGA) / Genomic Data Commons (GDC) is one way to identify and prioritize genes for biological validation and targeted therapies. Our group has built a resource, the Sleeping Beauty Cancer Driver Database (SBCDDB), to identify sets of cancer driver genes associated with specific tumor types or genetic aberrations underling cancer initiation. Using the SBCDDB, in combination with human mutation, copy-number alteration, and mRNA-seq datasets from the Broad Firehose, we have prioritized infrequently mutated driver genes in human tumors that are recurrently altered in SB tumors. Cancer drivers defined in both human tumors and in related mouse models of cancer, using distinct but complimentary methods to discover and catalog mutations, provide strong evidence for a causal role of detectable alteration in tumor initiation and/or progression. This work is an important step in a larger, comparative pan-cancer approach to better define molecular signatures that make up the tumor landscape.

#2357

Utilizing biological and protein structure-guided features to improve driver mutation discovery.

Sohini Sengupta,1 Adam Scott,1 Amila Weerasinghe,1 Dan C. Zhou,1 Matthew A. Wyczalkowski,1 Reyka G. Jayasinghe,1 Ken Chen,2 Gordon Mills,2 Mike C. Wendl,1 John Dipersio,1 Li Ding1. 1 _Washington Univ. St. Louis, Saint Louis, MO;_ 2 _MD Anderson Cancer Center, Houston, TX_.

Distinguishing between driver and passenger somatic mutations to pinpoint genetic alterations leading to oncogenesis still presents significant challenges. To meet these challenges, computational tools have been developed as effective filters, pruning most of the somatic mutations to a shortlist of high-priority, functional candidates for experimental validation. Most tools include searching for genes or pathways having mutation rates higher than explained by chance, mutations in conserved regions, or genes with neighboring mutations on the linear DNA or protein sequence. Recently, there has been a shift to utilize tertiary/quaternary protein structures to identify mutations clustering proximal to each other in 3D space. Such enrichment of mutations can indicate specific domains critical to normal protein function and when mutated, can drive tumor initiation and progression. HotSpot3D, a protein structure-based tool, identifies clusters enriched with proximal mutations within proteins. Though HotSpot3D has been valuable in identifying clusters of residues that are important to cancer, it does not distinguish the driving potential or structural impact of different mutations within a cluster nor does it consider the physical impact of different amino acid substitutions at the same site. The prediction power of HotSpot3D in distinguishing driver mutations from passenger mutations can be improved if spatial clustering considers physical/biological features proximal to mutations in significant clusters as well as the specific amino acid substitutions of mutations. We have created a machine learning algorithm that further prioritizes putative driver mutations found in HotSpot3D clusters by incorporating structural/biological features such as proximity of mutations to functional sites (active sites, phosphorylation sites, disulfide bonds, etc.), solvent accessibility, physiochemical property change of mutations, free energy change of mutations, conservation of residue sites, secondary structure state of residue sites, and expression/phosphorylation changes of samples containing mutations. We have curated experimentally validated mutations identified as neutral or oncogenic from various databases to serve as our training sets. This algorithm can be trained on the curated mutations in various protein subclasses such as homologous proteins, oncogenes, tumor suppressors, etc. to identify distinct structural feature signatures per subclass specific to driver mutations. This tool will aid in revealing putative driver mutations in genes not previously linked with cancer and help pinpoint mutations in known cancer genes that are driving cancer. Specifically, we are interested in applying the algorithm to druggable protein families such as G-Protein Coupled Receptors, Kinases, and Nuclear Hormone Receptors to better understand their role in tumor initiation and progression.

#2358

Uncovering novel mechanisms of resistance in AML using integrative functional genomics.

Azucena Ramos,1 Luis R. Millan Barea,2 Alexandre Puissant,3 Nina Fenouille,3 Gabriela Alexe,3 Kimberly Stegmaier,3 Michael T. Hemann2. 1 _Harvard Medical School/MIT M.D. Ph.D. Program, Boston, MA;_ 2 _MIT, Cambridge, MA;_ 3 _Dana Farber Cancer Institute, Boston, MA_.

Treatment options that effectively cure patients diagnosed with acute myeloid leukemia (AML) continue to represent an area of unmet need in oncology clinical care. While remission rates in AML patients can reach upwards of 90% under the current frontline therapy paradigm, nearly all patients relapse with treatment refractory disease less than 5 years after diagnosis. Relapse driven by therapy resistant cells that persist in the body after treatment (defined as minimal residual disease, or MRD) is the principal source of fatality in AML patients. Therefore, understanding how and where these leukemic cells survive treatment in vivo may help advance the rational development of highly synergistic combination therapies for the treatment of AML. Using a functional genomic approach (in vivo RNAi) and a new mouse model of AML chemoresistance (ChemoR) generated in our labs, we have identified several putative mediators of therapy resistance. Transcriptional profiling of the ChemoR model allowed us to generate a chemoresistance gene signature that we overlapped with the results of the shRNA screen to identify high-confidence genes of interest. The top genes from a ranked list of the most highly overexpressed (OE) genes in ChemoR cells and the top depleted genes from the shRNA screen in the context of therapy treatment were selected as high interest hits and subsequently tagged for individual follow-up experiments. Early validation studies suggest that some of our hits protect leukemic cells resident only at specific anatomical niches from therapy, while other proteins appear to be general chemosensitizers both in vivo and in vitro, and in both murine and human AML. Subsequent work will focus on elucidating the mechanisms by which these proteins promote resistance to frontline therapies in AML.

#2359

Adults with heterozygote carrier of immune deficiency variants have increased risk of cancer.

Daeyoon Kim,1 Murim Choi,2 Moon-woo Seong,3 Sung-Soo Yoon,4 Youngil Koh,4 Hongseok Yun2. 1 _Seoul National Univ. Cancer Research Inst., Seoul, Republic of Korea;_ 2 _Seoul National Univ. College of medicine, Seoul, Republic of Korea;_ 3 _Seoul National Univ. College of medicine, Department of Laboratory Medicine, Seoul, Republic of Korea;_ 4 _Seoul National Univ. College of medicine, Department of internal Medicine, Seoul, Republic of Korea_.

Introduction: Relationship between several mendelian disorders and cancer has been reported. Although correlation between immune-deficiency disorder and cancer development is well known, most studies have been conducted on patients with established immunodeficient diseases. Rate of tumor development in recessive heterozygous carriers of immunodeficiency genes is not clear yet.

Methods: Using whole-genome sequence data from International Cancer Genome Consortium (ICGC) Pan-Cancer Analysis of Working Group (PCAWG) and 1000 Genome projects, we analyzed 110 genes which are associated with immunodeficiency disorder and cancer risk. The 110 genes are reported previously as pathogen of immunodeficiency disorder and present in OMIM database. We selected a pathogenic variant of the 110 genes based on the clinical database such as HGMD and Clivar.

Results: Total of 461 pathogenic variants were detected in both cohorts. The prevalence of pathogenic variants of Immunodeficiency disorder was 4.7% in the ICGC-PCAWG cohort and 3.3% in 1000 Genome cohort. Pathogenic variants were observed more frequently in ICGC-PCAWG than in 1000 Genome cohort (p=0.00267) (odds ratio 1.55, 95% confidence interval 1.16-2.06), implying that immune deficiency disorder increases cancer risk. Gallbladder cancer patients most frequently had pathogenic variants (25%, 3/12) followed by ovarian cancer patients (8%, 9/110). The gene with the strongest cancer-evoking potential was TYK2 (p= 4.978 × 10-7). In addition, seven genes were uncovered to be significantly associated with cancer development (RORC, ATM, RTEL1, CTSC, RAG1, PMS2 and IRAK4).

Conclusion: In conclusion, using large whole-genome sequencing global projects, we statistically prove the previous notion that immunodeficiency provokes cancer development. We identified specific germline variants associated with cancer, which would be considered as cancer-predisposing genes.

#2360

Deregulated APA and cancer specific APA isoforms.

Oguzhan Begik, Melda Ercan, Harun Cingoz, Tolga Can, Merve Oyken, Ayse Elif Erson-Bensan. _Middle East Technical Univ., Ankara, Turkey_.

Certain aspects of diagnosis, prognosis, and treatment of cancer patients are still important challenges to be addressed. We developed a pipeline to uncover patterns of alternative polyadenylation (APA), a hidden complexity in cancer transcriptomes, to further accelerate efforts to discover novel cancer genes and pathways. Here, we found a significant shift in usage of poly(A) signals in six common tumor types compared to normal tissues. We further defined specific subsets of APA events to efficiently classify cancer types/subtypes. Triple negative breast cancers, for example, have specific 3'UTR length alterations where the significant majority are shortening events (70%, 113 of 165) of mostly proliferation-related transcripts compared with normal breast tissue. Such shortening events correlate with increased protein levels and relapse free survival of patients, suggesting functional significance of isoform variability. In line with this isoform diversity, we also detected deregulated expression of mRNA polyadenylation complex proteins in breast cancer cells. Of note, APA proteins are responsive to proliferative signals including estrogen and epidermal growth factor, suggesting a potential explanation to 3'-end isoform diversity in cancer cells. Overall, our study offers a computational and experimental approach for use of APA in novel gene discovery and classification in common tumor types, with important implications in basic research, biomarker discovery, and precision medicine approaches.

#2361

Big data analysis allows the identification of long non-coding RNAs with therapeutic potential against hepatocellular carcinoma.

Juan P. Unfried,1 Guillermo Serrano,1 Beatriz Suárez,1 Valeria Ferretti,1 Paloma Sangro,2 Celia Prior,1 Loreto Boix,3 Jordi Bruix,3 Bruno Sangro,2 Victor Segura,1 Puri Fortes1. 1 _Center for Applied Biomedical Research, Pamplona, Spain;_ 2 _University of Navarra Clinic, Pamplona, Spain;_ 3 _Institut d'Investigacions Biomèdiques August Pi i Sunyer, Barcelona, Spain_.

Hepatocellular carcinoma (HCC) is a disease associated with high morbidity and mortality for which there is currently no curative treatment and therapeutic targeting remains ineffective. To search for new therapeutic targets, we have evaluated the coding and non-coding transcriptome of all RNA-Seq data from TCGA, comprising over 10000 patients and 34 different tumors with HCC among them. To this, we have added the transcriptomes of additional 10000 samples from 53 human tissues from the GTEx project. Using neural networks and bioinformatics analyses we have identified coding and non-coding genes with preferential and differential expression and obtained a more precise location of HCC in the human transcriptional landscape. The analyses of tumor-deregulated gene expression indicated that coding genes have a high tumor promiscuity. Non-coding genes, on the contrary, showed higher tissue specificity and their expression was altered in a more tumor-specific manner, predictive of a therapeutic target with fewer secondary effects. We have identified 7321 long non-coding RNAs (lncRNAs) deregulated in at least one tumor type. In HCC, we found 1128 deregulated lncRNAs of which 138 were specific of HCC. Bioinformatic functional predictions indicated a positive relationship of deregulated lncRNAs with cell proliferation and motility while immune response was negatively associated. Ten lncRNAs with oncogenic potential were selected and their expression was validated in two independent cohorts of HCC patients. Most tumor samples showed high expression levels of these lncRNAs when compared to their paired peritumoral sample. Furthermore, several candidates were found to be expressed at very low levels in healthy liver. When expression of 2 of them was inhibited with antisense drugs in HCC cells, a drastic stall in proliferation was observed. Proliferation was recovered after restoring lncRNA expression. We think these candidates may play a key role in the proliferation and motility of cancer cells and show great potential as therapeutic targets. Our studies suggest that big data analyses can give a much-needed boost to HCC target search and to personalized medicine against cancer and other diseases.

#2362

Comprehensive portrait of canonical and non-canonical splicing in cancer.

Reyka G. Jayasinghe,1 Song Cao,1 Qingsong Gao,1 Matthew A. Wyczalkowski,1 Sohini Sengupta,1 Matthew J. Walter,1 Christopher Maher,1 Michael C. Wendl,1 Feng Chen,1 Eduardo Eyras,2 Alexander J. Lazar,3 Ken Chen,4 Ilya Shmulevich,5 Li Ding,1 The Splicing Analysis Working Group. 1 _Washington University in St. Louis, Saint Louis, MO;_ 2 _Catalan Institution of Research and Advanced Studies (ICREA), Barcelona, Spain;_ 3 _The University of Texas MD Anderson Cancer Center, Houston, TX;_ 4 _University of Texas M.D. Anderson Cancer Center, Houston, TX;_ 5 _Institute for Systems Biology, Seattle, WA_.

Current annotation methods typically classify mutations as disruptors of splicing if they fall on either the consensus intronic dinucleotide splice donor, GT, or the splice acceptor, AG. As a group, splice site mutations have been presumed to be invariably deleterious because of their disruption of the conserved sequences that are used to identify exon-intron boundaries. While this classification method has been useful, increasing evidence suggests that mutations outside of the canonical splice site can lead to transcriptional changes beyond disruption of the nearest junction. In this study, we have developed the MiSplice pipeline to determine the local and global effects of genomic mutations on splicing across 33 cancer types. To evaluate the local effects of mutations on splicing, we applied MiSplice to identify splice-disrupting mutations (SDMs) and splice-creating mutations (SCMs), genome-wide. We identified 1,964 novel SCMs, of which 26% and 11% were mis-annotated as missense and silent mutations and validated 10 of 11 genes in a mini-gene splicing assay. SDM identification predicted complex splicing patterns associated with canonical splice site mutations and identified a handful of mutations in proximity to the canonical junction that disrupt splicing factor binding sites. Interestingly, further investigation of the novel neoantigens produced by SCMs and SDMs are likely several folds more immunogenic than missense mutations. To explore the global impact of mutations on splicing we focused on recurrent and adjacent mutations disrupting the spliceosomal complex and related splicing factor genes from over 400 curated splice related genes. In addition, we applied HotSpot3D to identify splice-factor mutations (SFMs) that are significantly proximal to one another. Our analysis has identified novel SFMs that disrupt the spliceosomal complex and globally impact downstream splicing targets creating novel peptide sequences and alter key cancer genes. The current study has greatly extended the insight into the transcriptional ramifications of genomic alterations by integrating DNA and RNA sequencing data and painting the portrait of alternative splicing across cancer genomes.

#2363

Novel CDH1 mutations in breast invasive lobular carcinoma.

Garrett M. Frampton, Ryan Hartmaier, Ethan Sokol, Anika Gupta, Joel Greenbowe, Steven Roels, Laurie Gay, Philip J. Stephens. _Foundation Medicine, Cambridge, MA_.

Breast invasive lobular carcinoma (ILC) is the second most common type of breast cancer, making up approximately 10% of all invasive breast cancers. Breast ILC is characterized by loss of e-cadherin protein expression (CDH1), which is usually caused by loss of function mutations in the CDH1 gene. A subset of breast ILC cases have no identifiable CDH1 mutation, but presumably inactivate CDH1 through currently unknown mechanisms.

Tumors from more than 150,000 unique patients including more than 15,000 breast cancers were examined by comprehensive genomic profiling to detect base substitutions, indels, copy number alterations, and genomic rearrangements in the full coding regions of up to 465 genes. Statistical significance of the disease distributions was calculated using Fisher's exact test with false discovery rate multiple hypothesis testing correction.

Of 760 breast ILC cases, 583 (76.7%) had a known loss-of-function mutation in the CDH1 gene. To identify previously unknown mutations that result in or substitute for CDH1 loss-of-function we performed statistical analysis on all mutations, in any gene, occurring more than once in the 177 ILC cases without an identifiable CDH1 mutation. The most statistically significant mutation was very near to a splice site of CDH1 (chr16:68863554 c.2296-3A>G), occurring in 7 CDH1 negative ILC cases as well as 9 other cases in cancer types that frequently harbor CDH1 mutations (7 breast, 2 gastroesophageal junction) and 1 colorectal cancer. Based on tumor-only test based zygosity modeling, this mutation is only observed as somatic and usually homozygous in the tumor. Four other positions near to splice sites of CDH1 (chr16:68844247 c.832+3A>G, chr16:68849667 c.1565+5G>A, chr16:68856133 c.1936+5G>A, chr16:68857532 c.2164+3A>C), mutated in 13 CDH1 negative ILC cases, were also statically significant, somatic, and usually homozygous. No statistically significant mutations were found in genes other than CDH1.

Previously unappreciated somatic, non-canonical splice site mutations in CDH1 occur in ~10% (20/177) of breast ILC cases with no known CDH1 mutation. These mutations occur almost exclusively in tumor types known to harbor frequent CDH1 mutations and are usually homozygous in the tumor, strongly indicating that they are CDH1 loss-of-function mutations. 

### Kinases and Phosphatases

#2364

The phosphatase PRL-3 is a pro-survival oncogene in acute lymphoblastic leukemia.

Min Wei, Kristin O'Leary, Jinpeng Liu, Chi Wang, Jessica Blackburn. _University of Kentucky, Lexington, KY_.

PRL-3, phosphatase of regenerating liver 3, is one member of a subfamily of protein tyrosine phosphatases that belongs to the dual-specificity phosphatases (DSPs). Accumulating evidence shows that PRL-3 is a biomarker of tumor progression and metastasis and high PRL-3 expression is associated with reduced overall and disease-free survival in a variety of solid tumors, suggesting it's importance in tumor recurrence. However, the role for PRL-3 in leukemia relapse has not yet been described.

We have found that PRL-3 contributes to Acute Lymphoblastic Leukemia (ALL) malignancy by acting as a pro-survival gene, both in vitro and in vivo. Expression of PRL-3 in a MYC-induced zebrafish T-cell ALL model showed that PRL-3 expressing ALL had shorter latency and higher penetrance, compared with the fish injected with rag2:Myc; rag2:GFP alone. Growth after transplantation at low cell number was also significantly faster in the PRL3 expressing T-ALL, suggesting that PRL-3 plays an important role in ALL development and progression.

We found PRL-3 to be highly expressed in a majority of human ALL compared to normal peripheral blood mononuclear cells (PBMNC), which suggests clinical relevance. Analysis of gene expression microarray data of 119 patient samples reveals that PRL-3 was highly expressed in 58% of the patients. In addition, PRL-3 had the highest expression in the most aggressive and treatment resistant subgroups of T-ALL, the TAL/LMO and Immature subtypes. PRL-3 is also expressed by human T-ALL cell lines using quantitative RT- PCR and immunoblotting. PRL-3 knockdown leads to significant apoptosis in T-ALL cells. The treatment of T-ALL cells using small molecule drugs including 2-cyano-2-ene-ester compound (Analog 3) and thieno [3,2-b] pyridine 1-oxide (thienopyridone) , also resulted in significant growth inhibition of T-ALL cells in part through >4-fold increase in apoptosis, measured by annexin V staining.

In summary, our study provides evidence for a role of PRL-3 in T- ALL malignancy, and suggests that PRL3 may be a useful drug target in the treatment of this disease. Our future studies are focused on identifying the mechanisms by which PRL3 promotes T-ALL progression and survival.

#2365

A chemical genetics approach identifies PTP4A3 as a regulator of colon cancer cell adhesion.

Kelley E. McQueeney,1 Joseph M. Salamoun,2 Jennifer Ahn,1 Paula Pekic,1 Isabella K. Blanco,1 Heather Struckman,1 Elizabeth R. Sharlow,1 Peter Wipf,2 John S. Lazo1. 1 _University of Virginia, Charlottesville, VA;_ 2 _University of Pittsburgh, Pittsburgh, PA_.

Dysregulation of the tightly controlled protein phosphorylation networks that govern cellular behavior causes cancer. The membrane-associated, intracellular protein tyrosine phosphatase PTP4A3 is overexpressed in human colorectal cancer and contributes to cell migration and invasion. To interrogate further the role of PTP4A3 in colorectal cancer cell migration and invasion, we deleted the Ptp4a3 gene from murine colorectal tumor cells. The resulting PTP4A3-/- cells exhibited impaired colony formation, spheroid formation, migration, and adherence compared to the isogenic PTP4A3fl/fl cells. We replicated these phenotypic changes using the new small molecule allosteric PTP4A3 inhibitor, JMS-053. A related structure, JMS-038, which lacked phosphatase inhibition, displayed no cellular activity. Reduction in cell viability and colony formation by JMS-053 occurred in both mouse and human colorectal cell lines and required PTP4A3 expression. Ptp4a3 deletion increased the expression of extracellular matrix and adhesion genes, including the tumor suppressor Emilin 1. JMS-053 also increased Emilin 1 gene expression. Moreover, the human TCGA genomic database revealed colorectal tumors with high Ptp4a3 expression had low Emilin1 expression. These chemical and biological reagents reveal a previously unknown communication between the intracellular PTP4A3 phosphatase and the extracellular matrix and support efforts to pharmacologically target PTP4A3.

#2366

The tyrosine phosphatase, SHP-2, participates in regulating PD-L1 expression in non-small cell lung cancer.

Keller Toral, Brent Harris, Jarrod Creameans, Penni Black. _University of Kentucky, Lexington, KY_.

Immune checkpoint inhibitors, specifically those which target T cell surface receptor Programmed Cell Death-1 (PD-1) and its ligand, Programmed cell death ligand-1 (PD-L1), have demonstrated clinical benefit in patients with non-small cell lung cancer (NSCLC). Currently, tumor expression of PD-L1 serves as the FDA-approved companion diagnostic for response to anti-PD-1/PD-L1 therapy. However, a fraction of patients who express PD-L1 exhibit a response to treatment, and in some cases, patient tumors that do not express PD-L1 demonstrate response to therapy, making this a poor predictive biomarker. Further, the mechanism by which PD-L1 expression is controlled remains under-studied.

In this study, we seek to determine the impact of the protein tyrosine phosphatase, SHP-2, on PD-L1 expression through Jak/STAT- mediated signaling in NSCLC cell lines. SHP-2 can harbor activating mutations which prevent auto-inhibition of the catalytic phosphatase domain, and that may be pro-tumorigenic in some tumors. To understand the impact of SHP-2 activity on PD-L1 expression, we first assessed activation of STAT3 in NSCLC tumor cells after treatment with IFNγ and other mitogens. We then transfected NSCLC cells with wildtype and hyperactive SHP-2 mutants or ablated SHP-2 with siRNA specific for SHP-2 and measured both STAT3 and PD-L1 expression. Cell lines A549 and H661 exhibit low endogenous expression of both SHP-2 and PD-L1, and thus were chosen for transfection with hyperactive SHP-2 mutants (E76K and G503V), which also altered PD-L1 expression by western blot analysis. Quantitative real-time PCR (qPCR) and western blot analysis revealed that siRNA ablation of SHP-2 led to increased PD-L1 expression in the H460 cell line. Cells transfected with the SHP-2 activating mutants will also be assessed alteration of cell proliferation. Finally, using data obtained from The Cancer Genome Atlas project, STAT3 and PD-L1 expression levels are being evaluated in NSCLC tumor samples in which SHP-2 is mutated (E76K or G503V). With these data, we expect to gain a more complete understanding of the role of SHP-2 within the tumor microenvironment and the extent to which it controls PD-L1 expression, leading to new drug targets or combinations of drugs and better predictive biomarkers of response to PD-1 and PD-L1 inhibitors.

#2367

Identification of p-AKT as a PD marker for MER kinase in human G361 cells.

Yaoyu Chen. _Incyte, Wilmington, DE_.

Abnormalities in receptor tyrosine kinases (RTKs) have been shown to be involved in a wide range of important biological activities in cancer, including cell proliferation, differentiation and drug resistance. The TAM family of RTKs includes three family members: TYRO3, AXL, and MER. Binding of ligands to the these receptors was reported to lead to activation of the TAM kinases, as well as PI3K/AKT, RAS/RAF/MAPK and JAK/STAT signaling pathways in human cancer cells. Among these three TAM kinases, the MER signaling pathway represents an attractive target for the treatment of human cancers. MER phosphorylation is dependent on the ligand GAS6, however, ligand activated phospho-MER is often not stable and is very difficult to detect without pervanadate pretreatment in human cancer cells and this has been a hurdle for developing a selective MER kinase inhibitor. Therefore, it is important to identify a specific pharmacodynamic marker to monitor MER kinase activity in human cancer cells. Here, we report the development of a phospho-AKT assay for characterization of MER kinase inhibitors in human G361 cells. We profiled the expression of MER among multiple human cancer cells and demonstrated that MER and TYRO3, but not AXL show high levels of protein expression in human G361 melanoma cells. In G361 cells, phospho-AKT is induced by GAS6 treatment and the induction of phospho-AKT can be reversed by AXL/MER tool compounds. To determine the role MER and TYRO3 play in the activation of phospho-AKT, either MER or TYRO3 was selectively depleted by siRNA knockdown. We demonstrate that GAS6 induced phospho-AKT is only dependent on MER kinase, but not TYRO3 in human G361 melanoma cells. In addition, using phospho-AKT as readout, a high-throughput cell-based assay was established in the G361 human melanoma cells for evaluation of compound potency in inhibiting MER pathway activation. Further, we observed a correlation in compound potency between inhibition of phospho-AKT in G-361 cells and phospho-MER in MER-overexpressing BAF3 cells. In summary, we have demonstrated that GAS6 induced phospho-AKT can be a potential pharmacodynamic marker for inhibition of MER kinase and have successfully developed a cell-based functional assay for screening small molecule inhibitors of MER kinase for potential therapeutic utility in treating GAS6/MER-deregulated human cancers.

#2368

The phosphatase PRL3 as a novel therapeutic target in acute lymphoblastic leukemia.

Anna K. O'Leary, Min Wei, Jessica Blackburn. _University of Kentucky, Lexington, KY_.

Acute lymphoblastic leukemia (ALL) is one of the most prevalent cancers that is diagnosed in adolescents. The survival rate for ALL is good during the initial diagnosis but is much less optimistic for patients who have relapsed ALL, due to limited treatment options. Additionally, the high-dose cytotoxic chemotherapies that ALL patients are given have long-term adverse consequences. New and less toxic targeted therapies are needed for the treatment of ALL. We previously performed a large-scale transplantation screen in zebrafish to identify genes associated with high relapse potential in ALL, and a top hit from this screen was the protein tyrosine phosphatase 4A3, PTP4A3 or PRL3. Upregulation of PRL3 is correlated progression of colorectal, breast, and gastric cancers, and is associated with poor patient prognosis. We have found that PRL3 is highly expressed in a subset of patient ALL samples, and I am examining the role of PRL3 in ALL progression. We have found that PRL3 expression enhances ALL proliferation, and promotes leukemia survival after cytotoxic chemotherapy treatment. Small-molecule inhibition of PRL3 induces apoptosis in ALL, and synergizes with common ALL chemotherapies. I am currently examining the localization of PRL3 and identifying signaling pathways that it may be involved with to promote ALL progression. If my project is successful, it will provide scientific data that support our hypothesis that PRL3 is related to acute lymphoblastic leukemia. If there is a correlation between PRL3 and ALL, PRL3 could be used as a more effective and less toxic treatment for patients with ALL.

#2369

AMPK regulation of apoptosis through OSBP.

Kai Li Ong, Trever Thurgood, Michael Christensen, Jonny Malmrose, Denise Ng, Julianne Grose. _Brigham Young University, Provo, UT_.

Alterations in lipid metabolism have been frequently reported in tumor progression, with impaired PI(4,5)P2 homeostasis in the plasma membrane implicated in various cancers and metabolic disorders. Oxysterol binding protein (OSBP) and its related proteins (ORPs) constitute a large family of ubiquitously expressed, evolutionarily conserved lipid binding proteins that play critical roles in intracellular lipid trafficking. OSBP and ORPs associate with a wide range of cellular activities, however their functions and regulations are largely unclear. Emerging evidence has supported an essential role of OSBP in cell signaling pathways and cancer development. We recently identified yeast oxysterol binding protein Osh7 as a true substrate of yeast AMPK (Snf1). AMPK's involvement in cancer has been extensively studied in the past decade, and is an important mediator of cellular energy homeostasis. Despite many downstream targets identified, AMPK is not known to regulate oxysterol binding protein activities. We found that Snf1 may be responsible for the degradation of Osh7 and cells deficient in Osh7 and its homolog Osh6 display higher rates of apoptosis. Understanding the regulation of OSBP by AMPK may aid in better targeting AMPK for cancer treatment and prevention, at the same time provide a better understanding to the roles of OSBP in cancer development.

#2370

miR-19b enhances proliferation and apoptosis resistance via the epidermal growth factor receptor signalling pathway by targeting PP2A and BIM in non-small cell lung cancer.

Erik Vassella. _University of Bern, Bern, Switzerland_.

Background: Epidermal growth factor receptor (EGFR) mutations enable constitutive active downstream signaling of PI3K/AKT, KRAS/ERK and JAK/STAT pathways, and promote tumor progression by inducing uncontrolled proliferation, evasion of apoptosis and migration of non-small cell lung cancer (NSCLC). In addition, EGFR mutations increase the susceptibility of patients with NSCLC to tyrosine kinase inhibitor (TKI) therapy, but treated patients will invariably relapse with resistant disease. A global understanding of underlying molecular mechanisms of EGFR signaling may improve the management of NSCLC patients. Results: Here, we defined 17 miRNAs that are regulated by the PI3K/AKT branch of the EGFR signaling pathway. Bioinformatics analysis revealed that miRNAs that are dysregulated following PI3K/AKT inhibition act in a concerted manner to enhance the activity of the EGFR signaling pathway. These findings were closely mirrored by attenuation of miR-19b expression in NSCLC cell lines, where cell cycle progression, clonogenic growth and migration were reduced and apoptosis was enhanced. Conversely, miR-19b overexpression resulted in enhanced phosphorylation of ERK, AKT and STAT in EGFR mutant NSCLC cells. Co-treatment of NSCLC cells with the tyrosine kinase inhibitor (TKI) gefitinib and anti-miR-19b construct reduced migration and clonogenic growth in a synergistic manner suggesting that EGFR and miR-19b act together to control oncogenic processes. Serine/threonine phosphatase PP2A subunit PPP2R5E and BCL2L11 encoding BIM were identified as major targets of miR-19b. Consistent with this finding, PP2A activity was strongly enhanced in NSCLC transduced with anti-miR-19b, but not in cells co-transduced with anti-miR-19b and shPPP2R5E, suggesting that PPP2R5E is a major constituent of the PP2A complex. Accordingly, proliferation enhancement by miR-19b was due to targeting PPP2R5E. In contrast, apoptosis resistance was mainly due to targeting BCL2L11. Conclusion: Our results provide insight into the importance of targeting PPP2R5E and BCL2L11 by miR-19b in oncogenic processes of NSCLC. Attenuation of miR-19b expression could potentially be exploited in adjuvant therapy of EGFR mutant NSCLC.

#2371

Entospletinib, a potent SYK inhibitor, blocks constitutive and FCgRI activated signaling in FLT3-ITD cell lines.

Stacey L. Tannheimer, Adam Kashishian, Rick Sorensen, Kathleen S. Keegan. _Gilead Sciences, Inc., Foster City, CA_.

Spleen tyrosine kinase (SYK), a non-receptor protein tyrosine kinase, is an important mediator of immune receptor signaling in B cells, mast cells, neutrophils, and macrophages. SYK is expressed in 90% of AML blasts and has been reported to be is constitutively activated (Hahn et al., Cancer Cell 2009). High expression of phosphorylated SYK (pSYK) is associated with unfavorable outcomes, independent of other AML prognostic predictors such as age, cytogenetics, and white blood cell count (Boros et al., Oncotarget 2015). Entospletinib (ENTO) is a potent and selective inhibitor of SYK currently in clinical trials for the treatment of AML as monotherapy and in combination with standard-of-care therapies. We investigated the effects of ENTO on SYK phosphorylation and signaling in FLT3-ITD mutant AML cells. We found that ENTO blocked constitutive phosphorylation of pSYKY352, pSTAT5Y694, and MYC protein expression in FLT3-ITD-driven AML cell lines, MV4-11 and MOLM14. The concentration of ENTO required to inhibit pSYKY352 and pSTAT5Y694 correlated with inhibition of cell viability. Phosphorylation at these sites was inhibited by the selective FLT3 inhibitor, quizartinib; but not inhibited by Src family kinase inhibitor (dasatinib) or JAK inhibitors (ruxolitinib or tofacitinib). Further, a second-generation, highly selective SYK inhibitor, GS-9876, failed to inhibit the constitutive phosphorylation of these sites. Comparison of the biochemical and cellular kinase activities of the SYK and FLT3 inhibitors suggests that the observed inhibition of constitutive phosphorylation of pSYKY352 and pSTAT5Y694 and MYC expression was mediated by the direct activity of ENTO against FLT3-ITD, rather than SYK. Activation of FcγRI has been previously shown to activate SYK (Oellerich et al., Blood 2013). We demonstrate SYK activation at pSYKY348 and pSYKY323 after stimulation of the FCγRI with human IgG in the FLT3-ITD mutant cell line, MV4-11. Downstream signaling molecules pERK1/2T202/Y204, pVAV1Y160, and pLYNY507 were also activated by FCγR stimulation. ENTO inhibited phosphorylation of pSYKY348, pSYKY323 and pVAV1 Y160. The second-generation SYK inhibitor, GS-9876, also inhibited FCγRI-mediated activation of pSYKY348,pSYKY323 and pVAV1 Y160, suggesting that these phosphorylation events were mediated by SYK activity. In conclusion, ENTO inhibited constitutive and FCγRI-activated SYK phosphorylation and signaling pathways in FLT3-ITD mutant cell lines, resulting in inhibition of cell viability. Inhibition of constitutive SYK phosphorylation is mediated by direct activity of ENTO against FLT3-ITD; however, inhibition of FCγRI activated SYK results from direct inhibition of SYK.

#2372

**Kinome profiling of non-germline, genetically engineered mouse models of glioblastoma driven by** Cdkn2a **,** Egfr **, and/or** Pten **mutations reveals genotype-dependent kinase targets.**

Erin Smithberger,1 Alex R. Flores,1 Madison K. Butler,1 Harshil D. Dhruv,2 Gary L. Johnson,1 Michael E. Berens,2 Frank B. Furnari,3 C. Ryan Miller1. 1 _Univ. of North Carolina at Chapel Hill, Chapel Hill, NC;_ 2 _Translational Genomics Research Institute, Phoenix, AZ;_ 3 _Ludwig Institute for Cancer Research and University of California San Diego, San Diego, CA_.

The most attractive target for personalized medicine of glioblastoma (GBM) remains epidermal growth factor receptor (EGFR), due to its frequency in and specificity for the disease and the number of drugs that target its tyrosine kinase domain. Yet several EGFR tyrosine kinase inhibitors (TKI) have failed clinically, in part due to multiple molecular mechanisms of resistance. The most common oncogenic mutation in GBM is EGFRvIII, while the most common tumor suppressors lost are CDKN2A and PTEN. Mutations in these 3 genes frequently co-occur. To identify potentially attractive kinase targets for use in combination regiments with an EGFR TKI, we used multiplex inhibitor bead/mass spectrometry (MIB-MS) and RNA-seq to examine the baseline kinomes and transcriptomes of non-germline genetically engineered mouse (nGEM) models of GBM, specifically cultured Cdkn2a-null murine astrocytes (C) engineered to harbor human EGFRvIII (CEv3), Pten deletion (CP), or both (CEv3P). Among these 4 lines, 5.2-9.7% of the transcriptome was differentially expressed using DESeq2 at Q<0.001. Gene set analysis showed progressive loss of astrocyte and gain of stemness signatures in more heavily mutated cells (CEv3P>CEv3> CP>C), suggesting that EGFRvIII and Pten deletions cooperate to induce astrocyte de-differentiation into glioma stem cells (GSC). Principal components analysis showed a significant influence of both EGFRvIII (component 1, 44-48% variance) and Pten (component 2, ~33% variance) status on the transcriptome and kinome. Of the 228 expressed kinases detected using MIB-MS, 86 (38%) were differentially expressed in 1 or more genotypes. Integrated transcriptome and kinome analysis showed that Egfr was significantly over-expressed and hyperactive in EGFRvIII-mutated cells. Akt1 showed a non-expression driven increase in kinase activity in Pten-deleted cells, consistent with known effects of Pten on PI3K signaling. Pairwise genotype comparisons revealed 5-20 additional kinases that were differentially activated. Some, including Pdgfrb, Fgfr2, Lyn, Ddr1, and several members of the Ephrin family, represent potential targets for dual therapy with EGFR TKI. Clinically-curated human GBM patient-derived xenograft (PDX) models matched to CEv3P, CEv3, CP, and C nGEM models will afford comparisons in a patient-based preclinical setting for translational support. Functional kinome analysis using targeted EGFR TKI and MIB-MS in nGEM and PDX will help define the kinase networks required for EGFRvIII-driven GBM pathogenesis and may aid in the identification of novel treatment combinations.

#2373

ADA-07 suppresses solar ultraviolet-induced skin carcinogenesis by directly inhibiting TOPK.

Tianshun Zhang, Qiushi Wang, Ge Gao, Bode Ann, Zigang Dong. _Hormel Institute University of Minnesota, Austin, MN_.

Cumulative exposure to solar ultraviolet (SUV) irradiation is regarded as the major etiologic factor in the development of skin cancer. The activation of the mitogen-activated protein kinase (MAPK) cascades occurs rapidly and is vital in the regulation of SUV-induced cellular responses. The T-LAK cell-originated protein kinase (TOPK), an upstream activator of MAPKs, is heavily involved in inflammation, DNA damage, and tumor development. However, the chemopreventive and therapeutic effects of specific TOPK inhibitors in SUV-induced skin cancer have not yet been elucidated. In the current study, ADA-07, a novel TOPK inhibitor, was synthesized and characterized. Pull-down assay results, ATP competition and in vitro kinase assay data revealed that ADA-07 interacted with TOPK at the ATP-binding pocket and inhibited its kinase activity. Western blot analysis showed that ADA-07 suppressed SUV-induced phosphorylation of ERK1/2, p38, and JNKs, and subsequently inhibited AP-1 activity. Importantly, topical treatment with ADA-07 dramatically attenuated tumor incidence, multiplicity, and volume in SKH-1 hairless mice exposed to chronic SUV. Our findings suggest that ADA-07 is a promising chemopreventive or potential therapeutic agent against SUV-induced skin carcinogenesis that acts by specifically targeting TOPK.

#2374

Oncogenic kinases-induced PKM2 tyrosine 105 phosphorylation converts nononcogenic PKM2 to a tumor promoter and induces cancer stem-like cells.

Lin Zhang, Zhifen Zhou, Min Li, Dihua Yu. _UT MD Anderson Cancer Ctr., Houston, TX_.

The role of pyruvate kinase M2 isoform (PKM2) in tumor progression has been controversial. Previous studies showed that PKM2 promoted tumor growth in xenograft models; however, depletion of PKM2 in the Brca1-loss-driven mammary tumor mouse model accelerated tumor formation. Since oncogenic kinases are frequently activated in tumors and PKM2 phosphorylation promotes tumor growth, we hypothesized that PKM2 phosphorylation by activated kinases in tumor cells confers PKM2 oncogenic function, whereas non-phosphorylated PKM2 is non-oncogenic. Indeed, we found that PKM2 was phosphorylated at tyrosine 105 (Y105) in MDA-MB-231 breast cancer cells; whereas PKM2 was largely unphosphorylated in non-transformed MCF10A cells. Multiple kinases that are frequently activated in different cancer types were identified to phosphorylate PKM2-Y105 in our tyrosine kinase screening. Introducing the PKM2-Y105D phospho-mimetic mutant into MCF10A cells induced colony formation and the CD44hi/CD24neg cancer stem-like cell population partly by increasing YAP protein nuclear localization. ErbB2, a strong inducer of PKM2-Y105 phosphorylation, boosted YAP nuclear localization and cancer stem-like cell population. Lapatinib, an ErbB2 kinase inhibitor, decreased PKM2-Y105 phosphorylation and cancer stem-like cells, impeding PKM2 tumor-promoting function. Taken together, in non-transformed cells, unphosphorylated PKM2 is non-oncogenic; but in tumor cells, phosphorylated PKM2-Y105 by activated kinases gains oncogenic functions partly by activating YAP downstream signaling to increase cancer stem-like cell properties.

#2375

Concurrent HER or PI3K inhibition potentiates the anti-tumor effect of ERK inhibitor BVD-523 (ulixertinib) in preclinical pancreatic cancer models.

Hongmei Jiang,1 Mai Xu,1 Lin Li,1 Maureen Highkin,1 Daoxiang Zhang,1 Qiong Li,2 Andrea Wang-Gillam,1 Kian-Huat Lim1. 1 _Washington University School of Medicine, St. Louis, MO;_ 2 _Renji Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai, China_.

To date, effective treatments for inoperable pancreatic ductal adenocarcinoma (PDAC) remain elusive. Targeting KRAS, the gene that is mutated in >95% of PDAC, is a heavily pursued strategy, but unsuccessful in the clinic. Therefore, targeting key effector cascades of KRAS oncoprotein, particularly the mitogenic RAF-MEK-ERK represents the next best strategy. However, RAF or MEK inhibitors have not shown promising clinical efficacy in PDAC. Several studies have shown that cancer cells treated with RAF or MEK inhibitors adopt multiple mechanisms to re-activate ERK signaling. Therefore, development of ERK-specific inhibitors carries the promise to effectively abrogate this pathway. BVD-523 (ulixertinib) is a first-in-class ERK-specific inhibitor that has demonstrated anti-tumor activity in clinical trials. In this study, we showed that BVD-523 effectively inhibits transformed growth of multiple PDAC lines and potentiates the cytotoxic effect of gemcitabine. Moreover, using reverse-phase protein array analysis, we identified potential mechanisms by which PDAC cells may adopt to tolerate ERK inhibition. On this basis, we proposed and tested two rational combinatorial approaches with BVD-523 that showed promising preclinical efficacy in vitro and in mouse xenografts. Overall, we provide the first evidence that PDAC cells may readily adapt to pharmacologic ERK inhibition, and provide combinatorial therapeutic strategies that could benefit patients.

#2376

AXL receptor tyrosine kinase stimulates cell softening and cancer progression via Rac signaling pathway.

Masami Suganuma,1 Keisuke Iida,2 Shota Yokoyama,1 Anchalee Rawangkan,1 Kozue Namiki,1 Pattama Wongsirisin1. 1 _Saitama University, Saitama, Japan;_ 2 _Chiba University, Chiba, Japan_.

Cancer cells exhibit different biophysical properties, such as cell stiffness and elasticity, compared with normal cells. It is now well accepted that cell stiffness measured by atomic force microscope (AFM) is a quantitative indicator of cancer progression and metastasis. To study the role of cell softening in malignant progression, Using Transwell assays and AFM, six human non-small cell lung cancer cell lines were classified into two groups: a high motility-low stiffness (HMLS) group and a low motility-high stiffness (LMHS) group. We found a significant difference in expression of the AXL receptor tyrosine kinase, which belongs to the TAM (Tyro3, AXL, Mer) family, in the stimulation of motility and cell softening. HMLS cells expressed higher AXL and phosphorylated AXL levels than LMHS cells. Exogenous AXL gene expression in H1703 LMHS cells exhibited increased motility and decreased stiffness, along with lower levels of actin stress fibre formation. Conversely, the AXL-specific inhibitor R428 and AXL-targeting siRNA reduced motility and increased stiffness in H1299 HMLS cells. Knockdown of AXL gene stimulated both actin stress fibre formation and focal adhesion kinase phosphorylation, which inhibited tumour formation in a mouse xenograft model. Treatment with Ras/Rac inhibitor SCH 51344, which blocks disruption of actin stress fibres, exerted similar effects to AXL inactivation in H1299 and H1703-AXL cells, but not in H1703 cells (no AXL expression). Furthermore, treatment with an ROCK inhibitor, Y27632, did not show any effect in two AXL expressing cells, while it stimulated motility in H1703 cells. Considering all these results, we propose that the Ras/Rac pathway operates downstream of AXL and suppresses the Rho/ROCK pathway. Cell softening induced by AXL activation is a key biophysical property of cancer cells, and AXL is thus a new target for inhibition of malignant progression in non-small cell lung cancer.

#2377

Design, synthesis, and characterization of 4-aminopyrazole quinazolines as potent inhibitors of G protein-coupled receptor kinase GRK6 for the potential treatment of multiple myeloma.

David Uehling,1 Babu Joseph,1 Carly Griffin,1 Ratheesh Subramaniam,1 Ayome Abibi,1 Richard Marcellus,1 Michael Prakesch,1 Gennadiy Poda,1 Methvin Isaac,1 Chungyee Leung-Hagesteijn,2 Rodger Tiedemann,1 Rima Al-awar1. 1 _Ontario Institute for Cancer Research, Toronto, Ontario, Canada;_ 2 _University Health Network, Toronto, Ontario, Canada_.

Multiple myeloma (MM) is one of the most common hematological malignancies, but current therapeutic options are limited to high-dose chemotherapy or high-risk stem-cell transplantation. In a kinome-wide RNAi study by Tiedemann and colleagues (2010), the G-protein coupled receptor kinase GRK6 was identified as a critical kinase required for survival of MM cells. This study also suggests that MM cells, but not other cell types, are dependent on GRK6; and that gene silencing by shRNA or siRNA of GRK6, but not other members of the GRK family, results in decreased survival. Through gene silencing techniques, we determined that a functional GRK6 kinase domain is required for survival of MM cells. These findings helped validate that the kinase domain of GRK6 is a promising target for MM, and therefore encouraged us to embark on an effort to identify potent small molecule kinase inhibitors of GRK6. Toward that end, by screening a focused kinase-directed library of small molecule inhibitors, compounds with moderate potency against GRK6 in biochemical assays were identified. Through this exercise, we discovered that two structurally distinct aminopyrazoles AZ-960 and ASC-082 had modest inhibition of GRK6. By combining structural features of these hits and further optimization we identified the quinazoline analogue OICR9945, a potent GRK6 inhibitor with good selectivity against a panel of diverse kinases and anti-proliferative activity against MM cell lines. Herein, we describe the design, synthesis and early pharmacological characterization of OICR9945 along with structure activity relationships (SAR) of this series against GRK6 and other kinases.

#2378

Histone H4 tyr88-phosphorylation mediated by ACK1 promotes castration-resistant prostate cancer by epigenetic reprogramming of androgen receptor.

Nupam P. Mahajan, Kiran Mahajan, Nicholas Lawrence, Harshani Lawrence. _Moffitt Cancer Center, Tampa, FL_.

Androgen receptor (AR) plays a paramount role in the onset and progression of prostate cancer (PC). This very facet underlies androgen deprivation therapy (ADT), a preferred treatment to negate AR transcriptional activity. While ADT provides immediate palliative benefits, it is ineffective long term, as the recalcitrant disease recurs within 2-3 years and progresses to a lethal stage, referred to as the metastatic castration-resistant prostate cancer (mCRPC). The AR gene (AR) is amplified or mutated in >50% of mCRPCs, suggesting that PC cells may have reinvigorated AR transcription as a response to the loss of existing AR activity by ADT. Consequently, resistance to ADT has become one of the most vexing problems in PC therapy. AR antagonists are often ineffective due to their inability to repress the expression of AR or its splice variant, AR-V7. Here, we report that the tyrosine kinase ACK1 (TNK2) phosphorylates histone H4 at tyrosine 88 upstream of the AR transcription start site. The WDR5/MLL2 complex reads the H4-Y88-phosphorylation marks and deposits the transcriptionally activating H3K4-trimethyl marks promoting AR transcription. We developed a new class of ACK1 kinase-specific small molecule inhibitor (R)-9bMS that suppresses ACK1 kinase activity at IC50= 48 nM. Reversal of the pY88-H4 epigenetic marks by the ACK1 inhibitor (R)-9bMS-sensitized naive and enzalutamide-resistant prostate cancer cells and reduced AR and AR-V7 levels to mitigate CRPC tumor growth. Thus, a feedforward ACK1/pY88-H4/WDR5/MLL2/AR epigenetic circuit drives CRPC and is necessary for maintenance of the malignant state.

#2379

PTPRT pseudo-phosphatase domain is a denitrase that contributes to its tumor suppressor function.

Yiqing Zhao, Zhenghe Wang. _Case Western Reserve Univ., Cleveland, OH_.

Department of Genetics & Genome Sciences and Case Comprehensive Cancer Center, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, Ohio 44106, USA.

In addition to phosphorylation, tyrosine residues in proteins can have nitration at the 3-carbon position on the phenol ring (Y-NO2). Protein tyrosine nitration occurs under both physiological and pathologic conditions. However, enzymes that add or remove this protein modification remain to be identified. Protein tyrosine phosphatase receptor T (PTPRT) is one of the twelve receptor protein tyrosine phosphatases (RPTPs) that have two catalytic PTP domains in their intracellular parts. While the membrane proximal PTP domains (D1) are protein tyrosine phosphatases, it has been thought that the C-terminal PTP domains (D2) are pseudo-phosphatase that lack enzymatic activity. Here we report that the pseudo-phosphatase domain (D2) of PTPRT is a denitrase that removes nitro-groups from tyrosine residues in paxillin. PTPRT normally functions as a tumor suppressor and is frequently mutated in a variety of human cancers including colorectal cancer. Interestingly, a fifth of tumor-derived mutations of PTPRT are located in the D2 pseudo-PTP domain. We demonstrate that some of the tumor-derived mutations located in the pseudo-phosphatase domain impair the denitrase activity. Moreover, PTPRT mutant mice that inactivate the denitrase activity are susceptible to carcinogen-induced colon tumor formation. Our study uncovers a novel enzyme that functions as a tumor suppressor.

#2380

Induction of gap junctional chemotherapy bystander effect in breast cancer cells through regulation of the protein kinase A pathway.

Prarthana Pradeep, Alexander E. Urban, Jennifer C. Jones, Thomas M. Bodenstine. _Midwestern University, Downers Grove, IL_.

Gap junctional intercellular communication (GJIC) mediates the regulated transfer of cellular metabolites between cells and represents an integral component of homeostasis in epithelial cells. The effects of dysregulation of GJIC in cancer has proven to be complex and context dependent, however, a lack of GJIC between cancer cells and a causative role in cancer progression have been demonstrated. In the context of treatment, delivery of therapeutics is often hindered by a lack of vascularization in hypoxic solid tumors. Transfer of small molecular weight therapies between cells through gap junctions, a process known as bystander effect, represents a potential mechanism for transfer of therapies within a tumor in the absence of a blood supply. Previous evidence has shown that upregulation of the Protein Kinase A (PKA) pathway can restore GJIC in cancer cells by promoting the assembly of gap junction channels. As a proof-of-principle experiment, we hypothesized that stimulation of the PKA pathway in breast cancer cells could facilitate the intercellular transfer and subsequent cellular effects of chemotherapy. Using the MDA-MB-231 and T47D breast cancer cell lines which exhibit low GJIC, we utilized multiple PKA activators (8-Br-cAMP, forskolin, LY294002) and the PKA inhibitor H89 to regulate the activity of PKA. Pathway activation was confirmed by western blot analysis of phosphorylated CREB at serine 133 and immunofluorescent localization of PKA catalytic subunits. A corresponding increase in GJIC was assessed by following the transfer of the fluorescent gap-junction permeable dye calcein from labeled donor cells to non-labeled acceptor cells. Results were recorded by live-cell fluorescence microscopy and quantified using flow cytometry. The Hs578T breast cancer cell line which exhibits high levels of GJIC was used as a positive control in these experiments. To assess the effects of chemotherapeutic spread, cells were treated with doxorubicin and subsequently co-cultured with non-treated cells. Transfer of doxorubicin was traced by its autofluorescent properties and induction of apoptosis in doxorubicin treated and untreated cells was assessed using annexin V staining by flow cytometry. Transfer of doxorubicin to non-treated cells did not occur under control conditions. Following co-culture in the presence of PKA activators, transfer of doxorubicin into non-treated cells was evident and resulted in the induction of apoptosis. These results demonstrate that upregulation of GJIC by mediating changes to PKA promote the spread of chemotherapeutic effects and indicate a possible role for modulation of PKA in breast cancer cells to improve the transfer of chemotherapeutics.

#2381

Loss of S6K2 perturbs redox balance and fatty acid metabolism, leading to oxidative cell death.

Hsin-Yi Chen,1 Minu Samanta,1 Patricia Reyes-Uribe,1 Andrew V. Kossenkov,1 Xiangfan Yin,1 Yiling Lu,2 Mark Axelrod,3 Michael J. Weber,3 Qin Liu,1 Gordon B. Mills,2 Jessie Villanueva1. 1 _The Wistar Institute, Philadelphia, PA;_ 2 _The University of Texas MD Anderson Cancer Center, Houston, TX;_ 3 _University of Virginia, Charlottesville, VA_.

Oncogenes often upregulate nutrient sensing and utilizing pathways, leading to altered metabolism in tumor cells. RAS-driven tumors typify cancers undergoing marked metabolic reprogramming. In melanoma, RAS signaling is often deregulated; while activating mutations in NRAS are present in >25% of tumors, RAS-driven tumors account for almost 50% of cases. However, there are no effective therapies for this type of tumors. Although oncogenic NRAS activates the MAPK pathway, inhibition of this pathway alone has limited antitumor efficacy and most NRAS-mutant tumors are resistant to MAPK inhibitor (MAPKi) monotherapy. We discovered that NRAS-mutant melanomas resistant to MAPKi are highly dependent on the ribosomal protein S6 kinase 2 (S6K2). We demonstrate that loss of S6K2, a signaling effector of the mTORC1 nutrient-sensing pathway, triggers cell death selectively in NRAS-mutant melanoma resistant to MAPKi. Acute depletion of S6K2 enhanced ROS production, lipid synthesis and accumulation of intracellular unsaturated fatty acids. ROS susceptible (poly)-unsaturated fatty acids sensitized cells to ROS, resulting in lipid peroxidation and oxidative cell death. We further determined that S6K2 depletion was coupled to increased expression of markers of apoptosis and ferroptosis, suggesting that S6K2 blockade could trigger multiple forms of cell death, including apoptotic and a ferroptotic-like cell death. Notably, co-inhibition of S6K1 (a S6K isoform that is co-regulated by mTORC1) together with S6K2 diminished the effects of S6K2 blockade, suggesting that selective inhibition of S6K2 is required to induce cell death. Indeed, while silencing of S6K2 triggered lipid peroxidation and oxidative cell death and S6K1 silencing had negligible effects, concomitant depletion of S6K1 and S6K2 attenuated lipid peroxidation and cell death. Mimicking the effects of S6K2 loss by combining an ROS-inducing agent with unsaturated fatty acids or treating tumor cells with lipid peroxidation inducers restrained NRAS-mutant melanoma growth in vitro and in vivo. Taken together, our studies have identified a critical vulnerability of NRAS-mutant melanoma by uncoupling S6K1 and S6K2 to trigger metabolic dysfunction and tumor cell death. This strategy is remarkably distinct from using PI3K/mTOR inhibitors, which concurrently suppress S6K1 and S6K2 and are often cytostatic. We propose that this paradigm could be exploited to develop therapeutic approaches targeting NRAS-mutant tumors and overcome resistance to MAPKi in the context of oncogenic NRAS.

#2382

In silico/in vitro studies of novel hot spot mutations in BCR-ABL1: A multidisciplinary approach to shed a new light on TKI resistance.

Erik Laurini,1 Suzana Aulic,1 Natasa Skoko,2 Sulena Polez,2 Marco Baralle,2 Maurizio Romano,1 Sabrina Pricl1. 1 _Univ. of Trieste, Trieste, Italy;_ 2 _International Center for Genetic Engineering and Biotechnology (ICGEB), Trieste, Italy_.

Acquisition of mutations in the BCR-ABL1 kinase domain (KD) is frequently associated with tyrosine kinase inhibitor (TKI) failure in CML. Recently, we revealed a novel mutation "hot- spot" in the BCR-ABL1 KD region (residues 295 - 312), associated with high resistance and poor clinical outcomes [1]. Some of these new BCR-ABL1 variants locate in a protein region, which might not be directly involved in TKI interaction. Therefore, we hypothesized that some of these mutations might be linked to TKI resistance via a direct mechanism while others might exert an indirect effect on drug resistance. The present contribution integrates molecular, computational, and structural biology techniques to understand the eventual role of the newly reported hot-spot mutations of the BCR-ABL1 KD in TKI resistance observed in CML patients using a "two-tier level" investigation: isolated KDs and SH2-linker-SH3-KD BCR- ABL1 constructs. Molecular Modeling will assess in silico whether the clinically observed "hot-spot" BCR-ABL1 mutations might be directly related to a change in the thermodynamic stability and/or protein structure and how these modifications could be involved in the BCR-ABL1 binding profile to specific TKIs (e.g., imatinib, dasatinib, and ponatinib) compared with wild-type (WT) systems and "prototypical" BCR-ABL1 mutations (e.g. T315I). In parallel, in vitro analysis will evaluate the effects exerted by the selected mutations on the intrinsic activity of the kinase and to determine the inhibition activity of the selected drugs on these BCR-ABL1 variants. Specifically, direct drug binding to both KD and SSK constructs will be measured using isothermal titration calorimetry and in vitro kinase assays will monitor the ability of these mutant isoforms to auto-phosphorylate and to phosphorylate a substrate peptide. Finally, structural biology (e.g., SAXS and far-UV CD) evidences of WT and mutant isolated KDs and SH2-linker-SH3-KD (SSK) constructs (per se and in complex with TKIs) will be discussed to support in silico predictions. [1] Gibbons DL, Pricl S et al., Molecular dynamics reveal BCR-ABL1 polymutants as a unique mechanism of resistance to PAN-BCR-ABL1 kinase inhibitor therapy. Proc Natl Acad Sci U S A. 2014 Mar 4;111(9):3550-5. doi: 10.1073/pnas.1321173111.

#2383

EGFR expression increases following decline in activity of N-acetylgalactosamine 4-sulfatase (ARSB) and SHP2 (PTPN11) and increases in chondroitin 4-sulfate and JNK in prostate.

Sumit Bhattacharyya, Leo Feferman, Joanne K. Tobacman. _University of Illinois at Chicago, Chicago, IL_.

The enzyme arylsulfatase B (ARSB; N-acetylgalactosamine 4-sulfatase) removes 4-sulfate groups from the non-reducing end of dermatan sulfate and chondroitin 4-sulfate (C4S) and is required for degradation of these sulfated glycosaminoglycans. Decline in ARSB and the resultant increase in C4S was previously reported to inhibit the activity of the enzyme SHP2 (PTPN11), a ubiquitous, non-receptor tyrosine phosphatase, and to lead to transcriptional events mediated by the sustained phosphorylation of phospho-ERK1,2 and phospho-p38. Previous experiments had shown that decline in ARSB was associated with increased expression of the epidermal growth factor receptor (EGFR; Her1/ErbB1) in human prostate malignant tissue and in human prostate cells, but the mechanism for this increase was unknown. The current experiments were performed to determine how decline in ARSB leads to increased expression of EGFR. Experiments were performed in human prostate stem and epithelial cells and normal and malignant human prostate tissues. Measurements included: EGFR promoter activity, chromatin immunoprecipitation assay, and binding studies of chondroitin sulfates with SHP2. Materials used included: SR11302, an inhibitor of c-fos binding with nuclear DNA; c-Jun peptide that acts as a c-Jun mimetic and inhibits c-Jun phosphorylation by JNK; JNK-selective inhibitor peptide that inhibits JNK phosphorylation; SHP2 dominant negative and constitutively active DNA constructs; PHPS1, an inhibitor of SHP2; ARSB siRNA, and galectin-3 siRNA. Increased EGFR promoter activation and EGFR expression followed decline in ARSB, decline in activity of SHP2 (PTPN11), enhanced activity of c-Jun N-terminal kinase (JNK), increased nuclear DNA binding of c-Jun and c-Fos, and EGFR promoter activation. These effects demonstrate an impact of ARSB and chondroitin 4-sulfation on the activation of a critical dual phosphorylation threonine-tyrosine kinase and on the mRNA expression of an important tyrosine kinase receptor in prostate cells. Restoration of ARSB activity may provide a new therapeutic approach to malignancies in which there is activation of EGFR-mediated tyrosine kinase signaling pathways.

#2384

Preclinical evaluation of PD and efficacy of novel potent selective and orally bioavailable CDK12 covalent inhibitors in TNBC model.

Ramulu Poddutoori, Sujatha Rajagopalan, Subhendu Mukherjee, Sivapriya Marappan, Samiulla D S, Sasirekha Sivakumar, Shilpa S. Nayak, Ravindra M. V, Hadianawala Murtuza, Devaraja T. S, Srinivas Kondela, Suraj Tgore, Amit A. Dhudashiya, Charamanna K. B, Thomas Antony, Girish Daginakatte, Sanjeev Giri, Shekar Chelur, Murali Ramachandra, Chetan Pandit, Susanta Samajdar. _Aurigene Discovery Technologies Ltd., Bangalore, India_.

Cyclin-dependent kinase 12 (CDK12) is a transcription-associated kinase that participates in various cellular processes such as DNA damage response, splicing and pre-mRNA processing. In association with Cyclin K (CycK), CDK12 regulates transcription elongation by phosphorylating RNA polymerase II (RNAP II) at Serine 2 in the C-terminal domain (CTD). Overexpression of CDK12 in various tumor types suggests the possibility that CDK12 has oncogenic properties, similarly to other transcription-associated kinases. Considering its critical role in transcription and RNA processing CDK12 is emerging as a potential therapeutic target for cancer. Multiple series of potent and selective CDK12 covalent inhibitors were identified by iterative medicinal chemistry efforts and SAR-based approaches. Early compounds were optimized towards attaining good physicochemical properties, high potency, good selectivity and desirable pharmacokinetic profile to achieve anti-tumour activity. Very potent and highly selective CDK12 inhibitors have been identified from two distinct chemical series. The covalent mode of action for these biochemically potent compounds has been confirmed by CDK12 target engagement assay in the cellular context. These selective inhibitors showed significant anti-proliferative activity in TNBC and other cancer cell lines, which correlated with inhibition of pS2 (RNAP II), a bonafide CDK12 substrate and target engagement. In vivo target engagement, PD and efficacy data for optimized compounds with good oral bioavailability in a TNBC (HCC-70) xenograft model along with will be presented.

#2385

Evaluation of GCN2 chemical probes: Activity in leukemia.

Lea Lough, Dan Sherman, Eric Ni, Palaniraja Thandapani, Ioannis Aifantis, Timothy Cardozo. _New York School of Medicine, New york, NY_.

Cellular stress signals activate adaptive signaling pathways of the mammalian integrated stress response (ISR), which converge at the phosporylation of eIF2α. Drug-like chemical inhibitors targeting major ISR kinases have been identified, with the exception of GCN2. We synthesized and evaluated a series of GCN2 inhibitors based on a triazolo[4,5-d]pyrimidine scaffold. Several compounds potently inhibited GCN2 in vitro in orthogonal assays and displayed good selectivity over the related kinases PERK, HRI, and IRE1. The compounds had low solubility at pH 7.4, but sufficient permeability to inhibit phosporylation of eIF2α in HEK293T cells with an IC50 < 150 nM. In a screen of the NCI-60 cancer cell line panel, the leukemia cells were the only group of cancer cell lines to uniformly exhibit growth inhibition. Given the previously reported genetic association of Gcn2 with resistance to asparaginase, which is a first line clinical leukemia therapy, pharmacologic inhibition of Gcn2 may overcome resistance to asparaginase treatment in leukemia.

#2386

Identification of SIK1 as a potential therapeutic target for desmoplastic small round cell tumor.

Alifiani B. Hartono, Sean B. Lee. _Tulane University School of Medicine, New Orleans, LA_.

Desmoplastic Small Round Cell Tumor (DSRCT) is a rare and aggressive malignant cancer caused by the chromosomal translocation of t(11;22)(p13;q12) that produces a novel, aberrant transcription factor, EWS-WT1. EWS-WT1 is essential in the formation of DSRCT as well as for maintaining DSRCT cell growth. However, current research has not elucidated how EWS-WT1 leads to the oncogenesis of DSRCT. Through our integrative gene expression data, we identified Salt Inducible Kinase 1 (SIK1) as one of direct target genes of EWS-WT1 in JN-DSRCT-1 cell line. SIK1 is a member of the AMPK related kinases and is involved in gluconeogenesis and lipogenesis regulation, skeletal myocytes development, p53 dependent anoikis, and cell cycle regulation. Through our ChIP analysis, we showed that EWS-WT1 directly binds to a 2kb proximal promoter region of SIK1. Following SIK1 depletion, JN-DSRCT-1 cell proliferation sharply decreased, similar to the growth inhibition observed when EWS-WT1 is depleted. We further showed that cells do not transit to S phase when SIK1 is depleted, suggesting a critical role of SIK1 in cell cycle regulation in DSRCT. Taken together, we have established that EWS-WT1 directly activates SIK1 expression and promotes cell proliferation through SIK1. Therefore, our work identified SIK1 as a new potential therapeutic target in DSRCT.

#2387

The oncogenic role and therapeutic potential of PTPN11 in melanoma.

Kristen Suzanne Hill,1 Evan Roberts,1 Ellen Marin,1 Xue Wang,1 Jamie Teer,1 Jane Messina,1 Jerry Wu,2 Minjung Kim1. 1 _Moffitt Cancer Ctr., Tampa, FL;_ 2 _University of Oklahoma Health Sciences Center, OK_.

The RAS/RAF/MEK/ERK signaling pathway, which is intricately regulated by multiple proteins including PTPN11 (Tyrosine-Protein Phosphatase Non-Receptor Type 11, encoding SHP2), is frequently activated in melanoma. Although implicated as an oncogene in multiple cancer types, the oncogenic role of PTPN11 has not been established in melanoma. Recently, we preformed whole exome sequencing on tumors generated by a mouse model driven by loss of PTEN and CDKN2A (INK4A/ARF) and identified several conserved cross-species orthologous mutations in Kras, Erbb3, and Ptpn11. In this study, we addressed the functional roles of PTPN11 in melanoma tumorigenesis and tumor maintenance; as well as, PTPN11's effect on the RAS/RAF/MEK/ERK signaling pathway and its activation status in human melanoma.

PTPN11 can be activated by receptor tyrosine kinases (RTKs) or by point mutations. We observed activating phosphorylation on Tyr 542 of PTPN11 in 40% (n=15/38) of melanoma specimens and the majority of human melanoma cell lines (n=14), supporting frequent activation of PTPN11 in human melanoma even though the mutation rate is low (1~3%). PTPN11 knock-down suppressed ERK activation in NRAS mutant and BRAF/NRAS wild-type melanoma cells, but not in BRAF mutant cells. Moreover, we have shown that expression of active PTPN11E76K mutant drives soft-agar colony growth in vitro, tumor growth in nude mice, RAS/RAF/MEK/ERK activation, and resistance to MEK inhibition. Alternatively, knock-down of Ptpn11 reduces colony growth and ERK activation. We generated a tet-inducible, melanocyte-specific, PTPN11E76K transgenic mouse model in a PTEN and CDKN2A null background and observed melanoma formation. Implantation of melanoma cells derived from this model showed doxycycline dependent tumor growth in nude mice. Doxycycline withdrawal and subsequent extinction of PTPN11E76K caused regression of established tumors, supporting the tumor maintenance role of PTPN11. Subsequently, tumor tissue from this model underwent phosphor-tyrosine proteomic analysis to identify downstream effectors of PTPN11's protein tyrosine phosphatase activity. The proteins identified in this analysis are currently being confirmed. These data support the oncogenic roles of PTPN11 in melanoma by regulating RAS/RAF/MAPK pathway activation and the value of PTPN11 as a novel and actionable therapeutic target.

#2388

IC50 profiling against 320 protein kinases: Improving the accuracy of kinase inhibitor selectivity testing.

Daniel Mueller, Frank Totzke, Thomas Weber, Marcel Pathe, Christoph Schaechtele, Michael H. Kubbutat. _ProQinase GmbH, Freiburg, Germany_.

Protein kinases belong to one of the largest families of evolutionary related proteins. More than 500 distinct kinases are encoded by approximately 2% of all human genes. Over the past decades, numerous protein kinases have been described of which deregulation contributes to many human diseases. Today, more than 30 protein kinase inhibitors have been approved for the treatment of cancer emphasizing the significance of kinases as one of the most versatile drug targets.

Due to the structural similarity especially within the ATP-binding site, many kinase inhibitors show limited selectivity. Still, sufficient selectivity within the human kinases is of critical importance e.g. to reduce the risk of adverse side effects during treatment.

Therefore, measuring and improving selectivity of a compound within the kinome is of pivotal importance during drug discovery and optimization phase in the development of therapeutically relevant kinase inhibitors.

Broad profiling of kinase inhibitors in biochemical activity assays of several hundred kinases is nowadays well established. Commonly, kinase profiling is done using one or two concentrations of a test compound and measurement of the relative inhibition of the kinase activity compared to a high and low control.

However, due to the limited dynamic range of this approach, and the challenge to select the most appropriate compound concentration, this profiling approach gives often limited information with respect to the differences in the potency of compounds against On-target- and Off-target kinases.

We set up an IC50 kinase profiling approach that consists of measuring the effect of a compound on the activity of 320 human protein kinases at six different concentrations. We will present data showing the effect of compound concentration on the selectivity score in traditional profiling setting. IC50 kinase profiles of different approved and clinical stage kinase inhibitors will be presented demonstrating that an IC50 based profiling allows the accurate determination of selectivity of a compound based on the comparison of the IC50 values against the On-target- in relation to the IC50 values of the Off-target kinases providing significantly improved guidance in the further optimization of the test compound.

#2389

Role of MET in head and neck cancer.

Thomas Schlange,1 Martin Khan,2 Sami Khaznadar,3 arndt schmitz,4 Thomas Krahn,4 Oliver von Ahsen4. 1 _Bayer Pharma AG, Wuppertal, Germany;_ 2 _Dahme Spree Clinics, Königs-Wusterhausen, Germany;_ 3 _University Bonn, Bonn, Germany;_ 4 _Bayer Pharma AG, Berlin, Germany_.

Introduction c-MET is a well-known target due to its amplification and overexpression in gastric cancer and NSCLC. Overexpression has also been reported for Head and Neck Cancer (HNSCC), but initial clinical trials with MET inhibitors in HNSCC have not been successful.

Methods We investigated the expression level of MET in HNSCC and also characterized the molecular activity level based on the phosphorylation of the intracellular adaptor protein docking site, Tyrosine-1349. In a second larger cohort, we tested expression of MET and correlate it with follow-up data in order to test the prognostic relevance of MET expression in gastric cancer. All clinical samples were all obtained in compliance with clinical regulations and informed consent of every patient.

Results We found MET clearly overexpressed in HNSCC. However, the signaling activity of MET was not elevated compared to normal adjacent tissue. To test the relevance of MET for growth of HNSCC cells, we tested the activity of BAY 853474 in a panel of HNSCC cell lines. In contrast to gastric cancer control cell lines which had low nanomolar IC50 values, none of the 12 HNSCC cell lines was sensitive to MET inhibition. This was in contrast to their sensitivity to Cisplatin/Fluoruracil as positive control. Compared to MET dependent cell lines from gastric cancer, HNSCC cell lines had 10 fold less MET expression. The phosphorylation was two orders of magnitude below that of responder cell lines. On the molecular level, we also compared the properties of the HNSCC cell lines with that of fresh frozen tumor biopsies from 50 patients. In this analysis, it became evident that clinical samples had strikingly lower MET expression and phosphorylation even compared to the HNSCC cell lines. Based on these findings, a clinical response to MET inhibitors cannot be expected. In a larger cohort of several hundred patients, we are currently testing whether the elevated MET expression may still be prognostic for progression and/or survival. It appears possible that MET expression may give a growth advantage to tumor cells although it is clearly not fulfilling the criteria for an oncogenic driver in this indication. The significant overexpression of MET in tumor tissue and the very strong overexpression in cell lines compared to clinical samples show that MET expression gives a selective advantage to the tumor cells. We will show whether this advantage results in shorter time to progression or overall survival.

Conclusion We show that MET expression in HNSCC cell lines is not representative for the clinical situation. HNSCC does not overexpress Met and the molecular activity is low. MET is devalidated as therapeutic target in HNSCC. An analysis of the prognostic value of MET expression in HNSCC will follow and presented at the AACR meeting in 2018.

#2390

When MLK3 meets PAK1: Its implication in breast cancer tumorigenesis.

Subhasis Das,1 Rakesh Sathish Nair,1 Rajakishore Mishra,2 Tanmoy Bhowmik,1 Basabi Rana,1 Ajay Rana1. 1 _University of Illinois at Chicago, Chicago, IL;_ 2 _Central University of Jharkhand, Ranchi, India_.

Mixed Lineage Kinase-3, also known as MAP3K11 is a member of a larger family of MAP3Ks, called MLKs. MLK3 has been reported to play an important role in pro-apoptotic signaling, however its role in tumorigenesis is still not fully elucidated. Previously, we reported that MLK3 is down regulated by estrogen and HER2 amplification and this down regulation provided a survival advantage to ER+ and HER2+ breast cancer cells. To further understand the role of MLK3 signaling in breast cancer pathogenesis, we aimed to identify the upstream kinase(s) that might regulate MLK3 activity and perhaps tumorigenesis.

Based on available information from yeast, where Ste20 members are upstream of Ste11 (i.e. MAP3K), we hypothesized that mammalian homologue of Ste20, PAK1 could be upstream of MLK3. PAK1 kinase has been reported to be a major oncogene in many cancers, including breast cancer. Our results were however, counterintuitive to our hypothesis. We observed that instead, MLK3 was able to associate, and activate PAK1 Kinase activity. These results suggested that perhaps MLK3 is either upstream or parallel to PAK1 in the mammalian signaling network. We also observed that PAK1 was phosphorylated by MLK3 on Ser133 and Ser204 sites. Interestingly, Alanine substitution of S133 site constitutively activated PAK1 kinase activity, whereas Alanine substitution of the S204 site completely blocked MLK3-induced PAK1 activation. Constitutive active MLK3 was not able to phosphorylate and activate double mutant PAK1 S133A, S204A in vitro or in HEK293 cells, clearly indicated that PAK1 S204 site is important for MLK3-induced PAK1 activation. Since PAK1 is an oncogene in breast cancer, we determined the biological effect of S204 phosphorylation on breast cancer progression. Stable breast cancer cell line, MDA-MB-468, expressing PAK1 S204A, inhibited migration, invasion and showed significantly smaller tumors, compared to WT PAK1-expressing cells. Taken together, our results suggest that MLK3-induced modulation of PAK1 activity plays a major role in breast cancer tumorigenesis.

First two authors are equally contributed

#2391

Intramolecular regulation of ERK3's kinase activity and migration/invasion-promoting ability by the C-terminus.

Lobna Elkhadragy, Hadel Alsaran, Weiwen Long. _Wright State Univ., Dayton, OH_.

Extracellular signal-regulated kinase 3 (ERK3) is an atypical MAPK that has recently gained interest for its role in promoting cancer cell migration and invasion. However, little is known about the functions of ERK3's domains in its oncogenic signaling. In contrast to most of the conventional MAPKs, ERK3 has an elongated C-terminus extension beyond its N-terminal kinase domain. The first region of this extension (aa 341 - 481) is highly conserved between ERK3 and ERK4 and is called the C34 domain, whereas the remaining C-terminal tail (aa 482-721) is unique for ERK3. Here, we have investigated the roles of the C-terminal extension in ERK3's kinase activity and functions in cancer cells. We generated two C-terminal deletion mutants of ERK3: ΔCT1, which contains the kinase domain and the C34 domain, and ΔCT2 which comprises the kinase domain alone. By expressing and purifying ERK3, ΔCT1 and ΔCT2 from 293T cells and performing in vitro kinase assays, we found that ERK3's kinase activity towards the substrates myelin basic protein (MBP) and steroid receptor co-activator 3 (SRC-3) decreases gradually with the deletion of the C-terminal tail and the whole C-terminal extension. Next, we wanted to test whether the C-terminus extension was important for ERK3's interaction with SRC-3, an oncogenic protein recently demonstrated to be downstream of ERK3 signaling in cancer. By co-immunoprecipitation experiments, we found that neither the C34 domain nor the C-terminal tail is required for ERK3's interaction with SRC-3. Hence, we hypothesized that the C-terminus extension rather regulates ERK3's kinase activity via an intramolecular interaction with the kinase domain. Indeed, by co-expressing ERK3's kinase domain and the C-terminus extension, we revealed an interaction between the N- and C-termini of ERK3. To test the role of the C-terminal extension on ERK3's ability to promote cancer cell migration and invasion, we overexpressed ERK3, ΔCT1 and ΔCT2 in lung cancer cells and performed transwell cell migration and invasion assays. As compared to ERK3, both the deletion mutants similarly showed a significant reduction in migration and invasion. To test if the effect of the deletion mutants on migration/invasion was due to their decreased kinase activity, we also tested the ability of an ERK3 kinase-dead (KD) mutant (K49/50A) to promote cancer cell invasiveness. Interestingly, while the migration/invasion-promoting ability of ERK3-KD mutant was significantly lower than that of ERK3, it was significantly higher than that of the vector control and those of ΔCT1 and ΔCT2 mutants, suggesting a kinase-independent role for the C-terminal tail in ERK3's invasiveness-promoting ability. Taken together, our study unravels the importance of the C-terminus extension for ERK3's kinase activity and ability to promote migration and invasion.

#2392

Evaluation of synergistic PI3K/Akt and MEK5/ERK5 inhibition in triple-negative breast cancer.

Thomas D. Wright,1 Christopher Raybuck,1 Nathan Gartland,1 Katy Wendekier,1 Darlene Monlish,1 Suravi Chakrabarty,1 Patrick T. Flaherty,1 Matthew E. Burow,2 Jane E. Cavanaugh1. 1 _Duquesne Univ. School of Pharmacy, Pittsburgh, PA;_ 2 _Tulane Univ. School of Medicine, New Orleans, LA_.

Triple negative breast cancers (TNBCs) represent 15-20% of all breast cancers and are often associated with a poor prognosis. The lack of targeted therapies for TNBCs contributes to higher mortality rates. Therefore, there is a need to identify survival pathways that may be targeted in TNBCs. Aberrations in the Phosphoinositide-3-kinase (PI3K) and Mitogen Activated Protein Kinase (MAPK) pathways have been linked to increased breast cancer proliferation and survival. It has been proposed that these survival characteristics are enhanced through compensatory signaling and crosstalk mechanisms. The crosstalk between PI3K/Akt and MEK1/2/ERK1/2 has been characterized in several systems. However, new evidence suggests that MEK5/ERK5, a member of the MAPK family, is a key component in the proliferation and survival of several aggressive cancers. In this study, we examined the effects of dual inhibition of PI3K/Akt and MEK5/ERK5 in three TNBC cell lines: MDA-MB-231, BT549, and MDA-MB-468. We achieved Akt inhibition by using the clinically relevant inhibitors MK-2206 and Ipatasertib. We used a novel compound synthesized in our lab, SC-1-181, and the research tool, XMD8-92, to inhibit MEK5 and ERK5, respectively. Our results indicate that the dual inhibition strategy was more effective than single inhibition due to the loss of crosstalk between the two pathways. In particular, a loss of Bad phosphorylation at two distinct sites was observed with dual inhibition. Interestingly, this signaling pattern was observed without disturbing the ERK1/2 pathway. Furthermore, the inhibition of both pathways led to p21 restoration, decreased cell proliferation, and induced apoptosis. Additionally, the dual inhibition strategy was determined to be synergistic in TNBCs and was nontoxic in non-neoplastic cell lines. Lastly, we evaluated the contributions of Bromodomain 4 (BDR4), an off-target effect of XMD8-92, to the synergy mechanism. In summary, the results from this study provide a unique prospective into the utility of a novel dual inhibition strategy for targeting TNBCs.

#2393

Next-generation CSox-based sensors for continuous, homogeneous, and quantitative monitoring of protein kinase activity.

Erik M. Schaefer,1 Susan Cornell-Kennon,1 Barbara Imperiali2. 1 _AssayQuant Technologies, Hopkinton, MA;_ 2 _Massachusetts Institute of Technology, Cambridge, MA_.

Introduction: Protein kinases are a diverse group of 518 enzymes whose dysregulation lies at the center of many diseases across therapeutic areas and especially oncology. Currently, 30% of all drug development efforts are focused on protein kinases. Although many drugs are approved or in clinical trials, these are predominantly ATP-competitive inhibitors. More recently, there has been a surge in the generation of kinase inhibitors with different modes of action, where new tools are needed to effectively characterize inhibitor mechanism of action, predict drug potency, and drive decisions earlier in the drug development process. We developed a simple yet powerful method for the generation of sensors that can be used for the continuous, quantitative, and homogenous detection of kinase activity with recombinant enzymes and crude lysates to enable target discovery and drug development.

Experimental Procedures: We harnessed chelation-enhanced fluorescence by integrating the sulfonamido-oxine (Sox) chromophore into high-throughput peptide synthesis methods to identify optimized sequences based on physiologic substrates for Ser, Thr or Tyr kinases. Kinase reactions were monitored using fluorescence intensity in kinetic mode (excitation 360 nm, emission wavelength of 485-505 nm).

Results: We demonstrate the ability to rapidly identify novel optimized substrates, where performance measures included higher reaction rates, lower Kms, higher signal/background, increased sensitivity (down to low pM levels), and increased specificity. We identified highly generic substrates (for robust detection of 80 tyrosine kinases) and highly selective substrates (for quantitative detection of targeted kinases in crude cell or tissue lysates for profiling, potency assessments and SAR). We highlight recently developed novel substrates to monitor activity of high-profile tyrosine kinases, including the EGFR and clinically relevant mutants, DDR1/2, JAK kinases (JAK1-3, Tyk2), MET, SYK, Tec-kinases (BTK, ITK, TEC, TXK, BMX), and serine/threonine kinases, including Aurora, CDKs, MAPK pathway (MAP4Ks, Raf, MEK1/2, ERK1/2, RSK), PKR/EIF2AKs and PIM1.

Conclusions: The generation of robust activity-based assays opens new areas of the kinome for effective drug discovery. The Sox-based assay technology is ideal for elucidating drug mechanism of action, potency, kinase activation and profiling, and therefore can be applied across the entire target discovery and drug development workflow. These developments provide a quantum improvement in performance and productivity that is needed to address the challenges and opportunities of next-generation protein kinase and phosphatase inhibitors. These assays run on commonly available microplate instruments, providing access across the cancer research and drug development community.

### Metabolic Regulation

#2395

Acid-induced autophagic protein products are stored as adiposomes in breast cancer cells.

Smitha R. Pillai,1 Jonathan W. Wojtkowiak,1 Jonathan Nguyen,1 Mehdi Damaghi,1 Marilyn M. Bui,1 Timothy Garrett,2 Robert J. Gillies1. 1 _Moffitt Cancer Center, Tampa, FL;_ 2 _University of Florida, Gainesville, FL_.

Malignant tumors exhibit altered metabolism and consume higher levels of glucose compared to surrounding normal tissue, resulting in an acidic extracellular microenvironment. Adaptation to acidic conditions is a pre-requisite for tumor cells to survive and to out-compete the stroma into which they invade. Our previous studies demonstrated that acid adaptation is associated with survival mechanisms like chronic activation of autophagy and redistribution of the lysosomal proteins to the plasma membrane. When grown under acidic pH, breast cancer cells accumulate lipids as revealed by staining with Nile Red and perilipin 2, a protein that coats lipid droplets (adiposomes). Adiposomes are dynamic organelles that store neutral lipids surrounded by a shell of proteins and phospholipid monolayer. The lipids stored in adiposomes are produced de novo, as acid-induced lipogenic phenotype is maintained, even if cells are grown with de-lipidated serum. Fatty acid synthesis inhibition was selectively toxic under acidic conditions as compared to neutral pH and attenuated acid-induced adiposome accumulation. Using 13C isotopomer analysis, we observed a major shift in glucose metabolism from Embden Meyerhof to the Pentose Phosphate Pathway, resulting in increased production of NADPH, necessary for de novo lipid synthesis. To identify the carbon source of the lipid precursors in adiposomes, we employed 13C tracer analysis using [UL] 13C Glucose, 13C3 Leucine (labeled to steady-state) 13C2 Acetate and [UL] 13C glutamine as sources of lipids in the media. 13C label incorporation was determined by LC-MS/MS from adiposomes isolated from cells grown in media containing delipidated serum at pH 6.5. Label incorporation to glycerol backbone and acyl chains of lipids was observed when 13C glucose was used as the source. We could detect (M+1 ~6% of total) labeled 13C in triglycerides from adiposomes isolated from 13C3 leucine labeled MCF7, T47D and ZR75.1 cells. These data indicate that ketogenic amino acids arising from the autophagic breakdown of proteins are the major source of carbons in adiposomal lipids. Additionally, adiposome accumulation was significantly inhibited when cells were treated with autophagy inhibitors indicating that acid induced adiposomogenesis depends on autophagy. Further, we investigated the role of various acid-sensing GPCRs such as OGR1 and TDAG8 in transducing the acid signal. CRISPR/Cas9 mediated depletion of these receptors demonstrated that only OGR1 depletion abrogated acid induced adiposome accumulation and were defective in autophagy. In addition, acid-induced adiposomogenesis required PI3K and pAkt signaling. Hence, accumulation of adiposomes is a highly regulated metabolic process related to storing autophagic products, and appears to be important in cell survival under acidosis. This increased dependence on lipid metabolism reveals novel therapeutic vulnerabilities.

#2396

Short-term calorie restriction alters expression of tumor suppressor p21 in HER2-overexpressing breast cancer.

Magdalena A. Rainey, Laura A. Smith, Ciara H. O'Flanagan, Stephen D. Hursting. _University of North Carolina at Chapel Hill, Chapel Hill, NC_.

Breast cancer (BC) is the most common noncutaneous cancer among US women, and ~20% of these cancers overproduce the growth-promoting tyrosine kinase receptor, HER2. Although there are targeted treatments available for HER2-overexpressing BC, there remains an urgent need to identify new treatment strategies since this disease is associated with poor prognosis, resistance to therapy, and high risk of recurrence. We have established that calorie restriction (CR; 30% reduction in total energy relative to ad libitum-fed controls) has significant tumor suppressive effects across all breast cancer subtypes and has potential to be utilized as an adjunctive therapy. This study investigates the mechanisms by which CR decreases HER2-overexpressing BC progression. To mimic CR in vitro, murine MMTV-neu cells that overexpress neu, the rodent homolog of HER2, were treated with media containing reduced serum (1%), reduced glucose (1mM), or reduced serum and reduced glucose (1%/1mM) compared to control media (10% serum, 25mM glucose). MTT assays demonstrated that both serum restricted medias (1% and 1%/1mM) significantly decreased cellular viability (p<0.05) compared to control. To investigate the mechanism of this alteration, western blotting analysis of proteins associated with HER2 BC progression was conducted. In MMTV-neu cells treated with serum restricted medias (1% and 1%/1mM), there was a significant decrease in activity of tumor suppressor protein p21. Both p21 and phospho-21 expression were decreased with serum restriction, as well as the ratio of phospho-p21 relative to p21 (p<0.05). Phosphorylated p21 is localized to the cytoplasm and allows BC cells to evade apoptosis and proliferate uncontrollably. These results suggest that short-term CR, achieved specifically through serum restriction alone or in combination with glucose restriction, is associated with decreased phosphorylation and cytoplasmic localization of p21, which may be responsible for modulating anti-proliferative activities. This finding is significant because clinical studies have found that increased cytoplasmic p21 in HER2-overexpressing BC predicted reduced survival in patients at 5 years. Utilizing CR or pharmacologic regimens that mimic CR in conjunction with existing therapies may prevent or reverse cytoplasmic p21 localization in this cancer subtype, highlighting the importance of this investigation. Studies are ongoing in MMTV-neu cells transfected with plasmids encoding mutated p21 proteins that are constitutively expressed in either the cytoplasm or the nucleus. This will confirm the extent to which localization of p21 is responsible for CR-induced alterations in HER2-overexpressing BC cell proliferation.

#2397

Intracellular cholesterol regulates the DNA damage response in inflammatory breast cancer.

Shane R. Stecklein, Adam R. Wolfe, Bisrat G. Debeb, Richard A. Larson, Wendy A. Woodward. _MD Anderson Cancer Center, Houston, TX_.

Purpose: Inflammatory breast cancer (IBC) is a highly aggressive form of breast cancer, and patients with IBC remain at high risk of locoregional recurrence after radiotherapy. We previously demonstrated that depleting intracellular cholesterol induces radiosensitivity in IBC cells in vitro, and that IBC patients with high levels of high-density lipoprotein (HDL) and those taking a statin, which inhibits de novo cholesterol biosynthesis, have improved locoregional control after radiotherapy. These results suggest that targeting cholesterol metabolism may improve the radiotherapeutic management of IBC. Here, we aimed to understand the molecular mechanism(s) linking cholesterol metabolism and radiation-induced DNA damage and DNA repair and to evaluate statin pharmacotherapy as a DNA repair inhibitor and radiotherapeutic adjunct in an in vivo pre-clinical model of IBC.

Methods: KPL4, SUM149, SUM190, and IBC3 IBC cell lines were used to examine the effect(s) of simvastatin and human serum lipoproteins on radiation-induced DNA damage induction, DNA damage signaling, DNA damage-associated G1, intra-S, and G2/M cell cycle checkpoint activation, and DNA double strand break (DSB) repair by homologous recombination (HR) and non-homologous end joining (NHEJ).

Results: We have generated multiple IBC cells lines with an integrated Traffic Light Reporter (TLR) that is capable of measuring HR and NHEJ, and demonstrate that depletion of intracellular cholesterol abrogates HR-mediated repair of DNA DSBs. We also show that intracellular cholesterol has pleiotropic effects on cell cycle distribution and DNA damage-associated cell cycle checkpoints. We also report a novel polycistronic dual bioluminescence reporter system that can robustly visualize and quantitate DNA DSB induction and repair in vitro and in vivo.

Conclusions: Cholesterol is an important determinant of intrinsic radiosensitivity in IBC, and appears to regulate repair of radiation-induced DNA DSBs. Targeting cholesterol metabolism is a potential strategy to overcome IBC radioresistance and improve the prognosis for this aggressive disease.

#2398

Tumor cell-adipocyte gap junctions activate lipolysis in breast cancer.

Roman Camarda,1 Jeremy Williams,1 Serghei Malkov,1 Lisa J. Zimmerman,2 Suzanne Manning,2 Dvir Aran,1 Andrew Beardsley,1 Daniel Van de Mark,1 Jeffrey van Haren,1 Yong Chen,1 Charles Berdan,3 Sharon Louie,3 Celine Mahieu,1 Juliane Winkler,1 Elizabeth Willey,1 John D. Gagnon,1 Kosaku Shinoda,1 K. Mark Ansel,1 Zena Werb,1 Daniel C. Nomura,3 Shingo Kajimura,1 Torsten Wittmann,1 Atul J. Butte,1 Melinda E. Sanders,2 Daniel C. Liebler,2 Gregor Krings,1 John A. Shepherd,1 Andrei Goga1. 1 _University of California San Francisco, CA;_ 2 _Vanderbilt University, TN;_ 3 _University of California Berkeley, CA_.

During mammary tumorigenesis, a cell-cell interface exists between adipocytes and cancer cells. Several studies have demonstrated that breast tumor cells can secrete cytokines that induce lipolysis in adjacent adipocytes. However, evidence of tumor-adjacent lipolysis in clinical samples has been lacking. We therefore assayed for lipolysis in normal tissue adjacent to breast tumors (NAT) using (1) the three-component breast composition measure, a radiographic imaging method derived from dual-energy mammography that allows lipid content of a tissue to be quantified, on breast tumors and NAT from 46 patients, (2) a publically available dataset of gene expression in primary breast tumors and NAT from 9 patients, (3) laser capture microdissection and proteomics on primary breast tumors, stroma and NAT from 75 patients, and (4) immunoblot analysis of NAT from several patient-derived and transgenic mouse models of breast cancer. We found strong evidence in all cases that lipolysis is activated in breast cancer-adjacent adipose tissue. We next set out to model the breast cancer-adipocyte interface and determine the contribution of cell-cell contact to induced lipolysis. Gap junctions are cell-cell junctions formed by proteins called connexins, which are known to transport a variety of small molecules (<1kD) including cAMP, a critical pro-lipolytic signaling molecule. Using established dye transfer assays, we determined that gap junctions form between breast cancer cells, and between breast cancer cells and adipocytes. Using biochemical assays, we demonstrated that cAMP is a substrate of breast cancer cell gap junctions, that transfer of cAMP from breast cancer cells to adipocytes occurs, and that breast cancer cells activate lipolytic signaling, all in a gap junction-dependent manner. Finally, we found that gap junction communication in this context is dependent upon connexin 31 (Cx31), and we establish the importance of Cx31 for breast tumor growth and activation of lipolysis in tumor-adjacent adipose tissue in vivo.

#2399

Loss of ubiquitin-specific peptidase 18 (USP18) destabilizes the regulator of thermogenesis uncoupling protein-1 (UCP-1) and represses lung cancer growth.

Xi Liu,1 Yun Lu,2 Weiguo Hu,1 Zibo Chen,1 Lisa M. Mustachio,1 Jason Roszik,1 Lin Zheng,1 Masanori Kawakami,1 Yulong Chen,1 Sarah J. Freemantle,3 Ethan Dmitrovsky1. 1 _UT MD Anderson Cancer Ctr., Houston, TX;_ 2 _Geisel School of Medicine at Dartmouth, Hanover, NH;_ 3 _University of Illinois at Urbana-Champaign, Urbana, IL_.

USP18 is the ISG15 (Interferon-Stimulated Gene 15) deconjugase that removes ISG15 from substrate proteins. We found USP18 null mice are markedly cold sensitive versus their wild-type littermates. USP18 null mice had significantly (P < 0.05) greater temperature declines than wild-type mice. These mice also had lower lipolysis rates and altered fat to body weight ratios. This implicated USP18 as a regulator of lipid and fatty acid metabolism. Protein expression profiles of thermogenic regulators were examined in brown fat of USP18 null versus wild-type mice. Strikingly, the thermoregulatory protein UCP-1 was substantially repressed in brown fat of USP18 null mice. To establish that UCP-1 repression was caused by loss of USP18, stable USP18 knock-down was independently achieved in a panel of murine lung cancer cell lines using transfected small hairpin RNAs (shRNAs). USP18 down-regulation by different shRNAs reduced UCP-1 levels as compared to controls. Engineered gain of USP18 expression stabilized UCP-1 expression in these lung cancer cell lines. UCP-1 destabilization followed complex formation with the ubiquitin-like protein ISG15. Immunoprecipitation assays established that complexes formed between ISG15 and UCP-1. Prior work found that USP18 regulates lung tumorigenesis. We therefore examined UCP-1 levels in human lung adenocarcinomas using The Cancer Genome Atlas (TCGA). Lung adenocarcinomas with undetected UCP-1 expression had significantly improved survival versus cases that expressed UCP-1. Notably, gain of UCP-1 expression in both human and murine lung cancer cell lines promoted their growth. In marked contrast, UCP-1 knockdown reduced proliferation. We explored whether UCP-1 expression affected fatty acid metabolism. The beta-hydroxybutyrate (ketone body) colorimetric assay confirmed that UCP-1 overexpression promoted fatty acid beta-oxidation. Gain of UCP-1 expression also increased fatty acid beta-oxidation using seahorse assays. Thus, a direct link exists between USP18 and expression of the thermoregulator UCP-1. This affects UCP-1 stability and lung cancer growth by altering fatty acid metabolism. Together, these findings implicate the deubiquitinase USP18 as a novel molecular pharmacologic target that controls fatty acid metabolism as an energy source for lung cancer growth.

#2400

Complex gut microbiota modulate rat colon adenoma susceptibility, metabolites, and host gene expression.

Susheel Bhanu Busi, Zhentian Lei, Lloyd Sumner, James Amos-Landgraf. _Univ. of Missouri, Columbia, MO_.

We used a multi-omics approach to identify signatures of differential susceptibility in the F344/NTac-Apc+/Pirc (Pirc) rat model of familial adenomatous polyposis. We assessed how different gut microbiomes (GMs) effect the metabolome and host transcriptome to reveal mechanisms of susceptibility. We previously found that differences in the GM are causative determinants of cancer susceptibility using complex microbiota targeted rederivation (CMTR), in which isogenic Pirc rat embryos are transferred into surrogate dams each harboring distinct specific pathogen free gut microbiota: GM-1 and GM-2. Fecal samples collected from CMTR Pirc rats at 1 month of age were analyzed using ultra-high performance liquid chromatography mass spectrometry (UHPLC/MS) to assess the impact of bacterial metabolites on disease susceptibility. At 6 months of age animals were sacrificed, tumor burden assessed, and normal colonic epithelium (NE) and tumor (T) tissues were collected for RNASeq analysis. Metabolomic analysis identified several features that were unique to either GM-1 or GM-2, and principal component analysis revealed a significant separation of the groups. Based on mass-charge ratios, 117 features were significantly different between the groups, of which 28 were putatively identifiable metabolites through the HMDB (Human Metabolite Database) and METLIN databases. Hierarchical cluster analysis of the 1 month metabolome data based on Euclidean distance measurements (Ward's algorithm) showed separation of samples based on tumor burden at sacrifice. Metabolites present prior to observable disease onset at 1 month were correlated with adenoma burden 5 months later. RNAseq between GM-1 and GM-2 tissues identified 2173 differentially expressed genes (DEGs) in the normal epithelium, and 3406 DEGs between tumor groups (FDR < 0.05). Ordinate analysis confirmed both NE and T samples were significantly distinct between rats harboring the two GM profiles. Integrated pathway (IP) analysis of metabolome and RNASeq data based on detected features showed that bile acid biosynthesis was enriched in GM-1, identifying 14 target genes affecting this pathway. Metabolome data from serum samples from an independent group of 1 month old Pirc and wildtype rats with GM-1 also identified metabolites from bile acid biosynthesis elevated in Pirc serum. Six out of the 14 target genes from the IP analysis showed significant differences (FDR < 0.05) between GM-1 and GM-2 in the NE and T tissues indicating a potential role of metabolites modulating both NE and T gene expression. DEG pathway analysis indicated increased fatty acid metabolism and mucin biosynthesis in the high susceptibility group, and apoptosis and calcium signaling pathways increased in the resistant group. These data show the utility of using metabolomics for early detection of adenomas and provide insights into the mechanism of how the GM modulates colon cancer.

#2401

Effects of carcinoma-associated fibroblasts on cancer metabolism.

Tu Nguyen, Jimmy Kirsch, Karim Nabi, Christos Sazeides, Addison Quinones, Jessica Tan, Marjorie Antonio, Felipe Camelo, Jin Jung, Anne Le. _Johns Hopkins University, Baltimore, MD_.

Rationale: In previous studies, it has been found that cancer-associated fibroblasts (CAFs) interaction with carcinoma cells and helped cancer cell grow which contributed to cancer progression and aggressiveness. However, how CAFs helped cancer cells grow, specifically what nutrition CAFs provides to tumors has not yet been fully understood.

Objectives: We are studying cancer metabolism to identify pathways to target for cancer therapy. Therefore, in this study, we sought to determine which key metabolites in glucose metabolism, specifically glycolysis and the tricarboxylic acid (TCA) cycle metabolites that CAFs feed cancer cells in order for pancreatic cancer cells (P198), to grow better.

Measurement Methods and Main Results: We first assessed pancreatic cancer cell proliferation (P198 cells) with and without the presence of CAFs through the co-culture technique. We assessed P198 cell number after the co-culture. We found that P198 cells grew better when co-cultured with CAFs as compared to P198 that was grown alone. We next investigated which metabolites produced from the CAFs cells are feeding P198 cells. In order to determine which metabolites from glucose were provided from CAFs to P198 cells, we first grew CAFs in 13C6-labeled glucose, removed the labeling medium, then co-cultured P198 with CAFs. The collected metabolites were assessed using Agilent 6520 Quadrupole-Time-of-Flight (Q-TOF) mass spectrometer with Agilent 1260 HPLC and a 6490 triple-quadrupole (QQQ) mass spectrometer. Metabolites were identified by retention time using in-house compound standard databases. In parallel, metabolites were identified using MS/MS fragmentation data under identical conditions. Metabolic pathways were reconstructed and analyzed in three categories: bioenergetics, biosynthesis and redox homeostasis of glucose metabolism to study these metabolic pathways exchanged between CAF and P198. Metabolite peak intensities were normalized to the protein concentration and cell number. We observed an increase in intensities of metabolites in the TCA cycle and glycolysis in P198 after co-culture with CAFs.

Conclusion: These data supports growth benefits of pancreatic cancer in the presence of CAFs. We also found evidence of the metabolic exchange between CAFs and pancreatic cancer cells, which potentially explains the growth benefit.

#2402

Autocrine and paracrine role of tumor derived lactic acid in tumor growth and metastasis and in tumor cell nutrition.

Sabarish Ramachandran, Timothy Brown, Vadivel Ganapathy. _Texas Tech University, Lubbock, TX_.

Cancer cells display a unique phenomenon where, even in the presence of oxygen, cells switch from oxidative phosphorylation to glycolysis as the primary source of ATP with the production of lactic acid - called the Warburg Effect. Lactic acid, the end product of this metabolic switch, is effluxed out of tumor cells to prevent intracellular acidification. Recent evidence suggests that lactate and the excess protons in the tumor microenvironment play an active role in tumor growth. In particular, lactate has been shown to function as an agonist for GPR81, a G-protein-coupled receptor expressed on the surface of tumor cells. This autocrine signaling of lactate promotes tumor growth and metastasis, as well as angiogenesis and immune evasion. The present study assesses whether tumor cell-derived lactate has any paracrine role via its receptor in non-cancer cells present in the tumor microenvironment, and also if the lactic acid-induced extracellular acidification has any role in tumor-cell nutrition. To address the first issue, we generated MMTV-PyMT-Tg mice, a spontaneous model for breast cancer, on Gpr81+/+ and Gpr81-/- backgrounds. The absence of Gpr81 reduced the mammary tumor incidence, delayed mammary tumor progression, and reduced lung metastasis. These data demonstrate the essential role of GPR81 in breast cancer growth and metastasis; but does not differentiate between Gpr81 in tumor cells versus Gpr81 in the tumor microenvironment. We then used the syngeneic transplant of the mouse mammary tumor cell line AT-3 into the mammary fat pads of wild type and Gpr81-/- mice to assess the involvement of Gpr81 in the microenvironment. The growth of the transplanted tumor cells was significantly reduced in Gpr81-/- mice than in wild type mice. To address the second issue, we monitored the expression of GPR81 in human breast cancer cell lines and in normal mammary epithelial cell lines. The expression was many-fold higher in cancer cell lines than in normal cell lines. Hypoxic stress and nutritional deprivation further up-regulated GPR81 expression in tumor cells. Treatment of the ER-positive cancer cell line MCF-7 with lactate (endogenous agonist for GPR81) and 3-chloro-5-hydroxy benzoic acid (pharmacological agonist for GPR81), up-regulated the proton-coupled amino acid transporter PAT1 as evident from the increase in PAT1 mRNA. The function of PAT1, assessed by the uptake of glycine in the presence of an acidic extracellular pH, was also increased. We conclude that tumor-cell derived lactic acid functions in two different capacities to promote tumor growth. First, lactate is an autocrine as well as a paracrine signal via GPR81 expressed on tumor cells and on non-cancer cells in the tumor microenvironment to promote tumor growth and metastasis. Second, the lactic acid-induced acidic pH in the external milieu of tumor cells serves as a driving force to enhance amino acid entry into tumor cells via PAT1.

#2403

SGK-1-mediated ATP generation: A novel metabolic pathway that supports ECM-detached cell survival.

Jordan A. Cockfield, Joshua A. Mason, Zachary T. Schafer. _Univesity of Notre Dame, Notre Dame, IN_.

Successful metastasis requires cancer cells to overcome both anoikis -caspase dependent cell death triggered by extracellular matrix (ECM) detachment—and ECM-detachment-induced metabolic defects that compromise cell survival. While studies have begun to elucidate signal transduction cascades responsible for anoikis evasion, less is known about the precise signals cancer cells use to overcome ECM-detachment induced metabolic deficiencies. Previously, we discovered that oncogenic Ras utilizes a PI(3)K/SGK-1 signaling cascade in order to promote glucose-mediated ATP generation and survival of ECM-detached cells. We have expanded these studies and found that SGK-1 signaling is required in a variety of cell types and oncogenic backgrounds (during ECM-detachment) for glucose-derived ATP production and anchorage independent growth. Our data demonstrate that SGK-1 is required for glucose uptake due to its regulation of the GLUT1 transporter. SGK-1 appears to not only promote GLUT1 localization at the plasma membrane, but also to dramatically promote its transcription. Once inside the cell, SGK-1 promotes the flux of glucose into the glycolytic and pentose phosphate pathways (PPP). When further examining the mechanism by which SGK-1 promotes ATP generation, we surprisingly found that uncoupling the mitochondria did not impact the ability of SGK-1 to promote ATP generation. These data suggest that the TCA cycle is not required for SGK-1 mediated ATP generation. Intriguingly, ATP generation instead requires flux through the PPP and consequent production of glyceraldehyde-3-phosphate (G3P). PPP-derived G3P is shuttled back to glycolysis where ATP production robustly occurs. This metabolic pathway appears to be critical for the anchorage-independent growth of cancer cells as genetic or pharmacological disruption of glucose flux through the PPP significantly abrogates colony formation in soft agar in a variety of distinct cell lines. Overall, these data uncover a novel metabolic pathway downstream of SGK-1 that is highly conserved across multiple epithelial cancer cell lines during ECM detachment.

#2405

P53-dependent mitophagy controls glycolytic shift in radioresistant head and neck cancer cells.

Hyo Won Chang,1 Ji Won Kim,2 Mi Ra Kim,3 Hae Yun Nam,4 Myungjin Lee,1 Won Hyeok Lee,5 Song Hee Kim,5 Da Seul Seong,5 Myung Woul Han,5 Jong Cheol Lee,5 Jung Je Park,6 Ji-hyun Seo,6 Seong Who Kim,4 Sang Yoon Kim1. 1 _Asan Medical Center, University of Ulsan College of Medicine, Seoul, Republic of Korea;_ 2 _College of Medicine Inha University, Incheon, Republic of Korea;_ 3 _Inje University College of Medicine, Haeundae paik Hospital, Pusan, Republic of Korea;_ 4 _University of Ulsan College of Medicine, Seoul, Republic of Korea;_ 5 _Ulsan University Hospital, University of Ulsan college of medicine, Ulsan, Republic of Korea;_ 6 _Gyeongsang National University School of Medicine, Jinju, Republic of Korea_.

P53 is an important biomarker in response to genotoxic stress, but it is also known to plays a key role in the regulation of metabolic homeostasis. The loss of p53 is a well-established contributor to the malignant transformation and glycolytic phenotype acquisition of cells during cancer development. However, the role of p53 in genotoxic therapy-induced metabolic shift in cancers remains unclear. Here, we attempted to elucidate how p53 participates in the glycolytic shift of head and neck cancer cell lines following irradiation. We established a stable radioresistant head and neck cancer cells (HN30-R; p53 wild type and UMSCC1-R; p53 null type) through cumulative irradiation and then analyzed their glucose metabolic profiles and mitochondria respiration. As a result, the metabolic analysis revealed no changes glycolysis of HN30-R cells, but UMSCC1-R cells exhibited increased glycolysis through increased glucose uptake and lactate production and glycolytic intermediates as well as related glycolytic enzymes, compared to UMSCC1 cells. Also, we confirmed that the mitochondrial respiration was reduced by the maximal respiration parameters of oxygen consumption rate (OCR) and that abnormal mitochondria were accumulated by electron microscopy in UMSCC1-R cells. Thus, UMSCC1-R cells exhibited an increased sensitivity to glycolysis-targeting drugs such a hexokinases inhibitor (2-deoxy-D-glucoes; 2-DG) and a lactate dehydrogenase-A inhibitor (AT101), but HN30-R cells did not shown any changes. Moreover, we identified that mitophagy limits glycolytic shift through the p53-dependent clearance of abnormal mitochondria. Taken together, these results suggest that p53 null type cells increased aerobic glycolysis to overcome the accumulation of abnormal mitochondria in radioresistnat cells. Conversely, p53 wild type cells inhibited the glycolytic shift by regulating a integrity through p53-dependent mitophagy. Thus, glycolysis-targeted drugs could be an alternative strategy for overcoming recurrent cancers after radiotherapy, and p53 status could be a biomarker for selecting participants for clinical trials.

#2406

Metabolic phenotype and metastasis in patient-derived ovarian cancer xenografts.

MariaRosa Bani,1 Carmen Ghilardi,1 Alessandra Decio,1 Alessia Anastasia,1 Martina Verza,2 Stefano Indraccolo,2 Raffaella Giavazzi1. 1 _MarioNegri Inst.for Pharmacological Research, Milano, Italy;_ 2 _Istituto Oncologico Veneto - IRCCS, Padova, Italy_.

Introduction and Aim

Metabolic re-programming is an emerging hallmark of cancer. Alterations of the metabolism of glucose (i.e. glycolysis) and oxidative phoshorylation (OxPhos) are frequently observed in cancer cells.

Ovarian adenocarcinoma (OC) is the third most common gynecologic malignancy in the developed countries and the most lethal one, which has shown little improvement in overall survival for the last 20 years. OC is a widely metastatic disease disseminating to the organs of the abdomen through ascitic effusion. Understanding the mechanisms facilitating metastatic spread and disease recurrence is needed to develop therapeutic strategies.Our aim is the comprehension of the metabolic perturbations favoring the aggressiveness and metastatic dissemination of OC.

Experimental methods

We characterized a panel of OC-patient derived xenografts (OC-PDX) for their malignant behavior (ascites production, abdominal metastasis formation and survival time). Their metabolic features were depicted as i) glucose and lactate levels measured in ascites, ii) the monocarboxyate transporters MCT1 and MCT4 of the abdominal tumor masses (by IHC), iii) glycolysis- and OxPhos- related enzymes, as well as glucose and lactate transporters (by gene expression) in abdominal masses and ascites.

Results

We found a direct correlation between the amount of lactate and the expression of its transporters, thus OC-PDX showing higher expression of MC1/MCT4 and higher amount of lactate were categorized as "high glycolytic" and those with lower MCT1/MCT4 and lactate "low glycolytic". The low glycolytic OC-PDXs developed more abdominal metastasis than the highly glycolytic ones, indicating an inverse correlation between the glycolytic phenotype and the capability of the OC-PDX to disseminate, invade and grow into the organs of the peritoneal cavity. Interestingly, we found that the abdominal masses express lower levels of the glucose transporters (i.e. GLUT1), glycolysis-related enzymes (i.e. HK, LDHA) and MCT4 compared to cell aggregates from abdominal effusion; suggesting that ovarian cancer cells are able to modulates their metabolism (glycolysis/OxPhos) to best fit the needs favoring metastatic spread. Further studies are needed to fully understand this association.

Conclusions

Nowadays, metabolism is potentially "targetable". Our results strongly indicate that perturbing tumor's metabolic re-programming might affect metastatic dissemination.

M R B and C G contributed equally. Supported by Italian Association for Cancer Research (AIRC) IG2016 18853 to RG.

#2407

Extracellular acidosis inhibits glucose starvation-induced lung cancer cell death by suppressing overall metabolic rate.

Yuki Iwai,1 Ryota Kikuchi,1 Yasutaka Watanabe,2 Nobuyuki Koyama,2 Koichi Hagiwara,3 Hiroyuki Nakamura,1 Kazutetsu Aoshiba1. 1 _Tokyo Medical University Ibaraki Medical Center, Ibaraki, Japan;_ 2 _Tokyo Medical University Hachioji Medical Center, Tokyo, Japan;_ 3 _Jichi Medical University, Tochigi, Japan_.

Bioenergetic tumor metabolism is essential to meet ATP requirements for energy homeostasis. Cancer cells utilize aerobic glycolysis and proliferate continuously beyond the capacity of blood supply. These phenotypic alterations lead to various microenvironmental stresses, such as hypoxia, nutrient (glucose) deprivation, and extracellular acidosis, to which cancer cells must metabolically adapt in order to survive and proliferate. However, compared to hypoxia, relatively little is known about the metabolic adaptations in response to glucose deprivation and acidosis. In the present study, we explored the effects of extracellular acidosis on cancer metabolism under glucose deprivation stress, as extracellular acidosis and insufficient glucose supply usually co-exist in the tumor microenvironment. To this aim, A549 lung cancer cells were treated with acidosis, glucose deprivation, or a combination. Then, cell survival, total cellular ATP turnover, relative glycolytic and mitochondrial dependency and capacity, and de novo rRNA and protein synthesis were determined. We found that acidosis (pH 6.8) inhibited ATP depletion and cell death induced by glucose deprivation. The cytoprotective effect of acidosis against glucose deprivation-induced cell death was mediated by two distinct mechanisms. First, acidosis shifted cellular ATP production from glycolysis to OXPHOS that became independent of glucose uptake but dependent on glutaminolysis and fatty acid β-oxidation. Second, acidosis reduced ATP consumption by inhibiting de novo rRNA and protein synthesis that utilizes ATP. In contrast, the protective effect of acidosis on glucose starvation-induced cell death was independent of AMPK, PI3K, ERK, and p38 MAPK. Based on this mechanistic knowledge of acidosis-dependent metabolic alterations, we tested the effect of the mitochondrial complex I inhibitor metformin on death of lung cancer cells. Metformin and a combination of rotenone and AICAR mimicking an action of metformin induced ATP depletion and cell death in glucose-free medium, but under acidosis the cytotoxic effect of metformin was significantly attenuated because overall ATP turnover was reduced under low pH. These results suggest that during acidosis, cells are forced to a reduced glycolytic energy production, but the decrease in protein synthesis and ATP requirement serves as an adaptive response to glucose deprivation stress. This acidosis-dependent, glucose starvation-resistant phenotype may provide an adaptive strategy in an energy-restricted tumor microenvironment. Our findings highlight the importance of considering the two factors, acidosis and nutrient starvation in combination to understand their contributions to tumor metabolism.

#2408

LKB1 and KEAP1/NRF2 pathways cooperatively promote glutamine dependence and vulnerability to glutaminase inhibitors in KRAS-mutant lung adenocarcinoma.

Ana Galan-Cobo, Piyada Sitthideatphaiboon, Xiao Qu, Jeffrey J. Kovacs, Alissa Poteete, Pan Tong, Sungnam Cho, Varsha V. Gandhi, Ferdinandos Skoulidis, Jing Wang, Timothy P. Heffernan, John V. Heymach. _UT MD Anderson Cancer Center, Houston, TX_.

KRAS is the most commonly mutated oncogenic driver in non-small cell lung cancer (NSCLC) and other solid tumors. Recently we conducted an integrative analysis and found three major subgroups of KRAS-mutated cancer defined by co-occurring genomic events with distinct biology, molecular vulnerabilities, and therapeutic sensitivities. One of these genes, the serine/threonine kinase STK11 (LKB1), represents the second most commonly altered tumor suppressor in NSCLC and there are currently no treatment strategies tailored for LKB1-deficient NSCLC. KRAS-mutant/LKB1-deficient (KL) tumors are characterized by high co-occurrence of KEAP1 mutational inactivation. Inactivation of KEAP1 protects cells against REDOX stress via upregulation of NRF2 target genes, in part by production of glutathione. We evaluated the effects of blocking glutamine metabolism using an isogenic series of NSCLC cell lines harboring mutations in STK11 and KEAP1. Through sequential silencing or overexpression of LKB1, KEAP1, or NRF2 we demonstrated that glutaminase inhibitors (GLSi) can block cell proliferation while increasing energetic and REDOX stress specifically in LKB1 deficient cells with hyperactivation of the KEAP1/NRF2 pathway driven by KEAP1 mutations (KLK subtype). In KLK models, overexpression of LKB1 or KEAP1 partially reduced GLSi sensitivity, while siRNA-mediated down-regulation of NRF2 showed a similar effect. Furthermore, the combination of LKB1 add back coupled with down-regulation of NRF2 conferred even greater resistance to GLSi. To confirm the LKB1/KEAP1-driven response to GLSi, we performed in vivo experiments examining the response of subcutaneous xenografts of an A549 isogenic series; A549 (KLK), A549 LKB1 add back (KK) or A549 KEAP1 add back (KL); to GLS inhibition. These experiments demonstrated that GLSi impaired tumor growth in A549 (KLK) tumors, exhibiting significant statistical differences compared with the vehicle group from 18 days of treatment. Conversely, GLS inhibition did not significantly affect the growth of A549/LKB1 (KK) or A549/KEAP1 (KL) tumors. Collectively, our data indicate that in KLK tumors both pathways, LKB1 and KEAP1/NRF2, cooperatively drive a glutamine-addicted metabolic program, making KLK tumors selectively vulnerable to GLSi treatment. These findings have immediate clinical implications and support the future clinical testing of GLS inhibitors in KLK NSCLC.

#2409

The effect of heme on mitochondrial biogenesis and function of NSCLC cells.

Sagar Shashikant Sohoni, Chantal Vidal, Li Zhang. _UT Dallas, Richardson, TX_.

Heme is a central molecule for mitochondrial function and for many processes involved in oxygen utilization. Heme serves as a prosthetic group or a cofactor for a number of oxidative phosphorylation enzymes and other oxygen-utilizing hemoproteins. Heme also directly regulates the synthesis, translocation, and assembly of these enzyme complexes. Most, if not all, human cells can synthesize and uptake heme from the circulation. A number of epidemiologic studies have shown that high heme intake is associated with increased risk of cancer, including lung cancer. To assess the status of heme metabolism in lung cancer cells, we performed a series of experiments in non-small cell lung cancer (NSCLC) cell lines and compared the results with an immortalized normal lung cell line, HBEC30KT. We observed significant increase in rates of heme synthesis and heme uptake in NSCLC cells compared to HBEC30KT. Previous studies carried out in our lab showed intensified mitochondrial respiration and increased levels of heme as well as hemoproteins in NSCLCs. NSCLCs were also found to exhibit significantly high expressions of proteins involved in mitochondrial biogenesis such as SIRT-1, PGC1α, NRF-1, NRF-2, and TFAM. Expression levels of these proteins correlate with oxygen consumption and total heme content in NSCLCs, suggesting a possibility of role of heme in mitochondrial biogenesis. Immunofluoresence studies showed that heme depletion and add-back significantly affected localization and expression levels of these proteins. Studies are currently under way to determine how heme depletion affects the interactions of various transcription factors involved in mitochondrial biogenesis and function.

#2410

RIPK1-mediated induction of mitophagy compromises the viability of extracellular matrix-detached cells.

Mark A. Hawk, Zachary T. Schafer. _University of Notre Dame, Notre Dame, IN_.

For cancer cells to survive during ECM-detachment, they must inhibit anoikis and rectify metabolic deficiencies that cause non-apoptotic cell death. Previous studies in ECM-detached cells have linked non-apoptotic cell death to ROS generation, although the mechanistic underpinnings of this link remain poorly defined. Here, we uncover a novel role for receptor-interacting protein kinase-1 (RIPK1) in the modulation of mitochondrial ROS levels and cell viability during ECM-detachment. We find that RIPK1 activation during ECM-detachment results in the induction of PINK1-dependent mitophagy through a mechanism requiring the mitochondrial phosphatase PGAM5. As a consequence of mitophagy induction, ECM-detached cells experience diminished IDH2-mediated NADPH production in the mitochondria and the subsequent elevation in mitochondrial ROS levels leads to non-apoptotic cell death. Furthermore, we find that antagonizing RIPK1 or PGAM5 enhances tumor formation in vivo. Intriguingly, upon mining a meta-analysis of published lung cancer patient microarray data sets, we find that low RIPK1 expression in lung cancer patients correlates with decreased overall and progression-free survival. Thus, RIPK1-mediated induction of mitophagy may be an efficacious target for novel therapeutics aimed at specifically eliminating ECM-detached cancer cells and deterring tumor progression.

#2411

Reactive Oxygen Species regulates tumor stem cell survival in medulloblastoma via mitochondrial biogenesis.

Anshu Malhotra, Abhinav Dey, Anna M. Kenney. _Emory University, Atlanta, GA_.

Sonic hedgehog (Shh) signaling is closely coupled with the bioenergetics of medulloblastoma, the most common malignant pediatric solid tumor. We have previously reported that Shh causes deregulation of mitochondrial biogenesis by suppressing mitofusins. This led to fragmented mitochondria in vitro in primary cultures of mouse cerebellar progenitor cells, putative cells-of-origin of the SHH molecular subclass of medulloblastoma. This phenomenon was also observed in electron micrographs of SmoA1 mouse medulloblastomas in vivo. Ectopic expression of mitofusins in both proliferating progenitor cells and primary mouse medulloblastoma cell cultures restored mitochondrial fusion and led to a non-proliferative phenotype (1).

We have observed high levels of Reactive Oxygen Species (ROS) in SmoA1 mouse medulloblastoma cells in vitro. When total ROS in primary cultures of mouse medulloblastoma cells was scavenged by N-Acetyl Cytosine (NAC) treatment, the mitochondria regained their fused morphology, leading to a reduction in cell proliferation. NAC treatment of organotypic cerebellar slice cultures implanted with SmoA1 neurospheres also resulted in a reduction in proliferation as well as impaired spread of tumor cells within normal cerebellar tissue. Medulloblastoma tumor repopulating cells, which are responsible for radiation resistance and tumor recurrence, are primarily located in the perivascular niche (PVN) of Medulloblastoma (2). We therefore focused our attention on the role played by ROS in regulating mitochondrial structure of tumor repopulating cells, to decipher if this affects the survival of these cells. Indeed we have observed that NAC treatment combined with irradiation restores mitochondrial morphology, leading to enhanced cell death within the PVN of Medulloblastoma.

We further carried out gene expression analysis of ROS-regulating genes in the Shh subgroup of Medulloblastoma. NADPH Oxidase 4 (NOX4), which is a known producer of cytoplasmic ROS in cells (3), was found to be upregulated as compared to NOX1 and 3. This elevation was observed across all subgroups of medulloblastoma, but was most evident in the Shh subgroup. We have also found that NOX4 co-localizes in the PVN of medulloblastoma. Treatment with Apocynin, a NOX2/4 inhibitor, led to enhanced cell death in the PVN, similar to what we observed after NAC treatment. Taken together, our results show that anti-oxidant treatment restores the fused morphology of mitochondria in the PVN and leads the cells to apoptosis, thereby preventing tumor recurrence post-irradiation, and they implicate Nox4 as a potential therapeutic target.

1. Malhotra A, Dey A, Prasad N, Kenney AM. 2015. Mol Cancer Res 2. Hambardzumyan D, Becher OJ, Rosenblum MK, Pandolfi PP, Manova-Todorova K, Holland EC. 2008. Genes Dev 22: 436-48 3. Bedard K, Krause KH. 2007. Physiol Rev 87: 245-313

#2412

The nuclear homeoprotein NKX3.1, has a novel function in the mitochondria in regulation of prostate cancer initiation.

Aditya Dutta, Cory Abate-Shen. _Columbia University Medical Center, New York, NY_.

An emerging hallmark of cancer is reprograming of energy metabolism, in which mitochondrial plasticity plays a central role. Mitochondrial plasticity, particularly in response to oxidative stress, is coordinately orchestrated by both mitochondrial- and nuclear-encoded genes. Recent reports highlight the association of mitochondrial dysfunction and prostate cancer, one of the leading causes of cancer and cancer-related death for men. One of the earliest events in prostate cancer etiology is loss of functional NKX3.1, a homeoprotein that is known to play essential roles in prostate differentiation and prostate cancer by regulation of nuclear-encoded genes. We now show that in response to oxidative stress, NKX3.1 is localized to the mitochondria where it directly protects against reactive oxygen species (ROS). NKX3.1 regulates mitochondrial function by directly binding to the D-loop (mitochondrial DNA control region) to specifically regulate mitochondrial genes coding for the electron transport chain. Moreover, we show that aberrant NKX3.1 function in the mitochondria leads to enhanced reactive oxygen species and DNA damage, both of which are reversed by metformin, a biguanide class of anti-diabetic drug that targets the mitochondrial electron transport chain. This uncovers the potential for metformin administration to individuals with NKX3.1 loss as a precision prevention approach against prostate cancer initiation. These observations highlight novel functions for homeoproteins as direct regulators of mitochondrial DNA, and demonstrate the critical role of the mitochondrial stress response in enabling prostate cancer initiation with potential novel therapeutic implications. >

#2413

TP53 gene mutations differently regulate ovarian cancer metabolism: Ex vivo and in vitro studies.

Stephanie Antoun, David Atallah, Roula Tahtouh, Malak Moubarak, Nada Alaeddine, George Hilal. _Saint. Joseph Univ., Beirut, Lebanon_.

Background: Even in the presence of oxygen, cancer cells tend to metabolize glucose into lactate instead of undergoing oxidative phosphorylation. Knowing that glucose is crucial for tumor's proliferation, new patient's treatment strategies tend to combine chemotherapy to hypoglycemic drugs. p53 is a tumor suppressor and key regulator of glycolysis in cancer cells, however highly mutated in tumors. In ovarian cancer, the majority of studies concerning p53 mutations focus on the DNA binding domain since the majority of hotspot mutations affects this region of the gene. However, mutations affecting other regions such as the proline rich domain linked directly to apoptosis may also affect the protein's expression and activity. The aim of this study is to investigate the effect of various positions of mutations in TP53 gene on glycolysis, apoptosis and transcription of p53 target genes.

Material and Methods: this study was divided into two parts. The in vitro study was done using 3 ovarian cancer cell lines SKOV-3 (p53 null), Igrov-1 (WT p53) and Ovcar-3 (DNA domain mutant p53), along with the transfection of SKOV-3 cells by three different vectors: WTp53, P72R p53 and R249S p53. The ex vivo part was performed on 30 ovarian cancer biopsies. The biopsies were subjected to Tp53 immunohistochemistry and sequencing to study the mutations and their effect on p53 expression. The effect of Tp53 mutations on glucose metabolism was assayed by glucose consumption measurements, lactate production and mRNA expression of various glycolytic enzymes. To study the effect of mutations on the transcriptional activity of p53, p21 and MDM2 expression was quantified in these cells. Annexin V assay was performed on transfected SKOV-3 to study the apoptotic activity of WT and mutant p53.

Results: DNA binding domain mutant cells (Ovcar-3 and R249S SKOV-3) presented a 30% increase in glucose consumption and lactate production when compared to IGROV-1 and WT SKOV-3. P72R SKOV-3 exhibited the same pattern as WTp53 cells. The increase in the mRNA expression of glucose transporters GLUT-1 and 3 and of the glycolytic enzymes PK, PFK, G6PDH and aldolase in cells with DNA binding domain mutations varied from 20 to 30%, whereas the levels of PDHa and TIGAR decreased significantly (40%). The transcriptional activity of DNA binding domain mutant cells showed a 50% decrease in comparison to WT and P72R mutant cells. The apoptotic activity of p53 was unaltered in P72R transfected SKOV-3 cells. The ex vivo results revealed a high frequency of the P72R variant in the ovarian biopsies (69%), associated with an overexpression of the p53 protein. The glycolytic and transcriptional patterns of cells extracted from these biopsies were similar to those obtained in transfected cells.

Conclusion: Though both DNA-binding and proline-rich domains mutations lead to p53 overexpression, only the first type promotes Warburg effect and could have the worst patient's prognosis.

#2414

Interrogating cellular metabolism reveals mTORC2 as a new biomarker to stratify epidermal growth factor receptor-mutant non-small cell lung cancer.

Chun-Te Chiang, Nolan Ung, Alexandra N. Demetriou, Chi-Li Chiu, Niha Choudhury, Cosimo Arnesano, Scott E. Fraser, David B. Agus, Naim Matasci, Dan L. Ruderman, Shannon M. Mumenthaler. _University of Southern California, Los Angeles, CA_.

Most non-small cell lung cancer (NSCLC) patients with epidermal growth factor (EGFR) mutations initially respond to EGFR tyrosine-kinase inhibitors (TKIs), but these inhibitors are only effective for approximately one year, thus limiting the benefits to patients' survival. Stratification of patients with EGFR-mutant NSCLC would help physicians personalize and optimize patient treatment plans. To this end, we hypothesized that factors residing in the tumor microenvironment play a pivotal role in the evolutionary dynamics of therapeutic response to anti-EGFR therapies. We used the Operetta® High Content Imaging System to track cell dynamics over time under different microenvironmental stresses, and we found that the growth of TKI-resistant cells is more sensitive to glucose starvation than TKI-sensitive cells. To further interrogate cellular metabolism in TKI-sensitive and resistant cells, we utilized both seahorse metabolic assays and fluorescence lifetime imaging microscopy to monitor the changes in glycolysis and oxidative phosphorylation of living cells. We found TKI-resistant cells have higher glycolysis and lower oxidative phosphorylation rates compared to TKI-sensitive cells. Such metabolic reprogramming rendered TKI-resistant cells with lower metabolic plasticity to cope with metabolic stress. Using both genetic and pharmacologic approaches, we then demonstrated that mTORC2 activation contributes to the different metabolic phenotypes between TKI-sensitive and resistant cells. Furthermore, gene set enrichment analysis showed that the oxidative phosphorylation gene set is significantly enriched in patients with low mTORC2 activation, and the glycolysis gene set is upregulated in patients with high mTORC2 activation, which is correlated with shorter progression-free and overall survival time in patients. Overall, these data suggest that TKI-resistant cells use and require distinct metabolic programs that can be employed to stratify EGFR-mutant NSCLC patients for treatment with EGFR-TKIs.

#2415

HEY1 counteracts hypoxia-induced oxidative stress via transcriptionally repressing PINK1 in hepatocellular carcinoma.

David Kung-Chun Chiu, Iris Ming-Jing Xu, Robin Kit-Ho Lai, Aki Pui-Wah Tse, Dicky Cheuk-Ting Law, Vincent Wai-Hin Yuen, Larry Lai Wei, Hui-Yu Koh, Chun-Ming Wong, Irene Oi-Lin Ng, Carmen Chak-Lui Wong. _Univ. of Hong Kong, Hong Kong, Hong Kong_.

Background and Objective: Excessive accumulation of oxidative stress/reactive oxygen species (ROS) can be harmful to cancer cells. Hypoxia or O2 deprivation, which is commonly found in hepatocellular carcinoma (HCC), is a crucial factor that contributes to elevated ROS level in HCC cells as hypoxia causes inefficient transfer of electrons in the mitochondria. To survive, HCC cells need to devise strategies to counteract and balance hypoxia-induced oxidative stress. While it is known that hypoxia inducible factors (HIFs) are essential to metabolic reprogramming in HCC cells under hypoxia, there are significant gaps in knowledge about underlying mechanisms and transcriptional targets of HIFs.

Experimental Procedures: Gene profiling of HCC cell lines (exposed to 20% and 1% O2) was analyzed by transcriptome sequencing to identify novel candidate responsible for counteracting hypoxia-induced oxidative stress. ShRNA-mediated gene silencing and gene activation by CRISPR-dCas9 system were used to modify transcriptional expression of HEY1 for different functional assays. Transmission electron microscopy was used to visualize the mitochondrial structure. Orthotopic and subcutaneous HCC implantation models were used to evaluate the role of HEY1 in HCC progression. Transcriptome sequencing and ChIP assay were performed to identify novel transcriptional targets of HEY1.

Results: We showed that transcriptional repressor HEY1 was induced under hypoxia and directly regulated by HIF-1α. Overexpression of HEY1 was associated with poor overall survival in HCC patients. Importantly, we identified PINK1 as a novel repression target of HEY1. PINK1 is known to protect cells against mitochondrial dysfunction. We demonstrated that HEY1 actively repressed PINK1 and downregulation of PINK1 led to loss of mitochondrial mass and impaired mitochondrial cristae formation, subsequently decreasing intracellular ROS level. Downregulation of PINK also associated with poor overall survival and decrease-free in HCC patients. Genetic ablation of HEY1 in HCC cells profoundly reduced tumor growth and lung metastasis while genetic ablation of PINK1 in HCC cells reversely promoted HCC growth. Strikingly, HEY1 and PINK1 expressions reversely correlated in human HCC tissues.

Conclusion: This study unprecedentedly identifies an upstream regulatory mechanism of PINK1, which controls the oxidative stress in HCC cells. It also reveals a novel molecular mechanism by which ablation of HEY1 leads to elevation of oxidative stress, making HCC cells more vulnerable. Targeting HEY1 represents an attractive therapeutic approach against HCC.

#2416

CREB orchestrates metabolic reprogramming in PDGFRA-addicted lung squamous cell cancer.

Jun Xu, Nan Jin, Min Huang, Jian Ding, Meiyu Geng. _Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China_.

Aberration of platelet-derived growth factor receptor A (PDGFRA) gene has been increasingly identified in multiple types of human cancers, including hematologic malignancies, gastrointestinal stromal tumor (GIST), medulloblastomas and gliomas. In particular, amplification of 4q12 that contains PDGFRA locus has been reported in 3% to 7% of lung adenocarcinomas and 8% to 10% of lung squamous cell carcinomas. However, it remains unclear how amplified PDGFRA drives malignancy of lung squamous cell cancer. To address this question, we used a lung squamous cell cancer cell line NCI-H1703, which has been previously reported to have 24-fold amplification of the 4q12 region. Either siRNA knockdown or small-molecule inhibition of PDGFRA significantly inhibited cell growth, suggesting that PDGFRA was required for driving cell proliferation. We further showed that both oxidative phosphorylation and aerobic glycolysis were significantly affected followed by interference of PDGFRA, by measuring of oxygen consumption rate (OCR) and extra cellular acidification rate (ECAR). In particular, ATP generation level and maximum respiratory capacity, the key parameters of mitochondrial function, were significantly decreased in mitochondrial pressure testing. These observations suggested that oncogenic PDGFRA drove the metabolic reprogramming and promoted tumor malignancy in lung squamous cell cancer. To further explore the regulatory mechanism of the observed metabolic phenotype, we took the approach of genetic sequencing technology to analyze the transcriptome in PDGFRA-driven NCI-H1703 cells treated with or without PDGFRA inhibitor. It revealed that PDGFRA-driven transcriptional regulation of metabolic genes underpinned the specific metabolic phenotypes of mitochondrial function. In addition, analysis of transcriptome as well as a phosphorylated kinase array revealed that a transcription factor, cAMP response element-binding protein (CREB), regulated the metabolic gene expression in PDGFRA-addicted cells. Knockdown of CREB disrupted metabolic gene expression and the mitochondrial function, consistent with aforementioned findings of PDGFRA, suggesting that transcriptional regulation of metabolic genes was orchestrated by CREB. Together, we herein firstly provide the molecular link between oncogenic PDGFRA and metabolic vulnerability and reveal the important role of CREB in orchestrating metabolic reprogramming in PDGFRA-addicted lung squamous cell cancer.

#2417

eIF4E phosphorylation licenses Myc-driven metabolic and stress reprogramming.

Hang Ruan,1 Xiangyun Li,1 Brian Leibowitz,1 Jingshan Tong,1 Xinghua Lu,1 Nahum Sonenberg,2 Xiang Xu,3 Lin Zhang,1 Jian Yu1. 1 _University of Pittsburgh School of Medicine, Pittsburgh, PA;_ 2 _McGill University, Montreal, Quebec, Canada;_ 3 _Army Medical University, China_.

eIF4E, Eukaryotic Translation Initiation Factor 4E, regulates the rate limiting cap-binding step in mRNA translation. Oncogenic pathways converge to increase eIF4E expression, as well as phosphorylation of eIF4E (S209) and its inhibitor 4EBPs, which are believed to facilitate the translation of "weak'' mRNAs including cell type-specific and oncogenic targets. eIF4E phosphorylation, but not eIF4E, is dispensable for normal development. Pharmacologic inhibition of either p-eIF4E or p-4EBP has limited antitumor efficacy. Our current study aims to better define the role of p-eIF4E in colon cancer. We find that p-eIF4E, not eIF4E transcript or protein is elevated in human colon tumors and precursors. Knockin (KI) of phosphorylation-defective eIF4E mutant allele (4ES209A) in human colon cancer cells strongly suppresses Myc translation and cell growth under stress or in mice, while minimally impacting Cap binding or global translation. Surprisingly, p-eIF4E is found to be required to maintain p-4EBP1 levels, dissociate eIF4E from 4E-BP1, and stimulate glutamine metabolism via integrated stress response (ISR). Further, p-eIF4E and p-4EBP1 are highly elevated in the polyps in APC min/+ mice compared to adjacent normal intestinal crypts, and 4ES209A KI significantly inhibits intestinal polyposis and glutamine metabolism gene signatures. Finally, p-eIF4E renders CRC cells addicted to glutamine and glutaminolysis by preventing Myc- and ISR-driven metabolic crisis and cell death. In summary, we demonstrate that eIF4E phosphorylation is central to oncogenic translation, and is required to license Myc-driven metabolic adaptation and addiction via ISR-dependent transcriptional programs in colon cancer. These novel findings help reconcile the long-standing and context-dependent roles of ISR in cancer cell growth, death, plasticity and heterogeneity, and provide new ways for its therapeutic exploitation.

#2418

PKM2 methylation by CARM1 activates aerobic glycolysis to promote tumorigenesis.

Fabao Liu, Fengfei Ma, Yuyuan Wang, Ling Hao, Hao Zeng, Chenxi Jia, Yidan Wang, Peng Liu, Irene M. Ong, Baobin Li, Guojun Chen, Jiaoyang Jiang, Shaoqin Gong, Lingjun Li, Wei Xu. _UW-madison, Madison, WI_.

Metabolic reprogramming is a hallmark of cancer. Herein we discover that the key glycolytic enzyme pyruvate kinase M2 isoform (PKM2), but not the related isoform PKM1, is methylated by co-activator-associated arginine methyltransferase 1 (CARM1). PKM2 methylation reversibly shifts the balance of metabolism from oxidative phosphorylation to aerobic glycolysis in breast cancer cells. Oxidative phosphorylation depends on mitochondrial calcium concentration, which becomes critical for cancer cell survival when PKM2 methylation is blocked. By interacting with and suppressing the expression of inositol-1,4,5-trisphosphate receptors (InsP3Rs), methylated PKM2 inhibits the influx of calcium from the endoplasmic reticulum to mitochondria. Inhibiting PKM2 methylation with a competitive peptide delivered by nanoparticles perturbs the metabolic energy balance in cancer cells, leading to a decrease in cell proliferation, migration and metastasis. Collectively, the CARM1-PKM2 axis serves as a metabolic reprogramming mechanism in tumorigenesis, and inhibiting PKM2 methylation generates metabolic vulnerability to InsP3R-dependent mitochondrial functions

#2419

Lipid droplets promote glioblastoma cell growth through maintaining cholesterol homeostasis mediated by autophagy.

Feng Geng, Xiang Cheng, Chunming Cheng, Arnab Chakravarti, Deliang Guo. _Ohio Sate Univ., Columbus, OH_.

Lipid droplets (LDs) have been studied as the major storage site of neutral lipids for decades, especially in the progression of metabolic diseases. Recently, the functions of LDs in cancers have begun to gain significant attention. Here, we provide evidence that LDs promote glioblastoma (GBM) cell growth through autophagy mediated maintenance of cholesterol homeostasis. Our data showed that LDs in tumor tissues from GBM patients are engulfed by autophagic vacuoles and lysosomes. Cellular cholesterol deprivation or removal of plasma membrane cholesterol by methyl-β-cyclodextrin (MβCD) activates autophagy to hydrolyze LDs to support GBM cell survival, whereas inhibition of autophagy blocks transportation of cholesterol to plasma membrane, leading to cell death. Fluorescence imaging reveals direct trafficking of LD-stored cholesterol to the plasma membrane through autophagic liberation. Our data demonstrate that LDs serve as cholesterol reservoir for GBM cell growth, suggesting that targeting LD-autophagy-cholesterol homeostasis metabolic pathway represents a potential means to treat GBM and other malignancies.

### Metabolism and Cellular Functions 2

#2420

Skp2-mediated stabilization of MTH1 promotes survival of melanoma cells upon oxidative stress.

Lei Jin,1 Jiayu Wang,1 Guangzhi Liu,2 James S. Wilmott,3 Xu Guang Yan,1 Rick F. Thorne,1 Richard A. Scolyer,3 Xu Dong Zhang1. 1 _Univ. of Newcastle, Callaghan, Australia;_ 2 _Henan Provincial People's Hospital, Zhengzhou, China;_ 3 _Univ. of Sydney, Sydney, Australia_.

MTH1 helps prevent misincorporation of ROS-damaged dNTPs into genomic DNA, however, there is little understanding of how MTH1 itself is regulated. Here we report that MTH1 is regulated by polyubiquitination mediated by the E3 ligase Skp2. In melanoma cells, MTH1 was upregulated commonly mainly due to its improved stability caused by K63-linked polyubiquitination. While Skp2 along with other components of the Skp1-cullin-F-box (SCF) ubiquitin ligase complex were physically associated with MTH1, blocking the SCF function ablated MTH1 ubiquitination and expression. Conversely, overexpressing Skp2 elevated levels of MTH1 associated with an increase in its K63-linked ubiquitination. In melanoma cell lines and patient specimens, we observed a positive correlation of Skp2 and MTH1 expression. Mechanistic investigations showed that Skp2 limited DNA damage and apoptosis triggered by oxidative stress and that MAPK upregulated Skp2 and MTH1 to render cells more resistant to such stress. Collectively, our findings identify Skp2-mediated K63-linked polyubiquitination as a critical regulatory mechanism responsible for MTH1 upregulation in melanoma, with potential implications to target the MAPK/Skp2/MTH1 pathway to improve its treatment.

#2421

SOD1 is required for tumor maintenance in a genetically engineered KRAS/TP53 non-small cell lung cancer mouse model.

Xiaowen Wang,1 Justin Wong,2 Holly Van Remmen,3 Jessie Yanxiang Guo,4 Eileen White,5 X. F. Steven Zheng6. 1 _Rutgers University School of Graduate Studies Biomedical Sciences, Piscataway, NJ;_ 2 _Rutgers University School of Arts and Sciences, New Brunswick, NJ;_ 3 _Oklahoma Medical Research Foundation, Oklahoma City, OK;_ 4 _Rutgers Robert Wood Johnson Medical School, New Brunswick, NJ;_ 5 _Rutgers University, Piscataway, NJ;_ 6 _Rutgers Cancer Institute of New Jersey/Robert Wood Johnson Medical School, Rutgers , The State University of New Jersey, New Brunswick, NJ_.

Lung cancer is the second most common cancer and the primary cause of cancer-related mortality globally. Over half a million people are estimated living with lung and bronchus cancer with a 5-year survival rate of 18%. Identifying and studying new drug targets are important for improving lung cancer patient survival. 80% to 85% of lung cancers are Non-small cell lung cancer (NSCLC). Knocking down and small molecule inhibition of Superoxide dismutase 1 (SOD1) has been shown to be cytotoxic to cultured NSCLC cells and mouse tumors, suggesting that SOD1 is important for NSCLC survival, thus presenting a promising therapeutic strategy. SOD1 rapidly converts superoxide anion to hydrogen peroxide. It is a critical component of the cellular antioxidant defense system. In addition, our laboratory has recently demonstrated that SOD1 has a novel function as a transcription factor that regulates oxidative stress response genes. Currently, there are no selective SOD1 inhibitors available to study physiological functions of SOD1. In our study, we show that genetically knockout SOD1 resulted in reduced tumor burden in lung cancer mouse model. Using mouse tumor-derived primary cancer cells, we further show that SOD1 is critical for NSCLC cell proliferation and survival. This study reveals novel functions of SOD1 that are of biological and translational significance, and provide further support for SOD1 as a promising therapeutic target for NSCLC.

#2422

The lysine demethylase KDM1A inhibition attenuates hypoxic responses in glioblastoma.

Gangadhara R. Sareddy,1 Suryavathi Viswanadhapalli,1 Mei Zhou,1 Yiliao Luo,1 Xiaonan Li,1 Jinyou Liu,1 Aleksandra Gruslova,1 Takayoshi Suzuki,2 Andrew Brenner1. 1 _University of Texas Health, San Antonio, TX;_ 2 _Kyoto Prefectural University of Medicine, Kyoto, Japan_.

Background: Glioblastoma (GBM) is associated with poor survival (1 year-34.6% and 5 year-4.75%) and affects approximately 13,000 patients per year. Standard treatment consists of surgical resection, external beam radiation therapy, adjuvant chemotherapy with temozolomide and tumor treating fields. Nonetheless, despite a heavy investment in therapy, all patients eventually succumb to their disease. Hypoxic regions are common in GBM and are implicated in maintenance of glioma stem cells (GSCs), promoting angiogenesis and therapeutic resistance. Recently, we and others have shown that lysine-specific histone demethylase 1A (KDM1A) is overexpressed in GBM and GSCs. However, the role of KDM1A and its functions in hypoxic response remains unknown. The objective of this study is to elucidate the functional role of KDM1A in hypoxia and test the efficacy of novel KDM1A inhibitors on hypoxia-mediated functions in GBM.

Methods: The expression of KDM1A following hypoxia induction was determined in vitro by Western blotting and qRT-PCR. The expression of KDM1A in GBM tissue was examined by immunohistochemistry (IHC). KDM1A knockout cells were generated using CRISPR/Cas9 system and knockdown cells were generated using KDM1A shRNA lentiviral particles. The effect of KDM1A knockdown or treatment with KDM1A inhibitors NCL-1 and NCD-38 on stemness and self-renewal of primary patient-derived GBM cells was examined using neurosphere formation and extreme limiting dilution assays. Interaction of KDM1A with hypoxia inducible factor 1α (HIF-1α) was examined by immunoprecipitation (IP). The role of KDM1A on HIF target genes was determined using qRT-PCR and HRE-reporter activity. Mouse orthotopic xenografts models were used for preclinical evaluation of KDM1A inhibitors.

Results: Western blot analysis revealed that KDM1A expression is induced in primary GBM cells following hypoxia. IHC analysis demonstrated elevated expression of KDM1A in hypoxic regions in human GBM tissues as well as mouse GBM xenografts. IP analysis demonstrated that KDM1A interacts with HIF-1α. Cell viability assays revealed that NCD-38 is more potent in reducing the cell viability of primary GBM cells compared to other KDM1A inhibitors. KDM1A knockdown or KDM1A inhibitor treatment significantly reduced the HIF- target gene expression including VEGF, PGK, CAIX, and HRE-Luc reporter activity in GBM cells. Further, hypoxia mediated cell proliferation and self-renewal ability of GSCs was compromised in KDM1A knockdown and KDM1A inhibitor-treated cells. Further, knockdown of KDM1A or treatment with KDM1A inhibitor significantly reduced the in vivo tumor growth and improved survival in orthotopic models.

Conclusions: Our results demonstrate that KDM1A plays a critical role in hypoxia response and inhibition of KDM1A is a novel therapeutic approach for the treatment of GBM.

#2423

Hypoxia regulates tumor cell invasiveness through altered glycosylation.

Gordon Greville,1 Esther Llop,2 Rosa Peracaula Miró,2 Amanda McCann,3 Pauline M. Rudd,1 Radka Saldova1. 1 _NIBRT, Dublin, Ireland;_ 2 _University of Girona, Girona, Spain;_ 3 _University College Dublin, Dublin, Ireland_.

Glycosylation, one of the most fundamental post-translational modifications, is altered in many cancers. These alterations have been proven to impact on the progression of the tumor cell and promote survival. In literature published over the last half century, it is obvious there is a clear link between (a) chemo-resistance and hypoxia (b) hypoxia and epigenetics and more recently (c) glycosylation and epigenetics. We aim to bring these paradigms together with the remit of offering a more complete story and opening up new avenues of approach for the detection, diagnosis and treatment of breast and ovarian cancer.

Ovarian and breast cancer cells were exposed to differential hypoxic conditions (0.5%, 1.0%, 2.0%) and normoxia. Firstly, we compared the methylation status of hypoxia exposed cells to the normoxic controls. A combination of hydrophilic interaction liquid chromatography (HILIC) and statistical analyses allowed comparisons of the secreted N-glycans from cells exposed to differential hypoxic conditions. Western blots assessed apoptosis, senescence, autophagy and epithelial to mesenchymal transition. The OrisTM migration assay assessed the migration of the cells pre- and post-differential hypoxic exposure. RT-qPCR was used to measure gene expression of appropriate glycosyltransferases and possible transcription factors (TFs) in these samples to determine any associations with changes seen in N-glycosylation.

There were non-significant trends observed between the percentage oxygen that the cells were exposed to and resultant changes seen in branching and sialylation on secreted N-glycans from the breast and ovarian cancer cell lines. While only some of these changes could be explained by RT-qPCR data, GATA2/3 transcripts, identified in-silico as possible TFs, have been shown to significantly correlate with ST3GAL4 and MGAT5 glycosyltransferases, respectively. Hypoxia exposed cells also displayed increased migration with a greater effect seen in the 0.5% hypoxia exposed samples.

The GATA2 and GATA3 transcription factors are gaining popularity in recent years, with links to cancer stage, increased invasiveness and as possible therapeutic targets. Our recent data shows a strong correlation between GATA2 and GATA3 and the levels of glycosyltransferases involved in branching and sialyation. These glycan changes are known to be strongly involved in cancer cell survival and metastases. Here for the first time we may have a possible mechanism of action linking GATA2 and 3 and invasiveness of breast and ovarian cancer cells.

#2424

Hepatocellular carcinoma in the South Texas Latino population: Implications of STEAP2.

Carla Zeballos, Hakim Bouamar, Guixi Zheng, Xiang Gu, Yidong Chen, Francisco G. Cigarroa, Lu-Zhe Sun. _UTHSCSA, San Antonio, TX_.

Introduction: Hepatocellular carcinoma (HCC) is the most common type of liver cancer in adults and the third most common cause of cancer death worldwide; while incidence and mortality rates are two times higher in Latinos, incidence rates are the highest among Latinos in the South Texas region. The genetic and epigenetic events associated with the increased incidence of HCC in this population are largely unknown. We performed whole genome RNA sequencing in paired HCC tumor and adjacent non-tumor tissue total RNA from nine South Texas Latino patients. Analysis of differentially expressed genes revealed significant alterations in pathways associated with oxidative stress; most importantly, we found that the expression of STEAP2 (Six Transmembrane Epithelial Antigen of the Prostate 2) is increased five-fold in HCC tumor tissue compared to adjacent non-tumor tissue. In comparison to a non-Latino population, this finding was unique to South Texas Latinos. STEAP2 is a metalloreductase of iron and copper; reduced iron and copper ions can mediate the production of hydroxyl radicals resulting in increased oxidative stress, which can cause DNA damage and lipid peroxidation. We aim to prove that STEAP2 through regulation of iron and copper homeostasis, and an increase in oxidative stress, will lead to malignant transformation of hepatocytes resulting in tumor progression of HCC, including in obese hosts.

Material and Methods: Latino paired HCC and adjacent non-tumor tissues were collected for RNA sequencing, metal ion measurement and oxidative stress markers. STEAP2 RNA and protein expression levels in Latino and Caucasian samples were evaluated by RT-PCR, Western blot, and immunohistochemistry. HCC cell lines (SNU398 and HUH7) with knockdown (KD) and overexpression (OE) of STEAP2 were created to examine the proliferation, migration, anchorage independent growth, and oxidative stress in vitro.

Results: Analysis of RNA sequencing data demonstrated the overexpression of STEAP2 in HCC tumors in Latino patients, which were validated by RT-PCR and Western blot data. Lipid peroxidation product, 4-hydroxynonenal, and copper levels were higher in HCC tumor vs. adjacent tissue. KD of STEAP2 in the HCC cell lines decreased proliferation, migration and anchorage independent growth, while OE of STEAP2 increase migration and anchorage independent growth but not proliferation.

Conclusions: STEAP2 is specifically overexpressed in HCC tumors in Latinos in comparison to HCC tumors in non-Latino whites and appears to play a malignant-promoting role in HCC cells. Further studies on the role of STEAP2 as a novel tumor promoter in HCC and the mechanisms by which it promotes carcinogenesis are underway. The proposed studies will likely yield mechanistic insights into the molecular mechanisms that drive HCC development and progression in South Texas Latinos and potential therapeutic targets.

#2425

Characterization of VEGF-independent angiogenesis in an HIF1A knockdown model.

Marco Maruggi, Petrus De Jong, Robert Lemos, Brian James, Alejandro Campos, Garth Powis. _Sanford-Burnham Medical Research Inst., La Jolla, CA_.

Hypoxia inducible factor-1 (HIF-1) is the key transcription factor regulating the production of the vascular endothelial growth factor (VEGF), and is essential for the growth of solid tumors as a response to hypoxia due to poor tumor blood supply. We found that a pancreatic cancer cell line (MIAPaCa2) in which HIF-1 expression was targeted by lentiviral shRNA (shHIF1A) did not immediately form tumors when implanted in mice, compared with control xenografts (shEV). However, aggressive HIF-1-independent tumors with restored angiogenesis grew out from shHIF1A xenografts in a delayed manner. Determining what allows these tumors to grow without HIF-1 has significant implications in understanding the development of resistance in patients to anti-VEGF therapy, such as Avastin, and it could also provide clues why therapy of patients with macular degeneration is successful in only a subset of individuals. Using multispecies, bulk RNA analysis we have found that shHIF1A xenografts developed a senescence-associated secretory phenotype (SASP) signature, and had increased immune infiltration when compared to shEV tumors. To better dissect the mechanisms of tumor initiation in the absence of HIF-1, we performed single-cell RNA sequencing (scRNA-seq) of human MIAPaCa2 shEV and shHIF1A xenografts in mice. We used single-cell interpretation via multikernel learning (SIMLR) to identify cell populations and cell-specific gene signatures, gaining insight into how transcripts changed within populations following resistance. By focusing on population-specific transcript expression, we have identified multiple novel pro-angiogenic cytokines secreted by dendritic cells of the cDC2 lineage recruited to the tumor site. We are currently evaluating different therapeutic strategies to inhibit this mechanism, with the goal of overcoming anti-VEGF therapy resistance.

#2426

Tyrosine kinase and ERAD inhibitors promote oxidative stress-induced apoptosis through activation of ATF4 / KLF9 axis in medullary thyroid cancer.

Rozita Bagheri-Yarmand,1 Krishna M. Sinha,2 Ling Li,1 Yue Lu,1 Robert F. Gagel1. 1 _UT MD Anderson Cancer Ctr., Houston, TX;_ 2 _University of Texas Health Science Center at Houston, Houston, TX_.

Medullary thyroid carcinoma (MTC) is a neuroendocrine malignancy, caused by activating mutations of the RET proto-oncogene, with frequent lymph node and distant metastasis at diagnosis. Tyrosine kinase inhibitors (TKIs) that target the RET have proven effective for treatment of MTC, but resistance to TKIs evolves in half of the treated patients. We have previously shown that the expression levels of Activating Transcription Factor 4 (ATF4) which is a stress-induced transcription factor, significantly decreased or lost in half of the MTC tumors. We also demonstrated that forced expression of ATF4 or TKIs-induced level of ATF4 decreases survival of MTC cells by promoting degradation of RET, blocking the activation of RET downstream signaling pathways and subsequently induces apoptosis. More importantly, ATF4 knockdown by shRNA decreased the sensitivity to tyrosine kinase inhibitor-induced apoptosis. Further thyroid gland of Atf4-knockout mice showed C-cell hyperplasia, a precancerous lesion for MTC suggesting a potential tumor suppressor role of ATF4 in MTC. Stress-induced by the accumulation of unfolded proteins in the endoplasmic reticulum (ER) is a feature of specialized secretory cells, including MTC cells that secrete calcitonin. ATF4 promotes the induction of apoptosis under persistent stress conditions, although the mechanism is not clearly understood. We hypothesize that the combination of ATF4 inducer and TKIs causes an excessive cellular oxidative stress resulting in an activation of apoptosis, all of which may prevent resistance to TKIs. Here, we show that the ER-associated protein degradation (ERAD) inhibitor, eeyarestatin sensitizes MTC cells to the TKIs including sunitinib and vandetanib. The combination of eeyarestatin and TKIs causes a synergistic induction of ATF4 expression and its target genes, an accumulation of reactive oxygen species and subsequent cell death. Chromatin immunoprecipitation followed by sequencing assay (ChIP-seq) in MTC cells treated with eeyarestatin and immunoprecipitated with ATF4 and acetylated lysine 9 of histone 3, identified transcription Kruppel-like factor 9 (KLF9) gene as a transcriptional target of ATF4 activation. KLF9 plays a functional role in oxidative stress-induced cell death. Treatment with eeyarestatin and vandetanib alone or in combination increases the occupancy of ATF4 at the promoter of KLF9 gene and stimulates the expression of KLF9. Depletion of ATF4 by shRNA leads to downregulation of KLF9 expression and prevents oxidative stress-induced cell death. These findings suggest that induction of the ATF4/KLF9 axis causes oxidative stress leading to excessive ER stress, and subsequent cell death. Thus, combining TKIs and ERAD inhibitors that promote ATF4 levels could be effective therapeutic strategies for treating MTC and preventing resistance to TKIs.

#2427

Targeting intracellular pH regulation as a novel approach to overcome hypoxia-mediated drug resistance in FLT3/ITD mutated AML.

Fangli Chen, Adriana L. Rogozea, Xue Wu, George Sandusky, Mircea Ivan, Heiko Konig. _Indiana Univ. School of Medicine, Indianapolis, IN_.

Background: FLT3/ITD+ AML is a highly aggressive form of leukemia that carries a dismal prognosis. Evidence suggests that this poor outcome is due to the survival of a subset of leukemic cells (LCs) that elude therapy and survive in hypoxic niches of the bone marrow (BM). Effective targeting of LCs under hypoxic conditions is thus critical to cure AML. Our study examines the anti-leukemic potential of targeting Carbonic anhydrase-IX (CA9), a transmembrane protein which functions to maintain a neutral intracellular pH (pHi) under hypoxic stress conditions, against AML cells. Targeting CA9 is of particular interest as its expression is confined to only a few normal tissues and may therefore show relatively few side effects compared to standard therapies. Methods: Molm14 (M14) and primary cells (PC) from FLT3/ITD+ AML patients were incubated under normoxic (21% O2) and hypoxic (1% O2) conditions in the presence or absence of Quizartinib (Q), Cytarabine (Cy) or the CA9 inhibitor FC531 (FC). After 48h proliferation and apoptosis were determined per MTT assays, annexin V/PI staining and FACS analysis. Cells were assessed for CA9 mRNA expression as well as pHi levels via RT-PCR and fluorescent imaging, respectively. FLT3/ITD+ AML xenografts were generated by injecting M14 cells in the tail vein of NSG mice. Tissue samples were fixed in 10% NBF, embedded in paraffin and stained with H&E and CA9 for pathologic evaluation. Results: Cy and Q were significantly less effective against M14 cells under 1% compared to 21% O2. CA9 mRNA was upregulated in M14 cells (4.5±0.9-fold; n=4, p<.01) and in FLT3/ITD+ AML PCs from newly diagnosed (n=5) and relapsed/ refractory (n=3) patients (up to 529- and 73-fold, respectively) under 1% compared to 21% O2. Immuno-histochemical staining of the BM and spleen obtained from AML xenografts showed multifocal CA9 staining of LCs that was localized on the cell membrane. Under hypoxic conditions, FC but not Cy or Q, significantly induced apoptosis and showed dose-dependent growth inhibitory effects against M14 cells (n=3). In addition, treatment of M14 cells with FC under 1% O2 resulted in strong pHi acidification (ΔpH=-2.7±0.6, p<.05, n=3). Moreover, FC combined with Cy synergistically inhibited M14 cell growth in vitro and effectively eliminated LCs from the BM and spleen compared to Cy only treated mice. Accordingly, spleen size and weight were significantly reduced in mice treated with FC and Cy compared to single agent Cy (n=3). Conclusions: 1. Hypoxia blunts the effects Cy and Q in FLT3/ITD+ AML. 2. CA9 is induced in FLT3/ITD+ AML cells under hypoxic conditions. 3. The CA9 inhibitor FC confers anti-leukemic activity against FLT3/ITD+ AML cells under hypoxic conditions via pHi acidification and acts synergistically with Cy. 4. CA9 inhibition has the potential to target LCs residing in hypoxic niches and may be of value as an adjunct to chemotherapy for FLT3/ITD+AML therapy.

#2428

Redox regulation of β-catenin in colorectal cancer.

Alejandro D. Campos, Brian James, Petrus De Jong, Robert Lemos, Nikolas Marino, Garth Powis. _Sanford-Burnham Medical Research Inst., La Jolla, CA_.

Colorectal cancer (CRC) is the second most newly diagnosed cancer in the United States. β-catenin is the downstream effector of the Wnt/β-catenin signaling pathway. Loss of function (LOF) mutations on adenomatous polyposis coli (APC), a critical member of the β-catenin degradation complex leads to constitutively elevated β-catenin as an early oncogenic event in 85% of CRCs and is associated with decreased CRC patient survival. Increased level of β-catenin augments the nuclear accumulation of β-catenin, where it acts a co-activator of a number of transcription factors leading to the expression of downstream target genes that promote cancer cell proliferation, survival, and migration. There is no effective therapy for β-catenin as a cause of cancer. In addition to CRC, elevated levels of Wnt signaling have been reported in leukemia, melanoma, and breast cancers highlighting the need for novel therapies that attenuate aberrant β-catenin mediated transcription. Here we report on the redox dependency of the Wnt/β-catenin signaling pathway, identified through a forward genetic screen in Drosophila, and identify thioredoxin reductase-1 (TXNR1) as a potential target for the inhibition of aberrant β-catenin transcriptional activity through in vivo studies with the APCmin/+ mouse model. Drosophila harboring a mutation in their thioredoxin reductase (trxr-1) gene and a partial lethality phenotype were used in an enhancer/suppressor forward genetic screen. Increased lethality was observed when the trxr-1 mutation (trxr-1481) was combined with heterozygous mutations and deletions in wnt2, while wnt2 deletion and mutations alone had no effect on lethality. To test the translational value of these findings, APCmin/+ mice were treated with PX-12, a human TXNR1 inhibitor, daily by gavage from day 40 to time of death. We observed a dose dependent reduction in the number and size of intestinal polyps by PX-12 treatment, and an increased survival in treated mice. Furthermore, mechanistic studies utilizing human CRC cell lines harboring LOF mutations to the β-catenin degradation complex revealed β-Catenin mediated transcription, as measured by TOP Flash luciferase assay, was diminished when TXNR1 was silenced by siRNA. Additionally, we observed decreased β-catenin protein level in CRC cell lines treated with PX-12 or siRNA targeting TXNRD1 and its functional partner thioredoxin (TXN). The addition of the pan caspase inhibitor ZVAD but not the proteasome inhibitor MG132 was able to stabilize β-catenin protein upon treatment with PX-12 suggesting β-catenin destabilization is occurring via a caspase dependent mechanism. These results suggest a conserved mechanism for the redox regulation of β-catenin involving TXNR1; furthermore, inhibition of TXNR1 activity destabilizes β-catenin protein and decreases β-catenin mediated transcription in human CRC cell lines and inhibits intestinal neoplasia development in an experimental mouse model for CRC.

#2429

HIF-1α repressed glycerol-3-phosphate shuttle to reduce oxidative stress in liver cancer.

Shuo ZHANG, Robin Kit-Ho LAI, David Kung-Chun CHIU, Iris Mingjing XU, Aki Pui Wah TSE, Chun-Ming WONG, Irene Oi Lin NG, Carmen Chak Lui WONG. _The University of Hong Kong, Hong Kong, Hong Kong_.

Liver cancer is the fifth most prevalent and third most common cancer globally. Hepatocellular carcinoma (HCC), arises from hepatocytes, accounts for more than 80% of liver cancer. HCC cells undergo extensive metabolic rewiring to support their uncontrolled growth. HCC cells encounter extremely high level of oxidative stress which is mostly generated in the electron transport chain. Reactive oxygen species (ROS) rapidly accumulate in the electron transport chain (ETC) during hypoxia due to inadequate electron transfer to O2 in the ETC. The glycerol phosphate (GP) shuttle plays an important role to transfer electron source (NADH) from cytoplasm to mitochondria to initiate the ETC. The GP shuttle is mediated by glycerol 3 phosphate dehydrogenase (GPD) which converts dihydroxyacetone phosphate to glycerol 3 phosphate (G3P) through the oxidation of NADH to NAD+. This contributes to ATP production in the mitochondria. Here, we reported that hypoxia-inducible factor 1 (HIF-1) acts as a transcriptional repressor instead of an activator to suppress transcription of an important ETC component of the glycerol phosphate shuttle (GPD) to reduce activity of the GP shuttle, thereby reducing oxidative stress. GPD expression was significantly reduced under hypoxic condition. Surprisingly, knockout, knockdown, or inhibition of HIF-1 restored GPD expression in hypoxia. GPD expression was significantly under-expressed in tumorous as compared to non-tumorous liver tissues in 91 HCC patients of our center by 2.67 fold. TCGA database containing 49 HCC patients echoed with our in-house data. Downregulation of GPD was associated with poor cellular differentiation and overall survival in HCC patients. Stable overexpression of GPD1 in multiple HCC cell lines increased the levels of G3P as well as NAD+, which is crucial to lactate dehydrogenase (LDHA) activity. GPD overexpression altered mitochondrial activity, ROS levels, and suppressed HCC cell proliferation. Taken together, this study showed that the HIF-1 negatively controls key component of the GP shuttle to maximize HCC growth.

#2430

Targeting Nrf2 in esophageal adenocarcinoma sensitizes cancer cells to cisplatin treatment.

Dunfa Peng, Tianling Hu, Shoumin Zhu, Wael El-Rifai. _University of Miami, Miami, FL_.

Background: The incidence rate of the esophageal adenocarcinoma (EAC) has increased rapidly among men since 1976 in the USA. EAC is considered to originate from premalignant Barrett's esophagus (BE) which results from chronic gastroesophageal reflux disease (GERD). It has been reported that oxidative stress is steadily increased in GERD-BE-EAC progression cascade. NFE2-related factor 2 (Nrf2) is a major regulator of cellular anti-oxidant properties that maintains cell viability and cellular homeostasis.

Methods and Results: Using Western blot analysis, we detected overexpression of Nrf2 protein in several EAC cell lines, as compared to BE cell lines (CPA, BAR10T) and normal esophageal squamous cell lines. Nrf2 overexpression was also confirmed in primary EAC samples, using immunohistochemistry on a tissue microarray that contained 76 EACs. Knockdown of Nrf2 by siRNA or CRISPR/CAS9 in EAC cells (FLO1 and OE33), that have constitutive high levels of Nrf2, enhanced the sensitivity of cancer cells to cisplatin (CDDP). We confirmed these results by using a Nrf2 specific inhibitor (Brusatol). Further studies demonstrated that dysfunction of Nrf2, either through knockdown of Nrf2 expression or using its specific inhibitor, depleted several Nrf2 target genes such as GR, HO-1, GSTM2, GPX3 and GPX4, which have anti-oxidative properties, leading to a significant increase in cellular ROS levels. The excessive ROS levels resulted in an increase in oxidative DNA damage (measured by 8-oxoguanine) and double strand breaks (measured by p-H2AX) which were further increased by treatment with CDDP with subsequent increase in cancer cell death.

Conclusion: These results indicate that constitutive overexpression of Nrf2 in EAC cells protects tumor cells from high level of ROS and favors tumor cell survival and drug resistance. Targeting Nrf2 through its specific inhibitor may have a therapeutic window in EAC. Additional studies are ongoing to determine the molecular mechanisms underlying NRF2 overexpression in EACs.

#2431

EFNA3, a key functional mediator of hypoxic microenvironment in hepatocellular carcinoma.

Abdullah Husain, Yung Tuen Chiu, Daniel Wai Ho, Karen Man Sze, Lo Kong Chan, Yu Man Tsui, Carmen Chak Wong, Irene Oi Ng. _The University of Hong Kong, Hong Kong, Hong Kong_.

Hepatocellular carcinoma (HCC) is the third leading cause of all cancer-related deaths. Its rapid proliferative nature and HCC specific therapy such as transarterial chemoembolization (TACE) that block arterial blood supply to the tumor frequently induce hypoxic microenvironments i.e. regions with oxygen deprivation. Hypoxia has emerged as a major driving force to promote aggressive phenotypes and stemness of cancer cells in solid tumors. We aimed to identify the key contributors to HCC progression in hypoxic microenvironment. From the analysis of transcriptomic profiles of human HCCs in both our in-house RNA-sequencing (RNA-seq) data and TCGA data as well as HCC cells treated with hypoxia and normoxia, we identified Ephrin-A3 (EFNA3) as a potential hypoxia-inducible oncogene. EFNA3 is a membrane-bound ligand which can activate multiple receptor tyrosine kinases of the Eph family. This family of ligands and receptors are frequently dysregulated in multiple cancer types and have implications in stem cell plasticity. We further validated that EFNA3 was significantly overexpressed in HCCs as compared with their non-tumorous liver samples using qRT-PCR in a separate cohort of HCCs (p<0.0001, n=97). Upon clinicopathological correlation, overexpression of EFNA3 was significantly correlated with the presence of venous invasion (p=0.004) and more advanced tumor stage (p=0.026). In concordance, TCGA-LIHC dataset revealed significantly higher expression of EFNA3 in HCC samples with vascular invasion (p=0.0094) and advanced tumor stage (p=0.0002). Furthermore, we confirmed the upregulation of EFNA3 upon hypoxia treatment in multiple HCC cell lines. Functionally, stable knockdown of EFNA3 using short hairpin RNA approach significantly reduced proliferation and migratory rates of HCC cells. In addition, orthotopic liver injection model of the EFNA3 knockdown HCC cells demonstrated a significant reduction of the growth of the primary tumors. There was also a reduced incidence of distant metastasis to lungs in vivo. Using sphere formation assay to test self-renewal ability in vitro, EFNA3-knockdown HCC cells showed significantly reduced self-renewal ability. In addition, HCC cells positive/high for stemness markers (EpCAM, CD13, CD24, CD44, CD47, CD133) isolated from our PDTX model showed a consistently higher EFNA3 mRNA expression as compared with the negative/low cells. By flow cytometric analysis, the EFNA3 KD MHCC-97L demonstrated a decreased expression of CD47.

Conclusion: We identified EFNA3 as a potential hypoxia-inducible oncogene in HCC and demonstrated its critical role in tumor progression, metastasis and cancer stemness of HCC. Based on our results, EFNA3 may be an attractive therapeutic target to counteract hypoxia-specific disease progression.

#2432

The genomic consequences of tumor hypoxia in human cancers.

Vinayak Bhandari,1 Shadrielle M. Espiritu,2 Lydia Y. Liu,2 Emilie Lalonde,2 Takafumi N. Yamaguchi,2 Lawrence E. Heisler,2 Julie Livingstone,2 Vincent Huang,2 Yu-Jia Shiah,2 Veronica Y. Sabelnykova,2 Fouad Yousif,2 Melvin L. Chua,3 Michael Fraser,2 Theodorus van der Kwast,4 Paul C. Boutros,1 Robert G. Bristow5. 1 _University of Toronto; Ontario Institute for Cancer Research, Toronto, Ontario, Canada;_ 2 _Ontario Institute for Cancer Research, Toronto, Ontario, Canada;_ 3 _Division of Radiation Oncology, National Cancer Centre Singapore; Duke-NUS Graduate Medical School, Singapore;_ 4 _University of Toronto; Toronto General Hospital/University Health Network, Toronto, Ontario, Canada;_ 5 _University of Toronto; University of Manchester; The Christie NHS Foundation Trust; Manchester Cancer Research Centre, Manchester, United Kingdom_.

Introduction: Localized prostate cancers are classified into risk-groups using clinical measurements like grade and stage to inform treatment decisions. However, these groupings are imprecise: ~30% of intermediate-risk patients suffer relapse of their disease despite precision image-guided radiotherapy or radical prostatectomy. One reason for this variability in response to treatment is the underlying cellular and molecular heterogeneity of tumors. Prostate tumor cells exist within a microenvironment characterized by gradients of oxygen levels and prostate tumors with low levels of oxygen (hypoxia) have poor clinical outcomes.

Methods and Results: To understand the correlates of hypoxia in cancer we conducted a pan-cancer analysis of copy number alterations (CNAs) and single nucleotide variants (SNVs) across 19 cancer types. We measured hypoxia using multiple mRNA-based signatures and discovered numerous CNAs and SNVs enriched or depleted in hypoxic tumors, highlighting the role of hypoxia in shaping the genomic landscape of multiple tumor types. Next, we examined 548 patients with localized prostate cancer and statistically assessed the association of hypoxia with CNAs, SNVs, genomic rearrangements, focal genomic events (i.e. kataegis, chromothripsis), telomere length, clinical indices (i.e. grade, stage) and subclonal architecture. Tumor hypoxia is associated with specific CNAs and SNVs in prostate cancer driver genes. To translate these findings into a biomarker for prostate cancer precision medicine, we integrated tumor microenvironmental data with genomic and pathological information to stratify patients into distinct prognostic groups.

Impact: These data suggest that the aggressiveness of cancers is driven by the interplay of the tumor microenvironment and its genomic mutational profile.

#2433

**The hypoxic tumor microenvironment in vivo** **selects the tumor cells with cancer stem cell characteristics and increased resistance against genotoxic stresses.**

Hoon Kim, Qun Lin, Zhong Yun. _Yale University, New Haven, CT_.

Tumor hypoxia has been established as an independent prognostic factor associated with poor patient survival and resistance to conventional therapies. Emerging evidence suggests that hypoxia has the potential to maintain or enhance the stem cell phenotype and confers chemo- or radio-resistance to tumor cells. However, it remains to be determined whether cell fate and acquisition of therapy resistance is regulated in vivo by the hypoxic tumor microenvironment (TME). In this study, we have developed a hypoxia-sensing human breast cancer cell xenograft model using the hypoxia-response element (HRE)-driven enhanced green fluorescence protein (EGFP) as a hypoxia reporter. We have found that the hypoxic tumor cells isolated from xenografts contain increased subpopulations of tumor cells showing the cancer stem cell (CSC) characteristics. Upon re-implantation, the CSC characteristics of the hypoxic tumor cells continue to be further enriched during in vivo growth, whereas secondary xenografts derived from the non-hypoxic tumor cells remain similar to the primary xenografts. Consistent with this finding, the phenotypes exhibited by the hypoxic tumor cells are stably different from those of the non-hypoxic tumor cells isolated from the same tumor mass even when they are maintained under the same ambient culture conditions. Mechanistically, we find that the PI3K/AKT pathway is strongly potentiated in the hypoxic tumor cells and is required to maintain the CSC phenotype. Cell fate differences between hypoxic and non-hypoxic tumor cells are found only in tumor cells isolated from the hypoxic TME in vivo, and are not seen in tumor cells treated by hypoxia in vitro alone. In addition, we have found that naturally hypoxic tumor cells in a breast cancer xenograft model are moderately radio- and chemo-resistant compared to their neighboring non-hypoxic tumor cells in the same tumor. These naturally hypoxic tumor cells are proficient at repairing DNA damages and

resist apoptosis induced by genotoxic stresses. The ex vivo hypoxic tumor cells are capable of proficient activation of the DNA damage-sensing pathways, especially the ATM pathway. These previously unrecognized observations suggest that the hypoxic TME may promote malignant progression by coordinating induction, selection and preferential maintenance of the CSC phenotype in tumor cells. This presentation will review our results which strongly suggest that the hypoxic TME may induce adaptive responses, either by clonal evolution or selection, such that hypoxic tumor cells in vivo acquire new functional traits that confer resistance to genotoxic stresses and enhance survival. It can be postulated that previously hypoxic tumor cells may continue to exhibit their aggressive phenotypes long after they have migrated into non-hypoxic regions or after their native niche become normoxic.

#2434

Regulation of HIF1a stability by an antisense RNA.

Xin Huang, Huang Huang. _Univ. of Pittsburgh, Pittsburgh, PA_.

Hypoxia is a hallmark of solid tumors. Hypoxia-inducible factors (HIFs) are the main transcriptional factor that regulate the cellular response to hypoxic stress. HIFs are heterodimers consisting of an oxygen-sensitive alpha subunit (HIFα) and a constitutively active beta subunit (HIF1β, also called aryl hydrocarbon receptor nuclear translocator, ARNT). Among the three homologous HIFα genes, HIF1α, HIF2α, and HIF3α, the functions of HIF1α and HIF2α are best characterized. HIF1α is ubiquitously expressed in essentially all tissue types, while HIF2α expression is more tissue specific. Under normoxia, HIFα is hydoxylated by prolyl-4-hydroxylases (PHDs), targeting it for proteasome destruction mediated by the von Hippel Lindau (VHL) protein, an E3 ubiquitin ligase. Under hypoxic conditions, the activity of PHDs decreases and HIFα is stabilized and forms a dimer with HIF1β. The HIFα/HIF1β dimer is then translocated into the nucleus and binds specifically to the promoters of a large number of target genes involved in adaptation and protection against low oxygen conditions. It is well known that HIFα is stabilized by acute hypoxia and its level decreases under prolonged hypoxic conditions. However, the mechanism remains unknown. Now, we have identified an antisense RNA that regulates the interaction between HIFα and VHL. The induction of the anitsense RNA is responsible for the regulation of HIFα stability under prolonged hypoxia. Our study revealed a novel mechanism regulating HIFα pathway.

#2435

ST6Gal-I sialylation potentiates hypoxia adaptation by enhancing HIF-1a signaling.

Robert B. Jones, Kaitlyn A. Dorsett, Anita B. Hjelmeland, Susan L. Bellis. _Univ. of Alabama at Birmingham, Birmingham, AL_.

Aberrant surface glycosylation is a well-known hallmark of a tumor cell, however the functional role of glycans in cancer cell biology remains unclear. Tumor cells frequently display an increase in surface α2-6 sialylation, a modification added to N-glycosylated proteins by the ST6Gal-I sialyltransferase. ST6Gal-I sialylates a select cohort of surface receptors, which correspondingly modulates receptor-induced intracellular signaling cascades. Emerging evidence suggests that ST6Gal-I-mediated sialylation promotes the survival of tumor cells exposed to a variety of cell stressors. In the current study we identify a new function for ST6Gal-I in protecting against hypoxic stress. In response to low oxygen tension, the hypoxia inducible factor, HIF-1α, becomes stabilized in tumor cells. In turn, HIF-1α stimulates the transcription of genes important for cell survival. To interrogate a role for ST6Gal-I in hypoxic response, we evaluated HIF-1α accumulation in ovarian and pancreatic cancer cells with ST6Gal-I overexpression or knockdown. We find that ST6Gal-I activity augments HIF-1α accumulation in cells treated with chemical hypoxia mimetics (DFO and DMOG), or alternatively grown in a hypoxic environment. Furthermore, hypoxic cells with high ST6Gal-I expression have increased mRNA levels of HIF-1α transcriptional targets including the glucose transporters, GLUT1 GLUT3, and the glycolytic enzyme, PDHK1. Interestingly, cells with high ST6Gal-I expression also have an increased pool of HIF-1α mRNA, suggesting that ST6Gal-I may influence the biosynthesis of HIF-1α. Finally, cells grown in hypoxia for several weeks display an enrichment in ST6Gal-I expression, consistent with the concept that ST6Gal-I acts as a pro-survival factor. Taken together, these findings highlight a novel, glycosylation-dependent mechanism that facilitates tumor cell adaptation to a hypoxic milieu.

#2436

Anti-cancer effect by combination treatment of CDK and HSP90 inhibitors through HIF1α inhibition.

Shuai Zhao, Lanlan Zhou, David T. Dicker, Wafik S. El-Deiry. _Fox Chase Cancer Ctr., Philadelphia, PA_.

Hypoxia-inducible factor 1α (HIF1α) is the main mediator of the adaptive response to intra-tumoral hypoxia. HIF1α overexpression contributes to tumor angiogenesis, cancer cell survival and metastasis. Thus, targeting HIF1α provides new opportunities for cancer therapy. We have previously shown that cyclin-dependent kinase 1 (CDK1) stabilizes HIF1α through direct phosphorylation of its Ser668 residue in a Von Hippel-Lindau (VHL)-independent or p53-independent manner both under hypoxia and at G2/M under normoxia (Warfel et al., Cell Cycle, 2013). Because heat shock protein 90 (HSP90) is a VHL-independent HIF1α stabilizer, we hypothesized CDK and CDK inhibitor effects in the stabilization/destabilization mechanism. We found that CDK1 inhibition disrupts the interaction between HSP90 and HIF1α. Moreover, administration of the CDK1 inhibitor, Ro-3306, partially reversed a heat shock (40°C) response dependent increase in the level of HIF1α, which suggests that CDK1 activity may contribute to HSP90-mediated HIF1α stabilization. Combination treatment with both HSP90 inhibitor and CDK1 inhibition/knockdown decreased HIF1α level more robustly than either single treatment. Dual inhibition of CDK1 and HSP90 not only synergistically decreased cell viability but also remarkably reduced colony formation of HCT116 colon cancer cells. Previously we have also shown that similar to CDK1, CDK4 plays a role in HIF1α stabilization as well. This led us to investigate the effect of CDK4/HSP90 dual inhibition on colorectal cancer cells. The combination treatment of CDK4 knockdown/inhibition and HSP90 inhibition reduced the level of HIF1α in various cancer cell lines (e.g., colorectal, glioblastoma, prostate cancers). The FDA-approved CDK4 inhibitor palbociclib along with ganetespib synergistically inhibited cell viability in SW480 colon cancer cells under hypoxia or normoxia. No reduction in cell viability was noted in WI38 normal cells under normoxia at similar concentrations. In addition, we tested the therapeutic potential of the dual CDKi/HSP90i therapy in vivo. The combination treatment of palbociclib and ganetespib inhibited tumor growth in the HT29 colon cancer cell xenograft mouse model at relatively low doses. Immunohistochemistry staining of the tumor samples revealed higher levels of apoptosis, lower levels of VEGF expression and reduced microvessel formation in the combination treatment group. Our findings provide a compelling rationale for a therapeutic strategy to target HIF1α through the combination of CDK and HSP90 inhibitors in cancer therapy.

#2437

Novel metabolic adaptations support proliferation of African American prostate cancer cells under hypoxia.

Gati Panigrahi, Prakash Praharaj, Kiran Sai, Gargi Mahapatra, Taylor Peak, Sierra Patterson, Hakeem Oufkir, Anthony Molina, Steven Kridel, Gagan Deep. _Wake Forest Baptist Medical Center, NC_.

Hypoxia in prostate cancer (PCa) selects for aggressive clones and causes treatment failure, metastatic progression and mortality. Despite the key role of hypoxia in prostate carcinogenesis, little is known about the specific effect of hypoxia in African Americans, a race that faces a serious PCa-related health disparity with the highest incidence and mortality rate. We hypothesize that metabolic adaptation to hypoxia is one mechanism that contributes to PCa aggressiveness in African Americans. Therefore, in the present work we focused on the metabolic adaptations, both in vitro and in vivo, contributing to proliferation of African American PCa cells under hypoxic conditions. PCa cells from men of African American ancestry (E006AA-hT, MDA PCa 2b, WFCB17 and 22Rv1) showed proliferation and significantly higher clonogenicity under hypoxia (1% O2) compared to normoxia. In contrast, Caucasian PCa cells (LNCaP and PC3) showed significant growth inhibition under similar hypoxic condition. The proliferation under hypoxia in African American PCa cells was associated with higher beta-oxidation of lipids as measured by malate response in Oroboros Oxygraph system. In addition, [18F]-fluoro-4-thia-oleate ([18F]-FTO) uptake in African American PCa cells under hypoxia was more than 2-fold higher (p <0.001) compared to normoxic cells, confirming higher beta-oxidation in hypoxic PCa cells. Furthermore, African American PCa cells showed more than 2-fold (p <0.001) higher uptake of [11C]-acetate under hypoxia, suggesting increased fatty acid synthesis during hypoxia in African American PCa cells. This suggests that African American PCa cells may increase fatty acid synthesis under hypoxia to provide metabolic fuel. Fatty acid oxidation is associated with mitochondrial metabolism; accordingly, we observed higher oxidative phosphorylation, complex II activity and expression under hypoxia in African American PCa cells. Importantly, treatment with complex II inhibitor Atpenin A5 (1-2 microM) strongly reduced the clonogenicity (51%-79%, p <0.0001) of African American PCa cells under hypoxia. In vivo, mPET/CT imaging using [18F]-FTO and [18F]-fluoromisonidazole as PET tracers showed greater tumor uptake of both tracers in 22Rv1 tumors in male athymic nude mice, suggesting that hypoxia and fatty acid oxidation are associated with African American PCa growth. Furthermore, analyses of 22Rv1 tumor tissues by confocal microscopy showed higher level of carnitine palmitoyltransferase 1 (a key regulator of β-oxidation) and succinate dehydrogenase B (a subunit of complex II) in hypoxic areas (pimonidazole+) of the tumor. Taken together, these data demonstrate that proliferation of African American PCa cells under hypoxia is supported by high lipid beta-oxidation, lipid synthesis and complex II activity, providing a novel mechanism to promote disease aggressiveness.

#2438

**Reducing plate edge effect by controlling cell handling conditions for** in vitro **tumor hypoxia assays.**

Alicia Henn, Shannon Darou, Randy Yerden. _BioSpherix, Parish, NY_.

To reduce the impact of high variability in the edge wells of 96-well plates, the "edge effect", researchers often fill edge wells with buffer to try to insulate the rest of the plate. This removes over a third of the wells from the assay, adding cost, handling time, and space problems to in vitro assays. This is of particular concern for researchers working with cells in low oxygen, as handling more plates increases the time that cells are out of optimum conditions, risking HIF-1a modulation. For this project, we tested the idea that controlling the entire cell handling space to conditions that match the incubator, including the working space floor temperature and gas levels, may help reduce the edge effect seen in 96-well plates. The null hypothesis was that cell handling conditions would make no difference in the variability of cell density in 96-well plates loaded in traditional room air conditions compared to plates loaded in controlled conditions matching the incubator. To address cell loading specifically, A549 cells were harvested from cell culture flasks in traditional cell handling conditions (room temp, 20% O2, 0% CO2), then split. Half of the cells were transferred to an Xvivo System for controlled cell handling conditions. The cells in both conditions were transferred to media pre-conditioned to hypoxic incubator conditions and then plated in 96-well plates. Temperature mapping was performed with 16 thermal probes simultaneously in replicate plates loaded with medium only. The cells were incubated for 24 hours, then stained for cell density, and read on a plate reader. Cell viability was greater than 90% (n=4 plates/condition/trial x 4 trials). We found that edge well temperatures were more stable in plates handled in unbroken optimal conditions and edge effect on cell density was greatly reduced. In room temperature conditions, columns 1 and 12 cooled most rapidly. We concluded that full-time control of environmental conditions allowed for use of the whole plate with reduced edge effect. This helps reduce cost, time, number of plates and the risks to cells during cell handling.

#2439

The importance of cellular oxygenation measurements in the analysis of hypoxia-induced signaling and related metabolic adaptation.

Michelle Potter,1 James Hynes,2 Anke Nijhuis,3 Conn Carey,2 Christos Zois,1 Adrian Harris,1 Hector Keun,3 Karl Morten1. 1 _University of Oxford, Oxford, United Kingdom;_ 2 _Luxcel Biosciences, Cork, Ireland;_ 3 _Imperial College London, London, United Kingdom_.

There is a growing appreciation that typical cell culture conditions reflect a hyperoxic condition for the majority of cell types. In an effort to more accurately reflect the in vivo condition, a growing number of researchers are conducting in vitro measurements at what are deemed to be more physiologically relevant oxygen concentrations (1-8% O2). The importance of oxygen concentration in in vitro model design is underscored by observations that O2 levels influence a wide range of cellular phenomena, including metabolic poise, differentiation and senescence while the development of localised hypoxic environments are implicated in a variety of diseases including stroke, and cancer. Impaired O2 supply can reduce O2 concentrations to below normal physiological levels, initiating a graded series of complex interconnected adaptive responses mediated by regulators including the hypoxia-inducible factor (HIF). These processes are particularly relevant within solid tumours, where oncogene-driven proliferation causes nutrient and oxygen deprivation, aberrant angiogenesis, and the activation of O2-sensitive survival pathways.

Despite the importance of cellular oxygenation, research using in vitro models makes the assumption that oxygenation can be defined by simply altering the oxygen condition applied to the in vitro model. To test this key assumption, cellular oxygenation measurements are performed using a nanoparticulate intracellular oxygen probe (MitoXpress®-Intra), while tumour hypoxia/oygenation is modelled on a fluorescence plate reader equipped with an atmospheric control unit (CLARIOstar®). Data is presented illustrating that the depth of hypoxia experienced by the cell model is impacted significantly by respiratory activity, and that this additional oxygen deprivation is a dynamic process, effected by respiratory substrate availability and related metabolic poise. Data is also presented demonstrating that, in in vitro models, additional respiration-induced hypoxia directly impacts glycolytic flux and other relevant metabolic pathways. Together these data invalidate the assumption that cellular oxygenation can be inferred from ambient oxygen measurements. If ignored, the significant and dynamic deviations between ambient O2 and oxygenation at the cellular level leads to erroneous collusions regarding the relationship between oxygen concentration, HIF stabilisation and related metabolic adaptations. This in turn can impair the physiological relevance of experimental observations.

#2440

Potentiation of the action of chemotherapeutic drugs by the hypoxia-activated prodrug SN30000 in multicellular tumor spheroids.

Xinjian Mao, Sarah McManaway, William Robert Wilson, Kevin Owen Hicks. _University of Auckland, Auckland, New Zealand_.

Introduction: Chemotherapy is often limited by resistant subpopulations of tumor cells. Tumor hypoxia contributes to resistance of tumor cells to clinical drugs via multiple mechanisms. Hypoxia-activated prodrugs selectively target hypoxic cells, among which SN30000 is a second generation of tirapazamine analogue with superior diffusion coefficient and hypoxia selectivity. Here we hypothesize that SN30000/chemotherapy drug combinations will provide improved activity.

Methods: HCT116 and SiHa spheroids were used to screen schedules of SN30000 in combination with 4 clinical drugs (cisplatin, doxorubicin, gemcitabine, paclitaxel) under 5% O2 and 20% O2, with endpoints of spheroid growth delay and clonogenic cell survival. The schedules were tested on HCT116 and SiHa monolayers under 5% O2. HCT116 monolayers and spheroids exposed to SN30000 or the clinical drugs were dissociated for clonogenic survival assay. Tumor growth delay using the same schedules of the most promising combination (SN30000 + gemcitabine) were then tested in HCT116 xenografts in NIH-III mice. Body weight loss and core body temperature of mice in response to the treatments were monitored. Results: Exposing to 25 µM SN30000 and 25 µM cisplatin/5 µM doxorubicin/10 µM gemcitabine simultaneously for 2 hr, and SN30000 3 hr before gemcitabine under 5% O2 significantly delayed HCT116 and SiHa spheroid growth, but their combined activity was compromised under 20% O2. Dosing SN30000 and cisplatin simultaneously, and SN30000 3 hr before gemcitabine significantly delayed HCT116 and SiHa monolayer growth. Clonogenic survival assay demonstrated that HCT116 monolayers were more sensitive than spheroids to doxorubicin, but not the other 3 clinical compounds, consistent with the known limited diffusion of doxorubicin into spheroids.Dosing SN30000 3 hr before gemcitabine or at the same time, but not SN30000 3 hr after gemcitabine significantly delayed tumor growth, which was consistent with the spheroid data. Body weight measurements showed that the combinations of SN30000 and gemcitabine added little additional toxicity while SN30000/doxorubicin was more toxic when administered simultaneously. SN30000 caused hypothermia in mice with recovery after 3 hr.

Discussion: Screening schedules of SN30000 in combination with 4 clinical drugs in HCT116/SiHa spheroids and monolayers demonstrated schedule and oxygen dependence of the activity of SN30000 and gemcitabine combinations, which was further demonstrated in HCT116 xenografts. The mechanism of the promising interaction between gemcitabine and SN30000 is being further investigated by flow cytometry and immunohistochemical staining to explore the hypoxic fraction and cell proliferation following treatments, and by evaluating the plasma pharmacokinetics of SN30000 and gemcitabine to investigate the implications of SN30000-induced hypothermia.

#2441

Effects of hypoxia on Src family members' activity in prostate cancer cells.

Yao Dai, Dietmar Siemann. _Univ. of Florida College of Medicine, Gainesville, FL_.

Metastasis is the major reason of disease progression and poor prognosis in prostate cancer. Src family kinases (SFKs), including c-Src, Lyn and Fyn, are non-receptor tyrosine kinases that have been shown to play an essential role in castration resistance and metastasis in prostate cancer. Hypoxia, a typical hallmark of solid malignancies, is able to promote metastasis-associated functions. However, whether SFK members are upregulated under hypoxia is unclear. The current project will detect the impact of hypoxia on cell functions and molecular features in prostate cancer cells. For hypoxic exposure, prostate cancer cells were exposed to low oxygen tensions (1% O2) for varied durations (0, 2, 6, 24 h). To test cell migration, "wound-healing" assay was used. Cell invasion was detected by seeding cells into Matrigel-coated transwell chamber and cells on the bottom membranes were counted after treatment. Clonogenic assay was performed to test cell survival. At the molecular level, SFKs' phosphorylation in cancer cells and patient tissues was detected by Western blotting, and gene knockdown was accomplished by siRNA transfection. To detect the effects of Src inhibitors, cells were treated with saracatinib for indicated time periods. Phosphorylation of all three typical SFKs, e.g., c-Src, Lyn and Fyn, were highly expressed in prostate cancer patient tissues. At the cellular level, while short term hypoxic exposure (2-6 h) induced greater effects than prolonged hypoxia (24 h) in PC-3ML, these cellular functions were increased with 24 h hypoxic exposure in C4-2B cells. Further, hypoxia enhanced SFK phosphorylation in the pattern that was consistent with cell functions in both cell lines. Knockdown SRC, but not LYN or FYN, abolished hypoxia-induced invasion and p-SFK expression. Lastly, SFK inhibitor saracatinib showed stronger inhibition on functional behaviors facilitated under hypoxia than normal conditions. Our data show that hypoxia is able to enhance metastatic phenotypes by activating SFKs in prostate cancer cells. Interestingly, c-Src may be the most important signaling molecule for hypoxia-mediated behaviors. More importantly, SFK inhibitors are able to impair hypoxia-induced cell functions, suggesting their therapeutic potential by suppressing tumor metastasis that is driven under hypoxia in prostate cancer.

#2442

Detection of tissue hypoxia with EF5.

Kevin Su,1 Amy Noble,1 Nick Asbrock,1 Cameron Koch,2 Vi Chu1. 1 _MilliporeSigma, Temecula, CA;_ 2 _University of Pennsylvania, Philadephia, PA_.

Hypoxia, a condition of oxygen deprivation, is a salient feature of the tumor microenvironment. Hypoxia tumors are frequently resistant to conventional cancer therapies and are correlated with increased migratory and metastatic behavior. A number of detection modalities exist for the detection of hypoxia; however, only EF5 (2-[2-nitro-1H-imidazol-1-yl]-N-(2,2,3,3,3-pentafluoropropyl) acetamide) provides detailed information on the distribution of hypoxia in tissues at both the physiologic and pathologic levels. EF5 is a compound that, when injected into animal tissues, selectively binds to hypoxic cells and forms adducts. These adducts are detected by a mouse monoclonal antibody, clone ELK3-51, conjugated to fluorescent dyes including Cyanine 3, Alexa Fluor 488 and Cyanine 5. The use of EF5 and its specific fluorescent monoclonal antibody to selectively detect regions of hypoxia in mouse tissues is described. BALB/c mice between 7 to 10 weeks old and weighing between 18 to 30 g were used in the study. Mouse #1 was not treated with EF5 and its spleen was harvested as a negative control. Mouse #2 was injected with 10 mM EF5 (typically 1% of animal weight in mL) and allowed to run aerobically for 4 hours before euthanization. Mouse #3 was injected with EF5, euthanized after 10 minutes and kept warm at 37°C for 40 minutes to mimic the anaerobic hypoxic condition before the spleen was harvested (hypoxic control). Examination of spleen samples indicated highly specific binding of the EF5 adducts only in the anaerobic hypoxic mouse spleen. The use of EF5 with fluorescent conjugated antibodies provides a sensitive and quantitative method for the detection and measurement of tissue hypoxia in animal and human tumor tissues and cells.

#2443

Oxygen metabolism and hypoxia tolerance in organoid models of pancreatic ductal adenocarcinoma.

Ji Zhang,1 Qingquan Liu,2 Dan Cojocari,1 Mark Zaidi,2 Trevor McKee,2 Nikolina Radulovich,2 Ming-Sound Tsao,2 David Hedley,2 Marianne Koritzinsky,2 Bradly G. Wouters2. 1 _University of Toronto, Toronto, Ontario, Canada;_ 2 _Princess Margaret Cancer Centre, Toronto, Ontario, Canada_.

Background: Pancreatic Ductal Adenocarcinoma (PDAC) has extremely heterogeneous hypoxic microenvironments across patients and high levels of hypoxia are correlated with increased tumor aggressiveness and resistance to therapy. However, the underlying genetic contributors to variations in hypoxia and its importance to the disease is currently unknown. We hypothesize that genetic mutations in PDAC associated with two principal factors - oxygen metabolism and hypoxia tolerance - influence the steady state levels of hypoxia in individual tumors. The demand for oxygen, which is influenced by genetic driven changes in cellular metabolism, define the levels and steepness of hypoxia gradients around perfused vessels. Tolerance to hypoxia determines the time tumor cells can survive in severe microenvironments depleted of oxygen and other nutrients. Both factors are affected by the activation of adaptive hypoxia stress response pathways including the HIF, UPR, and autophagy pathways. Method: We developed patient-derived-organoids from PDAC tumors for in vitro studies of oxygen metabolism and glycolytic rates using the Seahorse XF96. We also characterized hypoxia tolerance through monitoring of organoid growth and secondary growth under defined levels of oxygenation. In addition, we have developed an immunofluorescence image analysis pipeline to evaluate in vivo oxygen demand/consumption through the quantification of oxygen and proliferation gradients around perfused blood vessels. Results: We observed significant heterogeneities in oxygen metabolism and hypoxia tolerance across our patient derived organoid models. We also demonstrated the importance of PERK/UPR pathway in mediating both oxygen metabolism and hypoxia tolerance through regulation of ULK1, a kinase involved in the initiation of autophagy. Inhibition or knockdown of ULK1 decreased cell survival and correspondingly sensitized cells to hypoxia in organoid and tumor models. This is accompanied by accumulation of mitochondria and a corresponding increase in oxygen consumption, resulting in increased development of hypoxic cells. Conclusion: These experiments demonstrate the dual importance of oxygen metabolism and hypoxia tolerance and set the stage for the evaluation of these parameters and identification of the underlying genetic drivers of the hypoxic microenvironment. These genetic markers would be used for patient-selection and development of hypoxia-targeted therapies.

#2444

Effects of GSH depletion and thioredoxin reductase inhibitors on the viability of MDA-MB-231 and MCF-7 cells.

Kaci Keleher,1 Emma Meetz,1 Collin Ellenbecker,1 Radek Buss,1 Emily Minton,1 Morris A. Johnson,2 Michael Smits,2 Russ Feirer1. 1 _St. Norbert College, De Pere, WI;_ 2 _BioChemical Solutions, LLC, Appleton, WI_.

The purpose of this study was to investigate the effect of altering the reducing environment in cancer cells by targeting both glutathione (GSH) and thioredoxin (Trx). This was achieved by administering low levels of buthionine sulfoximine (BSO) to deplete GSH and auranofin (AUR) to lower Trx by inhibiting thioredoxin reductase (TrxR). Methylglyoxal (MG), which alters cellular redox environment, was used in combination with BSO and AUR. The experiments were conducted using the breast cancer cells MDA-MB-231 and MCF-7, cell lines known to exhibit differences in metabolism and responses to chemotherapeutic drugs.

GSH was quantified using a luminescence-based assay. As expected, BSO led to a dramatic decrease of cellular GSH. Treatment with AUR and MG enhanced the decrease of GSH by BSO. Surprisingly, in the absence of BSO the combination of AUR and MG evoked an increase in GSH content in both cell lines. This increase might reflect a compensatory mechanism in which the inhibition of TrxR led to an increase in GSH synthesis in order to resist oxidative damage. Levels of reactive oxygen species (ROS) were quantified using redox-sensitive GFP (roGFP). After baculovirus transduction of the cells with roGFP, an increase in ROS was observed in both cell lines within 30 minutes of treatment with the combination of the three compounds.

Cellular viability was assessed using a colorimetric assay measuring the reduction of a tetrazolium salt in cells treated with various combinations and concentrations of BSO, AUR, and MG. BSO reduced viability of MDA-MB-231 cells at concentrations at or above 50 µM. AUR led to slight reductions in viability in this cell line, but when administered together, BSO at 100 µM and AUR at 1 µM caused an 85% decrease in viability. In the MCF-7 cell line, BSO caused no notable decrease in viability, while AUR likewise had no effect on viability. Administered together, however, BSO at 100 µM and AUR at 1 µM caused a 95% decrease in viability. MG enhanced the effects of BSO in both cell lines, but the reduction in viability was more pronounced in MDA-MB-231 cells. Similar to the effects of the compounds on viability, BSO alone had little effect on colony formation in both MDA-MB-231 and MCF-7 cells (as visualized by crystal violet staining). In addition, there was no effect on colony formation in either cell line when using AUR and MG unless initially treated with BSO. These effects were most significant when the concentration of BSO was greater than 50 µM.

Previous studies have reported metabolic differences between MDA-MB-231 and MCF-7 cells (Gatenby & Gillies, 2004). MDA-MB-231 cells rely on aerobic glycolysis while MCF-7 cells utilize mitochondrial oxidative phosphorylation. Therefore, MCF-7 cells have developed increased protection against ROS as compared to MDA-MB-231 cells (Theodossiou et al., 2017). These findings support the observed differences in response to BSO, MG, and AUR between the two cell lines.

#2445

Oxidative stress induction through TXNRD1 inhibition as a therapeutic strategy in hepatocellular carcinoma treatment.

Derek Lee, Iris Ming Jing Xu, David Kung Chun Chiu, Robin Kit Ho Lai, Chun Ming Wong, Irene Oi Lin Ng, Carmen Chak Lui Wong. _University of Hong Kong, Hong Kong_.

Hepatocellular carcinoma (HCC) is the most common form of primary liver cancer. It ranks third amongst cancer-related deaths worldwide owing to its late symptom presentation and lack of effective treatment regimens. Compared to normal cells, cancer cells experience distinctly higher amount of oxidative stress from increased reactive oxygen species (ROS) generation contributed by metabolic alterations, or its hypoxic microenvironment. Cancer cells also have a greater capacity of antioxidant production to maintain redox homeostasis. Immense generation of NADPH, a major metabolite and antioxidant is found in cancer cells to combat ROS as confirmed in our previous studies as we identified the folate cycle and pentose phosphate pathway (PPP) as major metabolic pathways responsible for NADPH production in human HCC. NADPH is an electron donor which can reduce and activate different antioxidant systems. The thioredoxin system is a ubiquitously-expressed mammalian antioxidant system activated by NADPH. The transmission of an electron from NADPH to TXN, the ROS-scavenging member of the thioredoxin system, is modulated by thioredoxin reductase 1 (TXNRD1). TXRND1 is the sole activating-enzyme of the thioredoxin system, playing a vital role in maintaining intracellular redox homeostasis. Furthermore, TXNRD1 was found to be significantly over-expressed in human HCC correlated with poor clinical prognosis and patient survival. Genetic inhibition of TXNRD1 via shRNA-knockdown significantly induced oxidative stress which suppressed HCC cell proliferation in vitro and liver tumor formation orthotopically implanted in vivo. Interestingly, TXNRD1-inhibition-induced oxidative stress rendered HCC cells more sensitive towards Sorafenib, its conventional therapeutic agent. This was highlighted by a dramatic induction of ROS-induced apoptosis in vitro and suppression tumor growth in vivo. NRF2, a transcription factor and master regulator against oxidative stress, was confirmed to bind with TXNRD1 using ChIP assay. Both genetic inhibition and pharmacological activation of NRF2 in HCC cells confirmed the dependent relationship between NRF2 and TXNRD1. Therapeutically, pharmacological inhibition of the thioredoxin system using the TXNRD1 inhibitor auranofin greatly sensitized HCC cells towards sorafenib. The synergism was observed through significant induction of ROS resulting in significant cell death in vitro and suppression of tumor formation in vivo. Our investigation demonstrated the induction of oxidative stress, as a result of thioredoxin system inhibition, rendered HCC cells more sensitive towards therapeutic treatment. Contrasting the widely-known, tumorigenic role of ROS, we now shed light on a paradigm-shifting strategy which ROS may be used as a therapeutic adjuvant to combat cancer.

#2446

1-Palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol (PLAG) attenuates gemcitabine-induced neutrophil extravasation.

Jinseon Jeong,1 Yong Jae Kim,1 Su-Hyun Shin,1 Do Young Lee,2 Byoung-Gon Moon,2 Ki-Young Sohn,2 Sun Young Yoon,2 Jae Wha Kim1. 1 _Korea Research Institute of Bioscience and Biotechnology, Daejeon, Republic of Korea;_ 2 _ENZYCHEM Lifesciences, Jecheon, Republic of Korea_.

Neutrophils are the most abundant leukocyte in circulation and are the first responder to foreign particles and/or injury. Some chemotherapeutics are known to induce depletion of blood neutrophils as its high cytotoxicity. However, it is also possible that circulating neutrophils undergo extravasation in response to chemotactic gradients induced by anticancer agents. Here, we found that 1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol (PLAG) attenuates gemcitabine-induced neutrophil extravasation via the inhibition of neutrophil-attracting chemokine expression in macrophages using in vivo and in vitro approaches. First, we observed that there were increased neutrophils infiltrated into the peritoneal cavity following gemcitabine treatment in 4T1 tumor-bearing mice. In addition, gemcitabine increased MIP-2 mRNA expression in the peritoneal cells as well as in other tissues of the mice. Likewise, gemcitabine induced the migration of circulating neutrophils into the peritoneal cavity in normal mice, and PLAG effectively decreased neutrophil migration by inhibiting the expression of adhesion molecules. Inhibition of CXCR2 by its antagonist, reparixin, abrogated gemcitabine-induced neutrophil migration, indicating that chemokines produced by gemcitabine mainly support neutrophil activation. In vitro experiments demonstrated that PLAG inhibited NADPH oxidase (NOX)-mediated reactive oxygen species production induced by gemcitabine, which is upstream of MIP-2 and/or CXCL8. Importantly, PLAG downregulated gemcitabine-induced membrane translocation of the cytosolic NOX subunit, Rac1, and phosphorylation of p47phox. We also demonstrated that 1-palmitoyl-2-linoleic-3-hydroxyl-rac-glycerol (PLH), the natural form of diacylglycerol, has no effects on gemcitabine-induced CXCL8 production and dHL-60 migration, suggesting that an acetyl group at the third position of the glycerol backbone may have a key role in the regulation of neutrophil activation. Altogether, this study suggests the potential of PLAG as a therapeutic strategy to modulate chemotherapy-induced neutrophil activation for cancer patients undergoing chemotherapeutic treatment.

#2447

Loss of mitochondrial function and increases in levels of oxidative stress pinpointed in human lung cell cultures exposed to particulate matter from September 11, 2001.

Ann Marie DiLorenzo,1 Lara Seder,2 Rebekah Madrid,1 Eric Tobias,1 Jonathan Ortiz,1 Vern Gatson1. 1 _Montclair State Univ., Upper Montclair, NJ;_ 2 _Montclair State Univ., Montclair, NJ_.

Not only did more than 2,753 individuals pass away the morning of September 11,2001, but there have been individuals suffering from mental and physical illness ever since. Thousands of individuals suffered from breathing complications, asthma, and lung cancer. Clinical trials on cancer patients, including a patient who was exposed to the World Trade Center Dust (WTCD), have seen a decrease in the progression of tumor growth with use of oral nicotinamide adenine dinucleotide (NADH). NADH is naturally occurring within a cell's mitochondria and aids in the production of adenosine triphosphate (ATP), the energy of the cell. NADH is an anti-aging, energy enhancing, substance that also reduces fatigue and successfully treats degenerative medical conditions. NADH is becoming a possible treatment for tumor regression but it is still unknown, on a cellular level, why NADH reacts with these cells to prevent further tumor growth. In this study, the amount of NADH produced by normal MRC-5 lung fibroblast cells will be compared to lung cells exposed to World Trade Center Dust (WTCD), using a Promega NAD/NADH-Glo Assay. The same cells will be evaluated to note the decline in Glutathione to determine the amount Reactive Oxidative Species ( ROS) brought on to lung cells by WTCD. ROS can decrease GSH levels by causing oxidative stress that leads to apoptosis. It is important for the cells to maintain high levels of reduced GSH and low levels of oxidized GSH. PROMEGA GSH Glo Glutathione assay will be used to determine if the toxic, mutagenic and apoptotic effects of WTC dust can be shown as the result of oxidative stress. The cells will be exposed to various concentrations of WTCD, ranging from 25-250 ppm. It is hypothesized that the cells with the highest concentrations of this toxic particulate matter will experience the most accumulation of oxidative stress, resulting in less NAD+, being reduced to NADH, within the mitochondria and simultaneous decrease in protective antioxidants as shown in changes in levels of Glutatione. This should allow correlations to be made on a more precise cellular mechanism between exposure to this WTCD, oxidative stress, and loss of mitochondrial function seen in the diseases of the exposed first responder population.

#2448

Chemotherapy drug-induced AXL activation and cell survival signaling via reactive oxygen species that can be inhibited to enhance drug efficacy in mesothelioma.

Derek B. Oien,1 Tamás Garay,2 Sarah Eckstein,3 Jeremy Chien1. 1 _University of New Mexico Comprehensive Cancer Center, Albuquerque, NM;_ 2 _Semmelweis University, Budapest, Hungary;_ 3 _The University of Kansas Medical Center, Kansas City, KS_.

Cellular oxidative stress is characterized by elevated reactive oxygen species (ROS), and is a frequent phenotype of cancer cells. The consequences of ROS are complex, where ROS can promote or inhibit tumorigenesis. In mesothelioma, asbestos exposure to serous membranes induces ROS through iron content and chronic inflammation, and ROS promote cell survival signaling in mesothelioma. On the other hand, a current chemotherapy regime for mesothelioma consisting of a platinum and antifolate agent combination also induce ROS. Mesothelioma is notoriously chemotherapy-resistant, and we propose that ROS induced by cisplatin and pemetrexed may promote cell survival signaling pathways, which ultimately may contribute to chemotherapy resistance. In The Cancer Genome Atlas datasets, we found AXL kinase expression is relatively high in mesothelioma compared to other cancer samples. We showed that ROS induce the phosphorylation of AXL, which was blocked by the selective inhibitor BGB324 in VMC40 and P31 mesothelioma cells. We also showed that cisplatin and pemetrexed induce the phosphorylation of AXL and Akt, which was also blocked by BGB324 as well as by N-acetylcysteine antioxidant. AXL knockdown in these cells enhances sensitivity to cisplatin and pemetrexed. Similarly, AXL inhibitor BGB324 also enhances sensitivity to cisplatin and pemetrexed. Finally, higher synergy was observed when cells were pretreated with BGB324 before adding chemotherapy. These results demonstrate cisplatin and pemetrexed induce ROS that activate AXL, and blocking AXL activation enhances the efficacy of cisplatin and pemetrexed. These results suggest AXL inhibition may represent an effective strategy to enhance the efficacy of chemotherapy in mesothelioma. We further hypothesize AXL signaling may be part of an adaptive response to chemotherapeutic agents, providing transient resistance to cisplatin and pemetrexed that may ultimately contribute to acquired chemotherapy resistance.

#2449

Superoxide regulates protumoral p16-driven senescence in the bone marrow microenvironment of acute myeloid leukemia.

Charlotte Hellmich,1 Amina Abdul-Aziz,1 Christopher Marlein,1 Yu Sun,1 Manar Shafat,1 Angela Collins,2 Rachel Piddock,1 Judith Campisi,3 Kristian Bowles,1 Stuart Rushworth1. 1 _Univ. of East Anglia, Norwich, United Kingdom;_ 2 _Norwich and Norfolk University Hospital, Norwich, United Kingdom;_ 3 _Buck Institute for Research on Aging, Novato, CA_.

Acute myeloid leukemia (AML) is primarily a disease of the elderly and depends on the bone marrow microenvironment. The aging bone marrow accumulates senescent cells, characterized by an irreversible arrest of cell proliferation and the secretion of numerous pro-inflammatory cytokines, chemokines and growth factors, termed the secretary associated senescence profile (SASP). Previously we showed that AML is capable of reprogramming bone marrow stromal cells (BMSC) and induces oxidative stress in the BMSC (via superoxide) to support blast survival and proliferation. Here, we hypothesize that AML derived superoxide induces a senescent aging phenotype in the BMSC, generating a SASP in the BMSC which then feed back to promote AML blast proliferation in the bone marrow niche. We isolated primary AML cells and BMSCs from the bone marrow of patients attending our clinic. To assess senescence of BMSC we measured p16 mRNA and protein levels and senescence associated β-Galactosidase activity. Primary AML was engrafted into NSG mice and CD105+ / CD45- cells (BMSC) were sorted and analysed for SASP. We transplanted syngeneic AML blasts (generated from lineage negative cells transduced with the oncogene MN1) into p16-3MR, WT C57Bl/6 and NOX2 (CYBB) deficient mice. Patient samples were taken in accordance with the Declaration of Helsinki and animal studies performed under approval of the UK Home Office. We report that human AML cells induce SASP in vivo as measured by RT-PCR of sorted BMSC cells from NSG mice engrafted with human AML. Next, we engrafted p16-3MR mice with MN1 cells and flow cytometry analysis of isolated BSMC (CD105+ CD45-) showed AML cells induce micro-environment senescence in vivo. Furthermore, depletion of p16 positive stromal cells through treating MN1 engrafted p16-3MR animals with ganciclovir significantly increased survival of MN1 engrafted mice compared with animals treated with vehicle control. In addition, we show that MN1 AML cells, transplanted into NOX2 deficient mice, generate high levels of superoxide in the bone marrow, which results in oxidative stress in the BMSC. Finally NOX2-KD in MN1 AML reduces BMSC senescence in the BM of p16-3MR recipient mice and results in improved animal survival. Here we show that the AML derived superoxide induces a senescent phenotype in the bone marrow micro-environment which then feeds back to enhance the growth capacity of AML. Targeting the senescent microenvironment may therefore provide novel therapeutic approaches for the treatment of this disease in the future. 

### Noncoding RNAs and Cancer

#2450

The importance of a family of long noncoding RNAs from the Y chromosome in radiotherapy and chemotherapy response of male non-small cell lung cancer.

Tayvia Brownmiller, Jamie Barr, Abby Harold, Erik Bey, Ivan Martinez. _West Virginia University, Morgantown, WV_.

Lung cancer is the number one cause of cancer-related deaths in the United States. The 5-year overall survival rate for patients with non-small cell lung cancer (NSCLC) stands at 18%, with women having a twice better overall survival rate than men. Currently, there is no solid evidence to explain the disparity between gender survival rates. The protein coding genes of the X and Y chromosomes have been relatively well characterized, and previous studies have implicated phenomena such as the dysregulation of X inactivation and the loss of the Y chromosome in a number of cancers. However, the regions of these chromosomes responsible for producing noncoding RNAs (ncRNAs) are not well understood. One such class of ncRNAs are long noncoding RNAs (lncRNAs), which are primarily classified as larger than 200 nucleotides and carry out functions in nearly every cellular process via a variety of mechanisms. In this study, we have identified a novel family of Y chromosome-linked lncRNAs that we hypothesize play a role in NSCLC cell survival. Our preliminary data show a time- and dose-dependent response in expression of the Y chromosome lncRNA family after radiation in radiosensitive NSCLC cell lines but not in radioresistant NSCLC cell lines, and we see a similar trend in response to cisplatin. Additionally, upon knockdown of this family in a sensitive cell line using two different shRNAs, we immediately observed an increase in proliferation of the knockdown cells when compared to shCtrl as well as increased cell viability and resistance to apoptosis in response to radiation, and increased resistance to cisplatin. We plan to further characterize these lncRNAs using methods such as qRT-PCR, virtual Northern blotting, traditional Northern blotting, direct RNA sequencing, cellular fractionation, and fluorescent in situ hybridization and determine their function by shRNA knockdown and subsequent assays to measure cellular processes in response to radiation and chemotherapy. The Identification of a male-specific component of lung cancer would fill a knowledge gap that could provide potential therapeutic targets and new prognostic factors, thereby improving the response rate of men with NSCLC.

#2451

Long noncoding RNA OVAAL promotes melanoma cell proliferation through translational suppression of p27.

YUNA YUAN ZHANG, Xu Guang Yan, Margaret Farrelly, Hamed Yari, Yuchen Feng, Ting La, Hessam Tabatabaee, Lei Jin, Xu Dong Zhang. _The University of Newcastle, Newcastle, Australia_.

Evading apoptosis and limitless replicative potential are two hallmarks of malignant cells. Here we report that melanoma cells surviving the induction of apoptosis by TNF-related apoptosis-inducing ligand (TRAIL) or the Mcl-1 inhibitor UMI-77 have even higher proliferative potential, and that this is associated with the increased expression of the long noncoding RNA (lncRNA) OVAAL which competes with p27 mRNA for binding to polypyrimidine tract binding protein 1 (PTBP1) and thus represses p27 mRNA translation. Prolonged exposure to TRAIL or Mcl-1 resulted in stable melanoma cell populations with increased proliferation activity. Comparative RNA-seq analysis identified OVAAL as the most significantly upregulated lncRNA in melanoma cells resistance to TRAIL and Mcl-1. ShRNA knockdown of OVVAL reversed the increased proliferative potential of resistant cells, and also inhibited proliferation in a subset of parental melanoma cell lines that constitutively expressed relatively high levels of OVAAL. RNA pulldown followed by Mass Spectrometry analysis revealed that among proteins that were physically associated with OVAAL was a protein called PTBP1 that bound to and enhanced the activity of the internal ribosomal entry site (IRES) in the 5' untranslated region (5'-UTR) of p27 mRNA. Indeed, OVAAL knockdown resulted in an increase in the association between PTBP1 and p27 IRES leading to upregulation of the p27 protein, whereas overexpression of OVAAL caused reduction in binding of PTBP1 to the IRES of p27. Furthermore, co-knockdown of PTBP1 abolished upregulation of p27 triggered by OVAAL knockdown. These results suggest that OVAAL competes with p27 mRNA for binding to PTBP1 and thus inhibits p27 expression and promotes cell proliferation. This was substantiated by binding assays using in vitro-synthesized biotin-labelled OVAAL, p27 IRES and purified PTBP1 in a cell-free system. Our results showed that the increased c-Myc activity was responsible for upregulation of OVAAL in melanoma cells selected for resistance to apoptosis induced by TRAIL or UMI-77. Collectively, these results identify the expression of OVAAL as an important mechanism promoting melanoma cell proliferation, with potential implications for targeting OVAAL in combination with apoptosis-inducing therapeutics in the treatment of melanoma.

#2452

**Dysregulated expression of the human long noncoding RNA** GHSROS **may influence prostate cancer progression and resistance to docetaxel.**

Patrick B. Thomas,1 Penny L. Jeffery,1 Gahete D. Manuel,2 Eliza J. Whiteside,3 Michelle Maugham,1 Carina Walpole,1 Jennifer H. Gunter,1 Elizabeth D. Williams,1 Colleen C. Nelson,1 Adrian C. Herington,1 Raul M. Luque,2 Rakesh N. Veedu,4 Lisa K. Chopin,1 Inge Seim1. 1 _QUT, Brisbane, Australia;_ 2 _University of Cordoba, Cordoba, Spain;_ 3 _University of Southern Queensland, Toowoomba, Australia;_ 4 _Murdoch University, Perth, Australia_.

Long noncoding RNAs (lncRNAs) play key regulatory roles in cancer progression and are novel therapeutic targets. We recently discovered the lncRNA gene, GHSROS (GHSR opposite strand), on the antisense DNA strand of the ghrelin receptor gene (GHSR). Here, we studied the expression and function of GHSROS in prostate cancer. Interrogation of microarray and RNA-seq data sets revealed that (similar to other lncRNA oncogenes) GHSROS is actively transcribed, although expressed at very low levels in cancer cell lines and tissues. By quantitative RT-PCR we demonstrate that GHSROS is highly expressed in a subset of high-grade prostate cancers (~11.4%). Moreover, the lncRNA is upregulated in high Gleason-score prostate tumors in two clinical data sets. Forced GHSROS overexpression significantly increased in vitro cell proliferation and migration of PC3, DU145, and LNCaP prostate cancer cell lines (P ≤ 0.05, Student's t-test). Increased cell proliferation observed in GHSROS-overexpressing prostate cancer cell lines was recapitulated in PC3, DU145, and LNCaP prostate cancer xenografts in NOD/SCID mice. Cell survival was significantly increased in GHSROS-overexpressing LNCaP cells treated with the cytotoxic drug docetaxel (P ≤ 0.05, Student's t-test). Docetaxel treatment also increased GHSROS expression in native LNCaP and PC3 cells in a dose-dependent manner (P ≤ 0.05, Student's t-test). These data suggest that GHSROS mediates tumor survival and resistance to docetaxel. To identify fundamental drivers of the observed tumorigenic phenotype of GHSROS-overexpressing cell lines, high-throughput RNA-seq data from in vitro cultured PC3 cells and LNCaP xenografts were examined. A quarter of the genes differentially expressed by GHSROS-overexpressing PC3 cells were also differentially expressed by GHSROS-overexpressing LNCaP xenografts. These 101 genes include several transcription factors with established roles in prostate cancer (including the androgen receptor) and genes associated with metastasis and poor prognosis. Finally, we developed two distinct antisense oligonucleotides (ASOs) targeting GHSROS, achieving >60% knockdown, and their function was assessed in vitro. ASO inhibition of GHSROS expression reciprocally regulated cell growth and migration and the expression of a range of genes. These ASOs are currently being assessed in preclinical animal models. Our findings suggest that the long noncoding RNA GHSROS reprograms prostate cancer cells toward a more aggressive phenotype and that the lncRNA represents a promising therapeutic target.

#2453

Eradication of neuroblastoma by suppressing the expression of a single noncoding RNA.

Andrew E. Tee,1 Pei Y. Liu,1 Giorgio Milazzo,2 Kate M. Hannan,3 Jesper Maag,4 Nenad Bartonicek,4 Renhua Song,5 Chen C. Jiang,6 Xu D. Zhang,6 Murray D. Norris,1 Michelle Haber,1 Glenn M. Marshall,1 Jinyan Li,5 Jo Vandesompele,7 John S. Mattick,4 Pieter Mestdagh,7 Giovanni Perini,2 Ross D. Hannan,3 Marcel E. Dinger,4 Tao Liu1. 1 _Children's Cancer Institute Australia, Sydney, Australia;_ 2 _Department of Pharmacy and Biotechnology, Bologna, Italy;_ 3 _Australian Cancer Research Foundation Department of Cancer Biology and Therapeutics, The Australian National University, Canberra, Australia;_ 4 _Garvan Institute of Medical Research, Sydney, Australia;_ 5 _Advanced Analytics Institute, Sydney, Australia;_ 6 _School of Medicine and Public Health, University of Newcastle, Australia;_ 7 _Center for Medical Genetics Ghent, Ghent, Belgium_.

N-Myc gene amplification occurs in one quarter of human neuroblastoma tissues, and is a marker for poor patient prognosis. We performed RNA sequencing experiments, and identified 5 transcripts, including RP1NB1, which were most considerably differentially expressed between N-Myc gene amplified and nonamplified human neuroblastoma cell lines. Affymetrix microarray studies revealed that DEPD was one of the few genes considerably downregulated in neuroblastoma cells after RP1NB1 depletion. Chromatin immunoprecipitation assays showed that knocking down RP1NB1 expression reduced histone H3 lysine 4 trimethylation, a marker for active gene transcription, at the DEPD gene promoter. Luciferase assays demonstrated that knocking down RP1NB1 decreased DEPD gene promoter activity. Depletion of RP1NB1 or DEPD with two independent siRNAs or shRNAs significantly reduced ERK protein phosphorylation, N-Myc protein phosphorylation at Serine 62, N-Myc protein stabilization, neuroblastoma cell proliferation and survival. Clonogenic assays showed that knocking down RP1NB1 with doxycycline completely abolished colony formation capacity of neuroblastoma cells stably transfected with doxycycline-inducible RP1NB1 shRNAs. Importantly, treatment with doxycycline in mice xenografted with neuroblastoma cells stably transfected with doxycycline-inducible RP1NB1 shRNA led to tumor eradication. In human neuroblastoma tissues from 600 neuroblastoma patients, high levels of RP1NB1 gene expression correlated with DEPD gene expression and poor patient prognosis. In conclusion, this study identifies the novel long noncoding RNA RP1NB1 as an important regulator of N-Myc protein stability and neuroblastoma tumorigenesis.

#2454

Identification of a novel lncRNA family that is required for efficient cellular transformation by Bcr-Abl oncogene.

Jilong Chen,1 Xuefei Wang2. 1 _Fujian Agriculture and Forestry University, Fuzhou, China;_ 2 _Chinese Academy of Sciences, Beijing, China_.

Human genome transcribes abundant long noncoding RNAs (lncRNAs) that are defined as the transcripts longer than 200 nt without protein-coding capacity. Recently, lncRNAs are being continuously recognized as critical regulators for various biological processes. Aberrant expression of lncRNAs has been shown to be a cornerstone in diverse human diseases and cancer development. Chronic myeloid leukemia (CML) is a hematological malignancy associated with a reciprocal translocation between chromosome 9 and 22 that generates the chimeric Bcr-Abl oncogene. About 10% of acute lymphoblastic leukemia (ALL) patients also contain Bcr-Abl oncogene. However, the functional relevance of lncRNAs in Bcr-Abl-mediated leukemia remains obscure. In this study, we perform a comprehensive analysis of lncRNAs in Bcr-Abl-positive K562 cells using a microarray of cDNAs encoding lncRNAs. This analysis revealed 987 upregulated lncRNAs and 1479 downregulated lncRNAs in response to treatment with imatinib, a Bcr-Abl inhibitor. Importantly, we identified a conserved, imatinib-inducible lncRNA (IIR) family, named lncRNA-IIR37. Upregulation of lncRNA-IIR37 has been detected in both human and mouse Abl-transformed cell lines after imatinib treatment. Interestingly, lncRNA-IIR37 levels are significantly lower in leukemic cells derived from Bcr-Abl-positive ALL patients than those in normal control group. Furthermore, altering lncRNA-IIR37 expression remarkably affects survival of Abl-transformed leukemic cells, and tumorigensis induced by leukemic cells in xenograft mouse model. Knockdown of lncRNA-IIR37 in transgenic mice significantly promotes Bcr-Abl-mediated primary bone marrow transformation. These results indicate that lncRNA-IIR37 functions as a suppressor gene in Bcr-Abl-induced tumorigenesis. In addition, we demonstrated that lncRNA-IIR37 directly bound and affected the phosphorylation of STAT5 to regulate leukemic cell survival. Together, our observations suggest that lncRNA-IIR37 is critically involved in Bcr-Abl-mediated leukemogenesis. These results provide novel insights into complicated mechanisms underlying cellular transformation by Bcr-Abl oncogene.

#2455

**The long non-coding RNA (lncRNA)** DANCR **in acute myeloid leukemia (AML) stem cells (LSC).**

Marius Bill,1 Malith Karunasiri,1 Jessica Kohlschmidt,1 Allison E. Walker,1 Zachary Brannan,1 Stefano Volinia,2 Clara D. Bloomfield,1 Ramiro Garzon,1 Adrienne M. Dorrance1. 1 _The Ohio State University, Columbus, OH;_ 2 _University of Ferrara, Ferrara, Italy_.

Introduction: Prognosis for AML patients (pts) is inadequate. Therapeutic strategies are needed to target not only bulk blasts but also the distinct LSCs, which are more resistant to chemotherapy & cause relapse. lncRNAs are non-coding RNA transcripts longer than 200 nucleotides that regulate cellular processes. A functional role in LSCs has yet to be elucidated.

Methods: We derived a LSC-specific lncRNA profile using whole transcriptome sequencing (RNA-seq) data of 377 pts (<60 years) with cytogenetically normal (CN) AML. These data were correlated with a 'core enriched' (CE) gene expression signature representing 44 genes deregulated in LSCs (Eppert, Nat.Med. 2011). DANCR expression in the LSC-enriched compartment was validated using qRT-PCR. Transferrin/antibody conjugated nanoparticle (NP) knock down (KD) was used as an efficient & less toxic avenue to deliver siRNA against DANCR or scramble (SCR) in vitro & in vivo. LSC features (self-renewal, quiescence, engraftment) were analyzed & frequency determined in 3 primary AML samples and a CN-AML (Flt3ITD/WT/MllPTD/WT) mouse model.

Results: 161 lncRNAs were consistently deregulated in CEhigh pts with a p<.001 & a correlation coefficient >.5 (Spearmen correlation test). We chose DANCR to further analyze because it was among the top lncRNAs upregulated in CEhigh pts, is highly conserved between human & mouse, & was previously shown to have a role in promoting stemness in hepatocellular carcinoma (Yuan, Hepatology 2016). We confirmed that DANCR expression was significantly higher in functionally validated LSC enriched populations (p=.05). To assess the role in self-renewal, we KD DANCR in pts cells & found a decrease in colony numbers after replating (average decrease vs SCR: 38.9%, p=.03). Using membrane labeling assays, we found a decrease of quiescent cells after DANCR KD (average decrease: 36.1%, p=.04). Long term colony-initiating cell cultures were performed to determine the LSC frequency & we found significant decreases in the number of LSCs in primary pts samples after DANCR KD (average decrease of LSC frequency: 79.2%, p=.001). Mice transplanted with murine AML cells (CD45.2+) were treated with NP (n=3 per group) for 5 days (d). 2d later, bone marrow was harvested & retransplantated (Tx) using limiting dilutions (2x106, 1x106, 5x105) into irradiated BoyJ (CD45.1+) recipients (n=30). 14d post-Tx, anti-Dancr recipient mice showed a lower engraftment (CD45.2/CD45.1: 37% vs 47%, p=.03) and also a concomitant improvement in survival (p<.001).

Conclusion: We show for the first time that LSCs have a distinct lncRNA profile. Among the highly expressed lncRNAs associated with LSCs we identified & validated DANCR. Our data show that DANCR has an impact on LSC function (self-renewal, quiescence, engraftment) & regulates LSC frequency. DANCR is the first lncRNA shown to have a functional role in LSCs & represents a novel target for AML treatment.

#2456

The PIWI-interacting RNA pathway in prostate is regulated by vitamin D and altered in cancer.

Bethany Baumann,1 Shang Gao,1 Giovanni Lugli,2 Larisa Nonn1. 1 _University of Illinois at Chicago, Chicago, IL;_ 2 _bioMerieux, Marcy-l'Étoile, France_.

Low serum vitamin D is a risk factor for aggressive prostate cancer. piRNAs and their interacting partners, PIWI-like (PIWIL) proteins, are aberrantly expressed in many cancers, but few studies have focused on piRNAs in the prostate. We used laser-capture microdissected (LCM) human prostate epithelium from frozen samples collected for a clinical trial of high dose vitamin D to examine the small non-coding RNA population by next-generation sequencing. The 8 patients for this study were selected for having either high (avg=48.7pg/ml) or low (avg=21.4pg/ml) prostatic vitamin D (1,25(OH)2D) levels. VDR ChIP-seq was performed as well, using human primary epithelial cells treated for 3hr with EtOH or 50nM vitamin D. In LCM-collected prostate epithelium unexpectedly piRNAs represented ~20% of the mapped reads. Of all the small RNA species investigated, piRNAs were the most affected by vitamin D status, increasing in abundance in tissues wish high vitamin D (p=0.036, t-test). The LCM data were compared to small-RNA sequencing data from benign prostate available from The Cancer Genome Atlas (TCGA), which represent whole prostate tissue and may contain stromal bias. The LCM-collected samples had a distinct, more consistent profile of small RNAs than TCGA samples. Two unambiguous piRNAs that were highly expressed in LCM data, but not in TCGA data, were verified as epithelial-specific in prostate by in situ hybridization. Several piRNA-biogenesis genes lie within 100kb of VDR ChIP peaks, including TDRKH, which was found to be vitamin D-regulated experimentally. The expression of piRNA-interacting PIWIL proteins was assessed by RT-qPCR, immunoblot and immunofluorescence on a tissue microarray containing paired benign and cancer tissues (N=23). Of the 4 human PIWIL proteins, PIWIL1, 2 and 4 were present in normal prostate epithelial cells by PCR. PIWIL1 protein was also detected in PrE and RWPE-1 benign cells, but PIWIL1 was not detected in the PC3 prostate cancer cell line. By immunofluorescence, PIWIL1 protein in prostate epithelium significantly decreased in prostate cancer (p=0.016, paired t-test). In conclusion, vitamin D status associated with the expression of piRNA in prostate epithelium in clinical trial samples. PIWIL1 protein levels were lower in prostate cancer, suggesting a pro-differentiation role for the PIWI pathway that may be affected in vitamin D-deficient patients.

#2457

Epithelial-to-mesenchymal transition and stemness: Key targets of HOTAIR-miR-203 interaction in renal cell carcinoma.

Pritha Dasgupta, Priyanka Kulkarni, Shahana Majid, Varahram Shahryari, Yutaka Hashimoto, Nadeem S. Bhat, Marisa Shiina, Guoren Deng, Sharanjot Saini, Soichiro Yamamura Yamamura, Yuichiro Tanaka, Rajvir Dahiya. _University of California San Francisco, San Francisco, CA_.

Background: Tumor metastasis and recurrence are major obstacles in renal cell carcinoma (RCC) treatment. RCC aggressiveness is highly associated with stemness and epithelial-to-mesenchymal transition (EMT) which involves increased cellular migration, invasion, inhibition of apoptosis and senescence. HOX transcript antisense RNA (HOTAIR), a lncRNA, is reported to be over-expressed, whereas miR-203 has low expression and is a tumor suppressor in various cancers. However, their association and role in RCC is not well understood. The aim of the present study was to examine the interaction of HOTAIR and miR-203 in the regulation of stemness and EMT in RCC.

Experimental Procedure: ACHN, Caki-1 (human RCC cell lines), normal renal epithelial cells HK-2 and clinical specimens were used for this study. Profiling of HOTAIR and miR203 expression were done by quantitative real-time PCR and luciferase assay was performed to confirm their interaction. Attenuation of HOTAIR (25nM siRNA) and overexpression of miR-203 (10nM mimic) for 72 hours were used for functional studies. The biological role of miR-203 and its interaction with HOTAIR was also investigated using nude mouse models.

Results: HOTAIR was observed to be overexpressed in RCC cell-lines and clinical specimens whereas, miR-203 was significantly under-expressed when compared to normal cells and tissues. Overexpression of miR-203 altered the cell cycle, inhibited epithelial-to-mesenchymal transition (EMT) and decreased cell proliferation along with induction of epithelial marker proteins and decrease in mesenchymal and stemness marker proteins. Conversely, knockdown of miR-203 in non-malignant HK-2 cells induced pro-cancerous characteristics. Direct binding of miR-203 to HOTAIR was shown by RNA immunoprecipitation and luciferase assay. Attenuation of HOTAIR expression reduced cell migration, invasion, and induced apoptosis, mimicking the effects of miR-203 overexpression. Administration of miR-203 mimic to established tumors in nude mice significantly suppressed tumor growth compared to controls.

Conclusion: Our results show that HOTAIR-miR-203 interaction inhibits EMT and stemness to regulate RCC progression. Statistical analysis of the clinicopathological data from kidney cancer patients suggests that HOTAIR and miR-203 may be useful in RCC diagnostics and therapeutics.

#2458

**The androgen receptor-regulated lncRNA landscape reveals a role for** ARlnc1 **in prostate cancer progression.**

Yajia Zhang,1 Sethuramasundaram Pitchiaya,1 Marcin Cieślik,1 Yashar S. Niknafs,1 Jean C. Tien,1 Yasuyuki Hosono,1 Lisha Wang,1 Yuanyuan Qiao,1 Xuhong Cao,1 Mats Ljungman,1 Hui Jiang,1 Rohit Mehra,1 Shuling Guo,2 Rohit Malik,3 Arul M. Chinnaiyan1. 1 _University of Michigan, Ann Arbor, MI;_ 2 _Ionis Pharmaceuticals, Carlsbad, CA;_ 3 _Bristol-Myers Squibb Co., Princeton, NJ_.

The androgen receptor (AR) signaling plays a key role in the development of the normal prostate as well as prostate cancer. While substantial efforts have been undertaken to study AR-regulated protein-coding genes in primary prostate cancer and castration-resistant prostate cancer, few studies have investigated the role of long noncoding RNAs. In this study, we employed transcriptome sequencing to delineate long noncoding RNAs (lncRNAs) associated with AR signaling in prostate cancer progression. ARlnc1 (AR-regulated lncRNA 1) was identified as being the top AR-induced, cancer-associated lncRNA in an integrative analysis of prostate cancer cell lines and tissues. Not only was ARlnc1 induced by AR, but ARlnc1 also was shown to sustain AR signaling by stabilizing the AR transcript via interaction with the AR 3' UTR. Knockdown of ARlnc1 suppressed AR expression, global AR signaling, and prostate cancer growth in vitro and in vivo. Taken together, these data support a role for ARlnc1 in maintaining a positive feedback loop that potentiates AR signaling during prostate cancer progression, and identifies ARlnc1 as a novel therapeutic target.

#2459

Clinicopathological subgroups of glioblastoma patients are characterized by specific lncRNA expression patterns.

Marek Vecera,1 Romana Butova,1 Radim Lipina,2 Stefan Reguli,2 Martin Smrcka,3 Radim Jancalek,4 Michal Filip,5 Marketa Hermanova,6 Leos Kren,7 Pavol Mojak,5 Jaroslav Juracek,1 Tana Machackova,1 Natalia A. Gablo,1 Jiri Sana,1 Ondrej Slaby1. 1 _CEITEC - Central European Institute of Technology, Masaryk University, Brno, Czech Republic;_ 2 _Department of Neurosurgery, University Hospital Ostrava, Ostrava, Czech Republic;_ 3 _Department of Neurosurgery, University Hospital Brno, Faculty of Medicine, Masaryk University, Brno, Czech Republic;_ 4 _Department of Neurosurgery, St. Anne's University Hospital, Brno, Czech Republic;_ 5 _Department of Neurosurgery, Tomas Bata Regional Hospital, Zlin, Czech Republic;_ 6 _First Department of Pathological Anatomy, St. Anne's University Hospital, Brno, Czech Republic;_ 7 _Department of Pathology, University Hospital Brno, Faculty of Medicine, Masaryk University, Brno, Czech Republic_.

Introduction: Glioblastoma (GBM) is the most frequent primary brain tumor of astrocytic origin. The prognosis is unfavourable with the median overall survival (OS) being between 12 and 15 months from diagnosis. Identification of new therapeutic targets, as well as new prognostic and predictive biomarkers for more accurate stratification of patients presents significant unmet medical needs. Long non-coding RNAs (lncRNAs) are regulators of gene expression playing important roles in the molecular pathology of GBM, indicating their potential as biomarkers and therapeutics targets.

Material and Methods: Our study included 80 GBM patients treated with Stupp protocol and 16 patients with non-dominant anterior temporal cortexes resected during surgery for intractable epilepsy. Informed consent approved by the local Ethical Commission was obtained from each patient. IDH1 mutations and MGMT methylation status were evaluated in all GBMs. RNA (RIN > 8) from 96 specimens was used for next-generation RNA sequencing (RNAseq). rRNA depletion and cDNA library preparation were performed with RiboCop rRNA Depletion Kit V1.2 (Lexogen) and NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB), respectively. RNAseq was done using NextSeq 500 High Output Kit and NextSeq 500 instrument (both Illumina). 24,087 protein-coding and 8,414 non-coding RNAs and their sequential variants with non-zero RPKM at least in one sample underwent statistical evaluation. CLC genomic workbench was used for the alignment and target counts.

Results: Statistical analysis revealed 84 (P < 0.001) dysregulated lncRNAs in GBMs compared to non-tumor brain tissue samples. The results also showed 485 dysregulated protein-coding RNAs with P < 0.001 and 24 protein-coding RNAs with P < 0.000001. 35 lncRNAs showed significant dysregulation when lncRNA profiles of GBM tissues with methylated MGMT promoter (≥ 25% methylation) were compared to those with unmethylated promoter (P < 0.01). When lncRNA patterns of GBM samples with mutated IDH1 were compared to those with wild-type IDH1, 60 lncRNAs were found to be significantly dysregulated (P < 0.001). Correlating lncRNA expression patterns with OS uncovered 6 lncRNA signature which enabled identifying patients with significantly worse prognosis (OS < 6 months).

Conclusion: We described significant dysregulation of lncRNAs and protein-coding RNAs in GBM tissue compared to non-tumor brain tissue and specific lncRNA patterns linked to MGMT methylation and IDH1 mutation status. We also identified 6 lncRNA signature allowing sensitive prognostic stratification of GBM patients. Our study indicates that lncRNAs could serve as promising diagnostic and prognostic biomarkers in GBM. This work was supported by Ministry of Health of the Czech Republic, grant nr. 15-33158A, 15-34553A, 15-31627A, 15-34678A, 16-31314A, 16-31765A and by grant of Czech Grant Agency nr. 16-18257S.

#2461

Prognostic impact of telomeric repeat containing RNA expression on long-term oncologic outcomes in colorectal cancer.

SungUk Bae, Woon Kyung Jeong, Seong Kyu Bae, Jae-Ho Lee. _Keimyung University Dongsan Medical Center, Daegu, Republic of Korea_.

Purpose: Telomere shortening is associated with colorectal carcinogenesis and transcription from subtelomeres produces telomeric repeat containing RNA (TERRA), a natural inhibitor of telomerase activity (TA). Previous studies on TERRA expression demonstrated downregulation in advanced stages of human tumors.

Materials and Methods: We evaluated the 2p TERRA expression and telomere length using quantitative real-time PCR in 60 patients who underwent surgical resection for colorectal cancer between June 2008 and November 2010.

Results: Patients were grouped according to 2p TERRA expression, with 27 (45%) and 33 (55%) in the low and high TERRA expression groups, respectively. The median follow-up period was 80 months (range 2-103). 2p TERRA expression was marginally significantly associated with preoperative CEA (P = 0.082) and significantly associated with telomere length (P < 0.05). Five-year overall survival rates in the high 2p TERRA expression group and the low expression group were 73.6% and 78.2%, respectively (p = 0.322), and five-year disease-free survival rates in the high 2p TERRA expression group and the low expression group were 66.1% and 75.1%, respectively (p = 0.317).

Conclusions: 2p TERRA expression was marginally significantly associated with preoperative CEA and significantly associated with telomere length; however, it was not prognostic on long-term oncologic outcomes in colorectal cancer.

#2462

Epstein-Barr virus-encoded microRNAs regulate the expression of BRCA1 in nasopharyngeal carcinoma.

Ka-Hei Lam,1 Raymond Wai-Ming Lung,1 Pok-Man Hau,1 Kevin Yuk-Lap Yip,1 George Sai-Wah Tsao,2 Ken Hung-On Yu,1 Joanna Hung-Man Tong,1 Kwok-Wai Lo,1 Ka-Fai To1. 1 _The Chinese University of Hong Kong, Hong Kong, Hong Kong;_ 2 _University of Hong Kong, Hong Kong, Hong Kong_.

Nasopharyngeal carcinoma (NPC) is an aggressive epithelial malignancy and is endemic in southern China and Southeast Asia. It is well-known to be associated with Epstein-Barr virus (EBV) infection. The miRNAs derived from EBV BamH1-A rightward transcripts (miR-BARTs) are abundantly expressed in NPC and have been suggested that can contribute to cancer development by regulating several gene expression. In this study, we established comprehensive expression profile of miRNAs in an EBV positive NPC cell line and 5 xenografts by small RNA sequencing. By using the prediction algorithm of miRanda and RNAhybrid, in silico analysis identified that several abundantly expressed miRNAs from viral and cellular genomes could negatively regulate BRCA1 expression via the 3' untranslated region (3'UTR). Notably, the regulatory role of miR-BART17-5p, miR-BART19-3p, miR182-5p on endogenous BRCA1 expression was demonstrated by transient transfection assays in HeLa cells and the direct recognitions were verified by luciferase reporter assays, while the downregulation of BRCA1 in primary NPCs was substantiated in the immunohistochemical (IHC) staining in a cohort of 61 histologically normal nasopharyngeal (NP) and 37 primary NPC cases (p<0.001). Taken togethher, we showed that the highly expressed miRNAs could work co-operatively to modulate BRCA1 activity. It has been reported that BRCA1 deficient tumor cells are defective in repairing the DNA double-strand breaks (DSBs) via error-free homologous recombination (HR). The impaired DSB repair by EBV latent infection may induce genome instability during NPC tumorigenesis and also contribute to sensitizing the tumor cells to ionizing radiation and DNA-damaging agent treatments.

Acknowledgement:

Theme-based Research Scheme (T12-401/13-R) and GRF (14104415, 14138016), Research Grant Council, Hong Kong

#2463

Long noncoding RNAs in tumor responses toward lovastatin.

Entkhab M. Alanisi, Jiuhui Wang, Yande Guo, Daotai Nie. _SIU School of Medicine, Springfield, IL_.

Background: Lovastatin is one of FDA-approved drugs used to treat hypercholesterolemia to reduce cardiovascular diseases. It is a compound of the statin family derived from type of species of Aspergillus terruses. Lovastatin targets hepatocytes and blocks the cholesterol synthesis pathway by inhibiting the rate-limiting enzyme 3-hydroxy-3-methyl glutaryl CoA reductase (HMG-CoA reductase). This leads to decreased production of cholesterol and many other biochemical end products, including ubiquinone and dolichol. These biochemical end products are important for cancer progression. When lovastatin blocks their production, it leads to increased apoptosis of cancer cells. Therefore, lovastatin has promising anticancer activities but the detailed mechanisms remain poorly understood. Long noncoding RNAs are originally thought as nonfunctional. Emerging evidence suggest that lncRNAs have crucial roles in many biologic functions like apoptosis and cell cycle, translation control, epigenetic regulation and splicing regulation. In this study, we set out to identify and determine Lnc-RNAs that can regulate tumor cell responses towards lovastatin treatment.

Methods: PC3, Heya8, HCT116 cells were obtained from ATCC. HCT116 p53-/- cells were gifts from Dr. Yin-Yuan Mo. The cytotoxic effects of lovastatin on those cell lines were evaluated by several approaches like MTS assays and morphologic changes. The qPCR array was used to identify lncRNAs whose levels are changed by lovastatin treatment. The regulation of lncRNA by lovastatin was further confirmed in multiple cell lines.

Results and Conclusion: Lovastatin treatment had cytotoxic effects in multiple cell lines we evaluated. Through profiling array, we have identified NEAT1, MALAT1, and RMST as a responder to lovastatin. The lncRNAs identified had a significant increase in the expression compared to the nontreated cells in a dose- and time-dependent manner. The RMST mRNA expression in the HCT116 p53-/- cell line was about 25-fold more than its level in the HCT116 wild-type cells after treatment of 20 μM lovastatin for 48 hours, suggesting a role of tumor suppressor p53 gene in regulating the RMST expression. We knocked out RMST using CRISPR-Cas9 approach and found that RMST is an important mediator for lovastatin to downregulate mTOR activities and reduce colony formation. Taken together, our data suggest that lncRNAs can be stimulated by lovastatin to elicit cellular responses.

#2464

**Divergent lncRNA** MYMLR **regulates MYC by eliciting DNA looping and promoter-enhancer interaction.**

Taisuke Kajino,1 Teppei Shimamura,1 Shuyi Gong,1 Kiyoshi Yanagisawa,1 Masahiro Nakatochi,2 Sebastian Griesing,1 Yukako Shimada,1 Keiko Kano,1 Motoshi Suzuki,1 Satoru Miyano,3 Takashi Takahashi1. 1 _Nagoya University Graduate School of Medicine, Nagoya, Japan;_ 2 _Center for Advanced Medicine and Clinical Research, Nagoya University Hospital, Nagoya, Japan;_ 3 _Human Genome Center, Institute of Medical Science, University of Tokyo, Tokyo, Japan_.

Long non-coding RNAs (lncRNAs), longer than 200 nucleotides in length, function in a wide range of processes by diverse mechanisms. lncRNAs are attracting research interest in the field of cancer research though their roles in regulation of oncogenes and/or tumor suppressors remain rather elusive. In order to search for MYC-modulating lncRNA, we performed a global search for lncRNAs affecting MYC activity using a systems biology-based approach with a K supercomputer and the GIMLET algorism based on local distance correlations. Consequently, MYMLR was identified and experimentally shown to maintain MYC transcriptional activity despite its low levels of expression and cell cycle progression. A proteomic search for MYMLR-binding proteins identified PCBP2, which transcriptionally regulates MYC expression in cooperation with MYMLR. Mechanistically, MYMLR places a 557-kb upstream enhancer region to make the MYC promoter and enhancer regions reside in the proximity. These findings implicate a crucial role for MYMLR in regulation of the archetypical oncogene MYC, and warrant future studies regarding the involvement of low copy number lncRNAs in regulation of other crucial oncogenes and tumor suppressor genes in cancer development.

#2465

Long non-coding RNA ZNF-366-6 predicts lack of response to neoadjuvant chemoradiotherapy in locally advanced rectal adenocarcinoma and presents meaningful biological associations with potential coding cancer driver transcripts related to gastrointestinal cancer pathways.

Lorenzo Ferrando,1 Gabriella Cirmena,1 Stefano Scabini,2 Federica Grillo,2 Luca Mastracci,2 Edoardo Isnaldi,1 Ciro Marrone,1 Roberto Murialdo,2 David Norman Brown,3 Roberto Fiocca,2 Emanuele Romairone,2 Alberto Ballestrero,2 Gabriele Zoppoli2. 1 _University of Genoa, Genova, Italy;_ 2 _IRCCS - Ospedale Policlinico San Martino, Genova, Italy;_ 3 _Hôpital Universitaire Multidisciplinaire Jules Bordet, Bruxelles, Belgium_.

Standard treatment for clinical stage II/III rectal adenocarcinoma (READ) includes a combination of chemotherapy with pyrimidine analogues, such as capecitabine, and radiation therapy, followed by surgery after 6 to 12 weeks. Currently no predictors of benefit from neoadjuvant chemoradiotherapy (NACT) exist, and transcriptomic studies, prevalently focused on coding transcripts or miRNAs have so far failed to find clinically useful gene signatures. In the present analysis we assessed the expression levels of 8,127 long non-coding RNAs (lncRNAs), poorly studied in the context of READ, to infer whether they could stratify pre-treatment READ samples according to patients' pathological complete response (pCR).

Methods: We collected and analysed with Agilent SurePrint G3 Human v2 8x60K microarrays, a consecutive series of 49 specimens from READ patients undergoing NACT with a combination of capecitabine (825 mg/m2) and radiation (50.4 Gy) cycles for 6 weeks. We performed a regularized variable selection regression ("glmnet" R package) to identify potential lncRNAs predicting pCR. We then analysed, with a bivariate correlation, the top lncRNAs predictors with the expression of 20,560 coding transcripts represented in the microarrays we used, and performed pathway enrichment analysis using Ingenuity® Pathway Analysis (Qiagen, Inc.).

Results: Out of 49 patients for whom we could obtain snap-frozen biopsies with complete clinical and pathological data, we analysed 30 patients, 12 with minor or absent pathological response and 18 with major or complete response to NACT. We identified a signature of 11 lncRNAs able to successfully stratify responder vs. non-responder patients. By investigating the correlation of these 11 lncRNAs with coding transcripts, we identified one of them, lnc-ZNF-366-6, presenting an unusually high correlation with more then 1136 coding genes (FDR < 0.01). By gene enrichment analysis we found an enrichment in cancer-related pathways, especially alterations in solid and colon adenocarcinoma pathways (p < 0.01), as well as mitochondrial dysfunction, oxidative phosphorylation and sirtuin signalling pathways (p < 0.00001), defined by the positively and negatively correlated variables with lnc-ZNF-366-6. Moreover, we identified potential drivers of disease or therapeutic targets, either correlated or anti-correlated with lnc-ZNF-366-6.

Conclusions: LncRNAs have the potential to classify responder versus non-responder READ patients undergoing NACT. Lnc-ZNF-366-6 deserves further experimental validation and functional analyses to clarify its exact contribution to resistance to DNA damaging agents, such as radiation, and to inhibitors of DNA/RNA synthesis, such as capecitabine.

#2466

A comprehensive genome-wide analysis of small noncoding RNAs in T-cell prolymphocytic leukemia.

Leticia G. Leon, Steven C. Koetzier, Fabiënne van Opstal, Joyce Schilperoord-Vermeulen, Anton W. Langerak, Stefan J. Erkeland. _ErasmusMC, Rotterdam, Netherlands_.

T-cell prolymphocytic leukemia (T-PLL) is a rare type of neoplasm with an adverse prognosis due to poor responsiveness to conventional chemotherapies. Accurate and rapid diagnosis of T-PLL is problematic due to the heterogeneous nature of the disease. Therefore, further molecular insights of T-PLL are needed. Most of the T-PLL cases (80%) harbor chromosome 14 inversions and translocations or activating mutations in the JAK-STAT pathway. Small non-coding RNAs (sncRNA) are defined as single stranded RNAs that are smaller than 200nt in length and include e.g. microRNAs (miR), small nucleolar RNA (snoRNA) and PIWI-interacting RNAs (piRNA). To increase the molecular understanding of T-PLL, we performed sncRNA expression profiling with the Illumina platform on a well-characterized panel of 21 T-PLL samples. Notably, detailed molecular, cytogenetic, morphologic and clinical data are available of all T-PLL patients included in this study. Of the selected samples, we performed gene expression profiling by microarrays, cytogenetics, VDJ-recombination and multi-color flow cytometric analysis. In short, we sorted CD4+ and CD8+ naïve (CD45RA+, CD27+), effector (CD45RA+, CD27-) and memory (CD45RO+, CD27+) T-cells, and TCRγδ+ T-cells from Ficoll separated PBMCs of buffy coat blood. Total RNA of leukemic cells isolated from the blood was extracted and cDNA libraries for sequencing sncRNAs were generated. For the annotation of the sequence reads we used the DASHR database of sncRNAs and miRbase database of mature miRs. SnoRNA (16%) and ribosomal (r) RNAs (25%) were the most abundant, whereas miRs were only 12% of the total sncRNA fraction. Next, we performed a correlation study using the expression profiles of the different types of sncRNAs and found a clear separation of T-PLL samples from the normal T-cell fractions when miRNA, piRNA and snoRNA expression profiles were used in the analysis. In addition, based on these data we could already identify potential subgroups of T-PLL, indicating that T-PLL is not a homogeneous disease. Finally, we noted aberrant expression of different types of sncRNAs compared to normal T-cell fractions, which may be indicative for essential roles of sncRNA species in the pathogenesis of T-PLL. Currently, we are in the process of characterizing sncRNA species that are aberrantly regulated specifically in T-PLL. Finally, we are selecting candidate sncRNAs for further functional characterization and for testing their diagnostic and/or prognostic value.

#2467

Role of PCA3 in prostate cancer cell proliferation and progression.

Bethanie Russel,1 Jiuhui Wang,2 Yuanqin Zhang,2 Daotai Nie2. 1 _University of Illinois at Springfield, Springfield, IL;_ 2 _Southern Illinois Univ. School of Medicine, Springfield, IL_.

Background

PCA3 is a long non-coding RNA used as an important biomarker in early prostate-cancer diagnosis but its biological functions are not understood. The aim of the study is to investigate the potential role of PCA3 in prostate cancer (PCa) initiation, growth and progression.

Methods

Human genomic DNA was used as templates for cloning of PCA3 gene. Primers were designed and polymerase chain reactions were performed. The 3 exons of PCA3 were cloned and confirmed by sequencing. To determine the impact of PCA3 and its individual exon in PCa cell growth and survival, we over-expressed them respectively in PC-3 and DU145 cell lines via transduction with lentiviral vector. qPCR was performed to examine the expression level of PCA3. To investigate the effects of PCA3 on prostate cancer cells, cell proliferation assay was performed by using trepan blue staining. Colony formation assays were performed to measure the clonogenic capacity of the cells after PCA3 overexpression and treatments with docetaxel.

Results and conclusions

Sequencing of PCA3 gene amplified from human tumor cells did not find mutations when compared to RefSeq. We overexpressed PCA3 in prostate cancer cells and found that increased PCA3 expression did not have significant impacts on DU145 or PC-3 cell proliferation. However, PCA3 exon 3 did increase the clonogenic capacity of DU145 cells when compared to the controls. DU145 cells with increased PCA3 exon 3 also presented increased resistance to docetaxel. No significance was observed in clonogenic capacity in transduced PC-3 cells, but PCA3 increased the motility of PC-3 cells. Our studies suggest that PCA3 can modulate prostate cancer cell clonogenic capacity, docetaxel resistance, and motility. Further studies are ongoing to determine the mechanisms involved.

#2468

Increased expression of snoRNA in the tumor vasculature.

Klarke M. Sample,1 Yingjie Nie,1 Roy Bicknell2. 1 _Guizhou Provincial People's Hospital, Guiyang, China;_ 2 _University of Birmingham, Birmingham, United Kingdom_.

The tumor microenvironment has long been known to cause the differential expression of many genes within the endothelial cells that comprise the tumor vasculature. Yet, the differential expression of short nuclear RNA (snoRNA) has not previously been observed within tumor associated endothelial cells. A differential gene expression analysis was conducted on total RNAseq data derived from primary endothelial cell isolates and validated by qPCR. By these means, SNORD75, SNORD76, SCARNA7 and SNORA81 were demonstrated to be expressed at a higher level in liver, lung and colon associated tumor endothelial cells when compared to patient matched endothelial cells from healthy adjacent tissue. This data suggests that these four snoRNA molecules could be contributing to pathological angiogenesis and therefore tumor growth. Further work is currently being conducted to investigate this possibility.

#2469

Coding and non-coding RNA in papillary thyroid cancer - markers for disease progression.

Sina Dadafarin,1 Anvita Gupta,1 Katharine Dermigny,1 Melanie Jones,1 Timmy O'Connell,1 JK Rasamny,1 Nina Suslina,2 Iacob Iacob,2 Monica Schwarcz,,1 Ameet Kamat,1 Cameron Budenz,1 Craig Berzofsky,1 Deya Jourdy,1 Tali Lando,1 Stimson Schantz,2 Sarnath Singh,1 Edward Shin,2 Augustine Moscatello,1 Raj Tiwari,1 Jan Geliebter1. 1 _New York Medical College, Valhalla, NY;_ 2 _New York Eye and Ear Infirmary, New York, NY_.

Thyroid cancer is among the most common endocrine malignancies, with papillary thyroid cancer (PTC) accounting for approximately 80% of new thyroid cancer cases in 2017 (ACS). Despite the high sensitivity (95%) of ultrasound-guided fine needle aspiration biopsies (FNAB), approximately 20% of FNA biopsies are indeterminate, which require resection, despite the fact that about 50% are benign. The outcome is that many patients undergo surgical resection of benign disease resulting in avoidable iatrogenic morbidity, and about $700 million in health care costs. Thus, identifying diagnostic/prognostic molecular signatures of PTC would greatly reduce the number of costly, unnecessary resections following indeterminate biopsies. Following consenting of NYEEI patients, surgery, and diagnosis by the pathologist, RNA was prepared from PTC and matched-normal tissue samples, rRNA eliminated and RNA-Seq performed (100bp, paired-end). STARv2.5.2b/ htseq-countv0.6.1 and DESeq2 were used to align raw sequences, and measure transcript abundance. Preliminary bioinformatics analysis was performed with Advaita's iPathway Software. Over 1500 protein-coding transcripts, and 386 lincRNAs achieved 1.5 fold level differential expression (p= 0.05). Gene Ontology enrichment analysis indicated that locomotion, cell motility, signaling, cell differentiation and cell communication were among the most statistically significantly biological processes altered between PTC and matched-normal tissue. Cytokine, signal transducer, ion channel activity, receptor and growth factor activities were among the most statistically significantly molecular functions altered between PTC and matched, normal tissue. Additionally, Pathway Analysis indicated that cell adhesion molecules, cytokine-cytokine receptor, ECM-receptor, cancer, proteoglycans and Jak-Stat signaling were significantly altered. Linc01614, linc00475, linc01510, RP11-244M2.1, linc00511, linc01314 and linc00973 were the most overexpressed lncRNAs. Patients who had lymph node metastasis showed differential expression compare to patients with localized disease. These same patients had statistically significantly alterations in the negative regulation of adhesion-dependent cell spreading and cell-matrix adhesion, establishment of cell polarity, and protein kinase C signaling according to Gene Ontology enrichment analysis. In addition to capturing protein coding transcripts, our RNA processing technique has enriched our RNASeq database for novel, noncoding RNAs that are known to play critical roles in cell homeostasis. The biological significance of differentially expressed RNA transcripts is currently being investigated. We anticipate that the detailed bioinformatics analysis of our coding and noncoding databases will yield new diagnostic/prognostic biomarkers, and therapeutic targets.

#2470

Identification of long noncoding RNAs regulated by ZEB1 and epithelial-mesenchymal transition in lung cancer cells.

Jeong Seon Kim, Young-Ho Ahn. _College of Medicine, Ewha Womans University, Seoul, Republic of Korea_.

Long non-coding RNA (lncRNA) are known to have various physiological and pathological functions. Some lncRNAs also show differential expression between cancer and normal tissues and play important roles in cancer development. In this study, we tried to identify novel lncRNAs that regulate epithelial-mesenchymal transition (EMT), invasion, tumorigenesis, and metastasis of lung cancer cells. We performed an NGS-based RNA sequencing analysis in murine lung cancer cells overexpressing ZEB1, an EMT-inducing transcription factor. ZEB1 overexpression changed the expression of total 194 lncRNAs: increased 80 and decreased 114 lncRNAs (fold change≥2, p≤0.01). We then selected 32 lncRNAs with high basal levels and confirmed their differential expression by qRT-PCR. In addition, we measured their expression changes after ZEB1-knockout in mesenchymal-like cells, TGFβ-treatment in epithelial-like cells, and ZEB1-overexpression in human lung cancer cells. Through these experiments, we found several lncRNAs regulated by ZEB1 or EMT in lung cancer cells. Expressions of these lncRNAs showed correlation with that of ZEB1 in human lung adenocarcinoma TCGA data. Further investigation will unveil the effects of these lncRNAs on EMT, cancer development, and metastasis of lung cancer cells.

#2471

Clinical significance of noncoding circHIPK3 RNA in human hepatocellular carcinoma.

Gyeonghwa Kim,1 Jun Sik Yoon,2 Se Young Jang,2 Soo Young Park,2 Won Young Tak,2 Young-Oh Kweon,2 Keun Hur1. 1 _Kyungpook National University School of Medicine, Republic of Korea;_ 2 _Kyungpook National University Hospital, Republic of Korea_.

Background and Aim: Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide, with poor prognosis and high risk of recurrence. Previous studies have shown that various microRNAs (miRNAs) are frequently dysregulated in HCC, which contributes to cancer development and progression. Nonetheless, the regulation mechanism of miRNAs is still unclear. Circular noncoding RNA (circRNA) is highly conserved and stable covalently closed RNA circles with gene-regulatory potential. Recently, one abundant circRNA from HIPK3 gene, circHIPK3, has been identified, and its function has been elucidated as multiple miRNAs sponge. Thus, we aimed to investigate the clinical relevance of circHIPK3 in HCC.

Materials and Methods: We analyzed clinical specimens from 152 pairs of HCC and corresponding normal liver (NL) tissues. Total RNAs were isolated from clinical tissues using Qiagen kit. CircHIPK3 expression levels were determined by quantitative real-time PCR (qRT-PCR), and its expression was normalized with GAPDH. In addition, we analyzed the correlation between circHIPK3 expression and various clinicopathologic features of HCC patients.

Results: CircHIPK3 expression was significantly downregulated in HCC tissues compared to corresponding NL tissues (P=0.036). In HCC patients, circHIPK3 expression was strongly suppressed in more advanced tumors (P<0.001). Further correlation analysis showed that circHIPK3 expression was significantly associated with T stage (P<0.001), TNM stage (P<0.001), BCLC stage (P<0.001), and alpha-fetoprotein (AFP) expression (P=0.033).

Conclusions: We have determined clinical significances of circHIPK3 expression in HCC, which may provide clinical evidence for the potential of circHIPK3 as novel markers for diagnosis and predicting prognosis in HCC patients.

#2472

Benchmark of lncRNA quantification in RNA-Seq of cancer samples.

Hong Zheng,1 Mikel Hernaez,2 Kevin Brennan,1 Olivier Gevaert1. 1 _Stanford University, Stanford, CA;_ 2 _University of Illinois at Urbana-Champaign, Champaign and Urbana, IL_.

Introduction

Long non-coding RNAs (lncRNAs) emerge as important regulators of various biological processes. Many lncRNAs with tumor-suppressor or oncogenic functions in cancer have been discovered. While many studies have exploited public resources such as RNA-Seq data in The Cancer Genome Atlas (TCGA) to study lncRNAs in cancer, it is crucial to choose the optimal method for accurate lncRNAs expression quantification. Multiples tools for processing RNA-Seq data have spurred in recent years, however, there is no accepted gold standard pipeline yet for optimal quantification of lncRNAs. Therefore, we aim to evaluate the performance of popular RNA-Seq analysis tools and recommend the best practice for RNA-Seq analysis of lncRNAs.

Methods

In this benchmarking study, we compared the performance of pseudoalignment methods Kallisto and Salmon, and alignment-based methods HTSeq, featureCounts, and RSEM, by applying them to a simulated RNA-Seq dataset with 63 samples, and a pan-cancer RNA-Seq dataset with 210 samples from TCGA. GENCODE release 25 were used as transcriptome reference. All the scripts were put in GitHub (zhengh42/RNASeq_pipeline).

Results

Pseudoalignment methods Kallisto and Salmon detect more lncRNAs than alignment-based methods and correlate highly with simulated ground truth. On the contrary, alignment-based methods tend to underestimate lncRNA expression or even fail to capture lncRNA signal in the ground truth. These underestimated genes include several cancer-relevant lncRNAs such as TERC and ZEB2-AS1.

Besides the high concordance with ground truth, pseudoalignment methods take less CPU time per sample. They are also flexible with both gene-level and transcript-level quantification, while HTSeq and featureCounts are suitable for gene-level, but not transcript-level analysis.

Overall, 10-16% of lncRNAs can be detected in the samples, with antisense and lincRNAs the two most abundant categories. A higher proportion of antisense RNAs are detected than lincRNAs. Moreover, among the expressed lncRNAs, more antisense RNAs are discordant from ground truth (Spearman's correlation less than 0.7) than lincRNAs when measured by alignment-based methods, indicating that antisense RNAs are more susceptible to mis-quantification. In addition, the lncRNAs with fewer transcripts, less than three exons, and lower sequence uniqueness tend to be more discordant.

Finally, incomplete annotation overestimates expression of both lncRNAs and protein-coding genes. Full transcriptome annotation, including both protein coding and noncoding RNAs, greatly improves the specificity of lncRNA expression quantification.

Conclusions

In summary, considering the concordance with ground truth, flexibility with both genes and transcripts analysis, and the running time, pseudoalignment methods Kallisto or Salmon in combination with the full transcriptome annotation is our recommended strategy for RNA-Seq analysis for lncRNAs.

#2473

Association of functional genetic variants of long noncoding RNA HOTAIR with oral cancer susceptibility.

Shun-Fa Yang,1 Shih-Chi Su,2 Chiao-Wen Lin3. 1 _Institute of Medicine, Chung Shan Medical University, Taichung, Taiwan;_ 2 _Whole-Genome Research Core Laboratory of Human Diseases, Chang Gung Memorial Hospital, Keelung, Taiwan;_ 3 _Institute of Oral Sciences, Chung Shan Medical University,, Taichung, Taiwan_.

Genetic and acquired factors are thought to be interrelated and imperative to estimate the risk and prognosis of oral squamous cell carcinoma (OSCC). Polymorphisms in the HOX transcript antisense intergenic RNA (HOTAIR) have been recently linked to the predisposition to diverse malignancies. In the present study, we aimed to evaluate the influences of HOTAIR gene polymorphisms, combined with environmental triggers on the susceptibility to oral tumorigenesis. Four single-nucleotide polymorphisms (SNPs) of the HOTAIR gene, rs920778, rs1899663, rs4759314, and rs12427129 were tested in 1200 normal controls and 907 OSCC patients. We detected a significant association of rs1899663 with the risk of OSCC (AOR, 2.227; 95% CI, 1.197-4.146; p=0.012) after adjustment for three potential confounders, smoking, betel nut chewing, and alcohol consumption. In the further analyses where habitual exposure to each of three environmental factors was individually excluded, we found that, in addition to rs1899663, non-betel nut users who carried the polymorphic allele of rs920778 were more prone to develop OSCC than were those homozygous for wild-type allele (TC: OR, 1.472; 95% CI, 1.069-2.029; p=0.018; TC+CC: OR, 1.448; 95% CI, 1.060-1.977; p=0.020). This genetic association was not seen while comparing subjects who do not smoke or consume alcohol between the case and control cohort. Moreover, as exploring the relationship between HOTAIR gene polymorphisms and clinical status of only OSCC patients who were non-betel nut chewers, besides the association with the advanced clinical stage, rs920778 and rs4759314 were correlated with the development of large-size tumors (OR, 1.891; 95% CI, 1.027-3.484; p=0.04) and increased lymph node metastasis (OR, 4.140; 95% CI, 1.785-9.602; p=0.001), respectively. Further functional assessments link rs920778 to the regulation of HOTAIR expression and epigenetic status. Our results reveal an interactive effect of HOTAIR gene polymorphisms and betel nut chewing on the development and progression of oral cancer.

#2474

Identification of lncRNAs involved in regulation of chromosome instability-associated genes in a prostate cancer model.

Rogelio Montiel Manriquez,1 Cristian Gabriel Oliverio Arriaga Canon,1 Fernanda Cisneros Soberanis,1 Carlo César Cortés,1 Inti Alberto De La Rosa Velazquez,2 Luis Alonso Herrera Montalvo1. 1 _Instituto Nacional de Cancerología, Mexico City, Mexico;_ 2 _Instituto Nacional de Ciencias Médicas y Nutrición, Mexico City, Mexico_.

In this study we aim to identify new lncRNAs, using RNA-seq data, and study whether those transcripts could be involved in chromosomal instability in prostate cancer and propose new possible RNA-based biomarkers. Using RNA-seq data from two prostate cell lines, neoplastic and non-neoplastic, we have identified a new lncRNA adjacent to CEP55, a gene associated with chromosomal instability. This lncRNA, which we have named lncRNA-CEP55, is 1.6 kb long and it is downregulated in the neoplastic cell line; this is also seen in the adjacent coding gene, CEP55, where the expression decreases in the neoplastic line, suggesting an important role of this transcript in carcinogenesis. This new lncRNA could be regulating CEP55 expression in these cell lines. Changes in expression of CEP55 have been associated to chromosomal instability and cancer development in colon cancer cell lines. Another lncRNA with differential expression in both cell lines has been identified, lncRNA-RFC4. This lncRNA has been reported before; it is an annotated gene in the human genome GRCh38/hg38, but it has not been studied in cancer. This lncRNA is highly expressed in the neoplastic cell line whereas its expression is very low in the non-neoplastic cell line. This lncRNA is 4.3 kb away from RFC4 (Replication Factor C Subunit 4), a protein coding gene related to mismatch repair and DNA double-strand break repair, which has been associated with chromosomal instability. lncRNA-RFC4 could be playing an important role in the cis regulation of RFC4 gene. lncRNA-CEP55 and lncRNA-RFC4 are important targets of study because of their differential expression in prostate cell lines (neoplastic and non-neoplastic). These lncRNAs could be playing an essential role in regulating the expression of the adjacent genes related to chromosomal instability, a enabling characteristic that facilitates the acquisition of the hallmarks of cancer.

#2475

Long noncoding RNA NNT-AS1 sponges miR-424 to promote the progression and metastasis of gastric cancer.

Beibei Chen,1 Qingfang Zhao,1 Lulu Guan,1 Huifang Lv,1 Liangyu Bie,1 Jinxi Huang,1 Jitian Li,2 Jianying Zhang,2 Xiaobing Chen1. 1 _The Affiliated Cancer Hospital of Zhengzhou University, Henan Cancer Hospital, Zhengzhou, China;_ 2 _University of Texas at El Paso, El Paso, TX_.

Background: Long noncoding RNAs (lncRNAs) have been illustrated to function as important regulator in carcinogenesis and cancer progression. However, the roles of lncRNA NNT-AS1 in gastric cancer remain unclear. In present study, we investigate the expression level of NNT-AS1 in gastric cancer tissue and the role on gastric cancer cells tumorigenesis.

Methods: NNT-AS1 expression level was measured using RT-PCR. The prognosis and survival rate of gastric cancer patients with high/low expression of NNT-AS1 was tracked and analyzed. Cellular experiments were performed to test the role of NNT-AS1 on gastric cancer phenotype, including proliferation analysis, transwell assay, EMT-related marker blots and xenograft mice assay. Bioinformatics online program and luciferase assay were performed to predict and verify the potential miRNAs targeting NNT-AS1.

Results: NNT-AS1 expression level was significantly upregulated in 55 cases of gastric cancer tissue samples compared with adjacent normal tissue. Besides, the aberrantly enhanced NNT-AS1 expression predicted poor prognosis and lower survival rate. In vitro, NNT-AS1 knockdown suppressed the gastric cancer cells proliferation, invasion and EMT-related marker (N-cadherin and vimentin) expression. In vivo, lentivirus mediated NNT-AS1 silencing decreased the tumor growth. MiR-503 was verified to be the target miRNA of NNT-AS1 using bioinformatics online program and luciferase assay. Furthermore, miR-424 could reverse the role of NNT-AS1 on gastric cancer cells.

Conclusion: In conclusion, our study indicates that NNT-AS1 sponges miR-424 to facilitate gastric cancer cells invasion and metastasis, revealing the oncogenic role of NNT-AS1 on gastric cancer cells.

#2476

Discovery of an androgen-responsive long noncoding RNA that associates with progression of ERG-overexpressing prostate cancers.

Annika Kohvakka,1 Anastasia Shcherban,1 Kati K. Kivinummi,1 Matti Annala,1 Alfonso Urbanucci,2 Matti Nykter,1 Tapio Visakorpi1. 1 _Univ. of Tampere, Tampere, Finland;_ 2 _Univ. of Oslo, Oslo, Norway_.

Androgen receptor (AR) signaling pathway has an important role in the growth and development of normal prostate, but also in tumorigenesis and progression of prostate cancer (PC). Although the mechanisms of AR signaling have been widely studied and utilized for treatment in advanced PC, the exact role of AR in development of primary PC is unclear. Former studies have found that AR cistrome is reprogrammed during tumorigenesis to bind novel genomic loci by master regulators, including the ETS family transcription factor ERG. While many AR-induced target genes are known, the effect of AR signaling on regulation of long noncoding RNAs (lncRNAs) is poorly understood, especially in the context of PC progression. Previously, we discovered multiple novel PC-associated transcripts (PCATs) to be aberrantly expressed in PC. Here, we evaluated the expression of 39 Tampere PCATs (TPCATs) in 87 radical prostatectomy specimens using high-throughput real-time PCR, and studied their association with time to PSA progression after prostatectomy. Six TPCATs were significantly associated with time to PSA progression, and four of them also associated with extracapsular extension. In addition, we assessed the expression of TPCATs in the TCGA prostate adenocarcinoma cohort, and found many to be correlated with ERG expression. Moreover, publicly available AR ChIP-seq data from PC tumors indicated that several ERG-associated TPCATs had AR-binding sites on their promoters, some of which overlapped with ERG binding sites. Most notably, we found one progression-associated TPCAT that was regulated by AR in an androgen-sensitive manner according to AR siRNA knockdown and DHT stimulation experiments in vitro. The same TPCAT was also highly associated with overexpression of ERG, and further validated to be a highly PC-specific lncRNA that was abundantly expressed in primary PCs. Taken together, these findings give more insight into the role of AR cistrome in the regulation of lncRNAs in primary PC, and introduce a potential novel prognostic marker to be used in early detection of aggressive PC.

#2477

Exosomal RNA mediates transformation of nontumorigenic human bronchial lung epithelial cells.

Sarunya Kitdumrongthum, Sean Humphrey, Hana Kubo, Feng Tian, Sarah Allen, Nadia Atallah, Humna Hasan, Andrea L. Kasinski. _Purdue University, West Lafayette, IN_.

Exosomes are nanovesicles with a diameter between 30-150 nm. Exosomes are produced and released by all cell types into body fluids and, thus, play a major role in cell-cell communication through the transfer of proteins, lipids, and nucleic acids into recipient cells. Uptake of bioactive cargo into recipient cells can impart physiologic and biologic changes in the recipient cell. Especially in cancer, exosome released from cancer cells can reprogram cells, driving various hallmarks of cancer, such as proliferation, migration, invasion, and angiogenesis. In this study we evaluated the effects of exosomes, isolated from non-small cell lung cancer (NSCLC) cells, on nontumorigenic human bronchial epithelial cells (HBECs). Firstly, we determined that cancer cells generate more exosomes than nontumorigenic cells and that cancer cell-derived exosomes are readily taken up by nontumorigenic cells. The nontumorigenic cells exposed to NSCLC-derived exosomes gained the ability to migration and invade. In parallel, we observed that NSCLC-derived exosomes promoted changes in epithelial-to-mesenchymal gene profiles, supporting the conclusion that HBECs were becoming transformed. Exosomes were also capable of permeablizing a monolayer of human endothelial cells, suggesting that NSCLC-derived exosomes may disrupt the vasculature enhancing metastatic capabilities. Importantly, the transformative effects of the exosomes were mediated mostly by exosomal RNA. Comparing the RNA profile from a panel of cancerous exosomes to RNAs contained inside of exosomes isolated from nontumorigenic, one long noncoding RNA (lncRNA) was highly elevated. Knockout of this lncRNA greatly diminished the capability of the cancerous exosomes to promote the observed hallmarks of cancer, suggesting that a single lncRNA is responsible for the majority of the tumorigenic capacity of the exosomes. In vivo mouse modeling and patient-based evidence are currently under way to support these cell-based findings.

#2478

miR-182-5p suppresses progression of renal cancer through cell cycle arrest by targeting lncRNA MALAT-1.

Priyanka Kulkarni, Pritha Dasgupta, Shahana Majid, Varahram Shahryari, Marisa Shiina, Yutaka Hashimoto, Nadeem Bhat, Guoren Deng, Sharanjot Saini, Soichiro Yamamura, Yuichiro Tanaka, Rajvir Dahiya. _University of California, San Francisco, San Francisco, CA_.

Background: Renal cell carcinoma (RCC) is a leading cause of death, accounting for nearly 14,000 (~2.4%) deaths in the United States in 2017, with clear cell carcinoma (ccRCC) being the most common histologic subtype. New and precise disease progression biomarkers are needed for early detection and follow-up. Characterization of these new biomarkers offers a new approach for the identification of novel therapeutic targets and drugs for RCC treatment. MicroRNAs (miRNAs) act as either onco-miRs or tumor suppressors in cancer. Here we show a tumor-suppressor role for miR-182-5p in ccRCC and novel regulation of cell proliferation through MALAT-1.

Methods: Profiling of miR-182-5p and MALAT-1 was performed in microdissected renal cancer tissues, matched adjacent normal regions and in human renal cancer cell lines by quantitative real-time PCR. To assess the functional significance of miR-182-5p in RCC, we overexpressed miR-182-5p/control miRNA (miR-CON) in RCC cell lines (ACHN, Caki-1) followed by functional assays. We also examined the therapeutic potential of synthetic miR-182-5p mimics in vivo using a renal cancer xenograft mouse model. We performed luciferase reporter assay and Ago2-RIP assay to investigate the interaction between miR-182-5p and MALAT-1. In addition, we assessed the effects of miR-182-5p overexpression and MALAT-1 downregulation on cell cycle progression in ccRCC cell lines.

Results: Expression analyses in a cohort of renal cancer clinical specimens showed that miR-182-5p expression is frequently downregulated in ccRCC. We observed that overexpression of miR-182-5p inhibited cell proliferation, colony formation, apoptosis and led to G2/M arrest, supporting an antiproliferative role for this microRNA. Overexpression of miR-182-5p led to decreased expression of CDC20 and AURKA, drivers of the cell cycle mitotic phase. Also, overexpression of miR-182-5p directly lowered the expression of MALAT-1 and knockdown of MALAT-1 mimicked the effects of miR-182-5p overexpression. Downregulation of MALAT-1 led to upregulation of p53 that ultimately downregulated CDC20, AURKA. In vivo studies demonstrated that administration of miR-182-5p caused regression of established renal tumor xenografts.

Conclusions: Collectively, these data suggest that miR-182-5p plays a tumor-suppressive role in ccRCC. These findings offer new insight into role of miR-182-5p in the inhibition of ccRCC tumor growth through MALAT-1 downregulation. This study may provide rationale for development of new strategies targeting MALAT-1 through miR-182-5p for treatment of ccRCC.

#2479

**LncRNA** GAS8-AS1 **directs epigenetic modulation of the tumor suppressor** GAS8 **via MLL1/WDR5 to suppress hepatocarcinogenesis.**

Ming Yang. _Shandong Cancer Hospital and Inst., Jinan, China_.

Long noncoding RNAs (lncRNAs) are vital players in hepatocellular carcinoma (HCC). We previously identified a significantly mutated lncRNA GAS8-AS1 locating in intron 2 of GAS8. However, its involvement in HCC is still largely unknown. In this study, we thoroughly functional evaluated its critical role in regulating chromatin dynamics, gene expression, cancer development and sorafenib multi-kinase target therapy in HCC. We for the first time declare that GAS8-AS1 and its host gene GAS8 act as novel HCC tumor suppressors employing a series of in vitro and in vivo assays. Consistent with this notion, a significantly decreased GAS8-AS1 or GAS8 expression exists in HCC tissues and is associated with poor prognosis of HCC patients. Interestingly, lncRNA GAS8-AS1 could evidently promote GAS8 transcription. Although GAS8-AS1 does not impact promoter DNA methylation of GAS8, we successfully annotated two lncRNA interacting proteins, Mixed Lineage Leukemia 1 (MLL1), a histone 3 Lys4 (H3K4) methyltransferase, and its partner WD-40 Repeat Protein 5 (WDR5) based on bioinformatics methodology. Chromatin immunoprecipitation and RNA immunoprecipitation assays elucidate that lncRNA GAS8-AS1 is required to maintain the GAS8 promoter in an open chromatin state. In accord with this, knockdown of GAS8-AS1 results in reduced MLL1/WDR5 binding, decreased H3K4me3 levels, diminished RNA Pol II functions and gene silencing of GAS8. GAS8-AS1 may function as part of a surveillance mechanism that keep activation of GAS8 promoter and transcription, which thus prevents hepatocarcinogenesis. Our results highlight the prevalent involvement of regulatory lncRNAs in cancers and provide pathogenic insights into HCC development and treatment.

### Nuclear Oncoproteins and Tumor Suppressor Genes

#2480

Olig2-dependent reciprocal shift in PDGF and EGF receptor signaling regulates tumor phenotype and mitotic growth in malignant glioma.

Fanghui Lu, Mei Xin, Qing Richard Lu. _Cincinnati Children's Hospital Medical Center, Cincinnati, OH_.

Malignant gliomas exhibit extensive heterogeneity and poor prognosis. Diverse molecular characteristics in gliomas represent a significant hurdle for effective therapy. We uncover that a population of Olig2+ cells function as glioma-propagating cells and is critical for tumor initiation of primary gliomas in mice. Elimination of Olig2+ mitotic progenitors blocks tumor initiation and progression. Intriguingly, deletion of Olig2 resulted in tumors that grow, albeit at a decelerated rate. Genome occupancy and expression profiling analyses reveal that OLIG2 directly activates cell proliferation machinery to promote tumorigenesis. Olig2 deletion causes a tumor phenotypic shift from an oligodendrocyte precursor-correlated proneural toward an astrocyte-associated gene expression pattern, manifest in downregulation of PDGF receptor-alpha and reciprocal upregulation of EGFR. Olig2 deletion further sensitizes glioma cells to EGFR inhibitors and extends animal lifespans. Our observations reveal that OLIG2 is a molecular arbiter of tumor phenotype plasticity that may underlie drug resistance, suggesting new strategies for enhancing EGFR drug sensitivity in glioma treatment.

#2481

Loss of FUBP1 impairs terminal neuronal differentiation and predisposes neural progenitors for transformation.

Inah Hwang,1 Dongqing Cao,2 Do-Yeon Kim,3 Tuo Zhang,1 Jian Hu,4 Yu Yao,2 Jihye Paik1. 1 _Weill Cornell Medicine, New York, NY;_ 2 _Fudan University, Shanghai, China;_ 3 _Kyungpook National University, Daegu, Republic of Korea;_ 4 _MD Anderson Cancer Center, Houston, TX_.

Loss of terminal differentiation capacity through genetic mutations is a selected genetic event in tumorigenesis. 1p19q co-deletion associated recurrent mutations of far upstream element-binding protein 1 (FUBP1) have been reported from genomic analysis of low- grade oligodendroglioma. It is frequently overexpressed in many human cancers and strongly correlated with disease progression. However, left unanswered is how FUBP1 serves uniquely as a tumor suppressor in the central nervous system. Here we show that expression of FUBP1 is dynamically regulated during neurogenesis, marking specific stages of neuronal differentiation. Loss of FUBP1 in neural stem/progenitor cells (NPC) increased the number of cells fail to terminally differentiate. Knockdown of FUBP1 further blocks differentiation and promotes tumorigenesis collaboratively with the expression of neomorphic isocitrate dehydrorgenase 1 mutant IDH1R132H. Mechanistically FUBP1 is necessary for mini exon 8a splicing of neurospecific isoform of histone demethylase LSD1 (nLSD1). It binds to intron 8 of LSD1 together with a neuron-specific splicing complex containing NOVA1 and 2 and works synergistically with SRRM4 for the inclusion of mini exon 8a. The expression level of nLSD1 is decreased upon loss of FUBP1 in neural progenitors, impairing the terminal neuronal differentiation and maturation. Reinforcing nLSD1 expression in FUBP1-downregulated NPC restores terminal differentiation, establishing nLSD1 as an obligatory effector of FUBP1-dependent neuronal differentiation. Together, these findings establish a direct role for FUBP1 in neuronal differentiation and also uncover the mechanism for its tumor suppressor function in the nervous system.

#2482

RanBP17 retards the epithelial-mesenchymal transition (EMT) and malignant progression of glioblastoma cells by regulating the exportation of beta-catenin from cell nucleus.

Yi Wang,1 Penyi Guo,2 Xiaozai Xie,2 Sina Zhang,1 Haitao Yu,2 Lijun Wu,2 Ziyan Chen,2 Gang Chen2. 1 _Department of Preventive Medicine, Wenzhou Medical University, Wenzhou, China;_ 2 _The First Affiliated Hospital of Wenzhou Medical Univ., Wenzhou, China_.

Background: Glioblastoma multiforme (GBM) is the most common and lethal primary human brain tumor and exhibits multiple molecular aberrations. In this study, we attempted to explore the role of a nuclear transport receptor RanBP17 in carcinogenesis and its underlying mechanisms in GBM.

Methods: The relevant information retrieved from the Human Integrated Protein Expression Database (HIPED), TCGA, and Rembrandt was analyzed to evaluate and compare RanBP17 expression in various tissues and cancer types. Next U87 and U251 were used to investigate the effects of RPS15A on epithelial-mesenchymal transition (EMT) and malignant behaviors of glioma cells. Further, RPS15A-associated signaling pathways were screened based on the data from the Kyoto Encyclopedia of Genes and Genomes (KEGG) database and verified by using RT-PCR and Western blot (WB) assays.

Results: RanBP17 was highly expressed in normal brain tissue, and loss or downregulation of RanBP17 was observed in GBM tissue (83/105, 79.05%) and associated with worse survival of patients. Overexpression of RanBP17 suppressed proliferation, migration and invasion of GBM cells in vitro and remarkably inhibited tumor growth in vivo. In addition, RanBP17 silencing promoted EMT of glioma cells via activating the expression of transcriptional factor Snail. Further experiments confirmed that RanBP17 overexpression promoted the export of β-catenin from cell nuclei and retarded wnt/β-catenin signaling. Conversely, knockdown of RanBP17 gene resulted in accumulation of β-catenin in glioblastoma cell nuclei, which, in turn, promoted expression of EMT-related proteins and malignant progression of glioblastoma by combining other transcript genes.

Conclusions: RanBP17 expression is significantly lower in GBM compared to normal brain tissue. Silencing RanBP17 accelerates the EMT process and malignant progression of glioblastoma due to the suppressed export of β-catenin from glioblastoma cell nuclei and the resulting inhibition of wnt/β-catenin signaling. Therefore, loss or downregulation of RanBP17 can be considered a negative prognostic factor for survival of GBM patients.

#2483

Estrogen receptor beta enhances chemotherapy response in GBM.

Mei Zhou, Gangadhara R. Sareddy, Jinyou Liu, Mengxing Li, Suryavathi Viswanadhapalli, Xiaonan Li, Rajeshwar R Tekmal, Andrew Brenner, Ratna K. Vadlamudi. _UT Health Science Ctr. at San Antonio, San Antonio, TX_.

Background: The incidence of developing glioblastoma (GBM) is greater in men than in women, and women of reproductive age have a survival advantage over men and postmenopausal women. These correlative findings suggest that estrogens play a significant role in suppression of GBM, but how they might do so is poorly understood. GBM cells express the estrogen receptor beta (ERβ) that functions as a tumor suppressor. However, the in vivo significance of endogenous ERβ and its role in GBM progression are incompletely understood. The objective of this study is to examine the role of endogenous ERβ in the progression and chemotherapy response of GBM.

Methods: To study the functions of endogenous ERβ in GBM cells, we have generated ERβ knockout (ERβKO) GBM cells using CRISPR/Cas9 system and lentivital-ERβshRNA transfected primary GBM cells (ERβKD). As a second model, we generated multiple ERβ overexpressing GBM model cells (both established and primary GBM cells) using lentiviral transduction. Effect of ERβ-KO, -KD or -overexpression was studied using cell proliferation, invasion and apoptosis using established in vitro assays. Mechanistic studies were conducted using RNA-seq, RT-qPCR and signaling analysis. In vivo role of ERβ was studied using orthotopic models of GBM and mouse survival was determined using Kaplan-Meier survival curves.

Results: ERβKO or ERβKD increased, while ERβ overexpression reduced the proliferation of GBM cells. To determine the effect of ERβKO on mice survival, U251-WT and U251-ERβKO cells were implanted intracranially into immunocompromised mice. Compared to control mice, ERβKO mice had significant reduction in survival. Further, IHC analysis of tumor sections from these mice revealed that ERβKO tumors had more expression of the proliferation marker Ki-67 than the control tumors. RNA-seq analyses using U87WT and U87ERβKO cells revealed that the ERβKO-modulated genes were related to DNA damage response, p53 pathway, NF-κB signaling, JAK-STAT, and mTOR pathways. Further, RNA-seq studies using U87 and U87ERβ overexpression models revealed downregulation of a number of genes involved in DNA repair, DNA damage response (DDR), ATM signaling and cell cycle checkpoint control. Mechanistic studies showed that ERβ modulates expression of genes involved in apoptosis and DDR, thus enhancing the response to chemotherapy drugs. Compared to control, ERβKO cells exhibited resistance to DNA-damaging agents including cisplatin and temozolomide, while ERβ-overexpressing cells were highly sensitive to these DNA-damaging agents. We also validated ERβ sensitization to temozolomide using orthotopic GBM models.

Conclusion: Using ERβ-KO and -KD GBM model cells, we have provided strong evidence demonstrating tumor suppressor and therapy sensitization potential of endogenous ERβ. Our results suggest that upregulation of ERβ expression/functions is an attractive therapy for treating GBM.

#2484

Structural analysis of features contributing to the oncogenic phenotype conferred by the zinc finger nuclear body protein Zc3h8.

John A. Schmidt, Emily R. Duffner, Emily M. Harris, Tyler Doan, Emanuel Irizarry, Keith G. Danielson, Janice E. Knepper. _Villanova Univ., Villanova, PA_.

The zinc finger protein Zc3h8 is overexpressed in many mouse and human solid tumors. We have shown that increased expression of Zc3h8 in cultured mouse mammary cells is associated with increased proliferation rate, increased motility and invasiveness, increased capacity for growth in three dimensions, and increased capacity for tumor formation. Partial knockdown of Zc3h8 expression from mouse mammary tumor cells has the opposite effect. The Zc3h8 protein localizes to both PML bodies and Cajal bodies in the nucleus and features of this localization are altered by treatment of cells with a casein kinase 2 inhibitor, TBB. We have embarked upon an analysis of the contributions of structural features of the Zc3h8 protein through mutagenesis of the gene at key positions. The casein kinase 2 phosphorylation site at position T32 was mutated to either an alanine to prevent phosphorylation, or to glutamic acid to mimic constitutive phosphorylation. Each of the three zinc fingers in the C-terminal region was mutated to replace one cysteine residue. The mutations were introduced into a variant of the zc3h8 gene that has 6 mutations introduced at synonymous positions in order to escape knockdown by RNAi, although the protein sequence generated is identical to the original Zc3h8. Each of the mutant constructs was introduced into mouse mammary tumor cells with knockdown of the endogenous Zc3h8. Mutation of the phosphorylation site T32A led to the formation of fewer, although larger PML bodies, with continued ZC3h8 co-localization. The T32E mutant, with constitutive negative charge at this position led to the formation of a large number of small nuclear foci. Mutant constructs were stably transfected into tumor cells with knockdown of endogenous Zc3h8, and behavior of the mutants was compared to the behavior of cells transfected with the synonymously mutated gene construct. Disruption of each of the zinc fingers individually led to decreased proliferation rate, decreased growth as spheroids or in soft agar, and decreased invasion through Matrigel relative to the synonymous Zc3h8. Tumor cells with Zc3h8 knockdown failed to form tumors in syngeneic BALB/c mice within 10 weeks, while cells transfected with a negative control vector formed tumors in all animals. Rescue of Zc3h8 expression with the synonymous mutant led to tumor formation in 50% of the animals, while no tumors formed from cells transfected with zinc finger mutants. We conclude that the zinc fingers contribute to aggressive tumor cell behavior while the T32 phosphorylation site is essential for proper localization to nuclear bodies.

#2485

CYP24A1-induced vitamin D insufficiency promotes breast cancer growth.

Makoto Osanai, Yusuke Ono, Akira Takasawa, Kumi Takasawa, Masaki Murata, Norimasa Sawada. _Sapporo Medical Univ., Sapporo, Japan_.

Vitamin D plays a critical role in the maintenance of tissue homeostasis by regulating the expression of genes affecting cell proliferation, differentiation, and apoptosis. The vitamin D 24-hydroxylase CYP24A1 functions in vitamin D target tissues to degrade the hormonal form of vitamin D. Existing knowledge regarding dysregulated CYP24A1 expression supports its candidacy as a putative oncogene. In our presentation, we demonstrate that the suppression of constitutive CYP24A1 expression by CYP24A1-specidic shRNA conferred target cells with increased susceptibility to apoptosis and consequently inhibited anchorage-independent growth in breast carcinoma cells. In addition, suppression of vitamin D metabolism following knockdown of CYP24A1 significantly reduced tumor growth in vivo. Since these data provide substantial evidence for a pro-survival and stimulatory oncogenic effect of CYP24A1 in breast carcinoma cells, we will discuss the potential feasibility of CYP24A1-inhibitory cancer therapies.

#2486

PTEN fusion gene promotes growth in cancer.

Yanping Yu, Silvia Chang, Baoguo Ren, George Michalopoulos, George Tseng, Jianhua Luo. _Univ. of Pittsburgh, Pittsburgh, PA_.

Deletion or mutation of PTEN is frequent in a variety of human cancers and is an underlying mechanism for cancer development. Here, we report a PTEN-involved fusion gene and the corresponding genome breakpoint in human cancers. Pten fusion is highly recurrent in cancers and their matched lymph nodes and is located nucleoli. Targeted knockout of this fusion gene in Du145 and MCF7 cells impeded tumor cell growths, retarded S phase entry, and reduced invasiveness, and was prone to the UV- induced apoptosis. Knockout of PTEN-fusion blocked xenografted-tumor growth in mice, while forced-expression of PTEN -fusion through hydrodynamic injection into mice led to hepatocellular carcinoma. These findings indicate that the genome rearrangement of PTEN-fusion results in an oncogenic fusion protein in addition to the loss of Pten as an underlying mechanism in driving cancer development.

#2487

Novel ZNF-fusions in NUT carcinoma.

Hitoshi Shiota,1 Janine E. Elya,1 Kristina Danga,1 Kelly Becht,1 Pauline Chou,2 Artyom A. Alekseyenko,1 Valentina Nardi,3 Christopher A. French1. 1 _Brigham andWomen's Hospital, Harvard Medical School, Boston, MA;_ 2 _Feinberg School of Medicine, Northwestern University, Chicago, IL;_ 3 _Massachusetts General Hospital, Harvard Medical School, Boston, MA_.

INTRODUCTION. NUT Carcinoma (NC) is a rare, aggressive squamous carcinoma characterized by chromosome translocations involving rearrangement of the NUT gene (also known as NUTM1) on chromosome 15. While the BRD4-NUT fusion oncoprotein drives the majority of NCs, approximately 25% of NCs harbor non-BRD4 fusion partner genes, including BRD3-, NSD3-, and ZNF532-NUT. All of these partner genes encode proteins that are key components of the BRD4-NUT oncogenic complex and have led to greater understanding of the oncogenic mechanism of NC. Despite these advances, several NCs harbor NUT-fusions with genes of unknown identity, here termed NUT-variant, offering further opportunity to discover additional NUT-fusion partners that may play a key role in BRD4-NUT complex function.

METHODS. In this study, we identified 7 NCs with NUT-variant fusions. Archer® FusionPlex®, a next generation sequencing-based derivation of rapid amplification of cDNA ends (RACE), was used to identify the NUT-fusions using RNA extracted from formalin-fixed paraffin-embedded (FFPE) archival tumor tissue. Confirmatory studies included RT-PCR, Sanger sequencing, and fluorescence in situ hybridization (FISH).

RESULTS. Of the 7 NCs with unknown NUT- fusion partners, Archer® FusionPlex® identified two tumors with classic BRD4-NUT fusions, two BRD3-NUT, one non-fusion, one technical failure, and one novel ZNF592-NUT fusion. The tumor harboring the ZNF592-NUT fusion was biopsied from a pelvic tumor in an 18 year old female patient. She was initially diagnosed with NC based on positive NUT immunohistochemical staining of her pelvic tumor, and died 13 months later. RT-PCR and Sanger sequencing confirmed the ZNF592-NUT fusion detected in Archer® FusionPlex® analysis. The ZNF592-NUT fusion was further confirmed by DNA-FISH performed on the same tissue. ZNF592 is known to form a "Z4" coregulator complex with ZNF532 (a NUT-fusion partner), ZNF687 and ZMYND8. Interestingly, we have previously reported that all Z4 members co-purify with the BRD4-NUT chromatin complex. Here we show that wild type ZNF532 and wild type ZNF592 co-localize with BRD4-NUT by immunofluorescence in the BRD4-NUT NC cell line TC-797. Furthermore, knockdown of ZNF532 and ZNF592 reduced cell viability/growth of TC-797 cells, indicating these are critical factors for NC growth and/or viability.

CONCLUSION. Here we report, through the identification of a novel NUT-fusion partner, ZNF592, evidence that supports a critical role of Z4 factors in BRD4-NUT complex oncogenic function, providing the opportunity for future mechanistic insights and targeted therapeutic inhibition. Secondly, the findings suggest that Archer® FusionPlex® may in certain circumstances have a greater sensitivity for the detection of NUT-fusions than conventional FISH, and should be considered as an alternative approach for fusion-partner detection and discovery in NC.

#2488

Functional characterization of nuclear TYRO3 in colorectal cancer.

Shaw-Jenq Tsai, Chun-Wei Chien, Sih-Yu Chen, Shao-Chieh Lin, Bo-Wen Lin, Jenq-Chang Lee. _National Cheng Kung University, Tainan, Taiwan_.

Colorectal cancer is one of most common death-related cancers worldwide. Aberrant expression of oncogenes is regarded as a predominant factor in colorectal cancer development. TYRO3 belongs to TAM (TYRO3, AXL, and MER) family, which is a subfamily of receptor tyrosine kinases. Our previous study discovered TYRO3, an oncogene overexpressed in colorectal cancer, promotes cell proliferation, metastasis, and tumor progression. Herein, we further identified that TYRO3 is present in nucleus of colon cancer cells using immunofluorescence staining in conjunction with confocal microscopy. By staining with three different antibodies recognizing N-terminal, central region, and C-terminal of TYRO3, respectively, we identified that central and C-terminal of TYRO3 is the fragment to be translocated to the nucleus. Transfection of exogenous Myc-TYRO3-GFP fusion protein further confirmed this finding. By bioinformatic tool prediction, we found there is a nuclear localization signal (NLS) and a nuclear export signal (NES) in TYRO3. To evaluate the function of nuclear TYRO3, we mutated NLS of TYRO3 and performed several functional studies. Results of immunofluorescent staining showed that after NLS mutation, TYRO3 cannot be translocated to nucleus, which was accompanied by distorted morphology. Furthermore, cancer cells transfected with NLS-mutated TYRO3 increased activated caspase-3 and number of apoptotic cells, suggesting nuclear localization of TYRO3 is essential for cell survival. Clinical data further support that levels of nuclear TYRO3 are a poor prognostic marker that is positively associated with colon cancer treatment failure and malignancy. These data support that nuclear TYRO3 is important for colon cancer cell survival and is a valuable biomarker for prognosis.

#2489

Sensitivity of liver cancer cell lines to B-catenin knock-down correlates with pathway activation.

Zhihu (Jeff) Ding,1 Chaomei Shi,1 Lan Jiang,1 Tatiana Tolstykh,1 Hui Cao,1 Dinesh Bangari,1 Susan Ryan,1 Taiguang Jin,1 Mikhail Levit,1 Karl Mamaat,1 Qunyan Yu,1 Hui Qu,1 Joern Hopke,1 May Cindhuchao,1 Dietmar Hoffmann,1 Fangxian Sun,1 Mike Helms,1 Kerstin Jahn-Hofmann,1 Sabine Scheidler,1 Douglas Fang,2 Liang Schweizer,1 Jack Pollard,1 Christopher Winter,1 Dmitri Wiederschain1. 1 _Sanofi, Cambridge, MA;_ 2 _Wuxi, Cambridge, China_.

Introduction: Hepatocellular carcinoma (HCC) is the 3rd leading cause of cancer-related mortality. Currently there are few treatment options available for advanced HCC patients. Multi-kinase inhibitors sorafenib and regorafenib are the only approved systemic HCC therapies and are only marginally effective in extending survival. HCC is largely driven by difficult-to-drug oncogenes, including β-catenin, and none of the current HCC therapeutics on the market or in development addresses these major genetic drivers of disease. β-catenin is an integral part of the WNT signaling pathway and plays a major role in regulation of cell survival, apoptosis and developmental processes. The goal of this study was to systematically interrogate contribution of β-catenin to HCC pathogenesis.

Methods: The extent of genomic alterations in β-catenin itself or components of its regulatory network was interrogated in two large HCC data sets, The Cancer Genome Atlas and HCC cohort from Asan Medical Center. β-catenin in vitro credentialing (target knock-down, PD modulation and cell growth inhibition) was carried out in cultured HCC cell lines using multiple siRNAs against β-catenin. Stably expressed doxycycline-inducible shRNAs targeting β-catenin were used to establish in vivo dependency in HCC tumor xenografts.

Results: Building on previous observations, we detected high frequency of somatic mutations in CTNNB1/β-catenin itself and in constituent members of its regulatory network in two independent cohorts of HCC patients. Furthermore, significant upregulation in β-catenin protein levels was detected in the overwhelming majority of HCC patient samples, patient-derived xenografts and established cell lines. Using genetic tools validated for target specificity through phenotypic rescue experiments, we discovered that dependency on β-catenin in human HCC cell lines generally tracks with its activation status. HCC cell lines that harbored activating mutations in CTNNB1/β-catenin or displayed elevated levels of non-phosphorylated (active) β-catenin were significantly more sensitive to β-catenin siRNA treatment than cell lines with wild-type CTNNB1/β-catenin and lower active β-catenin. Finally, significant therapeutic benefit of β-catenin knock-down was demonstrated in established HCC tumor xenografts using doxycycline-inducible shRNA system. β-catenin downregulation and tumor growth inhibition was associated with reduction in PD marker AXIN2, and decreased cancer cell proliferation.

Conclusions: β-catenin is an important therapeutic target in HCC that could potentially be targeted using therapeutic siRNA suitably formulated for delivery to human liver tumors. Genomic alterations in CTNNB1/β-catenin, as well as increased abundance of non-phosphorylated (active) β-catenin, may serve as predictive biomarkers for patient selection.

#2490

Characterization of the role of DPPA3 in stemness regulation in hepatocellular carcinoma by DNA methylation.

Qian Yan, Yu Zhang, Dandan Yu, Ngar Woon Kam, Xin-Yuan Guan. _The University of Hong Kong, Hong Kong, Hong Kong_.

Hepatocellular carcinoma (HCC) is one of the most common cancers in the world with a very poor prognosis, which is closely associated with cancer stem cell (CSC). Global RNA-sequencing was used in different developmental stages of hepatocytes, including embryonic stem cell, endoderm, liver progenitor, and premature hepatocyte, as well as two pairs of nontumor and tumor tissues from HCC patients. We found that Developmental Pluripotency Associated 3 (DPPA3) was highest expressed in the stage of liver progenitor cells, and was down-regulated along with development. In HCC patients, DPPA3 was expressed in less than 1% tumor cells, while no staining was detected in normal counterparts. Functional assays revealed that DPPA3 overexpression increased HCC cells proliferation rate, the ability of foci formation and colony formation in soft agar, as well as migration and invasion. Furthermore, DPPA3 significantly increased HCC cells sphere formation frequency and sensitivity to chemotherapeutic agent including cisplatin, 5-Fu, and Sorafenib. In order to investigate the role of DPPA3 in CSC maintenance and stemness regulation in HCC, whole genome methylation sequencing was applied to compare methylation status between DPPA3- and empty vector-transfected cells. Results showed that DPPA3 reduced CG methylation level within functional regions in the genome. Of the 541 differentially methylated regions, the majority of them were involved in developmental process and cell differentiation. Overall, our findings give a novel insight into how methylation regulation controls the fate of cancer stem cells.

#2491

Nuclear exporter protein XPO1 a novel prognostic and therapeutic target in gastric cancer.

Irfana Muqbil,1 Zaid Mahdi,2 Rahman Choudhary,3 Erkan Baloglu,4 William Senapedis,4 Yosef Landesman,4 Sharon Shacham,4 Michael Kauffman,4 Steve Kim,5 Rafic Beydoun,5 Richard N. Berri,6 Anthony Shields,5 Ramzi M. Mohammad,5 Asfar S. Azmi5. 1 _University of Detroit Mercy, Detroit, MI;_ 2 _University of Michigan, Ann Arbor, MI;_ 3 _Wayne State Univ. School of Medicine, DMC Residency Council Board Member, Detroit, MI;_ 4 _Karyopharm Therapeutics Inc, MA;_ 5 _Wayne State Univ. School of Medicine, Detroit, MI;_ 6 _St. John Hospital and Medical Center Van Elslander Cancer Center, Detroit, MI_.

The high mortality rate associated with Gastric Cancer (GC) indicates the urgent need for actionable therapeutic targets. The nuclear exporter protein exportin 1 (XPO1/CRM1) is the exclusive exporter of many tumor suppressor proteins (TPSs) and growth regulators. XPO1 is often over-expressed in different malignancies leading to aberrant cytoplasmic localization of TSPs and subsequent inactivation. A detailed analysis on the correlation of XPO1 with inflammation-metaplasia-dysplasia-carcinoma sequence progression was performed using immunohistochemistry in 70 GC cases: (a) 10 cases from normal gastric mucosa, (b) 10 cases of stomach with intestinal metaplasia with and without inflammation, (c) 10 cases of mucosa with low-grade dysplasia (d) 10 cases of mucosa with high-grade dysplasia, (e) 10 cases of gastric adenocarcinoma and (f) 20 cases of metastatic gastric carcinoma. A correlation between XPO1 expression, the pathological and clinical features of the disease as well as survival were analyzed. Gastric cancer cell lines were exposed to the Selective Inhibitors of Nuclear Export (SINE) compounds (selinexor, KPT-8602, KPT-185, or the natural agent, leptomycin B (LMB), then analyzed using cytotoxicity and molecular assays. In addition, the activity of selinexor was evaluated in a sub-cutaneous xenograft of gastric cancer cell line NCI-N87. XPO1 served as a prognostic marker for poor outcome as positive staining of XPO1 in GC correlated with aggressive behavior of the disease. Targeting XPO1 using SINE compounds or LMB resulted in inhibition of GC cellular growth (IC50<200 nM), induction of apoptosis and suppression of colony formation (p<0.01). Molecular analysis revealed nuclear retention of several important TSPs, induction of pro-apoptotic proteins and suppression of pro-survival factor Bcl-2. Selinexor given orally at doses of 15 mg/kg twice a week for three weeks caused statistically significant reduction of NCI-N87 tumors in mice (p<0.05). Efficacy studies of SINE compounds in patient derived models of GC are ongoing. Our findings strongly demonstrate the potential of XPO1 to serve not only as a prognostic marker but also as a therapeutic marker in GC that warrants further clinical investigations.

#2492

Down-regulation of AR splice variants through XPO1 suppression contributes to the inhibition of prostate cancer progression.

Irfana Muqbil,1 Yiwei Li,2 Erkan Baloglu,3 William Senapedis,3 Yosef Landesman,3 Christian Argueta,3 Michael Kauffman,3 Sharon Friedlander,3 Hua Chang,3 Sharon Shacham,3 Elisabeth Heath,2 Asfar S. Azmi2. 1 _University of Detroit Mercy, Detroit, MI;_ 2 _Wayne State Univ. School of Medicine, Detroit, MI;_ 3 _Karyopharm Therapeutics Inc, Newton, MA_.

Increased XPO1 (a key nuclear exporter of many tumor suppressor proteins [TSPs]) expression in prostate cancer (PCa) has been found to be associated with a high Gleason score and bone metastasis. However, it is unknown whether aberrant XPO1 activity regulates AR and AR splice variants (Arv) signaling in PCa progression. In this study, using 22Rv1 and VCaP prostate cancer cells which harbor full length AR and ARv, we conduct in vitro cellular and molecular biological experiments and in vivo animal studies to elucidate the role XPO1 inhibition plays in PCa progression through regulation of XPO1/AR/ARv signaling. We found that high expression of AR splice variant 7 (AR-v7) was correlated with increased XPO1 expression. Silencing of XPO1 by RNAi or treatment with Selective Inhibitor of Nuclear Export (SINE) compounds (selinexor and KPT-8602/eltanexor) down-regulated the expression of AR, AR-v7 and ARv567es at the mRNA and protein levels. Mechanistic studies showed that XPO1 silencing also inhibited the expression of AR/ARv regulators including FOXA1, Src, Vav3, MED1 and Sam68, leading to the suppression of ARv and AR target genes, UBE2C and PSA. SINE compound treatment of cells retained the eIF4E protein (translation initiation factor and nuclear transporter of capped depended mRNAs) in nuclear compartment, leading to nuclear retention of AR-v7 and PSA mRNAs. The nuclear localization of AR-v7 mRNA resulted in the reduction in the ARv protein. Furthermore, SINE compound treatment retained TSPs including Rb, p21, p53, APC and SMAD4 in the nucleus leading to inhibition of cell proliferation and induction of apoptosis. Moreover, by targeting XPO1/ARv signaling, SINE compounds suppressed prostate cancer growth in vitro and in vivo and potentiated the anti-cancer activity of conventional chemotherapeutic agent docetaxel and anti-AR agents (enzalutamide and abiraterone) through the inhibition of AR, AR-v7, FOXA1, PSA and UBE2C. From these results, we conclude that there is a causal relationship between XPO1 and AR splice variants. High expression of both XPO1 and AR-v7 could lead to constitutively activated AR signaling, CRPC development and progression, and anti-AR drug resistance. By targeting XPO1/AR/ARv signaling and increasing anti-AR sensitivity, SINE compounds could be novel agents used in combination with conventional chemotherapeutics and AR-targeted therapy for the treatment of PCa, especially CRPC.

#2493

Inactivation of ARID1A-SWI/SNF complex alters chromatin compactness at enhancer regions and affects transcription of key tumor signaling circuitry.

Yohan Suryo Rahmanto,1 Xu Shi,2 Wenjing Shen,1 Tsutomu Miyamoto,1 Yu Yu,1 Xi Chen,2 Meng-Horng Lee,3 Vivek Singh,1 Michele I. Vitolo,4 Denis Wirtz,3 Ronny Drapkin,5 Jianhua Xuan,2 Ie-Ming Shih,6 Tian-Li Wang1. 1 _Johns Hopkins University School of Medicine, Baltimore, MD;_ 2 _Virginia Polytechnic Institute and State University, Arlington, VA;_ 3 _Johns Hopkins University, Baltimore, MD;_ 4 _University of Maryland, Baltimore, MD;_ 5 _University of Pennsylvania, Philadelphia, PA;_ 6 _Johns Hopkins Medical Institutions, Baltimore, MD_.

Somatic mutations in ARID1A, a SWI/SNF chromatin remodeling gene, are prevalent in human cancer and represent an emerging mechanism by which a molecular genetic alteration reprograms epigenomes and propels tumorigenesis. Through comprehensive sequencing of chromatin immunoprecipitation and chromatin accessibility, we found chromatin binding regions for ARID1A/BRG1-containing SWI/SNF remodeling complexes, which were enriched at enhancers and corresponded to euchromatin state. ARID1A deletion caused global re-distribution of BRG1-containing complexes in chromatin. Integrative analyses of ATAC-seq, ChIP-seq, and transcriptome data obtained from endometrial epithelium and human endometrioid carcinomas identified high-confidence ARID1A-regulated genes that participate in myriad key tumor and developmental signaling circuitry tissue. Deletion of Arid1a was found to inactivate the TGF-β pathway and to accelerate tumor progression from pre-cancerous lesions to endometrioid carcinomas. This study provides compelling evidence that Arid1a loss is required for developing endometrial cancer from its precursor. Collectively, targeting epigenomic modifications may hold promise for suppressing invasive behaviors of neoplasms carrying ARID1A mutations.

#2494

A novel tool for monitoring endogenous progesterone receptor expression by mCherry using CRISPR-Cas9 genome editing technique.

Yiyang Li, Xiangbing Meng, Kuo-kuang Wen, Meng Wu, Adam Dupuy, Kimberly Leslie, Shujie Yang. _Univ. of Iowa, Iowa City, IA_.

Expression of progesterone receptor (PR) is a favorable prognostic marker for multiple solid tumors. However, PR expression is reduced or lost in malignant tumors which hinders response to progesterone therapy. Thus, monitoring and restoring functional PR expression is of utmost importance to sensitize progesterone therapy in endometrial cancer. We developed a stable cell line expressing the endogenous PR gene tagged a reporter gene (Hygromycin and mCherry) with CRISPR/Cas9-mediated genome editing. This allows efficient monitoring of PR expression in its native epigenetic landscape. The reporter gene expression faithfully reflects and amplifies PR expression following treatment with histone deacetylase inhibitor, romidepsin. This reporter gene transfected cells were successfully used to screen the FDA-approved 1018 library by high throughput screening in searching of potential novel drugs that increase PR expression. This strategy provides an efficient drug screening tool that can be used to screen novel PR inducers in multiple cancer cell lines.

#2495

RUNX1T1 **is amplified in combined small cell lung cancer.**

Tian He, Karen McColl, Alyssa Savadelis, Yanwen Chen, Gary Wildey, Afshin Dowlati. _Case Western Reserve University, Cleveland, OH_.

Small cell lung cancer (SCLC) is a highly aggressive malignancy that expresses neuroendocrine (NE) genes and represents approximately 15% of all lung cancers; the remaining histological subtype is non-small cell lung cancer (NSCLC). Clinically, there are two types of SCLC, termed 'pure' and 'combined'. Combined SCLC presents as a mixture of SCLC and NSCLC components within the same tumor mass. We analyzed 88 SCLC patient tumors by targeted exome sequencing and found that four SCLC patients demonstrated RUNX1T1 amplification. Interestingly, among these 88 SCLC patients there were two with combined SCLC and both demonstrated RUNX1T1 amplification only in the small cell component. Neither MYC (8p24) nor FGFR1 (8p11), two genes commonly amplified in SCLC that are nearby the RUNX1T1 locus (8p22), were co-amplified. RUNX1T1, also called ETO or MTG8, was first identified in the oncogenic fusion transcript AML1/ETO in acute myelogenous leukemia (AML) and was previously reported as a transcriptional co-repressor. Based on our observation we hypothesized that: 1) RUNX1T1 may be a specific marker for SCLC and may play a role in determining the SCLC phenotype, including NE expression, and 2) RUNX1T1 may play a role in transforming NSCLC to SCLC in the early stages of combined SCLC tumor formation. First we examined RUNX1T1 mRNA expression among cancer cell lines using the CCLE database and found that RUNX1T1 is highly expressed in SCLC cells compared to other cancer cells, especially NSCLC cells. This was also true in tumors using RNAseq data. Consistent with these mRNA profiles, our western blot results showed higher RUNX1T1 protein expression in SCLC compared to NSCLC cell lines. These findings validate that RUNX1T1 expression is specifically upregulated in SCLC compared to NSCLC. To investigate a role for RUNX1T1 in determining the SCLC phenotype, we overexpressed RUNX1T1 in SCLC cell lines by lentiviral transduction and observed marked growth suppression in H82, H2171, and H841 cells. Conversely, transient knock down of RUNX1T1 expression in the SCLC cell line H446 using siRNA decreased the expression of two NE proteins (NEUROD1 and TTF1). To explore a potential role of RUNX1T1 in NSCLC to SCLC transformation, we overexpressed RUNX1T1 in three NSCLC cell lines (PC-9, H1650 and A549) but observed no obvious phenotypic changes and no increase in NE gene expression. In conclusion, we have demonstrated that RUNX1T1 expression is upregulated in SCLC compared to NSCLC. Moreover, the results from RUNX1T1 overexpression and knockdown experiments in SCLC cell lines indicates a potential role of RUNX1T1 in regulating the SCLC phenotype and NE expression.

#2496

YY1 regulates the germinal center reaction by inhibiting apoptosis.

Sally E. Trabucco, Hong Zhang. _Univ. of Massachusetts Medical School, Worcester, MA_.

The germinal center (GC) reaction produces high-affinity antibodies for a robust adaptive immune response. If dysregulated, the same processes cause GC B cells to become susceptible to lymphomagenesis. It is important to understand how the GC reaction is regulated. In this study, we show that transcription factor YY1 is required to maintain a robust GC reaction in mice. Selective ablation of YY1 significantly decreased in the frequency and number of GC B cells during the GC reaction. This decrease of GC B cells was accompanied by increased apoptosis in these cells. Further, we found that loss of YY1 disrupted the balance between dark zones and light zones, leading to a preferential decrease in dark zone cells. Collectively, these results indicate that YY1 plays an important role in regulating the balance between dark zone and light zone cells in GCs and between survival and death of GC B cells.

#2497

Inhibitors of RNA Pol I and an aminoacyl-tRNA synthetase result in MYC and MYCN downregulation in MYC family protein-driven neuroblastoma cells.

Naohiko Ikegaki,1 Jasmine Zeki,1 Bill Chiu,1 Hiroyuki Shimada2. 1 _Univ. of Illinois College of Med. at Chicago, Chicago, IL;_ 2 _CHLA, Los Angeles, CA_.

We have previously reported histologic variants among unfavorable histology neuroblastomas (NB), having large cell appearance with vesicular nuclei and or prominent nucleolar (PN) formation, indicating hyperactive rRNA synthesis and protein translation. These features appear to be associated with dismal outcome and high-level MYC family protein expression. PN formation could provide critical prognostic and risk-stratification information for NB. At the same time, this observation could open up an opportunity for innovative therapy for aggressive NB. To assess this possibility, we examined the effect of two potent and specific small-molecule inhibitors of RNA Pol I activity and an aminoacyl tRNA synthetase (CX-5461 and Halofuginone, respectively) on growth and MYC family protein expression in NB cell lines. These small-molecule inhibitors inhibited growth of NB cell lines at low to submicromolar concentrations in vitro in 48 hours. Moreover, the inhibitors destabilized MYC and MYCN proteins in NB cells. Interestingly, halofuginone showed a rapid effect on the stability of MYC family proteins at 500 nM-1 uM (<3 hours). In contrast, CX-5461 at 1 uM was less effective at the 3-hour time. At the concentration of 100 nM-250 nM for halofuginone and 1 uM for CX-5461 for up to 48 hours of the drug treatments, MYC and MYCN expression were both downregulated. We further examined the preclinical efficacy of halofuginone in orthotopic Kelly human NB xenografts in mice using a silk film as the drug delivery device. Halofuginone showed a significant growth-suppressive effect on the xenografts, as indicated by the time periods to reach certain tumor sizes being significantly longer in the halofuginone-treated group than those in the control group. Moreover, histologic examination of the xenografts showed a marked suppression of MYCN protein expression in the halofuginone-treated xenograft at day 17 of the drug treatment. Specific inhibition of hyperactive rRNA synthesis and protein translation was shown to be an effective way to suppress MYC/MYCN protein expression and NB growth. Together, MYC-family protein overexpression and PN formation should be included in new NB risk stratification and considered for potential therapeutic targets.

#2498

Over-expression of dual-specificity phosphatase 4 (DUSP4) in multiple myeloma.

Tian Xia, Kin-Mang Lau, Chi Keung Cheng, Nelson CN Chan, Margaret H. L. Ng. _The Chinese University of Hong Kong, Hong Kong, Hong Kong_.

Super-enhancers are unique genomic domains with dense binding of transcription factors (TFs)/cofactors that drive expression of genes defining cell identity. They also contribute to over-expression of oncogenes. The abundance of multiple TFs and coactivators at the super-enhancers favors their cooperative binding and synergistic gene activation. Thus, super-enhancers are more sensitive to perturbation of TF levels than typical enhancers, providing a therapeutic window for preferential targeting oncogenic expression in cancer cells. Recently, a list of super-enhancer associated genes (SEAG) was identified in a multiple myeloma (MM) cell line MM1S and inhibition of transcriptional coactivator BRD4 by JQ1 selectively repressed MYC oncogene expression through reduction of BRD4 binding at the associated super-enhancer. Significances of SEAG other than MYC in myeloma cells remains unclear. By unsupervised hierarchical clustering analysis of expression of 682 identified SEAG in 74 MM, 28 relapsed MM and 15 normal plasma cells (NPC) (GSE6477), distinct expression pattern of SEAG was associated with MM as compared to NPC, indicating their importance in MM. Among the upregulated genes, DUSP4 (Dual Specificity Phosphatase 4), an inducible phosphatase of MAPK (Mitogen-Activated Protein Kinases), was significantly overexpressed in MM and further upregulated in the relapsed MM. High expression was associated with inferior patient outcomes. The overexpression was confirmed in our patient cohort which includes CD138-immunosorted myeloma cells from 30 untreated and 3 relapsed MM patients, 6 smoldering MM patients and 5 MGUS patients, 5 sorted normal plasma cells (NPC) and 7 MM cell lines. Using the cutoff for over-expression as higher than the mean level of NPC plus 2X Standard Deviation, 10 out of 30 MM and all the 3 relapsed samples showed significant DUSP4 over-expression. Functional significance of DUSP4 in MM was explored by siRNA knockdown and the knockdown inhibited myeloma cell growth, implying an oncogenic role of DUSP4 in MM. To study the significances of super-enhancer in regulation of DUSP4 expression, JQ1 was used and inhibition of BRD4 by JQ1 in MM1S, L363 and OPM2 repressed both DUSP4 expression and MM cell growth. By ChIP-PCR, we demonstrated BRD4 specifically bound onto the proximal region of DUSP4 promoter and the binding was blocked by JQ1. Taken together, the oncogenic role of DUSP4 as SEAG in MM was revealed and the preferential targeting SEAG by BRD4 inhibitor could provide insights into further developments of effective therapeutic approaches for MM patients.

#2499

Identification of functional proteins interacting with NSD2 in multiple myeloma.

Jing Yuan Chooi,1 Zhigang Xie,2 Hannah Lee-Foon Swa,3 Jayantha Gunaratne,3 Dennis Kappei,2 Wee Joo Chng1. 1 _National University of Singapore, Singapore, Singapore;_ 2 _Cancer Science Institute of Singapore, Singapore;_ 3 _Institute of Molecular and Cell Biology, A*STAR, Singapore, Singapore_.

Multiple myeloma (MM), characterized by the uncontrolled proliferation of malignant plasma cells, occurs mainly in the elderly population. Recurrent chromosomal translocations are central to the pathogenesis of MM, with t(4;14) being the second most common and associated with poor prognosis. The histone methyltransferase (HMTase) NSD2 is overexpressed in MM due to the t(4;14) translocation and has been suggested to play an important tumorigenic role in t(4;14) MM. However, the detailed molecular mechanism or signaling pathways describing how deregulation of NDS2 contributes to MM cell growth and survival are still uncertain. In this study, stable isotope labeling of amino acid in cell culture (SILAC)-based mass spectrometry analysis was used to determine NSD2-interacting proteins. Mass spectrometry analysis indicated that there were 75 proteins identified in the immunoprecipitation of NSD2 protein complexes. Gene ontology (GO) enrichment analysis showed that these proteins were enriched in processes related to chromatin organization, RNA processing and translation. Five high potential MMSET-interacting proteins from the most enriched functions were selected and verified by Immunoprecipitation and Western blot assays. The results indicated that there were interactions between NSD2 and SMARCA2, topoisomerase IIa, ADAR1, DDX5 and RPS6. In silico analysis with STRING database showed that SMARCA2 interacts with NSD2. SMARCA2 is the ATPase subunit of the SWI/SNF chromatin remodeling complex, which harnesses the energy from ATP to remodel the position of nucleosomes, making the DNA accessible during transcription, replication and DNA repair. Double immunofluorescence showed NSD2 and SMARCA2 co-localized in the cell nucleus. SMARCA2 was knocked down (KD) by shRNAs in t(4;14) MM cells. CellTiter-Glo Luminescent Cell Viability Assay (CTG assay) indicated that SMARCA2 KD by shRNA reduced t(4;14) MM cell growth. SMARCA2 KD also significantly inhibited the capacity to form colonies in t(4;14) MM cells on methylcellulose colony formation assay. Inhibiting SMARCA2 with active DNA-dependent ATPase A domain inhibitor (ADAADi) also reduced t(4;14) MM cell growth. In addition, SMARCA2 KD reduced cell cycle S phase. Western blot analysis showed downregulation of PRL3 and CCND1 protein level upon NSD2 or SMARCA2 KD. This study reveals a protein-protein interaction between NSD2 and SMARCA2. SMARCA2 KD showed SMARCA2 might be a novel therapeutic target for t(4;14) MM.

#2500

Identification and characterization of a bipartite nuclear localization signal reveals non-canonical 'oncogenic' role for cytoplasm-restricted ARID1B.

Srinivas Animireddy,1 Padmavathi Kavadipula,1 Viswakalyan Kotapalli,1 Swarnalata Gowrishankar,2 Satish Rao,3 Murali D. Bashyam1. 1 _Ctr. for DNA Fingerprinting & Diagnostics, Hyderabad, India; _2 _Apollo Hospitals, Hyderabad, India;_ 3 _Krishna Institute of Medical Sciences, Hyderabad, India_.

The AT-rich interaction domain-containing protein 1B (ARID1B/ BAF250b) is a component of SWItch/Sucrose Non-Fermentable (SWI/SNF) chromatin remodeling complex that functions as the primary chromatin remodeler during ontogeny and adult life. ARID1B is classified as a tumor suppressor in melanoma as well as in cancers of the uterus, ovary, lung, breast, colon and pancreas. Though human ARID1B is a nuclear protein, the molecular basis of its nuclear import is not understood. Using protein truncation analysis, in silico prediction and sequence comparison within forty three metazoan species, we identified a putative bipartite Nuclear localization signal (NLS) sequence, further confirmed by immunofluorescence analyses and cell fractionation followed by immunoblotting. An immunoprecipitation-mass spectrometry based screen for identifying ARID1B interacting proteins revealed members of the importin family (IPO5, KPNA2 and KPNB1). In addition, ARID1B nuclear import was inhibited by Importazole (general importin β inhibitor). Transcription activation function of the cytoplasm-restricted ARID1B-NLS mutant, as measured by quantifying CDKN1A and CDKN1B transcript and protein levels as well as promoter activity following ectopic expression in cancer cell lines, was significantly compromised when compared to wild type ARID1B. The NLS-mutant was also restricted in its ability to induce senescence. Surprisingly however, cytoplasmic localization of ARID1B appeared to result in an 'oncogenic' gain of function as it boosted cell proliferation, promoted ERK signaling and activated β-catenin/TCF-LEF function. A detailed analysis of the oncogenic role of cytoplasm-restricted ARID1B is ongoing. A careful scrutiny of various cancer specific somatic mutation databases revealed several mutations located within the ARID1B NLS sequence. In addition, ARID1B immunohistochemistry on breast cancer tissue microarrays (TMAs) revealed significant cytoplasmic localization. These observations suggest possible clinical relevance of cytoplasm-restricted ARID1B. Functional validation of NLS specific mutations and TMA analysis of other cancers is currently underway. Non-canonical oncogenic gain of function due to aberrant cytoplasmic localization has been shown previously for classical tumor suppressors such as pRB, p27, p21 and p53. Our results have revealed a novel 'oncogenic' facet of ARID1B resulting from its aberrant cytoplasmic localization.

#2501

Involvement of Notch signaling pathway in a panel of human cancer cell lines.

Lucile Astorgues-Xerri,1 Mathieu Martinet,1 Jinan Abdullah,2 Sandrine Faivre,3 Eric Raymond,2 Annemilaï Tijeras-Raballand1. 1 _AFR Oncology, Paris, France;_ 2 _Saint Joseph Hospital, Paris, France;_ 3 _Beaujon University Hospital, Clichy, France_.

Background. Notch pathway has been involved in cell fate determination, cell differentiation, cell proliferation, apoptosis, angiogenesis and drug resistance, as well as epithelial-to-mesenchymal transition (EMT). Our team has demonstrated the involvement of the PlGF/VEGFR1/Notch4 axis in the angiogenesis of HCC, but what about the role of Notch in cancer cells? In this study, we will focus on Notch1 and Notch4 since they have been extensively described in tumor angiogenesis. The aim of this work is to characterize the expression and the role of basal Notch1 and Notch4 activation in a panel of human cancer cell lines.

Materials and Methods. We characterized a panel of 8 pancreatic, 5 cholangiocarcinoma (CK), 5 colorectal, 8 head and neck (H&N), and 5 hepatocellular (HCC) human cancer cell lines for Notch1, Notch4, E-cadherin, and Vimentin expressions by Western Blot. In each cancer type, 2 cell lines were chosen (one each with high and low Notch expression, respectively) to further assess the role of Notch basal expression in cellular properties such as migration, invasion, and vasculogenic mimicry, by using wound-healing test, Boyden chamber assay, and Ibidi plates, respectively. We also studied the effects of Notch inhibition on those cellular activities.

Results. Our first results showed that Notch1 and Notch4 are activated in most of cancer cell lines at basal state. Notch4 is highly activated in 6 out of 8 pancreas cell lines, 7 out of 8 H&N cell lines, 2 out of 3 HCC cell lines, 3 out of 5 CK cell lines, and 2 out of 5 colon cell lines. In contrast, Notch1 is activated in all the CK cells, 3 out of 8 H&N cell lines, and 2 out of 3 HCC cell lines. Interestingly, in pancreatic and HCC cell lines, Notch4 activation is correlated with high expression of Vimentin. These results raised the question of the implication of Notch basal activation in the aggressiveness of tumor cells. Thus, we will further investigate the role of Notch1 and Notch4 in migration and invasion properties of tumor cells. Furthermore, since Notch1 and Notch4 are widely described in angiogenesis, we will also assess the vasculogenic mimicry properties of tumor cell with high versus low basal activation of Notch1 and Notch4. These results will be displayed at the conference.

Conclusions. In this study, we show the high prevalence of Notch1 and Notch4 basal activation in a large panel of cancer cells lines, suggesting its role in cellular activities of cancer cells. Since Notch inhibition is currently an interesting topic for antitumor therapy, this study could help to discriminate the tumor types or tumor characteristics that are good candidate for Notch inhibition in the clinics. 

### Receptors and Growth Factors

#2502

Role of TMEM97/σ2 receptor in breast cancer cell proliferation and responses to chemotherapy.

Yuanqin Zhang, Jiuhui Wang, Daotai Nie. _Southern Illinois Univ. School of Medicine, Springfield, IL_.

Introduction:

Breast cancer is the most prevalent cancer for women both in the developed and the developing countries. In 2017, about 40,610 women are expected to die from breast cancer in the U.S. The sigma receptors (σRs) are transmembrane receptors with two subtypes, termed sigma-1 receptor and sigma-2 receptor. Sigma-2 receptor has been found overexpressed in numerous human cancer tissues, and compound targeting it are now in clinical trials for breast cancer diagnosis. But it's biological role in breast cancer is unknown because the encoded gene has not been clearly identified. Recently, it has been demonstrated that TMEM97, not progesterone Receptor Membrane Component 1 (PGRMC1), is a bona fide sigma-2 receptor. The identification of sigma-2 (σ2) receptor will make it possible to define its biological functions in breast cancer and develop modulator of σ2 receptor as potential cancer therapeutics.

Methods:

The expression of TMEM97/σ2 receptor in multiple cancer cells was evaluated by RT-qPCR. Regulation of P53 on TMEM97/σ2 receptor was determined by Western Blot after 48 hours Doxorubicin treatment of MCF7 cell and HCT116 wild type and P53-/- cells. PB28, an agonist of σ2 receptor and an antagonist of σ1 receptor, was used (0, 1μM, 10μM and 20μM for 4 days) to test its effects on MCF7 cell growth and colony formation. TMEM97/σ2 receptor was cloned and sequenced. We overexpressed TMEM97/σ2 receptor in MCF7 cells to characterize its role in cell proliferation, survival, and colony formation, with or without treatment of its agonist PB28.

Results and conclusion:

TMEM97/σ2 receptor is expressed in various cancer cell lines including lung, breast and prostate cancer, albeit at different levels. TMEM97 expression was reduced in MCF7 cells when p53 expression was stimulated by doxorubicin. PB28 reduced the number of viable cells and also reduced colony formation of MCF7 cells in a dose dependent manner. Meanwhile overexpression of TMEM97/σ2 receptor retarded cell growth in MCF7 cells and sensitized them to PB28 as evidenced by reduced IC50 when compared with control cells. The data suggest that TMEM97/σ2 receptor is expressed in breast cancer cells, with its expression suppressed by p53, and activation of this receptor led to reduced tumor cell proliferation.

#2503

Rational design of small-molecule immune checkpoints' inhibitors.

Khaled Barakat. _University of Alberta, Edmonton, Alberta, Canada_.

Blocking the PD-1/PD-L1 and CTLA-4 pathways recently emerged as a 'game changer' in cancer immunotherapy, leading to the selection of monoclonal-antibodies (MABs) targeting PD-1 as 'drug of the year' for 2013. Although these antibodies restored exhausted T cells' function to recognize and kill tumor cells, these MABs have numerous disadvantages. These include their very high cost and very severe side effects. Our team has been focused on designing small molecule inhibitors for this pathway. Compared to available MAB therapies, our small molecules may offer a more affordable, more easily administered and better controlled treatment for a variety of cancers. Here, we demonstrate our efforts toward this goal and summarize our data on our promising compounds targeting the PD-1/PD-L1 and CTLA-4 pathways.

#2504

Sialylation of EGFR by the glycosyltransferase ST6Gal-I results in receptor activation and resistance to gefitinib mediated apoptosis.

Colleen Britain, Andrew Holdbrooks, Susan Bellis. _Univ. of Alabama at Birmingham, Birmingham, AL_.

The Epidermal Growth Factor Receptor (EGFR) is one of the most commonly altered receptors in cancer. In the present study, we report that sialylation of EGFR by the tumor-associated sialyltransferase ST6Gal-I increases receptor activation resulting in resistance to the EGFR tyrosine kinase inhibitor gefitinib. ST6Gal-I adds an α2-6 sialic acid (a bulky, negatively charged sugar) to select cell surface receptors, thereby modulating receptor function. Previously our group has shown that ST6Gal-I activity confers hallmark cancer stem cell characteristics such as invasion, chemoresistance, and resistance to microenvironmental stressors such as growth factor deprivation and hypoxia. Kinomics data collected for this study reveal that in OV4 ovarian cancer cells, where ST6Gal-I has been overexpressed using a lentiviral construct, ST6Gal-I overexpressing cells have an increase in overall tyrosine kinase activity in comparison to cells without ST6Gal-I expression, whereas the overall serine/threonine kinase activity is largely unaffected by ST6Gal-I expression. Specifically, the receptor tyrosine kinase, EGFR, is one of the most significantly altered kinases in ST6Gal-I expressing cells, with ST6Gal-I expressing cells having an increase in overall activity in comparison to non-expressing cells. Based on these findings, we further interrogated the role of ST6Gal-I on EGFR function. To this end, we first confirmed the sialylation status of EGFR in cells with either forced ST6Gal-I overexpression or shRNA knockdown. Using OV4 and Skov3 cells, ovarian cancer cells, as well as BxPC3 pancreatic cancer cells, we conclude that not only is EGFR a substrate of ST6Gal-I, but that sialylation of EGFR by ST6Gal-I results in basal activation of the receptor, independent of EGF ligand treatment. Further, upon stimulation with EGF, receptor activation is increased in ST6Gal-I expressing versus non-expressing cells. To determine the functional significance of EGFR sialylation by ST6Gal-I, we treated cells with differential ST6Gal-I expression with gefitinib, an EGFR tyrosine kinase inhibitor widely used as an anti-cancer therapy. These studies revealed that ST6Gal-I expressing cells were protected from gefitinib mediated apoptosis. Finally, given that EGFR activation has been shown to promote epithelial to mesenchymal transition (EMT), we evaluated ST6Gal-I expressing cells treated with EGF for the EMT marker Slug. We report that while ST6Gal-I expressing cells inherently have elevated Slug expression versus cells without ST6Gal-I, treatment with EGF greatly enhances Slug expression in ST6Gal-I expressing versus non-expressing cells. Collectively, these results reveal a novel functional role for sialylated EGFR and suggest that the sialylation of EGFR may facilitate EMT as well as provide cells a mechanism to overcome chemoresistance.

#2505

Baicalein reverses chronic stress-induced ovarian cancer promotion through GABA receptors.

Ke Li, Yu Kang, Yinhua Yu. _Fudan University Affiliated Obs & Gyn Hospital, Shanghai, China_.

Studies suggest that chronic stress accelerate the progression of ovarian cancer, and the promotion effects can be abrogated by β-blockers. However, β-blockers should be assessed and tailored to each cancer patient as they will exert toxic effects in overdose. Baicalein, a widely used Chinese herbal medicine, identified as a new type of anticancer drug with an obvious function of anti-anxiety in animal experiments and whose mechanism may be mediated by GABA receptors. The focus of the current study was to determine whether baicalein could block the effects of chronic stress on tumor growth and investigate the underlying mechanism that relates to GABA receptors. Two kinds of ovarian cancer cell lines (OVCAR-8 and CAOV-3) were selected to be investigated in the inhibitory effect of baicalein on cancer cells. Cell CCK-8 assay, wound healing assay, transwell assay and flow cytometry were used for in vitro experiments. Chronic stress was induced by a restrained stress model in nude mice carrying xenografts in the presence and absence of treatment with baicalein. Xenograft sizes and weight were measured after 4 weeks. Norepinephrine was measured by HPLC and GABA was measured by immunoassays in blood and tumor tissues. Expression of GABA receptors in the xenografts was analyzed by real-time quantitative PCR, western blotting and immunohistochemistry (IHC). Our results showed that ovarian cancer cell lines exhibited significant promotion in the presence of NE concentrations of 10Μm and baicalein concentrations of 10μM could reverse its effects by Cell CCK-8 assay and wound healing assay. Daily restraint stress resulted in significantly increased tumor growth in tumor model of ovarian cancer in nude mice. And this increase was completely blocked with daily baicalein treatment. Chronic stress attenuated expression of GABA receptors in vivo and in vitro; conversely, baicalein increased expression of GABA receptors. Overall, our data suggest that baicalein stimulated GABA receptors activation to reduce tumor growth in ovarian cancer. Considering the stimulatory effects of chronic stress on cancer growth, our study implicates baicalein as a potential medicine for blocking the deleterious effects of chronic stress.

#2506

Flotillin 2 negatively regulates EGFR phosphorylation and Cbl-mediated ubiquitination and degradation.

Mariya Liyasova, Xu Zhang, Udayan Guha, Stanley Lipkowitz. _National Cancer Inst., Bethesda, MD_.

EGF receptor (EGFR) signaling is dysregulated by gene amplification or activating mutations of the EGFR in a variety of human malignancies. Activation of the EGFR by EGF leads to recruitment of the ubiquitin ligase Cbl, which ubiquitinates EGFR targeting it for degradation and acts as an adaptor, recruiting additional proteins required for EGFR internalization and trafficking to the lysosome. However, the detailed molecular mechanisms remain elusive. Complete characterization of proteins involved in EGFR trafficking and degradation may uncover additional mechanisms of EGFR dysregulation in cancer. To characterize the EGFR degradative pathway, we investigated protein complexes formed on Cbl with Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) mass spectrometry. By labeling proteins with stable isotopes in culture, we compared complexes in 3 groups: 1) complexes formed on Cbl without EGF stimulation (control), 2) complexes formed on Cbl with EGF stimulation for 30 min and 3) complexes formed on Cbl with EGF stimulation in the presence of YM201636, a trafficking inhibitor, which was added to prevent degradation of the Cbl/EGFR complex and to enrich it for other proteins important for EGFR trafficking. This approach identified over a hundred novel Cbl interactors, whose abundance in the complex with Cbl changed significantly with the treatments. Fifty of these proteins were selected for RNAi knockdown to examine their effects on EGFR degradation. We found that knockdown of Flotillin 2 (FLOT2) protein, a major scaffold protein within caveolar lipid rafts, led to increased phosphorylation and accelerated degradation of EGFR upon EGF stimulation compared to negative control siRNA in HeLa cells. Conversely, overexpression of FLOT2 in HEK293T inhibited EGFR phosphorylation and formation of the EGFR/Cbl complex upon EGF stimulation and led to decreased EGFR ubiquitination and degradation. CRISPR knockout of FLOT2 in HeLa cells led to decreased steady-state levels of EGFR, increased phosphorylation of EGFR and ERK1/2 compared to non-targeting CRISPR control. Stable overexpression of FLOT2 in HeLa cells increased steady-state levels of EGFR, but decreased EGFR phosphorylation and ubiquitination upon EGF stimulation. FLOT2 is frequently amplified and overexpressed in breast, endometrial and lung cancers. In a lung adenocarcinoma cell line H441, in which FLOT2 gene is amplified, knockdown of FLOT2 increased EGFR phosphorylation and ubiquitination, as well as EGFR signaling, as detected by the phosphorylation of ERK1/2. Our data indicate that FLOT2 negatively regulates EGFR phosphorylation, ubiquitination and degradation. Although it is not clear how FLOT2 amplification could contribute to cancer pathogenesis, we speculate that studying how FLOT2 regulates EGFR signaling could lead to a better understanding of its role in normal physiology and cancer.

#2507

Axl suppression inhibits cell migration, invasion and survival in breast and prostate cancer cell lines.

Mai Tanaka, Samantha S. Dykes, Dietmar W. Siemann. _University of Florida, Gainesville, FL_.

Cancer is the second leading cause of death in the United States with approximately 90% of cancer-related deaths resulting from metastatic disease. In tumor cells, upregulation of certain signaling pathways promote metastatic phenotypes characterized by enhanced invasion, migration, survival, proliferation and induction of angiogenesis. One pathway of considerable interest involves the receptor tyrosine kinase Axl that is expressed in a variety of tumor types and is associated with poor prognosis and metastasis. Furthermore,

Axl's ligand, Growth arrest specific 6 (Gas6) protein, can activate a number of downstream signaling pathways that promote metastatic phenotypes. The purpose of the current study was to characterize Axl and Gas6 in a number of human tumor cell lines and to determine the effect of Axl suppression on the metastatic phenotypes, specifically cell migration, invasion and survival. A panel of human tumor cell lines, including prostate, breast, colorectal, renal, and osteosarcoma, were characterized for Axl expression by Western blot and qPCR. Gas6 expression was assessed by qPCR and Gas6 secretion was determined in conditioned media by ELISA. Axl was genetically inactivated by shRNA

in prostate (DU145) and breast (MDA-MB-231) cancer cell lines. The efficiency of knockdown was determined by Western blot. The effect of Axl knockdown on the migratory and invasive potentials of these cell lines was evaluated by transwell migration and invasion assays. A clonogenic cell survival assay was used to assess the impact of Axl knockdown on tumor cell viability. Axl-expressing tumor cells expressed and secreted its ligand Gas6, suggesting that Axl is co-expressed with Gas6, and may be mediated by autocrine regulation. Genetic inactivation of Axl (shRNA) inhibited cell migration, invasion and reduced clonogenic cell survival in both the MD-MB-231 breast and DU145 prostate cancer cell lines. These findings suggest that Axl may be a novel therapeutic target to inhibit the dissemination of tumor cells. Ongoing experiments are evaluating the impact of the Axl-targeting agent R428 on Axl signaling and metastatic phenotype.

#2508

Effect of ALK-inhibitors in synovial sarcoma (SS) cell line HS-SYII.

Hiroshi Gyotoku. _Second Department of Internal Medicine (Respiratory Medicine) Nagasaki University Hospital, Sakamoto, Nagasaki-shi, Nagasaki, Japan_.

Synovial sarcoma (SS) accounts for approximately 8% of all soft tissue sarcomas (STS). The prognosis is poor because of a few effective chemotherapy regimens for STS such as Doxorubicin, Eribulin, and Trabectedin. Pazopanib, a multi-targeted tyrosine kinase inhibitor of PDGFR, VEGFR and c-Kit, is the first molecular targeted drug approved by FDA for STS. Recently, a preclinical efficacy of MET-inhibitor and ALK-inhibitor for a synovial sarcoma cell line was reported. We found that the response to ALK-inhibitors (Alectinib, Crizotinib and Lorlatinib) for SS cell line HS-SY-II different from Aska-SS cell line in a previous report. In addition, a combination of ALK-inhibitor and Pazopanib, previously reported that single-agent is effective in HS-SY-II, potentiates a cytotoxic effect by MTT assay than each single agent. Western blotting revealed that both of ALK-inhibitor single agent and the combination of Pazopanib and ALK-inhibitor suppress the phosphorylation of ERK and AKT, downstream of MAPK and PI3K pathway. Our data suggests that the combination therapy Pazopanib and ALK-inhibitor may be effective to SS patients clinically.

#2509

Blockage of angiotensin II type 1 receptor (AGTR1) inhibits epithelial-mesenchymal transition (EMT) and tumor growth in breast cancer.

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

The angiotensin II type I receptor (AGTR1) has been implicated in diverse aspects of human disease, from the regulation of blood pressure and cardiovascular homeostasis. A recent study has reported pathogenic overexpression of the angiotensin II type 1 receptor (AGTR1) in breast cancers. We sought to investigate the effect of losartan, an angiotensin II receptor blocker on cell proliferation, epithelial-mesenchymal transition (EMT), angiogenesis and tumor growth in breast cancer. Overexpression of AGTR1 was associated with accelerated cell proliferation, concomitant with increased expression of survival factors including poly(ADP-ribose) polymerase (PARP) and X-linked inhibitor of apoptosis (XIAP), as well as extracellular signal-regulated kinase (ERK) activation. AGTR1-overexpressing MCF7 cells were more aggressive than their parent line, with significantly increased activity in migration and invasion assays. These observations were associated with changes in EMT markers, including reduced E-cadherin expression and increased p-Smad3, Smad4 and Snail levels. Treatment with the AGTR1 antagonist losartan attenuated these effects in vitro. To confirm the physiological relevance of our in vitro observations, we examined the effect of losartan on tumor growth using a mammary fat pad xenograft model with the AGTR1-overexpressing cells. Losartan administration resulted in a significant reduction in tumor growth and a significant decrease in the number of Ki-67-positive cells in the AGTR1-overexpressing xenograft tumors. Losartan administration notably upregulated E-cadherin and suppressed vimentin expression. Furthermore, microvessel density analysis revealed that the number of CD31-positive microvessels in both the peritumoral and intratumoral areas was significantly decreased in the losartan-treated groups. Our findings support the notion that AGTR1 is a potentially useful diagnostic marker and its inhibition may provide an effective therapeutic strategy for breast cancer treatment.

#2510

Clinicopathologic significance of fibroblast growth factor 2 type IIIb and type IIIc in gastric cancer.

Kenji Kuroda, Masakazu Yashiro, Go Masuda, Yuichiro Miki, Tomohisa Okuno, Shingo Togano, Takahiro Toyokawa, Hiroaki Tanaka, Kazuya Muguruma, Kosei Hirakawa, Masaichi Ohira. _Osaka City University Graduate School of Medicine, Osaka City, Japan_.

[Background]Fibroblast Growth Factor Receptor 2(FGFR2) amplification occuers flequently in undifferentiated gastric cancer(GC), and accounts for 3-10% of primay GC. FGFR2 shows two isoforms, IIIb-type expressed in epithelial cells, and IIIc-type expressed in mesenchymal cells. The aim of this study is evaluating clinicopathological significance of FGFR2-IIIb and IIIc expression on GC cells.

[Method]562 patients who had undergone GC surgery were retrospectively examined. Immunohistochemistry of resected specimen was used to investigate the expression of FGFR2-IIIb and IIIc.

[Result]FGFR2-IIIb and FGFR2-IIIc were positive in 28 cases (4.9%) and 4 cases (0.7%) of 562 gastric cancers. All of FGFR2-IIIc positive tumors were also positive for FGFR2-IIIb in a same tumor, but both were not positive in same cells. 5-year overall survival rate of FGFR2 positive group is significantly poorer (p=0.0011, log-lank) than that of FGFR2 negative. Moreover, the prognosis of FGFR2-IIIc positive group was significantly poorer (p=0.003, log-lank) than that of FGFR2-IIIb positive group. In univariate analysis, the overall survival was significantly correlated to FGFR2-IIIb positive, FGFR2-IIIc positive, invasion depth, macroscopic type, histological type, lymph node metastasis, peritoneal dissemination, lymphatic invasion, and cytology. In multivariate analysis, FGFR2IIIc was significantly correlated to overall survival (p=0.045)

[Conclusion] The expression of FGFR2IIIc and/or FGFR2IIIb was observed in 5% of gastric cancer. FGFR2 amplification is associated with poor overall survival. As a result, treatment targeting FGFR2 amplification has the potential to provide clinical benefit for FGFR2 positive GC.

#2511

ErbB2 receptor levels in patient-derived spheroids vs. solid tumors reveal high ovarian cancer cell plasticity.

Mara P. Steinkamp, Ashley Remy, Irina Lagutina, Carolyn Y. Muller, Diane S. Lidke, Bridget S. Wilson. _University of New Mexico, Albuquerque,, NM_.

Although ErbB receptors are often expressed in primary ovarian tumors, targeted therapy against these receptors has been minimally successful. Since therapies typically target EGFR or ErbB2 that are present in 25-35% of cases, variability in receptor expression among patients may mask effects. ErbB3 and ErbB4 are more ubiquitous, but targeting these receptors is less common. The timing of treatment with targeted therapies may also be crucial. Clinical trials test these therapies in recurrent platinum-resistant disease, but they might be more effective at preventing recurrence after surgery by eliminating free-floating cancer spheroids that give rise to new solid tumors. Since cancer spheroids are not routinely characterized in ovarian cancer, we isolated spheroids from the malignant ascites of 22 ovarian cancer patients undergoing cytoreductive surgery and analyzed their ErbB receptor signatures by immunofluorescence. Surprisingly, cancer spheroids from nearly all samples (85%) were positive for ErbB2. This differs markedly from the 35% of primary ovarian cancer tumors that were ErbB2 positive in our previous study. NSG mice injected intraperitoneally with cancer spheroids to model disseminated disease developed a combination of solid tumors and malignant ascites. Spheroids obtained from these patient-derived xenograft (PDX) models were also ErbB2 positive. However, solid tumors from the PDX models showed little to no ErbB2 labeling. This altered receptor expression demonstrates the plasticity of ovarian cancer cells as they transition from free-floating spheroids to solid tumors. In the ErbB receptor family, this change may be ErbB2-specific. Analysis of ErbB3 receptor status found no change between spheroids and solid tumors. One implication of this finding is that ErbB2 targeted therapies may be effective in ovarian cancer cases where patients are optimally debulked with only microscopic residual disease. Because spheroids are ErbB2 positive, while solid tumors tend to be ErbB4 and/or ErbB3 positive, we explored the effect of a pan-ErbB inhibitor, afatinib, on tumor burden in our mouse models. Cells were inoculated into the peritoneal cavity and tumors were allowed to develop before treatment. To measure tumor burden in PDX models, anti-HLA-ABC antibody was conjugated to CF750 near-infrared dye and injected IP 24 hours prior to imaging. Tumor burden of fluorescently labeled tumors was measured on the IVIS Spectrum using fluorescence tomography. A decrease in tumor burden and a reduction in ascites was seen in groups treated with afatinib or paclitaxel alone, but particularly with the combined treatment of afatinib and paclitaxel. Supplementing current first-line treatments with ErbB targeted therapies could potentially destroy chemoresistant cells while limiting increased toxicity.

#2512

Agonist-induced heterodimerization between CXCR4 and CB2 inhibits Gα13/RhoA-mediated cell migration.

Kisha Scarlett,1 Elshaddai White,2 Christopher Coke,2 Jada Carter,2 LaToya Bryant,1 Cimona V. Hinton1. 1 _Center for Cancer Research and Therapeutic Development; Clark Atlanta University, Atlanta, GA;_ 2 _Clark Atlanta University, Atlanta, GA_.

Heterodimerization of G protein-coupled receptor exemplifies that GPCRS can change intracellular signaling that would result from each individual receptor. Yet, how heterodimers affect receptor pharmacology remains unknown. We've demonstrated that agonist-bound C-X-C Chemokine Receptor 4 (CXCR4) and Cannabinoid Receptor 2 (CB2) formed a non-functional heterodimer on the membrane of cancer cells, inhibiting their migratory potential in vitro. The impact on the signaling entities responsible for reduced migration upon heterodimerization remain elusive, and therefore, we investigated whether the physical association of CXCR4 and CB2 resulted in decreased signaling from the CXCR4-mediated Gα13/RhoA signaling axis, which leads to phenotypic changes involved in migration. To depict receptor specificity in the role of heterodimer formation on downstream signaling events, we used human siRNA against CXCR4 prior to assaying for Gα13/RhoA immune-complexes to demonstrate that heterodimerization of CXCR4/CB2 decreased RhoA activation with the same potency as genomic knockdown of CXCR4 (PC3). To demonstrate that agonist-induced CXCR4/CB2 heterodimerization attenuated RhoA-dependent cell migration, we immunoblotted for PRG protein expression to determine the heterodimer reduced expression of PDZ-RhoGEF (PRG) which is required for RhoA-dependent cell migration and immunoblotted for LKB1, an establish regulator of RhoA-dependent cell polarity. Finally, to characterize the reduction of RhoA-mediated signaling, we performed both wound healing and transwell migration assays in PC3 cells. In PC3 cells transfected with CXCR4-siRNA, we observed a significant reduction in Gα13/RhoA immune-complexes, which was analogous to samples where CXCR4 and CB2 heterodimerized, suggesting that the physical heterodimer is a viable strategy for antagonizing CXCR4. Immunoblotting techniques revealed that the heterodimer decreased PRG protein expression levels in PC3 cells, compared to cells where CXCR4 signaling was active. The reduction in RhoA signaling upon heterodimerization is directly linked to the propensity of cells to migrate as we saw reduced transformation, reduced wound healing, and reduced migration compared to cells where CXCR4 signaling was active. Our results indicate mechanistic insight into our previous observation that a physical heterodimer reduces cell migration via antagonism of the RhoA pathway. Considering clinical and social support for medicinal cannabinoids in cancer treatment, our current and previous studies mechanistically demonstrate cannabinoid applications and efficiency of using agonists instead of antagonists which currently result in severe immune dysfunction due to the inhibition of CXCR4. This heterodimer can be used as a target for metastasis prevention due to CXCR4 in metastasis and cannabinoids in cancer metastasis treatment and pain management.

#2513

FGFRL1 deficiency reduces motility and tumor growth of cells derived from esophageal squamous cell carcinoma.

Yoshinori Takei,1 Takafumi Matsumura,1 Kazuaki Watanabe,1 Hirokazu Nakamine,2 Tetsuo Sudo,1 Kazuharu Shimizu,1 Yutaka Shimada1. 1 _Kyoto Univ., Kyoto, Japan;_ 2 _The Japan Baptist Hospital, Kyoto, Japan_.

Fibroblast growth factor receptor-like 1 (FGFRL1) belongs to the FGFR protein family. We previously reported that FGFRL1 is highly expressed in esophageal squamous cell carcinoma (ESCC) patients with lymph node metastasis and with the depth of ESCCs. Furthermore, transient inhibition of FGFRL1 expression induces cell cycle arrest and apoptosis of ESCC cells. However, the intracellular domain of FGFRL1 lacks the tyrosine kinase domain. Thus the mechanisms of which FGFRL1 contributes to the development of ESCC are unclear. Two cell lines (Clone 15 and 21) deficient for FGFRL1 expression were established from an ESCC cell line (KYSE520) using the CRISPR-Cas9 method. We evaluated the characteristics of these two cell lines in vitro and in vivo. In these FGFRL1-deficient KYSE520 cells, actin filaments around the nucleus were observed sparsely, whereas the filaments along the plasma membranes were observed as strong as those in the parent KYSE520 cells. Depleting FGFRL1 reduced cell motility in vitro and, decelerated tumor growth in a mouse xenograft model. Microarray analysis of mRNA expression revealed that FGFRL1-depletion resulted in reduced expression of MMP-1 and FGFBP1. In conclusions, FGFRL1-deficiency reduced both cell motility and tumor growth of KYSE520 cells. This is consistent with that high expression of FGFRL1 coincident with lymph node metastasis and poor prognosis. These results demonstrate that FGFRL1 expressed in ESCC cells has a role in aggressiveness of ESCCs.

#2514

Most clinically approved anti-EGFR antibodies fail to neutralize EGFRvIII, explaining their lack of efficacy in high-grade glioma.

Sameer A. Greenall,1 Timothy E. Adams,2 Terrance G. Johns1. 1 _Hudson Institute of Medical Research, Clayton, Australia;_ 2 _CSIRO, Parkville, Australia_.

The epidermal growth factor receptor (EGFR), a cell-surface receptor tyrosine kinase, is amplified or overexpressed in 60% of gliomas. Furthermore, in 30% of gliomas, an autoactive, disulphide-bonded truncation mutant, EGFRvIII, is co-expressed. Both the wild-type (wt) and EGFRvIII are central to driving gliomagenesis. Multiple anti-EGFR antibodies have been developed to neutralise these receptors; however, despite promising preclinical results, their efficacy against glioma in the clinic has been disappointing for reasons that are not understood. We have tested the efficacy and mechanism of action of the majority of known FDA-approved or preclinical anti-EGFR antibodies [panitumumab, cetuximab, necitumumab, nimotuzumab, matuzumab and chimeric 806 (ch806)] against five primary EGFRvIII-bearing glioma lines. We find that cetuximab, necitumumab, nimotuzumab and matuzumab all fail to fully neutralize EGFRvIII activity, leading to sustained downstream signalling and modest antiproliferative effects. ch806 neutralized EGFRvIII activity but did not engage and neutralize wtEGFR. Only panitumumab was able to completely neutralize both wtEGFR and EGFRvIII activation, resulting in a superior antiproliferative response. Not all EGFRvIII-bearing cell lines were responsive to panitumumab (3 out of 5), suggesting that the expression of EGFRvIII is not indicative of sensitivity to anti-EGFR therapy and that additional mechanisms of resistance are present when EGFRvIII is expressed. Only when cetuximab or necitumumab was combined with ch806, to neutralize both wtEGFR and EGFRvIII, was an antiproliferative response similar to panitumumab achieved. Mechanistically, panitumumab neutralized p-ERK but not p-AKT or p-STAT3 signaling. Panitumumab, cetuximab and ch806 induced EGFRvIII internalization and recycling by Rab11-positive endosomes back to the cell surface, while wtEGFR was degraded. Subsequent wtEGFR translation replaced the degraded wtEGFR, ensuring receptor pool replenishment. Finally, panitumumab does not exert its effects by breaking the active EGFRvIII disulphide-bonded dimer; rather, it may work by trapping EGFRvIII into an inactive high molecular weight receptor cluster, an effect that was not observed for cetuximab. Animal trials to compare the efficacy of these antibodies against one another in intracranial orthografts of patient-derived cell lines are ongoing. Collectively, our results challenge the existing premise that EGFRvIII is neutralized and degraded by EGFR antibodies and demonstrate that both wtEGFR and EGFRvIII must be neutralized for maximum antitumor effect. These results may help explain why existing antibodies such as cetuximab and nimotuzumab have failed in clinical trials in glioma and provide rationale that panitumumab may offer a superior and effective candidate for a further clinical trial in glioma.

#2515

Aberrant expression of CSF1R in melanoma is driven through an endogenous viral promoter and it contributes to malignant growth and BRAF-inhibitor resistance.

Orsolya Giricz,1 Yongkai Mo,1 Kimberly B. Dahlman,2 Xiomaris M. Cotto-Rios,1 Chiara Vardabasso,3 Hoa Nguyen,4 Bernice Matusow,4 Matthias Bartenstein,1 Veronika Polishchuck-Lee,1 Douglas B. Johnson,2 Tushar B. Bhagat,1 Rafe Shellooe,4 Elizabeth Burton,4 Gaston Habets,4 John M. Greally,1 Yiting Yu,1 Gideon Bollag,4 Paraic A. Kenny,1 Kith Pradhan,1 E. Richard Stanley,1 Emily Bernstein,3 Evripidis Gavathiotis,1 Brian L. West,4 Jeffrey A. Sosman,2 Amit Verma1. 1 _Albert Einstein College of Medicine, Bronx, NY;_ 2 _Vanderbilt University Medical Center, Nashville, TN;_ 3 _Icahn School of Medicine at Mount Sinai, NY;_ 4 _Plexxikon, Berkeley, CA_.

Epigenetic changes in cancer are thought to contribute to the regulation of invasion and metastasis. To study this at a genome-wide level in melanoma, we analyzed the methylome of 44 cases of malignant melanoma. We saw widespread demethylation occurring preferentially outside of CpG islands. Comparison of primary and metastatic lesions showed demethylation occurs early during carcinogenesis with few additional alterations in advanced tumors. The colony stimulating factor-1 receptor was aberrantly expressed and hypomethylated in nearly all cases. Its expression was validated by IHC and RNA-FISH on primary tumors and by qPCR, Western blotting and FACS in BRAF mutant and WT cell lines. CSF1R can be aberrantly expressed via an upstream LTR element in Hodgkin's lymphoma. After analyzing our patient samples and cell lines, we have found this aberrant transcript may be the dominant form in melanoma as well. Expression of one of its ligands IL34 was also shown in the cell lines by both ELISA and qPCR pointing to a potential autocrine regulatory loop. The effects of a small molecule inhibitor, PLX3397 as well as shRNA-mediated knockdown of the receptor were investigated in 2D and 3D cell culture. We saw inhibition of cell growth, smaller colony size, increased apoptosis and decreased invasiveness suggesting a functional role for CSF-1R in melanoma. Treatment of melanoma with BRAF-V600E inhibitors is effective for a time, but resistance invariably develops. The feedback activation of EGFR, BRAF amplification, BRAF splice variants and others are known to aid in the acquisition of resistance and the rebound activation of the MAPK-pathway. We are suggesting a role for CSF1R in this process. In Western experiments, the rebound of phospho-ERK after BRAF inhibitor treatment was accelerated with the addition of CSF1R ligands, or delayed with PLX3397, also attenuating AKT phosphorylation. Melanoma cells stably expressing shRNA against CSF1R recapitulated the effects of the inhibitor. Assaying the cells at different time points during a long-term V600E inhibitory experiment, we saw increasing levels of the transcription factor RUNX1, followed by increasing levels of IL34 and of the receptor, as well as its maturation, and presentation on the cell surface. shRNA-mediated knockdown of RUNX1 resulted in lower levels of the CSF1R and IL34 transcripts and delayed the rebound. Analysis of primary RNA-Seq data showed an increase in RUNX1, CSF1R and IL34 expression in resistant tumors. Co-inhibition of CSF1R and BRAF was also tested and resulted in synergistic blockade of cell growth in vitro and xenograft growth in vivo.The CSF1R inhibitor, PLX3397 is currently in clinical trials for glioblastoma, prostate, breast cancers and other cancers. These data present a preclinical rationale for its study in malignant melanoma.

#2516

Inhibition of Β-catenin/CBP signaling in oral cancer alters EGFR N-glycosylation and abundance.

Kevin B. Chandler, Khalid Alamoud, Vinay K. Kartha, Khikmet Sadykov, Stefano Monti, Maria A. Kukuruzinska, Catherine E. Costello. _Boston Univ., Boston, MA_.

Head and neck cancer is a debilitating malignancy, with the majority of cases arising in the oral cavity as oral squamous cell carcinoma (OSCC). A major driver of OSCC is the epidermal growth factor receptor (EGFR), whose activity is aberrantly upregulated in >90% of tumors. EGFR is highly modified with N-linked glycans; fucosylation of N-linked glycans interferes with EGFR dimerization and activation. Thus, post-transcriptional changes may govern EGFR activity. In OSCC, EGFR signaling converges on Wnt/ Β-catenin activity, known to play pivotal roles in the pathobiology of this malignancy through the interaction of nuclear Β-catenin with the histone acetyltransferase CREB-binding protein (CBP). We have shown that a small-molecule inhibitor of Β-catenin-CBP interaction, ICG-001, interferes with OSCC proliferation and aggressive features in cellular, zebrafish and murine models. Also, OSCC-cell line derived mouse tumor xenografts exhibit reduced EGFR abundance, and genomic analyses show a positive correlation between ICG-001 and EGFR inhibition. Given that modification of EGFR with N-glycans impacts its cell-surface localization and signaling, we hypothesized that ICG-001 affected EGFR N-glycosylation. We immunoprecipitated EGFR from indolent CAL27 and metastatic HSC-3 cells after treatment with ICG-001 or vehicle control and determined the effect of inhibition of Β-catenin/CBP activity on its N-glycosylation status. We subjected immunoprecipitated EGFR to proteolysis, performed glycopeptide enrichment via hydrophilic interaction liquid chromatography (HILIC), analyzed glycopeptides with an Agilent 6550 Quadrupole Time-of-Flight (Q-TOF) MS using collision-induced dissociation, and compared site-specific glycoform patterns for the two cell types +/- ICG-001. At specific N-glycosylation sites, EGFR from indolent CAL27 cells had highly fucosylated N-glycans, while EGFR from metastatic HSC-3 cells displayed N-linked glycans with a paucity of fucose. Treatment of HSC-3 cells with ICG-001 revealed higher fucosylation at sites N151, N420, suggesting that ICG-001 promoted modification with terminal fucose, potentially inhibiting EGFR signaling. Parallel analyses of gene expression signatures in response to ICG-001 treatment in HSC-3 cells showed increased transcriptional expression of fucosyltransferases, FUT2 and FUT3 that fucosylate residues on the outer arms of N-linked glycans. Our studies suggest that the Β-catenin/CBP axis promotes EGFR signaling by inhibiting its fucosylation through downregulation of FUT2 and FUT3 expression and activity. Thus, inhibition of Β-catenin/CBP signaling with ICG-001 may serve as a therapeutic approach to downregulate EGFR protumorigenic activity in OSCC.

Supported by NIH grants P41 GM104603 (CEC), F32 CA196157 (KBC), and by the Evans Center for Interdisciplinary Biomedical Research ARC #9950000118 (MAK).

#2517

The role of endocytic traffic regulator EHD1 in EGFR traffic and breast cancer.

Insha Mushtaq,1 Eric C. Tom,1 Priyanka Arya,1 Timothy Bielecki,1 Bhopal Mohapatra,1 Sameer Mirza,1 Matthew D. Storck,1 Dena Ahmad,2 Rokaya El Ansari,2 Emad Rakha,2 Vimla Band,1 Hamid Band1. 1 _University of Nebraska Medical Center, Omaha, NE;_ 2 _University of Nottingham & Nottingham University Hospitals NHS Trust, City Hospital Campus, Nottingham, United Kingdom_.

Epidermal growth factor receptor (EGFR) is a prototype receptor tyrosine kinase and serves as an oncogene in a variety of solid tumors. Its surface expression is dynamically regulated, and display of an activation-competent cell surface pool is an essential pre-requisite for cellular responses to EGF and other ligands, as well as for its oncogenic function. While mechanisms that regulate the post-activation endocytic fate of EGFR have received much attention, little is known about the mechanisms that control the basal, pre-activation, surface display of EGFR. Here, we identify a novel role of the endocytic recycling regulator EHD1 in the cell surface display of EGFR and EGF-driven functional responses. We demonstrate that inducible knockdown of EHD1 in human mammary epithelial cells impairs the EGFR delivery to the cell surface, resulting in reduced EGFR cell surface expression and accumulation in the Golgi, a phenotype rescued by exogenous EHD1. Reduced surface display of EGFR by depletion of EHD1 impaired EGF-induced proliferation mammary epithelial cells and a breast cancer cell line, demonstrating that the new molecular pathway we describe is functionally important. Given the role of EGFR and its family members in breast cancer, we carried out IHC screening of EHD1 expression in human breast cancer using tissue microarrays from a large (>800 samples) and well-annotated breast cancer cohort. Of the 757 evaluable samples, EHD1 was overexpressed in 115 (15%) patients. Notably, a substantially higher percentage of HER2/ErbB2+ patients (25%) showed EHD1 overexpression. Survival analysis revealed that EHD1 overexpression was associated with a significantly shorter overall survival over 15 years of follow up. These studies reveal novel roles of EHD1 in regulating EGFR traffic and suggest its potential roles in regulating other EGFR family members, especially HER2/ErbB2. Our patient sample analyses support a role for EHD1 overexpression in promoting breast tumorigenesis. Future studies will be directed at directly testing the role of EHD1, and its family members, in HER2 traffic and the role of EHD1 in breast tumorigenesis. As EHD proteins possess ATPase activity, which is required for their function, our studies raise the prospect of developing inhibitors of these enzymes as potential anti-cancer agents.

#2518

Folate hydrolase-1 is a novel target for J591-brachytherapy in Merkel cell carcinoma.

Barbara Meier,1 Marigdalia K. Ramirez-Fort,2 Kristina Lachance,3 Candice D. Church,3 Sean S. Mahase,4 Joseph M. Jenrette,2 Christopher S. Lange,5 Lars E. French,1 Paul Nghiem,3 Neil H. Bander4. 1 _University Hospital Zurich, Zurich, Switzerland;_ 2 _Medical University of South Carolina, Charleston, SC;_ 3 _University of Washington, Seattle, WA;_ 4 _Weill Cornell Medicine, New York, NY;_ 5 _SUNY Downstate Medical Center, Brooklyn, NY_.

Folate hydrolase-1 (FOLH1) is a type II transmembrane protein. Oncologically, FOLH1 is upregulated throughout prostate cancer cells; it is also luminally expressed by the neovasculature of most solid tumors but not by normal vessels. J591, a monoclonal antibody (AB), is specific to, and is effectively endocytosed after extracellular binding to, FOLH1. J591 is presently being developed in clinical trials as a vehicle for AB-based brachytherapy in FOLH1+ cancers. Merkel cell carcinoma (MCC) is a rare, neuroendocrine tumor; metastatic (m) MCC is associated with poor survival. We characterized FOLH1 expression in MCC to determine its target potential for J591-brachytherapy. Paraffin sections from primary (p) and mMCC were deparaffinized and rehydrated. Samples were stained with 3E6 (DAKO), a mouse IgG1 monoclonal anti-human FOLH1. Mouse IgG1 (10 ug/mL in 1% bovine serum albumin) was used as an isotype-matched negative control. Anti-CD31 (IgG1) was used as a positive control. Kaplan-Meier survival curves were calculated based on patient outcome data and FOLH1 expression. 81 MCC tumors were evaluated. 67% (54/81) of all cases with 77% (24/31) of pMCC and 60% (30/50) of mMCC tumors demonstrated FOLH1+ neovessels. No cellular staining of tumor cells was identified. 34 patients with FOLH1 +/- MCC were demographically homogeneous in terms of sex, age, immunosuppression status, prior therapies, stage at diagnosis, and local or distant recurrences. No significant differences were detected based on FOLH1 status, in regards to MCC specific survival (P=0.905), or overall survival (P=0.687), as measured from time of diagnosis. FOLH1 is expressed in the majority of pMCC and mMCC cases and is nonprognostic. Our findings support further investigation of targeted therapy with J591-brachytherapy for the management of MCC.

#2519

Identification of novel MET fusion transcript amplification in glioblastoma.

Oluwademilade Nuga, Ana deCarvalho, Yuling Meng, Laura Hasselbach, Kevin Nelson, Susan Irtenkauf, Andrea Transou. _Henry Ford Hospital, Detroit, MI_.

Glioblastoma (GBM) is the most common and aggressive primary CNS malignant tumor with a 15.2% 2 year survival rate for tumors diagnosed between 2008 and 2012. Amplification of MET proto-oncogene has been identified in 4% of glioblastomas, leading to high expression and ligand independent activation in some cases. Our goal was to develop MET-amplified GBM models to study signaling and investigate response to MET inhibitors as a therapy for GBMs. We identified 2 GBM patients (HF3035 and HF3077) with MET gene amplifications after low-pass whole genome DNA sequencing of 13 cases. Fluorescent in situ hybridization (FISH) analyses confirmed heterogeneous MET amplification in HF3035 and HF3077 tumors, in 63.5% and 83.0% of nuclei, respectively. In vitro neurosphere cultures derived from these tumors showed drastic depletion of MET amplicons, to 15.5% MET in HF3035 (P7) and 1.5% in HF3077 (P11). FISH in the metaphase neurosphere spreads showed that MET amplification was extrachromosomal. Interestingly, MET-amplified neurospheres were strongly selected for after intracranial (IC) implant in immunocompromised mice. HF3035 and HF3077 PDX presented MET amplified in high frequency: 79.5-86.5% for HF3035 and 47-65% for HF3077. Met expression levels by RNAseq were congruent with the oscillating gene amplification pattern. In depth RNA sequencing analysis using PRADA has revealed genomic rearrangements involving MET, yielding three novel MET-CAPZA2 fusion transcripts. For both cell lines exon 1 of CAPZA2 was fused to exon 2 of MET, resulting in full length MET coding region, with altered 5' cis-regulatory sequences. For HF3035 samples, we observed an additional in frame fusion of exon 1 of CAPZA2 to exon 6 of MET, resulting in a truncated MET transcript with 13 codons from CAPZA2. Co-expression of the wild-type and fusion MET transcripts in the tumors and PDXs were validated using PCR. MET and p-MET levels were high thoughout the parental and PDX tumors. Capmatinib, which is a selective c-MET inhibitor was administered to the PDXs orally 5days/week. The treatment was effective in improving survival of HF3077 IC PDXs (p=0.028) and decreasing subcutaneous tumor size to 30% of the controls after 2 week treatment (t-test, p=0.017). However, treatment of HF3035 IC PDXs did not significantly improve survival (p=0.313). Kaplan-Meier survival curves were compared by log-rank (Mantel-Cox) test, sig. set at p<0.05. MET and p-MET detection by IHC of control and capmatinib treated xenografts show complete inhibition of p-MET, but did not affect MET overexpression in HF3035 PDX.

Our results show that highly amplified regions are susceptible to genomic arrangements and the formation of fusion genes. Under investigation, is the basis for the strong selection for MET expressing cells in vivo and potential novel roles for MET in tumor progression.

#2520

FGFR2 and miR-671-5p as key participants involved in the progression of human esophageal squamous cell carcinoma.

Xiaojia Chen,1 Xiaoyan Li,1 Baoqing Tian,1 Xuan Tan,1 Wei Han,1 Jiakang Wang,2 An Hong1. 1 _Institute of Biomedicine, Jinan University, Guangzhou, China;_ 2 _Cancer Center of Guangzhou Medical University, Guangzhou, China_.

Esophageal cancer is the world's tenth common malignant tumor. Esophageal cancer is divided into esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma (ECA). ESCC is the dominant histological subtype. The overall 5-year survival rate for people with advanced-stage of ESCC is only about 10%-20%, but ESCC is still not well-studied. Therefore, it is very important to study the mechanism of cancer progression in ESCC in order to develop new therapies for this deadly disease.

Fibroblast growth factor receptor2 (FGFR2) is one of the RTK members and generally regarded as an oncogene, but little is known about its function in ESCC. In our study, we proved FGFR2 played the key role in ESCC progression. From In vitro studies, overexpression of FGFR2 promoted the ESCC cells' migration, invasion, and proliferation, which was confirmed through knocking down FGFR2 in ESCC cells. In vivo xenograft tumor mouse model studies showed the tumor size from ESCC cells with FGFR2 over-expression larger than that of the control group. Conversely, the tumor size from ESCC cells with FGFR2 knock-down was significantly smaller compared with the control group. Molecular mechanism studies showed that overexpression of FGFR2 activated the ERK and AKT signal pathways and altered the cell cycle in ESCC.

In addition, decreased expression of miR-671-5p was identified by microRNA array and QPCR verify assay in ESCC–FGFR2+ tissue. Using a dual influence report system, we confirmed miR-671-5p could regulate expression of FGFR2. It was also found that FGFR2 was highly expressed whereas miR-671-5p was expressed at a low level in most of the FGFR2-positive ESCC clinic samples. Furthermore, we demonstrated that miR-671-5p could inhibit the proliferation and metastasis of ESCC cells in vitro and it could suppress growth of ESCC in vivo. The miR-671-5p suppressed MAPK and PI3K signal pathways through down-regulation of phosphorylation of FGFR2. Therefore, miR-671-5p was seen as a tumor suppressor in ESCC.

Our results demonstrated that FGFR2 and miR-671-5p are potential therapeutic targets in ESCC. They both participate in the progression of ESCC, but also play the contrasting roles. Antagonists of FGFR2 or activators of miR-671-5p could be therapeutics against ESCC.

#2521

Patient GBM cell originated secretome analysis identifies cytokine A, as a potent therapeutic target.

Suji Han, Kayoung Shin, Kyoungmin Lee, Sungsoo Kim, Hyunju Kang, Jin-Ku Lee, Hyun Nam, Kyeung Min Joo, Do-Hyun Nam. _samsung medical center, Seoul, Republic of Korea_.

GBM is the most aggressive human primary brain tumor and leads to poor clinical outcomes. According to previous studies, exogenous factors secreted from primary brain tumor cells can change the growth pattern of normal cells. We analyzed the ingredients of cell culture media from two patient glioblastoma cells which can grow without growth factors. As a result, we selected a significant factor for tumor cell growth which is called cytokine A, the Heparin-binding growth factor. Because of the cell viability maintenance ability of cytokine A, we tried to investigate detailed mechanism of cytokine A and applied the result to the combination therapy. Cytokine A is critical for tumorigenesis of GBM by modulating DNA damage signaling and cell cycle through ROS. For that reason, cytokine A is effective therapeutic target and can be used in combination therapy.

#2522

Luminal epithelial cell expression of amphiregulin is required for mammary gland development and facilitates mutant p53-driven tumorigenesis.

Kristopher A. Lofgren,1 David R. Meier,1 Megan A. Girtman,1 Esther A. Peterson,2 E. Charles Jenkins,3 Paraic A. Kenny1. 1 _Gundersen Medical Foundation, La Crosse, WI;_ 2 _University of Puerto Rico, Rio Piedras, San Juan, Puerto Rico;_ 3 _Icahn School of Medicine Mount Sinai, New York, NY_.

Amphiregulin (AREG) is an estrogen-responsive ligand of the epidermal growth factor receptor. Mice with germline deletion of AREG are defective in pubertal mammary expansion. Using shRNA, we have previously reported that the estrogen-responsive growth of MCF7 xenograft tumors in immunocompromised mice is also AREG-dependent. Here we use a series of genetically engineered mouse AREG models (lacZ-knock-in, conditional knockout and germline knockout) to address two outstanding questions: (1) Among the numerous AREG-expressing cell types in the mammary gland and stroma, does the requirement for AREG in mammary gland development reside specifically in the luminal epithelial cells? (2) Is AREG required for mammary tumorigenesis in immunologically-intact mice? An initial characterization of mammary gland development using an AREG-lacZ knock-in allele identified AREG promoter activity in the luminal epithelial but not the myoepithelial compartment. We generated AREGfl/fl mice and achieved luminal epithelial cell specific AREG deletion using MMTV-Cre. Mice lacking AREG in the luminal epithelial compartment (but otherwise proficient in AREG expression) phenocopied the deficit in outgrowth seen in the AREG germline deletion, indicating that luminal epithelial AREG expression is essential for mammary gland development. To determine the extent to which AREG deletion affects mammary tumor latency and multiplicity in immune-intact animals, we used a Cre-regulated model of p53 mutation in mammary tissue (MMTV-Cre x p53LSL-R172H). We used this p53-dependent model to generate at-risk cohorts with AREG deficient (AREG-/-) and AREG proficient (AREG+/+ & AREG+/-) genotypes. Although the cohorts are still maturing, the median age of mammary tumors observed in AREG proficient mice is 258 days (median tumor multiplicity is 2 per animal), while only a single tumor has been detected in the AREG deficient cohort (at 415 days). Together, these data point to two temporally distinct roles for amphiregulin--an essential function in mammary ductal expansion during puberty and a subversion of this developmental role during tumorigenesis later in life.

### Transcription Factor Crosstalk and Aberrant Transcriptional Control

#2523

Cooperativity of HOXA5 and STAT3 is critical for HDAC8 inhibition-mediated transcriptional activation of PD-L1 in human melanoma cells.

Chen Chen Jiang,1 Yu Fang Wang,2 Simonne Sherwin,1 Margaret Farrelly,1 Fen Liu,1 Xu Guang Yan,1 Amanda Croft,1 Tao Liu,3 Lei Jin,1 Xu Dong Zhang1. 1 _University of Newcastle, Callaghan, Australia;_ 2 _Sichuan University, Sichuan, China;_ 3 _University of New South Wales, Sydney, Australia_.

While the expression of programmed death-ligand 1 (PD-L1) is an important mechanism by which cancer cells evade the immune system, PD-L1 expression in cancer cells are commonly associated with patients' responses to treatment with anti-PD-1/PD-L1 antibodies. However, how PD-L1 expression is regulated in melanoma cells remains to be fully elucidated. Here we report that the class I histone deacetylase (HDAC) HDAC8 controls transcriptional activation of PD-L1 by a transcription complex consisting of transcription factors homeobox A5 (HOXA5) and signal transducer and activator of transcription 3 (STAT3). Inhibition of HDAC8 upregulated PD-L1 in melanoma cells. This was due to an increase in the activity of a fragment of the PD-L1 gene promoter that is enriched with binding sites for both HOXA5 and STAT3. Indeed, knockdown of HOXA5 or STAT3 abolished upregulation of PD-L1 by HDAC8 inhibition. Moreover, HOXA5 and STAT3

were physically associated and appeared interdependent in activating PD-L1 transcription. Functional studies showed that HDAC8-mediated regulation of PD-L1 expression participated in modulating anti-melanoma T cell responses. Collectively, these results identify HDAC8 as an important epigenetic regulator of PD-L1 expression, with implications for better understanding of the interaction between melanoma cells and the immune system.

#2524

A new mTOR-independent effect of rapamycin: Transcriptional regulation of TRIB3.

Bojana Stefanovska, Cécile Vicier, Véronique Scott, Guillaume Meurice, Fabrice André, Olivia Fromigué. _Gustave Roussy, Villejuif, France_.

Background: Mechanistic Target of Rapamycin (mTOR), a key component of the PI3K/Akt signaling pathway, coordinates various cellular processes such as metabolism, growth, cell proliferation and cell cycle progression. Dysregulations affecting mTOR frequently occur in cancer, and mTOR inhibitors/rapalogs are used as monotherapy or a part of combination therapy in breast or kidney metastatic cancer. Despite this, response rate is modest. Several mechanisms of resistance have been reported, but do not fully characterize insensitive tumor cells. Purpose: This study investigated new target genes of rapamycin/rapalogs. Methods: Expression levels of target genes were evaluated in a panel of cancer cell lines by transcriptomic analyses, real-time qPCR and western blot. Transcriptional regulation was investigated by in silico analyses of the promoter region, gene reporter assays using site-directed mutagenesis, and ChIP. Stably silenced cell lines were established by lentiviral transduction of shRNA sequences. Results: We identified one gene: TRIB3 (Tribbles Pseudokinase 3), whose expression is downregulated in the presence of rapamycin in a panel of cancer cell lines and in PBMCs isolated from cancer patients. This gene encodes for a pseudokinase involved in a glucose-induced insulin resistance in Type 2 Diabetes by inhibiting the activation of Akt1/2. We observed that silencing mTOR did not mimic rapamycin downregulation of TRIB3, suggesting an mTOR-independent effect of rapamycin. We identified a binding site for the transcriptional repressor GCF2/LRRFIP1 in the promoter region of TRIB3, and confirmed that the presence and the binding of GCF2 to TRIB3 promoter are required for TRIB3 downregulation by rapamycin. Finally, we identified FKBP3 (FK506-Binding Protein 3), a protein that can bind rapamycin with high affinity, as a GCF2 partner, confirming the direct effect of rapamycin on TRIB3 promoter. Conclusions: Taken together, our investigations identified a novel transcriptional target gene of rapamycin: TRIB3. We also characterized the molecular mechanism of regulation by rapamycin through a FKBP3/GCF2-dependent repression of TRIB3 promoter activity, and independently of mTOR signaling. Such variations in TRIB3 expression level in cancer cells could be linked to rapamycin sensitivity, and may represent a valuable therapeutic target.

#2525

Identification of differentially expressed myeloma-specific pathways in the absence of VLA-4.

Deep K. Hathi, Chantiya Chanswangphuwana, Michael P. Rettig, Samuel Achilefu, John F. DiPersio, Monica Shokeen. _Washington University in St Louis, St Louis, MO_.

Multiple myeloma (MM) is a cancer of terminally differentiated plasma B-cells in the hematopoietic bone marrow (BM). Despite advances in MM therapy and stem cell transplantation, MM accounts for approximately 20% of all mortalities resulting from hematologic malignancies. One of the major proteins involved in modulating myeloma cell survival and adhesion within the BM is the very late antigen-4 (VLA-4; also known as α4β1 or CD49d/CD29). VLA-4 is a bidirectional integrin overexpressed in myeloma cells that is implicated in key MM tumorigenesis pathways, including cell proliferation, survival, and adhesion-mediated drug resistance.

Previous studies have demonstrated that VLA-4 is inconsistently overexpressed in myeloma cell lines and clinical studies. This heterogeneous expression of VLA-4 may affect disease progression, localization of myeloma lesions, and efficacy of myeloma therapies. Here, we generated VLA-4 knockout (KO) versions of the murine myeloma cell line 5TGM1-GFP to study changes in disease kinetics and identify clinically relevant proteins and pathways that are influenced by VLA-4 expression in myeloma cells.

VLA-4 KO 5TGM1-GFP cell lines were generated using CRISPR/Cas9 to induce a single base pair deletion in the ITGA4 (α4) gene. Lack of α4 surface expression and VLA-4 functionality was verified in KO cell lines in vitro with fluorescence activated cell sorting (FACS) of anti-CD49d and recombinant soluble vascular cell adhesion protein-1 staining, respectively. Implantation of the KO clones in the immunocompetent C57Bl/KaLwRij model via intravenous dissemination of myeloma cells resulted in minimal, but heterogeneous, uptake in the BM and spleen. FACS confirmed the absence of VLA-4 expression in the excised MM tumors, suggesting the differences in disease kinetics and tumor localization between the KO cell lines were induced by changes in MM pathways affected by the knockdown of VLA-4 function.

To identify proteins and pathways affected by knockdown of α4, we performed RNA sequencing on over 100,000 genes in 5TGM1-GFP wild-type (WT) and KO cell lines. Hierarchical clustering on statistically significant differentially expressed genes (false discovery rate-corrected p<0.001) identified 591 overlapping genes, and 975 non-overlapping genes. Comparison with clinical gene expression data from the MIT Broad Institute MM database identified endoglin, myc, and estrogen receptor-1 as prominent genes with high mutation rates in patients. Finally, myeloma-specific differentially expressed genes were analyzed using graph-based network analysis and gene ontology to identify key proteins and pathways. Our results demonstrate the impact of VLA-4 on BM localization and oncologic pathways that may be exploited in the development of novel therapeutic strategies.

#2526

Modulation of metastatic factors in prostate cancer cells by DAX-1 (NR0B1), an androgen-induced orphan nuclear receptor.

Roxxana Valeria Beltran Valencia, Christina Tzagarakis-Foster. _University of San Francisco, San Francisco, CA_.

DAX-1, a member of the nuclear hormone receptor superfamily, has a pivotal role in adrenal and gonadal development. However, recent studies have shown that DAX-1 acts as a transcriptional repressor influencing the progression of many types of cancers. We hypothesize that DAX-1 has key roles in regulating metastatic genes during prostate cancer progression. We found that introducing DAX-1 into prostate cancer cells lacking endogenous expression of DAX-1 leads to a decrease in metastatic rates. Consequently, we analyzed DAX-1 transcriptional regulation over different markers associated with metastasis. Since the androgen receptor is a key regulator of prostate cancer progression, we used CRISPR to knockout DAX-1 expression in hormone-dependent prostate cancer cells, allowing us to understand the role DAX-1 has as a negative regulator of metastatic genes after androgen receptor activation. We have shown that treatment of hormone-dependent prostate cancer cells with a non-aromatizable androgen leads to upregulation of DAX-1 expression. Here we use the same mechanism to induce DAX-1 expression and analyze the effects on metastatic markers. Results have shown that DAX-1 is sufficient to decrease the expression of metastatic markers and inhibit metastatic rates in prostate cancer cells. These results further explain some of the intrinsic mechanisms involved in prostate cancer metastasis and could reveal new therapeutic routes in the treatment of metastatic prostate cancer.

#2527

Regulation of mitotic progression in T-cell acute lymphoblastic leukemia by the Ikaros tumor suppressor.

Jonathon L. Payne,1 Elanora Dovat,2 Mario Soliman,2 Chunhua Song,2 Sinisa Dovat2. 1 _Loma Linda University School of Medicine, Loma Linda, CA;_ 2 _Pennsylvania State University College of Medicine, Hershey, PA_.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy that represents a therapeutic challenge. Next-generation sequencing revealed that a subset of high-risk T-ALL which is associated with poor prognosis harbor inactivating mutations or deletion of one allele of the IKZF1 tumor suppressor. The IKZF1 gene encodes the Ikaros protein that functions as a regulator of transcription and a tumor suppressor. However, the molecular mechanism of Ikaros' tumor suppressor function in T-ALL is unclear. The use of quantitative chromatin immunoprecipitation (qChIP) determined that Ikaros binds to the promoter regions of Anaphase Promoting Complex Subunit 1 (ANAPC1) and Anaphase Promoting Complex Subunit 7 (ANAPC7) cell cycle genes in T-ALL primary cells in vivo. ANAPC1 and ANAPC7 genes encode proteins whose function is essential for progression through mitosis, as well as through the G1 phase of the cell cycle. Ikaros overexpression in T-ALL via retroviral transduction, results in reduced expression of ANAPC1and ANAPC7, as evidenced by quantitative RT-PCR (qRT-PCR) and Western blot. The luciferase reporter assay further confirmed Ikaros' function as a transcriptional repressor of ANAPC1 and ANAPC7. The knock-down of Ikaros with shRNA in T-ALL resulted in increased transcription of ANAPC1 and ANAPC7, as evidenced by qRT-PCR. These data suggest that Ikaros can regulate mitotic progression in T-ALL by repressing transcription of ANAPC1 and ANAPC7 genes. Next, we studied the mechanisms that regulate Ikaros' ability to repress ANAPC1 and ANAPC7 in T-ALL. Ikaros' function as a transcriptional repressor is regulated by Casein Kinase II (CK2). CK2 is overexpressed in hematopoietic malignancies and increased expression of CK2 results in T-ALL in murine models. We tested the effect of CK2 inhibition on Ikaros' ability to regulate transcription of ANAPC1 and ANAPC7 in human T-ALL. Molecular inhibition of CK2 with shRNA against the CK2 catalytic subunit resulted in reduced transcription of ANAPC1 and ANAPC7, as evidenced by qRT-PCR. This was associated with increased DNA-binding of Ikaros to the promoters of ANAPC1 and ANAPC7, as evidenced by qChIP. These data suggest that CK2 impairs Ikaros' ability to transcriptionally repress ANAPC1 and ANAPC7 and to regulate mitotic progression in T-ALL. Inhibition of CK2 enhances transcriptional repression of ANAPC1 and ANAPC7 by Ikaros, resulting in the cell cycle arrest of T-ALL. In conclusion, our results show that mitotic progression in T-ALL is controlled by transcriptional regulation of ANAPC1 and ANAPC7 by Ikaros. Overexpression of CK2 impairs Ikaros' ability to repress ANAPC1 and ANAPC7 expression, which contributes to deregulation of the control of mitotic progression in T-ALL. Results suggest the novel therapeutic mechanism of CK2 inhibitors for treatment of T-ALL.

#2528

**Determining gene expression variability between** in vitro **and** in vivo **cancer models: Monolayer, spheroids, and mouse allografts.**

Nicholas R. Hum, Wei He, Aimy Sebastian, Elizabeth K. Wheeler, Matthew A. Coleman, Gabriela G. Loots. _Lawrence Livermore National Laboratory, Livermore, CA_.

An ideal pre-clinical environment that recapitulates in vivo growth conditions ex vivo is an essential pre-requisite for effective drug screening. Conventional monolayer culturing of cancer cells as a pre-clinical model have repeatedly failed to recapitulate responses seen clinically. While 3D culturing methods are able to generate some level of tumor heterogeneity including hypoxic core, cell-cell interactions, gradients of drug penetration, as well as cancer stem cell differentiation, these culturing methods fail to incorporate the complex heterogenous cell composition and transient fluxes in nutrients or drugs.

To investigate the effects of culturing conditions on gene expression of cancer cells, RNA sequencing was performed on a mouse mammary carcinoma (4T1) cell line grown in a variety of culture conditions: 2D (monolayer) or 3D (spheroid). Additionally, gene expression analysis was performed on tumors derived from 4T1 cells injected subcutaneously (SQ) into the murine flank or orthotopically (OT) into the mammary fat pad of BALB/c mice.

Pairwise analysis of RNA sequencing data identified 235 down- and 1029 up- regulated genes differentially expressed between the 2D and 3D culture methods. Differential expression identified genes involved in cell migration, extracellular matrix organization, cell adhesion, angiogenesis, hypoxic response, cell differentiation, as well as key cancer related pathways including TNF, Jak-STAT, and PI3K-AKt primarily upregulated in 3D culture. Similar differential expression was found for genes encoding extracellular matrix proteins, which may be reflective of the 3D environment. Both in vivo allograft models produced highly similar gene expression profiles with only 31 up- and 20 down- regulated genes differentially expressed between OT and SQ tumors. Down-regulated genes were enriched in transcriptional regulatory gene networks and up-regulated genes were enriched for signaling/secreted proteins. The gene expression profiles of in vivo tumors were significantly different when compared to the 2D (973 down regulated, 1971 upregulated genes); and 99 of these transcripts were only expressed in in vivo tumors, highlighting the increased heterogeneity of cell composition found in allografts.

3D culturing of cancer cells upregulates pathways known to be critical to tumor progression including genes known to be essential for adhesion, differentiation, and ECM remodeling however the gene expression profile of spheroids is significantly different than that of cancer cells expanded in vivo. Future culturing methods incorporating immune cells, cancer supporting cells such as fibroblasts and other stromal components are more likely to improve the phenotype of 3D cultured tumor cells.

This study received funding from LLNL LDRD grant 17-ERD-121. This work was conducted under the auspices of the USDOE by LLNL (DE-AC52-07NA27344).

#2529

GENT 2.0, a newly updated database: Gene expression across normal and tumor tissues.

Seung-Jin Park. _Korea Research Institute of Bioscience and Biotechnology, S.Korea, Republic of Korea_.

We previously developed GENT1.0 (Gene Expression Database of Normal and Tumor tissues), a web-accessible database that provides gene expression patterns across diverse human cancer and normal tissues. GENT shows gene expression patterns in various tissues easily and has more than 40,000 samples for 25 normal-cancer paired tissues. Here, we made updated database called GENT2.0 based on 60,000 microarray data for 36 normal-cancer paired tissues with various functions. GENT2.0 1) has collected over 60,000 amounts of data for 36 normal cancer paired tissues; 2) can analyze histologic or molecular subtypes or tumor stages for 19 tissues; 3) provides all sorts of plots in svg (Scalable Vector Graphics) format with a high-resolution figure; 4) has twice the amount of data compared to GENT1.0, and the web page loading speed is almost the same as GENT1.0; and 5) data will be accessible on the web. We hope GENT2.0 will be a useful resource for cancer researchers in many stages from target discovery to target validation.

#2530

An analysis of correlative interactions between Plk1, Notch1 and FoxM1 in melanoma.

Shengqin Su, Chandra K. Singh, Mary A. Ndiaye, Vijayasaradhi Setaluri, Nihal Ahmad. _University of Wisconsin-Madison, Madison, WI_.

Melanoma is one of the most aggressive forms of skin cancer, and existing therapies have not been effective in managing this neoplasm. Therefore, a deeper understanding of the mechanisms of melanoma development/progression is required. The goal of this study was to define the potential interactions between several key melanoma-linked proteins, namely Polo-like kinase 1 (Plk1; a cell cycle regulator involved in G2/M transition), Notch (Notch1; a type I transmembrane protein that decides cell fate during development), and forkhead box M1 (FoxM1; a transcription factor regulating S and G2/M phases of cell cycle) in human melanoma. Previously, we have shown that Plk1 is overexpressed in melanoma, and its inhibition results in G2/M phase arrest and apoptosis in multiple melanoma cells. Recent studies have shown that both Notch1 and FoxM1 may be involved in the pathogenesis of certain cancers, including melanoma. In this study, employing an in-house human tissue microarray (TMA) coupled with high-throughput, multispectral Vectra scanning and inForm analysis, we analyzed 126 clinical tissue specimens of normal skin and localized cutaneous melanoma. The TMA was co-immunostained for Plk1, Notch1, FoxM1, and the melanoma biomarker S100, followed by Vectra scanning and analyses with inForm software. Using this system, we determined the levels of Plk1, Notch1, and FoxM1 in both the nucleus and cytoplasm of melanoma cells which were positive for S100 staining. By applying statistical tools of Wilcoxon Signed-Rank Test and Pearson's Correlation Coefficient in R programming, we found that while all the proteins are detected in both nucleus and cytoplasm, FoxM1 and Notch1 are enriched in the nucleus, and Plk1 is enriched in the cytoplasm. Visualization of the correlation coefficients on a heat-map showed that all 3 sets of proteins (Plk1:Notch1, Plk1:FoxM1, and Notch1:FoxM1) are positively correlated, with Plk1:Notch1 having the strongest correlation, and FoxM1 having a weaker correlation with the other two proteins. The associative relationship between Plk1 and Notch1 leads to the hypothesis that Plk1 and Notch1 may be positively correlated in melanoma. We further studied how Breslow thickness, a measure of melanoma aggressiveness, is related to Plk1, Notch1 and FoxM1. By analyzing the TMA cores with available clinical data, we found a positive correlation between Breslow thickness and Notch1, as well as Plk1. Interestingly, although Breslow thickness was significantly correlated with FoxM1 in the nucleus, no correlation was found in the cytoplasm, suggesting that FoxM1 levels may be independent of Plk1 and Notch1. Overall, our study found novel correlative interactions between Plk1 and Notch1 in melanoma, which may have relevance to melanoma progression. Additional studies are needed to validate our findings and dissect the possible interaction between Plk1 and Notch1 during melanoma development and progression.

#2531

Diagnostic evaluation of indeterminate pulmonary nodules via rna-seq of bronchial epithelium.

Xingyi Shi,1 Ehab Billatos,2 Jiarui Zhang,1 Jennifer Beane,1 Elizabeth Moses,1 Gang Liu,2 Christopher Stevenson,3 Marc E. Lenburg,2 Avrum Spira2. 1 _Boston University, Boston, MA;_ 2 _Boston University School of Medicine, Boston, MA;_ 3 _Janssen pharmaceutical Inc., Philadelphia, PA_.

RATIONALE: Diagnosis of the estimated 1.5 million indeterminate pulmonary nodules found annually on chest CT in the US poses a significant clinical challenge. Prior work from our group has identified alterations in the bronchial epithelial cell microarray-derived transcriptome which are associated with lung cancer and which can serve as a clinically useful biomarker for the evaluation of patients undergoing bronchoscopy for suspect lung cancer. Given that RNA sequencing has a broader measurement range and allows for unbiased detection of novel transcripts, we evaluated the ability of bronchial epithelial gene-expression from RNA sequencing to identify patients with lung cancer among individuals with indeterminate pulmonary nodules.

METHODS: Bronchial epithelial brushings were collected from current and former smokers (n=93; age>45, pack-year>20) undergoing diagnostic work up for indeterminate pulmonary nodules (7-30 mm in diameter) at 4 military and 7 VA hospitals within the DECAMP consortium. Patients were followed clinically for up to two years after sample collection until a final diagnosis of lung cancer (n=52) or benign disease (n=41) was made. We performed total RNA sequencing using the Illumina TruSeq Stranded Total RNA Sample Preparation kit. Individual samples were sequenced to generate paired-end reads per sample. Reads were aligned to the human assembly Genome Reference Consortium Human Build 37 (GRCh37) in Ensembl using Spliced Transcripts Alignment to a Reference (STAR) and summarized into count data via RNA-Seq by Expectation Maximization (RSEM).

RESULTS: We found no differences in age, gender, or smoking status between patients with malignant vs. benign nodules. Using a generalized linear model accounting for smoking status, we identified 34 differentially expressed genes (DEGs, FDR q < 0.2) from the bronchial epithelium associated with lung cancer. Of these 34 DEGs, 28 were up-regulated in individuals with lung cancer and enriched for pathways related to the response to hypoxia and p53. The 6 down-regulated DEGs were enriched for immune cell activation, supporting an immunosuppressive field effect of lung cancer. Concordant enrichment of the previous microarray-based bronchial gene-expression classifier was observed using Gene Set Enrichment Analysis (GSEA, FDR q < 0.05), supporting the cross-platform and cross-cohort validity of the microarray-based classifier.

CONCLUSION: Taken together, findings from this pilot study suggest that there are distinct lung-cancer associated airway epithelial gene-expression alterations detected by RNA-sequencing which replicate a previously published microarray-based signature. Additional alterations identified by our RNAseq studies may be leveraged to refine and further develop an RNA-sequencing based airway biomarker for the early detection of lung cancer in high-risk smokers.

#2532

Wild-type p53 represses MELK expression by blocking the recruitment of FOXM1 to MELK promoter.

Lakshmi Reddy Bollu, Jonathan Shepherd, Dekuang Zhao, Yanxia Ma, Abhijit Mazumdar, Powel H. Brown. _UT MD Anderson Cancer Ctr., Houston, TX_.

Background: Triple-negative breast cancer (TNBC) is the most aggressive form of breast cancer with poor prognosis due to frequent distant metastasis and lack of successful targeted therapies. To identify potential druggable targets, previously we performed a gene expression analysis and identified protein kinases that are highly expressed in TNBCs. In this study, we investigated the molecular mechanism through which wild-type p53 represses MELK expression in TNBC cells. Through these studies, we discovered that the expression of Maternal Embryonic Leucine zipper Kinase (MELK) is highly elevated in TNBC when wild-type p53 is lost or mutated. We also show that overexpression of wild-type p53 represses MELK expression without binding to MELK promoter.

Experimental Design and Methods: To identify the p53-responsive region in MELK promoter, we have made multiple promoter deletions using PCR deletion mutagenesis. The p53-responsive region was determined by measuring the activity of these promoter constructs in the presence of wild-type p53 using luciferase assays. TransFac software was used to identify potential transcription factors in the p53-responsive region. The effect of overexpression of p53 and FOXM1 on MELK expression was examined through Western blotting analysis. Using co-immuno precipitation (Co-IP) studies, we determined the protein-protein interactions between wild-type p53 and FOXM1. The effect of wild-type on FOXM1 recruitment was determined using chromatin immuno precipitation (ChIP) assays.

Results: Through promoter deletion analysis, we identified that deletion of the region between -1.69kb to -0.69kb in MELK promoter significantly reduced p53-dependent suppressive effect on MELK promoter activity. Screening this p53-responsive region using TransFac software, we identified a binding site for FOXM1, a previously reported MELK transcription factor. Similar to the regulation of MELK by wild-type p53, overexpression of wild-type p53 repressed FOXM1 and knockdown of wild-type p53 increased FOXM1 expression. Overexpression of FOXM1 increased MELK expression in p53-null and p53-mutant cells but not in p53 wild-type cells. Co-IP studies revealed that wild-type p53 interacts with FOXM1. ChIP studies revealed that overexpression of wild-type p53 significantly reduced the recruitment of FOXM1 to MELK promoter in TNBC cells.

Conclusion: These studies elucidated the regulation of MELK expression by wild-type p53 and demonstrated that wild-type p53 represses MELK expression by inhibiting expression and recruitment of FOXM1 to the MELK promoter. In this study, we identified a novel mechanism for upregulation of MELK expression in TNBC, which is associated with poor prognosis of breast cancer patients.

This work was supported by Susan G Komen Promise Grant (PB, SH, GM), SAB Komen grant (PB) and Young Foundation grant (PB).

#2533

**Regulation of proliferation and metastasis gene expression by DAX-1 (** NR0B1 **) in human breast cancer cells.**

Christina Tzagarakis-Foster. _University of San Francisco, San Francisco, CA_.

The orphan nuclear receptor DAX-1 (Dosage Sensitive Sex Reversal, Adrenal Hypoplasia Congenita, critical region on the X chromosome, gene 1) is known to serve an important role early on in mammalian sex determination as well as in the regulation of expression of steroid hormones. While it is unclear whether DAX-1 functions independently, DAX-1 has been demonstrated to act as a repressor of many nuclear hormone receptors, including ER, AR, PR, LRH-1 and SF-1. DAX-1 plays a role in select adult tissues, including gonadal tissue. However, it has also been shown to play a role in several types of cancer, though its specific function is not well understood. For example, in some types of cancer DAX-1 expression is upregulated, while in other cancers it is highly downregulated or completely absent. In an effort to better understand DAX-1 function both in normal and disease states, we examined the actions of DAX-1 in MCF7 breast cancer cells, which do not express the DAX-1 gene. We probed three different PCR arrays (breast cancer genes, proliferation genes and metastatic genes) to determine which genes were influenced by DAX-1 expression. With the introduction of DAX-1 in ERα positive breast cancer cells (MCF-7), we find that DAX-1 inhibits cell proliferation and blocks estradiol activation of key cell cycle regulatory genes, such as cyclin D1, as well as important metastatic genes. We show that DAX-1 binds directly to ERα and the ERα-DAX-1 complex binds to the cyclin D1 promoter. The outcome of DAX-1 expression in MCF7 cells is cell cycle arrest through the repression of key cell cycle regulatory genes. These results demonstrate that DAX-1 functions as a corepressor of ERα in MCF7 breast cancer cells by preventing the activation of growth-promoting genes and decreasing cell proliferation in response to estrogens. These findings could have significant implications for future drug development strategies specifically aimed at ER-interacting proteins such as DAX-1.

#2534

Single-cell RNA sequencing reveals lung cancer-associated alterations in bronchial cell subpopulations in the mainstem bronchus.

Xingyi Shi,1 Grant Duclos,1 Joshua Campbell,2 Yaron Gesthalter,2 Patrick Autissier,1 Yves M. Dumas,1 Robert Terrano,1 Gang Liu,2 Marc E. Lenburg,2 Avrum Spira,2 Jennifer Beane2. 1 _Boston University, Boston, MA;_ 2 _Boston University School of Medicine, Boston, MA_.

RATIONALE: We have previously shown that gene expression alterations in the mainstem bronchus epithelium reflects a physiologic response to cigarette smoke and can serve as a diagnostic biomarker for lung cancer. Furthermore, we have shown that single cell RNA sequencing (scRNA-Seq) can be used to detect smoking-associated gene expression alterations within specific airway epithelial and immune cell types. In this study, we use scRNA-Seq to profile the transcriptomes of single cells procured from the airways of patients with or without lung cancer in order to detect cancer-associated alterations within specific cell types.

METHODS: We obtained bronchial brushings in the mainstem bronchus from ever smokers undergoing diagnostic bronchoscopy for suspect lung cancer. Patients were followed post-procedure until a final lung cancer diagnosis (n=5; 3 adenocaricinomas, 1 squamous cell carcinoma and 1 large cell carcinoma) or benign disease (n=7) was made. Single cells were isolated using FACS (n=190/donor) and RNA library preparation was performed using the CEL-Seq protocol followed by RNA-seq (75nt paired-end) on an Illumina Hiseq 2500. Latent Dirichlet allocation was used to identify transcriptomically distinct cellular subpopulations specific to the airways of patients with and without lung cancer.

RESULTS: We detected distinct subpopulations of bronchial cells expressing known markers of basal (KRT5), ciliated (FOXJ1), club (SCGB1A1), and goblet (MUC5AC) epithelial cells. Cell type distributions differed between cancer status and within cancer sub-types. Specifically, patients with cancer had an increase in FOXJ1+ cells. Many of the genes in the previously reported diagnostic biomarker such as ATP12A and NKX3-1 are expressed in specific cell types.

CONCLUSION: The scRNA-Seq data of the normal appearing airways of subjects with and without lung cancer suggests that the epithelial and immune cell types present and their relative proportions change with cancer status. Additionally, previously described lung cancer biomarker genes are expressed in a cell type specific manner indicating that future biomarker improvements may be made by examining subpopulations of cells. Analysis of a greater number of samples is needed to make robust conclusions regarding the changes associated with smoking, cancer, and cancer subtype in this sample population.

#2535

Single-cell transcriptome analysis on lymphocytes of GATA2 deficiency patients.

Elina A. Hirvonen,1 Inari Natri,1 Pirkko Mattila,2 Jenni Mattila,2 Esa Pitkänen,3 Kimmo Porkka,4 Outi Kilpivaara,1 Ulla Wartiovaara-Kautto4. 1 _Genome-Scale Biology, Research Programs Unit and Department of Medical and Clinical Genetics, University of Helsinki, Helsinki, Finland;_ 2 _Institute for Molecular Medicine Finland (FIMM), University of Helsinki, Helsinki, Finland;_ 3 _Genome Biology Unit, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany;_ 4 _Department of Hematology, Helsinki University Hospital Comprehensive Cancer Center, University of Helsinki, Helsinki, Finland_.

GATA2 is an essential transcription factor in the proliferation and maintenance of hematopoietic stem and progenitor cells. Germline GATA2 mutations cause hematological malignancies, especially myelodysplastic syndrome and acute myeloid leukemia. Pre-leukemic phenotype of GATA2 deficient patients varies markedly. However, peripheral blood (PB) lymphopenia is characteristic for almost all the patients and encounters most prominently B and natural killer (NK) cells. We aimed to gain understanding on the effects of GATA2 deficiency in hematopoiesis by comparing expression patterns in lymphocytes in GATA2 wild type and GATA2 deficient individuals.

We conducted single-cell transcriptomic analysis using 10x Genomics Chromium Single Cell 3′ Solution on PB samples from three healthy controls and three GATA2 deficient patients (p.Q328X, p.T354M, and one haploinsufficiency due to unidentified reason). Erythrocytes were lysed prior to washing, filtering and resuspending white blood cells in 0.04% BSA/PBS (1x106 cells/mL). The gel beads in emulsion were generated using Chromium Single Cell 3' v2 Reagent Kit. The sample libraries, prepared according to 10x Genomics instructions, were sequenced in Illumina HiSeq 2500. Cell Ranger v1.3 analysis pipelines (10x Genomics) were used to demultiplex and convert Chromium single cell 3' RNA-sequencing barcode; read data to FASTQ files; and generate align reads and gene-cell matrices. The data were analyzed using Seurat, an R package designed for single cell analysis. Cell type clusters were identified through known markers from Seurat's graph-based clustering.

Significant gene expression differences in lymphocytes (GATA2 deficient patients vs. controls) were observed (Wilcoxon rank sum test, Bonferroni-corrected p <0.05). In NK cells the distinction was found e.g. in KLRB1, CD74, CCL5, STAT1, and LAG3, all linked to immune responses, and BTG1 and DDIT4, regulating cell proliferation and differentiation. Expression changes in immune system-associated genes were also noted in T cells e.g. in STAT1, IL7R, CD48, and CXCR4, as well as in NPM1, TPT1, and TXNIP, genes identified to participate in diverse cellular processes. Also, several genes encoding ribosomal proteins were differentially expressed between patients' and controls' T cells. Comparison of B cells revealed changes in e.g. GZMH, PPBP, and LCN2, which, too, play roles in the immune system.

We observed significant dissimilarities in gene expression pattern in GATA2 deficient patients' vs. controls' lymphocytes. These were reflected in several genes involved in immune system and cellular processes. Connection between our findings and patients' clinical manifestations is intriguing and may have an impact in the development of future patient care. We will extend our material and include also bone marrow sample single-cell transcriptomes and healthy mutation carriers to the analysis.

#2536

Unique HPRT1 upregulation in malignant tissue: Potential use as diagnostic biomarker.

Michelle H. Passey, Abigail M. Felsted, Zachary E. Ence, Stephen R. Piccolo, Kim L. O'Neill, Richard A. Robison. _Brigham Young Univ., Provo, UT_.

The aim of this study is to examine the gene expression of the purine salvage pathway enzyme Hypoxanthine Guanine Phosphribosyltransferase (HPRT) in malignant and normal tissue to determine potential upregulation. Due to the critical role HPRT plays in the cell cycle, it was hypothesized that in a rapidly proliferating malignant tissue there may be differential gene expression. In breast, colon, lung, and prostate cancer the 5-year survival rates decrease by nearly 72% when diagnosed in stage 3 or 4 as opposed to an early stage diagnosis. We focused our investigation on evaluating if HPRT could serve as an early stage diagnostic agent for the four most commonly diagnosed cancers (lung, colorectal, breast, and prostate cancer) which together make up 42.5% of all cancer diagnoses. Initially, we evaluated differences in HPRT expression levels via RNA-sequencing data in 3,147 tumor and 316 normal samples from The Cancer Genome Atlas (TCGA). Samples from 1119 breast invasive carcinoma (p-value= 1.66x10-42), 483 colon adenocarcinoma (p-value= 9x10-18), 541 lung adenocarcinoma (p-value=3.16x10-32), 502 lung squamous carcinoma (p-value= 1.49x10-59), and 502 prostate adenocarcinoma (p-value= 1.53x10-4) patients were compared to healthy individuals and showed significant HPRT over-expression shifts in malignant tumors. To continue this investigation we obtained histological tissue from 52 breast cancer patients, 54 lung cancer patients, 100 colorectal cancer patients, and 56 prostate cancer patients with a varying level of cancer stage and tumor type. Healthy tissue, margins of carcinoma, and pre-cancerous tissues were also stained to determine stage dependence of HPRT expression. Briefly, tissues were treated with a monoclonal anti-HPRT antibody along with a GAPDH positive control and an isotope control. Tissues were incubated with an HRP-polymer conjugated anti-HPRT antibody and followed by a diaminobenzidine (DAB) substrate which, when oxidized, results in antigen labelling. Tissues were imaged and analyzed using ImageJ software, which converted images to a grayscale. From there, we set a parameter for tissues with "HPRT high" expression, and "HPRT low" expression. Overall, we found 33-55% of malignant tissues to have a significant upregulation of HPRT (Lung-33%, Breast-55%, Colon-33%, Prostate-47%). These findings were consistent with our examination of HPRT expression in TCGA and our findings also indicated that the protein over-expression is not dependent on stage. These findings indicate that HPRT may potentially be a valuable biomarker in detecting early cases of malignancy in all of the four major cancers.

#2537

Regulation of LMO2 oncogene expression in high-risk B-cell acute lymphoblastic leukemia.

Yali Ding,1 Jonathon L. Payne,2 Shriya Kane,1 Elanora Dovat,1 Mario Soliman,1 Chunhua Song,1 Sinisa Dovat1. 1 _Pennsylvania State University College of Medicine, Hershey, PA;_ 2 _Loma Linda University School of Medicine, Loma Linda, CA_.

LIM domain only protein 2 (LMO2) is an oncogene that is overexpressed in a subset of B-cell acute lymphoblastic leukemia (B-ALL). The mechanisms that regulate LMO2 expression in B-ALL are still unknown. Here, we show that transcription of LMO2 in B-ALL is regulated by Ikaros, a tumor suppressor protein, which is encoded by the IKZF1 gene, and whose deletion is associated with development of high-risk B-ALL. The use of global chromatin immunoprecipitation coupled with the next-generation sequencing (ChIP-seq) studies in primary human B-ALL cells and in cell lines revealed a strong Ikaros occupancy at the promoter of the LMO2 gene. Ikaros binding to the LMO2 promoter was confirmed by quantitative chromatin immunoprecipitation (qChIP). We tested the role of Ikaros in regulating LMO2 transcription in B-ALL using gain-of-function and loss-of-function experiments. A luciferase reporter assay with the LMO2 promoter showed that Ikaros directly represses LMO2 transcription. Overexpression of Ikaros in B-ALL via retroviral transduction was associated with strongly reduced expression of LMO2. Furthermore, Ikaros knock-down with shRNA, resulted in increased transcription of LMO2 in B-ALL cells. These results suggest that Ikaros represses transcription of LMO2 in B-ALL. Since Ikaros' function in B-ALL is negatively regulated by pro-oncogenic Casein Kinase II (CK2), we tested whether CK2 inhibition affects Ikaros-mediated repression of LMO2. Both molecular inhibition with shRNA, and pharmacological inhibition of CK2 with a specific CK2 inhibitor, CX-4945, resulted in reduced expression of LMO2. Inhibition of CK2 was also associated with increased Ikaros occupancy at the LMO2 promoter. In high-risk B-ALL that have deletion of a single copy of the IKZF1 gene, Ikaros does not bind to the LMO2 promoter. Treatment of primary B-ALL cells that have IKZF1 haploinsufficiency restores Ikaros binding to the LMO2 promoter and results in reduced LMO2 expression. In conclusion, our data demonstrate that Ikaros and CK2 regulate transcription and overall expression of the LMO2 oncogene in B-ALL. Our results identify a novel mechanism of therapeutic action of CK2 inhibitors in high-risk B-ALL—repression of LMO2 expression via restoration of Ikaros' tumor suppressor function.

#2538

The interaction of IGFBP3 with HIF2α in the invasion regulation of ovarian epithelial cell cancer.

Ho-Jun Shih. _National Taiwan Univ. College of Medicine, Taipei, Taiwan_.

Ovarian cancer is the most lethal gynecological cancer among women worldwide. The majority of malignant ovarian tumors are epithelial ovarian cancer and metastasis occurred in most patients at the time of diagnosis with worse outcome. New target therapy such as anti-angiogenesis therapy was proved to be effective to prolong progression free survival in epithelial ovarian cancer (EOC) patients. In our previous study, we have established an ovarian endometrioid carcinoma cell line OVTW59. By cDNA microarray analysis and further functional analysis, we have identified Insulin-like Growth Factor Binding Protein-3 (IGFBP-3), Calreticulin (CRT) and Thrombospondin-1(THBS1) correlated with tumor invasion in OVTW59. IGFBP3 and CRT was further found related with poor patient survival outcome. In this study, we focused on the mechanism of IGFBP3 in cancer invasion and metastasis. First we focused on the THBS1 related angiogenesis mechanism which was induced by hypoxia. Through this study, we identified HIF-2α, but not HIF-1α, playing as a significant role in EOC. By q-RT-PCR, Western blotting and immunohistochemical staining, HIF-2α showed opposite expression with IGFBP3, and high IGFBP3 was correlated with high THBS1 expression and low CRT expression. Using other cell lines, the IGFBP3/THBS1/HIF-2α/CRT expression patterns were similar in 293T, H1299 and A549. Furthermore, by Luciferase promoter assay, we verified that IGFBP3 could induce THBS1, inhibit HIF-2α expression, but does not regulate the expression of CRT. IGFBP3 is known as an invasion suppressor gene, THBS1 is an endogeneous inhibitor of angiogenesis, HIF-2α is a regulator of the hypoxia response and angiogenesis inducer, and CRT as a cell adhesion and invasion related protein. Overall, our study shows that IGFBP3 could induce tumor progression and metastasis through direct regulation of THBS1, and HIF-2α and indirect regulation of CRT in EOC.

#2539

Antiapoptotic gene expression signature reveals a combined talk to prevent apoptosis: A model to choose the proper BH-3 mimetic drug.

Jacopo Nanni,1 Giorgia Simonetti,1 Samantha Bruno,1 Giovanni Marconi,1 Maria Chiara Fontana,1 Cristina Papayannidis,1 Maria Chiara Abbenante,1 Lorenzo Montanaro,2 Michele Cavo,1 Giovanni Martinelli3. 1 _University of Bologna, DIMES, Seragnoli Institute, Bologna, Italy;_ 2 _University of Bologna, DIMES, Bologna, Italy;_ 3 _Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, Meldola, Italy_.

BACKGROUND: Apoptotic regulation involves several actors which can have a positive and negative effect and may represent a target for personalized therapy. Nowadays, BCL-2 inhibitors have successfully passed preliminary phases of clinical experimentation, however the involvement of other essential proteins could represent a resistance mechanism to BCL-2 inhibitors and reveal novel therapeutic targets in acute myeloid leukemia (AML). Our study aim to investigate the expression of anti-apoptotic genes in AML patients.

METHODS: We performed Human Transcriptome Array 2.0 (Affymetrix) in a cohort of 59 newly diagnosed AML. K-means clustering has been used to categorize patients in different cluster. A pool of 7 healthy donors was used to normalize expression data. Survival analysis was conducted with Kaplan-Meyer method and differences in survival were assessed with Log-Rank test.

RESULTS: BCL-2, MCL-1 and BCL2L1 gene expression values in 59 AML patients allowed us to establish 5 clusters (Fig. 1a): cluster 1 with the overexpression of MCL1, cluster 2 with low expression of all analyzed genes; cluster 3 with overexpression of BCL2L1, cluster 4 with overexpression of BCL-2 and cluster 5 with high values of expression of all 3 genes. Clusters 2 and 5 included the majority of patients (40/59, 68%). Furthermore, the 3-D Scatter Plot allowed us to observe how patients out of the central cloud (Clusters 2-5) always presented an overexpression of one out of the three anti-apoptotic genes. Moreover, when BCL-2 level is low (Cluster 1-2-3), MCL-1 and BCL2L1 levels are high, respectively. In term of overall survival (OS), there were no statistically significant difference between Cluster 1-3-4, where one different anti-apoptotic gene is always overexpressed. Finally, analyzing the impact of BCL-2 expression on OS in a cohort of 36 young patients treated with chemotherapy, those with high expression of the three antiapoptotic genes had worse outcome (p=0.07).

CONCLUSION: BCL-2, MCL-1 and BCL2L1 expressions are quite balanced in AML patients evaluated. One out of the three genes is often overexpressed, confirming the key role of anti-apoptotic genes in leukemogenesis. Even though BCL-2 expression influence patients chemosensitivity, MCL-1 and BCL2L1 overexpression has been documented when BCL-2 is normally or downregulated. In conclusion, we suppose that BCL-2 inhibitors resistance may be caused by MCL-1 and BCL2L1 overexpression. Finally, a combined approach should be realized to switch-off leukemic anti-apoptotic mechanisms. Gene expression profile could provide a synthetic lethal instrument to choose the proper BH3-mimetic drug or combination.

Supported by: ELN, AIL, AIRC, project Regione-Università 2010-12 (L. Bolondi), FP7 NGS-PTL project, HARMONY project, Fondazione del Monte BO e RA project.

#2540

TRIM24 is an oncogenic transcriptional co-activator of STAT3 in glioblastoma.

Deguan Lv,1 Yanxin Li,1 Weiwei Zhang,1 Angel A. Alvarez,2 Jianming Tang,1 Bo Hu,2 Shi-Yuan Cheng,2 Haizhong Feng1. 1 _Shanghai Jiao University School of Medicine, Shanghai, China;_ 2 _Northwestern University Feinberg School of Medicine, Chicago, IL_.

Aberrant amplification and mutations of epidermal growth factor receptor (EGFR) are the most common oncogenic events in glioblastoma (GBM), but the mechanisms by which they promote aggressive pathogenesis are not well understood. Here, we determine that non-canonical histone signature acetylated H3 lysine 23 (H3K23ac)-binding protein tripartite motif-containing 24 (TRIM24) is upregulated in clinical GBM specimens and required for EGFR-driven tumorigenesis. In multiple glioma cell lines and patient-derived glioma stem cells (GSCs), EGFR signaling promotes H3K23 acetylation and association with TRIM24. Consequently, TRIM24 functions as a transcriptional co-activator and recruits STAT3, leading to stabilized STAT3-chromatin interactions and subsequent activation of STAT3 downstream signaling, thereby enhancing EGFR-driven tumorigenesis. Our findings uncover a pathway in which TRIM24 functions as a signal relay for oncogenic EGFR signaling and suggest TRIM24 as a potential therapeutic target for GBM that are associated with EGFR activation.

#2541

Combined androgen and glucocorticoid receptor (AR/GR) activity drives TNBC progression.

Deniz N. Dolcen, Eva Tonsing-Carter, Ryan Harkless, Caroline Kim, Kathleen Bowie, Gini Fleming, Suzanne Conzen. _University of Chicago, Chicago, IL_.

Background: A recent effort to distinguish early-stage TNBC based on outcome and to expose driver pathways using gene profiling/cluster analysis divided TNBC into four subtypes- basal-like, mesenchymal, immunomodulatory, and luminal androgen receptor (LAR). The clinical AR+ TNBC subtype (>10% AR by IHC) comprises up to 35% of metastatic disease. The discovery of this TNBC subtype and the concomitant development of potent new anti-androgens for prostate cancer have led to several clinical trials evaluating AR antagonists in TNBC. In parallel, glucocorticoid receptor (GR) expression and activation have been shown to mediate anti-apoptotic gene expression in TNBC. However, to our knowledge, no one has examined the coordinate expression of GR and AR activity and the potential crosstalk downstream of dual GR and AR activation. We hypothesized that AR/GR cooperative gene expression pathways may induce TNBC therapy resistance and tumor progression. Methods and Results: We analyzed GR+ TNBC primary tumor gene expression from TCGA samples and found a wide range of AR expression. To examine whether GR-associated gene expression differed in high versus low AR+ TNBC, we divided TCGA TNBC primary tumors (N=123) at the AR median into "high" versus "low" AR expression. We then identified relative GR target gene expression levels in those genes identified from a previous TNBC meta-analysis. Invasion, motility and proliferation pathways were found to be most significantly expressed among GR-associated genes in AR high TNBC (top 5 significant pathways). Similarly, we found that GR and AR co-activation in AR+ MDA-MB-453 TNBC cells significantly increased cell migration compared to GR or AR activation alone. In addition, when MDA-MB-453 cells were exposed to the AR antagonist enzalutamide (enza) for more than 30 days (long-term, LT), the magnitude of increase in cell migration following GR activation increased, suggesting that increased GR activity may compensate for long-term AR blockade. Furthermore, long-term enza exposure (LTenza) in AR+ MFM-223 and MDA-MB-453 cells resulted in increased GR mRNA and protein levels. The magnitude of GR-mediated target gene expression was also significantly increased in LTEnza versus control-treated cells. In vivo, LTenza-treated MDA-MB-453 xenografts were relatively insensitive to paclitaxel chemotherapy treatment, consistent with increased GR activity compared to control cells. Conclusions: We conclude that coordinate GR and AR expression in TNBC contributes to a particularly poor outcome in TNBC. Downstream AR/GR gene expression favoring cell survival and invasive phenotypes may contribute to this outcome. Consideration of dual AR/GR inhibition in TNBC may increase the effectiveness of anti-androgen therapy.

#2542

c-Jun and FOXO1 play key roles in the overexpression of oncogenic PKC-ἱ in human prostate and melanoma cell lines.

Andre H. Apostolatos, Wishrawana S. Ratnayake, Anisul Islam, Christopher Apostolatos, Tracess Smalley, Mildred Acevedo-Duncan. _University of South Florida, Tampa, FL_.

Protein Kinase C-ἱ (PKC-ἱ) is an anti-apoptotic oncogene overexpressed in multiple cancers including prostate, melanoma, ovarian, breast, and glioma. We have previously shown that PKC-ἱ is part of a cycle that helps cancer cells avoid senescence by releasing the transcription factor NF-kB. Additionally, PKC-ἱ has an anti-apoptotic effect while promoting epithelial-mesenchymal transition in melanoma {Ratnayake, et al. Int. J. Oncol. 51(5), (2017), 1370-1382}. PKC-ἱ is activated externally by factors like loss of PTEN and TGFβ stimulation. However treatment with PKC-ἱ specific inhibitors downregulate expression of both PKC-ι and phosphorylated PKC-ι, suggesting PKC-ἱ plays a role in regulating its own expression. A previous study showed the ELK1 transcription factor to be a regulator of PKC-ἱ (Gustafson, et al. J. Biol. Chem 279(10), (2003), 9400-8}. In this study, transcription factors c-Jun, ISGF3, PAX3, EGR1, and FOXO1 which bind on or near the promoter sequence of the PRKCI gene, were analyzed for their role in PKC-ἱ regulation in prostate cancer cell lines (DU145 and PC3) and melanoma cell lines (SK-MEL-2 and MeWo). Each transcription factor was systematically silenced. Western blots revealed that treatment with either c-Jun or FOXO1 siRNA significantly altered expression of PKC-ἱ. Results showed c-Jun followed by FOXO1 are the two major transcription factors which involve in PKC-ι expression in prostate cells compared to FOXO1 in melanoma cells. This suggests that these transcription factors act as activators that regulate PKC-ι expression and can be downstream targets of PKC-ι. We found that regulation of PKC-ι expression was governed mainly through NF-κB pathway. siRNA knockdown of NF-κB p65 and application of NF-κB inhibitors confirmed the above observation. We have also used immuno-paired-antibody detection assay to determine which pathways were affected with PKC-ι inhibitor treatments other than NF-κB pathway. qPCR and microarray were performed to analyze the transcriptome of treated cells to match protein levels with mRNA levels. We have analyzed multiple targets both up and downstream of PKC-ἱ to determine the mechanism of PKC-ἱ self-regulation. Overall results suggest that PKC-ι inhibition downregulates its own expression making it a novel target in prostate and melanoma cancer treatment. 

## CLINICAL RESEARCH:

### Adoptive Cell Therapy 2

#2543

Concurrent chemotherapy and temozolomide-resistant CAR-T immunotherapy enhances glioblastoma clearance in experimental animals.

Sadhak Sengupta,1 Mansour Gergi,2 Prajna Guha,2 Sudarshana Sengupta,2 Richard P. Junghans,3 Prakash Sampath1. 1 _Alpert School of Medicine, Brown University, Providence, RI;_ 2 _Roger Williams Medical Center, Providence, RI;_ 3 _Tufts University School of Medicine, Boston, MA_.

Dismal long-term progression-free survival of glioblastoma (GBM) patients, who are concurrently treated with radiation and chemotherapy, demands development of alternative strategies for management of this disease. Recent clinical success of chimeric antigen receptors expressing T cells (CAR-T) in GBM patients, interest in tumor-directed immunotherapy has been reinvigorated. However as per standard care, immunotherapy must be concurrent with or following the standard care protocol for glioma therapy, which includes Temozolomide (TMZ) chemotherapy. Concurrent chemotherapy with TMZ reduces the efficacy of adoptive T cell immunotherapy by inducing severe lymphopenia, while post-chemotherapy treatment minimizes the window of opportunity for effective immunotherapy, because of inherent tumor heterogeneity resulting in an altered antigenic profile in recurrent GBMs. To address this, we have created a TMZ-resistant CAR-T cell directed against IL13Rα2+ GBM tumors by introducing a mutated O6-methylguanine-DNA methyltransferase (MGMT) moiety in the existing IL13CAR backbone. This specific mutation in MGMT (P140KMGMT) blocks the interaction of TMZ to MGMT and therefore protects the rapidly proliferating anti-GBM CAR-T cells from TMZ-induced lymphopenia. Activated P140KMGMT-IL13CAR-T cells were protected from TMZ-induced cytotoxicity in vitro without any loss of anti-tumor effects of these modified CAR-T cells. Experimental xenograft animals treated with P140KMGMT-IL13CAR-T cells and concurrent TMZ therapy showed 2-fold increase in median survival over groups that were treated with TMZ alone, and 2.8-fold increase in overall survival over groups that were treated with TMZ-sensitive second generation IL13CAR-T cells and concurrent TMZ. These encouraging results point us strongly in the direction of developing this CAR-T technology towards a promising future of concurrent chemotherapy and immunotherapy of GBM.

#2544

Anti-MUC1* CAR T for solid tumors.

Cynthia C. Bamdad, Andrew K. Stewart, Benoit J. Smagghe, Pengyu Huang, Luke T. Deary, Nelson D. Glennie. _Minerva Biotechnologies, Waltham, MA_.

Purpose: The purpose is to develop a CAR T therapeutic that will effectively treat solid tumors by targeting cleavage product MUC1*, which is the growth factor receptor, rather than full-length MUC1, and by including a novel element that is only expressed by activated CAR T cells that overcomes problems of tumor heterogeneity and microenvironment. This will be a first-in-human trial since every other MUC1-targeting therapeutic tested in humans targeted full-length MUC1. Studies show that MUC1 cleavage and release from tumor surface increase as tumor stage increases. MUC1* has no sites for O-glycosylation. Therapeutics that target aberrant O-linked glycans, such as 5E5, only enrich for the tumor-promoting MUC1* form.

Experimental Procedures: Anti-MUC1* CARs were generated using standard lentiviral vector procedures and transduced into human T cells. 60 novel CARs were tested in vitro to confirm cytokine release and specific cell killing upon exposure to MUC1*-positive cancer cells, but not to cells expressing full-length MUC1 or MUC1-negative cells. Co-culture killing was measured by video, IF, FACS and xCELLigence. In vivo, 500K MUC1*-positive tumors were implanted s.c. or i.p. Human anti-MUC1* CAR T cells were injected into tumor or tail vein. Tumors were measured weekly by IVIS for 60 days. Post sacrifice, tumors and blood were analyzed for presence of CAR T cells, target cells and apoptotic markers.

New Data: Human tissue studies for IND have been completed (n>3,000) and indicate our anti-MUC1* CAR T, huMNC2-CAR44, should be safe and effective. huMNC2-scFv-Fc robustly stained over 90% of breast, 83% ovarian, 78% pancreatic and 71% lung tumors, yet showed no staining on most normal tissues, including critical organs. huMNC2-CAR44 was selected from 60 novel CARs that we developed and tested. Selection was based on extensive human tissue studies, in vitro testing against a wide range of human cancer cells and in vivo testing. In vivo huMNC2-CAR44 T cells were very effective in inhibiting tumor growth. Kaplan-Meier curves show that at Day 61, there were no tumor-related deaths for HCT-MUC1* mice in the CAR T treated group, whereas untransduced T cells and PBS control animals were all dead by Day 20. At Day 18 the average IVIS measurement for CAR T treated group was 0.5 e7 compared to 87 e7 for untransduced T-cell treated group. At present (Day 56), ovarian cancer control group animals have been ordered sacrificed. Average IVIS measurement for the control group is 743 e7 compared to 38 e7 for the CAR T treated group, with 2 mice with no detectable tumor and 2 more under 6 e7.

Conclusions: Subtle differences between MUC1* expressed on healthy stem-like cells and MUC1* expressed on cancerous cells have allowed development of cancer-specific MUC1* antibodies, especially suited for cancer immunotherapies. Tissue studies indicate huMNC2-CAR44 T cells should be safe and effective for treatment of breast and ovarian cancers. Conditional on IND approval, human testing will begin Q2/Q3 2018.

#2545

Antitumor efficacy of Type I polarized dendritic cells in combination with immune checkpoint blockade in a preclinical model of breast cancer.

Krithika N. Kodumudi, Doris Wiener, Amrita Basu, Brian Czerniecki. _H.Lee Moffitt Cancer Center, Tampa, FL_.

Breast cancer is the most commonly diagnosed cancer and a major cause of cancer death among women. Our group has shown progressive loss of the anti-HER2 Th1 immune response in HER2 positive breast cancer patients and administration of peptide-pulsed Type I polarized dendritic cell (DC1) vaccine induced strong anti-HER2 immune response with pathologic complete response rate (pCR) in DCIS (ductal carcinoma in situ) and early invasive breast cancer (IBC). Within the tumor microenvironment, presence of multiple factors, including expression of co-inhibitory immune checkpoint signals has been shown to inhibit anti-tumor immune responses and preventing complete tumor regression. Hence, there is a need to develop strategies that disrupt these negative regulators in the tumor microenvironment in order to achieve robust anti-tumor immune responses. In this study, we investigated whether blockade of immune checkpoints, PD-1 or PD-L1 in combination with DC1 vaccine enhance anti-tumor immune response in a preclinical model of HER2 positive TUBO breast cancer. Balb/c mice received TUBO cells subcutaneously on day 0. Two different treatment regimens were followed to examine anti-tumor efficacy. For regimen-1, TUBO- bearing mice received three doses of p66 (MHC class I) peptide-pulsed DC1 vaccine starting on day 7 at weekly intervals concurrently with intraperitoneal injection of anti-PD-1 or anti-PD-L1 monoclonal antibodies. For regimen-2, starting on day 7 mice received six doses of HER2 specific p66 peptide- pulsed DC1 vaccine twice a week. Monoclonal checkpoint antibodies were given after the last dose of DC1 vaccine. Mice that received DC1 vaccine followed by treatment with anti-PD-1 or anti-PD-L1 monoclonal checkpoint antibodies had significant delay in tumor growth compared to the mice that received DC1 or checkpoint antibodies alone. However, mice that received DC1 in combination with anti-PD-1 antibody had superior efficacy in delayed tumor growth and improved survival rate. TUBO- bearing mice that received regimen-1, with DC1 vaccine and checkpoint antibodies concurrently had no significant delay in the tumor growth compared to mice treated with DC1 alone. Induction of anti-tumor immune response using HER2 specific MHC class II peptide- pulsed DC vaccine in combination with immune checkpoint blockade is ongoing. Overall, these findings suggest that optimal scheduling of immune checkpoint blockade is critical in improving anti-tumor efficacy of DC1 vaccine. This could be a potential combinatorial approach for the treatment of HER2 positive breast cancer in clinical settings.

#2546

Regulation of CAR-T cell therapy in real time using bispecific small molecule adaptors and monospecific competitors.

Haiyan Chu,1 Yingjuan Lu,1 Yong Gu Lee,2 Leroy Wheeler,1 Mellissa Nelson,1 Elaine Westrick,1 Marilynn Vetzel,1 Patrick J. Klein,1 Philip S. Low,2 Christopher P. Leamon1. 1 _Endocyte, West Lafayette, IN;_ 2 _Purdue University, West Lafayette, IN_.

Chimeric antigen receptor modified T cell (CAR-T) therapy has revolutionized personalized cancer treatment. The recent FDA approval of two CAR-T therapies for hematological cancers has marked a pivotal milestone in this new era of cellular therapies. CAR-T cells are genetically modified to become activated and proliferate in the presence of tumor antigens. In patients with high tumor burden and rapid T cell expansion, however, severe side effects including severe cytokine release syndrome (sCRS) and life-threatening cerebral edema may occur.

To "fine-tune" CAR-T cell activity in real time, we devised a three-pronged therapeutic strategy by using (1) 4-1BB-CD3ζ CARs expressing anti-fluorescein (anti-FITC) scFv instead of an anti-tumor ligand/self-antigen scFv, (2) low-molecular-weight bi-specific adaptors comprised of a FITC hapten linked to a tumor-specific ligand, and (3) competitors that can interfere with the adaptor/CAR-T interaction with tumor cells. Using folate-FITC (EC17) as the bi-specific adaptor molecule, we evaluated the performance of FITC-CAR-T cells in various folate receptor (FR)-positive tumor models. Our results show that CAR-T activation and proliferation in-vivo was EC17- and tumor FR-dependent. Treatment-related toxicity (sCRS) was observed but could be easily controlled, or even prevented, by adjusting the concentration, dosing frequency or timing of bi-specific adaptor administration. Importantly, animals experiencing sCRS could be rescued by administrating competitors of the bi-specific adaptor. In the case of EC17/CAR-T therapy, improved animal behavior was observed as early as a few hours after dosing with a rescue agent and a rapid reduction of interferon-γ was also detected. Circulating CAR-T cells remained functional, and could be re-activated following subsequent EC17 doses.

Taken together, our bi-specific adaptor/CAR-T approach offers a unique combination of a man-made hapten (FITC), flexible dosing control, and sCRS prevention/rescue without compromising antitumor activity or permanently removing CAR-T cells. Further studies are warranted to provide a rational basis for translating such therapeutic practice to the clinic.

Footnotes

HC and JL contributed equally to this work.

#2547

A potential new autologous mast cell cancer immunotherapy.

Jesse D. Plotkin,1 Michael G. Elias,1 Tracy R. Daniels-Wells,2 Anthony Dellinger,1 Manuel L. Penichet,2 Christopher L. Kepley1. 1 _UNC Greensboro, Greensboro, NC;_ 2 _UCLA, Los Angeles, CA_.

Mast cells (MC) are important immune cells that reside in tissue and are most widely recognized for their role as mediators of Type I hypersensitivity reactions (allergies). In certain cancers, such as breast cancer, the presence of MC in the tumor microenvironment has been associated with favorable prognosis, which may be due to their ability to release, through activation of the high affinity IgE receptor FcεRI, anti-tumor mediators such as tumor necrosis factor alpha (TNF-α) and granulocyte-macrophage colony-stimulating factor (GM-CSF). Thus, developing approaches to target autologous MC to tumor antigens is a potential new form of cancer immunotherapy. However, this concept has not been realized given that MC reside in tissue; thus, efficient removal, expansion ex vivo, and targeting to tumor sites has not yet been feasible. Previous attempts to generate MC from human progenitor cells have yielded cells that are often immature and not optimally functional. We report here, for the first time, the generation of functional human MC from adipose tissue in numbers sufficient for clinical applications. The adipose derived MC (ADMC) are phenotypically similar to connective tissue MC as evidenced through their expression of tryptase, chymase, c-kit, and FcεRI. The ADMC release several anti-tumor mediators through FcεRI activation. The ADMC, sensitized with an anti-HER2/neu IgE with human constant regions, bound to and were activated by HER2/neu-positive human breast cancer cells (SK-BR-3). ADMC sensitized in this way were observed to induce apoptosis in breast cancer cells when co-cultured. Importantly, breast cancer cell apoptosis was also observed after addition of media containing mediators released from activated ADMC. In conclusion, we present a novel method for the production of functional human MC from adipose tissue and demonstrate that these cells can be directed to target and eliminate breast cancer cells. It may now be possible to use them in conjunction with IgE antibodies targeting cancer cells for autologous mast cell cancer immunotherapy (AMCIT) as a new way to treat solid tumors such as those overexpressing HER2/neu for which new therapies are urgently need.

#2548

An image-based method to detect and quantify T cell mediated cytotoxicity of 2D and 3D target cell models.

Brad Larson,1 Diane Kambach,1 Wini Luty,2 Glauco Souza3. 1 _BioTek Instruments, Inc., Winooski, VT;_ 2 _BioreclamationIVT, Baltimore, MD;_ 3 _N3D Biosciences, Houston, TX_.

T cell-mediated cytotoxicity plays an important role in a suite of new methods being developed with the goal of incorporating a patient's immune system to combat cancer. The purpose of this study was to develop a sensitive, image-based in vitro method to evaluate and optimize adoptive T cell immunotherapies. Experiments were performed using MDA-MB-231 and fibroblast target cells co-cultured in 2D and 3D models, along with T cell to target cell ratios of 20:1, 10:1 and 5:1. T cell activation procedures were also tested where T cells were activated in the presence of anti-CD3 and anti-CD28 antibodies plus an IL-2 superkine only, or in a directed fashion with antibodies, superkine, and varying concentrations of target breast cancer cells. Experimental data show that directing the activation of T cells to recognize surface antigens on specific target cancer cells enhances the killing ability of the cells when compared to results from nondirected cells. In addition, increased T cell to target cell ratios induce higher levels of target cell cytotoxicity. Finally, it was evident that assay performance using 2D cultured target cells could be reliably carried out through a 72-hour incubation period. However, it exhibited limited utility beyond this point due to increasing numbers of uninduced necrotic cells within wells, negating the effect of test conditions. This phenomenon was unseen from 3D cultured target cells, indicating their ability to maintain a higher level of viability when cultured in this manner. Necrotic induction can then be reliably attributed to cell treatments using multiday incubations. The combination confirms the ability of the image-based method to deliver dependable results to assess T cell-mediated cytotoxicity using 2D and 3D target cell models.

#2549

Development of CAR T-cell therapy targeting glypican-3 in liver cancer.

Dan Li,1 Nan Li,1 Yifan Zhang,1 Haiying Fu,1 Madeline B. Torres,1 Qun Wang,2 Tim F. Greten,1 Mitchell Ho1. 1 _National Institutes of Health, Bethesda, MD;_ 2 _East China Normal University, Shanghai, China_.

Background: Glypican-3 (GPC3) is a candidate therapeutic target in hepatocellular carcinoma (HCC). We have generated the HN3 and hYP7 antibodies that recognize the N-terminus and C-terminus of GPC3, respectively. Here, we engineered human T cells that express GPC3-specific chimeric antigen receptors (CARs) and evaluated their potential for the treatment of HCC.

Methods: The GPC3-specific CARs (CAR.HN3 and CAR.hYP7) contain a CD3ζ chain and the 4-1BB co-stimulatory endodomain along with a truncated human EGFR polypeptide (huEGFRt) that retains the cetuximab epitope. A set of GPC3 positive-liver cancer cell lines including Hep3B, HepG2 and Huh7 were engineered to express the firefly luciferase gene. These cells were co-cultured with GPC3 CAR T cells at various ratios for 24 hours. A bioluminescent luciferase reporter assay was then used to evaluate the cytolytic activity of effector CAR T cells. We also evaluated the anti-tumor activity of CAR T cells in xenograft models with intraperitoneal injection of luciferase-expressing HCC cells into NOD/SCID/IL2gnull (NSG) mice. The tumor growth was measured via bioluminescence tumor imaging.

Results: We found that the CAR.hYP7 cells targeting the C-terminus of GPC3 showed the highest cytolytic activity against GPC3-positive HCC cells. In two HCC intraperitoneal xenograft models (Hep3B and HepG2), luciferase-expressing HCC cells preferentially grew on the mouse liver. More importantly, we found that a single intraperitoneal treatment with a high dose of CAR.hYP7 cells (40 million per mouse) exhibited sustainable antitumor efficacy in the Hep3B model and all of the mice survived after 9 weeks post CAR T-cell treatment without recurrence. The serum levels of alpha fetoprotein (AFP) in mice treated with a high dose of CAR.hYP7 cells were less than 50 ng/ml, the threshold value for HCC diagnosis. Additionally, a single intraperitoneal treatment with a low dose of CAR.hYP7 cells (10 million per mouse) caused regression of liver tumor xenografts. In the low dose CAR.hYP7 group, Hep3B tumors in the mice grew locally and restricted to the fat tissues far from the mouse liver, indicating that CAR T cells prevented tumors from seeding and growing in the liver. Although CAR.HN3 cells targeting the N- terminus of GPC3 also exhibited high cytotoxicity to HCC cells in vitro, no significant Hep3B tumor growth inhibition was seen in the mice treated with CAR.HN3 cells.

Conclusion: Our work suggests that GPC3-targeting CAR T cells, in particular CAR.hYP7, is a promising therapeutic intervention for liver cancer that can be translated to human use.

#2550

Generation of CAR-iPS-T cells expressing CD8β.

Tatsuki Ueda,1 Shoichi Iriguchi,1 Yohei Kawai,1 Atsutaka Minagawa,1 Hiroyuki Miyoshi,2 Seitaro Terakura,3 Yasushi Uemura,4 Knut Woltjen,1 Yuzo Kodama,5 Hiroshi Seno,5 Yasumichi Hitoshi,6 Tetsuya Nakatsura,4 Koji Tamada,7 Shin Kaneko1. 1 _Center for iPS Cell Reserch and Application, Kyoto University, Kyoto, Japan;_ 2 _Keio University, Tokyo, Japan;_ 3 _Nagoya University, Nagoya, Japan;_ 4 _National Cancer Center, Kashiwa, Japan;_ 5 _Kyoto University, Kyoto, Japan;_ 6 _Thyas Co. Ltd., Kyoto, Japan;_ 7 _Yamaguchi University, Yamaguchi, Japan_.

Chimeric antigen receptor (CAR) is an artificial protein that provides HLA-independent antigen specificity to T cells. CAR-T therapy has shown remarkable clinical responses especially in hematologic malignancies. But this therapy needs cell preparation for each patient. That limits its applicability. We have reported regeneration of T cells from iPSCs (Cell Stem Cell 2013). This technology provides unlimited number of T cells, so CAR-T therapy using iPSCs can broadens its applicability. Regenerated T cells previously described in some reports including ours have some different characters from peripheral T cells. In particular, they express only CD8α, and do not express CD8β. We successfully generate CAR-iPS-T cells expressing CD8β with some modification of differentiation protocols. We assayed the function of CAR-iPS-T cells in view of the difference between CD8β positive and negative iPS-T cells. We found CD8β positive CAR-iPS-T cells showed enhanced function to suppress tumor progression compared with CD8β negative CAR-iPS-T cells in subcutaneous xenograft model. In vivo kinetics study revealed that CD8β positive CAR-iPS-T cells have superior function to traffic to target expressing tumor site and enhanced sustainability in vivo compared with CD8β negative CAR-iPS-T cells. These findings indicate that CD8β positive CAR-iPS-T cells may be a potent cell source for iPSC-based cancer immunotherapy.

#2551

Allogeneic CRISPR engineered anti CD70 CAR T cells demonstrate potent preclinical activity against both solid and hematological cancer cells.

Zinkal Padalia,1 Ashley Porras,1 Mary Lee Dequeant,1 Jason Sagert,1 Thao Nguyen,1 Matthias John,1 Melanie Allen,1 Henia Dar,1 Daniel Henderson,1 Seshidar Police,1 Dakai Mu,1 Kelly Maeng,1 Elaine Huang,1 Sarah Spencer,1 Nickolaus Lorson,2 Paul Gonzales,2 Chelsea Holmquist,2 Gregg Hirschfeld,2 Jonathan A. Terrett,1 Demetrios Kalaitzidis1. 1 _Crispr Therapeutics, Cambridge, MA;_ 2 _TD2, Scottsdale, AZ_.

Autologous CAR T therapeutics have recently been approved for use in B-cell malignancies. While responses have been impressive using CD19 directed CAR T cells there has been a lack of comparable success for CAR T cells directed at solid tumor antigens. In an effort to address the need for effective and durable off-the shelf therapies for both hematologic and solid tumors we have developed allogeneic CAR T cells targeting the CD70 antigen. CD70 is expressed in both hematologic malignancies as well as in solid cancers such as renal cell carcinoma (RCC), while its expression in normal tissues is restricted to a subset of lymphoid cell types. CAR-T cells expressing a CD70 targeting CAR were generated by CRISPR Cas9 genome editing. T cells from healthy donors were edited to express a CD70 CAR by knocking this construct into the concurrently knocked out TCR alpha constant region (TRAC). Loss of the TCR reduces the risk of graft versus host disease enabling an allogeneic therapeutic. CD70 CAR T cells displayed potent cell killing function in vitro against CD70 expressing lymphoid and renal cancer derived cell lines across a broad range of antigen expression levels. CD70 CAR T cells also secreted IFNg, released granzyme B and proliferated in a CD70 specific manner. Furthermore, the CD70 targeting CAR T cells were able to eliminate established ccRCC tumor xenografts in mice

#2552

Preclinical development of T-cell receptor therapy targeting the 5T4 tumor antigen on renal cell carcinoma.

Yuexin Xu, Alicia J. Morales, Michael J. Cargill, Andrea Towlerton, Edus H. Warren, Scott S. Tykodi. _Fred Hutchinson Cancer Research Ctr., Seattle, WA_.

Purpose: The cancer-testis tumor antigen 5T4 is expressed on ~ 95% of primary RCC tumors as well as a variety of other tumor types but not on healthy tissue. Consequently, 5T4 has been targeted by antibody and vaccine therapies in cancer patients. Our group has previously isolated high-avidity human CD8+ T-cell clones specific for 5T417-25 and HLA-A2 that are capable of recognizing RCC tumor in vitro and as a murine xenograft. These clones provide the source for T-cell receptor alpha (TRA) and beta (TRB) genes to develop engineered T cells with re-directed cytotoxic specificity for tumors expressing the 5T4 antigen.

Experimental Procedures: Targeted single-cell RNA-seq was performed on 5T417-25 specific T-cell clones to sequence the highly variable complementarity-determining region 3 (CDR3) of TRA and TRB genes. Full length 5T417-25 specific TRA and TRB sequences were then re-constructed and modified by codon optimization and introduction of transmembrane domain cys-cys covalent crosslinking to augment correct alpha and beta chain pairing. TRA and TRB genes were cloned into third generation lentiviral vectors and transduced into CD8+ T cells from healthy donors. Re-directed effector function for target cells expressing 5T4 was measured by chromium release assay and IFN-gamma ELISA. CRISPR/Cas9 gene editing techniques to augment effector activity of engineered T cells by suppressing native TCR expression and disrupting inhibitory co-receptor genes for PD-1, LAG-3, or TIM-3, are also under development.

Results: Seven unique TRA-TRB pairs were identified from a panel of 38 5T417-25 specific CD8+ T cell clones. Lentiviral mediated expression of all seven 5T417-25 specific TCR transgene constructs was detectable by 5T417-25/HLA-A2 tetramer immunostaining with transduction efficiency from 30% to 80%. 5T4-TCR transduced CD8+ T cells demonstrated re-directed cytotoxic activity to 5T4+/HLA-A2+ RCC, breast, and colorectal tumor cells, but not to 5T4-/HLA-A2+ normal cells. CRISPR/Cas9 targeting of a conserved sequence in the native TRA constant region was sufficient to suppress native TCR expression on polyclonal CD8+ T cells derived from healthy donors. Characterization of the proliferation kinetics, differentiation phenotypes and effector functions of 5T4-TCR transduced T cells modified by gene editing inhibition of native TRA or inhibitory co-receptors (PD-1, LAG-3 or TIM-3) is ongoing.

Conclusions: Transgenic T cell products can be re-directed to recognize a broadly shared epitope of the 5T4 tumor antigen and to kill 5T4+ tumor lines in vitro. Further augmentation of effector activity and specificity by CRISPR/Cas9 gene editing techniques is a developmental priority. Transgenic 5T4-reactive T cells are of interest for future testing as a therapeutic cellular immunotherapy product in patients with 5T4+ tumors.

#2553

**PSMA-specific, TGF-ß-insensitive CD8** + **T cells derived from metastatic castration resistant prostate cancer (mCRPC) patients induce apoptosis of PSMA positive prostate cancer (PCa).**

Qiang Zhang,1 Brian Helfand,2 Ximing Yang,1 Benedito Carneiro,1 Francis J. Giles,1 Timothy Kuzel,3 Chung Lee,1 Massimo Cristofanilli1. 1 _Northwestern Univ., Chicago, IL;_ 2 _NorthShore University HealthSystem, Chicago, IL;_ 3 _Rush University, Chicago, IL_.

Introduction: Manufacture of tumor specific CD8+ T cells has been an obstacle for developing immunotherapies for PCa, especially in patients with mCRPC. Here we report a new immunotherapeutic approach using adoptive transfer of patient-derived PSMA-specific, TGF-ß-insensitive human CD8+ T cells to induce the apoptosis of PCa. Methods: Peripheral blood CD8+ T cells were collected from mCRPC patient by leukapheresis, and cultured in FDA approved Cell Processing Work Station with CD-3/CD28 beads. We developed a TßRIIDN-TK-IRES-PZ1 chimeric T cell receptor retroviral construct using an anti-PSMA IgTCR(ζ) gene (PZ1) and a dominant negative TGF-ß type II receptor (TßRIIDN), that could induce CD8+ T cells to be PSMA reactive and insensitive to TGF-ß. PC-3 cells (PSMA negative) or PSMA positive PC-3-PSMA cells were used for target cells for in vivo study. Subcutaneous injection of PC-3 and PC-3-PSMA cells (2x105 cells/each) into the left and right flank region respectively in each of 32 immunodeficient RAG-1 mice was performed. One week later, the animals were randomly assigned to one of three adoptive transfer groups (16 mice /each group, 2x106 CD8+ T cells/each mice): Group 1: PSMA-specific, TGF-ß-insensitive CD8+ T cells infected with TßRIIDN-TK-IRES-PZ1 (71.1% positive); Group 2: Naïve CD8+ T cells. The animals were provided 2 weekly adoptive transfer treatments and sacrificed after 3 weeks. The infiltration of CD8+ T cells and apoptosis of tumor tissue was evaluated by immunofluorescence staining and TUNEL assay. Results: In Group 1, the average tumor weight was significantly lower in the PC3-PSMA tumor (0.413g) compared to the PC3 tumor (2.75 g). There was no difference between the PC3 tumor (2.36g) compared to PC3-PSMA tumor (2.45g) in Group 2. H&E staining showed large amount of nuclear fusion, fragmentation and necrosis were found in PC3-PSMA tumors in Group 1 compared to Group 2. In Group 1, tumor apoptosis (72.5/1,000 μm2) and CD8\+ T cell infiltration (45.5/1,000 μm2) in PC3-PSMA tumor parenchyma was significantly higher compared to PC3 tumor (6.7/1,000 μm2 and 3.1/1,000 μm2 respectively). There was no significant apoptosis or CD8\+ T cells infiltration observed in either PC3 or PC3-PSMA tumor in Group 2 and Group 3. This result indicated that PSMA-specific, TGF-ß-insensitive CD8+ T cells can infiltrate into the PSMA positive tumor parenchyma and induce tumor apoptosis. Conclusion: This study demonstrated that our approach combines TGF-ß insensitive with PSMA selectivity can significantly enhance the specificity and anti-tumor ability of mCRPC patient's naïve CD8+ T cells, and simultaneously suppress the tumor by induce significant tumor apoptosis. Therefore, these PSMA-specific, TGF-ß-insensitive CD8+ T cells may offer a novel therapeutic intervention for both primary PCa treatment as well as for disease recurrence.

#2554

To clone tumor-specific TCRs from a cell-based immunotherapy-benefit cervical cancer patient.

Xiaoling Liang, Shudan Ou, Minjun Ma, Xihe Chen, Xiangjun Zhou, Yanyan Han. _HRYZ Biotech. Co., Shenzhen, China_.

Adoptive transfer of tumor-specific T cell receptor (TCR)-engineered T cells, TCR-T therapy, has shown great efficacy in clinical trials against solid tumors. A cervical cancer patient with bone metastasis was treated with a cell-based immunotherapy, named Multiple-Antigens Stimulating Cellular Therapy (MASCT), which is a combination of multiple tumor antigen peptides loaded dendritic cells (DCs) and autologous T cells stimulated by these DCs. After repeated treatment, the patient showed partially response and remained stable disease for 20 months. Moreover, boosted specific immune responses were detected in patient's periphery blood by IFNγ-Elispot assay against various tumor antigens, such as CEA, RGS5 and HPV18/58. The clinical benefits of this patient indicated that tumor-specific T cells were expanded in vivo and played an important role to control tumor progression. These T cells may be the good sources to isolate tumor-specific TCRs for TCR-T therapies. Given that, patient's PBMCs were stimulated in vitro with selected tumor peptides respectively, such as CEA, RGS5 and HPV18 E7. Tumor-specific T cells were defined as IFNγ-secreting T cells upon peptide stimulation and were further enriched by using magnetic beads. Part of the enriched T cells were analyzed for TCR alpha/beta repertoire via NGS, the other part were sorted into single T cells and analyzed for TCR alpha/beta pairing. The TCR repertoire NGS data revealed that the tumor-specific T cells harbor several hundred unique TCR alpha/beta clonotypes. Some unique TCR alpha/beta clonotypes were considered as tumor-specific TCR candidates, since the copy numbersof these clonotypes were significantly and repeatedly increased after stimulation with CEA, RGS5 or HPV18 E7, and further increased after IFNγ enrichment. After TCR alpha/beta pairing confirmation by single T cell sequencing, alpha and beta chains of these TCRs were sent to synthesis for further investigation. In conclusion we have successfully identified specific TCR clonotypes targeting selected tumor antigens from a MASCT-benefited cervical cancer patient. After functional validations, including epitope specificity, HLA restriction as well as tumor recognition, these tumor-specific TCRs may be good candidates for developing safer and more effective TCR-T therapy targeting multiple tumor antigens.

#2555

Engineering adoptive T cell therapy to co-opt Fas ligand-mediated death signaling in solid tumors.

Kristin Anderson,1 Shannon Oda,1 Breanna Bates,1 Edison Chiu,2 Christopher Morse,1 Nicolas Garcia,1 Philip Greenberg1. 1 _Fred Hutchinson Cancer Research Ctr., Seattle, WA;_ 2 _University of Washington, Seattle, WA_.

Over 20,000 women are diagnosed with ovarian cancer annually, and over half will die within 5 years. This rate has changed little in the last 20 years, highlighting the need for therapy innovation. A promising new strategy with the potential to control tumor growth without toxicity to healthy tissues employs immune T cells engineered to target proteins uniquely overexpressed in tumors. Mesothelin (MSLN) contributes to the malignant and invasive phenotype in ovarian cancer, and has limited expression in healthy cells, making it a candidate immunotherapy target in these tumors. Using patient-derived cell lines and the ID8 mouse cell line, we found that T cells engineered to express a human or mouse MSLN-specific high-affinity T cell receptor (TCRMSLN) can kill respectively human or murine ovarian tumor cells in vitro. In a disseminated ID8 tumor model, adoptively transferred TCRMSLN T cells preferentially accumulated within established tumors, delayed ovarian tumor growth, and significantly prolonged mouse survival. However, our data also revealed that elements in the tumor microenvironment (TME) limit engineered T cell persistence and ability to kill cancer cells.

To identify immunosuppressive features active in the ovarian TME in both human and murine disease, we performed gene expression analyses on whole tumor tissue (Thermo Fisher) or sorted cell populations (Nanostring). Deep transcriptome profiling confirmed the expression of similar gene signatures in human cancers and the preclinical ID8 model, including immunosuppressive pathways. RNA sequencing revealed consistently high expression of Fas ligand (FasL), notable because FasL signaling can play a significant role in the generation and persistence of tumors. Fas/FasL signaling can mediate T cell death, including activation-induced cell death, an apoptotic mechanism responsible for regulating T cell expansion, suggesting tumor cells may upregulate FasL for protection from tumor-infiltrating lymphocytes. By flow cytometry and immunohistochemistry, we and others previously detected FasL in the tumor vasculature and TME of human ovarian cancer. To overcome this potential T cell evasion mechanism, we generated a panel of immunomodulatory fusion proteins (IFP) containing the Fas receptor (Fas) extracellular binding domain fused to a CD28 or 4-1BB co-stimulatory domain rather than the natural death domain. Relative to T cells modified with only TCRMSLN, T cells engineered to express both TCRMSLN and a Fas IFP better infiltrate tumors, preferentially expand/persist, and retain function in the TME when transferred into tumor-bearing mice. Moreover, adoptive immunotherapy with IFP+ T cells significantly prolonged survival in tumor-bearing mice, relative to TCRMSLN T cells lacking an IFP. As many solid tumors overexpress FasL, the use of IFPs may provide an opportunity to enhance engineered adoptive T cell therapy in many other malignancies.

#2556

Expanded ovarian cancer patient NK cells represent a novel, cytotoxic phenotype and reduce autologous tumor in a patient-derived xenograft ovarian cancer model.

Sophie M. Poznanski,1 Tina Nham,1 Marianne V. Chew,1 Amanda J. Lee,1 Isabella Y. Fan,1 Dean A. Lee,2 Hal Hirte,1 Ali A. Ashkar1. 1 _McMaster University, Hamilton, Ontario, Canada;_ 2 _Ohio State University, Columbus, OH_.

Introduction: Ex vivo expanded NK cells are emerging as a promising cancer immunotherapy and have recently shown clinical effectiveness in treating hematological malignancies. NK cell therapy has encountered challenges against solid tumors, such as ovarian cancer, due to the immunosuppressive tumor environment which inhibits NK cell function. In addition, autologous NK cell therapy has been limited due to the diminished function of cancer patient NK cells and inhibition by self-MHC. In the present study, we assessed whether expansion of ovarian cancer patient NK cells overcomes limitations of autologous NK cell therapy against high grade serous ovarian cancer (HGSOC).

Experimental Procedures: NK cells were expanded ex vivo from peripheral blood (PB) and ascites of HGSOC patients or PB of healthy donors using K562-mb-IL-21 feeder cell expansion. We compared expanded and unexpanded NK cell cytotoxicity against ovarian cancer cells in vitro. Given recent evidence that CD56brightCD16- NK cells have greater anti-tumor potential than CD56dimCD16+ NK cells, we assessed relative expression of CD56 and CD16 on expanded NK cells. To study autologous NK cell therapy, we established a translational patient-derived xenograft (PDX) model of ovarian cancer. We adoptively transferred expanded ovarian cancer patient PB- and ascites-NK cells into cell-line xenograft and autologous PDX ovarian cancer models. Tumor burden, ascites progression, and survival of xenograft mice were assessed.

Results: We report that expanded NK cells express a novel CD56bright/brightCD16+ phenotype and have greater in vitro cytotoxicity than unexpanded NK cells against ovarian cancer cells. Adoptive transfer of expanded ovarian cancer patient PB- and ascites-NK cells reduced tumor burden of well-established ovarian cancer in cell-line xenograft mice to levels comparable to no tumor controls. Furthermore, expanded NK cells improved median survival by 3.4-5.2 times compared to untreated xenograft mice. Importantly, expanded ovarian cancer patient NK cells exerted a therapeutic effect in autologous PDX mice: expanded NK cells delayed ascites progression and inhibited development of peritoneal tumors.

Conclusions: Our results support an emerging concept that CD56bright NK cells have greater anti-tumor capacities than CD56dim NK cells. The therapeutic effects of expanded ovarian cancer patient NK cells on tumor burden and survival indicate that expansion converts immunosuppressed cancer patient NK cells to a cytotoxic subset that remains highly functional in an autologous tumor environment. The effectiveness of expanded ascites-NK cells identifies tumor-associated NK cells as a potential NK cell source for therapy. This study indicates that expanded autologous NK cells may be a promising therapy for ovarian cancer and other solid tumors.

#2557

Assessing the therapeutic efficacy of disease-specific T-cell biofactories.

Claire E. Repellin, Puja Patel, Lucia Beviglia, Harold Javitz, Lidia C. Sambucetti, Parijat Bhatnagar. _SRI International, Menlo Park, CA_.

Current protein-based cancer therapies have several disadvantages, which include general toxicity, lack of response at the administered dose and differing responses from patients to patient. To circumvent these issues, we propose a platform in which engineered T-cells specifically recognize tumors and secrete therapeutic peptides directly at the disease site leading to targeted cancer cell killing. This approach will increase the specificity of the therapy and decrease toxicity to healthy cells.

We transformed acute T-cell lymphoma cells into biofactories for site-specific synthesis of therapeutic proteins upon stimulation by antigen-presenting disease cells. The effector T-cell line was engineered by stably introducing a chimeric T-cell receptor to recognize Folate-Receptor alpha (FRα) or Mesothelin (MSLN) protein. Upon binding of the effector T-cells to the receptor, an intracellular cascade directed expression of non-human proteins is induced. Specific T-cell binding to human ovarian cancer cell lines and signaling was measured by in vitro co-culture using luciferase production as a surrogate for therapeutic peptide secretion.

We demonstrated that T-cells can be genetically programed to synthesize and secrete proportionate amounts of engineered proteins upon engaging the tumor-associated antigens (FRα or MSLN) on a human ovarian cancer cell line, OVCAR3 (FRα+MSLN+). A FRα-MSLN\- ovarian cancer cell line, A2780cis, was used as the non-targeted negative control. The difference in protein secretion following stimulation by the two cell lines, as measured by luciferase activity, was statistically significant within 1 hour. It reached ~35-fold within 1 to 3 days, and we observed stable expression for at least 10 days. The luminescent signal was proportionate to the number of OVCAR3 cells. To further validate the specificity of target engagement, we generated A2780cis-FRα positive and A2780cis-MSLN positive cell lines and demonstrated selective binding and activation of the corresponding effector cells in co-culture assays. No binding was detected to the A2780cis-vector control cells. In vivo results for T-cell biofactories targeting OVCAR3 tumors 24 hours post-stimulation validated the in vitro results. Currently, we are engineering T-cell biofactories to release cytotoxic peptides and are assessing their therapeutic efficacy against cancer cells in vitro using co-culture assays and supernatant transfer.

Our results show that T-cells can be genetically reprogrammed to serve as biofactories for the synthesis of therapeutic proteins upon stimulation by antigen-presenting disease cells. Importantly, these studies demonstrate the feasibility of developing the next generation of adoptively transferred T-cell therapies to target tumors that express FRα (e.g., ovarian, breast, lung) and/or MSLN (e.g. ovarian, lung, pancreatic) on their cell surfaces for cancer therapy.

#2559

Generation and evaluation of an FLT3 CAR-T cell therapy for the treatment of acute myeloid leukemia.

Tara L. Arvedson,1 Alice Bakker,1 Herve Lebrec,1 Gregor Adams,2 Armen Madiros,2 Priya Koppikar,3 Mercedesz Balazs,1 Mei Gong,1 Yan Zheng,1 Rebecca Goldstein,1 Tony Polverino,2 Lawren Wu,1 Angela Coxon1. 1 _Amgen, South San Francisco, CA;_ 2 _Kite Pharma, Santa Monica, CA;_ 3 _UT MD Anderson Cancer Center, Houston, TX_.

Background: FLT3 (CD135) is a receptor tyrosine kinase expressed by hematopoietic progenitor cells, and when activated by ligand binding, FLT3 signaling can induce survival, proliferation and differentiation. Acute myeloid leukemia (AML), an indication with high unmet need, derives from hematopoietic progenitor lineage cells; consequently, many AML patient samples retain FLT3 expression on circulating leukemic cells. In comparison to the wide expression seen in AML patient samples, normal tissue expression of FLT3 is restricted to the hematopoietic compartment: a subset of hematopoietic stem cells, a subset of lineage committed progenitor cells and dendritic cells. Cell surface FLT3 protein was not detectable in those solid tissues that express quantifiable FLT3 mRNA (cerebellum, pancreas). As a result of this favorable expression profile, targeting AML cells using T cells expressing a chimeric antigen receptor (CAR) directed against FLT3, is expected to provide benefit to patients.

Methods: A panel of fully-human, anti-human FLT3 antibodies were generated, screened for specific FLT3 binding, converted to single chain variable fragments (scFv) and re-evaluated for binding and stability. Three anti-FLT3 scFv sequences were selected for incorporation into CAR constructs. T cells transduced with these CAR constructs were evaluated in vitro for cytotoxicity, proliferation and cytokine secretion, and in vivo in a mouse xenograft model. The lead anti-FLT3 scFv was combined with an anti-CD3 scFv (bispecific T cell engager (BiTE®) format) for evaluation in non-human primates (NHP).

Results: Human T cells engineered to express anti-FLT3 CAR constructs were evaluated for cytotoxicity, proliferation and cytokine secretion in the presence of three FLT3-positive cell lines, each with a different number of surface-expressed FLT3 receptors, and one FLT3-negative cell line. While all constructs were selectively active against FLT3-positive cells, one construct was more active than the others against cells expressing very low levels of FLT3; 90% depletion of target cells with ~1600 receptors/cell was observed after 40 hours of co-culture with CAR T cells in a 1:1 ratio. This lead construct was further evaluated in a mouse xenograft model, where FLT3 CAR-T cells provided a survival advantage. The anti-FLT3 scFv used in the lead CAR construct was evaluated in NHP in the form of a BiTE®. In this 16-day, multi-dose study, the FLT3 BiTE® induced elimination of FLT3+ cells as assessed by depletion of FLT3 mRNA-expressing cells (97% reduction in FLT3 mRNA) in the circulation and increased levels of soluble FLT3 ligand in serum.

Conclusions: These results demonstrate the potential of using FLT3 CAR-T cell therapy for the treatment of AML.

#2560

Preclinical efficacy of allogeneic anti-CD123 CAR T-cells for the therapy of blastic plasmacytoid dendritic cell neoplasm (BPDCN).

Tianyu Cai,1 Kathryn L. Black,2 Ammar Naqvi,2 Roman Galetto,3 Agnès Gouble,3 Julianne Smith,4 Antonio Cavazos,1 Lina Han,1 Qi Zhang,1 Vinitha Kuruvilla,1 Sergej Naumovich Sergej Konoplev,1 Sattva S. Neelapu,1 Andrew A. Lane,5 Monica L. Guzman,6 Hagop Kantarjian,1 Andrei Thomas-Tikhonenko,2 Naveen Pemmaraju,1 Marina Konopleva1. 1 _UT MD Anderson Cancer Center, Houston, TX;_ 2 _The Children's Hospital of Philadelphia, Philadelphia, PA;_ 3 _Cellectis SA, Paris, France;_ 4 _Cellectis Inc, New York, NY;_ 5 _Dana-Farber Cancer Institute, Boston, MA;_ 6 _Weill Cornell Medical College, New York, NY_.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare, aggressive hematologic malignancy with historically poor outcomes and no established standard of care. Nearly 100% of patients with BPDCN overexpress CD123, and targeting CD123 emerged as an attractive therapeutic target given its differential expression on BPDCN cell surface.

UCART123 product (Cellectis) uses genetically modified allogeneic T-cells (derived from healthy donors, so-called "off the shelf") containing an anti-CD123 CAR and a RQR8 depletion ligand that confers susceptibility to rituximab. The expression of the T-cell receptor (TCR) is abrogated through the inactivation of the TCRα constant gene, using Cellectis' TALEN® gene-editing technology.

We have previously reported the selective in vitro anti-tumor activity of UCART123 cells against CD123+ primary BPDCN samples using cytotoxicity assays, T-cell degranulation assay and the secretion of IFNγ and other cytokines (IL2, IL5, IL6, IL-13 and TNF-α) by UCART123 cells when cultured in the presence of BPDCN cells (Tianyu Cai, 2017 ASH). However, UCART123 had minimum toxicity against normal bone marrow cells.

To evaluate in vivo anti-tumor activity of UCART123 cells, we established two patient-derived xenografts (PDX1-2) from patients with relapsed BPDCN in NSG-SGM3 mice. In PDX-1 model, all mice in vehicle-treated group died by D53, with high tumor burden in peripheral blood, spleen and bone marrow. Three out of 9 (33%) mice treated with 3×106 UCART123 and Six out of 9 (67%) mice treated with 10×106 UCART123 were alive and disease-free at the end of the study (D299).

In PDX-2 model, while UCART123 similarly extended survival of the mice (D104-241), relapses occurred in all treatment cohorts at D90-155. Flow cytometric analysis showed that all of the relapses were associated with emergence of CD123- BPDCN clones (95-96% CD123-). To understand the molecular basis for loss of CD123 surface expression, we isolated RNA from two CD123 positive samples from vehicle group and two CD123 negative samples from 1×106 UCART123 group. RT-PCR and RNA-seq detected the presence of full-length transcripts containing exons 1-12 in both CD123 positive samples. In one of the two CD123 (-) samples, CD123 transcripts were completely absent, along with loss of transcripts of neighboring genes. In another CD123 (-) sample, CD123 transcripts containing only exons 1-9 were detected, indicating the presence of a truncation. Interestingly, if translated, this transcript would produce a protein isoform lacking the transmembrane domain (Ex 10).

In summary, UCART123 therapy results in BPDCN eradication and long-term disease-free survival in a subset of primary BPDCN PDX models. However, loss of CD123 through diverse genetic mechanisms could lead to escape from UCART123 therapy and cause relapses. A phase I trial of UCART123 in BPDCN is opened for enrollment (NCT03203369).

#2561

Fully human immunoglobulin heavy chain only-derived CD33 CAR for the treatment of acute myeloid leukemia.

Dina Schneider,1 Ying Xiong,1 Weizao Chen,2 Zhongyu Zhu,2 Darong Wu,1 Jennifer Hwang,2 Dimiter S. Dimitrov,2 Boro Dropulic,1 Rimas J. Orentas1. 1 _Lentigen Technology Inc., a Miltenyi Biotec Company, Gaithersburg, MD;_ 2 _Protein Interactions Group, CCRNP, NCI, NIH, Frederick, MD_.

CD33 antigen is a promising target expressed on non-solid cancers, including acute myeloid leukemia (AML). Present investigative approaches to treatment of CD33-positive AML include antibody drug conjugates (My96, Mylotarg®) and CART cells incorporating CD33-targeting domains derived from a humanized scFv. Here, we designed a chimeric antigen receptor utilizing targeting domain derived from a fully human CD33 fragment variable heavy chain sequence, termed CAR33VH, and examined its in vitro and in vivo potency against AML.

Primary human CD4+ CD8+ T cells derived from three healthy donors were transduced with lentiviral constructs (LV) encoding CAR33VH, or control CAR construct based on scFv My96 (My96CAR). Flow cytometric analysis revealed expression of CAR33VH at 32%-45%, and expression of My96CAR at 77% - 86%. When challenged with CD33+ AML tumor lines HL60 and MOLM-14 in vitro, both constructs demonstrated efficient target killing. CD33- tumor lines K562 and Reh were not sensitive to CAR killing, underscoring CAR specificity to CD33 antigen. Pro-inflammatory cytokines IFN gamma, TNF alpha and IL-2 in culture supernatants of CART cells incubated with CD33+ HL-60 and MOLM-14 tumors, but not with CD33- K562 cells overnight, were induced, as measured by ELISA. Long-term co-incubation assay of CART cells with HL-60 leukemia at E:T ratios 1:5 to 1: 0.04, suggested similar killing potency and persistence of CAR33VH to the positive control scFv-based CAR. In in vivo AML model, NSG mice engrafted with MOLM-14 cells stably expressing firefly luciferase, both CAR33VH, and My96CAR control were equally efficient in tumor elimination.

In conclusion, CAR33VH, comprised of a fully human heavy-chain variable fragment only antigen binding domain, was efficient in tumor killing in vitro and in vivo, and may be used clinically for treatment of CD33+ hematologic malignancies. To our knowledge, this is one of the first instances demonstrating the feasibility of employing heavy chain only binder sequence in CART design.

#2562

Affinity-enhanced T-cell receptor (TCR) for adoptive T-cell therapy targeting MAGE-A4.

Joana Senra,1 Pamela Villalobos,2 Barbara Mino,2 Luisa Solis,2 Carmen Behrens,2 Joseph P. Sanderson,1 Manoj Saini,1 Andrew B. Gerry,1 Nicholas J. Pumphrey,1 Miguel Maroto,1 Boris Sepesi,2 Patrick Hwu,2 David S. Hong,2 Elizabeht A. Mittendorf,2 George S. Blumenschein,2 Ignacio I. Wistuba,2 Gwendolyn Binder-Scholl,1 Rafael Amado3. 1 _Adaptimmune, Oxford, United Kingdom;_ 2 _MD Anderson Cancer Center, Houston, TX;_ 3 _Adaptimmune, Philadelphia, PA_.

Melanoma-associated antigens-A (MAGE-A) are members of an extensive family of cancer/testis (CT) antigens. Although emerging studies highlight the potential role of MAGE-A family members in promoting cancer growth, the biological function of MAGE-A4 remains poorly understood. MAGE-A4 is an attractive target because it has a high frequency of expression in multiple solid tumors according to The Cancer Genome Atlas (https://cancergenome.nih.gov/). In an attempt to further characterize its expression in solid tumors, immunohistochemistry analysis was performed in 534 resected non-small cell lung carcinoma (NSCLC) cases, stage I to IV with clinicopathological information including overall survival and recurrence. MAGE-A4 expression was observed in 24% of all NSCLC cases, with higher frequency observed in squamous cell carcinoma (SCC) (51%) versus adenocarcinoma (8%). MAGE-A4 expression was observed in 23% of the primary tumors and 48% of the recurrent tumor samples. Log-rank (Mantel-Cox) analysis showed that histology diagnosis, pathological stage, and MAGE-A4 expression are independent prognostic factors in NSCLC. However, no MAGE-A4 prognostic value was observed when analyzing the SCC subtype. An HLA-A*02-restricted MAGE-A4 TCR specific for MAGE-A4230-239 peptide GVYDGREHTV was identified from a pool of parental TCRs and engineered to optimize specificity and activity (MAGE-A4c1032). TCR fine specificity was assessed initially by mapping the response of the MAGE-A4c1032T-cells to panels of synthetic peptides with combinatorial substitutions at each amino acid with every other possible amino acid (X-Scan), to identify potentially cross-reactive peptides in the human and common pathogen proteomes. Secondly, MAGE-A4c1032T-cells were screened against a wide panel of normal primary cells from multiple organ systems, induced pluripotent stem cell-derived cells (iCells), and autologous whole blood to test for off-target reactivity, and against a panel of EBV-derived B-lymphoblastic cell lines expressing a wide range of HLA molecules to assess the risk of alloreactivity. Alloreactivity was observed in antigen negative cells expressing HLA-A*02:05, and therefore this allele is exclusionary. MAGE-A4c1032T-cell potency was assessed by a variety of in vitro assays, including proliferation, IFN-γ release and cytotoxicity in response to antigen-positive tumor lines in 2D and 3D culture, and cytokine release in response to freshly prepared antigen-positive primary tumor material. In conclusion, MAGE-A4 is a promising target for SPEAR T-cell therapy, and clinical trial opened this year to treat patients with inoperable or metastatic (advanced) NSCLC (SCC, adenosquamous, or large cell carcinoma), ovarian, head and neck SCC, gastric, esophageal (SCC or adenocarcinoma), urothelial tumors and melanoma (NCT03132922).

#2563

Macrophage Toll-like receptor-chimeric antigen receptors (MOTO-CARs) as a novel adoptive cell therapy for the treatment of solid malignancies.

Edwin J. Velazquez, John E. Lattin, Taylor D. Brindley, Zachary Z. Reinstein, Roger Chu, Lu Liu, Evita G. Weagel, Michelle H. Townsend, Kiara V. Whitley, Eliza L. Lawrence, Brandon T. Garcia, Scott Weber, Richard A. Robison, Kim L. O'Neill. _Brigham Young University, Provo, UT_.

Recent clinical trials using chimeric antigen receptors (CAR) T cells have demonstrated tremendous success in eradicating hematologic malignancies. Notwithstanding the excitement generated by CAR T cell therapy, its clinical efficacy has not been effectively translated to the context of solid tumors; the physical barriers of solid malignancies and the immunosuppressive conditions at the tumor site hinder the efficacy of CAR T cells. Macrophages have the ability to infiltrate almost every tissue and frequently are recruited into tumors. Therefore, macrophages are an attractive vehicle for CAR therapy and could help solve current challenges that CAR T cells face in the treatment of solid tumors. MOTO-CAR cells are monocyte-derived human macrophages that are genetically modified by a lentiviral or adenoviral approach to express a synthetic tumor-targeting receptor and to secrete cytokines, ligands or chemokine receptors. MOTO-CAR receptors are composed of a single-chain variable fragment (ScFv) that binds to a specific tumor target, a hinge to link it to a transmembrane domain, and an engineered Toll/Interleukin-1 receptor (TIR) signaling domain. When the ScFv binds to the tumor cell via its tumor target, an activation signal is transmitted. Myd88 dependent and independent signaling cascades are elicited, activating the macrophage and polarizing it towards a proinflammatory phenotype to eliminate cancer cells in a selective way. We previously reported the expression of Thymidine Kinase 1 (TK1) on the cell membrane of the non-small cell lung carcinoma NCI-H460 and A549 cell lines. The in vitro function of TK1 MOTO-CAR cells was evaluated against these cancer cell lines, using GFP-based phagocytosis and killing assays. Additionally, cell migration and interaction was recorded using time-lapse video with a confocal microscope. Upon co-culturing, with its target TK1 specific MOTO-CARs showed a nearly 4-fold increase in killing activity when compared with the controls (p<0.01). MOTO-CAR cells were produced through a lentiviral approach with around 30% of the cells expressing MOTO-CARs and with an adenoviral approach using the Ad5f35 vector with an efficiency of 70-80% of cells being transduced. Furthermore, after transduction MOTO-CAR cells showed a consistent M1 phenotype expressing high levels of CD14, CD80, CD206 and low levels of CD163. Time-lapse videos showed migration and clustering of MOTO-CAR cells around H460 GFP + cells. Moreover, cell death was observed upon contact of MOTO-CAR cells with target cells as well as phagocytic activity. In vivo testing using an orthotopic NOD scid gamma mice model is in progress. Our preclinical data show evidence that human macrophages are a suitable vehicle for CAR therapy and have the potential to successfully extrapolate the clinical efficacy of CAR therapy to the context of solid tumors.

#2564

Selection of affinity-enhanced T-cell receptors for adoptive T-cell therapy targeting MAGE-A10.

Ellen Border,1 Joseph P. Sanderson,1 Thomas Weissensteiner,1 Natalie Hyland,1 Tom Holdich,1 Francine Brophy,2 Rafael Amado,2 Andrew Gerry,1 Nicholas Pumphrey1. 1 _Adaptimmune, Oxford, United Kingdom;_ 2 _Adaptimmune, Philadelphia, PA_.

Circulating T-cells, bearing T-cell receptors (TCRs) that have passed thymic selection, generally have low affinity for self-protein-derived cancer antigens and therefore have a limited ability to detect and eliminate tumor cells. Engineering TCRs to enhance and optimize their affinity, and therefore potency, for cancer targets is a promising strategy for adoptive immunotherapy in cancer patients. Modification of TCRs can potentially generate cross-reactivity to antigens expressed by normal tissue. Such cross reactivity might not be detected by in vivo animal studies, due to species differences in the antigenic repertoire. To mitigate the risk of such toxicities in clinical trials, we developed a comprehensive in vitro testing strategy. This strategy involves systematic substitution at each position of the antigenic peptide sequence using all natural amino acids to generate a profile of peptide specificity ("X-scan"). The likelihood of off-target reactivity was investigated by searching the human proteome for sequences matching this profile and testing against a panel of cell lines. Starting from a panel of parental TCRs with diverse sequences, we engineered several affinity-enhanced TCRs specific for the cancer-testis antigen MAGE-A10. The properties of two of these TCRs, which had the optimal balance of potency and specificity, could not be distinguished effectively with conventional biochemical and cellular assays. The X-scan method permitted us to select the most specific and potent candidate for further pre-clinical testing. This MAGE-A10ᶜ⁷⁹⁶TCR is now being studied in clinical trials to treat HLA-A2+ patients with non-small cell lung cancer (NCT02592577), urothelial cancers, melanoma, or head and neck cancers (NCT02989064).

#2565

EBV peptide-derived vaccine significantly enhanced in vitro cytotoxicity against EBV-positive B-cell lymphoma (EBV-BL) treatment using TMV-based delivery system.

Aradhana Awasthi Tiwari,1 Alison McCormick,2 Dina Edani,1 Aaron Newman,2 Christeen Azmy,1 Janet Ayello,1 Christian Klein,3 Mitchell S. Cairo1. 1 _New York Medical College, Valhalla, NY;_ 2 _Touro University California, California, CA;_ 3 _Roche, Zurich, Switzerland_.

Background: EBV latent proteins (LMP-1/2) increase the chemoresistance in Burkitt Lymphoma (BL) cells by constitutively activating the CD40 receptor. It has also been demonstrated that B- cell lymphoma resistant to rituximab is a result of LMP-1 induced miR-155 expression and survival mediated by AKT phosphorylation (Hong et al.Gene Ther, 2012). EBV peptides delivered by a virus carrier (Tobacco mosaic virus;TMV) has the potential to boost immunity and stimulate potent T cell responses against EBV-associated antigens and induce a virus specific T cell response against EBV infected malignant cells (Miles/Cairo et al. BJH,2012, Kemnade/McCormick et al. Vaccine, 2014).We previously observed that obinutuzumab (Anti-CD20 mAb) significantly enhanced cell death and increased overall survival against BL (Awasthi/Cairo et al. BJH, 2015) in xenografted NSG mice. However, synergistic effects of obinutuzumab in-combination with LMP1/2-peptide vaccine against mature-B-NHL are unknown.

Objective: To determine the efficacy of peptide specific cytotoxic T-lymphocytes (CTL) or the EBV vaccine alone/ in-combination with obinutuzumab against mature-B-NHL

Methods: Raji4RH (provided by M. Barth, MD, Roswell Park Cancer Institute) and Raji cells (ATCC, USA) were cultured in 10% RPMI. Tumor cells were incubated with TMV-RIED IgG (LMP2) with obinutuzumab (100ug/ml) for 4 hrs with NK cells. Cytotoxicity was determined by DELFIA cytotoxicity assay at 10:1 E:T ratio. Further, to generate LMP1/LMP2 peptide specific cytotoxic T cells (CTL), mature dendritic cells were pulsated with TMV-conjugated LMP1/LMP2 peptides for 24 hrs. PBMC isolated T cells were mixed with APC (LMPs-mature -DCs) at 1:20 ratio for activation and maturation. LMP-peptide specific CTLs were investigated in-vitro for cytotoxicity efficacy against BL cell lines.

Results: LMP1 and LMP2 peptide were successfully conjugated with TMV. TMV conjugated and purified serum vaccine was tested against BL cell lines. Conjugated serum vaccine (LMP2-(RIED-TMV) +obinutuzumab+NK, compared to obinutuzumab+NK or vaccine+NK alone significantly enhanced in-vitro cytotoxicity 62.67±7.82% vs. 53.66±6.43% vs. 50.8±5.2% (p=0.04 and 0.02), respectively against BL (Raji). Furthermore, cytotoxicity of CTL (LMP1-MSD-TMV) or (LMP2-TYG-TMV) cells were significantly enhanced compare to IL2 treated T cells only, 58.5±8.6% or 48.56±15.18% vs. 21.42±13.3% in Raji (P=0.01, 0.02), and 57.8±7.9% or 45.52±19.4% vs. 14.48±4.2% in Raji4RH (P=0.003, 0.002), respectively.

Conclusion: Our preliminary data indicates that LMP1 (MSD-TMV) and LMP2 (TYG-TMV) successfully conjugated with TMV and TMV-conjugated peptide specific CTL cells significantly enhanced cytotoxicity against BL cell lines compared to T cells only.

#2566

A TCL1-specific T-cell receptor redirects T cells against B-cell lymphomas and non-hematological tumors.

Jinsheng Weng, Kelsey E. Moriarty, Yong Pan, Flavio Egidio Baio, Hua Wang, Hiroki Torikai, Sourindra N. Maiti, Fuliang Chu, Xiaoyun Cheng, Swathi Karri, Deepshika Medapalli, Laurence J. N. Cooper, Sattva S. Neelapu. _UT MD Anderson Cancer Center, Houston, TX_.

T-cell leukemia/lymphoma Antigen1 (TCL1) is a B-cell oncoprotein that is overexpressed in multiple forms of B-cell lymphoma. In our previous study, we identified a HLA*A0201-restricted T cell epitope from TCL1 that stimulated CD8+ T cells to specifically lyse lymphoma cells. In this study, we have cloned the TCL170-79 peptide-specific T-cell receptor (TCR) gene that after lentivirus transduction into donor T cells can redirect the specificity of T cells to lyse T2 cells pulsed with TCL170-79 but not control peptide. The TCR-transduced CD8+ T cells efficiently lysed HLA-0201+, TCL1-expressing human lymphoma cell lines and primary lymphoma cells, but not normal B cells. Adoptive transfer of TCR-transduced T cells into a mantle cell lymphoma xenograft mouse model inhibited the tumor growth and significantly extended the survival of mice. In addition, we found that TCL1 is expressed in multiple solid tumors. The TCR-transduced T cells lysed colon and liver tumor cells but not normal human cells in vitro as well as in a mouse xenograft model. Collectively, our data demonstrate that TCL1-specific TCR-based immunotherapy is highly effective against human B-cell lymphomas and non-hematological tumors.

#2567

Novel therapeutic interventions to alleviate CAR T cell-induced cytokine release syndrome.

Theodoros Giavridis, Sjoukje J. van der Stegen, Justin Eyquem, Mohamad Hamieh, Michel Sadelain. _Memorial Sloan Kettering, New York, NY_.

CD19 chimeric antigen receptor (CAR) therapy is highly effective for some refractory B cell malignancies. Its broad implementation is limited in part by the occurrence of cytokine release syndrome (CRS), which is characterized by fever, hypotension and respiratory insufficiency that may require intensive care to overcome. CRS is associated with elevated serum cytokines, including interleukin-6 (IL-6), the blockade of which abates CRS in some but not all patients. The biological mechanisms involved in the pathogenesis of CRS are currently unknown, including the specific role of CAR T cells, tumor cells and other host cells. The development of relevant animal models is imperative in order to study the biology of CRS, analyze the role of different cell populations and ultimately identify interventions that not only treat but prevent CRS without reducing the therapeutic efficacy of CAR T cells.

Here we present a novel mouse model in which CD19 CAR-induced CRS is predictably and reproducibly elicited and mirrors the rapid onset and acuteness of clinical CRS. Symptoms present within 24 hours post CAR T cell administration, including rapid weight loss and eventual mortality. Survival correlates negatively with pro-inflammatory human (T cell-derived) and murine (host-derived) cytokine levels. A comparison between the cytokine fingerprint in patients with CRS and our mouse model revealed remarkable similarity between the two profiles. Importantly, IL-6 is elevated, as seen in patients receiving CAR therapy, but of mouse origin. In accordance with the demonstrated clinical benefit of tocilizumab, we demonstrate that CRS-associated mortality can be abrogated by murine IL-6 receptor blockade.

We further show that CAR T cell - tumor interaction is required for the robust recruitment of myeloid cells to the tumor site, including neutrophils, eosinophils, macrophages, monocytes and dendritic cells. Cytokine measurements and the transcriptomic analysis of myeloid populations from multiple sites reveal that IL-6 is of myeloid cell origin, dependent on CAR T cell - myeloid proximity and interactions at the tumor site. Furthermore, we probe the impact of myeloid involvement by activating or suppressing macrophage functions to show that CRS outcomes are largely determined by macrophage engagement.

Probing into the functions of macrophages led us to identify multiple therapeutic interventions that can ameliorate the toxicities of CRS. We have also designed a novel CAR construct that can prevent CRS-associated mortality while maintaining intact antitumor efficacy in a mouse model. Overall, our results suggest an indispensible role for the myeloid system in severe CRS, especially macrophages, representing a cellular compartment that has been largely overlooked in pre-clinical models to date. Moreover, our model should be useful to guide the rational design of novel CAR T cells that can autonomously ameliorate or prevent CRS.

#2568

CAR T cells secreting an immune checkpoint blockade scFv have enhanced anti-tumor efficacy.

Sarwish Rafiq,1 Oladapo Yeku,1 Hollie Jackson,1 Terence purdon,1 Dayenne van Leeuwen,1 Su Yan,2 Pei Wang,2 Jingyi Xiang,2 Cheng Liu,2 Venkatraman Seshan,1 Renier Brentjens1. 1 _Memorial Sloan Kettering Cancer Center, New York, NY;_ 2 _Eureka Therapeutics, Emeryville, CA_.

T cell therapies have had valuable clinical responses in patients with cancer. Chimeric antigen receptor (CAR) T cells are genetically engineered to recognize tumor cells and CAR T cell therapy has had impressive results in the setting of B cell acute lymphoblastic leukemia but has been less effective in treating other types of hematologic and solid tumors. The inhibitory tumor microenvironment (TME), including expression of ligands that bind inhibitory receptors on T cells, e.g. programmed death receptor 1 (PD-1), can dampen CAR T cell responses. To prevent PD-1-mediated dampening of CAR T cell function, we have co-modified CAR T cells to secrete PD-1 blocking single chain variable fragments (scFv). We first designed mouse constructs with which we could investigate the scFv-secreting CAR T cells in the context of a syngeneic immune-competent intact TME. CAR constructs were engineered directed against either human CD19 or MUC-16 (ecto) and an anti-mouse PD-1 scFv. Mouse T cells transduced with these constructs expressed the CAR on the surface and secreted detectable amounts of scFv that bound to mouse PD-1. The scFv-secreting CAR T cells were cytotoxic and produced IFN- γ when co-cultured with PD-L1 expressing tumors in vitro. We utilized a syngeneic mouse model to study scFv secreting CAR T cells in a model with an intact TME. In tumor-bearing mice treated with CAR T cells, scFv-secreting CAR T cells enhanced survival as compared to second generation CAR T cells. The survival benefit achieved with scFv-secreting CAR T cells was comparable to that achieved with systemic infusion of PD-1 blocking antibody, but with localized delivery of PD-1 blockade. Mice treated with scFv-secreting CAR T cells had detectable scFv in vivo in the TME. Lastly, long term surviving mice had detectable CAR T cells in the bone marrow by PCR, demonstrating persistence and suggesting an immunological memory. We next aimed to translate PD-1 blocking scFv CAR T cells to a clinically relevant human model utilizing a novel anti-human PD-1 blocking scFv. Human T cells modified with the CAR constructs express the CAR on the surface and secrete detectable amounts of PD-1 blocking scFv. The scFv binds to human PD-1 and scFv-secreting CAR T cells are cytotoxic to PD-L1 expressing tumors. Expression of PD-1-blocking scFv enhances CAR T cell function against PD- L1 expressing tumors in xenograft models of hematological and solid tumors by enhancing survival in tumor-bearing mice as compared to second generation CAR T cells. Furthermore, scFv-secreting CAR T cells exhibit in vivo bystander T cell enhancement of function, suggesting scFv-secreting CAR T cells can reactivate endogenous TILs in the TME. These data support the novel concept that localized delivery of scFv by CAR T cells can successfully block PD-1 binding to PD-L1 to enhance the overall anti-tumor efficacy of CAR T cell therapy.

#2569

CAR19 T cells secreting antigen-retargeting fusion proteins have remarkable potency against diverse tumor types.

Paul Rennert, Fay Dufort, Lihe Su, Lan Wu, Alyssa Birt, Roy Lobb, Christine Ambrose. _Aleta Biotherapeutics, Natick, MA_.

Introduction. CAR-CD19 T cell therapeutics are approved to treat B cell leukemias and lymphomas. Previously we showed that providing CAR19 T cells with a retargeting fusion protein (FP), consisting of soluble CD19 protein linked to an scFv, redirects CAR19 T cell cytotoxic activity to other tumor antigens. Further, exposure of CAR19 T cells to normal or malignant B cells expressing CD19 improves effector function and phenotype, due to the provision of costimulatory signals. Such signals are missing when CAR-T cells engage antigens on solid tumor cells. In addition, lack of sufficient antigen for expansion and persistence is a critical issue for CAR T therapeutics targeting solid tumors in the clinical setting. We have redirected CAR19 T cells to other tumor antigens via novel FP and bispecific FP (biFP) expression cassettes. CAR19 T cells that express FP or biFP solve critical issues in the cell therapy field by 1) promoting CAR19 T cell expansion, efficacy and persistence by engaging CD19 on B cells; 2) addressing antigen heterogeneity/escape in hematologic malignancies and solid tumors. FP- and biFP-mediated cytotoxicity is extremely potent at pM concentrations. Here we provide examples of in vitro and in vivo modeling to characterize the activity of the CAR19 T cells that are retargeted to kill other tumor types.

Experimental Procedures. A CAR-CD19 construct was created as the recipient for FP and biFP expression cassettes, in a lentiviral vector with an MCSV promoter. The CAR consists of the FMC63 scFv, a stalk, TM and CD28 and/or 4-1BB cytoplasmic signaling domains, and the CD3ε cytoplasmic signaling domain. FP and biFP cassettes were designed to encode the extracellular domain of the CD19 protein, followed by an scFv or VHH to one (FP) or two (biFP) antigens, separated from the CAR sequence by a P2A cleavage site.

Results. Flow cytometry and binding analyses demonstrated FP and bi-FP-mediated bridging between target tumor cells and CAR19 T cells. In vitro cytotoxicity studies showed robust redirected killing of antigen+ (CD19-) tumor cells by CAR19 T cells. This cytotoxicity is specifically mediated by the secreted FP or biFP and correlated with antigen affinity. The efficiency of cytotoxicity was enhanced by the presence of CD19+ B cells. Tumor xenograft models using Her2+ and CD38+ tumor lines demonstrated effective tumor killing in vivo, with or without the presence of CD19+ cells.

Conclusions. Redirected CAR19 T cells potently kill diverse tumor cells in vitro and in vivo. The strategy of redirecting CAR19 T cells to other tumor cell types by encoding novel FP and biFP expression cassettes solves critical issues in cell therapy by supporting CAR19 T cell activation and expansion and by providing a simple and modular multi-antigen targeting system. Further technology refinements include the creation of inducible promoters for FP expression, adding anti-PD-L1 activity, and providing cytokine support.

#2570

OFF-switch CAR T cell for safety-enhanced cancer immunotherapy.

Elise F. Gray-Gaillard,1 Greta Giordano Attianese,1 Pablo Gainza-Cirauqui,2 Sabrina Vollers,2 Sailan Shui,2 Bruno Correia,2 Melita Irving,1 George Coukos1. 1 _Ludwig Institute for Cancer Research at the University of Lausanne and Canton of Vaud Teaching Hospital, Lausanne, Switzerland;_ 2 _École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland_.

CARs are hybrid molecules comprising a tumor antigen-targeting moiety, typically a scFv, followed by a linker, transmembrane (TM) domain, and various endodomains (EDs) involved in T-cell activation. First generation CARs include the ED of CD3ζ only, required for 'signal 1' of T cell activation, while 2nd and 3rd generation CARs also have one or more co-stimulatory EDs, respectively, such as CD28 and 41BB, to provide 'signal 2'. CAR T cells have shown robust clinical responses against advanced haematologial malignancies, leading to two recent FDA approvals for CD19-targeting CARs. Solid tumors, however, remain an important challenge to CAR therapy, in part due to the fact there are limited cell surface-expressed antigens (protein, carbohydrate, etc.) that are tumor-restricted, thus running the risk of severe on-target/off-tumor toxicity. Here we describe our progress in the development of a split OFF-switch CAR for which a small molecule is used to stop signaling.

We designed a split CAR in which CD3ζ and the co-stimulatory ED are found on separate, co-expressed components. Briefly, Component A includes the scFv, a linker, TM domain, Protein A, and CD28, while the second one, Component B comprises a short extracellular region having no tumor-binding capacity, a TM domain, Protein B, a high affinity binding partner for Protein A, and CD3ζ. Proteins A and B were computationally designed from human proteins that normally do not interact, and upon the design of a novel protein-protein interaction (PPI) they form a heterodimer with an experimentally determined KD of 400 pM. Notably, this novel PPI was rationally designed to be rapidly disrupted by a clinically approved small molecule (KD of 10 pM for Protein B).

Lentiviral constructs were built encoding the two components separately, or together on the same vector, and used to engineer both a Jurkat NFAT promoter-mCherry reporter line, and primary human T cells following activation with anti-CD3/anti-CD28 beads. Flow cytometry was used to assess CAR cell-surface expression, and AMNIS imaging to visualize co-localization. Both Jurkat and T cells could co-express Components A and B, but when transduced on their own, neither A nor B could be detected on the cell-surface. However, we observed instability of Component B over time. Finally, we demonstrated co-localization of the chains in Jurkats, as well as functionality of the CAR, as measured by mCherry and IL2 production, in the presence of target cells. Ongoing work includes the titration of the OFF-switch small molecule with target cells in vitro, and in vivo. We have also re-designed Component B to include a larger extracellular domain in an attempt to stabilize its expression.

In conclusion, we used computational design to engineer a split-architecture CAR that can specifically activate engineered cells but further optimization is required to enhance its stability. We believe that such OFF-switch CARs hold important promise for increasing patient safety. 

### Biomarker Discovery 3

#2571

**An integral genomic/expression analysis reveals a cooperative role of** APC **and** TP53 **mutations in identifying cetuximab-sensitive colorectal cancer.**

Mingli Yang,1 Michael J. Schell,2 Andrey Loboda,3 Michael V. Nebozhyn,3 Jiannong Li,2 Jamie K. Teer,2 W. Jack Pledger,1 Timothy J. Yeatman1. 1 _Gibbs Cancer Ctr. and Research Inst., Spartanburg, SC;_ 2 _Moffitt Cancer Center & Research Institute, Tampa, FL; _3 _Merck, Sharp and Dohme, West Point, PA_.

The epidermal growth factor receptor (EGFR) is a major therapeutic target in metastatic colorectal cancer (CRC). Mutations in KRAS/NRAS have been identified as the resistance mechanisms to anti-EGFR antibody therapy (cetuximab/pantiumab). However, genomic understanding of the sensitivity to the EGFR-targeted therapy is lacking. A pre-specified, 203-gene cetuximab sensitivity (CTX-S) expression signature score was first validated on two previously published datasets: (1) 77 CTX-treated CRC tumors (progression free survival (PFS)>7 days), and (2) 147 in vitro CTX-treated CRC cell lines. The CTX-S signature was found to be consistently correlated with a newly developed 24-gene APC (truncating) mutations-specific Wnt/β-catenin score on four independent CRC datasets that also include Moffitt (n=468) and TCGA (n=624) CRC datasets. This promoted us to carry out a variety of analyses that revealed a cooperative role of mutant APC + mutant TP53 in identifying cetuximab sensitive subpopulations, which was further evidenced by additional validation analysis on two CRC PDX datasets (n=52 and n=98). While debating or inconclusive, other potential predictive markers of EGFRi therapies have been reported, including BRAF(V600E), PIK3CA mutations, loss of PTEN, and overexpression of HER2 as negative indicators, as well as increased EGFR copy number, and overexpression of EGFR ligands epiregulin and amphiregulin as positive indicators. Here, for the first time, we report that the mutant genes may cooperatively act as positive indicators of EGFRi response. Our study suggests that addition of the routine sequencing of APC and TP53 to extended RAS testing may refine the patient selection strategy to improve cetuximab/pantiumab response and potentially expand the therapeutic opportunity to previously-excluded mutant KRAS/NRAS patients who have "APC + TP53" mutations.

#2572

HSD3B1 **genotype and abiraterone metabolism in patients with prostate cancer.**

Mohammad Alyamani,1 Hamid Emamekhoo,2 Sunho Park,1 Jennifer Taylor,1 Nima Almassi,1 Sunil Upadhyay,3 Allison Tyler,1 Michael P. Berk,1 Tae Hyun Hwang,1 Petros Grivas,1 Brian Rini,1 Jorge Garcia,1 Richard J. Auchus,3 Nima Sharifi1. 1 _Cleveland Clinic, Cleveland, OH;_ 2 _University of Wisconsin Carbone Cancer Center, Madison, WI;_ 3 _University of Michigan Medical School, Ann Arbor, MI_.

Background The common HSD3B1 (1245C) germline variant encodes for a 3βHSD1 missense that increases enzyme activity that allows tumors to utilize extragonadal androgens and is a predictive biomarker of resistance to ADT and sensitivity to CYP17A1 inhibition by the nonsteroidal drug ketoconazole. However, 3βHSD is also the first enzyme necessary in the conversion from abiraterone, a steroidal CYP17A1 inhibitor, to 5α-abiraterone, which stimulates the androgen receptor (AR) and prostate cancer progression. The HSD3B1 (1245C) variant might therefore increase 5α-abiraterone synthesis in patients on abiraterone therapy, possibly limiting clinical benefit.

Significance This work aims to characterize the pharmacokinetics of steroidal metabolites of abiraterone and to determine the association between HSD3B1 genotype and the generation of 5α-abiraterone metabolites.

Patients and Methods Part 1: 15 healthy male volunteers received a single oral dose of 1000 mg abiraterone acetate plus 240 mg of the AR antagonist apalutamide, steroidal metabolites of abiraterone were quantitated at 12 time points from 0.5-96 hours post-dose. Part 2: 5α-abiraterone metabolites and HSD3B1 genotype was determine in 30 patients with castration-resistant prostate cancer (CRPC) on abiraterone therapy. Metabolite concentrations were normalized to the 8 hour time point of the pharmacokinetic study and the association between HSD3B1 genotype and 5α-abiraterone metabolites was determined.

Results There were 8, 19, and 3 pts with homozygous wild-type, heterozygous, and homozygous variant HSD3B1 genotypes. Patients who inherit 0, 1 and 2 copies of the HSD3B1 (1245C) variant have a stepwise increase in serum concentrations of 5α-abiraterone metabolites (p=0.002).

Conclusion The generation of 5α-abiraterone preferentially in patients with the HSD3B1 (1245C) variant might negate the benefits of abiraterone specifically in a patient subset that is otherwise more likely to benefit from CYP17A1 inhibition by the nonsteroidal drug ketoconazole.

#2573

Selection of candidate biomarkers for aggressive prostate cancer based on targeted proteomics.

Yuqian Gao,1 Hui Wang,1 Jennifer Cullen,2 Yongmei Chen,2 Athena Schepmoes,1 Gyorgy Petrovics,2 Thomas Fillmore,1 Tujin Shi,1 Wei-Jun Qian,1 Richard Smith,1 Sudhir Srivastava,3 Jacob Kagan,3 Albert Dobi,2 Karin Rodland,1 Shiv Srivastava,2 Tao Liu1. 1 _Pacific Northwest National Laboratory, Richland, WA;_ 2 _Uniformed Services University of the Health Sciences, Bethesda, MD;_ 3 _National Cancer Institute, Bethesda, MD_.

Mass spectrometry based targeted proteomics such as selected reaction monitoring (SRM) provides an effective, antibody-independent strategy for sensitive, specific and multiplexed verification of genomics biomarker candidates at the protein level. In order to identify a panel of proteins with the potential to predict prostate cancer progression, we have selected 52 protein candidates from existing prostate cancer genomics data sets and validated cancer drivers, and performed quantitative proteomics analysis in tissue samples using the highly sensitive PRISM (high-pressure, high-resolution separations coupled with intelligent selection and multiplexing)-SRM approach. PRISM-SRM assays have been developed for the 52 prostate cancer biomarker candidates including: prostate cancer relevant genes and common cancer drivers. One set of 105 formalin-fixed paraffin-embedded (FFPE) whole mount prostate tissue specimens were analyzed using PRISM-SRM with heavy isotope-labeled synthetic peptides as internal standards: 20 primary tumors from patients showing metastatic progression, 37 primary tumors from patients who showed biochemical recurrence (BCR), and 48 primary tumors from patients with no BCR or metastatic progression after more than ten years of follow-up after radical prostatectomy. Overall, PRISM-SRM analyses of the FFPE tissue samples enabled the detection of 42 out of 52 biomarker candidates; in comparison regular LC-SRM without the front-end chromatographic enrichment could detect only 21 of these candidates at the protein level. Kruskal-Wallis test of the PRISM-SRM results provided a statistical evaluation of comparison of relative protein levels among the "no progression", BCR and "metastatic progression" groups. Prostate differentiation/AR signaling related proteins (FOLH1, PSA and NCOA) or tumor progression (TGFB1, CCND1 and SPRC) were significantly different between the three groups. These promising biomarker candidates for early detection of aggressive prostate cancer are being further evaluated, individually and in panels, in an independent cohort of 234 samples for their potential prognostic applications. In summary, PRISM-SRM provides a highly sensitive method for quantification and rapid screening of promising cancer biomarker candidates defined by multiomics platforms.

#2574

Biglycan, tumor endothelial secreting proteoglycan, as possible biomarker for lung cancer.

Hirofumi Morimoto,1 Nako Maishi,1 Yasuhiro Hida,2 Hiroshi Nishihara,3 Yutaka Hatanaka,4 Yoshihiro Matsuno,4 Toru Nakamura,5 Satoshi Hirano,5 Kyoko Hida1. 1 _Vascular Biology, Hokkaido University IGM Institute for Genetic Medicine, Sapporo, Japan;_ 2 _Department of Cardiovascular and Thoracic Surgery, Graduate School of Medicine, Hokkaido University, Sapporo, Japan;_ 3 _Genomics Unit, Keio Cancer Center, Keio University School of Medicine, Sapporo, Japan;_ 4 _Department of Surgical Pathology, Hokkaido University Hospital, Sapporo, Japan;_ 5 _Department of Gastroenterological Surgery II, Graduate School of Medicine, Hokkaido University, Sapporo, Japan_.

[Backgroud and Purpose]: Tumor endothelial cells (TECs) have been reported to have various abnormalities. For example, TECs demonstrate higher expression levels of several molecules (TEC markers) compared with normal endothelial cells (NECs). Recently we identified biglycan (Bgn) as one of TEC markers. Bgn has been reported to be associated with tumor progression in various cancers. In addition, we have found that Bgn secreted by TEC promoted intravasation of tumor cells and lung metastasis in vivo mouse tumors. Lung cancer, of which non-small-cell lung cancer (NSCLC) is the most common form, remains the leading cause of cancer-related mortality worldwide, with many patients presenting with advanced disease at initial diagnosis. Moreover, in spite of recent advances in chemotherapy and targeted therapy, 5-year survival for patients with NSCLC is still disappointingly low at less than 20%. Therefore, it is urgent need to develop effective biomarkers for early diagnosis or new therapeutic target. In this study, we analyzed the association between Bgn expression in TEC in lung cancer and the cancer progression of these patients to evaluate the possibility of Bgn as a biomarker. [Method]: A total 46 patients with lung cancer, who had undergone thoracic surgery at Hokkaido university hospital from February 2015 to March 2017 , were enrolled in this study. First, we performed immunohistochemistry of CD31 and Bgn with serial sections of resected lung tumor tissues, and analyzed the correlation between Bgn positive vessel density in primary lung tumors and clinicopathological factors. Second, we measured the Bgn levels in preoperative serum by ELISA,and the correlation between Bgn positive vessel density in lung tumor tissue and blood Bgn levels. [Result]: The Bgn positive vessel density in primary lung tumor tissue was significantly correlated with primary tumor invasion (p=0.036) and pleural invasion (p=0.036). The preoperative serum Bgn level was also significantly correlated with primary tumor invasion (p=0.0017), pleural invasion (p=0.046) and preoperative blood c-reactive protein level (p=0.012). The Bgn positive vessel density in primary lung tumors was significantly correlated with preoperative serum Bgn level (p<0.0001). In case of lung adenocarcinoma, average of serum Bgn level in Ground Glass Opacity (GGO) positive cases (178ng/ml) was significantly lower than that in GGO negative cases (473ng/ml) (p=0.043). The serum Bgn level was significantly higher in the patients classified into high-grade group (1082ng/ml) by IASLC/ATS/ERS classification than that in intermediate-grade or low-grade groups (264ng/ml, p=0.037). [Conclusion]: In conclusion, these results suggested that Bgn expression in TEC could be a biomarker of lung cancer

#2575

NKX6.1 **hypermethylation predicts the outcome of stage II colorectal cancer patients undergoing chemotherapy.**

Ya-Wen Lin, Sou-Yi Chang, Chih-Chi Kuo, Cheng-Wen Hsiao, Chih-Hsiung Hsu, Yu-Ching Chou, Yu-Lueng Shih. _National Defense Medical Ctr., Taipei, Taiwan_.

Background: Colorectal cancer (CRC) is a common malignancy worldwide. CRC patients in the same stage often present with dramatically different clinical scenarios. Thus, robust prognostic biomarkers are urgently needed to guide therapies and improve treatment outcomes. The NKX6.1 gene has been identified as a hypermethylation marker in cervical cancer and functions as a metastatic suppressor by regulating epithelial-mesenchymal transition (EMT). Here, we investigated whether hypermethylation of NKX6.1 might be a prognostic biomarker for CRC.

Results: In this study, we analyzed the methylation and expression of NKX6.1 in CRC tissues and CRC cell lines. We quantitatively examined the NKX6.1 methylation levels in 151 pairs of CRC tissues by using methylation-specific polymerase chain reaction (MS-PCR) analysis and found that NKX6.1 was hypermethylated in 35 of 151 CRC tissues (23.18%). NKX6.1 gene expression was inversely correlated with the DNA methylation level in CRC cell lines in vitro. Then, we analyzed the association of NKX6.1 methylation with clinical characteristics of these CRC patients. NKX6.1 methylation was positively associated with tumor size [odds ratio (OR): 2.84; 95% confidence interval (CI): 1.24 to 6.52]. These data demonstrated that patients with NKX6.1 methylation presented poorer 5-year overall survival (p=0.0167) and disease-free survival (p=0.0083) than patients without NKX6.1 methylation after receiving adjuvant chemotherapy. Most importantly, our data revealed that stage II CRC patients with NKX6.1 methylation had poorer 5-year disease-free survival (p=0.0322) than patients without NKX6.1 methylation after adjuvant chemotherapy.

Conclusions: Our results demonstrated that methylation of NKX6.1 is a novel prognostic biomarker in CRC and may be used as a predictor of the response to chemotherapy.

#2576

Dclk1 can be a predictor for poor survival in patients with resected pancreatic cancer.

Yoshihiro Takemoto, Arata Nishimoto, Yukari Hironaka, Yuki Suehiro, Naruji Kugimiya, Atsushi Suga, Eijiro Harada, Kimikazu Hamano. _Yamaguchi University, Ube, Japan_.

Background: Doublecortin-like kinase-1 (Dclk1) shows higher expression levels in human pancreatic cancer compared with human normal pancreatic tissue. Dclk1 positively regulates tumor growth, invasion, metastasis, pluripotency factors, angiogenic factors, and epithelial-mesenchymal transition (EMT)-related genes in pancreatic cancer cells. We previously reported that Dclk1 inhibition in combination with gemcitabine (GEM) treatment offers a novel approach to treat pancreatic cancer. However, few studies focusing on the role of Dclk1 in pancreatic cancer using clinical specimens have been reported. Objective: To investigate the expression of Dclk1 in resected specimens of pancreatic cancer and its prognostic significance. Methods: Tumor specimens were obtained from 26 patients with pancreatic cancer (stage 3 and 4) who underwent resection between April 2005 and March 2015 in our department. Dclk1, Ki67, and phosphorylated MEK expression levels were examined using immunohistochemical staining. The expression levels of these markers were evaluated using Histoscore (Intensity [0-3] x Extent [0-4]). In order to identify proteins phosphorylated by Dclk1, we analyzed a protein microarray using a pancreatic cancer cell line. Results: In total, 42.3% (11/26) of samples expressed high levels of Dclk1 (Histoscore≧3.3) and were classified into the high Dclk1 group. There were no significant differences between patients in the high Dclk1 group and the low Dclk1 group in terms of clinical characteristics and surgical findings. Patients in the high Dclk1 group exhibited significantly shorter survival times than those in the low Dclk1 group (P < 0.05). The expression of phosphorylated MEK, which is observed on a mitogen-activated protein kinase (MAPK) pathway, decreased by >50% in Dclk1-inhibited pancreatic cancer cells by protein microarray. Interestingly, the expression of pMEK and Ki67 in resected specimens tended to increase in samples in the high Dclk1 group. Therefore, Dclk1 might contribute to the progression of pancreatic cancer through the phosphorylation of MEK. Conclusion: Dclk1 may be a marker for poor prognosis in patients with resected pancreatic cancer.

#2577

Predictive value of multigene assay for chemotherapy benefit in patients with HR+/HER2- early breast cancer.

Mi Jeong Kwon. _Kyungpook National Univ., Daegu, Republic of Korea_.

Our risk score based on a combination of multigene expression and clinical variables was validated to predict the risk of distant metastasis in hormone receptor-positive and human epidermal growth factor 2-negative (HR+/HER2-) early breast cancer in our previous study. In addition to its prognostic value, we investigated the predictive value of the risk score for adjuvant chemotherapy benefit in lymph node-negative (LN-), HR+/HER2- breast cancer. 346 patients who treated with hormone therapy (n = 203) alone or hormone therapy plus chemotherapy (n = 143) were classified as low-risk and high-risk group according to the risk score. There was a significant improvement in 10-year distant metastasis-free survival (DMFS) by the addition of adjuvant chemotherapy to hormone therapy in patients with a high risk score (26.5%, P = 0.003), whereas no benefit of chemotherapy was observed in those with a low risk score. The test for interaction between chemotherapy treatment and risk score was significant. The risk score also identified patients who will not benefit from chemotherapy among clinical high-risk group. Our study suggests that the risk score predicts the benefit of adjuvant chemotherapy in LN-, HR+/HER2- breast cancer and the addition of the risk score in the traditional cliniopathological factors may aid the improved treatment decisions.

#2578

Expression of epiregulin/amphiregulin associates with prognosis in recurrent/metastatic head and neck cancer treated with cetuximab.

Yasunao Kogashiwa, Hitoshi Inoue, Kiyomi Kuba, Masanori Yasuda, Mitsuhiko Nakahira, Masashi Sugasawa. _Saitama Medical University International Medical Center, Hidaka, Japan_.

Objectives: The anti-EGFR antibody cetuximab, combined with chemotherapy, is the standard therapy for recurrent/metastatic (R/M) head and neck squamous cell carcinoma (HNSCC). However, the response rate and survival benefit are limited. The purpose of this study was to investigate the expression of the epidermal growth factor receptor ligands epiregulin (EREG) and amphiregulin (AREG), as predictive prognostic markers for the efficacy of cetuximab combined with chemotherapy in R/M HNSCC.

Materials and methods: We conducted a retrospective analysis of 37 patients diagnosed with R/M HNSCC. Tumor samples from biopsy specimens or surgical specimens were analyzed for EREG and AREG mRNA gene expression, and clinical outcomes evaluated. Survival rates were estimated by Kaplan-Meier analysis. Cox proportional hazard regression was used to test the potential association between ligand expression, overall survival (OS), and progression free survival (PFS).

Results: Values of EREG ≤ 15.87 and AREG ≤ 11.80, determined by receiver operating characteristic (ROC) curve analysis, were associated with significantly longer OS (p=0.03 and p=0.01, respectively), as well as PFS (p=0.04 and p=0.04, respectively). EREG and AREG expression levels were significantly correlated (r=0.73, p<0.01). Multivariate analysis revealed that AREG expression was significantly associated with OS and PFS.

Conclusion: R/M HNSCC patients with higher EREG and AREG gene expression appear to benefit more from cetuximab combined with chemotherapy than those with lower expression. The potential for these ligands as prognostic biomarkers for cetuximab-containing regimens requires further evaluation.

#2579

Loss of chromosome 18q11.2-18q12.1 is predictive for progression-free survival in metastatic colorectal cancer patients treated with bevacizumab.

Erik van Dijk,1 Hedde Biesma,1 Martijn Cordes,1 Dominiek Smeets,2 Maarten Neerincx,1 Sudipto Das,3 Verena Murphy,4 Anna Barat,5 Orna Bacon,3 Jochen H.M. Prehn,3 Johannes Betge,6 Gaiser Timo,6 Bozena Fender,7 Gerrit A. Meijer,8 Deborah A. McNamara,5 Rut Klinger,9 Miriam Koopman,10 Matthias P.A. Ebert,6 Elaina W. Kay,5 Bryan T. Hennessey,3 Henk M.W. Verheul,1 William M. Gallagher,9 Darran P. O'Connor,3 Cornelis J.A. Punt,11 Fotios Loupakis,12 Diether Lambrechts,2 Annette Byrne,3 Nicole C.T. van Grieken,1 Bauke Ylstra1. 1 _VUmc University Medical Center, Amsterdam, Netherlands;_ 2 _KU Leuven, Leuven, Belgium;_ 3 _Royal College of Surgeons in Ireland, Dublin, Ireland;_ 4 _Cancer Trials Ireland, Dublin, Ireland;_ 5 _Beaumont Hospital, Dublin, Ireland;_ 6 _University Hospital Mannheim, Mannheim, Germany;_ 7 _OncoMark Ltd, Dublin, Ireland;_ 8 _Netherlands Cancer Institute, Amsterdam, Netherlands;_ 9 _University College Dublin, Dublin, Ireland;_ 10 _University Medical Center Utrecht (UMCU), Utrecht, Netherlands;_ 11 _Academic Medical Center, Amsterdam, Netherlands;_ 12 _Veneto Institute of Oncology IOV, Padova, Italy_.

Introduction: Most patients with metastatic colorectal cancer (mCRC) have a limited benefit from the addition of bevacizumab to standard combination chemotherapy. However, a subset of patients benefits substantially, highlighting an unmet clinical need for a predictive biomarker of response to bevacizumab. Previously, we demonstrated that losses in chromosome 5q34, 17q12 and 18q12.1-21.1 have a significant correlation with progression free survival (PFS) in patients treated with, but not without bevacizumab. These data warrant extensive validation. Patients: For validation, two cohorts of patients that received bevacizumab were analysed, one of 121 mCRC patient samples of the European multicenter study Angiopredict and one of 81 mCRC patient samples from the Italian multicenter study MoMA. A third cohort was included of 90 mCRC samples from patients that did not receive bevacizumab. Genome wide copy number aberrations were correlated with PFS, Results: Loss of chromosome 18q11.2-18q21.1 most significantly associated with PFS in both cohorts that received bevacizumab (Angiopredict: HR=0.61, p=0.016; MoMa: HR=0.55, p=0.019). No significant association with PFS was observed in the cohort that did not receive bevacizumab (HR=0.85, p=0.67). Patients of all three cohorts without an 18q11.2-21.1 loss had similar PFS regardless of treatment (Angiopredict, median PFS 211 days; MoMa, median PFS 232 days; no bevacizumab, median PFS 189 days). No significant associations with PFS were found for chromosome 5q34 and 17q for any of the cohorts. Conclusion: We conclude that loss of chromosomal region 18q11.2-18q21.1 is consistently predictive for PFS in patients receiving bevacizumab. 18q11.2 loss provides an increase in median PFS of 83 days, compared to patients without this loss. No significant increase was found for patients that did not received bevacizumab.

#2580

A mosaic ERG oncoprotein staining pattern predicts upstaging and poor pathologic features in prostate cancer patients.

William Gesztes,1 Shahnoza Dusmatova,1 Justin Mygatt,2 Jennifer Cullen,1 Denise Yong,1 Huai-Ching Kuo,1 Yongmei Chen,1 Lauren Hurwitz,1 Inger Rosner,2 Shiv Srivastava,1 Isabell Sesterhenn3. 1 _Center for Prostate Disease Research (CPDR), Rockville, MD;_ 2 _Center for Prostate Disease Research (CPDR) and the Walter Reed National Military Medical Center, Bethesda, MD;_ 3 _The Joint Pathology Center, Silver Spring, MD_.

Purpose: Building on previous work, this study examined the independent role of ERG staining pattern in the index prostate cancer (CaP) tumor, on probability of upstaging and biochemical recurrence (BCR) following radical prostatectomy (RP).

Methods: A retrospective cohort study of patients enrolled at the Walter Reed National Military Medical Center who underwent RP between 1993 and 2016 was conducted. ERG oncoprotein expression was assessed in CaP index tumors (i.e., largest and poorest grade) using immunohistochemistry (IHC) staining with 9FY monoclonal antibody of representative sections of formalin-fixed paraffin-embedded whole-mounted RP specimens. ERG status was classified as positive (ERG+), negative (ERG-) and a pattern of alternating +/- regions within the index tumor (ERG "Hybrid" or ERG-h). Kaplan Meier (KM) estimation curve analysis was used to examine BCR-free survival across ERG status. Multivariable (MV) logistic regression (LR) modeling was used to determine whether ERG status predicted upstaging (cT1-2 to pT3-4).

Results: Among 1,385 eligible patients, mean age at RP and follow up time were 58.8 and 7 years, respectively. Twenty-nine percent of patients self-classified as African-American (AA), and 18% experienced BCR. The ERG-h pattern was observed in 81 patients (5.8%). Pathologic stage T3a-T4a was observed in 51.3%, 32.2% and 30.5% of patients with ERG-h, ERG+ and ERG- tumors, respectively. In unadjusted KM analysis, 10-year BCR-free survival probabilities were 78%, 75%, and 82% for ERG-h, ERG-, and ERG+ patients, respectively (p=0.08). A trend of incrementally poorer BCR-free survival was observed for ERG-h, ERG- versus ERG positive patients. MV LR analysis revealed the greatest odds of upstaging for ERG-h vs. ERG- patients (OR=2.8, p=0.0008), controlling for age at RP (p<.0001), biopsy Gleason score (p<.0001), PSA at diagnosis (p=0.0054) and patient race (p=0.07).

Conclusions: In this racially diverse cohort of RP patients, a mosaic-like pattern of ERG staining predicted greater upstaging and poorer pathologic features. There may be a role for examination of this "hybrid" mosaic staining pattern in patient risk stratification during treatment decision making.

#2581

Plasma HPV cell free DNA as an earlier predictor of treatment response in advanced oropharyngeal cancer.

Glenn J. Hanna, Julianna G. Supplee, Yanan Kuang, Kate Geary, Pasi A. Janne, Robert I. Haddad, Cloud P. Paweletz. _Dana-Farber Cancer Institute, Boston, MA_.

Purpose: Human papillomavirus (HPV) is associated with the majority of oropharyngeal squamous cell carcinomas (OPSCC), with a rising incidence of OPSCC predominantly among middle-aged men. Tumor-derived HPV cell-free (cf)DNA can be detected and quantified in circulation with high sensitivity and specificity. Although cfDNA has limited clinical application in monitoring locoregional spread of these tumors, up to 10% of HPV+ OPSCC patients present or recur with distant, metastatic disease. The potential of HPV DNA monitoring in this setting remains largely unexplored.

Methods: We present a proof-of-concept prospective observational cohort of recurrent, metastatic (R/M) HPV+ OPSCC patients treated with standard systemic therapies. We utilized droplet digital (dd)PCR to identify and quantify HPV cfDNA (strains 16, 18, 31, 33 and 45) at multiple time points throughout treatment. We then compared HPV cfDNA concentration at various timepoints with clinical parameters such as disease burden and treatment response.

Results: Clinicopathologic data from 12 R/M patients revealed a predominantly male cohort (11/12, 92%) with a median age of 55 at diagnosis. Eight (67%) were on immunotherapy treatments and the other four on standard chemotherapy during the study period (2.5 months). Plasma HPV cfDNA was detected in 6/12 (50%) samples (range 0-4460 copies/mL) in at least one timepoint during the study. All patients with undetectable HPV cfDNA had evidence of stable, low burden disease or no measurable lesions (near-complete response) on their current therapy. HPV cfDNA levels strongly correlated with both disease burden (p < 0.01) and distant disease sites (p < 0.01). In three cases, HPV DNA levels declined prior to contrast enhanced imaging findings which later confirmed treatment response (up to 14 days prior to planned imaging).

Conclusion: Our results suggest that high sensitivity plasma HPV cfDNA levels can be an early indicator of treatment response. HPV cfDNA monitoring could significantly impact clinical decision-making and improve patient outcomes by informing treatment response or failure. Further studies are underway to validate these findings and determine how early cfDNA can detect metastatic disease.

#2582

A novel PI3K/Akt-pathway activation biomarker using comprehensive genomic profiling (CGP) for clinical trial assay.

Yuting He,1 Matthew Wongchenko,2 Joel Skoletsky,1 Christine Burns,1 Yali Li,1 Paula Maness,1 Doris Kim,2 Doron Lipson,1 Philip Stephens,1 Vincent Miller,1 Jeffrey Ross,1 Steven Gendreau,2 James Sun1. 1 _Foundation Medicine Inc., Cambridge, MA;_ 2 _Genentech Inc., South San Francisco, CA_.

Introduction Patients with PI3K/Akt-pathway activation may be sensitive to selective Akt-inhibitors that are currently under development. We have developed a novel next-generation sequencing (NGS)-based composite biomarker assay that identifies patients with PI3K/Akt-pathway activated tumors by identifying activating PIK3CA and AKT1 alterations, and inactivating alterations in PTEN. This assay was analytically validated, and applied to triple-negative breast cancer (TNBC) patients in the LOTUS trial (NCT02162719), a placebo-controlled phase II clinical trial to assess the safety and efficacy of adding ipatasertib to paclitaxel treatment in patients with metastatic TNBC (Kim et al., 2017).

Methods DNA extracted from FFPE tumor tissue underwent whole-genome shotgun library construction and hybridization-based capture, followed by sequencing using Illumina HiSeq 4000. Sequence data were processed using a proprietary analysis pipeline designed to detect base substitutions, indels, copy number alterations, genomic rearrangements, microsatellite instability, and tumor mutational burden. The assay further evaluated the PI3K/Akt-pathway activation biomarker status that consists of six features: 1-2) AKT1 and PIK3CA activating mutations, 3) PTEN homozygous deletion, 4) PTEN heterozygous deletion (HE), 5) PTEN dominant negative mutations, and 6) bi-allelically inactivated (BI) PTEN mutations defined as mutation plus loss of heterozygosity (LOH). We evaluated the limit of detection (LoD) and the precision of the biomarker for two novel genomic features: HE and BI, with the other four features previously validated.

Results Analytical validation of novel biomarker features: The LoD of detecting PTEN HE and BI was determined to be 30%, the lowest tumor content at which the features can be detected at 90% probability. In the precision study, 100% (81/81) agreement was achieved across different replicates within the same sequencer run and across different sequencer runs for biomarker positive samples, demonstrating high reproducibility in calling PTEN HE and BI.

Conclusions We developed and analytically validated an NGS-based assay that identifies complex and novel genomic alterations (heterozygous deletion and bi-allelic inactivation) in PTEN that is part of a composite PI3K/Akt-pathway activation biomarker. This assay identified patients that appeared to derive greater benefit in the Phase II LOTUS study as compared to using PTEN IHC to only identify patients with PTEN protein loss (Kim et al., 2017). This assay could be generalized to identify other biomarkers with similar types of genetic alterations. It also demonstrates that NGS-based CGP can broaden the intent to treat population to be more specifically related to the mechanism of action of a drug, while also being more selective to patients with potential to respond.

#2583

Imaging flow cytometry assay development and validation for the detection of histone H4 acetylation in white blood cells.

Suriyon Uitrakul, Gareth James Veal, Claire Hutton, David Jamieson. _Newcastle University, Newcastle upon Tyne, United Kingdom_.

Introduction: Histone deacetylases have been identified as oncogenes in several cancer types, providing an attractive target for anticancer treatment. In this respect, the histone deacetylase inhibitor valproic acid has been shown to inhibit the growth of multiple paediatric tumor types and is well tolerated in children with refractory solid or CNS tumors.

Objective: The aim of the current study was to develop and validate a novel imaging flow cytometry method for the detection of histone H4 acetylation in lymphoid and myeloid cell populations, and to assess the applicability of the method to a clinical trial setting.

Method: HL-60 cells and whole blood samples from healthy volunteers were incubated with valproic acid (0.5-8 mM) for 0.5-24 hours, followed by RBC lysis for the whole blood samples and fixed with cold methanol. Additional blood samples were collected from patients with ependymoma who were receiving valproic acid as part of the SIOP Ependymoma II clinical trial. An imaging flow cytometry method was developed using an ImageStreamχ flow cytometer, collecting WBCs with excitation of PE conjugated acH4 antibody and DAPI. Data were collected using Inspire™ software and further analysed by Ideas™ software 6.2. Both in vitro and ex vivo experiments were repeated on at least 3 occasions.

Result: In the HL-60 cell line the mean percentage of acH4 positive cells was 1.98% in the vehicle control sample, increasing to 10.9-77.9% when treated with 0.5-8 mM valproic acid for 6 hours, with percentages of 8.7-49.0% observed following incubation with 4 mM valproic acid for 0.5-24 hours. Comparable data were generated in lymphoid and myeloid WBC populations following ex vivo incubation of whole blood samples with valproic acid. Increases in the percentage of acH4 positive cells were observed in samples collected at 4 hours post-administration in patients receiving valproic acid as compared to pre-treatment samples. Myeloid cells appeared to have a smaller proportion of acH4 positive cells than observed in the lymphoid population but a greater fold increase above basal levels.

Conclusion: A new assay for detection of histone H4 acetylation in WBCs by imaging flow cytometry has been developed and optimised. The assay showed increases in acH4 positivity in both in vitro and ex vivo experiments following exposure to valproic acid. The method can be used for the measurement of acH4 as a pharmacodynamic biomarker for histone deacetylase inhibitors in drug development and monitoring of drug efficacy in clinical trials.

#2584

**Mutations in low-grade serous ovarian cancer and response to** BRAF **and** MEK **inhibitors.**

Tania Moujaber,1 Dariush Etemadmoghadam,2 Cristina Mapagu,1 Catherine Kennedy,1 Yoke-Eng Chiew,1 Casina Kan,1 Nikilyn Nevins,1 Sivatharsny Srirangan,1 Sian Fereday,2 Nadia Traficante,2 Australian Ovarian Cancer Study group, David Bowtell,2 Rosemary Balleine,3 Paul Harnett,4 Anna deFazio1. 1 _Westmead Institute for Medical Research, University of Sydney, Australia;_ 2 _Peter MacCallum Cancer Centre, University of Melbourne, Australia;_ 3 _Children's Medical Research Institute, University of Sydney, Australia;_ 4 _Crown Princess Mary Cancer Centre, Westmead Hospital, University of Sydney, Australia_.

Low-grade serous ovarian cancer (LGSC) responds poorly to platinum based chemotherapy and is characterized by activating RAS-MAPK pathway mutations, including oncogenic BRAF. Drugs that target this pathway are effective in BRAF-mutant melanoma but other cancer types, such as colorectal cancers, are less sensitive. Early phase trials report a 15% response rate to MEK inhibitors in LGSC patients, however it is not known which features may predict response.

We aimed to determine clinical characteristics and treatment response in women with LGSC and to determine whether response to targeted pathway inhibition is associated with specific mutation profiles in LGSC cell lines.

Tumor samples from a cohort of grade 1 and 2 serous ovarian cancer patients were analyzed using targeted, exome or whole genome sequencing. Patient characteristics, treatment and clinical outcome were assessed. Cell lines derived from patients with LGSC with known mutation profiles, AOCS2 (KRAS/BRAF/NRAS wild-type), MPSC1 (BRAFV600L, NRASQ16R), VOA1056 (NRASQ16R) and HCC5075 (KRASG12V), were treated with BRAF (dabrafenib) or MEK inhibitors (trametinib, pimasertib and binimetinib) and response was compared to cell lines derived from high-grade serous cancer (HGSC) and BRAF-mutant melanoma.

Women diagnosed with grade 1 or 2 serous carcinoma between 1994-2015 were identified from 1654 invasive ovarian serous cancer cases in the Australian Ovarian Cancer Study and the GynBiobank. HGSC cases were excluded following histopathology review and TP53 mutation screening. Amongst 65 confirmed LGSC patients, 18 (27.7%) had a KRAS mutation, 9 (13.8%) had a BRAF mutation and 7 (10.8%) had a NRAS mutation. Women with advanced stage LGSC and residual disease following debulking surgery had a similarly poor progression-free and overall-survival compared with HGSC patients. We saw a dramatic response to BRAF inhibition in a patient with BRAFV600E-positive LGSC, however, the LGSC cell lines did not respond to dabrafenib. This is not surprising as the cell lines did not harbour the hot-spot BRAFV600E mutation. MPSC1 has a BRAFV600L and a NRASQ16R mutation, but dabrafenib did not inhibit growth. HCC5075 (KRASG12V) was sensitive to all three MEK inhibitors, but response to MEK inhibition in the other LGSC cell lines was modest. In conclusion, LGSC are generally chemotherapy resistant and molecular analyses can identify targetable mutations. However, LGSC are heterogenous with respect to underlying mutations and response to pathway inhibitors is likely to depend on which mutations and pathways are activated. BRAF mutations are not uncommon in patients with LGSC and should be routinely tested as BRAF inhibitors can be an effective treatment for these patients. MEK inhibitors may also be effective in a subset of cases. The results highlight the need for novel clinical trial design, as traditional clinical trials are unlikely to be effective in rare ovarian cancer sub-types.

#2585

Immunoseroproteomic profiling in autoantibodies to ENO1 as potential biomarkers in immunodiagnosis of osteosarcoma by serological proteome analysis (SERPA) approach.

Jitian Li,1 Manyu Huang,2 Liping Dai,3 Zongchang Han,2 Xiaofei Qin,2 Wen Xie,2 Wei Qian,1 Wuyin Li,2 Jianying Zhang1. 1 _The University of Texas at El Paso, El Paso, TX;_ 2 _Henan Luoyang Orthopedic-Traumatological Hospital, Henan Institute of Orthopedic and Traumatology,, Luoyang, China;_ 3 _Zhengzhou University, Zhengzhou, China_.

Background: Osteosarcoma (OS) is the most common highly malignant primary solid bone-tumor. Despite its relatively low incidence rate among overall cancers, it remains one of the most harmful primary malignant tumors in childhood and adolescence. It is now evident that serum autoantibodies against tumor-associated antigens (TAAs) could be used as serological cancer biomarkers in types of cancers.

Methods: Serological proteome analysis (SERPA) approach was applied to profile anti-TAA autoantibody responses in sera from patients with OS, and Normal Human, and explore differences of these responses. This approach can detect autoantibodies to TAAs that could serve as clinical biomarkers and immunotherapeutic agents. Sera from OS, Osteochondroma (OC) and Normal Human Sera (NHS) were probed by immunoblotting against cellular proteins extracted from U2-OS and Saos-2 cell lines, with OS sera showing stronger immunoreactivity. MALDI-TOF/TOF Mass Spectrometry (MS) analysis of immunoreactive protein spots revealed that several OS sera contained autoantibodies to a number of proteins, particularly to alpha-enolase (ENO1).

Results: Analysis of 172 serum samples from patients with OS, OC and NHS by ELISA showed higher frequency of anti-ENO1 autoantibodies in OS sera compared to others. Interestingly, descant of ENO1 immunoreactivity was observed in most patients after treatments, which may imply a potential association between anti-ENO1 autoantibody titers and disease progression. Nine of twelve sera reacted strongly against purified ENO1, but 3 weakly against purified ENO1, which indicates 75.0% sera with positive optimal density values from ELISA were consistently positive in Western blotting. The expression of ENO1 in Osteosarcoma tissues was evaluated by immunohistochemistry in tumor microarray.

Conclusion: Our intriguing findings firstly demonstrate that ENO1 is one of autoantigens that elicit autoimmune responses in OS and can be used as biomarkers in immunodiagnosis and progression of OS.

Key words: Osteosarcoma (OS), Tumor-associated antigen (TAA), Serological proteome analysis (SERPA), ENO1, Immunodiagnosis.

#2586

PI3K/AKT signaling pathway is transcriptionally elevated in prexasertib-resistant TNBC PDX models.

Yan Ding, Jiangang Liu, John N. Calley, Hui-Rong Qian, Philip W. Iversen, Philip J. Ebert, Richard P. Beckmann, Gregory P. Donoho, Ricardo Martinez, Wenjuan Wu, Aimee Bence Lin, Emma Bowden, Amit Aggarwal. _Eli Lilly, Indianpolis, IN_.

Triple-negative breast cancer (TNBC) is a heterogeneous disease characterized by poor clinical outcomes and limited treatment options. Transcriptional analysis has divided TNBC into different molecular subtypes (BL1, BL2, M and LAR) that were characterized with different signaling pathways and differed in response to similar neoadjuvant chemotherapies [1]. In a sporadic TNBC phase II study, prexasertib monotherapy showed modest single agent activity, which suggested that combination therapy is needed to maximize the benefit from prexasertib (LY2606368), a selective CHK1 inhibitor [2]. Here we leveraged a large cohort of TNBC patient-derived xenograft (PDX) models developed with nude mice to find biomarker/pathways/combination strategies related with prexasertib. Parametric Gene Set Enrichment Analysis (PGSEA) has been shown as a powerful sample-level analysis to generate enrichment scores for pathways [3]. First we confirmed that our collection of PDX models captured the heterogeneities of TNBC disease, with similar compositions of molecular subtypes as the TCGA dataset. The different molecular subtypes of PDX models also showed similar pathway changes as TNBC disease, such as increase of cell-cycle pathway in BL1-subtype, Glycolysis in BL2-subtype and ECM pathway in M-subtype. Molecular subtype analysis of a single-mouse (n=1) PDX trial evaluating antitumor activity of prexasertib showed that majority of M-subtype showed resistance to prexasertib and the other subtypes showed differential sensitivity. Within the BL1-subtype, the resistant models also showed high expression of stem-cell like features. Further PGSEA analysis based on KEGG signaling pathways revealed that the PI3K/mTOR pathway was transcriptionally elevated in resistant models, which was consistent with the additive effect of the combination therapy of prexasertib and PI3K/mTOR inhibitor (LY3023414) in this PDX trial. These data provide rationale for the development of combination therapy of prexasertib and PI3K/mTOR inhibitor for TNBC.

1. Lehmann, B.D., et al., Refinement of Triple-Negative Breast Cancer Molecular Subtypes: Implications for Neoadjuvant Chemotherapy Selection. PLoS One, 2016. 11(6): p. e0157368.

2. Karzai, F., et al., A phase II study of the cell cycle checkpoint kinases 1 and 2 (CHK1/2) inhibitor (LY2606368; prexasertib) in sporadic triple negative breast cancer (TNBC). Annals of Oncology, 2016. 27.

3. Kim, S.Y. and D.J. Volsky, PAGE: parametric analysis of gene set enrichment. BMC Bioinformatics, 2005. 6: p. 144.

#2587

Imbalances in the disposition of estrogen and naphthalene in Taiwanese breast cancer patients.

Po-Hsiung Lin,1 Dar-Ren Chen,2 Wei-Chung Hsieh,3 Mao-Huei Ko,1 Yi-Lun Liao1. 1 _National Chung Hsing Univ., Taichung, Taiwan;_ 2 _Changhua Christian Hospital, Changhua, Taiwan;_ 3 _Da-Chien General Hospital, Miaoli, Taiwan_.

The aims of this study were to measure the background level of quinone metabolites of 17β-estradiol (E2), including E2-2,3-Q and E2-3,4-Q, and naphthalene quinone (1,2-NPQ and 1,4-NPQ)-derived protein adducts in human albumin (Alb) derived from breast cancer patients (n=143) and healthy controls (n=122) in Taiwanese women and to explore the relationships between these adducts with risk factors of breast cancer. Results from the analysis indicated that Alb adducts of estrogen quinones, including E2-2,3-Q-4-S-Alb and E2-3,4-Q-2-S-Alb were detected in all subjects with mean levels at 400 and 676 pmole/g in cancer patients and 204 and 67 pmole/g in controls, respectively. In addition, the mean levels of Alb adducts of 1,2-NPQ and 1,4-NPQ on human serum albumin were estimated to be 209 and 73.6 pmole/g in cancer patients and 113 and 174 pmole/g in controls, respectively. Levels of E2-2,3-Q-4-S-Alb correlated significantly with those of E2-3,4-Q-2-S-Alb. Similarly, levels of 1,4-NPQ-Alb correlated significantly with those of 1,2-NPQ-Alb. Additionally, we noticed that E2-2,3-Q-4-S-Alb and E2-3,4-Q-2-S-Alb positively correlated with 1,4-NPQ-Alb (p<0.001) whereas levels of E2-2,3-Q-4-S-Alb associated with 1,2-NPQ-Alb (p<0.01). Furthermore, we observed that ratio of E2-3,4-Q-2-S-Alb to E2-2,3-Q-4-S-Alb in breast cancer patients were ~5 fold greater than those of healthy controls whereas ratio of 1,2-NPQ-Alb to 1,4-NPQ-Alb in cancer patients were ~4 fold greater than those of healthy controls. This evidence suggests that the cumulative body burden of estrogen quinones is a significant indicator of the risk of developing breast cancer and that breast cancer risk is associated with the imbalances in the disposition of estrogen and naphthalene in Taiwanese women.

#2588

Label-free classification of live cells using quantitative phase microscopy images for negative selection of circulating tumor cells.

Hirotoshi Kikuchi,1 Yusuke Ozaki,1 Amane Hirotsu,1 Hidenao Yamada,2 Shigetoshi Okazaki,1 Tomohiro Murakami,1 Tomohiro Matsumoto,1 Yoshihiro Hiramatsu,1 Kinji Kamiya,1 Takanori Sakaguchi,1 Hiroyuki Konno,1 Hiroya Takeuchi1. 1 _Hamamatsu Univ. School of Medicine, Hamamatsu, Japan;_ 2 _Hamamatsu Photonics K.K., Hamamatsu, Japan_.

Background: Circulating tumor cells (CTCs) are used as a liquid biopsy target in the treatment of cancer patients and are important for personalized medicine. Most CTC detection technologies are based on their surface markers such as EpCAM and cytokeratin. However, those positive selection methodologies appear to count only some CTCs because invasive tumor cells tend to change their surface markers during progression. Because the vast majority of nucleated cells circulating in the bloodstream are white blood cells (WBCs), eliminating WBCs from circulation is an efficient method to purify CTCs by negative selection. In this study, we demonstrate the development of cell recognition by computer vision technologies for pattern recognition based on the morphological features of live cells.

Materials and methods: Blood samples were obtained from healthy volunteers. Five cancer cell lines, SW480, DLD-1, HCT116, Panc-1, and HepG2 were used as CTC models. We applied quantitative phase microscopy (QPM) that detects the small delay of the phase and image optical path lengths and provides quantitative morphological information of live cells with high contrast without staining. WBC recognition algorithm was developed using computer vision technologies for pattern recognition based on the histogram-oriented gradient (HOG) features extracted from QPM images (QPIs) for negative selection of CTCs.

Results: We observed WBCs and five cell line cells by QPM, extracted certain features from the obtained images as training data for pattern recognition, and created an algorithm to differentiate WBCs from cell lines. The obtained algorithm successfully differentiated WBCs from cell lines (AUC=0.98). To simulate WBCs and cell lines with a small and strong change, respectively, in optical thickness (OT) at the center of the cell, we created a homogeneous hemi-ellipsoid model and a heterogeneous hemi-ellipsoid model, respectively. In these models, OT gradually increased from the edge to the center, but fluctuated around the center to simulate intracellular heterogeneity of a cancer cell. Based on these simulations, the classifier was interpreted to recognize intracellular heterogeneity, especially in the center of the cell. Finally, we applied our image recognition methods to QPIs of the cells flowing in the chamber, and properly differentiate WBCs from cell line cells flowing in the chamber (AUC=0.99).

Conclusions: We developed a novel method for label-free image identification of live cells by computer vision technologies for pattern recognition based on the features of QPIs. Our study makes it possible to sort CTCs in a non-cytotoxic manner, thus providing good opportunities for molecular biological approaches to isolate CTCs.

#2589

Novel predictor of FGFR1 inhibition efficacy in non-small cell lung cancer.

Álvaro Quintanal-Villalonga,1 Irene Ferrer,1 Ángela Marrugal,1 Laura Ojeda-Márquez,1 Jon Zugazagoita,2 Laura García-Redondo,1 Fernando López-Ríos,3 Luis Montuenga,4 Sonia Molina-Pinelo,5 Sonia Molina-Pinelo,5 Amancio Carnero,5 Luis Paz-Ares2. 1 _H12O-CNIO, Madrid, Spain;_ 2 _Hospital Universitario 12 de Octubre, Madrid, Spain;_ 3 _Laboratorio de Dianas Terapéuticas, Hospital Universitario HM Sanchinarro, Madrid, Spain;_ 4 _Center for Applied Medical Research (CIMA), Pamplona, Spain;_ 5 _Instituto de Biomedicina de Sevilla, Sevilla, Spain_.

Fibroblast growth factor receptor (FGFR)1 has been associated with tumorigenesis in a variety of tumors, including lung cancer. FGFR1 amplification is detected in up to 20% of lung squamous cell carcinomas, and was initially related to tumorigenesis and to response to FGFR inhibitors in preclinical models of this pathology. These data encouraged the establishment of FGFR1 amplification as the main eligibility criterion for inclusion of patients in FGFR inhibitors clinical trials. However, the use of FGFR inhibitors in FGFR1-amplified tumors has shown limited success, with only 5-10% of selected patients showing partial response, suggesting that this genomic aberration may not be a good predictive biomarker for the efficacy of these inhibitors. In this work, we provide in vitro and in vivo data showing that the oncogenic effects of FGFR1 expression depend on the expression of the adhesion molecule N-cadherin in non-small cell lung cancer (NSCLC). In line with these results, FGFR1 expression correlates with poorer prognosis only in tumors with high N-cadherin levels in two independent NSCLC patient cohorts. We observed that high FGFR1 expression alone is not sufficient to predict FGFR inhibition efficacy in lung cancer cell lines and patient-derived xenografts, with only high FGFR1- and high N-cadherin-expressing models responding to selective FGFR inhibitors. Altogether, our data show that the determination of the expression of FGFR1 alone is not sufficient to predict FGFR inhibition efficacy. The co-determination of N-cadherin expression may optimize patient selection for this therapeutic strategy.

#2590

Race-specific differential expression of CCL25 and CCR9 in triple-negative breast cancer.

Jeronay King, Neeraj Kapur, Hina Mir, Dominique N. Gales, James W. Lillard, Shailesh Singh. _Morehouse School of Medicine, Atlanta, GA_.

Breast cancer (BrCa) is the second leading cause of cancer-related deaths among women. Therapeutic intervention of the disease is impeded due to the heterogeneity contributed by inter- and intra-tumoral diversity, as well as difference among cancer-bearing individuals. Triple-negative breast cancer (TNBC) is a hormone-independent and highly aggressive subtype that disproportionally affects African-American (AA) women. However, the molecular basis of racial disparity in therapeutic response and clinical outcome in AA and Caucasian (CA) patients is still obscure. Our group was the first to show the involvement of CCR9 and its natural ligand, CCL25 in cancer progression and therapeutic response, including breast cancer. In this study, we have shown the association of CCR9 and CCL25 with TNBC. Both CCR9 and CCL25 are highly expressed in TNBC cells (MDA-MB-468 and MDA-MB-231) compared to other BrCa subtypes (MCF-7, MDA-MB-361, SKBr-3). Expression of this receptor-ligand pair was significantly elevated in TNBC cells derived from AA compared to those derived from CA patients. RNA-Seq based transcriptome data obtained from The Cancer Genome Atlas (TCGA) confirmed these results. Further, phospho-proteomic profiling of cancer signaling molecules showed race specific hyper-activation of GSK-3α, GSK-3β and NF-κB in AA derived cells. These molecules are known to provide the survival advantage to cancer cells. These findings, therefore, highlight significance of CCR9 and its association with the disparity in observed in TNBC. Hence, CCR9 targeted approach to address the disparity in the therapeutic outcome of TNBC seems to be promising.

#2591

Role of EGFR-AS1 lncRNA in growth factor addiction and treatment response in squamous cell cancers.

Daniel SW Tan,1 Fui-Teen Chong,1 Hui-Sun Leong,1 Shen Yon Toh,1 Dawn P. Lau,1 Xue Lin Kwang,1 Xiaoqian Zhang,2 Gek San Tan,3 Anders J. Skanderup,2 Tony KH Lim,3 Ramanuj Dasgupta,2 N Gopalakrishna Iyer1. 1 _National Cancer Ctr. Singapore, Singapore, Singapore;_ 2 _Genome Institute of Singapore, Singapore, Singapore;_ 3 _Singapore General Hospital, Singapore, Singapore_.

Squamous-cell cancers (SCCs) represent one of the commonest lethal malignancy worldwide. Despite evidence demonstrating that majority are addicted to Epidermal Growth Factor Receptor (EGFR)-signaling, targeting with monoclonal antibodies or tyrosine-kinase inhibitors (TKIs) has been met with limited success. We recently demonstrated that TKI-sensitivity is modulated by a long non-coding RNA (lncRNA) situated in the antisense strand of EGFR (EGFR-AS1). 'Synonymous' alterations in EGFR-AS1 affected TKI-response in patient-derived models, and could be used to predict response in a small patient cohort. The lncRNA likely functions in cis by altering EGFR splicing, favouring expression of a truncated EGFR isoform (isoform D), with previously unknown function. Importantly, mediation of this effect requires EGFR ligand derived from paracrine or autocrine sources. Remarkably, even modest EGFR-AS1 knockdown showed profound effects on TKI sensitivity and tumor growth in vivo; the latter suggesting lncRNA-addiction. TKI-resistance models were established from patient-derived lines, through serial passaging with sub-lethal drug doses. Detailed analyses of four of these models demonstrate alterations that affect each of the steps involved in this addiction pathway: ligand binding, EGFR-AS1 expression/amplification and isoform D expression. Based on our mechanistic insights, we propose that patient-response to EGFR inhibitors is contingent on establishing a comprehensive suite of inter-related biomarkers as delineated here. This data also supports a novel therapeutic approach that involves modulating a lncRNA and/or splice variants of a well-established oncogenic driver.

#2592

Whole-exome sequencing cell free DNA analysis documents new tumor specific alterations at relapse of high-risk pediatric cancers.

Mathieu Chicard, Adrien Danzon, Nathalie Clémént, Irene Jimenez, Eve Lapouble, Gaelle Pierron, Angela Bellini, Nada Leprovost, Sylvain Baulande, Paul Fréneaux, François Doz, Daniel Orbach, Isabelle Aerts, Hélène Pacquement, Jean Michon, Franck Bourdeaut, Olivier Delattre, Gudrun Schleiermacher. _Inst. Curie, Paris, France_.

Background : Pediatric cancers are characterized by few recurrent genetic alterations, but genetic heterogeneity and clonal evolution can play a role in tumor progression. Liquid biopsies now enable monitoring of tumor-specific genetic alterations in sequential samples by analysis of cell free DNA (cfDNA).

Methods : We have enrolled 28 consecutive patients with newly diagnosed high risk pediatric cancer (neuroblastoma n=8; rhabdomyosarcoma n=7; Ewing sarcoma n=3; other cerebral or extracerebral high risk cancers n=10) in a prospective clinical study NGSkids (clingov trial : NCT02546453) with an aim to study clonal evolution based on sequential cfDNA analysis. With a median follow up of 22 months, 7 patients have experienced relapse. Molecular analysis consisted of standardized Illumina© 100PE 100x whole exome sequencing (WES) of tumor tissue and paired germline material , and WES of cfDNA extracted from plasma samples at diagnosis, during treatment and follow up, with 2-9 sequential samples available per patient. cfDNA analysis was performed using an in-house procedure, with 20-200 ng of cfDNA subjected to WES following modified library construction and capture approaches to account for cfDNA molecule size (target depth 100x). Following filtering on germline to focus on tumor cell specific alterations, SNVs were called using GATK-UnifiedGenotyper, GATK-HaplotypeCaller, Samtools and Mutect. Copy-number profiles were generated using Varscan and DNAcopy.

Results:: At diagnosis, cfDNA quantities were higher in advanced stages of disease (localized stages - mean 64 ng/ml of plasma (range 24-172); metastatic stages - mean 505 ng/ml of plasma (range 20-2,782)). CfDNA WES analysis yielded satisfactory depth in all cases. At diagnosis, a mean of 9 tumor cell specific SNVs common to both primary tumor and corresponding cfDNA was observed (range 1 - 57), with a mean of 11 and 6 specific to the primary and cfDNA respectively, indicating spatial heterogeneity. Whereas cfDNA samples obtained at follow-up in patients without evidence of disease revealed no or few tumor cell specific SNVs, interestingly, cfDNA samples obtained at relapse harbored additional, new relapse-specific SNVs (mean 10; range 2-42) in all cases with relapse, targeting genes such as MAPK and MLL4. Deep sequencing (10,000X) capture techniques with a panel encompassing all identified SNVs is currently being applied to all cfDNA samples, including 1-8 intermediate samples per patient, with an aim to develop models of clonal evolution.

Discussion and Conclusion: CfDNA WES proves to be an extremely powerful tool to study spatial and temporal heterogeneity in pediatric high risk cancers, providing further proof of the importance of clonal evolution in cancer progression. Full characterization of cfDNA at relapse, which might represent more aggressive clones, might orient towards targeted treatment approaches.

#2593

AR-V7 and AR-V9 expression is not predictive of response to AR-axis targeting agents in metastatic castration-resistant prostate cancer.

Sarah Q. To,1 Edmond Kwan,1 Heidi Fettke,1 Andrew Mant,2 Maria Docanto,1 Luciano Martelotto,1 Patricia Bukczynska,1 Nicole Ng,3 Lisa-Jane Graham,4 Phillip Parente,2 Carmel Pezaro,2 Kate Mahon,4 Lisa Horvath,4 Tilman Todenhöfer,5 Arun Azad1. 1 _Monash University, Melbourne, Australia;_ 2 _Eastern Health, Melbourne, Australia;_ 3 _Monash Health, Melbourne, Australia;_ 4 _Chris O'Brien Lifehouse, Sydney, Australia;_ 5 _Eberhard-Karls-University, Tuebingen, Germany_.

In 2014, a landmark study was published demonstrating that androgen receptor splice variant (AR-V) AR-V7 expression was a negative predictive biomarker for response to abiraterone acetate and enzalutamide in metastatic castration-resistant prostate cancer (mCRPC) patients. However, these results were not supported by the recently reported ARMOR3-SV phase III clinical trial, which employed an identical circulating tumor cell assay to assess AR-V7 expression. Therefore, the predictive utility of AR-V7 expression in mCRPC remains uncertain, as does any potential association between other AR splice variants and treatment response. To further investigate, we designed a highly sensitive and specific whole blood assay for detecting AR-V7 and AR-V9. We then examined for a correlation between baseline AR-V7/V9 status and treatment outcome in 37 mCRPC patients commencing abiraterone or enzalutamide. Blood was taken in a PAXgene RNA tube at baseline prior to the commencement of therapy and at end of treatment (if applicable). 24% (9/37) of patients were AR-V-positive. Notably, PSA response rates did not significantly differ between AR-V-positive (6/9) and AR-V-negative (18/28) patients (66% vs. 64%, p=0.896). Likewise, median PSA progression-free survival was not significantly different between AR-V-positive and AR-V-negative patients (9.2 months vs. not reached; p=0.894). Additionally, we identified 2 patients who were AR-V7 negative at baseline but had converted to AR-V7 positive in their end-of-treatment sample. This was associated with much shorter PSA-PFS than those that remained negative (2.58 months vs. no reached), suggesting gain of AR-V expression may be a marker of acquired resistance. These data, which support the findings of the pivotal ARMOR3-SV clinical trial, suggest that baseline AR-V expression does not predict outcomes in mCRPC patients receiving abiraterone or enzalutamide.

#2594

A multigene recurrence signature identifies highly proliferative tumors that escape immune surveillance in early stage lung and pancreas adenocarcinoma.

Nathaniel Weygant,1 Jiannan Yao,2 Dongfeng Qu,1 Parthasarathy Chandrakesan,1 Guangyu An,2 Courtney W. Houchen1. 1 _University of Oklahoma Health Sciences Center, Oklahoma City, OK;_ 2 _Beijing Chao-Yang Hospital, Beijing, China_.

Background: Accurately predicting recurrence in early stage cancer patients may be used to avoid unnecessary therapies with significant side effects or deliver early interventions that can improve outcomes. Both early stage lung (LAc) and pancreatic adenocarcinoma (PDAc) are high-recurrence cancers for which biomarkers that can quantify this risk could benefit patients. In stage I LAc only a small percentage of stage IB patients receive chemotherapy, while more patients may derive additional benefit if identified. In PDAc, patients with the most aggressive stage I-II disease could derive benefit from early aggressive combination chemotherapy if identified.

Methods: Training and test sets (n=10) were selected by random sampling RNA-seq expression data into groups of 65% and 35% respectively. Cox proportional hazards and Kaplan-Meier analysis were used to predict RFS and OS. Maximally selected rank statistics were used to designate low and high-risk signature groups. Subgroup analysis was performed using multivariate Cox methods. Pathway analysis was performed with Webgestalt. RNA-seq expression data for LAc and PDAc was obtained from the cancer genome atlas and gene microarray data from NCBI GEO. All statistical analyses and plots were generated in R v3.2.

Results/Discussion: An unbiased analysis of RNA-seq data identified a 19-gene consensus signature (19-GCS) that is upregulated in tumors and able to predict LAc stage I RFS. Multivariate analysis demonstrated its independence as a prognostic factor and efficiency in independent validation sets. Pathway analysis identified proliferation and immune surveillance as altered pathways in 19-GCS high-risk (sHR) compared to low-risk (sLR) tumors. In confirmation of this finding Cyclin D was downregulated while Cyclin E/A/B were upregulated in sHR tumors. Moreover, the 19-GCS was correlated with proliferation markers Ki-67 and PCNA and showed universal correlation with G2/M phase-specific genes. The expression of MHC Class I and II subunit markers were lost in sHR tumors as were specific MHC-related genes. Moreover, the expression of tumor-infiltrating immune cell-specific markers were largely lost in sHR tumors. The 19-GCS was better able to predict RFS and OS in PDAc than in LAc, and consistent with PDAc's severity, more patients were identified as high-risk. Gene expression analysis identified the same molecular characteristics in sHR PDAc as in sHR LAc. Analysis of clinical outcomes for both LAc and PDAc demonstrated that sHR predicted progressive disease in response to both primary and followup therapy. Overall, the 19-GCS identifies highly proliferative early stage lung and pancreatic tumors with impaired MHC mechanisms that shield them from immune-mediated destruction. This deadly combination leads to recurrence and poor clinical outcomes that might be avoided if identified early.

#2595

Role of VEGF/VEGFR autocrine in mediating EGFR TKI resistance in non-small cell lung cancer.

Chike Osude, Leo Lin, Brad Foster, Neelu Puri. _University of Illinois College of Medicine at Rockford, Rockford, IL_.

The treatment paradigm for late stage non-small cell lung carcinoma (NSCLC) has shifted towards molecular targeted therapy like epidermal growth factor receptor (EGFR), one of the most studied targets. Tyrosine kinase inhibitors (TKIs) against EGFR have proven to be effective, however TKI resistance is common due to the acquisition of the T790M mutation in the EGFR kinase domain. There are currently very few studies suggesting the role of VEGFR in mediating erlotinib resistance. We suggest that over-expression of vascular endothelial growth factor receptors (VEGFRs) in tumors may create an autocrine loop that may mediate TKI resistance. The purpose of this study is to investigate the VEGF/VEGFR autocrine loop as a potential mechanism of TKI resistance via alternative signaling. H2170 NSCLC parental cells were obtained from ATCC. The resistant cells were obtained by treating parental cells with increasing concentrations of erlotinib. DNA sequencing was performed to exclude the presence of the T790M mutation. Quantitative real-time PCR was carried out for target genes coding for VEGF, VEGFR-2, and neuropilin-1 (NP-1). Fluorescence-activated cell sorting (FACS) analysis and immunofluorescence (IF) analysis were performed with antibodies against VEGFR-2 and NP-1. The viability of resistant cells was assessed after being treated with increasing concentrations of a VEGF inhibitor (VEGF neutralizing antibody) in medium with or without erlotinib using a cell proliferation assay (MTT). Thirty paraffin embedded tumor biopsies were immunostained (IHC) with an antibody against VEGFR-2. On the transcriptional level, real-time PCR revealed a 13.1, 3.4 and a 2.9 fold increase in gene expression for VEGF, VEGFR-2 and NP-1 respectively in the resistant cells compared to the parental cells. When looking at membrane bound receptors, FACS analysis showed a 2.1 and a 3.8 fold increase in the expression of VEGFR and NP-1 in resistant cells, respectively. IF studies correlated with the FACS results, showing significantly higher expression of both VEGFR-2 and NP-1 in the resistant cells. MTT assays demonstrated that the VEGF inhibitor was able to decrease cell death from 10 to 80% (0.1-10ug/ml) as evidenced by decreasing viability of resistant cells treated with increasing concentrations of VEGF inhibitor compared to the untreated control. Kaplan-Meier analysis revealed shorter median survival with high vs low expression of total VEGFR-2 expression (7mo vs 25mo, p=0.05) in NSCLC tumors in patients diagnosed with late stage NSCLC. Our results suggest that by up-regulating the VEGF/VEGFR autocrine system as an alternative signaling pathway to promote proliferation, tumor cells may bypass the inhibitory effect of TKI's on the EGFR pathway, leading to resistance. Furthermore, IHC demonstrated the presence of high expression of VEGFR-2 in tumors, suggesting its potential as a prognostic biomarker for lung cancer.

#2596

Spliced messenger RNA of tumor-educated platelets yields a new diagnostic prospective for pancreatic cancer.

Tessa Ya Sung Le Large,1 Laura L. Meijer,1 Sjors G. In 't Veld,1 Nik Sol,1 Myron G. Best,1 Giulia Mantini,1 Ewoud van der Lelij,1 Francois Rustenburg,1 Heleen Verschueren,1 Niccola Funel,2 Ingrid Garajova,3 Maarten F. Bijlsma,4 Marc G. Besselink,4 Elisa Giovannetti,1 Tom Wurdinger,1 Geert Kazemier1. 1 _VU Univ. Medical Ctr., Amsterdam, Netherlands;_ 2 _University Hospital of Pisa, Pisa, Italy;_ 3 _University Hospital of Bologna, Bologna, Italy;_ 4 _Academic Medical Center, Amsterdam, Netherlands_.

Pancreatic ductal adenocarcinoma (PDAC) is often diagnosed at advanced disease stages and invasive procedures are needed to confirm diagnosis. Innovative liquid biopsy-based approaches are warranted to diagnose early stages and prevent unnecessary treatment for advanced disease or misdiagnosed benign diseases. CA19.9 is the only clinically implemented biomarker, but its sensitivity is inadequate and it lacks specificity. Spliced messenger RNA (mRNA) of tumor-educated platelets (TEPs) emerged recently as new potent pan-cancer biomarker (Best et al, Cancer Cell 2015&2017). The aim of this study was to evaluate PDAC-specific TEP profiles as diagnostic and staging biomarker. Samples were collected at four international academic centers. Firstly, TEPs were isolated from blood of 135 PDAC patients including early disease (stage I-IIb), advanced disease (stage III) and metastatic patients (stage IV). In addition, 250 age-matched healthy controls (HC) and 72 patients with a benign disease in the pancreaticobiliary tract were included to create a heterogeneous control group that reflects the real patient population at the outpatient clinic. After total RNA was isolated, SMARTer amplification was performed and whole transcriptome mRNA was sequenced on the Illumina platform. Intron-spanning platelet mRNA reads were selected for further differential expression analysis. PDAC patients had a significant different mRNA TEP profile compared to HC (p-value <0.0001). Unsupervised clustering with our gene panel robustly separated HCs from of PDAC. In order to train our swarm-enhanced classification algorithm, all samples were allocated to either the training, evaluation or validation cohorts to determine and evaluate the optimal algorithm and gene panel. In our preliminary first validation cohort, our algorithm predicted PDAC from our control group with an accuracy over 90%. Moreover, we classified early stage disease correctly as PDAC, proving the diagnostic power of our method. This study underlines the value of profiling spliced mRNA of TEPs for cancer diagnostics. Here we showed the capability of TEPs to diagnose PDAC. By the swarm-enhanced classification algorithm, our method showed robust differences in PDAC and non-cancer spliced mRNA profiles, even at early stage disease. Future analyses including other hepatopancreaticobiliary cancers are needed to establish the discriminative power of our algorithm. Evaluation of premalignant lesions and treated patients is ongoing to prove its monitoring value, and large-scale multicenter validation is warranted to facilitate implementation in the clinic.

#2597

Development and characterization of novel non-small cell lung cancer (NSCLC) circulating tumor cells (CTCs)-derived xenograft (CDX) models.

Vincent Faugeroux,1 Emma Pailler,1 Olivier Deas,2 Judith Michels,1 Laura Mezquita,1 Laura Brulle-Soumare,2 Stefano Cairo,2 Jean-Yves Scoazec,1 Virginie Marty,1 Pauline Queffelec,1 Maud Ngo-Camus,1 Claudio Nicotra,1 David Planchard,1 Patricia Kannouche,1 Benjamin Besse,1 Jean-Gabriel Judde,2 Françoise Farace1. 1 _Institut Gustave Roussy, Villejuif, France;_ 2 _XenTech, Evry, France_.

Background: CDX models are expected to provide crucial information on mechanisms involved in metastatic progression, tumor-initiating properties of CTCs and the development of drug resistance. However, excepted for small-cell lung cancer (SCLC), CDX models are very difficult to develop. Here, we report the establishment, phenotypic and molecular characterization of four NSCLC CDX models and three in vitro cell lines derived from these CDXs.

Methods: CTCs were enriched by RosetteSep from 30 ml blood samples and implanted subcutaneously into Nod/Scid-IL2Rγ-/- (NSG) mice. CDXs were phenotypically and genetically characterized by immunofluorescence, immunohistochemistry and whole-exome sequencing (WES). CDX-derived cell lines were established after mouse fibroblast depletion using classical culture medium. Standard conditions were used for IC50 determination.

Results: Between January 2014 and June 2017, CTCs from 58 NSCLC patients with advanced metastatic disease were implanted into NSG mice resulting in the establishment of four CDXs. All had an epithelial phenotype. Based on CellSearch® counts, median and mean numbers of engrafted CTCs were 9 and 693 respectively (range, 0-17,694). GR-CDXL1, GR-CDXL2, GR-CDXL3, GR-CDXL4 were established starting from 3500, 35, 330, and 1102 CTCs respectively. Measurable tumors were obtained between 100 and 200 days after CTC implantation and were maintained by successive transplantations in NSG mice. Three in-vitro cell lines were established from GR-CDXL1, GR-CDXL3 and GR-CDXL4 tumors, and expressed an epithelial phenotype and CSC-markers such as ALDH, CD133 and CD90. Immunohistochemistry with epithelial and neuroendocrine markers, TTF1 and Ki67 indicated that CDXs and CDX-derived cell lines were representative of the corresponding patient tumor specimens (available in three patients). WES indicated 86%, 93%, 82% mutational similarity between GR-CDXL2, GR-CDXL3 and GR-CDXL4 and the corresponding tumor biopsies. The mutational similarity of GR-CDXL1, GR-CDXL3 and GR-CDXL4 and their corresponding in vitro cell lines was 24%, 83% and 84% respectively. WES of individual CTCs isolated at the time of CTC implantation is ongoing. In in vitro cytotoxicity assays, CDX-derived cell lines mirrored the patient's responsiveness to cisplatin and paclitaxel chemotherapy. The results of ongoing in vivo drug efficacy assays and of mutational tree analyses reconstructing the phylogenic evolution of tumor biopsies, CTCs, CDX and cell lines will be presented.

Conclusion: This study revealed considerable similarities between CDXs and their corresponding patient tumor biopsies. These NSCLC CDX models represent unique tools to identify clonal mutations associated with the tumor-initiating capacity of CTCs and explore the genetic and phenotypic basis of metastasis and drug resistance associated with advanced NSCLC.

#2598

In silico **gene expression analysis of PTHrP and its association with molecular subtypes and organ-specific metastasis in human triple-negative breast cancer.**

Gloria Assaker, Anne Camirand, Siham Sabri, Richard Kremer. _McGill University, Montreal, Quebec, Canada_.

Triple-negative breast cancer (TNBC) represents 10-20% of all BC cases, and is characterized by an aggressive clinical course with high risk of metastasis and lack of targeted therapy. Gene expression profiling revealed the heterogeneity of TNBC enabling its classification into distinct molecular subtypes with distinct susceptibilities to chemotherapies; including basal-like (BL), mesenchymal (M), and luminal androgen receptor (LAR) subtypes. Overexpression of the parathyroid hormone-related protein (PTHrP) in breast cancer has been extensively linked to its progression with increased propensity for bone metastasis. However, its expression and implication in organ-specific metastasis in the TNBC subtype remains largely unknown. In this study, we conducted in silico analyses to examine the association of PTHrP with various molecular BC subgroups and with organ-specific metastasis in TNBC.

Breast Cancer Gene-Expression Miner Version 4.0 (bc-GenExMiner v4.0) online database was used to evaluate the relative mRNA expression levels of the PTHLH gene (which encodes PTHrP) with respect to other markers. This microarray-based tool uses 36 public datasets and 5861 patients, and allows the expression, correlation, and prognostic analyses of genes in BC. Using the gene expression correlation analysis module of bc-GenExMiner v4.0, we assessed Pearson's correlation coefficient between PTHLH expression and gene signatures characteristic of TNBC molecular subtypes or representative of organ-specific metastasis in BC.

We found that PTHLH expression displays significant positive correlations with components of signalling pathways enriched in the mesenchymal subtype, and with key luminal markers and AR signalling genes characteristic of the LAR subtype. While PTHLH expression presents significant positive correlations with signature genes involved in bone and lung metastases in all BC subtypes, we identified for the first time a correlation between PTHLH expression and brain metastasis specifically in TNBC patients. Interestingly, PTHLH correlates with the brain metastatic genes HBEGF (Heparin-binding EGF-like growth factor) and ANGPTL4 (Angiopoietin-like 4) selectively in both TNBC and BL subtypes as opposed to other BC subtypes, which is in line with studies reporting their common morphological and genetic features and increased rate of brain metastasis.

In conclusion, our in silico analysis reveals for the first time a strong association between PTHrP expression and specific TNBC molecular subtypes' markers, as well as its potential role in the mechanisms of TNBC brain metastasis and the identification of a novel gene signature with a prognostic value for brain progression in TNBC.

Funding: Department of Defense (DoD, USA) Award No. W81XWH-15-1-0723

#2599

Circulating nucleic acids as biomarkers of prognosis and chemorefractory status in metastatic pancreatic cancer.

Vincent Bernard, Dong U. Kim, F. Anthony San Lucas, Jonathan Castillo, Kelvin Allenson, Feven C. Mulu, Bret M. Stephens, Jonathan Huang, Eugene Koay, Cullen M. Taniguchi, Milind Javle, Robert A. Wolff, Matthew H. Katz, Gauri R. Varadhachary, Hector A. Alvarez, Anirban Maitra. _MD Anderson Cancer Center, Houston, TX_.

Background: Prognostic and therapeutic stratification of pancreatic ductal adenocarcinoma (PDAC) patients remains elusive due to a lack of effective biomarkers, and a predilection towards metastatic disease. Peripheral blood-based liquid biopsies for tumor markers has emerged as a potential minimally invasive strategy for tumor monitoring. We have implemented a liquid biopsy assay utilizing DNA derived from vesicles known as exosomes (exoDNA) and circulating tumor DNA (ctDNA) in the metastatic setting to determine the potential utility of these liquid biopsy compartments in tumor management.

Methods: A total of 318 plasma samples from 123 metastatic pancreatic patients were prospectively collected. ExoDNA and ctDNA were then extracted from matched plasma samples. Digital PCR, was used to identify codon 12/13 KRAS gene mutations. We assessed clinical endpoints in relation to progression free survival (PFS) and overall survival (OS) using univariate and multivariate analyses. In a metastatic PDAC patient, six serial liquid biopsies and seven tissue biopsies taken throughout disease progression underwent whole genome sequencing for detection of copy number events.

Results: Detection rates of KRAS mutations in exoDNA and ctDNA at baseline treatment naïve staus were 61.0% and 52.9%, respectively. On multivariate COX regression analysis, exoDNA KRAS mutant allelic fraction (MAF) ≥ 5% was a significant predictor of poorer PFS (HR 2.28, 95% CI 1.18-4.40, P=0.014) and OS (HR 3.46, 95% CI 1.40-8.50, P=0.007) in metastatic patients. Among 34 patients, liquid biopsy tumor monitoring was performed across 127 serial blood draws during a median followup time of 11.1 months. The presence of an exoDNA MAF peak ≥ 1% during tumor monitoring was significantly correlated to radiological progression (p=0.0003). Specifically, detection of an exoDNA MAF peak ≥ 1% preceded radiological progression by a median of 50 days compared to a median of 0 days for CA19-9 (p=0.03). CtDNA did not emerge as a significant predictor of survival outcomes in our cohort. In a patient with multiple longitudinal liquid biopsies, comprehensive genomic profiling of exoDNA further demonstrated our ability to capture additional mutational events as they emerged during therapy and correlated to progression including amplifications in ERBB2 and MYC and deletions in CDKN2A and SMAD4.

Conclusions: Liquid biopsies in PDAC provide direct evidence of those patients likely to experience poorer outcomes allowing for more effective therapeutic stratification. Liquid biopsies also demonstrate utility in characterization of putative emerging driver events during disease progression.

### Biomarkers of Therapeutic Response in Clinical Trials

#2600

Resistance biomarkers in patients with head and neck squamous cell carcinoma treated with cetuximab.

Maud Kamal, Olivia Leblanc, Sophie Vacher, Charlotte Lecerf, Emmanuelle Jeannot, Jerzy Klijanienko, Frederique Berger, Caroline Hoffmann, Valentin Calugaru, Nathalie Badois, Anne Chilles, Maria Lesnik, Samar Krhili, Thomas Jouffroy, Ivan Bieche, Christophe Le Tourneau. _Institut Curie, Paris, France_.

BACKGROUND: Cetuximab, a monoclonal antibody targeting EGFR, improves overall survival in the locally advanced setting in combination with radiotherapy and in first-line recurrent setting in combination with chemotherapy in HNSCC patients. No predictive biomarker of efficacy of cetuximab in these settings. We aimed at identifying biomarkers of sensitivity/resistance to cetuximab.

PATIENTS AND METHODS: HNSCC patients treated with cetuximab at Institut Curie, for which complete clinicopathological data and formalin-fixed, paraffin-embedded (FFPE) tumor tissue were available and collected before cetuximab treatment, were included in this retrospective study. Immunohistochemistry analyses of PTEN and EGFR were performed to assess expression levels. PIK3CA and KRAS mutations were analyzed using high resolution melting (HRM) and Sanger sequencing. The predictive value of these alterations on progression-free survival (PFS) was evaluated.

RESULTS: PIK3CA and KRAS/HRAS mutations were associated with poor PFS in HNSCC patients treated with cetuximab in first-line recurrence setting but not in patients treated with cetuximab in combination with radiotherapy. PTEN loss of expression had a negative prognostic impact in HNSCC patients treated with cetuximab and radiotherapy, while EGFR high expression was not found to be a sensitivity biomarker for cetuximab in our patient population.

CONCLUSIONS: PIK3CA and RAS mutations were resistance biomarkers of cetuximab in first-line recurrent setting, whereas PTEN loss was a resistance biomarker of cetuximab when given in combination with radiotherapy.

#2601

Clinical outcomes of pts with advanced solid tumors treated according to NGS guided-therapy in a Brazilian cancer center.

Marcos Andre Costa, Marcelo Santos, Roberto Abramoff, Renata D'alpino, Carlos Teixeira, Ariel Kann, Jacques Tabacof, Riad Younes. _Hospital Alemão Oswaldo Cruz, São Paulo, Brazil_.

Introduction: Next Generation Sequencing (NGS) is a key tool since it unreveals genetic alterations (GA) with potential for targeted-therapy, frequently, not detected for conventional methods done previously. Nevertheless, avaiability of these drugs is a major concern and, additionally, oncologists are challenged to deal with such a huge amount of findings whose clinical significance and sensibility to those drugs are, many times, uncertain. Is broadly unknown, except for isolated initiatives, the correlated clinical outcomes of targeted-therapy guided by NGS.

Methods: In this retrospective study, we describe clinical outcomes of 34 pts with advanced solid tumors (tu) treated in a single Cancer Center in Brazil according to therapies guided by NGS. All tests were performed using Illumina HiSeqs of Foundation Medicine (FM). The f/u was obtained from our electronic charts.

Results: From apr/14 to oct/17, 34 pts were identified, 13M/21F, mean 58y/o. Histologies were: 7 NSCLC, 10 mCRC, 3 pancreatic, 3 breast and 11 varied. In 26 / 34 pts (76%), druggable GA were identified and 17 (50%) were treated with NGS-guided therapies - 14 with TKi and 3 with immunotherapy (imm). Importantly, 8/ 17 pts (47%) experienced clinical benefit (DE, PR, CR) by RECIST 1.1 (1 melanoma, 1 colon, 6 lung adeno). Seven (41%) had PD in the first control in 4-8w (mCRC treated with cobimetinib because of TP53 G12D; mCRC-trastuzumab-HER2 R678Q; breast-olaparibe-BRCA-2 T431fs*20; breast-EVE-PIK3CA E545K; endometrium-EVE-PIK3CA C604R/PIK3CA E81K and PTEN R233*/PTEN S229; mCRC-trastuzumab+lapatinib-HER2 amplification V777L; HCC-EVE-IKBKE amplification and PTEN loss exon 2-9), 1 had just started the therapy (BRCA-2 mutated pancreatic adeno with olaparib) and 1 died before restaging (HER2-mut mCRC treated with chemo+trastuzumab). The DoR was better in those with anti-ALK (3 pts, 18 mo) and anti-EGFR (1 pt, 6 mo ongoing response) and in those with TMB-I/high treated with imm (1 melanoma with TMB27 and 2 lung adeno with TMB 18 and 8). Amongst the 10 pts with evaluable TMB values, 6 were TMB-I/high and, in those, was found a high frequency of mutations: mean of 7). Curiously, 1 pt with mCRC with Kras G12V mutation treated with Trametinib, according to FM recommendations, experienced SD for 12 mo. Finally, 7 pts had samples tested by local companies using other methods (IHC, FISH, PCR) and in 5 (14% of all 34) it did not identified the GA observed by NGS.

Conclusion: NGS identified a significant amount of GA otherwise not detected by conventional tools, changed management and resulted in improved clinical benefit, especially for those with lung adeno treated by anti-ALK/EGFR and those with TMB-I/high treated by imm. Addionally, this study suggests that tu with higher amount of GA in NGS are prone to harbor TMB-I/high and also confirms the low responsiveness of solid tumors to EVE even with driver-mutations in the PI3K-Akt-mTor pathway.

#2602

Clinical implication of mutation load in patients with HER2-positive refractory metastatic breast cancer.

Yeon Hee Park,1 Kyunghee Park,1 Song Ee Park,1 Eunjin Lee,1 Ji-Yeon Kim,1 Jin Seok Ahn,1 Young-Hyuck Im,1 Hun Jung,2 Chooghoon Lee,3 Woong-Yang Park,1 Razvan Cristescu4. 1 _Samsung medical center, Seoul, Republic of Korea;_ 2 _Samsung Bioepis, Suwon, Republic of Korea;_ 3 _MSD Korea Ltd, Seoul, Republic of Korea;_ 4 _Merck & Co., Inc., Kenilworth, NJ_.

Background and Purpose Studies have suggested that the antigenicity of tumor cell is highly correlated with response to immune checkpoint inhibitors. Checkpoint blockade has been found to be particularly effective in tumors with high tumor mutational burden (TMB) in certain indications such as melanoma and NSCLC, while cancers with lower TMB have been shown to be less responsive to checkpoint inhibitors. In HER2-positive metastatic breast cancer (MBC), TMB has potential to be a candidate prognostic or predictive marker for conventional treatments including HER2 targeting agents. This research was conducted to explore the clinical implication of somatic TMB in a well-defined HER2-positive MBC population who were previously treated but progressed.

Methods Forty-six patients with HER2-positive MBC who have progressed from more than two HER2-directed therapies were enrolled from 2007 to 2016. Whole exome sequencing was performed on FFPE tumor samples and matched normal tissue. Sequencing reads were aligned to reference human genome using BWA-MEM followed by standard pre-processing steps and GATK to generate analysis ready BAM files. MuTect suite was used to generate somatic single nucleotide variants (SNV) calls using default parameters by comparing BAM files from tumor and matched normal samples. TMB for a subject was defined as the sum of somatic non-synonymous SNVs that passed all the filters described.

Results Among 46 patients, 13 (28.3%) patients were determined to estrogen receptor (ER)-positive and 9 (19.6%) patients were determined to progesterone receptor (PR)-positive in immunohistochemistry (IHC) analysis. The median age 48 years (range: 29-68) and all (100%) patients were female. 20 patients (43.5%) were recurrent MBC compared to de novo (n = 26, 56.5%). 16 (34.6%) patients demonstrated more than 100 mutations (and defined as high TMB in this population). The median follow up of duration was 57.5 months (95% CI: 55.4 - 60.6). The median overall survival (OS) according to low or high TMB status (44.9 months vs. 85.8 months) was significantly different (p = 0.016). In a multivariate Cox regression analysis, TMB was the only independent prognostic factor for good OS when adjusted for age and recurrence (Hazard ratio = 0.32, 95% CI, 0.103 - 0.998, p = 0.049).

Conclusions Data implicated that high TMB can be a prognostic marker that predicts good OS outcomes with conventional HER2-directed treatments and chemotherapy. Furthermore, future trials using TMB as a stratification marker could identify the right population with potential to respond to immune checkpoint inhibitor after recurrence following HER2-directed treatments. To our knowledge this is the first report that tumor mutation burden (TMB) associate with response to standard of care therapy in HER2+ breast tumors.

#2603

Exploratory analysis of Janus kinase 1 (JAK1) loss-of-function (LoF) mutations in patients with DNA mismatch repair-deficient/microsatellite instability-high (dMMR/MSI-H) metastatic colorectal cancer (mCRC) treated with nivolumab + ipilimumab in CheckMate-142.

Scott Kopetz,1 Theirry Andre,2 Michael J. Overman,1 Vittorina Zagonel,3 Sara Lonardi,3 Massimo Aglietta,4 Fabio Gelsomino,5 Ray McDermott,6 Ka Yeung Mark Wong,7 Alain Hendlisz,8 Pilar Garcia Alfonso,9 Heinz-Josef Lenz,10 Alice Walsh,11 Rebecca A. Moss,11 Danielle Greenawalt,11 Z. Alexander Cao11. 1 _MD Anderson Cancer Center, Houston, TX;_ 2 _Hôpital Saint Antoine and Sorbonne Universités, UPMC Paris 06, Paris, France;_ 3 _Istituto Oncologico Veneto IOV-IRCSS, Padova, Italy;_ 4 _University of Torino, Turin, Italy;_ 5 _University Hospital of Modena, Modena, Italy;_ 6 _St Vincent's University Hospital, Dublin, Ireland;_ 7 _University of Sydney, Sydney Medical School, Sydney, Australia;_ 8 _Institut Jules Bordet, Brussels, Belgium;_ 9 _Hospital General Universitario Gregorio Marañón, Madrid, Spain;_ 10 _University of Southern California Norris Comprehensive Cancer Center, Los Angeles, CA;_ 11 _Bristol-Myers Squibb, Princeton, NJ_.

Background JAK1 LoF mutations were reported as a mechanism of resistance to anti-PD-1 therapy in patients with metastatic melanoma (n = 2) or mCRC (n = 1).1,2 Here, we report an exploratory analysis of CheckMate-142 (NCT02060188) investigating whether JAK1 LoF mutations were present in patients with dMMR/MSI-H mCRC who achieved clinical benefit with nivolumab + ipilimumab treatment.

Methods Patients received nivolumab 3 mg/kg + ipilimumab 1 mg/kg Q3W × 4 doses and then nivolumab 3 mg/kg Q2W. Whole-exome sequencing was performed on pretreatment biopsies preserved in RNAlater and germline DNA from peripheral blood mononuclear cells. Single nucleotide variants and insertions/deletions were called by 2 variant callers in paired normal and tumor samples, and the union of mutations was included in the downstream analysis as previously described.3 Best overall response was assessed by investigators (RECIST v1.1).

Results JAK1 LoF mutations were identified in 4 of 40 patients evaluated in this analysis. Of these 4 patients, 1 had an ongoing partial response of 9.7 months at data cutoff, and 3 had stable disease (Table); 2 patients with stable disease were still on treatment (12 and 17 months) at data cutoff, and 1 discontinued treatment after 2.9 months due to an unrelated adverse event and had not progressed after 10 months off treatment.

Conclusions Clinical benefit was achieved with nivolumab + ipilimumab in patients with dMMR/MSI-H mCRC who harbored a JAK1 LoF mutation. Additional analyses are needed to assess the effect of concurrent JAK1 loss of heterozygosity and other genetic alterations in patients with dMMR/MSI-H mCRC. 1. Zaretsky JM et al. N Engl J Med. 2016;375:819-829. 2. Shin D et al. Cancer Discov. 2017;7:188-201. 3. Carbone DP et al. N Engl J Med. 2017;376:2415-2426.

Table. Best overall response in patients with dMMR/MSI-H mCRC identified as having wild-type JAK1 or a JAK1 LoF mutation

|

---|---

|  | JAK1 LoF mutant

n = 4 | JAK1 wild type

n = 36

Best overall response, n | |

|

Complete response | |

0 | 1

Partial response | |

1a | 18

Stable disease | |

3b | 11

Progressive disease | |

0 | 4

Not evaluable | |

0 | 2

fs, frameshift. a JAK1 mutation: I975*. b JAK1 mutations: D899Gfs*10, K860Nfs*16, and K142Rfs*26. | |

|

#2604

PD-L1 rs2282055 is associated with opposite treatment effect between platinum-based chemotherapy and nivolumab treatment.

Takashi Nomizo, Hiroaki Ozasa, Takahiro Tsuji, Tomoko Funazo, Yuto Yasuda, Hironori Yoshida, Yuichi Sakamori, Toyohiro Hirai, Yong Hak Kim. _Kyoto University, Kyoto, Japan_.

Treatment of lung cancer is improved recently, especially immune-check point inhibitors prolong progression-free survival (PFS) and overall survival (OS), however, Lung cancer is still leading cause of death worldwide. This is because of response rates in the treatment of non-small cell lung cancer (NSCLC) with combination platinum-based chemotherapy vary from 20% to 40%, and response rates of the treatment of NSCLC with nivolumab is around 20%, whereas with leaving a large number of patients with either stable or progressive disease. It is currently difficult to predict treatment response to chemotherapy and nivolumab since there are no precise biomarkers for it. Recently, we have reported that PD-L1 single nucleotide polymorphisms (SNPs) are associated with response to nivolumab treatment. This study was intended to determine the efficacy of platinum-based combination chemotherapy either in a doublet or triplet, and nivolumab treatment respect to PD-L1 SNPs among patients with NSCLC. A total of 139 patients with NSCLC were treated with platinum-based doublet or triplet chemotherapy, 73 patients treated with nivolumab and were also evaluated for PD-L1 SNPs from plasma DNA. We investigated the association among PD-L1 SNPs, objective response rate (ORR) and PFS. PD-L1 rs2282055 was associated with ORR and PFS in the patients treated with pulatinum-based chemotherapy and nivolumab. In the patients treated with nivolumab, the ORR was 24%, 12%, and 0% for the G/G, G/T and T/T genotypes of PD-L1 rs2282055, respectively. The G allele of PD-L1 rs2282055 was significantly associated with better clinical response compared with the T allele (P = 0.0056 [Cochran-Armitage trend test]). The median PFS time was 2.1 months (95% confidence interval [CI], 1.8 months to 3.9 months) for the G/G and G/T genotypes and 2.2 months (95% confidence interval [CI], 0.9 months to 2.6 months) for the T/T genotype (P = 0.0210). On the other hand, The T allele of PD-L1 rs2282055 was significantly associated with better clinical response compared with the G allele in the patients treated with platinum-based combination chemotherapy either in a doublet or triplet (P = 0.0080 [Cochran-Armitage trend test]). The median PFS time was 11.0 months (95% confidence interval [CI], 6.2 months to 16.3 months) for the T/T genotypes and 7.3 months (95% confidence interval [CI], 6.0 months to 8.2 months) for the G/T and G/G genotype (P = 0.0284). In conclusion, these results suggest that the T/T genotype of PD-L1 SNP rs2282055 associated with the better treatment effect of platinum-based combination chemotherapy, on the contrary, the T/T of rs2282055 negatively associated with response to nivolumab treatment. It might be used as a biomaker for selection of the regimen of NSCLC treatment.

#2605

Androgen receptor (AR) anomalies and efficacy of apalutamide (APA) in patients (pts) with nonmetastatic castration-resistant prostate cancer (nmCRPC) from the phase 3 SPARTAN study.

Matthew R. Smith,1 Shibu Thomas,2 Simon Chowdhury,3 David Olmos,4 Jinhui Li,5 Paul N. Mainwaring,6 Stéphane Oudard,7 Felix Y. Feng,8 Michael Gormley,2 Deborah S. Ricci,2 Brendan Rooney,9 Angela Lopez-Gitlitz,10 Margaret K. Yu,10 Eric J. Small8. 1 _Massachusetts General Hospital Cancer Center and Harvard Medical School, Boston, MA;_ 2 _Janssen Research & Development, Spring House, PA; _3 _Guy's, King's and St. Thomas' Hospitals, Great Maze Pond, London, United Kingdom;_ 4 _Spanish National Cancer Research Centre (CNIO), Madrid and Hospitales Universitarios Virgen de la Victoria y Regional, Institute of Biomedical Research in Málaga, Malaga, Spain;_ 5 _Janssen Research & Development, San Diego, CA; _6 _Paul Mainwaring Pty Ltd, Queensland, Australia;_ 7 _Georges Pompidou Hospital, Paris, France;_ 8 _Helen Diller Family Comprehensive Cancer Center, University of California San Francisco, San Francisco, CA;_ 9 _Janssen Research & Development, High Wycombe, United Kingdom; _10 _Janssen Research & Development, Los Angeles, CA_.

Introduction The next-generation AR inhibitor APA improves median metastasis-free survival by 2 yrs and second progression-free survival (PFS2; time from randomization to progression during the first mCRPC therapy or death) in men with nmCRPC and prostate-specific antigen doubling time ≤ 10 mos. AR anomalies (splice variants, ligand binding mutations, amplification) have been linked to AR signaling-targeted therapy resistance in mCRPC, but their relevance in nmCRPC is unknown. We assessed the frequency of AR anomalies after APA ± androgen deprivation therapy (ADT) and their effect on PFS2.

Methods Pts received APA (240 mg QD) or placebo (PBO), with continuous ADT. ARv7 expression was tested using real-time PCR in 200 whole blood samples (end of treatment [EOT], first progression). Next-generation sequencing was performed on 240 EOT plasma samples to detect 5 clinically relevant AR mutations (L702H, W742C, H875Y, F877L, T878A) and AR amplification. Kaplan-Meier methods were used to estimate median PFS2, and Cox proportional hazard models to estimate HR and 95% CI.

Results At EOT, 9.4% (9/96) of pts in the APA group and 12.5% (13/104) in the PBO group expressed ARv7. AR mutations were seen in 8.4% (10/118) and 6.5% (8/122) of pts in the APA and PBO groups, respectively. AR amplification occurred in 15.2% (18/118) and 14.7% (18/122) of pts in the APA and PBO groups, respectively. Although AR anomaly positivity had minimal impact on PFS2 with respect to APA, shorter median PFS2 was observed in AR anomaly-positive vs -negative pts, irrespective of treatment (Table).

Conclusions APA treatment in nmCRPC pts did not increase the frequency of AR anomalies common in AR signaling-targeted therapy-resistant mCRPC. AR anomaly positivity was associated with shorter median PFS2 in the PBO group but not in the APA group. These preliminary findings suggest adding APA may circumvent mechanisms of resistance to standard ADT. | |  | |  | |

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

|

Median PFS2, mos* | Events*

Group | AR anomaly positive | AR anomaly negative | HR (95% CI) | P value | AR anomaly positive | AR anomaly negative

Overall | 23.3 | 27.1 | 1.57 (0.94-2.61) | 0.080 | 23/43 | 42/116

APA | 19.7 | 25.4 | 1.17 (0.54-2.51) | 0.693 | 10/19 | 20/49

PBO | 23.3 | 29.7 | 1.99 (1.0-3.97) | 0.046 | 13/24 | 22/67

*Includes only pts who received abiraterone acetate plus prednisone or enzalutamide as the first mCRPC therapy.

#2606

Biological interpretation of circulating miRNA biomarkers predicting regorafenib clinical benefit in patients with hepatocellular carcinoma (HCC) in the RESORCE trial.

Henrik Seidel,1 Karl Köchert,1 Michael Teufel2. 1 _Bayer AG, Berlin, Germany;_ 2 _Bayer AG, Whippany, NJ_.

Regorafenib is a small molecule multi-kinase inhibitor approved for the treatment of patients with hepatocellular carcinoma (HCC) who have previously received sorafenib based on an improvement in overall survival versus placebo in the phase 3 randomized, double-blind RESORCE trial (NCT01774344; Bruix et al., Lancet, 2017).

Expression of 776 plasma miRNAs was quantified by qPCR in baseline plasma samples collected from 343 of 573 patients (60%) from RESORCE (Teufel et al., ESMO2017, abs 705P). Predictive and prognostic effects of miRNAs were evaluated using a Cox proportional hazards model. Nine miRNAs showed significant predictive association with overall survival. A survival benefit in favor of regorafenib was associated with increased plasma levels of miR-30a, miR-122, miR-125b, miR-200a and miR-374b, and decreased plasma levels of miR-15b, miR-107, miR-320 and miR-645. Here, we focus on the biological interpretation of these statistical results.

Expression data from The Cancer Genome Atlas was used to bridge from miRNAs via mRNAs to biological function. Gene set enrichment analysis of mRNAs based on correlation with expression of predictive miRNAs identified a list of the top 142 gene sets concordantly found for at least 6 of the 9 predictive miRNAs at a FDR of 0.01. Top gene sets showed consistent patterns related to HCC subtypes and several processes including liver cancer progression, metabolism of lipids, amino acids, bile acids and xenobiotics, glucuronidation, and doxorubicin resistance. Results suggested that response to regorafenib was improved in G4/G5/G6 versus G1/G2/G3 subtypes of the Boyault classification and in the CTNNB1 and Polysom7 subtypes of the Chiang classification which both coincide with the S3 subtype of the Hoshida classification (Hoshida et al., Seminars in liver disease, 30:35, 2010), a tumor subclass that is generally characterized by a retained hepatocyte-like phenotype, with well-differentiated and smaller tumors, and better survival. This is consistent with known roles of the predictive miRNAs in epithelial-mesenchymal transition, fibrogenesis, glycolysis, proliferation, and enhancement of apoptosis via several mechanisms. For example, miR-122 targets IGF1R, PDK4, LDHA and GALNT10, thereby regulating RAS/RAF/ERK signaling and glycolysis, which has been reported to overcome resistance to sorafenib.

In summary, our results suggest that response to regorafenib is favorable in tumors with properties that are consistent with the S3 subtype of the Hoshida classification. Conclusions from this hypothesis-generating investigation require confirmation in follow-up studies with molecular characterization of tumor biopsies and regorafenib response data.

#2607

Blood based biomarkers and association with clinical outcome (CO) in advanced stage patients (pts) treated with immunotherapy (IO).

Dylan J. Martini,1 Yuan Liu,2 Colleen Lewis,3 Hannah Collins,3 Mehmet Akce,3 Haydn Kissick,2 Bradley C. Carthon,3 Walid L. Shaib,3 Olatunji B. Alese,3 Rathi Pillai,3 Conor E. Steuer,3 Christina Wu,3 David H. Lawson,3 Ragini Kudchadkar,3 Bassel El-Rayes,3 Viraj A. Master,2 Suresh Ramalingam,3 Taofeek K. Owonikoko,3 R Donald Harvey,3 Mehmet Asim Bilen3. 1 _Emory University School of Medicine, Atlanta, GA;_ 2 _Emory University, Atlanta, GA;_ 3 _Winship Cancer Institute of Emory University, Atlanta, GA_.

Background: Optimal biomarkers for cancer pts treated with IO are currently lacking. Markers of inflammation, such as neutrophil-to-lymphocyte ratio (NLR), monocyte-to-lymphocyte ratio (MLR), and platelet-to-lymphocyte ratio (PLR) are readily available and associated with poor outcomes. We investigated the association between these markers and CO in pts treated with IO.

Methods: We conducted a retrospective review of 90 pts with advanced cancer treated on IO-based phase I trials at the Winship Cancer Institute of Emory University between 2009-2017. Baseline NLR, MLR, and PLR were treated as continuous variables and rescaled by their own standard deviation. Overall survival (OS) was measured from the first dose of IO to date of death or hospice referral. Progression-free survival (PFS) was determined from first dose of IO to clinical or radiographic progression or death. We defined clinical benefit (CB) as complete response, partial response, or stable disease. Univariate association (UVA) and multivariable analysis (MVA) were carried out using Cox proportional hazard model or logistic regression model. Baseline covariates included race, gender, ECOG PS, # of prior therapies, Royal Marsden Hospital (RMH) risk group, IO indication, and # of metastatic sites.

Results: The median pt age was 63 years and most (59%) were men. The most common histologies were melanoma (33%) and GI cancers (22%). The majority (81%) were RMH good risk. 46% of pts had CB on IO. The median NLR, MLR, and PLR was 3.63, 0.48, and 182.65, respectively. Increased NLR, MLR, and PLR were all associated with worse OS, PFS, and chance of CB (Table 1). NLR, MLR and PLR are highly correlated to each other (Pearson correlation coefficients ≥ 0.8, all p < 0.0001).

Conclusion: NLR, MLR, and PLR are strongly associated with CO in pts treated with IO. Prospective validation of these findings are warranted.

Table 1: UVA and MVA of NLR, MRL, and PLR with CO | OS  | PFS | CB

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

|

UVA | MVA | UVA | MVA | UVA | MVA

|

HR (CI) | p-value | HR (CI) | p-value | HR (CI) | p-value | HR (CI) | p-value | OR (CI) | p-value | OR (CI) | p-value

NLR | 1.37 (1.11-1.70) | 0.003* | 1.30 (1.02-1.66) | 0.031* | 1.42 (1.18-1.70) | <0.001* | 1.32 (1.06-1.63) | 0.011* | 0.47 (0.23-0.94) | 0.033* | 0.57 (0.26-1.27) | 0.169

MLR | 1.38 (1.14-1.67) | <0.001* | 1.22 (0.98-1.52) | 0.07 | 1.39 (1.18-1.63) | <0.001* | 1.26 (1.03-1.55) | 0.026* | 0.45 (0.23-0.89) | 0.021* | 0.71 (0.32-1.57) | 0.398

PLR | 1.40 (1.15-1.69) | <0.001* | 1.27 (1.03-1.56) | 0.027* | 1.40 (1.19-1.63) | <0.001* | 1.27 (1.06-1.53) | 0.01* | 0.35 (0.17-0.74) | 0.006* | 0.28 (0.11-0.67) | 0.005*

*statistically significant

#2608

Predictive values of serum protein levels in advanced non-small cell lung cancer patients treated with nivolumab.

Jun Oyanagi, Yasuhiro Koh, Hiroaki Akamatsu, Kuninobu Kanai, Atsushi Hayata, Nahomi Tokudome, Keiichiro Akamatsu, Keiichiro Akamatsu, Masanori Nakanishi, Hiroki Ueda, Nobuyuki Yamamoto. _Wakayama Medical University, Wakayama, Japan_.

Background: Though PD-ligand 1 (PD-L1) expression on tumor tissue has been established as companion diagnostics in non-small-cell lung cancer (NSCLC) for anti-PD-L1 treatment, additional biomarkers to enrich the patients likely to benefit from the therapy are critically needed. Here, we conducted a serial evaluation of multiple serum proteins relevant to the modulation of immune system in NSCLC patients treated with nivolumab.

Patients and Methods: Advanced NSCLC patients after failure of at least one prior chemotherapy regimen received nivolumab monotherapy (3mg/kg, q2W) until progressive disease (PD) or unacceptable toxicity. Serum samples were collected at baseline and at week 4. Best response was classified into partial response (PR), stable disease (SD), or progressive disease (PD) according to RECIST v1.1. Using LuminexTM xMapTM technology, serum levels of 54 proteins consisting of cytokines, chemokines, growth factors, and angiogenesis factors were measured. All statistical analyses were carried out using JMP Pro software (ver. 13.0) and Mann-Whitney U test and Spearman's test were performed accordingly. A p value <0.05 was considered as significant.

Results: Thirty-eight patients were registered in the study between January 2016 and March 2017 at Wakayama Medical University Hospital and 34 were included in the final analysis. Demographics of the patients were as follows: median age 68 (range, 49 to 86); male 73 %; stage IV, 100 %; squamous/non-squamous, 30/70 %. Overall response rate was 22% (7/34), and disease control rate was 53% (18/34). Among 54 serum proteins measured serially, the level of serum TNF-α was significantly lower at baseline in non-PD patients than PD patients (p < 0.05). The level of TNF-α was also correlated with longer progression free survival (PFS) (r= -0.5693). Serum IL-8 level at week 4 in PR patients were significantly lower than those in non-PR patients (p <0.01) though no difference was observed at baseline between the two, supporting the relevance of serum IL-8 level to the efficacy of nivolumab. Correlation between IL-8 levels at week 4 and longer PFS turned out to be more significant (r=-0.6864). In addition, serum levels of VEGF-A, TGF-β1 and PDGF-AB/BB were significantly lower in PR patients than those in non-PR patients at week 4 (p <0.05). However, these protein levels were not correlated with PFS.

Conclusions: We identified that the serum levels of IL-8, TNF-α, VEGF-A, TGF-β1 and PDGF-AB/BB as potential biomarkers to predict clinical benefit from nivolumab treatment in advanced NSCLC by multi-analyte protein-based assay. Incorporating additional serum protein levels may have potential to improve the patient enrichment besides previously reported potential biomarkers such as IL-8 and VEGF levels. Further evaluation is warranted in a larger cohort to validate the findings.

#2609

Overexpression of YAP1 of EGFR-mutant lung adenocarcinoma before tyrosine kinase inhibitor is associated with poor survival.

Soon Auck Hong,1 Jin-Hyoung Kang,2 Sook Hee Hong2. 1 _Soonchunhyang University Cheonan Hospital, Cheonan, Republic of Korea;_ 2 _Seoul St. Mary's Hospital, The Catholic University of Korea, Seoul, Republic of Korea_.

Purpose: EGFR tyrosine kinase inhibitor (EGFR TKI) was approved as a first line treatment for EGFR mutant lung adenocarcinoma (LADC) with advanced stage. YAP1 (Yes-associated protein 1) is a main effector of hippo pathway, related with adverse prognosis and EGFR TKI modulation of non-small cell lung cancer. This study aimed to clarify a prognostic role of YAP1 in EGFR mutant LADC and efficacy for EGFR TKI through the course of EGFR TKI.

Materials and Methods: 41 patients with paired lung cancer specimen before and after EGFR TKI were enrolled in this study. The expression of YAP1 protein was evaluated by immunohistochemistry.

Results: 15 cases (36.6%) with high YAP1 expression was found in pre-EGFR TKI LADC, while high YAP1 expression in 21 cases (52.5%) was detected after EGFR-TKI. The transitional level of YAP1 between pre- and post-EGFR TKI was significantly upregulated (P=0.002). High YAP1 before EGFR TKI was related with shorter OS (P=0.023) and PFS (P=0.041). In addition, high YAP1 before EGFR TKI in T790M mutant LADC was related with poor OS (P<0.001).

Conclusion: YAP1 burden before EGFR TKI was crucial role in prognosis of EGFR mutant LADC treated by EGFR TKI.

#2610

Consequences of a high incidence of microsatellite instability (non-Lynch) and BRAF mutated tumors in a population based cohort of metastatic colorectal cancer.

Kristine Aasebø,1 Anca Dragomir,2 Magnus Sundström,2 Per Pfeiffer,3 Per-Henrik Edqvist,2 Geir Eide,1 Fredrik Ponten,2 Camilla Qvortrup,3 Bengt Glimelius,2 Halfdan Sørbye4. 1 _University of Bergen, Bergen, Norway;_ 2 _University of Uppsala, Uppsala, Sweden;_ 3 _Odense Univsersity Hospital, Odense, Denmark;_ 4 _University Hospital of Bergen, Bergen, Norway_.

Background: Clinical trials in metastatic colorectal cancer (mCRC) have shown few patients with microsatellite instable high (MSI-H) tumors and BRAF mutations (mutBRAF). Recent data show that patients with MSI-H tumors benefit from immunotherapy and there are promising data of adding BRAF inhibitors to standard treatment in patients with mutBRAF. It is therefore important to know the frequency of these molecular changes and their importance for outcome in unselected populations of mCRC.

Methods: A prospectively collected population-based cohort of 798 mCRC patients from three geographic areas in Scandinavia was studied. Gene analysis was available for 446 cases for BRAF and KRAS analysis, MSI analysis was done for mutBRAF only. Immunohistochemistry was performed for BRAF and MMR in 611 patients. Kaplan-Meier method, logistic regression and Cox proportional hazards models were used for statistical analyses.

Results: Totally 8 % (46/589 patients) were MSI-H and 20 % (119/586 patients) were mutBRAF. MSI-H tumours harboured mutBRAF in 76 % of cases and 29 % of mutBRAF was MSI-H. MSI-H correlated to female sex, age >75 years, right-sided tumor, mutBRAF, lymph node metastasis and less often lung and liver metastasis. Patients with MSI-H received less often chemotherapy compared to MSS patients; 46 % vs. 64 % received 1st line, 13 % vs. 38 % received 2nd line and 2 % vs. 17 % received 3rd line chemotherapy, respectively. Patients with MSI-H had poorer prognosis, however only significant in BRAF wildtype (wtBRAF) patients, and the negative prognostic potential of mutBRAF was only valid in MSS tumors. Median overall survival (OS) was 4 months in MSI-H vs. 11 months in MSS tumors (p < 0.001). After multivariate analysis of OS in patients given 1st line chemotherapy MSI-H and mutBRAF was independent poor prognostic factors with HR 2.2 (95 % CI: 1.09, 4.60, p = 0.028) and HR 1.9 (95 % CI: 1.24, 2.97, P = 0.004) respectively. Median progression-free survival (PFS) during 1st line chemotherapy was 4 months for MSI-H vs. 8 months for MSS tumors (p = 0.087), and multivariate analysis of PFS showed HR 2.21 (95 % CI: 1.10, 4.44, p = 0.027). MSI-H and wtBRAF patients (n=10) had the worst prognosis with median OS of 1 month (p <0.001) and median PFS after 1st line chemotherapy of 2 months (p = 0.015).

Conclusion: In unselected patients with mCRC, MSI-H and mutBRAF are more common than previously reported, and consequently more patients could benefit from immunotherapy and BRAF inhibitor treatments. Most MSI-H patients harbored mutBRAF (non-Lynch) in contrast to patients in recent mCRC immunotherapy trials, and further studies are needed to evaluate the effect of immunotherapy in this subgroup. Chemotherapy had minimal effect in MSI-H patients and fewer received 2-line palliative chemotherapy indicating that these patients could be considered for immunotherapy as 1st line treatment.

#2611

Evaluation of ANG/TIE/hypoxia pathway genes and signatures as predictors of response to trebananib (AMG 386) in the neoadjuvant I-SPY 2 TRIAL for Stage II-III high-risk breast cancer.

Denise M. Wolf,1 Christina Yau,1 Lamorna Brown-Swigart,1 Gillian Hirst,1 I-SPY 2 TRIAL Investigators, Smita Asare,2 Richard Schwab,3 Don Berry,4 Laura Esserman,1 Kathy S. Albain,5 Brian Leland-Jones,6 Laura van 't Veer1. 1 _UCSF, San Francisco, CA;_ 2 _QuantumLeap Healthcare Collaborative, San Francisco, CA;_ 3 _UCSD, San Diego, CA;_ 4 _Berry Consultants, LLC, Austin, TX;_ 5 _Loyola University, Chicago, IL;_ 6 _Avera Cancer Institute, Sioux Falls, SD_.

Background: The angiogenesis (ANG1/2) inhibitor trebananib (TR) was one of the experimental agents evaluated in I-SPY 2. In I-SPY 2, all patients received at least standard chemotherapy (paclitaxel followed by doxorubicin/cyclophosphamide: T->AC). HER2- patients were randomized to receive TR+T->AC vs. T->AC. For HER2+ patients, TR was administered with trastuzumab (TR+H+T->AC vs. H+T->AC). We hypothesized that genes/signatures in the ANG/TIE signaling axis may specifically predict response to TR, and tested expression levels of 11 genes: TIE1/2, ANGPT1/2/4, AGPTNL1/3, VEGFA, ICAM1, PECAM1 and MMP2. We also evaluated angiogenesis and hypoxia expression signatures, based on the hypothesis that hypoxic tumors with a fragile blood supply may be vulnerable to drugs in this class.

Methods: Data from 266 patients (TR: 134 and concurrent controls: 132) were available. Pre-treatment biopsies were assayed using Agilent 44K (32627) or 32K (15746) expression arrays; and these data were combined using ComBat. All I-SPY 2 qualifying biomarker analyses follow a pre-specified analysis plan. We use logistic modeling to assess biomarker performance. A biomarker is considered a specific predictor of TR response if it associates with response in the TR arm, and if the biomarker x treatment interaction is significant (likelihood ratio test, p<0.05). This analysis is also performed adjusting for HR and HER2 status as covariates, and within receptor subsets. Additional exploratory global transcriptomic analysis was performed using DAVID. Our statistics are descriptive rather than inferential and do not adjust for multiplicities of other biomarkers outside this study.

Results: ANGPT1, a direct target of trebananib, associates with pCR in the TR arm but not the control arm, and shows a significant interaction with treatment that retains significance in a model adjusting for HR and HER2. In addition ICAM1, expressed on endothelial and immune cells, strongly associates with response in the TR arm, but also in the control arm in the population as a whole. In the HR+HER2- subset, both ICAM1 and PECAM1 associate with pCR in the TR arm and not the control arm, with a trend toward treatment interaction. Interestingly, in the TN subset, where pCR rates were highest in the TR arm relative to control, these mechanism-of-action biomarkers did not appear to predict response. Rather, in exploratory whole genome analysis, response of TN's strongly associates with immune related genes (e.g. HLA's, IL21R, CCL13).

Conclusion: Following our pre-specified analysis, ANGPT1 succeeds as a specific predictor of response to trebananib in I-SPY 2. In addition, ICAM1 and PECAM1 associate with response in the HR+HER2- subset; and in exploratory analysis immune signaling predicts response in the TN subset. These biomarkers may merit further evaluation in future trials.

#2612

BluePrint Luminal subtype predicts non-response to HER2-targeted therapies in HR+/HER2+ I-SPY 2 breast cancer patients.

Pei Rong Evelyn Lee,1 Zelos Zhu,1 Denise Wolf,1 Christina Yau,1 William Audeh,2 Annuska Glas,2 Lamorna Brown-Swigart,1 Gillian Hirst,1 Angela DeMichele,3 I-SPY 2 TRIAL Investigators, Laura Esserman,1 Laura van 't Veer1. 1 _UCSF, San Francisco, CA;_ 2 _Agendia Inc., Irvine, CA;_ 3 _University of Pennsylvania, Philadelphia, PA_.

Background: Previous studies suggest that within the triple positive HR+HER2+ subtype, patients classified as BluePrint (BP) Luminal subtype are more responsive to pertuzumab and trastuzumab (P/H) as opposed to trastuzumab (H) alone. In the I-SPY2 TRIAL, HER2-targeted treatment arms include H, P/H, neratinib (N), and T-DM1/Pertuzumab (T-DM1/P); and patients were classified by BP molecular subtyping in addition to conventional receptors. We evaluated BP subtype as a predictor of response in HR+HER2+ patients and assessed pathway differences between BP molecular subtypes.

Methods: 125 HR+HER2+ patients (N: 42; P/H: 29, T-DM1/P: 35; H: 19) with pre-treatment Agilent microarrays and BP subtype assignments were considered. We assess association between BP subtypes and pCR using Fisher's exact test. To identify genes associated with BP Luminal vs. BP HER2 subtype, we (1) apply a Wilcoxon rank sum test and (2) fit a logistic model, with the Benjamini-Hochberg (BH) multiple testing correction (BH p<0.05 from both tests). We then perform pathway enrichment analysis using DAVID. Our study is exploratory and does not adjust for multiplicities of other biomarkers in the trial outside this study.

Results: Of the 125 HR+HER2+ patients, 71 were BP HER2-type and 50 were BP Luminal-type. The distribution of pCR rates in BP Luminal/ HER2 subtypes are as follows:

Distribution of pCR rates in BP Luminal/ HER2 subtypes by treatment arm

---

|

N (n = 40) | P/H (n = 29) | T-DM1/P (n = 34) | H (n = 18) | All arms (n = 121)

BP Luminal (n = 50) | 0 / 9 (0%) | 1 / 12 (8.3%) | 2 / 16 (12.5%) | 1 / 13 (7.7%) | 4 / 50 (8%)

BP HER2 (n = 71) | 12 / 31 (38.7%) | 13 / 17 (76.5%) | 15 / 18 (83.3%) | 1 / 5 (20%) | 41 / 71 (57.7%)

Odds Ratio | 6.1; p = 0.127 | 30.2; p = 0.000484 | 29.6; p = 8.60E-05 | 2.8; p = 0.490 | 15.3; p = 1.04E-08

In a whole transcriptome analysis, 1725 genes were differentially expressed. By DAVID enrichment analysis, the most significantly enriched pathways were related to immune function, with the BP HER2 subgroup showing higher expression.

Conclusion: Our analysis suggests that HR+HER2+ BP Luminal subtype is associated with lower response rates to HER2-targeted agents, including P/H, and may need an alternative strategy. BP HER2 subtype appears associated with higher expression of immune-related genes, relative to BP Luminal; and suggests that immune signaling may contribute to HER2-targeted therapy sensitivity.

#2613

Predictive impact of low-frequency pretreatment T790M mutation in patients with EGFR-mutated non-small cell lung cancer treated with EGFR tyrosine kinase inhibitors.

Yoshiya Matsumoto,1 Kenji Sawa,1 Jun Oyanagi,2 Mitsuru Fukui,1 Naoki Yoshimoto,1 Tomohiro Suzumura,1 Shigeki Mitsuoka,1 Kazuhisa Asai,1 Tatsuo Kimura,1 Nobuyuki Yamamoto,2 Tomoya Kawaguchi,1 Kazuto Hirata,1 Yasuhiro Koh2. 1 _Osaka City University, Osaka, Japan;_ 2 _Wakayama Medical University, Wakayama, Japan_.

Background: T790M mutation in EGFR accounts for nearly 50% of the acquired mechanism of resistance to EGFR-tyrosine kinase inhibitors (TKIs). Previous studies suggested that EGFR T790M mutation was also detected in a considerable number of EGFR-TKI-naïve non-small-cell lung cancer (NSCLC) patients utilizing ultrasensitive detection methods such as droplet digital PCR (ddPCR). Here we investigated the significance of low-frequency pretreatment EGFR T790M mutation (preT790M) and its association with cancer molecular heterogeneity in the efficacy of EGFR-TKIs.

Materials and methods: Fifty-two advanced NSCLC patients harboring activating EGFR mutations treated with first-line EGFR-TKIs at Osaka City University Hospital between August 2013 and July 2016 were enrolled in the study. DNAs from tumor biopsies at diagnosis were available from 44 patients for detecting preT790M by the cobas® EGFR Mutation Test v2 (cobas) and ddPCR (RainDance Technologies) and those from 33 were available for assessing the actionable mutations in 50 genes by next-generation sequencing (NGS). NGS was performed on the Ion PGM using Ion AmpliSeq Cancer Hotspot Panel v2 (Life Technologies). Paired biopsies before EGFR-TKIs treatment and at disease progression (PD) were obtained from 15 patients to assess the T790M mutation by cobas.

Results: The overall detection rate of preT790M by ddPCR was 40.9% (18/44), while not detected in any case by cobas. The median progression free survival (mPFS) was 10.0 months for the patients with preT790M and 13.5 months for those without preT790M (p=0.288), respectively. When divided into 3 categories based on the frequency of preT790M such as high with T790M allele frequency (AF) >0.3%, low with AF ≤0.3% and undetected, 12 patients with high AF had a relatively shorter mPFS than 6 with low AF (p=0.090) and 26 without detectable preT790M (p=0.046) (7.7 vs 17.1 vs 13.5 months, respectively). NGS revealed 18 additional coexisting actionable mutations in 15 out of 33 patients: TP53 (n=7), PIK3CA (n=5), CTNNB1 (n=3) and uncommon EGFR (n=3). There was a trend for patients with higher preT790M AF to harbor less additional coexisting mutations (27% with high AF, 60% with low AF and 53% without preT790M, p=0.320). In 15 paired biopsies, T790M mutation was detected in 60% (9/15) at PD by cobas in the clinical setting; 67% (2/3) with high AF, 50% (1/2) with low AF and 60% (6/10) without detectable preT790M, suggesting T790M mutation after first-line EGFR-TKIs developed from both clonal selection and secondary acquisition models.

Conclusion: Results of our study indicated that EGFR-mutated NSCLCs with higher AF preT790M >0.3% had significantly shorter duration of response to EGFR-TKIs. In addition, the AF of preT790M in EGFR-mutated NSCLC may be associated with coexisting actionable mutation load potentially affecting the efficacy of EGFR-TKIs.

#2614

Macrophage erythroblast attacher (MAEA) polymorphisms are associated with clinical outcome in TRIBE study mCRC patients treated with 5-fluorouracil/bevacizumab-based therapy.

Shivani Soni,1 Yuji Miyamoto,1 Wu Zhang,1 Martin D. Berger,1 Shu Cao,1 Elizabeth Melendez,1 Alberto Puccini,1 Madiha Naseem,1 Ryuma Tokunaga,1 Francesca Battaglin,1 Michelle Mcskane,1 Chiara Cremolini,2 Alfredo Falcone,2 Fotios Loupakis,3 Heinz-Josef Lenz1. 1 _University of Southern California, Los Angeles, CA;_ 2 _Istituto Toscano Tumori, Pisa, Italy;_ 3 _Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS), Padova, Italy_.

Background: Macrophage erythroblast attacher (MAEA) also known as Emp (Erythroblast macrophage protein) plays indispensable role in erythroblast and macrophage development and targeted disruption of Emp is embryonically lethal (Soni et al., JBC 2006). Recently, down-regulation of Emp was implicated in abnormal cell motility and higher expression of mitogen-activated protein kinase 1 (MAPK 1) and thymoma viral proto-oncogene 1 (Akt 1) (Gulnaz et. al., BCMD 2016). Our preliminary studies, shows that higher expression of MAEA is associated with longer colorectal cancer (CRC) patient survival. We tested two MAEA polymorphisms (rs13149952 and rs1128427), whether these are correlated with clinical outcome in mCRC treated with 5-Fluorouracil / Bevacizumab based therapy. Methods: Genomic DNA was isolated from blood samples of two cohorts of mCRC patients. Cohort 1 included 227 patients treated with FOLFIRI/BEV (TRIBE trial Arm A, RAS wildtype/mutant=55/116, median age=60 years old, median follow-up period=49.3 months). Cohort 2 had 229 patients treated with FOLFOXIRI/BEV (TRIBE trial Arm B, RAS wildtype/mutant=60/115, median age=60 years old, median follow-up period=46.6 months). PCR-based direct Sanger sequencing was used to determine polymorphism. Results: Our results showed that MAEA rs1128427 in TRIBE arm A patients with RAS mutant tumors having any C genotype was significantly associated with longer overall survival as compared to TT genotype in univariate analysis {25.9 vs 18.8 months, HR(95% CI)=0.61(0.38,0.98), p=0.036}. In TRIBE arm B cohort, MAEA rs1128427 in patients with RAS mutant tumors carrying CC genotype showed significantly longer overall survival than patients with any T allele in both univariate and multivariable analyses {33.4 vs 25 months, uni: HR(95% CI)=0.57(0.34,0.95), p=0.028; multi: HR(95% CI)=0.56(0.32,0.96), p=0.034}. Conclusion: Our findings suggest for the first time, that polymorphisms in MAEA may predict the clinical outcome in Bevacizumab based therapies. Further prospective studies are warranted.

#2615

Heterogeneity of HER2 expression in gastric cancer (GC) leads to high deviation rates between local and central testing and hampers efficacy of anti-HER2 therapy: Survival results from the VARIANZ study.

Florian Lordick,1 Ivonne Haffner,1 Birgit Luber,2 Dieter Maier,3 Elba Raimundez,4 Jan Hasenauer,4 Albrecht Kretzschmar,5 Ludwig Fischer von Weikersthal,6 Miriam Ahlborn,7 Jorge Riera Knorrenschild,8 Gabriele Siegler,9 Beate Rau,10 Stefan Fuxius,11 Thomas Decker,12 Katrin Schierle,1 Christian Wittekind1. 1 _University Medicine Leipzig, Leipzig, Germany;_ 2 _Technische Universität München, Munich, Germany;_ 3 _Biomax Informatics AG, Planegg, Germany;_ 4 _Helmholtz Zentrum München, Munich, Germany;_ 5 _MVZ Mitte, Leipzig, Germany;_ 6 _Klinikum St. Marien, Amberg, Germany;_ 7 _Klinikum Braunschweig, Braunschweig, Germany;_ 8 _University Hospital Marburg, Marburg, Germany;_ 9 _Paracelsus Medizinische Privatuniversität Nürnberg, Nürnberg, Germany;_ 10 _Charité Universitätsmedizin Berlin, Berlin, Germany;_ 11 _Onkologische Schwerpunktpraxis Heidelberg, Heidelberg, Germany;_ 12 _Studienzentrum Onkologie Ravensburg, Ravensburg, Germany_.

Background: HER2 is a membrane-bound receptor tyrosine kinase that belongs to the epidermal growth factor receptor family. The anti-HER2 directed monoclonal antibody trastuzumab is approved for 1st-line treatment of stage IV HER2+ GC, while other drugs addressing HER2 (pertuzumab, TDM1, lapatinib) failed to improved survival outcomes. Until today, resistance against HER2-directed treatment of GC is poorly understood. The VARIANZ study aims to assess the biologic background of resistance to anti-HER2 therapy in GC.

Methods: This academic network study funded by the German Federal Ministry of Education and Research (BMBF 01ZX1610E) recruited patients (pts) who received medical treatment for stage IV GC in 34 sites. HER2 expression was verified centrally by two dedicated GI pathologists using immunohistochemistry (DCS, HI608C0I) and chromogenic in situ hybridization (Zytomed Systems, C-3022-40). Treatment and survival outcomes were reported by investigators.

Results: 502 pts were enrolled from May 2014 to Oct 2017. At present, 442 samples were fully characterized for HER2. According to criteria from the ToGA study, 73 of 442 samples were found HER2+ in central testing. In 57 samples with a HER2+ status diagnosed by local pathologists, HER2 positivity could not be confirmed centrally. Deviation rate between local and central testing was 23%. Centrally confirmed HER2-positive GC displayed a higher percentage of tumor cells staining positive for HER2 (49.72 ± 28.58% [SD] vs. 11.35 ± 17.74% [SD]; p<0.001) and a higher HER2/CEP17 ratio (5.78 ± 2.55 [SD] vs. 1.43 ± 0.41 [SD]; p<0.001). Survival outcomes indicate that only pts with centrally confirmed HER2+ status benefit from trastuzumab with an overall survival of 30.7 months (95%-CI 10.7 - 50.8; n=28) versus 7.9 months (95%-CI 2.8 - 12.9, n=33); HR for death was 3.9 in pts with unconfirmed vs. confirmed HER2+ GC receiving trastuzumab plus chemotherapy; p<0.0001.

Conclusions: Variability between local and central HER2 assessment in GC is significant. Pts with centrally unconfirmed HER2+ status have no benefit from anti-HER2 therapy. HER2 status should be assessed in highly qualified laboratories and cut-offs for defining HER2+ should be reconsidered. Alternative treatment options--other than trastuzumab--should be investigated in pts with unconfirmed or borderline HER2 expression.

#2616

Baseline blood lymphocytes are associated with improved clinical outcome in atezolizumab-treated patients across multiple indications.

Yijin Li, Ching-Wei Chang, Marcella Fasso, Carol O'Hear, Priti S. Hegde, Luciana Molinero. _Genentech, South San Francisco, CA_.

Background: The PD-L1/PD-1 inhibitors activate the immune system both in the tumor microenvironment and peripheral blood. Here we investigated the relationship between peripheral immune system and clinical outcome of metastatic cancer patients treated with atezolizumab (anti-PDL1) across five tumor types.

Methods: 426 patients with melanoma (n=45), non-small cell lung cancer (NSCLC, n=89), kidney cancer (RCC, n=72), triple-negative breast cancer (TNBC, n=116) and bladder cancer (UBC, n=104) were treated with atezolizumab in the phase I clinical trial PCD4989g (NCT01375842). Normal/abnormal blood neutrophils, monocytes, eosinophils, basophils and lymphocytes were measured by local laboratories, while T (CD3, CD4 and CD8), B (CD19), and NK (CD56) lymphocyte subsets were centrally analyzed by flow cytometry. Neutrophil:lymphocyte ratio (NLR) and relative lymphocyte counts (RLC) were also evaluated. Association of the immune cell subsets and clinical outcome (ORR, PFS and OS) was assessed in multivariate analyses considering liver metastases, LDH, line of therapy and ECOG performance status.

Results: The prevalence of hematologic abnormalities across tumors was: lymphopenia (27-57%), followed by neutrophilia (2-24%), monocytosis (0-15%), eosinophilia (1-13%), eosinopenia (0-14%), basophilia (2-7%) and basopenia (0-6%). Neutrophilia, monocytosis, lymphopenia and low/high eosinophils were associated with worse PFS and OS, although with a different imprint depending on tumor ontogeny: neutrophilia was linked to reduced OS in melanoma and PFS for UBC, monocytosis was associated with lower OS in TNBC and UBC. Lymphopenia was associated with reduced PFS and OS in NSCLC, TNBC and reduced PFS in melanoma. NLR>=5 was associated with reduced PFS in melanoma, NSCLC, RCC, UBC and reduced OS in UBC, NSCLC and TNBC. RLC>=17.5% was linked to longer PFS in melanoma, RCC, UBC and to increased OS in TNBC, NSCLC and UBC. Lymphocyte subset analysis showed lymphopenia in B cells (40-61%), CD3 T cells (32-71%), CD4 T cells (28-66%), CD8 T cells (25-46%) and NK cells (16-23%). CD3 lymphopenia was associated to reduced OS in NSCLC, and CD4 lymphopenia was associated to reduced OS in NSCLC and TNBC. NK lymphopenia was associated to decreased OS and PFS in TNBC.

Conclusion: This is the first study showing that higher pretreatment relative lymphocyte counts is associated to improved clinical outcome in patients from different tumor etiologies treated with atezolizumab. The association of peripheral T cell counts to improved outcome in some indications suggests that the local antitumor response may be linked to pre-existing systemic T cells.

#2617

**A multicenter project to test the validity and logistics surrounding the testing of** AR-V7 **mRNA expression in circulating tumor cells.**

Anieta M. Sieuwerts,1 Bianca Mostert,1 Michelle van der Vlugt-Daane,1 Jaco Kraan,1 Corine Beaufort,1 Mai Van,1 Wendy J. Prager,1 Bram De Laere,2 Nick Beije,1 Paul Hamberg,3 Hans M. Westgeest,4 Metin Tascilar,5 Luc Y. Dirix,2 Wendy Onstenk,1 Ronald de Wit,1 Martijn P. Lolkema,1 Ron H. Mathijssen,1 John W. Martens,1 Stefan Sleijfer1. 1 _Erasmus MC Cancer Institute and Cancer Genomics Netherlands, Rotterdam, Netherlands;_ 2 _University of Antwerp, Antwerp, Belgium;_ 3 _Franciscus Gasthuis & Vlietland, Rotterdam, Netherlands; _4 _Amphia Ziekenhuis, Breda, Netherlands;_ 5 _Isala Ziekenhuis, Zwolle, Netherlands_.

Introduction:

The presence of the androgen receptor splice variant 7 (AR-V7) in circulating tumor cells (CTCs) has shown to be associated with resistance to anti-AR treatment with abiraterone or enzalutamide, but not to chemotherapy containing taxanes such as cabazitaxel in patients with metastatic castration-resistant prostate cancer (mCRPC). The primary objective of this project was to set up a validated multi-center pipeline to measure AR-V7 by RT-qPCR in RNA isolated from CellSearch-enriched CTCs. This pipeline included a) the pre-analytical phase (appropriate anonymized sample collection and sample preservation), b) the analytical phase (assay performance with high analytical sensitivity, precision and specificity and c) the post-analytical phase (data evaluation and reporting).

Materials and Methods:

CellSearch-enirched CTCs from metastatic castration-resistant prostate cancer (mCRPC) patients were characterized by RT-qPCR. To validate the pipeline, we determined the proportion of blood samples from mCRPC patients having three or more CTCs per 7.5 mL of blood for whom an AR-V7 status can be assayed and returned to the clinician within 11 days (design PRELUDE study protocol).

Results:

In the range of the RNA equivalent of 0.2-12 AR-V7 positive VCaP cells the coefficient of variation (CV) for AR-V7 was 9% (n=37). The limit of detection (LOD) was 0.3 and the limit of quantification (LOQ) 3 cells in the final RT-qPCR. In none of 17 healthy blood donors an AR-V7 signal was detected, showing high diagnostic specificity (100%). No differences were observed between AR-V7 data generated by four different technicians or in two different laboratories.

For the 45 patients in the PRELUDE study, thirteen patients were not eligible due to poor RNA quality (n=1) or because the blood sample did not contain enough epithelial signal (n=12). Twenty-two patients were AR-V7 negative and ten AR-V7 positive. The median, 75th and 90th percentile reporting times from blood draw to dissemination of the test results were 7, 8 and 9 days, respectively.

Conclusion:

This AR-V7 test with validated cut-offs, a strong intra- and inter-laboratory performance and the ability to report the outcome within a clinically acceptable time frame for the large majority of patients, met our pre-specified objectives. The AR-V7 test is now ready to be used in prospective studies (including randomized controlled trials) to test its predictive value for outcome on post-docetaxel (anti-AR of cabazitaxel) treatment.

#2618

The thymidine kinase 1 (TK1) protein concentration in sera from Hodgkin lymphoma patients: A marker for cell death and disruption of malignant cells.

Jagarlamudi Kiran Kumar,1 Staffan Eriksson,1 Johan Mattsson Ulfstedt,2 Per Venge,3 Daniel Molin2. 1 _AroCell AB, Uppsala, Sweden;_ 2 _Department of Immunology, Genetics and Pathology, Section of Oncology, Akademiska Hospital, Uppsala, Sweden;_ 3 _Department of Medical Sciences, Uppsala University and Diagnostics Development AB, Uppsala, Sweden, Uppsala, Sweden_.

TK activity measurements have been used for many years to monitor cancer disease activity. However, what the blood concentrations of TK1 protein reflect is still uncertain. A TK1 immunoassay, the AroCell TK 210 ELISA, has been developed based on specific monoclonal antibodies against the C-terminal region of TK1. We utilized this assay to monitor the blood concentrations of TK1 in a cohort of patients with Hodgkin lymphoma (HL) before and after conventional treatment. TK1 concentrations in serum or plasma were measured using the AroCell TK 210 ELISA. For comparison, a large number of other biomarkers and cells were measured in blood such as C-reactive protein (CRP), Lactate dehydrogenase (LDH), hemoglobin, WBC, platelet counts, erythrocyte sedimentation rates (ESR), albumin, liver enzymes, creatinine. Fifty-eight patients with HL were included before start of treatment. Seventy three percent had the nodular sclerosis subtype and 52% stages I-II and 48% stages III-IV disease. Blood was sampled before treatment, during treatment and twice after completion of treatment. The upper normal TK1 concentration of healthy subjects (n=250) was 0.45 μg/L with a median of 0.25 μg/L (range 0.15-0.66 μg/L) as compared to HL, which was 0.26 μg/L (range 0.03-17 μg/L). The differences in ranges were significant (p<0.0001, F-statistics). Nodular sclerosis patients had higher TK1 levels as compared to the other subtypes (p=0.03). TK1 increased after treatment (p=0.001) in patients with normal concentrations from start i.e. <0.45 μg/L, whereas the concentrations decreased in 5/6 patients with TK1 >0.45 μg/L. TK1 concentrations before start of treatment was correlated to high CRP (p=0.01), low Hemoglobin (p=0.03), high WBC (p<0.001) and high platelet counts (p<0.01), but not to albumin or ESR or any other of the measured biomarkers. In a multivariate regression analysis, TK1 was correlated to LDH as the only independent variable (p<0.0001). In Hodgkin lymphoma patients, serum or plasma concentrations of TK1 may reflect malignant cell death and cell disruption rather than cell proliferation during treatment. Our results and hypothesis may help to interpret TK1 protein measurements as clinical tools in cancer management.

#2619

Genetic landscape of KRAS-NRAS-BRAF-PIK3CA wild type metastatic colorectal cancer patients enrolled in the CAPRI clinical trial.

Anna Maria Rachiglio,1 Matilde Lambiase,1 Francesca Fenizia,1 Alessia Iannaccone,1 Cristin Roma,1 Claudia Cardone,2 Antonella De Luca,1 Erika Martinelli,2 Evaristo Maiello,3 Fortunato Ciardiello,2 Nicola Normanno1. 1 _National Cancer Institute, Naples, Naples, Italy;_ 2 _Università della Campania "L. Vanvitelli", Naples, Italy;_ 3 _IRCCS Casa Sollievo della Sofferenza, Italy_.

Introduction: The Cetuximab After Progression in KRAS wild type colorectal cancer patients (CAPRI) study enrolled KRAS exon 2 wild-type (wt) metastatic colorectal cancer (mCRC) patients who received first-line FOLFIRI-cetuximab. Retrospective analysis showed that mCRC patients with KRAS/NRAS/BRAF/PIK3CA wt (quadruple-wt) tumors had an excellent prognosis when treated with anti-EGFR agents. In order to identify additional mechanisms that might allow to better select patients who benefit of anti-EGFR monoclonal antibodies, we analyzed quadruple-wt tumors from the CAPRI clinical trial with a large targeted sequencing panel and correlated results with patients' outcome.

Materials and methods: NGS analysis was performed using the Oncomine Comprehensive v.2 panel from Thermofisher on Ion Torrent PGM. The panel covers 143 cancer genes providing information on hotspot mutations of 73 oncogenes, copy number variation (CNV) of 49 genes, full-length sequence of 26 tumor suppressor genes, and sequence of 22 fusion driver genes. Data analysis was carried out using Ion Reporter™ Software v5.0.

Results: Complete data of the first cohort of 17 patients are summarized. These patients had a median progression-free survival (mPFS) of 12,3 months (m) and median overall survival (mOS) of 34,03 mos. The analysis revealed in all 17 tumors the presence of at least one mutation, and in 9/17 showed the presence of at least one CNV. Variants were detected in the following genes: TP53 (17 mutations), APC (16), FBXW7 (2), MAP2K1 (1), PTPN11 (1), PTCH1 (1), ATM (1), CTNNB1 (1), PIK3R1 (1), PTEN (1), CDKN2A (1), KRAS (1). CNVs were found in APC (2 deletions), TP53 (1 deletion), PIK3R1 (1 deletion), BCL2L1 (4 amplifications), GAS6 (3 amplifications), MYC (2 amplifications), ZNF217 (2 amplifications), FLT3 (1 amplification), ERBB2 (1 amplification), APEX 1 (1 amplification). The 3 patients with the shortest PFS and OS had peculiar genetic alterations that might be associated with resistance to EGFR targeting drugs. Case #980 (PFS 3,93 m; OS 9,2 m) carried a KRAS p.Gln61His variant at a low allelic frequency (0,38%) that was confirmed by plasma testing with BEAMing. Case #4120 (PFS 5,77 m; OS 25,33 m) had a loss of function PTCH1 variant (p.Tyr1316fs) leading to activation of the sonic hedgehog (SHH) pathway. Case #1491 (PFS 6,63 m; OS 19,03 m) carried a p.Lys57Glu mutations in the MAP2K1 gene that causes constitutive activation of the corresponding protein.

Conclusions: These preliminary data show that quadruple-wt CRC carry genetic alterations that potentially can drive resistance to anti-EGFR monoclonal antibodies. While the significance of the identified variants needs to be explored in experimental models, these findings do suggest that wide genetic profiling of CRC might improve our ability to select patients who are highly sensitive to EGFR inhibition.

#2620

Aquaporin 11 as a new predictive biomarker of overall survival and platinum-based chemotherapy response in lung adenocarcinoma patients.

Michael Sharpnack, David P. Carbone, Mikhail M. Dikov, Elena E. Tchekneva. _Ohio State Unibversity, Columbus, OH_.

Background: Biomarkers predictive of response to chemotherapy are critically needed for the precise selection of treatment protocols in lung adenocarcinoma (LUAD). Aquaporins (AQPs), transmembrane water channels, are emerging targets in cancer. Recently discovered, non-ubiquitous family member aquaporin 11 (AQP11), a tissue-specific endoplasmic reticulum (ER) resident, was identified as a cellular pro-survival factor implicated in the maintenance of ER homeostasis. AQP11 is mapped to 11q13-q14 amplicon harboring oncogenic drivers and associated with poor prognosis in cancer patients. We recently showed that high AQP11 expression is an in-vitro therapeutic biomarker of cisplatin therapy, which directly interferes with AQP11 functional structure. In addition, silencing of AQP11 expression in human lung cancer cell lines significantly increased response to cisplatin treatment. We hypothesized that LUAD tumors expressing high levels of AQP11 depend on AQP11-mediated cytoprotection and would be more responsive to platinum-based chemotherapy targeting AQP11. Method: We downloaded and curated matched mRNA expression, survival, and drug response data from The Cancer Genome Atlas (TCGA) LUAD dataset (N=369). Results: Analysis of PRECOG and TCGA databases showed that high AQP11 mRNA expression is negatively prognostic in patients with LUAD. TCGA LUAD cases were categorized by AQP11 mRNA levels into high and low expression (high = AQP11 mRNA expression mean + 1 standard deviation). Patients with high tumor AQP11 mRNA expression (10.3%) had significantly worse overall survival (OS) compared to patients with low AQP11 expression (p=0.0015). An analysis of patients treated with platinum-based chemotherapy (N=74) showed that patients with high AQP11 mRNA expression (7%) had significantly higher OS then patients with LUAD expressing low levels of AQP11 (p=0.0263). Conclusions: This study identifies AQP11 as a new biomarker of OS and chemotherapy in LUAD patients. It is conceivable that lung tumors expressing high levels of AQP11 are dependent on its function and elevated AQP11 expression renders tumor resistance to microenvironment and therapy-induced stress and associates with lower OS of patients. At the same time, as cisplatin efficiently targets AQP11 functional multimeric structure, these AQP11-depending tumors are more prone to platinum based chemotherapy. This study provides a rationale for combination anti-AQP11 and chemotherapy in LUAD tumors with high AQP11 expression.

#2621

Genomic determinants of progression and response to therapy in prospectively characterized glioma patients.

Philip Jonsson, Andrew L. Lin, Shahiba L. Ogilvie, Shweta S. Chavan, Andrew T. McKeown, Natalie M. DiStefano, Marc Rosenblum, Lisa M. DeAngelis, Ingo K. Mellinghoff, Barry S. Taylor. _Memorial Sloan Kettering Cancer Center, New York, NY_.

While the genomic landscape of low- and high-grade gliomas has been characterized and incorporated into further disease classification, its relationship to disease progression and treatment response remains poorly understood. To address this question, we integrated prospective clinical sequencing of 1,007 primary and recurrent tumors from 924 glioma patients with clinical and treatment phenotypes. Genomic alterations in effectors of cell cycle progression were a biomarker of aggressive disease in 1p19q intact IDH-mutant disease, arising preferentially in enhancing and recurrent tumors. When present at recurrence, the lesions were associated with rapid disease progression. These alterations were also present in all astrocytic tumors that ultimately relapsed with alkylating therapy-associated hypermutation. Analysis of matched pre-treatment and post-progression tumors indicated that cell cycle alterations preceded the emergence of hypermutation. In total, 391 patients (42%) harbored a potentially therapeutically actionable lesion in their tumor of which 73 patients (19%) received a diversity of targeted therapies. Both the type of mutation and its clonality were associated with therapeutic sensitivity in this cohort. For instance, RAF dimer-dependent BRAF hotspot mutations arose predominantly in IDH-wildtype glioblastomas, were subclonal, and affected patients did not respond to MAPK-directed therapy. In contrast, BRAF V600 hotspot mutations arose clonally in histologically distinct gliomas, both low- and high-grade, and responded to MAPK-directed therapy (either RAF, MEK or ERK inhibition). Collectively, these data reveal previously unrecognized genomic determinants of disease progression and treatment response in diverse types of glioma and serve as a rationale for utilizing genomic information in clinical care of patients with glioma. 

### Novel Immunotherapies and Laboratory Models in Pediatric Cancer

#2622

T cell content of lymphatic fluid in pediatric patients is biased towards naïve and early memory cells: Implications for immunotherapy.

David M. Barrett, Julie Storm, Jessica Perazzelli, Yoav Dori. _Children's Hospital of Philadelphia, Philadelphia, PA_.

Lymphatic fluid originating from tissue and organs drains back into the venous system usually near the innominate vein on the left side. T lymphocytes make up a large majority of the cells in lymphatic fluid, but little is known about their functional status. Cellular therapies such as chimeric antigen receptor T cell (CAR T) therapy appear to depend on collecting large numbers of naïve or early memory T cells, which may not be available in peripheral blood. We have prospectively characterized peripheral blood and lymphatic fluid T cells from 25 pediatric patients receiving therapeutic thoracic duct access procedures and identified the phenotype and CAR T cell potential. Patients were treated at the Children's Hospital of Philadelphia for congenital or acquired lymphatic flow disorders, such as chylothorax, during which the thoracic duct was cannulated for therapeutic purposes. Excess fluid as well as a concomitant peripheral blood sample was collected and de-identified under an IRB-approved protocol. We quantified the CD3+ population using flow cytometry, and expanded these T cells using CD3/CD28 stimulatory beads as in CAR T cell manufacturing. We found an average cell concentration of 1.48e6 cells/mL of lymphatic fluid, with an average of 44% CD3+T cells versus 18% in blood (p<0.05). The lymphatic fluid to blood percent of naïve T cells was 36% v. 17% (p<0.01), Stem Central Memory 13% v. 7%(p<0.01), Central Memory 14% v. 27%(p<0.01), Effector Memory 7% v. 5% (ns) and Terminal Effector 30% vs 43% (ns). This bias towards naïve and SCM T cells was correlated with fewer negative checkpoint regulators (PD-1, LAG3 and Tim3) in lymphatic fluid T cells (7%) versus blood (30%). The T cells from lymphatic fluid expanded more robustly to CD3/28 stimulation as in CAR T manufacture, with an average of 32-fold expansion versus 9-fold for blood (p<0.01). In a xenograft model of pediatric leukemia, the CAR T cells derived from lymphatic fluid produced a deeper and more sustained remission than matched CAR T cells made from peripheral blood. This is the most detailed description of T cells from the lymphatic fluid of children to date. The shift in naïve and SCM T cells in lymphatic fluid versus peripheral blood of the same patient is a confirmation of expectations. The fact that lymphatic fluid is rich in cells that are potentially better suited to CAR manufacture raises the possibility of collecting cells from this source if peripheral blood is not suitable. Further studies on the functional potential of lymphatic fluid T cells are planned.

#2623

CHP-IL12 combined with surgery suppresses tumor metastasis and prolongs survival.

Qingnan Zhao, Jiemiao Hu, Jeffry J. Cutrera, Abhisek Mitra, Xueqing Xia, Shulin Li. _University of Texas MD Anderson, Houston, TX_.

Interleukin 12 (IL-12) is the most promising cytokine for treating tumors by itself or arming immunogenic cells but its toxicity and moderate response limit its clinical use. To avoid or minimize the toxicity issue and boost the anti-tumor immune response, our lab has discovered a comprehensive tumor-targeting carcinoma homing peptide (CHP). Fusion of this peptide encoding DNA sequence to IL12 encoding sequence in frame yields the proprietary product—CHP-IL12 (US patent 9,657,077 B2), or tumor-targeting IL12, which increased the accumulation of IL12 in tumor environment and decreased liver toxicity compared to wild-type IL12 (wt-IL12) gene therapy. Here, we report the therapeutic efficacy of this CHP-IL12 on metastasis and to comprehend the underlying mechanism description between CHP-IL12 and wildtype (wt)-IL12 for boosting anti-tumor response, two orthotopic and spontaneous tumor metastasis models (one adult and one pediatric tumor) were utilized. CHP-IL12 gene therapy showed greater efficacy in controlling growth of established tumors compared to wt-IL12. CHP-IL12 gene therapy, in combination with surgery, reduced number of metastatic nodules in lungs, livers and bones. CHP-IL12, compared to wt-IL12, conferred a potential immunostimulatory effect involving 1) reduction of T-reg cells, myeloid-derived suppressor cells (MDSC) and tumor associated macrophages (TAM); 2) recruitment of activated CD8+ T cells to the tumor. Moreover, CHP-IL12 increased IL12, IFNγ level in tumor environment and decreased IL12, IFNγ level in serum, which is the major cause of toxicity. Furthermore, in combination with surgery, CHP-IL12 gene therapy led to long-term survival and enhanced immunological memory development of tumor bearing mice compared to wt-IL12 gene therapy.

These results collectively indicate that CHP-IL12 fusion gene therapy coupled with surgery has a powerful potency in inhibition of tumor metastasis and prolonging survival and a low potency in inducing toxicity. Therefore, CHP-IL12 fusion gene therapy in combination with surgery may translate into clinic as a promising anti-tumor strategy for both adult and pediatric tumors.

#2624

Dual PLK1 and BRD4 inhibition has synergistic therapeutic effects against high-risk rhabdomyosarcoma.

Natalie Timme,1 Shuai Liu,2 Hailemichael Yosief,2 Heathcliff Dorado García,1 Ian MacArthur,1 Annabell Szymansky,1 Constantin Dohna,1 Georg Seifert,1 Patrick Hundsdörfer,1 Andrej Lissat,1 Angelika Eggert,1 Johannes Schulte,1 Wei Zhang,2 Anton Henssen1. 1 _Charité University Hospital, Berlin, Germany;_ 2 _University of Massachusetts Boston, Boston, MA_.

Concomitant inhibition of multiple therapeutic targets is an established strategy to improve the durability of clinical responses to targeted therapies. Rhabdomyosarcoma is the most common childhood soft tissue solid tumor and cure rates for patients with high-risk rhabdomyosarcoma remain dismal. Resistance to intensive chemotherapy is common, and few molecular therapies have been introduced clinically. High-risk rhabdomyosarcoma is driven by expression of the oncogenic fusion protein PAX3-FOXO1, which acts as a transcription factor. Recently, many efforts have been made to identify pharmacological means of inhibiting PAX3-FOXO1, given its central role in rhabdomyosarcoma cell line survival. The BET protein BRD4 has been shown to be required for transcription of PAX3-FOXO1 and its target genes and pharmacological inhibition via BET protein inhibitors exhibits anti-rhabdomyosarcoma effects. Phosphorylation of PAX3-FOXO1 by the polo like kinase 1 (PLK1) increases the stability of the oncoprotein. Based on these previous observations, we hypothesized that simultaneous inhibition of BRD4 and PLK1 could synergistically target PAX3-FOXO1 stability and activity, leading to increased anti-rhabdomyosarcoma effects. Indeed, combination treatment with PLK1 and BRD4 inhibitors exhibited synergistic effects specifically against PAX3-FOXO1-driven rhabdomyosarcoma cell lines. Dual PLK1-BRD4 inhibition suppressed proliferation and induced apoptosis in rhabdomyosarcoma cell lines at low nanomolar concentrations. Our results demonstrate a new strategy towards achieving dual synergistic targeting of BRD4 and PLK1 with a single agent which may lead to clinical translation for therapy of refractory rhabdomyosarcoma.

#2625

Patient derived organoids to guide personalized neuroblastoma treatment.

Rachele Rosati,1 Hallie A. Swan,2 Laura Scolaro,3 Kateryna Krytska,3 John M. Maris,3 Franz X. Schaub,1 Carla Grandori1. 1 _Cure First, Seattle, WA;_ 2 _SEngine Precision Medicine, Seattle, WA;_ 3 _Children's Hospital of Philadelphia, Philadelphia, PA_.

Goal: Neuroblastoma (NBL) is the second most frequent pediatric solid tumor. We have seen an increase in survival for children with NBL, however treatment still consists of many cycles of chemotherapy and radiation. We reasoned, that in vitro high throughput drug screening (HTDS) of patient derived organoids using a library of targeted therapies, would highlight novel targeted and personalized treatment options.

Methods: We optimized an in vitro 3D culture for high-risk NBL samples, initially established as patient derived xenografts (PDX) and screened a total of 10 patient samples with genomic information using two different drug libraries: Informer Set (320 drugs) compiled by the Cancer Target Discovery and Development network which is focused on cancer pathways and SEngine Library (123 drugs) focused on clinically relevant therapies. The screen was performed in a 384 well-format and cell viability was measured. Organoids were characterized by immunohistochemistry and genomic and pathway alterations were confirmed.

Results: Here we highlighted unique results from 4 cases. The first sample showed sensitivities to CDK inhibitors in concordance with amplification of CDK4. However, for this patient additional drug sensitivities were not directly linked to a genetic alteration, such as AZD7762 (CHK inhibitor) and MK-1775 (WEE1 inhibitor) indicating sensitivities in the cell-cycle checkpoints, and an exceptional response to Enzalutamide, an androgen receptor antagonist. The second case had unique sensitivities to IGF1R and Insulin Receptor inhibitors (Linsitinib and BMS-754807) indicating a specific addiction to this growth-factors/survival signal. The third sample had multiple sensitivities linked to genomic alterations, such as

Dabrafenib, concordant with the presence of a BRAF activating mutation. In addition, sensitivity to Alisertib was detected as previously reported for MYCN amplified NBL, but this was not true for other MYCN amplified cases. Interestingly, this third sample also showed profound sensitivities to PARP inhibitors, most likely related to a mutation in the BRP1 gene a BRCA1 interacting protein gene. The fourth case showed a strong sensitivity to nutlin-3 (MDM2 inhibitor) and Oxaliplatin.

Conclusion: A newly established NBL 3D patient organoid culture system coupled with personalized HTDS and genomic analysis offers an effective platform for the discovery of novel personalized therapies and drug combinations tailored to individual patients. In this NBL cohort, we discovered both drugs predicted by the patient-specific genomic alterations, as well as novel patient-specific drug sensitivities without a known genomic marker. More in depth genomic analysis is underway, but epigenetic alterations could explain these exceptional responses. In summary, this investigational study of 10 NBL highlights the need of personalization of targeted therapies in NBL.

#2626

Combination of chemotherapy and cytokine facilitates adoptive transferred effector T cells homing to the core of metastatic tumors.

Jiemiao Hu, Qingnan Zhao, Xueqing Xia, Richard Gorlick, Shulin Li. _UT MD Anderson Cancer Ctr., Houston, TX_.

Metastatic disease accounts for most cases of cancer mortality, due to its systemic nature and resistance to existing therapeutics for primary tumors. Immune therapy has become a viable alternative for treating drug-resistant tumors. In fact, increased immune density in metastasis lesions is associated with beneficial clinical outcomes. Notably, tumor infiltrating lymphocyte (TIL) infusions extended the overall survival of refractory melanoma patients. However, whether or not T cell therapy could be used to successfully limit the metastatic progression largely depends on the complexity of T cell penetration and tumor-immune cell interaction in distant sites. During the past years, our group has identified a simple combination of an immune stimulatory signal (interleukin-12) and chemotherapy (doxorubicin) that persistently induces NKG2D ligand on tumor cells in vivo across tumor types and developed a novel CD28 stimulation based approach for fast induction of NKG2D receptor on CD8+ T cells. We found in breast carcinoma model (4T-1) and pediatric osteosarcoma models (K7M3 and LM8), adoptive transfer of NKG2D+CD8+ T cells after the combination of IL-12 plus doxorubicin treatment dramatically reduced the numbers and sizes of metastatic nodules, leading to significantly prolonged survival time. Mechanically, the treatment of IL-12 plus doxorubicin widely upregulated the expression of NKG2D ligands exclusively on tumor cells in metastatic lesions, and allowed high density of NKG2D+CD8+ T cell accumulation at both the core and invasive margin areas of metastatic tumors. Furthermore, this combination therapeutic led to reduced regulatory T cell infiltration, resulting in increased CD8+ T cell to regulatory T cell ratios in metastatic tumors. Collectively, here we reported that a simple combination treatment of IL-12 plus doxorubicin with subsequent NKG2D+ CD8+T cell adoptive transfer may transform the metastatic tumor microenvironment to enable the influx of NKG2D+CD8+ T cells which effectively kill tumor cells via NKG2D-NKG2D ligand interaction. This promising therapeutic approach greatly improved the effectiveness and potential of adoptive T cell therapy in treating cancer patients with metastatic diseases.

#2627

A mathematical model to predict prognosis in breast cancer survivors following an exercise intervention.

Christina M. Dieli-Conwright,1 Jean-Hughes Parmentier,2 Steven D. Mittelman,3 Nathalie Sami,1 Kyuwan Lee,1 Stacey Finley1. 1 _USC, Los Angeles, CA;_ 2 _Children's Hospital Los Angeles, Los Angeles, CA;_ 3 _UCLA, Los Angeles, CA_.

BACKGROUND

Metabolic syndrome (MetS) and obesity are associated with increased risk of cardiovascular disease, type 2 diabetes, and cancer recurrence, and are higher in breast cancer survivors than age-matched postmenopausal women. We previously reported that exercise improves MetS and reduces pro-inflammatory biomarkers in obese breast cancer survivors. Whether these exercise-induced changes impact cancer outcomes is currently unknown. In this pilot study, we apply a robust mathematical analysis to identify biomarkers of MetS and obesity associated with clinical outcomes in obese breast cancer survivors following participation in a 16-week exercise intervention.

EXPERIMENTAL DESIGN

Eleven obese postmenopausal breast cancer survivors were randomized to either the exercise or control group. The exercise group participated in 16 weeks of supervised aerobic and resistance exercise sessions 3 times/week. Fasting blood and adipose tissue samples were analyzed for cytokine secretion, macrophage phenotype, and MetS. Prognostic outcomes included disease-free survival (DFS), overall survival (OS), distant DFS (DDFS), and recurrence-free interval (RFI). Partial least squares regression (PLSR) was used to quantify the importance of specific patient measurements in predicting the clinical response. PLSR is a multivariate regression analysis that quantifies the relationships between the participant characteristics and tissue and plasma measurements (inputs) and the clinical response (outputs) and can be used to identify which patient measurements most significantly associate with specific clinical outcomes.

RESULTS

MetS, macrophage phenotype, and inflammatory biomarkers were significantly improved in the exercise group compared to the control group (p<0.01) and these biomarkers were used to build the PLSR model. An accurate and predictive PLSR model could be constructed for two clinical responses: distant DFS and RFI. Overall, the percentage of type 1 macrophages (M1) is predicted to significantly contribute to predicting the clinical responses of distant DFS and RFI, where patients with a lower percentage of M1 have a better clinical response. Other patient measurements were identified as significantly contributing to distant DFS (prolactin, serum amyloid A, type 2 macrophages, brain-derived neurotrophic factor, interleukin-6, and insulin-like growth factor binding protein-1) which impact the clinical response to different extents.

CONCLUSIONS

Exercise improves biomarkers related to MetS, obesity, and inflammation. The PLSR model quantifies patient characteristics and measurements associated with clinical outcomes. Our analysis provides quantitative insight into potential biomarkers to predict response to exercise and prompts the need for an in depth longitudinal study to determine the effects of exercise on long-term survival in obese breast cancer patients.

#2628

Synthetic lethal targeting of ATR in alternative lengthening of telomeres-dependent rhabdomyosarcoma.

Heathcliff Dorado García,1 Jennifer von Stebut,1 Ian MacArthur,1 Koshi Imami,2 Natalie Timme,1 Kerstin Schoenbeck,1 Annabell Szymansky,1 Georg Seifert,1 Patrick Hundsdoerfer,1 Andrej Lissat,1 Matthias Selbach,2 Angelika Eggert,1 Johannes Schulte,1 Anton Henssen1. 1 _Charité University Hospital, Berlin, Germany;_ 2 _Max Delbrueck Center for Molecular Medicine, Berlin, Germany_.

Synthetic lethal dependencies have recently emerged as tumor-specific vulnerabilities which provide broad therapeutic windows and have successfully been used for rational therapeutic target discovery. Despite intense international efforts over many decades, cure rates for patients with high-risk rhabdomyosarcoma remain dismal. Resistance to intensive chemotherapy is common, and targets for molecular therapies are largely undefined. Telomere maintenance is a hallmark of cancer and requires either activation of telomerase (TERT) or alternative lengthening of telomeres (ALT). ALT is active in a large subset of rhabdomyosarcomas and not active in healthy tissues suggesting it is a unique, tumor-specific process. ALT has recently been shown to depend on DNA damage response activation through the ATR kinase, and studies in osteosarcoma have revealed that ALT renders cancer cells hypersensitive to ATR inhibition. This defines ALT as a tumor-specific synthetic lethal determinant for susceptibility to ATR inhibition. Consistent with previous reports, we observed that ALT-dependent rhabdomyosarcoma cells are hypersensitive to ATR inhibition via AZD6738, a small molecule ATR inhibitor currently being tested in clinical trials. Intriguingly, we found that treatment with ATR inhibitors not only induced DNA damage but also resulted in aneuploidy and subsequent apoptosis in ALT-dependent rhabdomyosarcoma cells, suggesting loss of telomere protection and inadequate chromosomal segregation occur during mitosis upon ATR inhibition. To test this, we measured unrepaired DNA double-strand break formation using TUNEL. ALT-dependent cells, but not TERT-dependent cells, showed increased TUNEL labeling, consistent with an increase in double-stranded DNA breaks and unprotected DNA ends. Consistent with reduced telomere maintenance, ATR inhibition significantly reduced the number of C-circles in rhabdomyosarcoma cells relying on ALT. Furthermore, AZD6738 treatment reduced phosphorylation of known ATR targets, and differentially phosphorylated peptides were enriched for proteins involved in alternative lengthening of telomeres as measured using SILAC labeling and LC-MS/MS phospho-proteomic analysis. Finally, treatment with AZD6738 reduced tumor burden in patient-derived primary rhabdomyosarcoma xenografts, which was potentiated by combined treatment with cisplatin. Our findings delineate a therapeutically actionable DNA repair dependency induced at least in part by ALT and may lead directly to clinical translation of ATR inhibitors for therapy of refractory rhabdomyosarcoma.

#2629

Genome-informed therapy for osteosarcoma.

Leanne Sayles,1 Marcus Breeese,1 Amanda Koehne,1 Bogdan Tanasa,1 Stanley Leung,1 Alex Lee,1 Avanthi Shah,1 krystal Straessler,1 Kim Hazard,2 Mi-Ok Kim,1 Alejandro Sweet-Cordero1. 1 _University of California San Francisco, San Francisco, CA;_ 2 _Stanford University, Stanford, CA_.

Osteosarcoma (OS) is characterized by numerous copy-number alterations (CNAs) and structural variations (SVs) in cancer-relevant genes. In contrast, recurrent point mutations are not seen. Thus, OS is a "C-class" (copy number driven) rather than an "M-class" (mutation driven) cancer. However, little is known with regards to whether copy-number alterations can be used to select therapies for aggressive cancers such as OS. The genomic heterogeneity of OS suggests that there may be different oncogenic drivers in subsets of patients. Thus, a systematic effort to identify targetable, patient-specific key driver genes (likely CNAs) is required. We established a clinically annotated patient derived tumor xenograft (PDTX) bank of 16 OS samples obtained at diagnosis, after surgical resection and from metastasis, thus representing the full spectrum of disease. Comparison between PDTXs with a corresponding matched primary tumor demonstrated high correlation in copy number (by WGS for 12 samples) and gene expression (by RNAseq for 13 samples), suggesting that PDTXs are faithful preclinical models for OS. To identify recurrent CNAs, we analyzed this WGS dataset together with a public dataset of OS WGS samples. With this combined dataset of 69 samples from 52 patients, we searched for recurrent CNAs across an actionable cancer gene list and identified genes amplified at least 4-fold in at least 2 samples. The two most frequently amplified genes in OS are CCNE1 and MYC. Other frequent alterations were those in the PI3K pathway (PTEN loss and/or AKT amplification), AURKB amplification, CDK4 amplification and VEGFA amplification. Importantly, all of these CNAs were reflected in at least one PDTX model. We hypothesized that in OS some of these CNAs are key cancer drivers that can be targeted for cancer treatment. To test this hypothesis, we rank-ordered the CNAs in 9 PDTXs by the amplitude of the copy number gain. We used this simple heuristic to identify candidate drivers for individual samples. We then identified 6 drugs that could be used to target specific amplified genes and tested these drugs in corresponding CNA-matched PDTX. In all cases, we saw significant growth inhibition in "matched" PDTXs whereas the effect was minimal in PDTXs treated with "unmatched" therapies. These results support the hypothesis that specific genes within CNA serve as oncogenic drivers in OS and thus outline a feasible approach to personalized, genome-informed therapy for this disease. This work could serve as the necessary pre-clinical proof of principle for development of a targeted therapy "basket" trial for OS.

#2630

Preclinical identification of Venetoclax combination strategies with Idasanutlin, CUDC-907, Prexasertib or Talazoparib for neuroblastoma treatment.

M. Emmy M. Dolman,1 Laurel T. Bate-Eya,1 Lindy Vernooij,1 Bianca Koopmans,1 Lindy K. Alles,1 Anke H. Essing,1 Daphne Lelieveld,2 David A. Egan,2 Mark Kerstjens,3 Ronald W. Stam,1 Huib N. Caron,4 Jan J. Molenaar1. 1 _Princess Maxima Center for Pediatric Oncology, Utrecht, Netherlands;_ 2 _University Medical Center Utrecht, Utrecht, Netherlands;_ 3 _Erasmus MC, Sophia Children's Hospital, Rotterdam, Netherlands;_ 4 _Emma Children's Hospital, Amsterdam Medical Center, Amsterdam, Netherlands_.

BACKGROUND: The anti-apoptotic protein B cell lymphoma/leukaemia 2 (BCL-2) is highly expressed in the majority of all neuroblastomas. In previous preclinical studies, we have shown that treatment of BCL-2-dependent neuroblastoma with BCL-2 inhibitors leads to programmed cell death. These results have contributed to the initiation of a phase I trial to study the safety and pharmacokinetics of Venetoclax in children with relapsed or refractory neuroblastoma.

AIM: The current study aims to identify and validate targeted combination strategies to prevent or overcome neuroblastoma resistance to Venetoclax.

METHODS: Targeted drug candidates for combination treatment with Venetoclax were identified by high-throughput drug screening of non-resistant and Venetoclax-resistant BCL-2-dependent neuroblastoma cell lines. Top hits were subsequently tested more extensively in vitro and in vivo to validate the screening results.

RESULTS: High-throughput drug screens identified the MDM2 inhibitor Idasanutlin as one of the strongest re-sensitizers for Venetoclax in Venetoclax-resistant BCL-2-dependent neuroblastoma cells with wild-type p53. Subsequent in vitro validation showed that Idasanutlin induced cyclin dependent kinase inhibitor 1 (p21)-mediated growth arrest in non-resistant neuroblastoma cells, but induced a BCL-2-associated X protein (BAX)-mediated apoptotic response in Venetoclax-resistant neuroblastoma cells in the presence of Venetoclax. In vivo combination of Venetoclax with Idasanultin resulted in a remarkably improved anticancer effect compared to single agent therapy, with very good partial and complete responses in BCL-2-dependent neuroblastoma xenografts. Combination screens additionally revealed that the clinically studied targeted inhibitors CUDC-907 (PI3K/HDAC), Prexasertib (CHK1) and Talazoparib (PARP1/2) improved the efficacy of Venetoclax more potently than ALK inhibitors in BCL-2-dependent neuroblastoma cells harboring ALK mutations.

CONCLUSION: Our findings suggest that the clinical use of Venetoclax for the treatment of children with BCL-2-dependent neuroblastoma tumors can be improved by combination therapy with targeted inhibitors. The presence of additional neuroblastoma driving genomic events is not always predictive of the optimal combination strategy for BCL-2-dependent neuroblastoma subgroups. These findings should guide the design of combination Phase 2 trials of Venetoclax with other targeted compounds such as Idasanutlin, CUDC-907, Prexasertib and Talazoparib.

### Prognostic Biomarkers

#2631

Cancer associated macrophage-like (CAMLs) cells in blood predict progression and survival for all stages of solid tumors.

Daniel L. Adams,1 Steven H. Lin,2 R. Katherine Alpaugh,3 Thai Ho,4 Jeffrey R. Marks,5 Stuart S. Martin,6 Susan Tsai,7 Saranya Chumsri,8 Cha-Mei Tang,9 Massimo Cristofanilli,10 Raymond Bergan11. 1 _Creatv MicroTech, Inc., Monmouth Junction, NJ;_ 2 _MD Anderson Cancer Center, Houston, TX;_ 3 _Fox Chase Cancer Center, Philadelphia, PA;_ 4 _Mayo Clinic Cancer Center, Pheonix, AZ;_ 5 _Duke University, Durham, NC;_ 6 _University of Maryland School of Medicine, Baltimore, MD;_ 7 _Medical College of Wisconsin, Milwaukee, WI;_ 8 _Mayo Clinic Cancer Center, Jacksonville, FL;_ 9 _Creatv MicroTech, Inc., Rockville, MD;_ 10 _Northwestern University, Chicago, IL;_ 11 _Oregon Health and Science University, Portland, OR_.

Background: We previously demonstrated that cancer associated macrophage-like cells (CAMLs) are cancer specific giant polyploid cells circulating in the blood of patients with solid tumors. Building on our initial discovery, others have shown that these hyperploidy cells are an innate immune response that is associated with decreased survival. However to date, no studies have been done to elucidate their clinical significance as in relation to the various stages of malignant disease. We established a 4 year prospective study of 315 patients from a variety of solid tumors (breast, prostate, lung, renal cell, pancreas, and esophageal) comparing the morphological properties of CAMLs in both early and late stage disease as they relate to progression free and overall survival (PFS/OS). These data suggest that CAML size appears have a strong negative correlation with progression and survival of cancer patients, irregardless of stage of disease, indicating their possible use as a non-invasive blood based biomarker in solid tumors.

Methods: A prospective multi-institutional study used anonymized peripheral blood from 315 cancer patients [stage I (n=62), stage II (n=72), stage III (n=69) & stage IV (n=106)] from subjects with breast (n=59), esophageal (n=27), lung (n=59), renal cell carcinoma (n=37), prostate (n=74), pancreas cancers (n=59). CAMLs were isolated by the CellSieve™ microfiltration technique at 5 institutions and stained for cytokeratin 8, 18, & 19, CD14 and CD45. After imaging, a size based threshold ≥50µm was used to separate the patient cohorts, based on previously published assessments.

Results: At least one CAML was found in 92% of cancer patients (n=289/315), with the lowest sensitivity in stage 1 (86%), followed by stage 2 (90%), stage 3 (97%) and stage 4 (95%). The property of CAML size was then evaluated. Overall, CAMLs of <50µm in size had superior PFS (HR: 3.8; 95% CI 2.8-5.3; p<0.001) and OS (HR=3.8; 95% CI 2.6-5.7;p<0.001) than those with ≥50 µm CAMLs. By each stage individually, stage 1 (PFS HR=7.5; 95% CI 3.2-17.7; p<0.001), stage II (PFS; HR=4.1; 95% CI 1.9-8.8; p<0.001), stage III (PFS HR=3.1; 95% CI 1.7-5.7; p=0.007), and stage IV (PFS HR=2.7; 95% CI 1.6-4.4; p<0.001).

Conclusions: In this first large scale prospective study of the clinical utility of CAMLs, it appears that they are present in every stages of invasive solid tumor malignancies, with prevalence increasing with stage. Interestingly decreased CAML size was found to be associated with improved outcome and longer PFS. These data suggest that CAML detection and size assessment constitute a new real time predictor of progression, and survival in both early and late stage disease indicating the need for additional validation studies.

#2632

Identification of PTHrP as a biomarker of short survival & brain metastasis in a tissue microarray retrospective analysis of triple-negative breast cancer.

Gloria Assaker,1 Anne Camirand,1 Bassam Abdulkarim,1 Atilla Omeroglu,2 Jean Deschenes,3 Leon Van Kempen,4 Richard Kremer,1 Siham Sabri1. 1 _McGill University & Research Institute of McGill University Health Centre, Montreal, Quebec, Canada; _2 _McGill University & McGill University Health Centre, Montreal, Quebec, Canada; _3 _Cross Cancer Institute and University of Alberta, Edmonton, Alberta, Canada;_ 4 _McGill University, Montreal, Quebec, Canada_.

Triple-negative breast cancer (TNBC) represents 10-20% of all BC cases, and is characterized by aggressive clinical course, frequent relapse, poor patient outcome, and lack of targeted therapy due to the lack of expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER-2). Identifying TNBC molecular drivers and biomarkers would thus be highly beneficial to develop efficient targeted therapies. The parathyroid hormone-related protein (PTHrP) is known for its role in mammary gland and bone development, in addition to breast cancer progression. The aim of this study is to investigate the role of PTHrP as a potential prognostic biomarker in human TNBC.

We assessed PTHrP expression using immunohistochemical analysis of a tissue microarray (TMA) constructed for 523 patients newly diagnosed with TNBC between January 1998 and December 2008 in a single-center series with centralized ER, PR and HER-2 testing and standardized treatment and follow-up. We evaluated the correlation between PTHrP expression and TNBC patients' clinico-pathological features as well as progression and survival outcomes for a subset of 314 patients with available clinical data.

We show that PTHrP is overexpressed in 55.2% of TNBC tumors and high PTHrP expression is significantly associated with higher propensity for brain progression compared to other sites of distal progression (p=0.0458). Univariate analysis revealed that high PTHrP expression is significantly associated with decreased overall survival (OS) (p=0.0055), but not with progression-free survival (PFS) (p=0.1270). To further investigate the prognostic value of PTHrP with respect to different TNBC molecular subtypes, we analysed expression of markers known to stratify different TNBC subtypes. Multivariate analysis of PTHrP as an independent prognostic factor of survival with respect to different TNBC molecular subtypes is currently underway.

In conclusion, we provide for the first time evidence that increased PTHrP expression is significantly associated with shorter OS and higher propensity of brain progression in patients diagnosed with TNBC. Consequently, stratification of this disease based on PTHrP expression might identify patients with relatively higher risk of aggressive disease with brain progression. Additional studies investigating the role of PTHrP in organ-specific metastasis and using PTHrP-targeting strategies are warranted to improve the therapeutic outcome for patients diagnosed with TNBC.

Funding: Alberta Cancer Research Institute (ACRI) grant and Department of Defense (DoD, USA) Award No. W81XWH-15-1-0723

#2633

The effects of aberrant global methylation in hepatocellular carcinoma.

Daniel C. Edelman,1 Fatma Shebl,2 Yonghong Wang,1 Holly Stevenson,3 Lawrence Sternberg,4 Joshua Sampson,1 Baiyu Yang,5 Paul S. Meltzer,1 Katherine McGlynn1. 1 _NIH/National Cancer Institute, Bethesda, MD;_ 2 _Yale University, New Haven, CT;_ 3 _Texas University at Austin, Austin, TX;_ 4 _NIH/National Cancer Institute, Frederick, MD;_ 5 _Stanford University, Stanford, CA_.

A better understanding of the molecular pathology of hepatocellular carcinomas (HCC), particularly in the United States of America (USA), could provide more effective biomarkers for early detection, diagnosis and treatment for this aggressive cancer. From two SEER Residual Tissue Repositories (Hawaii and Iowa), 483 surgical samples were obtained (223 cases of matched tumor/adjacent-normal); 30 were of OCT origin with the remaining FFPE. Purified genomic DNA was tested by the Illumina Infinium HumanMethylation450K BeadChip array according to the manufacturer's recommendations. Data were processed by using Lumi, methylumi, and other relevant R packages and also the Partek Genomics Suite. ANOVA results with FDR<0.01 between tumor and normal samples plus absolute beta value differences greater than 0.2 in FFPE or OCT, respectively, were compared and the overlapping 7279 targets generated the final gene list. In tumor, the genes MYH7 and TPO are hypomethylated, whereas OTX1 and C1orf70 are hypermethylated. Methyl2CGH aggregated patterns (FFPE only), showed that tumor was enriched for large chromosomal losses in Chr 8p and 17p with possible focal losses in Chr 4, 6, 8, 10, 12, 13, 17, 18 and 22. Large aberrations in chromosomal gains were seen in Chr 1q and 8q with possible focal gains seen in most chromosomes (excluding 9, 16, 21, 22). Importantly, 41 tumor suppressor genes are associated with copy number loss; these include RB1, TP53, MAP2K4 and CBFA2T3. However, identification of differentially methylated regions by using a segmentation method (NEXUS) suggested that tumor samples were more likely to be hypermethylated across the genome than hypomethylated (1q only). A possible limitation is that combining OCT and FFPE data is suspect due to at least 40 genes that specifically are hypermethylated in OCT samples but not FFPE. We previously reported that relative telomere length is prognostic for survival in HCC (Yang, et al., 2016). In this study we detected one gene, ZSCAN4, in the telomere maintenance pathway that is hypomethylated in tumor samples; the biological significance of this needs to be explored. In conclusion, for the under-studied USA HCC population, we have established strong preliminary methylation and copy number data indicating that there are specific epigenetic and chromosomal abnormalities that upon verification, could prove useful as biomarkers for improved understanding of the pathogenesis, early detection, and diagnosis of HCC.

#2634

**Predictive value of** 18 **F-FDG PET/CT scanning in combination with clinical parameters in patients with newly diagnosed multiple myeloma.**

Honglei Tu,1 Fuling Zhou,1 Yunbao Pan,1 Francois X Claret2. 1 _Zhongnan Hospital, Wuhan, China;_ 2 _Department of Systems Biology, The University of Texas MD Anderson Cancer Center, Houston, TX_.

Objective: To evaluate the association of 18F-2`-deoxy 2`-fluorodeoxyglucose (18F-FDG) PET/CT with clinical parameters in predicting patients with newly diagnosed multiple myeloma (MM).

Methods: One hundred and twenty MM patients undergoing 18F-FDG PET/CT scanning were analyzed in a retrospective cohort study.

Results: Based on multivariate analysis, β2M, LDH, number of focal lesions (FLs) and SUVmax were significantly correlated with overall survival (OS). These 4 variables were used to construct a new staging system (NSS) based on the number of risk factors. NSS provided a better discrimination of risk between stages III and II than International staging system (ISS) (p < 0.001 vs. p = 0.086). For OS, there was no significant difference among risk groups in Durie-Salmon (DS) stage (p > 0.05). Based on Spearman correlation analysis, presence of lesions in appendicular skeleton, number of FLs and SUVmax appeared to indicate advanced stage of MM. ROC curves which showed the combination of β2M with Calcium got a specificity of 96.3% for the presence of lesions in appendicular skeleton, and LDH alone had 100% specificity in predicting the number of FLs, although the sensitivity was only 50%.

Conclusions: 18F-FDG PET/CT in combination with clinical parameters provided an accurate and simple method for risk stratification of patients with newly diagnosed MM.

#2635

Differential prognostic implications of programmed death-ligand 1 expression on tumor cells and tumor-infiltrating immune cells in patients with locally advanced esophageal squamous cell carcinoma treated with preoperative chemoradiotherapy.

Ta-Chen Huang,1 Cher-Wei Liang,2 Chia-Chi Lin,1 Ya-Jhen Chen,3 Kuan-Ling Lin,1 Chih-Hung Hsu1. 1 _National Taiwan Univ. Hospital, Taipei, Taiwan;_ 2 _Fu Jen Catholic Univ. Hospital, New Taipei City, Taiwan;_ 3 _National Taiwan Univ., Taipei, Taiwan_.

Introduction

Previous studies have reported inconsistent results on the prognostic implications of program death-ligand 1 (PD-L1) expression for patients with locoregional esophageal squamous cell carcinoma (ESCC). We evaluated the differential prognostic impacts of the PD-L1 expressions on tumor cells and on tumor infiltrating immune cells in a cohort of locally advanced ESCC patients, receiving neoadjuvant chemoradiotherapy (CRT).

Patients and Methods

A cohort of 66 patients of locally advanced ESCC have been enrolled to a phase II clinical trial of a single-cycle chemotherapy (a combination of weekly paclitaxel, cisplatin and 24 hour infusional 5-fluorouracil, 2 week on plus one week off, followed by paclitaxel/cisplatin (TP) -based preoperative CRT and radical surgery. Primary tumor tissues were retrospectively collected for PD-L1 immunohistochemical stain by the Clone SP142 antibody (Spring Bioscience, Pleasanton, CA, USA). The expression of PD-L1 was scored as previously reported. Tumor cells (TCs) were scored according to percentage of positively-stained cells: TC3≥50%, TC2≥5% and <50%, TC1≥1% and <5%, and TC0<1%; Immune cells (ICs): IC3≥10%, IC2≥5% and <10%, IC1≥1% and <5%, and IC0<1%.

Results

PD-L1 expression was evaluable on tumor cells in 49 patients and on immune cells in 51 patients. TC0, TC1, TC2, and TC3 were scored in 27 (55%), 9 (18%), 11 (22%), and 2 (4%) patients, respectively; and IC0, IC1, IC2, and IC3 were in 25 (49%), 9 (18%), 15 (29%), and 2 (4%) patients, respectively. Regression analysis revealed that PD-L1 expressions on TC and IC exhibited different impacts on overall survival (OS): higher PD-L1 expression on TC trended to correlate with worse OS, whereas higher PD-L1 expression on IC trended to correlated with better OS. The median OSs of patients with TC0 and those with TC 1-3 were 52 months and 21 months, respectively, with a hazard ratio (HR) 1.5 (95% confidence interval [CI]: 0.7~3.3; P= 0.27). According to the PD-L1 expressions on TC and IC, patients with TC(+)/IC(+),

TC(+)/IC(-), TC(-)/IC(+), TC(-)/IC(-) exhibited median OSs of 26, 7, 42, and 43 months, respectively. Patients with TC(+)/IC(-) PD-L1 expression had significantly worse OS than other patients (HR: 2.6, P=0.03). Among patients with TC(+) PD-L1 expression, those with IC(-) had significantly worse OS than those with IC (+), too (HR: 2.5, P=0.04).

Conclusion

High PD-L1 expression on TC and low PD-L1 expression on IC associate with inferior prognosis in locally advanced ESCC patients treated with neoadjuvant CRT. (The study was supported by the grant NTUH 104-M2891 and the grant MOST 105-2314-B-002-186-MY3)

#2636

Combined MRI and molecular signatures of prostate cancer: Association with biochemical recurrence.

Stephanie A. Harmon,1 William Gesztes,2 Denise Young,2 Sherif Mehralivand,3 Brad J. Wood,3 Peter A. Pinto,1 Gyorgy Petrovics,2 Albert Dobi,2 Inger L. Rosner,2 Baris Turkbey,3 Shiv Srivastava,2 Peter L. Choyke,3 Isabelle A. Sesterhenn4. 1 _Leidos Biomedical Research, Bethesda, MD;_ 2 _Uniformed Services University of the Health Sciences, Bethesda, MD;_ 3 _National Cancer Institute, Bethesda, MD;_ 4 _Joint Pathology Center, Silver Spring, MD_.

Purpose: To assess the correlation of histologic and prostate cancer associated gene oncoprotein expression and imaging features in MRI-detected lesions of prostate cancer patients with biochemical recurrence (BCR) after definitive treatment.

Methods: Patients underwent multiparametric MRI (mpMRI) at 3T at a single institution prior to TRUS/MRI fusion-guided biopsy of MRI-defined index lesions and prior to definitive therapy (radical prostatectomy), for which clinical follow-up was available. All mpMRI lesions were prospectively scored low, moderate, moderate-high, or high for suspicion of harboring clinically significant cancer, as determined by positivity on individual MRI sequences and indication of extraprostatic extension (EPE) on imaging. The expression of prostate cancer drivers (ERG, PTEN, AR and p53) and PSA were examined on targeted-biopsy specimens by immunohistochemistry (IHC). All histological assessments were completed blinded to imaging findings and treatment outcomes. Cox proportional hazard regression was used to evaluate associations between imaging, molecular, and surgical characteristics with interval to BCR.

Results: In a preliminary cohort of 56 patients, 29 patients met clinical criteria for BCR (median interval 12.46 months, range 1-55 months) and 27 patients remain recurrence-free (median follow-up 30.75 months, range 1-72+ months). Median interval between mpMRI and targeted biopsy was 29 days (range 12-167 days) and radical prostatectomy was 4.13 months (range 1.3-8.0 months). Pre-operative serum PSA was the strongest correlate with time to BCR (HR: 1.63, 95%-CI: 12.-2.1, p<0.0001). The p53 staining intensity 3+ in >1% of cells or 2+ in >10% of cells was associated with poor BCR interval (HR: 3.35, 95%-CI: 1.5-7.2, p=0.002), remaining so in multivariate model with pre-operative MRI, PSA, and post-surgical findings on pathology. p53 positivity was observed more frequently in Gleason≥4+3, for both biopsy (fisher's exact p=0.006) and final pathology grading (fisher's exact p=0.01). Moderate/high to high index MRI suspicion was found to correlate with EPE at surgery (fisher's exact p=0.03) and showed modest correlation to time-to-BCR in univariate analysis (HR: 2.11, 95%-CI: 0.99-4.5, p= 0.052) and remained so in multivariate analysis. PTEN loss was observed in 8 patients (14%). PSA+ expression by IHC was observed in 49/56 patients, with remaining 7 patients showing variable/heterogeneous (PSA+/-) expression.

Conclusions: MRI impression and p53 status from MRI index lesion were independent predictors of time to biochemical recurrence, along with pre-operative PSA levels. These results are part of an ongoing study to identify histo-radiological features of aggressive prostate cancers.

#2637

Expression of SALL4 in prostate cancer.

Dhafer A. Alghezi,1 Paul Whitley,1 Mark Beresford,2 Rebecca Bowen,2 John Mitchard,2 Andrew Chalmers1. 1 _Bath university, Bath, United Kingdom;_ 2 _Bath Royal United Hospitals NHS, Bath, United Kingdom_.

Few prognostic biomarkers for Prostate cancer have been identified and there are clinical difficulties in distinguishing between relapsing and non-relapsing disease. The aim of our study is to investigate the hypothesis that proteins that have been linked to stem cells might be potential biomarkers, that could distinguish between aggressive tumors requiring radical intervention and those that have a good prognosis. One of the potential biomarkers that are being investigated is Sall4, a zinc finger transcriptional factor which has an important role in stem cell development and oncogeneses, including a possible role in prostate cancer progression and relapse. However, its role in prostate cancer remains unclear.

Sall4 nuclear staining has been evaluated by immunohistochemistry using two sources of patient samples, a tissue microarray group and a Bath cohort. The tissue microarray group consists of 96 cases including normal prostate, adjacent normal prostate, and prostate cancer samples. The Bath cohort consists of 22 samples, including samples from patients that had recurrent disease and those who remained disease free.

Sall4 nuclear staining was found to be decreased in prostate cancer samples compared to benign tissue from the tissue microarray group. Sall4 nuclear staining was also negatively associated with poorly differentiated grades of prostate cancer in the tissue microarray group. The Bath cohort did not have enough benign tissues to confirm the relationship between cancer and benign tissues but showed a negative association with the grade. Sall4 nuclear staining in tissue from both sets of samples was not associated with prostate cancer stages. It can be seen from this preliminary data that Sall4 might play an important role in tumor formation and/or aggressiveness and warrants further investigation to understand its function and establish if it could represent a potential diagnostic biomarker for prostate cancer.

#2638

Overexpressions of cortisol, annexin V and Lost in expression of PD-L1 and DARC proteins are associated with aggressive prostate cancer phenotypes in Black men: A CaPTC Cohort Study.

Faruk Mohammed,1 Folakemi T. Odedina,2 Sani Ibrahim,1 Abdulmumini Hassan Rafindadi,1 Ahmed Adamu,1 Abdullahi Adamu,1 Ahmad Bello,1 Surajo Mohammed Aminu,1 John Idoko,1 Aishatu Maude Suleiman,1 Solomon O. Rotimi,3 Ernie Kaninjing,2 Getachew A. Dagne,4 Clayton Yates,5 Yawale Iliyasu,1 Nissa Askins,2 Iya Eze Bassey,6 Renee Reams,7 Abdullahi Mohammad,1 Hussaini Yusuf Maitama,1 Dauda Maigatari,1 Mohammed Sani Shehu,1 Cheh Augustine Awasum,1 Abdulkadir Lawal Rafinadadi,1 Danladi Amodu Ameh,1 Serah Adewunmi,8 Ruth Agaba,9 Haruna Mohammad Muktar,1 Ahmad Mai,1 Saad Aliyu Ahmed,1 Ahmad Bello Kumo,1 Kasimu Umar Adoke,1 Ahmad Tijjani Lawal,1 Ahmad Muhammad,1 Omolora Fatiregun,10 Sunday Atawodi,1 Shehu Akuyam,1 Yusuf Abdulrashid,1 Mubarak Liman,11 Aliyu Muhammad,1 Abidemi Omonisi,12 Rebecca Gali,13 Hassan Dogo,14 Nkegoum Blaise,15 Anthonia Sowunmi,16 Titilola Akinremi,17 Emeka J. Iweala,3 Jigo Dangude Yaro,1 Badejo Adebukola Catherine,18 Akinwumi Oluwole Komolafe19. 1 _Ahmadu Bello University, Zaria, Nigeria;_ 2 _University of Florida, Orlando, FL;_ 3 _Covenant University, Ota Ogun State, Nigeria;_ 4 _University of South Florida, Tampa, FL;_ 5 _Tuskegee University, Tuskegee, AL;_ 6 _University of Calabar, Calabar, Nigeria;_ 7 _Florida A &M University, Tallahassee, FL; _8 _University of Ilorin, Zaria, Nigeria;_ 9 _Covenant University, Zaria, Nigeria;_ 10 _Lagos State University Teaching Hospital, Lagos, Nigeria;_ 11 _Nuhu Bamalli Polytechnic, Zaria, Nigeria;_ 12 _Ekiti State University, Ekiti, Nigeria;_ 13 _University of Maiduguri, Zaria, Nigeria;_ 14 _University of Maiduguri, Maiduguri, Nigeria;_ 15 _University Hospital Center Yaounde, Yaounde, Cameroon;_ 16 _Lagos University Teaching Hospital, Lagos, Nigeria;_ 17 _Federal Medical Center, Abeokuta, Nigeria;_ 18 _University of Agriculture, Abeokuta, Nigeria;_ 19 _Obafemi Awolowo University, Ile-Ife, Nigeria_.

Prostate cancer (CaP) is the most common male gender cancer and present with a 5-year prevalence, incidence and mortality rate in Nigerian Black men. There is disproportionate prevalence and poor understanding of CaP in Black men globally. The Prostate Cancer Transatlantic Consortium (CaPTC) has documented significant CaP burden among Nigerian black men in Nigeria and in the Diaspora. Notably, African ancestry is one of the most important risk factors of CaP globally. Increased levels of cortisol, a stress-related catabolic hormone is significantly associated to prostate specific antigen and poor prognosis in prostate cancer patients. Cortisol causes degradation of muscle proteins into amino-acids. Amino acids such as leucine, serves as a source of fuel for aggressive prostate cancer cells in a Warburg effect model through the L-amino acid transporters (LATs). The annexins are calcium and phospholipid binding proteins implicated in cancer development and progression. Overexpression of annexin 1 is one of the mechanisms by which cortisol inhibit inflammation. Annexin V has a high affinity to phosphatidylserine and play important role of inhibiting engulfment of apoptotic cells by macrophages to increase the immunogenicity of tumor cells undergoing apoptosis. PD-L1 is an important membrane-bound costimulatory molecule that inhibits immune responses through its receptor, PD-1. Overexpression of PD-L1 results to cancer cells aggressively evading the host immune system. The Duffy Antigen/Receptor for Chemokines (DARC) is a 7-transmembrane protein atypical chemokine receptor expressed on erythrocytes and vascular endothelial cells that binds to and clears angiogenic chemokines. Expression pattern of DARC is associated to cancer growth and metastasis. In this study, we report the expression pattern of tissue Cortisol, Annexin V, PD-L1 and DARC proteins in relationship to Gleason score in Nigerian Black men with Prostate cancer using immunohistochemistry and immunofluorescence. In addition, we assessed salivary cortisol levels by enzyme immunoassay and physical activity by a standardized CaPTC-AC3-MADCaP (C.A.M) CaP measures from 500 subjects recruited by via CaPTC cohort. Results showed significant expression of tissue and salivary cortisol in the CaP patients compared to the normal subjects (p > 0.05). The Tissue cortisol protein expression was higher in CaP cases with Gleason score 8. There was significant overexpression of Annexin protein in virtually the prostate cancer samples studied. We found a negative expression of PD-L1 and DARC proteins in all the CaP tissues studied. Cortisol and Annexin may serve as an important biomarker for prostate cancer diagnosis and prognosis in Nigerian Blacks.

#2639

Prognostic significance of non-small cell lung cancer and response to EGFR-TKI of EGFR mutated lung cancer besed on PD-L1 expression.

Kenichi Kobayashi, Masahiro Seike, Rintaro Noro, Shinobu Hemmi, Akihiko Gemma. _Nippon Medical School, Tokyo, Japan_.

Background: Recent clinical trials have shown that immune checkpoint blockades that target either PD-1 or PD-L1 yield remarkable responses in a subgroup of non-small cell lung cancer (NSCLC) patients.

Materials and Methods: We retrospectively examined, by immunohistochemical analysis, 211 NSCLC patients who had undergone complete surgical resection. Using 32 independent samples, we also evaluated PD-L1 expression in 32 NSCLC patients with epidermal growth factor receptor (EGFR) mutations treated by EGFR-tyrosine kinase inhibitors (EGFR-TKIs).

Results: Overall survival of PD-L1 positive surgical NSCLC patients with stages I-III and stage I disease were significantly shorter than those of PD-L1 negative NSCLC patients (P <0.01 and P = 0.02, respectively). In 86 squamous cell carcinoma (SQ) cases, PD-L1 positive patients showed significantly poorer survival than PD-L1 negative patients (P = 0.01). In stage I NSCLC patients, gender and PD-L1 expression were found to be independent predictors of death after multivariate analysis (Hazard ratio [HR] 3.57, P <0.01 and HR 2.05, P = 0.04, respectively). In SQ patients, p-stage and PD-L1 expression were independent predictors of death (HR 2.53, P <0.01 and HR 2.26, P = 0.02, respectively). Response to EGFR-TKIs, progression-free survival and overall survival were not significantly different between PD-L1 positive and negative NSCLC patients with EGFR mutations.

Conclusion: PD-L1 positive expression in a tumor was a significant independent predictor of poor outcome in patients with lung SQ and in stage I NSCLC patients. Based on PD-L1 expression, the clinical response to EGFR-TKIs and prognosis did not differ in EGFR-mutated NSCLC patients.

#2640

Detection of androgen receptor (AR) and AR-V7 in small cell prostate carcinoma by immunohistochemistry.

Pei Zhao,1 Yezi Zhu,1 Liang Cheng,2 Jun Luo1. 1 _Johns Hopkins University, Baltimore, MD;_ 2 _Indiana University School of Medicine, Indianapolis, IN_.

Background: Small cell prostate carcinoma (SCPC) is a rare and highly malignant subtype of prostate cancer. SCPC only accounts for 0.5%-2% of untreated primary prostate cancer, but can present in up to 25% lethal metastatic prostate cancer autopsies. Small cell prostate carcinoma frequently lacks androgen receptor (AR) and prostate specific antigen (PSA) expression, and often responds poorly to androgen deprivation therapy (ADT). However, the expression pattern of different AR isoforms in SCPC after ADT remains poorly characterized. In this study, we have validated and developed a new immunohistochemical assay for full length AR (AR-FL) and AR splice variant-7 (AR-V7) protein expression in prostate cancer specimens. The expression of AR-FL and AR-V7 protein were determined in post-ADT radical prostatectomy samples from twelve SCPC patients. In this study, we developed and validated a new immunohistochemical assay for the full-length AR (AR-FL) and AR splice variant-7 (AR-V7) proteins. We conducted an IHC study in post-ADT surgical specimens from twelve SCPC patients treated with neoadjuvant ADT.

Methods: AR-FL, AR-V7, and total AR proteins were specifically detected using AR C-terminal domain antibody (Sigma-Aldrich), AR-V7 antibody (RevMab), and AR N-terminal domain antibody (AR441, Santa Cruz), respectively. These antibodies were validated using prostate cancer cell lines with known AR profiles, including LNCaP95 cells which express both AR-FL and AR-V7 and the AR-negative PC3 cells. The IHC protocols were optimized for each individual antibody, and applied to the 12 SC/NE tissue specimens.

Results: Twelve surgical (RP or TURP) prostate small cell carcinoma specimens were collected from patients after neoadjuvant hormonal therapy at Indiana University School of Medicine. Seventy five percent (9/12) of the specimens showed positive staining for the full-length AR with various intensities. Thirty three percent (4/12) of the specimens showed positive staining for AR-V7. Among the specimens with positive AR-V7 staining, two samples displayed very weak staining, one sample showed weak to moderate staining, and one sample showed strong staining. All positive specimens displayed a heterogeneous pattern of AR/AR-V7 staining. All specimens positive for AR-V7 were also positive for the full length AR.

Conclusion and future direction: Our preliminary results indicate that small cell prostate carcinoma may continue to express AR and AR-V7. The functional and clinical relevance of AR/AR-V7 detection in this setting remains unknown. Future investigation will focus on clinical correlation in an expanded patient cohort.

#2641

The presence of Cyclooxygenase 2, tumor-associated macrophages, and collagen features as prognostic markers for invasive breast carcinoma patients.

Karla Esbona, Yanyao Yi, Menggang Yu, Rachel Van Doorn, Matthew Conklin, Douglas Graham, Kari Wisinski, Suzanne Ponik, Kevin Eliceiri, Lee Wilke, Patricia Keely. _Univ. of Wisconsin-Madison, Madison, WI_.

Inflammation and the organization of collagen in the breast tumor microenvironment is an important mediator of tumor progression. The objective of this study was to assess whether the tissue localization of COX-2 and tumor-associated macrophages were associated with clinicopathological features of invasive carcinoma, including collagen deposition and patient survival outcome. A tumor microarray (TMA) of 371 biopsy specimens from patients with invasive breast carcinoma was analyzed for expression levels of COX-2, the macrophage marker CD68 and activated macrophage marker CD163 in either the tumor nest (TN) or the tumor-associated stroma (TS). The TMA cohort included females; age 18 to 80, with a median follow up of 8.4 years. Biomarkers were correlated against clinicopathological and collagen features. Additionally, survival curves were calculated according to the Kaplan-Meier method. We found that high expression of COX-2 was associated with tumor size (P = 0.006), grade (P < 0.0001), proliferation (P < 0.0001), ER\+ (P = 0.001), collagen deposition (P < 0.0001) and density (P = 0.001). One possible mechanism for this, is COX-2 dependent recruitment of macrophages to the tumor microenvironment. This is supported by our finding of high infiltration of both macrophage markers that were associated with tumor size and proliferation; however, only high levels of stromal CD163 were associated with collagen deposition. In order to better analyze patient survival data, samples were divided into quartiles based on their levels of COX-2 expression and/or macrophage infiltration. COX-2 localization in the TN (P = 0.016, HR = 1.71), perpendicular alignment of collagen to the tumor boundary (TACS-3) (P = 0.049, HR = 1.65) and macrophage recruitment were predictors of poor overall survival (OS). Furthermore, patient survival was worsened if patients had a high CD163 macrophage infiltration (TN: P < 0.0001, HR = 2.86; TS: P = 0.002, HR = 2.54). This notion is further established by the finding of our multivariate analysis that high numbers of CD163+ macrophages in the TS as an independent prognostic factor (P = 0.001, HR = 2.90). These results suggest that in invasive carcinoma the localization of inflammatory markers within the tumor are biomarkers for patient survival outcome. Therefore, we propose that these patients may benefit from a selective COX-2 inhibitor and/or immune modulation therapies.

#2642

CBLC is a potential biomarker for the susceptibility to paclitaxel in lung adenocarcinoma.

Yi-Ping Lin,1 Shiao-Ya Hong,2 Yu-Rung Kao,2 Cheng-Wen Wu2. 1 _Institute of Microbiology and Immunology, National Yang-Ming University, Taipei, Taiwan;_ 2 _Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan_.

CBL family proteins have been reported as E3 ubiquitin ligases of receptor tyrosine kinases. CBLC is the most divergent member of CBL family in human due to lack of the distal part of C terminus found in other two proteins, CBL and CBLB. By data mining from TCGA database, CBLC expression was frequently upregulated in lung adenocarcinoma. However, very little is known about the tumorigenic functions of CBLC compared to other CBL family proteins. Although patients with lung adenocarcinoma harboring EGFR mutations can be treated with tyrosine kinase inhibitors (TKIs), there is no good target therapy for treating lung adenocarcinoma patients with EGFR wild-type or TKIs resistance. Here, this study is to investigate if CBLC can be a good target for treatment to benefit these patients. In lung adenocarcinoma cells, CBLC knockdown significantly decreased cell viability, while CBLC overexpression increased the ability to form colonies in soft agar. Flow cytometry analyses revealed that CBLC depletion decreased the cell population of G2/M phase and increased cell apoptosis. Our study also showed that CBLC knockdown in EGFR wild-type cells increased the drug resistance to paclitaxel, which is widely used as a first-line chemotherapy drug in lung adenocarcinoma patients with EGFR wild-type or TKI resistance. Taken together, these findings indicate that CBLC might play a role in promoting cell mitosis and could be a potential biomarker of paclitaxel susceptibility for patients with EGFR wild-type lung adenocarcinoma.

#2643

**Increased copy number of** EGFR **gene is an independent outcome predictor in resected non-small cell lung cancer patients treated by adjuvant chemotherapy.**

Vladimira Koudelakova,1 Radek Trojanec,1 Jana Potockova,1 Jiri Drabek,1 Jana Stranska,1 Petra Smickova,2 Pavla Kourilova,1 Ivona Grygarkova,2 Vitezslav Kolek,2 Jozef Skarda,1 Marian Hajduch1. 1 _Palacky University Olomouc, Olomouc, Czech Republic;_ 2 _University Hospital, Olomouc, Olomouc, Czech Republic_.

Background: Lung cancers are the leading cause of cancer-related deaths worldwide. Surgical resection alone remains preferred treatment for early stage patients with non-small cell lung cancer (NSCLC), whereas surgery plus platinum-based adjuvant chemotherapy is recommended for stages II - IIIa. Despite wide spectrum of known genetic alterations, there is a lack of biomarkers predicting response to adjuvant therapy. The aim of our study was to determine the predictive role of selected gene alterations. Methods and patients: Formalin-fixed paraffin embedded tumor samples were obtained from 209 NSCLC patients with stage I to IIIa. Of these, 59 patients were treated by surgical resection only, the follow-up adjuvant chemotherapy was indicated in remaining 150 cases. There were 146 males and 63 females (mean age 66; range 29-82 years), tumor histology revealed 79 adenocarcinomas, 105 squamous cell carcinomas, 21 large cell carcinomas, two adenosquamous carcinomas and two sarcomatoid carcinomas. Fluorescent in situ hybridization (FISH) was used for evaluation of EGFR, CMYC, MET and FGFR1 gene copy numbers and ALK and ROS1 copy numbers and rearrangements, respectively. EGFR, KRAS and BRAF mutations were screened using Cobas EGFR Mutation Test (Roche), TheraScreen: KRAS Mutation kit (Qiagen) and BRAF p.Val600Glu kit (IntellMed). Results: In the group of 150 resectable NSCLC patients treated with platinum-based adjuvant therapy, increased copy number of EGFR gene predicted poor overall survival (OS) (p = 0.00001, hazard ratio (HR) 1.66, 95% confidence interval (CI) 1.32-2.10) as well as disease-free survival (DFS) (p = 0.0001, HR 1.54, 95% CI 1.23-1.91) in univariate analysis. In multivariate analysis adjusted to age and stage, the EGFR copy number was independent predictor of OS (p = 0.001) as well as DFS (p = 0.0004). In the group of 59 patients treated by surgical therapy only, no correlation of tested markers with clinical-pathological data was identified. Conclusions: Increased copy number of EGFR gene was found to be an independent predictor of outcome in NSCLC patients treated by adjuvant platinum-based therapy. Acknowledgment: This work was financially supported by NPU LO1304, TE02000058 and EATRIS-CZ.

#2644

Relative T regulatory cell populations are indicative of HCC in patients with underlying liver disease.

Nicholas J. Skill, Mary A. Maluccio. _Indiana University, Indianapolis, IN_.

Hepatocellular carcinoma continues to be an increasing burden to patients and the health care system despite recent advances in treatment of viral hepatitis. Since the vast majority of cancers develop in patients with underlying liver disease, we would expect that efforts to cull out the patients at highest risk for cancer emergence would be possible, yet we have struggled to do so. The difficulty lies in the complex microenvironment of the diseased liver that leads to striking aberrancies, many of which are related to inflammation and can overlap with biomarkers felt to be related to cancer. Our goal with lymphocyte profiling is to stratify patients with underlying liver disease into those with and without cancer. Improved surveillance modalities for patients at highest risk of developing HCC are required. Moreover, there is an increasing push to include immunology in cancer detection and treatment. To address these issues, we performed lymphocyte profiling in patients with underlying liver disease with and without HCC and living related renal donors (LRD) by selective cell marker staining and flow cytometry, in order to determine if lymphocytes, which are key to cancer immunology, are modulated concordant with HCC. Specifically, whole blood was stained in order to quantitate T helper (Th), T cytotoxic (Tctx), B cells, natural killer (NK), and T regulatory cells (Treg) by flow cytometry. Th, Tctx and B cells were significantly reduced and NK cells were significantly increased in patients with underlying liver disease. These changes were independent of HCC diagnosis. In contrast, Treg cell levels were dependent on HCC diagnosis. In patients with underlying liver disease without HCC, Treg levels, when compared to LRD (30±6cells/mm3), were significantly reduced (15±3), whereas in patients with underlying liver disease and HCC, Treg levels were increased when compared to patients without HCC and unchanged when compared to LRD (28±6). To correct for the observed lower T-cell levels associated with underlying liver disease, lymphocyte populations were expressed as a ratio when compared to total T-cell numbers. This conversion identified proportional Treg cells population as a marker of HCC. In patients without HCC the Treg/T cell ratio was unchanged when compared to living related donors (0.02±0.002, 0.02±0.002, P=0.3). In contrast, Treg/Tcell ratio was significantly increased in patients with HCC (0.08±0.02, P=0.001). Consequently, given that the primary function of Treg cells is to inhibit T cytotoxic cells, a proportional increase in Treg cells would be associated with immunologic escape for HCC cells. Therefore, surveillance of lymphocyte populations, in particular T cells and Treg, would evaluate changes in immunologic capacity and increased Treg/Tcell ratio would be indicative of an increased risk for HCC in patients with underlying liver disease.

#2645

Intratumoral assessment of candidate colorectal cancer prognostic markers using RNA in situ hybridization.

Arthur Morley-Bunker,1 John Pearson,1 Margaret Currie,1 Helen Morrin,1 Martin Whitehead,2 Tim Eglinton,3 Logan Walker1. 1 _University of Otago, Christchurch, Christchurch, New Zealand;_ 2 _Canterbury Health Laboratories, Christchurch, New Zealand;_ 3 _Canterbury District Health Board, Christchurch, New Zealand_.

Genome-wide expression studies using microarrays and RNAseq have increased our understanding of colorectal cancer development. Translating potential gene biomarkers from these studies for clinical utility has typically relied on PCR-based technology and immunohistochemistry. However, these techniques often suffer from limitations because of tumor sample heterogeneity, or a lack of correlation between protein and corresponding mRNA transcript levels. RNAscope® (Advanced Cell Diagnostics) is an RNA in situ hybridization method that overcomes these limitations by enabling single-molecule detection, while preserving cellular and tissue morphology. The aim of this research was to investigate the clinical utility of RNAscope to measure gene expression of two potential prognostic markers (GFI1 and TNFRSF11A) that were identified from a previously reported study from The Cancer Genome Atlas (TCGA) Network. We analyzed 112 consecutively collected archival formalin-fixed, paraffin-embedded tumors from a collection of well-characterized colorectal cancer cases. Consistent with TCGA Network data, we found significant associations for reduced GFI1 and TNFRSF11A mRNA expression and clinicopathologic features in our cohort. Reduced GFI1 expression measured by RNAscope was associated with high-grade tumor (P = 0.02) and left-sided tumor (P = 0.01). Reduced TNFRSF11A expression was associated with metastasis (P = 0.04) and high nodal involvement (P = 0.02). RNAscope was combined with semiautomated image analysis to provide quantification of mRNA expression at the single-cell level, allowing for cell-type determination of mRNA expression levels within a tumor. These data showed that GFI1 and TNFRSF11A were expressed at a significantly higher level in carcinoma cells compared to noncarcinoma cells (lymphocytes, stromal cells and normal cells). Thus, we were able to demonstrate that reduced expression measured in patients with poorer prognosis was not due to contamination of tumor samples with noncarcinoma cells. Limited information is available regarding TNFRSF11A's involvement in colorectal cancer. Gene knockdown of TNFRSF11A in colorectal cell line models showed changes in cell growth properties. To our knowledge, this is the first study to assess the intercellular expression patterns of GFI1 and TNFRSF11A as candidate prognostic markers in colorectal tumor. Furthermore, we show that intratumoral expression analysis of candidate genes by RNAscope is an informative technology for validating results from genome-wide expression studies.

#2646

The liquid biopsy network in advanced breast cancer (ABC): Benchmarking the prognostic role of circulating tumor DNA (ctDNA) to the goal standard circulating tumor cells (CTCs) enumeration.

Lorenzo Gerratana,1 Andrew A. Davis,1 Giovanna Rossi,2 Kimberly Strickland,3 Zhaomei Mu,1 Lisa Flaum,1 William J. Gradishar,1 Cesar Santa-Maria,1 Hushan Yang,3 Massimo Cristofanilli1. 1 _Northwestern University Feinberg, School of Medicine, Chicago, IL;_ 2 _European Institute of Oncology, Milan, Italy;_ 3 _Thomas Jefferson University, Philadelphia, PA_.

Background: Liquid biopsy technologies, including CTCs and ctDNA, may provide prognostic information for advanced breast cancer (ABC) patients. We evaluated quantification and molecular characterization of ctDNA-based testing for prognostic stratification and predictive analysis.

Methods: This retrospective study analyzed a cohort of 91 patients (pts) with ABC, which was longitudinally characterized for CTCs and ctDNA. CTCs were isolated through the CellSearch immunomagnetic kit (Menarini Silicon Biosystems, PA), and ctDNA was analyzed using the Guardant360 NGS-based assay (Guardant Health, CA). A receiver operating characteristic (ROC) analysis was performed to identify the best ctDNA percentage (%ctDNA) cut-off capable to separate pts with ≥ 5 CTC/7.5 ml of blood. Survival analyses for both CTC and %ctDNA were investigated by log-rank test. Associations between %ctDNA stratification and detectable ctDNA mutations were explored through Fisher's exact test, and multiplicity was corrected through Bonferroni-Holm method (Q value).

Results: 40 pts (44%) were diagnosed with luminal-like ABC, 25 pts, (27%) with HER2-positive ABC, and 26 pts (29%) with triple negative ABC. 84 pts (92%) had stage IV disease and 26 pts (31%) were de novo metastatic. 22 pts (35%) had a CTC count ≥ 5, while among pts with CTC < 5, 21 (34%) had no detectable CTCs. The median %ctDNA was 1.65 (IQR: 0.3 - 9.8), and 11 pts had no detectable ctDNA. A cut-off of 5.7 was selected (AUC: 0.7533) by ROC analysis. A significant association for both PFS and OS was observed for the selected threshold (log-rank test P=0.024 and P=0.0015, respectively). Pts with a %ctDNA < 5.7 had a median PFS and OS of 6.2 ms (IQR: 2.1 - 12.1) and 21.7 ms (IQR: 12.82 - not reached), respectively. For pts with a %ctDNA ≥ 5.7, median PFS and OS were 3 ms (IQR: 2.1 - 7) and 7.2 ms (IQR: 7.2 - not reached), respectively. The prognostic impact of CTCs was confirmed for PFS and OS (P=0.024 and P<0.0001, respectively). Notably, the 5.7 %ctDNA cut-off retained its significance also in pts without detectable CTCs (P=0.0209), and subgroup analysis highlighted a significantly different prognostic impact across breast cancer subtypes (P for interaction = 0.029). Interestingly, pts with ≥ 5.7 %ctDNA had a higher frequency of ctDNA detected MET and PIK3CA mutations (P=0.0007 and P=0.003 respectively; Q=0.0126 and Q=0.051, respectively) and higher number of detectable mutations (P<0.0001).

Conclusions: The CTCs-derived %ctDNA cut-off is capable of prognostic stratification in ABC. Interestingly, ctDNA can stratify pts also without detectable CTCs, providing valuable information on prognosis, mutational tumor burden and disease monitoring and characterization. Further prospective studies are needed to validate these findings.

#2647

Bromodomain-containing protein 7 (BRD7) is a candidate tumor suppressor in breast carcinoma and prognostic biomarker.

Cristiana Lo Nigro,1 Daniela Vivenza,1 Ornella Garrone,1 Rodolfo Brizio,1 Fiamma Mantovani,2 Giannino Del Sal,2 Marco Carlo Merlano,1 Tim Crook3. 1 _S. Croce & Carle Teaching Hospital, Cuneo, Italy; _2 _Area Science Park, Trieste, Italy;_ 3 _Royal Surrey County Hospital, Guildford, United Kingdom_.

Background Breast cancer (BC) is a multifactorial disorder in which multiple genetic and epigenetic factors changes drive tumor initiation and progression. Bromodomain-containing protein 7 (BRD7) is a member of the bromodomain-containing protein family mainly localized in the nuclear compartment of the cell. BRD7 is a transcriptional co-activator for p53 modulating histone acetylation, p53 acetylation and promoter activity in p53 target genes. Previous studies show that BRD7 is down-regulated in a number of cancer types and this may correlate with clinical outcomes. However, the potential involvement of changes in BRD7 and prognostic importance in BC are poorly understood.

Purpose of the study We have investigated the expression, CpG island methylation and sub-cellular localization of BRD7 in BC cell lines and clinical cases and thereby assessed its prognostic significance by correlating with clinicopathologic features and time-dependent clinical outcomes.

Materials and methods BRD7 expression was analysed by qRT-PCR and IHC in 13 BC cancer cell lines. Expression was also assessed by IHC in 50 cases of primary breast cancer and 14 paired metastatic lesions. CpG island methylation was determined quantitatively by pyrosequencing. BC cases were divided for nuclear and non-nuclear BRD7 antigen localization and then stratified for high and low BRD7 expression. The effects of BRD7 expression and BC clinical characteristics data on OS and PFS were evaluated using univariate and multivariate Cox regression analyses.

Results BRD7 was expressed at levels similar to normal breast epithelium and the CpG island unmethylated in all 13 cell lines. Methylation analysis was successful in 42 FFPE BC samples collected, with 26,2% of them methylated (11/42). IHC analysis on primary tissues revealed that BDR7 is mainly localized in the nucleus (34/50: 68%). Patients with BRD7 nuclear localization (N=34) showed smaller tumors (p=0.012), a longer OS (p=0.002) and a lower relapse rate (p=0.03). Moreover, nuclear BRD7 expression is associated with wild-type TP53 (p=0.04) and unmethylated BRD7 (p=0.04), compared to the non-nuclear cases (N=16). No difference was seen in nodal status, grading, clinical stage, histological type, ER, PR, HER2 and Ki-67 expression. Patients (N=12) showing both nuclear localization and high BRD7 expression presented the smallest tumors (10/12 = 83%) and both a longer OS (p=0,005) and PFS (p=0,001). Among the 4/14 patients with nuclear localization in their primary tumors, 3 (75%) presented a diffuse or negative BRD7 in the paired metastasis.

Conclusion BRD7 is a strong candidate for breast cancer tumor suppressor and prognostic biomarker.

#2648

Prognostic significance of thyroid hormone receptor-alpha-2 (THRα2) expression in triple-negative breast cancer: A TCGA study.

Katarzyna J. Jerzak,1 Anna Dvorkin-Gheva,2 Jessica G. Cockburn,2 Anita Bane,2 John A. Hassell2. 1 _University of Toronto, Toronto, Ontario, Canada;_ 2 _McMaster University, Ontario, Canada_.

Background: Thyroid hormones promote breast cancer cell proliferation and expression of their cognate nuclear receptors has shown prognostic potential in small cohort studies. Among two isoforms of thyroid hormone receptor alpha (THRα), the alpha1 splice variant (THRα1) promotes thyroid hormone mediated cell proliferation whereas the alpha2 variant (THRα2) opposes it. Hence, THRα2 expression may be a favorable prognostic biomarker in breast cancer.

Methods: A publicly available database of breast tumors archived by The Cancer Genome Atlas (TCGA) was employed for this study. We analyzed RNA expression of THRα1 and THRα2 in 106 triple negative breast cancers (TNBCs) and correlated it with tumor stage (I vs II vs III) and nodal involvement (positive vs negative). Tumor grade was not uniformly reported. Univariate Cox proportional hazards regression models were fitted to determine the prognostic impact of THRα1 and THRα2 expression on overall survival (OS) and multivariate models were adjusted for age, tumor stage and radiation treatment.

Results: The median age of women was 54 (range 29-90) and 12.3% died. The majority (62.3%) of patients presented with stage II disease; 16.0% were stage III and 17.9% were stage I at diagnosis. There was no significant correlation between THRα1 or THRα2 expression and tumor stage or nodal involvement. Expression of THRα2 was associated with improved OS in both uni- and multi-variate models (Table).

Conclusions: In this study, THRα2 expression was independently prognostic for improved OS in TNBC. We previously demonstrated similar results in 158 TNBCs via immunohistochemistry but differentiation between RNA (as opposed to protein) splice variants is more precise. These results support investigation of THRα2 up-regulation or THRα1 inhibition as therapeutic strategies.

Table. Prognostic associations of THRα2 expression in TNBC

---

Variable | Univariate HR (95%CI) | p value | Multivariate HR (95%CI) | p value

Log (THRα2) | 0.46 (0.26-0.81) | <0.01 | 0.28 (0.09-0.84) | 0.02

Log (THRα1) | 0.54 (0.31-0.94) | 0.03 | 1.38 (0.53-3.62) | 0.51

Age (years) | 0.98 (0.94-1.02) | 0.37 | 0.92 (0.84-1.01) | 0.10

Stage (I vs II vs III vs IV) | 6.14 (2.19-17.22) | <0.01 | 26.03 (4.05-167.14) | <0.01

Radiation therapy (yes vs no) | 0.23 (0.04-1.22) | 0.08 | 0.10 (0.02-0.73) | 0.02

#2649

Expression and prognostic value of EGR1 and EGR3 in gliomas.

Arnon M. Knudsen,1 Ida Eilertsen,1 Susanne Kielland,1 Henning B. Boldt,2 Caspar S. Mellegaard,1 Sune Munthe,2 Bjarne W. Kristensen1. 1 _University of Southern Denmark, Odense C, Denmark;_ 2 _Odense University Hospital, Odense C, Denmark_.

Introduction

Gliomas are the most frequent primary brain tumors. For the most malignant glioma - the glioblastoma - the median survival is below 15 months. Since only few prognostic biomarkers are of benefit in daily practice, new markers are urgently needed. EGR1 and EGR3 are transcription factors involved in the regulation of cell-cycle and cell differentiation, but they have also been associated with migration of cancer cells. Studies have shown prognostic potential of EGR1 and EGR3 in breast-, gastric-, colorectal-, and prostate cancer. The purpose of this study was to investigate the expression and potential prognostic value of EGR1 and EGR3 in gliomas.

Methods

Samples of 214 gliomas were stained with anti-EGR1 and -EGR3 antibodies respectively. The stainings were assessed with a software-based digital classifier and a semi-quantitative scoring system. Kaplan-Meier estimates and Log-rank tests were used to compare survival distributions, and ROC-curve analysis with dichotomizing was applied to identify optimal cut-off values. Cox proportional hazards regression was performed to further investigate and identify independent prognostic variables.

Results

Both EGR1 and EGR3 were expressed in all glioma samples, but with considerable variation between tumors. EGR1 expression ranged from 1-83% positive cells and increased with WHO grade, while EGR3 expression ranged from 5-95% positive cells independent of WHO grade. High EGR1 levels were associated with improved survival in glioblastomas, while low EGR3 levels were associated with improved survival in WHO grade II and III gliomas. In glioblastomas, the EGR1 high/EGR3 low group was associated with improved patient survival when compared to the EGR1 low/EGR3 high group. Similar results were found in MGMT-promoter methylated glioblastomas, but not in MGMT-unmethylated glioblastomas. Cox proportional hazards regression showed that a high EGR1 fraction remained a significant prognostic variable when adjusted for confounders, both in all gliomas as one group (P=0.048) and glioblastomas exclusively (P=0.011). The combination of EGR1 high/EGR3 low in glioblastomas also remained significant after adjustment for confounders (P=0.030).

Conclusion

Both EGR1 and EGR3 proteins were expressed in all investigated gliomas. High levels of EGR1 seemed to be associated with improved patient survival in glioblastomas. In MGMT-promoter methylated glioblastomas, the EGR1 high/EGR3 low group was associated with improved patient survival when compared to the EGR1 low/EGR3 high group. Validation of the results in an additional patient cohort is needed, including analysis of prognostic value in different types of grade II and III gliomas.

#2650

Clinical implication of microsatellite instability in early gastric cancer.

Yoon Young Choi, Dong Gyu Kim, Taeil Son, Hyoung-Il Kim, Jae-Ho Cheong, Woo Jin Hyung, Sung Hoon Noh. _Yonsei University Health System, Seoul, Republic of Korea_.

Purpose: The role of microsatellite instability is well established in advanced gastric cancer. However, its clinical implication has not been well addressed in early gastric cancer. The purpose of this study is to evaluate the clinical characteristics of microsatellite instability in early gastric cancer.

Methods:

Patients who underwent gastrectomy with curative intent in Severance hospital from January, 2005 to December, 2010 and diagnosed as early gastric cancer (pT1a/b) were enrolled in this study. Remnant gastric cancer and patients who received chemotherapy before operation were excluded. Microsatellite instability status was evaluated by two mononucleotide repeat markers (BAT25 and BAT26) and three dinucleotide repeat markers (D5S346, D2S123, and D17S250) and instability in two or more markers was defined as miscrosatellite instability high (MSI-H), otherwise classified as microsatellite stable (MSS).

Results: Of 1156 tumors that included the final analysis, 85 (7.4%) were MSI-H. MSI-H cancer was related to frequent female gender (54.1% vs. 32.8%, p<0.001), older age (63.4 ± 10.65 vs. 56.7 ± 11.50, p<0.001), lower body (81.2% vs. 59.7%, p=0.002), intestinal histology (63.5% vs. 48.2%, p=0.005), lympho-vascular invasion (25.9% vs. 13.3%, p=0.001), submucosal invasion (63.5% vs. 48.3%, p=0.007), and trend of lymph node metastasis (17.6% vs. 10.8%, p=0.056) compared to MSS type. Lymph node metastasis and lymph-vascular invasion was not differed by MSI status in mucosal gastric cancer (3.2% vs.

2.3%, p=0.755 and 3.2% vs. 2.3%, p=0.755, respectively). In sumbucosal gastric cancer, however, lympho-vascular invasion was frequently observed in MSI-H tumor (38.9% vs. 25.0%, p=0.027) but there was no difference in lymph node metastasis (25.9% vs. 19.9%, p=0.298). When we compared the disease free survival by MSI status, the prognosis of MSI-H tumor was similar compared to that of MSS tumor (log-rank test p=0.797, adjusted Hazard ratio of MSI-H by age, sex, pTstage and number of metastatic LNs: 0.932 [95% confidence interval: 0.423-2.054, p=0.861]).

Conclusions: MSI status could not be a useful biomarker in early gastric cancer to predict prognosis of it. However, frequent lympho-vascular invasion of MSI-H early gastric cancer would be a warning that careful patient's selection for endoscopic treatment or limited lymph node dissection for surgery is necessary.

#2651

A mesenchymal-associated transcriptomic signature has a prognostic and predictive potential in stage II and III colorectal cancer patients.

Takatoshi Matsuyama,1 Raju Kandimalla,2 Xuan Wang,3 Toshiaki Ishikawa,1 Naoki Takahashi,4 Yasuhide Yamada,4 Masamichi Yasuno,1 Yusuke Kinugasa,1 Hiroyuki Uetake,1 Ajay Goel5. 1 _Tokyo Medical and Dental University, Tokyo, Japan;_ 2 _Baylor Scott & White Research Institute and Charles A Sammons Cancer Center, Dallas, TX; _3 _Baylor Research Institute, Baylor University Medical Center, Dallas, TX;_ 4 _National Cancer Center Hospital, Tokyo, Japan;_ 5 _Baylor Scott & White Research Institute and Charles A Sammons Cancer Center, Baylor University Medical Center, Dallas, TX_.

Purpose: Clinical decision making for adjuvant chemotherapy, which includes selection of appropriate patients and optimal treatment regimen, remains the most pressing challenge in the management of stage II and III colorectal cancer (CRC) patients. In this study, we attempted to address this issue by developing a clinically actionable mesenchymal-associated transcriptomic signature (MATS) that can accurately predict prognosis and identify CRC patients who could realistically benefit from adjuvant chemotherapy.

Experimental Design: To develop a mesenchymal-associated transcriptomic signature, we first performed gene expression profiling in 152 laser capture microdissected CRC tissues, followed by validation of this signature in 1365 CRC patients from various publicly available cohorts (GSE41258, GSE39582, GSE33113, GSE17536 and TCGA). Subsequently, this signature was trained (N=142) and validated (N=286) in two independent clinical cohorts of stage II and III CRC patients, in which Cox's regression analysis was performed to evaluate the recurrence predictive power of this signature. Furthermore, we investigated the clinical significance of MATS in its ability to identify patients who can benefit from fluoropyrimidine-based adjuvant chemotherapy.

Results: Through a comprehensive genome-wide expression analysis, we have identified an eight-gene mesenchymal-associated transcriptomic signature that can significantly predict recurrence-free survival (RFS) and identify a mesenchymal CRC subtype with high accuracy. To evaluate its translational potential, we further trained and validated this signature in two independent CRC cohorts consisting of 428 stage II and III CRC patients. Interestingly, MATS successfully stratified patients into low- and high-risk groups with 5-year RFS rates ranging from 87% and 54% in the training cohort [HR: 4.11 (CI: 2.72-15.43)] and 82% and 56% in the validation cohort [HR: 2.66 (CI: 1.66-3.98)], respectively. While the 48 patients with MATS low-risk stage III CRC showed a substantial improvement in survival, the 77 patients with MATS high-risk stage III CRC did not benefit from the fluoropyrimidine-based adjuvant chemotherapy. Furthermore, in multivariate analysis, MATS was significantly associated with RFS along with T-stage and lymphovascular invasion in both training as well as validation cohorts with HRs of 3.80 (CI: 1.85-7.82, p=0.0003) and 2.09 (CI: 1.28-3.42, p=0.003), respectively.

Conclusions: Our novel MATS could robustly identify RFS, and this classifier was superior to various clinicopathologic risk factors used in the clinic for risk stratification in CRC patients. In addition, MATS could also predict which stage III CRC patients might benefit from fluoropyrimidine-based adjuvant chemotherapy, which is a significant step forward in the clinical management of these patients.

#2652

Metabolomics biomarkers for endometrial cancer and its recurrence after surgery in postmenopausal women.

Yannick Audet-Delage,1 Lyne Villeneuve,1 Jean Grégoire,2 Marie Plante,2 Chantal Guillemette1. 1 _Pharmacogenomics Laboratory, Centre Hospitalier Universitaire de Québec (CHU) Research Center and Faculty of Pharmacy, Laval University, Quebec, Quebec, Canada;_ 2 _Centre Hospitalier Universitaire de Québec (CHU) and Faculty of Medecine, Laval University, Quebec, Quebec, Canada_.

Endometrial cancer (EC) is the most frequent cancer of the female genital tract in developed countries. Most EC occurs after menopause and are diagnosed as endometrioid type I carcinomas that exhibit a favorable prognosis especially for grades 1 and 2. In contrast, type II non-endometrioid carcinomas such as serous tumors have a poor prognosis. The use of small molecule biomarkers may facilitate accurate diagnosis and prognostic predictions. Our goal was to identify novel blood-based markers associated with EC subtypes and recurrence after surgery in postmenopausal women. Using mass spectrometry (MS)-based metabolomics, we examined preoperative serum metabolites of healthy women (n=18), non-recurrent and recurrent cases with type I endometrioid (n=24) and type II serous (n=12) carcinomas, matched for pathological characteristics, body mass index and age. Biochemicals including complex lipids were analyzed by gas chromatography-MS and ultrahigh performance liquid chromatography-tandem MS. A total of 1592 compounds of known identity and 14 different lipid classes were assessed. When comparing EC cases to healthy women, 137 metabolites were significantly different, with spermine as the most altered biochemical, identifying this metabolite as a potential biomarker of EC. Furthermore, recurrent cases of both histological types had higher levels of the monoacylglycerol 1-oleoylglycerol than non-recurrent cases. Type I recurrent cases were also characterized by much lower levels of bile acids and elevated concentrations of phosphorylated fibrinogen cleavage peptide, whereas type II recurrent cases displayed higher levels of ceramides and their derivatives. Our findings provide a first detailed metabolomics study in EC and identify potential non-invasive biomarkers to define clinically relevant risk groups.

#2653

Cell proliferation and Cathepsin L expression are inversely correlated in bladder cancer progression and patient survival.

Larry E. Douglass,1 Michelle C. Robillard,1 Dan Stelzer,1 Mariah Dooley,1 James A. Deddens,2 Julia H. Carter1. 1 _Wood Hudson Cancer Research Lab., Newport, KY;_ 2 _University of Cincinnati, Cincinnati, OH_.

In 2017 there will be an estimated 79,000 new cases of bladder cancer (UBC) resulting in 16,870 deaths. A better understanding of UBC progression and prognostic factors could improve patient management. UBC anatomic stage is determined by depth of invasion. Stage 0 is non-invasive, Stage 1 invades the sub-mucosa, Stage 2 invades the detrusor muscle, Stage 3 invades perivesical fat, and Stage 4 invades local or distant organs. 70-80% of UBCs are superficial, but can recur locally and be multifocal. Muscle invasive bladder cancers (MIBCs) can metastasize to pelvic lymph nodes or distant organs. To better understand the molecular mechanisms of UBC progression we measured cell proliferation utilizing the molecular marker of proliferating cells Ki 67 (Dako) and immunohistochemically detected expression of the lysosomal cysteine proteinase Cathepsin L (Abcam) in archived surgical specimens from 145 UBC patients. A board certified pathologist diagnosed and graded each histologic specimen and marked three areas for photography and image analysis. Clinical information and follow-up was obtained from existing medical records and the Kentucky Cancer Registry. IRB approval for the study was obtained from St. Elizabeth Healthcare who also donated the FFPE surgical specimens. We hypothesized that the % of proliferating tumor cells would increase with loss of cell differentiation and that Cathepsin L expression would increase with depth of tumor invasion into and beyond the detrusor muscle. Patients with high grade tumors (Grades 3 and 4) had a poor survival compared to patients with low grade tumors (p < 0.0001) and patients with MIBC (Stages 2-4) had a poorer survival that patients with superficial tumors (Stages 0 - 1) (p < 0.0001, HR = 18.63). Cell proliferation as determined by % of Ki 67 labeled nuclei was highly correlated with stage and grade (p <0.0001). In contrast Cathepsin L expression measured either semi-quantitatively by histoscore (area X intensity of stain) or quantitatively by computer assisted image analysis was negatively correlated with stage, grade, and Ki 67 labeling (p <0.0001). Recently multiple subtypes of MIBC have been identified, including basal tumors with squamous differentiation and luminal tumors. The highest rate of cell proliferation and the lowest expression of Cathepsin L in our data set were found in MIBC with squamous differentiation. Ki 67 labeling in UBC was highly prognostic of poor patient survival (p = 0.0003), whereas Cathepsin L expression as measured either by histoscore or image analysis was highly prognostic of good patient survival (p < 0.0001). Since Cathepsin L is encoded by the CTSL gene located on chromosome 9, these data are consistent with published reports of frequent genetic loss and mutations of chromosome 9 in UBC. These data also strongly support the value of UBC cell proliferation as a prognostic marker for stratifying UBC patients.

#2654

Immunologic and prognostic correlates of WISP1 in prostate cancer.

Pierre-Olivier Gaudreau,1 Sylvie Clairefond,2 Pierre-Luc Boulay,3 Pavel Chrobak,2 Bertrand Allard,2 Sandra Pommey,2 Fred Saad,2 Marian Young,4 John Stagg2. 1 _MD Anderson Cancer Center, Houston, TX;_ 2 _CHUM Research Center, Montreal, Quebec, Canada;_ 3 _Institut de Recherche en Immunologie et en Cancerologie (IRIC), Montreal, Quebec, Canada;_ 4 _National Institute of Dental and Craniofacial Research, National Institutes of Health (NIH), Bethesda, MD_.

Background: Avoidance of immune destruction and tumor-promoting inflammation are equally important cancer hallmarks. In the context of prostate cancer, inflammatory markers and high levels of immune infiltrates have been associated with shorter biochemical recurrence (BCR)-free survival. WNT1 Inducible Signaling Pathway (WISP1) has been implicated in prostate cancer metastasis and the regulation of inflammation in diverse benign diseases. Thus, the objectives of this study were: 1) to assess the prognostic value of WISP1 in human prostate cancer, and 2) to determine the association of WISP1 to the inflammatory landscape specific to this disease.

Methods: A tumor microarray (TMA) was constructed with radical prostatectomy specimens of 285 prostate cancer patients. Multicolor manual immunofluorescence (IF) was performed to simultaneously detect WISP1, CD8 and cytokeratins 8 and 18. WISP1 expression levels were determined by the mean fluorescence intensity (MFI) in stromal, epithelial, cytoplasmic and nuclear (DAPI) areas in each core, and CD8+ cell density was determined for each compartment by dividing cell count by the percentage of the core occupied by the compartment. Finally, the prostate cancer TCGA dataset (n = 548) was used to validate the prognostic value of WISP1 mRNA expression, as well as its association to CD8+ lymphocytes using previously validated gene signatures (Becht et al., 2016).

Results: IF analyses of our TMA revealed that high levels of WISP1 in normal adjacent epithelium are significantly associated with shorter BCR-free survival in Kaplan-Meier (log-rank = 4.246, p = 0.039) and univariate Cox regression analyses (hazard ratio = 1.477; p = 0.042), but not in multivariate Cox regression analyses (hazard ratio = 1.381; p = 0.101). Furthermore, a significant correlation was found between WISP1 expression and CD8+ cell density. Gene expression analyses further showed that WISP1-high prostate tumors are associated with a CD8+ lymphocyte gene enrichment profile, and confirmed that patients with WISP1-high prostate tumors have reduced BCR-free survival (Wilcoxon rank, p = 0.003).

Conclusions: Overall, our results support a negative prognostic association for WISP1 as well as a proinflammatory role. WISP1 may represent a relevant target for the improvement of prostate cancer immunotherapy.

#2655

HYAL4: A molecular driver and potential marker of invasive bladder cancer.

Sarrah S. Lahorewala, Daley S. Morera, Andre R. Jordan, Vinata B. Lokeshwar. _Augusta University, Augusta, GA_.

INTRODUCTION AND OBJECTIVES: Muscle invasive bladder cancer (MIBCa) causes the majority of morbidity and mortality in BCa patients. Prognosis of MIBCa patients may be improved by ide ntification of novel prognostic biomarkers and therapeutic targets. Chondroitin sulfate proteoglycans are known to promote tumor growth and metastasis. However, a Chondroitinase (Chase) that degrades chondroitin sulfate has not been identified. HYAL4, a member of the glycosaminoglycan (GAG) degrading enzyme-family, potentially has Chase activity; however, HYAL4 (HY4) has not be studied in any biological system, normal or disease. The objective of this study was to evaluate the expression of all six members of the GAG-degrading enzyme family in bladder cancer (BCa) specimens and to investigate HY4 functions in preclinical models of BCa.

METHODS: Q-PCR was performed to measure mRNA levels of GAG-degrading enzyme family genes in 59 bladder tissues (normal (NBL) = 25; tumor (TBL) = 38). HY4 expression was also measured in 40 cystectomy specimens from MIBCa patients who later were treated for metastatic disease with Gemcitabine plus cisplatin (G+C) chemotherapy. HY4 was either stably expressed or knocked-down in immortalized urothelial and BCa cell lines. Transfectants were assayed for Chase activity, anchorage independent growth, motility, invasion, molecular signaling. Tumor growth and metastasis was monitored by bioluminescence imaging.

RESULTS: Among the 6 genes, HYAL1 and HY4 mRNA levels were significantly (6-13-fold) elevated in TBL tissues when compared to NBL tissues (P<0.001). HY4 levels were 7-fold elevated in muscle invasive BCa. In univariate and multivariate analyses, HY4 predicted metastasis and death due to BCa (chi-sq: 6.93; P=0.0089; risk-ratio: 6.9). In metastatic BCa patients, high HY4 levels significantly correlated with G+C treatment failure (χ2 = 7.5; P=0.0062) with > 80% accuracy. HY4-expressing transfectants secreted HY4 and Chase activity in their conditioned media. Overexpression of HYAL-4 in normal urothelial and BCa cells significantly increased, while its knockdown abrogated, anchorage-independent growth, invasion, and chemotactic motility (> 3-fold). HY4 expression induced Gemcitabine resistance (IC50: vector: 3.1 nM; HY4: 126 nM). HY4 expression upregulated a stem cell signature. Urothelial cells expressing HY4 formed tumors in NOD/SCID mice. HY4-expressing BCa cells metastasized to lung and spleen in an orthotopic BCa model.

CONCLUSION: This first study on HY4 shows that it is a novel molecular determinant of MIBCa and Gemcitabine resistance and a potential marker for clinical outcome.

#2656

Estrogen receptor pathway activity in endometrial carcinomas and its relation to tumor grade and recurrence.

Louis J. van der Putten,1 Anne van Brussel,2 Willem Jan van Weelden,3 Márcia A. de Inda,2 Leon F. Massuger,3 Henk van Ooijen,2 Anja van de Stolpe,2 Johanna M. Pijnenborg3. 1 _Radboud university medical center, Nijmegen, Netherlands;_ 2 _Philips Research, Eindhoven, Netherlands;_ 3 _Radboud University Medical Center, Nijmegen, Netherlands_.

Background

Immunohistochemical (IHC) loss of expression of the estrogen (ER) and progesterone receptor (PR) from endometrial carcinomas is associated with high tumor grade, and with recurrent disease. However, expression of these receptors is not always indicative of functional pathway activity. A novel approach to predict signaling pathway activity, based on Knowledge-based Bayesian computational models interprets quantitative transcriptome data as the functional output of an active signaling pathway by using expression levels of transcriptional target genes. The objectives of this study were to compare ER pathway activity to ER and PR IHC results with respect to endometrial carcinoma grade and disease recurrences.

Materials and Methods

Originally for Affymetrix, the pathway analysis was adapted for RT-qPCR enabling use on FFPE tissue. Calibration and threshold definition was performed using tissue with known pathway activity. Pathway analysis was performed on: (1) A public dataset (GSE56026) containing Affymetrix expression microarray data from 62 patients with grade 1 (n=23),2 (n=17) and 3 (n=11) endometrioid and serous (n=12) carcinomas, and (2) formalin fixed, paraffin embedded (FFPE) tissue from endometrioid (n=70) and serous carcinomas (n=15), treated at the Radboud university medical center, which were immunohistochemically stained for ER and PR, and analyzed for pathway activity using qPCR.

Results

(1)

Analysis of the public dataset showed an inverse relation between ER pathway activity score and cancer grade, with the lowest ER activity in serous carcinomas. The Mann-Whitney U test showed significant differences between grade 1 and 2 (p=0.015), grade 1 and 3 (p=0.004), and grade 1 and serous carcinomas (p<0.0001).

(2)

These results were confirmed in the FFPE tissue. The ER pathway was active in 30% (n=18), and inactive in 70% (n=41) of the samples. In advanced stage endometrioid and all serous carcinomas, ER pathway was active in 8% (n=2) and inactive in 92% (n=24) of the samples (Fisher p=0.03). Endometrial carcinoma related death (ECD) rate was 20% (n=17). While individually, ER pathway activity, ER and PR IHC were all significantly correlated with ECD, (logistic regression odds ratio, OR = 0.53, 0.12, 0.15, and p=0.0004, 0.0005, 0.002, respectively). In a multivariate analysis only ER pathway activity and disease stage remained significantly correlated with ECD (OR=0.52, p=0.03).

Conclusion

In two independent patient cohorts, the ER pathway model analysis, performed on data from Affymetrix microarrays and qPCR measurements showed an inverse relationship between the pathway activity and increasing tumor grade. While ER activity and ER/PR staining were related, only ER activity and cancer stage remained significant in a multivariate analysis with respect to immunohistochemistry alone when trying to predict recurrent disease.

#2657

Immune checkpoint status does not impact overall survival in NSCLC.

Donovan Watza, Valerie Murphy, Chrissy Lusk, Angie S. Wenzlaff, Lonardo Fulvio, Christine Neslund-Dudas, Gerold Bepler, Ann G. Schwartz. _Barbara Ann Karmanos Cancer Inst., Detroit, MI_.

Checkpoint blockade has altered standard of care for non-small cell lung cancer (NSCLC) expressing PD-L1, with trials demonstrating favorable prognosis in patients whose tumors were PD-L1 positive and were subsequently paired with anti-PD-1 therapy. However, the published literature remains controversial on whether PD-L1 expression confers prognostic significance independent of immunotherapy in NSCLC. Recent meta-analyses suggest that PD-L1 expression is associated with worse outcomes in NSCLC although other such studies associate PD-L1 expression with favorable prognosis. Moreover, few studies in NSCLC have simultaneously assessed PD-1 expression on tumor-infiltrating leukocytes (TILs), an important contributor to tumor checkpoint status. To assess the prognostic significance of PD-L1/PD-1 status in NSCLC we assembled 280 NSCLC FFPE tissues from lung cancer cases with detailed clinical and questionnaire data available who were recruited from the Metropolitan Detroit region. Tissues were compiled into duplicate tissue-microarrays and were assayed for PD-L1 (223C), PD-1 (NBP1-88104), and whole-transcriptome mRNA expression. PD-L1 staining was assessed by an experienced pathologist and was reported as the percent of tumor cells with membrane positive 223C staining to mirror clinical reporting of PD-L1 status. PD-1 staining was also assessed by the pathologist and was reported as the percent of TILs with membrane positive NBP1-88104 staining. RNA was extracted from FFPE samples and assayed on the Affymetrix Whole-Transcriptome 2.1 array. Array data was batch normalized and Log2 transformed prior to analysis. Immune marker effects on overall survival (OS) were measured with a Cox proportional hazards model, adjusting for tumor stage and histology. 36% of NSCLC tumor samples were PD-L1 positive, defined as ≥ 1% of tumor cells demonstrating positive 223C membrane staining. PD-L1 status was not associated with tumor stage or histology but was strongly correlated with PD-L1 (CD274) mRNA expression (OR=2.37, p=1.1x10-7), adjusting for stage and histology. PD-L1 positive NSCLC were found to have similar OS when compared to tumors with no detectable PD-L1 tumor staining (HR=0.94, p=0.78). Likewise, tumors containing PD-1 positive TILs did not differ in OS when compared to tumors with no detectable PD-1 positive TILs (HR=0.68, p=0.14). Furthermore, there were no detectable differences in OS when combined PD-L1/PD-1 status was assessed (PD-L1+/PD-1+ HR=0.72 p=0.36, PD-L1+/PD-1- HR=1.39 p=0.49, PD-L1-/PD-1+ HR=0.79 p=0.49, PD-L1-/PD-1- Referent). Our findings suggest that neither PD-L1 tumor or PD-1 TIL status are significant prognostic markers in NSCLC independent of immunotherapy.

#2658

Molecular characterization of renal cell carcinoma: A potential three microRNA prognostic signature.

Soum D. Lokeshwar,1 Andre R. Jordan,2 Sarrah S. Lahorewala,3 Vinata B. Lokeshwar3. 1 _University of Miami Miller School of Medicine, Miami, FL;_ 2 _Medical College of Georgia at Augusta University, Augusta, GA;_ 3 _Medical College of Georgia at Augusta University, Augsuta, GA_.

Introduction and Objective: With 10% survival at 5-years, metastatic disease presents poor prognosis for renal cell carcinoma (RCC) patients. Prognosis can be improved if biomarkers that predict disease outcome are identified and validated. Aberrantly expressed microRNAs (miRs) promote (RCC) growth and metastasis and are potentially useful biomarkers for metastatic disease. However, consensus of a clinically significant miRNA signature has not been established. The objective of this study was to identify a miRNA signature for predicting clinical outcome in RCC patients using a four-pronged interconnected discovery and validation approach.

Methods: Differentially expressed miRs were identified and analyzed in 113 specimens (Normal kidney: 59; tumor: 54). In the discovery phase, miRNA-profiling was initially performed in matched normal and tumor specimens from 8 patients, followed by validation in 16 additional specimens. Seven miRs that were significantly aberrantly expressed in miRs in both discovery phases were analyzed by qPCR and their levels were correlated with RCC subtypes and clinical outcome. miRNA signature was confirmed in a TCGA dataset of 241 RCC specimens.

Results: Discovery phase identified miR-21, miR-142-3p, miR-142-5p, miR-150 and miR-155 as significantly upregulated (2-4-fold) and miR-192, miR-194 as downregulated (3-60-fold) in RCC; miR-155 distinguished small tumors (< 4 cm) from benign oncocytomas. miR-142-5p levels were significantly up-regulated in patients with lymph node positive disease and metastasis (P=0.0002). A 3-miR combination, miR-21+142-5p+194 significantly but negatively correlated with metastasis (P=0.0017; OR: 0.53) and disease-specific mortality (P=0.018; OR: 0.37). The signature had high sensitivity (86.7 - 93.3%) and specificity (82 - 87%) to predict metastasis and disease-specific mortality. In the TCGA dataset, the 3-miR signature significantly and negatively associated with metastasis (P=0.019; OR: 0.54) and overall survival (P=0.0919; OR: 0.37).

Conclusion: The interconnected discovery-validation approach identified a novel signature of 3 aberrantly expressed miRs as a potential prognostic indicator for disease outcome among RCC patients. Early detection of metastasis using the validated qPCR assay for the 3-miR signature may allow early intervention for improving patient prognosis.

#2659

GLUT1 **variants and the prognosis of patients with NSCLC after surgery.**

Shin Yup Lee,1 Sook Kyung Do,1 Jin Eun Choi,1 Michael Neugent,2 Justin Goodwin,3 Jung-whan Kim,2 Jae Yong Park1. 1 _Kyungpook National Univ. School of Med., Daegu, Republic of Korea;_ 2 _The University of Texas at Dallas, TX;_ 3 _Yale School of Medicine, CT_.

Background This study was conducted to investigate whether polymorphisms of glucose transporter 1 (GLUT1) gene are associated with the prognosis of patients with non-small cell lung cancer (NSCLC) after surgical resection.

Methods Five single nucleotide polymorphisms (SNPs) in GLUT1 were investigated in a total of 354 patients with NSCLC who underwent curative surgery. The association of the SNPs with overall survival (OS) was analyzed.

Results Among the five SNPs investigated, two SNPs (GLUT1 rs3820589T>A and rs4658G>C) were significantly associated with OS in multivariate analyses. GLUT1 rs3820589T>A was associated with significantly better OS (adjusted hazard ratio [aHR] = 0.57, 95% confidence interval [CI] = 0.34-0.94, P = 0.03, under dominant model), and rs4658G>C was associated with significantly worse OS (aHR = 1.91, 95% CI = 1.09-3.33, P = 0.02, under recessive model). In the stratified analysis by tumor histology, the effect of these SNPs on OS was only significant in squamous cell carcinoma, but not in adenocarcinoma. When the two SNPs were combined, OS decreased as the number of bad genotypes increased (Ptrend = 4 × 10-3).

Conclusion This study suggests that genetic variation in GLUT1 may be useful in predicting survival of patients with early stage NSCLC.

#2660

Glycosylation of ascites-derived exosomal CD133 is a potential prognostic biomarker in patients with advanced pancreatic cancer.

Takahiko Sakaue,1 Hironori Koga,1 Hideki Iwamoto,1 Yasuko Imamura,2 Toru Nakamura,1 Mitsuhiko Abe,1 Fumitaka Wada,1 Atsutaka Masuda,1 Toshimitsu Tanaka,1 Masaru Fukahori,1 Tomoyuki Ushijima,1 Yoshinobu Okabe,1 Tatsuyuki Kakuma,3 Takuji Torimura1. 1 _Department of Medicine, Kurume University School of Medicine, Kurume City, Japan;_ 2 _Liver Cancer Research Division, Kurume University Research Center for Innovative Cancer Therapy, Kurume City, Japan;_ 3 _Biostatistics Center, Kurume University, Kurume City, Japan_.

Background: It is known that cancer cells surviving in ascites show cancer stem cell (CSC)-like features. The CSC-like cells abundantly secrete extracellular vesicles called exosomes, which may play important roles in maintaining tumor-specific microenvironment in the abdominal cavity. We have demonstrated that exosomes were rich in malignant ascites of patients with advanced pancreatic cancer, and that the exosomes contained much CD133 among the CSC-associated proteins examined. Of note, the expression level of exosomal CD133 was highest in pancreatic cancer patients compared with that in gastric cancer patients and those with liver cirrhosis. Thus, the aim of this study was to assess whether expression levels of CD133 in the ascites-derived exosomes obtained from pancreatic cancer patients inversely correlate with their prognosis.

Methods: 133 patients with pancreatic cancer who visited the Center for Multidisciplinary Treatment of Cancer in Kurume University Hospital from June, 2014 to June, 2017 were enrolled into this study. Informed consent was obtained from all patients in accordance with the principles stated in the Declaration of Helsinki and the guidelines of the Ethical Committee of Kurume University. Exosomes derived from consecutive 19 patients with malignant ascites of 133 patients were purified by using ExoQuick kit (System Biosciences). Western blotting and densitometry were performed to detect CD133 and to determine its expression levels, respectively. We focused on possible relationship between band intensity of highly glycosylated CD133 (defined as "upper band") and overall survival of the patients.

Results: Median overall survival of 19 patients with malignant ascites was 256 days (45-1031 days), while that of 133 patients was 225 days (4-1617 days). Then, the 19 patients showing worse prognosis were subjected to densitometric analysis of intensity of the upper band for CD133, reflecting glycosylation of the molecule. As a result, there was an equilateral correlation between the upper band density of CD133 and overall survival (p=0.0309). The finding suggested that longer survival was predicted even for the advanced pancreatic cancer patients with malignant ascites when increased density in the upper band of ascites-derived exosomal CD133 was seen.

Conclusions: Although the number of patients was limited and glycosylation profile of the upper band was under investigation in this study, it is suggested that glycosylation of CD133 derived from exosomes in malignant ascites may be useful in selecting patients with better prognosis who are feasible to receive further chemotherapeutic treatment for advanced pancreatic cancer.

## CANCER CHEMISTRY:

### Antitumor Agents

#2661

Novel albumin-binding maytansinoids inducing long-term partial and complete tumor regressions in several human cancer xenograft models in nude mice.

Friederike I. Nollmann, Patricia Perez Galan, Javier Garcia Fernandez, Heidi K. Walter, Johannes P. Magnusson, Federico Medda, Felix Kratz, Stephan D. Koester, Khalid Abu Ajaj, Lara Pes, Serghei Chercheja, Anna Warnecke. _CytRx Corporation, Freiburg, Germany_.

Introduction and objectives: Maytansine and its analogs (DM1 and DM4) are potent microtubule-targeting compounds that inhibit proliferation of cells during mitosis. Their potent anticancer activity made them attractive for drug development.1 Unfortunately, their narrow therapeutic window prevents the clinical application of these molecules. So far only T-DM1, an antibody-maytansinoid conjugate targeting the HER2 receptor, has been approved for the treatment of Herceptin®-resistant breast cancer. Previous work2 showed that by direct esterification at the C3-OH-position of maytansinol, in vitro cytotoxicity may be enhanced compared to the parent drug maytansine. To make these highly potent compounds available for cancer treatment, novel maytansinoids were attached to a water-solubilizing hydrazone linker which selectively binds to the Cys-34 position of human serum albumin upon i.v. injection. The active drug is designed to be liberated in the acidic environment of the tumor as well as in acidic cellular compartments of tumor cells.3 Based on stability and release data in vitro, albumin-binding maytansinoids were selected for further in vivo evaluation. Herein, we present the data from a head-to-head comparison of novel albumin-binding maytansinoids with their parent maytansine in different cell- and patient-derived human tumor xenograft models.

Methods: Patient-derived human tumor xenografts (LXFE937 etc.) or tumor cells (A2780, MDA-MB 231, MDA-MB 468 etc.) were transplanted subcutaneously to female NMRI nu/nu mice (n=7-8 per group). Therapy with maytansine (0.4 or 0.5 mg/kg) and albumin-binding maytansinoids (2-3 mg/kg maytansine eq) was initiated after tumors reached a mean volume of 80-380 mm3. Animals were treated by i.v. injection once per week over 4 weeks.

Result and conclusion: Treatment with novel albumin-binding maytansinoids was better tolerated than with maytansine and MTDs were 4-7 times higher showing no significant body weight loss (< 5%). The albumin-binding maytansinoids demonstrated a significantly higher antitumor activity compared to both the control group and the group treated with maytansine and induced long-term partial and complete tumor regressions in all experiments.

1) W. C. Widdison et al, J. Med. Chem., 49: 4392-4408 (2006); K. A. Poon et al, Toxicology and Applied Pharmacology, 273: 298-313 (2013); H. L. Perez et al, Drug Discovery Today, 19:869-881 (2014)

2) Abstract XXX (Structure-activity relationship studies and biological evaluation of novel maytansinoids, a class of highly selective tubulin inhibitors)

3) F. Kratz et al., ChemMedChem, 3:20-53 (2008); US 7, 387, 771; F. Kratz, J. Control. Release, 132:171-183 (2008), F. Kratz, U. Beyer, Drug Delivery, 5: 281-299 (1998).

#2662

Indenoisoquinoline topoisomerase I inhibitors target the DNA G-quadruplex formed in the c-Myc promoter and cause MYC downregulation.

Kaibo Wang, Mohamed S. A. Elsayed, Guanhui Wu, Mark Cushman, Danzhou Yang. _Purdue University, West Lafayette, IN_.

The inhibition of DNA topoisomerase I (Top1) has proven to be a successful approach toward the design of anticancer agents. Indenoisoquinoline derivatives have been designed and developed as Top1 inhibitors and can overcome some shortcomings associated with the camptothecin Top1 inhibitors, resulting in three indenoisoquinoline compounds, indotecan (LMP 400), indimitecan (LMP776), and LMP744 entering Phase I clinical trials at the National Cancer Institute. However, some active indenoisoquinolines did not show potent Top1 inhibition, suggesting a separate mechanism of action. c-Myc is one of the most commonly deregulated genes in human cancers; a DNA G-quadruplex (G4) formed in the proximal promoter region of c-Myc has been found to function as a transcriptional silencer and be amenable to small molecule targeting. As indenoisoquinolines share structure similarity to our c-Myc G-quadruplex (MycG4)-interactive compounds, we hypothesized that indenoisoquinolines could stabilize MycG4 and downregulate MYC transcription. Using a fluorescence resonance energy transfer (FRET) assay, we examined the MycG4 stabilization activity of 56 indenoisoquinoline compounds that we previously developed as Top1 inhibitors. Notably, 26 of the 56 compounds decreased the fluorescence intensity by at least 50%, and the 10 most potent compounds reduced fluorescence intensities by over 70%, indicating that these indenoisoquinolines are effective MycG4 stabilizers. The identified compounds were further characterized by fluorescence anisotropy, circular dichroism, and nuclear magnetic resonance for MycG4 interactions, followed by western blot assays for MYC downregulation. Significantly, the compounds with good MycG4 binding activity and affinity exhibited significant dose-dependent reduction of MYC expression, indicating that targeting the c-Myc promoter G-quadruplex is a likely mechanism of action for these active anticancer compounds. Furthermore, some active indenoisoquinolines exhibited both MYC downregulation and Top1 inhibition, suggesting that dual targeting c-Myc G-quadruplex and Top1 may represent a novel mechanism of action for anticancer drug design.

#2663

CR42-24, a novel colchicine derivative, a therapeutic for bladder cancer.

Clayton Bell, Kyle Potts, Desmond Pink, John Lewis, Jack Tuszynski. _University of Alberta, Edmonton, Alberta, Canada_.

Colchicine is an anti-mitotic drug that targets unpolymerized tubulin, and inhibits microtubule polymerization. It is primarily used to treat gout but has been investigated in numerous clinical trials to treat conditions such as leukemia (ALL), prostate cancer, Behcet's disease, etc.. However, due to its narrow therapeutic window colchicine has had limited clinical translation. A possible approach to reduce general is to produce a derivative with an increased cancer specificity and selectivity by a narrower molecular target selection. Previously, our lab has designed and synthesized a novel colchicine derivative (CR42-24) with a more favorable pharmacological profile compared to colchicine. This was accomplished by designing a structure with an increased affinity for βIII tubulin. βIII tubulin is a β-tubulin isotype that is incorporated into tubulin dimers that make up microtubules. βIII is an excellent target for novel therapies as it has low expression in healthy tissue, is overexpressed in metastatic cancers, and is a clinical marker of poor prognosis. Using cell line screening we demonstrate that CR42-24 is highly toxic to a variety of cancer types with IC50 values at low nanomolar concentrations. More specifically CR42-24 is shown to be highly effective against bladder cancer. Current chemotherapy for bladder cancer is a combination of gemcitabine and cisplatin (Gem/Cis). Although marginally successful, many patients develop resistance to Gem/Cis leaving them with limited options for a second line therapy, thus development of alternative therapies is highly desired. Using in vitro assays we demonstrate that CR42-24 kills aggressive bladder cancer cell types, and is more effective than gemcitabine and cisplatin. Additionally, CR42-24 doubles survival rates of mice with bladder cancer xenografts. We also show that CR42-24 is highly synergistic with other chemotherapies, thereby increasing its therapeutic potential. Through our studies we have shown that CR42-24 is effective in treating aggressive bladder cancer and thus may serve as an alternative or second line therapy.

#2664

Identification of two small molecules with small cell lung cancer growth inhibition response profiles different from etoposide/topotecan.

Allison A. Mootz,1 Michael Peyton,2 Paul Yenerall,2 Kimberley Avila,2 Kenneth Huffman,2 Tomohiro Haruki,2 Mahboubeh Papari-Zareei,2 Victor Stastny,2 Luc Girard,2 Elizabeth McMillan,3 Michael Roth,2 John MacMillan,4 Bruce Posner,2 Michael White,2 Gazdar Adi,2 John Minna2. 1 _Texas Tech University Health Sciences Center, El Paso, TX;_ 2 _University of Texas Southwestern Medical Center, Dallas, TX;_ 3 _The Rockefeller Univeristy, New York, NY;_ 4 _University of California, Santa Cruz, Santa Cruz, CA_.

Small Cell Lung Cancer (SCLC) is an aggressive, highly metastatic cancer with an overall 5-year survival rate of <5% whose treatment has not changed in the past 20 years and has been designated by the US Congress and the National Cancer Institute as a "recalcitrant cancer." SCLCs are characterized by high initial response rates to chemotherapy (platin + etoposide) and radiation therapy but relapse is almost universal within 1 year. A NCI screen of 526 clinical or clinical investigational agents in 63 SCLC lines found large response phenotype differences (Polley, JNCI (2016) 108(10): djw122) but, in general, SCLC lines were either "sensitive" or "resistant" to all drugs tested and there was no correlation with tumor molecular biomarkers, or prior chemotherapy treatment. Thus, new drugs are needed (preferably with tumor associated molecular biomarkers) that have different profiles than available agents. As part of an NCI Cancer Treatment Discovery and Development Network (CTD2N) consortium we have screened large chemical libraries and identified chemical compounds that kill subsets of non-small cell lung cancers (NSCLCs) but not normal lung epithelial cells thus displaying a "therapeutic window" and tumor specificity. We first tested 116 of these compounds against 4 SCLC lines and found 30 that displayed dose dependent SCLC toxicity. We have so far tested 2 (SW034510, SW140154) compounds against a panel of 22 SCLC lines, which were also in the NCI screen, along with etoposide and topotecan as comparators, using 8-point dose response curves, for effect on SCLC growth inhibition. The SCLC lines (all with p53 and Rb mutations) were also phenotyped for expression of Myc family members (c-, N-, and L-), lineage transcription factors ASCL1 and NeuroD1 and neuroendocrine mRNAs. As found by Polley et al, etoposide and topotecan response phenotypes were correlated with each other (r2 = 0.7). By contrast, our two compounds, SW034510 and SW140154, response phenotypes did not correlate with etoposide/topotecan (r2 values of <0.08, and 0.1 respectively) and did not correlate with each other. Both agents cause cell death as assessed by the CellTox Green Assay suggesting efficacy across the ASCL1/NeuroD1, Myc family expression profiles. Cleaved caspase-3 western blot shows SW034510 causes caspase induced apoptosis. Cell cycle analysis suggests SW140154 causes a G1 cell cycle arrest. SARM1 gene expression anti-correlates with SW140154 IC50s. In conclusion, starting with chemical compounds that have selectivity for NSCLC, we have identified two new chemical compounds with specificity for subsets of NSCLC over normal lung epithelial cells, and which also inhibit the growth/kill subsets of SCLC in patterns different from the established therapy. Thus, these two new compounds provide potentially new therapeutic opportunities for SCLC patients resistant to current chemotherapies.

#2665

**Bioassay approach isolation and crystal structure elucidation of isoglabratephrin: A prenylated flavonoid from** Tephrosia apollinea **and its anticancer property.**

Loiy E. Ahmed Hassan,1 Muhammad Adnan Iqbal,2 Aman Shah Abdul Majid,3 Amin M. Abdul Majid,4 Zarina Thasneem Zainudeen5. 1 _Omdurman University, Omdurman, Sudan;_ 2 _universiti Sains Malaysia, Penang, Malaysia;_ 3 _Quest International University Perak, Ipoh, Malaysia;_ 4 _Australian National University, Australia;_ 5 _University of Oxford, Oxford, United Kingdom_.

Cancer is characterized by uncontrolled cell division caused by dysregulation of cell proliferation. Therefore, agents that impair cancer cell proliferation could have potential therapeutic value. Higher plants are considered to be a good source of anticancer agents, and several clinically tested chemotherapeutic agents have been isolated from plants or derived from constituents of plant origin. In the present study, a prenylated flavone (isoglabratephrin) was isolated from aerial parts of Tephrosia apollinea using a bioassay-guided technique. Chemical structure of the isolated compound was elucidated using spectroscopic techniques (NMR, IR, and LC-MC) and confirmed using single crystal X-ray analysis. The antiproliferative effect of isoglabratephrin was tested using three human cancer cell lines (prostate (PC3), pancreatic (PANC-1), and colon (HCT-116) and one normal cell line (human fibroblast). Isoglabratephrin displayed selective inhibitory activity against proliferation of PC3 and PANC-1 cells with median inhibitory concentration values of 20.4 and 26.6μg/ml, respectively. Isoglabratephrin demonstrated pro-apoptotic features, as it induced chromatin dissolution, nuclear condensation, and fragmentation. It also disrupted the mitochondrial membrane potential in the treated cancer cells. Thus, isoglabratephrin could be a new lead for development of cancer chemotherapies to treat human prostate and pancreatic malignancies.

#2666

Subtype-selective lethal molecules disrupt the regulatory module that drives high-risk neuroblastoma.

Michael E. Stokes, Alessandro Vasciaveo, Jonnell Candice Small, Presha Rajbhandari, Gonzalo Lopez, Andrea Califano, Brent R. Stockwell. _Columbia University, New York City, NY_.

Neuroblastoma (NBL) is a tumor that is derived from the developing neural crest, resulting in approximately 15% of pediatric cancer deaths. This cancer is characterized by multiple tumor subtypes that vary in their aggressiveness and clinical prognosis. Though MYCN amplification is associated with an aggressive subtype and poor prognosis, recent molecular classification identified a novel aggressive subtype driven by a mesenchymal gene signature. To understand the molecular mechanisms underpinning each subtype, network-based analysis identified key transcriptional regulators for each subtype that act in concert to drive tumor pathology. We hypothesize that chemical inhibitors disrupting this regulatory module will have greater chance of success in vivo, by targeting the specific drivers of the tumor subtype. In an effort to identify subtype-selective lethal molecules that could be developed into targeted therapies, we screened ~5500 bioactive molecules across a panel of four NBL cell lines representing either MycNAMP or the newly identified mesenchymal subtype (MESN). A number of subtype selective compounds were identified, and their effect on the regulatory module was evaluated using high-throughput transcriptome analysis (PLATE-Seq) (Bush EC et al., 2017) followed by Virtual Inference of Protein activity by Enriched Regulon (VIPER) analysis (Alvarez MJ et al., 2016). This analysis revealed 15 compounds that exhibited greater potency in MycNAMP cells, and inverted the MR signature that characterizes this subtype. Compounds that reverted the signature caused destabilization of MycN through diverse mechanisms, highlighting new therapeutic approaches to targeting the MycNAMP subtype. This screening strategy also identified MESN-selective compounds, including multiple statin drugs that inhibit mevalonate biosynthesis in cells. The subtype-selectivity of these well-studied drugs is being used to define their lethal mechanisms in the MESN subtype, and to understand how MycNAMP cells evade their inhibitory effects. Our multifaceted approach, including cell line-selective chemical screening coupled with high-throughput transcriptome and network analysis, can serve as a framework for future drug discovery in a variety of cancers.

#2667

Patient-derived lymphoma cell lines and high-throughput screening enable identification of potential cytotoxic therapeutic agents for dogs and cats with lymphoma.

Garrick M. Moll, Vilma Yuzbasiyan-Gurkan. _Michigan State Univ. College of Veterinary Med., East Lansing, MI_.

Treatment success in lymphoma, the most common hematopoietic cancer seen in dogs and cats, continues to be elusive. The clinical arsenal is limited to a few conventional chemotherapeutic drug protocols that result in short-term responses. In this study, dog and cat patient-derived tumor cells were tested via high-throughput screening (HTS) against a pool of over 100 FDA-approved compounds for human cancer treatment as a means to identify potential treatment strategies that can be relatively easily adapted to the dog and cat, as well as over 500 protein kinase inhibitors as a means to identify potential oncogenic driver pathways. Cells from lymph node fine needle aspirates, pleural effusions, or peripheral blood mononuclear cells from over 20 lymphoma patients were placed in cell culture media with and without growth factors. Cells from two cases, ZS15 from a Boxer dog and MT16 from a domestic shorthair cat, have continued to expand for over 12 and 6 months, respectively. Characterization by flow cytometry for detection of CD3 and CD21 identified these patient-derived cells to be T-cell lymphocytes. HTS and follow-up dose response curve (DRC) assays were performed, identifying multiple drugs effective in inhibiting growth of MT16 and ZS15 cells at nanomolar concentrations in vitro; these included anthracenedione/anthracycline-class and actinomycin-class drugs, as well as mRNA synthesis, microtubule/spindle formation, proteasome, histone deacetylase, or insulin growth factor-1 receptor inhibitors, many of which are currently not used in veterinary species. Notably, ZS15 cell growth was inhibited by mammalian target of rapamycin (mTOR) inhibitors, corroborating previous exome sequencing data of Boxer T-cell lymphomas that identified recurrent driver mutations in the PTEN-mTOR pathway. In addition, many of these drugs showed much higher IC50s in HTS and DRC assays on primary canine and feline fibroblasts, used as surrogates for normal tissue, revealing the specificity of their action on the cancer cells. These results advocate use of HTS and DRC assays as a means for identification of oncogenic driving mechanisms and for identification of drugs novel to veterinary medicine that warrant further investigation as alternative treatments for lymphoma in companion animals, which in turn can serve as valuable and accessible models for human lymphomas.

#2668

Regio- and stereospecific synthesis of oridonin D-ring aziridinated analogues for the treatment of triple-negative breast cancer via mediated irreversible covalent warheads.

Ye Ding,1 Dengfeng Li,2 Chunyong Ding,1 Zhiqing Liu,1 Eric A. Wold,1 Na Ye,1 Haiying Chen,1 Mark A. White,1 Qiang Shen,2 Jia Zhou1. 1 _UT Medical Branch, Galveston, TX;_ 2 _The University of Texas MD Anderson Cancer Center, TX_.

The covalent drugs dramatically resurge in recent years due to the comprehensive optimization of the structure-activity relationship (SAR) and structure-reactivity relationship (SRR). Natural product oridonin with an impressive pharmacological profile through its covalent enone warhead on the D-ring has attracted substantial SAR studies to appreciate its potential in the development of new molecular entities for the treatment of various human cancers and inflammation. Herein, for the first time we report the excessive reactivity of this covalent warhead and mediation of the covalent binding capability through a Rh2(esp)2-catalyzed mild and concise regio- and stereospecific aziridination approach. Intriguingly, new analogue YD0514 with a more drug-like irreversible covalent warhead has been identified to significantly induce apoptosis and inhibit colony formation against triple-negative breast cancer with comparable to enhanced antitumor effects in vitro and in vivo, while displaying lower toxicity to normal human mammary epithelial cells in comparison with oridonin.

#2669

Identification of a novel small-molecule inhibitor for treatment of human colon cancer patients.

Chun-Ho Park,1 Sun-Chul Hur,1 Joseph Kim,1 Dae Hee Lee,1 Yoon Sun Park,1 Jae-Sik Shin,1 Seung-Woo Hong,1 Jai-Hee Moon,2 Hyojin Kim,3 So Hee Lee,3 Hyebin Park,3 Joonyee Jung,2 Mi Jin Kim,2 Sang Soo Park,2 Jun Ki Hong,2 Ji Hee Gong,3 Jieun Kim,3 Il-Whea Ku,4 Hyun Ho Lee,3 Dong-Hoon Jin5. 1 _Wellmarkerbio Co., Ltd, Seoul, Republic of Korea;_ 2 _Ulsan College of Medicine and Asan Institute for Life Science, Asan Medical Center, Seoul, Republic of Korea;_ 3 _Wellmarkerbio Co.,Ltd, Seoul, Republic of Korea;_ 4 _Wellmarkerbio Co.,Ltd and Center for Drug Discovery Support, Asan Institute for Life Science, Asan Medical Center, Seoul, Republic of Korea;_ 5 _Wellmarkerbio Co.,Ltd, and Department of Convergence Medicine, Ulsan College of Medicine, Asan Medical Center,Asan Institute for Life Science, Asan Medical Center, Seoul, Republic of Korea_.

As the representative targeted anticancer drug for colon cancer patients, Erbitux is the EGFR targeted therapeutic antibody and used for treatment of KRAS wild-type colon cancer patients. Even some patients with KRas wt gene did not respond to Erbitux. However, there is no treatment available for Erbitux-resistant patient group with KRAS WT gene, which is almost 50% of KRAS WT gene holders. Recently, our team identified Erbitux primary resistant related proteins named as CRG (Cetuximab-Resistant Gene) by array analysis based Erbitux responder or nonresponder colon cancer patients derived tissues and confirmed by in vitro and in vivo assay system. Based on these results, we synthesized a novel series of CRG targeted inhibitor. CRG inhibitor (CRG i;WM compound) is a lead compound for treating colon cancer patients who do not respond to Erbitux and have KRAS wild-type gene. CRG i has potent in vitro enzyme activity and high anticancer activity against various colon cancer cell lines with good selectivity in in vitro and in vivo system. Furthermore, the compound has good potent ADME/Tox. profiles for optimized lead. CRG i also displays strong anticancer effect in in vivo xenograft models and patient-derived xenograft (PDX) models. In addition, CRG i shows promising signs of overcoming Erbitux resistance in CRG knockout cells-derived xenograft model. We continue to discover improved preclinical candidates with better selectivity and ADME/Tox. profiles and validate predictive biomarker in colon cancer patients. In these efforts, we found some compounds with better profiles than CRG i. We are also trying to develop small molecules having highly potent activity against mutant CRG. In conclusion, active CRG is a promising biomarker and target for Erbitux-resistant KRAS wt colon cancer patients. Our compounds can be promising therapeutic agents for Erbitux-resistant KRAS wt colon cancer patients.

#2670

Natural compound tetrocarcin-A downregulates junctional adhesion molecule-A in conjunction with erα, her2 and inhibitor of apoptosis proteins and inhibits tumor cell growth.

Sri Harikrishna Vellanki,1 Lance Hudson,1 Hanne Jahns,2 Rodrigo G.B. Cruz,1 Giovanni Sette,3 Adriana Eramo,3 Ann M. Hopkins1. 1 _RCSI, Dublin 9, Ireland;_ 2 _University College Dublin, Dublin 4, Ireland;_ 3 _Istituto Superiore di Sanità, Rome, Italy_.

Overexpression of the tight junction protein Junctional Adhesion Molecule-A (JAM-A) has been linked to aggressive disease in breast and several other cancers, but JAM-targeting drugs remain elusive. Screening of a natural compound library identified the cytotoxic antibiotic Tetrocarcin-A as a novel downregulator of JAM-A protein expression in breast cancer cells. Specifically, Tetrocarcin-A-induced cytotoxicity was accompanied by downregulation of JAM-A protein expression in conjunction with that of estrogen receptor alpha (ERα) and human epidermal growth factor receptor 2 (HER2) in MCF7-HER2-overexpressing breast cells. Lysosomal inhibition partially rescued the downregulation of JAM-A, HER2 and ERα caused by Tetrocarcin-A, and attenuated its cytotoxicity. Interestingly, Tetrocarcin-A repressed the transcriptional activity of c-FOS by inhibiting ERK1/2-mediated phosphorylation at Threonine-232, and downregulated protein expression of the inhibitor of apoptosis proteins (IAP). Investigation of cell death pathways revealed that Tetrocarcin-A induced caspase-dependent apoptosis. To begin testing the potential clinical relevance of our findings, we extended our studies into various other models. Encouragingly, Tetrocarcin-A downregulated JAM-A expression and caused cytotoxicity in primary breast cancer and lung cancer stem cells, and inhibited the growth of model human breast tumors in an in vivo model involving invasion across the chicken egg chorioallantoic membrane. Taken together, our data suggest that Tetrocarcin-A merits future evaluation as a novel cancer therapeutic by virtue of its ability to downregulate JAM-A expression, induce apoptosis and reduce tumorigenic signalling. This work was financially supported by Science Foundation Ireland (grant 13/IA/1994 to AMH). Tetrocarcin-A was provided by the NCI/DTP Open Chemical Repository https://dtp.cancer.gov. (NSC 333856)

#2671

17beta-hydroxywithanolides inhibit the proliferation of castration-resistant prostate cancer cells by reducing levels of cFLIP.

Alan D. Brooks,1 Nicola E. Wright,2 Ya-ming Xu,3 Kithsiri Wijeratne,3 Poonam Tewary,1 Neil Cross,2 A. A. Leslie Gunatilaka,3 Thomas J. Sayers1. 1 _Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc., Frederick, MD;_ 2 _Sheffield Hallam University, Sheffield, United Kingdom;_ 3 _College of Agriculture and Life Sciences, The University of Arizona, Tucson, AZ_.

Using a high throughput gene expression profiling assay directly targeting genes of the androgen receptor pathway, we previously identified the 17beta-hydroxywithanolide (17-BHW), physachenolide C (PCC), as a potent inhibitor of prostate cancer cell growth. Using a panel of prostate cancer cell lines, PCC at low concentrations (IC50 20-50 nM) reduced prostate cancer cell numbers under both standard and 3D in vitro growth conditions. This reduction in cell number was due to growth inhibition and not apoptosis, with PCC causing reductions in the levels of cyclin D, an increase in p21 and arrest of cells in the G1 phase of the cell cycle. Subsequently over 150 natural and semi-synthetic withanolides were evaluated for their ability to inhibit prostate cancer cell growth. All highly active withanolides were 17-BHWs, and some were up to 4-fold more active than PCC. Preliminary structure-activity relationship (SAR) studies suggested that the enone moiety in ring A was essential for activity. In addition, acetoxylation at C-18, an alpha orientation of the side-chain lactone group and the double bond at C-24(25) of the lactone ring played important roles in determining the activity of 17-BHWs as inhibitors of prostate cancer cell growth. Interestingly highly active 17-BHWs rapidly reduced levels of both antiapoptotic cFLIPL and cFLIPS proteins in 22Rv1 prostate cancer cells. However, this reduction did not sensitize 22Rv1 cells to apoptosis. Nonetheless specific reduction of cFLIPL in the castration-resistant 22Rv1 cells using siRNA significantly inhibited their proliferation. This suggests that cFLIPL plays a critical role in the proliferation of some castration-resistant prostate cancer cells. Since the 17-BHW scaffold is amenable to optimization by a medicinal chemistry approach, this could lead to the identification of highly active natural product-based inhibitors of castration-resistant prostate cancer cell proliferation. The cellular molecular target(s) of active 17-BHWs that promote cFLIP reduction are currently under further investigation.

Funded by FNLCR Contract HHSN261200800001E and the University of Arizona.

#2672

Phyllanthusmins induce apoptosis and reduce tumor burden in high grade serous ovarian cancer by late-stage autophagy inhibition.

Alexandria N. Young,1 Denisse Herrera,1 Andrew Huntsman,2 Daniel D. Lantvit,1 Melissa A. Korkmaz,1 Leslie N. Aldrich,1 A. Douglas Kinghorn,2 James R. Fuchs,2 Joanna E. Burdette1. 1 _University of Illinois at Chicago, Chicago, IL;_ 2 _Ohio State Univeristy, Columbus, OH_.

High grade serous ovarian cancer (HGSOC) is a lethal gynecological malignancy with a need for new therapeutics. Many of the most widely used chemotherapeutic drugs are derived from natural products or their semi-synthetic derivatives. We developed potent synthetic analogues of a class of compounds known as the phyllanthusmins, inspired by natural products isolated from Phyllanthus poilanei Beille. The most potent analogue, PHY34, had the highest potency in HGSOC cell lines in vitro and displayed cytotoxic activity through activation of apoptosis. PHY34 exerts its effects by initially inhibiting autophagy at a late stage in the pathway, involving the disruption of lysosomal function. The autophagy activator, rapamycin, combined with PHY34 eliminated apoptosis, suggesting that autophagy inhibition was required for apoptosis. PHY34 was readily bioavailable through intraperitoneal administration in vivo where it significantly reduced cancer cell lines grown in hollow fibers as well as ovarian tumor burden. We show that PHY34 is a new late-stage autophagy inhibitor with nanomolar potency and significant antitumor efficacy as a single-agent against HGSOC in vivo. This class of compounds holds promise as a potential, novel chemotherapeutic and demonstrates the effectiveness of targeting the autophagic pathway as a viable strategy for combating the disease.

#2673

Securidaca1 and 2 induced apoptosis: A PI3K-AKT dependent pathway in cervical cancer.

Titus C. Obasi, Cornelia Braicu, Ioana Berindan-Neagoe, Radu N. Oprean. _University of Medicine and Pharmacy, Cluj-Napoca, Romania_.

Background: Securidaca1 and 2 are plant-derived substances isolated from African medicinal plant S. longipedunculata, using new bioactivity-guided method partly described in Obasi et al. (2017). Our previous report suggested that certain fractions from this plant root were able to activate apoptosis in cervical cancer cell lines. Generally, over 90% of cervical cancer has been associated with high-risk human papilloma virus (HPV), whose etiologic molecule E6 oncoprotein is involved in the process of carcinogenesis. The purpose of this study, therefore, is to investigate the proapoptotic potentials of these substances and their ability to block cell survival mechanism, via antiapoptotic effectors of PI3K-AKT downstream.

Experimental Procedure: Two cervical cancer cell lines (Caski and BU25TK), positive with HPV were used for the investigation. Cells were treated with the plant-derived substances and the IC50 was determined by MTT assay. Fluorescence microscopy (Annexin V-FITC, MPC and PI staining) was used to investigate autophagy/apoptosis, while relevant genes were analyzed by RT-qPCR.

Results: Reduced cell proliferation was earlier achieved in dose- and time-dependent manner by fractions retaining Securidaca1 and 2, with IC50 of 7.03 and 16.39 (μg/mL) respectively on Caski cells. Late apoptosis was also activated on cells treated with securidaca1 fraction, while the securidaca2 in contrast indicated early apoptosis. Analysis by RT-qPCR revealed a fold-change expression of antiapoptotic proteins, MCL-1 and BCL2L1, more pronounced in securidaca1. Interestingly, the AKT-3 was significantly inhibited, correlating with low expression of MCL-1, BCL2L1, VEGFA and CDH-1. Data analysis suggests the most likely activation of apoptosis via PTEN regulated PI3K-AKT pathway and respondent downstream (mTOR/ NF-kB).

Conclusion: The ability of Securidaca1 and 2 to kill tumor cells and induce apoptosis in manner involving multiple regulatory pathways enhances their potential against possible resistance and makes them attractive as candidates for anticancer development.

#2674

Novel tubulin polymerization inhibitors repress tumor xenografts in nude mice and leukemia in zebrafish.

Yanqi Xie,1 Liliia Kril,2 Wen Zhang,1 Mykhaylo Frasinyuk,3 Svitlana Bondarenko,3 Elizabeth Hausman,2 Zachary Martin,2 Przemyslaw Wyrebek,2 Tianxin Yu,1 Changguo Zhan,2 Vitaliy Sviripa,1 Jessica Blackburn,2 David Watt,2 Chunming Liu1. 1 _Univ. of Kentucky Markey Cancer Ctr., Lexington, KY;_ 2 _Univ. of Kentucky, Lexington, KY;_ 3 _National Academy of Science of Ukraine, Ukraine_.

In an effort to screen natural compounds and their derivatives as novel agents for cancer treatment, we developed two semisynthetic nature compounds, A14 and A52, which have potencies in an in vitro cell proliferation studies against LS174T and PC-3 cells in the mid-nanomolar range. A14 inhibited more than 90% of in vitro cancer cell proliferation around 300 nM and also inhibited in vivo PC-3 prostate cancer xenografts in nude mice. Cell morphology studies and comparisons with known antineoplastic agents suggested that A14 disrupted microtubule organization. In vitro tubulin polymerization assay suggested that A14 inhibited this process. Based on molecular docking, A14 fits very well in the colchicine-binding site at the interface between the alpha-tubulin and beta-tubulin. Further analyses suggest that A14 sensitivity may correlate with CDK/cyclin activity. A14 inhibited PC-3 prostate cancer xenografts in nude mice. In addition to solid tumors, A14 and its analog A52 strongly inhibited a panel of human leukemia cancer cell lines. Furthermore, these compounds inhibited Myc-induced T cell acute lymphoblastic leukemia (T-ALL) in a zebrafish model. Although many tubulin inhibitors were available, there is no compound that targeting the colchicine-binding site has been approved by FDA for cancer treatment. This new family of tubulin polymerization inhibitors could be further developed for the treatment of both solid and liquid tumors.

#2675

Quinacrine-mediated activation of JNK1 inhibits Nrf2 and causes cell death in human colon cancer.

Eun-Taex Oh, Chan-Woo Kim, Ha Gyeong Kim, In-Seon Lee, Chae Won Lee, Heon Joo Park. _Inha University College of Medicine, Incheon, Republic of Korea_.

Up-regulation of Nrf2 is associated with poor prognosis in skin, breast, prostate, lung, head and neck, endometrial, and colorectal cancer, and increases chemoresistance and radioresistance by inducing antioxidant and detoxification genes. Nrf2 inhibitors are not currently available for clinical use. Here, we investigated the feasibility of using quinacrine, an old antimalarial drug, as a novel Nrf2 inhibitor for treating colorectal cancer. Immunohistochemical analysis of colorectal cancer samples from 60 patients was used to assess the correlation between Nrf2 expression levels and clinicopathological features. Using data from tumors in the Cancer Genome Atlas (n=290 normal, n=295 cancer) and publicly available data sets, we validated the correlation between Nrf2 expression and poor prognosis in colorectal cancer. The effect of quinacrine on the activation of JNK1-mediated Nrf2 degradation was analyzed with co-immunoprecipitation, immunoblotting, and luciferase reporter assays. To confirm that quinacrine-mediated Nrf2 degradation caused cancer cell death, we used Nrf2 gain-of-function and loss-of-function experiments. The cytotoxicity of quinacrine against colorectal cancer cells under both normoxia (20% O2) and hypoxia (0.5% O2) was assessed by clonogenic survival assays. Finally, a xenograft mouse model was used to confirm the results of our in vitro experiments in an in vivo setting. High Nrf2 levels were correlated with increased expression of its target genes and poor survival among colorectal cancer patients in immunohistochemical analysis and analysis for publicly available data sets. The quinacrine-mediated activation of JNK1 induced Nrf2 degradation by increasing the interaction between Keap1 and Nrf2, thereby causing cancer cell death under normoxia and hypoxia and suppressing tumor growth in our in vivo model. Overexpression of Nrf2 effectively prevented quinacrine-mediated Nrf2 degradation and cancer cell death regardless of oxygen tension. Additionally, siRNA-mediated JNK1 knockdown inhibited quinacrine-mediated Nrf2 degradation. Luciferase reporter assays and qPCR revealed that quinacrine inhibited Nrf2-mediated transcription in cancer cells under normoxia and hypoxia. Finally, the quinacrine-mediated degradation of Nrf2 was found to increase the sensitivity of colorectal cancer in in vivo model and cancer cells under both normoxia and hypoxia in vitro to ROS-inducing drugs and anticancer drugs commonly used against colorectal cancer. Quinacrine caused colorectal cancer cell death by promoting activation of JNK1-mediated Nrf2 degradation.

#2676

Radiotherapy-assisted tumor selective metronomic oral chemotherapy.

Youngseok Cho, Seung Woo Chung, Youngro Byun. _Seoul National University, Seoul, Republic of Korea_.

Chemotherapy have commonly been used in maximum tolerated dose (MTD) to completely eradicate the cancer. However, such treatments often failed due to the complex and dynamic nature of cancer. Therefore, it has been suggested that cancer should be treated as a chronic disease, controlling its growth by providing continuous therapeutic pressure for long-term. Such an approach, however, requires a therapy that is non-toxic and orally available with sufficient potency. Herein, we propose a radiotherapy-assisted orally available metronomic apoptosis-targeted chemotherapy, which delivers doxorubicin continuously to the irradiated tumor with high selectivity while causing minimal toxicities to the normal tissues. DEVD-S- DOX/DCK complex is the anticancer prodrug for our strategy that could selectively release doxorubicin in the irradiated tumor tissue with sufficient oral bioavailability. The prodrug was completely inactive by itself, but displayed potent anticancer activity when coupled with radiotherapy. Consequently, the daily oral administration of DEVD-S-DOX/DCK in combination with the low-dose radiotherapy effectively suppressed the growth of tumor in vivo with no significant systemic toxicities despite that the accumulated dose of doxorubicin exceeded 150 mg/kg. Therefore, our novel therapy using DEVD-S-DOX/DCK complex is considered as an outstanding treatment option for treating cancer for long-term attributed to its oral availability and low-toxicity profile as well as the potent anticancer effect.

#2677

A natural product-derived Compound A effectively targets both cancer stem and non-stem cells in NSCLC.

Seung Yeob Hyun, Huong T. Le, Jaebeom Cho, Hye-Young Min, Ho-Young Lee. _Seoul National Univ., Seoul, Republic of Korea_.

Cancer stem-like cells (CSCs) contribute to tumor recurrence and chemoresistance. Hence, strategies eradicating CSCs are crucial for effective anticancer therapies. Here, we demonstrate transcriptional upregulation of the heat shock proteins (Hsp90 and Hsp70) and their co-chaperones and enhanced activities of the Hsp system in both non-CSCs and CSCs in non-small cell lung cancer (NSCLC) cells. Genetic and pharmacologic strategies targeting Hsps eliminated both CSCs and non-CSCs of NSCLC, suggesting the functional role of the Hsp system in the two populations in NSCLC. We further identified Compound A, a natural product-derived compound, as a novel Hsp70 inhibitor eliminating both the CSC and non-CSC populations in NSCLC. Compound A inhibited the sphere-forming ability of NSCLC CSCs by inducing apoptosis. Compound A also suppressed the viability and colony-forming abilities of NSCLC cells (non-CSCs) and their sublines carrying acquired chemoresistance by inducing apoptosis with minimal toxicity in normal cells derived from various organs. Mechanistically, Compound A disrupted Hsp70 function by binding to the ATP-binding pockets of Hsp70. These data suggest the rationale to target the Hsp system for controlling both CSCs and non-CSCs in NSCLC and the potential of Compound A as a natural Hsp70 inhibitor to eliminate the two populations with limited toxicities.

#2678

Antitumor effect against 67 kDa laminin receptor expressed on high-grade tumor cells.

Ikumi Sugiyama, Yasuyuki Sadzuka. _Iwate Medical University, Iwate, Japan_.

[Purpose] In chemotherapy for cancer, liposomes as drug delivery system (DDS) carriers are expected to have a useful effect. The aim of this study was, therefore to evaluate the antitumor effects of, and elucidate the mechanisms underlying, (-)-epigallocatechin-3-O-gallate (EGCG) and polyethyleneglycol (PEG)-modified liposomes. EGCG functions as a target ligand of the 67 kDa laminin receptor (67LR), which is expressed on high-grade tumor cells. An EGCG derivative was synthesized for binding to the end site of PEG structure. In this study, the antitumor effects of EGCG-modified liposomes as active-targeting liposome were evaluated.

[Methods] Liposome was loaded with doxorubicin (DOX) as an antitumor agent. C57BL/6 mice were subcutaneously inoculated with B16F10 mouse melanoma cells (5x105 cells/animal). Each sample was administered intravenously with 2.5 mg/kg DOX. EGCG solution (EGCG sol.) was also administered intravenously at 11.3 mg/kg, based on the amount of EGCG modification in DOX-loaded EGCG-PEG-modified liposomes (EPL). Caspase-3 was evaluated as 7-amino-4-trifluoromethyl coumarin (Ex: 400 nm, Em: 505 nm), following a reaction with the fluorescent substrate DEVD-AFC. Caspase-8 was determined reacted with DEVD-pNA and detected as chromophore p-nitroanilin (λ: 400nm).

[Results and Discussion] EPL significantly decreased tumor size against B16F10 mouse melanoma cells, in mice bearing 67LR-high-expression tumors. The tumor weight of the EPL group was significantly lower than that of the control (p<0.01). Typical passive-targeting liposomes (PL) accumulated into the tumors due to improved blood circulation and an enhanced permeability and retention (EPR) effect, and decreased tumor weight. On the other hand, EPL disappeared from the blood immediately, compared with PL or PL + EGCG sol. DOX concentration of liver and spleen in EPL group did not increase, indicating that the rapid disappearance of EPL from the blood was not due to reticuloendothelial system (RES) trapping. Caspase-3 activity, which indicates apoptosis induction, was also elevated only in the EPL group, and the caspase-3 activity of the PL + EGCG sol. group was the same as that of the PL group. Hence, it was important that EGCG was included to modify the liposomes via PEG, as soluble EGCG did not accumulate at a high enough concentration to exert an apoptotic effect. Moreover, EPL significantly increased caspase-8 activity. It was suggested that EPL-induced apoptosis occurred due to caspase-8 activity induced following the binding of EGCG to 67LR as a cell-death ligand. In conclusion, it was suggested that EPL was a superior antitumor agent in high 67LR-expressing tumor cells as the liposomes had dual effects, namely antitumor effects due to the loaded DOX and apoptosis induced by the bound EGCG.

#2679

Cyanobacterial trypsin-3 inhibitor inhibits prostate cancer cell invasion.

Hannu Koistinen,1 Matti Wahlsten,1 Muhammad N. Ahmed,1 Kaarina Sivonen,1 Matthias Nees,2 Ulf-Håkan Stenman,1 David P. Fewer1. 1 _University of Helsinki, Helsinki, Finland;_ 2 _University of Turku, Turku, Finland_.

Trypsin-3 is a highly active protease that has recently been identified as a potential therapeutic target for the reduction of tumor growth and metastasis in prostate, breast and pancreatic cancers. Current trypsin inhibitors typically inhibit a broad range of different trypsin-like enzymes. Thus, novel approaches to identify trypsin-3 selective inhibitors are needed. Cyanobacteria produce a vast diversity of natural products with complex chemical structures many of which are potent protease inhibitors.

We screened extracts of 140 cyanobacteria strains and discovered numerous strains showing selectivity towards trypsin-2 and -3 inhibition. We isolated a complex glycopeptide from one of these strains, which selectively inhibited human trypsin-2 and -3 with IC50 values of about 100 nM, while trypsin-1 was not inhibited. Importantly, we also found that this peptide inhibited invasion of aggressive and metastatic PC-3M prostate cancer cells though ECM preparation, while it did not affect the proliferation of the cells.

Our results suggest that microbial natural products may offer a viable alternative source of potent and selective trypsin-3 inhibitors. Such inhibitors may be suitable, after further development, for targeting the mechanisms associated with the invasion and metastatic dissemination of cancer cells, i.e., for treatment of aggressive cancers.

#2680

**Antitumor promoting activity of the phytotherapic** Polypodium leucotomos **in oral cancer.**

Pammela A. Borges,1 Gabriela S. Teixeira,1 Nathalia N. Pantarotto,1 Luan Oenning,1 Liliane O. Marinho,1 Lucilene Lopes,1 Eduardo J. Caldeira,1 Pablo A. Salvador,2 Ricardo D. Colleta,3 Clóvis A. Pinto,1 Gustavo Simões Augusto,4 Taize M. Augusto,1 Carine E. Oliveira,5 Nilva K. Cervigne1. 1 _Faculty of Medicine of Jundiai (FMJ), Jundiai, Brazil;_ 2 _Universidade de Sao Paulo (USP), Sao Paulo, Brazil;_ 3 _Piracicaba Dental School (FOP-Unicamp), Piracicaba, Brazil;_ 4 _Campinas State University (UNICAMP), Campinas, SP, Brazil;_ 5 _Universidade Federal de Alfenas (UNIFAL), Alfenas, Brazil_.

The oral squamous cell carcinoma (OSCC) is the eighth most prevalent malignant neoplasm and representing an important health problem worldwide. OSCCs have a highly variable clinical course, and due to its late diagnostic in advanced stages, the overall survival rate is less than 50% in 5 years. Despite the advances and last recent discoveries, the prognosis of this disease is still unpredictable. The search for new therapeutic strategies of OSCC is still needed to improve patient survival. From this point of view, oscc treatment and adjuvant methods represent a promising and challenging field for researchers. In the last few years, the use of some types of complementary medicines, such as phytotherapeutic products, has significantly increased among cancer patients. Recently, a phytotherapeutic compound, Polypodium Leucotomos (PL), was particularly introduced on the market for the treatment of inflammatory changes and other skin diseases caused by UV radiation, due to its anti-inflammatory, anti-oxidant and immunomodulatory properties. It is possible that PL also demonstrates antitumor action, and therefore seems a plausible target for further investigation regarding its therapeutic potential on oral cancer. The present study has characterized the biological effects of the extract of PL on proliferation, migration and invasion of oral cancer cell lines; and investigated the antitumor potential of PL in vivo, by treating oral tumors chemically induced on the tongue of Balb/C mice. Our results showed that oral cancer cells proliferations measured by MTT assay has decreased up to 60% and 78% for a period of 48hrs PL treatment for the SSC9 and LN1 cells, respectively. The scratch assay did not demonstrate a significant reduction on the migration potential these cells. As for the invasion assay, we verified that both lineages, but mainly LN1, a more aggressive oscc cell line, diminish its breach capacity, after previous incubation with PL (p<0.01). We further investigated whether PL extract would have any role in protecting the cellular DNA of the damages caused by UV radiation, often associated with cancer. The comet assay data showed that normal primary gingival fibroblasts previously treated with PL, and then submitted to UVR, had a reduction on DNA damage in about 40%, compared to the fibroblasts non treated (p<0.05). Our preliminary in vivo assays suggest that PL extract has an antitumor activity. Majority of animals with 4NQO-induced tongue tumor recovered weight after 6 weeks treatment, with significant reduction of tumor size and decreased Ki67 positivity in the injured oral tissue, compared the mice not treated with PL extract. Altogether, our results demonstrated, for the first time, the potential of the extract Polypodium Leucotomos as an effective phytotherapeutic agent for prevention and/or adjuvant treatment of oral cancer.

#2681

Antiproliferative effects of the naturally occurring solamargine and related glycoalkaloids against liver cancer cells.

Mostafa I. Fekry,1 Shahira M. Ezzat,1 Maha M. Salama,1 Ahmed M. Al-Abd2. 1 _Cairo University, Cairo, Egypt;_ 2 _King Abdulaziz University, Jeddah, Saudi Arabia_.

Hepatocellular carcinoma (HCC) is progressively increasing tumor with lack of accurate prognosis and inadequate systemic treatment approaches. Solanum sp. (such as Solanum melongena) is a folk herb which is reported to possess anticancer properties. In a continuity for our interest in pursuing the anticancer activity of compounds isolated from the fruit peels of Solanum melongena, the HPLC profiling and ESI-MS assessment for the methanolic extract evidenced the presence of bioactive glycoalkaloids (solasonine, solasodine and solamargine). These glycoalkaloids were isolated, purified and proved to possess in vitro cytotoxicity against human liver cancer cell lines (Huh7 and HepG2). Herein, we investigated the potential mechanism of action of these compounds using DNA content flow-cytometry and apoptosis/necrosis differential anaylsis using annexin-V/FITC staining. Solasonine, solasodine and solamargine inducd significant antiproliferative effect against liver cancer cells (Huh7 and HepG2) which was attributed to cell cycle arrest at S-phase. Solamargine, solasodine and solasonine induced significant apoptosis in Huh7 cells. Only solamargine-induced cell cycle arrest, was reflected as apoptotic cell killing effect against HepG2 cells. In conclusion, glycoalkaloids derived from Solanum melongena and particularly, solamargine are promising antiproliferative agents with potential anticancer effects.

#2682

Global transcriptomic profiling reveals anticancer role of oligomeric proanthocyanidins from grape seeds in colorectal cancer.

Preethi Ravindranathan, Divya Pasham, Uthra Balaji, Shusuke Toden, Ajay Goel. _Baylor Scott & White, Dallas, TX_.

Background: Grape seed extract contains an abundance of oligomeric proanthocyanidins (OPCs) (monomers, dimers and trimers) and a small fraction of larger polymers. Although the anti-cancer properties of OPCs from grape seeds have been well recognized, the molecular mechanisms by which they exert anti-cancer effects are poorly understood.

Purpose: Herein, we cognize the underlying molecular mechanisms of the anti-cancer properties of OPCs by identifying their downstream transcriptional targets.

Methods: First, we assessed the anti-tumorigenic properties of OPCs from grape seeds in colorectal cancer (CRC) cells by proliferation assay, cell cycle analysis and cell migration assay. We then examined the global changes in gene expression induced by OPCs by RNA-sequencing based gene-expression profiling in a panel of CRC cell lines, including HCT116, SW480, SW620, RKO and HT29. We thereafter validated the RNA-sequencing results in HCT116 and SW480 cells, in a xenograft animal model, and in patient-derived organoid models.

Results: We observed that OPCs from grape seeds inhibited cell proliferation, induced cell cycle arrest and inhibited cancer cell migration in CRC cell lines. Additionally, OPCs downregulated EMT-inducing genes ZEB1, ZEB2 and SNAI1, and upregulated epithelial marker E-cadherin. The RNA-sequencing identified differentially expressed genes following OPC treatment, and the KEGG pathway analysis revealed that genes involved in the cell cycle (p=3.63e-09) and DNA replication (p=1.49e-09) were the most significantly modulated pathways across all cell lines. A closer look at these pathways revealed the downregulation of well-characterized oncogenes, including TGFβ3, E2F1 and CCNE2, and the upregulation of tumor suppressor genes SFN, CDKN1A and MAD1L1. We subsequently validated the modulation of these genes independently in cell lines, mice xenografts and patient-derived tumor organoids. Intriguingly, our in vivo experiments revealed that OPCs more potently decreased xenograft tumor growth vs. the unfractionated grape seed extract (GSE) that includes larger polymers of proanthocyanidins, and inhibited organoid formation from patient-derived tumors more robustly than GSE.

Conclusions: We, for the first time, illuminated the genome-wide effects of OPCs from grape seeds in CRC. First, we showed that OPCs modulated multiple cancer-associated cellular pathways, especially the cell cycle and DNA replication. In addition to mice models, we validated the anti-cancer effects of OPCs in a pre-clinical patient-derived tumor organoid models. Secondly, we showed that OPCs have higher efficacy in tumor inhibition in mice xenografts than GSE, possibly due to better absorbability. Consequently, as OPCs block various oncogenic pathways, it could prevent the emergence of acquired resistance in cancer patients undergoing targeted therapies.

#2683

The olive-based oleocanthal as a dual HER2-MET inhibitor for the control of breast cancer recurrence.

Abu Bakar Siddique, Hassan Ebrahim, Mohamed Mohyeldin, Seetharama D. Jois, Khalid A. El Sayed. _Univ. of Louisiana College of Pharmacy, Monroe, LA_.

Dysregulation of the receptor tyrosine kinases (RTKs) HER2 and c-Met correlate well with poor breast cancer (BC) prognosis and invasive aggressive profile. c-Met amplification proved to be the HER2-dependent tumors' inevitable escape mechanism from the anticancer effects of targeted therapies including trastuzumab, cetuximab, and small-molecule RTK inhibitors like lapatinib, gefitinib, and erlotinib. Dual HER2-Met inhibition is highly expected to be effective and less likely to develop resistance. This study reports the novel dual HER2-Met inhibitory molecular mechanism for the extra virgin olive oil-derived phenolic secoiridoid (-)-oleocanthal (OC). OC showed typical type-I binding mode at c-Met ATP kinase domain. OC aldehydes, ester, and phenol groups showed critical interactions with the critical activation loop ASP1222/TYR1230 and the hinge region PRO1158/MET1160. In addition, OC overlapped two out of the three lapatinib critical interactions at the HER2 kinase domain, the hinge region MET801 and PHE864; besides, OC uniquely targeted the hinge region's THR862 and SER783, justifying unique binding mode. OC showed low-µM level inhibitory activities against both c-Met and HER2 kinases in cell-free Z-LYTE assays. In vitro, OC induced dose- and time-dependent inhibition of the proliferation and migrations of several BC cells including BT-474, SK-BR-3, MDA-MB-231, MCF-7, MDA-MB-468 at low μM IC50 dose range. This effect was associated with selective blockade of HER2 and Met receptors activation in response to their natural ligands HGF and EGF, respectively, confirmed by Western blotting, flow cytometry and immunocytochemistry studies. OC potently induced autophagy via the upregulation of LCA/B, Atg-3, Atg-7, Atg-16L within 6-12 h of SKBR-3 cells treatment. OC had no effect on the viability of the nontumorigenic human MCF-12A mammary epithelial and neuronal Schwann RSC 96 cells. In vivo, 5-10 mg/kg oral or ip dose range of OC potently inhibited 65-90% of the growth of the HER2-dependent BT-474 and the c-Met-dependent MDA-MB-231 BC cells in female athymic nude mice xenograft models. Orally, 10 mg/kg dose of OC prevented over 90% of local and regional recurrences of both tumor phenotypes after primary tumor surgical excision in orthotopic xenograft mice models. This was further confirmed by significant reductions of Ki-67, CD31, pHER2 and p-c-Met levels in treated animal tumors by IHC studies. OC is a novel dual c-Met-HER2 inhibitory dietary supplement lead with excellent potential for translational use to prevent and control breast malignancies with aberrant c-Met or HER2 activities.

Supported by NCI Award R15CA167475 and Louisiana Board of Regents ITRS Award LEQSF (2017-20)-RD-B-07.

#2684

Drug sensitivity profiling of BT-474 breast cancer cell line for identification of novel therapies targeting HER2-positive breast cancer.

Jenni H. Mäki-Jouppila,1 Tiina E. Kähkönen,1 Mari I. Suominen,1 Jussi M. Halleen,1 Jenni Bernoulli,1 Jani Saarela,2 Katja Fagerlund1. 1 _Pharmatest Services, Turku, Finland;_ 2 _Institute for Molecular Medicine Finland FIMM, University of Helsinki, Helsinki, Finland_.

Developing targeted therapy for breast cancer expressing hormone receptors such as estrogen receptor (ER) and progesterone receptor (PR) as well as human epidermal growth factor receptor 2 (HER2) is essential. However, breast cancer has high susceptibility to spread despite the targeted therapies and metastases often turn resistant to the used treatment. Therefore, new therapies are needed especially at the stage when cancer becomes resistant to hormonal therapy. The aim of the study was to investigate drug sensitivity of BT-474 breast cancer cell line and to recognize possible vulnerabilities especially in HER2+ breast cancer.

BT-474 cell line (ER+, PR+, HER2+) is originally derived from human solid, invasive ductal carcinoma of the breast. Drug sensitivity of the cell line was assessed by screening of a large compound library consisting of many clinically available and emerging anti-cancer drugs including e.g. conventional chemotherapy, metabolic modifiers, kinesin inhibitors, apoptotic modulators, immunomodulators and kinase inhibitors such as HER2, pan-HER2 and EGFR-HER2 inhibitors. A panel of 525 compounds was tested in five concentrations covering a 10.000-fold drug-relevant concentration range in 384-well format. Cells were seeded to pre-drugged plates, followed by cell viability measurements (CellTiter-Glo) after 72 hours. Maximal and minimal responses to drugs were analyzed, the EC50 values were calculated and Drug Sensitivity Score (DSS) was calculated for each drug as a measure of reduced viability. A selective DSS (sDSS) was calculated to identify the selective drug response pattern.

DSS analysis of BT-474 cell line showed sensitivity to conventional chemotherapy including paclitaxel and docetaxel and to kinase inhibitors such as EGFR and HER2 inhibitors neratinib, afatinib, mubritinib, poziotinib, dacomitinib and lapatinib with nanomolar EC50 values. Moreover, BT-474 cells showed sensitivity to PI3K and mTOR inhibitors, such as copanlisib and omipalisib, and to HSP90 and HDAC inhibitors, such as tanespimycin and abexinostat.

Screening of large compound libraries combined with DSS and sDSS analysis enables drug sensitivity profiling of BT-474 breast cancer cells for discovery of novel therapies against HER+ breast cancer. Furthermore, drug sensitivity profiling enables repurposing of existing drugs to new indications and identification of vulnerabilities in different types of cancer cells.

#2685

Targeting TGFβ signaling pathways in bone metastatic breast cancer cells to limit metastatic progression using curcuminoids, a turmeric-derived natural product.

Andrew Kunihiro, Julia A. Brickey, Jen B. Frye, Janet L. Funk. _University of Arizona, Tucson, AZ_.

Tumor-cell TGFβ signaling has been demonstrated to drive osteolytic breast cancer bone metastases (BrCa BMET) in mouse models. This established an important link between the microenvironment and tumor cells in bone, wherein osteoclast-mediated bone resorption stimulates the release of TGFβ from bone matrix, which induces tumoral secretion of osteolytic factors, such as parathyroid-related protein (PTHrP) that drive further osteoclast-mediated bone destruction in a positive feedback loop. These findings have prompted interest in the use of pharmacologic TGFβ inhibitors, currently under development, for the treatment of BrCa BMET. Interestingly, studies have demonstrated antagonistic effects of a natural product, turmeric-derived polyphenols called curcuminoids (CURC), on BrCa BMET progression and TGFβ-inducible, Smad-dependent signaling in a TGFβ-dependent human xenograft model (human MDA-MB-231 [MDA-SA]).The studies described here were undertaken to determine whether this inhibitory effect is generalizable to other BrCa cells that form TGFβ-dependent BMETs (human MDA-1833 and MDA-2287; murine 4T1) and to begin to elucidate the mechanism by which CURC block BrCa cell TGFβ signaling. Analogous to prior findings using MDA-SA cells forming BMET that are dependent on TGFβ-inducible tumoral secretion of PTHrP via Smad and non-Smad (specifically, p38) dependent pathways, CURC inhibited TGFβ inducible activation (phosphorylation) of receptor Smad2 (Western) in all TGFβ-dependent BrCa cell lines tested.In MDA-SA cells, CURC inhibition of TGFβ-stimulated Smad2 phosphorylation was time- and dose-dependent (Western), and associated with decreased expression of a Smad-dependent luciferase reporter gene in transiently transfected cells. In both MDA-SA and 4T1 cells, these changes were accompanied by decreased TGFβ-stimulated PTHrP secretion (RIA). These effects in MDA-SA cells were not associated with changes in the expression of canonical TGFβ signaling proteins, such as Smad anchor for receptor activation (SARA), which is involved in Smad phosphorylation, or Smad4, a required cofactor for Smad-mediated gene transcription. Also unchanged were noncanonical TGFβ-stimulated, Smad-independent signaling pathways (e.g., p38, ERK1/2 or JNK). Of note, however, in all cell lines tested, CURC inhibition of Smad2 phosphorylation was accompanied by a decrease in constitutive Smad2 levels, with variable decreases in the ratio of phosphorylated vs. total Smad levels. These results suggest that CURC can specifically and uniformly inhibit Smad-regulated TGFβ signaling in bone metastatic BCa cells forming TGFβ-dependent osteolytic lesions, an effect that may be mediated, at least in part, by changes in constitutive levels of receptor-regulated Smad expression.

#2686

Polyphenolic triptorelin and leuprorelin derivatives as anticancer prodrugs.

Yousef Beni, Samiyah Alhamed, Jawzah Alnakhli, Kaleh Karim, William Boadi. _Tennessee State University, Nashville, TN_.

Triptorelin (TRP) and Leuprorelin (LEP) are two synthetic analogue of gonadotropin-releasing hormone (GnRH), first was approved by the FDA for treatment of advanced prostate cancer and endometriosis in mid 80s. The surge of testosterone, known as a flare effect, is the major side-effect of cancer chemotherapy using TRP and LEP in a combination regiment. Therefore, improving of the biological activity of TRP and LEP by increasing the cellular uptake and retention is a remedy to this end.In this research, polyphenolic derivatives were linked to TRP and LEP through hydrophobic linkers to enhance and optimize the hydrophobicity of these drugs with the expectation to improve the cellular uptake. In this regard, several TRP and LEP conjugates of tris(4-methoxyphenyl)methanol (TPM) derivatives with optimized hydrophobicity were synthesized by the reaction of methoxy benzenes (e.g. methyl 2-methoxybenzoate, 1,2-dimethoxybenzene, methoxy-2-methylbenzene, methoxy-2-nitrobenzene, chloro-2-methxyenzene, anisole, 2-fluoroanisole or 2-methylanisole and 1,3,5-trioxane, followed by the conjugation with TRP or LEP and decanedioic acid or dodecanedioic acid in the presence of HBTU/DIPEA/DIC in moderate yields.Comparative antiproliferative assays between TPM-TRP conjugates and the corresponding non-covalent physical mixtures of the TPM derivatives and TRP were performed against human acute lymphoblastic leukemia (CCRF-CEM), human ovarian adenocarcinoma (SK-OV-3), and mouse pre adipocytes (3T3-L1) cells. TPM-TRP conjugates inhibited the cell proliferation of CCRF-CEM, SK-OV-3 and 3T3-L1 cells by 55-92%, 24-73%, 37-56%, respectively, at a concentration of 10-50 µM after 24-72 h of incubation. Similar antiproliferative assays between TPM-LEP conjugates and the corresponding non-covalent physical mixtures of the TPM derivatives and LEP were performed against human Caucasian prostate adenocarcinoma (PC3), human breast cancer cell line (BT549) and mouse pre-adipocytes (3T3-L1) cells and indicated moderare to high inhibition of the cell proliferation at a concentration of 5-100 µM after 24-72 h of incubation. These data suggest that TPM-TRP and TPM-LEP derivatives with optimized hydrophobicity can be used to improve the biological activity of TRP or LEP.

#2687

Cypripedin inhibits non-small cell lung cancer migration through suppression of epithelial-to-mesenchymal transition.

Surassawadee Treesuwan, Varisa Pongrakhananon. _Chulalongkorn University, Bangkok, Thailand_.

Lung cancer is one of the most common cause of cancer-related death worldwide because of high metastasis rate. Cancer migration is a prerequisite event among multi-complex processes of metastasis, being one of the interested target for cancer therapy. Accumulative evidence emphasizes that epithelial-to-mesenchymal transition (EMT) plays an important behavior exerting cell motility and survival, and relevant to cancer metastasize. Even the therapeutic approaches have been developed, lung cancer metastasize remains challenge for drug discovery and development. Recent studies highlight the potent anticancer properties of the compounds derived from Orchid. However, the underlying mechanism was unrevealed. This study demonstrated that cypripedin, a phenanthrenequinone extracted from Dendrobium densiflorum, possess an ability to inhibit in vitro cell migration in non-small cell lung cancer H460 and H23 cells. The number of motile cells, examined by transwell migration and wound healing assay, were gradually reduced in a dose-dependent concentration of cypripedin, along with the reduction of colony number and size formed under an anchorage-independent growth. Mechanistic investigation shows that the mesenchymal markers including N-Cadherin, Vimentin and Slug were significantly down-regulated following cypripedin treatment. Our finding suggests that cypripedin potentiates anti-migrative activity in non-small cell lung cancer through the attenuation of EMT process, which support further extensive studies against lung cancer.

#2688

ZB716 is orally efficacious in blocking tumor growth in mouse xenograft models.

Shanchun Guo,1 Changde Zhang,1 Jiawang Liu,1 Shilong Zheng,1 Lin Yang,2 Guangdi Wang1. 1 _Xavier University of Louisiana, New Orleans, LA;_ 2 _Chongqing Medical and Pharmaceutical College, China_.

Advances in oral SERDs development so far have been confined to nonsteroidal molecules such as those containing a cinnamic acid moiety, which are in early-stage clinical evaluation. ZB716 was previously reported as an orally bioavailable SERD structurally analogous to fulvestrant. In this study, we examined the binding details of ZB716 to the estrogen receptor alpha (ERα) by computer modeling to reveal its interactions with the ligand binding domain as a steroidal molecule. The ability of ZB716 to inhibit cell growth and downregulate ER expression in endocrine resistant, ERα mutant breast cancer cells was demonstrated. Moreover, in the MCF-7 xenograft and two patient derived xenograft models, orally administered ZB716 showed superior efficacy in blocking tumor growth when compared to fulvestrant. Importantly, such enhanced efficacy of ZB716 was shown to be attributable to its markedly higher bioavailability, as evidenced in the final plasma and tumor tissue concentrations of ZB716 in mice where drug concentrations were found significantly higher than in the fulvestrant treatment group.

#2689

Nanoformulated mellitin from bee venom exhibits excellent anti-prostate cancer efficacy.

Islam R. Abdelaal,1 Imtiaz A. Siddiqui,1 Vaqar M. Adhami,1 Hazem F. Abdelaal,1 Hadir Mohamed,1 Mohamad Rady,2 Hasan Mukhtar1. 1 _University of Wisconsin-Madison, Madison, WI;_ 2 _Al Azhar University, Cairo, Egypt_.

Melittin, a major peptide component of bee venom, has shown chemopreventive as well as chemotherapeutic effects against many cancers both in cell culture and preclinical animal model systems. However, several issues including nonspecificity, degradation, inefficient systemic delivery, bioavailability, and more importantly its enhanced hemolytic activity have hampered its translation to humans. We recently reviewed the literature on bee venom (Cancer Letters, 2017;402:16-31) and suggested that the use of nanotechnology mediated delivery of melittin can enhance significantly its systemic delivery to target cancer cells and with minimal hemolytic effect. We employed nanotechnology to improve the therapeutic outcome of bee venom by using perfluorocarbon nanoemulsion vesicles to deliver melittin, hereafter referred to as 'NanoBees'. The nano carriers were synthesized as an oil-in-water emulsion composed of a liquid perfluorooctyl bromide core having a monolayer of phospholipid forming a stabilizing interface with the aqueous media. We concentrated our studies on prostate cancer (PCa) because it is an attractive candidate and suitable target because of its long latency which offers a relatively long period of time for pharmacological interventions. We assessed the effectiveness of delivery of melittin, encapsulated in nanoformulation in vitro against PCa cells and in vivo in animal models. Treatment of PCa cells DU-145, C42 and PC3 PCa cells with formulated NanoBees (0-40 μg/ml) inhibited cell viability and proliferation and induced apoptosis. We observed an increase in the levels of proapototic Bax and concomitant decrease in the levels of antiapoptotic Bcl-2 resulting in a shift in the ratio of these molecules that favored apoptosis. We also observed an increase in poly(ADP-ribose) polymerase (PARP) cleavage and significant increase in the activation of cleaved caspases 3,7,8,9 in all tested PCa cell lines. These proteins were more than two fold up-regulated in the cells treated with NanoBees when compared to native melittin. Treatment of PCa cells with NanoBees inhibited cell proliferation in vitro through inhibition of the PI3K/Akt pathway and simultaneous downregulation in the expression of CyclinD1 and CDK4. We next tested the effect of NanoBees in a xenograft mouse model implanted with PCa PC3 cells. Intraperitoneal administration of NanoBees (5, 10 and 15 mg/Kg) significantly decreased tumor volume and growth rate compared to saline and void nanoparticle treatment. Incorporation of melittin in nanoemulsion prevented side effects associated with native melittin such hemolysis. These data support our hypothesis that nanotechnology could enhance the outcome of cancer therapy of melittin derived from bee venom through boosting bioavailability and limiting toxicity thus leading to a significant potential for possible clinical outcome.

#2690

**Inhibitory effects of a** Ganoderma lucidum- **derived fraction on triple-negative breast cancer (TNBC) cell lines.**

Camille Santiago-Negron,1 Ivette J. Suarez-Arroyo,2 Ricardo Vallejo-Calzada,2 Taotao Ling,3 Fatima Rivas,3 Michelle M. Martinez-Montemayor2. 1 _University of Puerto Rico Bayamon Campus, Bayamon, PR;_ 2 _Universidad Central del Caribe - School of Medicine, Bayamon, PR;_ 3 _St. Jude Children's Research Hospital, Memphis, TN_.

Breast cancer (BC) is known as the most common type of cancer among the United States female population. Triple-negative breast cancer (TNBC) accounts for 10–15% of these cases and it is defined as tumors lacking estrogen and progesterone receptors, as well as the epidermal growth factor receptor 2 (HER2). Treatment options for this disease is limited to chemotherapeutic agents, and its patient's long-term prognosis is greatly affected due to its recurrence pattern. Unfortunately, there are no targeted therapies for this type of BC. Therefore, new drugs and strategies are needed to improve the clinical responses and outcomes of these patients. The medicinal mushroom Ganoderma lucidum extract (GLE), has bioactive compounds with anti-cancer properties such as polysaccharides, triterpenes, phenols, and steroids. Accordingly, our earlier studies propose GLE as a promising treatment option for BC due to its non-toxic and selective anti-cancer activities. However, in this study we report the effects of GLE derived fractions after 72h on TNBC (SUM-149, MDA-MB-231) and MCF-10A non-cancerous mammary epithelial cell viability. Furthermore, we evaluated apoptosis using Annexin V immunofluorescence assays on both TNBC cell lines. For fraction development, pulverized GLE (10.0g) was dissolved in 150mL of isopropanol and refluxed for 24h. The resultant extract was filtered, and the solvent evaporated. The crude oil was fractionated via silica gel column chromatography. Based on MS and NMR analysis, 8 fractions were prepared for biological evaluation. Further purification via preparatory HPLC and structure elucidation of active fractions is currently under investigation. Additional GLE (10.0g) was extracted with isopropanol for 24h, and the crude compound was purified by silica gel column chromatography (hexane/ethyl acetate, 1:9) to generate 14 fractions. Further assessment of the fractions resulted in similar chemical composition so they were combined into 8 unique fractions based on MS, where F3 proved to have the greatest efficacy. Our results demonstrate that F3 causes a dose-dependent decrease in TNBC viability, with an IC50 of 12.7 and 14.7µM, in 231 and SUM-149 cells respectively. Moreover, Annexin V assays indicate that F3 induces apoptosis on both TNBC cell lines. Interestingly, F3 was not cytotoxic to non-cancerous cells at the concentrations tested. These findings might be of great importance to the research and medical community, because they advance the understanding of GLE derived compounds as a potential adjuvant treatment of TNBC. 

### Cancer Biology Insights Emerging from Proteomic Investigations

#2691

Discovery of novel markers in Barrett's-related esophageal adenocarcinoma.

Joe Abdo,1 Chris Wichman,2 Nick E. Dietz,3 John Fleegel,1 Pawel Ciborowski,2 Sumeet K. Mittal,4 Devendra K. Agrawal1. 1 _Creighton University School of Medicine, Omaha, NE;_ 2 _University of Nebraska Medical Center, Omaha, NE;_ 3 _CHI Health Creighton University Medical Center Bergan Mercy, Omaha, NE;_ 4 _St Joseph's Hospital and Medical Center - Dignity Health, Phoenix, AZ_.

Despite the fact there are currently a dozen treatment options for esophageal adenocarcinoma (EAC), durable patient responses to anticancer therapies are hard to achieve in EAC patients. The rate of incidence of EAC is increasing faster than any cancer in our country, and the survival rates have remained very low for decades, even in the current era of molecular medicine. Therefore, our group designed an ambitious, large scale mass spectrometric experiment to cast a molecular dragnet across our patient samples to detect overexpression and downregulation patterns that contribute to the aggressiveness of EAC. We used FFPE tissue samples from esophagectomy surgery before any chemotherapy or radiation had been administered, analyzing unadulterated EAC tissue, Barrett's esophagus (BE) tissue, and normal esophageal squamous epithelium from 50 patients. Our platform consisted of pathologist-guided microdissection, a Liquid Tissue® process turning FFPE tissue into a digested lysate, discovery mass spectrometry (TripleTOF 6600) and extensive biostatistics. We discovered a perfect storm of pathogenic processes which explains from several perspectives the aggressiveness of this disease as well as EAC's proclivity to resist chemotherapeutic agents. We found 20 relevant proteomic events in which tumor suppressors are turned off, growth factors are overexpressed, chemoresistance markers are upregulated, chemosensitivity markers are downregulated as well as revealing eight new potential drug targets in which therapeutic antagonism could slow tumorigenesis in EAC. Of these 20 proteins we robustly investigated four markers (DAD1, ISG15, S100P and UBE2N) which all have mutual intersections with prognostic, diagnostic and therapeutic aspects of esophageal pathogenesis. Overexpression of DAD1 (9 out of 10), ISG15 (10/10), S100P (10/10) and UBE2N (10/10) was discovered in BE tissue. In the adjacent EAC tissue, we found significant overexpression of DAD1 (20 out of 20), ISG15 (20/20), S100P (20/20) and UBE2N (19/20) proteins. These expression levels spiked when transitioning from normal squamous epithelium to highly dysplastic BE tissue demonstrating that BE starts to act like cancer by using more glucose for energy (Warburg effect), increasing proliferative measures, and suppressing apoptosis. Also, overexpressed DAD1 protein levels contributes to cisplatin resistance which is striking since 95% of the 123 patients treated at CUMC for EAC received cisplatin in the first-line setting. S100P expression has been found to increase resistance to 5-FU in colorectal cancer. 5-FU was prescribed to 72% of the patients in our cohort (n=123), so if there is innate resistance to the two most prescribed drugs for EAC patients then alternative patient management strategies are needed. Here we've discovered four new prognostic and therapeutic biomarkers with potential clinical utility for EAC.

#2692

Elucidation of the pre-B-cell receptor signaling network in acute lymphocytic leukemia cells.

Roland Walter,1 Khouloud Kouidri,1 Jessica Peter,1 Martine Pape,1 Laura Merschen,1 Bjoern Haeupl,1 Carmen Doebele,1 Hubert Serve,1 Yanlong Yi,2 Henning Urlaub,2 Thomas Oellerich1. 1 _University Hospital, Frankfurt, Germany;_ 2 _Max Planck Institute for Biophysical Chemistry, Göttingen, Germany_.

Antigen receptors transduce complex signals and thereby control important cell fate decisions of the B-lineage including differentiation, proliferation and B-cell survival. The initial steps of B-cell development take place in the bone marrow, and the first dependency of a developing B-cell on antigen receptor signaling occurs at the pre-B-cell stage where expression of the pre-BCR is essential for cell survival. In contrast to B-cell-receptor (BCR) signaling, the nature of pre-BCR signals is far less characterized. It is believed that pre-BCRs transduce a constitutive baseline signal that is essential for survival and differentiation of pre-B-cells into immature B-cells. Since pre-BCRs are crucial regulators of pre-B-cell survival and differentiation, it is not surprising that dysregulation of pre-BCR signaling is observed in acute lymphocytic leukemia (ALL) cells where it contributes to survival of the malignant cell clones. To identify drug targets in the pre-BCR signaling network, we have systematically characterized pre-BCR signaling in two pre-BCR-dependent ALL cell lines by quantitative phosphoproteomics. Because pre-BCRs transduce a constitutive baseline signal, we interfered with pre-BCR signaling by either Syk inhibitor treatment or by an inducible shRNA-mediated knock-down of the pre-BCR component Ig-α to monitor pre-BCR-dependent signals. We identified and quantified more than 15,000 phospho-sites. Among these sites, more than 500 were regulated upon interference with pre-BCR signaling. Although the upstream components are shared between pre-BCRs and BCRs, the wiring of their downstream signals occurred to be different which is reflected by our finding that the majority of the identified pre-BCR signaling effectors have not been described in the context of BCR signaling in mature B-cells or lymphomas. Finally, we have identified vulnerabilities in the pre-BCR signaling network by intersecting our phosphoproteomic data with data from loss-of-function CRISPR-Cas9 screens for essential genes in ALL cell lines. Taken together, this study provides a comprehensive dataset for pre-BCR signaling and identifies effector proteins that are relevant for the regulation of ALL cell survival and proliferation.

#2693

Mass spectrometric approach to identify N-glycosylation of light chain in patients with immunoglobulin light chain amyloidosis (AL).

Sanjay Kumar,1 Angela Dispenzieri,1 Surendra Dasari,1 Paolo Milani,2 Giampaolo Merlini,2 MeLea Hetrick,1 David Barnidge,1 Benjamin Madden,1 David Murray1. 1 _Mayo Clinic, Rochester, MN;_ 2 _University of Pavia, Pavia, Italy_.

AL is a rare plasma cell disorder, resulting from clonal bone marrow plasma cells secreting immunoglobulin light chains (LC), which aggregate abnormally into fibrils, and leads to amyloid deposits in different organs. Previous studies using LC immunopurification and MALDI-TOF/MS (MASS-FIX) on patients with known plasma cell disorders (PCDs), demonstrated that monoclonal LC from AL patients display unusually complex mass to charge spectra (m/z) for their LCs at much higher frequencies than other PCDs such as multiple myeloma. We hypothesized that these were post-translational modification, presumably glycoforms. Here, we report investigations into LC glycosylation using MASS- FIX. Serum from 212 lambda and 72 kappa AL patients were collected following Mayo Clinic IRB guidelines. LC was purified from serum using nanobody enrichment method, followed by MASS-FIX. The LCs spectra acquired on MASS-FIX were visually analyzed and were classified into different patterns. The kappa and lambda LC mass distributions with abnormal complex patterns were interrogated for the presence of glycosylation by exposure to the LC to a deglycoslylating enzyme (PNGase F) and analysis of LC mass distribution using MASS-FIX and by LC-ESI-Orbitrap. Monoclonal LCs demonstrating significantly mass shifted peak outside the normal kappa and lambda mass distribution were observed in 31% of kappa and 6% of lambda patients with AL. In order to determine if this characteristic mass shift was due to glycosylation, 16 kappa and 6 lambda LCs were treated with a PNGase F and LC spectrum were re-examined by MASS-FIX and LC-ESI-Orbitrap. The monoclonal LCs whose masses were significantly shifted beyond normal kappa and lambda mass distributions were found to shift to lower molecular weight after PNGase F treatment, confirming the presence of N-glycosylation. Moreover, an additional 5 kappa and 23 lambda samples had other complex patterns, typically with multiple additional peaks at increments of 162 Da. These samples did not shift with PNGase F suggesting either glycation or O-glycosylation. This work is significant as it confirms that N-glycosylated LCs are over represented in patients with AL in comparison to other PCDs. In patients with AL, the frequency of N-glycosylation of kappa LC clones was 5 times higher than that of lambda LC clones. More work is needed to explore the mechanism for this finding, the role of glycosylation in fibril formation, and the other complex patterns observed. In addition, MASS-FIX method used in this work is currently being validated in the clinical lab such that rapid detection of N-glycosylation in patients with monoclonal gammopathies will be possible routinely, potentially leading to earlier clinical suspicion for AL among patients with monoclonal gammopathies and hence to earlier detection of AL.

#2694

Receptor-interacting protein kinase 2 promotes prostate cancer progression by activating the MAX:MYC pathway.

Yiwu Yan,1 Bo Zhou,1 Xiaopu Yuan,1 Michael E. Doche,1 Zhaoxuan Ma,1 Colm Morrissey,2 Arkadiusz Gertych,1 Sungyong You,1 Beatrice Knudsen,1 Michael R. Freeman,1 Wei Yang1. 1 _Cedars-Sinai Medical Center, Los Angeles, CA;_ 2 _University of Washington, Seattle, WA_.

Background: Prostate Cancer (PCa) is the most common non-skin cancer among American man. Currently, major challenges in the clinical management of PCa include 1) a lack of biomarkers that can reliably distinguish aggressive from indolent PCa and 2) a shortage of therapeutic strategies that can prevent or suppress the progression of PCa into aggressive PCa.

Methods: Label-free quantitative proteomics was performed on three widely used PCa cell lines with different levels of aggressiveness (i.e., LNCaP, DU145 and PC3). Upstream regulator analysis and network modeling were conducted to identify candidate key promoters of PCa aggressiveness. The levels of gene amplification, mRNA expression, and protein expression of a key candidate promoter, receptor-interacting protein kinase 2 (RIPK2), were compared by analyzing publicly accessible PCa genomic and transcriptomic datasets and by immunohistochemistry (IHC) staining of PCa tissue microarrays (TMA). Cell proliferation and invasion assays were performed to compare PC3 cells with or without CRISPR/Cas9 knock-out of RIPK2. Quantitative proteomics and bioinformatics analysis were conducted to identify the underlying mechanisms, followed by validation via western blotting, quantitative real-time PCR (qRT-PCR) and in vitro functional assays.

Results: Quantitative proteomics analysis of the three PCa cell lines identified over 6,500 proteins, among which 341 are significantly (adjusted p-value < 0.01) differentially expressed. Bioinformatic analysis and network modeling revealed RIPK2 as a novel candidate key promoter of aggressive PCa. Consistently, the levels of RIPK2 DNA amplification, mRNA expression, and protein expression are significantly higher in more aggressive PCa than in less aggressive PCa. Moreover, cell functional assays showed a significant inhibition of cell proliferation and invasion after the CRISPR/Cas9 depletion of RIPK2 in PC3 cells. Mechanistically, quantitative proteomics identified MAX (Myc-associated factor X) as one of the most prominent candidate downstream effectors of RIPK2. Consistently, upstream regulator analysis suggested that Myc activity was significantly downregulated in RIPK2 deleted PC3 cells, compared with control cells. These findings were further validated by RT-PCR, western blotting, and MYC luciferase reporter assay. In addition, pathway enrichment analysis identified the EIF2 signaling pathway, a key pathway regulated by MAX:MYC, as the most significantly downregulated pathway after RIPK2 knock-out.

Conclusion: Taken together, our data elucidate that RIPK2's amplification and overexpression in PCa cells may activate the MAX:MYC-EIF2 signaling axis and thus in turn promote PCa cell proliferation and invasion. The aberrant activation of the RIPK2-MAX:MYC-EIF2 pathway may serve as a valuable biomarker and novel drug target of PCa progression.

#2695

**Quantitative phosphoproteome analysis identifies novel functional pathways of** **tumor suppressor** **DLC1** **in estrogen receptor-positive breast cancer.**

Yesim Gökmen-Polar, Jason D. True, Edyta Vieth, Yuan Gu, Xiaoping Gu, Guihong D. Qi, Amber L. Mosley, Sunil Badve. _Indiana Univ. School of Medicine, Indianapolis, IN_.

Deleted in Liver Cancer-1 (DLC1), a member of the RhoGAP family of proteins, functions as a tumor suppressor in several cancers including breast cancer. However, its clinical relevance is unclear in breast cancer. In this study, expression of DLC1 was correlated with prognosis using publicly available breast cancer gene expression datasets and quantitative polymerase chain reaction (qPCR) in cohorts of Estrogen Receptor-positive (ER+) breast cancer. Mutation and methylation status of DLC1 were assessed in these datasets including The Cancer Genome Atlas (TCGA). To seek further insights in understanding the role of DLC1 in ER+ breast cancer, we developed a knock-in model of DLC1 in T47D breast cancer cells. Label-free global proteomic and TiO2 phosphopeptide enrichment assays were performed and validated using Western blotting. Data are available via ProteomeXchange with identifier PXD008220. Here, we report that low expression of DLC1 correlates with poor prognosis in patients with ER+ breast cancer with further decrease in metastatic lesions. Analysis of the TCGA data showed that down regulation of DLC1 is not due to methylation or mutations. Stable knock-in of DLC1-full-length inhibits cell colony formation significantly in vitro compared to its control counterpart. 6726 phosphopeptides were quantified by phosphoproteomics analysis in both conditons, whereas 205 and 122 were unique to DLC1 knock-in and T47D-control cells, respectively. Pathway analysis using DAVID showed the top three significant clusters of differentially identified phosphopeptides involving cell-cell adhesion, mRNA processing and splicing, and transcription regulation. Decreased phosphorylation of epithelial cell transforming sequence 2 (ECT2) at the residue T359, critical for its active conformational change, was validated. In conclusion, this data suggests that high expression of DLC1 reduces cell growth and is associated with favorable prognosis. Our results document an inverse relation between ECT2 phosphorylation and DLC1 expression, promising a novel strategy for treating ER+ breast cancer.

#2696

Deciphering the interactome of the metastasis-suppressor protein RAI2.

Stefan Werner, Kai Bartkowiak, Pascal Steffen, Katharina Besler, Lena Böttcher, Hartmut Schlüter, Klaus Pantel, Harriet Wikman. _Univ. Medical Ctr. Hamburg-Eppendorf, Hamburg, Germany_.

We have identified low RAI2 expression as a novel metastasis-associated genetic alteration especially related to the presence of disseminated tumor cells in the bone marrow of breast cancer patients and poor outcome (Werner et al, Cancer Discovery, 2015). In hormone-dependent breast and prostate cancer cell lines RAI2 depletion induces down-regulation of hormone receptor expression. Furthermore, RAI2 depletion is associated with chromosomal instability. Nevertheless, to date the exact molecular mechanisms behind these observations remain largely elusive. To better define the RAI2 interactome we performed SILAC-immunoprecipitation quantitative proteomics. HEK293T cells were stably isotope labeled with amino acids in cell culture and transfected with a plasmid for expression of a HA-tagged RAI2 protein. Whole protein extracts were used for co-immunoprecipitation and possible interacting proteins of RAI2 were determined by mass spectrometry based on their SILAC ratios in enriched cell lysates from RAI2 over-expressing and control cells. Subsequently, the DAVID functional annotation tool was used for annotation of co-precipitated proteins. For validation of newly identified potential interactions, we applied combined co-immunoprecipitation, immunoblot and microscopic imaging analysis. The quantitative proteomics approach revealed high confidence interactions of the RAI2 protein with nine gene products (CTBP1, CLTC, HSPA8, NONO, HSPA9, HSPA1A, TNRC6B, UPF1, AP2B1). Additionally, eleven low confidence interactions (HSPA5, PARP1, SFPQ, NUDT21, KRT8, DDX24, FXR1, HSPB1, COL17A1, SLC25A11, FARSA) were identified. The highest SILAC ratio was obtained for CTBP1. Functional annotation showed significant enrichment for the GO terms mRNA processing (GO:0006397, p=0.018), paraspeckles (GO:0042382, p<0.001) and polyA RNA binding (GO:0044822, p<0.001). We validated molecular interactions of RAI2 with CTBP1 and the paraspeckles proteins NONO and SFPQ by combined co-immunoprecipitation and immunoblot analysis. Immunofluorescence staining showed that the RAI2 protein is predominantly co-localized with CTBP1 in nuclear speckles, which is dependent on an intact non-consensus bipartite CTBP-binding domain of the RAI2 protein. In conclusion, the RAI2 protein interacts with different scaffolding proteins and various RNA binding proteins that are involved in mRNA processing, i.e. post-transcriptional regulation. Based on these findings we conclude that the tumor suppressive function of the RAI2 protein is mainly associated with post-transcriptional regulation probably by orchestrating protein scaffolds and subnuclear compartmentalization.

#2697

Alterations in the HSP70 interactome are induced by overexpression of MUC1 in pancreatic cancer.

. Nitika,1 Donald J. Wolfgeher,2 Pinku Mukherjee,1 Andrew W. Truman1. 1 _University of North Carolina, Charlotte, NC;_ 2 _The University of Chicago, Chicago, IL_.

Heat Shock Protein 70 (HSP70) is an evolutionarily well-conserved molecular chaperone involved in several cellular processes such as folding of proteins, modulating protein-protein interactions and transport of proteins across membranes. HSP70 works synergistically with a series of chaperones, co-chaperones, adaptors, and folding enzymes known as the HSP70 "interactome". Previous work has demonstrated that HSP70 interactome changes can be promoted by overexpression of oncoproteins. An important "client" of HSP70 is the oncoprotein MUC1. MUC1 is overexpressed and promotes drug resistance in pancreatic cancer. We applied quantitative affinity-purification mass spectrometry (AP-MS) to characterize the HSP70 interactome differences between PC3/NEO and PC3/MUC1 cell lines. 439 HSP70-interacting proteins were identified and quantified via LC-MS/MS. Interestingly while 53% of HSP70 interactions remained constant upon MUC1 overexpression, 29% significantly increased and 18% decreased. Many of the altered proteins were key co-chaperones that regulate and maintain HSP70 activity. High levels of MUC1 promoted HSP70 association with 15 co-chaperones (HSPA5, DNAJB1, STIP1, TXNDC5, HSPB1, CCT2, TCP1, P4HB, CCT6A, CCT8, PDIA3 and HSP90B). Conversely, MUC1 overexpression triggered the loss of HSP70 interaction with only 2 co-chaperones (PPIB and DNAJA2). We are currently validating these altered interactions using an array of biochemical techniques. Our hope is that by understanding these HSP70 interactome changes, we may be able to uncover novel ways to inhibit MUC1 function in pancreatic cancer.

#2698

Phosphoproteomics-guided anticancer drug combination design with a novel small-molecule PP2A activator.

Danica D. Wiredja,1 Peter Liao,1 Jaya Sangodkar,2 Daniela Schlatzer,1 Mark R. Chance,1 Goutham Narla1. 1 _Case Western Reserve University, Cleveland, OH;_ 2 _Icahn School of Medicine at Mt. Sinai, New York, NY_.

Significance: Phosphatases are cellular antagonists of kinase signaling and have a diverse range of substrates, many of which are ideal targets for small molecule-based anticancer treatment. Our lab and collaborators have generated a novel series of compounds that bind to and activate the serine/threonine protein phosphatase 2A (PP2A), and these small-molecule activators of PP2A (SMAPs) induce apoptosis and inhibit tumor growth in numerous cancer models. Based on these findings, this project aimed to identify drug combinations with our SMAP that could drive optimal anticancer activity.

Methods: We first turned to global shotgun phosphoproteomics to compare the signaling response signatures of our SMAP against a combination of a MEK inhibitor (MEKi) and an AKT inhibitor (AKTi). We then applied Kinase-Substrate Enrichment Analysis (KSEA) to extract key diverging biologic patterns between these two arms, with the goal of identifying distinct pathways that are inhibited in each condition. These results, in turn, could provide insight to differences between phosphatase activation versus kinase inhibition.

Results: KSEA profiling revealed distinct signaling responses between the two arms. Notably, the kinase inhibitors caused robust downregulation of the MAPK signaling pathway, whereas SMAP treatment showed limited involvement of that cascade. Thus, we hypothesized that a combination of SMAP + MEKi would yield enhanced anticancer response compared to either single agent. Subsequent murine xenograft testing demonstrated superior tumor response with SMAP + MEKi versus either compound alone.

Conclusion: This project offers a phosphoproteomics-level glimpse to the signaling effects between phosphatase activation compared to kinase inhibition. The data, in turn, could be used for rational drug combination design to improve antitumor therapy.

#2699

Proteomic landscape of de novo pediatric acute myeloid leukemia.

Fieke W. Hoff,1 Yihua Qiu,2 Wendy Hu,3 Amina A. Qutub,3 Alan S. Gamis,4 Richard Aplenc,5 E Anders Kolb,6 Todd A. Alonzo,7 Eveline SJM de Bont,1 Terzah M. Horton,8 Steven M. Kornblau2. 1 _University Medical Center Groningen, Netherlands;_ 2 _UT MD Anderson Cancer Ctr., Houston, TX;_ 3 _Rice University, Houston, TX;_ 4 _Children's Mercy Hospitals and Clinics, Kansas City, MO;_ 5 _Children's Hospital of Philadelphia, Philadelphia, PA;_ 6 _Nemours Center for Cancer and Blood Disorders, Wilmington, DE;_ 7 _University of Southern California, Los Angeles, CA;_ 8 _Texas Children's Cancer Center, Houston, TX_.

Background: Despite substantial increases in therapy intensity, the overall survival of pediatric acute myeloid leukemia (AML) is still guarded, with survival rates of approximately 60%. This indicates the need for new therapeutic strategies, as well as improved risk stratification. Chemotherapies target proteins rather than genetic events, yet little is known about the proteomic landscape in pediatric AML. This study provides a global assessment of pediatric AML protein expression and correlates protein expression with outcome.

Methods: A reverse phase protein array (RPPA) probed with 298 validated antibodies was performed to determine protein expression in ''bulk'' (CD3-/19-) AML cells from 505 diagnostic pediatric AML patients who participated in the Children's Oncology Group AAML1031 phase 3 clinical trial. Proteomic profiling was applied in the context of 31 protein functional groups (PFG) (e.g., cell cycle, apoptosis) to analyze their expression in relation to related proteins. Progeny clustering was performed to identify patients with correlated protein expression patterns within each PFG (protein cluster). Block clustering searched for protein clusters that recurrently co-occurred (protein constellation), and for subgroups of patients that expressed similar combinations of protein constellations (patient signatures). Signatures were correlated with patient and disease characteristics.

Results: For each PFG, protein clusters (n=120) could be discerned that showed different protein expression states. From this we constructed 11 protein constellations and 10 patient signatures. Signatures were correlated with event-free survival (EFS) when we combined signatures into favorable (Sig. 4, 8), intermediate (Sig. 6, 7, 9) and unfavorable (Sig. 1-3, 5, 10) groups (p=0.01). Other significant clinical correlations between signatures included CEPBA (40% in Sig. 6, vs. 9% overall, p<0.001), MRD status (high in Sig. 2 vs. low in Sig. 6+7, p=0.006) and several laboratory features. Proteins that were significantly altered compared to normal CD34+ cells were identified for each signature. From this list, 20 proteins were recognized as universally downregulated (CDKN1A, PPP2R2A) and only PIK3CA was universally upregulated. Many druggable proteins showed association with specific protein signatures: high KIT (Sig. 1, 2, 6), high BCL2 (Sig. 1, 2, 6, 9) and high NPM1 (Sig. 1, 2, 6, 9).

Conclusion: We studied the proteomic landscape in 505 pediatric AML patients, and identified 10 protein signatures based on 11 protein constellations. We identified signatures that did well with ADE therapy vs. signatures that did not. Recognition of deregulated proteins could help to select drugs that could potentially improve individualized therapies for the latter signatures.

#2700

Specific cell populations from the primary tumor invasive front and lymph nodes show protein signatures associated with local metastasis in head and neck cancer.

Ariane F. Busso-Lopes,1 César Rivera,1 Carolina C. Macedo,2 Barbara P. Mello,3 Luisa L. Villa,3 Wilfredo A. González-Arriagada,4 Adriana F. Paes Leme1. 1 _Brazilian Center for Research in Energy and Materials - CNPEM, Campinas, Brazil;_ 2 _University of Campinas, Piracicaba, Brazil;_ 3 _University of Sao Paulo, Sao Paulo, Brazil;_ 4 _Valparaiso University, Valparaiso, Chile_.

Lymph node metastasis is the main prognostic factor in patients with head and neck squamous cell carcinoma (HNSCC), but no molecular markers are currently used in clinical practice. Considering that the invasive front (IF) of the tumor has a prognostic value due to the invasive and metastatic potential of the cells, we explored the mechanisms associated with lymph node metastasis in the primary tumor IF and its paired lymph nodes from HNSCC patients. For that, the proteomic profile was determined for formalin-fixed, paraffin-embedded (FFPE) tissues using mass spectrometry in a 29-patient cohort. Specific cell populations from primary tumor IF (tumor and stroma cells) and lymph nodes (microenvironment cells) were microdissected and HPV genotyping was performed. In total, 27.5% of the cases were positive for HPV16 and 2,154 ± 285 proteins were identified for the three cell populations analyzed. Eighty-one proteins from tumor cells, 36 proteins of stroma cells and 149 proteins of lymph node microenvironment cells were differentially expressed between HNSCC patients positive and negative for lymph node metastasis (Student´s t-test; P-value ≤ 0.05). Specific processes were enriched for the differentially expressed proteins from tumor, stroma and lymph node cells, including post-translational modification, splicing and translation, respectively. Interestingly, differential proteins isolated from primary tumor cells are mainly transported by exosomes. Therefore, our data provide protein signatures related to lymph node metastasis that are expressed by specific cell populations and candidates as prognostic markers in HNSCC.

Financial support: FAPESP (Process number 2015/19191-6).

#2701

Proteogenomic analysis of endoscopy biopsy identifies alterations in apoptotic pathways in trastuzumab-resistant gastric cancers.

Jeong Won Kang, Il Ju Choi, Hark Kyun Kim. _National Cancer Center, Goyang, Republic of Korea_.

Although the combination of trastuzumab with cisplatin and fluoropyrimidine is standard therapy for metastatic HER2-positive gastric cancers, predictive biomarkers for this therapy need to be further refined. Given the multifactorial nature of clinical trastuzumab resistance, we performed nano-liquid chromatography (LC)-tandem mass spectrometry (MS/MS) proteomic analyses on 13 pairs of frozen HER2-positive tumors and adjacent normal tissue counterparts collected from patients who were treated with trastuzumab in combination with cisplatin and fluoropyrimidine (TCF). We quantified >6,000 protein groups per tumor on the average, and this number was almost comparable to those obtained by NCI's Clinical Proteomic Tumor Analysis Consortium projects, which characterized resected tumors using similar nano-LC-MS/MS technologies. Nano-LC-MS/MS data for differentially expressed proteins were successfully validated by our immunostaining analyses. When we correlated proteomic data with clinical outcome, underexpression of apoptosis pathway proteins was the prominent proteomic signature in TCF-resistant tumors, as compared with TCF-sensitive tumors. Proapoptotic proteins in FAS signaling and mitochondrial apoptosis pathways were less abundant in TCF-resistant tumors than in sensitive tumors. When iTRAQ reporter ion ratios were compared between pretreatment and acquired-resistant tumors from clinical responders, apoptosis pathways were significantly enriched in proteins that were overexpressed in the acquired-resistant tumors. Assuming that the pretreatment tumors of clinical responders were mostly composed of chemosensitive clones, these antiapoptotic proteins may confer survival advantage on resistant clones, resulting in the repopulation of resistant clones overexpressing these antiapoptotic proteins. For the first time to our knowledge, therefore, our proteogenomic study of frozen endoscopic biopsy samples revealed that alterations in apoptotic pathway proteins were most significantly associated with clinical trastuzumab resistance. Our study is also the first to demonstrate that nano-LC-MS/MS technology can be applied to small biopsy tissue samples for precision medicine research.

#2702

The mTOR complex 2-associated interactome and cell migration ability determination in brain cancers.

Naphat Chantaravisoot,1 Piriya Wongkongkathep,1 Narawit Pacharakullanon,1 Fuyuhiko Tamanoi,2 Joseph A. Loo,2 Trairak Pisitkun1. 1 _Chulalongkorn University, Bangkok, Thailand;_ 2 _University of California, Los Angeles, Los Angeles, CA_.

The mechanistic target of rapamycin complex 2 (mTORC2) is a multiprotein complex driving several cellular processes to promote metabolic activities, cell survival, cell proliferation, DNA damage control, and cell motility. It has been implicated as a critical player in tumorigenesis, metabolic reprogramming, and promotion of cancer aggressiveness. Since the functions of this particular complex have not been well understood, the characterization of mTORC2 and its signaling pathway is necessary for better understanding of known mTORC2-mediated function, to define new roles, and to identify more mTORC2 interacting partners in cancers, particularly ones with hyperactivated mTORC2 such as glioblastoma multiforme. GBM has been claimed as one of the most aggressive type cancers. In GBM cells, mTORC2 was reported to be regulating glioblastoma migration and invasion through the control of actin cytoskeleton reorganization and filamin A. Previously, we characterized mTORC2 by proteomic analysis using affinity purification coupled to mass spectromytry (AP-MS) technique. We were able to identify protein-protein interactions between RICTOR, a specific component of mTORC2, and associated proteins and revealed a number of mTORC2 interacting partners that have never been reported. These proteins include low-abundance proteins and less-characterized proteins. In this work, we further investigated more insights about the differences of mTORC2-associated interactomes under various conditions affecting cancer cell migration. We found the comparison between low-grade and high-grade glioma cell lines shows distinct groups of RICTOR-bound proteins. In addition, we have carried out migration assays and immunofluorescence staining experiments to determine cancer cell migration and localization of RICTOR and some of identified proteins under each culture condition. The results suggested that the information from proteomic analysis correlated with intracellular RICTOR localization and cell migration ability. EMT proteins and more cytoskeletal proteins were found in more motile and more invasive cells while nuclear proteins were identified when using low-grade glioma cells. Furthermore, we were able to use this approach to investigate cancer cell lines from other organs and found some relevance between mTORC2 complex localization and cell migration. Ultimately, these results will be analyzed together with the expression of other potential cancer markers to help determine the characteristics of brain cancers.

#2703

Proteomic identification of Galectin-1 in murine primary and metastatic triple-negative breast tumors.

Kassondra Balestrieri, Kim Kew, Mohamed Ramez, Keith Pittman, Nasreen Vohra, Kathryn Verbanac. _East Carolina Univ. School of Medicine, Greenville, NC_.

The purpose of this study was to identify proteins associated with Triple Negative Breast Cancer (TNBC) metastases using a proteomics approach, in order to gain insight into pathways and mechanisms that could help identify biomarkers or targets for intervention. For our investigations, we used a murine claudin-low tumor model that mirrors human claudin-low TNBC molecular profiles, T11. Cultured T11 cells were lysed, trypsin-digested, and analyzed by liquid chromatography mass spectrometry (LC/MS). A total of 104 proteins were identified with >95% confidence using Protein Pilot software. Among these proteins was Galectin-1 (Gal-1); seven distinct Gal-1 peptides were identified with protein coverage of 58%. Galectins are a family of ß-galactoside-binding proteins that share homology in the carbohydrate binding domain. Gal-1 was selected for further investigation due to its reported roles in cancer progression and immune evasion. Gal-1 expression was next examined in naïve murine lung tissue and in lung metastases from mice orthotopically implanted with T11. Lung tissue was homogenized, trypsin-digested and analyzed by LC/MS. Although Gal-1 was not identified in naïve lungs, three Gal-1 peptides (28% protein coverage) were identified with >95% confidence in lungs with macrometastases. LC/MS analysis did not identify any other galectin family members in primary tumor or lung metastases. Matrix-assisted laser desorption ionization (MALDI) imaging of trypsin-digested tissue sections revealed two distinct Gal-1 peptides localized to tumor regions within metastatic lung tissue. Immunohistochemistry (IHC) analysis showed higher Gal-1 expression in orthotopic T11 primary tumors compared to naïve mammary fat pad, and also higher Gal-1 expression in T11 lung metastases compared to naïve lung. Within tumors, Gal-1 was detected in extracellular matrix, in the cytoplasm of tumor cells and associated with tumor cell membranes. These findings are supported by our analysis of both murine and human genomic data sets, which showed Gal-1 overexpression in claudin-low TNBC compared to other breast cancer subtypes and normal breast tissue. There are several reports that exogenous Gal-1 induces an M2-like phenotype in myeloid cells, consistent with our prior findings that primary tumors, pre-metastatic and metastatic lungs exhibit significant M2-like myeloid cell infiltrates. Future investigations in our lab will focus on determining the role of tumor-produced Gal-1 in the regulation of myeloid cell function to promote cancer progression.

#2704

Mass spectrometry-based profiling of lysine acetylation and arginine methylation for biomarker discovery in triple negative breast cancer.

Alissa J. Schunter,1 Hongbo Gu,2 Jian Min Ren,1 Vicky Yang,1 Matthew P. Stokes1. 1 _Cell Signaling Technology, Danvers, MA;_ 2 _Takeda Pharmaceuticals, Cambridge, MA_.

(a) Introductory sentence

Antibody enrichment and liquid chromatography mass spectrometry (LC-MS) were used to profile lysine acetylation and arginine mono-methylation in triple negative breast cancer patient sera without prior immunodepletion of abundant proteins.

(b) Brief description of experiments

Triple negative breast cancer (TNBC) is the most aggressive type of breast tumor and there are currently no approved targeted therapies. Better biomarkers are needed for early detection and for therapeutically informative subtyping of this genetically heterogeneous disease. We recently reported a methodology to enrich post-translationally modified peptides from serum (Gu et al, Mol Cell Proteomics 2015) and found acetyl lysine (AcK) and mono-methyl arginine (RMe) to be some of the most abundant post-translational modifications (PTMs) in the sera of cancer patients. This method has the advantage of profiling serum samples without prior depletion of abundant serum proteins, a major limitation of current proteomic methods. To develop a biomarker signature of TNBC, these two PTMs were profiled in the sera of 10 patients with stage I-IIA TNBC and 10 healthy female controls. Serum proteins were trypsin digested prior to immunoaffinity enrichment of the modified peptides with PTM-specific antibodies. The enriched peptides were analyzed by LC-MS and relative abundance of peptides across samples was measured using label-free quantification.

(c) Summary of new, unpublished data

PTM enrichment quantified hundreds of AcK and RMe sites across samples. AcK levels decreased globally in patients compared to controls, suggestive of increased lysine deacetylase or decreased acetyltransferase activity. Of these, a number of sites were found significantly altered between TNBC patient samples and normal controls at the p<0.05 level by two-sided t-tests, including novel sites not previously reported in the PhosphoSitePlus database or in previous publications. Some of the regulated sites reside on proteins with important immune functions and cancer-related pathways.

(d) Statement of conclusions

AcK and Rme peptide signatures were identified that correlate with disease status, tumor grade, and stage. Patient follow-up over time will allow for investigation of markers of treatment response and disease progression as well. AcK and Rme are known to affect gene transcription profiles in cancer. Drug molecules targeting multiple families of AcK interacting proteins, including KATs, HDACs, and BET proteins, have entered clinical trials for breast and other cancers. AcK was globally downregulated in the samples surveyed, which suggests that these tumors may be responsive to some of these drug classes. Further analysis will correlate the AcK and RMe profiles to identify a PTM signature of therapeutically vulnerable epigenetic regulators in TNBC.

#2705

Functional proteomics to reveal distinct signature for acute myeloid leukemia subtypes.

Patrick Pirrotte,1 Victoria David,1 Ritin Sharma,1 Daniel H. Wai,2 Cristina Tognon,3 Brian Druker,3 Eiman A. Aleem4. 1 _Translational Genomics Research Institute, Phoenix, AZ;_ 2 _Instiute of Molecular Medicine, Phoenix Children's Hospital, University of Arizona College of Medicine-Phoenix, Phoenix, AZ;_ 3 _OHSU Knight Cancer Institute, Portland, OR;_ 4 _University of Arizona College of Medicine-Phoenix, Phoenix, AZ_.

Patients with relapsed/ refractory AML have an overall survival rate <30%. Genomic approaches are being used for patients' stratification, but they have not been sufficient at predicting response to therapy. We propose an integrative approach to identify distinctive phosphorylated protein biomarkers in AML, map them to signaling networks, and compare these to pathways identified through analysis of gene expression. This study aims at identifying distinct phosphoproteomic signatures by mass spectrometry (MS) in different AML subtypes. Methods: Four AML cell lines and 40 bone marrow samples from patients with AML representing different AML subtypes are used. Samples were prepared using a MOAC-TiO2 stationary phase for phosphopeptide enrichment, and analysis on a Thermo LTQ Orbitrap Velos MS. Identified phosphorylated STY sites were cross-referenced against iPTMnet. Phosphoproteins were further matched to potential pathways using Ingenuity Pathway Analysis (IPA). Gene-expression profiling data from cell lines, available on HG-U133 Plus 2 arrays, were obtained from GEO and ArrayExpress. ANOVA was performed to identify significantly differentially expressed (fold-change ≥2) probe sets. The probes were mapped to their genes to identify over-represented canonical pathways. Results: 873 different phosphoproteins were identified among the 4 AML cell lines. Nearly 3,000 phosphopeptides were identified (868 in Kasumi-1, 861 in KG-1a, 681 in MV-4-11, and 917 in UT-7epo), of which 2,600 were matched to known sites in iPTMnet and nearly 200 identifications were novel phosphosites. 580 phosphosites were previously reported as known kinase targets (e.g., PRKCB, CDK2 and AKT1). From the top five most significant pathways identified by IPA, Telomerase Signaling was significant in 4 cell lines. DNA Methylation and Transcriptional Repression Signaling was significant in 3 out of 4 cell lines. The numbers of differentially expressed probe sets were: 1,788 mapped to 99 pathways in Kasumi-1; 1,310 mapped to 66 pathways in KG-1a cells; 2,699 mapped to 191 pathways in MV-4-11 cells; and for UT-7epo cells, 2,778 differentially expressed probe sets were mapped to 147 over-represented pathways. Conclusions: We identified phosphoproteomic signatures in AML subtypes. Ongoing studies are directed towards similar profiling of bone marrow of 40 AML patients. Phosphoproteomic profiling will greatly contribute to (1) stratify patients into risk groups, (2) better select targeted therapy based on activated protein pathways, and (3) better predict response to therapy.

#2706

Effects of germline and somatic mutations on protein expression in tumor and adjacent normal tissues in breast, ovarian, and colorectal tumors.

Matthew H. Bailey,1 Daniel C. Zhou,1 Yige Wu,1 Matthew A. Wyczalkowski,1 Liang-Bo Wang,1 Fernanda Martins Rodrigues,1 Gordon Mills,2 Samuel Payne,3 David Fenyo,4 Li Ding1. 1 _Washington University in St. Louis, St. Louis, MO;_ 2 _The University of Texas MD Anderson Cancer Center, Houston, TX;_ 3 _Pacific Northwest National Laboratories, Richmond, WA;_ 4 _New York University, New York, NY_.

The National Cancer Institute's Clinical Proteomic Tumor Analysis Consortium (CPTAC) provides proteomic data that accurately quantify protein expression in tumors. This work improves upon previous estimates of expression, i.e., RNA-seq and or microarray, because it directly measures protein quantity using mass spectrometry. These data are critical for establishing more accurate estimates of a mutation's impact on tumor cell microenvironment and molecular processes. Here we address the association of both germline and somatic mutations with protein expression in three cancer types: breast cancer (BRCA), ovarian cancer (OV) and colorectal cancer (CO). Additionally, we analyze both primary tumors and adjacent normal pairs that provide insight into tumor etiology. CPTAC2 prospective proteome and phosphoproteome established a dataset of ~450 samples. For analysis we require samples to have reliable protein and phosphoprotein measures. This filtering strategy resulted in a dataset with measurements for 8,000-12,000 proteins and over 30,000 phosphosites for these three cancer types. We first performed analysis on tumors with likely predisposition germline mutations. We defined germline-predisposed samples as tumor samples with germline mutations in one or more of the following DNA repair genes: BRAC1/2, MSH2/6, PMS2. When analyzing samples with and without germline predisposition, an altered protein expression profile was found, albeit less extreme. We observed overexpressed genes in predisposed samples that include C9orf16, PRDX5, SERPINB8, and CMPK1, and found genes to have lower expression that include TULP1, MAEL, KMT2B, and HIST1H1D. Additionally, we performed differential gene expression analysis using samples with adjacent normal tissue biopsies. We examined both the proteome and phosphoproteome levels, comparing tumor vs. adjacent normal samples, with and without germline predisposition mutations. Preliminary analysis for BRCA between tumor and normal samples showed altered protein expression profile in tumors, with about 60% of the genes showing higher or lower protein expression in the tumor. This pattern is recapitulated when restricting our analysis to known cancer driver genes. Genes found to be overexpressed in tumor samples include GNB1, SERPINA1, CDKN2C, IGF1, ERG, AZGP1, and H3F3A, while genes found to have lower expression include PRKDC, DDX5, NUP93, CTCF, ATRX, MYD88, SMARCA4, KDM6A, SF3B1, and MED12. Proteomic/phosphoproteome data deliver reliable results on both cis and transmutational effects on protein expression at both the germline and somatic level. Furthermore, these data provide a glimpse into the tissue microenvironment of adjacent normal tissue and indicate biologic stresses of germline mutations on tissues.

#2707

Integrated analysis of colorectal carcinoma by co-extraction of RNA, DNA and protein from FFPE tumor samples.

Vishal Patel, Ji Gao, Mingyi Liu, Aiqing He, Xi-Tao Wang, Kathryn Vanderlaag, Ariella Sasson, Stefan Kirov, Sunil Kuppasani, Omar J. Jabado, Kandasamy Ravi, Ashok Dongre, Julie Carman, Heidi LeBlanc. _Bristol-Myers Squibb, Princeton, NJ_.

Background: Integrated analysis of multi-omic data is important in understanding oncogenesis, pathology, and drug mechanism of action. Formalin-fixed, paraffin-embedded (FFPE) tissues comprise the bulk of archival specimens in hospitals and clinical trials. Commercial kits are commonly used to extract RNA, DNA, or protein for biomarker studies; however, high-quality extractions are challenging due to crosslinking. We explored the feasibility of performing proteomic, transcriptomic, and genetic analysis from limited FFPE samples collected in clinical trials through a pilot study in colorectal cancer (CRC) and normal tissue.

Methods: Qiagen kits were employed, with some modifications, to extract nucleic acid and protein from FFPE sample lysates that would be amenable to next-generation sequencing and liquid chromatography/mass-spectrometry (LC/MS) proteomic profiling. Commercially sourced FFPE slides from CRC biopsies and normal colon, heart, and skin samples were processed (n = 10 each; ~600-mm2 by 5-μm/slice). RNA-seq library preparation was performed using the Illumina® TruSeq Access kit. Label-free LC/MS proteomic analysis was carried out using trypsin/Lys-C-digested protein fragments on an EASY-nLC™ 1200 coupled to a Q Exactive™ Plus (Thermo Fisher). MS data were processed with MaxQuant to estimate protein abundance (Cox and Mann, Nat Biotech 2008).

Results: Of 40 samples tested, 38 yielded quantifiable nucleic acid and protein of sufficient quality for transcriptional and proteomic analyses. Mean RNA yield was ~300 ng (150 ng-1.2 µg). RNA degradation was significant, with a mean DV>200 of 45% (Agilent Bioanalyzer). Mean DNA yield was 530 ng, with a mean fragment size of 2 kb. Mean protein yield was 50 µg (1 µg-300 µg). RNA libraries had a mapping rate range of 85%-90% and a coding rate range of 70%-80%; ~16,000 genes were reliable detected (log2 TPM > 1). The number of unique proteins detected ranged from 3,000 to 4,000 for CRC, normal colon, and heart samples; skin samples averaged 1,000 proteins and had the lowest overall yield. Correlation of RNA and protein expression for the same genes was weak (Spearman's rho ~0.3-0.4) at the per-sample level but statistically significant. Using linear models, we identified differentially expressed transcripts and proteins between the CRC and normal colon samples. Pathway enrichment analysis of both modalities indicated changes in cell cycle, which is consistent with rapid growth of tumor cells. Changes in nucleoside metabolism, extracellular matrix remodeling, and innate immune response were more apparent at the protein level.

Conclusion: We found that multi-omic analysis was feasible with FFPE samples, and proteomics can be used to validate RNA results. Additionally, proteomics reveal post-translational events, such as extracellular matrix remodeling, that provide unique insights into cancer pathology.

#2708

Imputation-free analysis of high throughput TMT proteomics of 116 lung squamous samples.

Eric A. Welsh, Paul A. Stewart, Matthew C. Chambers, Guolin Zhang, Bin Fang, Steven A. Eschrich, John M. Koomen, Eric B. Haura. _Moffitt Cancer Center, Tampa, FL_.

Introduction: Chemical labeling of peptides using tandem mass tags (TMT) is a "barcoding" strategy, enabling relative protein quantification across a single panel of samples (as opposed to each run separately). Each multiplex assay is, effectively, its own "batch" of samples, and thus direct comparison of intensities between TMT multiplexes is problematic. Additionally, although there is relatively little missing data within a single plex, there can be large differences in missingness across plexes, with the two types of missingness exhibiting different behavior (infrequent and biased towards low abundances within-plex; more frequent and more stochastic between-plex). We have addressed these issues by developing new pipelines for data normalization, protein-level rollup, and downstream clustering, which seek to minimize the negative impact of missingness. This method development was driven by, and applied to, a set of 116 human lung squamous (SQLC) tumors, with the aim of improving the strength of down-stream biological signal and interpretation.

Experiment: TMT analysis was performed on 116 SQLC samples. Each 6-plex contained 4 tumors and 2 pool replicates. The shared pool of 116 tumors was assayed on every multiplex to allow for controlling for variability between plexes, with one pool in ch-126 and the other varying channel between plexes. IDPicker was used for spectral quantification. Spectra abundances were normalized within-plex, and ratios calculated for each channel against the ch-126 pool. Spectra-level ratios were rolled up into protein-level ratios using the geometric mean of ratios within each protein group. Geometric mean protein-level abundance rollup was performed on abundances for each ch-126 pool, normalized across pools, and the geometric mean calculated for each protein group across pools. These mean protein-level abundances were then used to scale the ratios back into final normalized abundances. Average linkage hierarchical clustering was performed on abundance z-scores using a novel distance metric, calculated as the root mean squared deviation (RMSD) of points present in both vectors, divided by a binary presence/absence similarity coefficient such as Ochiai similarity.

Results: After normalization, principal component analysis showed no batch effect due to differences between plexes. Heat maps generated using the novel distance metric exhibited improved biological signal over RMSD alone. Tumors cluster into 3 major groupings: high immune + low transcriptional/translational activity, low immune + high transcriptional/translational activity, and samples with medium levels of both.

Conclusion: Missingness-aware methods of shared-pool TMT normalization and clustering minimize the negative impact of missingness and yield strong biological signal. Preliminary results suggest that immune response is a major source of differences between lung squamous tumors.

## IMMUNOLOGY:

### Immune Checkpoints 2

#2709

TTI-622 (SIRPα-IgG4 Fc), a CD47-blocking innate immune checkpoint inhibitor, suppresses tumor growth and demonstrates enhanced efficacy in combination with antitumor antibodies in both hematologic and solid tumor models.

Gloria H. Y. Lin,1 Natasja N. Viller,1 Marilyse Chabonneau,1 Laura Brinen,1 Tapfuma Mutukura,1 Karen Dodge,1 Simone Helke,1 Vien Chai,1 Violetta House,1 Vivian Lee,1 Hui Chen,1 Alison O'Connor,1 Debbie Jin,1 Rene Figueredo,2 Saman Maleki Vareki,2 Mark Wong,1 Emma Linderoth,1 Lisa D. Johnson,1 Xinli Pang,1 James Koropatnick,2 Jeff Winston,1 Penka S. Petrova,1 Robert A. Uger1. 1 _Trillium Therapeutics Inc., Mississauga, Ontario, Canada;_ 2 _Lawson Health Research Institute, London, Ontario, Canada_.

Tumor cells frequently evade macrophage-mediated destruction by increasing cell surface expression of CD47, which delivers an anti-phagocytic ("do-not-eat") signal by binding the inhibitory signal regulatory protein α (SIRPα) receptor on macrophages. Previous studies have shown that blockade of the CD47-SIRPα pathway using TTI-621, a soluble SIRPα-IgG1 Fc fusion protein, triggers macrophage phagocytosis of tumor cells in vitro, and potently inhibits tumor growth in vivo. In the current study, the in vitro and in vivo efficacy of TTI-622, a soluble SIRPα-Fc variant protein containing an IgG4 Fc tail, was evaluated in multiple model systems.

Unlike CD47-blocking antibodies, TTI-622 binds minimally to human erythrocytes, and does not induce hemagglutination in vitro. Therefore, it avoids a large circulating antigen sink, and is less likely to cause anemia in patients. Additionally, TTI-622 potently induces phagocytosis of a broad panel of tumor cells derived from patients with both hematological and solid tumors. Although in vitro phagocytosis of human platelets is also observed, TTI-622 preferentially induces phagocytosis of tumor cells over platelets in a competitive phagocytosis assay.

The in vivo efficacy of TTI-622 monotherapy and/or combination therapy was evaluated in different tumor models. In a DLBCL (Toledo) xenograft tumor model, both early and delayed treatments resulted in statistically significant decreases in tumor growth, and improved survival relative to treatment with control Fc. In the Burkitt lymphoma (Daudi) and multiple myeloma (MM.1S) xenograft tumor models, the potential of combining TTI-622 with daratumumab (anti-CD38 antibody) was also explored. In both models, TTI-622 monotherapy demonstrated partial tumor growth inhibition. However, the therapeutic efficacy was further enhanced when TTI-622 was combined with daratumumab. Intriguingly, a TTI-622 non-responsive head and neck cancer (FaDu) xenograft tumor model became responsive by combining TTI-622 with suboptimal doses of cetuximab (anti-EGFR antibody). The combination of TTI-622 with cetuximab resulted in a statistically significant decrease in tumor growth, and improved survival relative to monotherapy treatments.

Collectively, these results demonstrate that TTI-622 induces potent, tumor-specific macrophage phagocytosis across a range of hematological and solid tumors, and is efficacious as a monotherapy agent in a DLBCL xenograft tumor model. Furthermore, TTI-622 potentiates the efficacy of daratumumab and cetuximab in hematological and solid xenograft tumor models, respectively. These data support the clinical evaluation of TTI-622 in combination with anti-tumor antibodies in cancer patients with both solid and hematological malignancies.

#2711

Development of a new innovative multifunctional immune checkpoint inhibitor.

Hiroki Nagase,1 Keiko Fukushima,2 Asuka Hattori,1 Mayu Shinohara,1 Atsushi Takatori,1 Takayoshi Watanabe,1 Nobuko Koshikawa,1 Takahiro Inoue,1 Jason Lin,1 Yoshinao Shinozaki1. 1 _Chiba Cancer Ctr. Research Inst., Chiba, Japan;_ 2 _Zenyaku Kogyo Co., Ltd, Tokyo, Japan_.

Blockade of PD-1, PD-L1 or CTLA-4 is an attractive strategy for Immuno-Oncology (I-O) therapy. Moreover, combination approaches, which inhibit non-redundant pathways of PD-1/PD-L1 and CTLA-4, have been reported to show enhanced anti-tumor activity as well as improved progression-free survival in clinical trials of melanoma. The use of immune checkpoint blockade (ICB) drugs, however, can lead to severe or life-threatening complications of autoimmune disorders such as diabetes, thyroiditis, pulmonary fibrosis and vitiligo. Therefore, new innovations are critical to overcome possibilities of unintended immunoactivation and minimize drug cross-reactions and complications. Here, we designed and synthesized a novel alkylating Pyrrole-Imidazole (PI) polyamide with indole-seco-CBI capable of disrupting expressions of human PD-1, PD-L1 and CTLA-4 genes (CCC07-01) by binding to a common motif. Our initial evaluation process confirmed that the induction of tumor- and tumor-environment-specific combinatorial ICB therapy was possible by only a single agent. CCC07-01 administration significantly suppressed PD-1, PD-L1 and CTLA-4 mRNA and protein expressions at low nanomolar doses (15, 10, 5 nM respectively) in several cell lines. Furthermore, logP characterization experiments found CCC07-01 to be sufficiently lipophilic that enhanced the molecule's capability to accumulate and be retained in tumor tissues. Additionally, elevated doses of CCC07-01 induced substantial cytotoxicity in SW480, RKO and A2058 cancer cells (IC50=27, 12 and 11 nM, respectively) but primary cells derived from normal human tissues were not affected. These results illuminated the therapeutic possibility of CCC07-01 through, at low doses, its anti-tumor effect in tumor microenvironments and generalized cytotoxicity at increased dosage levels. CCC07-01's enhanced retention in tumor may also contribute to lower risks of triggering adverse autoimmune events compared to other ICB approaches. Preliminary results from human RKO colon carcinoma xenograft NGS mice, following the engraftment of human HLA-matched peripheral blood mononuclear cells (PBMC), showed significant tumor reduction 14 days after the injection of CCC07-01 (2.3 mg/kg, p < 0.05) and displayed detectable amounts of predominant effector memory CD8 T cells in the spleen 14 days post-administration (9.2 mg/kg, p < 0.05). The approach of simultaneously targeting elements in immune checkpoints, as demonstrated by CCC07-01, can lead to new therapeutic opportunities and reduce healthcare costs by curbing undesired autoimmune complications. Together with Zenyaku Kogyo, we envision a breakthrough in the field of current antibody-based ICB therapy as we continue to evolve and improve the use of PI polyamides as a viable candidate for ICB.

#2712

Naptumomab Estafenatox induces T cell recognition, turning anti-PD-1 unresponsive "cold" tumors into "hot" responsive tumors.

Meir Azulay,1 Sveta Lifshits,1 Adam Fridman,1 Gunnar Hedlund,1 Marie Törngren,2 Michal Shahar1. 1 _NeoTX Therapeutics LTD, Rehovot, Israel;_ 2 _Active Biotech, Lund, Sweden_.

Tumor recognition is a key factor in checkpoint inhibitors (CPI) efficacy and acquired resistance. Lack or loss of tumor antigens expression or inefficient presentation prevents tumor cell recognition by T cells, inhibiting CPI anti-cancer effect. Naptumomab estafenatox (Nap) is a tumor-targeted superantigen (TTS) protein that increases tumor recognition by both coating tumor cells with bacterial-derived superantigens (SAg) as well as selectively expanding T cells lineages that can recognize it. Nap consists of a genetically engineered SAg, staphylococcal enterotoxin A (SEA/E-120), linked to a fragment antigen binding (Fab) moiety directed to the 5T4 oncofetal tumor-associated antigen expressed on many tumors. Here we present new pre-clinical results of synergistic anti-tumor effect of Nap (or its murine equivalent TTS) with anti-PD1, under conditions which mimic poor tumor recognition. In vitro studies with Nap investigated the effect of co-culturing SAg activated T cells with HCC827 NSCLC or MDA-MB 231 Triple-Negative Breast Cancer cells. Co-cultures of T cells with either cancer cell line resulted in an increase in tumor staining for PDL1, which was further elevated in the presence of Nap, probably due to increase in IFNꝩ secretion. The viability of HCC827 or MDA-MB-231 cells co-cultured with the SAg activated T cells was examined in the presence or absence of the PD1 inhibitor, Nap or the combination. Anti-PD1 alone had no effect on T cell mediated cytotoxicity, whereas Nap induced a significant cytotoxic effect, with the combination of anti-PD1 plus Nap producing the most significant reduction in tumor cell viability. Similar to the in vitro studies, the in vivo mouse studies using low immunogenic tumor models showed limited or no effect of anti-PD1 monotherapy. Although TTS alone increased T cells activation and infiltration into the tumor, the combination with anti-PD1 was significantly more effective in increasing serum cytokines, increasing the CD8:CD4 ratio in the tumor, reducing tumor burden and prolonging median survival. In summary, our studies show that combination of anti-PD1 with tumor-targeted superantigens overcomes the limited effect of anti-PD1 monotherapy in low immunogenic tumor models in vitro and in vivo. This novel combination may offer clinical benefit for CPI unresponsive patients, particularly in cases where tumor recognition is lost or restricted. Clinical phase 1-2 trials are planned with the combination of naptumomab estafenatox and anti-PD1/PDL1 blockade.

#2713

Preclinical development of first-in-class antibodies targeting Siglec-9 immune checkpoint for cancer immunotherapy.

Olivier Bénac,1 Marion Gaudin,1 Mélody Ors,1 Aude Le Roy,2 Hélène Rispaud Blanc,1 Caroline Soulas,1 Stéphanie Chanteux,1 Benjamin Rossi,1 Laurent Gauthier,1 Carine Paturel,1 Yannis Morel,1 Ivan Perrot,1 Stéphanie Cornen1. 1 _Innate Pharma, Marseille, France;_ 2 _CRCM, Inserm, U1068; Institut Paoli-Calmettes; Aix-Marseille Université, UM 105; CNRS, UMR7258, F-13009, Marseille, France_.

Siglec-9 is a MHC class I-independent inhibitory receptor expressed on NK and myeloid cells (including dendritic cells, monocytes and neutrophils). Its ligands are sialic acid-containing carbohydrates which are over-expressed on various tumor types compared to normal tissues (1). Sialylation of tumor cells is involved in tumor cell malignancy and is reported for decades as a mechanism of escape from immune surveillance (2). The loss of beta-2-microglobulin, an essential component of MHC class I antigen presentation, was recently described as a common mechanism of resistance to checkpoint blockade in clinical trials and revealed the need for MHC class I-independent therapies (3-4). Thus, Siglec-9-sialic acid interaction disruption may promote anti-tumor immunity independently of MHC class I expression by tumors. Here, we describe the discovery and characterization of first-in class anti-human Siglec-9 antibodies as new checkpoint blockade therapy in a wide range of cancers. Antibodies were discovered that efficiently block the interaction between Siglec-9 and its ligands. Epitope mapping revealed that antibodies bind to distinct epitopes on Siglec-9 near the sialic acid binding site. In vitro assays showed that they potently reverse inhibitory functions of Siglec-9 on NK cells leading to subsequent sialic acid-expressing tumor cell killing. Interestingly, Siglec-9 is enhanced on both CD4+ and CD8+ T cells from RCC, melanoma and NSCLC PBMC patients suggesting a putative additional role on adaptive immunity. Siglec-9 was also co-expressed with other inhibitory receptors on NK cells and combination with other immune checkpoint blockers in in vitro assays is ongoing. The antibodies displaying the most interesting features were successfully humanized. (1) Pathol Oncol Res. 2016 Jul;22(3):443-7 (2) Nature. 1968 Jun 29;218(5148):1254-5 (3) Nat Commun. 2017 Oct 26;8(1):1136 (4) N Engl J Med. 2016 Sep 1;375(9):819-29

#2714

Combination of monalizumab and durvalumab as a potent immunotherapy treatment for solid human cancers.

Caroline Soulas,1 Romain Remark,1 Vedran Brezar,1 Julie Lopez,1 Elodie Bonnet,1 Flavien Caraguel,1 Ana Lalanne,2 Caroline Hoffmann,2 Caroline Denis,1 Thomas Arnoux,1 Clarisse Caillet,1 Arnaud Dujardin,1 Guillaume Habif,1 Olivier Lantz,2 Cécile Bonnafous,1 Eric Vivier,3 Pascale Andre1. 1 _Innate Pharma, Marseille, France;_ 2 _Institut Curie, Paris, France;_ 3 _Centre d'Immunologie de Marseille Luminy, Marseille, France_.

Inhibitory CD94-NKG2A (Natural Killer Group 2A) receptors are expressed on subsets of natural killer (NK) cells, γδ and CD8+ T cells. HLA-E (Human Leukocytes Antigen-E), the ligand for CD94-NKG2A, is upregulated on cancer cells of several solid tumors, providing a negative regulatory signal to tumor-infiltrating lymphocytes (TILs). Blockade of this immune checkpoint pathway with anti-NKG2A monoclonal antibodies (Ab) enhances NK cell responses to tumor cells in vitro and in humanized mice. Here, we describe NK and CD8\+ T cell infiltrates in several human solid tumors by immunohistochemistry (IHC) and multicolor flow cytometry. We then studied the effects of in vitro targeting both pathways on primary human NK and CD8+ T cells. By using IHC on formalin-fixed paraffin-embedded samples on cohorts of solid tumors i.e. non-small cell lung cancer (NSCLC, n=45), stomach tumor (n=76), colorectal cancer (CRC, n=103), head and neck tumor (HNSCC, n=68), pancreatic tumors (n=77) and renal cell carcinoma (RCC, n=75), we observed NKp46+ NK cells in the large majority of cancer types (especially in RCC, HNSCC and stomach cancers). CD8+ T cells were found at significantly higher densities in all tumor types. In another cohort of HNSCC samples (n=60), higher proportions of CD94+ lymphocytes were found within the tumor islets both in the primary tumors and in the metastatic lymph nodes. In addition, in CRC patients with liver metastasis (n=101), high CD94+ cell densities were associated with poor overall survival. These data suggest that NKG2A blockade might unleash NK and T cells that are present in close contact to tumor cells. As previously shown, we confirmed that CD94-NKG2A ligand, HLA-E, was expressed by tumor cells in the large majority of solid tumor. In addition, high PD-L1 expressions were observed in NSCLC, stomach tumors, CRC and HNSCC. Flow cytometry analyses revealed that NK and CD8+ T cells co-expressing NKG2A and PD-1 were more numerous in tumor compared to matched peripheral blood and adjacent tissues of NSCLC and HNSCC patients. To mimic the "NKG2A+PD-1+" TILs phenotype, chronic stimulation of peripheral blood cells from healthy volunteers was performed using IL-15. The conditions led to the induction of NKG2A and PD-1 on NK and antigen-specific CD8+ T cell subsets. These effector cells were then co-cultured with tumor cell lines expressing HLA-E +/- PD-L1. We showed that monalizumab, a first-in class anti-NKG2A Ab, combined with durvalumab resulted in higher responses (CD107 mobilization and IFN-γ production) compared to each Ab alone. These data were confirmed in a syngeneic mouse model where both NK and CD8+ T cells were found to be involved in tumor rejection. Together, these data confirm that blocking NKG2A can potentiate the anti-tumor efficacy of PD-1 inhibitors and support the rationale for ongoing clinical trials investigating the monalizumab/durvalumab combination (NCT02671435 and NCT03088059).

#2716

**Development of 4-1BB agonists: Ligand fusion shows greater anti-tumor activity than antibodies** in vivo **.**

Eric Tam, Galina Craig, Sara Ghassemifar, Alexander Koshkaryev, Sara Movassaghian, Cormac Cosgrove, James Sampson, Daryl Drummond, Andreas Raue. _Merrimack Pharmaceuticals, Inc., Cambridge, MA_.

Members of the tumor necrosis factor (TNF) superfamily of costimulatory receptors have emerged as promising targets for modulation of tumor infiltrating lymphocytes. Among these, 4-1BB (CD137) has a prominent role in promoting survival and expansion of cytotoxic CD8 T cells as well as activation of CD4 T cells, NK cells, and antigen presenting cells. The clinical relevance of 4-1BB agonism is currently being evaluated for two antibodies: urelumab (IgG4) and utomilumab (IgG2). However, it has been established that for the trimeric TNF receptor family, the homotrimeric ligands are superior activators compared to bivalent agonistic antibodies. Here we describe a prototype 4-1BB agonist consisting of three human 4-1BB ligands linked together as a single polypeptide chain and fused to the C-terminus of human IgG1 Fc (STIM-41BBL). Compared to a clinically relevant human 4-1BB antibody, STIM-41BBL shows increased activation of 4-1BB in a NF-kappa B reporter cell line. STIM-4-1BBL also shows augmented cytokine release compared to the antibody in CD8 T cells. Furthermore, we have developed a mouse STIM-41BBL and like the human counterpart, the ligand fusion induced greater cytokine release than an agonistic mouse 4-1BB antibody (IgG2a) in CD8 T cells. We have also introduced mutations (D265A/N297A) to the mouse 4-1BB antibody to make it effectorless in order to better approximate the clinical 4-1BB antibodies. Subsequently, we demonstrate that the murine STIM-41BBL had greater anti-tumor activity than the 4-1BB antibody in a MC38 tumor model. These results support the continued development of a 4-1BB ligand based therapeutic and suggests the greater potential of a STIM-41BBL fusion to a targeting antibody for more precise activation of immune system.

#2717

Antitumor effects are augmented in mouse models of interstitial-lung disease caused by immune-checkpoint inhibitors.

Masashi Arita, Satoshi Watanabe, Naohiro Yanagimura, Yuko Mishina, Miyuki Sato, Satoshi Shoji, Kosuke Ichikawa, Rie Kondo, Junta Tanaka, Takuro Sakagami, Toshiyuki Koya, Toshiaki Kikuchi. _Niigata University, Niigata City, Japan_.

In recent years, cancer immunotherapy has been emerged as a major therapeutic modality in several malignancies. Immune-checkpoint inhibitors (ICI), such as anti-programmed death 1 (PD-1) / programmed death -ligand 1 (PD-L1) antibodies and anti-cytotoxic-T-lymphocyte associated protein-4 (CTLA-4) antibodies have demonstrated durable antitumor effects. In phase III trials, PD-1 / PD-L1 blockade therapies have shown better survival benefits compared to standard chemotherapies in patients with non-small lung cancer. However, the mechanisms of anti-PD-1 treatments have not been fully understood. Immune-checkpoint molecules play an important role to regulate autoimmune reactions by T cells. Thus, PD-1 / PD-L1 blockade therapy and anti-CTLA-4 treatment can cause immune-related adverse events including interstitial-lung disease (ILD). ILD is a serious and life threatening adverse event. On the other hand, better outcomes have been reported in lung cancer patients who developed ILD during anti-PD-1 treatment. These findings suggest that effector T cells attack similar antigens presented on both tumor cells and normal lung tissues. In order to elucidate the mechanisms of development of ILD and augmentation of anti-tumor effect by ICI, we established mouse model of ILD. In this study, C57BL/6J mice were injected intravenously with MCA 205 fibrosarcoma or B16 melanoma to make lung metastases. These mice were administered intraperitoneally with anti-PD-1 antibodies and/or anti-CTLA-4 antibodies. EGFR-TKI-ILD mice, which have been previously reported elsewhere, were also established to compare the pathological findings with those of ICI-induced ILD. Also, we administered lipopolysaccharide (LPS) intratracheally before injection of ICIs to test whether lung inflammation exacerbate ILD caused by ICIs. The infiltration of inflammatory cells and thickening of interstitial wall in the lung tissue were observed in mice treated with the combination of anti-CTLA-4 antibodies and anti-PD-1 antibodies. The antitumor effects of anti-PD-1 treatment against lung metastases were enhanced in mice which had ILD caused by anti-PD-1 antibodies and injury of bronchial epithelium. High density of infiltration of lymphocytes in tumor tissues was observed in ILD mice. Further study is warranted to determine effector cells which are responsible for establishment of ILD and augmentation of antitumor effects.

#2718

Preclinical development of humanized CD39 and CD73 blocking antibodies targeting the ATP/adenosine immune checkpoint pathway for cancer immunotherapy.

Ivan Perrot,1 Marc Giraudon Paoli,1 Séverine Augier,1 Marilyne Royannez Blemont,1 Marion Gaudin,1 Frédéric Bosco,1 Rachel Courtois,1 Stephane Delahaye,1 Diana Jecko,1 Nicolas Gourdin,1 Maryline Salin Agu,1 Cyril Perrier,1 Paul Ricaut,1 Aurélie Docquier,2 Stéphanie Chanteux,1 Benjamin Rossi,1 Agnès Représa,1 Caroline Denis,1 Romain Remark,1 Cécile Bonnafous,1 Laurent Gauthier,1 Ariane Morel,1 Nathalie Bonnefoy,2 Jérémy Bastid,2 Yannis Morel,1 Carine Paturel1. 1 _Innate Pharma, Marseille, France;_ 2 _Orega Biotech, Ecully, France_.

Within the tumor microenvironment, adenosine causes immune suppression and dysregulation of immune cell infiltrates resulting in tumor spreading. Adenosine accumulation results from the hydrolysis of extracellular immunoactivating ATP and ADP into AMP by the CD39 (ENTPD1) ectonucleotidase. AMP can be further hydrolyzed into immunosuppressive Adenosine (Ado) by CD73 (NT5E), a cell membrane ectonucleotidase of the NTPDase family.The immunosuppressive role of CD39 expressed on both Tregs and tumor cells has been demonstrated in several reports. Conversely, CD73 expression in the tumor environment has been associated with poor disease outcome and/or with a pro-metastatic phenotype. Blockade of CD39 and CD73 may promote anti-tumor immunity directly by accumulating immunostimulating ATP for CD39 and indirectly by reducing adenosine accumulation for both targets.In cancer tissue, using IHC and flow cytometry analyses, we observed that while CD73 is often expressed by tumor cells, CD39 is more frequently up-regulated on tumor infiltrating cells compared to PBMC or adjacent non-tumor tissue. We next describe the discovery and preclinical development of a unique anti-huCD39 blocking antibody and of an anti-human CD73 antibody for cancer immunotherapy. These anti-CD39 and anti-CD73 antibodies specifically bind with high affinity to huCD39 and huCD73 proteins, respectively. They potently inhibit enzyme activity of their respective targets under their soluble and membrane-associated forms, without inducing down-modulation of these enzymes expressed at the cell surface. Innate's antibodies efficiently reverse Ado-mediated T cell suppression in vitro in presence of ATP and both CD39- and CD73-expressing immune cells and additionally exhibit unique features. The anti-CD39 Ab maintains high concentration of ATP in the extracellular compartment that enhances DC activation and subsequent T cell proliferation in vitro. The anti-CD73 blocking Ab exhibits a more potent ability to block soluble and membrane-associated CD73 enzyme activity than benchmark Abs currently in clinical development. Finally, in vivo blockade of ATP/Ado pathway in CD39ko mice resulted in improved anti-tumor efficacy of immunogenic cell death inducer chemotherapy and of immune checkpoint therapies, including PD1 and CTLA4.Taken together, these data support the clinical development of anti-CD39 and anti-CD73 neutralizing Abs for cancer immunotherapy, potentially in combination with chemotherapy or Immune Checkpoint therapy. The humanized anti-huCD39 and anti-huCD73 monoclonal antibodies are currently in preclinical development.The research leading to CD73 results were obtained within the TumAdoR collaborative consortium that received funding from the European Community's Seventh Framework Program (FP7/2007-2013) under grant agreement n°602200.

#2719

Dual blockade of PD-L1 and LAG-3 with FS118, a unique bispecific antibody, induces CD8+ T-cell activation and modulates the tumor microenvironment to promote antitumor immune responses.

Matthew Kraman, Natalie Fosh, Katarzyna Kmiecik, Katy Everett, Carlo Zimarino, Mustapha Faroudi, Mateusz Wydro, Alexander Koers, Lesley Young, Daniel Gliddon, Michelle Morrow, Jacqueline Doody, Mihriban Tuna, Neil Brewis. _F-star Biotechnology Limited, Cambridge, United Kingdom_.

Background

Despite advances with therapies targeting the PD-1/PD-L1 pathway, many patients are refractory or relapse following treatment. LAG-3 expression on exhausted T cells and T-regulatory cells (Tregs) in the tumor may be responsible for this resistance and provides a rationale for co-treatment with antibodies targeting LAG-3 and PD-L1. An alternative approach is the development of a bispecific antibody encompassing binding sites for two antigens. FS118 is a bispecific antibody targeting LAG-3 and PD-L1 that provides dual pathway blockade with the potential to drive unique biology via co-binding of PD-L1 and LAG-3.

Methods

FS118 was evaluated in vitro for antigen binding and de-repression of LAG-3 and PD-L1 function in both a D011.10 T-cell activation system and a super-antigen stimulated peripheral blood mononuclear cells (PBMC) assay. FS118 was also assessed in a human CD8 specific MHC I restricted antigen recall assay. Anti-tumor activity of a murine-specific molecule, mLAG-3/PD-L1 mAb2, was evaluated in vivo in the MC38 mouse tumor model and associated immunophenotypic changes were evaluated using flow cytometry.

Results

In murine in vitro assay systems, mLAG3/PD-L1 mAb2 recapitulates the function of FS118 in human systems. Furthermore, FS118 was shown to provide increased activation of human CD8+ T-cells compared to a PD-L1 mAb alone in response to stimulation with MHC Class I restricted peptides. In vivo, studies performed in MC38 tumor-bearing mice studies indicated that mLAG-3/PD-L1 could result in significant anti-tumor activity equivalent to a combination of antibodies targeting LAG-3 and PD-L1. Pharmacodynamic assessment demonstrated changes in the immunophenotype of tumor-infiltrating lymphocytes in the tumor of mLAG3/PD-L1 mAb2 treated mice. These changes were observed in cohorts which received the anti-mouse LAG-3/PD-L1 mAb2 and revealed a both a loss of LAG-3 surface expression on CD4+ and CD8+ T cells, as well as an increase in the CD8:Treg ratio.

Conclusions

Dual blockade of LAG-3 and PD-L1 with a bispecific antibody results in T-cell activation at least comparable to a combination of antibodies targeting LAG-3 and PD-L1 in primary T-cell assays and murine tumor models. Taken together, the human PBMC based assays and mouse tumor model data demonstrate that a LAG-3/PD-L1 mAb2 can not only potently suppress the checkpoint inhibitor LAG-3 at the tumor site, but does this in part through stimulating a CD8+ T cell mediated response. These data provide evidence to support the rationale for clinical development of FS118, a LAG-3/PD-L1 mAb2, for the treatment of human cancers.

#2720

The CD47-blocking innate immune checkpoint inhibitor, TTI-621, triggers CD47-mediated tumor cell apoptosis.

Julia Bershadsky Izrailit, Natasja Nielsen Viller, Xinli Pang, Penka S. Petrova, Robert A. Uger, Jeff Winston, Emma Linderoth. _Trillium Therapeutics Inc, Mississauga, Ontario, Canada_.

CD47 is a cell surface glycoprotein of the immunoglobulin superfamily that is utilized by tumor cells to avoid immune destruction. Overexpression of the CD47 ligand on tumor cells provides a "do-not-eat" signal when bound to its receptor, signal regulatory protein α (SIRPα), on macrophages, resulting in inhibition of phagocytosis and tumor survival. TTI-621 is a soluble SIRPα recombinant fusion protein with an IgG1 Fc tail that triggers macrophage phagocytosis of tumor cells in vitro, and potently inhibits tumor growth in vivo. It is currently being evaluated in two clinical studies in patients with hematologic and solid cancers (NCT02663518 and NCT02890368). TTI-621's pro-phagocytic effect has been shown to be a result of blockade of the CD47-SIRPα pathway as well as activation of Fc gamma receptors (FcγR) on macrophages. However, engagement of CD47 has also been shown to directly induce apoptosis in tumor cells. Therefore, the objective of this study was to further examine the pro-apoptotic potential of TTI-621.

The induction of apoptosis in tumor cells by TTI-621 was evaluated using flow cytometry and protein expression assays. Ligating CD47 with immobilized TTI-621 efficiently induced apoptosis in malignant T-ALL and DLBCL cells but had no effect on apoptosis in normal peripheral blood mononuclear cells. Immobilized TTI-621 or anti-CD47 mAb promoted caspase-independent but PLCγ-1-dependent apoptosis. Under conditions of cellular stress, both soluble and immobilized TTI-621 induced apoptosis in T-ALL cells. In contrast to immobilized TTI-621, treatment of stressed tumor cells with soluble TTI-621 triggered caspase-dependent but PLCγ-1-independent apoptosis. In addition, anchoring the IgG1 Fc tail of TTI-621 to FcγR-expressing cells enhanced tumor cell apoptosis in a dose-dependent and CD47-dependent manner.

These results suggest that in addition to blocking the anti-phagocytic "do-not-eat" signal on tumor cells and activating FcγR on macrophages, binding of TTI-621 to FcγRs on tumor-infiltrating immune cells may provide a cross-linking scaffold to enhance TTI-621-mediated apoptosis via CD47 on cancer cells. Additionally, under conditions of cellular stress, TTI-621 may induce tumor cell death directly via binding to CD47 on malignant cells, in a cell-autonomous, cytotoxic mechanism. Thus, TTI-621 may employ multiple mechanisms to elicit its anti-tumor effects.

#2721

Selective FcγR engagement by CTLA-4 antibodies results in increased functional activity.

Jeremy D. Waight,1 Dhan Chand,1 Sylvia Dietrich,1 Randi Gombos,1 Thomas Horn,1 Ana M. Gonzalez,1 Mariana Manrique,1 Antoine Tanne,1 Christopher Dupont,1 Lukasz Swiech,1 Ben A. Croker,2 Jennifer S. Buell,1 Robert Stein,1 Alex Duncan,1 David A. Savitsky,1 Nicholas S. Wilson3. 1 _Agenus, Lexington, MA;_ 2 _Boston Children's Hospital, Harvard University, Lexington, MA;_ 3 _Gilead Sciences, Inc, Foster City, CA_.

Therapeutic antibodies targeting T cell co-inhibitory pathways, such as cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed cell death protein-1 (PD-1), have emerged as an important class of cancer therapies. Insights into how different IgG isotypes modulate biological activities of antibodies have opened new avenues to enhance their therapeutic effects. For example, Fc-FcγR interactions have been shown to enhance antibody-directed effector cell activities, as well as antibody-dependent forward signaling into target cells via receptor clustering. Here, we describe a novel FcγR-dependent mechanism for antibodies targeting CTLA-4. Our findings suggest that selective Fc-FcγR binding dramatically improves the quality of the immune synapse, which in turn modifies apical T cell receptor signaling events to increase effector T cell activity. Our data also suggest that subsets of antigen-presenting cells (APCs), expressing FcγRIV in mice and FcγRIIIA in humans are important mediators of this effect. Importantly, we find this mechanism to be independent of regulatory T cell (Treg) depletion. Altogether, we describe a novel mechanism of action that provides a foundation for a new class of Fc-engineered antibodies to enhance antitumor immune responses.

#2722

Investigation of the expression profile and functional role of PD-1, TIM-3 and LAG-3 in human tumors.

Srimoyee Ghosh, Jon Travers, Kristen McEachern, Sujatha Kumar, Sridhar Ramaswamy, David Jenkins. _TESARO Inc., Waltham, MA_.

While immunotherapies directed against PD-1 and PD-L1 have proven effective across multiple indications, there is still a large unmet medical need for therapies for patients who do not respond or who develop acquired resistance during the course of treatment. There are several emerging hypotheses to explain the lack of response, one of which is the upregulated expression of additional checkpoint receptors, such as TIM-3 and LAG-3, that limit the ability of PD-1 pathway blockade to re-establish effective anti-tumor immunity.

To investigate the potential role of TIM-3 and LAG-3 relative to PD-1, we set out to evaluate the expression and function of these receptors in several systems. Firstly, we used a flow cytometry approach to enumerate immune cell populations in a panel of human tumor samples, including non-small cell lung cancer (NSCLC). In these samples, PD-1, TIM-3 and LAG-3 expression was observed in both T-cell and non-T-cell populations. PD-1 was found to be co-expressed with TIM-3 and LAG-3 in CD8+ T-cells and we also found expression of TIM-3 and LAG-3 in other cell types, including regulatory T-cells and myeloid cells.

Building on the expression data, we explored the functional effects of targeting these receptors, evaluating the effect of single agent and combination effects of anti-PD-1, anti-TIM-3 and anti-LAG-3 antibodies in mouse models. In these studies, we found not only that dual-blockade of PD-1 with either anti-TIM-3 or anti-LAG-3 resulted in improvements in efficacy over monotherapy, but also that the triple combination of all 3 inhibitors was associated with further anti-tumor activity. In a humanized mouse model of NSCLC, the triple combination was associated with pharmacodynamic effects not only in T-cells, but also reductions in Tregs and macrophages.

Taken together, these studies provide further evidence to suggest that in addition to PD-1, LAG-3 and TIM-3 are important emerging immunotherapy targets and provide rationale for not only doublet but also triplet combinations as an approach to cancer therapy.

#2723

In vivo activity of combinations of cytotoxic regimens with anti-PD1 and anti-PDL1 in various syngeneic cancer models.

Chloé Grasselly,1 Morgane Denis,1 Aurore Bourguignon,1 Nolan Talhi,2 Anne Tourette,2 Doriane Mathe,2 Laurent Serre,3 Lars Petter Jordheim,1 Charles Dumontet1. 1 _Anticancer Antibodies, CRCL, INSERM U1052, CNRS UMR 5286, CLB, UCBL, Lyon, France;_ 2 _Antineo, Lyon, France;_ 3 _Univ Lyon, Université Claude Bernard Lyon 1, INSERM 1052, CNRS 5286, Centre Léon Bérard, Centre de recherche en cancérologie de Lyon, Lyon, France_.

Immune checkpoint inhibitors (ICIs) show impressive response rate in multiple cancer types. Nivolumab and atezolizumab has already been approved by the FDA. However, a large proportion of patients do not show any benefits or will develop acquired resistance phenomenon. In order to increase long term survival, alternative strategies are focused on rationale combination of immunotherapies with well-known therapeutic agents such as chemotherapies. In this present project, we described the impact of various chemotherapies regimen in combination with anti-PD1 and anti-PDL-1 on preclinical bladder, colorectal and metastatic breast cancers models.

We performed a study of PD-1 or PDL-1 blockade in combination with reference chemotherapies in four fully immunocompetent mouse models of cancer: we used the MVAC (methotrexate, vinblastine, doxorubicin, cisplatin) regimen as standard chemotherapy in bladder cancer MBT-2 and MB49, cyclophosphamide and doxorubicin in breast cancer 4T1, and capecitabine with oxaliplatin in colorectal cancer MC38. We analyzed the in vivo antitumor response and collected tumor samples four days after first treatment administration to analyze the immune infiltrate in tumors.

In vivo phenotype study reveals variable responsiveness to ICP blockade. We found strong effects of the combinations in two of the models studied: bladder cancer MB49 and colorectal cancer MC38, a lack of effect in breast cancer 4T1, and an inhibition of immune checkpoint antibody activity in MBT-2 preclinical model. Analysis using flow cytometry showed strong differences both at baseline between mice models as well as after exposure to ICIs, chemotherapy or the combination of both types of treatment. All the preclinical models studied at baseline were highly infiltrated with cells considered as « immunosuppressive », particularly MDSCs. We showed that the balance between antitumor effector cells and immunosuppressive cells could be inverted with some of the treatment combinations. As an example, in the MB49 model the most impressive modifications induced by the combination was an increase of the M1/M2 ratio. However, profound alterations of tumors microenvironment were not systematically correlated with a favorable in vivo phenotype. Indeed, we observed no benefit of the combination in the 4T1 model, although the combination profoundly modified the immune microenvironment with a strong decrease in MDSCs and a large increase in the CD4 and CD8 infiltrates.

Our results suggest that modulation of immune cells infiltrate by cytotoxic agents could impact on the potency of ICIs when these are co-administered, with either an enhance or a reduced antitumor activity of the combination. However this impact appears to be model- and combination-dependent. The present investigations should help to select appropriate chemotherapies to combine with target monoclonal antibodies for clinical trials.

#2724

YIV906 (PHY906) enhanced the antitumor activity of immune checkpoint blockade therapy: Anti-PD1 against liver cancer.

Wing Lam,1 XiaoChen Yang,1 Zaoli Jiang,1 Xue Han,1 Fulan Guan,1 William Cheng,1 Shwu-Huey Liu,2 Lieping Chen,1 Yung-Chi Cheng1. 1 _Yale Univ. School of Medicine, New Haven, CT;_ 2 _Yiviva Inc, New York, NY_.

Immune checkpoint blockade therapy has recently been recognized as a breakthrough in cancer treatment. Recently, FDA has approved Nivolumab (anti-PD1) for the treatment of hepatocellular carcinoma (HCC) patients previously treated with sorafenib. However, the percentage of patients with a complete response was only 1.9% that is much lower than Nivolumab used for treating melanoma. Diarrhea, nausea and fatigue are common side effects for HCC patients who received Nivolumab. YIV906(PHY906) is inspired by the Huang Qin Tang, which was first described in Chinese texts 1800 years ago for the treatment of numerous gastrointestinal symptoms. Consistent preparations of PHY906 can be manufactured apart 10 years. Our clinical results suggest that YIV906 has potential to increase the therapeutic index of capecitabine or sorafenib for HCC patient by improving quality of life and prolonging life. In animal studies, YIV906 can increase the anti-tumor activity of a broad spectrum of chemotherapies through multiple mechanism of actions. We believe that YIV906 could have potential to improve the efficacy of Nivolumab, either by increasing its anti-tumor activity or decreasing its side effects. Here, we studied the effect of YIV906 on the anti-tumor activity of anti-PD1 using BDF1 mice bearing with Hepa 1-6 tumors. Results indicated that YIV906 alone had only moderate effects on the Hepa 1-6 tumor growth. Anti-PD1 alone could stabilize 80% tumor growth and caused 20% tumor shrinkage following 8-day injection. YIV906 plus anti-PD1 made all tumors disappear following 7-day treatment and no tumor rebound was found for one month. When YIV906 and different dosages of anti-PD1 ( 70ug or 200ug per animal) were used, YIV906 plus anti-PD1 (70ug) had an stronger anti-tumor effect than anti-PD1 (200ug) alone. The above results suggested that YIV906 could reduce the usage of anti-PD1 by 3-fold while having better antitumor effects than anti-PD1 alone. qRT-PCR results recovered that many genes related to M1-like macrophages and T cell activation were strongly up-regulated in the tumor tissues following YIV906 plus anti-PD1 treatment. More macrophages were found in tumor tissues associated with an upregulation of MCP1 protein following YIV906 plus anti-PD1 treatment. Biostatical analysis, based on the mRNA expression of M1 and M2-like macrophage signature genes, suggested the tumor microenvironment was favorable for M1 status following YIV906 plus anti-PD1 treatment. In addition, YIV906 could decrease LPS-induced PD1 protein of macrophages in culture. In conclusion, YIV906 might enhance the anti-tumor activity of anti-PD1 by changing the tumor microenvironment favorable to M1-like macrophages. Our results provide supportive information to initialize HCC clinical trial with YIV906 plus Nivolumab. This work was supported by grant (PO1CA154295-01A1) from NCI, USA. Dr. Y.-C. C is a fellow of NFCR, USA.

#2725

Galectin-9 drives TIM-3 mediated immune suppression.

Amit M. Deshpande, Rachel Fontana, Yue Zhang, Mary Ruisi, Dong Zhang, Tilo Senger, P. Alexander Rolfe. _EMD Serono, Billerica, MA_.

Purpose: Following recent Immunotherapy successes by targeting the checkpoints PD1, PD-L1, and CTLA-4, additional checkpoint inhibitors and their counter-structures are being explored as therapeutic targets to reverse T cell exhaustion and increase T-cell anti-tumor activity. One such inhibitory receptor, TIM3, has been reported to be expressed on severely dysfunctional T cells in preclinical cancer models and in multiple human cancers. While TIM3 expression is significantly upregulated on TILs, including exhausted CD8+ T cells and Tregs, constitutive expression of TIM3 on myeloid cells has also been shown to negatively regulate anti-tumor activity of T cells. Inhibitory signaling in T-cells is activated by engagement of TIM3 by Galectin 9 (GAL9, LGALS9) and putatively by ligands CEACAM1, HMGB1, and Phosphatidylserine. GAL9 has also been shown to induce T cell apoptosis in the TME. Given the complex nature of TIM3 biology, we sought to characterize expression of TIM3 and its ligands across multiple tumor indications.

Experimental Design: TIM3, LGALS9, CEACAM1, HMGB1, and signatures capturing T and myeloid populations were examined in TCGA RNASeq data. TIM3 and LGALS9 in various cell subsets was evaluated using public single-cell RNASeq. Multiplex immunofluorescence (mIF) staining was performed on tumor TMAs procured from commercial sources.

Results: Analysis of TCGA data showed that TIM3 expression strongly correlates with macrophages and with T cells. TIM3 expression strongly correlates with LGALS9 but not with CEACAM1 or HMGB1 across essentially all TCGA indications. LGALS9 expression was observed to correlate more strongly with T cells, macrophages, and IFNγ expression signatures compared to HMGB1 and CEACAM1, suggesting it may be the most relevant ligand as adaptive mechanism of resistance to cytotoxic activity. Interestingly, the genes most correlated with TIM3 and its ligands were largely distinct between cell types in the single-cell data. This allows for cell type-specific expression signatures to predict levels of these genes from bulk tumor expression data. Such signatures combined with overall survival data from TCGA suggest that TIM3+ T cells are active in immune response and that TIM3+ macrophages have a negative predictive value. Experiments to confirm findings for TIM3 and its ligands from TCGA using mIF analysis are ongoing. Results will be reported in detail in the poster.

Conclusions: These results suggest that interaction of TIM3 and GAL9, as opposed to its other ligands, may be an important mechanism of immune suppression in the TME. Thus, blocking TIM3/GAL9 interaction would potentially reactivate anti-tumor activity by modulating both T cell intrinsic and extrinsic functions involving multiple immune cells subsets. Future correlative and clinical investigations should consider the potential combined role of TIM3 and Gal9 for development of next generation IO therapeutics.

#2726

In vitro functional activity of OMP-336B11, a GITRL-Fc fusion protein, on primary human immune cells.

Ivan H. Chan, Ming-Hong Xie, Andrew Lam, Fumiko T. Axelrod, Jennifer Elechko, Angie I. Park, Austin Gurney. _OncoMed, Redwood City, CA_.

Glucocorticoid-induced TNFR-related protein (GITR) is a member of the TNF receptor superfamily. GITR, a co-stimulatory surface receptor, is activated upon binding to GITR Ligand (GITRL) and mediates co-stimulation of T-cell and NK responses and inhibits the suppressive activity of regulatory T-cells (Tregs). As such, GITR is an attractive immuno-oncology target for activation via GITR agonists. OMP-336B11 is a single-gene recombinant fusion protein consisting of two trimeric human GITRLs and a human immunoglobulin (IgG1) Fc domain. Murine preclinical studies using a surrogate GITRL-Fc fusion protein demonstrated robust antitumor efficacy. Functional characterization of OMP-336B11 in various human immune cell assays is presented here. In human peripheral blood mononuclear cells (PBMC), OMP-336B11 stimulated IL-2 cytokine release in a dose-dependent manner. In activated human T-cells, OMP-336B11 enhanced cell proliferation in a dose-responsive fashion. OMP-336B11 also augmented IL-2 induced IFNγ from human NK cells. To elicit NK mediated cytotoxicity of high GITR expressing cells (i.e., Tregs), OMP-336B11 is designed with an IgG1 Fc domain. Co-incubation of primary human NK cells (effector) and GITR expressing cells (target) resulted in an OMP-336B11 dependent dose-titratable increase in target cytotoxicity. Furthermore, OMP-336B11 agonistic activity was compared to anti-GITR agonist antibodies. By measuring cell proliferation and IFNγ from activated T-cells, OMP-336B11 demonstrated superior activity compared to the other agonist anti-GITR antibodies. In conclusion, OMP-336B11 is designed to induce effective GITR activation and also to mediate depletion of GITR-high cells. OMP-336B11 is currently undergoing phase I clinical study.

#2727

The discovery and characterization of PTZ-201, a fully-human, high affinity, antagonistic anti-TIGIT monoclonal antibody.

Sandra Abbott,1 Ana C. Anderson,2 Heather Brodkin,1 Daniel J. Hicklin,1 Nesreen Ismail,1 Nels Nielson,3 Christopher Nirschl,1 Andres Salmeron,1 Cynthia Seidel-Dugan,1 Philipp Steiner,1 William Winston1. 1 _Potenza Therapeutics, Cambridge, MA;_ 2 _Harvard Medical School, Boston, MA;_ 3 _Adimab, Lebanon, NH_.

The costimulatory molecule CD226 and the coinhibitory receptor TIGIT (T cell immunoreceptor with immunoglobulin and immunoreceptor tyrosine-based inhibitor motif (ITIM) domains) function as a checkpoint pathway regulating immune function with similarity to the CD28-CTLA4 pathway. CD226 and TIGIT bind to the same ligands, PVR (poliovirus receptor, also known as CD155) and PVRL2 (poliovirus receptor-related 2, also known as CD112). CD226 is a positive regulator of T cell responses, while TIGIT is an inhibitor of these signals. TIGIT is highly expressed on memory T cells and Tregs and is co-expressed with other immune checkpoint molecules on exhausted T cells. TIGIT activation has been demonstrated to regulate CD4+ and CD8+ T cell activity, regulatory T cell function, and NK cell activity. Blocking the binding of TIGIT to its ligands has been demonstrated to activate immune function in the tumor microenvironment, and TIGIT antagonists have anti-tumor activity in murine syngeneic tumor models. TIGIT's function suggests it may be a promising target for clinical development of antagonistic monoclonal antibodies to increase the anti-tumor immune response. PTZ-201, developed by Potenza Therapeutics, is a fully human monoclonal IgG4 anti-human TIGIT antagonistic antibody which blocks the binding of TIGIT to its ligands PVR and PVRL2. PTZ-201 binds to human TIGIT with high affinity, binds to Jurkat cells engineered to express TIGIT, and binds to human primary lymphocytes. PTZ-201 increases interleukin (IL)-2 production in functional cell assays using an engineered Jurkat cell assay. In addition, PTZ-201 increases IFNγ and TNF production in cultured peripheral blood mononuclear cells and CD4+ T cells sub-optimally stimulated with anti-CD3/anti-CD28 antibodies. In a cytomegalovirus (CMV)-specific immune recall assay, PTZ-201 increases the activation PBMCs (increased TNF production) and increases the frequency of cytokine producing CD4+ and CD8+ T cells, compared to an isotype control. In this CMV recall assay, the combination of PTZ-201 with an anti-programmed death (PD-1) antibody results in a statistically significant increase in TNF production when compared to either agent alone. Finally, tumor infiltrating lymphocytes (TILs) isolated from multiple human tumor types express high levels of TIGIT, with a majority of CD8+ and Tregs co-expressing PD-1 and TIGIT. These data support the continued development of PTZ-201 as a novel checkpoint inhibitor for the treatment of cancer.

#2728

Circulating levels of Resokine, a soluble modulator of the immune system, are upregulated in both experimental cancer models and in patients across multiple tumor types.

Ryan Adams, Elisabeth Mertsching, Leslie Nangle, Kathy Ogilvie, Steven Crampton, John Bruner, Samantha Tyler, Sanna Rosengren, Andrea Cubitt, David King, John Mendlein. _aTyr Pharma, San Diego, CA_.

Resokine is a newly identified regulator of immune cell activity, and circulating levels of Resokine in normal individuals may represent a soluble set-point control to modulate T cell activity. Resokine activity is a non-canonical function arising from the tRNA synthetase gene family, and the activity is effected by a 60 amino acid N-terminal domain arising from the gene for histidyl-tRNA synthetase which is present in the full-length protein as well as multiple splice variants that have lost their original tRNA synthetase functionality. Resokine is secreted from cells, including tumor cell lines, and in vitro studies have demonstrated that Resokine can inhibit the activation of immune cells. In vitro, for example, Resokine addition during T cell activation induced by antibodies to CD3 and CD28, can result in reduced levels of inflammatory cytokines such as IL-2, interferon gamma, and TNF alpha; inhibition of the up-regulation of cell-surface activation markers, such as CD69, CD40L and 4-1BB; and inhibition of release of the cytotoxic mediator granzyme B.

We have tested levels of circulating Resokine in both mice with syngeneic tumors as well as >300 cancer patients across multiple tumor types. In normal C57Bl6 mice serum levels of Resokine ranged from 70-250pM (n=10) whereas in mice bearing B16F10 tumors, levels were significantly higher (450-3000pM, p<0.001) and correlated with tumor size. Resokine levels in normal human volunteers exhibit a more variable range, from 8pM to >2333pM (n=148), with 18% of individuals having levels <30pM, which was set as the active threshold level based on the concentration required to inhibit T cell activation in vitro. In contrast, samples across >300 cancer patients with different tumor types, exhibited higher circulating levels with only 4% of individuals having levels below the activity threshold of 30pM. This data is consistent with the hypothesis that tumors secrete Resokine as an additional mechanism to down-regulate immune activity, and suggests further investigation of the utility of Resokine levels as a new biomarker of immune activity in patients.

#2729

Quantitative cell-based bioassays to advance individual or combination immune checkpoint immunotherapy.

Jamison Grailer, Julia Gilden, Pete Stecha, Denise Garvin, Jim Hartnett, Frank Fan, Mei Cong, Zhi-Jie Jey Cheng. _Promega Corp., Madison, WI_.

Immune checkpoint receptors play a critical role in maintaining immune homeostasis and are genetically and functionally associated with autoimmune disease, cancer and persistent viral infections. Blockade of immune checkpoints has emerged as a promising new approach to enhance anti-tumor immune responses. After initial successes with PD-1 and CTLA-4-targeted therapies, immunotherapy research has broadened to include additional immune checkpoint receptors targeted individually or in combination. A significant challenge in the development of biologics that target immune checkpoints is access to quantitative and reproducible functional bioassays. Existing methods rely on primary cells and measurement of complex functional endpoints. As an alternative to primary cell assays, we have developed a panel of cell-based reporter bioassays that can quantitatively determine the potencies of biologics targeting the immune checkpoints PD-1, CTLA-4, LAG-3, TIM-3, TIGIT, and BTLA, or bispecific antibody or antibody cocktails targeting two immune checkpoint receptors such as PD-1+TIGIT, PD-1+LAG-3, and PD-1+CTLA-4. These assays consist of engineered T effector cells that express luciferase reporters driven by specific response elements integrating signaling from the T cell receptor and immune checkpoint target(s), and engineered artificial antigen presenting cells. These mechanism of action-based bioassays demonstrate high specificity, sensitivity and reproducibility. Therefore they can serve as valuable tools for immunotherapy drug development.

#2730

Preclinical characterization of the anti-PD-L1 monoclonal antibody LY3300054.

Carmine Carpenito, Yiwen Li, George X. Wang, Maria S. Malabunga, Jaafar N. Haidar, Amelie Forest, Mary Y. Murphy, Gerald E. Hall, Cindy Wang, Leyi Shen, Andreas Sonyi, Darin Chin, Anthony L. Pennello, Ivan V. Inigo, David Surguladze, Yung-mae Yao, Douglas Burtrum, Ruslan D. Novosiadly, Kris Persaud, Dale L. Ludwig, Michael D. Kalos. _Eli Lilly and Company, New York, NY_.

Modulating the PD-L1/PD-1 axis in man through function blocking antibodies can release potent anti-tumor immunity, leading to durable objective responses across multiple tumor types. Here we describe the discovery and preclinical characterization of LY3300054, a fully human IgG1λ antibody capable of binding human PD-L1 with high affinity and inhibiting binding to its two cognate receptors, PD-1 and CD80.

LY3300054 is an antagonist monoclonal antibody recognizing human PD-L1 with high affinity (KD 0.08 nM), selected and derived from a scFv phage library. LY3300054 was engineered and expressed as an IgG1-Fc null monoclonal antibody to ablate immune effector function. Multiple in vitro assays, including mixed leukocyte reaction (MLR) and tetanus toxoid recall assay were utilized to demonstrate LY3300054 potent functional activity in enhancing the activation of primary human T cells in culture. The biological activity of LY3300054 on T cells was also shown to be enhanced by co-administration of anti-CTLA4 mAb (ipilimumab) in MLR. Further, we evaluated LY3300054 activity in vivo using xenograft mouse tumor models reconstituted with human immune cells. LY3300054 demonstrated anti-tumor activity in both NCI-H292 xenografts co-implanted with human PBMCs and HCC827 xenograft model upon infusion of human T cells. LY3300054 in vivo activity was also tested in HCC827- and Ov79-bearing immunodeficient NSG/NOG mice reconstituted with human hematopoietic stem cells. In this setting, LY3300054 displayed robust anti-tumor and immunomodulatory effects exemplified by T cell inflamed phenotype in the tumor and peripheral tissues. High-content molecular profiling identified distinct gene expression changes indicative of T cell activation in all models tested. A mutational strategy based on integrating the PD-1/PD-L1 structure data with the orthologous sequence data of PD-L1 has identified a residue on PD-L1, which is part of the PD-L1/PD-1 interface, that is critical for the species specificity of LY3300054.

This study demonstrates that LY3300054 is novel anti-PD-L1 monoclonal antibody, capable of potently enhancing human T cell function both in vitro and in vivo, and provides previously not described insights into the effects of PD-L1 blockade on the intra- and extra-tumoral immune response. LY3300054 is currently under clinical evaluation in monotherapy and combination with other therapeutic modalities in multiple tumor types (NCT02791334; NCT03099109; NCT02791334; NCT02791334)

#2731

Checkpoint inhibitor therapy in combination with the implantation of agarose encapsulated cancer cells inhibits tumor growth in a mouse model of osteosarcoma.

Pradeep R. Dumpala,1 Prithy C. Martis,1 Melissa A. Bemrose,1 Atira Dudley,1 Barry H. Smith,2 Lawrence S. Gazda1. 1 _The Rogosin Institute, Xenia, OH;_ 2 _The Rogosin Institute, New York, NY_.

We have previously shown that murine renal adenocarcinoma (RENCA) cells encapsulated in agarose macrobeads (MB) secrete factors that inhibit the proliferation of freely growing tumor cells outside the MB, both in vitro and in vivo. This effect is being investigated in ongoing clinical trials with colorectal patients who underwent laparoscopic intraperitoneal implantations of RENCA MBs (NCT01053013, NCT02046174). In the current study, we report the effect of immune checkpoint inhibitors (anti-PD-1 and anti-PD-L1 monoclonal antibodies) in combination with RENCA MBs on tumor burden in BALB/cJ mice induced with K7M2 murine osteosarcoma tumors. Mice that developed tumors (2-5 mm) on subcutaneous injection of K7M2 cells were randomly enrolled in the following study groups: sham, RENCA MBs only, anti-PD-1 only, RENCA MBs + anti-PD-1, anti-PD-L1 only, RENCA MBs + anti-PD-L1. Anti-PD-1 and anti-PD-L1 antibodies were administered ip bi-weekly for 3-6 weeks. RENCA MBs were implanted in the peritoneal cavity on the day of enrollment. Mean tumor volumes (mm3) throughout days 0-24 were: RENCA MB (199.17 ± 125.57), anti-PD-1 only (278.87 ± 180.34), anti-PD-L1 only (332.46 ± 185.13) and sham treated mice (512.61 ± 230.12). RENCA MB and anti-PD-L1 combination therapy did not inhibit tumor growth (375.00 ± 511.61). PD-L1 expression was absent in primary tumors as observed by immunostaining. Treatment with RENCA MBs plus anti-PD-1 treatment demonstrated an additive effect (116.94 ± 83.77) compared to RENCA MB only or anti-PD-1 alone. Moreover, tumors in 4 out of 16 mice from the RENCA MB plus anti-PD-1 group receded and were undetectable (3 in less than 1 week and 1 at 39 days) following treatment. Tumors also regressed and were not palpable in 1 mouse each in the anti-PD-1 and RENCA MB plus anti-PD-L1 groups at day 14 and 9, respectively. Discontinuation of anti-PD-1 and anti-PD-L1 treatment after 3 weeks of treatment resulted in loss of tumor growth regulation. Preliminary data suggests that continued treatment with these immune checkpoint inhibitors beyond 3 weeks prolongs the regulation of tumor growth in this model. These data support the use of anti-PD-1 in combination with the implantation of RENCA macrobeads in this mouse model of osteosarcoma, and the possibility of similar approaches in the clinic.

#2732

Cell-based reporter bioassays to evaluate the Fc gamma receptor-dependent agonistic activities for therapeutic antibodies targeting immune costimulatory receptors.

Jun Wang, Michael Beck, Jamison Grailer, Pete Stecha, Jim Hartnett, Frank Fan, Mei Cong, Zhi-Jie Jey Cheng. _Promega Corp., Madison, WI_.

Immune costimulatory receptors synergize with TCR-CD3 signaling to promote cell cycle progression, cytokine production, T cell survival and effector functions. They belong to immunoglobulin super-family (CD28, ICOS) and tumor necrosis factor receptor (TNFR) superfamily (4-1BB, OX40, CD27, CD40, GITR, DR3, and HVEM). Preclinical and clinical evidence shows that agonist antibodies against costimulatory receptors can stimulate antitumor immunity and are emerging as a promising approach for cancer immunotherapies. Furthermore, FcγR engagement, especially FcγRIIB due to its bioavailability, has been shown to provide a crosslinking scaffold for the antibody IgG to facilitate TNFR clustering/activation and become requisites for some agonist antibody activities. Here, we report the development of a suite of cell-based reporter bioassays to quantitatively measure the potencies of therapeutic antibodies designed to target costimulatory receptors. For this, we developed engineered effector cells that express each costimulatory receptor and a luciferase reporter driven by response element that specifically responds to costimulatory receptor signaling. We also developed a FcγR-expressing cell line to evaluate the involvement of FcγR in agonist antibody-mediated costimulatory signaling. The assays are specific for each costimulatory receptor, and can be used to detect both agonistic and blocking activities for the antibodies. When co-incubated with the engineered FcγR-expressing cells, they are able to show the Fcγ receptor-dependent or independent agonist activities. In summary, these reporter bioassays can serve as powerful tools in immunotherapy drug development for antibody screening and potency determination.

#2733

Developing novel anti-OX40 antibodies on Biocytogen's in vivo drug screening platform.

Chaoshe Guo,1 Yanan Guo,1 Yuelei Shen,1 Benny Yang2. 1 _Beijing Biocytogen Co., Ltd, Beijing, China;_ 2 _Eucure (Beijing) Biopharma Co., Ltd, Beijing, China_.

OX40, also known as CD134 or TNFRSF4, is a member of the TNFR superfamily of receptors. OX40 is a co-stimulatory receptor, and it interacts with its ligand to providepositive signal for T cell activation. OX40 is not expressed on resting naïve T cells, however its expression goes up after 24 to 72 hours following activation. Disruption of OX40 pathway led to defective T cell responses and overexpression of OX40 caused massive immune activation. Due to its importance in keeping immune homeostasis, strategies modulating the OX40 pathway hold great promise in fighting against cancer and autoimmune diseases. We have interests in developing novel OX40 antibodies for boosting cancer patients' own immunityinlight of other successful immune checkpoint modulators. We developed a cohort of OX40 specific antibodies using the classic hybridoma technology. In order to screen their efficacy to stimulate anti-tumor activity in live animals, we managed to rank these antibodies using Biocytogen'shumanized mouse platform of immune checkpoints. Specifically, we utilized humanized OX40 mice (h-OX40) and implanted syngeneic tumors subcutaneously, followed by treating mice with purified testing antibodies. Via this approach, we are able to discern several lead antibodies that effectively inhibited tumor growth without prior knowledge of their in vitro activities. Furthermore, we identified that one clone works in concert with Keytruda in the dual humanized model of h-OX40 and h-PD1. We are currently investigating the mechanisms of action underlying these potentially useful antibodies.

#2734

Novel CTLA4 antibodies demonstrate potent antitumor activity in a humanized mouse model.

Jie Xiang,1 Yanan Guo,2 Yuelei Shen,2 Benny Yang3. 1 _Biocytogen LLC., Worcester, MA;_ 2 _Beijing Biocytogen Co., Ltd, Beijing, China;_ 3 _Eucure (Beijing) Biopharma Co., Ltd, Beijing, China_.

CTLA4 (cytotoxic T-lymphocyte-associated protein 4), is a member of the immunoglobulin superfamily. It is expressed in T cells upon activation and transmits an inhibitor signal to T cells. Also, it is constitutively expressed in regulatory T cells (Tregs) which is associated with their immunosuppressive phenotype. CTLA4 is homologous to the co-stimulatory protein CD28, and both receptor binds to CD80 and CD86. Cancer cells can be recognized and destructed by the host's immune system. In this setting, CTLA4 functions as a brake to dampen anti-tumor T cell responses, which promote cancer progression. Antibodies targeting CTLA4 was expected to subvert T cell inhibition. Moreover, regulatory T cells residing in the tumor microenvironment can be killed by anti-CTLA4 antibody since these cells have higher expression level of CTLA4 comparing with the activated T cells. Yervoy, an anti-CTLA4 antibody, was the first FDA-approved antibody targeting the immune checkpointfamily, and it protects a fraction of cancer patients when either used alone or in combination with other drugs. We asked whether the efficacy of anti-CTLA4 antibodies could be evaluated in humanized mouse models. And if so, whether we can use in vivo assay to guide the development of potent antibodies targeting CTLA4. Here, we describe that using the platform established at Biocytogen, we successfully discovered two antibodies whose efficacy is equivalent or exceed that of Yervoy. The clinical evaluation of these new entities are wanted.

#2735

**Cancer immunotherapy with agonistic anti-4-1BB scFv producing and secreting** Bifidobacterium **in syngeneic mouse model.**

Tomio Matsumura,1 Koichiro Shioya,1 Yasuyoshi Kanari,1 Yuko Shimatani,1 Shiro Kataoka,1 Shun'ichiro Taniguchi,2 Takaaki Nakamura1. 1 _Anaeropharma Science, Inc., Matsumoto, Japan;_ 2 _Shinshu University, Matsumoto, Japan_.

Agonistic antibodies against immune checkpoint molecules, such as 4-1BB, reportedly demonstrate antitumor effects at the early clinical phase, whereas immune-related adverse events hamper further development. A single chain variable fragment (scFv), which is expected to have better penetration into tissues, is clinically being investigated, however, poor distribution into tumor tissues due to rapid clearance is a serious issue.

In order to improve drug delivery and decrease adverse events, we have developed in situ Delivery and Production System (i-DPS) by genetically engineering a non-pathogenic anaerobic Bifidobacterium. We previously reported that the modified Bifidobacteria localized and proliferated only in the hypoxic area of the tumor engrafted in the mouse post i.v. injection, thus producing anti-tumor molecules persistently and selectively at the tumor site (AACR2010).

Here we present i-DPS with agonistic anti-murine 4-1BB scFv. We created a recombinant Bifidobacterium producing and secreting anti-murine 4-1BB scFv. The anti-murine 4-1BB scFv increased IFN-γ production by 171% over the control group (PBS(-)) in the mouse splenocytes activated by ant-CD3 antibody in vitro. Anti-tumor effects of Bifidobacteria producing the anti-murine 4-1BB scFv were demonstrated in the syngeneic model of CT-26 in Balb/c mouse (61.7% tumor growth inhibition (TGI) at 1×109 cfu/mouse, i.v. on day 14). We further evaluated the anti-tumor effects of i-DPS for anti-murine 4-1BB scFv (1×109 cfu/mouse, i.v.) in combination with PD-1 antibody (i.p.), and confirmed that the combination therapy significantly suppressed the tumor growth on day 21 (TGI: combination, 60.7%; anti-murine 4-1BB scFv, 38.8%; anti-mPD-1 mAb, 8.9%). In conclusion, i-DPS for anti-4-1BB scFv will provide a new promising modality for the immune-therapy targeting hypoxic solid tumors.

#2736

Preclinical pharmacologic and pharmacodynamic studies of a novel and potent IDO1 inhibitor D-0751.

Yaolin Wang, Zhe Shi, Zixing Han, Zhenwu Wang, Yueheng Jiang, Xing Dai. _InventisBio Inc., Shanghai, China_.

Indoleamine 2,3-dioxygenase 1 (IDO1) is a heme-containing enzyme that catalyzes the degradation of the essential amino acid L-tryptophan to kynurenine. It plays an important role in the initial and rate-limiting step in the breakdown of tryptophan. It has been reported that IDO1, an enzyme induced by IFN-gamma, is one of the central regulators of immune responses in various physiologic and pathologic settings and the enzyme is frequently expressed in cancers. Kynurenine produced by IDO1 suppresses T-cell function in the tumor microenvironment, resulting in tumor cell evasion from immune cells. IDO1 inhibitors, such as epacadostat and BMS-986205, have shown preliminary clinical therapeutic response in melanoma and other solid tumors when combined with anti-PD-1 antibody. Here, we report the discovery of a novel and selective IDO1 inhibitor with potent activity in various cell-based assays. Our lead molecule D-0751 inhibits IDO1 activity in HeLa cell and human whole blood assay with IC50 of 0.6 nM and 13 nM, respectively. It activates the human T-cell activity in a HeLa co-culture study with EC50 at 9 nM. D-0751 is optimized to have good PK profile in rodent, dog and monkey as well as slow clearance when incubated with human hepatocyte. Pharmacodynamic (PD) effect has been observed in mouse models (blood and tumor samples) as well as in dog (blood samples) with efficient kynurenine reduction observed at optimal dose level. Taken together, these results support the advancement of D-0751 as a clinical candidate in combination with anti-PD-1/L1 antibody in various solid tumor indications.

#2737

Does androgen attenuation of PD-L1 expression in thyroid cancer modulate disease incidence.

Timmy J. O'Connell,1 Anvita Gupta,1 Melanie Jones,2 Tali Lando,1 Deya Jourdy,1 Edward Shin,3 Augustine Moscatello,1 Raj Tiwari,1 Jan Geliebter1. 1 _New York Medical College, Valhalla, NY;_ 2 _United States Military Academy Preparatory School, West Point, NY;_ 3 _New York Eye and Ear Infirmary of Mount Sinai, New York, NY_.

Thyroid cancer (ThCa) is the most rapidly increasing cancer in the US with 56,870 new cases expected in 2017. Interestingly, there is a disparity in incidence between females and males, with women developing ThCa three times more often than men. Immune elimination of nascent tumor cells may explain the difference in disease incidence between sexes. To address this hypothesis, the effect of androgen on immune checkpoint molecule expression in an androgen responsive-thyroid cancer cell line was examined. The undifferentiated ThCa cell line, 8505C, was transfected with androgen receptor (AR) yielding 84E7, a clone that constitutively expresses AR. RNASeq was performed on 8505C and 84E7, treated without and with the AR ligand, 5α-dihydrotestosterone (DHT). PD-L1 was the sole immune checkpoint molecule that exhibited a significant expression change with a 72% reduction in mRNA content. Additional studies provided evidence of qualitative (immunofluorescence) and quantitative (flow cytometry, western blotting) DHT-mediated decreases in PD-L1 expression, with the PD-L1 decreases being both time- and dose-dependent. To further confirm that the effect was specific to AR and PD-L1, we pre-treated 84E7 with flutamide, a selective AR antagonist, to pharmacologically inhibit DHT-mediated AR signaling. This resulted in a dose-dependent and >90% restoration of cell surface PD-L1 expression, evidencing a definitive link between AR activation and PD-L1 signaling. Preliminary investigation into the mechanistic link between AR and PD-L1 shows that NFkB signaling is the likely intermediary. AR activation increases IkBα expression thereby preventing NFkB translocation into the nucleus, resulting in decreased PD-L1 transcription. Furthermore, in human tissue samples from TCGA (n=55), we found female thyroid tissue is significantly enriched with PD-L1 as compared to male thyroid tissue (p=0.0073). Finally, an inverse relationship exists in which AR expression is decreased and PD-L1 expression is increased in human thyroid tumor tissue samples as compared to normal thyroid tissue samples in both TCGA (n=55, p<0.005) and NYMC (n=56, p<0.005) RNAseq datasets. These results are significant in that PD-L1 is expressed by tumor cells as a strategy for evading the immune response allowing for continued tumor growth and metastases. In the thyroid, the presence of androgen-activated AR could lead to an environment that is more favorable for immune system activation and may help eliminate nascent ThCa cells. Thus, men experience a decreased incidence of ThCa due to an immunologically enhanced anti-tumor microenvironment. Alternatively, higher expression levels of PD-L1 observed in female thyroid cancer patients help explain the increased incidence of ThCa and may indicate an opportunity for sex-based therapeutics.

#2738

PD-L1/PD-1 checkpoint inhibition in anaplastic thyroid cancer and enhancement of ICAM1-targeted chimeric antigen receptor (CAR)-T cell tumor lysis.

Katherine D. Gray,1 Yogindra Vedvyas,2 Olivia Kalloo,1 Enda Shevlin,2 Theresa Scognamiglio,1 Marjan Zaman,2 Andrew Tassler,1 Moonsoo M. Jin,2 Rasa Zarnegar,1 Thomas J. Fahey,1 Irene M. Min1. 1 _New York Presbyterian Hospital - Weill Cornell Medicine, NEW YORK, NY;_ 2 _Weill Cornell Medicine, NEW YORK, NY_.

Poorly-differentiated thyroid cancer (PDTC) and anaplastic thyroid cancer (ATC) are associated with a rapidly progressive course and poor median survival. Recently we published promising results in a murine model showing efficient clearance of metastatic tumor burden of human ATC cell line 8505c and an anaplastic patient-derived cell line using healthy donor T cells transduced with a third-generation chimeric antigen receptor (CAR) construct targeting intercellular adhesion molecule-1 (ICAM1). However, patient-derived T cells using an identical ICAM1-targeted CAR construct demonstrated slower tumor-killing in vitro. To investigate the role of immune checkpoint proteins PD-L1 and PD-1 in thyroid cancer and as potential inhibitors of ICAM1-CAR T, we stained formalin-fixed paraffin-embedded archived tissue samples for PD-L1 (papillary thyroid cancer, PTC, n = 17; ATC, n = 13) using immunohistochemistry (IHC) and extracted peripheral blood mononuclear cells (PBMCs) from patient blood samples and healthy donors to evaluate PD-1 expression on CD3+/CD8+ T cells using flow cytometry (healthy donor, n = 2; PTC, n = 7; PDTC/ATC, n = 6). Twelve percent of PTC samples were PD-L1+ (threshold >1% positivity) versus 69% of PDTC/ATC samples (Chi-square, p = 0.012); PD-L1 positivity was associated with circumferential ICAM1 staining on IHC (Chi-square, p = 0.003). Similarly, median percentage of PD-1 expression on PBMC's was higher in PDTC/ATC than PTC (58.1% versus 5.2%, Wilcoxon rank sum test; p = 0.03). Interestingly, expression of elevated PD-1 was seen in PBMC samples from patients in the absence of PD-L1 positivity of the primary tumor (IHC) in 4 of 5 matched tumor samples. This suggests that PD-1 in circulating CD8+ cells may be an important predictor of response to checkpoint inhibitor therapy. Finally, effector:target assays with patient-derived ICAM1-CAR T effector cells (baseline PD-1 expression 96%) against 8505c (PD-L1 100%/ICAM1 68%) and matched patient-derived cell line (PD-L1 >50%/ICAM1 88%) showed enhanced tumor killing in the presence of anti-PD-1, which approached the level of tumor cell death seen with healthy donor ICAM1-CAR T. Our results show that PD-1 is increased in the peripheral blood of patients with poorly differentiated and anaplastic thyroid cancer and is a potential therapeutic target. Furthermore, anti-PD-1 may be used to counter PD-L1+ tumor-induced T cell suppression (or exhaustion) to increase the efficacy of CAR T cells in ATC. 

### Immune Mechanisms Invoked by Therapies 1

#2739

**The ketone body β-hydroxybutyrate alters expression of PD-L1 on malignant glioma cells but does not directly affect T cells** in vitro **.**

Eric C. Woolf,1 Kavita R. Manhas,2 Deborah R. Healey,1 Joseph N. Blattman,2 Adrienne C. Scheck1. 1 _Barrow Neurological Inst., Phoenix, AZ;_ 2 _Arizona State University, Tempe, AZ_.

Patients with newly diagnosed glioblastoma multiforme (GBM), the most aggressive grade of glioma, have an average life expectancy of ~15 months, and less than 6% survive five years, despite the current standard of care, which consists of aggressive surgery followed by radiation and chemotherapy. Advances in the survival of GBM patients require the design of new therapeutic approaches. One novel strategy being explored in our laboratory and others involves using ketogenic therapy (KT). This approach involves shifting the body's metabolic physiology by increasing the production of ketone bodies such as β-hydroxybutyrate (BHB) and decreasing blood glucose. We have demonstrated that nutritionally-induced KT enhances survival and potentiates standard therapy in a mouse model of malignant glioma. Our previous study shows that KT works in part as an immune adjuvant, boosting tumor-reactive immune responses in the microenvironment by alleviating immune suppression in vivo. In particular we found that KT reduced expression of programmed cell death-1 (PD-1) on tumor-infiltrating T cells and its ligand PD-L1 on tumor cells. The PD-1/PD-L1 axis represents a crucial mechanism of glioma-mediated immunosuppression and is a major focus of immunotherapy in GBM and other cancers. The current study aims to determine the role of BHB in the anti-tumor and immune-enhancing effects of KT. We show that in vitro treatment with BHB induces a variety of molecular changes in glioma cells including reduced protein expression of PD-L1. Given that our in vivo data suggests that KT enhances T cell function against tumors we also sought to determine the impact of BHB on T cell function in vitro. Activated CD8+ and CD4+ T cells were cultured with varying doses of BHB prior to flow cytometric analysis. There were no significant changes in expression of immune checkpoint receptors (PD-1, CTLA-4 and LAG-3), production of anti-tumor promoting cytokines (IFN-y, TNF, IL-2) or viability in either T cell population following treatment with any dose of BHB. This suggests that BHB and KT may indirectly impact T cell function by influencing the way tumor cells manipulate the microenvironment. Taken together this data helps us better understand the anti-tumor mechanisms underlying KT and warrants further exploration of combinational treatment strategies using this approach and immunotherapies for GBM.

#2740

PEGylated recombinant hyaluronidase PH20 (pegvorhyaluronidase alfa PEGPH20) converts HA-rich tumors from resistant to sensitive to anti-PD-L1 immunotherapy in murine syngeneic breast cancer models.

Renee Clift, Xiaoming Li, Barbara Blouw, Curtis B. Thompson, Yujun Huang. _Halozyme Therapeutics, Inc., San Diego, CA_.

Hyaluronan (HA) is an extracellular glycosaminoglycan that accumulates in the tumor microenvironment (TME) of many solid tumors. In preclinical studies, enzymatic degradation of TME HA by intravenous PEGylated recombinant human hyaluronidase PH20 (PEGPH20) remodels the TME, reduces tumor interstitial fluid pressure, decompresses tumor blood vessels, and facilitates delivery of chemotherapeutics. PEGPH20-mediated HA degradation increases anti-PD-L1 efficacy in HA-rich EMT-6 tumors and enhances CD8+ T-cell infiltration in tumors. Cancer patients can be resistant to anti-PD-L1; strategies to increase T cell infiltration may lead to anti-PD-L1 effectiveness in nonresponders. We hypothesized that PEGPH20 may convert HA-rich tumors with low T cell infiltration from resistant to sensitive to anti-PD-L1 immunotherapy. 4T1 breast cancer cells transduced with HAS3 to generate HA-rich 4T1/HAS3 cells averaged ~2.9×104 CD8+ T cell/g tumor on day 18 post-tumor inoculation and were resistant to anti-PD-L1 monotherapy. PEGPH20 treatment of 4T1/HAS3 tumors significantly increased CD8+ T cell infiltration to ~7.2×104/g tumor (p=0.0007) and 4T1/HAS3 tumors became sensitive to anti-PD-L1 treatment. PEGPH20 + anti-PD-L1 led to 64.0% tumor growth inhibition (TGI; p<0.0001 vs vehicle + isotype antibody [IgG2b]) vs PEGPH20 alone (10.7% TGI) or anti-PD-L1 alone (-5.1% TGI). PEGPH20 significantly increased infiltration of CD4+ T cells and NK cells, and decreased the percentage of CD11b+GR1+ myeloid derived suppressive cells among CD45+ cells, vs vehicle + IgG2b in 4T1/HAS3 tumors. EMT-6 tumors were sensitive to anti-PD-L1 when starting tumor size was small (~133 mm3) but became resistant with larger starting tumor size (~325 mm3). PEGPH20 + anti-PD-L1 converted larger EMT-6 tumors to sensitive to anti-PD-L1 with 64.9% TGI (p=0.049 vs vehicle + IgG2b), whereas neither PEGPH20 alone nor anti-PD-L1 alone significantly inhibited tumor growth. Small EMT-6 tumors (~136 mm3) were resistant to a lower dose of anti-PD-L1; however, PEGPH20 + low-dose anti-PD-L1 converted EMT-6 tumors from resistant to sensitive to low-dose anti-PD-L1 (65.4% TGI, p<0.0001 vs vehicle + IgG2b). Imaging studies using fluorescence-labeled anti-PD-L1 antibody showed PEGPH20 enhanced the accumulation of anti-PD-L1 in TME. Our data suggest that PEGPH20-mediated HA removal increases infiltration of lymphocytes and immunotherapeutic antibody accumulation in HA-rich tumors. PEGPH20 converts HA-rich tumors from resistant to sensitive to anti-PD-L1. Clinical trials are ongoing to evaluate PEGPH20 + anti-PD-L1 in patients with cholangiocarcinoma/gallbladder cancer (NCT03267940) and gastric cancer (NCT03281369), and to evaluate PEGPH20 + anti-PD-1 in patients with non-small cell lung cancer and gastric cancer (NCT02563548).

#2741

Combination of rapamycin with immune checkpoint blockade in a syngeneic breast carcinoma model.

Erin Trachet, Chris Elders, Sumithra Urs, Sarah Krueger, Alden Wong, Maryland Rosenfeld Franklin. _MI Bioresearch, Ann Arbor, MI_.

Many advances in the treatment of breast cancer have been driven by the development of targeted therapies that inhibit signal transduction pathways, as well as the development of therapies that activate a patient's immune system to unleash antitumor immunity. The choice of an animal model that mimic aspects of human breast cancer is crucial for evaluating new immunotherapeutic combination strategies. An excellent syngeneic model of breast cancer is 4T1. 4T1-luc2 is poorly immunogenic and shares many characteristics with human breast cancer. To understand the potential benefit from combining a targeted therapy with an immune modulator we utilized the orthotopic 4T1-luc2 model. The mTOR pathway plays an important role in metabolism, cell growth and survival. Targeting mTOR has been an active area of oncology drug discovery and clinical development for breast cancer and other malignancies. In addition, clinical success through blockade of the cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) has resulted in a paradigm shift for drug development within the oncology community. To this end, we designed a series of experiments to evaluate rapamycin, a first generation mTOR inhibitor, in combination with the immune checkpoint inhibitor antibody against CTLA-4. We find that 4T1-luc2 tumors have very few T cells but a significant MDSC (myeloid derived suppressive cells) population. Treatment with anti-CTLA-4 (10mg/kg) produced a slight increase in T cells and a slight decrease in MDSCs. However, these changes in the immune profile did not manifest into meaningful changes in tumor burden. Treatment with low dose (8, 5 or 3mg/kg) rapamycin was well tolerated and produced a modest tumor growth inhibition (~50%) and modulation on the immune profile. The overall impact on the immune profile was like what we've seen with anti-CTLA-4 treatment, with a ~5% decrease in MDCSs but a relatively untouched T cell population. This made the paring of these agents appealing as we hypothesized that the combination may result in further reduction of MDSCs which could make the tumor microenvironment more susceptible to therapeutic intervention. To test this, we looked at rapamycin (8mg/kg) given either simultaneously with, or 1 week prior to, anti-CTLA4. Single agent activity was as expected with %T/C on day 22 at 63% with rapamycin and 84% with anti-CTLA-4. Combination therapy was well tolerated but we found that neither dosing schedule resulted in improved anti-tumor activity over monotherapies with 52% T/C following simultaneous treatment and 53% following sequential treatment. A study to evaluate the impact of the drug combinations on the immune profile is ongoing and will be presented. While both rapamycin and anti-CTLA-4 were capable of modulating the immune profile, improvements in efficacy were not observed. This model provides a system with which preclinical hypotheses can be efficiently tested.

#2742

Additional mechanisms of action of ladiratuzumab vedotin contribute to increased immune cell activation within the tumor.

Anthony T. Cao, Shaylin Higgins, Nicole Stevens, Shyra J. Gardai, Django Sussman. _Seattle Genetics, Bothell, WA_.

Ladiratuzumab vedotin (LV, SGN-LIV1A) is an antibody-drug conjugate directed against the LIV-1 protein that is currently under investigation for treatment in metastatic breast cancer. This ADC consists of a monoclonal antibody conjugated to monomethyl auristatin E (MMAE), a potent microtubule-disrupting agent. LV antitumor activity is thought to primarily be the result of intracellular payload release, leading to mitotic arrest and apoptotic cell death. While LV-induced cell death has been extensively studied, its potential immune modulatory activity has yet to be explored. Whereas normal apoptosis is nonimmunogenic, multiple chemotherapeutics have been shown to elicit a unique form of immunogenic cell death (ICD), characterized by the exposure of various proinflammatory intermediates prior to cell death. We have recently demonstrated that a CD30-directed ADC containing MMAE (brentuximab vedotin) was able to elicit ICD as a consequence of microtubule disruption and subsequent induction of endoplasmic reticulum stress. Induction of ICD in preclinical models of Hodgkin lymphoma results in directed antitumor immune responses, which is further potentiated by anti-PD-1 therapy. In this study, we provide evidence that ladiratuzumab vedotin induces apoptosis of tumor cells in a manner that is consistent with ICD. Treatment of LIV-1+ cells with LV resulted in the activation of all arms of the ER stress response, via the activation of ATF6 and phosphorylation of IRE1 and eIF2a. Severity of ER stress was confirmed by phosphorylation of the downstream effector Jun N-terminal kinase (JNK), and induction of the unfolded protein response. Critically, induction of C/EBP homologous protein (CHOP) indicated irreparable ER stress and subsequent apoptosis. Concurrent with ER stress induction but prior to cell death, LV-treated cells also released ATP and HMGB1 into the supernatant, hallmarks of ICD that are important for immune cell activation and recruitment into the tumor microenvironment. Critically, treatment of subcutaneously engrafted LIV1+ MCF7 cells generated a proinflammatory cytokine response within the tumor. Furthermore, intratumoral dendritic cells and macrophages displayed an inflammatory phenotype, highlighted by increased MHCII expression, providing evidence for improved immune activation and engagement. Via induction of ER stress, tumor cells killed by ladiratuzumab vedotin may initiate an antitumor immune response and provide a rationale for exploring therapeutic strategies that combine ADCs with other immune stimulatory regimens.

#2743

Accumulation of predicted neoantigens by MMR deficiency triggered by temozolomide treatment of human colorectal cancer.

Ludovic Barault,1 Giovanni Germano,1 Simona Lamba,2 Giuseppe Rospo,2 Alessandro Magri,1 Federica Maione,2 Mariangela Russo,1 Giovanni Crisafulli,1 Chiara Falcomatà,1 Carlotta Cancelliere,2 Alice Bartolini,2 Giulia Lerda,2 Giulia Siravegna,1 Benedetta Mussolin,2 Roberta Frapolli,3 Monica Montone,2 Giovanni Randon,4 Filippo de Braud,4 Nabil Amirouchene-Angelozzi,2 Silvia Marsoni,2 Maurizio D'Incalci,3 Armando Orlandi,5 Enrico Giraudo,1 Andrea Sartore-Bianchi,6 Salvatore Siena,6 Filippo Pietrantonio,4 Alberto Bardelli,1 Federica Di Nicolantonio1. 1 _University of Turin, Candiolo, Italy;_ 2 _Candiolo Cancer Institute – FPO, IRCCS, Candiolo, Italy;_ 3 _IRCCS-Istituto di Ricerche Farmacologiche Mario Negri, Milan, Italy;_ 4 _Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy;_ 5 _Policlinico Universitario A. Gemelli, Rome, Italy;_ 6 _Niguarda Cancer Center, Grande Ospedale Metropolitano Niguarda, Milan, Italy_.

Recent clinical trials have reported that alkylating agents, such as dacarbazine and temozolomide (TMZ) are effective in 10-15% metastatic colorectal cancer (mCRC) patients. In a retrospective analysis of 105 mCRC patients enrolled in clinical trials with alkylating agents, we saw that O6-methylguanine-DNA- methyltransferase (MGMT, the enzyme responsible for repairing drug induced DNA adducts), promoter methylation and protein status could identify a patient subgroup more likely to benefit from these drugs. However, the time to progression of patients treated with alkylating agents was limited by the onset of resistance. In order to identify determinants of response to these agents, we tested TMZ in a collection of 47 molecularly annotated CRC cell lines. We found that only cells displaying mismatch repair (MMR) proficiency as well as promoter methylation and transcriptional silencing of MGMT were sensitive to the treatment. Sensitive cells were chronically exposed to TMZ until the emergence of resistance occurred in seven out of ten models. We identified the mechanisms of acquired drug resistance to be either re-expression of MGMT and/or mutations in MMR genes. Biopsies of eight patients, relapsing upon TMZ based regimen after a long lasting clinical benefit (>3 months), were analyzed for MGMT re-expression and for exome or target panel next generation sequencing. These analyses confirmed the results found in preclinical models: five biopsies showed MGMT re-expression, two demonstrated mutations in the MMR genes (i.e. MSH6 or MSH2), and one displayed a mutation in BRCA2. Interestingly, biopsies and cell lines in which resistance was associated to alterations in DNA repair genes displayed increased mutational loads compared to pre-treatment samples. Despite the absence of the microsatellite switch commonly found in MMR deficient driven cancer, follow up over time in absence of drug of a subset of resistant cell line models demonstrated that those which displayed a mutation in the MMR had an accumulation of predicted neo-antigens, suggesting an increased immunogenicity. This work has implications for the design of future trials incorporating TMZ as part of a multi-modality strategy for treating MGMT deficient and MMR proficient CRC.

#2744

Telomelysin-induced immunogenic cell death synergizes with anti-PD-1 antibody in non-immunogenic gastrointestinal tumors.

Nobuhiko Kanaya,1 Shinji Kuroda,1 Toshiaki Morihiro,1 Yoshihiko Kakiuchi,1 Tetsushi Kubota,1 Satoru Kakiuchi,1 Masahiko Nishizaki,1 Yasuo Urata,2 Hiroshi Tazawa,1 Shunsuke Kagawa,1 Toshiyoshi Fujiwara1. 1 _Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Kita-ku Okayama, Japan;_ 2 _Oncolys BioPharma, Inc., Tokyo, Japan_.

Background: Telomelysin (OBP-301) is a telomerase-specific oncolytic adenovirus, in which the telomerase promoter controls its replication and induction of cell death in human tumor cells. A FDA-approved phase I study has confirmed the safety and biological activity of intratumoral administration of Telomelysin in patients with advanced solid tumors in the US. Anti-programmed death-1 antibody (PD-1 Ab) has dramatically improved clinical outcomes for patients with melanoma or lung cancer; however, the clinical benefit is limited to small population of patients with certain degree of tumor infiltrating lymphocytes (TILs). Therefore, more effective strategy to enhance the immunogenicity of non-immunogenic tumors is needed. Here, we show the potential of our oncolytic adenovirus as an immunogenic agent sensitizing non-immunogenic gastrointestinal tumors to PD-1 Ab.

Methods: A murine colon cancer cell line CT26 (BALB/c) and a murine pancreatic cancer cell line PAN02 (C57BL/6) were used in this study. An OBP-301 variant (OBP-502) that expresses the RGD mutant fiber to infect murine tumor cells via interaction with integrin was used as an oncolytic adenovirus. PD-1 Ab (clone 4H2) was obtained from Ono Pharmaceutical Co. Ltd. As immunogenic cell death markers, extracellular ATP and high mobility group box 1 (HMGB1) levels were measured following OBP-502 infection. In vivo subcutaneous tumor models, CD8-positive TILs, vaccination efficiency of OBP-502-treated cells and combination therapy of OBP-502 with PD-1 Ab were evaluated.

Results: OBP-502 efficinetly killed CT26 and PAN02 cells, and also significantly increased extracellular ATP and HMGB1 on both cell lines in vitro, suggesting that OBP-502-induced cell death might be immunogenic. Indeed, in the CT26 and PAN02 subcutaneous tumor models, OBP-502 induced massive accumulation of TILs compared to control. Vaccination of mice with OBP-502-infected CT26 cells rejected tumors in three of 10 vaccinated mice (30%) and also significantly suppressed the growth of remaining seven CT26 tumors. The combination therapy of OBP-502 and PD-1 Ab significantly suppressed the growth of CT26 subcutaneous tumors and successfully eradicated tumors in four of 12 mice (33%) while either single treatment failed to eradicate in most mice. When these 4 tumor-free mice treated with the combination therapy were re-challenged by inoculation of CT26 cells, two of 4 mice (50%) remained tumor-free. Similar antitumor effects of the combination therapy were observed in the PAN02 tumor model.H.E staining of major organs harvested after each treatment demonstrated no serious systemic toxicity that PD-1 Ab has been reported to cause sometimes in clinical practice.

Conclusion: Our telomerase-specific oncolytic adenovirus has the potential to activate antitumor immunity, leading to sensitization of non-immunogenic gastrointestinal tumors to PD-1 Ab.

#2745

EpCAM targeted immunotoxin prolongs survival of metastatic colorectal cancer patients by a previously unknown immunostimulatory mechanism.

Øystein Fodstad, Svein Dueland, Kjersti Flatmark, Else Marit Inderberg, Gunnar Kvalheim, Yvonne Andersson. _Norwegian Radium Hospital, Oslo, Norway_.

A first-in-man study of the anti-EpCAM immunotoxin MOC31PE with and without parallel immunosuppression with Cyclosporin A (CsA) was performed in metastatic colorectal cancer (CRC) patients. The safety and maximum tolerable doses in patients with EpCAM-positive tumors were determined, and only mild and reversible dose-limiting liver toxicity was seen, with no subjective side effects to the patients. Although no complete or partial responses were observed on CT scans at eight weeks, upon evaluation of the overall survival (OS) of the patients we surprisingly found that CRC patients treated with immunotoxin alone (n=15) had a median OS of 16.3 months (95% CI=5.6-27.0) compared to 6.0 months (CI=5.8-6.2) (p<0.001) for the combination group (n=18) (hazard ratio (HR): 0.248, 95% credible interval (CrI): 0.109-0.564). The 6.0 months OS for the patients treated with CsA in addition to the immunotoxin is comparable to OS of patients with progressive disease on last line of standard chemotherapy. The Th1 cytokine profile in sera from patients treated with MOC31PE alone indicated that the immuntoxin induces an immunogenic cell death resulting in T-cell activation, and that this effect disappeared in the patients who had received simultaneous treatment with CsA. Further, we demonstrate in in vitro experiments that the immunogenic cell death factors ATP and HMGB1 were secreted from CRC cell lines treated with MOC31PE alone. Finally, conditioned media from MOC31PE treated CRC cells induced maturation of dendritic cells and promoted activation of killer (CD8+) and helper (CD4+) T cells, showing that MOC31PE treatment induces release of immune stimulating factors. The present results demonstrate the novel and exciting finding that MOC31PE induces an immunogenic effect resulting in prolonged overall survival of patients with metastatic CRC, an effect obliterated by the parallel immunosuppression of CsA.

#2746

Synergistic immunotherapeutic effects of TLR7/8 agonist on low-dose cyclophosphamide-treated C26 model.

Jing Yang,1 Patrick Z. Li,2 Xiangyun Yin,3 Xiaohua Nie,3 Jingshu Wang,3 Walter Lau,1 Liguo Zhang,3 Lixin Li1. 1 _Birdie Biopharmaceuticals, Inc., Beijing, China;_ 2 _Ren Da Fu Zhong, Beijing, China;_ 3 _Institute of Biophysics, CAS, Beijing, China_.

In recent years, low dose cyclophosphamide has been used clinically either as a single agent or as combination therapy to treat lymphomas, and breast and ovarian cancers. The efficacy of low dose cyclophosphamide is primarily due to its ability to immune-modulate in addition to its direct anti-tumor effects. This ability highlights the potential for synergism between conventional chemotherapy and novel immunotherapy. Toll-like receptors (TLRs) are a crucial part of the innate immunity and present the first line of defense against pathogens. Resiquimod is the ligand for TLR7 and 8 and directly activate innate immune cells, including myeloid dendritic cells, plasmacytoid dendritic cells, and monocytes/macrophages. This activation may result in activation of co-stimulatory molecules, production of antiviral cytokines, and stimulation of cell-mediated NK and T cell immune responses. The aim of this study was to analyze whether immunopotentiating TLR7/8 agonist might synergize with low doses of cyclophosphamide in the treatment of colorectal carcinoma. The effect of combining a weekly dosing of cyclophosphamide intraperitoneally with systemic administration of Resiquimod was evaluated in an in vivo murine C26 synergistic model. Systemic administration of Resiquimod in mice induced significant systemic immune responses as evidenced by upregulating IFNα inducible gene clusters. The combined therapy with Resiquimod and cyclophosphamide was superior to each single treatment and increased efficacy of tumor inhibition and prolonged their survival significantly. Importantly, the combined treatment generated a significant numbers of tumor-specific T cell infiltration in tumor microenvironment. Our data suggest that systemic administration of TLR7/8 agonist enhances anti-tumor effect of low dose of cyclophosphamide. The data supports the use of a combination of TLR7/8 agonist and cyclophosphamide as a novel therapeutic strategy against colorectal carcinoma.

#2747

Anti-glypican-3 monoclonal antibody (codrituzumab/GC33/RO5137382) treatment enhances tumor infiltration of PD-L1-positive macrophages, and combination therapy with anti-PD-L1 monoclonal antibody promotes antitumor effects.

Mika Endo, Yasuko Kinoshita, Kenji Adachi, Yoshinori Narita, Jun Amano, Atsuhiko Kato, Takeshi Watanabe, Yoko Kayukawa, Yoko Miyazaki, Toshihiko Ohtomo. _Chugai Pharmaceutical Co., Ltd., Japan_.

Introduction: Codrituzumab/GC33/RO5137382 (GC33) is a humanized monoclonal antibody that targets glypican-3 (GPC3), an oncofetal protein expressed on the cell surface of hepatocellular carcinoma (HCC). GC33 interacts with CD16/FcγR3 and triggers antibody-dependent cellular cytotoxicity. Because anti-PD-L1/PD-1 agents have shown marked antitumor effect in various cancer types including HCC, we investigated if GC33 plus anti-PD-L1 mAb combination can augment antitumor efficacy in a mouse hepatoma syngeneic model transfected with human GPC3, named Hepa1-6/hGPC3.

Methods: The Hepa1-6/hGPC3 cells were subcutaneously inoculated into C57BL/6J mice. After tumor mass was established, anti-mouse GPC3 mAb (mGC33; once-weekly), anti-mouse PD-L1 mAb (anti-mPD-L1 mAb; once-weekly), or a combination was administered on the first day of treatment (Day 0). Tumor tissues were collected on Day 21 for immunohistochemical (IHC) of F4/80 and PD-L1. To analyze tumor infiltrating lymphocytes (TILs), mGC33, anti-mPD-L1 mAb, or combination was administered to the Hepa1-6/hGPC3 mice. After 3 and 8 days from the 2nd dosing, TILs were analyzed to quantify the CD45-, CD3ε-, CD4-, or CD8α-positive TILs and CD11b+F4/80+ macrophages by flow cytometry.

Results: In the Hepa1-6/hGPC3 model, combination therapy demonstrated a marked antitumor effect compared to the corresponding dose of mGC33 or anti-mPD-L1 mAb alone. Pathological complete responses were observed only in combination groups. The necrosis was more marked with combination therapy than with mGC33 or anti-mPD-L1 mAb alone. Though F4/80-positive cells existed mainly in the stroma in the vehicle group, these cells infiltrated the tumor periphery after mGC33 treatment. Most tumor-infiltrating immune cells, including macrophages and multinucleated giant cells, were PD-L1-positive. The combination increased CD45-, CD3ε-, and CD8α-positive T lymphocytes, but not CD4-positive T lymphocytes on Days 3 and 8 after the 2nd dosing. TILs were not increased in mice treated with either mGC33 or anti-mPD-L1 mAb.

Conclusions: In this mouse model, mGC33 plus anti-mPD-L1 mAb combination therapy showed more potent antitumor efficacy than either monotherapy. mGC33 treatment enhanced tumor infiltration of PD-L1-positive immune cells, such as macrophages and multinucleated giant cells. Because anti-mPD-L1 mAb can block the binding between PD-L1 on macrophages and PD-1 on T cells, the CD8-positive T lymphocytes may be increased by combination therapy. These results suggest that the combination therapy of GC33 and anti-PD-L1 mAb may be clinically useful as a treatment for HCC.

#2748

Antitumor activity of HDAC inhibition in bladder cancer mouse models correlates with enhanced immune response.

Kyle G. Stewart, Andrew S. Truong, Bhavani Krishnan, Mi Zhou, Ryoichi Saito, Jordan Kardos, Ujjawal Manocha, Sean T. Bailey, William Y. Kim. _University of North Carolina at Chapel Hill, Chapel Hill, NC_.

Aberrant chromatin remodeling by epigenetic modifier proteins such as histone deacetylases (HDACs) is common within many types of cancer, including muscle-invasive bladder cancer (MIBC). The removal of acetyl groups from the lysine residues of histones leads to transcriptional silencing that promotes tumor growth and potentially enhances cancer cells' ability to evade the host immune response. Therefore, HDACs present themselves as attractive targets for cancer therapy. Histone deacetylase inhibitors (HDACi) demonstrate broad anti-cancer activity. Many proposed mechanisms exist to explain their effects, including those that involve the immune system. Here, we evaluated the efficacy and transcriptional effects of entinostat, a selective class 1 HDAC inhibitor (HDAC1 and HDAC3), in a novel preclinical murine model of high-grade MIBC.

BBN963 cells were derived from a primary bladder cancer GEM, cultured, and implanted subcutaneously into immunocompetent C57BL/6 mice and immunodeficient NOD scid gamma (NSG) mice. Animals were randomized to control or entinostat treatment. Tumor volume was recorded weekly and fresh tissue collected for RNAseq and Ingenuity Pathway Analysis (IPA). Entinostat exhibited in vivo activity in the BBN963 model in both C57BL/6 and NSG mice, however the anti-tumor response in B6 mice was significantly greater than the observed response in NSG mice. RNAseq analysis on tumor tissue collected from NSG mice indicated that entinostat treated tumors had distinct gene expression changes. Comparison of control to entinostat treated tumors revealed 4987 genes were significantly upregulated and 4112 genes were significantly downregulated (p-value < 0.05). IPA contextualized these changes as an enhanced inflammatory response, as indicated by increased expression of upstream regulators TGFβ, LPS, and IFNγ. Additionally, hierarchical clustering using established immune gene signatures stratified entinostat treated and untreated tumors into two distinct groups.

Our results suggest that the anti-cancer properties of entinostat are in part immunologically mediated. BBN963 tumors in B6 mice displayed a more robust therapeutic response compared to their NSG counterparts. Transcriptional analysis of the tumor tissue indicated significant differences in immune gene signature levels as well as inflammatory pathway activation. Therefore, an intact immune system appears critical to achieve a maximal therapeutic response observed with HDAC inhibition. Together these results lay the foundation for further elucidating entinostat's mechanism of action in the context of high-grade MIBC.

#2749

Effects of subtype specific chemotherapeutic immunomodulation in bladder cancer.

Jordan Kardos, Lisa Bixby, Andrew Truong, Bhavani Krishnan, Kyle Stewart, Benjamin Vincent, William Kim. _University of North Carolina-Chapel Hill, Chapel Hill, NC_.

In patients with bladder cancer, programmed death-ligand-1 (PD-L1) and programmed death-1 (PD-1) inhibitors have been shown to be effective in around twenty percent of patients, and there is evidence indicating that the level of immune infiltration and immune suppression within the tumor microenvironment correlates with response to these treatments. We have previously shown that there are intrinsic subtypes of bladder cancer, with the basal subtype characterized by high levels of immune infiltration, and the luminal subtype characterized by immune exclusion. Here we show that treatment with current standard of care chemotherapeutic agents has a subtype specific effect on the tumor microenvironment. Cisplatin-based chemotherapeutic treatment of luminal tumors induces a mesenchymal phenotype, immune infiltration, and a transition to a more basal-like tumor, while not altering these characteristics of the tumor microenvironment in basal tumors. Two of the most widely used standard of care chemotherapeutic regiments are Cisplatin-Gemcitabine (GemCis) and Methotrexate-Vinblastine-Doxorubicin-Cisplatin (MVAC), and we show that MVAC treatment induces significant immune infiltration within the luminal subtype while GemCis treatment does not, indicating there are treatment specific effects on the immune microenvironment. Furthermore, using mouse models of bladder cancer previously developed by our lab that accurately reflect the basal and luminal subtypes of bladder cancer, treatment with a representative chemotherapeutic regimen induced immune infiltration in the luminal mouse model while not affecting the tumor immune microenvironment in the basal mouse model. These results indicate chemotherapeutic regimens have subtype specific effects on the tumor microenvironment which could potentially be used to increase the efficacy of immune checkpoint inhibitors.

#2750

An epigenetic and immunologic approach to optimize therapy for HNSCC.

Rachel Goldberg, Masahiro Shibata, Evgeny Izumchenko, Luigi Marchionni, David Sidransky, Mohammad O. Hoque. _Johns Hopkins Medical Insts., Baltimore, MD_.

Head and Neck Squamous Cell Carcinoma (HNSCC) is the sixth most prevalent cancer worldwide, and despite advances in treatment options, the five-year survival rate for patients with HNSCC is dismal. Monotherapy (such as surgery or radiation) can be effective if detected early, however more advanced cases require treatment with various combinations of surgery, radiation and chemotherapy. Recently, interest in the utilization of immunotherapy for advanced HNSCC has increased, including the FDA approval of pembrolizumb and nivolumab (checkpoint inhibitors) for patients with recurrent or metastatic HNSCC. However, about 80% of patients remain unresponsive to checkpoint inhibitors. Numerous pre-clinical and clinical attempts aim to increase the sensitivity of immuno-agents. One such attempt is combination of immune-agents with epigenetic therapy. Epigenetic alterations in tumor cells modulate gene expression patterns that may facilitate evasion of immune recognition. The efficacy of epigenetic modulating agents to reverse these alterations is documented in various cancer types including HNSCC. Here, we sought to explore whether the utilization of combination therapy of epigenetic drugs (DNA methyltransferase inhibitor, 5azacytidine and the histone deacetylase inhibitor, romidepsin) with checkpoint inhibitors to increase the efficacy of immune-agents in a pre-clinical model of HNSCC.

To explore the gene expression alterations induced by epigenetic drugs in HNSCC, we treated six HNSCC cell lines in vitro with 5azacytidine and romidepsin, and analyzed gene expression patterns by microarray and quantitative RT-PCR (QRT-PCR). We found significant differences in immune-related gene expression patterns in epigenetic drug-treated cell lines compared to untreated controls. Further, to explore the immune-associated gene expression changes, we performed QRT-PCR using a Taqman low density (TLDA) human immune array. By this analysis, we found changes in expression patterns of several immune related genes including HMOX1, NFKB2, HLA-DRA, STAT3, IL12a, CSF1, CSF2, FAS, and IL-18 in epigenetic drug-treated cells. Additionally, by QRT-PCR analysis of several candidates of the viral defense pathway, we found significant upregulation of Interferon type 1 (IFN 1) related genes STAT1, OASL, IFI6 and IRF7 in epigenetic drug-treated cell lines. These results indicate that HNSCC cell lines acquire a new immune signature following treatment with epigenetic agents. Next, we used an in vivo syngeneic model of HNSCC, to compare tumor growth following treatment with mono or combination therapy. Our preliminary data suggests that combination treatment with epigenetic drugs and an immune-agent more effectively reduces tumor growth than either of these drugs alone. In conclusion, our results highlight the importance of considering combination of epigenetic and immune therapy for HNSCC patients that respond poorly to immune therapy alone.

#2751

Regorafenib attenuates IFN-γ-induced PD-L1 expression and enhances antitumor immunity.

Xiao-Feng Zhu. _Sun Yat-sen Univ. Cancer Ctr., Guangzhou, China_.

Immune checkpoint blocked therapy (ICB) targeting the PD-1/PD-L1 axis induce durable tumor regressions in a sizeable minority of cancer patients. To enhance the efficacy of existing immunotherapies, we implemented a screen of kinase inhibitors capable of increasing the activity of T cells suppressed by PD-L1. Here, we identified regorafenib, which was approved to treat patients with advanced gastrointestinal stromal tumors and metastatic colorectal cancer, as a potent agent significantly attenuating IFN-γ-induced PD-L1 expression without affecting MHC-I expression, contributing to enhanced anti-tumor effects with ICB in vitro and in vivo, due in part to inhibit JAK1/2-STAT1 signaling and the activation of ERK by suppressing the tyrosine kinase receptor RET-Src axis. Additionally, our analysis of GEO database and TCGA database revealed that RET and Src co-high expression was an independent unfavorable prognosis factor in both melanoma patients with or without ICB via inhibiting anti-tumor immune response. Further, gene set enrichment analysis (GSEA) demonstrated, compared to RET and Src co-high expression group, anti-tumor immune response related gene signatures as prominent modules in other group. Collectively, our data unveiled a new mechanism of reducing IFN-γ-induced PD-L1 expression and provided a rationale to explore a new combination of immunotherapies and regorafenib in melanoma treatment.

#2752

EGFR tyrosine kinase inhibitor limits tumor relapse through enhancing anti-tumor T cell responses.

Zhida Liu, Chuanhui Han, Aijun Shen, Chunbo Dong, Jian Qiao, Yang-Xin Fu. _UT Southwestern Medical Center, Dallas, TX_.

EGFR tyrosine kinase inhibitors (TKIs) have been clinically considered as the first line therapy for EGFR-driven tumor patients. Although the initial outcome of patients has significantly improved, almost all patients eventually relapse. Fully understanding the mechanisms of the anti-tumor effect of EGFR TKIs will help promote the clinical development of more efficient therapeutic strategies. The traditional view of the main mechanism is that the inhibitors can directly inhibit EGFR downstream signaling pathways to induce tumor cell death. Whether and how EGFR TKIs affect the host immune system is largely unknown. In current study, we have established syngeneic murine tumor models that are sensitive to EGFR TKIs treatment. To our surprise, we revealed that EGFR TKI treatment could control tumor growth and limit tumor relapse in T cells dependent manner. Mechanistically, EGFR TKI triggers type I IFN and its stimulation gene CXCL10 production through the Myd88 signaling pathway to enhance tumor specific T cells infiltration and activation. However, EGFR TKI cannot efficiently limit tumor relapse in advanced tumor models because of PD-L1 upregulation in the tumor microenvironment after treatment. We have observed that anti-PDL1 blockade can synergize with EGFR TKI to control advanced tumors. Our study will challenge the current regimen of EGFR TKI in clinic and provide new efficient therapeutic strategies for EGFR dependent cancer patients.

#2753

Translation to the clinic of EVT801: A novel immune-oncology agent for addressing innate-driven immunosuppression into the tumor microenvironment and expanding patient population responding to immune checkpoint therapies.

Pierre Fons,1 Michael Esquerre,1 Julien Mazieres,2 Philippe Rochaix,3 Anne Gomez-Brouchet,2 Antoine Alam,4 Florie Bertrand,1 Celine Poussereau-Pomie,1 Jerome Meneyrol,1 Anne Pradines,5 janick selves,2 Isabelle Rouquette,2 Isabelle Blanc,4 Francoise Bono,6 Donogh P. O'Brien,7 Michael Paillasse,1 Joanna Lisztwan,1 Mark Whittaker1. 1 _Evotec, TOULOUSE, France;_ 2 _Institut Universitaire du Cancer Toulouse-CHU de Toulouse, TOULOUSE, France;_ 3 _Institut Universitaire du Cancer Toulouse-Institut Claudius Regaud, TOULOUSE, France;_ 4 _sanofi, Marcy l'étoile, France;_ 5 _Institut Universitaire du Cancer Toulouse-Centre de recherche en cancérologie, TOULOUSE, France;_ 6 _Onxeo, Paris, France;_ 7 _Donogh O'Brien BioConsulting, Les Poirioux, France_.

Amongst resistance mechanisms of immune checkpoint therapies (ICTs), expansion and recruitment of immunosuppressive innate cells and in particular Myeloid Derived Suppressor Cells (MDSCs) can be a major cause of resistance to ICTs. Here we describe joint efforts of clinicians and researchers to translate the promising EVT801 action on MDSCs into the clinic. We have identified a highly selective drug candidate targeting VEGFR3, EVT801, showing intermediate in vivo activity on tumours, accompanied by a decrease of MDSCs, when VEGFR3 is expressed in the tumour microenvironment. To evaluate the potential to combine with ICTs, the 4T1 orthotopic mammary carcinoma mouse model, was used. We have demonstrated that EVT801 increases the therapeutic activity of an anti-PD-1 antibody (Ab) on primary tumours as compared to single agent treatment but also results in a decrease of lung metastasis. Furthermore, the decrease of MDSCs in the blood in response to EVT801 is correlated with tumor shrinkage and therapeutic efficacy. In parallel, Immuno Histo Chemistry analyses have shown that treatment with EVT801 increases CD8+ T-cells infiltration inside the tumor. Taken together, these results indicate that EVT801 represents an innovative drug for cancer immunotherapy that improves the frequency of response to ICT by controlling MDSCs immunosuppressive activity and in turn by unleashing tumor-specific T-lymphocytes. To translate these promising results into the clinic, we have evaluated different biomarkers to properly select patients and monitor efficacy of EVT801: (1) We refined an antiPD1 Ab resistance gene signature and correlated it with VEGFR3 pathway gene overexpression. (2) We investigated VEGFR3 expression in the tumor microenvironment (TME) for different cancer indications. Taken together, these results will facilitate patient stratification as based on the PD1 resistance gene signature associated with VEGFR3 expression in the TME. (3) In non-small cell lung cancer patients, we validated that a high-level baseline of circulating M-MDSCs is associated with poor survival. In correlation with EVT801 activity on circulating MDSC as deciphered on preclinical mouse models, we propose that circulating MDSC levels could be a biomarker of activity for EVT801 in non-responder patients to ICTs. EVT801 represents a novel agent for cancer immunotherapy for non-responder patients to ICT. Patient stratification strategy, target engagement biomarker and biomarkers of activity have been identified and will enable the initiation of a planned Phase I clinical trial with EVT801.

#2754

NT219, a novel dual inhibitor of STAT3 and IRS1/2, converts immuno-oncology resistant tumors to responders.

Lana Kuperschmidt,1 Hadas Reuveni,2 Shani Carmi,1 Neta Moskovits,1 Netta R. Shraga,1 Evgeny Solomonov,3 Ohad Ronen,4 Salomon Stemmer,5 Izhak Haviv1. 1 _Bar Ilan University, Zfat, Israel;_ 2 _Tyrnovo LTD, Tel Aviv, Israel;_ 3 _Ziv Hospital, Zfat, Israel;_ 4 _Gallilee Medical Center, Naharia, Israel;_ 5 _Clalit, Petach Tikva, Israel_.

Feedback activation of STAT3 and IGF1R/IRS plays a prominent role in mediating drug resistance to a broad spectrum of targeted cancer therapies and chemotherapies. Both the IRS1/2 and STAT3 are major signaling junctions regulated by various oncogenes, and altered during EMT and drug resistance. STAT3 has also been known to play an active role in immune-evasion of tumors and inhibition of STAT3, both in the tumor, as well as in the tumor's microenvironment, may therefore potentiate immune attack on the tumor. NT-219 is a dual inhibitor of STAT3 and IRS1/2 developed by TyrNovo Ltd. to overcome cancer drug resistance. NT-219 inhibits STAT3 phosphorylation and eliminates IRS1/2 in a unique 3-step mechanism: dissociation of IRS1/2 from the IGF1 receptor, induction of IRS1/2 serine phosphorylation, and subsequent degradation by the proteasome. We recently demonstrated that the inhibition of both IRS and STAT3 are required and essential for overcoming drug resistance. NT-219 efficacy was demonstrated in Patient-Derived tumor Xenograft (PDX) models of multiple cancer types: melanoma, sarcoma, pancreatic, colon, lung, and head & neck, when added-on to the approved therapies. In these models, NT-219 overcame acquired resistance to several oncology drug families: inhibitors of EGFR (Tarceva®, Erbitux®, Tagrisso®), MEK (Mekinist®), mutated-BRAF (Zelboraf®), mTOR (Afinitor®) as well as with chemotherapy agents (Gemzar®, 5FU, Oxaliplatin). We recently demonstrated that NT-219 works in synergy also with immune-oncology therapies. By using double autologous PDX models we demonstrated that NT-219 converted non-responding tumors to responders to Keytruda®. It also enhanced the immunotherapeutic potential of Cetuximab. The unique mode of action of NT219 may open a new avenue of combined targeted therapies in a wide range of malignancies, and has the potential to expand response duration and target patient population to the applicable drugs.

#2755

NKTR-262: Prodrug pharmacokinetics in mice, rats, and dogs.

Myong Lee. _Nektar Therapeutics, San Francisco, CA_.

Background: TLR7/8 agonists are currently under investigation in clinical trials to determine their anti-tumor effect. Systemic exposures to the TLR7/8 agonists have resulted in severe adverse effects, thereby preventing the TLR7/8 agonists from achieving therapeutic plasma concentrations. NKTR-262 is a novel polymer-modified TRL7/8 agonist prodrug which, upon intratumoral delivery, slowly liberates the TLR7/8 agonist to provide sustained intratumoral engagement of the TLR7/8 pathway to promote an immune stimulatory environment and tumor antigen release.

Method: In vivo PK parameters were obtained in bilaterial tumor-bearing (CT26 and EMT6) and naïve Balb/c mice, SD rats, and Beagle dogs following a single dose (i.t., i.v. and s.c., respectively) of NKTR-262. Samples were analyzed by LC/MS/MS. Non-compartmental analysis was used to obtain PK parameters. In vitro studies with NKTR-262 and the corresponding non-polymer modified TLR7/8 agonist were performed to assess plasma release, agonist CLint in liver microsomes and hepatocytes, plasma protein binding, blood-to-plasma ratios and to identify CYP isoform(s) responsible for oxidative metabolism. PK/PD modeling was performed in NONMEM7 using a semi-mechanistic model described by Simeoni et al 2004.

Results: NKTR-262 exhibited a low volume of distribution and low systemic clearance. NKTR-262 showed a prolonged elimination t½, with an order of dog > rat > mouse. NKTR-262 was eliminated renally (~50% total clearance) and by prodrug conversion. Release of the non-polymer modified TLR7/8 agonist from NKTR-262 was confirmed in vitro and in vivo in all pre-clinical species and in vitro in plasma from humans. The PK profiles of the released TLR7/8 agonist closely followed NKTR-262 PK profiles. The absolute bioavailability of NKTR-262 following i.t. and s.c. injection was low to moderate. I.t. administration of NKTR-262 generated high and sustained exposure to the TLR7/8 agonist in the treated tumor, which was two orders of magnitude greater than in plasma. Contralateral tumor and plasma exposures to the agonist were similar. The tumor type had no impact on plasma PK profiles. The agonist exhibited low plasma protein binding and distributed evenly between blood cells and plasma. The in vitro CLint of the agonist was low, and oxidative metabolism was mostly CYP3A4-mediated. The Simeoni model captured tumor volume change over time with an estimated EC50 1.34 ng/mL.

Conclusion: The TLR7/8 agonist was released from NKTR-262 in vivo and in vitro in all pre-clinical species and in vitro in plasma from humans. Intratumoral administration of NKTR-262 resulted in pharmacologically active levels of the released TLR7/8 agonist in the tumor and reduced unwanted systemic exposure. Released agonist PK profiles followed the PK profiles of NKTR-262. NKTR-262 was cleared via prodrug conversion and renal clearance. The prodrug-generated TLR7/8 agonist is a low clearance compound with low protein binding.

#2756

Synergistic tumor-suppressive effect of apatinib, a selective VEGFR-2 inhibitor, in combination with immunotherapy in a syngeneic murine lung cancer model.

Bumjin Kim, Arlo N. McGinn, Cheol Hee Park, Sung Chul Kim. _LSK BioPharma, Salt Lake City, UT_.

Anti-angiogenesis therapy is considered one of several promising combination strategies with immunotherapy. The potential for synergistic combination is based on more than just the disparate mechanisms of action of limiting new vessel formation and circumventing cancer immune evasion. Beyond limiting sprouting of new vessels, angiogenesis inhibition is known to normalize existing tumor vasculature and ameliorate the immune-suppressive tumor microenvironment. These effects respectively increase the number of tumor-infiltrating lymphocytes in the tumor milieu and allow these immune agents to kill cancer cells more efficiently. So far, a number of preclinical studies have supported the benefit of combining anti-angiogenesis therapy with immunotherapy. Monoclonal antibodies targeting VEGF and VEGFR-2, as well as multi-kinase small-molecule inhibitors targeting VEGFR have shown synergistic efficacy in various tumor models. Those studies also revealed that this combination limits tumor growth through increasing anti-tumoral T-cell populations (e.g. CD4+ and CD8+ T-cells) as well as decreasing immune-suppressive immune cell populations (e.g. Treg and MDSCs). Moreover, clinically, the combination treatments have reached phase III development for hepatocellular, renal cell, non-small cell lung carcinoma, and ovarian cancer patients. Apatinib is an orally-available, small-molecule tyrosine kinase inhibitor of VEGFR-2 with high selectivity and potency (IC50 = 1.0 nM) as well as a low toxicity profile proven in multiple clinical trials. Apatinib has been approved in China for third-line gastric cancer and many clinical trials have been conducted worldwide for multiple tumor types including an ongoing global phase III gastric cancer study. Herein, we present preclinical outcomes that apatinib synergistically suppressed tumor growth when combined with anti-PD-1 immunotherapy. In this study, a LL/2 murine lung carcinoma syngeneic mouse model was used to test the tumor growth inhibition (TGI) activities of single-agent apatinib and anti-muPD-1 treatment as well as a combination of both agents. Over a 20-day period, the anti-muPD-1 antibody was dosed as a single agent at 10 mg/kg IP, BIW and apatinib was tested as a single agent at 75, 150, and 300 mg/kg, PO, QD and again at the same concentration in combination with anti-muPD-1 at 10 mg/kg. Apatinib mono-treatment (300 mg/kg) and anti-muPD-1 mono-treatment showed only 22% and 37% of TGI, respectively; however, the 300 mg/kg apatinib + anti-muPD-1 group showed the highest and most significant TGI of 55% (p<0.0001). Collectively, we determined that apatinib treatment played a synergistic role on suppressing tumor growth when combining with anti-PD-1 treatment, which is encouraging for upcoming clinical trials of apatinib in combination with anti-PD-1 therapies planned for early 2018.

#2757

Antibody-drug conjugate payloads induce markers of immunogenic cell death in cancer cells.

Xingzhi Tan, My-Hanh Lam, Shoba Ragunathan, Keziban Unsal-Kacmaz, Frank Loganzo. _Pfizer, Inc., Pearl River, NY_.

Activating the immunologic destruction of cancer cells is a clinically validated therapeutic approach. Immunogenic cell death (ICD) is a reported mechanism of activating the immune system wherein anti-cancer treatments induce specific tumor cell markers, followed by dendritic cell activation and T-cell recruitment. Antibody-drug conjugates (ADCs) are targeted biotherapeutics which deliver cytotoxins to cancer cells. Our hypothesis is that ADCs also may have immune-modulating properties and that the payloads delivered by ADCs may induce ICD. We developed three assays to evaluate ICD in cultured tumor cell lines: (1) HMGB1 extracellular protein levels, (2) cell surface calreticulin translocation, (3) and ICD-induced dendritic cell activation (ICD-DCA). First, cytototoxity was assessed for all compounds, then ICD studies were conducted at dose-multiples relative to each compound's anti-proliferative IC50. Next, cells were treated with various chemotherapeutic agents and extracellular HMGB1 protein detected by ELISA. However, HMGB1 levels strongly correlated with cytotoxicity, and did not differ among treatments. We next assessed calreticulin (CRT), an ER-resident protein reportedly translocated to the cell surface upon ICD. We evaluated the specificity of commercial antibodies to detect CRT in human (A549) or murine (EMT6) cell lines. Cells were incubated with CRT siRNA specific for murine or human sequences. Flow cytometry (for cell surface CRT) and immunoblots (for total CRT) confirmed that a commonly used CRT antibody detects CRT in murine cells, but not human cells. Murine EMT6, 4T1, B16F10, or CT26 cells were then treated with compounds of varied mechanisms-of-action. Our assays confirm previous reports that anthracycline chemotherapeutics (e.g., doxorubicin) induce CRT surface translocation. We also discovered that analogs of both the calicheamicin and cyclopropylpyrroloindolone (CPI) DNA inhibitor class of ADC payloads robustly induced CRT surface translocation in several murine cancer lines within 24 hr, even at relatively low subtoxic doses. Some DNA inhibitors did not induce CRT. Microtubule inhibitor auristatins and mertansine (DM1) induced CRT translocation, but at high cytotoxic doses and after 48 hr incubation. Next, tumor cells were treated with compounds, washed, then co-cultured with murine bone-marrow derived dendritic cells (BMDC). The DNA inhibitors which induced CRT on tumor cells also increased levels of CD86 activation marker and cytokine release from BMDC in these co-cultures. Hence, we have demonstrated that calreticulin, a reported marker of ICD, is activated in cultured murine tumor cells by unconjugated payloads typically delivered by ADCs. These compounds can also induce DC activation in tumor:BMDC co-cultures. These data suggest the potential for immune system activation using cytotoxic ADC payloads.

#2758

Transcriptomics analysis of SNX-2112 on immune and mitochodrial genes.

Everardus Orlemans, Steven E. Hall. _Esanex Inc., Indianapolis, IN_.

Introduction: SNX-2112 is a potent anti-tumor compound with effects on epigenetics (hypomethylation) and onco-metabolism. SNX-5422 is the orally active prodrug of SNX-2112, a highly selective inhibitor of heat-shock protein 90 (Hsp90) that has shown anti-tumor activity in clinical trials. Hsp90 is commonly elevated in tumor tissues and plays an important role in immune responses. Previous investigations have shown that SNX-2112 is highly active in TP53 null tumors and has good activity in combination with checkpoint inhibitors in syngeneic mice studies. Preclinical studies have suggested that Hsp90 inhibitors enhance antigen-specific T-cell recognition of cancer cells, increase the expression of both differentiation and MHC Class I antigens, and do not interfere with T-cell function.

Methods: In two in vitro studies, the effects of SNX-2112 on immune (A375 melanoma cell line and the TP53 mutant chronic myelocytic leukemia K562 cell line) and mitochondrial genes (K652) using transcriptomics were investigated. A375 and K562 cells were cultured in media (GIBCO or Sigma, USA) supplemented with 10% fetal bovine serum at 37°C, 5% CO2, and 95% humidity. Data from transcriptomics were obtained, and affected genes analyzed.

Results: In A375 cells, transcriptomics analysis revealed that SNX-2112 downregulated multiple overexpressed immune-related genes (including NFKB1, NFKB2, BRAF, HLA-DMA, HLA-DMB, HLA-DOB, HLA-DQB1, and HLA-DRB6) and upregulated multiple suppressed immune-related genes (including IFIT1, IFIT2, IFIT3, MLANA, DDX58, PMEL (gp100), DCT (TRP-2), MR1, MICA, and MICB).

Transcriptomics analysis of SNX-2112 on K562 immune genes revealed that IFIT1-3, IFI6, IFI44, ISG15, DDX-58, and HLA-DRA were all upregulated. Genes downregulated by SNX-2112 included NFKB and IKBKB (Ikk), HLA-DOB, HLA-DPA1, HLA-DPB1, HLA-DQB1, PSMB8, and TAP1.

In both the A375 and K562 cells lines, SNX-2112 reduced the expression of MYB oncogene, (reported to control regulatory T-cell proliferation) and caused increases in RB1CC1 (fip200) and STAT1, crucial survival factors for naïve T-cells. Additionally, tryptophan metabolism genes IDO1 and TDO2 were downregulated by SNX-2112.

K562 mitochondrial genes downregulated by SNX-2112 included the following:

•TP53 and p53 family members-controlled markers: HK2, GLS2, TIGAR

•Glutamine addiction/Warburg effect: SLC1A5, GLUD1

•Folate pathway: SLC19A1, TYMS, SHMT1, AS3MT, DHFR2

•OXPHOS: CISD1, CHCHD4

Conclusion: SNX-2112 increased multiple suppressed immune-related genes and downregulated multiple overexpressed immune-related genes in A375 and K562 cell lines. Anti-tumor activity of SNX-2112 appears to be, in part, the result of interference with onco-metabolic pathways. Inhibition of IDO1 and TDO2 suggests possibly enhanced anti-tumor activity in combination with checkpoint inhibitors.

#2759

Rational combinations for immune checkpoint blockade using β-catenin RNAi therapy.

Shanthi Ganesh, Serena Shui, Kevin Craig, Weimin Wang, Bob D. Brown, Marc Abrams. _Dicerna Pharmaceuticals, Inc, Cambridge, MA_.

Recent research has shown that Wnt/β-catenin signaling drives resistance to cancer immunotherapy by promoting the exclusion of T-cells from the tumor microenvironment. DCR-BCAT is an RNAi-based experimental medicine targeting β-catenin, formulated in a tumor-selective nanoparticle. We had previously reported that systemic administration of DCR-BCAT increased tumor-associated cytotoxic T-cells and dramatically improved responses to immunotherapy agents, in murine syngeneic models and GEM models. In this new work, we explore the mechanisms by which Wnt/β-catenin signaling promotes resistance to immune checkpoint blockade, and propose context-specific therapeutic combinations using RNAi therapy. We will present novel in vivo experimentation demonstrating that DCR-BCAT significantly enhances sensitivity to immunotherapy using different drug regimens in multiple tumor types.

#2760

Therapeutic ultrasound increases CD8+ lymphocyte infiltration of pancreatic tumors.

Petros Mouratidis,1 Gail ter Haar2. 1 _The Institute of Cancer Research, London, United Kingdom;_ 2 _The Institute of Cancer Research London, London, United Kingdom_.

Objectives: The clinical benefit of immunotherapy has not yet been realized in pancreatic cancer, which is characterized by a low antigenicity and dense stroma profile. Focused ultrasound can be used in the treatment of solid tumors, either by inducing necrosis (using ablative temperatures), or by creating cavitation which results in mechanical disruption of the stroma surrounding cancer cells. Both processes may increase the immunogenicity of tumors, and there is anecdotal clinical evidence to that effect. In a pre-clinical study, orthotopic pancreatic tumors have been subjected to a number of different ultrasound exposure regimes (both thermal & cavitational) to explore whether increased T cell infiltration and immune cell activity in the tumor can be induced.

Methods: Syngeneic orthotopic pancreatic KPC tumors (KrasLSL.G12D/+; p53R172H/+; PdxCre tg/+) were grown in murine C57BL/6 subjects (> 12 weeks old). Tumor growth was monitored using imaging ultrasound. Tumors were exposed to ultrasound using the Alpinion VIFU 2000 Therapeutic Ultrasound system, with exposure parameters designed to result in either heating (power = 65 W, exposure duration = 2 seconds, duty cycle = 100 %, 6 repeats) or cavitation (power = 200 W, duty cycle = 1 %, pulse repetition frequency = 1, 60 repeats) in the target tissue. Cavitation was detected using a passive, weakly focused, broadband (0.1 to 20 MHz) sensor. The effect of ultrasound on the viability of tumor cells, and their extracellular matrix, was investigated using histology of formalin-fixed, paraffin-embedded control and treated tumors. Immunohistochemical analysis of CD4+ and CD8+ cells in the tumor, and flow cytometry of the blood and spleen, were used.

Results: Therapeutic ultrasound treatments resulted in cell and collagen depleted regions inside the tumor. Significant broadband signal (suggestive of cavitation) was seen only in the 'cavitation' ultrasound exposures. Skin damage, where seen, was limited. Within a week of therapeutic ultrasound exposures a) an increase in the infiltration of CD8+ cells in the tumor volume, b) an increase in the percentage of the tumor area covered by CD8+ cells, c) an increase in the ratio of the percentage of the tumor area covered by CD8+ cells to that covered by CD4+ cells, and d) a decrease in the percentage of the tumor area covered by CD4+ cells were seen. Immune system regulation was also evident in the blood and spleen of treated subjects.

Conclusions: Therapeutic ultrasound exposure parameters that could regulate the immune response of subjects carrying orthotopic pancreatic tumors have been identified. These results provide a rationale for the use of Therapeutic ultrasound in combination with current immunotherapeutic approaches to improve the control of pancreatic tumor growth.

#2761

Antibody and T cell response profiling in pancreatic cancer patients before and after chemotherapy identify tumor associated antigens suitable for immunotherapy.

Sara Bulfamante,1 Giorgia Mandili,1 Moitza Principe,1 Daniele Giordano,1 Emanuela Mazza,1 Claudia Curcio,1 Laura Follia,1 Giulio Ferrero,1 Andrea Evangelista,2 Maria Antonietta Satolli,2 Paola Cappello,1 Francesco Novelli1. 1 _University of Turin, Turin, Italy;_ 2 _Hospital Città della Salute e della Scienza di Torino, Turin, Italy_.

Background: Pancreatic ductal adenocarcinoma (PDA) is one of the most lethal cancer, both for lack of effective screening method and for resistance to chemotherapy (CTX) and radiotherapy. However, some chemotherapeutic agents, such as gemcitabine, have immune modulatory effects. We started from the hypothesis that more immunogenic antigens can be induced by CTX and targeted by passive or active immunotherapy. To discover TAAs that might be selected for immunotherapy, antibody response in PDA patients' sera was analyzed before and after CTX. TAAs mostly recognized after CTX were selected and used to evaluate whether PDA patients' T cells have an increased TAA-specific response after CTX and if the immune checkpoint blockade (ICB) could enhance T cells activation.

Material and methods: Antibody response in sera of PDA patients, before and after CTX treatments, was analyzed by Serological Proteome Analysis (SERPA) and the antigens recognized were identified by mass spectrometry. T cell proliferation and IFNγ and IL10 production were evaluated on patients' PBMCs (peripheral blood mononuclear cells) stimulated with TAAs in presence or not of ICB.

Results: After CTX the level of antibody recognition increased, both in term of intensity and number of recognized TAAs. For some of these TAAs the increased antibody recognition showed a positive correlation with patients' survival. Of note, after CTX an increased complement dependent cytotoxicity against PDA cell lines was demonstrated in 48% of PDA patients' sera. In most cases after CTX PDA patients' T cells stimulated in vitro with recombinant selected TAAs proliferated more. Moreover, about 50% of TAA-specific T cell responses switched from a protumor regulatory to an antitumor effector phenotype after CTX. Interestingly, this shift was better elicited by ICB after CTX than before.

Conclusions: These data indicate that in PDA patients CTX induces an increase of TAAs-specific antibodies. Furthermore, CTX can switch several TAA-specific T cell responses from regulatory to effector phenotype and ICB could potentiate this polarization. Based on these results the combination of DNA vaccination against TAA and CTX is currently investigated in a PDA mouse model.

#2762

Identification of novel immunotherapeutic targets in anaplastic thyroid cancer.

Sanjukta Chakraborty, Rachana R. Maniyar, Neha Y. Tuli, Ghada Ben Rahoma, Sarnath Singh, Ameet Kamat, Craig Berzofsky, Cameron Budenz, Augustine Moscatello, Jan Geliebter, Raj K. Tiwari. _New York Medical College, Valhalla, NY_.

Thyroid cancer is the most rapidly increasing cancer in the US with anaplastic thyroid cancer (ATC) being the rarest and most aggressive form. ATC patients display a higher mutational burden and are refractory to current mainstream treatments including second generation kinase inhibitors like vemurafenib (PLX4032). BRAFV600E is one of the most common mutations associated with this phenotype. Presence of BRAFV600E has been associated with an immunosuppressive microenvironment in thyroid cancer that presumably facilitates immune evasion. With recent advancements in immunotherapy, well defined targeted treatment plans can address the unmet medical need of ATC. In an effort to define potential immunotherapeutic targets that can be combined with small molecule inhibitors in this subtype, we evaluated the expression of immune checkpoint molecules in four thyroid cancer cell lines TPC-1 (papillary), BCPAP (BRAFV600E positive papillary), 8505C (BRAFV600E positive anaplastic) and CGTH-W-1 (follicular) by qRT PCR at the basal level and after treatment with vemurafenib and mTORC1 inhibitor rapamycin. Prominent co-stimulatory molecules like CD27, CD30, 4-1BB and DR3 were significantly downregulated at the transcript level in ATC (8505C) as compared to the other thyroid cancer subtypes. We observed a higher baseline expression of co-inhibitory molecules like full length CTLA4 (mCTLA4), soluble CTLA4 (sCTLA4), LAG3, 2B4, PD-1 and PD-L1 in ATC (8505C) compared to the other subtypes of thyroid cancer. Although vemurafenib treatment decreased the expression of PD-L1 as expected, interestingly, it enhanced the expression of co-inhibitory molecules LAIR1, 2B4, mCTLA4 and PD-1 by approximately 1.5 folds. These results suggest an inherent redundancy in inhibitory immune checkpoint molecules that presumably compensate for each other, functionally, making them novel targets in ATC. With our study we identified a distinct group of co-inhibitory molecules in ATC that can be targeted by antagonistic antibodies in a combinatorial treatment regimen with small molecule inhibitors like vemurafenib.

#2763

Tryptophan metabolism contributes to radiation-induced immune checkpoint reactivation in glioblastoma.

Pravin Kesawani,1 Antony Prabhu,1 Shiva Kant,1 Praveen Kumar,1 Stewart F. Graham,1 Katie Buelow,1 George Wilson,1 C Ryan Miller,2 Prakash Chinnaiyan1. 1 _Beaumont Health, Royal Oak, MI;_ 2 _Lineberger Comprehensive Cancer Center and Neurosciences Center, University of North Carolina School of Medicine, Chapel Hill, NC_.

Glioblastoma (GBM) is an aggressive brain tumor with limited treatment options. Immune checkpoint inhibitors designed to revert tumor-induced immune suppression have emerged as potent anti-cancer therapies. We performed cross-platform analyses coupling global metabolomic and gene-expression profiling in patient-derived gliomas. We identified aberrant tryptophan metabolism as a metabolic node in glioblastoma, which represents an emerging immune checkpoint. Specifically, GBM demonstrated an accumulation of tryptophan and kynurenine when compared to low-grade glioma (LGG), while kynurenate, a metabolite immediately downstream of kynurenine, was significantly lower in glioblastoma, resulting kynurenine/kynurenate ratio demonstrating >90% accuracy in discriminating between GBM and LGG. These metabolic findings were corroborated by increased expression of indoleamine-2,3-dioxygenase-1 (IDO1) in GBM, a key enzyme involved in tryptophan metabolism and kynurenine production. Cross-platform analysis using gene expression arrays allowed for molecular subtyping of GBM, demonstrated that aberrant tryptophan metabolism was specific to classical and mesenchymal subtypes, while kynurenate accumulation, the metabolite elevated in LGG, was only evident in the proneural subtype. This metabolic phenotype was recapitulated in GBM preclinical models, which demonstrated robust IFNγ-induced IDO1 pathway activation and kynurenine production. The novel IDO1 inhibitor GDC-0919 demonstrated potent inhibition of tryptophan metabolism in our model and importantly, effectively crossed the blood-brain barrier. To explore the immune consequence of aberrant tryptophan metabolism in GBM, we extended investigations to an adult astrocytic, genetically engineered mouse (GEM) cell line to allow for in vivo studies using immune competent mice. Using this orthotopic mouse model, we demonstrated that although GDC-0919 as a single agent did not have anti-tumor activity, it had strong potential for enhancing radiation response in glioblastoma, which was further augmented when using a hypofractionated regimen. The immunological evaluation demonstrated that radiation response in glioblastoma involves immune stimulation, reflected by an increase in activated and cytotoxic T-cells, which is balanced by immune checkpoint reactivation, reflected by an increase in IDO1 expression and immunosuppressive regulatory T cells. GDC-0919 mitigated radiation-induced immune suppression and enhanced immune activation. These findings support clinical efforts designed to combine IDO1 inhibition with hypofractionated radiation in glioblastoma, offering the promise of harnessing a patient's immune system to attack these otherwise recalcitrant tumors.

#2765

Combination ONC201 and radiation therapy in the treatment of breast cancer.

Sachin R. Jhawar,1 Devora Schiff,1 Hao Wu,1 Aditya Thandoni,1 Suemair Hassan,1 Joshua Allen,2 Martin Stogniew,2 Rohinton Tarapore,2 Wolfgang Oster,2 Mark Stein,1 Ann W. Silk,1 Sharad Goyal,3 Bruce G. Haffty,1 Andrew Zloza1. 1 _Rutgers Cancer Institute of New Jersey, NJ;_ 2 _Oncoceutics, Inc., PA;_ 3 _George Washington University School of Medicine and George Washington University Cancer Center, DC_.

Purpose: Metastatic breast cancer patients continue to have poor outcomes on current treatments, and thus, novel therapies in combination with standard treatments are needed. Towards this we studied the pre-clinical effects of combining ONC201, a first-in-class imipridone (which works via activation of ER stress, upregulation of TRAIL, and activation of death receptor 5 [DR5]) with radiation therapy (RT). Since the DR5 pathway is also required for RT-induced apoptosis, we hypothesized that addition of ONC201 could increase the effectiveness of RT. Pertinent Experimental Procedures: Human and mouse breast cancer cell lines were used to study in vitro cancer cell death (by AlamarBlue and clonogenic assays). Patient-derived breast cancer tissue and mouse cell lines were used to study in vivo tumor growth kinetics. Cell lines and mice were administered radiation (0-8 Gy) delivered via a Gammacell 40 exactor. ONC201 was given at does 0-10 µM. Mechanisms of tumor cell death were elucidated using western blots of cleaved PARP (cPARP) and caspase 3 (cC3). Immune response mechanisms were elucidated using in vivo cell-specific depletion. Summary of Data: Multiple breast cancer lines (MB231, MB468, and 4T1) exhibited decreased cell viability after combination therapy compared to either single therapy. In particular, ON201 (1 µM) followed by RT (8 Gy) resulted in a decreased MB468 breast cancer cell viability compared to no treatment (36.9%, P<0.0001), RT alone (30.5%, p<0.0001) and ONC201 alone (26.9%, p<0.0001). A clonogenic assay using MB468 revealed a decreased surviving fraction at every RT dose (2-8 Gy). Western blot analysis revealed increasing cPARP and cC3 with increasing ONC201 (2-8 Gy). In vivo studies utilizing 4T1 cells demonstrated that combination RT (6 Gy) and ONC201 (48 mg/kg given 3x weekly) decreases tumor growth and prolongs host survival compared to either treatment alone (P<0.01). Depletion of NK or CD8 T cells (but not CD4 or B cells) abrogated the positive effects of ONC201 + RT. Similar efficacy of ONC201 + RT was achieved in NSG mice surgically transplanted breast cancer patient tumor sections and immune reconstituted with autologous patient immune cells. Conclusions: Treatment with combination ONC201 + RT results in decreased in vitro breast cancer cell viability and increased in vivo cancer control. Future studies are aimed at translating this combination therapy for clinical use towards improving and saving cancer patient lives.

#2766

Resistance to radiotherapy and PD-L1 blockade is mediated by TIM-3 upregulation in anti-PD-L1 refractory head and neck cancer.

Ayman J. Oweida, Mohammad Hararah, Andy Phan, Shilpa Bhatia, Shelby Lennon, David Binder, David Raben, Lynn Heasley, Eric Clambey, Raphael Nemenoff, Sana D. Karam. _University of Colorado Denver - Anschutz Medical Campus, Aurora, CO_.

Background: Clinical trials targeting the programmed-death ligand axis (PD-1/PD-L1) show that a majority of head and neck squamous cell carcinoma (HNSCC) patients are resistant to PD-1/PD-L1 inhibition. Strategies aimed at sensitizing tumors to immunotherapy can provide durable response compared to conventional therapies. We previously reported that local radiation to the tumor can transform the immune landscape and render poorly immunogenic murine orthotopic HNSCC tumors sensitive to PD-L1 inhibition. However, the response to combined radiotherapy (RT) and PD-L1 inhibition was transient. In this work, we characterize the immune landscape of HNSCC tumors during RT and PD-L1 treatment and interrogate mechanisms of resistance.

Methods: We employed time-of-flight mass cytometry (CyTOF) for characterizing the tumor immune microenvironment at early and late stages of response to RT and anti-PD-L1. For studying the effect of dual checkpoint blockade and RT, poorly immunogenic murine squamous cell carcinoma cells (LY2 and MOC2) were injected into the right buccal mucosa. Mice were randomized to IgG, anti-PD-L1, anti-TIM-3, RT or combinations of RT, PD-L1 and TIM-3. Mechanistic experiments were performed on tumors harvested 72 hours after treatment. Tumors were assessed for levels of activated T cells (CD44+IFNg+) and regulatory T cells (FoxP3+CD4+) by flow cytometry and T-cell infiltration by immunohistochemistry.

Results: Tumors treated with RT and PD-L1 blockade significantly upregulated TIM-3 expression on CD4 and CD8 T cells. Furthermore, the proportion of regulatory T cells expressing TIM-3 was significantly increased during the tumor regrowth phase with RT and anti-PD-L1 treatment. Targeting TIM-3 concurrently with PD-L1 and RT led to significant tumor growth retardation, enhanced T-cell cytotoxicity and improved survival. In addition, dual targeting of PD-L1 and TIM-3 in combination with RT significantly decreased the proportion of regulatory T cells (4.1-fold decrease relative to IgG control and 2.8-fold decrease relative to RT+anti-PD-L1). However, the response to dual checkpoint blockade and RT was not durable and all tumors eventually relapsed. Analysis of relapsed tumors revealed decreased T-cell infiltration, re-emergence of regulatory T cells and decreased proportion of cytotoxic and helper T cells.

Conclusion: Our study shows upregulation of TIM-3 in response to RT and PD-L1 inhibition. However, dual targeting of TIM-3 and PD-L1 with RT did not provide a durable response in two genetically distinct HNSSCC tumor models. In light of the increased number of clinical trials employing dual immune checkpoint blockade, our findings reveal the complexity of tumor immune evasion mechanisms and underscore the need for multimodality targeting of HNSCCs.

#2767

Phosphatidylserine targeting and radiation improves survival in a mouse tumor model resistant to checkpoint blockade.

Rachel Giese,1 Sadna Budhu,1 Christopher Barker,1 Aditi Gupta,1 Steve King,2 Joseph Shan,2 Jedd Wolchok,1 Taha Merghoub1. 1 _Memorial Sloan Kettering Cancer Center, New York, NY;_ 2 _Peregrine Pharmaceuticals, Inc., Tustin, CA_.

Background: Despite the excitement surrounding checkpoint blockade, many tumors remain resistant thus there is a need to sensitize tumors to immunotherapy and augment tumor rejection. Phosphatidylserine (PS) is a phospholipid expressed on the outer surface of apoptotic cells and a variety of immune cell types. PS signaling polarizes macrophages to the M2 phenotype, inhibits TLR signaling and is upregulated on the surface of tumor cells by radiotherapy (RT). RT also promotes infiltration of CD8 T-cells to the tumor microenvironment. Accordingly, RT may improve CD8 T-cell mediated tumor rejection in the context of checkpoint blockade and sensitize tumors to PS antagonism. We hypothesize PS-targeting antibody, mch1n11, in combination with local RT, may be an effective adjuvant or alternative treatment in patients with tumors resistant to checkpoint blockade such as anti-PD-1 (aPD-1). Furthermore, we hypothesize the combination of aPD-1 with mch1n11 and RT will synergize to improve the effector function of CD8 T-cells and enhance tumor elimination.

Methods: B16-F10 murine melanoma was injected into the hind limb of C57BL/6 mice. One cohort received triple combination therapy comprised of aPD-1, mch1n11, and RT given simultaneously at an early time point. Another cohort that showed continued tumor growth after therapy with aPD-1 was used to model checkpoint blockade resistance. These mice received delayed treatment with intra-peritoneal mch1n11 in combination with a one-time 15 Gy RT dose targeted to the tumor-bearing hind limb. The aPD-1 resistant cohort was subdivided into two groups: in one cohort aPD-1 was administered prior to and continued with mch1n11+RT therapy. In the other cohort, aPD-1 was discontinued prior to mch1n11+RT administration. Untreated mice, mice treated with aPD-1 alone, mice treated with isotype antibodies, and mice treated without initial aPD-1 therapy that received delayed mch1n11+RT therapy served as controls.

Results: Early triple combination therapy results in almost complete tumor elimination and leads to statistically significant prolonged survival. In the aPD-1 resistant cohort, both prior and continuous aPD-1 treatment with mch1n11+RT improved survival compared to all four controls. There was no significant difference in tumor size or survival between the adjuvant and alternative experimental groups. Experiments are ongoing to characterize immune infiltrates of tumors and the individual effects of aPD-1, RT and mch1n11.

Conclusion: This preclinical model suggests a phosphatidylserine targeting antibody combined with single dose RT presents an alternative or adjuvant therapy for tumors resistant to checkpoint blockade. The data from this preclinical model will be used to develop a clinical trial for patients with tumors resistant to checkpoint blockade.

### Therapeutic Antibodies, Including Engineered Antibodies 2

#2768

Potentiating the action of antibody-dependent cell-mediated cytotoxicity with immunocytokines.

Patrizia Murer, Jonathan D. Kiefer, Louis Plüss, Dario Neri. _ETH Zürich, Zürich, Switzerland_.

Intact antibodies in the IgG format typically display only moderate in vivo activity against solid tumor masses, when their mechanism of action relies on antibody-dependent cellular cytotoxicity (ADCC). The therapeutic activity is mainly limited by the lack of immune effector cells (and most notably NK cells) in the neoplastic mass.

Our group had previously reported that the therapeutic activity of Rituximab and other intact immunoglobulins could be strongly potentiated by the antibody-based targeted delivery of interleukin-2 (IL2) to the tumor environment. The combination regimen induced complete remissions of established localized lymphomas and provided long-lasting protection from disseminated lymphoma, in mouse models that featured functional NK cells.

In this study, we examined the ADCC activity of TA99, a monoclonal antibody which specifically recognizes gp75, a conserved tumor-associated antigen over-expressed in both murine and human melanoma cells. In a preventive setting TA99 in murine IgG2a format completely inhibited the formation of lung metastases in mice, which had been injected intravenously with B16F10 melanoma cells. However, when the B16F10 melanoma tumor was grown subcutaneously in immunocompetent mice, the ADCC activity was not potent enough to eradicate the pre-established tumors and only a modest tumor growth inhibition was observed. A microscopic tissue distribution analysis revealed that TA99-IgG2a had selectively accumulated in the tumor mass compared to normal organs and that NK cells within the neoplastic lesions were scarce.

In order to increase NK cell density at the tumor site and potentiate ADCC activity, we developed fusion proteins of the TA99 antibody with murine TNFα, IL2 and IL12. The antibody-cytokine fusion proteins were used alone and in combination with TA99-IgG2a. Combination treatment led to a marked increase of anti-cancer activity against established subcutaneous tumors. Our findings indicate that the targeted delivery of certain pro-inflammatory cytokines to the tumor site potentiates the anti-cancer properties of intact antibodies, enhancing their ability to induce ADCC.

#2769

Targeting malignant mesothelioma with an antibody against the tumor extracellular matrix protein fibulin-3.

Mohan Sobhana Nandhu,1 Chandra Goparaju,2 Sharon L. Longo,1 Harvey I. Pass,2 Mariano S. Viapiano1. 1 _SUNY Upstate Medical University, Syracuse, NY;_ 2 _NYU Langone Medical Center, New York, NY_.

Malignant mesothelioma (MM) is an aggressive tumor of mesothelial tissues that has poor prognosis and limited therapeutic options. Pass et al. (N Engl J Med 2012) identified the extracellular matrix protein fibulin-3 as an accurate biomarker of MM progression, which can be detected in the tumor parenchyma and in pleural effusions. Here, we report a systematic study of the mechanisms of fibulin-3 in MM and its value as a molecular target. Fibulin-3 activated canonical NFkB signaling and regulated the expression of NFkB-downstream genes, including the proteases MMP9 and MMP13, and the proinvasive proteins TNC and MLCK. Fibulin-3 knockdown downregulated NFkB signaling, reduced MM cell viability and potentiated the cytotoxicity of cisplatin in vitro. We next developed a function-blocking antibody ("mAb428.2") against an epitope of fibulin-3 required for NFkB activation (Nandhu et al., Clin Cancer Res 2017). This antibody blocked fibulin-3 signaling in vitro and reduced the expression of this protein, as well as NFkB-regulated MMP9, in subcutaneous (sc) MM tumors implanted in nude mice. Next, we treated mice carrying sc or intrapleural models of MM with mAb428.2 or control IgG1 (8x 30 mg/kg, q24h) delivered IV. Our anti-fibulin-3 antibody caused a significant reduction of tumor burden and extension of overall survival in both models. Overall, downregulation and inhibition of fibulin-3 proved a successful approach to enhance MM therapy. Given the value of this protein as a MM biomarker, we expect that detection of fibulin-3 may serve to identify patients with the highest chance to respond to combination therapies including anti-fibulin-3 approaches, achieving maximum therapeutic against these aggressive tumors.

#2770

Dual-targeting mesothelin/CD47 bispecific antibodies for tumor-directed blockade of CD47 in solid cancer.

Valéry Moine, Lucile Broyer, Xavier Chauchet, Eric Hatterer, Stefano Majocchi, Vanessa Buatois, Limin Shang, Gérard Didelot, Giovanni Magistrelli, Yves Poitevin, Ulla Ravn, Marie H. Kosco-Vilbois, Nicolas Fiischer, Walter G. Ferlin, Krzysztof Masternak. _Novimmune SA, Plan-les-Ouates, Switzerland_.

Mesothelin (MSLN) is a lineage restricted cell surface protein with unknown biological function, expressed at low levels on mesothelial cells in healthy tissue. MSLN is also a tumor differentiation antigen as it is highly expressed across a wide range of solid tumors with the highest prevalence in mesothelioma, pancreatic, biliary, ovarian, lung and gastric cancers. Most solid and hematological cancers also upregulate the expression of CD47, a ubiquitous innate immune checkpoint receptor. CD47 interacts with signal-regulatory protein alpha (SIRPα) on myeloid cells, which leads to the inhibition of tumor cell phagocytosis and anti-tumor immune responses. Accordingly, elevated levels of CD47 on tumor cells are associated with cancer's immune evasion capacity and correlate with poor clinical prognosis, all of which makes CD47 a relevant target for therapeutic blockade. We generated a series of dual-targeting bispecific CD47/MSLN antibodies (biAbs) selectively binding to MSLN-positive tumor cells, but not MSLN-negative healthy cells expressing physiological levels of CD47 (e.g., all blood cells). These CD47/MSLN biAbs block CD47-SIRPα interaction in a MSLN-dependent, tumor-specific manner, thus permitting to bypass tolerability and "antigen sink" issues related to ubiquitous CD47 expression in healthy tissues. An array of CD47/MSLN biAbs with anti-MSLN arms targeting different MSLN epitopes was tested in vitro, in antibody dependent cellular phagocytosis (ADCP) and antibody dependent cellular cytotoxicity (ADCC) assays, as well as for anti-tumor activity in vivo using mouse xenograft models. With various MSLN-positive human cancer cell lines, MSLN/CD47 biAbs demonstrate significantly enhanced cancer cell killing by ADCC and ADCP as compared to the corresponding anti-MSLN monoclonal antibody format (mAbs) as well as to amatuximab, a therapeutic anti-MSLN mAb (currently in Phase II clinical trials for mesothelioma). Correspondingly, the MSLN/CD47 biAbs also display superior efficacy in controlling tumor growth in the xenograft models in vivo. Taken together, we conclude that MSLN/CD47 biAbs should allow for efficacious, yet safe, targeting of CD47 in multiple solid tumor indications in the clinic. More generally, our data support the concept of tumor-directed blockade of CD47 with biAbs as a novel way of improving the efficacy of antibody-based cancer therapies.

#2771

Harnessing novel immune escape mechanisms for cancer therapeutics: OXAB1 target validation, proof of concept and preclinical development.

Livija Deban,1 Wei Cao,2 Yaoyao Fu,2 Angelo Kaplan,2 Rachel L. Duesk,2 Arnima Bisht,2 Sudha Swaminathan,1 San Lin Lou,2 Manal Amoury,2 Bukky Adelegan,1 Joanne Berry,1 James E. Ackroyd,1 Chuck Hannum,2 Robert Boyd,1 Christian Rohlff,1 Haining Huang2. 1 _Oxford BioTherapeutics LTD., Abingdon, United Kingdom;_ 2 _Oxford BioTherapeutics Inc, San Jose, CA_.

The evasion of the host's immune system by tumor cells is a well-established mechanism of tumor establishment and progression - a notable example is the upregulation of PD-L1 on tumor cells to inhibit the activation of PD-1-expressing T cells. Identification of this immune escape mechanism has led to numerous therapeutics targeting the PD-1/PD-L1 axis; nevertheless, there remain a significant majority of patients who are unable to benefit from current therapies. Here we present our findings of (i) an active novel immune escape mechanism mediated via OX001R/OX001L receptor-ligand axis, and (ii) an antibody targeting this axis with potential therapeutic benefit. OX001R/OX001L is a newly identified T cell modulatory axis involved in cancer immune escape. The receptor (OX001R) is expressed on activated CD8 T cells and functions as a co-stimulatory molecule. The ligand (OX001L) is expressed on various cancer cells. The downregulation of OX001L is reported to correlate with poorer prognosis and increased risk of metastasis in multiple cancer indications. Our analysis of NSCLC samples showed the degree of tumor T cell infiltration to be significantly correlated with OX001L downregulation (p=0.0001) and PDL1 upregulation, suggesting that the co-stimulatory ligand OX001L may be involved in cancer immune escape. Our further in vitro experiments showed that (i) tumor cells downregulate OX001L and upregulate PDL1 upon T cell engagement and (ii) OX001L downregulation requires direct cell-cell contact and therefore involves a distinct mechanism when compared with the IFN gamma mediated PDL1 upregulation. To apply our discovery to therapeutics, we conducted a series of in vitro and ex vivo assays with agonistic antibodies targeting OX001R. We found that anti-OX001R-specific agonist antibodies promote T cell activation resulting in enhanced IL-2 and IFN gamma production. Moreover, anti-OX001R-specific agonist antibodies efficiently activate patient TILs in ex vivo 3D tumor explants. When tested in a humanized animal model of lung cancer, agonistic anti-OX001R antibody demonstrated significant anti-tumor activity with tumor growth inhibition comparable to that seen with pembrolizumab. Furthermore, additive effect was seen when the combination of anti-OX001R and anti-PD-1 antibodies was tested. Guided by these data, we have developed OXAB1, an agonistic humanized mAb targeting OX001R. The OXAB1 antibody shows enhanced activity in both in vitro and ex vivo studies. Collectively, our results extend the research into cancer immune escape mechanisms associated therapeutics by discovering, and providing evidence of the importance of, the OX001R/OX001L axis; and support the clinical translation of an agonistic antibody to OX001R in cancer therapy, either as monotherapy or in combination with PD-1/PD-L1 blockers.

#2772

Preclinical development of a novel antibody-drug conjugate targeting "cold" tumors.

Angelo Kaplan,1 Nickolas Attanasio,1 To Uyen T Do,1 Sudha Swaminathan,1 Arnima Bisht,1 San Lin Lou,1 Jason Allen,2 Robert Boyd,2 James E. Ackroyd,2 Gleb Feldman,2 Christian Rohlff,2 Rachel L. Dusek1. 1 _Oxford BioTherapeutics Inc, San Jose, CA;_ 2 _Oxford BioTherapeutics LTD., Abingdon, United Kingdom_.

The promise of immunotherapy for cancer is underscored by the recent efficacy of checkpoint inhibitors, which hinder the ability of tumors to escape attack by the immune system. Patients most likely to benefit from such therapy include those whose tumors are inflamed and who express the PD-L1 checkpoint protein. It is imperative that alternative therapies are developed for patients whose tumors do not exhibit these characteristics. Using our proprietary OGAP® system, we identified a novel membrane cancer target, OX001L. OX001L expression in certain cancers is associated with poor prognosis and reduced survival. IHC studies showed substantial prevalence of OX001L across multiple tumor types, with a majority of the OX001L positive samples scoring negative for PD-L1. In non-small cell lung cancer, OX001L expression was significantly increased in tumors lacking abundant intratumoral PD-1 positive T-cell infiltrate or PD-L1 expression compared to inflamed or PD-L1 positive tumors (p<0.00001). This finding suggests that OX001L may play a role in cancer immune escape. In order to effectively target OX001L positive tumor cells, we generated a human OX001L therapeutic antibody which exhibits target selectivity and cross-reactivity to the cynomolgus monkey OX001L orthologue, as demonstrated by FACS. Using immunofluorescence microscopy, we found that the antibody-antigen complex exhibits efficient internalization from the plasma membrane. Glycoengineering improved the antibody's ability to mediate potent antibody dependent cellular cytotoxicity (ADCC) activity in vitro and in vivo. Furthermore, when conjugated to a DNA alkylating toxin, the OX001L antibody promoted highly potent in vitro cytotoxicity of histotypically distinct cancer cell lines and also effected substantial anti-tumor activity in vivo. Importantly, the OX001L antibody and antibody-drug conjugate (ADC) were well tolerated in cynomolgus monkeys. These data indicate that OX001L ADC is a distinctive therapeutic molecule which could act on OX001L positive tumors via both ADC and ADCC mechanisms of action. The ability of the ADC to also promote recruitment and activation of cytotoxic T lymphocytes in OX001L positive tumors, similar to other cytotoxic payloads, will be further explored. This represents a unique therapeutic opportunity to target "cold"/PD-L1 negative, OX001L positive tumors which are less likely to respond to conventional checkpoint inhibitor therapy. In addition, a combination of the OX001L ADC and checkpoint inhibitors could be used to treat OX001L positive, "hot"/PD-L1 positive tumors. Targeting such tumors with both agents (and through two distinct, non-overlapping mechanisms of action) may yield a higher degree of success and/or a decreased propensity for relapse in patients than would checkpoint inhibitor monotherapy. We plan to test this exciting therapeutic opportunity in humanized in vivo models.

#2773

A DNA-damaging lupus autoantibody synergizes with PARP inhibitors against DNA repair-deficient tumor cells.

Zahra Rattray,1 Jaymin M. Patel,2 Philip W. Noble,1 Valentina Dubljevic,3 Deanne L. Greenwood,3 James A. Campbell,3 James E. Hansen1. 1 _Yale University, New Haven, CT;_ 2 _Harvard Medical School, Boston, MA;_ 3 _Patrys Ltd, Melbourne, Australia_.

The lupus anti-DNA autoantibody 3E10 is a compelling candidate for development as a targeted therapy for DNA repair-deficient malignancies. 3E10 has previously been shown to localize to tumors due to its attraction to DNA released by dying cancer cells, penetrate into cell nuclei, inhibit DNA repair, and kill cancer cells with defects in homology-directed repair (HDR) of DNA double-strand breaks. A more potent derivative of 3E10 with increased affinity for DNA has been developed (referred to here as 3E10EN), and identification of optimal combination therapies with 3E10EN is needed to facilitate planning for upcoming clinical trials. In the present study, we found that 3E10EN increases the activity of the DNA repair enzyme poly (ADP-ribose) polymerase (PARP) in HDR-deficient cells and hypothesized that combination treatment with 3E10EN and PARP inhibitors (PARPi) would yield synergistic effects on HDR-deficient cancer cell survival.

PARP content and activity in HDR-deficient and proficient cells prior to and following treatment with 3E10EN was evaluated. 3E10EN did not impact PARP protein content but yielded a significant increase in pADPr signal in HDR-deficient cells, which suggests a compensatory increase in PARP activity in response to DNA damage accumulation in HDR-deficient cells. Combinations of 3E10EN and the PARPi olaparib were tested on a panel of HDR-deficient cells, and a matched pair of BRCA2-deficient and proficient DLD1 cells. Olaparib inhibited the increase in pADPr caused by 3E10EN, and colony formation assays analyzed by the Chou-Talalay method confirmed that 3E10EN and olaparib synergized against HDR-deficient cancer cells. Conversely, HDR-proficient cells were resistant to 3E10EN and olaparib combination treatment.

The original 3E10 is a murine antibody isolated from a lupus mouse model, and in preparation for its further development as a new drug we have recently designed Deoxymab 1 (DX1), a humanized version of 3E10EN. DX1 exhibits improved activity relative to the 3E10EN prototype, and when tested on a panel of HDR-deficient and proficient cells, DX1 and olaparib exhibited synergistic effects similar to that observed with the 3E10EN prototype.

In conclusion, we have found that both the prototype 3E10EN and humanized DX1 synergize with PARPi against HDR-deficient tumor cells. These findings provide the rationale for further studies to determine the potential for this approach to be translated into a clinically relevant therapeutic strategy.

#2774

The triple combination of the FAP-IL2v immunocytokine with PD-L1 checkpoint inhibitory and CD40 agonistic antibodies results in long-term tumor control in the orthotopic PancO2 model.

Valeria Nicolini, Inja Waldhauer, Anne Freimoser, Emily Corse, Jahad Charo, Pablo Umana, Christian Klein. _Roche Pharma Research & Early Development, Schlieren, Switzerland_.

Introduction: We have previously described the FAP-targeted immunocytokine FAP-IL2v and its combination with PD-L1 checkpoint inhibition. Here we show that the anti-tumoral efficacy of FAP-IL2v can be strongly enhanced by combination with a CD40 agonistic antibody as well as the triple combination of FAP-IL2v with a CD40 agonistic and a PD-L1 checkpoint inhibitory antibody in the syngeneic PancO2 model. Methods: Anti-tumoral efficacy was assessed in the orthotopic intra-pancreatic PancO2 model transfected with Luciferase in C57BL/6 mice. Mice were injected intra-pancreatically on study day 0 with 1x10E5 Panc02-Fluc cells, randomized and weighed. One week after tumor cell injection mice were injected i.p. with the murine specific surrogate immunocytokine muFAP-muIL2v (muIgG1 DAPG) (2 mg/kg, q1wx3), anti-muPD-L1 surrogate antibody (muIgG1 DAPG) (10 mg/kg, q1wx3) and/or the anti-muCD40 surrogate antibody FGK4.5 (muIgG1) (10mg/kg, q1wx1). Bioluminescence imaging (BLI) was performed after ip injection of 150 mg/kg D-Luciferin 10 min before acquisition using IVIS® SPECTRUM. An analogous experiment was performed in C57BL/6 B cell knockout mice. Results: In the orthotopic PancO2-Fluc model the triple combination of FAP-IL2v plus anti-PD-L1 plus anti-CD40 showed superior outcome compared to vehicle and the respective monotherapies in terms of median survival and overall survival in the majority of animals followed by the combination of FAP-IL2v with anti-CD40 and the combinations of anti-PD-L1 with anti-CD40 and FAP-IL2v with anti-PD-L1, respectively. Bioluminescence imaging confirmed the survival outcome of the respective groups by decrease in/absence of bioluminescence signals in the tumor area. Tumor re-challenge experiments showed that the animals with long-term survival were protected both in the C57BL/6 and the C57BL/6 B cell knockout mice. Conclusions: These data show that the anti-tumoral efficacy of FAP-IL2v in the syngeneic PancO2 model can be strongly enhanced by combining it with a CD40 agonistic antibody on top of a PD-L1 checkpoint inhibitory antibody. Most notably, the triple combination of FAP-IL2v with CD40 agonism and PD-L1 checkpoint inhibition resulted in long term survival of the majority of animals. These data provide the preclinical rationale for further clinical investigation of the combination of FAP-IL2v (RG7461) with the PD-L1 antibody atezolizumab and the CD40 agonistic antibody selicrelumab (RG7876).

#2775

A novel immunostimulatory OX40/PD-L1 bivalent bispecific antibody (MEDI1109) for the treatment of patients with cancer.

Michael D. Oberst,1 Srinath Kasturirangan,1 Clifford Sachs,1 Catherine Auge,1 James Moynihan,1 Raymond Rothstein,1 James Hair,2 Francis Neal,2 Srinivas Mamidi,2 Shino Hanabuchi,1 Amanda Watkins,2 Yanan Zheng,3 Kim Rosenthal,1 Daniel J. Freeman,1 Scott Hammond1. 1 _MedImmune, MD;_ 2 _MedImmune, United Kingdom;_ 3 _MedImmune, CA_.

Targeted immunotherapy utilizing agonist antibodies (Ab) to OX40 shows great promise in preclinical mouse models alone or in combination with various therapies including PD1/PD-L1 antagonist Abs. Ab-mediated signaling through OX40 requires higher-order clustering by Fcγ receptors to co-stimulate antitumor-specific T cells and attenuate regulatory T cell (Treg) immunosuppression, leading to antitumor activity. To date, OX40 agonists are well-tolerated but show limited activity alone or in combination with checkpoint blockade in clinical trials. We hypothesize that the antitumor activity of OX40 agonists in patients may be limited by insufficient intratumoral exposure and an inadequate amount of clustering by Fcγ receptors within the tumor microenvironment. To overcome these limitations, we engineered MEDI1109, a bivalent bispecific Ab composed of an OX40 agonist Ab molecularly fused to two single-chain variable fragments derived from a PD-L1 antagonist. The resulting bispecific Ab retained the functional properties of each parent Ab that included binding and agonizing OX40, binding and full blockage of the PD-L1/PD-1 pathway, depletion of OX40+ Tregs and overcoming Treg suppression. Emerging data suggest that the bispecific Ab also drives novel biology including (1) increased OX40 agonism through clustering by either PD-L1 and Fcγ receptors, (2) improved intratumoral exposure through PD-L1 targeting, (3) elimination of PD-L1 positive tumor cells by NK cell mediated antibody-dependent cellular cytotoxicity and (4) enhanced antitumor immunity through concurrent PD-L1/PD-1 blockade on tumor cells and OX40 co-stimulation of antitumor T cells. Intravenous administration of OX40/PD-L1 bispecific Abs to cynomolgus monkeys resulted in the proliferation of peripheral blood CD4+ and CD8+ total memory T-cell populations, and in the reduction of soluble PD-L1 levels in the plasma. These results demonstrate that MEDI1109 is a unique and potent immune modulating agent and may be utilized to enhance antitumor immunity in patients with cancer.

#2776

MEDI5752: A novel bispecific antibody that preferentially targets CTLA-4 on PD-1 expressing T-cells.

Simon J. Dovedi,1 Yariv Mazor,2 Matthew Elder,1 Sumati Hasani,2 Bo Wang,2 Suzanne Mosely,1 Desmond Jones,1 Anna Hansen,2 Chuning Yang,2 Yanli Wu,2 Ikbel Achour,2 Nick Durham,2 Gareth Browne,1 Thomas Murray,1 James Hair,1 Michelle Morrow,1 Godfrey Rainey,2 Maria Jure Kunkel,2 John Gooya,2 Daniel Freeman,2 Ronald Herbst,2 Robert Wilkinson1. 1 _MedImmune, Cambridge, United Kingdom;_ 2 _MedImmune, Gaithersburg, MD_.

Studies have demonstrated that the clinical benefit of PD-1 blockade can be further improved by combination with an αCTLA-4 mAb in some indications. However, this increased activity is commensurate with significant immune related adverse events (irAE's). Therefore, novel approaches are required to uncouple toxicity from anti-tumour efficacy and realise the full potential of this combination. MEDI5752 is a monovalent bispecific human IgG1 monoclonal antibody (mAb) with an engineered fragment crystallisable (Fc) domain to reduce Fc effector function, that specifically binds two clinically validated negative T cell regulators; PD-1 (programmed cell death 1) and CTLA-4 (cytotoxic T-lymphocyte-associated protein 4). MEDI5752 has been designed to suppress the PD-1 pathway and provide modulated CTLA-4 inhibition to uncouple CTLA-4 dependent peripheral toxicity from tumour efficacy. PD-1 expression is a defining feature of tumour infiltrating lymphocytes (TILs). We show that MEDI5752 can saturate CTLA-4 on PD-1+ cells at orders of magnitude lower concentrations than required to saturate CTLA-4 on PD-1- cells. Moreover, our data demonstrate that monovalent targeting of CTLA-4 with MEDI5752 is significantly less potent (15 fold) than bivalent targeting with a parental αCTLA-4 mAb in reporter assays. In contrast, the switch to monovalent targeting of PD-1 has limited effect on potency (within 3-fold compared to a parental αPD-1 mAb) in a PD-1/L1 reporter assay. Together these data demonstrate the potential for MEDI5752 to inhibit CTLA-4 on TILs whilst sparing peripheral T cell populations and reducing toxicity. Furthermore, profiling of MEDI5752 in a range of primary T cell activation assays reveals equivalent activity to a combination of parental PD-1 and CTLA-4 antibodies. MEDI5752 is rapidly internalised upon target binding with kinetics similar to the parental αCTLA-4 mAb reflecting the rapid recycling of this receptor. However, in contrast to an αCTLA-4 mAb (or an αPD-1 mAb), MEDI5752, by tethering CTLA-4 to PD-1, leads to the internalisation and subsequent degradation of PD-1. This novel mechanism of action further differentiates MEDI5752 from a combination of mAb's targeting PD-1 and CTLA-4. MEDI5752 is a novel monovalent bispecific which may provide an improved therapeutic index when compared to a combination of bivalent αPD-1 and αCTLA-4 mAb's, and could provide benefit in cancer indications.

#2777

Antigen Seeding Technology by Engineered Toxin Bodies provides a targeted immuno-oncology approach for treatment of cancers.

Brigitte Brieschke, Sangeetha Rajagopalan, Garrett L. Robinson, Jack P. Higgins, Erin K. Willert, Hilario J. Ramos. _Molecular Templates, Austin, TX_.

Engineered Toxin Bodies (ETBs) are a distinct class of targeted immunotoxins in development by Molecular Templates as anti-cancer therapeutics. ETBs drive a potent and targeted response mediated by antibody-like binding, cellular internalization, and enzymatic ribosomal inhibition via the delivery of a Shiga-like toxin subunit A (SLTA) that has been proprietarily modified to avoid innate and adaptive immune recognition. In addition to this primary mechanism of action (MOA), the intrinsic ability of ETBs to induce internalization in a targeted fashion, present a unique delivery platform for next generation MOAs in the immuno-oncological (IO) space. Recent advances in IO such as checkpoint inhibitors, bi-specific T cell engagers, and CAR-T cells now provide direct clinical evidence that restoration of T cell functionality or re-direction of fully functional T cells drive significant clinical benefit to patients that have failed traditional standard of care. Molecular Templates has developed the Antigen Seeding Technology (AST) platform, rendering next generation ETBs with the unique ability to deliver foreign protein antigen to the cytoplasm of targeted populations of tumor cells. This MOA allows for the intracellular processing of antigen and subsequent surface MHC-I presentation required for activation of a re-directed T lymphocyte response and the capacity to restore a functional immune clearance program against the tumor. Our first generation AST molecule has been engineered as a PD-L1 targeted ETB carrying the immunodominant HLA:A02 restricted peptide (NLVPMVATV) from the Cytomegalovirus (CMV) tegument protein-pp65. This design takes advantage of targeting of PD-L1 expressing tumor cells, which are components of multiple cancers responsive to current IO modalities. Furthermore, the incorporation of an immunodominant CMV-peptide allows for the appropriation of a highly functional and non-exhausted CMV-specific lymphocyte response that is highly prevalent in the human population. Pre-clinical data demonstrate a potent ability for AST to trigger ETB-mediated T cell responses and tumor cell clearance of multiple PD-L1+ tumor targets. This response is highly specific, as treatment of PD-L1+ positive but not negative cell lines leads to detectable surface expression of NLVPMVATV in an HLA:A02 dependent manner. Furthermore, this result correlates with activation of effector T cells and subsequent eradication of multiple PD-L1/A02+ tumor targets after co-culture with healthy T cells from A02/CMV+ donors. Together, our POC data shows that using our AST-platform, we can deliver a targeted antigenic response to re-direct CMV-T cell responses to various tumor cell lines, trigger a T cell-mediated response and targeted-tumor cell clearance. Molecular Templates intends on extending the AST platform to clinical lead programs in 2018.

#2778

Discovery and preclinical characterization of BAY 1905254 a novel immune checkpoint inhibitor for cancer immunotherapy targeting the immunoglobulin-like domain containing receptor 2 (ILDR2).

Julia Huetter,1 Uwe Gritzan,2 Ilona Gutcher,1 Sven Golfier,1 Wolf-Dietrich Doecke,1 Merlin Verena Luetke-Eversloh,1 Helge Roider,1 John Hunter,3 Andrew Pow,4 Spencer Liang,3 Zurit Levine,5 Ofer Levy,5 Ilan Vaaknin,5 Bertolt Kreft,1 Lars Roese1. 1 _Bayer AG, Berlin, Germany;_ 2 _Bayer AG, Cologne, Germany;_ 3 _Compugen, CA;_ 4 _New Paradigm Biosciences, MA;_ 5 _Compugen, Israel_.

ILDR2 is a type I transmembrane protein belonging to the B7 family of immunomodulatory receptors with 98 % sequence identity between the extracellular domains of human and murine orthologues. The exact physiological role of ILDR2 is still unclear, but it has been implicated in the development of type 2 diabetes and the formation of tri-cellular junctions. Our detailed analyses reveal that ILDR2 is expressed in kidney, testis, liver, and lymph node fibroblastic reticular cells (FRC). The latter is particularly interesting since FRC belong to the CD45- stromal population, a specialized cell subset located in the T cell zone. These cells are essential for recruitment of naïve T cells and activated dendritic cells to the lymph node and have been reported to exhibit immuno-regulatory properties.

Due to its structural similarity to members of the B7 family and due to its expression in FRCs, we speculated that ILDR2 might play a role e.g. in T cell priming and the initiation of antigen-specific T cell responses. In line with this, we and our collaborators from Compugen were able to show that an ILDR2 -Fc fusion protein is able to a) bind to activated (but not naïve) T cells, b) suppress TCR-stimulated cytokine secretion and c) exhibit immunomodulatory effects in several models of autoimmune diseases, i.e. multiple sclerosis, rheumatoid arthritis, and type 1 diabetes.

As a consequence, we set out to generate antibodies against this novel immuno-oncology target and describe here for the first time the characterization of BAY 1905254, a human/mouse cross-reactive IgG2 antibody blocking the immunosuppressive activity of ILDR2. BAY 1905254 specifically binds to ILDR2 but not to the closely related family members ILDR1 and ILDR3/LSR. It enhances antigen-specific T cell proliferation in vivo in an ovalbumin vaccination model with OT-I transgenic T cells. BAY 1905254 exhibits anti-tumor activity in different syngeneic mouse models with efficacy correlating with increasing mutational load. Furthermore, additive/synergistic anti-tumor effects can be observed in combination with either an anti-PD-L1 Ab, an immunogenic cell death inducing chemotherapeutic (Docetaxel) or tumor antigen immunization. Ex vivo analysis reveal that BAY 1905254 treatment results in increased intra-tumoral IFN-y levels and enhanced infiltration of CD45+ cells, e.g. of CD8α\+ dendritic cells (DCs). Interestingly, this DC population is known to play a crucial role in cross-presentation, thus, in the initiation of CD8+ T cell responses.

In summary, BAY 1905254 is a function-blocking Ab able to counteract the immuno-suppressive function of the newly discovered immuno-oncology target ILDR2. The Ab exhibits in vivo efficacy both in monotherapy as well as in combination with various other approaches, and it is planned to advance BAY 1905254 into FiM trials in 2018.

#2779

**Molecular mechanistic and** in vivo **efficacy studies of Fn14-targeted fusion constructs containing human granzyme B.**

Ana Alvarez de Cienfuegos,1 Lawrence A. Cheung,1 Khalid A. Mohamedali,1 Landon J. Inge,2 Timothy Whitsett,2 Jeffrey A. Winkles,3 Louis DePalatis,4 Linda Paradiso,4 Michael G. Rosenblum1. 1 _MD Anderson Cancer Center, Houston, TX;_ 2 _Joseph's Hospital and Medical Center, Phoenix, AZ;_ 3 _University of Maryland. School of Medicine, Baltimore, MD;_ 4 _Mirata Biopharma, Spring, TX_.

The past 15 years have seen impressive preclinical and clinical efforts to develop smart therapeutics with enhanced specificity for tumor cells and with much reduced "off-target" or nonspecific toxicity to normal tissues. For the antibody-drug conjugate approach, these constructs are composed of a targeting antigen binding domain, which delivers the cytotoxic domain into the cell. The majority of the cytotoxic payloads used in these constructs are categorized as either microtubule inhibitors or DNA-damaging drugs. We have developed a fusion protein, GrB-Fc-IT4 (MRT-101), that contains a humanized scFv binding domain targeting the cell surface receptor Fn14, an antigen highly expressed in a variety of solid tumors, and containing the human serine protease granzyme B (GrB) as the cytotoxic payload. The construct includes the IgG hinge Fc domain linker for efficient dimerization and an overall high molecular weight thereby designed to provide a prolonged serum half-life (~40 h). This unique format mimics human immune effector cell function and induces target cell death through activation of a variety of well described pro-apoptotic cascade signals. Western blot studies on human TNBC cells (MDA-MD-231) have shown that intact MRT101 is translocated into the cytosol in less than 1 h after exposure and is detectable in the cytosol for at least 8 h. The free GrB component is also detected by Western blot 4 h after treatment and persists for up to 8 h. Both of these agents trigger apoptotic cascades through activation of various caspases and induction of mitochondrial damage. Studies demonstrating cytochrome C release and mitochondrial depolarization are ongoing and will be reported. Incubation with the lysosomotropic agent chloroquine did not alter the IC50 of MRT-101, suggesting that the fusion protein is not appreciably held in the endosomal compartment. In vivo studies have shown that MRT-101 is well tolerated in BALB/c mice after intravenous administration of 5 doses at 20 mg/kg/dose. This dose level showed no evidence of toxicity in any of the major organs such as liver and kidneys. In vivo efficacy studies conducted on NSG-NOD scid mice demonstrated significant tumor growth inhibition of established orthotopic breast tumors (MDA-MB-231), with no tumor growth for up to 30 days after implantation. Treatment of nude mice bearing lung PDX tumors showed a 60% tumor growth inhibition when compared to the vehicle control group. These results, in combination with previous in vitro and in vivo studies, demonstrate that the completely human MRT-101 construct is a selective, highly potent, non-toxic and effective antigen-driven drug with significant potential for the treatment of Fn-14 positive tumors that acts through a new and unique mechanism of action. Research supported by Mirata Biopharma LLC and conducted, in part, by the Clayton Foundation for Research.

#2780

Engineering human fusion constructs for targeted delivery of granzyme B to CD33 and CEA antigens.

Lawrence H. Cheung, Khalid A. Mohamedali, Walter N. Hittelman, Michael G. Rosenblum. _UT MD Anderson Cancer Ctr., Houston, TX_.

Our laboratory has developed a unique platform for the delivery of the highly pro-apoptotic serine protease granzyme B (GrB). The construct contains enzymatically active GrB and therefore does not require release from the targeting carrier by a fragile linker in contrast to conventional antibody-drug conjugates (ADCs). We employed two scFvs targeting the CD33 antigen found on AML and the juxtamembrane epitope of CEA found on numerous solid tumors. These human scFvs (designated HU33 and CEA respectively) were fused to human GrB through an engineered IgG heavy-chain fragment (hinge to Fc and designated Fc) which contains a dimerization domain. The GrB-Fc-HU33 and GrB-Fc-CEA constructs were cloned into the pSECTag vector containing an upstream 5kDa purification tag with an enterokinase (EK) cleavage site for facile removal. The molecules were expressed in transiently-transfected HEK-293E suspension cells and grown at 37°C for 3 days in 5% CO2. The molecules were isolated from culture media and purified by IMAC followed by EK cleavage and SP chromatography to remove the free tag. The yields were 6-10 mg/L for both molecules. Specific binding of each molecule to the antigen target is undergoing Biacore and/or ELISA assessment. The cytotoxicity of the GrB-Fc-HU33 against HL-60 cells showed a specific cytotoxic effect with an IC50 of 120 nM. Tests of this agent against a panel of cells expressing various levels of CD33 are ongoing. The cytotoxic effects of the GrB-Fc-CEA were assessed against HT29, HT1080-CEA and A431 cells and demonstrated IC50 values of 80, 110 and 95 nM respectively. Based on IC50 values, these data demonstrate that the fusion construct targeting CD33 is efficiently internalized and effectively delivers active serine protease to the cytosol even at relatively low antigen expression levels. In addition, the juxtamembrane epitope of the CEA antibody also allows efficient internalization for delivery of GrB to the cytosol. Immunofluorescence and confocal studies to examine the time course of internalization and the pro-apoptotic events triggered by these agents are under examination and will be presented. Research conducted, in part, by the Clayton Foundation for Research.

#2781

"Wheelz": A novel engineered human antibody for possible CAR T-cell therapy.

Kiara V. Whitley, Edwin J. Velazquez, Kelsey B. Bennion, Brianne M. Kingery, Scott K. Weber, Kim L. O'Neill. _Brigham Young University, Provo, UT_.

The purpose of this study is to present a novel engineered human antibody specific to thymidine kinase 1 (TK1). Recent developments in chimeric antigen receptor (CAR) T-cell therapeutic approaches have rocketed into the spotlight in the field of cancer immunotherapy. One of the limitations to CAR T-cell therapy is the need for novel targets on cancer cells. The upregulation of serum TK1 has been shown to be an important biomarker in cancer detection and prognosis. Furthermore, we have previously reported the cell surface expression of TK1 on multiple cancer cell lines and in clinical samples. This data merits an investigation of the use of TK1 as a target for CAR T-cells. We have created an engineered human antibody against TK1. We present "Wheelz," a single chain fragment variable monoclonal antibody with a constant region (scFv-Fc) to potentially target cancer cells expressing surface TK1.

Our engineered antibody was isolated from a yeast display library (donated by the Wittrup lab at MIT) using magnetic sorting and flow cytometry to find the top 0.1% of TK1-binding single chain fragments. We fused the resulting scFv region to a constant region (Fc) fragment through restriction digestion and ligation. We then transformed the construct with a secretion vector into yeast and induced the transformed yeast to produce the antibody, which we called "Wheelz." We purified the antibody using His-tag. In order to confirm specificity to TK1, we used dot blotting and saw specific binding to purified TK1. We performed a western blot using chemiluminescence imaging. This indicated that the engineered human scFv-Fc binds to TK1 present in normal serum, cancer serum, and cancer cell extract in TK1's monomeric, dimeric, and tetrameric forms and did not bind to any other human cellular proteins.

In the past, anti-TK1 scFv antibodies of murine origin were used in CAR experiments with some cytotoxic effect. Because "Wheelz" is an engineered human antibody, it could improve previous models by minimizing immune rejection in the patient. Future research will explore the cytotoxic-inducing effects of "Wheelz" before inserting the antibody into a CAR. We will also explore the specificity and cytotoxicity of eight other scFv regions isolated from yeast display that preliminarily appear to be specific for TK1. The specific binding of the engineered scFv-Fc antibody to cell-surface biomarker TK1 makes it a promising approach in cancer immunotherapy. "Wheelz" has the potential to expand the repertoire of tumor targets in CAR T-cell therapy.

#2782

Examining the dynamic regulation of OX40 following receptor agonism and T-cell activation: Implications for antibody-mediated enhancement of T-cell function.

Marie-Claude Gaudreau, Christina Milburn, Chan Gao, Alla Pritsker, Mark Fereshteh, Zheng Yang, Bryan Barnhart, Alan Korman, Michael Quigley. _Bristol-Myers Squibb, Princeton, NJ_.

Background: Following the clinical success of checkpoint blockade with CTLA-4 and PD-1 pathway-targeted agents, the field of cancer immunotherapy is rapidly expanding. Costimulatory molecules from the tumor necrosis factor receptor superfamily (TNFRSF), including OX40, may be promising targets to enhance the benefits of immunotherapy. While the functional consequences of signaling through OX40, either via OX40 ligand or agonistic antibodies, have been well documented, little attention has been paid to the temporal regulation of the OX40 receptor itself following T-cell activation and receptor agonism. Here we present functional characterization of BMS-986178, a fully human IgG1 monoclonal OX40- agonistic antibody.

Methods: We developed in vitro assays using healthy donor human CD4+ effector T (Teff) and regulatory T (Treg) cells to (1) characterize the functional activity of BMS-986178 across a wide range of concentrations (0.0001-200 nM), (2) address how OX40 receptor expression is modulated following T-cell activation, and (3) determine whether antibody treatment affects receptor regulation. Analysis of murine tumor-infiltrating lymphocytes (TILs) allowed for the comparison of receptor regulation across species.

Results: BMS-986178-mediated enhancement of human CD4+ Teff responses and reversal of Treg suppression was dependent on antibody cross-linking, as activity was lost in the absence of FcγR- or secondary antibody-mediated cross-linking in the assays. Maximal increases in activation markers (CD25, ICOS), proliferation, and cytokine production mediated by BMS-986178 treatment occurred at ≈20% receptor occupancy (RO). This functional enhancement was lost at relatively high antibody concentrations (>1 nM) associated with full RO. Lower concentrations of BMS-986178 (0.001-0.3 nM) were sufficient to drive upregulation of OX40, both on the cell surface and the soluble receptor; however, as RO approached 100%, a loss in surface and soluble OX40 was observed. Loss of receptor at the point of full RO was also observed in murine TILs. The downregulation of OX40 was specific to treatment with BMS-981678, as it was not observed with anti-CD28 or antibodies against other TNFRSF members. Internalization of the receptor:antibody complex as well as epigenetic regulation of the OX40 locus were examined for their role in receptor downregulation.

Conclusions: These results demonstrate a clear relationship between RO and the ability of BMS-986178, an agonist OX40 antibody, to enhance T-cell responses. Furthermore, these findings provide insight into antibody-mediated receptor modulation in vitro, with potential implications for defining the optimal dose and schedule of agonist OX40 antibodies and, perhaps more broadly, for agonists targeting other costimulatory molecules.

#2783

Empowering therapeutic monoclonal antibodies with IFN-alpha for cancer immunotherapy.

Jun Guo, Yu Xiao, Ramesh B. Lyer, Marc R. Lake, Uri S. Ladror, Xin Lu, Bill Pappano, John E. Harlan, Medha J. Tomlinson, Gail T. Bukofzer, Cherrie K. Donawho, Alexander R. Shoemaker, Tzu-Hsuan Huang. _AbbVie Inc., North Chicago, IL_.

Type 1 IFNs stimulates secretion of IP-10 (CXCL10) which is a critical chemokine to recruit effector T cells to the tumor microenvironment and IP-10 knockout mice exhibit a phenotype with compromised effector T cell generation and trafficking. Type 1 IFNs also induces MHC class 1 upregulation on tumor cells which can enhance anti-tumor CD8 T cell effector response in the tumor microenvironment. Although type 1 IFNs show great promise in potentiating anti-tumor immune response, systemic delivery of type 1 IFNs is associated with toxicity thereby limiting clinical application. In this study, we fused tumor targeting antibodies with IFN-alpha and showed that the fusion proteins can be produced with high yields and purity. IFN fusions selectively induced IP-10 secretion from antigen positive tumor cells, which was critical in recruiting the effector T cells to the tumor microenvironment. Further, we establish a real time in vitro antigen specific CTL killing assay using IncuCyte Zoom and tested the anti-tumor efficacy of anti-PDL1-IFN-alpha fusion using this system. We found anti-PDL1-IFN-alpha as low as 10 pM exhibits potent activity in potentiating OT1 CD8 T cells killing against OVA expressing tumor cells, while control IFN fusion did not exhibit any activity in the same experiment. Furthermore, IFN-alpha fusion antibody was well tolerated in vivo and demonstrated anti-tumor efficacy in an anti-PD-L1 resistant syngeneic mouse tumor model. Our data supports the hypothesis of targeting type 1 IFN to the tumor microenvironment may enhance effector T cell responses in non-inflamed tumors.

#2784

Simultaneous checkpoint-checkpoint or checkpoint-costimulatory receptor targeting with bispecific antibodies promotes enhanced human T cell activation.

Michael Hedvat, Christine Bonzon, Matthew J. Bernett, Gregory L. Moore, Kendra Avery, Rumana Rashid, Alex Nisthal, Suzanne Schubert, Rajat Varma, Sung-Hyung Lee, Liz Bogaert, Irene W.L. Leung, Seung Chu, Umesh Muchhal, John Desjarlais. _Xencor Inc., Monorvia, CA_.

Combination checkpoint blockade promotes productive anti-tumor clinical responses often associated with an increase in immune-related adverse events. We developed optimized bispecific antibody candidates that simultaneously engage PD1 and CTLA4 (XmAb20717), CTLA4 and LAG3 (XmAb22841), and PD1 and ICOS (XmAb23104), that preferentially bind to cells co-expressing the targeted receptors. Because tumor infiltrating lymphocytes (TILs) typically express multiple immune checkpoints and costimulatory receptors, we hypothesized that these bispecific antibodies will enable selective targeting of tumor-reactive TILs, leading to safer and more cost-effective combination checkpoint blockade. The PD1 x CTLA4 bispecific antibody XmAb20717 and the PD1 x ICOS bispecific antibody XmAb23104 promoted superior in vitro T cell activation when compared to an anti-PD1 bivalent antibody. The CTLA4 x LAG3 bispecific antibody XmAb22841 combines productively with an anti-PD1 bivalent antibody achieving additive T cell activation through triple checkpoint blockade. Treatment with bispecific antibodies caused superior engraftment and activation of human T cells in NSG mice compared to an anti-PD1 bivalent antibody. Bispecific antibodies also potentiate allogeneic anti-tumor activity against human cancer cells in vivo. Each bispecific antibody promoted a unique RNA gene signature suggesting distinct mechanisms of action. These bispecific antibodies demonstrate compelling immune modulatory activity suggesting clinical development is warranted for the treatment of human malignancies.

#2785

Potency-matched dual cytokine antibody fusion proteins in combination with PD-L1 blockade.

Roberto De Luca, Dario Neri. _ETH Zürich, Zürich, Switzerland_.

Among the different pharmacological approaches for cancer treatment, antibody-cytokine fusion proteins (also called "immunocytokines") represent an emerging class of biopharmaceutical products, capable of boosting the immune system to attack tumor cells. These products promote the selective accumulation of pro-inflammatory molecules at the site of disease, mediated by the antibody fragment. These fusion proteins have been designed to reduce the systemic toxicity of traditional cytokines, currently approved for certain malignancies.

Immunocytokine products based on interleukin-2 (IL2), interleukin-12 (IL12) or tumor necrosis factor (TNF) have been extensively studied in mouse model of cancer and in patients.

When used as single agents, immunocytokines are often not able to cure mice and patients. However it as been shown by our group that the combination of IL2- and TNF- based products was capable to completely eradicate tumors in mouse models and to induce complete responses in patients.

The development of combination products at the industrial level leads to a duplication of activities and costs. On the other hand, the opportunity to develop products with two payloads fused into a single molecular entity may facilitate development. This approach is limited by the fact that different cytokines often show different maximal tolerated doses (MTDs), and their relative potency need to be tuned. For example, TNF is administered at a dose, which is ten times lower compered to the one of IL2.

We have recently described a novel class of immunocytokine products, termed "potency-matched dual cytokine antibody fusion proteins" consisting of a tumor-targeting antibody (for example F8, specific to the alternatively spliced EDA domain of fibronectin), simultaneously fused to both IL2 and TNF. To match biological activity of the two payloads a single-point mutation was inserted in the TNF sequence and the resulting product, named IL2-F8-TNFmut was able to selectively localize to lesions with an excellent tumor-to-organ ratios and it was found to completely eradicate soft-tissue sarcomas in immunocompetent mice, which did not respond to standard chemotherapy. We now report that complete responses were observed in a variety of mouse models of cancer and that the therapeutic efficacy of IL2-F8-TNFmut was further improved when used in combination with an anti-PD-L1 antibody.

Potency-matched dual cytokine antibody fusion proteins may find clinical applications for various types of malignancies, used either as single agents or in combination with immune-checkpoint inhibitors.

#2786

Pharmacokinetics and in vitro/in vivo characterization of high-affinity bispecific EGFR/CD16A NK cell engagers for the treatment of EGFR-expressing tumors.

Michael Kluge, Michael Tesar, Uwe Reusch, Stefan Knackmuss, Torsten Haneke, Kristina Ellwanger, Ivica Fucek, Thomas Mueller, Ute Schniegler-Mattox, Martin Treder. _Affimed GmbH, Heidelberg, Germany_.

The epidermal growth factor receptor (EGFR) is a validated target for the treatment of several solid tumor types. Current EGFR-targeting monoclonal antibodies (mAbs) and tyrosine kinase inhibitors function mainly through blocking of signal-transduction. Moreover, treatment with these agents is dependent on the receptor's mutational status which may cause treatment resistance in a large number of patients. In addition, EGFR-targeting therapies have been associated with side effects considered to impact prescription rates, in particular in the U.S. A natural killer (NK) cell-based EGFR-targeting approach has the potential to widen the therapeutic window and overcome intrinsic and acquired resistance. Therefore, it offers a promising and well differentiated therapeutic option. We describe the engineering of different bispecific NK cell engagers designed to redirect NK cell-mediated killing to EGFR-positive tumors. Different bispecific EGFR/CD16A antibody constructs targeting human and cynomolgus EGFR and CD16A were designed using novel human Fv antibody binding domains from a toolbox of formats. The Fv domains were evaluated in terms of their pharmacokinetic (PK) properties. Furthermore, the antibody constructs were characterized in antigen and cell binding assays, as well as in in vitro cytotoxicity assays against tumor cell lines expressing Raswt or mutated Ras. PK parameters were determined in CD1 mice and in vivo efficacy of selected antibodies was investigated in an A-431 tumor model in humanized mice. We generated antibody constructs with varying half-lives, including antibodies with a PK profile which allows for dosing comparable to IgG-based antibodies, while aiming at an improved safety profile compared to other EGFR-targeting therapies. Different high affinity tetravalent, bispecific antibodies binding to CD16A and the extracellular domain of EGFR were characterized. While binding to CD16A of classical mAbs is impaired by serum IgG, no substantial changes in binding affinity of our constructs to NK cells were observed. The antibodies showed superior potency and efficacy compared to mAbs or Fc-enhanced antibodies when tested in cytotoxicity assays. In vivo efficacy of selected antibodies was demonstrated in an A-431 tumor model in humanized mice. We have successfully designed and characterized several novel, highly potent and differentiated tetravalent bispecific antibody constructs to redirect NK cell-mediated cell killing to EGFR-positive tumor cells. These drug candidates are suitable for the treatment of EGFR-expressing malignancies with the potential to overcome resistance to other EGFR-targeting agents and offering a potentially improved safety profile. These candidates are currently in development as monotherapy and are also explored in combination with immune activating agents.

#2787

Generation of first-in-class anti-CD25 antibodies depleting Treg without interfering with IL2 signalling for cancer therapies.

Josephine C. Salimu,1 Mark Brown,1 Pascal Merchiers,1 Beatriz Goyenechea,1 Kevin Moulder,1 Robert Dejonge,1 Aghiles Boughetane,1 Isabelle Solomon,2 Frederick Arce Vargas,2 Karl S. Peggs,2 Anne Goubier,1 Sergio A. Quezada2. 1 _Tusk Therapeutics Ltd, Stevenage, Hertfordshire, United Kingdom;_ 2 _University College London Cancer Institute, London, United Kingdom_.

Regulatory T cells (Treg) are key players of the suppressive tumour microenvironment (TME). Their presence is correlated with a bad prognosis in multiple cancers, while a greater ratio of effector T cells (Teff) to Treg is associated with improved outcome. Studies demonstrating high expression of CD25 on Tregs but not Teff in human tumors have underscored its relevance as a target for Treg depletion. However, clinical trials conducted to target Treg in cancer patients with existing anti-CD25 (aCD25) antibodies have shown contradicting results, and no aCD25 antibody is in clinical development for this application. Importantly, aCD25 antibodies tested to date block IL2 signalling via CD25. We demonstrated (Solomon et al. AACR2018) that such blocking drastically reduces the therapeutic activity of aCD25 antibodies and that a single administration of a depleting antibody targeting CD25 but not blocking the IL2 signalling effectively depletes Treg, increases Teff activity and promotes eradication of established tumors in several mouse models of cancer.

With this rationale in mind, we have generated a panel of fully human aCD25 IgG1 antibodies, which we characterized for their binding to human and cynomolgus monkey CD25. The antibodies were then screened for their impact on IL2 binding, using a sandwich binding assay on Octet, and on IL2 signalling, using a STAT5 phosphorylation assay. Antibodies were also tested for their ability to deplete CD25 positive cell lines and in vitro-derived human Treg in ADCC and ADCP assays. Finally, the impact of selected antibodies on Treg within the TME and on Teff responses was evaluated in human samples.

Among the antibodies binding to human and cynomolgus CD25, we have selected a panel interfering with neither IL2 binding to CD25 nor IL2 signalling. Interestingly, epitope binning assays demonstrated that none of the selected antibodies bind to epitopes overlapping with those of the existing clinical antibodies, Daclizumab and Basiliximab. Among the non-IL2 blocking antibodies, those showing the maximum target cell lysis via ADCC and highest phagocytosis via ADCP represent potential lead clinical candidates.

Most interestingly, contrary to the existing clinical aCD25 antibodies, which block IL2 signalling, our candidates do not inhibit Teff proliferation and function, confirming the specificity of action toward Treg and the importance of preserving IL2 signalling. Finally, we have shown that our clinical candidates efficiently deplete Treg in the TME.

We present the first anti-human CD25 antibodies selected for their capacity to deplete human Treg while preserving IL2 signalling and activity of Teff. These antibodies provide a novel therapeutic approach to alleviate immune suppression in the TME, which would provide an ideal combination partner for existing standard of care and IO treatments, and could potentially be used as a monotherapy.

#2788

VB6-845d tumor cell killing elicits biologic features of immunogenic cell death.

Rachelle L. Dillon,1 Shilpa Chooniedass,1 Arjune Premsukh,1 Glen C. MacDonald,2 Jeannick Cizeau,1 Gregory P. Adams2. 1 _Viventia Bio Inc, Winnipeg, Manitoba, Canada;_ 2 _Eleven Biotherapeutics, Cambridge, MA_.

VB6-845d is a Targeted Protein Therapeutic, TPT, that comprises a Fab fragment specific for the Epithelial Cell Adhesion Molecule (EpCAM) genetically fused to deBouganin via a furin protease sensitive linker. DeBouganin (deB) is a de-immunized variant of bouganin, a ribosome inactivating protein (RIP), that when internalized blocks protein synthesis thereby leading to cell death. While all cytotoxic molecules induce tumor cell killing, only some are capable of inducing biological manifestations indicative of immunogenic cell death (ICD). ICD is characterized by the collective appearance of distinct cellular changes termed damage associated molecular patterns, or DAMPs, which play a role in immune cell activation by triggering pro-inflammatory processes. Key DAMPs necessary for defining ICD are cell surface translocation of calreticulin, an endoplasmic reticulum chaperone protein, ATP secretion and release of HMGB1 (high mobility group box 1). To evaluate whether VB6-845d cell killing elicits the hallmark features of ICD, VB6-845d treated tumor cell lines were assessed for the presence of these distinct signaling molecules. In vitro studies showed that VB6-845d cytotoxicity induces the translocation of calreticulin to the cell surface as well as the release of ATP and HMGB1. The expression of other potential immune regulators following VB6-845d treatment was also examined. In summary, the data presented suggests that VB6-845d mediates tumor cell killing by an ICD pathway. The potential cross-priming effect initiated by VB6-845d-induced ICD suggests the use of VB6-845d in combination with immune checkpoint inhibitors may enhance their effectiveness in EpCAM-positive epithelial cancers.

#2789

Development of highly potent T-cell receptor bispecifics with picomolar activity against tumor-specific HLA ligands.

Sebastian Bunk, Martin Hofmann, Felix Unverdorben, Leonie Alten, Meike Hutt, Claudia Wagner, Oliver Schoor, Mathias Ferber, Jens Fritsche, Toni Weinschenk, Harpreet Singh-Jasuja, Dominik Maurer, Carsten Reinhardt. _Immatics Biotechnologies GmbH, Tübingen, Germany_.

T-cell receptor (TCR)-based immunotherapy has emerged as a promising treatment modality for malignant diseases. TCRs naturally recognize human leucocyte antigen (HLA)-bound peptides from foreign and endogenous antigens regardless of their source proteins' extracellular or intracellular location. Using its proprietary discovery engine XPRESIDENT®, Immatics can identify, quantify, and validate Tumor-Associated Peptides (TUMAPs) exclusively presented in cancer tissues. Immatics has established state-of-the-art technology to discover and affinity maturate TUMAP-specific TCRs originating from human T-cell repertoire. The maturated single-chain TCRs (scTv) are used to build a pipeline of highly potent T-cell engaging bispecific TCR molecules directed against TUMAPs. In brief, we use artificial antigen-presenting cells to selectively expand TUMAP-specific T-cells from which the coding TCR sequence is retrieved by 5'RACE after highly sensitive flow cytometry-based single cell sorting. About 50-150 TCRs per TUMAP are transiently re-expressed on human T-cells and extensively characterized for their functional properties. TCRs exhibiting highly active and specific TUMAP recognition are selected for yeast surface display to generate stabilized and affinity maturated scTv. The maturated scTv are engineered into Immatics' proprietary bispecific TCR scaffold comprising a humanized T-cell recruiting antibody domain for potent redirection and activation of T-cells against TUMAPs and an effector function-silenced IgG1 Fc domain. Here we present data of our bispecific TCR program targeting the TUMAP Ag008-01 bound to HLA-A*02. We confirmed the abundant presence of Ag008-01 in several cancer indications and its absence in human normal tissues by using XPRESIDENT® technology combining quantitative mass spectrometry, transcriptomics and bioinformatics. Based on the parental TCR showing highly active and specific recognition of Ag008-01, we generated stabilized and affinity maturated scTv variants with significant improvement in binding affinity towards Ag008-01 in the range of 1000-fold and higher. The maturated scTv variants showed no or minimal binding to off-target peptides selected from the XPRESIDENT® normal tissue database based on the criteria of highest sequence similarity to Ag008-01. By incorporating the maturated scTv into our proprietary bispecific TCR format, which outperformed five alternative TCR bispecific format designs, we obtained highly potent bispecific TCR molecules with picomolar activity. We observed half-maximal lysis of Ag008-01 expressing tumor cell lines at TCR bispecific concentrations below 100 picomolar while no reactivity was observed towards a panel of cell lines lacking Ag008-01 expression. Our data support proof-of-concept for the design of our novel class of T-cell engaging bispecific TCR-antibody fusion proteins.

#2790

The anti-TGFβ neutralizing antibody, SAR439459, blocks the immunosuppressive effects of TGFβ and inhibits the growth of syngeneic tumors in combination with anti-PD1.

Richard C. Gregory, Rita Greco, Hongjing Qu, Natalia Malkova, Mikhail Levit, Keli Perron, Waldemar Racki, Fangxian Sun, Gary Shapiro, Christopher Winter, Dmitri Wiederschain, Tun Tun Lin, Jack Pollard. _Sanofi Oncology, Cambridge, MA_.

TGFβ may act as a tumor suppressor early in tumorigenesis; however, TGFβ is also known to support tumor growth via multiple mechanisms, including promotion of epithelial-mesenchymal transition, fibroblast recruitment, ECM deposition, neovascularization, and immunosuppression of both adaptive and innate immune cells. SAR439459 is pan-neutralizing TGFβ antibody that is being developed for the treatment of solid tumors. In this study, we investigated the effect of SAR439459 on the immune system in primary human immune cells in vitro and in murine syngeneic tumor models in vivo. Exogenous TGFβ inhibited proliferation of CD8pos primary human T cells and suppressed the production of inflammatory cytokines such as IFNγ. The addition of SAR439459 blocked these effects. Additionally, TGFβ enhanced the development of inducible Tregs by approximately 4-fold, while the inclusion of SAR439459 abrogated this effect. The effects of exogenous TGFβ and SAR439459 were next examined in the context of T Cell Receptor signaling in a co-culture system of Jurkat cells expressing luciferase reporter under the control of NFAT-responsive promoter and PD-L1pos APCs. The addition of anti-PD1 stimulated TCR signaling in this system, while the addition of TGFβ inhibited anti-PD1-mediated response. SAR439459 restored the ability of anti-PD1 to stimulate the TCR signaling. These data support the notion that TGFβ neutralization could positively impact T effector cells and inhibit Tregs development. Therefore, combining a TGFβ-neutralizing antibody with anti-PD1 could enhance T cell responsiveness. Compared to checkpoint inhibitors, which are suggested to cause the reactivation and de-repression of exhausted T cells, the effects of TGFβ on the immune system are thought to be distinct. To test this, the combination of anti-PD1 and SAR439459 was examined in syngeneic mouse models. Established MC38 tumors were treated with anti-PD1, SAR439459 or the combination of both antibodies. In tumor-bearing mice treated with either anti-PD1 or SAR439459 alone, neither Ab resulted in significant tumor growth inhibition. However, the combination of anti-PD-1 and SAR439459 led to tumor regression in the majority of tumor-bearing mice. These results provide the rationale for combining TGF-β neutralization with checkpoint inhibitors, specifically anti-PD1, for the treatment of human cancers.

#2791

Physiologically based pharmacokinetic modeling and simulations to estimate the efficacious dose of the CEACAM6 function-blocking antibody BAY 1834942.

Sabine Wittemer-Rump,1 Christoph Niederalt,2 Joerg Willuda,1 Mark Trautwein,3 Merlin Luetke-Eversloh,1 Wolf-Dietrich Doecke,1 Clemens Guenther,1 Christian Scheerans1. 1 _Bayer AG, Berlin, Germany;_ 2 _Bayer AG, Leverkusen, Germany;_ 3 _Bayer AG, Wuppertal, Germany_.

BAY 1834942 is an immunostimulatory function-blocking (fb) antibody (Ab) against the target carcinoembryonic antigen related cell adhesion molecule 6 (CEACAM6) expressed on tumor cells in multiple cancer indications. The suggested mode of action of BAY 1834942 is the blockade of the immunosuppressive effect of CEACAM6 on activated T cells which restores the immune response against cancer cells.

Available preclinical pharmacokinetic (PK) and in vitro pharmacodynamic (PD) data, target receptor density information and tumor (patho-)physiology were used to create a model framework taking into account the three most essential 'pillars' (target exposure, target binding and drug activity) to estimate the human efficacious dosing of BAY 1834942. For this purpose, a physiologically-based pharmacokinetic (PBPK) model considering target binding of BAY 1834942 in tumor tissue and on blood granulocytes has been developed. The aim of the PBPK model was to estimate the dose range and regimen for humans leading to exposure level at the tumor site that allows sufficient target binding. The PBPK simulations were based on 1) an analysis of PD in vitro data in order to estimate the degree of saturation needed for maximum drug activity, 2) the assessment of CEACAM6 receptor numbers on tumor cells and blood granulocytes and 3) in vivo PK data in order to develop and evaluate the PBPK model. For the latter, plasma PK data of BAY 1834942 in monkeys were used as well as known tumor concentration-time profiles of the antibodies MOPC21 (non-targeting Ab) and ZCE025 (anti-CEA Ab) in mice and humans. Uncertainty of parameters which are relevant for CEACAM6 target saturation was considered by stochastic in silico simulations to estimate the CEACAM6 saturation at the tumor vs. dosing. This analysis revealed that the predicted human efficacious dose strongly depends on CEACAM6 density. Thus, a low CEACAM6 density scenario (25,000 CEACAM6/tumor cell) and a high CEACAM6 density scenario (250,000 CEACAM6/tumor cell) were simulated and used to support dose selection for the first-in-man (FIM) study of BAY 1834942. The FIM study is currently under preparation.

#2792

The combination of immune checkpoint blockers with the anti-ICOS KY1044 antibody results in a strong tumor response.

Richard C. Sainson, Matthew McCourt, Anil Thotakura, Nahida Parveen, Miha Kismac, Gwenoline Borhis, Joana Carvalho, Tracey Myers, Robert Rowlands, Hanif Ali, Hannah Craig, Vivian Wong, Qi Liang, Volker Germaschewski. _Kymab, Cambridge, United Kingdom_.

The last few years have shown a strong paradigm shift in cancer therapies with the approval of antibodies targeting immune checkpoints (CTLA-4, PD-1 and PD-L1). These immune checkpoint blockers (ICBs) are associated with a strong and long-lasting response in patients suffering from different malignancies. However, there is still a high proportion of patients showing intrinsic or acquired resistance to ICBs. Although the mechanisms associated with the lack of immune response are multiple, the presence of highly anti-inflammatory regulatory T cells (TRegs) in the tumour microenvironment (TME) is known to negatively affect the response to these therapies. Similarly, high TReg levels in the TME have been associated with poor prognosis in several cancers. Together, this highlights the potential of targeting and depleting TRegs to enhance anti-tumour responses.

The Inducible T-cell costimulator (ICOS/CD278) is related to the CD28 superfamily and is induced when T cells get activated. ICOS expression levels vary in different immune cell subtypes and in different tissues. In preclinical mouse tumour models, TRegs (CD4+/FOXP3+) constitutively express ICOS on their surface and the expression of ICOS on TRegs is significantly higher than that on effector T cells (TEffs). In addition, ICOS expression on TRegs is higher in the TME than in the blood or spleen, which makes it a strong candidate for preferential depletion of TRegs in tumours. By immunizing Kymice™ in which the endogenous Icos gene has been knocked out, we identified a novel, fully human antibody called KY1044 that cross-reacts with mouse ICOS. KY1044 is an anti-ICOS subclass G1 kappa monoclonal antibody that selectively binds to dimeric ICOS (Fc fusion) with an affinity of less than 2nM. Using in vitro and in vivo approaches we demonstrate that KY1044 has a dual mechanism of action: (1) it promotes the preferential depletion of intratumoural ICOShigh TRegs resulting in an increase in the TEff:TReg ratio in the TME; and (2) it stimulates ICOSLow TEff cells. Using the mouse effector enabled version of KY1044 (mIgG2a) we confirm, using syngeneic models, a strong anti-tumour efficacy as monotherapy or in combination with surrogates of ICBs. We also demonstrated a tumour antigen specific immunity, as highlighted by the rejection of the original tumour cells in animals cured of the disease and re-challenged by the same cell line. Noteworthy, Pharmacodynamic studies demonstrate long-term depletion of TRegs and a significant increase in the TEff:TReg ratio in response to KY1044. In summary, our data demonstrate that targeting ICOS with KY1044 is a valid approach for manipulating the immune system and for inducing a strong anti-tumour response in several indications. The data presented here also warrant the assessment of KY1044 in cancer patients in a clinical trial.

#2792A

Preclinical activity of ADCT-601, a novel pyrrolobenzodiazepine (PBD) dimer-based antibody-drug conjugate (ADC) targeting AXL-expressing tumors.

Francesca Zammarchi,1 Karin Havenith,1 Simon Chivers,1 Paul W. Hogg,1 Charlie Britten,1 Sandamali Dissanayake,1 Peter Tyrer,2 Francois Bertelli,2 Ian Hutchinson,2 Luke Masterson,2 Phil Howard,2 John A. Hartley,3 Patrick H. van Berkel1. 1 _ADC Therapeutics, London, United Kingdom;_ 2 _Spirogen, Medimmune, London, United Kingdom;_ 3 _University College of London, London, United Kingdom_.

AXL, a member of the tyrosine kinase receptor family TAM, is a transmembrane receptor overexpressed in many solid (e.g. lung, breast, pancreas, glioma and esophageal) and hematological malignancies (acute myeloid and chronic lymphocytic leukemia) and its overexpression is maintained in both primary tumors and metastasis. Moreover, expression and activation of AXL is associated with poor clinical prognosis and several studies suggest that overexpression of AXL results in resistance to both conventional chemotherapy and targeted therapies. All these features make AXL an attractive target for the development of an ADC to treat AXL-expressing cancers.

ADCT-601 is an ADC composed of a humanized IgG1 antibody against human AXL, site-specifically conjugated using GlycoconnectTM technology to PL1601, which contains a valine-alanine cleavable linker and the PBD dimer cytotoxin SG3199.

The purpose of this study was to characterize the in vitro and in vivo anti-tumor activity of ADCT-601 in human cancer cell lines and xenograft models and to determine its safety and tolerability in the rat.

In vitro, ADCT-601 demonstrated potent cytotoxicity in a panel of cell lines of different origin and levels of AXL, while its potency was strongly reduced in AXL-negative cell lines.

In vivo, ADCT-601 showed potent anti-tumor activity in the human triple-negative breast cancer-derived MDA-MB-231 xenograft, expressing moderate levels of AXL (≈36,000 copies/cell). In this model, a single dose of ADCT-601 at 1 mg/kg resulted in strong and sustained anti-tumor activity and resulted in 5/10 partial responders (PR) and 4/10 tumor-free survivors (TFS) at the end of the study on day 60. In the SN12C model, a human renal cell carcinoma-derived xenograft with high level of AXL expression (≈88,000 copies/cell), single doses of ADCT-601 at 0.3, 0.6 or 1 mg/kg showed dose-dependent anti-tumor activity compared to the vehicle- and isotype control ADC-treated mice. At the highest dose, ADCT-601 resulted in 7/8 and 6/8 complete responder (CR) and TFS, respectively, at the end of the study on day 60. Moreover, ADCT-601 showed potent and durable anti-tumor activity in a pancreatic cancer patient-derived xenograft model expressing heterogeneous levels of AXL where each single dose of ADCT-601 tested (0.3, 0.6 and 1 mg/kg) resulted in complete eradication of the tumors at the end of the study on day 43. Conversely, none of the mice in the vehicle and isotype control ADC groups had any PR, CR or TFS. ADCT-601 was stable, well tolerated and showed a favorable PK profile in the rat with a half-life of 9 days and a MTD of 6 mg/kg.

In conclusion, ADCT-601 demonstrated potent and specific in vitro and in vivo anti-tumor activity in various cancer-derived models with different levels of membranous AXL. ADCT-601 was stable and well tolerated in the rat, warranting further development of this ADC into the clinic.

## EXPERIMENTAL AND MOLECULAR THERAPEUTICS:

### Design, Structure/Activity, and Modeling

#2793

Design, synthesis and evaluations of novel class of orally bioavailable eIF4A3-selective inhibitors.

Ryo Mizojiri, Daisuke Nakata, Yoshihiko Satoh, Daisuke Morishita, Sachio Shibata, Misa Iwatani-Yoshihara, Yohei Kosugi, Mai Kosaka, Junpei Takeda, Shigekazu Sasaki, Kazuaki Takami, Koichiro Fukuda, Masahiro Kamaura, Shinobu Sasaki, Ryosuke Arai, Douglas R. Cary, Yasuhiro Imaeda. _Takeda Pharmaceutical Co Ltd, Fujisawa, Kanagawa, Japan_.

Eukaryotic initiation factor 4A3 (eIF4A3) is a member of the ATP-dependent RNA helicase protein family and controls RNA metabolism, including nonsense-mediated mRNA decay (NMD) and RNA localization, as a core component of the exon junction complex (EJC). Cancer cells often harbor a wide variety of mutations introducing PTCs, and cancer cells are thought to be highly dependent on the NMD pathway to avoid the accumulation of aberrant proteins. Therefore, targeting eIF4A3 inhibition could potentially represent a promising approach for anticancer therapy, however, the detailed physiological functions of eIF4A3 are not clear. To enable the further biological and pharmacological evaluation of eIF4A3, we recently discovered several classes of both ATP-competitive and allosteric eIF4A3 inhibitors which showed potent and selective eIF4A3 inhibition. Among these, the allosteric eIF4A3 inhibitors exhibited significant NMD inhibition in a cellular reporter assay along with eIF4A3 inhibitory potency and were examined for understanding the function of eIF4A3 in RNA homeostasis. Although the allosteric inhibitors showed potent enzymatic activity as well as cellular potency, the physicochemical properties such as solubility, metabolic stability, and pharmacokinetics were insufficient for conducting further biological and pharmacological studies. To identify improved probe molecules for the study of eIF4A3 function, we continued our chemistry efforts on allosteric inhibitors using the novel 3-(4-chlorophenyl)-1,4-diacylpiperazine derivative as a lead compound. Although many of them showed improved solubility and metabolic stability while maintaining eIF4A3 inhibitory potency, further testing indicated it to be a P-gp substrate. To address this issue, a pyridin-2(1H)-one derivative was prepared based on the calculated tPSA value for reducing polarity, which led us to the discovery of the novel class of orally bioavailable eIF4A3-selective inhibitors. Herein, we demonstrated their biological and pharmacological evaluations and showed anti-tumor efficacy without severe body weight loss in xenograft mice. Thus, our novel series of compounds represent promising tool compounds for the in vivo pharmacological study of selective eIF4A3 inhibition.

#2794

A novel bis-aspirinyl selenazolidine compound AS-10 as potential colon cancer therapeutic.

Deepkamal N. Karelia,1 Manoj Pandey,2 Daniel Plano,1 Shantu Amin,1 Arun K. Sharma1. 1 _Penn State Univ. College of Medicine, Hershey, PA;_ 2 _Cooper Medical School of Rowan University, Camden, NJ_.

Despite rigorous screening and aggressive surgical and chemotherapeutic treatments, colorectal cancer (CRC) remains the third leading cause of cancer-related deaths in women and the second leading cause in men in the United States. It is expected to cause about 50,260 deaths during 2017 (American Cancer Society). The 5-year survival rate for patients with colorectal cancer (CRC) is 64%, although for patients who present with distant metastases at diagnosis the 5 year survival is only 12%. Therefore, there is an urgent need to develop effective therapeutic strategies that will enhance the survival and improve quality of life of CRC patients. Non-steroidal anti-inflammatory drugs (NSAIDs), particularly aspirin, have recently been shown to be effective in reducing colon cancer incidences and are proposed to be effective CRC chemopreventive agents. Through extensive structure activity relationship (SAR) studies based on designing selenium-NSAID hybrid small molecules, we have identified a small drug-like molecule, AS-10, which contains two aspirinyl groups flanked to a selenazolidine ring. AS-10 effectively inhibited the viability of CRC cells (HT29, RKO, and HCT116) with an EC50 in the range of 1 to 2.5 µM. Interestingly, AS-10 showed more selectivity towards killing cancer cells than normal mouse embryonic fibroblasts (MEFs) and was >50 times more potent than current standard of care, 5-FU, for in killing CRC cells. Cell cycle studies revealed AS-10 treated CRC cells to have G1 and G2/M phase cell cycle arrest. Further, increasing protein expression of cell cycle inhibitory protein p21 confirmed our cell cycle findings in AS-10 treated CRC cells. Since these CRC cell lines are p53 mutant cells, it was noteworthy to see p53 independent expression of p21. Moreover, we explored the possibility of apoptosis in these cell cycle arrested cells and found that AS-10 treatment increased casapse 3/7 activity, and cells positive for phosphatidylserine on the outer cell membrane surface indicative of apoptosis. In silico evaluation shows that AS-10 with cLogP 2.2 and MW 473 fits perfectly into the requirements of Lipinski's Rule-of-Five, formulated to determine the drug-likeness of a small molecule. In addition, in nude mice, AS-10 exhibited a high maximum tolerated dose (MTD) (~80 mg/kg, IP, three times per week). Furthermore, AS-10 (45 mg/kg, IP, three times per week) effectively inhibited tumor growth by ~70% in a nude mouse colon cancer xenograft model without any apparent systemic toxicity. Overall, AS-10 appears to be a promising small molecule worthy of further development as a therapeutic for CRC patients.

#2795

Screening and characterization of drugs that inhibit the ubiquitin-proteasome system.

Karthik Selvaraju,1 Arjan Mofers,1 Paola Pellegrini,1 Ellin-Kristina Hillert,2 Padraig D'Arcy,1 Stig Linder1. 1 _Linköping University, Linköping, Sweden;_ 2 _Karolinska Institute, Stockholm, Sweden_.

Pharmacological inhibition of proteasomal cysteine deubiquitinase (DUB) activity (i.e., USP14 and UCHL5 DUBs) has been shown to be particularly cytotoxic to cancer cells and to result in inhibition of tumor growth in several in vivo models. Most proteasome DUB inhibitors contain α, β-unsaturated ketones with the potential to covalently bind DUB active site cysteines. We here performed a screen of 5000 compounds containing α, β-unsaturated ketones in order to identify novel proteasome DUB inhibitors and to examine the question of the specificity of such compounds. Surprisingly, only ~3% of 5000 compounds containing α, β-unsaturated ketones showed anti proliferative activity at 5 uM. Almost one third of these cytotoxic compounds were found to block proteasome processing. Treatment of cells with the hit compounds induced the accumulation of proteasome bound high molecular weight polyubiquitin conjugates, induction of the chaperone HSP70B' and the oxidative stress marker heme oxygenase-1 (HO-1) and to induce an apoptotic response. A subset of 10 compounds were studied in detail and found to inhibit proteasome DUB activity in vitro. Importantly, hit compounds did not show inhibitory activity on a panel of recombinant non-proteasome DUBs. Inhibition of proteasome processing by hit compounds was not due to inhibition of 20S proteasome enzymatic activity. Exposure of multiple myeloma cells to the hit compounds resulted in thermostabilization of USP14 at the relevant concentrations using CETSA. These results suggest that α, β-carbonyls-containing compounds are promising candidates for development of drugs targeting proteasome DUBs and that drugs showing unexpected selectivity can be identified.

#2796

Predicting in vivo efficacy from in vitro data: Quantitative systems pharmacology modeling for an epigenetic modifier drug in cancer.

Mehdi Bouhaddou,1 Li J. Yu,1 Serena Lunardi,2 Spyros K. Stamatelos,1 Fiona Mack,1 James M. Gallo,3 Marc R. Birtwistle,4 Antje-Christine Walz5. 1 _Hoffman-La Roche, Inc., New York, NY;_ 2 _Oryzon Genomics S.A., Barcelona, Spain;_ 3 _Albany College of Pharmacy and Health Sciences, Albany, NY;_ 4 _Icahn School of Medicine at Mount Sinai, New York, NY;_ 5 _Hoffman-La Roche, Inc., Basel, Switzerland_.

Epigenetic modulators are increasing in prominence as potential cancer therapies. These drugs achieve their effectiveness by inducing transcriptional changes that can inhibit cancer progression. Here, we focus on a potent and selective covalent small molecule inhibitor of LSD1 (RO7051750/ORY-1001), a lysine-specific histone demethylase enzyme. LSD1 inhibition causes epigenetic reprogramming of cancer cells by downregulating pro-proliferative neuroendocrine genes, inducing a pro-differentiation, cytostatic effect. Furthermore, the durability of this response is enhanced by covalent drug binding and the resulting dependence on target turnover. To explore the impact of dose and scheduling on the pharmacodynamic profiles underlying treatment efficacy, we developed a predictive model of drug effect, which incorporates experimentally-derived pharmacokinetic and pharmacodynamic data.

This mechanistic mathematical model was calibrated based on in vitro cell-type specific kinetic data, which possesses high dimensionality across time points and dose ranges. The model describes drug-induced target engagement of LSD1, which acts as a transcriptional regulator of neuroendocrine gene, GRP. GRP levels drive reversible epigenetic "switching" between a proliferating (denoted P) and a quiescent (denoted Q) cell population (low GRP: P to Q; high GRP: Q to P), capturing the cytostatic effects of the drug. The model was trained with in vitro measurements of target engagement, GRP mRNA levels, and cell growth inhibition (including drug-free cell growth) across various time points and dosages from a small cell lung cancer cell line (NCI-H510A). Importantly, GRP mRNA and cell viability dynamics were also captured upon drug withdrawal, which enables the modeling of drug effect durability. The resulting model quantitatively describes the relationship between drug dose, target engagement, biomarker and cell growth dynamics in vitro.

To scale to the in vivo setting, we integrated the in vitro-trained pharmacodynamic model with a pharmacokinetic model describing drug distribution and clearance in mouse. This hybrid model accurately predicted in vivo tumor growth dynamics in the NCI-H510A mouse xenograft across a range of doses and schedules. We used the model to explore the target engagement and biomarker profiles underlying treatment efficacy, both in vitro and in vivo.

In conclusion, by training our model on key cell-type specific in vitro kinetic data we were able to accurately predict anti-tumor growth effects in vivo. This quantitative modeling framework highlights the biological profiles underlying efficacy for this latest generation of target-selective epigenetic modifying drugs.

#2797

Synthesis and evaluation of derivatives of selective inhibitor ERGi USU, for ERG-positive prostate cancer cells.

Charles Peter Xavier,1 Ahmed A. Mohamed,1 Nishat Seraj,2 Vineet Kumar,3 Taduru Sreenath,1 Inger L. Rosner,1 Gyorgy Petrovics,1 Meera Srivastava,4 Clifton L. Dalgard,4 Sanjay V. Malhotra,3 Nicole A. LaRonde,2 Albert Dobi,1 Shiv Srivastava1. 1 _Center for Prostate Disease Research, Department of Surgery, Uniformed Services University of the Health Sciences and the Walter Reed National Military Medical Center, Rockville, MD;_ 2 _Department of Chemistry and Biochemistry, University of Maryland, College Park, MD;_ 3 _Department of Radiation Oncology, Stanford University School of Medicine, Stanford, CA;_ 4 _Department of Anatomy, Physiology and Genetics, Uniformed University of Health Sciences, Bethesda, MD_.

Introduction and objectives:

Prostate cancer is the most frequently diagnosed non-skin malignancy and third leading cause of cancer related deaths among men in the United States. Currently, early detected organ confined prostate cancer (CaP) is managed by active surveillance, surgery or radiation therapy. A significant subset of patients (20% to 40%) experience biochemical recurrence after definitive treatment. New AR axis inhibitors (abiraterone and enzalutamide) are leading to significant improvements in treatment of late stages of CaP. However, sustained therapeutic response remains a challenge due to high mutation load at late stages of the disease. Thus, there is an urgent need for evaluating new therapeutic targets for early stages of CaP. ERG oncoprotein and ERG dependent pathways are promising targets for early stage cancer therapy. Previously identified ERGi-USU from our laboratory, demonstrated remarkable exclusivity for inhibiting ERG protein and cell growth of ERG positive tumor cells in both in vitro and in vivo. With comprehensive structure activity relationship (SAR) studies, we generated new derivatives with substituents around the core structure to further enhance efficacy.

Methods: Cell growth inhibition of small molecules was validated with established prostate cancer cell lines and normal prostate/endothelial derived cell panel. Preferential species-specific binding of ERGi-USU to human RIOK2 was confirmed by tryptophan fluorescence quenching assay.

Result: Based on SAR of the parental ERGi-USU, 90 new ERGi-USU derivatives were designed using structure based predictions. Of these, 37 compounds were prioritized for chemical synthesis and biological evaluations using assays that were developed earlier in our laboratory. We completed primary screen of these compounds in cell culture models. Among these one compound (ERGi-USU-6) inhibited the growth of ERG positive prostate cancer cells with remarkable improvement IC50=70 nM. This value is in the range of current FDA approved drugs. The result also confirmed the high selectivity of ERGi-USU-6 for the inhibition of ERG positive cancer cell growth. Further, mechanistic studies revealed ERGi-USU binding and disruption of the atypical RIOK2 kinase basic function of ribosome biogenesis, due to initiation of ribosomal stress, cell cycle arrest and apoptosis in ERG positive VCaP cells. We also demonstarte preferential binding of ERGi-USU to human RIOK2 by tryptophan fluorescence quenching assay.

Conclusion: The ERGi-USU-6, derivative of ERGi-USU showed improved efficacy in selectively inhibiting the growth of ERG positive cancer cells. Since ERG is a prostate cancer causing oncogene that affects approximately one third of CaP patients world-wide, early therapeutic intervention with ERGi-USU derivatives may prevent the development of late stage disease in prostate cancer patients.

#2798

**Folate receptor-targeted cancer chemotherapy with novel 6-substituted thieno[2,3-** d **]pyrimidine compounds with nitrogen and fluorine containing aromatic side chains as de novo purine biosynthesis inhibitors.**

Nian Tong,1 Aleem Gangjee,1 Adrianne W. Povrik,2 Carrie O. Connor,2 Jianjun Hou,2 Larry Matherly2. 1 _Duquesne Univ., Pittsburgh, PA;_ 2 _Wayne State University, Detroit, MI_.

Targeted cancer chemotherapy inhibits the growth of cancer cells by interfering with critical pathways required for tumor progression. Three major transporters are involved in membrane transport of folates into mammalian cells and include the reduced folate carrier (RFC), folate receptors (FRs) α and β, and proton-coupled folate transporter (PCFT). The clinically used agents methotrexate, pemetrexed, raltitrexed and pralatrexate are all transported by the ubiquitously expressed RFC into both tumor cells and normal tissues, and hence are non-selective. The lack of tumor selectivity results in dose-limiting toxicity. We reported 6-substituted thieno[2,3-d]pyrimidine (6TP) compounds with a phenyl side-chain and bridge lengths of 2-8 carbons as potent inhibitors that are selectively transported by FRs over RFC and inhibit proliferation of FR-expressing KB tumor cells with low nanomolar IC50s. The most potent compound (AGF50), a thieno[2,3-d]pyrimidine with a 4-carbon bridge and a phenyl side chain, showed FR selectivity over RFC and potent inhibition of de novo purine biosynthesis at least in part due to inhibition of glycinamide ribonucleotide formyltransferase (GARFTase). In this report, fluorine was strategically placed on the aryl side chain which could induce conformational restriction due to possible intramolecular fluorine-hydrogen bonding interactions. We found that fluorine substitution on the phenyl side-chain, ortho (o-) to L-glutamate (AGF304) improved potency and selectivity for FRα (4.5-fold) and FRβ (20-fold) compared to AGF50, as reflected in inhibition of proliferation of Chinese hamster ovary cells expressing human FRs. The pyridine nitrogen can also form H-bonds with NH of the L- glutamate amide, which could improve the potency and selectivity of 6TPs with pyridine side-chains. Analogs with systematic pyridine side-chain modifications, as well as ortho fluorine substitution on the pyridine ring, were designed, modeled, and synthesized. The 2',5'-pyridine analog AGF132 was ~9-fold more active toward FRα-expressing cells but approximately equipotent toward FRβ. The fluorinated 2',5'-pyridine analog AGF305 was ~5-fold less potent for FRα and was >50-fold more potent for FRβ compared to AGF132. AGF132, AGF304 and AGF305 inhibited growth of KB human tumor cells approximately equivalent to AGF50. Whereas adenosine completely protected cells from growth inhibition by the 6TPs, protection with 5-aminoimidazole-4-carboxamide (AICA) was incomplete, suggesting inhibition at AICA ribonucleotide formyltransferase (AICARFTase), along with GARFTase. Our 6TPs with aryl side-chain modifications are slated for further preclinical studies to identify potential clinical candidates that target the de novo purine biosynthesis pathway.

#2799

New-generation glucose transporter inhibitors targeting non-small cell lung cancer and triple-negative breast cancer.

Pratik Shriwas, Yanrong Qian, Xuan Wang, Dennis Roberts, Stephen Bergmeier, Xiaozhuo Chen. _Ohio University, Athens, OH_.

Cancer is a top killer second only to cardiovascular diseases. Cellular metabolic reprogramming and altered energetics are key features of cancer. Opportunistic uptake of extracellular nutrients has recently been named as an emerging hallmark of cancer metabolism. Glucose is a key nutrient taken in by cancer cells to meet their high energy and biosynthesis needs. Drastic upregulation of glucose uptake and glycolytic activity, a phenomenon called the Warburg effect, is prevalent in most cancers, including non-small cell lung cancer (NSCLC) and triple-negative breast cancer (TNBC). Cancer cells overexpress specific glucose transporters (GLUTs), particularly GLUT1, on their membranes to transport glucose via facilitated diffusion. Considering the addiction of cancer cells for glucose and their sensitivity and vulnerability to changes in glucose supply, GLUTs, especially GLUT1, have been recognized as an attractive anticancer target. We previously reported the identification of first-generation lead GLUT1 inhibitor WZB117. WZB117 treatment of human NSCLC A549 cells resulted in inhibition of glucose uptake, glycolysis and induction of apoptosis and necrosis (1). In addition, WZB-117 reduced tumor growth by more than 70% in an A549 tumor mouse model (1). Through a structure activity relationship (SAR) study of WZB117, a second-generation lead, DRB18, has been identified. DRB-18 is much more stable and 5 to 10 times more potent than WZB117 in inhibiting growth and proliferation in more than 90% of the 60 different cancer cell lines in 9 major cancer types as revealed by NCI-60 screenings. The compound shows particularly higher potency against cell lines with more metastatic potential such as HOP92 (NSCLC) and MDA-MB-231 (TNBC) with IC50 values in high nM range. DRB-18 also demonstrates a favorable liver microsomal metabolic profile and a maximal tolerance dose (MTD). All these indicate that DRB-18 is a potential therapeutic candidate for treating NSCLC and TNBC.

Reference: 1. Liu Y, Cao Y, Zhang W, et al. A small-molecule inhibitor of glucose transporter 1 downregulates glycolysis, induces cell-cycle arrest, and inhibits cancer cell growth in vitro and in vivo. Mol Cancer Ther 2012;11(8):1672-82.

#2800

**Design, synthesis, selection, and evaluation of a potent multi-kinase targeting** N4 **-phenylsubstituted-7** H **-pyrrolo[2,3-** d **]pyrimidin-4-amine derivative with significant anticancer activity in P53 mutant and wild type models of upper gastrointestinal cancers.**

Amruta Samant,1 Tanvi Visal,1 Priya Pancholi,1 Dhvanir Kansara,1 Tanmay Dichwalkar,1 Robert Senones,2 Sonali Kurup,2 Vikas Sehdev3. 1 _Arnold & Marie Schwartz College of Pharmacy and Health Sciences, Long Island University, Brooklyn, NY; _2 _College of Pharmacy, Roosevelt University, Schaumburg, IL;_ 3 _Presbyterian College School of Pharmacy, Presbyterian College, Clinton, SC_.

Introduction: Upper Gastrointestinal Cancers (UGCs) exhibit resistance to conventional chemotherapy due to variable P53 status and constitutive overactivity of EGFR, ERBB2/HER-2, Aurora kinases, and JAK2 oncogenes. UGC is a leading cause of cancer related deaths worldwide and development of multi-kinase targeting inhibitors could pave the way for improved chemotherapy and better therapeutic outcomes. In this study, we investigated a series of N4-phenylsubstituted-7H-pyrrolo[2,3-d]pyrimidin-4-amines and selected a promising novel investigational derivative (RS-41) that targets multiple kinases and exhibits significant anticancer activity in P53 mutant and wild type models of UGC. Methods: Target kinase inhibition and selectivity screening (T-KISS) and MTT-cell viability assays were performed to determine potency and selectivity of kinase inhibition and anticancer efficacy of primary derivatives, respectively. RS-41 was identified as the most promising agent following analysis via T-KISS and MTT-cell viability assay. Next, clonogenic cell survival assay, cell cycle analyses, and western blot analyses were done to evaluate the effect of RS-41 treatment on cell survival, cell cycle progression, and expression of apoptotic markers in P53 mutant (FLO-1) and P53 wild type (AGS) UGC cells, respectively. The initial T-KISS and cell-viability data aided in the design of second-generation derivatives of RS-41. Results: The initial T-KISS and cell viability data aided in identification of structural moieties that were modified to make the second generation of derivatives with improved multi-kinase inhibition. Evaluation with T-KISS showed that RS-41 derivative selectively inhibits AURKA (IC50-0.96±0.03µM), JAK2 (IC50-1.21±0.17µM), and EGFR (IC50-5.92±0.75µM) kinases, respectively. The cell viability and clonogenic cell survival data indicate that treatment with RS-41 mediates significant (P≤0.05) inhibition of FLO-1 and AGS UGC cell viability and survival. The cell cycle data and western blotting data also exhibited a marked increase (P≤0.05) in the percentage of sub-G1-phase (cell death) and expression of various apoptotic markers in FLO-1 and AGS cells following treatment with RS-41 for 24 and 72 hrs. Conclusions: Our T-KISS data indicate that multi-targeted inhibition of EGFR, AURKA and JAK2 can be achieved with the pyrrolo[2,3-d]pyrimidine scaffold. In addition, we demonstrate that inhibition of various oncogenic kinases with RS-41 is an effective therapeutic strategy for inducing apoptosis in both P53 mutant and wild type UGC cells.

#2801

Novel selenodiazoles as antitumor and radical scavenging agents.

Ana Carolina Ruberte,1 Daniel Plano,1 Ignacio Encio,2 Arun K. Sharma,3 Carmen Sanmartin1. 1 _University of Navarra, Pamplona, Spain;_ 2 _Universidad Pública de Navarra, Pamplona, Spain;_ 3 _Penn State Univ. College of Medicine, Hershey, PA_.

Heterocyclic compounds with selenium atom in endo position, such as ebselen (EBS), have achieved very promising activity against cancer and hold an excellent pharmacological profile. In fact, EBS is currently under Phase II clinical trial for the evaluation of its therapeutic safety and efficacy. The purpose of this study was to make structural modifications over this scaffold to improve its anticancer and antioxidant characteristics.

Twenty-eight novel benzo[c][1,2,5]selenodiazole-5-carboxamide derivatives were designed and synthesized. Cytotoxicity of these structures, using the MTT assay, was tested in vitro against a panel of five human cancer cell lines, including prostate (PC-3), colon (HT-29), leukemia (CCRF-CEM), lung (HTB-54) and breast (MCF-7). The selectivity of all the compounds was further examined in two non-malignant cell lines derived from breast (184B5) and lung (BEAS-2B). For MCF-7 cells, the apoptotic status and cell cycle analysis of the cells were based on the TUNEL technique. Likewise, their radical scavenging activity was determined using the DPPH assay.

Interestingly, three compounds exhibited higher cytotoxic activity than EBS in solid tumors, along with higher selectivity indexes. The cytotoxic activity of lead compound 8 was remarkable in MCF-7 cells. The antioxidant capacity of four compounds was greater than EBS.

The toxicity of derivative 8 in the non-tumoral cell line 184B5 was low (lethal dose (LD50) > 100 µM) and it exhibited promising antiproliferative values in MCF-7 cells with a growth inhibition of 50% (GI50) of 3.7 µM. The radical scavenging capacity of four compounds was greater than EBS.

Given the low toxicity and moderate antioxidant activity, these compounds can serve as an excellent scaffolds to achieve new and potent antioxidant compounds useful for several diseases such as cancer, neurodegenerative and heart diseases.

#2802

Discovery of the first selective and cell-active eIF4A3 inhibitors.

Masahiro Ito,1 Misa Iwatani-Yoshihara,1 Toshio Tanaka,1 Douglas R. Cary,1 Yusuke Kamada,1 Yoshihiro Ishibashi,1 Hideyuki Oki,1 Satoshi Sogabe,1 Shoichi Nakao,1 Daisuke Morishita,1 Takashi Ito,1 Hiromichi Kimura,1 Tomohiro Kawamoto,1 Samuel Aparicio,2 Atsushi Nakanishi,1 Yasuhiro Imaeda1. 1 _Takeda pharmaceutical company, Fujisawa, Kanagawa, Japan;_ 2 _BC Cancer Agency, Vancouver, British Columbia, Canada_.

Background: Eukaryotic initiation factor 4A3 (eIF4A3) is a member of the Asp-Glu-Ala-Asp (DEAD) box RNA helicase family. There are three subtypes of eIF4A, eIF4A1, 2, and 3. eIF4A1 and eIF4A2 are required for translation initiation as their name suggest, however eIF4A3 is functionally distinct and one of the core components of the exon junction complex (EJC). The EJC is known to be involved in a variety of RNA metabolic processes typified by nonsense-mediated RNA decay (NMD), which is the surveillance mechanism that recognizes mRNAs containing premature termination codons to prevent the accumulation of truncated proteins. It is known that eIF4A3 is an ATP-dependent RNA clamp that can serve as a nucleation center to recruit other EJC components, and siRNA-mediated knockdown of eIF4A3 leads to a defect in NMD. In order to investigate the functions of eIF4A3 further and evaluate the therapeutic potential, we conducted a search for molecular probes of eIF4A3.

Methods: Using an RNA dependent ATPase assay as a guide, intensive structure activity relationship (SAR) study and chemical optimization of high-throughput screening hit were conducted. Thereafter, NMD inhibitory activity, selectivity, helicase inhibitory activity, and physicochemical properties of optimized compounds were confirmed. Additionally, direct binding of inhibitors to eIF4A3 and their binding mode were analyzed using biophysical methods.

Results: Optimized compounds showed high selectivity for eIF4A3 and exhibited significant NMD inhibitory activity in HEK293T cells. The drastic difference in eIF4A3 inhibitory activity between eutomers and distomer revealed the importance of stereochemistry at the 3-position of the piperazine ring for eIF4A3 inhibition. The lack of significant NMD inhibition by distomers suggests that NMD inhibition of eutomers is mediated by eIF4A3 inhibition. A surface plasmon resonance (SPR) biosensing assay and hydrogen/deuterium exchange mass spectrometry (HDX MS) provided strong evidence for the direct binding of the inhibitors to eIF4A3 and implications for their binding site.

Conclusion: We discovered the first selective eIF4A3 inhibitors exhibiting cellular NMD inhibitory activity. These novel inhibitors not only represent novel molecular probes for investigation of RNA biology but also could be promising lead compounds for drug discovery research. References: [1] Ito, M.; Tanaka, T.; Cary, D. R.; Iwatani-Y, M.; Kamada, Y.; Kawamoto, T.; Aparicio, S.; Nakanishi, A.; Imaeda, Y. Discovery of novel 1,4-diacylpiperazines as selective and cell-active eIF4A3 inhibitors. J. Med. Chem. 2017, 60, 3335-3351. [2] Iwatani-Y, M.; Ito, M.; Ishibashi, Y.; Oki, H.; Tanaka, T.; Morishita, D.; Ito, T.; Kimura, H.; Imaeda, Y.; Aparicio, S.; Nakanishi, A.; Kawamoto, T., Discovery and characterization of a eukaryotic initiation factor 4A-3-selective inhibitor that suppresses nonsense-mediated mRNA decay. ACS Chem. Biol. 2017, 12, 1760-1768.

#2803

Novel auristatins with high activity on efflux-positive models and demonstrable bystander activity.

Philip N. Moquist,1 Tim D. Bovee,1 Andrew B. Waight,1 Sarah Owen,2 Jamie A. Mitchell,1 Margo Zaval,1 Marsha Quick,1 Sharsti Sandall,1 Kim K. Emmerton,1 Nicole Blesie,1 Robert P. Lyon,1 Peter Senter,1 Svetlana Doronina1. 1 _Seattle Genetics, Bothell, WA;_ 2 _University of Michigan, Ann Arbor, MI_.

Auristatins are a class of clinically validated antimitotic agents utilized as payloads in antibody-drug conjugates (ADCs). Auristatins display many of the desirable properties necessary for ADC cytotoxins, such as low nanomolar potency, cell permeability, and activity across multidrug-resistant (MDR+) cell lines. Herein, we report the development of novel auristatins, which have a unique combination of these favorable properties. The hydrophilic monomethyl auristatin F (MMAF) was chosen as the parent structure, and a medicinal chemistry campaign was undertaken to functionalize various sites of the auristatin with hydrophobic moieties in an effort to improve membrane permeability. The structure-activity relationships (SAR) of the new auristatins demonstrated clear trends correlating hydrophobicity, structure, and polarity with permeability and in vitro cytotoxicity. The highest-performing molecules showed a preference for hydrophobic functionalization at the N-terminal dolavaline. The payloads were linked to the C-terminal position of the auristatin with a lysosomally cleavable maleimido-dipeptide linker. Examination of the ADCs revealed low ng/mL activity in CD30+ and CD19+ cell lines in vitro. Anti-CD70 ADCs demonstrated high in vivo efficacy in a 786-O xenograft and complete remissions (CRs) in the efflux-positive renal cell carcinoma model. Anti-CD30 ADCs were dosed in a CD30+ and CD30- admixed Karpas/Karpas-35R xenograft model to demonstrate proof-of-concept in vivo bystander activity. In summary, these novel auristatins showed potential across multiple indications as ADC payloads.

#2804

Identifying an optimal site for linker conjugation of the taccalonolides, novel microtubule stabilizing drugs.

April L. Risinger,1 Lin Du,2 Antonius Ola,1 Robert H. Cichewicz,2 Susan L. Mooberry1. 1 _UT Health San Antonio, San Antonio, TX;_ 2 _University of Oklahoma, Norman, OK_.

The taccalonolides are a structurally distinct class of microtubule stabilizers purified from the roots and rhizomes of plants of the genus Tacca. The covalent binding of taccalonolides bearing a C-22,23 epoxide to β-tubulin generates a distinct profile of microtubule stabilization that results in a high level of cellular persistence and the ability to circumvent multiple mechanisms of clinically relevant drug resistance, including expression of P-glycoprotein. The biological effects of 121 structurally distinct taccalonolides, including 23 isolated natural products and 98 semi-synthetic derivatives have been evaluated. These compounds range in potency from 0.6 nM to over 50 µM, allowing for detailed structure activity relationship studies.

Some of the most potent taccalonolide derivatives include semi-synthetic modifications at C-6, C-7, or C-15 in combination with C-22,23 epoxidation. While these compounds did not have an acceptable therapeutic window for in vivo antitumor activity by systemic administration, they demonstrated highly effective and exquisitely persistent antitumor activity in a drug-resistant tumor model when delivered directly into the tumor. Additional studies were undertaken to see whether large chemical moieties could be added at these sites to facilitate targeted delivery of these compounds. In contrast to C-7 and C-15 derivatives, which are cleaved, C-6 modifications are stable and retain microtubule stabilizing and cytotoxic activities. In particular, fluorescent C-6 taccalonolide conjugates allowed, for the first time, direct visualization of these compounds in live cells, which co-localized with bundled microtubules. Further probing of the functionality at taccalonolide C-6 will allow for more detailed mechanistic studies of drug transport and distribution as well as optimization of a linker that could be used for antibody-based drug delivery.

#2805

Combretastatins as highly cytotoxic natural compounds.

Petr Dzubak,1 Jiři Rehulka,1 Sona Gurska,1 Uwe Riener,2 Marian Hajduch1. 1 _Palacky University in Olomouc, IMTM, Olomouc, Czech Republic;_ 2 _University of Vienna, Viena, Austria_.

Combretastatins originally isolated from the South African willow tree Combretum cafrum are highly cytotoxic natural products, but their use is limited because of the cis/trans isomerization of the cis-stilbene moiety under physiological conditions. Herein we report the preparation of cis-restrained carbocyclic analogs of CA-4. The compounds, which differ by the size and hybridization of the carbocyclic ring have been evaluated for cytotoxic activity in vitro against a panel of human cancer and two non-tumor fibroblast cell lines and for their ability to inhibit tubulin polymerization. Biological data, supported by molecular docking studies, identified a cyclobutenyl derivative of the natural product as a promising drug candidate with selective nanomolar cytotoxic activity towards cancer cell lines, showing high potency against multidrug-resistant cells and inhibiting tubulin polymerization. This work was supported by Ministry of Health of the Czech Republic (15-31984A), internal grant of Palacky University (IGA_LF_2017_026) and by the Czech Ministry of Education, Youth and Sports LO1304, CZ-OPENSCREEN, (LM2015063) and EATRIS (LM2015064).

#2806

Inhibition of melanoma development by Isatin analogs.

Dhimant H. Desai, Raghvendra Gowda, Krishne Gowda, Saketh S. Dinavahi, Gavin P. Robertson, Shantu G. Amin. _Penn State Univ. College of Medicine, Hershey, PA_.

Despite major breakthroughs in melanoma drug development, there remains a need for new drugs and novel approaches. Of the three forms of skin cancer, malignant melanoma has the most significant impact on human health carrying the highest risk of mortality from metastasis. An estimated 87,110 new cases of invasive melanoma will be diagnosed and 9,730 people will die of melanoma in the USA in 2017. Melanoma accounts for less than one percent of skin cancer cases, but the vast majority of skin cancer deaths. The vast majority of melanomas are caused by the sun. In fact, one UK study found that about 86 percent of melanomas can be attributed to exposure to ultraviolet (UV) radiation from the sun. The estimated 5-year survival rate for patients whose melanoma is detected early is about 98 percent in the USA, whereas, the survival rate falls to 62 percent when the disease reaches the lymph nodes, and 18 percent when the disease metastasizes to distant organs. Thus, novel effective therapies are urgently needed to treat this disease.

The Isatin (1H-indole-2,3-dione) is found as an endogenous molecule in humans and other mammals and its analogs display diverse types of biological activities including anti-cancer activities. Earlier our group synthesized a novel series of 5,7-dibromoisatin analogs. Various melanoma cells were treated with several Isatin derivatives having functional groups like selenocyanate, thiocyanates, thiourea, and selenourea for 72 hours and the cell survival was estimated by MTS assay. Agents were treated with melanoma cell lines UACC 903, 1205 Lu or normal fibroblast cell line FF2441. Treatment with KS99, a thiocyanate analog and KS101, a selenourea analog of 5,7-dibromoisatin effectively killed melanoma cells after 72 h treatment. Overall, IC50 values of Isatin derivatives on melanoma cell lines were 3.0-5.7μM and 2.1-5.7μM, respectively. IC50 value on normal fibroblast cells with these Isatin derivatives was 5.4-20.7μM. KS101 was toxic to the xenograft mice at 1mg/kg body weight, animals developed vein inflammatic symptoms after 20 days of treatment. However, liposomal formulation of KS101 was safe up to 30mg/kg body weight in 1205 xenografted nude mice when treated alternate days by tail vein injection. The melanoma tumor burden was reduced by 47%. We will discuss structural activity relationship (SAR) of Isatin derivatives, and its in vitro and in vivo inhibitory effects against melanoma.

#2807

Cytotoxic activity of novel organotin compounds against different cancer cell lines.

Paulina Cherek,1 Michaela Balogova,1 Krishna Damodaran,2 Helga M. Ögmundsdottir1. 1 _Univ. of Iceland Faculty of Medicine, Reykjavik, Iceland;_ 2 _Univ. of Iceland Faculty of Physical Sciences, Reykjavik, Iceland_.

Cisplatin has been a successful drug in oncology for decades. Its use is however limited by toxicity and development of resistance. Efforts for improvement by screening a large number of different platinum compounds have only yielded to new drugs to the clinic, oxaloplatin and carboplatin. An alternative approach is therefore to explore the potential of other metal-containing compounds. The toxic properties of organic tin compounds have been exploited in industry for a long time. More recently such compounds have been studied for potential therapeutic cytotoxic effects yielding promising results. Here we describe the first screening of two novel organotin compounds, based on the same basic structure, containing bromide (SNO7) or chloride (SNO8). The compounds are being screened against the following cancer cell lines: T47-D (breast), A549 (lung), OVCAR-3 (ovarian), Aspc-1 (pancreatic). IC50 was determined from survival analysis by Crystal violet, and induction of apoptosis by Annexin V staining, monitored by IncuCyte. Cisplatin was used as reference. IC50 values (µg/ml) for T47-D and Aspc-1 were, respectively: 44.5 and 13.8 (SNO7), 13.4 and 26.3 (SNO8) and 34.9 and 34.5 (Cisplatin). SNO7 induced apoptosis starting after 24 hours in OVCAR-3 and A549 at 10 µg/ml and also T47-D at 25 µg/ml; SNO8 in T47-D at 25 µg/ml (OVCAR-3 and A549 not tested) and Cisplatin in OVCAR-3 at 10 µg/ml (A549 not tested). These early results indicate differences in sensitivities between compounds and cell lines implying some specificity in mode of action and suggest that tin-containing compounds could be active against cancer cells that are resistant to cisplatin.

#2808

Simultaneous inhibition of SHP2 phosphatase at two allosteric sites.

Michelle Fodor,1 Edmund Price,1 Ping Wang,1 Hengyu Lu,1 Andreea Argintaru,1 Zhouliang Chen,1 Meir Glick,1 Huai-Xiang Hao,1 Mitsunori Kato,1 Robert Koenig,1 Jonathan R. LaRochelle,2 Gang Liu,1 Eric McNeill,1 Dyuti Majumdar,1 Gisele Nishiguchi,1 Lawrence Perez,1 Greg Paris,1 Christopher Quinn,1 Timothy Ramsey,1 Martin Sendzik,1 Michael Shultz,1 Sarah Williams,1 Travis Stams,1 Stephen C. Blacklow,2 Matthew J. LaMarche,1 Michael G. Acker1. 1 _Novartis Institutes for BioMedical Research, Cambridge, MA;_ 2 _Harvard Medical School, Boston, MA_.

SHP2 is a cytoplasmic non-receptor tyrosine phosphatase involved in the propagation of extracellular signaling through receptor tyrosine kinases. Aberrant SHP2 activity has been identified as a driver in multiple cancers and SHP2 has also been implicated in the PD-1/PD-L1-mediated exhaustion of effector T-cells, leading to immune system evasion of tumors. Recently, we reported the identification of SHP099, an allosteric inhibitor of SHP2 with in in vivo efficacy against multiple RTK-driven tumor xenograft models. Here we report the use of alternate screening paradigms to identify a novel allosteric inhibitor which binds to a previously uncharacterized pocket on SHP2. Like SHP099, the second allosteric inhibitor stabilizes a closed conformation of SHP2, which blocks access to the phosphatase active site. Structure based drug design led to improvements in potency, and combination studies in biochemical, biophysical and cellular assays confirm dual occupation of SHP099 and the second allosteric molecule, resulting in improved potency. This work highlights a rare opportunity for dual occupation of inhibitors for a single target and provides additional tools for the exploration of SHP2 biology.

#2809

Novel 2,5,8-trihydro-6,7-disubstituted-4-(3'-alkylsulfonamidoanilino) quinazoline derivatives as RAF inhibitor.

Shih Chieh Yen, Shao-Zheng Peng, Hung-Jyun Huang, Ju-Ying Yang, Yen-Hsi Liu, Sian-Yi Ciou, Mann-Yan Kuo, Chu-Bin Liao. _DEVELOPMENT CENTER FOR BIOTECHNOLOGY, NEW TAIPEI CITY, Taiwan_.

The oncogenic mutations of BRAF which occur most frequently in V600E, leading to constitutive activation of the MAPK signaling pathway, are common in a variety of human cancers. Recently, drugs targeting RAF kinase have been approved as an effective treatment for human malignancies that rely on this target for their growth. Thus, suppressing RAF kinase activity is a clinically meaningful approach to treat cancer patients harboring RAF driven oncogene. In this study, a series of 2,5,8-trihydro-6,7-disubstituted-4-(3'-alkylsulfonamidoanilino)quinazoline was synthesized and evaluated for their RAF kinases inhibition activity. Several potent compounds displayed double-digit nanomolar IC50 values for RAF kinases including CRAF, BRAF, and BRAFV600E. Preliminary profiling of one of the most active compounds in a panel of protein kinases revealed its selectivity for RAF kinases. In cells, this compound exhibited selective cytotoxicity against cancer cells harboring BRAF V600E mutation and dose-dependent inhibition of phosphorylation of RAF downstream effectors MEK and ERK. Moreover, the compound was oral active in mice bearing A375 BRAFV600E‐mutant melanoma xenograft. Therefore, the novel quinazoline derivative proved to be active as RAF inhibitor.

#2810

Computationally predicted sensitivity of clinical cohorts identifies drug relationships and biomarkers associated with response to PCM-075, a PLK1 selective inhibitor.

Penn Whitley,1 Peter J. Croucher,1 Barbara Valsasina,2 Dario Ballinari,2 Italo Beria,2 Jeffrey N. Miner,1 Mark G. Erlander1. 1 _Trovagene, San Diego, CA;_ 2 _Nerviano Medical Sciences, Nerviano, Italy_.

Polo-like kinase 1 (PLK1) is a serine-threonine kinase which regulates various cellular processes, including mitosis, DNA replication, and the stress response, and is over-expressed in many malignancies. PCM-075, a PLK1 selective inhibitor, is currently in a phase1b/2 clinical trial in AML in combination therapy with low dose cytarabine (LDAC) (NCT03303339). To identify potential drug relationships and biomarkers associated with response to PCM-075, a two-step computational approach was conducted. Using a modification of the 'imputed drug-wide association study' (IDWAS) method(1), we first performed ridge regression model training on IC50 values and gene expression signatures for PCM-075 and 138 other drugs from the Genomics of Drug Sensitivity in Cancer (GDSC) database . In the second step, we applied these models to clinical patient datasets (The Cancer Genome Atlas (TCGA) populations containing 32 Cancer types and 9,968 total patients), generating a predicted sensitivity value to all drugs for each patient.

Hierarchical clustering of imputed sensitivities across all patients showed that the PCM-075 activity profile is most closely related to cytarabine, etoposide and cisplatin, all of which are compounds that impact DNA replication and lead to apoptosis. Correlation of predicted response values was significant both within and between cancer types, with the acute myeloid leukemia (AML) cohort showing highest increased sensitivity to PCM-075, cytarabine and etoposide relative to the other 31 cancer types examined. Interestingly, other PLK1 inhibitors present in the GDSC (BI-2536 and GW843682X) do not closely cluster with PCM-075. Association of imputed TCGA sensitivity values with somatic variant data shows a high degree of concordance between predicted biomarkers, including TP53 mutation status, further suggesting a functional relationship.

Given that this computational analysis suggests that similar pathways are affected by both cytarabine and PCM-075, we tested this experimentally by assessing the level of interaction between these two compounds within an AML cell line (HL-60). The combination resulted in statistically significant synergy (BRAID, k~2.7(1.31-4.25)) for anti-proliferative activity. The mechanistic cause of synergy may be related to DNA damage caused by cytarabine which is known to inhibit and degrade the PLK1 protein.

In conclusion, we have identified a pathway signature that clusters PCM-075 with a subset of current oncology therapeutics. Synergy between these compounds in combination suggests the potential for use in treating AML and potentially other cancer types.

1.Genome Res. 27: 1743-1751 (2017). 

### Modulation of DNA Damage and Repair

#2811

High IFN/STAT-related gene expression is associated with sensitivity to PARP inhibitors of triple negative breast cancer.

Stefano Cairo, Olivier Déas, Truong-an Tran, Jean-Gabriel Judde. _XenTech, Evry, France_.

Triple negative breast cancer (TNBC) comprises 15-20% of all breast cancers, and carries increased risk of distant metastasis, early relapse and short post-recurrence survival. TNBC shares clinical and pathological features with hereditary BRCA1/2-related breast cancers. In TNBCs, besides mutation-associated BRCA inactivation occurring by loss of heterozygosity (LOH) in about 15% of the cases, dysregulation in the homologous-recombination (HR)-dependent DNA-repair pathway has been attributed to a number of mechanisms. PARP1 and PARP2, two important regulators of the DNA base-excision-repair pathway, have emerged as therapeutic targets for TNBC. Indeed, a subset of TNBC patients is responsive to PARP inhibitors (PARPi), a class of novel therapeutic agents that inhibits the function of several PARP family members. Pathogenic BRCA1/2 mutations, and other genomic markers have been proposed as molecular markers to identify tumors with homologous recombination deficiency (HRD) that are more likely to be sensitive to PARPi. However, these tests lack specificity as less than half of TNBC patients harboring BRCA-mutated or HRD-positive tumors are responsive to PARP inhibitors, and a number of TNBC patients without BRCA1/2 mutations are responsive to PARP inhibitors.

In the present study, we investigated the antitumor activity of PARPi in a panel of 28 TNBC patient-derived xenograft PDX models. Responses were correlated with BRCA1/2, with HRD status and with other HRD markers, and GSEA analysis was run to compare gene expression profile of PARP inhibitor-sensitive vs resistant TNBC PDXs.

Among the gene signatures differentially expressed we identified the IFN/STAT signature as upregulated in tumors sensitive to PARPi. We previously described that TNBC PDXs that respond to a genotoxic stress induce activation of the IFN/STAT pathway. Therefore we hypothesized that a significant fraction of tumors scoring HRD positive (by BRCA mutation or genomic scarring assay) may have reverted back to a phenotype of HR competency, making them refractory to PARP inhibition. In this scenario, IFN/STAT-related gene expression might be a feature of HRD-positive tumors in which HR deficiency has not been compensated and is still effective, resulting in PARP inhibitor sensitivity. Inclusion of this parameter strongly ameliorated the predictive power of the HRD assay, with decreased rate of false-positives (28.6% for IFN/STAT signature + HRD versus 52.9% for HRD alone) and overall misclassification reduced to only 5/28 PDXs (17.6% for IFN/STAT signature + HRD versus 32.1% for HRD alone).

This study suggests that high IFN/STAT-related gene expression can be a strong marker of persistent tumor instability and improve the performance of HRD-based PARPi efficacy predictive scores.

#2812

Status of TGFbeta signaling determines PARP inhibitor sensitivity in head and neck cancer.

Qi Liu,1 Lin Ma,1 Luis Palomero,2 Miquel Àngel Pujana,2 Trevor Jones,1 Patrick Ha,1 John Murnane,1 Mary Helen Barcellos-Hoff1. 1 _UCSF, San Francisco, CA;_ 2 _Catalan Institute of Oncology, Bellvitge Institute for Biomedical Research (IDIBELL), Spain_.

Although human papilloma virus (HPV) status is the most informative prognostic marker in head and neck squamous cell carcinoma (HNSCC), the mechanisms that provide the survival advantage are poorly understood. Here we demonstrate that defects in transforming growth factor β (TGFβ) signaling confer a profound vulnerability in DNA repair for HPV+ HNSCC. We interrogated HNSCC profiled by The Cancer Genome Atlas (TCGA) with a 77 gene chronic TGFβ signature, defined as those genes that were significantly and reciprocally altered in MCF10A cultured for 7 days with TGFβ or a small molecule inhibitor of TGFβ type 1 receptor kinase (TBRIi). The 50 TGFβ-upregulated genes and 27 TGFβ-downregulated genes were negatively correlated (P<0.0001; R2=-0.68) in single-sample gene set enrichment analysis confirmed that the signature is informative in HNSCC. Unsupervised clustering of 294 HNSCC TCGA patient specimens using the TGFβ signature showed that HPV+ cancers were clustered by loss of TGFβ regulation. Patients whose cancers exhibit this profile had significantly better overall survival (P=0.002), independent of HPV status. We previously found that TGFβ signaling suppresses miR-182, which is a reported inhibitor of homologous recombination (HR). HPV+ cell lines increased expression of miR-182 compared to HPV- cell lines. Thus we evaluated radiation induced RAD51 foci formation, a biomarker of HR, in viable explants of patient-derived xenografts (PDX) treated with or without TBRIi. HPV+ PDX showed significantly fewer RAD51 foci than HPV- samples (P<0.005). HPV- PDX specimens treated with TBRIi also reduced RAD51 foci formation. We then established an HPV- cell line containing reporters to measure the frequency of DNA double strand break (DSB) repair with HR or alternative end-joining (Alt-EJ) following DSB induction with I-SceI endonuclease. TBRIi decreased HR events. In contrast, Alt-EJ was significantly increased (P<0.05). Poly (ADP-ribose) polymerase 1 (PARP1) is required for DSB repair with Alt-EJ. TCGA data shows dramatically increased expression of PARP1 in HPV+ HNSCC (P<0.0001). Hence, we speculated that loss of TGFβ signaling increase reliance on PARP1 mediated Alt-EJ to cope with DSB. Consistent with this, HPV+ cell lines were more sensitive to olaparib; while olaparib in combination with TGFβ inhibitor significantly increased cell death in HPV- cell lines by flow cytometry analysis of annexin V positive cells, but not in HPV+ cell lines. Notably, antagonizing miR-182 in HPV+ cells significantly reduced olaparib sensitivity (P<0.05). Together these data indicate HPV suppression of TGFβ signaling in turn shifts DNA repair from error-free HR to hazardous Alt-EJ through upregulated miR-182. Importantly, TGFβ inhibitors can recapitulate this DNA repair deficiency in hard to treat HPV- HNSCC.

#2813

**Efficacy and pharmacokinetics of niraparib in** BRCA **-mutant and wild-type intracranial triple negative breast cancer murine models.**

Maria Sambade,1 Amanda Van Swearingen,1 Kaiming Sun,2 Jing Wang,2 Kevin Mikule,2 Carey K. Anders1. 1 _Univ. of North Carolina at Chapel Hill; Lineberger Comprehensive Cancer Center, Chapel Hill, NC;_ 2 _Tesaro, Inc., Waltham, MA_.

Introduction: Brain metastases arising from triple negative breast cancer (TNBC BM) are common and typically occur in the setting of progressive extracranial metastases. No targeted systemic therapy is approved to control both intra- and extracranial TNBC. Poly (ADP-ribose) polymerase (PARP) inhibition has emerged as a promising anti-cancer agent to treat BRCA-associated TNBC, as well as TNBC with homologous recombination deficiency (HRD). Niraparib is a brain permeable and selective PARP-1/-2 inhibitor that has shown activity across many solid tumor types, including TNBC. We sought to define the efficacy and intracranial exposure of niraparib in several BRCA-mutant (mut) and wild-type (WT) intracranial mouse models of TNBC.

Experimental Procedures: Niraparib (50mg/kg by oral gavage daily) was tested in 2 BRCAmut (SUM149, MDA-MB-436) and one BRCA-WT (MDA-MB-231BR) intracranial models of TNBC. Efficacy was assessed by serial in vivo bioluminescence and overall survival (OS). Whole cell lysates of intracranial tumors were used to determine drug penetrance and PARP inhibition. Niraparib concentrations were measured after protein precipitation and HPLC with positive ion electrospray mass spectroscopy (Q1/Q3:321.09/304.10 Da). PARP inhibition was measured using a PAR ELISA (HT PARP in vivo Pharmacodynamic Assay II, #4520-096-K, Trevigen).

Results: Treatment with niraparib compared with vehicle control resulted in improvement in OS in the MDA-MB-436 model (undefined at day 80 v. 50 days, p<0.001), but not the SUM149 (31 v. 32.5 days, p=0.3) or MDA-MB- 231BR (22 v. 23 days, p=0.47) models. Concurrent reductions in intracranial tumor burden by bioluminescence was observed for the MDA-MB-436 model, but not the SUM149 or 231BR models. Intracranial tumor PK has been evaluated in the SUM149 and 231BR models illustrating niraparib brain penetration (553ng/g and 658ng/g, respectively). PD studies illustrate intracranial tumor reductions in PAR in response to niraparib (94% for SUM149, p<0.05; 25% for MDA-MB-436, p=0.4; 24% for MDA-MB-231BR, p=0.7).

Conclusions: Niraparib illustrates intracranial exposure and target inhibition in several models of TNBC BM. Efficacy was variable across BRCAmut models, which could be explained by the known inherent resistance of the SUM149 cell line to PARP inhibition via RAD51 (Liu et al, CCR 2017). Thus, studies evaluating mechanisms of response and resistance of TNBC, beyond BRCA1 and RAD51, to niraparib are warranted.

#2814

Antitumor effects of WEE1 inhibitor, AZD1775 alone or in combination with PARP inhibitor in triple negative breast cancer cells.

Dong Hyeon Ha,1 Arrum Min,1 Seongyeong Kim,1 So Hyeon Kim,1 Hyemin Jang,1 Yu Jin Kim,1 Daeun Jung,1 YoonJung Park,1 Koung Jin Suh,2 Kyung-Hun Lee,2 Tae-Yong Kiim Kim,2 Seock-Ah Im2. 1 _Cancer Research Institute, Seoul National University College of Medicine, Seoul, Republic of Korea;_ 2 _Biomedical Research Institute, Seoul National University Hospital, Seoul, Republic of Korea_.

Background: Wee1 is a critical component of the G2/M cell cycle checkpoint control and mediates cell cycle arrest by regulating the phosphorylation of CDC2. Inhibition of Wee1 has been reported to enhance the cytotoxic effect of DNA damaging agents in different types of carcinomas. Especially, recent reports have shown that DNA replication stress and double strand breaks are induced by the activation of CDK1 following WEE1 inhibitor treatment. We investigated the effects and underlying mechanisms of WEE1 inhibitor, AZD1775, in Triple Negative Breast Cancer (TNBC). In addition, we examined whether AZD1775 would enhance the anti-tumor effect of PARP inhibitor in TNBC cells by modulating DNA repair activity. Methods: MTT assay was performed to investigate the antitumor effect of WEE1 inhibitor, AZD1775 alone or with PARP inhibitor in breast cancer cell lines. The regulation of DNA damage response activity by AZD1775 or/and PARP inhibitor was accessed by the comet and western blotting. The status of the S phase was evaluated by EdU-BrdU dual pulse labeling, and the drug effect was also confirmed in vivo by mouse xenograft model of TNBC. Results: AZD1775 showed heterogeneous antitumor effects among TNBC cells regardless of TP53 mutation statues. WEE1 inhibition led to accelerated S phase progression and mitotic entry in the sensitive MDA-MB-231 cells. In addition, increased DNA damage and caspase-3 dependent apoptosis were observed in sensitive cells following AZD1775 treatment. Furthermore, AZD1775 enhanced cellular sensitivity to PARP inhibitor through increase apoptosis and DNA damage accumulation. In a MDA-MB-231 xenograft model, AZD1775 impedes tumor growth as well. Conclusions: In the TNBC, the WEE1 inhibitor has a heterogeneous cytotoxic effect regardless of the p53 mutation status. In MDA-MB-231 cell, which is a sensitive TNBC cells to WEE1 inhibitor AZD1775, the progression of the cell cycle was accelerated and the aberrant DNA content was increased. This resulted in DNA damage, which led to cell death, and this effect could be applied in vivo. In addition, we investigated that WEE1 inhibition can increase PARP inhibitor sensitivity in TNBC cells via increasing accumulation of DNA damage. Our results provide a rationale for the future clinical trials of PARP inhibitor combined with WEE1 inhibition in triple negative breast cancer.

#2815

Ganoderma lucidum **chemosensitizes triple negative breast cancer to carboplatin mediated by DNA damage response.**

Ivette J. Suarez-Arroyo, Michelle M. Martinez-Montemayor. _Universidad Central del Caribe School of Medicine, Bayamon, PR_.

Management of triple negative breast cancer (TNBC) is challenging because of a lack of targeted therapy, its aggressive behavior and poor prognosis. Interest in using platinum agents, such as carboplatin as a line of treatment emerged from data suggesting frequent DNA repair defects in TNBC. Carboplatin resistance may occur due to multiple factors including increased activation of DNA repair mechanisms. Studies show that Ganoderma lucidum extract (GLE) sensitizes cancer cells to different anti-cancer therapies. Thus, we aim to investigate the effects of GLE in combination with carboplatin in the DDR signaling and in tumorigenesis. To study whether GLE sensitizes TNBC cells to carboplatin therapy, we treated SUM-149 and MDA-MB-231 cells with several concentrations of carboplatin, GLE, or both treatments simultaneously for 72h. Our results demonstrate that when carboplatin and GLE are used in combination the IC50s decreased in both breast cancer cell lines in comparison with treatments alone. Immunobloting assays revealed that GLE and/or the combination decrease the expression of 53BP1, p-CHK2, CHK2, p-CHK1, CHK1, p-ATR, p-ATM and p-BRCA1 in SUM-149. Accordingly, immunofluorescence studies to monitor the recruitment of DNA repair proteins 53BP1 and phosphorylated γH2AX at the lesion sites after treatments suggest that the GLE and/or carboplatin combination impair DDR. To determine tumor progression, severe combined immune deficient mice were injected with SUM-149 cells and then treated with carboplatin/GLE or carboplatin for 10 weeks. The combination significantly decrease tumor volume in comparison with the vehicle. Taken together, our results provide evidence of GLE's potential in chemosensitizing TNBC cells to carboplatin therapy affecting the DDR. These results highlight GLE's anti-breast cancer therapeutic potential.

#2816

Patient derived ovarian cancer xenograft (OC-PDX) to study the response of the PARP inhibitor olaparib.

Francesca Bizzaro,1 Alessia C. Marchetti,1 Alessandra Decio,1 Francesca Ricci,1 Mark J. O'Connor,2 Molly A. Taylor,2 Zhongwu Lai,3 Simon T. Barry,2 Maria R. Bani,1 Raffaella Giavazzi1. 1 _IRCCS-Mario Negri Institute for Pharmacological Research, Milan, Italy;_ 2 _Astrazeneca, Cambridge, United Kingdom;_ 3 _Astrazeneca, Waltham, MA_.

Up to 50% of High Grade Serous (HGS) ovarian cancer patients exhibit homologous recombination deficiency (HRD) through various mechanisms including germline and somatic mutations in BRCA-1 or 2. Olaparib, a poly(ADP-ribose)polymerase (PARP) inhibitor, was recently approved for the treatment of patients with germline BRCA-mutated-advanced ovarian cancer. We have analyzed our panel of patient derived ovarian cancer xenografts (OC-PDX)1 to investigate the association of the somatic BRCA1/2 mutational status with the response to olaparib.

Whole Exome Sequencing (Illumina HiSeq4000) was performed on a panel (n=26) of OC-PDX to identify mutations in BRCA1/2. Next Generation Sequencing (NGS) results were confirmed by Sanger sequencing on tumor DNA and RNA. Thirteen OC-PDX were selected and treated with 100mg/kg olaparib for 4 weeks or as a maintenance regimen until progression. Cisplatin was used as reference drug. Efficacy was evaluated as the best T/C% (best growth inhibition) for subcutaneous (s.c) tumors. To mimick patient disease, OC-PDX were also established as intra-peritoneal models (i.p.) and best ILS% (best increment of lifespan) calculated.

Response to olaparib varied among the models with distinct responsive and non-responsive groups associated with BRCA mutational status.

OC-PDX with a homozygous frameshift mutation in BRCA1/2 (n=5), loss of BRCA1 (n=1) or no expression of BRCA1/2 (n=1) showed a sustained response to olaparib after 4 week treatment (T/C values from 2% to 40%; ILS of one representative OC-PDX = 74%), with complete responses following a longer term maintenance treatment. One BRCA1 mutated OC-PDX model was resistant, progressing rapidly under treatment. In this orthotopic model ILS% and tumor dissemination score of treated mice were similar to vehicle. Models responsive to olaparib were in general also sensitive to cisplatin. BRCA1/2 wild type OC-PDX models (n=4 s.c and n=1 i.p) did not respond to olaparib, even following a longer term treatment (T/C from 41% to 86%; ILS = 5%). NGS revealed a heterozygous mutation in BRCA2 in one OC-PDX. Sanger sequencing of the RNA confirmed the presence of both the wild type and the mutated BRCA2 transcripts. This OC- PDX was poorly responsive to olaparib (T/C = 57%).

Our data showed that in general tumors with a homozygous mutation, loss or no expression of BRCA1/2 responded to olaparib; the drug was not active on those with mutation in heterozygosis (BRCA2+/-) or wild type. These findings indicate that tumor somatic mutations play a role in the response to olaparib. Models of OC-PDX moderately responsive to olaparib offer the opportunity to assess the potential of combination treatments.

1 Ricci, F. et al. Patient-derived ovarian tumor xenografts recapitulate human clinicopathology and genetic alterations. Cancer Res 74, 6980–90 (2014)

#2817

RAD6 promotes acquired chemoresistance and a poor prognostic marker in ovarian cancer.

Chinnadurai Mani, David Clark, Ranganatha Somasagara, Sebastian Spencer, Kaushlendra Tripathi, Komariah Palle. _Mitchell Cancer Institute, Mobile, AL_.

Ovarian cancer (OC) is deadly and incurable for patients who relapse after primary cancer is treated with surgery and platinum-based chemotherapy. Unfortunately, OC has a high rate of disease recurrence (70-80%) and most recurrent tumors are chemoresistant, causing deaths of nearly 15,000 women from this disease each year in U.S.A., which highlights the need for new therapeutic strategies. Platinum drug resistance and refractory disease pose major challenges in treating this disease and are major factors contributing to the poor survival rate of OC patients. Our studies demonstrated that RAD6 (an E2 ubiquitin conjugating enzyme) signaling is stimulated upon chemotherapeutic treatment and promotes expression of DNA repair proteins including Fanconi anemia (FA) pathway. Analysis of clinical samples revealed upregulation of RAD6 in ovarian tumors compared to normal ovarian tissues and RAD6 inhibition reduces cancer cell survival. In a pilot clinical study (n=26) comparing matched OC tumors from patients before and after carboplatin chemotherapy regimen, we found that RAD6 was upregulated after treatment and correlated with both chemoresistance and poor progression-free survival. Knockdown or inhibition of catalytic activity (small-molecule inhibitor) of RAD6 attenuated carboplatin-induced monoubiquitination of target proteins such as FANCD2, PCNA and histone 2B, thereby sensitizing OC cells to carboplatin. Interestingly, inhibition of RAD6 alone in OC cells induced replication stress and reduced cell survival and proliferation by arresting cells in the G2/M phase. Moreover, RAD6 plays an important role in the activation of the trans-lesion synthesis (TLS) pathway by monoubiquitinating PCNA and in the activation of the FA DNA repair pathway. These are critical mechanisms for cells to repair DNA crosslinks induced by platinum drugs. Together with these observations, our data suggest that inhibition of RAD6 could be a viable therapeutic target for overcoming platinum resistance and disease recurrence induced by FA pathway in ovarian cancer.

#2818

AsiDNA and HDAC inhibitors: A cross-potentiation team working to kill tumor cells.

Wael Jdey,1 Hélène Toussaint,1 Coraline Cassegrain,1 Laura Bellassen,1 Richard Tripelon,1 Vincent Hayes,1 Perrine Pivette,1 Véronique Trochon-Joseph,1 Jean-Louis Labernardière,1 Marie Dutreix,2 Françoise Bono Colombie1. 1 _Onxeo, Paris, France;_ 2 _Institut Curie, Orsay, France_.

Purpose: while being registered and used in restricted subset of T-cell lymphoma, HDAC inhibitors (HDACi) have shown limited antitumor effect as single agents. Recent studies have shown an effect of HDACi on DNA damage accumulation, rationalizing their combination with DNA repair inhibitors. In the current study, we propose a novel therapeutic strategy, based on drug combination of HDACi with the pan-DNA break repair inhibitor AsiDNA to promote their antitumor activity.

Experimental design: AsiDNATM is a double stranded DNA molecule (decoy oligo-nucleotide) that mimics double stranded DNA breaks (DSBs) to interfere with DNA repair, redirecting repair enzymes away from sites of tumor DNA damage. Belinostat is a pan-HDACi displaying a better safety profile compared to other HDACi. We characterized the effects of each drug on DNA break accumulation and genetic instability by ɣH2AX analysis, COMET assay and micronuclei detection. We further studied the antitumor efficacy of the combination of the two drugs. Finally, we assessed the effect of AsiDNA on the occurrence of acquired resistance after long term treatment with belinostat.

Results: Molecular analyses of DNA damage after treatment demonstrate that belinostat paves the way for AsiDNA efficacy by inducing DNA DSBs as measured by ɣH2AX accumulation and tail moment increase on COMET assay. Moreover, continuous treatment with belinostat induced an increase of basal genetic instability in tumor cells measured by micronuclei accumulation, a prerequisite for AsiDNA antitumor efficacy1. On the other hand, AsiDNA enhances the effects of belinostat on histone acetylation, demonstrating a high potentiation of belinostat activity on its targets by AsiDNA. This mechanism-based cross-potentiation between AsiDNA and belinostat results in a high synergistic antitumor efficacy of the combined treatment in different tumor models. This synergistic effect was further confirmed with several HDACi belonging to different classes. Importantly, the combined treatment do not induce any DNA damage increase and/or lethality in non-tumor cells. Finally, repeated treatments allowed the emergence of resistance to belinostat, which is abrogated in presence of AsiDNA, indicating an unlikely tumor escape to this combined therapy.

Conclusion: Altogether these results indicate a cross potentiation between AsiDNA and belinostat, and support the rational to investigate the clinical activity of this novel synergistic combination in different tumor types. As belinostat has obtained FDA conditional approval, and AsiDNA is already tested in a first-in-man clinical trial, a potential exists for a rapid clinical confirmation of the interest of this new combination.

#2819

Targeting histone chaperone FACT complex sensitizes medulloblastoma cells to chemo and radiation therapy.

Sutapa Ray, Heyu Song, Shrabasti Roychoudhury, Pranjal Biswas, Sutapa Ray, Kishor Bhakat. _University of Nebraska Medical Center, Omaha, NE_.

Medulloblastoma (MB) is the most common malignant brain tumor in children. Post-surgical radiotherapy in combination with adjuvant chemotherapy are considered as standard treatment of care for MB. However, this long-term treatment often leads to harmful neurologic effects to the developing brain in children. Therefore, identification of novel therapeutic target to increase efficacy and reduce toxicity in MB represent an unmet clinical need.

Apurinic/apyrimidinic (AP)-endonuclease (APE1) plays a central role in the base excision repair (BER) pathway by repairing AP sites and single-strand breaks, the downstream cytotoxic BER intermediate that occurs following radiation and chemotherapy. Elevated levels of APE1 or its activity has been shown to be associated with response to radiation and chemotherapy in MB. However, how APE1 repairs AP sites in the context of nucleosome in cells and how APE1-dependnt BER pathway can be manipulated to increase the therapeutic efficacy of chemo and radiation therapy remain an open question. We have recently shown that APE1 is acetylated (AcAPE1) at AP site damage in chromatin by p300 and that acetylation enhances AP-endonuclease activity of APE1. Here, we show that APE1 interacts with the nucleosome remodeling histone chaperone Facilitates of Chromatin Transcription (FACT) complex (a heterodimer of SSRP1 and SPT16 proteins) and that FACT complex is required for binding and acetylation of APE1 at AP site damages in highly aggressive MB cell lines HD-MB03 and ONS-76. Furthermore, we have mapped genome-wide binding of AcAPE1 in HDMB cells and our ChIP-seq data analysis revealed ~68154 (p<.001) AcAPE1 binding predominantly located in the transcribed gene regions corresponding to 16370 genes. We aligned ChIP-Seq with RNA-Seq of HD-MB03 cells and found a significant (p<0.0001) correlation of AcAPE1 binding with gene expression. Furthermore, our data show that inhibition of FACT with small molecule inhibitor CBL0137/curaxin, reduces SSRP1 and SPT16 levels from soluble fraction and trap them in chromatin. We found that CBL0137/curaxin treatment results in inhibition of APE1 acetylation, and that MB cells cannot repair DNA damages in the presence of FACT inhibitor. FACT has been shown to play an important role in removal of cisplatin-induced DNA adducts via nucleotide excision repair pathway (NER). Of importance, downregulation of APE1 levels or inhibition of FACT with CBL0137/curaxin sensitizes a panel of highly aggressive MB cell lines to cisplatin and alkylating drug temozolomide.

Together, our study suggest that co-suppression of both BER and NER pathways via targeting FACT complex can greatly sensitize MB tumors to chemo and radiation therapy. A major implication of our studies is that combination of curaxin with low dose of cisplatin may have considerable therapeutic potential to improve efficacy and reduce cytotoxic side effects of cisplatin to MB patients.

#2820

Synergistic antileukemic activity of the antibody-drug conjugate (IMGN779) combined with PARP inhibition in preclinical human acute myeloid leukemia models.

Claire Fritz, Scott M. Portwood, Julie Adams, Eunice S. Wang. _Roswell Park Cancer Inst., Buffalo, NY_.

Background: Acute myeloid leukemia (AML) is an aggressive hematologic malignancy of immature myeloid cells. Antibody-drug conjugates (ADC) comprising humanized anti-CD33 monoclonal antibodies linked to cytotoxic payloads are known to exert clinical activity in AML patients. However, dose-dependent effects of ADC therapy, specifically hepatotoxicity, myelosuppression and veno-occlusive disease, have limited their clinical utility in patients. Inhibitors of the DNA repair enzyme poly (ADP-ribose) polymerase (PARP), so-called PARP inhibitors (PARPi), enhance tumor death by preventing repair of single-stranded DNA breaks. We have previously shown (Portwood S et al., ASH 2016) that the PARPi olaparib improves the activity of the anti-CD33 ADC (IMGN779) in preclinical AML models. Here we assessed the efficacy of multiple PARPi (olaparib, niraparib, rucaparib, veliparib, and talazoparib) alone and in combination with IMGN779 for AML therapy.

Materials and Methods: Human AML cell lines (HEL, HL60) were characterized for CD33 expression using flow cytometry after staining with antibody-linked fluorescent QuantiBrite Beads. Cells were treated with varying doses of IMGN779 (100pM-1nM) and PARPi (10nM-100uM) alone and in combination for 96h. Viability was measured after treatment utilizing a WST-1 colorimetric assay. Induction of apoptosis, cell cycle effects, and DNA damage were characterized by flow cytometry following 72h of treatment. Synergy reports were generated using Compusyn software.

Results: All PARPi demonstrated dose-dependent inhibition of AML (HEL, HL60) growth with the following IC50 ranges: talazoparib 200-400nM, niraparib 2.5-7.3 μM, olaparib 7.4-8.9 μM, rucaparib 39-75 μM, and veliparib 88-100 μM. Combination therapy with IMGN779 and four PARPi (veliparib, niraparib, olaparib, talazoparib) resulted in synergistic in vitro antileukemic effects with CI values <1. Statistically significant improvements in antiproliferative effects, apoptosis induction, and DNA damage as measured by γH2AX phosphorylation (p<0.05) were observed with combination PARPi and 779 therapy as compared with monotherapy. While single-agent PARPi largely induced G2 cell cycle arrest, combination IMGN779 and PARPi resulted in prominent S phase cell cycle arrest.

Conclusions: Our results demonstrate that PARP inhibitors as a class exert dose-dependent antitumor activity against human CD33+ AML cells. Moreover, PARPi consistently induce in vitro synergistic antileukemic effects against human AML cells when combined with the anti-CD33 ADC (IMGN779). Studies evaluating the mechanisms of action and the efficacy of combinatorial therapy in primary AML cells are under way. These findings support further clinical studies of PARP inhibition as a potential means of enhancing the activity of ADCs for acute myeloid leukemia therapy.

#2821

Tousled like Kinase 1 binds to and regulates the protein kinase Nek1: Its implication in prostate cancer.

Vibha Singh, Praveen K. Jaiswal, Hari K. Koul, Xiuping Yu, Arrigo Benedetti. _LSUHSC Shreveport, Shreveport, LA_.

Introduction: Prostate Cancer (PCa) is one of the most common urological malignancies in men in the United States. Tousled Like kinases (TLKs) are involved in numerous cellular functions, including the DNA Damage Response (DDR). Through a novel proteomic approach, we have identified that NIMA kinase NEK1 strongly interacts and co-localizes with TLKs and has a role in the DDR, upstream of ATR and Chk1. In the present study we have tested Thioridazine (THD), an anti-psychotic drug and inhibitor of TLKs in inhibiting the activity of NEK1 in prostate cancer cell lines and LNCaP cell derived xenografts in NOD/SCID mice and the subsequent role of ATR-Chk1 axis following DNA damage.

Material & Methods: We have used a prostate cancer cell line viz. LNCaP, C4-2, C4-2b, 22Rv1, DU145 and PC3 and normal prostate cell line RWPE-1. Clonogenic activity was measured by colony formation assay. Immunoblotting was done for TLKs, NEK1, p-ATR and p-Chk1 protein in PCa cell line and tumor xenografts tissues. LNCaP xenografts in NOD/SCID mice were used in present study. We have used THD (alone) or in combination with the anti-androgen drug bicalutamide in PCa cell lines as well as in xenografts mice model.

Results: Treatment with THD suppresses the outgrowth of androgen-independent (AI) colonies of LNCaP cells cultured with bicalutamide. Moreover, THD significantly inhibited the colony formation efficiency of several PCa cells in vitro (even of androgen independent lines). Intra-Peritoneal (I.P.) administration of THD or Bicalutamide long-term was not very effective in inhibiting tumor growth. In contrast, combination therapy of THD and Bicalutamide remarkably inhibited the tumor growth in LNCaP xenografts model. Further p-ATR and p-Chk1 axis was inhibited by treatment with THD and in combination with bicalutamide as evident by immunoblotting of residual tumors. In cells, following DNA damage, addition of the TLK inhibitor THD impaired ATR and Chk1 activation, indicating the existence of a TLK1>NEK1>ATR>Chk1 pathway.

Conclusions: Our data indicated that THD repressed growth of PCa cells in vitro and in vivo, and for Androgen-dependent (LNCaP) cells, in combination with bicalutamide it was very effective at preventing outgrowth of AI cells and hence as a targeted ADT adjuvant therapy. Moreover, THD impaired DDR. Collectively, these results strongly suggest that THD might be a novel anti-tumor agent for use in prostate cancer.

#2822

ATR inhibitors are active as single agents and in combination with PARP1 and ATM inhibitors in molecularly distinct subsets of small cell lung cancer models.

Carl M. Gay, Pan Tong, Lerong Li, C. Allison Stewart, Triparna Sen, Bonnie S. Glisson, John V. Heymach, Jing Wang, Lauren Averett Byers. _UT MD Anderson Cancer Ctr., Houston, TX_.

Small cell lung cancer (SCLC) is an aggressive form of lung cancer, notable for rapid emergence of drug resistance following initial chemotherapy. Rates of five-year overall survival are only 7% across all stages and only one drug, topotecan, is approved by the FDA for recurrent SCLC. As a result, the National Cancer Institute has named identifying novel vulnerabilities in SCLC as an urgent area of need. Increased expression, relative to non-small cell lung cancer (NSCLC), of numerous components of the DNA damage response (DDR) pathway, including poly (ADP-Ribose) polymerase 1 (PARP1) and ataxia telangiectasia mutated (ATM), is observed in SCLC. Thus, targeting DDR has emerged as an attractive therapeutic strategy in SCLC, bolstered by recent data demonstrating activity of PARP1 inhibitors (PARPi) in SCLC patients. Interestingly, data suggest that PARPi resistant models from other tumors may rely on another DDR component, ataxia telangiectasia and Rad3 related protein (ATR), for survival. ATR/ATR is also highly expressed in SCLC compared to NSCLC and normal lung tissue. Preclinical data have shown that treatment with ATR inhibitors (ATRi) is especially effective in p53- and ATM-deficient tumor models, a notable fact given that SCLC is universally p53-mutant and that ATM-mutant and ATM-deficient SCLC is a small, but significant proportion of all SCLC. We treated 22 human-derived SCLC cell line models with two clinically relevant ATRi, VX-970 (formerly VE-822) and AZD-6738, and observed single agent activity of both ATR inhibitors in a significant number of cell lines, with half-maximal inhibitory concentrations (IC50s) as low as 30 nM and >100-fold difference in IC50s between the most and least sensitive cell lines. Utilizing extensive genomic, transcriptomic and proteomic characterization of these cell lines, we then identified predictive biomarkers of response to ATRi in SCLC, including low ATM expression. As low ATM was associated with ATRi sensitivity, we tested whether the addition of an ATM inhibitor (AZD-0156) may further sensitize SCLC models to ATRi. We treated 12 SCLC cell lines with AZD-6738 and AZD-0156 in combination and identified a subset of lines in which synergy is observed between the two agents. Similarly, as targeting ATR has been shown to overcome PARPi resistance in other cancer types, we treated 12 SCLC cell lines with the ATRi AZD-6738 and the PARPi olaparib in combination and again observed a subset of lines in which the two agents acted synergistically. Interestingly, the lines in which ATRi+ATMi and ATRi+PARPi synergy is observed are distinct and include lines that were the most resistant to single-agent AZD-6738. Together, these data support further investigation of ATRi in SCLC and suggest that via the use of ATRi alone or in combination with ATMi or PARPi, multiple molecularly distinct subsets of SCLC can be effectively targeted.

#2823

DNA methyltransferase inhibitors sensitize NSCLC cells to PARP inhibitors by induction of a double strand break repair defect.

Rachel Abbotts,1 Michael Topper,2 Daniel Fontaine,1 Christopher Biondi,1 Eun Yong Choi,1 Rena Lapidus,1 Stephen Baylin,2 Feyruz Rassool1. 1 _University of Maryland, Baltimore, MD;_ 2 _Johns Hopkins University, Baltimore, MD_.

Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related death in the US. Poly (ADP-ribose) polymerase inhibitors (PARPi) show efficacy as radiosensitizers in NSCLC, but arguably have gained greater importance in cancer therapeutics by exhibiting synthetic lethality (SL) in tumors with defects in the homologous recombination (HR) pathway of double strand break repair (DSBR). Mechanistically, PARPi cytotoxicity is partially mediated through trapping of PARP1 at sites of DNA damage, with next generation PARPis such as talazoparib (Tal) being potent PARP trappers. Our group has previously demonstrated that combining Tal with a DNA methyltransferase inhibitor (DNMTi) enhances PARPi cytotoxicity in acute myeloid leukaemia and breast cancer models. DNMTis are cytosine analogs that become incorporated into replicating DNA, where they covalently bind their target enzyme DNMT1. When combined with PARPi, DNMTis enhance PARP trapping at damage sites, which collapse into cytotoxic double strand breaks (DSB) when encountered by a replication fork.

In the current study, we demonstrate synergistic cytotoxicity of Tal in combination with the DNMTi 5-azacytidine (Aza) in NSCLC cell lines by combination index analyses and colony forming assays. Further potentiation of Tal+Aza cytotoxicity is observed following induction of DNA damage via IR. These results were substantiated in an A549 xenograft model (n=8 per group) where Tal+Aza+IR significantly reduced tumor volume and increased survival compared to vehicle (p<0.001) or Tal+Aza without IR (p<0.05). We and others have previously reported that low dose DNMTi results in stable transcriptional changes in the cancer epigenome, including DNA damage response pathways. Here, we show by microarray analysis that NSCLC cell lines (A549, H23, H1299, H460) treated with Aza exhibit up- and downregulation of multiple genes in the DNA repair reactome, including decreased expression of the key HR-related gene FANCD2, and the non-homologous end joining (NHEJ) gene Ku80. These results were validated by mRNA and protein quantification in Aza-treated NSCLC cell lines and xenograft tumor samples. In keeping with a FANCD2-associated HR defect, Aza exposure is associated with reduced accumulation of RAD51 foci following IR, and reduced HR activity in an extrachromosomal HR assay. Additionally, increased sensitivity to IR post Aza treatment can be explained by decreased expression of NHEJ factor Ku80. These results indicate that Aza treatment in NSCLC induces a DSBR deficiency that can be targeted for SL by PARPi and can further sensitize to IR. Given these agents are currently in use in the clinic, this combination offers a promising therapeutic strategy that could be rapidly translated into clinical practice.

#2824

Charting the landscape of genomic instability in acute myeloid leukemia: Interaction between G-quadruplexes and heme oxygenase-1 in leukemia.

Anna Konturek-Ciesla, Anette Radziszewska, Maciej Cieśla, Alicja Czmoczek, Monika Zukowska, Jozef Dulak, Alicja Jozkowicz, Karolina Bukowska-Strakova. _Jagiellonian University, Kraków, Poland_.

Genomic instability contributes to the development and expansion of acute myeloid leukemia (AML). Therefore, identification of factors pivotal for the genome integrity is essential for basic science and translational medicine. We propose that interplay between G-quadruplexes (G4) and heme oxygenase-1 (HO-1) affects the response to DNA damage and switch from normal to malignant hematopoiesis. AML is associated with chromosomal translocations as well as single point mutations. The risk of mutagenesis is increased by guanine-rich DNA sequences, folding into G-quadruplexes (G4), which can abrogate the replication. In inorganic solutions G4 are stabilized by heme. Whether such stabilization occurs in the living cells and how it affects mutagenesis' rate have not been tested so far. Cellular heme is removed by heme oxygenase-1 (HO-1), which evokes differential effects depending on subcellular localization. Here we assessed the impact of HO-1 in conjunction with G4 on genomic instability of AML. To address the function of HO-1 in leukemia we established the MOLM13 cell line engineered to knock-down (KD) HO-1. Rescue studies were performed with wild type (WT) and nuclear export signal (NES)-containing HO-1 constructs. Ex vivo assays were done with murine and human bone marrow cells. Cellular localization of HO-1 and G4 was determined by immunofluorescence in hematopoietic stem and progenitor cells (HSPCs). Co-localization of HO-1 and G4 was further assessed by proximity ligation assay (PLA). DNA damage and viability were determined at the steady state and in response to doxorubicin. Finally, association of HO-1 expression with mutational status and progression of AML was evaluated in cohort of 80 control and AML patients. We demonstrated that in HSPCs HO-1 localizes mainly in the nucleus. In stem cells its expression is low and increases upon differentiation, with the highest amount in the myeloid progenitors. Immunostaining confirmed the presence of G4, relatively scarce in stem cells but more numerous in granulocyte-macrophage progenitors. Importantly, we found for the first time that HO-1 co-localizes with G4 and that it can regulate the abundance of DNA damage at the steady state, with HO-1 KD resulting in increased phosphorylation of histone H2AX, marker of double strand DNA breaks. Strikingly, doxorubicin treatment increased not only DNA damage but also expression of HO-1. Accordingly, in AML patients we found a correlation between lower HO-1 expression, higher incidence of FLT3 mutations and the dismal prognosis. HO-1 protein is present in the nucleus of hematopoietic and leukemic cells, where it can co-localize with G-quadruplexes and evoke important functional effects. HO-1 expression is associated with clinical outcome of AML and with FLT3 mutation rate.

#2825

**PIN1-SCF** FBW7 **proteolytic signaling regulates the Fanconi anemia pathway.**

Jingming Wang, Bryan Chan, Michael Tong, Markus Seeliger, Hyungjin Kim. _Stony Brook University, Stony Brook, NY_.

Genome instability caused by defective DNA repair mechanisms is a hallmark of cancer and drives tumorigenesis. The Fanconi anemia (FA) DNA repair pathway contributes to the integrity of the genome by resolving DNA interstrand cross-links (ICLs) encountered during DNA replication. Deregulation of the FA pathway is associated with cancer predisposition and affects therapeutic outcomes against DNA-damaging cytotoxic chemotherapy. FANCD2 monoubiquitination by a multi-subunit ubiquitin E3 ligase, the FA core complex, is an essential gateway that connects the DNA damage response to enzymatic steps of DNA ICL repair. We have previously identified FAAP20 as a critical component of the FA pathway; and demonstrated that the phosphorylation of the Cdc4 phospho-degron (CPD) motif in FAAP20 by GSK3β is required for its proteasomal degradation mediated by the E3 ligase, SCFFBW7 complex. However, the upstream signaling that governs the FAAP20 phosphorylation status and its detailed mode of action for FAAP20 degradation remain elusive. Here, we show that PIN1, a phosphorylation-specific prolyl isomerase, is a key regulator of the integrity of the FA core complex and, therefore, FA pathway activity as a whole. By catalyzing a proline cis-trans isomerization of the phosphorylated backbone and thereby converting the substrate into a conformation that is favorable or refractory to downstream proteolytic signaling, PIN1 modulates the rate of protein turnover. We demonstrate that PIN1 interacts and catalyzes a phosphorylated Ser48-Pro49 motif of FAAP20 and by inducing a conformational change of FAAP20, PIN1 enhances the affinity of FAAP20 with PP2A (Protein phosphatase 2), the phosphatase of the CPD motif in FAAP20 and thereby prevents its degradation from the GSK3β-FBW7 mediated proteolytic signaling. Collectively, our studies uncover PIN1-dependent isomerization as a new regulatory mechanism for DNA ICL repair. Given that PIN1 overexpression is prevalent in diverse human cancers, identifying a way to regulate PIN1 activity in the FA pathway may help develop a PIN1 inhibitor as a chemosensitizer for cytotoxic chemotherapy to increase its therapeutic efficacy.

#2826

Differential activity of ATR and WEE1 inhibitors in DLBCL subtypes is linked to replication stress and differences in mode of action.

Lucy A. Young,1 Lenka Oplustil O'Connor,1 Zena Wilson,2 Corinne Reimer,3 Alan Lau,1 Jiri Bartek,4 Mark J. O'Connor1. 1 _AstraZeneca, Cambridge, United Kingdom;_ 2 _AstraZeneca, Alderley Park, United Kingdom;_ 3 _AstraZeneca, Waltham, MA;_ 4 _Danish Cancer Society Research Centre, Copenhagen, Denmark_.

The checkpoint kinases ATR and WEE1 protect cells from replication stress (RS), a hallmark of cancer that has potential to be exploited for cancer treatment. ATR and WEE1 inhibitors are in early clinical trials. Here we report selective anti-tumour activity of inhibitors to ATR (AZD6738) and WEE1 (AZD1775) in a subset of diffuse large B-cell lymphoma (DLBCL) cell lines. Using the cell-of-origin (COO) gene expression scoring system, we found enhanced sensitivity to AZD6738 in the activated B-cell like (ABC, n=6) and unclassified cell lines (n=6) with a combined median GI50 of 0.372 µM, compared to germinal centre B-cell like (GCB, n=7) cell lines, with median GI50 of 1.554 µM. Grouping the cells into AZD6738 sensitive (GI50 ≤ 0.5 µM) or insensitive (≥ 1 µM), we show that the ABC/unclassified COO score, together with high c-MYC protein or CDKN2A/B deletion identified 7/8 sensitive cell lines and 0/7 insensitive cell lines. Furthermore, markers of RS correlated with cell line sensitivity. Treatment of cells with 0.5 µM AZD6738 increased the quantity of 53BP1 nuclear bodies by 3-6 fold across the sensitive cell lines compared to only 2 fold across the insensitive cell lines. DNA fibre analysis also showed that sensitive cells lines had slower rates of replication fork progression (0.80 - 1.12 kbmin-1) compared to insensitive cell lines (1.22 - 1.41 kbmin-1). Across the same DLBCL cell line panel sensitivity to the WEE1 inhibitor AZD1775 was positively correlated with AZD6738 sensitivity, although at lower concentrations. However, using OCI-LY19 and DB cells to reflect high and low replication stress respectively, we show that AZD6738 and AZD1775 differ mechanistically in the extent of DNA damage and apoptosis induced within cell cycle phases. Treatment with 1 µM AZD1775 induced S-phase arrest and increased γH2AX+ cells in S/G2 from 4% to 20% in both cell lines at 8 h, indicating replication-associated DNA damage. However, earlier onset of cell death was observed in the OCI-LY19 cells, with 30% γH2AX+ cells undergoing apoptosis at 24 h compared to 25% γH2AX+ DB cells at 72 h. In contrast, AZD6738 only caused G1 arrest in OCI-LY19 daughter cells, with 20% apoptosis at 72 h. These findings are reflected in the anti-tumour activity of the inhibitors in xenograft DLBCL models in vivo. Once-daily, oral dosing of 50 mg/kg/day AZD6738 or 120 mg/kg/day AZD1775 in OCI-LY19 xenograft model showed efficacy with mean % tumour growth inhibition (%TGI) at day 8 of 104.1% (n=10) and 114.3% (n=10) respectively. In addition, the combination of AZD1775 (60 mg/kg/day; monotherapy %TGI=88.7, n=10) and AZD6738 (25 mg/kg/day; monotherapy %TGI=89.8%, n=10) dosed using a 5 day on, 9 day off dosing schedule was tolerated in mice and resulted in tumour regressions (combination %TGI=108.4%, n=8). These data suggest potential for both single agent and combination opportunities for ATR and WEE1 inhibitors in the DLBCL ABC subtype.

#2827

Alternative non-homologous end joining DNA repair as therapeutic target in multiple myeloma.

Daniele Caracciolo, Martina Montesano, Emanuela Altomare, Grazia Consolo, Nicola Amodio, Maria Teresa Di Martino, Marco Rossi, Cirino Botta, Eugenio Morelli, Giada Juli, Pierosandro Tagliaferri, Pierfrancesco Tassone. _Magna Graecia University, Catanzaro, Italy_.

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Alternative Non Homologous End Joining DNA repair as therapeutic target in Multiple Myeloma

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Daniele Caracciolo, Martina Montesano, Emanuela Altomare, Grazia Consolo, Nicola Amodio, Maria Teresa Di Martino, Marco Rossi, Cirino Botta, Eugenio Morelli, Giada Juli, Pierosandro Tagliaferri, Pierfrancesco Tassone. UMG of Catanzaro, Catanzaro, Italy

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Background- Multiple Myeloma (MM) is a hematologic malignancy strongly characterized by genomic instability. We previously demonstrated that LIG3-dependent Alt-NHEJ repair is significantly involved in the genomic instability which promote survival and drug resistance of MM cells. Based on this rationale, we aimed to elucidate the efficacy of available Alt-NHEJ pathway inhibitors as new therapeutic agents in MM.

Materials and methods- Cell proliferation and apoptosis were evaluated with CellTiter-Glo assay and Annexin V staining. Alt-NHEJ repair was evaluated using EJ2-GFP and EJ5-GFP plasmids. LIG3, LIG1, PARP1, Caspase 3 and p-H2AX, levels were analyzed by Western blot of whole protein extracts. In vivo anti-MM activity was evaluated in NOD-SCID mice bearing subcutaneous AMO-1 Bortezomib resistant (ABZB) xenografts, daily treated with Olaparib via oral gavage.

Results- By interrogating public available datasets, we found that higher mRNA expression of Alt-NHEJ core components was correlated with shorter survival of MM patients. Indeed, plasmid-based reporter assays confirmed a prevalent activation of Alt-NHEJ repair in MM cells as compared to normal lymphoblastoid cell lines, except for U266 cells which instead displayed a prevalence of Canonical-NHEJ repair activity. Next, we attempted to evaluate the effects of available Alt-NHEJ inhibitors on MM cell survival. Consistently, L67 (LIG3-LIG1 inhibitor) or Olaparib (PARP-inhibitor) induced a significant reduction of proliferation and clonogenic cell growth of different MM cell lines at low micromolar concentrations, while no significant effects on survival were observed in U226 as well as in normal lymphoblastoid cells. Importantly L67 or Olaparib impaired viability of MM cell lines or primary plasmacells co-cultured on stromal cells, thus overcoming the supporting role played by MM-microenvironment on plasma-cells survival. As result of Alt-NHEJ repair inhibition, anti-proliferative effects were correlated to increase of DNA double-strand breaks (DSBs), cell cycle arrest and finally apoptosis. Notably, we observed that Bortezomib resistant AMO-1 derivative cells (ABZB) exhibited significantly increased sensitivity to a combination of L67 and Olaparib that increased the number of DSBs. Moreover, Alt-NHEJ inhibitors synergistically sensitized primary cells as well as MM cell lines to Bortezomib. Finally, to demonstrate the in vivo relevance of our findings, we showed that clinically available Parp-inhibitor Olaparib, exerted a significant anti-MM activity on ABZB cells injected in immunocompromised mice.

Conclusion- Taken together, our findings indicate that MM cells are addicted to Alt-NHEJ repair parthway, which therefore represent a novel druggable target pathway in MM.

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#2828

MMR status affects efficiency of homologous recombination repair of F10-induced DNA DSBs.

William H. Gmeiner,1 Chinnadurai Mani,2 Komaraiah Palle2. 1 _Wake Forest Univ. School of Medicine, Winston-Salem, NC;_ 2 _University of South Alabama, Mobile, AL_.

Background: DNA mismatch repair (MMR) is an important stratification factor in colorectal cancer (CRC). 10-20% of sporadic CRC cases display high microsatellite instability (MSI-H) due to DNA mismatch repair (MMR) deficiency, and these are refractory to current fluoropyrimidine (FP) chemotherapy. Our studies with DNA-directed FP polymers, such as F10, indicate they are highly effective regardless of MMR-deficiency consistent with inducing MMR-independent cell death. We investigate the role of MMR-status on potency of F10 and induction of DNA damage and activation of the DNA damage response as part of an overall effort to determine if fluoropyrimidine polymers can be used effectively in the treatment of MMR-deficient CRC.

Methods: Studies were conducted using MMR-deficient HCT-116 CRC cells and HCT-116 3-6 cells. Potency was assessed by cell-titer glo and clonogenic assays. Replication stress was evaluated by DNA fiber combing. DNA damage and activation of the DNA damage response was evaluated by Western blots for gH2AX, Chk1 activation, and proteins important for homologous recombination repair.

Results: F10 is highly potent to CRC cells regardless of MMR status and is ~1,000-fold more potent than 5-FU to MMR-deficient HCT-116 cells. The increased potency of F10 parallels increased efficiency at causing replication fork slowing as assessed by DNA fiber analysis. F10 causes DNA double strand breaks (DSBs) and activates Chk1 consistent with causing replication fork collapse. DNA DSBs, Chk1 activation, and upregulation of FANCD2, which mediates HR-repair of DNA DSBs all show a marked MMR-dependence consistent with hMLH1 being a factor in HR-mediated repair of F10-induced DNA DSBs.

Conclusions: F10 is highly potent to CRC cells regardless of MMR status via a mechanism involving replication fork slowing and increased replication stress. MMR status affects basal levels of proteins involved in HR and the upregulation of these proteins in response to F10 treatment. Our studies indicate FP polymers are likely to be generally effective in CRC regardless of MMR status however MMR status may be an important factor in a personalized medicine approach to their use as combination with Chk1 inhibitors and other drugs modulating the DNA damage response may depend on MMR status.

#2829

Targeting the DNA damage response and DNA-PK signaling via small molecule Ku inhibitors.

John J. Turchi,1 Navnath S. Gavande,1 Pamela S. VanderVere-Carozza,1 Tyler Vernon,1 Katherine S. Pawelczak2. 1 _Indiana Univ School of Medicine, Indianapolis, IN;_ 2 _NERx Biosciences, Indianapolis, IN_.

Normal 0 false false false EN-US X-NONE X-NONE /* Style Definitions */ table.MsoNormalTable {mso-style-name:"Table Normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-parent:""; mso-padding-alt:0in 5.4pt 0in 5.4pt; mso-para-margin-top:0in; mso-para-margin-right:0in; mso-para-margin-bottom:10.0pt; mso-para-margin-left:0in; line-height:115%; mso-pagination:widow-orphan; font-size:11.0pt; font-family:"Calibri","sans-serif"; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin;} The DNA dependent protein kinase (DNA-PK) is a validated target for cancer therapeutics that drives the DNA damage response (DDR) and plays a critical role as a primary sensor in the non-homologous end joining (NHEJ) DNA double strand break (DSB) repair pathway. Various anti-cancer therapeutic strategies mediate their cytotoxic effects by inducing DSBs, including ionizing radiation (IR), and clinical outcomes are directly related to the repair of DNA damage. Modulating the pathway responsible for repairing DSBs will have a profound impact on the efficacy of DNA damaging agents in the clinic. To date, development of inhibitors for DNA-PK has focused on targeting the active site with ATP mimetics. We have taken the novel and innovative approach to inhibiting DNA-PK via blocking the Ku 70/80 heterodimer interaction with DNA, a necessary step in DNA-PK activation. Exploiting this unique mechanism of kinase activation, we have identified a series of highly potent and specific DNA-PK inhibitors that impart their inhibitory activity via disruption of the binding of Ku protein to DNA ends. This novel approach affords significant advantages to current approaches in kinase inhibition. Novel derivatives of our initial hit inhibit DNA-PK catalytic activity at nanomolar concentrations and potentiate cellular sensitivity to DSB-inducing agents like etoposide and bleomycin. Data demonstrate that the cellular effects observed are a function of Ku inhibition and that this novel class of DNA-PK inhibitors can be further developed as anti-cancer therapeutics that can be used as an adjuvant to, or concomitant with radiotherapy and other cancer therapies that induce DNA damage.

#2830

Understanding chemotherapy-induced replicative stress to identify rational combination therapies.

Ozan Alkan,1 Birgit Schoeberl,1 Millie Shah,1 Alexander Koshkaryev,1 Tim Heinemann,1 Daryl Drummond,1 Michael B. Yaffe,2 Andreas Raue1. 1 _Merrimack Pharmaceuticals, Cambridge, MA;_ 2 _Massachusetts Institute of Technology, Cambridge, MA_.

Cells respond to DNA damage by activating complex signaling networks that decide cell fate, promoting not only DNA damage repair and survival but also cell death. We have developed a multi-scale computational model using the U2OS osteosarcoma cancer cell line that quantitatively links chemotherapy-induced DNA damage response signaling to cell fate. The computational model was trained and calibrated based on an extensive data set that comprises cell cycle distribution of the initial cell population, signaling data measured by western blot, and cell fate data in response to chemotherapy treatment measured by time-lapse microscopy. The resulting mechanistic model can predict the cellular responses to chemotherapy alone and in combination with targeted inhibitors of the DNA damage response pathway, which we were able to confirm experimentally. Computational models, like the one presented here, can be used to understand the molecular basis that defines the complex interplay between cell survival and cell death, as well as to rationally identify chemotherapy-potentiating drug combinations.

#2831

Collateral lethality: A new target for personalized oncology.

Yu-Hsi Lin,1 Naima Hammoudi,1 Nikunj Satani,2 Jeffrey Ackroyd,1 Sunada Khadka,1 Dimitra Georgiou,1 Joe Marszalek,1 Yuting Sun,1 Marina Protopopova,1 Maria E. Di Francesco,1 Barbara Czako,1 Alan Y. Wang,1 Ronald A. DePinho,1 Florian L. Muller1. 1 _The University of Texas MD Anderson Cancer Center, Houston, TX;_ 2 _The University of Texas Health Science Center at Houston, Houston, TX_.

Large-scale genomic profiling efforts, such as The Cancer Genome Atlas (TCGA) have painted an unprecedentedly detailed picture of the genetic alterations that underlie carcinogenesis, yet the key challenge remains as to how to turn this information into actionable therapies. Genomic deletions are a frequent event in diverse cancers, which inactivate a limited number of tumor suppressor genes ("driver"-events) but frequently include many chromosomal neighbors as "passengers", some of which play critical but redundant roles in normal cellular housekeeping. The overall hypothesis we propose to test is that collateral deletions of such "passenger" genes can be utilized as novel targets of synthetic lethality, an idea which we term "collateral lethality." The large number of passenger deleted genes, playing diverse functions in cell homeostasis, offers a rich repertoire of pharmacologically targetable vulnerabilities presenting novel opportunities for the development of personalized anti-neoplastic therapies. We have provided in vitro proof-of-principal of a collaterally deleted glycolytic gene Enolase 1 (ENO1) at the 1p36 tumor suppressor locus in glioblastoma (GBM), that leads to dramatic sensitization to inhibition of the redundant paralogue, ENO2. The next step is to take this concept to the clinic. Our overall goal is to generate a clinical candidate Enolase inhibitor for tumors with ENO1-deletions, like GBM for which there is no other treatment option and the therapeutic benefit could be quite substantial.

#2832

Novel therapy for oral squamous cell carcinoma using non-thermal atmospheric pressure biocompatible plasma activated water.

Ihn Han,1 In Seok Song,2 Ying Li,1 Min Ho Kang,1 Jae Jun Ryu,2 Eun Ha Choi1. 1 _Kwangwoon University, Seoul, Republic of Korea;_ 2 _Korea University Anam Hospital, Seoul, Republic of Korea_.

Non-thermal atmospheric pressure biocompatible plasma (NBP) has been reported that was possible to induce apoptosis on various cancer cells including lung, ovary, brain, liver, breast, and pancreas cancer. We developed NBP device, which has been reported to have cytotoxicity on cancer cells induce DNA damage and apoptosis. In particular,reactive oxygen and/or nitrogen species (RONS) were generated by plasma device to give oxidative stress and depolarization of mitochondria membrane in cancer cells. Now we developed a microwave plasma jet system, which could generate nitric oxide (NO) and NO-plasma activated water (NO-PAW). Previously, we found that NO-PAW could induce apoptosis and accumulate ROS in human cervical cancer cells (1). In this study we investigated the efficacy of NO-PAW on oral squamous cancer cells (OSCC), a common malignant tumor of the head and neck (2, 3). We observed that cytotoxicity and apoptotic stimulation by NO-PAW were significantly increased. Also, expression level of caspase and PARP in OSCC was increased after treatment by NO-PAW. However, the apoptosis effect was not observed in human gingival fibroblasts (as normal human cells). Additionally, we found that NO-PAW inhibited cancer progression in vivo. In conclusion, NO-PAW could suppress the progress of OSCC by inducing cell apoptosis. This indicated NO-PAW as a novel tool for OSCC therapy, and probably will be also effective for other oral cancer.

Acknowledgments: This work was supported by the Leading Foreign Research Institute Recruitment Program through the National Research Foundation of Korea (NRF- 2016K1A4A3914113) funded by the Ministry of Science, ICT, and Future Planning (MSIP) of the Korean Government for EH. Choi, I. Han. The Korea government, and the Basic Science Research Program through the NRF of Korea, funded by the Ministry of Education (NRF-2015R1C1A2A01054137) for I. Han.

References:

1. Y. Li, M.H. Kang, H.S. Uhm, G.J. Lee, E.H. Choi, I. Han. Sci Rep 2017;7:1.

2. E. Majchrzak, B. Szybiak, A. Wegner, P. Pienkowski, J. Pazdrowski, L. Luczewski, M. Sowka, P. Golusinski, J. Malicki, W. Golusinski. Radiol Oncol 2014;48:1.

3. B. Wang, S. Zhang, K. Yue, X.D. Wang. Chin J Cancer 2013;32:614.

### Monitoring the Evolution of Therapeutic Resistance

#2833

Single-cell imaging reveals MAPK pathway reactivation in drug-tolerant melanoma cells.

Chen Yang, Chengzhe Tian, Sabrina L. Spencer. _Univ. of Colorado Boulder, Boulder, CO_.

Spontaneous genetic mutations allow an initially drug-sensitive population of cancer cells to acquire a drug-resistant phenotype. However, little is known about how drug-sensitive cells first evade drug action and survive in the presence of drug, referred to as "drug tolerance," a crucial step on the road to resistance. In this study, we combined single-cell time-lapse microscopy and MATLAB-based automated cell tracking to identify the molecular basis of drug tolerance. By simultaneously monitoring CDK2 and ERK activity in single cells, we found that melanoma cells harboring a BRAF-V600E mutation have high ERK activity and consecutively cycled with an inter-mitotic time of around 14 hr in full growth media. Upon treatment with dabrafenib, an ATP competitive B-Raf inhibitor, all cells entered a CDK2-low quiescence with reduced ERK activity. Strikingly, after 72 hr of dabrafenib treatment, a fraction of cells escaped drug action, re-entered the cell cycle, and divided once in presence of drug before re-entering a transient quiescence. Additional cells continued to escape and divide in the presence of drug, with a steady-state level of about 10% of the population engaged in the cell cycle at any point during the week-long observation period. We refer to cells that re-entered the cell cycle as "escapees" and cells that stayed in quiescence during the observation period as "non-escapees." Remarkably, we found that escapees showed significantly higher ERK activity than non-escapees after a few days of treatment, even though their initial ERK activities were indistinguishable. To test whether reactivation of the MAPK pathway is the cause of cell cycle re-entry, we co-treated cells with dabrafenib and the MEK inhibitor trametinib. This co-treatment resulted in even lower ERK activity and elimination of the escapee population. This result implies that dabrafenib, even at high doses, does not fully turn off the MAPK pathway, thereby enabling eventual escape from drug. By monitoring escaping cells at the molecular level in real time, our work thus suggests a possible mechanism for cells to develop drug tolerance in less than three days, long before acquiring genetic mutations.

#2834

Genomewide CRISPR studies of sequential treatment with CRBN-based degronimids: Insights into the molecular evolution of treatment resistance in myeloma and beyond.

Ryosuke Shirasaki, Sara Gandolfi, Ricardo De Matos Simoes, Geoffrey Matthews, Sondra Downey, Olga Dashevsky, Tang Huihui, Michal Sheffer, Eugen Dhimolea, Megan Bariteau, Jeffrey Sorrel, Nick Kwiatkowski, Thinghu Zhang, Nathanael Gray, Constantine Mitsiades. _Dana-Farber Cancer Institute, Boston, MA_.

Degronimids are bifunctional agents in which a thalidomide-like moiety brings the E3 ligase CRBN close to proteins which bind to the second functionality of these compounds, leading to ubiquitination and proteasomal degradation of these latter proteins. Our groups and others have examined BET bromodomain (BRD)-containing proteins BRD2,3, and 4 (dBET6) or CDK9 (ZZ1-33, referred to herein as dCDK9). We documented that both dBET and dCDK9 compounds trigger rapid induction of multiple myeloma (MM) cell death in vitro. To dissect the mechanisms regulating the response vs. resistance of MM cells to degronimids, we performed genome-wide CRISPR/Cas9-based gene editing screens using lentiviral particles of a pooled sgRNA library (Brunello; ~70,000 sgRNAs against ~20,000 genes; 4 sgRNAs/gene; plus additional non-targeting control sgRNAs). In screens with both CRBN-mediated "degraders", we observed consistent and pronounced enrichment of sgRNAs for CRBN itself, and also other upstream components or regulators of the complex that regulates CRBN function (e.g. DDB1, CAND1), E2 ubiquitin conjugating enzymes (e.g. UBE2G1) and multiple members of the signalosome complex (COPS7A, COPS7B, GPS1, et.c.). These results indicate that a primary mechanism of degronimid-resistance relates not to adaptation to the degradation of their respective targets, but to via loss of function (LOF) of CRBN or its positive regulators, to prevent degradation of the degronimid target(s). Also, we observed that dCDK9-resistant MM.1S cells which established from our CRISPR screen, upon more extensive treatment with either dCDK9 or dBET led to further enrichment of the CRBN sgRNA-containing tumor cells, with significant decrease in the fraction of the population with sgRNAs for other hits. These observations imply that LOF for positive regulators of CRBN activity may provide protection in short-term, but the role of several of these genes for MM cell proliferation/survival may limit the longer-term fitness of these populations, allowing cells with CRBN LOF to outcompete other potential forms of resistance. This finding may also indirectly explain why clinical resistance to thalidomide or its derivatives, CRBN-mediated "degraders of IKZF1/IKF3, are reported to involve LOF of CRBN, rather than most of its regulators of CRBN. Our CRISPR studies document the concordance in mechanisms of resistance to "degronimids" targeting different proteins for degradation; and how time-course analyses in these CRISPR screens can reveal the dynamics between sub-populations of tumor cells harboring sgRNA-mediated LOF for different candidate genes.

#2835

**Temporal sequencing of anticancer drugs,** ex vivo **, optimizes therapeutic effect by targeting drug-induced glucose-6-phosphate dehydrogenase.**

Baraneedharan Ulaganathan,1 Andrew Dhawan,2 Biswanath Majumder,3 Munisha Smalley,1 Saravanan Thiyagarajan,3 Gopinath S. Kodaganur,4 S Krishnamurthy,5 Mohammed Mamunur Rahman,6 Elizaveta Freinkman,7 Pradip Majumder,1 Mohammad Kohandel,8 Aaron Goldman9. 1 _Mitra Biotech, Integrative Immuno-Oncology Center, Woburn, MA;_ 2 _Oxford Univeristy, Oxford, United Kingdom;_ 3 _Mitra Biotech, Bangalore, India;_ 4 _Bangalore Institute of Oncology, Bangalore, India;_ 5 _Kidwai Memorial Hospital, Bangalore, India;_ 6 _MBL International, Woburn, MA;_ 7 _Whitehead Institute for Biomedical Research, Cambridge, MA;_ 8 _University of Waterloo, Waterloo, Ontario, Canada;_ 9 _Mitra Biotech, Integrative Immuno-Oncology Center, Brigham and Women's Hospital, Harvard Medical School, Woburn, MA_.

Background: Phenotypic cell state transitions are emerging as novel drivers of transient resistance to cancer chemotherapy. We recently demonstrated that non-cancer stem cells are able to undergo a phenotypic cell state transition that enables acquire a 'reversible' drug tolerant state. Consequently, we made two key discoveries, drug tolerance 1) results in a cross-resistance to other classes of anticancer drugs 2) coincidental to a switch in the metabolic behavior. Further, evidences prompted to investigate if drug-induced metabolic reprogramming contributes to combination therapy resistance in a population of cancer cells that have gained tolerance to a primary therapy. Methods: We investigated the metabolic phenotype of drug tolerant cells using 3-D in-vitro models of breast cancer. A systems biology approach was used to identify key, interconnecting proteins involved in metabolic dysregulation, drawing inferences among signaling networks and events in a temporal context. Using an in-silico simulation we perturbed glucose metabolism, and tested how timing, combination and drug order impact the therapeutic effect of combination therapy and validated our findings using in-vivo experiments. Finally, to provide a direct clinical translation, we tested temporally-sequenced 3-drug combinations using CANscriptTM, a human explant tumor assay that captures the entire tumor ecosystem. Results: We report that conventional chemotherapies used to treat breast cancer, results in an adaptive cross-tolerance against an unrelated chemotherapeutic agent via induction of both glycolytic and oxidative pathways. These drug-tolerant cells switch to a CD44Hi phenotypic cell state, and rely on both the Akt pathway and HIF1α-Glut1 axis in a reactive oxygen species-dependent manner, which temporally cooperate to remodel a glucose shunt towards the pentose phosphate pathway. Mathematically modeling these pathways, we demonstrate how a sequentially-applied, 3-drug combination that includes G6PD metabolic inhibitors and cytotoxic agents can improve therapeutic effect. The use of CANscriptTM demonstrated that pharmacodynamics, biomarkers of resistance, and temporal ordering of drugs can influence the phenotypic response to therapy, reflecting in-vitro and in-vivo evidence, in a patient-specific manner. Conclusions: Timing, sequence and order of drugs is emerging as a critical component of combination therapy for cancer. Our results demonstrate that timing the order of G6PD inhibitors in exquisitely sequenced combination with chemotherapy can emerge as a new paradigm in the treatment of cancer. Ex-vivo, human tumor models that fully capture the tumor microenvironment can contribute to and potentially uncover the mechanisms of action, phenotypic effect, and pharmacodynamics of anticancer drug combinations in distinct temporal sequences.

#2836

Spatially resolved single-cell analysis of cellular plasticity and mechanisms of drug resistance.

Sethuramasundaram Pitchiaya, Jeremy D'silva, Nicole Lee, Sathiyapandi Narayanan, Xia Jiang, Saravana M. Dhanasekaran, Arul M. Chinnaiyan. _Univ. of Michigan Comp. Cancer Ctr., Ann Arbor, MI_.

Cellular heterogeneity adversely affects clinical stratification, treatment decisions, and development of therapeutic resistance in cancer. Heterogeneity manifests as variability in gene expression and scales with the number of unique cell types and/or the extent of phenotypic plasticity. Therefore, an incisive tool that effectively quantifies heterogeneity, robustly identifies rare cell populations, efficiently predicts cell state transitions, and preserves the associated phenotypic manifestations will provide key insights into the mechanisms of plasticity and development of drug resistance. To this end, we developed High-Throughput Single-cell analysis using single-molecule Fluorescence In Situ Hybridization (HITS-FISH) - a completely automated imaging-based tool that provides absolute quantification of gene expression (mature and immature transcripts that are coding or non-coding), while still preserving spatial and morphological information. Using a combination of HITS-FISH and single cell RNAseq, which provides a high-throughput readout of gene expression signatures, we find that multiple, potentially plastic cell states (genetic, epigenetic, and cell cycle) coexist within a seemingly homogeneous population of cancer cells from various tissues. HITS-FISH suggests that one of the major contributors of such heterogeneity is pervasive aneuploidy, which is accentuated during treatment of cancer cells with chemotherapeutic agents and subsequent development of resistance against such drugs. Currently, we are further characterizing these plastic cells via lineage tracing and single-cell analysis to identify pre-resistant cell states and drug-induced gene-expression reprogramming.

#2837

Single-cell heterogeneity with transcriptional memory confers resistance to cancer therapy.

Sydney Shaffer, Benjamin Emert, Arjun Raj. _University of Pennsylvania, Philadelphia, PA_.

Targeted therapies for cancer are a promising class of drugs that inhibit the specific molecular alterations that underlie the uncontrolled proliferation seen in cancer. The primary shortcoming of targeted therapy is disease relapse, which is driven by a subpopulation of cells that are resistant to these drugs. This phenomenon is generally thought to be genetic in origin; however, our recent work on melanoma shows that nongenetic cellular plasticity may provide a mechanism of resistance to these therapies. Furthermore, we showed that through the addition of the drug itself, cells transition from this transient plasticity into a new, stably resistant cell state via cellular reprogramming, suggesting that the time an individual cell exists in a state is important for producing the divergent resistance phenotype. However, there are currently no methods available to quantify the timescale of these fluctuations for the whole transcriptome. Thus, broadly generalizing this concept of timescales for cellular plasticity, we developed a novel method for genome-wide quantification of the timescales of gene expression memory based on a modern version of the ingenious Luria-Delbrück fluctuation analysis. Specifically, we isolated individual cells, allowed them to expand, and then performed RNA sequencing on each of the samples. For genes with uniform expression, all the samples should have similar RNA sequencing counts; however, for genes that occasionally turn on in a subset of cells, the RNA sequencing counts across all samples should show high variance. In melanoma, this method revealed the gene expression state of rare cells resistant to targeted therapy. In a completely new model, triple-negative breast cancer, this method revealed a novel rare subpopulation of cells exhibiting resistance to chemotherapy. More generally, this method has the potential to reveal other new phenotypes associated with rare cell biology, including metastasis and the early stages of stem cell differentiation.

#2838

Discovery of adaptive resistance pathways and anti-resistance combination therapies in cancer from phosphoproteomic data.

Augustin Luna,1 Özgün Babur,2 Gonghong Yan,3 Emek Demir,2 Chris Sander,4 Anil Korkut3. 1 _Harvard Medical School, Boston, MA;_ 2 _Oregon Health & Science University, Portland, OR; _3 _University of Texas MD Anderson Cancer Center, Houston, TX;_ 4 _Dana-Farber Cancer Institute, Boston, MA_.

Drug resistance to targeted therapies, either intrinsic (existing before treatment) or acquired (resulting from adaptation to therapy), is a major challenge in patient care. Drug combinations offer a possible solution to prevent drug resistance by blocking multiple routes to tumor proliferation. Discovery of effective combinations remains a challenging task due to complexity of the underlying biological processes and inter-tumor heterogeneity. Here, we developed a statistical pathway analysis method that (i) reveals pathways involved in drug response and adaptive resistance and (ii) nominates combination targets and therapies to down-regulate the resistance pathways. The method is based on the rationale that (i) use of therapies targeting specific genomic aberrations can lead to compensatory responses (e.g. through feedback loop activation in the short term or the development of oncogenic alterations in the long term) leading to drug resistance, and (ii) collective changes in pathway activities are better predictors of resistance and mitigation strategies than individual responses.

We construct a pathway model of signaling interactions for measured molecular species through automated extraction of pathway information from Pathway Commons plus manual expert curation. Next, we combine the pathway model with the cell-type-specific drug response data to calculate a TargetScore (TS) for each protein. The score quantifies the adaptive pathway responses to a perturbation by integrating the change in the level of a (phospho)protein along with its pathway neighborhood in response to the single drug. A high TS corresponds to involvement in adaptive response (e.g. upregulation of RTK expression by MEK inhibitor via a feedback loop) and a low TS corresponds to the activity of the drug (e.g. inhibition of ERK phosphorylation by MEK inhibitor). Finally, by identifying the sub-pathways with enriched, high TS values, we determine the adaptive resistance pathways. We test the resulting predictions (combinations of the original single drug with drugs targeting members of the resistance pathways) experimentally.

TargetScore is amenable to calculations for hundreds of samples treated with individual (or combinations of) drugs in multiple doses and/or time points and assayed for hundreds to thousands of molecular entities (mRNA or proteomic). Analysis of longitudinal data may allow us to trace the evolution of drug resistance and, potentially, the optimum time points for intervention. The current protocol is defined with a focus on proteomic RPPA data, but can be adapted to other kinds of molecular data associated with adaptive responses. We applied our method to BET-BRD inhibition in ovarian cancer to compare resistance/response pathways in cells with varying degrees sensitivity. The analysis nominated cell-type-specific, anti-resistance combinations involving BET inhibitors.

#2839

Notch3-dependent beta-catenin signaling mediates EGFR TKI drug persistence in EGFR mutant NSCLC.

Rajeswara Rao Arasada,1 Konstantin Shilo,1 Jianying Zhang,1 Rashelle Ghanem,1 Tadaaki Yamada,2 Seiji Yano,2 David Carbone1. 1 _The Ohio State University, Columbus, OH;_ 2 _Kanazawa University Cancer Research Institute, Kanazawa, Japan_.

EGFR tyrosine kinase inhibitors (EGFR-TKIs) usually cause dramatic responses in EGFR-mutant lung cancer, but different patients with identical drivers have widely different progression-free intervals, and resistance universally develops. In those cases without pre-existing resistant clones, even though every cell expresses the driver, some tumor clonogens survive in order to acquire target reactivation or bypass resistance mechanisms. The involvement of β-catenin in EGFR TKI resistance has been previously reported however the precise mechanism by which β-catenin activation contributes to EGFR TKI resistance is not clear. Our transcriptome analysis identified that EGFR inhibition results in the activation of β-catenin signaling in a Notch3-dependent manner, which facilitates the survival of a subset of cells that we call "adaptive persisters". We have previously reported that EGFR-TKI treatment rapidly activates Notch3, and here our co-immunoprecipitation studies identified a physical association of Notch3 with β-catenin, which in turn leads to increased stability and activation of β-catenin. In vitro pulmosphere formation assays and in vivo limiting dilution assays reveled that adaptive persisters display characteristics of stem-like cells. Furthermore, tumor xenograft studies using HCC827 and HCC4006 demonstrate that the combination of EGFR-TKI and the β-catenin inhibitor, ICG-001 inhibits the development of these adaptive persisters, decreases tumor burden, improves recurrence free survival, and overall survival. Overall, Notch3 plays a pivotal role in the maintenance of lung cancer stem cells (CSCs) and the induction of EGFR TKI mediated drug persisters with cancer stem cell like characteristics, but is difficult to selectively target. Our study finds that this effect is driven by the Notch3-dependent activation of β-catenin signaling in a non-canonical fashion. A combination of EGFR TKI and β-catenin inhibitors decreases tumor take, delays tumor recurrence and increases overall survival in EGFR mutant models. Thus, our findings provide novel insights on role of drug persistence early after EGFR TKI therapy and presents a rationale for clinical testing of EGFR TKIs with β-catenin inhibitors for the treatment of EGFR mutant NSCLC.

#2840

Characterization of pazopanib resistance in renal cell carcinoma cell lines.

Bozhena Vynnytska-Myronovska, Joana Heinzelmann, Sebastian Hölters, Michael Stoeckle, Kerstin Junker. _Saarland University, Homburg, Germany_.

BACKGROUND: Together with Sunitinib (Sun), Pazopanib (Paz) is considered as a gold standard in metastatic renal cell carcinoma treatment (RCC). Similarly to other tyrosine kinase inhibitors (TKI), development of resistance constitutes the main problem of Paz-based therapy. This warrants the in vivo investigations of the processes related to Paz resistance development.

METHODS: Parental human RCC cell lines, 786-O/wt and CAKI-2/wt, were continuously treated with Paz at IC50 concentration and Paz-resistant 786-O and CAKI-2 cell subpopulations (786-O/res and CAKI-2/res) were developed. Parental and resistant cells were compared in terms of the malignant phenotype by WST-1 proliferation, cross-resistance to another TKI, migration wound healing assays and signaling pathways analysis by Western blot. MiRNA expression was analyzed using microarrays (Agilent Technologies).

RESULTS: The in vitro analysis of RCC cells growth under Paz treatment revealed that this drug inhibited proliferation of parental 786-O and CAKI-2 cells with IC50 (inhibitory concentration of 50%) of 14 and 11 µmol/L, respectively. The continuous treatment with this drug resulted in the gradual resistance development in both tested cell lines. At the end of the period of continuous treatment (32 passages, 3 months) the resistance level of 786-O/res and CAKI-2/res cells was increased 2-fold compared with 786-O/wt and CAKI-2/wt. The analysis of cell motility revealed that the short-termed exposure to Paz retarded the migration of both parental and Paz-resistant 786-O and CAKI-2 cells. However, no differences in the motility rates of parental and resistant cells were observed. The analysis of the signaling pathways that might be involved in the resistance development revealed no changes in the expression of epithelial-to-mesenchymal transition (EMT) markers N-cadherin, vimentin and β-catenin between parental and Paz-resistant subpopulations of both cell lines. Cell response to Paz treatment differs between the tested parental RCC cell lines CAKI-2 and 786-O in terms of the expression of phosphorylated forms of the growth-promoting molecules of Akt-mTOR and MAPK-ERK pathways. A significant change in miRNA expression was found in both cell lines at different time points of treatment compared to parenteral cells.

CONCLUSIONS: Our data suggest that the process of Paz resistance development is cell line specific. Akt-mTOR and MAPK-ERK signaling pathways seem to be related to Paz resistance development in RCC. Development of resistance is associated with specific miRNA alterations. Further studies have to analyze the functional effects of miRNAs on resistance and possible signaling pathways.

#2841

Her2/neu expression as potential marker of regorafenib resistance in CRC.

Loredana Vecchione,1 Stefania Napolitano,2 Valentina Belli,3 Erika Martinelli,3 Nunzia Matrone,3 Christophe Hapke,1 Ulrich Keilholz,1 Josep Tabernero,4 Fortunato Ciardiello,3 Teresa Troiani3. 1 _Charitè-Universitätsmedizin Berlin, Comprehensive Cancer Center, Berlin, Italy;_ 2 _University of Campania "Luigi Vanvitelli", Napoli, Italy;_ 3 _University of Campania, Napoli, Italy;_ 4 _Vall d'Hebron Institute of Oncology (VHIO), Spain_.

Nature Reviews Clinical Oncology, (2017). doi:10.1038/nrclinonc.2017.43 Jason J. Luke 0 0 2017-03-27T13:39:00Z 2017-11-29T14:35:00Z 1 312 1781 14 4 2089 14.0 Normal 0 false false false EN-US JA X-NONE /* Style Definitions */ table.MsoNormalTable {mso-style-name:"Table Normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-parent:""; mso-padding-alt:0cm 5.4pt 0cm 5.4pt; mso-para-margin-top:0cm; mso-para-margin-right:0cm; mso-para-margin-bottom:8.0pt; mso-para-margin-left:0cm; line-height:107%; mso-pagination:widow-orphan; font-size:11.0pt; font-family:Calibri; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-ansi-language:EN-US;} Regorafenib is a multikinase inhibitor currently approved for the treatment of chemorefractory mCRC patients. No predictive biomarkers of efficacy have been identified. We recently reported that amplification or mutations in HER-2/neu might predict resistance to regorafenib in a small cohort of patients treated with Regorafenib. We therefore sought to better investigate the role of HER-2/neu in response to Regorafenib in preclinical and clinical models. Short-term proliferation assay with Regorafenib was performed in a panel of CRC cell lines. Basal protein expression of total and phospho Her2 were analized by WB analysis. RPPA data from MD Anderson cell lines project were used for external validation of total and phospho HER-2/neu. Sperman correlation was applied to correlate the expression levels of total and phospo HER2/neu and IC50 to Regorafenib. HT29 CRC cell line (HT29) was made resistant to Regorafenib (HT29R). WB analysis of basal levels of proteins of the EGFR and Her2 axis were investigated in both HT29 and HT29R. Moreover, HT29 and HT29R were treated with EGFR inhibitors, Her2/neu inhibitors, MAPK and PIk3Ca inhibitors. Apoptosis was perfromed by FACs analysis by using Annexin-V staining. We found a negative correlation between pHer2 expression and response to Regorafenib in both our data set of CRC cell lines and in the MD Anderson panel. Overexpression of all the epitelial markers of the EGFR and HER2/neu axis was observed in HT29R as compared to HT29. This translated to an increased sensitivity and apoptosis to Trastuzumab in HT29R as compared to HT29 (IC50 with 50 times fold change) and PIk3Ca inhibitor (IC50 with 5 times fold change). No differences of IC50 were observed with other inhibitors. Moreover, combined treatment with Trastuzumab and PIk3Ca inhibitor induced significantly early and late apoptosis in HT29R as compared to HT29. Our preliminary data indicate pHer2 levels to be predictive of resistance to Regorafenib in CRC. Functional work and IHC analysis of CRC patients treated with Regorafenib is ongoing and will be presented.

#2842

The biological effects and therapeutic potential of directly targeting K-Ras.

Andrew L. Wolfe. _University of California San Francisco, CA_.

K-Ras is a small GTPase that is among the most frequently mutated oncoproteins in cancer, with alterations common in lung adenocarcinoma, colorectal cancer, and up to 90% of pancreatic cancers. Tumors with mutant K-Ras often become addicted to its presence and in inducible genetic mouse models the sudden removal of mutant K-Ras can trigger cancer regressions. However, these cancers remain among the most refractory to therapy and pharmacological agents capable of inhibiting many common K-Ras mutants have remained elusive. Here, we set out to evaluate the efficacy of recently developed inhibitors that directly target K-Ras in cancer and understand potential mechanisms of resistance to K-Ras based therapies. Treatment of K-Ras mutant cells with K-Ras inhibitors led to mutant-specific halting of cell growth. Upon extended treatment a subpopulation of drug-resistant cells emerged that displayed adaptation patterns leading to reversible drug resistance. To identify cooperating factors that modulate sensitivity to K-Ras inhibition, screens were performed in wild-type and isogenic K-Ras mutant cells using a genome-wide CRISPRi library and a pharmacological compound library in the presence or absence of K-Ras inhibitors. These strategies identified synthetically lethal pathways and favorable combination therapies, moving toward the goals of identifying the optimal patient populations that can most benefit from K-Ras inhibitors and enabling a personalized medicine approach to cancer therapy.

#2843

Calcium blockers modulate the steroid resistance in acute lymphoblastic leukemia.

Hyun Joo Jung. _Ajou University Hospital, Suwon, Republic of Korea_.

Background: Acute lymphoblastic leukemia (ALL) is one of common cancers in children and adults, and a fair number of ALL patients still die although the overall survival rate is approaching 90% with risk-directed therapy in many developed countries. Response to steroid is the most important prognostic factor of patients with ALL, and the development of steroid resistance is lethal to the ALL patients. To improve survival for steroid-resistant ALL patients, it is necessary to develop new concepts to overcome the steroid resistance of ALL.

Methods: We generated the steroid-adapted subclones through prolonged cultures of ALL cell lines with gradually increased steroid concentration.Also, we used biochemical and molecular methodologies to demonstrate the signaling axis of tissue transglutaminase (TG2)-nuclear factor-kappa B (NF-kappa B) and cell cycle to evaluate the effect of calcium blockers on the steroid-resistant ALL cells.

Results: Steroid-adapted ALL cells that were generated by culture with gradually increased steroid concentration were much more resistant to steroid than their parent ALL cells. We found that calcium blockers altered TG2 activity and nuclear factor-kappa B (NF-kappa B) expression in these steroid-resistant ALL cells. Also, calcium blockers induced the change of expression of NF-kappa B target genes in steroid-adapted ALL cells. In addition, calcium blockers induced the change of ABC transporter expression and the quiescence of steroid-resistant ALL cells. We also found that calcium blockers induced apoptosis of steroid-resistant ALL cells and reduced the steroid resistance of steroid-resistant ALL cells.

Conclusion: This study shows that calcium blockers improved the cytotoxicity of steroid on steroid-resistant ALL cells via manipulation of TG2 and NF-kappa B activities and cell cycle. The combination of calcium blockers with chemotherapeutic agents may be a new alternative strategy to overcome the steroid resistance of ALL.

#2844

Cisplatin response of NSCLC cells irresponsive to knocking down VRAC subunits LRRC8A or LRRC8D.

Nico O. Ruprecht,1 Martin Hungerbühler,1 Christof Granzow,2 Christoph M. Kempf,1 Johannes T. Heverhagen1. 1 _University Hospital Inselspital Bern, Bern, Switzerland;_ 2 _FLACOD GmbH, Germany_.

Platinum compounds represent the backbone of combination chemotherapy protocols for advanced lung cancer. Their efficacies are limited chiefly by either primal or chemotherapeutically acquired cellular platinum resistance whose complex mechanisms became more accessible through recent discoveries (Voets et al., 2015, EMBO J 34(24): 2985-2987). By stepwise increasing the cisplatin concentration in the culture medium, we generated cisplatin-resistant sublines of the wild type A240286S cell strain which derived from the suprarenal metastasis of a lung adenocarcinoma (Heuser et al., 2005, Cancer Lett 223(1): 57-66). All IC50 values were determined under flavin-protecting conditions (Granzow et al., 1995, Cancer Res 55(21): 4837-4843). For cisplatin, IC50 values were determined of 0.46±0.05 µM in the wild type A240286S cells, 6.03±1.39 µM in the Pt2.0 A240286S subline (growing in the presence of 2 µM cisplatin), 9.09±0.56 µM in the Pt4.0 A240286S subline (growing in the presence of 4 µM cisplatin), and 18.49±1.96 µM in the Pt8.0 A240286S subline (growing in the presence of 8 µM cisplatin). Testing for cross resistance to oxaliplatin of these tumor cell lines yielded respective IC50 values of 0.19±0.01 µM, 0.50±0.06 µM, 0.58±0.18 µM, and 0.78±0.11 µM oxaliplatin. Cultivation in the absence of cisplatin for seven months reduced the respective IC50 values for cisplatin to 0.99±0.07 µM in the Pt2.0 A240286S subline and to 4.02±0.23 µM in the Pt4.0 A240286S subline. As described by Planells-Cases et al., 2015, EMBO J 34(24): 2993-3008, the subunit composition of VRAC may determine the cellular resistance to platinum-based anti-cancer drugs in haploid KBM7 leukemia cells. In our study, the LRRC8D expression decreased with increasing cisplatin levels. In the Pt8.0 A240286S subline, LRRC8D expression was nearly abolished. However, knockdown of the LRRC8A or LRRC8D subunits of VRAC showed no effect on the cisplatin response in A240286S wild type cells. The respective IC50 values determined were 0.47±0.03 µM in the LRRC8A knockdown cells and 0.61±0.01 µM in the LRRC8D knockdown cells. In conclusion, in A240286S NSCLC cells, knockdown of the subunit composition of VRAC had no effect on cisplatin response. In addition, and in agreement with clinical experience (Stordal et al., 2007, Cancer Treat Rev 33(4): 347-357), the induction of cisplatin resistance in these cells was accompanied by cross resistance to oxaliplatin. Acknowledgments: We thank Rolf Jaggi and Irene Keller for contributing excellent technical support.

#2845

The mechanisms of resistance to temozolomide in glioma cells.

Shigeo Ohba, Yuichi Hirose. _Fujita Health University, Toyoake, Japan_.

Glioblastoma is one of the most aggressive tumor in the central nervous system tumors, with 5-year survival rates of less than 10%. The standard therapy for glioblastomas is maximal safe resection, followed by radiation therapy and chemotherapy with temozolomide (TMZ). One of the reasons of the worse prognosis of the tumor is the acquisition of resistance to TMZ in glioma cells. TMZ is a DNA-methylating agent. TMZ-induced O6-methylguanine adducts, in the absence of repair by O6-methylguanine DNA methyltransferase (MGMT), mispair with thymine during the next DNA replication cycle, leading to futile DNA mismatch repair (MMR) by the MMR system, formation of double strand breaks (DSBs) and eventual cell death of glioma cells. Several mechanisms have been proposed about the acquisition of resistance to TMZ in glioblastomas, such as changes of expression of MGMT, MMR, nucleotide excision repair and homologous recombination (HR). To clarify the mechanisms of resistance to TMZ and to find the way to overcome the resistance to TMZ, several clones of U251 cells resistant to TMZ were obtained and analyzed. FACS analyses showed several patterns of cell cycle distributions of the cells treated with TMZ. #3 resistant clone showed G2 arrest at day3 after TMZ exposure and this arrest was abrogated sooner compared to the G2 arrest of parental U251. TMZ did not induce G2 arrest in #8 resistant clone. Consistent with the results, TMZ-induced DSBs in #3 resistant cells were rapidly disappeared compared to those in U251 cells, and the DSBs were least induced by TMZ in #8 resistant cells. The expression of MGMT was not found in U251 parental cells, #3 cells nor #8 cells, which mean MGMT was not associated with the resistance to TMZ in those cells. The protein levels of MSH6, which was associated with MMR, was reduced in #8 cells. The inhibitor of PLK1 or PARP reduced the cell growth independent of MMR. The ability of HR was increased in #3 resistant clone. By suppression of Rad51, which was major factor associated with HR, the resistant #3 clone was re-sensitized to TMZ, however #8 was not. These results suggested that inhibition of HR may be a viable means to resensitize some kinds of temozolomide-resistant gliomas to TMZ.

#2846

Extracellular ATP induces different types of drug resistances in cancer cells through ATP internalization-mediated intracellular ATP level increase.

Xiaozhuo Chen, Xuan Wang, Haiyun Zhang, Yanrong Qian, Yunsheng Li, Yanyang Cao, Pratik Shriwas. _Ohio Univ., Athens, OH_.

Cancer cells are able to uptake extracellular ATP (eATP) via macropinocytosis to elevate intracellular ATP (iATP) levels, enhancing their survival from drug treatment (1). However, the involved drug resistance mechanisms are unknown. Here we investigated the roles of eATP as either an energy or a phosphorylating molecule in general drug resistance mediated by ATP internalization and iATP elevation. We report that eATP increased iATP levels and promoted drug resistance to various tyrosine kinase inhibitors (TKIs) and chemo-drugs in human cancer cell lines of five cancer types, including non-small cell lung cancer, breast cancer, colon, liver cancer, and pancreatic cancer (2). In A549 lung cancer cells, the resistance was downregulated by macropinocytosis inhibition or siRNA knockdown of PAK1, an essential macropinocytosis enzyme, strongly suggesting that the resistance is mediated at least in part by micropinocytosis (and therefore ATP internalization) (2). The elevated iATP upregulated the efflux activity of ABC transporters in A549 and SK-Hep-1 cells, as well as phosphorylation of PDGFRα and proteins in the PDGFR-mediated Akt-mTOR and Raf-MEK signaling pathways in A549 cells, increasing cell cycle progression and decreasing drug-induced apoptosis. Similar phosphorylation upregulations were found in A549 tumors. The resistance was not significantly affected by purinergic receptor-mediated signaling. These results demonstrate, for the first time, that eATP induces different types of drug resistance by eATP internalization and iATP elevation, implicating the ATP-rich tumor microenvironment in cancer drug resistance, expanding our understanding of the roles of eATP in the Warburg effect and offering new anticancer drug resistance targets.

References

1.Qian, Y. et al. Mol Cancer Res 14(11); 1087-1096 (2016)

2.Wang, X. et al. Oncotarget 8, 87860-87877 (2017).

#2847

Investigation of the combinatorial treatment strategy for BRAFmutant anaplastic thyroid cancer based on real time monitoring technology.

Hyung Kwon Byeon,1 Minhee Ku,2 Ji Hye Choi,2 Hyunjung Kee,2 Ji-Hoon Kim,3 Yoon Woo Koh,2 Jaemoon Yang2. 1 _Korea University Medical Center, Seoul, Republic of Korea;_ 2 _Yonsei University College of Medicine, Seoul, Republic of Korea;_ 3 _Yonsei University Wonju College of Medicine, Wonju, Republic of Korea_.

Purpose Previously, we have identified that c-Met-mediated reactivation of PI3K/AKT pathway contributes to acquired resistance to BRAF inhibitor in BRAF mutant (BRAFm) anaplastic thyroid cancer (ATC). So dual inhibition of BRAF and c-Met led to sustained treatment response. However, previous RTK array result in 8505C cells following PLX4032 treatment revealed increased expression of Src together with c-Met. Therefore further in-depth investigation is required to elucidate the optimal combinatorial strategy to maximize the treatment effects in BRAFm ATC. Using non-invasive, real-time monitoring in vitro and in vivo technologies, the best treatment strategy was investigated with consideration of not only the specific treatment regimens but also the mode of combinatorial treatment.

Methods Real-time cell analysis (RTCA), a cell-based label-free technology using electronic impedance sensors was adopted as a strategy for real-time screening of cellular response to drugs. Live wound healing assays with supporting live cell microscopy and confocal microscopy were conducted for in vitro analyses and both ectopic xenograft and metastatic mouse models were utilized for in vivo analyses. Tumor characteristics were evaluated using 9.4T MR diffusion weighted imagings.

Results Dual inhibition of BRAF and c-Met was not fully effective in overcoming BRAF inhibitor resistance in BRAFm ATC. Src-mediated signal has a greater contribution than c-Met-mediated signaling in conferring BRAF inhibitor resistance. Inhibition of Src exerted a more potent effect than c-Met inhibition. Simultaneous drug treatment was more effective than sequential therapy. Combinatorial treatment of BRAF inhibitor (PLX4032) and Src inhibitor (dasatinib) presented the maximal therapeutic effect against BRAFm ATC.

Conclusions Real-time/non-invasive monitoring assays sensitively provides more accurate information regarding drug response.

#2848

Radiographic and genomic evolution of individual metastases during HER2 blockade in colorectal cancer.

Giulia Siravegna,1 Luca Lazzari,1 Andrea Sartore-Bianchi,2 Giovanni Crisafulli,1 Benedetta Mussolin,1 Andrea Cassingena,2 Cosimo Martino,1 Richard Lanman,3 Rebecca Nagy,3 Giorgio Corti,1 Alice Bartolini,1 Pamela Arcella,1 Monica Montone,1 Francesca Lodi,1 Alice Vanzati,2 Emanuele Valtorta,2 Giovanni Cappello,1 Andrea Bertotti,1 Sara Lonardi,4 Vittorina Zagonel,4 Francesco Leone,1 Mariangela Russo,1 Antonella Balsamo,1 Mauro Truini,2 Federica Di Nicolantonio,1 Alessio Amatu,2 Erica Bonazzina,2 Silvia Ghezzi,2 Daniele Regge,1 Angelo Vanzulli,2 Livio Trusolino,1 Salvatore Siena,2 Silvia Marsoni,1 Alberto Bardelli1. 1 _Inst. for Cancer Research and Treatment, Candiolo, Italy;_ 2 _Grande Ospedale Metropolitano Niguarda, Milano, Italy;_ 3 _Guardant Health, CA;_ 4 _Istituto Oncologico Veneto - IRCCS, Padova, Italy_.

Targeting HER2 with trastuzumab and lapatinib is effective in ERBB2 amplified metastatic colorectal cancer (mCRC). Although at least 30% of the patients initially respond, secondary resistance occurs in most of the cases.

Since the drivers of secondary resistance to trastuzumab and lapatinib in ERBB2 amplified mCRC are unknown, we exploited longitudinal plasma collections and patient-derived cell models to define the molecular bases of resistance to HER2 blockade. Levels of ERBB2 amplification in plasma circulating tumor DNA (ctDNA) paralleled response and relapse. The emergence of EGFR, ERBB2, RAS, BRAF and PIK3CA variants in ctDNA was associated with resistance. Radiographic measurements of individual metastases coupled with longitudinal liquid biopsies unveiled lesion-specific patterns of heterogeneous response in several patients. Phylogenetic tracking and functional analyses on tissue samples and patient-derived cell models established from eight metastases of a single case revealed new druggable oncogenic dependencies and genomic evolution associated with resistance. These data highlight the relevance of coupling imaging and liquid biopsies analyses in precision oncology and provide the rationale for additional lines of therapies in HER2 positive mCRC relapsing upon HER2 blockade.

#2849

Biodynamic imaging predicts response of breast cancer patients to neoadjuvant chemotherapy.

John J. Turek,1 David D. Nolte,1 Ran An2. 1 _Purdue Univ., West Lafayette, IN;_ 2 _Animated Dynamics, Inc, Indianapolis, IN_.

The goal of this research was to determine if biodynamic imaging (BDI) and frozen breast cancer tumors from tissue banks could be used to retrospectively assess response to chemotherapy and identify the more chemoresistant patients. Neoadjuvant chemotherapy (NAC) is a common protocol for breast cancer patients prior to surgery. Many patients will respond to initial chemotherapy, but there is a significant percentage of patients that do not receive a benefit from NAC due to chemoresistance. Accurate prediction of chemoresistance prior to therapy, could enhance the effectiveness of NAC by allowing for better drug selection. BDI uses Doppler detection of light scattered by intracellular motion in living tissue, and several xenograft, and clinical studies have shown that this phenotypic profiling method is a highly accurate predictor of resistance to chemotherapy. Frozen, clinically annotated breast cancer samples were obtained from several tissue banks. All of the patients received adriamycin-cyclophosphamide and paclitaxel (AC-T) neoadjuvant chemotherapy prior to surgery. The samples were rapidly thawed and 1mm3 pieces were placed in tissue culture medium in a 96 well plate, sealed with a gas permeable membrane, and 6 replicates for each drug treatment assayed over an 18 hour period. Drugs tested were the AC combination, and also the individual drugs (adriamycin, 4-hydroxycyclophosphamide, and paclitaxel). A negative drug vehicle control was 0.1% DMSO in growth medium, and vincristine was used as a positive control. For the analysis, patients were classified as sensitive or resistant based upon the clinically annotated pathology reports. The BDI spectral data for each drug treatment was analyzed using a machine learning algorithm to identify patterns in the frequency vector biomarkers for each patient using a combination of a similarity matrix and a linear separability analysis using a neural net perceptron. The neural net perceptron and hold-out cross validation of BDI spectral data correctly identified all patients as sensitive or resistant. Frozen samples from tissue banks contain enough viable cells to evaluate drug response using BDI. This makes it possible to conduct retrospective studies on chemoresistance using frozen banked tissue samples and also makes it possible to study the relationship between genotype to chemo-phenotype. Supported by Animated Dynamics, Inc.

#2850

FGFR signaling facilitates recurrence of minimal residual disease post trastuzumab emtansine treatment in breast cancer.

Saeed Salehin Akhand, Connor Purdy, Michael Wendt. _Purdue Univ., West Lafayette, IN_.

The Her2 subtype of breast cancer (BC) accounts for about 20-25% of all the BC patients. This subtype is defined by the overexpression of human epidermal growth factor receptor 2 (Her2) which drives elevated downstream signaling promoting tumorigenesis. Trastuzumab emtansine (T-DM1) is an antibody drug conjugate in which an anti-Her2 antibody targets Her2 overexpressing tumor cells and delivers emtansine, a highly potent microtubule inhibitor. Metastatic BC patients who have progressed on Trastuzumab show improved overall survival with T-DM1 treatment. However, there is an unmet medical need in a group of patients where their initial response is followed by disease recurrence due to the development of acquired resistance. Therefore, it is important to investigate the molecular mechanisms of resistance to T-DM1. In this study, we established minimal residual disease (MRD) following T-DM1 treatment of HMLE-Her2 xenografts. Upon removal of the drug, tumors relapsed and were no longer responsive to T-DM1. In addition, immunohistochemistry staining revealed that these T-DM1 drug-resistant tumors lost their Her2 expression. In contrast to these in vivo results, continuous treatment of HMLE-Her2 cells with T-DM1 failed to show any acquired resistance in vitro. However, induction of epithelial-mesenchymal transition (EMT) via pretreatment with TGF-β facilitated acquisition of drug resistance to T-DM1. Similar to our in vivo resistant model, these in vitro resistant cells showed diminution in Her2 expression. Flow cytometry analysis suggested that TGF-β treatment may promote a heterogeneous expression of Her2, which helps the selection of a low Her2 expressing, T-DM1 resistant population. Next, we performed cell viability assays in the presence various kinase inhibitors. While the T-DM1 resistant cells were not sensitive to Lapatinib and Afatinib; they showed a dramatic response to FIIN-4, a covalent inhibitor of FGFR. Indeed, we observed elevated expression of FGFR1 at both the mRNA and protein levels in the T-DM1 resistant models. Moreover, we have found that ectopic overexpression of a truncated FGFR1 splice variant, FGFR1β, was sufficient to increase cell survival in response to T-DM1 treatment. Along these lines, orthotopic HMLE-Her2 tumors were less responsive to T-DM1 upon FGFR1β overexpression. Furthermore, overexpression of FGFR1β also leads to enhanced tumor recurrence following T-DM1 treatment. In line with these observations, we found that a patient-derived xenograft (PDX) from a Her2+ BC patient who had progressed on trastuzumab, were responsive to FIIN-4 while showing limited response to T-DM1. Currently, we are investigating potential mechanisms of FGFR mediated survival of T-DM1 resistant cells. Finally, we are exploring combination therapeutics using T-DM1 and FGFR inhibitors in various models of Her2 BC.

#2851

The evolution of tumor cells under AsiDNA treatment results in "autosensitization".

Maria Kozlac,1 Wael Jdey,2 Pierre-Marie Girard,1 Françoise Bono,2 Marie Dutreix1. 1 _Institut Curie, Orsay, France;_ 2 _Onxeo, Paris, France_.

Purpose: The Achilles' heel of all conventional and targeted anticancer treatments is intrinsic or acquired resistance. The era of precision medicine where tumors are selected for specific treatments based on their genetic alterations is revolutionary, but unfortunately prolonged responses are rarely observed due to rapid emergence of resistant clones. We recently developed a new concept of DNA repair inhibitor (Dbait) acting by activating enzymes involved in DNA damage signaling. We tested how such agonist activity would be prone to induce resistance to Dbait treatment.

Experimental design: We performed repeated cycles of treatment with various targeted therapy agents (Imatinib, Olaparib, 6-thioguanine and the clinical form of Dbait, AsiDNATM). We analyzed the specific sensitivity of independent cultures after each treatment cycle. The study was performed in different tumor cell lines ((MDAMB231, MDAMB468, HCC1143, THP1, U937, KBM7) and in two non-malignant breast cell lines (MCF10A, MCF12A). Extensive transcriptome and genome studies were performed on MDAMB231 breast cancer cell lines.

Results: Resistant clones appeared at a frequency of 1.45% for Olaparib, 0.62% for imatinib, and 1.66% for 6-thioguanine. Similar protocol did not select for resistance to AsiDNA. Unexpectendly, tumor cells became more sensitive to AsiDNA after each cycle of treatment. This behavior was specific of AsiDNA and was not observed with other treatments. The six tumor cell lines tested developed AsiDNA autosensitisation and no resistance. Non tumoral cells were not affected by repeated treatments. The acquired sensitivity of the treated tumor populations was conserved for months after end of treatment Transcriptional and genetic analysis of independently treated MDAMB-231 populations reveals that all evolved similarly. They display a few conserved genome modifications and a large deregulation of 1160 genes resulting in an overexpression of luminal associated genes and a decrease in mesenchymal associated genes.

Conclusion: Our results indicate a phenomenon of "autosensitisation", along treatment which has never been described for anticancer treatment and could prevent development of resistance during treatment.

#2852

Identifying circulating biomarkers of acute response and resistance to clinical ATR and Chk1 inhibitors.

Nicholas Bateman,1 Wei Ao,1 Domenic Tommarello,1 Kelly Conrads,1 Pang-ning Teng,1 Kathleen Darcy,1 Chad Hamilton,1 G. Larry Maxwell,2 Christopher Bakkenist,3 Thomas Conrads4. 1 _Gynecologic Cancer Center of Excellence, Annandale, VA;_ 2 _Department of Obstetrics & Gynecology, Falls Church, VA; _3 _University of Pittsburgh, Pittsburgh, PA;_ 4 _Inova Schar Cancer Institute, Falls Church, VA_.

Objectives: Clinical ATR kinase inhibitors (ATRi) are an emerging class of therapeutics being used to treat diverse solid tumor malignancies, including ovarian cancers. The development of circulating biomarkers indicating response or resistance to clinical ATRi will support the ongoing development of this emerging class of therapeutics. This study is focused on establishing isogenic models of ATRi-resistance in ovarian cancer cell lines and identifying conserved secreted protein alterations correlating with acute response or resistance to ATRi treatment in these models. Methods: We generated isogenic cell line models of ATRi-resistant, CCNE1-amplified (OVCAR3) and non-CCNE1 amplified (OV90) ovarian cancer cell by metronomic treatment of cell lines with the ATR inhibitor AZD6738 (Astra Zeneca). We characterized sensitivity of models to AZD6738 as well as inhibitors of checkpoint kinase 1 (Chk1, LY2606368), ataxia-telangiectasia mutated (ATM, KU55933) poly (ADP-ribose) polymerase (PARP, BMN-673) and cisplatin combination by dose response assay and further assessed impact of ATRi treatment on cell cycle. Lastly, we analyzed conditioned media by LC-MS/MS-based proteomic analyses from ATRi-resistant OVCAR3 cells or following acute treatment with ATRi. Results: Metronomic treatment of ovarian cancer cells with ATRi induced resistance to ATRi irrespective of CCNE1 status. ATRi-resistant cells were also resistant to Chk1i, but not to ATM, PARP inhibitors or combinations of ATRi and cisplatin. Cell cycle analyses revealed that ATRi-sensitive cells arrest in S-phase and continue active DNA replication, whereas ATRi-resistant cells arrest in G1/S phase and cease DNA replication following ATRi treatment. Comparison of proteins identified in conditioned media revealed > 30 proteins bearing signal peptide sequences potentially mediating cellular secretion as significantly elevated in the cellular secretomes of ATRi-resistant or ATRi sensitive cells treated with ATRi, with only a single protein co-altered between these sample, i.e. alpha-N-acetylgalactosaminidase (NAGA). Conclusions: Our findings show that metronomic treatment of ovarian cancer cells with ATR inhibitors induces resistance to ATRi as well as an inhibitor of Chk1, an immediate downstream effector of ATR. Our analyses show that ATRi-resistant cells remain sensitive to inhibitors of orthogonal DNA damage response signaling pathways, i.e. ATM and PARP, as well as to cisplatin sensitization induced by ATRi co-treatment. ATRi-resistant cells further exhibit a conserved G1/S-phase cell cycle arrest response to ATRi treatment. Lastly, our preliminary secretome analyses provide proof of concept data that identification of secreted biomarkers specific for acute response or resistance to ATRi (Chk1i)-treatment is feasible.

#2853

Development of novel preclinical models of secondary resistance to the anti-CD37 antibody drug conjugate (ADC) IMGN529/DEBIO1562 in diffuse large B-cell lymphoma (DLBCL).

Alberto J. Arribas,1 Luciano Cascione,1 Luca Aresu,1 Eugenio Gaudio,1 Andrea Rinaldi,1 Chiara Tarantelli,1 Murodzhon Akhmedov,1 Emanuele Zucca,2 Davide Rossi,1 Anastasios Stathis,2 Francesco Bertoni1. 1 _Università della Svizzera italiana, Institute of Oncology Research, Bellinzona, Switzerland;_ 2 _IOSI Oncology Institute of Southern Switzerland, Bellinzona, Switzerland_.

Background: CD37 is a membrane protein expressed almost exclusively in cells of the immune system (Bertoni & Stathis, Blood 2016). The IMGN529/DEBIO1562 ADC consists of an anti-CD37 antibody conjugated via a thioether-based linker to the cytotoxic anti-microtubule agent maytansinoid DM1. IMGN529/DEBIO1562 has shown preclinical (Deckert et al., Blood 2013; Gaudio et al., ASH 2016) and clinical antilymphoma activity (Stathis et al., ASH 2014). A better understanding of the resistance mechanisms could lead to rational combination strategies to prevent development of resistance and enhance efficacy. Hence, we generated DLBCL cell lines with secondary resistance to IMGN529/DEBIO1562.

Materials and Methods: IMGN529/DEBIO1562 resistance was generated through chronic treatment of cell lines for over 6 months. Proliferation of acquired stable resistance was tested by MTT assay (72 hrs) in resistant and parental cells after 2 weeks of drug-free culture. Multidrug resistance phenotype (MDR1 expression by real-time PCR) was ruled out. Resistant and parental cells underwent transcriptome profiling by RNA-seq and whole-exome sequencing (WES). CD37 surface expression was determined by FACS.

Results: The activate B-cell like SU-DHL-2 and the germinal center B-cell type SU-DHL-4 DLBCL cell lines were exposed to increasing concentrations of IMGN529/DEBIO1562 starting from IC50s for several months until they acquired resistance to the compound. Two SU-DHL-2 and 2 SU-DHL-4 resistant cell lines were obtained. In parallel, both cell lines were cultured upon the same conditions with no drug exposure (parental). SU-DHL-2 and SU-DHL-4 resistant lines showed IC50s that were approximately 10-fold higher than parental counterpart while maintaining a sensitivity similar to parental cells when exposed to the free DM1 toxin. CD37 expression was lost at both protein and mRNA levels in SU-DHL-2 resistant cells, and this appeared due to an homozygous deletion affecting the CD37 gene locus at 19q13.33, as identified by WES. CD37 loss was also associated to changes of the recently described CD37 signatures in DLBCL patients (Xu-Monette et al., Blood 2016). Besides the CD37 gene loss, the populations of resistant SU-DHL-2 cells shared 25 somatic mutations in genes coding for extracellular matrix components, protein kinases and transcription factors. The SU-DHL-4 resistant cells, which did not lose CD37 expression, harbored 48 shared mutations affecting kinases, cytokines, transcription factors and oncogenes and were enriched in specific pathways at RNA-seq.

Conclusions: We presented two novel DLBCL models of secondary resistance to the anti-CD37 ADC IMGN529/DEBIO1562. These models, apparently driven by different biologic processes, will help in clarifying mechanisms of resistance to the drug and developing combination therapeutic approaches.

#2854

**Reconstruction of BRAF** V600E **driven colorectal carcinogenesis and identification of novel drug combinations involving BRAF and RTK inhibitors.**

Nadine Reischmann,1 Ricarda Herr,1 Sebastian Halbach,1 Miriam Heizmann,1 Hauke Busch,2 Melanie Boerries,1 Tilman Brummer1. 1 _University of Freiburg, Freiburg, Germany;_ 2 _Univerity of Luebeck, Luebeck, Germany_.

BRAF mutations are found in approximately 10% of colorectal cancers (CRC) and are associated with an aggressive, less-differentiated and therapy-resistant phenotype. Inhibitors targeting BRAFV600E elicit only limited survival benefits when used as single agents. This unresponsiveness was mechanistically attributed to the relief of negative feedbacks on the epidermal growth factor receptor (EGFR) and initiated dual and triple combinatorial trials. These trials often involve the combination of BRAF inhibitors (BRAFi) and/or MEK inhibitors with anti-EGFR antibodies such as cetuximab or panitumumab. Although first results of these dual or triple therapies demonstrated improved efficacy, the response rates still were heterogeneous. Here, we show that BRAFi upregulate a variety of receptor tyrosine kinases (RTK) in CRC cell lines, including not only the EGFR, but also human epidermal growth factor receptor (HER) 2 and HER3. Importantly, combination of BRAFi (vemurafenib, dabrafenib or encorafenib) with inhibitors dually targeting the EGFR and HER2 (lapatinib, canertinib or afatinib) significantly reduced the metabolic activity and proliferation of CRC cells. Similarly, genetic depletion of HER2 and HER3 re-sensitized CRC cells to BRAF inhibition. Interestingly, BRAF inhibition also led to increased levels of the GRB2-associated binders (Gab) 1 and Gab2, two important mediators of RTK signaling. The Gab2 upregulation was directly dependent on the loss of BRAFV600E signaling and not caused by "off-target" effects, as demonstrated by allele-specific shRNA mediated BRAFV600E knockdown. These findings suggest new escape mechanisms for current treatment regimens and indicate that targeted therapy in BRAF mutant CRC could benefit from broad RTK pathway blockade.

Importantly, the BRAF/HER family inhibitor combination was also more effective in murine intestinal MouseT1 cells (originating from a Vil-Cre;BrafLSL-V637E/+;Tp53LSL-R172H/+ mouse model), which indicates a species-independent phenomenon. These novel and our previous findings that BRAFV600E suppresses features of epithelial differentiation and effector function prompted us to establish crypt organoid cultures from mice carrying conditional BraffloxV600E/+ and/or Tp53LSL-R172H/+ knock-in alleles, either singly or in combination. Using a Vil-CreERT2 transgene, we were able to induce expression of oncogenic BRAF and dominant-negative p53R172H in organoids by 4-hydroxy-tamoxifen mediated Cre activation. We demonstrate that the sudden expression of BRAFV600E and TP53R172H induce marked morphological and molecular changes in small and large intestinal crypts. This organoid model system represents an excellent tool to better understand key characteristics of BRAF mutant CRC such as intrinsic aggressiveness, poor differentiation and resistance to BRAFi.

#2855

Identification of optimal treatment sequence and acquired resistance mechanisms of ALK inhibitors using EML4-ALK transgenic mouse model.

Kyoung-Ho Pyo,1 Lim Sun Min,2 Jae Hwan Kim,1 Ji Min Lee,1 Ha Ni Jo,1 Jae Soek Cho,1 Mi-Ran Yun,3 Sung Eun Kim,1 Hye Ryun Kim,4 Chun-Feng Xin,1 Tae-Min Kim,5 Byoung Chul Cho4. 1 _Yonsei Univ., Seoul, Republic of Korea;_ 2 _CHA Bundang Medical Center, Seongnam-si, Gyeonggi-do, Republic of Korea;_ 3 _JEUK Institute for Cancer Research, Republic of Korea;_ 4 _Yonsei Cancer Center, Seoul, Republic of Korea;_ 5 _The Catholic University, Seoul, Republic of Korea_.

Introduction: EML4-ALK is a distinct molecular entity that is highly sensitive to ALK tyrosine kinase inhibitors (TKIs). While many trials have showed the superiority of ALK TKIs over cytotoxic chemotherapy, the optimal sequencing of ALK TKIs is still obscure. Acquired resistance to ALK TKIs remains a key challenge, and unknown mechanisms of resistance need to be resolved. In this study, we aimed to identify optimal treatment sequence and acquired resistance mechanisms of ALK TKIs using EML4-ALK transgenic mice model that recapitulates human EML4-ALK lung adenocarcinoma.

Methods: The tumorigenesis of EML4-ALK transgenic mice was based on Cre-ERT2/Lox system, and intraperitoneal injection with tamoxifen induced lung tumors. When tumor nodules were observed, EML4-ALK transgenic mice were treated with either crizotinib (150mg/kg) followed by ceritinib (75mg/kg) upon progression (N=13) or with ceritinib followed by crizotinib (N=12) upon progression. Tumor response was evaluated weekly using magnetic resonance imaging. Progression-free survival (PFS) was measured to compare the efficacy between two ALK TKIs. For the analysis of acquired resistance mechanism, whole-exome sequencing, RNA sequencing, and targeted sequencing of ALK gene were performed.

Results: Head-to-head comparison of crizotinib and ceritinib was performed in the first-line setting. A total of 13 mice were treated with upfront crizotinib, and mice that showed progressive disease (PD) were crossed over to ceritinib (n=9). A total of 12 mice were treated with upfront ceritinib, and mice that showed PD (n=8) were crossed over to crizotinib. Four mice in each group were sacrificed for analysis of resistance mechanism at the time of PD. The PFS of ceritinib was significantly longer than that of crizotinib [24 weeks (95% CI, 21.4-26.5) vs. 8 weeks (95% CI, 6.5-9.0), P < 0.001]. Subsequent treatment with ceritinib following crizotinib resulted in significantly longer PFS than crizotinib following ceritinib [7 weeks (95% CI, 4.1-9.9) vs 3 weeks (95% CI, 2.5-3.5), P < 0.001]. Altogether, treatment with ceritinib followed by crizotinib showed a prolongation of PFS, compared to crizotinib followed by ceritinib (27 weeks vs. 15 weeks, P < 0.001). We noted that ALK-TKI resistant tumors harbored several copy number variations (CNVs), and nonsynonymous oncogenic mutations, but found no previously reported resistant mechanisms including secondary mutations, or CNVs. Wnt signaling related gene set was increased in both crizotinib- and ceritinib-resistant tumors, and Hippo signaling related gene set was increased in ceritinib-resistant tumors on KEGG pathway-based analysis.

Conclusion: Our findings suggest that ceritinib is superior to crizotinib when used in the first-line setting. Novel mechanisms of acquired resistance such as increased Wnt and Hippo signaling need further validation.

#2856

Targeting OXPHOS with IACS-010759 to eliminate standard of care resistant tumor cells.

Joseph R. Marszalek, Sahil Seth, Denise Corti, Qi Zhang, Gloria V. Echeverria, Lina Han, Yuting Sun, Jennifer Molina, Sonal Gera, Edward Chang, Tin O. Khor, Mikhila Mahendra, Ningping Feng, Jason P. Gay, Timothy McAfoos, Virginia Giuliani, Xi Shi, Sabrina Jeter-Jones, Sarah Loponte, Chieh-Yuan Li, Christopher A. Bristow, Maria Emilia Di Francesco, Helen Piwnica-Worms, Marina Konopleva, Alessandro Carugo, Andrea Viale, Philip Jones, Timothy P. Heffernan, Giulio F. Draetta. _UT MD Anderson Cancer Center, Houston, TX_.

Tumors are comprised of heterogenous populations of tumor cells that rely on both glycolysis and oxidative phosphorylation (OXPHOS) for bioenergy and synthetic processes in support of cell proliferation. Over the past few years, we and others have reported that there is a subpopulation of tumors cells that are resistant to standard of care treatment or targeted therapies, and that these so-called persistent tumor cells possess stem cell like properties. Of note, these cells have elevated levels of mitochondria and are dependent on OXPHOS for survival. We have previously disclosed the discovery of IACS-010759, a potent, selective inhibitor of complex I of the electron transport chain, which is orally bioavailable and has excellent PK and physicochemical properties in preclinical species. IACS-010759 is currently in phase I clinical trials in relapsed/refractory AML and solid tumors where initial safety, pharmacokinetics, efficacy and pharmacodynamic impacts on tumor cell biology are being evaluated. As part of the development of IACS-010759, we were interested to explore the impact of the compound to target the persistent tumor cells, in particular by treating AML, TNBC and PDAC PDX models post-chemotherapy with IACS-010759. For all three contexts, IACS-010759 extended progression free survival, consistent with IACS-010759 targeting the recently described metabolically adapted residual tumor cells. For solid tumor indications, we have utilized innovative barcoding and clonal tracking strategies to confirm dependency of a specific subpopulation of tumor cells on OXPHOS. We show that OXPHOS inhibition extends survival and limits AML growth in secondary transplantation by stimulating terminal differentiation of putative stem cells. Taken together, these data provide rationale for multiple Phase II/III clinical trials where IACS-010759 will be used to target persistent tumor cell population and extend survival.

#2857

PI3K/AKT activation in de novo and acquired resistance to ibrutinib in lymphoid malignancies.

Yue Li,1 Isha Kapoor,2 Huayuan Zhu,1 Brian T. Hill,2 Wei Xu,1 Alex Almasan2. 1 _First Affiliated Hospital of Nanjing Medical University, Nanjing, China;_ 2 _Cleveland Clinic, Cleveland, OH_.

B-cell lymphoid malignancies, including CLL and DLBCL, are characterized by abnormal activation of Bruton's tyrosine kinase (BTK)-mediated B-cell receptor signaling that contributes to malignant transformation. Ibrutinib (IB) is an oral covalent BTK inhibitor that has shown impressive clinical activity. However, many IB-treated patients relapse over time with fulminant progression. We sought to define the molecular pathways that mediate Ib resistance by studying changes that occurred in vitro in cell lines and primary patient samples with prolonged exposure to IB. We found IB-increased apoptosis in a dose- and time-dependent manner, with upregulated levels of PTEN, BIM, and FOXO3a in Mec-1 and RIVA cells; followed by decreased phosphorylation (p) of Ser473-AKT and Ser253-FOXO3a. mRNA expression levels of Foxo3a and its targets Bim and Pten were significantly upregulated in a dose- and time-dependent manner. Interestingly, a subset of miRNAs, including miRNA-494 were significantly downregulated with upregulation of target transcripts Pten and Bim. Similar results were observed after IB treatment of primary CLL cells of naïve patients and those on new drug targets at high risk for relapse. To examine if miRNA-494-dependent PTEN regulation and activation of PI3K/AKT/FOXO3a signaling has a role in de novo and acquired resistance to IB in B-cell malignancies, we investigated the resistance mechanism that develops to IB therapy by generating IB-resistant (Ib-R) cells. Chronic exposure of RIVA cells to IB demonstrated AKT activation and increased pSer253-FOXO3a, in turn, leading to decreased PTEN and BIM levels; attributed to its decreased transcription as assessed by qRT-PCR in Ib-R RIVA cells. Interestingly, Ib-R RIVA cells had strikingly higher miRNA-494 expression and decreased Pten mRNA levels, indicating further regulation of PTEN/AKT/FOXO3a signaling. Moreover, unlike IB-sensitive CLL samples, patients with partial remission and Ib-R, before and after in vivo IB treatment, demonstrated significant upregulation of miRNA-494 and decreased Pten mRNA expression. The Ib-R cells were sensitized to treatment with IB by PI3K (idelalisib) and AKT (MK-2206) inhibition. MK-2206 downregulated pAKT and sensitized Ib-R cells through AKT-mediated PTEN activation. siRNA-mediated downregulation of AKT and FOXO3a significantly modulated cell survival, demonstrating the importance of these cell survival factors for Ib-R. Importantly, overexpression of miRNA-494 inhibitor increased Pten mRNA levels, further indicating regulation of apoptosis by AKT/FOXO3a signaling. Collectively, these findings reveal a novel mechanism of ibrutinib resistance in B-cell malignancies that modulates the expression and activity of tumor suppressor proteins and pro-apoptotic factors to confer drug resistance that could be effectively inhibited by a rational combination of therapeutic regimens.

#2859

Deep learning recognizes FTD-resistant isolated cancer cells of colon cancer.

Kiminori Yanagisawa,1 Masamitsu Konno,1 Masayasyu Toratani,1 Hirohiko Niioka,2 Ayumu Asai,1 Jun Koseki,1 Kenta Tsunekuni,1 Taroh Satoh,1 Kazuhiko Ogawa,1 Jun Miyake,2 Yuichiro Doki,1 Masaki Mori,1 Hideshi Ishii1. 1 _Osaka University Graduate School of Medicine, Suita city, Japan;_ 2 _Osaka University Graduate School of Information Science and Technology, Suita city, Japan_.

In recent years, innovative technologies that extract feature descriptions from the large volume of data on speech recognition, visual object recognition and detection as well as many other domains, such as drug discovery and DNA sequence annotations by deep learning techniques and applying them to automatic recognition etc. are drawing attention. As cancer research aiming at applying deep learning techniques to cases that are resistant to surgical therapy and drug therapy in metastatic colorectal cancer, we developed a fundamental technology that can predict the resistance of free cancer cells to fluorinated pyrimidine anticancer drugs by deep learning from the morphological image data taken from images. An experimental model was used in our investigation in order to clarify whether or not its image recognition ability can be applied to the determination of drug resistance of free cancer cells circulating in the peripheral blood. That is, a cell line established by inducing a resistance to FTD or 5 FU added to the cell culture solution was prepared over several months and the ability to recognize the tolerance of the drug was examined from a large volume of image data, and it was shown that it can be distinguished dominantly in a short-term culture system. Further, as a result of examination after separation at the single cell level, it was possible to distinguish fluorescent-labeled resistant strains dominantly. In addition, we were able to recognize the drug resistance character well by injecting resistant strains intravenously into the mice to prepare a model of free cancer cells and collecting circulating free cancer cells. Moreover, as a pre-clinical model, resistant strains were mixed with susceptible strains at various ratios and transplanted into mice and experimented. As a result, the nature of the resistance to treatment was predicted by image recognition, and death of the mice due to cancer was well correlated with the malignant trait of drug-resistant cancer cells. Then, by linking the feature expression obtained from the image and the Omics data, a detailed stratification of treatment resistance was possible. From the above, a technique in the mouse that can distinguish free cancer cells collected from the peripheral blood by deep learning of images was constructed, and a foundation to be applied to medical treatment and precision medical care in the future was established.

#2860

The chemosensitizing and oncolytic effects of Dihydrotanshinone in glioblastoma.

Varun Kumar, Donna Leonardi. _Bergen County Academies, Hackensack, NJ_.

Temozolomide is the primary chemotherapeutic agent used to treat glioblastoma. However, many tumors are initially resistant to or develop resistance to temozolomide, mainly due to high levels of O6-methylguanine DNA transferase (MGMT) which repairs DNA damage traditionally caused by temozolomide. Dihydrotanshinone (DHT) is extracted from Salvia miltiorrhiza, a Chinese medicinal plant, and has been shown to have antiproliferative effects on various cancer cell lines. DHT has also been to shown to induce apoptosis via induction endoplasmic reticulum stress, that can reportedly sensitize cells to temozolomide. MTS cellular proliferation assays or trypan blue viability assays were used to determine the effects of DHT/temozolomide combinatorial treatment. Enzyme-linked immunosorbent assay (ELISA) was used to determine effects on MGMT, P-glycoprotein, AKT, p65, and IlkB levels after singular and combinatorial treatment. DNA microarray was used to gauge gene expression and miRNA levels. Solute clearance of the blood-brain barrier was analyzed using a co-culture model system and confocal microscopy. DHT had a selectively cytotoxic synergistic oncolytic effect in a MGMT-deficient cell line and a sensitizing effect in a MGMT-expressing cell line. Cytotoxicity due to DHT was shown to be reactive oxygen species-dependent, while the combinatorial effect of DHT and temozolomide synergistically reduced MGMT and P-glycoprotein expression and protein levels. In addition, the NFkB complex was sensitive to combinatorial treatment, while AKT activity and gene expression was downregulated in response to treatment. DHT was shown to augment temozolomide efficacy, indicating that, since DHT can penetrate the blood–brain barrier, temozolomide in combination with DHT may represent a promising therapeutic option for glioblastoma.

#2860A

Evolution and characterization of carboplatin resistance at single-cell resolution.

Devora Champa, Si Sun, Cheng-Yu Tsai, Stephen Howell, Olivier Harismendy. _UCSD, La Jolla, CA_.

Acquired resistance to carboplatin is a major obstacle to the cure of ovarian cancer, but its molecular underpinnings are still poorly defined or inconsistently reported. Multiple cellular functions have been identified as being capable of modulating sensitivity to the platinum drugs; however, results from single-gene or transcriptome-wide studies have failed to disclose any one actionable gene or set of genes that are consistently altered across different cell lines or studies. We hypothesized that heterogeneity within the treated cell populations is responsible for this situation and that cloning and molecular analysis of single cells can capture the heterogeneity and dynamics of resistance acquisition, revealing cellular and molecular principles driving the phenotype. Following a rigorous experimental design, starting with a single ovarian cancer cell, we selected 8 subclones for resistance to carboplatin using repeated cycles of drug exposure. An additional 4 clones were left untreated for comparison. The resistant clones did not show significant mutations or gene copy number variations in common or reduced drug uptake, but displayed important phenotypic and gene expression heterogeneity. A gene set enrichment analysis revealed that each clone altered different molecular processes to become resistant. Suspecting that clonal heterogeneity was also present within each experimental replicate, we measured mRNA expression from 26,892 single cells from both sensitive and resistant clones, which resulted in the identification of 7 transcriptional states associated with resistance evolution and clone identity. Interestingly, the resistant states were not stable and continued to evolve after removal of the drug, without noticeable change in resistance level. Reduced proliferation and active interferon signaling were identified as two processes shared by most resistant states, becoming apparent early in the course of the resistance acquisition. Up to 12% of untreated cells were found to already be in a resistant state and this fraction diminished with each round of sub-cloning. Up to 18% of the cells from resistant clones showed extreme levels of key resistance genes and pathways, suggesting the importance of cellular heterogeneity in the development of resistance. Finally, six expression signatures derived from the in vitro resistant states distinguished primary from recurrent high-grade serous ovarian cancers and predicted both response to primary therapy and overall survival. The analysis of the biologic processes associated with each signature suggested functional differences between intrinsic and acquired resistance. This multidimensional, single-cell analysis decomposed a multigenic phenotype into tangible resistant states, offering new insights into the dynamics of the acquisition of resistance to carboplatin, a drug of major importance to the treatment of ovarian and other cancers. 

### New Agents and New Targets

#2861

PUMA induction by p73 mediates response and resistance of colorectal cancer to anti-EGFR antibody therapy.

Kyle Knickelbein,1 Jingshan Tong,1 Dongshi Chen,1 Yi-Jun Wang,1 Sandra Misale,2 Alberto Bardelli,3 Jian Yu,1 Lin Zhang1. 1 _University of Pittsburgh School of Medicine, Pittsburgh, PA;_ 2 _Memorial Sloan Kettering Cancer Center, New York, NY;_ 3 _Candiolo Cancer Institute-FPO, IRCCS, Candiolo (TO), Italy_.

Intrinsic and acquired resistance to anti-EGFR antibody therapy, frequently mediated by mutant or amplified KRAS oncogene, is a significant challenge in the treatment of colorectal cancer (CRC), the second-leading cause of cancer-related death in the United States. However, the mechanism of KRAS-mediated therapeutic resistance is not well understood. In this study, we found that clinically used anti-EGFR antibodies, including cetuximab and panitumumab, induce killing of sensitive CRC cells through p73-dependent transcriptional activation of the pro-apoptotic Bcl-2 family protein PUMA. Inhibition of EGFR and downstream AKT by cetuximab and panitumumab promotes Tyr99 phosphorylation of p73, which then binds to PUMA promoter to activate its transcription, resulting in cell death via the mitochondrial pathway. PUMA induction and p73 phosphorylation are abrogated in CRC cells with acquired resistance to anti-EGFR antibodies due to KRAS alterations and residual AKT activity. Upon screening a panel of anticancer drugs, we found that inhibition of aurora kinases preferentially kills mutant KRAS CRC cells. A combination with aurora kinase inhibitor overcomes KRAS-mediated resistance to anti-EGFR antibodies in vitro and in vivo by restoring PUMA induction and eliminating the residual AKT activity. Collectively, our results suggest that p73-dependent PUMA induction plays a critical role in mediating the sensitivity of CRC cells to anti-EGFR antibodies, and that restoration of PUMA induction and apoptosis is a promising approach to improve the efficacy of EGFR-targeted therapy.

#2862

Inhibition of DNA repair pathways in breast cancer is a potential mechanism of action of IT-139.

Isabel Conde,1 Yassi Fallah,1 Diane Demas,1 Suzanne Bakewell,2 Filipa Lynce,1 Ayesha N. Shajahan-Haq1. 1 _Georgetown University Medical Center, Lombardi Comprehensive Cancer Center, Washington, DC;_ 2 _Intezyne Technnologies, FL_.

IT-139 is a novel small-molecule drug that demonstrated a manageable safety profile at the maximum tolerated dose and modest antitumor activity as a monotherapy in a phase I clinical trial. A high affinity for albumin and unique chemistry allow increased uptake and activation of IT-139 within tumor cells, respectively. To date, IT-139 has been reported to inhibit the endoplasmic reticulum stress-sensing protein GRP78. However, different cancer cells show disparate responses to IT-139 and the precise mechanism of action in high-response versus low-response cancer cells remains unclear. In vitro studies have indicated that IT-139 induces cytostatic rather than cytotoxic effects as a monotherapy. In this study, to understand IT-139 mediated signaling mechanism in breast cancer cells, we used estrogen receptor-positive (ER+) MCF7 breast cancer cells to obtain gene-metabolite integrated models following IT-139 treatment (10 or 100 μM for 72 h). Our data showed that IT-139 significantly reduced expression of genes involved in the DNA repair pathway, including BRCA1 and RAD51, following treatment with IT-139 in MCF7 cells. Validation of our findings in other breast cancer cell lines including MDA-MB-175 (ER+), MDA-MB-231 and MDA-MB-468 (both ER-) cells also showed increased phosphorylation of H2AX (Ser139) following IT-139 treatment, suggesting lack of proper DNA surveillance and subsequent accumulation of DNA double-stranded breaks (DSB). Consequently, we investigated the effects of combination treatment approaches with IT-139 and other clinical anticancer therapies that interfere with nucleotide synthesis or DNA damage repair pathways, particularly in ER- breast cancers. Our data showed additive effects of IT-139 in combination with capecitabine (5-FU prodrug) or olaparib (PARP inhibitor) and a synergistic effect with carboplatin in ER- breast cancer cell lines. Further analysis of IT-139 mediated signal transduction in breast cancer cells will reveal whether it can be combined with aforementioned chemotherapies to circumvent resistance.

#2863

Dissecting the structural basis for inhibitors of RNA-binding proteins.

Lan Lan,1 Minli Xing,1 Xiaoqing Wu,1 Philip Gao,1 Justin T. Douglas,1 Yu Zhan,1 Gulhumay Gardashova,1 Jiajun Liu,1 Robert P. Hanzlik,1 Jeffrey Aubé,2 Kristi L. Neufeld,1 Berl R. Oakley,1 Roberto N. De Guzman,1 Liang Xu1. 1 _Univ. of Kansas, Lawrence, KS;_ 2 _University of North Carolina, NC_.

RNA-binding proteins (RBPs) are key regulators of cellular functions, especially in post-transcriptional regulations. Dysregulation of RBPs is implicated in many diseases including cancer. One of the RBPs that is overexpressed in a variety of human cancer is Musashi-2 (MSI2). Elevated MSI2 expression is associated with ectopic oncogenic pathways, including but not limited to NUMB/Notch, PTEN/mTOR, TGF-β/SMAD3, making MSI2 a promising therapeutic target for cancer. In the case of NUMB, MSI2 binds to and negatively regulates translation of NUMB, a negative regulator of Notch signaling. Protein structure is critical for drug discovery and structure-based rational design. However, so far there is no structure available for MSI2 protein. Recently, using nuclear magnetic resonance (NMR) we solved the first solution structure of MSI2 RNA-recognition Motif 1 (MSI2-RRM1) that forms the major long narrow RNA-binding pocket. Using a fluorescence polarization (FP) chemical libraries screening, we identified several groups of hit compounds that disrupt the binding of MSI2-NUMB RNA potently. These compounds induced apoptosis, inhibited cancer cell proliferation, invasion and metastasis in multiple tumor models tested in vitro and in vivo. They also interfered with cancer stem cell functions with reduced tumorsphere formation. Several lead compounds showed promising efficacy in animal tumor models of human breast, prostate and colon cancer. To investigate the specificity of the compounds towards other RBPs, the compounds were tested in biophysical binding assays such as FP, time resolved Fluorescence Resonance Energy Transfer assay (TR-FRET), NMR spectroscopy and cell-based assays. We compared the affinity of these MSI2 inhibitors towards other RBPs such as MSI1 and HuR. Using docking and molecular dynamics simulation, we analyzed the structure-activity relationship of these inhibitors towards different RBP targets. This work adds significant information to the structure of MSI2-RRM1 and the structural basis for designing more potent and specific inhibitors of RBPs.

#2864

Inhibition of the late stages of autophagy overcomes hypoxia-induced chemoresistance and targets leukemic stem cells in acute myeloid leukemia.

Kaitlyn M. Dykstra,1 Dirkje W. Hanekamp,2 Matthew Johnson,3 Monica L. Guzman,4 Eunice S. Wang1. 1 _Roswell Park Cancer Institute, Buffalo, NY;_ 2 _VU University Medical Center, Amsterdam, Netherlands;_ 3 _Roanoke College, Salem, VA;_ 4 _Weill Cornell Medicine, New York, NY_.

Background: Acute myeloid leukemia (AML) is an aggressive hematological malignancy which resides within an inherently hypoxic bone marrow (BM) microenvironment. Hypoxia has been linked to chemoresistance, particularly of leukemia stem cells (LSCs) responsible for relapsed disease. We hypothesized that upregulation of autophagy promotes survival and chemoresistance in AML cells (specifically LSCs) within the hypoxic BM. Targeted inhibition of specific stages of autophagy therefore may represent a novel means of eradicating disease.

Methods: Human AML cell lines (HEL-Luc, HL60, MOLM13) were treated with cytarabine (AraC) and multiple autophagy inhibitors (Spautin-1, MRT68921, chloroquine (CQ), bafilomycin (BafA1)) under normoxia (21% O2) or hypoxia (1% O2) and evaluated for apoptosis. MOLM13 cells were infected with lentivirus containing shRNAs against LC3B, Atg7 or control. Autophagy was measured using CytoID dye and immunoblotting for LC3 and p62. SCID mice systematically engrafted with HEL-Luc cells were treated with CQ (25 mg/kg ip for 5 days/week for 3 weeks) and evaluated for leukemia burden by bioluminescent imaging and time to morbidity. Irradiated NSG mice were injected with primary AML cells and treated with vehicle or Baf A1 (1mg/kg ip twice a week for 4 weeks). BM was then harvested and transplanted into secondary NSG recipients without additional treatment. Leukemia engraftment in mice was measured by flow cytometry for hCD45.

Results: Autophagy was upregulated in multiple human AML cell lines after prolonged exposure to hypoxia as well as in a subset of primary AML samples. Inhibiting the early stages of autophagy pharmacologically with Spautin-1 or MRT68921 or shRNA knockdown of autophagy proteins Atg7 or LC3B had no effect on chemosensitivity under hypoxia. However, inhibiting fusion of the autophagosome with the lysosome (late stages) with either CQ or Baf A1 did overcome hypoxia-induced resistance to AraC. CQ also reduced tumor burden in a systemic xenograft model of AML as measured by bioluminescent imaging and resulted in a slight overall increase in survival (17.5 vs. 19 days, p=0.02). In the in vivo LSC model, Baf A1 had no effect on leukemic burden in the primary recipients; however, mice that received secondary transplants from Baf A1-treated mice demonstrated significantly reduced leukemic burden as compared to the control, consistent with LSC specific targeting.

Conclusions: Our results demonstrate that treatment with the late stage autophagy inhibitors (chloroquine, bafilomycin A1) can both overcome hypoxia-induced chemoresistance and preferentially inhibit LSC growth in vivo. Taken together, these data support the development of late stage autophagy inhibitors for the treatment of AML and prevention of relapsed disease. In vivo studies to assess the efficacy of AraC/Baf A1 combination therapy are currently underway.

#2865

Ciclopirox olamine: A common antifungal agent that inhibits growth of esophageal tumor cells in vitro and in vivo.

Randi J. Ryan, Prabhu Ramamoorthy, Dharmalingam Subramaniam, Shrikant Anant, Scott Weir. _University of Kansas, Kansas City, KS_.

Esophageal carcinoma continues to carry a poor prognosis due presentation at advanced stages. Current chemotherapeutics often only prolong survival for months to a few years. Drug repositioning is an important pharmacologic strategy that involves investigation and implementation of known drugs for treatment of new diseases. This method significantly reduces the costs required for establishing the safety of a drug and is associated with higher rates of drug approval. Ciclopirox olamine (CPX) is an antifungal agent that has been on the market since the 1970s. The mechanism of action is believed to include disruptions of DNA repair and cell division signals. Recently, however, this drug has been studied for its antitumor properties demonstrated in human rhabdomyosarcoma, breast carcinoma, colon adenocarcinoma, bladder carcinoma, and hematologic malignancies. To date, there are no studies related to the effect of CPX on esophageal cancer. Here, we show that ciclopirox olamine causes growth inhibition of four esophageal cell lines: TE-10, SKGT4, FLO1 and ESO1. This growth inhibition is demonstrated by hexosaminidase assay performed after incubation with the drug for 24-72 h. The IC50 values range between 5-20µM depending on the cell line used. Cell death was also demonstrated by immunofluorescent staining with Hoschst and propidium iodide, which shows a decrease in the number of viable cells with increasing concentrations of CPX. Clonogenicity assay was also performed with each cell line and demonstrates a decrease in the ability of cells to form colonies after 24 and 48 hour treatment with CPX and subsequent incubation in standard media for 5-7 days. Cell cycle analysis demonstrates G0/G1 arrest in cells treated with CPX. The effects of CPX on the cell cycle are further supported by Western blot analysis showing a decrease in in CDK4 and CDK6, which are necessary for cell cycle progression from the G0/G1 phase. We have also found that treatment with CPX results in a decrease in β-catenin in TE10 cells, suggesting that the drug is affecting this pathway to cause growth inhibition of tumor cells. Finally, we used our ESO1 mouse esophageal squamous cell carcinoma cell line for a xenograft study in which tumor cells were injected into the flanks of mice. Mice treated with intraperitoneal injections of 300 mg/m2 of CPX had smaller tumor volumes compared to untreated controls. In summary, we have shown that CPX inhibits growth of esophageal tumor cells both in vitro and in vivo. Our data suggest that CPX induces cell cycle arrest of tumor cells. The mechanism of tumor cell inhibition may be related to downregulation of the WNT/β-catenin signaling pathway. In future studies, we plan to analyze the effects of CPX on other components in the WNT family from in vitro cells as well as tumor xenografts.

#2866

Anticancer effect of a new cyclic peptide ALOS4 is associated with its systemic anti-inflammatory activity.

Elimelech Nesher,1 Albert Pinhasov,1 Maria Becker,1 Michael Kirby,1 Raichel Cohen Harazi,1 Shiri Yakobovich,1 Oryan Agranyoni,1 Bar Shoval,1 Shany I. Elbaz,1 Olga Udartseva,2 Ilya Gitlin,2 Katerina Leonova,2 Katerina Gurova,2 Andrei Gudkov,2 Igor Koman1. 1 _Ariel University, Ariel, Israel;_ 2 _Roswell Park Cancer Institute, Buffalo, NY_.

The association of chronic inflammation with cancer development and progression has been well recognized. Here, we report a synthetic cyclic peptide, ALOS4, capable of suppressing tumor growth following systemic administration and possessing anti-inflammatory properties. Although ALOS4 was initially isolated by phage display for its ability to bind avb3 integrin and demonstrated tumor-suppressor activity in preclinical xenograft and syngeneic models of melanoma, it surprisingly showed no functional activity resembling RGD peptides in functional in vitro assays. Remarkably, ALOS4 displayed no signs of toxicity in treated animals, even at doses exceeding >100-fold beyond the efficacious dose. To identify the mechanisms underlying profound antitumor activity of this safe peptide, we used a large panel of cell-based repeater assays designed to detect modulators of a variety of signaling pathways including p53, NF-kB, and different types of stress responses including heat shock and hypoxia. We also tested the effects of ALOS4 on tumor cell growth, adhesion, clonogenicity, morphology and other in vitro properties. ALOS4 showed no detectable activity in any of these numerous assays with one exception: it suppressed the ability of treated cells to induce interferon type I signaling in response to mimics of double-stranded RNA (Poly I:C). Based on this observation, we hypothesized that the antitumor effect of ALOS4 is driven by a systemic anti-inflammatory effect rather than by a direct effect on tumor cells. Consistently, ALOS4 treatment dramatically altered both the abundance and content of the immunocyte population infiltrating subcutaneous melanomas in mice. These results suggest that ALOS4 may be an anticancer agent with a new mechanism of action that targets the tumor-supporting interferon-driven mechanism of tumor-host interaction.

#2867

Glycomimetic antagonist of E-selectin, GMI-1271, enhances therapeutic activity of the hypomethylating agent 5-azacitidine in the KG1 model of AML.

Theodore A. Smith, William E. Fogler, Ji-Won Lee, John L. Magnani. _Glycomimetics, Inc., Rockville, MD_.

Binding of AML blasts to E-selectin activates cellular survival pathways leading to chemoresistance. GMI-1271 is a novel E-selectin antagonist that when used in combination with chemotherapy results in improved survival in mouse syngeneic and xenogeneic AML tumor models. GMI-1271 in combination with two chemotherapy regimens is in early clinical trials for the treatment of AML. Azacitidine (5-AC) is a DNA-methyltransferase inhibiting cytosine nucleoside analog that at low doses induces DNA hypomethylation and transcriptional activation, while at higher doses is directly cytotoxic to neoplastic cells including AML blasts. 5-AC is approved in Europe for the treatment of limited populations with AML. We evaluated GMI-1271 in combination with 5-AC in the KG1 AML tumor model to assess the potential for therapeutic benefit of the combination.

NSG mice (10/group) received i.v. injections of KG1 cells, and were treated with saline, GMI-1271 alone, 5-AC alone, or the combination of GMI-1271 and 5-AC. The median survival time (MST) of mice treated with 5-AC was 88 days and statistically different (P<0.002) to groups treated with saline (MST=69.5 days) or GMI-1271 alone (MST=69 days). All mice treated with saline or GMI-1271 alone succumbed to progressive tumor growth. At study conclusion (Day 104 post tumor injection) 20% of mice treated with 5-AC remained alive. Importantly, the therapeutic activity of 5-AC was significantly enhanced when combined with GMI-1271 (MST>104 days, P=0.0140 compared to 5-AC alone) with 70% of mice surviving to study conclusion. These results indicate that E-selectin/AML blast interaction in the KG1 model protects from the anti-tumor activity of 5-AC and that GMI-1271 attenuates this protection.

To investigate the nature of the observed in vivo activity of GMI-1271 and 5-AC, KG1 cells were cultured for 96 h in a noncytotoxic concentration (100 nM) of 5-AC and the reactivity of the cells to HECA-452 (an antibody that recognizes an E-selectin carbohydrate ligand) and binding to E-selectin were determined by flow cytometry. Treatment with 5-AC resulted in a 28% increase in reactivity of cells to HECA-452 and a 32% increase in binding to E-selectin. Further in vitro assays of static adhesion revealed an increase in the adhesion of 5-AC-treated KG1 cells to E-selectin. Notably, the enhanced adhesion of KG-1 cells to E-selectin was reversed using GMI-1271. Collectively, these results demonstrate that 5-AC can lead to increased expression of E-selectin ligands on AML cells and that the therapeutic potential of 5-AC could be improved by combination with GMI-1271.

#2868

Preclinical evaluation of the multi tyrosine kinase inhibitor TAS-115 in genetically engineered mouse models of prostate cancer.

Marco A. De Velasco,1 Yurie Kura,1 Naomi Ando,1 Noriko Sato,1 Masahiro Nozawa,1 Kazuhiro Yoshimura,1 Kazuko Sakai,1 Kazuhiro Yoshikawa,2 Kazuto Nishio,1 Hirotsugu Uemura1. 1 _Kindai University Faculty of Medicine, Osaka-Sayama, Japan;_ 2 _Aichi Medical University, Japan_.

The tumor microenvironment (TME) is comprised of various cell types that can contribute to malignancy by promoting cancer cell proliferation, immune evasion, angiogenesis and metastasis. Here we use genetically engineered mouse models of prostate cancer to show that TAS-115, a multi-kinase inhibitor that targets c-MET, VEGF-R, and CSF1-R signal pathways, suppresses prostate cancer growth by targeting the TME. Conditional Pten-knockout (Pten-KO) and Pten/Trp53-double knockout (Pten/P53-DKO) mice were used to evaluate the pharmacodynamic activity of TAS-115 and efficacy studies were carried in 16-week-old Pten/P53-DKO mice. Four weeks of treatment with TAS-115 significantly suppressed prostate tumor growth in by 22.6%, P=0.041. Epithelial cancer cell proliferation in was reduced by 28.6%, (P=0.072) in TAS-115-treated mice, however, no changes were seen in the induction of apoptosis. Immunohistochemical (IHC) analysis revealed potent but focal inhibition of ERK phosphorylation in both epithelial and stromal cells. A tendency for reduced phosphorylation of AKT and STAT3 were also noted in mice receiving TAS-115. IHC analysis also revealed strong inhibition of CSF1-R phosphorylation in tumor infiltrating immune cells of mice treated with TAS-115. We also examined the effects of TAS-115 in a castration-resistant Pten/P53-DKO prostate tumor model. In this model, we did not note statistically significant reductions in tumor burden, however, 2 of 6 (33.3%) mice treated with TAS-115 had noticeably reduced tumor burden (>20% reduction relative to median overall tumor burden levels). No significant changes were observed in overall tumor proliferation, apoptotic rates or in AKT and ERK phosphorylation levels in the tumors of TAS-115-treated mice however, a significant reduction of STAT3 phosphorylation staining index (-39.3, P=0.0424) was observed, the prostates tumors of mice receiving TAS-115. Similar to castration-naïve treated tumors, CSF1-R phosphorylation was strongly inhibited in infiltrating immune cells of castration-resistant prostate tumors from TAS-115-treated mice. Unsupervised clustering from a qRT-PCR-based focused panel of immune-responsive genes showed greater consistency and a higher magnitude of response in TAS-115-treated mice in the castration-resistant tumor model. Further analysis of microvessel density showed a 20% reduction in tumor neovascularization in both intervention models. The direct antitumor effect of TAS-115 on cancer cells was examined in vitro using mouse prostate cancer cell lines. In these studies, TAS-115 showed moderate growth inhibition. Overall our studies show that TAS-115 is capable of suppressing prostate tumor growth by acting primarily on the TME and provide evidence to support further investigation of TME modulation using small molecule multi-kinase inhibitors.

#2869

Nintedanib abrogates the activation and tumor-promoting effects of fibroblasts from lung adenocarcinoma patients.

Jordi Alcaraz,1 Marta Gabasa,1 Rafael Ikemori,1 Frank Hilberg,2 Noemí Reguart3. 1 _Univ. of Barcelona, Barcelona, Spain;_ 2 _Boehringer-Ingelheim Inc, Vienna, Austria;_ 3 _Hospital Clínic de Barcelona, Barcelona, Spain_.

Nintedanib is a clinically-approved multikinase receptor inhibitor that, in combination with docetaxel, provides clinical benefits to advanced lung adenocarcinoma (ADC) patients but not to lung squamous cell carcinoma (SCC) patients. However, the mechanisms underlying the selective therapeutic effects of Nintedanib in ADC remain poorly understood. Of note, Nintedanib is also approved to treat patients with idiopathic pulmonary fibrosis (IPF), a rare disease characterised by an abundant desmoplastic stroma rich in pathologically activated fibroblasts. Since the tumor stroma in lung cancer is also desmoplastic, and we recently showed that tumor-associated fibroblasts (TAFs) derived from ADC and SCC patients exhibit different phenotypes in vitro, we hypothesized that TAFs may underlie the selective effects of Nintedanib in ADC. To test this hypothesis we activated TAFs in culture with the pro-fibrotic cytokine TGF-β1 in the presence of increasing concentrations of Nintedanib, and collected the corresponding conditioned medium. Remarkably SCC-TAFs showed very modest inhibition of a panel of fibrotic markers including α-SMA, P4HA2 and fibrillar collagens (COL1A1, COL3A) in response to Nintedanib, in striking contrast to ADC-TAFs and paired lung parenchyma fibroblasts, which were markedly affected. This was matched by a significant reduction in the abilities of the conditioned medium of ADC-TAFs but not SCC-TAFs to promote cancer cell growth and invasion in a panel of lung cancer cell lines after Nintedanib treatment. These results reveal that Nintedanib is an effective inhibitor of stromal fibrosis and its associated tumor-promoting effects in ADC, and that the poor antifibrotic response of SCC-TAFs to Nintedanib may contribute to the differential clinical benefit observed in both subtypes. Our findings also support that TGF-β signalling and aberrant TAF-carcinoma cross-talk are regulated by different mechanisms in ADC and SCC. In addition they support that preclinical models based on carcinoma-TAF interactions may help defining the mechanisms of the poor antifibrotic response of SCC-TAFs to Nintedanib and testing new combined therapies to further expand the therapeutic effects of this drug in solid tumors.

#2870

A specific pan-FGFR inhibitor has antitumor activity against prostate cancer patient derived xenografts, PDX, expressing high FGFR1.

Estefania Labanca, Jun Yang, Peter Shepherd, Xinhai Wan, Justin M. Roberts, Michael W. Starbuck, Nora M. Navone. _UT MD Anderson Cancer Ctr., Houston, TX_.

Prostate Cancer (PCa) is one of the most commonly diagnosed malignancies in men. Patients with advanced metastatic PCa have effective treatment options, but none of them are curative. Androgen deprivation is the most effective therapy, but growth of the cancer resumes over time in most cases, and the disease progresses to castration-resistant PCa (CRPC). Bone is the main site of CRPC progression. Acquired (or inherent) resistance mechanisms to second line therapy options for CRPC eventually lead to disease recurrence and, ultimately, death. The underlying mechanisms of PCa progression to first or second line therapy options are diverse and include fibroblast growth factor (FGF) axis activation. Indeed, we previously reported that blockade of FGFRs with dovitinib (TKI258) (Novartis Pharmaceuticals), a receptor tyrosine kinase inhibitor (TKI) with potent activity against FGFR1-3 and vascular endothelial growth factor receptor (VEGFR) has clinical activity in men with CRPC and bone metastases (PMID: 25186177), thus providing direction for therapy development of FGFR blockade in PCa. Because dovitinib was withdrawn from the clinic by Novartis, we seek to identify an alternative agent with activity against FGFR1 as a candidate for therapy development. With that goal, we tested the antitumor activity of a specific pan-FGFR TKI, JNJ-42756493 (JNJ) (Janssen Pharmaceutical Companies of Johnson&Johnson) against PCa patients derived xenografts (PDXs) expressing high (MDA PCa 118b) and low (MDA PCa 183) endogenous levels of FGFR1. Because bone is the primary site of CRPC progression we tested the antitumor activity of JNJ against these PDXs growing in the bone of mice. By assessing tumor volume by MRI, we found that JNJ has antitumor activity against MDA PCa 118b but not MDA PCa 183. Immunohistochemical analysis of FGFR1 expression exhibited reduction of FGFR1 in tumors of the treated group compared with vehicle treated group in MDA PCa 118b samples. Both these evidences suggest that FGFR1 is the main driver of PCa progression in this PDX and that JNJ is a potent agent against PCas with high FGFR1 expression.Due to the important role that FGF axis has in bone biology, we assessed the effect of JNJ in the bones of mice without tumors by micro-CT analysis. Interestingly, we observed a reduction in bone parameters including bone volume/ total volume (BV/TV) and trabecular thickness (Tb.Th) in the treated group compared with the vehicle treated group, suggesting FGF axis blockade reduces bone mass. However, we identified an increase in the bone surrounding the tumors in the MDA PCa 118b tumor-bearing bones of mice treated with JNJ. These results highlight the complex role of FGF axis in the PCa-bone interaction and warrant further studies to identify candidate patients for this therapy and markers of response in men treated with FGFR inhibition.

#2871

Diosgenin inhibits the proliferation of MCF-7 breast cancer cells through the demethylation of miR-145 gene.

Fengqin Shi,1 Lingeng Lu,1 Ya Li,2 Li Hou,2 Xinyi Chen,2 Han Rui,1 Chong Wang,2 Ya Yue2. 1 _Yale University, New Haven, CT;_ 2 _Dongzhimen Hospital, Beijing University of Chinese Medicine, Beijing, China_.

Background: Altered DNA methylation in microRNA (miRNA) host genes has been frequently associated with abnormal miRNA expression during the pathogenesis of cancer. miR-145 was identified as a tumor-suppressive miRNA that was down-regulated in several cancer types. Accumulating evidence has clearly demonstrated that a low expression of miR-145 in breast cancer was correlated with an aberrant hypermethylation in its host gene. Diosgenin is a steriod saponin, which is extracted from the tubers of Dioscorea wild yam. The anti-tumor mechanisms of diosgenin have been demonstrated via the modulation of multiple cell signaling pathways, which are associated with growth, differentiation, apoptosis and oncogenesis. However, there is little information regarding the epigenetic-based effects of Diosgenin in breast cancer. In our study, we hypothesize that Diosgenin has anti-proliferative effects on breast cancer cells through the demethylation of miR-145.

Material and Methods: MCF-10A breast epithelial cells and MCF-7 breast cancer cells were used to investigate the effects of Diosgenin on the expression and methylation of miR-145 using real time-PCR (RT-PCR) and methylation-specific PCR (MSP), respectively. An MTS proliferation assay was performed to assess the effect of Diosgenin on the proliferation of MCF-7 breast cancer cell.

Result: In comparison with MCF-10A breast epithelial cells, MCF-7 breast cancer cells showed the hypermethylation of miR-145, and significantly down-regulated miR-145 expression. Diosgenin treatment led to proliferation inhibition of MCF-7 breast cancer cells. In MCF-7 breast cancer cells, the Diosgenin treatment led to significantly up-regulated miR-145, whereas the methylation level of miR-145 significantly decreased.

Conclusion: Diosgenin, a natural compound, exhibits an anti-proliferative effect on breast cancer cells, and affects the methylation and expression of miR-145. These findings indicate that the demethylation of miR-145 may be a molecular mechanism underlying the anti-tumor of Diosgenin in breast cancer.

#2872

Mechanism of anti-proliferative and anti-invasive actions of ω3-polyunsaturated fatty acids in human hypopharyngeal carcinoma cells.

Seung-Hyeon Han,1 Soyeon Shin,2 Young-Joo Jeon,1 Jun-Young Heo,1 Gi-Ryang Kweon,1 Seung-Kiel Park,2 Jong-Il Park,2 Kyu Lim3. 1 _Dept. of Biochemistry, Department of Medical Science, School of Medicine, Chungnam National University, Daejeon, Korea, Daejeon, Republic of Korea;_ 2 _Dept. of Biochemistry, School of Medicine, Chungnam National University, Daejeon, Korea, Daejeon, Republic of Korea;_ 3 _Dept. of Biochemistry, Department of Medical Science, School of Medicine, Cancer Research Institute, Chungnam National University, Daejeon, Korea, Daejeon, Republic of Korea_.

Hypopharyngeal carcinoma is one of head and neck cancers. According to the latest statistics from the United States National Cancer Institute, it is estimated that about 17,000 Americans will be diagnosed with pharyngeal cancer in 2017. Although ω3-polyunsaturated fatty acids (ω3-PUFAs) have anti-tumorigenic properties in the several cancers, the anti-cancer effect of ω3-PUFAs on hypopharyngeal carcinoma has been not known yet. In this study, we report inhibitory mechanisms of ω3-PUFAs on cell growth and invasion in hypopharyngeal carcinoma. DHA and EPA inhibited the cell growth of FaDu and SNU1041 in a dose- and time-dependent manner; in contrast, arachidonic acid, a ω6-PUFA, had no significant effect. In flow cytometry analysis, subG1 population increased in DHA-treated FaDu and SNU1041. Moreover, apoptotic cell death was confirmed by TUNEL assay, and the induction of cleaved PARP and caspase-3. Furthermore, treatment of hypopharyngeal carcinoma cells with DHA resulted in a significant increase in autophagic activity, as revealed by increased LC3-II levels, GFP-LC3 puncta, and autophagic flux activation. EGF-induced phosphorylation of EGFR, which is frequently overexpressed in hypopharyngeal carcinoma, was also suppressed after DHA pretreatment, and levels of p-Akt (p- Aktthr308 and AktSer473), which is a downstream signal, was also inhibited in the FaDu and SNU1041 cells. The invasiveness of cells was significantly inhibited by DHA treatment in vitro transwell assay as well. The MMP-2 and MMP-9 promoter activities were inhibited after DHA treatment. Cox-2 and VEGF promoter activities were also suppressed by DHA. Furthermore, DHA decreased the levels of reporter activity of NF-κB, which is transcription factor that regulates MMPs, Cox-2 and VEGF expression. Fat1-stably expressing FaDu cells (fFaDu-sc) was established (fFaDu-sc express a Caenorhabditis elegans ω3-desaturase converting ω6- to ω3-PUFAs endogenously), and the proliferation of fFaDu-sc was more attenuated than that of cells expressing control vectors (fFaDu-cc). Moreover, fFaDu-sc showed reduction of cell invasion compared with fFaDu-cc. in transwell chamber. The MMPs promoter activities were also suppressed in fFaDu-sc. Taken together, these findings provide evidence that ω3-PUFAs may inhibit invasion as well as cell growth through suppression of p-EGFR and MMPs expression in hypopharyngeal carcinoma cells, indicating that the utilization of ω3-PUFAs may represent a potential effective therapy for the chemoprevention and treatment of human hypopharyngeal carcinoma. [This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (NRF-2015R1D1A1A01056887) and by the framework of international cooperation program managed by National Research Foundation of Korea (2015K2A2A6002008)].

#2873

Targeting RAGE for prostate cancer treatment.

Kyle J. Ernzen,1 Subramanyam Dasari,2 Gnanasekar Munirathinam3. 1 _Viterbo University, La Crosse, WI;_ 2 _Indiana University, Bloomington, IN;_ 3 _University of Illinois College of Medicine Rockford, Rockford, IL_.

Prostate cancer (PCa) is the second most common cancer among men and is the third leading cause of cancer-related death in the United States. In 2017 alone, an estimated 161,360 men will be diagnosed with PCa and approximately 26,730 deaths will result from this disease in the United States. The most common methods for PCa treatment include surgery, radiotherapy, and hormone therapy. These treatment options unfortunately have serious drawbacks such as the possibility of organ damage, adverse side effects, and eventual PCa resistance to treatment. Due to the severe threat that PCa poses across the male population, it is vital that more effective and safe treatment options are explored. Previous research has indicated that the receptor for advanced glycation end products (RAGE) plays a vital role in the survival of prostate cancer cells, indicating that RAGE could be a potential therapeutic target. In the present study, we focused on targeting RAGE using two different antagonists, RAGE aptamer and azeliragon as potential alternative therapies for PCa. Confocal microscopy assays initially confirmed that RAGE aptamer expression strongly binds to VCaP cells while azeliragon treatment decreased the expression of RAGE in these cells. MTT cell viability assays indicated that RAGE aptamer and azeliragon decrease PCa cell proliferation in a dose-dependent manner for VCaP and DU-145 cell lines respectively. Cell cycle analysis suggested that RAGE aptamer and azeliragon-treated DU-145 cells enforced cellular G0/G1 phase arrest in a dose-dependent manner. Annexin V FITC and ROS immunofluorescence assays showed that RAGE aptamer and azeliragon induce apoptosis potentially by inducing oxidative stress in PCa cells. Apoptosis activation in RAGE aptamer and azeliragon-treated VCaP cells was confirmed by the detection of PARP-1 cleavage through western blotting. Taken together, our findings suggest that RAGE aptamer and azeliragon may be a promising alternative form of therapy for PCa patients.

#2874

Benzaldehyde inhibits the multiple signals in cancer by suppressing the binding activity of overexpressed 14-3-3ζ and represses the pRb/E2F transcriptional pathway.

Jun Saitoh, Hideyuki Saya. _Keio University School Of Medicine, Shinjuku Ku Tokyo, Japan_.

Benzaldehyde (BA) is the simplest aromatic aldehyde composed of only benzene ring and aldehyde. It is constituents of many fruits and flavor essence. Anti-cancer effect of BA was first reported in 1980, and multi institutional clinical trials by using derivatives of BA were performed in 1980's in Japan. However, clinical study was aborted due to the policy and strategic change in the company. Only the safety of the compound was confirmed. Given that we had multiple effective clinical cases including pancreatic cancers and chemo-resistant non Hodgkin lymphoma, we attempted to elucidate the mechanism of anti-cancer effect of BA. We have previously reported that BA inhibits cancer-related multiple pathways such as PI3K/AKT/mTOR, STAT3, NFκB and ERK, leading to cell death and growth suppression in BA sensitive pancreatic cancer cell line BxPC3, but not in normal cells. We found that such mechanism is based on the BA-mediated inhibition of 14-3-3ζ binding to its client proteins. To further analyze the BA-mediated regulation of 14-3-3ζ, we generated HEK293 cells overexpressing 14-3-3ζ and confirmed that BA suppressed mTOR pathway in the 14-3-3ζ expressing HEK293 cells but not in parental HEK293 cells. Furthermore, we found that depletion of 14-3-3ζ suppresses mTOR signal in BxPC3 and BA-induced reduction of phosphorylation of mTOR signal proteins. Moreover, we performed microarray analysis on three pancreatic cancer cell lines (BxPC-3, AsPC1, PANC1) and found that stress response genes were upregulated in BxPC-3 cells by BA treatment. E2F2 and E2F targeted genes and histone cluster genes in 6p22 were suppressed in not only BxPC-3 but also BA low-sensitive PANC1. We confirmed that BA treatment suppresses the expression of E2F2 and E2F target genes. Of note, protein levels of pRb and cyclin D were also suppressed by BA treatment. Those data suggest that BA inhibits overexpressed 14-3-3ζ mediated multiple signaling pathway and consequently represses pRB/E2F and E2F target genes, resulting in inhibition of cell cycle progression, DNA replication and DNA repair. We propose that those mechanisms can explain why this safe compound is effective for refractory cancers.

#2875

Calycosin is a double-edged sword in the treatment of pancreatic cancer.

Zhu Zhang, Joshua KS Ko. _Hong Kong Baptist University, Hong Kong, Hong Kong_.

Calycosin is known for its suppression of breast cancer cell growth. We are the first group to report the effects of calycosin in pancreatic cancer development. Growth inhibition of the human pancreatic cell line MIA PaCa-2 was evaluated by MTT assay. Cell cycle distribution was determined by flow cytometry. Cell migratory activity was investigated by a wound healing assay. Subcutaneous xenografting of MIA PaCa-2 cells was conducted in Balb/C nude mice, while orthotopic inoculation of the mouse Pan01 pancreatic cancer cells was performed in C57BL/6 mice. Our results have shown that calycosin exhibited modest growth inhibition of MIA PaCa-2 cells, together with induction of S phase arrest and restoration of p53. Besides, promotion of both caspase-dependent apoptosis and autophagy implicate that the anticancer mechanism of calycosin strongly rely on programmed cell death. Alternatively, calycosin also inhibited the migration of MIA PaCa-2 cells in line with downregulation of epithelial–mesenchymal transition (EMT) and pro-metastatic biomarkers. The above in vitro benefits of calycosin was confirmed by a significant reduction of tumor mass in the MIA PaCa-2 xenograft. Nevertheless, when we look closely on the in vivo anticancer effect of calycosin in the Pan01 orthotopic mice model, although both attenuation of tumor growth and proapoptotic action were confirmed by histological findings, the tumor tissue expression of Snail, vimentin and CD31 was all elevated, indicating possible promotion of EMT and pro-angiogenic potential of the drug. These controversial findings suggest that calycosin could be an effective anticancer agent by promoting programmed cell death and inhibition of cell proliferation of pancreatic cancer cells, while it may also facilitate metastatic development of tumor, particular at the advanced stages. It would be essential to further dissect the conditions and factors that differentiate the dual actions of calycosin in pancreatic cancer therapy.

#2876

Targeting VCP to enhance pancreatic cancer response to DNA damaging agents.

Yuliana Astuti, Charlene Lam, Harpreet S. Wasan, Elaina N. Maginn. _Imperial College London, London, United Kingdom_.

Dysregulation of DNA damage response and repair (DDR) is common in pancreatic and other cancers, and impairs their sensitivity to DNA damaging agents. Targeting DDR mediators is therefore an attractive strategy to improve response to DNA damaging agents. We have identified the AAA ATPase VCP as a lead candidate for this via siRNA screening. VCP-chemotherapy synergistic combinations have not as yet been explored in solid tumours. To investigate the mechanism by which VCP modulates response to cisplatin-induced DNA damage, the effect of VCP functionality loss in decreasing cell viability was validated using two pharmacological VCP inhibitors, NMS-873 and CB-5083, in a panel of pancreatic cancer cell lines. Effects on ovarian cancer cell lines were also assessed for comparison, and IC50 values (72 hours) were found to range from 0.03 - 1.4μM. Greater sensitivity to both inhibitors corresponded with accumulation of K48-ubiquitinated protein levels, and increased expression of the unfolded protein response-related genes BiP, DDIT3, and the spliced form of XBP1. NMS-873 (10nM) or CB-5083 (50nM) were also found to increase cisplatin sensitivity (decreased IC50 values, 48 hours) in MIA-PaCa-2, SKOV-3, PEO1, and PEO4 cell lines, but not in AsPC-1 or PANC-1 cells. To investigate if cisplatin sensitization relates to changes in DDR activity, DNA double strand break repair (DSB) assays, using GFP-reporter constructs, were carried out but lower efficiency of homologous recombination (HR) and non-homologous end-joining pathways were found in both PANC-1 and SKOV-3 cells following VCP inhibition. Consistent with decreased HR activity, VCP inhibition reduced numbers of cisplatin-induced Rad51 foci in both cell lines. Unexpectedly, this was associated with increased proteasomal degradation of Rad51, as indicated by western blotting. Phosphorylation of VCP at serine 784 was detected following cisplatin treatment, and interestingly was found to be reduced following inhibition of DNA-PKcs, one of the main PI3K-related kinases (PIKKs) which directs DSB repair. To explore the role of this modification, a phospho-deficient VCP, with mutations in 4 predicted PIKK-targeted sites (including serine 784), was generated. A significant increase in apoptosis, as measured by Caspase-Glo®3/7 assay, was observed in cisplatin-treated SKOV-3 cells, as an initial model system, expressing this phospho-deficient VCP. Studies are underway to further elucidate the importance of each individual phosphorylation site on cisplatin response and DSB repair. Our findings suggest VCP could be a potential therapeutic target for pancreatic and other cancers. Confirming how VCP regulates DSB repair will allow development of novel strategies to enhance the efficacy of DNA damaging agents in cancer.

#2877

Synergistic antitumor interaction of valproic acid and simvastatin sensitizes prostate cancer to docetaxel by targeting cancer stem cells compartment via YAP-pathway modulation.

Federica Iannelli,1 Maria Serena Roca,1 Chiara Ciardiello,1 Simona De Rienzo,1 Rita Lombardi,2 Angela Sorice,2 Susan Costantini,2 Tania Moccia,1 Maria Rita Milone,2 Biagio Pucci,2 Alfredo Budillon,1 Francesca Bruzzese1. 1 _INT Fondazione G. Pascale Napoli, Naples, Italy;_ 2 _CROM- Centro Ricerche Oncologiche Mercogliano, Avellino, Italy_.

Introduction: docetaxel (DTX) represents the standard of care first line treatment of castration-resistant prostate cancer (PCa). However, the onset of systemic side effects hampers patient's compliance and DTX resistance invariably emerges, leading to disease relapse, suggesting the need of novel combination strategies. Cancer stem cells (CSC) drive PCa survival and metastasis and are thought to be responsible for the development of resistance to DTX. The mevalonate pathway (MVP) plays a critical role in PCa progression and is implicated in cell stemness, proliferation, and organ size regulation through the YAP-TAZ signaling axis. Here, we evaluate the combination treatment of valproic acid (VPA), an histone deacetylase (HDAC) inhibitor and the cholesterol-lowering drug simvastatin (SIM), an inhibitor of HMGCoA reductase (HMGCR), the rate limiting enzyme in MVP, alone and plus DTX in PCa models.

Method: synergistic antiproliferative effects were assessed on LNCAP, 22Rv1, DU145, PC3 and DU14580 SIM-resistant cell lines and normal epithelial prostate EPN cells, by calculating combination index (CI) according to Chou and Talalay method. Apoptosis was measured by FACS analysis and caspase assay. Tumor spheroids were obtained by low attach systems and scored with luminescence 3D-cell viability assay. Proteins and genes expression was assessed by western blot and real time PCR analysis. In vivo experiments were performed on xenograft models in athymic mice. Cholesterol content was evaluated by nuclear magnetic resonance (1H-NMR).

Results: synergistic antiproliferative and proapoptotic effect of VPA-SIM was observed in all PCa cell lines tested, except in EPN cells and was confirmed in PCa spheroids. SIM-dependent cholesterol content downmodulation was potentiated by VPA and mevalonic acid, the product of HMGCR activity, reverts all the antitumor combined effect, suggesting the involvement of MVP in the synergistic interaction. Mechanistically, the combination is able to induce a reduction of HMGCR mRNA expression and the inhibitory phosphorylation of HMGCR and YAP, followed by a reduction of CTGF, BRCA5 and Cyr61 YAP-target genes, as well as a reduction of a CSC-marker gene such as NANOg. Notably, the VPA-SIM combination, sensitizes PCa cells to DTX treatment in sensitive and DTX-resistant cells, as shown by CI calculation, apoptosis, and CSC enriched-spheroid experiments. The synergistic interaction of the triple combination was also confirmed in vivo in both DU145R80 and 22Rv1 xenograft models.

Conclusions: Overall, this study suggests that the combination of two safe generic drugs such as VPA and SIM, may improve the therapeutic index of DTX and revert DTX-resistance, representing an innovative and feasible antitumor strategy for the treatment of prostate cancer that warrants further clinical evaluation.

#2878

CWP232291A suppresses the ovarian cancer growth via inhibition of Wnt/β-catenin signaling pathway.

Untack Cho,1 Se Ik Kim,2 Yong Sang Song2. 1 _Seoul National University, Seoul, Republic of Korea;_ 2 _Seoul National University, College of Medicine, Seoul, Republic of Korea_.

Over the last decades, anticancer therapy options have expanded. Although many types of new drugs are consistently developed, acquired chemoresistance remains as a main obstacle for treatment of ovarian cancer. To overcome the chemoresistance, development of new effective drug is essential. CWP232291A (CWP291) was designated for targeting Wnt/β-catenin signaling pathway. Growth inhibition by CWP291 was tested using 12 ovarian cancer cell lines and 2 ascites-derived ovarian cancer cells. CWP 291 showed more growth-inhibitory capacity to ovarian cancer cells compared to cisplatin. Interestingly, there was no effect for growth inhibition in normal peripheral blood mononuclear cells (PBMC). Active form of β-catenin and GRP78 expression was determined in treatment of CWP291. Treatment of CWP291 significantly reduced the expression level of β-catenin active form and GRP78, but cisplatin did not affect the expression level of β-catenin active form and GRP78. Antiproliferative effect of CWP291 was also determined using ovarian cancer tissue derived-organoid model system. Ovarian cancer tissue derived-organoid was established from 10 ovarian cancer patient samples. Size and number of ovarian cancer tissue-derived organoid was reduced in treatment of CWP291. Taken together, CWP291 has more anticancer effect to ovarian cancer than standard chemo-agent, cisplatin. In conclusion, CWP291 provides a new therapeutic approach in ovarian cancer treatment.

#2879

Trifluoperazine, a novel autophagy inhibitor, prolongs the survival of experimental brain metastases.

Xin Zhang,1 Himalaya Parajuli,2 Synnøve Aasen,2 Heidi Espedal,3 Jian Wang,1 Rolf Bjerkvig,4 Xingang Li,5 Frits Thorsen3. 1 _Department of Neurosurgery, Qilu Hospital of Shandong University and Brain Science Research Institute, Shandong University, Jinan, China. Kristian Gerhard Jebsen Brain Tumour Research Centre, Department of Biomedicine, University of Bergen, Bergen, Norway;_ 2 _Kristian Gerhard Jebsen Brain Tumour Research Centre, Department of Biomedicine, University of Bergen, Bergen, Norway;_ 3 _Kristian Gerhard Jebsen Brain Tumour Research Centre, Department of Biomedicine, University of Bergen, Bergen, Norway. Molecular Imaging Center, University of Bergen, Bergen, Norway;_ 4 _Kristian Gerhard Jebsen Brain Tumour Research Centre, Department of Biomedicine, University of Bergen, Bergen, Norway. Department of Oncology, Luxembourg Institute of Health, Luxembourg;_ 5 _Department of Neurosurgery, Qilu Hospital of Shandong University and Brain Science Research Institute, Shandong University, Jinan, China_.

Brain metastasis is common in patients with melanoma and has been associated with a poor prognosis. Although recent advances with BRAF and immune checkpoint therapies have improved patient survival, the existence of blood-brain barrier (BBB) prevents many anti-tumour reagents from entering the brain, which significantly limits their use in the treatment of brain metastasis. Our previous study showed that trifluoperazine (TFP), an antipsychotic drug which has been used in the clinic for more than 50 years, could increase the radiosensitivity effects on glioblastoma (GBM) in vitro and in vivo1. In this study, we tested the anti-tumour effect of TFP in a well-established animal model of experimental melanoma brain metastases. Melanoma brain metastasis models were established by injecting a primary human melanoma brain metastasis cell line (H1), which was transduced with the genes for green fluorescent protein and luciferase, into the left cardiac ventricle of immunodeficient mice. Two weeks after tumour cells injection, mice were treated with PBS or TFP intraperitoneally. Magnetic resonance imaging was done to calculate the tumour numbers and volumes. Survival duration was also calculated for each group. In vitro, cell viability, cell proliferation and spheroid growth were tested in four melanoma metastasis cell lines (H1, H3, Melmet1 and Melmet5). Western blot analysis of autophagy-related proteins, RFP-GRP-LC3B, transmission electron microscopy, Lysotracker Red, DQ-BSA and immunofluorescence were used to test the autophagic status of melanoma metastasis cell lines after TFP treatment. From the results of MRI on Week 4 and Week 6 after tumour injection, we found that TFP decreased tumour numbers and volumes significantly, as compared to untreated animals. We also discovered that TFP prolonged animal survival, compared to the control group. In vitro, we found that TFP decreased cell viability, cell proliferation and spheroid growth. Further study showed that TFP inhibited autophagic flux by suppressing the fusion of autophagosomes with lysosomes, and impairing the function of lysosomes. In summary, as a novel late stage autophagy inhibitor, TFP showed efficient anti-tumour effects and increased survival in an experimental brain metastases model. 1. Zhang, X., et al., Trifluoperazine, a novel autophagy inhibitor, increases radiosensitivity in glioblastoma by impairing homologous recombination. J Exp Clin Cancer Res, 2017. 36(1): p. 118.

#2880

Targeting Rho GTPases in ovarian clear cell cancer.

Nicolai S. Arildsen, Ingrid Hedenfalk. _Lund University, Lund, Sweden_.

Background: Ovarian clear cell carcinomas (OCCC) constitute a rare subtype of epithelial ovarian cancer with distinct clinical features. Resistance to first-line platinum-based treatment is common. We recently reported deregulated Rho GTPase signaling in OCCC. Rho GTPases are small signaling G proteins, and a subfamily of the RAS superfamily. Rho GTPases are reported to regulate actin organization and cytoskeletal organization. We hypothesized that inhibiting Rho GTPases would have a potent cytotoxic effect on OCCC cell lines.

Materials and Methods: Sulphorhodamine B (SRB) assays were used to assess the antiproliferative effects of 72h of treatment with the cholesterol-lowering agent simvastatin, the pan GTPase inhibitor CID1067700 (CID) and carboplatin (as control) in the two OCCC cell lines JHOC5 and OVMANA. Single effects (IC50) and combination effects were calculated using the Chou-Talalay method. We assessed cytoskeletal integrity using phalloidin staining of actin and DAPI for cell death. Western blotting was used to assess the expression of cleaved PARP (cPARP) after 4h and 24h of treatment.

Results: All three drugs had similar IC50 values in both OCCC cell lines; however, they were significantly different from the high-grade serous ovarian cancer control cell line CAOV3. JHOC5 and OVMANA were more resistant to carboplatin (IC50: 65.3 µM and 76.2 µM, respectively) compared to CAOV3 (IC50: 16.4 µM). JHOC5 and OVAMANA were more sensitive to simvastatin and CID (JHOC5: 6.7 µM/69.2 µM, OVMANA: 10.3 µM/84.9 µM, simvastatin/CID1067700) compared to CAOV3 (27.7 µM/122.5 µM). The combination index (CI) for either CID or simvastatin in combination with carboplatin was moderately antagonistic for all cell lines (CI: 1.6-2.4). Combinations of CID and simvastatin were mildly to moderately synergistic (CI: 0.6-0.8) for all cell lines. Simvastatin and CID were found to interfere with actin organization, compared to carboplatin, with simvastatin also interfering with cell membrane integrity. A small increase in cPARP was observed after 24h of treatment with all drugs in single regimens.

Conclusion: Cytotoxic effects of simvastatin and CID both in single regimens or in combination were achieved with lower doses in OCCC cell lines than in the high-grade serous cells; however, combinations of simvastatin and CID were found to be mildly synergistic in all cell lines. Simvastatin was found to be more potent than CID. Interestingly, all combinations with carboplatin were found to be moderately antagonistic. Actin organization was more extensively disrupted with simvastatin than CID. An initial assessment of cell death mechanisms suggested a mechanism working through cleaved PARP. However, these findings provide a basis for further studies into the molecular mechanisms of simvastatin and CID in OCCC. These studies are currently under way, including an in-depth assessment of the underlying signaling and cell death mechanisms.

#2881

RET signaling in pancreatic cancer: A novel target by metformin to suppress cancer progression.

Huailong Chang,1 Zhiyong Xiao,2 Tao Li,1 Lanjing Zhang,3 Yong Lin,4 Darren Carpizo,1 Xianglin Tan1. 1 _Rutgers Cancer Institute of New Jersey, New Brunswick, NJ;_ 2 _Beijing Institute of Pharmacology & Toxicology, Beijing, China; _3 _University Medical Center of Princeton, Plainsboro, NJ;_ 4 _Rutgers the State University of New Jersey, Piscataway, NJ_.

Metformin has recently gained attention as an anti-cancer drug and/or a chemoprevention agent because of its roles in inhibiting mTOR, lowering hyperinsulinemia, modulating inflammatory responses, and selectively killing cancer stem cells. However, the key underlying molecular mechanisms for the inhibitory effects of metformin on pancreatic cancer progression remain largely unknown. RET (REarranged during Transfection), a single-pass transmembrane receptor tyrosine kinase and its ligand, glial cell-derived neurotrophic factor (GDNF), were strongly expressed in pancreatic cancer and correlated to invasion and worse survival after surgical resection. In this study, we investigated the roles of RET in the inhibitory effects of metformin on pancreatic cancer cell growth and migration. Real-time PCR and Western blot were used to determine mRNA and protein levels of molecular markers, respectively. siRNAs were adopted to specifically knockdown target genes. Boyden chamber assay was applied to assess cell migration in vitro. We observed that metformin treatment significantly reduced the mRNA and protein expression of RET in a dose-dependent manner in pancreatic cancer cells PANC-1 and MIA PaCa-2. The inhibitory effects of metformin on RET expression were not diminished when AMPK was knockdown by AMPK siRNA or the specific inhibitor, Compound C, suggesting that metformin may suppress RET in an AMPK-independent manner. Metformin treatment or RET knockdown by RET siRNA significantly decreased the phosphorylation of NF-κB and p70S6K, while modestly reduced the phosphorylation of the ERK, AKT, or STAT3. Furthermore, metformin treatment or RET knockdown significantly inhibited GDNF-induced cell migration. These data indicate that targeting RET with metformin may be an attractive and novel strategy for the prevention and treatment of pancreatic cancer progression and metastasis. Further in vitro and in vivo studies are warranted to investigate how metformin modulates RET signaling to inhibit the progression and metastasis of pancreatic cancer.

#2882

Elucidation of the mechanism of action of the multi-kinase inhibitor midostaurin in NSCLC.

Claudia Ctortecka, Brent Kuenzi, Bin Fang, Natalia Sumi, Victoria Izumi, Fumi Kinose, Eric Haura, John Koomen, Uwe Rix. _Moffitt Cancer Center, Tampa, FL_.

Background: Non-small cell lung cancer (NSCLC) constitutes a strong unmet medical need in spite of recent successes with targeted drugs and immunotherapy in some subsets. We had observed the recently FDA-approved drug midostaurin to exhibit potent anticancer activity in a subset of NSCLC cell lines that was unrelated to its known targets FLT3 or PKC. We here explored a new repurposing opportunity and investigated the mechanism of action (MoA) of midostaurin in NSCLC utilizing a multilayered functional proteomics approach.

Methods: Chemical proteomics identified kinase targets and global and tyrosine phosphoproteomics generated a network view of intracellular signaling upon midostaurin treatment. Functional validation was performed using RNA interference and selective small-molecule probes. Cell viability assays were carried out using CellTiterGlo and colony formation assays. Induction of apoptosis was determined by immunoblotting and fluorescent microscopy. Flow cytometry assessed cell cycle stages. Signaling changes were determined by immunoblotting.

Results: Viability screening confirmed anticancer activity in several midostaurin-sensitive NSCLC cell lines at clinically relevant concentrations independent of its canonical targets. Using chemical proteomics, we identified multiple midostaurin kinase targets. Network-based integration of the chemical proteomics and quantitative tyrosine and global phosphoproteomics data using STRING for protein-protein interactions and NetworKIN for substrate-kinase predictions revealed several nodes to be relevant for the MoA of midostaurin. Subsequent functional validation showed that simultaneous inhibition of TBK1, PDK1 and AURKA was required to mimic midostaurin's cellular effects in these cells. Immunoblotting for downstream targets of these kinases revealed PLK1 as a critical signaling effector. Rational combination of midostaurin with the more potent PLK1 inhibitor BI2536 caused strong synergy.

Conclusion: Midostaurin showed activity in a subset of NSCLC cells. Combination of complementary functional proteomics approaches and subsequent network-based integration revealed novel insight into the MoA of midostaurin and NSCLC vulnerabilities. We utilized this mechanistic knowledge for the rational combination of midostaurin with the clinical PLK1 inhibitor BI2536, which is highly synergistic and has strong potential for clinical translation.

#2883

Repurposing atovaquone, an antiprotozoal drug for management of breast cancer by inhibiting HER2/β-catenin signaling.

Nehal Gupta, Sanjay Srivastava. _Texas Tech Univ. Health Sciences Ctr., Amarillo, TX_.

Breast cancer is the second highest cause of cancer-related mortality in women. HER2 is an oncogene overexpressed in about 30% of breast cancer patients and β-catenin, a proto-oncogene plays a pivotal role in breast cancer metastasis and drug resistance leading to poor prognosis. In the current study, we evaluated the anti-cancer effects of an anti-protozoal drug, atovaquone against several breast cancer cell lines such as MCF-7, 4T1, HCC1806, CI66, SKBR3 and T47D. Our results showed that atovaquone treatment induced apoptosis as evaluated by PI/Annexin assay and cleavage of caspase-3 and PARP, and inhibited the growth of all the breast cancer cell lines tested. Similar observations were made in patient-derived cancer cell lines. In addition, atovaquone treatment significantly reduced the expression of HER2, β-catenin and its downstream molecules such as pGSK-3β, TCF-4, cyclin D1 and c-Myc in these cell lines. Treatment of HCC1806 cells with 5 µM atovaquone resulted in significant reduction in mammosphere forming efficiency (MFE) of breast cancer cells. Mammosphere-forming cells are known to exhibit cancer stem cell (CSC)-like properties together with drug resistance. Efficacy of atovaquone was evaluated in the in-vivo tumor model by orthotopic implantation of two highly aggressive 4T1 and CI66, triple negative breast cancer cells in the mammary fats of female mice. Our results demonstrate that oral administration of atovaquone suppressed the growth of CI66 and 4T1 tumors by 70% and 60% respectively. Atovaquone treated tumors exhibited reduced HER2, β-catenin and increased apoptosis when analyzed by western blot. We also developed resistance in 4T1 breast cancer cells towards paclitaxel, a first line chemotherapeutic agent for treatment of metastatic breast cancer. 4T1 paclitaxel resistant cells were orthotopically implanted in female balb/c mice and mice were treated with atovaquone for 20 days. Our results showed that atovaquone was able to suppress the growth of paclitaxel resistant tumors by 42%. Conclusively, our results indicate that atovaquone effectively reduces the growth of primary breast tumors as well as paclitaxel resistant tumors by inhibiting HER2/β-catenin signaling. Most importantly, atovaquone is already in clinical use with an established safety record therefore, any positive findings from our studies will prompt further clinical investigation into repurposing atovaquone for the treatment of advanced breast cancer patients. [Supported in part by R01 grant CA 129038 awarded by National Cancer Institute, NIH].

#2884

PELP-1 promotes adverse endocrine therapy response in ER+ breast cancer.

Michael Rees,1 Chris Smith,2 Peter Barrett-Lee,1 Stephen Hiscox2. 1 _Velindre Cancer Centre, Cardiff, United Kingdom;_ 2 _Cardiff University, Cardiff, United Kingdom_.

Introduction: Despite the benefits of endocrine therapy for the management of ER positive breast cancer, not all patients respond equally leading to resistance and associated disease relapse and progression. We previously reported that endocrine agents themselves may induce an invasive phenotype in ER positive breast cancers with low/aberrant expression of E-cadherin. Here we investigate this phenomenon further, and provide data supporting a role for the ER co-receptor PELP-1, in mediating an adverse response to endocrine agents.

Methods: The effects of tamoxifen ('tam'), fulvestrant ('fas') and estrogen withdrawal (as a model for aromatase inhibitor therapy) on the invasive and migratory capacity of endocrine-sensitive MCF-7 and T47D cells, in the presence or absence of functional E-cadherin and/or PELP-1 (using siRNA knockdown), was assessed via matrigel invasion and Boyden chamber migration assays. The effects of endocrine therapies alongside E-cadherin/PELP-1 modulation on cell proliferation were also assessed by growth assays, and western blotting using phospho-specific antibodies was performed to investigate signalling pathway changes associated with endocrine-induced changes in invasion and migration.

Results: Both tamoxifen and fulvestrant induced a pro-invasive and pro-migratory phenotype in ER positive breast cancer cells displaying a high basal expression of PELP-1 which was augmented in the context of poor cell-cell contact (mean fold increase in invasion with endocrine treatment was 3.1±1.2; p=0.002 [MCF7+tam] and 2.7±1.4; p=0.002 [MCF7+fas]. Mean fold increase in invasion with endocrine treatment + E-cadherin siRNA was 22.4±5., p<0.001 [MCF7+tam] and 18.6±2.2 [MCF7+fas]). This process occurred in a Src-dependent manner with Src inhibition reversing endocrine induced invasion/migration (mean fold increase in invasion with endocrine treatment + E-cadherin siRNA in the absence of Src activity was 1.3±0.5, p<0.001 [MCF7+tam] and 0.8±0.5; p<0.001 [MCF7+fas]). siRNA-mediated suppression of PELP1 also reduced endocrine treatment-induced invasion and migration to a similar extent as Src suppression. In contrast to tam and fas, estrogen withdrawal did not induce an invasive or migratory phenotype irrespective ot E-cadherin or PELP1 expression level.

Conclusion: Our data demonstrates that anti-estrogens exert pro-invasive effects on ER-positive breast cancer cell models, particularly in the absence of homotypic cell-cell contact through a mechanism involving PELP1 and Src. These results suggest that E-cadherin and PELP-1 may be potential biomarkers when deciding upon optimum adjuvant endocrine therapy, whereby tumors with high PELP-1 / low E-cadherin expression may benefit from estrogen withdrawal therapy via aromatase inhibition, instead of direct ER modulation/antagonism.

#2885

Dihydroceramide increase precedes golgi dispersal, pro-survival autophagy, ER stress, and UPR in fenretinide + safingol treated neuroblastoma cells.

Nikhil Vad, Dong Wang, Hwangeui Cho, Dattesh Verlekar, Charlie Linch, C Patrick Reynolds, Min Kang, Barry J. Maurer. _Texas Tech Univ. Health Sciences Ctr., Lubbock, TX_.

Introduction: We have reported that fenretinide (4-HPR) is active against high-risk neuroblastoma (NB) in vitro and in vivo; our Phase I trials of novel 4-HPR formulations evidenced clinical activity. Mechanisms of activity include increase of cytotoxic dihydroceramides. Fenretinide activity in vitro is enhanced by safingol (S), the L-threo diastereomer of sphinganine. Here, we delineate stress pathways activated in response to 4-HPR±S and identify ixazomib, a proteasomal inhibitor, and antimalarial, mefloquine, a disruptor of autophagy, as new potential synergizing agents for 4-HPR±S. Methods: Sphingolipids were assessed by LC/MS/MS; cytotoxicity by fluorescence-based plate assay in 2% and 5% oxygen; apoptosis by TUNEL assay. ER stress markers, unfolded protein response (UPR), and autophagy, were assessed using immunoblotting, immunoprecipitation, and electron/confocal microscopy. Target validation was by gene silencing. Results: 4-HPR rapidly increased D-erythro-dihydroceramides; safingol was catabolized to L-threo-dihydroceramides. Safingol (2-3 µM) caused multi-log cytotoxic synergy of 4-HPR in eight of ten GBM and five NB cell lines (CI<0.7). Treatments resulted in golgi fragmentation (+6-12h) without decrease of stack proteins, and preceded increase of ER stress transducer, GRP78, and pro-apoptotic CHOP protein; UPR was evidenced by increase of poly-ubiquitinated proteins and increased autophagic flux (+12-48h). Cell death was apoptotic (cleaved caspase-3/PARP; TUNEL-positive) and non-apoptotic (+12-48h). Disruption of autophagy by mefloquine, or siRNA-silencing of BECN1 or ATG7, temporally-accelerated cytotoxicity and increased total apoptosis (p<0.05) in GBM cells. Consistent with cytotoxicity being dependent on misfolded protein stress, ixazomib further increased ER stress markers and accelerated/increased cytotoxicity (p<0.05). Treatment produced early inhibition (+4h) of tyrosine phosphorylation of p97/VCP, an AAA+ ATPase critical for the fusion of tER membrane vesicles into golgi stacks. Knockdown of p97/VCP recapitulated features of 4-HPR±S treatment, including golgi fragmentation, increase of ubiquitinated proteins and autophagic vacuoles. Treatment with D-erythro-sphinganine plus dihydroceramide desaturase inhibitor, GT-11, plus safingol increased both D-erythro and L-threo-dihydroceramides and recapitulated morphological, biochemical, and cytotoxic effects. Conclusion: Delineation of response pathways allowed the identification of mefloquine and ixazomib as new potential co-drugs to synergize 4-HPR±S. Confirmatory studies in NB xenografts are in progress. Intravenous fenretinide is currently in a Phase 2 trial in adult refractory/relapsed Peripheral T-Cell Lymphoma. A Phase I trial of fenretinide + safingol is in progress in the South Plains Oncology Consortium.

#2886

ChIP-seq analysis to explore DNA replication profile in trifluridine-treated human colorectal cancer cells in vitro.

Takashi Kobunai, Kazuaki Matsuoka, Teiji Takechi. _Taiho Pharmaceutical Co., Ltd., Tokyo, Japan_.

Background: Trifluridine (FTD) is a key component of the novel oral antitumor drug trifluridine/tipiracil, which was approved for the treatment of patients with metastatic colorectal cancer refractory to standard chemotherapies. FTD, an antineoplastic thymidine analogue, is efficiently incorporated into the genomic DNA of tumor cells. However, the molecular mechanisms underlying its cytotoxic effects remain unclear. Therefore, the DNA replication profile in FTD-treated cells was comprehensively analyzed to explore the mode of FTD incorporation into the genomic DNA. Method: We generated the DNA replication profile in HCT-116 human colorectal cancer cells as follows: asynchronous cultures of HCT-116 were exposed to 5 μM 5-bromo-2′-deoxyuridine (BrdU), a non-cytotoxic control drug or FTD, for 4 h, harvested, and subjected to DNA immunoprecipitation using an anti-BrdU antibody. An input and immunoprecipitated DNA from each experiment were sequenced, and the sequence reads generated using Illumina sequencing were aligned uniquely to the human genome (hg19), allowing up to two mismatches using the BWA algorithm with default settings. The density of aligned reads along the genome was calculated and stored in a bigWig file for visualization in genome browsers. Peak finding and gene ontology analysis were performed using MACS and Genomic Regions Enrichment of Annotations Tool (GREAT). The 60-bp sequences surrounding the summits of the top 1000 peaks were analyzed for conserved motifs using the online program MEME. Results: In an antibody validation step, we observed significant FTD peaks over input DNA. We identified 6043 and 5080 high confident FTD and BrdU peaks in HCT-116 cells, respectively. Interestingly, 2911 of 6043 FTD peaks were uncommon to BrdU peaks, while 3232 peaks were common in FTD and BrdU. The FTD and BrdU peaks were not randomly distributed throughout the genome. Most FTD and BrdU peaks were ±5 kb or further away from transcription start sites. We identified the genes associated with distant peaks of FTD using GREAT. FTD peaks occurred near genes involved in extracellular matrix structural composition such as ACAN, BGN, CHAD, and COL12A1, while BrdU peaks were near genes involved in cysteine-type endopeptidase inhibitor activity such as ARRB1, BIRC7, LCN1, and CST1. Dinucleotide repeats such as TGTGTG were observed as conserved motifs in FTD-peak sequences. Conclusion: Global FTD incorporation patterns can delineate active replication loci, namely FTD preferentially incorporating loci, in human cancer cells. The DNA replication profile in FTD-treated cells differed from that in BrdU-treated cells. Thus, differences in the incorporation patterns of FTD and BrdU into the genomic DNA revealed the mechanisms of antitumor activities of FTD.

#2887

Evaluating the effectiveness of MAPK, AKT, and mTOR inhibitors in reducing proliferation in cellular models of papillary and follicular thyroid cancer.

Brianna LeBoeuf,1 Braxton Anderson,1 Margaret Young,1 Aime Franco,2 Laura MacDonald1. 1 _Hendrix College, Conway, AR;_ 2 _University of Arkansas for Medical Sciences, Little Rock, AR_.

Thyroid cancer is the most common endocrine malignancy, and incidence has been steadily increasing, suggesting occurrence will exceed that of colon cancer by 2030. Papillary and follicular thyroid cancer subtypes are most common, which are activated by BRAF and HRAS mutations, respectively. Interestingly, papillary and follicular thyroid cancers are associated with different pathologies and metastases. Currently, both papillary and follicular thyroid cancers are treated by surgical removal of the thyroid, followed by radioactive iodine treatment to eliminate any remaining tumor cells. Unfortunately, for individuals with progressive thyroid cancer, these treatment options are not effective, highlighting a need for increased investigation into mechanisms of drug sensitivity. In this study, we sought to evaluate mechanisms of differential drug sensitivity through RNA-seq analysis of cell lines derived from mouse models of papillary and follicular thyroid cancer. Additionally, we evaluated the effects of MAPK, AKT, and mTOR inhibitors on murine papillary and follicular thyroid cancer cell proliferation. To accurately assess drug efficacy, we calculated GR50 concentrations for each inhibitor in six papillary and follicular thyroid cancer cell lines. We discovered that papillary and follicular thyroid cancer cell lines responded differently to MAPK and AKT inhibitors, suggesting that treatment approaches should be tailored to subtype despite having mutations in the same signaling pathway.

#2888

DNA Repair Enzyme Signature as a biomarker of MAPK pathway inhibition by targeted therapies in melanoma cell lines.

Sylvie Sauvaigo,1 Manel Benkhiat,2 Florian Braisaz,2 Florence de Fraipont,2 Caroline Aspord,3 Stéphane Mouret,2 Joël Plumas,3 Fanny Bouquet,4 Marie-Thérèse Leccia2. 1 _LXRepair, Grenoble Cedex 9, France;_ 2 _CHU Grenoble Alpes, Grenoble, France;_ 3 _EMR EFS-UGA-INSERM U1209- CNRS, Grenoble, France;_ 4 _Institut Roche, Boulogne-Billancourt, France_.

About 50% of melanomas carry activating mutation in BRAF or NRAS genes. BRAF/MEK inhibitors elicit a transient effective response but resistance rapidly develops through various MAPK/PI3K/AKT pathway activating mechanisms. DNA Repair mechanisms are regulated by these signaling pathways. We hypothesized that effective inhibition of the MAPK pathway should translate into decrease of DNA Repair capacities.

To gain insights into this hypothesis we measured the DNA Repair capacities of 12 melanoma cell lines treated or not by BRAF and MEK inhibitors (respectively Vemurafenib V and Cobimetinib C, Roche laboratories), alone and in combination.

We evaluated various excision synthesis repair mechanisms using a multiplexed functional DNA Repair assay using cell extracts. We thus obtained a comprehensive overview of the cell lines DNA Repair capacities.

Significant qualitative and quantitative differences were observed between the DNA Repair profiles of the 3 mutation groups in non-treated cells.

Globally DNA Repair capacities of BRAFm cells significantly decreased following treatment by V and V+C confirming that excision synthesis repair mechanisms are under the control of the MAPK pathway. When the cell lines were examined individually, interesting features were observed. 3/7 showed drastic decrease of DNA repair with V and V+C treatment. On the contrary, C alone activated repair in 3/7, possibly reflecting a paradoxical activation of some pathways.

In addition, among the 3 NRASm cell lines, only 1 exhibited a marked decreased of DNA Repair after C treatment. Surprisingly V and V+C activated some repair activities in one WT cell line, possibly reflecting some paradoxical activation of the complex kinase network.

Our results suggest that mutations in signaling kinase pathways impact the so-called DNA Damage Response and translate into specific DNA Repair Enzyme Signatures. Crosstalk of tyrosine kinases with the DNA damage signaling pathway is well known. It is possible to take advantage of their intricate regulation through characterization of DNA Repair Enzymatic Signature to gain information on the inhibition efficacy of targeted drugs.

Interestingly this approach could be conducted on clinical samples and we propose to use the DNA Repair Enzyme Signature as a new tool to investigate effectiveness of targeted therapies in metastatic melanoma.

This study was sponsored by Institut Roche.

#2889

Aitongxiao (ATX) induces apoptosis and blocks exosomes release in human liver carcinoma cells.

Ming B. Huang,1 Jiang Leng2. 1 _Morehouse School of Medicine, Atlanta, GA;_ 2 _Guangxi University of Chinese Medicine, Nanning, Guangxi, China_.

Background: The role of exosomes in cancer development has become the focus of much

research, due to the many emerging roles possessed by exosomes. These contents of exosomes

could contribute to tumorigenesis. We discovered Aitongxiao (ATX), a group of 80% alcohol

extracts from 15 plants, has been shown to have an anti-tumor effect on liver carcinoma; however,

the mechanism for the anti-cancer effect of ATX on human liver carcinoma cells is still unclear.

Objective: Due its inhibitory effects on chemical carcinogenesis and inflammation, Aitongxiao

(ATX) has been proposed as an effective agent for the prevention or treatment of human

carcinoma. In this study, we aimed to explore the effect of ATX on human liver carcinoma cells.

Methods: The inhibitory effect of Aitongxiao (ATX) on proliferation of HepG2 and HKCL-C3 cells

was assessed by CCK-8 assay. The cell cycle distribution and cell apoptosis were determined by

flow cytometry. The expression of Alix and CD63 exosomes marker proteins, were detected by

Western Blotting. The exosomes protein concentration was measured by and fluorescent

antibody assays. Exosomes size and particle number were measured by NanoSight analysis.

Results: Aitongxiao (TAX) inhibited the growth of human liver carcinoma cells in a dose and time-

dependent manner. Flow cytometry analysis showed that ATX induced G2/M phase arrest and

cell apoptosis. ATX treatment promoted the cleavage of caspase 3. In addition, the ATX reduced

exosomes release from HepG2 cells, and lose-dose ATX significantly enhanced the growth

inhibition induced by 5-Fu.

Conclusions: Aitongxiao (ATX) could inhibit the growth of human liver carcinoma cells in vitro.

ATX induced both apoptosis and cell cycle arrest and blocked exosomes release, probably

through the inhibition of STAT3 function. Lose-dose ATX significantly enhanced the growth

inhibitory effect of 5-FU on breast cancer cells, further indicating the potential clinical values of

ATX for the prevention or treatment of human liver carcinoma.

Keywords: Aitongxiao, liver carcinoma, HepG2 cells, apoptosis, exosomes

#2890

Trifluridine efficacy in cancer stem cell.

Kenta Tsunekuni,1 Masamitsu Konno,2 Jun Koseki,2 Ayumu Asai,2 Yuichiro Doki,2 Masaki Mori,2 Hideshi Ishii2. 1 _Taiho Pharmaceutical Co.,Ltd, Tokushima, Japan;_ 2 _Graduate School of Medicine, Osaka University, Osaka, Japan_.

Background: Cancer stem cells (CSC) have been involved in disease recurrence, metastasis, and therapeutic resistance, but effective anti-cancer agent for these cells is not still currently available. Trifluridine (FTD) is a key component of the novel oral antitumor drug trifluridine/tipiracil, which was approved for the treatment of patients with metastatic colorectal cancer who have been previously treated with fluoropyrimidine, oxaliplatin and irinotecan based chemotherapy etc. and improved overall survival. Here we showed the efficacy of FTD in CD44 and CD133 high expressing (CD44high/CD133high) cancer cells which has CSC like property.

Method: CD44high /CD133high and CD44low /CD133low DLD-1 cells were separated by Fluorescence-Activated Cell Sorting (FACS) and subjected to sphere formation assay. Anti-proliferative and anti-sphere forming effects of FTD in CD44high /CD133high cells were measured. Then, 150 days FTD long exposed colorectal cell line's CSC markers CD44 and CD133 were measured by flow cytometry and its tumor initiating property in in vitro and in vivo were evaluated.

Results: CD44 high /CD133 high DLD-1 cells formed much more sphere than CD44low /CD133low cells significantly. Compared to unsorted cells, CD44high /CD133high cells displayed 5-FU resistance (IC50 value of 5-FU for unsorted and CD44 high /CD133 high cells were 2.5 μM and 5.5 μM respectively) but not FTD resistance (IC50 value of FTD for unsorted and CD44 high /CD133 high cells were 8.9 μM and 10.7 μM, respectively) in in vitro proliferation assay. In addition, FTD treatment in a subtoxic concentration 1 μM for DLD-1 cells disrupted sphere formation. FTD-long exposed DLD-1 cell lines showed decreased CD44high /CD133high population compared to its parental cell line from 11.3% to 0.7%, and showed decreased sphere-forming efficacy. In addition, FTD-long exposed DLD-1 cell lines decreased tumor forming potential in in vivo. These results suggest that FTD is effective for CSC like cells and that long exposure to FTD might lead CSC depletion.

Conclusion: FTD was proved to be effective for CSC like CD44high /CD133high cells. It suggests that FTD might be useful for CSC targeted chemotherapy in case of CD44 and CD133 are highly expressed subtype of tumors.

### New Targets 2

#2891

Molecular profiling of hormone-resistant prostate cancer cells with TRPV6 oncochannel knockout/knockdown.

Tyler Lutes,1 Michelle Davey,2 Christopher Rice,2 Tyson MacCormack,1 John M. Stewart,2 Dominique Dugourd2. 1 _Mount Allison University, Sackville, New Brunswick, Canada;_ 2 _Soricimed Biopharma Inc., Moncton, New Brunswick, Canada_.

TRPV6 calcium channel is a recognized oncochannel over-expressed in a number of epithelial cancers (e.g. breast, ovarian, prostate). TRPV6, a member of one of seven subfamilies of TRP channels, plays an important role in Ca2+ absorption from the intestinal lumen. TRPV6 over-expression is associated with a poor prognosis particularly with breast and prostate cancers. Although some TRPV6 mechanistic work is available and indicates that NFAT is involved, there is a need to better understand pathways involved in its mechanism of action (MOA). These data could allow the identification of clinically relevant biomarkers and identify potential for synergy of TRPV6-targeting therapies with other anti-cancer treatments. To determine which key pathways TRPV6 influences in prostate cancer, two TRPV6 knockouts (KO) were produced in a castration-resistant prostate cancer cell line (PC-3) using the CRISPR-Cas9 approach with two different gRNAs. In addition, knockdowns (KD) were produced by transfecting PC-3 cells with two TRPV6 siRNA targeting different regions of TRPV6 mRNA. The mRNA expression of 184 genes was analyzed using an RT-qPCR TaqMan array. The panel consisted of genes involved in cancer calcium signalling, genes that have been associated with the MOA of TRPV6 and genes directly or indirectly involved in NFAT signalling. Gene profiling results were generally consistent between the TRPV6 KD and KO cells. Results showed a dramatic reduction in expression of TRPV6 mRNA in the KO/KD cells (14 to 166-fold). TRPV6 KO/KD affected multiple genes involved in cancer cell proliferation (e.g. ESR1, CDH1), metastasis (e.g. SNAI1, MUC1/16), resistance to apoptosis (e.g. Bcl-2-type proteins, Fos) and angiogenesis (e.g. VEGFA/B, FLT4), as well as genes involved in the M2 immune tumor microenvironment (e.g. IL6 and VEGFA). The TRPV6 KO/KD data confirm the basic MOA of TRPV6, activating NFAT by increasing cytosolic calcium and provide information on the downstream pathways involved (e.g. NF-kB, estrogen and MAPK). The data allow for the identification of clinical efficacy biomarkers for TRPV6 inhibitors (e.g. a panel of CXCL12, FLT4, MUC16 and IL-6). Furthermore, results indicate TRPV6 inhibitors have the potential to modify the immune composition of the tumor microenvironment from a tumor promoting to a tumor control response, and thus may help trigger an anti-cancer immune defence. This study demonstrates a central role of TRPV6 and calcium signalling in cancer development.

#2892

**EGLN1 is a synthetic lethal target in** ARID1A **-mutant ovarian cancer.**

Kimberly J. Briggs, Chengyin Min, Hongxiang Zhang, Alan Huang. _Tango Therapeutics, Cambridge, MA_.

The successful use of PARP inhibitors for the treatment of BRCA-mutant ovarian cancer underscores the importance of synthetic lethality as a therapeutic opportunity for tumors with loss-of-function mutations in tumor suppressor genes. Ovarian cancer ranks as the fifth deadliest cancer among women, and one ovarian cancer subtype, clear cell carcinoma, is particularly underserved. The most frequent genetic alteration in ovarian clear-cell carcinoma is loss-of-function mutation of the SWI/SNF-A complex subunit, ARID1A. We interrogated the data published by Project Achilles to determine candidate synthetic lethal partners with ARID1A in ovarian cancer, and identified EGLN1, a member of the EglN-family of prolyl hydroxylases. Inhibition of EGLN1, either genetically or pharmacologically, leads to decreased viability in ovarian cancer cell lines. Pharmacological inhibition of EGLN1 is clinically achievable as evidenced by several well-tolerated, small molecule inhibitors currently in clinical trials for the treatment of anemia in the context of chronic kidney disease. The synthetic lethal interaction of ARID1A and EGLN1 may provide a therapeutic opportunity for patients diagnosed with ovarian clear-cell carcinoma.

#2893

Balancing the efficacy and toxicity of anti-CD47 antibodies by direct screening in humanized mouse models.

Yanan Guo,1 Chaoshe Guo,1 Yuelei Shen,1 Benny Yang2. 1 _Beijing Biocytogen Co., Ltd, Beijing, China;_ 2 _Eucure (Beijing) Biopharma Co., Ltd, Beijing, China_.

CD47 is a transmembrane protein found on the surface of many cells in the body. Its expression is often up-regulated in many cancer cells. The receptor of CD47 was identified as SIRPa, which is expressed on phagocytic cells. Engagement of SIRPa by CD47 serves as "do not eat me" signal, thus inhibiting the phagocytic activity of macrophages. Cancer cells often hijack this pathway by boosting CD47 expression on their surface, in order to prevent innate cell-mediated phagocytosis. Molecules blocking the CD47-SIRPa interaction will unleash such inhibition and promote tumor destruction. Accordingly, anti-CD47 antibodies offer new hope to successful cancer treatment. Despite people are enthusiastic about CD47 as a potential target, the side effects associated with CD47 blockade have emerged as a major concern. For example, treatment with anti-CD47 antibody greatly reduced the number of circulating red blood cells and platelets that also express CD47. Thus, the class of CD47 antibodies that stimulate tumor cell killing while sparing normal cells in vivois desirable for the cancer patients. We are interested in understanding whether humanized mouse models could aid the evaluation of a variety of monoclonal CD47 antibodies we generated. We first screened them using the CDX platformestablished at Biocytogen. Indeed, most antibodies were able to clear human tumor cells in B-NDG mice. However, when we screened the toxicity of these antibodies using humanized CD47 mice (B-hCD47), many antibodies caused rapid death of animals upon the first administration. Interestingly, we found two clonal antibodies bearing the least toxicity since the treated animals exhibited minimal weight loss. Thus, we concluded that humanized mice could expedite the development of safer CD47 antibodies that can be advanced to human clinical trials.

#2894

Neratinib effects significant changes in human brain endothelial cells, demonstrating that it may have a therapeutic use in cancers with brain metastasis.

Tracey A. Martin,1 Sioned Owen,1 Dafydd A. Dart,1 Francesca Avogadri Connors,2 Alshad S. Lalani,2 Richard P. Bryce,2 Wen G. Jiang1. 1 _Cardiff University, Cardiff, United Kingdom;_ 2 _Puma Biotechnology, Los Angeles, CA_.

Background: Brain metastases constitute a significant part of intracranial tumors and the majority of brain metastases originate from lung, breast cancers and malignant melanoma. Breast cancer patients who develop brain metastases tend to have poor prognosis with short overall survival. Moreover, human epidermal growth factor receptor 2 (HER2)-positive breast cancer have an increased propensity for brain metastases. Currently, patients with brain metastases have limited therapeutic options. The failure of many cancer therapeutics for the treatment of brain metastasis has been partly attributed to an intact blood-brain barrier (BBB) tightly controlled by endothelial tight junctions (TJ). Neratinib is an orally available tyrosine kinase inhibitor that irreversibly binds to and inhibits EGFR, HER2 and HER4 receptor tyrosine kinases. This study aimed to examine the effect of neratinib on protein expression and phosphorylation status in human brain endothelial cells.

Methods: Changes in protein phosphorylation in human brain (TY09 and CMEC D3) and venous endothelial cells (HECV) was assessed following neratinib treatment using protein microarrays (Kinexus, Canada). Z scores and percent changes from control (%CFC) were calculated between neratinib vs. control treatment samples at IC50. Alterations in gene expression were ascertained using AmpliSeq™ technology.

Results: Of over 800 proteins evaluated, neratinib caused significant %CFC (>50%) increase in 78 targets (such as EGFR, vimentin) and a %CFC decrease in 56 targets across all endothelial cells (including ROCKI). When comparing changes between brain and vascular cells, there was a significant change in %CFC for 36 proteins, including β-catenin and FYN. Of interest β-catenin is involved in TJ regulation. Differential expression of 21 genes associated with TJ including TJP1, JAM2, MABI1, CLDN7 and CLDN10 was also observed following neratinib treatment of brain or vascular endothelial cells I comparison to vascular cells treated with neratinib.

Conclusions: These results show that neratinib may alter both gene expression and phosphorylation status of a number of proteins linked to metastasis in human brain endothelial cells. Interestingly, several of these proteins are known to be involved in TJ regulation or function, suggesting that neratinib may modulate the activity of TJ in the BBB. Studies validating these findings are ongoing and may provide valuable insights into a new mode of action of neratinib for the treatment for cancers with brain metastasis.

#2895

Verteporfin targets p62 in prostate cancer therapeutics.

Lei Wang, Donghern Kim, James Wise, Xianglin Shi, Zhuo Zhang, Robert S. DiPaola. _University of Kentucky, Lexington, KY_.

To test the hypothesis that p62 is an optimal target for autophagy inhibition and Verteporfin, a clinically available drug approved by FDA to treat macular degeneration that inhibits autophagy by targeting p62 protein, can be developed clinically to improve therapy for advanced prostate cancer. Results showed that Verteporfin inhibited cell growth and colony formation in PC-3 cells. Verteporfin generated crosslinked p62 oligomers, resulting in inhibition of autophagy and constitutive activation of Nrf2 as well as its target proteins, Bcl-2 and TNF-α. In normal prostate epithelial cells, forced expression of p62 caused constitutive Nrf2 activation and development of apoptosis resistance and Verteporfin treatment exhibited inhibitory effects. Verteporfin treatments also inhibited starvation-induced autophagic influx of these cells. Verteporfin inhibited tumorigenesis of both normal prostate epithelial cells with p62 expression and prostate cancer cells and decreased p62, constitutive Nrf2, and Bcl-xL in xenograft tumor tissues, indicating that p62 can be developed as a drug target against prostate cancer. So p62 has a high potential to be developed as a therapeutic target. Verteporfin represents a prototypical agent with therapeutic potential against prostate cancer through inhibition of autophagy by a novel mechanism of p62 inhibition.

#2896

A genome-wide CRISPR/Cas9 pooled screen identifies EML4-ALK variant-specific synthetic lethal partners of HSP90 inhibitors.

Marco P. Licciardello, Paul A. Clarke, Paul Workman. _Institute of Cancer Research, London, United Kingdom_.

The chromosomal rearrangement generating the EML4-ALK fusion protein occurs in 5–7% of patients with non-small-cell lung cancer (NSCLC). Distinct breakpoints give rise to variants with different EML4 fragments and an invariable ALK kinase domain. Patients treated with ALK tyrosine kinase inhibitors, such as crizotinib or alectinib, inevitably develop resistance to the drugs and need alternative therapies. EML4-ALK v1 and v3a/b, the most common variants of the fusion oncogene, display distinct cellular localization and different sensitivity to the inhibition of HSP90 chaperoning activity. In the clinic, HSP90 inhibitors have shown encouraging results in EML4-ALK+ NSCLC patients, but the responses have been heterogeneous. Currently, there is no EML4-ALK variant-based classification of patients and this might have contributed to confounding results in clinical trials. Moreover, the identification of additional biomarkers of sensitivity could further improve patient stratification and suggest potential combinatorial regimes. As chaperones are molecular hubs enriched for synthetic lethal partners, we performed a CRISPR/Cas9-based pooled screen in human NSCLC cell lines carrying different EML4-ALK variants in the presence of non-lethal concentrations of an HSP90 inhibitor. In the screen, we found already described as well as previously unreported EML4-ALK variant-specific synthetic lethal interactions. We are currently dissecting the mechanism behind these interactions, and evaluating their therapeutic potential for EML4-ALK+ NSCLC patients.

#2897

Therapeutic efficacy of antibodies targeting domain 1 of HSPG2 in breast cancer.

Vidhi Khanna,1 Stephen Kalscheuer,1 Jayanth Panyam,1 Da Yang,2 Sihan Li2. 1 _University of Minnesota, Minneapolis, MN;_ 2 _University of Pittsburgh, Pittsburgh, PA_.

Significant advancements in chemotherapy have improved the survival rates of patients presenting with local or regional breast cancer to as high as 99% and 85%, respectively. On the contrary, patients with metastatic breast cancer have a dismal 5-year survival rate of 26%. Thus, there is an urgent need for research strategies directed towards treatment of metastasis. Our lab used a phage display-based cell panning procedure to develop two fully humanized antibodies (Clone 6 and AM6) that are able to bind specifically to breast cancer metastatic cells. Target deconvolution revealed HSPG2/Perlecan Domain 1 as the cell surface antigen bound by the antibodies. Immunohistochemistry studies revealed high HSPG2 expression across various tumor subtypes including melanoma, bladder cancer, glioblastoma and ovarian cancer. There was a significant correlation between high HSPG2 and with poor survival in bladder and ovarian cancers. Interestingly, we observed significant tumor growth inhibition in the triple-negative MDA-MB-231 breast cancer xenograft model. This efficacy was significantly reduced when the tumor growth inhibition studies were repeated in NSG mice, suggesting NK cell-mediated antibody dependent cellular cytotoxicity (ADCC) as the potential mechanism of action. The data presented here point to the relevance of HSPG2 as a novel target for not only breast cancer but also other malignancies. We also show the potential of Clone 6 and AM6 as therapeutic and targeting agents. Further studies are required to understand the significance of HSPG2 overexpression and its correlation with tumor progression as well as to confirm the mechanism of action with Clone 6 and AM6 antibodies.

#2898

Inhibition of HDAC6 as a novel targeted therapy in breast cancer.

Tizita Z. Zeleke,1 Preeti Putcha,1 Jiyang Yu,2 Andrea Califano,3 Jose M. Silva1. 1 _Icahn School of Medicine at Mount Sinai, New York, NY;_ 2 _St. Jude's Research Hospital, Memphis, TN;_ 3 _Columbia University, New York, NY_.

The identification and use of biomarkers is critical to guided clinical care. Cancer treatment has made use of many biomarkers to determine which therapeutic avenues are adequate for each patient. For example, the use of Trastuzumab is intimately linked to the presence of HER2/NEU amplification in breast cancers. Previous work from our group established the importance of HDAC6 in maintaining the homeostasis of inflammatory breast cancer cells (IBC). We also reconstructed the HDAC6 regulon using gene expression profile signatures from over 900 primary breast cancers to obtain a biomarker that guide the use of HDAC6 inhibitors towards the right patient group. This method identified a regulon of 162 transcripts as a set of transcriptional targets whose expression is affected by HDAC6 master regulator activity. Finally, the expression of all transcripts that are co-regulated by HDAC6 in the breast cancer samples were integrated into a single score termed as the HDAC6 score. Through this work, we established that HDAC6 score is associated with cancer cell response to HDAC6 inhibitor ACY1215.

HDAC6, one of eighteen deactylases, is distinct from all other HDACs as it is located mainly in the cytosol and has non-chromatin substrates such as α-tubulin and HSP90. HDAC6 inhibitors have gained a lot of momentum in the clinic for hematological malignancies and our computational analysis has also revealed that these tumors also exhibit a high HDAC6 score. Therefore, by identifying the HDAC6 score for tumors, we can widen the scope of using HDAC6 inhibitors to a wide range of cancers.

Our increasing understanding of tumor biology has led to the use of combinatorial therapies to fully benefit from cancer therapy. This project will encompass the use of combinatorial therapy containing HDAC6 inhibitors and paclitaxel to demonstrate an increased potential in treating breast cancer. These studies will closely mirror the clinical trial that we are undertaking with our collaborators from Acetylon where metastatic breast cancer patients are being treated using HDAC6 inhibitor ACY1215 in combination with nab-paclitaxel (NCT02632071). This project aims to provide the frame work to utilize for the first time the HDAC6 score as a marker for identifying tumors that will be most susceptible to HDAC6 inhibition in the clinic.

#2899

Antitumor activity of CIGB-300, a peptide CK2 inhibitor, in breast cancer models.

Maria F. Gottardo,1 Carla S. Capibianco,1 Johanna E. Sidabra,1 Yasser Perera,2 Silvio Perea,2 Daniel F. Alonso,1 Hernan G. Farina1. 1 _Quilmes National University, Bernal, Buenos Aires, Argentina;_ 2 _Center for Genetic Engineering and Biotechnology, Havana, Cuba_.

CK2 is a serine-threonine kinase that has been involved in growth, proliferation and cell apoptosis. CK2 has a constitutive expression and it has more than 300 substrates. In recent years, CK2 became an interesting target for anticancer therapies. Inhibition of CK2 showed antitumor activity in different types of cancer. Because breast cancer is one of the main tumor types in which CK2 is overexpressed, we focus on the effect of CK2 inhibition as a modulator of key features of breast cancer cell biology. The aim of the present study was to evaluate the role of inhibition of CK2 by CIGB-300 in murine and human mammary carcinoma cell lines. For this purpose, we evaluated the action of CIGB-300 on viability, apoptosis, cell cycle, clonogenic capacity and migration using three different breast tumor cell lines, MCF7 (positive for estrogen, progesterone and HER2-neu receptors), MDA MB 231 (representative of triple negative tumors) and F3II, a murine mammary carcinoma. As compared the potential of CK2 inhibition in these models we included another chemical inhibitor of the enzyme, CX-4945. Our results showed that CIGB-300 reduced the viability of MDA MB 231 (IC50=120 μM) and MCF-7 (IC50=140 μM) (p<0.05, ANOVA). We observed that in both human and murine cell lines CIGB-300 exerts a pro-apoptotic action (TUNEL assay, p<0.05, χ2) and arrest cell cycle. We also found that the inhibition of CK2 by CIGB-300 decreased the clonogenic capacity and migration of F3II, MCF-7 and MDA-MB-231 (p<0.05, ANOVA). In vivo studies using the syngeneic F3II breast cancer model in BalB/c mice showed a decrease in the number of lung metastases in mice treated systemically with CIGB-300 after primary tumor surgery (Mann Whitney test, p<0.05). However, CK2 inhibition did not affect the growth of local recurrences. Also, intravenous administration of CIGB-300 decreased lung metastasis development of F3II cells. These results demonstrate an antimetastatic effect of CIGB-300 in a systemic inoculation model after surgical removal of primary tumors. In this work we demostrated the antitumor effect of CIGB-300 in breast cancer models such as those reported in myeloid leukemia, lung and cervix and head cancer.

#2900

Evaluation of endoglin/CD105 as a tumor vasculature target with antibody drug conjugates.

Rui Wu, Cristina Gavrilescu, Yimao Liu, Valerie Chamberlain Santos, Katharine C. Lai, Luke Harris, Prerak Shah, Kerry Donahue, Ravi Chari, Richard Gregory, Thomas Chittenden, Cynthia Guidi, Thomas A. Keating. _ImmunoGen inc., Waltham, MA_.

Purpose: Solid tumor growth and metastasis requires continuous formation of new blood vessels. Endoglin/CD105 is a well-acknowledged endothelial cell proliferation marker, which is strongly expressed in tumor-associated vasculature. Vasculature targeting agents induce their anti-tumor effects through selective damage to the vasculature and reduction of tumor blood perfusion. Here, we evaluated endoglin as a potential vascular target for antibody-drug conjugate (ADC) therapy. Anti-endoglin ADCs were prepared with two classes of potent cytotoxic payloads, using either the microtubule disrupting maytansine derivatives DM1 and DM4, or the highly potent indolinobenzodiazepine DNA-alkylating payload DGN549. The pharmacokinetics, safety, and anti-tumor activities of the anti-endoglin ADCs were evaluated in rats.

Experimental Design: Humanized monoclonal antibodies against endoglin were generated and screened for binding to both human and rat endoglin. Payloads were conjugated to the lead antibody through lysine residues. Binding and cytotoxicity of ADCs were tested in vitro on human umbilical vein endothelial cells (HUVECs). The antibody was administered to Sprague Dawley rats, and the circulation half-life determined by ELISA of the plasma samples. In vivo efficacy of anti-endoglin-DM and anti-endoglin-DGN549 ADCs were tested in multiple xenograft tumor models in Taconic female rnu/rnu rats.

Results: A humanized anti-endoglin antibody, huRH105, which binds to both human and rat endoglin with high affinity was generated. The pharmacokinetic profile of huRH105 in rat demonstrated dose-dependency with the low dose (0.5 mg/kg) cohort having a high plasma clearance rate. ADCs of huRH105 were prepared with three different payloads: DM1, DM4 and DGN549. All three ADCs bound with similar sub-nanomolar affinity to endoglin-expressing HUVEC cells, and exhibited potent specific cytotoxicity against these cells in vitro. huRH105-SMCC-DM1 was tolerated at a single dose of 10-20 mg/kg in rats, with no discernable impact on body weight. This ADC displayed only modest anti-tumor activity against human A2780 ovarian tumor xenografts at 5 mg/kg in rats. To identify optimal drug linkers for more potent anti-tumor activity, two other ADC constructs, huRH105-sSPDB-DM4 and huRH105-DGN549 were evaluated against A2780 tumor xenografts grown subcutaneously. huRH105-sSPDB-DM4 was not active at 5 mg/kg; huRH105-DGN549 displayed modest anti-tumor activity at 0.33 mg/kg, eliciting tumor growth delay without notable toxicities.

Conclusion: Endoglin targeted ADCs, huRH105-DM and huRH105-DGN549 conjugates were prepared and evaluated for their in vitro cytotoxicity and in vivo anti-tumor activity/tolerability. The ADCs exhibited modest anti-tumor activity and therapeutic indices in rat models.

#2901

Identifying synthetic lethal interactions in castration-resistant prostate cancer using HSP40 and HSP70 inhibitors.

Michael A. Moses,1 Genesis Rivera-Marquez,1 Isabelle Taylor,2 Hao Shao,2 Jason Gestwicki,2 Jane Trepel,1 Len Neckers1. 1 _National Cancer Institute, Bethesda, MD;_ 2 _University of California, San Francisco, San Francisco, CA_.

Castration-resistant prostate cancer (CRPC) is frequently characterized by elevated expression of alternative nuclear receptors able to at least partially maintain the androgen receptor (AR) transcriptional program. These alternative receptors include ARv7, a ligand binding domain (LBD)-deficient, constitutively active splice variant that correlates with poor prognosis, reduced survival, and resistance to existing LBD-targeted standard of care therapy. As a nuclear hormone receptor, AR exists in a dynamic cycle with molecular chaperones (heat shock proteins, HSPs) HSP40/HSP70/HSP90 which foster proper folding and remodeling of the LBD to bind ligand. Notably, HSP90 inhibitors promote AR degradation and display efficacy in prostate cancer xenograft models. Although it has been shown that LBD-deficient ARv7 functions independently of HSP90, we have observed that loss of the LBD does not abrogate HSP40/HSP70 binding, as ARv7 interacts with HSP40/HSP70 in CRPC cells. As such, targeting either HSP40 (C86) or HSP70 (JG98/JG231) inhibits stability and function of AR/ARv7. However, this is complicated by the fact that over 40 isoforms of HSP40 (DnaJ proteins) and 13 HSP70 paralogs exist in humans. Therefore, we sought to identify the requirements for components of the HSP40/HSP70 network in mediating intracellular stability of AR/ARv7, and determine which chaperones orchestrate sensitivity to HSP40/HSP70 inhibitors. Using a doxycycline-inducible lentiviral proteostasis shRNA library, we identified genes that appear to be required for proliferation of 22Rv1 CRPC cells (expressing endogenous AR and ARv7) distinct from those found in ARv7-deficient LNCaP prostate cancer cells. These include genes from several chaperone families including DnaJA3 (HSP40), HSPA2 (HSP70-2), and HSPD1 (HSP60), which may be unique regulators of ARv7 stability. Synthetic lethal screens using C86 and JG231 were also performed to determine which genes may mediate sensitivity to these chaperone inhibitors. Knockdown of HSP40 isoforms (DnaJA1, DnaJA2), HSP70 paralog HSPA4, or stress factors (ATF6, HSF1) in combination with C86 lead to growth impairment in 22Rv1 cells. DnaJA1, DnaJA2, and DnaJA3 were all found in the cytosol and nucleus of 22Rv1 cells, and in a complex with AR/ARv7 after a biotin-C86 IP, suggesting they be required for AR/ARv7 stability, dimerization, and/or nuclear translocation. Other genes from chaperone families HSP40 (DnaJC7, DnaJC8, DnaJC16), HSP70 (HSPA5, HSPA8, HSPA9), and HSP90/HSP90 co-chaperones (HSP90AB1, Cdc37, PTGES3 [p23]) were found to be important mediators of sensitivity to JG231, revealing targets for possible combination therapies. Taken together, these data help identify HSP40/HSP70 subnetworks regulating AR/ARv7 and those mediating sensitivity to chaperone inhibitors to aid in identifying new drug targets for efficacy in CRPC.

#2902

Targeting the tight junction protein claudin-1 in colorectal cancer.

Sara Cherradi,1 Adeline Ayrolles-Torro,1 Nadia Vezzo-Vié,1 Eve Combes,1 Lucile Canterel-Thouennon,1 Fabien Gava,2 Valérie Lobjois,2 Bernard Ducommun,2 Celine Gongora,1 Maguy Del Rio1. 1 _Institut de Recherche de Cancérologie (IRCM), Montpellier, France;_ 2 _Institut des Technologies Avancées en Sciences du Vivant (ITAV), Toulouse, France_.

Colorectal cancer (CRC) is one of the major causes of cancer-related deaths in the Western world. When localized, CRC is often curable by surgery, but the prognosis of patients with metastatic disease remains very poor. The current treatment of metastatic CRC (mCRC) relies on therapy combining chemotherapy and targeted therapies. However, relapses are observed in most cases due to the occurrence of drug resistance. Therefore, more therapeutic options are required particularly by identifying new molecular targets that can be reached by antibodies.

We previously showed that Claudin-1 (CLDN1), a major constituent of tight junctions, is overexpressed at the membrane of CRC cells which makes it a good target for antibodies. Besides, CLDN1 is differentially expressed in the new CRC molecular subtypes. We have then developed a monoclonal antibody (mAb) against CLDN1, called 6F6, which targets the extracellular part of CLDN1. Results showed that the 6F6 mAb significantly decreased cell growth, survival, and migration in vitro as well as in vivo. Furthermore, we demonstrated the cytostatic effect of the 6F6 mAb through proliferation assays.

We are now deciphering the role of CLDN1 on 3D spheroids, a model that reflects the physiopathology. Using immunofluorescence staining on frozen sections we are exploring the impact of CLDN1 on spheroids proliferation. We are also investigating on the distribution of tight junction proteins implicated on cell proliferation in the presence or absence of CLDN1.

CLDN1 is a new promising therapeutic target in CRC, it is now interesting to elucidate its role in colorectal cancer cell proliferation.

Keywords: Colorectal, Cancer, Claudin-1, monoclonal antibodies, spheroids, proliferation

#2903

A novel compound induces synthetic lethality for p53 mutations in osteosarcoma cells.

Tomoo Iwakuma,1 Alejandro Parrales,1 Peter McDonald,2 Anuradha Roy,2 Mitchell W. Braun,1 Frank J. Schoenen,2 Jenna Wang,2 Steve Rogers,2 Melinda Broward,2 Tyce Bruns,2 Shrikant Anant,1 Dan A. Dixon,1 Fred Meyer,1 Katherine M. Chastain,3 Douglas H. Thamm,4 Scott J. Weir,1 Kathleen Neville,5 Joy M. Fulbright3. 1 _Univ. of Kansas Medical Ctr., Kansas City, KS;_ 2 _Univ. of Kansas, Lawrence, KS;_ 3 _Children's Mercy Hospital, Kansas City, MO;_ 4 _Colorado State University, Fort Collins, CO;_ 5 _Arkansas Children's Hospital, Little Rock, AR_.

Osteosarcoma is the second-highest cause of cancer-related death in children and adolescents. Despite advances in chemotherapy and surgery, the survival rate for metastatic osteosarcoma remains below 30% for the last three decades. Discovery of new chemotherapy agents would be crucial for improving outcomes of osteosarcoma patients. Here, using high-throughput screening, we have found a new compound, called 2-{2-[(3,5-dimethoxybenzyl)sulfanyl]-1,3-thiazol-4-yl}-N-(2-thienylmethyl)acetamide (referred to as KU0171032), as an inducer of apoptosis in various canine and human osteosarcoma cells. This compound shows minimal effects on non-transformed osteoblast and fibroblast cells. Intriguingly, KU0171032-induced apoptosis is more robust in osteosarcoma cells having mutant p53 or null for p53, as compared to cells with wild-type p53. Knockdown of wild-type p53 in U2OS and SJSA-1 cells significantly enhances sensitivity to KU0171032 with increase in DNA damage and caspase-3 cleavage. Moreover, KU0171032 significantly reduces in vivo tumor growth of osteosarcoma cells with p53 knockdown or carrying mutant p53. Our results strongly suggest that KU0171032 shows synthetic lethality with p53 mutations in osteosarcoma cells. Given that loss of p53 activity is frequent event in many cancer types including osteosarcoma while normal cells usually retain wild-type p53, this compound could be used to develop novel therapeutic strategies that capitalize on vulnerabilities in osteosarcoma and other types of cancer.

#2904

Awakening metastatic breast cancer cells to chemotherapy.

Ana Rita Lourenco, Jay Lopez, Michael Crowley, Vivek Mittal, Dingcheng Gao. _Weill Cornell Medical College, New York, NY_.

Breast cancer is the most common cancer in women with more than 316,000 new cases and expected 40,610 deaths in the US in 2017. Despite vast improvement in cancer management and treatment in the past years, not all subtypes of breast cancer have benefited equally. This includes the most aggressive form of invasive breast cancer, triple negative breast cancer (TNBC) subtype (ER-, PR- and HER2-), which accounts for about 20-25% of all breast cancer cases and presents a higher breast cancer-related mortality compared to other breast cancer subtypes. The poor survival rates attributed to TNBC is due to the lack of available biological targets and high rates of metastatic recurrence. Therefore, chemotherapy is the only therapeutic option for patients with metastatic TNBC. However, development of chemotherapy resistance is observed in the great majority of the cases and patients relapse within 5 years of a diagnosis. Given the unmet clinical needs, the discovery of novel therapeutic options that overcome chemoresistance of metastatic breast cancer is required. We have identified a group of genes which expression was enriched in chemotherapy resistant tumors cells, suggesting that these are potential therapeutic candidates to overcome chemotherapy resistance. Of these, CDKN1A (p21) was included. Furthermore, Immunostaining of metastatic lung tissue confirmed that surviving tumor cells under CTX treatment highly express p21. The enrichment of p21 in surviving tumor cells under chemotherapy, together with its well documented functions in cell-cycle arrest and regulation of apoptosis, provides a rationale to explore the underlying mechanisms that govern p21-induced chemoresistance and whether targeting p21 will re-sensitize the chemoresistant tumor cells in vivo.

#2905

Deciphering and blocking the mechanisms of cadmium induction of TCTP in prostate cancer cells.

Rama Saad, Gnanasekar Munirathinam, Theresa Kucynda, Marta Ribeiro, Paul Lindholm, Andre Balla. _University of Illinois at Chicago, Chicago, IL_.

While cadmium is a well-known lung carcinogen, its role in prostate cancer is yet to be understood. Recently, a meta-analysis study showed that cadmium did not increase the risk of prostate cancer. It is not known whether cadmium exposure at environmentally relevant concentrations increases aggressiveness of already existing prostate cancer. Although our lab previously showed that the 75th percentile of cadmium concentration in prostate cancer was associated with biochemical recurrence, more evidence is needed to substantiate this finding. We also showed evidence that cadmium promotes prostate cancer cells migration and invasion via inducing the expression of translationally controlled tumor protein (TCTP). Furthermore, it was demonstrated that cadmium induces the phosphorylation of p38, and that the p38 inhibitor SB203580 (SB) blocks the effects of cadmium on migration and invasion. In this study, we aimed to delineate the signaling events upon cadmium treatment; whether p38 or TCTP is upstream. We addressed this via inhibition of p38 using SB203580 and by inducing TCTP degradation via dihydroartemisinin (DHA) followed by measurement of TCTP expression and phosphorylation of p38 respectively. p38 inhibition led to the increased expression of TCTP on both mRNA and protein levels. Pretreatment with DHA led to less TCTP expression and less phosphorylation of p38. We also aimed to understand the effect of cadmium on cell proliferation, survival and migration. While DHA lowered the colony count and EdU incorporation, cadmium did not alter these functions. In addition, DHA abrogated the cadmium-mediated migration of PC3-Inv prostate cancer cell line. Furthermore, we aimed to determine the effect of nanomolar cadmium concentrations on the phosphorylation of PlK1, which is well established to be upstream of TCTP. Our results demonstrated that nanomolar concentrations of cadmium resulted in increased phosphorylation of PlK1 in both PC3 and LNCaP cells. Altogether, our results suggest that environmentally relevant exposures of cadmium induce TCTP expression through several pathways, and that there is a feedback mechanism between p38 and TCTP.

#2906

eEF1A2 interacts with and inhibits PKR to enhance cancer cell survival.

Juan F. Martínez-Leal, Alejandro Losada, Maria Jose Muñoz, Marta Martinez-Diez, Juan Manuel Dominguez, Carlos M. Galmarini. _PharmaMar S.A., Madrid, Spain_.

Human translation elongation factor 1α2, encoded by the eEF1A2 gene, is a pro-oncogenic protein absent from the majority of body tissues (with exception of brain, heart and skeletal muscle1), but expressed in many cancers1-3, where it provides tumor cells with improved fitness and survival. Though its canonical function is delivering aminoacyl-tRNAs to the ribosome, other "moonlighting" functions such as enhancing sphingosine kinase4,5 or antioxidant (most probably through peroxiredoxin-1 stimulation) activities6 have been described for the elongation factor. Recently, we have reported that eEF1A2 is the target for plitidepsin, a marine-derived cyclic depsipeptide currently under development for the treatment of relapsed or refractory multiple myeloma patients7. We have also confirmed that eEF1A2 interacted with previously described partners as PRDX1 and SPHK and enhanced their pro-survival activities8. Here we investigated the role of new "moonlighting functions" of eEF1A2 in the maintenance of the tumor phenotype and survival of cancer cells. Through co-immunoprecipitation and HPLC/MS we have uncovered the interaction between eEF1A2 and dsRNA-activated protein kinase (PKR, EIF2AK2). We have analyzed the kinase activity of PKR in the presence of eEF1A2, demonstrating that PKR activity is inhibited when complexed with eEF1A2. This complex is disrupted after plitidepsin binding to eEF1A2, rendering PKR active. Once activated, the kinase triggers a MAPK cascade and the NF-κB signaling pathway, leading to the activation of the extrinsic apoptotic pathway and the death of the tumor cell. Taken together, these results show that the fitness boost that the moonlighting functions of eEF1A2 provide to cancer cells, which are important for their growth and survival, constitutes an Achilles heel that can be purposely exploited in anticancer therapy. 1 Abbas, W., Kumar, A. & Herbein, G. Front Oncol 5, 75 (2015). 2 Anand, N. et al. Nature genetics 31, 301-305 (2002). 3 Li, Z. et al. PloS one 5, e10755 (2010). 4 Leclercq, T. M., Moretti, P. A., Vadas, M. A. & Pitson, S. M. J Biol Chem 283, 9606-9614 (2008). 5 Leclercq, T. M., Moretti, P. A. & Pitson, S. M. Oncogene 30, 372-378 (2011). 6 Chang, R. & Wang, E. J Cell Biochem 100, 267-278 (2007). 7 Losada, A. et al. Sci Rep 6, 35100 (2016) 8 Losada, A. et al. Abstract #1165, AACR Annual Meeting (2017)

#2907

Targeting the epigenetic regulator KAT2a in cancer.

Andrea Gunnell,1 Jessica Downs,2 Lewis Pennicott,1 Kay Osborn,1 Darren Le Grand,1 Katie Duffell,1 Hitesh Patel,1 Jessica Hudson,1 Jessica R. Booth,1 Simon Ward1. 1 _University of Sussex, Brighton, United Kingdom;_ 2 _Institute of Cancer Research, London, United Kingdom_.

Gene expression frequently requires the dynamic remodelling of the chromatin architecture to orchestrate the assembly of transcriptional protein complexes at the promoter sites of DNA. Chromatin remodelling complexes are multicomponent proteins that facilitate reorganisation of chromatin structure using the energy derived from ATP hydrolysis, and as a result play a pivotal role in controlling gene expression. Mammalian SWI/SNF complexes are examples of chromatin remodelling complexes with multiple roles in maintaining genome stability. It is estimated that 19.6% of all human tumours have mutations in SWI/SNF complexes, leading to the deregulation of gene expression as an early driver of carcinogenesis. Our interest in developing novel treatments for renal cancer focused on the role of the chromatin remodelling complex, PBAF. The BAF180 component of PBAF is mutated in 40 % of clear cell renal carcinomas and its loss is an early event in renal cell tumorigenesis. We performed an siRNA based high throughput screen to identify genes associated with genome stability that were synthetically lethal with the loss of BAF180 and identified KAT2a as a potential druggable target. KAT2a is a protein that also functions in the regulation of gene expression as a component of the SAGA histone modifying complex. It is a multifunctional protein that can both 'read' and 'write' acetylation marks on histone proteins through its bromodomain and a histone acetyltransferase domain respectively. We will describe our efforts to establish suitable assays to support the discovery of selective inhibitors of the bromodomain of KAT2a. The work will include the establishment of a high throughput assay to measure compound binding, crystallographic characterisation of small molecule inhibitors of the bromodomain, and our initial studies towards the target validation of the bromodomain to recapitulate the synthetic lethality observed with depletion of BAF180.

#2908

Characteristics of triple negative breast cancer that result in sensitivity to englerin A.

Corena V. Shaffer,1 April L. Risinger,1 John A. Beutler,2 Susan L. Mooberry1. 1 _University of Texas Health Science Center at San Antonio, San Antonio, TX;_ 2 _National Cancer Institute, Frederik, MD_.

New approaches are needed to improve long term survival for patients with metastatic triple negative breast cancers (TNBCs). Patients with metastatic TNBC have a poorer 3-year survival compared to patients with metastatic HER2+ or ER/PR+ disease. Identifying new therapeutic targets of TNBC subgroups could provide opportunities for more effective treatments. We conduct a screening program to identify compounds with selective activity against a panel of TNBC cell lines, reasoning that mechanism of action studies of selective cytotoxins could identify new therapeutic targets. Englerin A was identified as a compound with highly selective actions against two TNBC cell lines: BT-549 (>1800-fold selectively) and Hs578T (>600-fold selectivity) as compared to other TNBC cell lines. Previous studies showed that englerin A is an agonist of TRPC1/4/5 non-specific cation channels and PKCθ in renal cancer cells.1–4 RT-PCR analyses showed that BT-549 and Hs578T cells express more TRPC4 than either the insensitive MDA-MB-231 TNBC cells or the A498 renal cancer cells. If BT-549 and Hs578T cells are sensitive to englerin A due to high expression of TRPC4 channels, then treatment with TRPC4 channel inhibitors should decrease sensitivity to englerin A. These studies show that the TRPC1/4/5 channel inhibitor Pico1455 caused a rightward shift in the concentration response curve of englerin A in both BT-549 and Hs578T cells. These data are consistent with TRPC1/4/5 channel activation contributing to englerin A-induced cell death of BT-549 and Hs578T cells. TRPC channels are non-specific cation channels, therefore their activation is expected to cause an influx of cations including calcium and sodium. Live cell calcium imaging showed the influx of calcium into BT-549 cells following englerin A treatment, further supporting the hypothesis that englerin A-induced cell death in these cell lines is the result of TRPC channel activation. TRPC channel activation initiates disruption of intracellular cation homeostasis, therefore we tested ouabain, a Na+/K+ATPase inhibitor, to determine if BT-549 and Hs578T cells were also sensitive to TRPC-independent methods of intracellular cation homeostasis disruption. BT-549 and Hs578T cells were 12- and 2-fold more sensitive to ouabain as compared to other TNBC cell lines, suggesting that some TNBC cells lines are particularly sensitive to disruption of intracellular cation homeostasis. Ongoing work is aimed at identifying additional factors in englerin A's mechanism of action in BT-549 and Hs578T cells. Our studies suggest that effective treatments for this subgroup of TNBC may include agents which disrupt cation homeostasis.

1.Ludlow, M. J. et al. (2016).

2.Akbulut, Y. et al. (2015).

3.Carson, C. et al. (2015).

4.Sourbier, C. et al. (2013).

5.Rubaiy, H. N. et al. (2017).

#2909

Development of new mechanism based therapeutic antibodies in non-small cell lung cancer.

Jai-hee Moon,1 Dae Hee Lee,2 Jae-Sik Shin,2 Joseph Kim,2 Yoon Sun Park,2 Seung-Woo Hong,2 So Hee Lee,2 Mi Jin Kim,3 Joonyee Jung,3 Chun-Ho Park,2 Sun-Chul Hur,2 Hyojin Kim,2 Hyebin Park,2 Sang Soo Park,4 Jun Ki Hong,3 Ji Hee Gong,2 Jieun Kim,2 Hyun Ho Lee,2 Il-Whea Ku,5 Dong-Hoon Jin6. 1 _University of Ulsan College of Medicine, Department of Medical Science, Convergence Medicine, and Asan Institute for Life Science, Asan Medical Center, Seoul, Republic of Korea;_ 2 _Wellmarkerbio Co.,Ltd, Seoul, Republic of Korea;_ 3 _University of Ulsan College of Medicine, Department of Convergence Medicine and Asan Institute for Life Science, Asan Medical Center, Seoul, Republic of Korea;_ 4 _University of Ulsan College of Medicine,Department of Convergence Medicine and Asan Institute for Life Science, Asan Medical Center, Seoul, Republic of Korea;_ 5 _Wellmarkerbio Co.,Ltd, Center for Drug Discovery Support, Asan Institute for Life Science, Asan Medical Center, Seoul, Republic of Korea;_ 6 _Wellmarkerbio Co.,Ltd, Asan Institute for Life Science, Asan Medical Center, Department of Convergence Medicine, Ulsan College of Medicine, Asan Medical Center, Seoul, Republic of Korea_.

Lung cancer is second most common cancer in the world. Non Small Cell Lung Cancer (NSCLC) accounts approximately 80-85% of all lung cancer diagnosis. Traditional therapies of this disease were surgical resection, chemotherapy, and radiotherapy, alone or in combination. In addition, targeted therapeutic approach was based on the concept of discovering genetic alterations and the signaling pathways in cancer. Recently, to overcome the critical points for standard therapies, many groups were studied immunotherapeutic approaches, such as programed cell-death protein 1 (PD-1) antibody. However, rational use of these agents has been limited by the lack of a definitive predictive biomarker. Therefore, we identified new target, cancer immunotherapy related gene, CMG by shRNA libraries screening analysis on chemo-agents & target therapy resistant non-small cell lung cancer cells. First of all, we investigated CMG expression by immunohistochemistry in various tissue microarray (TMA). These results show that CMG highly expressed in Lung cancer, Liver cancer, and gastric cancer. We investigated target potentials on lung cancer, liver cancer, and gastric cancer cell lines using in vitro and in vivo assay system. Knockdown of CMG by CMG shRNA was induced cell death in various cancer cell lines. In addition, suppression of CMG was induced tumor size regression in CMG shRNA stable cell lines-derived xenograft model. Based on these results, we synthesized a novel series of CMG therapeutic antibody.

CMG therapeutic antibody is a lead antibody for treating Lung cancer patients who express CMG gene. These antibodies have anti-cancer effects and immunotherapeutic effects in lung cancer (NSCLC), liver cancer, and gastric cancer. In addition, the in vivo efficacy of CMG antibody was assessed in mouse lung cancer derived syngeneic mouse model. The CMG antibody was tri-daily i.p. injected and the tumor volume was measured and compared between groups. Dramatic tumor regression was observed in CMG antibody treated group. These results were shown that these antibodies have immunotherapeutic potentials. In conclusion, CMG is a promising target for Lung cancer patients (chemo-agents resistant or PD-1 resistant Lung cancer patienrts). Our antibodies can be promising therapeutic agents for lung cancer, Liver cancer, and gastric cancer.

#2910

Identification of URST1 as a novel prognostic biomarker and therapeutic target for various subtypes of breast cancer.

Masako Nakamura,1 Atsushi Takano,2 Phung Manh Thang,1 Yohei Miyagi,3 Yataro Daigo2. 1 _Shiga University of Medical Science, Otsu, Japan;_ 2 _Shiga University of Medical Science/Center for Antibody and Vaccine Therapy, Research Hospital, Institute of Medical Science, the University of Tokyo, Otsu/Tokyo, Japan;_ 3 _Molecular Pathology and Genetics Division, Kanagawa Cancer Center Research Institute, Kanagawa, Kanagawa, Japan_.

Breast cancer is one of the most common causes of cancer death for women worldwide. To identify diagnostic and therapeutic targets for breast cancer, we screened molecules that were highly expressed in the majority of breast cancers by our gene expression profile database. During this process, we identified URST1 (up-regulated in solid tumor 1) as a candidate. Immunohistochemical analysis showed that URST1 was expressed in 195 of 257 (75%) breast cancer cases (69% in luminal type, 88% in HER2-positive cancer, 86% in Triple-negative cancer) that had undergone curative surgery, whereas no staining was observed in adjacent normal breast tissues. URST1 expression was significantly related to poor clinical outcome for breast cancer patients (P = 0.01, Log-rank test) and multivariate analysis showed that it was an independent prognostic factor. Knockdown of endogenous URST1 expression by siRNAs against URST1 or treatment with URST1 inhibitor significantly inhibited the growth of breast cancer cells through cell cycle arrest at G2/M phase. Our data suggest that URST1 is likely to be a prognostic biomarker and therapeutic target for various subtypes of breast cancers.

#2911

The propitious dual roles of deferoxamine in head and neck cancer management.

Alexis Donneys, Chitra Subramanian, Jeremy Lynn, Kevin Urlaub, Kevin Kovatch, Halil S. Uygur, Mark S. Cohen, Steven R. Buchman. _University of Michigan, Ann Arbor, MI_.

Introduction: For head and neck cancer survivors, radiotherapy (XRT) improves survivorship at a cost of impaired quality of life. XRT-induced pathologic fractures (fxs) and associated nonunions are devastating iatrogenic sequelae associated with microvascular damage in bone, yet there are no pharmacologic strategies to prevent or treat these maladies. Deferoxamine (DFO), an FDA-approved iron chelator, has been extensively investigated for its ability to promote tissue vascularization through upregulation of the HIF-1α pathway. Our laboratory has previously demonstrated improved fx healing and prevention of nonunions in radiated bone utilizing localized DFO administration. However, the role of DFO in head and neck squamous cell carcinoma (HNSCC) tumorigenesis is currently unknown. While iron is essential for normal cell proliferation, excessive iron is associated with multiple oncogenic pathways. Thus, localized iron chelation may have beneficial antitumorigenic properties. Elucidating the role of iron metabolism and correlations between iron chelation and angiogenic signaling may offer opportunities for diagnostic and treatment purposes, while simultaneously improving tissue engineering efforts for HNSCC survivors.

Methods: We surveyed the UALCAN survivorship data base for iron-related genes in HNSCC. In vitro, established MDA1986 (HPV+) and UMSCC108 (HPV-) cell lines were grown in culture. The effects of DFO were probed on 3-D tumorspheres. HIF-1α was examined by IHC, and Western blot (WB) was used to elucidate iron mediators. In vivo, MDA-1986 buccal xenografts were developed in Nu/Nu mice. Control, DFO, and XRT groups were generated, and DFO and XRT were administered as previously described. IVIS imaging and tumor volumes were used to monitor growth. UALCAN data guided the selection of iron-regulatory proteins identified by WB in excised tumors.

Results: TFRC and FTH-1 were highly upregulated, suggesting that dysregulation of iron metabolism contributes to HNSCC development. In vitro, we observed a dose-dependent decrease in 3-D tumorsphere formation and no alterations in HIF-1α expression. WB demonstrated TFRC downregulation in response to 100µM DFO in both HPV+ and - cells. In vivo, a significant decrease in tumor growth and radiance was observed with DFO treatment (comparable to XRT) when compared to untreated controls. Excised tumor WB demonstrated downregulation of TFRC, identifying it as a pivotal indicator of response to iron-chelation therapy.

Conclusion: In vitro and in vivo studies reveal that DFO exhibits antitumorigenic effects, and suggest that HIF-1α is not affected by iron chelation in HNSCC cells. Moreover, TFRC was identified as a biomarker for indicating treatment response. Our findings signify that localized DFO may have antitumorigenic effects, in addition to the previously established regenerative therapeutic effects for the treatment of XRT-induced bone pathologies.

#2912

CRISPR-Cas9: A tool for rapid target discovery and validation.

Ceri M. Wiggins, David Walter, Paul Russell, Clare Sheridan, Chantelle Hudson, Joanne Yarker, Carlos le Sage, Nicola McCarthy, Jonathan D. Moore. _Horizon Discovery, Cambridge, United Kingdom_.

Genetic perturbations mediated by CRISPR-Cas9 are transforming the drug discovery process. In the hunt for novel drug targets, researchers finally have a screening tool with sufficient penetrance and power to identify new interactions, followed by a rapid gene editing technology to streamline validation approaches. At Horizon Discovery, we have now initiated more than 250 CRISPR-Cas9 knockout (KO) screens, including a significant effort to identify novel synthetic lethal interactions in colon and lung cancer genotypes. These screens have identified a reassuring number of known essential interactions, such as MDM2 and PPMID in TP53 wild type cancer cell lines. These results provide confidence in the ability of these screens to also discover novel targets that should make it through the validation process. Indeed, we have discovered novel targets for selective ablation of TP53 mutant colon cancers, several of which are strongly supported by the literature. In FBXW7 mutant colon cancers, CRISPR-Cas9 KO screens identified a set of potential novel druggable targets in addition to MCL1 - a known substrate of FBXW7, which showed a strong dependency in FBXW7 mutant lines. An important step in triaging primary hits ahead of extensive validation work has been secondary screens that target functional domains with high density sgRNA tiling. They allow more targets to be examined than would be feasible by any arrayed approach. Data from such screens, which test tens to hundreds of guides in unison, not only increases hit calling power, but can also yield information regarding important domains and mechanism of action. This was evident in the FBXW7 secondary screen data, where guides targeting the key functional BH domains of MCL1 proved to be the most effective. This information can be used to prioritise individual targets for further validation, using techniques that include an inducible CRISPR-Cas9 system to assess the effect of individual guides in an arrayed format on cell proliferation and survival, and CRISPRi. These data exemplify how CRISPR-Cas9 can enable the discovery of a new, more robust generation of drug and antibody targets.

#2913

Cancer growth under check: Anti-vacuolar ATPase "a2" antibody as novel therapy against ovarian cancer.

Arpita Kulshrestha, Gajendra K. Katara, Shayna Levine, Manoranjan Sahoo, Safaa A. Ibrahim, Alice Gilman-Sachs, Kenneth D. Beaman. _Rosalind Franklin University of Medicine and Science, North Chicago, IL_.

Tumor growth and metastasis involves complex crosstalk among cancer cells and immune infiltrates. The balance of pro- and antitumor infiltrating immune cells can impact the outcomes of tumor growth. Vacuolar-ATPase (V-ATPase) proton pump promotes cancer growth and metastasis by regulating the pH-associated cancer cell-signaling and the tumor microenvironment. V-ATPase is overexpressed on cancer cell surface in a wide array of tumor types including ovarian cancer (OVCA) as well as on inflammatory cells recruited to the tumor microenvironment. This makes V-ATPase an important target for anticancer therapy. We have created a monoclonal antibody that specifically recognizes the plasma membrane isoform of V-ATPase (a2v) present distinctly on malignant cells and absent on normal cells. The purpose of this study is to investigate the in vivo effect of anti-VATPase-a2v antibody (Mab; 2C1) on the growth of human ovarian cancer (OVCA). The antitumor effects were evaluated in female athymic nude mice xenograft model. Mice were injected with human OVCA cell line (A2780;0.4X106 cells; s.c in upper flank) and the tumor growth was compared to mice that were simultaneously given a single dose of MAb (2C1; 300µg in PBS) or an isotype control antibody(mouse IgG1A). The 2C1 treatment resulted in delayed tumor growth (2.4 fold, p< 0.05), with no apparent in vivo toxicity. Histologic staining of 2C1-treated tumors revealed a loose tumor matrix with higher immune-infiltration compared to control. There was a marked decrease in cancer cell numbers in 2C1-treated tumor tissues compared to control using CA125 cancer-antigen staining. In in vitro assays, there was no measurable cytotoxic effect of 2C1 on OVCA cell proliferation, indicating the role of tumor microenvironment in delayed tumor growth. We further investigated the infiltrated immune population in 2C1-treated tumors. Interestingly, we observed an overall increased total leukocyte population (CD45; p=0.028) and a higher expression of iNOS (IHC-score 2C1 treated tumor= 11.3, control =7.6; p=0.04), suggesting an antitumor response. The total F4/80 macrophage population was higher in anti-VATPase-a2v antibody treated cells (absolute numbers in 2C1 treated tumor=245.2, control tumor=193.1; p=0.04). In 2C1 treated mice, tumor-associated macrophages displayed antitumor properties with correlated iNOS expression. Further, a marked increase in the number of Ly6G positive cells (neutrophil marker) was observed in 2C1 treated tumors, further suggesting an elevated antitumor response. Studies are under way to characterize other immune infiltrated populations that may contribute to antitumor response in 2C1 treated tumor tissues. In conclusion, the study demonstrates that the anti-VATPase "a2" antibody is an effective form of treatment in ovarian cancer.

#2914

ITGA-2 is a novel therapeutic target for glioblastoma and high-grade gliomas.

Alexander Moses-Gardner, Peng Guo, Marsha Moses, Edward Smith. _Children's Hospital Boston, Boston, MA_.

High grade gliomas, including glioblastoma multiforme (GBM), are the most common brain cancers in adults, representing between 15 and 20% of all brain tumors diagnosed, including a significant fraction of pediatric cases. It is also among the most deadly tumor types, with only approximately 5% of diagnosed GBM patients surviving 5 years post-diagnosis. Treatment for this type of tumor involves brain surgery, typically paired with intense chemotherapy and radiation.

The ultimate goal of this study is to develop clinically safe, specific, and effective antibody-directed therapeutics for these devastating cancers. Our experimental approach used four established cell lines, consisting of three commercial GBM cell lines (A172, U87, and U118 MG) recommended by the American Type Culture Collection (ATCC) as representative GBM lines, and a control glial cell line (SVGP12) recommended by ATCC as a control. Each of these lines were cultured according to standard protocols and stained, followed by a quantitative and unbiased screening of 72 common cancer targets using comparative flow cytometry. Based on relative immunofluorescence, we found that the cell surface marker integrin alpha-2 (ITGA-2) was elevated thousandfold or more in all three of the tumor lines tested while showing minimal to no expression in the healthy glial control. All assays were conducted in triplicate and confirmed via immunofluorescence staining and confocal fluorescent microscopy.

When compared with the currently used molecular target for GBM, Epidermal Growth Factor Receptor (EGFR), ITGA-2 is both more strongly expressed on the three tumor cell lines and significantly less expressed on the healthy glial cells. These data suggest that ITGA-2 demonstrates significantly improved targeting specificity and efficiency potential compare d to EGFR. Potential therapeutic efficacy was also evaluated via antibody blockade. Significantly reduced growth and migration of these tumor cells following ITGA-2 neutralizing antibody blockade was confirmed via standard assays conducted over 24 and 48 hour periods with antibody titration curves. The presence of ITGA-2 was also confirmed in GBM tissue, with its absence noted in healthy brain tissue, via staining of tissue microarrays for ITGA-2.

In conclusion, we have discovered a novel cell-surface marker for GBM that is robustly expressed on multiple representative GBM cell lines but not on normal control glial cells. This finding may have significant clinical potential for the treatment of glioblastoma multiforme and other high grade gliomas and supports further research into the use of ITGA-2 as a therapeutic target for GBM. Our ultimate goal is to leverage these findings to develop precision-targeted nanomedicine with the capability to encapsulate and successfully deliver both radiosensitizing agents and chemotherapeutics, providing a novel method to target tumors and improve patient outcomes using tailored therapy.

#2915

Discovery and optimization of potent and selective inhibitors of USP7 to enhance anti-tumor immunity and target tumor growth.

Betty Abraham, Lavanya Adusumilli, Berenger Biannic, Delia Bradford, Martin Brovarney, David Chian, Gene Cutler, Xinping Han, Dennis Hu, Scott Jacobson, Sherra Johnson, Paul Kassner, Deepika Kaveri, John Ketcham, Andrea Kim, Paul Leger, Lisa Marshall, Sachie Marubayashi, Jack Maung, Jenny McKinnell, Cesar Meleza, Yamini Ohol, Akinori Okano, Leanne Peiser, Deepa Pookot, Payal Rana, Jacob Schwarz, Nick Shah, Grant Shibuya, Michael Sun, Silpa Suthram, Oezcan Talay, Angela Wadsworth, David Wustrow, Kyle Young, Andrew Napper. _FLX Bio, Inc., South San Francisco, CA_.

USP7 is a deubiquitinase (DUB) that has attracted much attention recently due to its multiple roles in promoting cancer progression. By removal of ubiquitin from protein substrates, USP7 stabilizes oncogenes such as MDM2 and Myc, destabilizes and inactivates the key tumor suppressors p53 and PTEN, and imparts resistance to DNA-damaging chemotherapy by enhancing DNA repair responses. USP7 plays an important role in suppression of immune responses in the tumor microenvironment by stabilizing the transcription factor FOXP3 and thereby enhancing the suppressive function of regulatory T cells. Thus, inhibition of USP7 is an appealing therapeutic strategy because it has the potential to impact important oncology targets such as transcription factors that have been widely viewed as undruggable. We employed structure-based and other medicinal chemistry techniques to enable the design of potent and selective USP7 inhibitors. Using a high-throughput assay of DUB activity employing rhodamine-labeled ubiquitin, we optimized several series of reversible USP7 inhibitors to sub-100 pM potency and selectivity of >10,000-fold over all other DUBs. Cellular activity was demonstrated using a luciferase reporter gene assay of p53 activation, revealing compounds with EC50 values ranging down to 20 nM. To assess the role of USP7 inhibition in enhancement of immune responses, we determined relief of suppression of effector T cells in vitro. Effector T cells (CD8+) were co-cultured with regulatory T cells (CD4+ FOXP3+) and antigen-presenting cells for 4 days, after which CD8+ cell proliferation was determined by flow cytometry. Treatment with USP7 inhibitors during co-culture resulted in relief of regulatory T cell suppression of CD8+ cell proliferation. In vivo enhancement of immune responses was assessed in rodent models of inflammation and tumor growth. Direct effects on tumor cell growth and viability were explored by profiling cytotoxicity of USP7 inhibitors as single agents and in combination with chemotherapeutic agents in a broad range of cancer cell lines. In preparation for future clinical development, compounds were modified to obtain desirable in vitro and in vivo ADME and toxicity profiles. Following extensive pre-clinical optimization, we have in hand orally bioavailable compounds with high permeability, low clearance, and minimal off-target activity.

#2916

Targeting lysophospholipid metabolism inhibits pancreatic cancer cell proliferation under nutrient-limiting conditions.

Petrus R. de Jong,1 Marco Maruggi,1 Alejandro D. Campos,1 Morgan A. Brand,1 Robert Lemos,1 David A. Scott,1 Sally A. Litherland,2 J. Pablo Arnoletti,3 Brian P. James,1 Garth Powis1. 1 _SBP NCI-Designated Cancer Center, La Jolla, CA;_ 2 _Florida Hospital Cancer Institute, Orlando, FL;_ 3 _Florida Hospital Institute for Surgical Advancement, Orlando, FL_.

Patients with pancreatic ductal adenocarcinoma (PDAC) have a poor prognosis, and more effective systemic treatments for patients with local progression or metastasis (85% of cases) are needed. The pancreatic tumor microenvironment provides a rich source for novel drug targets. We aimed to identify and validate novel metabolic drug targets that are unique to hypoxic PDAC cells. Using bulk RNA sequencing in combination with metabolomics analyses in vitro, we previously found that PDAC cells negate the loss of intracellular unsaturated fatty acids in hypoxia by orchestrating the release of lysophospholipids (lyso-PLs) by cancer-associated fibroblasts, which are then taken up and stored in intracellular lipid droplets in hypoxic cancer cells. To confirm the relevance of these findings in vivo, we performed 3' droplet based single-cell RNA sequencing (scRNA-seq) combined with metabolomics analyses of intracellular and extracellular (tumor interstitial fluid) metabolites of MIAPaCa2 and patient-derived xenografts (PDX). Identification of cell lineages and subpopulations with hypoxic gene signatures was performed to correlate changes in metabolite levels with metabolic gene expression in vivo. This approach confirmed differential expression of lipid droplet-associated enzymes in hypoxic areas of the tumor, including lyso-PL acyl transferases (LPCAT1, LPCAT3), and phospholipases (LYPLA1, PLA2G15). We found that resistance of PDAC cell lines to pharmacologic treatment with inhibitors of fatty acid desaturases (FADS), reminiscent of hypoxia and nutrient starvation in vivo, was mediated by uptake of lyso-PLs from the medium. Importantly, genetic knockdown of LPCAT and LYPLA isoforms reversed the resistance to FADS inhibitors in culture in vitro and in vivo. Clinical relevance was demonstrated by mRNA expression analysis of PDAC patients from The Cancer Genome Atlas (TCGA) database, which showed that the expression of lyso-PL metabolizing genes is correlated with a significant worse prognosis (log-rank test, P=0.008). We are currently developing pharmacologic approaches to target LPCAT and LYPLA enzymes in hypoxic cancer cells as a novel approach for PDAC patients with unresectable disease.

#2917

Differentiation therapy and the mechanisms that terminate malignant proliferation.

Francis O. Enane,1 Xiaorong Gu,2 Murray Korc,1 Yogen Saunthararajah2. 1 _Indiana University School of Medicine, Indianapolis, IN;_ 2 _Cleveland Clinic, Cleveland, OH_.

Historically, chemotherapy often attempts to activate apoptosis in tumor cells even when master regulators of apoptosis (e.g., TP53) are inactivated by genetic mutations, leading to multi-drug resistance. A potential form of non-toxic therapy is to engage p53-independent terminal differentiation. Exponential proliferation in lineage committed progenitors, coordinated by the transcription factor MYC, is succeeded by terminal differentiation, whereas MYC-coordinated aberrant proliferation in human malignancies is not succeeded by terminal differentiation. The mechanisms that suppress terminal differentiation are mostly unclear, impeding development of differentiation therapies.

We performed a systematic review of published literature between January 2007 – January 2017 to identify genetic alterations that produce candidate molecular targets for differentiation induction in three treatment-recalcitrant cancers: hepatocellular carcinoma (HCC), ovarian cancer (OVC) and pancreatic ductal adenocarcinoma (PDAC). We then validated candidate molecular targets in-vitro with FANA molecules and CRISPR-Cas9, and by in-vivo validation of small molecule drugs in HCC.

By systematic review, lineage tracing studies suggested master transcription factors of cell fate and their cofactors as links to suppression of cell cycle exit by differentiation. Indeed, sequence analysis of HCC, OVC and PDAC identified frequent genetic haploinsufficiency in lineage transcription factors (e.g., GATA4 67% hetloss HCC, n=442, FOXO1 63% hetloss, OVC n=302, PDX1 58% hetloss, PDAC n=109), while their coactivators (e.g. ARID1A/B, and ARID3A/C) were frequently inactivated by genetic mutation/deletion. Interestingly, corepressors (e.g., DNMT1, EED, BAZ1A/B, and SUZ12) that cooperate with these transcription factors to turn off genes were frequently amplified but not inactivated. By immunoprecipitation and mass spectroscopy, we identified in HCC that corepressors (e.g., DNMT1) were aberrantly recruited to wild-type hepatocyte transcription factors (FOXA1/2) and this interaction epigenetically suppressed hepatocyte differentiation genes (e.g., HNF4A). Pre-clinical trials evaluating DNMT1 therapeutic efficacy - by DNMT1-FANA or DNMT1-CRISPR-Cas9 - suppressed HCC proliferation but increased hepatocyte differentiation. Non-cytotoxic treatment (0.2µM) of decitabine (DEC) - DNMT1 inhibitor - terminated HCC proliferation without inducing apoptosis. This inhibition enhanced FOXA1/2 interaction with coactivators, decreased corepressors, increased hepatocyte differentiation by decreasing MYC protein, increasing p27 protein and activating HNF4A. Using 0.2mg/Kg of DEC in vivo - in obesity induced mouse model of HCC - DEC reduced HCC tumor burden, without apoptosis induction. Thus, aberrant corepressors are rational molecular targets that engage terminal differentiation in cancer cells.

#2918

**Decursin from** Angelica gigas **Nakai regulates CXCR7 expression and its downstream signaling in gastric cancer cells.**

Solbi Kim,1 Nayoung Kim,1 Hyewon Ryu,1 Gyu-Yong Song,2 Hyo Jin Lee2. 1 _Chungnam National University College of Medicine, Daejeon, Republic of Korea;_ 2 _Chungnam National University, Daejeon, Republic of Korea_.

Background C-X-C motif chemokine receptor 7 (CXCR7) is a member of the G protein-coupled receptor family and is reported to be involved in the invasion, migration and tumor progression of various cancers. However, few compounds regulate CXCR7 expression. Therefore, compounds that negatively regulate CXCR7 may inhibit tumor progression and thus serve as candidates for effective anti-cancer drugs. In this study, we investigated the regulation of CXCR7 by decursin derived from Angelica gigas and its downstream signaling.

Methods A CXCR7-overexpressing stable cell line was established in gastric cancer cells, and the expression of membrane CXCR7 was confirmed by flow cytometry and immunocytochemistry. Cell viability was examined using the CCK-8 assay. We investigated the potential of decursin to inhibit cell migration and invasion using a Transwell system. We also examined the mechanism of CXCR7 regulation by decursin by performing Western blot analysis of downstream proteins using phospho-specific antibodies.

Results CXCR7 overexpression significantly increased the proliferation, invasion and migration of gastric cancer cells. In addition, spheres were well formed under anchorage-independent conditions in CXCR7-expressing gastric cancer cells. Levels of phospho-Stat3 and -Akt, positive regulators of cancer progression, were significantly increased. Decursin treatment reduced CXCR7 expression at the cellular membrane, and significantly decreased cancer cell invasion and migration. When treated with decursin, the sphere formation capacity decreased in a dose-dependent manner. In addition, decursin decreased the phosphorylation of Stat3 and its downstream target, c-Myc; however, it had no effect on Akt phosphorylation.

Conclusion Decursin reduces the invasion, migration and tumor progression of cancer cells by inhibiting CXCR7. Decursin could be proposed as a new anti-cancer drug by decreasing the expression of CXCR7, which is believed to be a therapeutic target in gastric cancer.

#2919

Discovery of small-molecule compounds targeting c-MYC using a novel cell-based screen.

Biju Mangatt,1 Anthony D. Pietra,1 Anna Waszkiewicz,1 Jon-Paul Jaworski,1 Sonja Ghidelli-Disse,2 Thomas J. Berrodin,1 Christian S. Sherk,1 Derrick W. Meinhold,1 Anna Rutkowska-Klute,2 Shanker K. Sundaram,1 Gopinath Ganji,1 Wendy S. Halsey,1 George P. Livi,1 William Li,1 James Mack,1 Stuart P. Romeril,1 Elisabeth A. Minthorn,1 Rakesh Kumar,1 Gerard C. Drewes,2 Dirk A. Heerding,1 Lorena A. Kallal,1 Carolyn A. Buser,1 Jesus R. Medina1. 1 _GlaxoSmithKline, Collegeville, PA;_ 2 _GlaxoSmithKline, Heidelberg, Germany_.

Elevated expression of the c-MYC oncogene (either due to gene amplification, translocation, abnormality in upstream signaling pathways and/or protein stabilization) is one of the most common abnormalities in human cancers. Efforts to identify direct pharmacological inhibitors of c-MYC function have not yet yielded drug-like molecules. Therefore, we sought to pursue alternative screening strategies for this classically "undruggable" transcription factor. We developed a novel antibody-based high-throughput HTRF screening assay that specifically detects endogenous c-MYC protein levels in a MYC amplified cancer cell line. Taking advantage of the short half-life of c-MYC, both at the protein and mRNA level, we conducted a cell-based screen of the GSK screening collection to identify compounds that can rapidly decrease c-MYC protein levels. Elimination of false positive hits using stringent triage assays successfully identified two valid hit series exemplified by GSK970 and GSK417. Molecular mode of action studies revealed these molecules inhibit MYC transcription by binding to the minor groove of DNA with AT sequence specificity. Unfortunately, this mechanism of c-MYC inhibition demonstrated poor in vivo translatability as tissue DNA acts as a molecular sink, effectively sequestering compound, and limiting its pharmacodnyamic response.

#2920

Metabolic modulation of the tumor microenvironment using Synthetic Biotic™ Medicines.

Kip A. West, Adam Fisher, Dan Leventhal, Anna Sokolovska, Ning Li, Chris Plescia, Mary Castillo, Vincent Isabella, Starsha Kolodziej, Paul Miller, Jose M. Lora. _Synlogic, Cambridge, MA_.

Background: The immunosuppressive milieu found within the tumor microenvironment (TME) has long been understood to be a key driver of tumor initiation and progression. More recently it has been appreciated that metabolites derived from biosynthetic pathways are major components in forming this immune-privileged niche. For example, the conversion of tryptophan into kynurenine by indoleamine 2,3 dioxygenase (IDO) or the reduction of adenosine triphosphate to adenosine by the ectoenzymes CD39 and CD73 leads to T cell dysfunction and exhaustion, and a significantly blunted antitumor immune response. At Synlogic we are using synthetic biology in combination with natural probiotics to develop engineered bacteria or "Synthetic Biotic Medicines," which are programmed with precision to correct disease-causing and -promoting metabolic defects. Here we present results showing the development of two engineered bacterial strains that have been designed to consume either kynurenine or adenosine, two molecules known to play central roles in promoting tumor immune tolerance, with the goal of relieving TME-associated immunosuppression and promoting antitumor immunity.

Methods and Results: Synthetic biologic techniques were employed to generate the adenosine-consuming strain (SYN-Ade) or the kynurenine-consuming strain (SYN-Kyn) by introduction of genetic elements that were highly efficient in the metabolism of adenosine or kynurenine, respectively. In in vitro biochemical assays, SYN-Ade and SYN-Kyn were able to deplete test media containing levels of adenosine and kynurenine that are ~100-fold and 20-fold higher than the adenosine or kynurenine levels found in the tumors of cancer patients, (180uM of adenosine or 80uM of kynurenine, respectively) to undetectable levels within 2 hours. For the kynurenine-consuming strain, this in vitro kynurenine consumption translated to robust in vivo pharmacodynamic activity. In mice bearing subcutaneous CT26 tumors, the administration of SYN-Kyn by intratumoral (IT) injection led to significant decreases in tumor kynurenine levels, which was equivalent to small-molecule inhibition of the IDO enzyme. Importantly, the combination of SYN-Kyn with an anti-CTLA4 antibody in the CT26 tumor model or the combination of SYN-Kyn or SYN-Ade with a cocktail of anti-PD1/CTLA4 antibodies in MC38 tumor-bearing mice led to significant antitumor effects over those observed with the antibodies alone.

Conclusions: Taken together, these results demonstrate that the application of synthetic biology to engineer nonpathogenic bacteria is a viable approach to deliver profound efficacy in experimental models of cancer, and support the further development of these Synthetic Biotic medicines as potential immuno-oncology therapies. 

### Novel Experimental Combinations

#2921

Co-inhibition of BET and proteasome enhances ER stress and Bim-dependent apoptosis with augmented cancer therapeutic efficacy.

Guoqing Qian, Weilong Yao, Williams D. Hall, Mala Shanmugam, Sagar Lonial, Shi-Yong Sun. _Emory University, Atlanta, GA_.

Background: Agents that inhibit bromodomain and extra-terminal domain (BET) protein, a promising cancer therapeutic target which imposes epigenetic modulation of gene expression, have been actively tested in the clinic as potential anticancer drugs. Some proteasome inhibitors such as carfilzomib (CFZ) are FDA-approved drugs for the treatment of patients with advanced multiple myeloma. The current study focuses on studying the combination of a BET inhibitor (e.g., JQ1) and a proteasome inhibitor (e.g., CFZ) as a novel cancer therapeutic strategy.

Methods: Diverse solid and hematologic cancer cell lines were used to test the effects of the combinations on cell survival and apoptosis via SRB assay, colony formation assay, flow cytometric analysis, and Western blotting. Bim knockout cell lines were generated using a CRISPR-based method for mechanistic study. Transcriptional regulation of target genes was examined by quantitative RT-PCR. The anti-tumor effects were also examined in a xenograft tumor model.

Results: The tested combinations (e.g., JQ1 with CFZ) synergistically decreased cancer cell survival and enhanced apoptosis in vitro and inhibited tumor growth in vivo. The combinations dramatically induced apoptosis accompanying with enhanced elevation of Bim and several ER stress marker proteins. Bim knockout significantly blocked apoptosis induced by the combinations in both HCT116 and L363 cells. Transcriptional upregulation of Bim by the combinations was observed.

Conclusions: The enhanced anti-tumor effects of dual inhibition of BET and proteasome is likely due to augmented induction of ER stress and Bim-dependent apoptosis. Our findings warrant further investigation of this combinatorial strategy as an effective regimen against cancer in the clinic.

#2922

The utility of BET inhibition in the sensitization and re-sensitization of pancreatic cancer cells to paclitaxel.

Feda H. Hamdan, Ana P. Kutschat, Madhobi Sen, Xin Wang, Steven A. Johnsen. _University Medical Center Göttingen, Göttingen, Germany_.

While the mortality rates of cancer are generally declining, pancreatic cancer persists to be an exception with a 5-year-survival rate of less than 7%. As late diagnosis and resistance to conventional therapies are major contributors to high mortality rates, novel treatment options are needed to improve the prognosis of pancreatic cancer patients. Recent findings showed that inhibition of the Bromodomain and Extraterminal Domain (BET) family of epigenetic reader proteins is effective, both alone and in combination with conventional chemotherapy, in decreasing pancreatic tumor growth in patient-derived xenografts. Thus, we aim to evaluate the potential role of and mechanisms of action of BET inhibitors (BETi) as an adjuvant therapy option in pancreatic ductal adenocarcinoma. We established L3.6 pancreatic cells that are resistant to paclitaxel by maintaining them in incrementally higher concentrations for 3 months. Paclitaxel-resistant cells showed a half maximal inhibitory concentration (IC50) 100-fold higher than that of parental cells. Intriguingly, we report that low, non-cytostatic concentrations of the BETi, JQ1, not only sensitized cells to paclitaxel, but also induced significant re-sensitization of chemoresistant cells. In order to elucidate the mechanism by which BETi induces chemo-sensitization, we investigated the differential gene expression profiles of resistant and sensitive cells. Thereby, we uncovered that BETi reverses the regulation of transcriptionally-activated genes in resistant cells. Interestingly, these genes showed a major tendency to gain BRD4 at putative enhancer regions. We anticipate that enhancer RNAs (eRNAs) transcribed at these particular enhancers may provide us with novel biomarkers which can be used to predict chemotherapeutic resistance and the possibility of re-sensitization by BETi. In conclusion, we provide evidence that BETi can potentially be used as adjuvant agents in pancreatic cancer. However, approaches may likely be largely independent of their anti-proliferative effects that require higher concentrations and possibly lead to intolerable adverse effects, but rather to their transcriptional regulatory functions that attenuate the activated programs in chemoresistant cancers.

#2923

BET inhibitors synergize with HDAC inhibitors and downregulate expression of interferon response genes in glioblastoma.

Olga Gusyatiner,1 Minh Diêu Thanh Pham,1 Yvonne Lei,1 Jungyeon Park,1 Pierre Bady,1 Mauro Delorenzi,2 Monika Hegi1. 1 _University Hospital of Lausanne, Epalinges, Switzerland;_ 2 _University of Lausanne, Lausanne, Lausanne, Switzerland_.

Glioblastoma (GBM) is the most aggressive brain tumor in adults with a median overall survival of only 15 months. Despite multiple attempts, single agent therapies have failed in clinical trials and new strategies for combination treatments are urgently needed.

Here, our aim was to predict rational combination therapies with BET inhibitors (BETi) that target bromodomain and extra-terminal tail (BET) proteins and are currently evaluated as anti-cancer drugs. In order to identify BETi-induced vulnerabilities in cancer relevant pathways that may be targeted with a second drug, we obtained differential expression profiles of glioma sphere lines (GS-lines) treated with the BETi JQ1. Gene set enrichment analysis revealed several significantly disturbed pathways that included IFN-alpha response genes and signatures of response to histone deacetylase inhibitors (HDACi).

In order to validate the observed down regulation of the interferon response gene signature, we primed the GS-lines with IFN-alpha and confirmed that interferon-stimulated genes (ISGs), such as MX1, OAS1, and CD274 are down regulated after a 4-hour exposure to JQ1. Importantly, the levels of pSTAT1 in the nucleus remained unchanged upon JQ1 treatment, suggesting that JQ1 was acting directly on the transcriptional level of ISGs and not on the canonical JAK-STAT pathway. Similar results were obtained in adherent GBM cell lines that constitutively express ISGs. Moreover, in U87MG orthotropic xenografts in mice, a single i.p. injection of JQ1 downregulated OAS1 and CD274 expression. Currently we are studying the mechanism of how BETi represses transcription of ISGs by chromatin immuno-precipitation.

Finally, we show that HDACi and JQ1 synergize to reduce cell viability of GS-lines in vitro. Further experiments will test HDACi and BETi drug combinations in mouse orthotopic xenografts of GS-lines.

#2924

Preclinical analysis of synergistic antitumor effect of eribulin and HDAC inhibitor for triple-negative breast cancer cells.

Takaaki Oba,1 Hiroto Izumi,2 Ken-ichi Ito1. 1 _Shinshu University, Matsumoto, Japan;_ 2 _Occupational and Environmental Health University, Kitakyushu, Japan_.

Eribulin (ERI), an inhibitor of microtubule dynamics, is a key drug for metastatic breast cancer. While HDAC inhibitors have been shown to presents antitumor effect for various malignancies. Among HDAC family, HDAC6 is known as a deacetylase of tubulin. To develop a novel therapeutic strategy for triple negative breast cancer (TNBC), we tested the combinational effect of ERI and HDAC inhibitor for TNBC cells in vitro. Two TNBC cell lines (MDA-MB-231, Hs578T) and their ERI-resistant cells (MDA-MB-231/E, Hs578T/E) established in our laboratory were used in the study. Vorinostat (VOR), a pan-HDAC inhibitor, and ricolinostat (RICO), a selective HDAC6 inhibitor were used. The sensitivity to VOR or RICO was not different between the parental and the ERI-resistant cells. In the protein expression analysis, HDAC6 expression was not altered between parental and ERI-resistant cells. Prior treatment with VOR or RICO increased the expression of acetylatedα-tubulin in both parental and ERI-resistant TNBC cell lines. When low concentration of VOR (0.5 μM) or RICO (0.5 μM) was added simultaneously with ERI, the sensitivity to ERI was enhanced significantly in both parental MDA-MB-231 and Hs578T cell lines. Furthermore, when both parental cell lines were treated with VOR (5 μM) or RICO (5 μM) for 48 hrs prior to addition of ERI, the sensitivity to ERI was significantly enhanced. The enhancement of sensitivity to ERI both by the simultaneous addition with low concentration of VOR or RICO and by the prior treatment with VOR or RICO was observed in the ERI-resistant cells as well. Moreover, inhibition of HDAC6 by small interfering RNA partially enhanced the sensitivity to ERI in the both parental cell lines. Our study demonstrated the possibility that the combination of ERI with HDAC inhibitor might enhance the antitumor effect of ERI and overcome ERI resistance through acetylation of α-tubulin by HDAC6 inhibition in TNBC cells.

#2925

Tandem targeting of poly (ADP-ribose) polymerase (PARP) and epidermal growth factor receptor (EGFR) as a novel strategy for enhancing radio- and chemosensitivity of refractory tumors.

Martin Rupp, Zhor Senhaji Mouhri, Ursula Stochaj, Bertrand Jean-Claude. _McGill Univ., Montréal, Quebec, Canada_.

Poly (ADP-ribose) polymerase (PARP) inhibitors are a novel therapeutic approach for the selective targeting of tumours with BRCA1/2 hereditary deficiencies. BRCA1/2 are two key proteins involved in homologous recombination (HR) repair. Although the therapeutic benefit of PARP inhibitors has now been proven in the clinic in patients with BRCA1/2 mutations, much effort has been deployed to expand the use of PARP inhibitors beyond tumours harbouring inherited deficiencies in HR- mediated DNA repair. Several combination modalities with agents that can induce HR deficiency and exploit the concept of "contextual synthetic lethality" have been reported for PARP inhibitors. Here we exploited the contextual synthetic lethality between PARP and the epidermal growth factor receptor (EGFR) to design and synthesize a novel dual EGFR targeted PARP inhibitor, termed ZSMR06. The results showed that: a) ZSMR06 is capable of inhibiting EGFR and PARP in whole cells in the nanomolar range (36-63 nM); b) it was selectively potent against BRCA2 mutant and EGFR expressing isogenic cells; c) as monotherapy, ZSMR06 was extremely potent with growth inhibitory activities superior to that of an equimolar combination of olaparib (a clinical PARP inhibitor) and gefitinib (a clinical EGFR inhibitor) in a large panel of tumour cell lines; d) ZSMR06 strongly potentiated the effect of temozolomide (TMZ) and induced significantly stronger (p<0.05-0.001) growth inhibition than olaparib + TMZ combination in cells expressing O6-methylguanine-DNA methyltransferase (MGMT), an enzyme that confers a robust resistance to TMZ; e) ZSMR06 selectively potentiated TMZ on EGFR expressing cells; f) it was able to strongly sensitize bladder cancer cells to radiation (DMF50= 2.96-9.5). These results in toto showed that ZSMR06 due to its dual mechanism of action is capable of behaving as a strong potentiator of TMZ and radiation in resistant cells. Its potency in non-BRCA1/2 mutant tumour cells indicates that it may be the first prototype of hybrid molecules capable of expanding the use of PARP inhibition beyond BRCA1/2 mutations.

#2926

The glycogen synthase kinase-3 inhibitor, 9-ING-41, synergizes with chemotherapy to inhibit pancreatic tumor growth in vivo.

Li Ding,1 Jin-san Zhang,1 John R. Dube,1 Vijay S. Madamsetty,2 Daniel M. Schmitt,3 Debabrata Mukhopadhyay,2 Daniel D. Billadeau1. 1 _Mayo Clinic, Rochester, MN;_ 2 _Mayo Clinic College of Medicine and Science, Jacksonville, FL;_ 3 _Actuate Therapeutics, Fort Worth, TX_.

Glycogen synthase kinase-3 (GSK3) is a ubiquitously expressed serine-threonine protein kinase involved in multiple cellular functions ranging from the control of glycogen metabolism to transcriptional regulation. We have previously demonstrated that GSK-3β is overexpressed in human pancreatic ductal adenocarcinoma (PDAC) and aberrant nuclear accumulation of GSK-3β serves as a hallmark of poorly differentiated PDAC. Moreover, genetic ablation of GSK-3β or GSK-3 inhibition led to PDAC tumor cell killing, identifying GSK-3β as a therapeutic target in PDAC. Herein, we describe a novel GSK-3 inhibitor, 9-ING-41. The effect of 9-ING-41 on tumor cell growth was tested using several PDAC and patient-derived xenograft (PDX) cell lines in vitro and in vivo using an orthotopic pancreatic cancer animal model. Interestingly, 9-ING-41 treatment resulted in the stabilization of cyclin B levels as well as enhanced serine-10 phosphorylated histone H3 (pS10HH3) and consequently M phase arrest and blocked mitotic exit. The prolonged M phase arrest induced mitotic catastrophe in pancreatic cancer cell lines and inhibited in vitro colony formation. An in vivo orthotopic pancreatic cancer animal model study using PDX-derived cell lines demonstrated that 9-ING-41 hindered tumor growth in combination with several standard chemotherapeutic agents. Significantly, we found that 9-ING-41 treatment impaired the DNA damage response pathway activated by Gemcitabine in pancreatic cancer cell lines. In conclusion, our study indicates that GSK-3 plays an important role in pancreatic cancer progression and 9-ING-41 in combination with existing gemcitabine or irinotecan could be a new strategy for treating patients with PDAC.

#2927

Antitumor and antiangiogenesis activities of E7386, an orally active CBP/β-catenin modulator, as a single agent and in combination with lenvatinib in human HCC xenograft models.

Kazuhiko Yamada, Yusaku Hori, Hiroshi Kamiyama, Takayuki Kimura, Yasuhiro Funahashi, Junji Matsui, Yoichi Ozawa. _Eisai co., Ltd., Ibaraki, Japan_.

E7386 is a novel orally active CBP/β-catenin modulator that has showed anti-tumor activity in human tumor xenograft models with aberrant activation of Wnt/β-catenin signaling pathway. Hepatocellular carcinoma (HCC) is one of the leading causes of cancer deaths worldwide. In HCC, it has been reported that aberrant activation of the canonical Wnt/β-catenin signaling pathway frequently occurs (20-40%). In this study, we investigated anti-tumor activity of E7386 as monotherapy or in combination with lenvatinib in human HCC xenograft models. Lenvatinib is a multiple receptor tyrosine kinase inhibitor targeting vascular endothelial growth factor receptors and oncogenic kinases. A recent Phase III study demonstrated the treatment effect of lenvatinib on OS by statistical confirmation of non-inferiority as compared with sorafenib patients with unresectable HCC. We evaluated the anti-tumor activity of E7386 as a single agent and in combination with lenvatinib in 4 human HCC cell lines (SNU398, HepG2, Hep3B2.1-7 and Huh-7) xenograft models. These cell lines were injected subcutaneously into male nude mice, and tumors were allowed to grow to 150-200 mm3 in size. Then, the mice were given oral administration of E7386 and/or lenvatinib for 14 days. Single treatment of E7386 demonstrated statistically significant anti-tumor effect in HepG2 and SNU398 xenograft models with β-catenin mutation; delta T/C value (dT/C) of 60% at 100 mg/kg qd in HepG2 model, dT/C of 74% and at 50 mg/kg qd in SNU398 model. On the other hand, no tumor growth inhibition was observed in Hep3B2.1-7 and Huh-7 xenograft models with wild type β-catenin. Lenvatinib at 10 mg/kg showed the anti-tumor effect in all 4 xenograft models, dT/C of 68%, 43%, 52% and 70% in SNU398, HepG2, Hep3B2.17 and Huh-7, respectively. E7386 at 50 mg/kg in combination with 10 mg/kg of lenvatinib showed more potent anti-tumor effects compared with each single treatment irrespective of β-catenin mutation status, dT/C of 35%, 14%, 9% and 17% in SNU398, HepG2, Hep3B2.17 and Huh-7, respectively. Similar combined activities were observed at the range of 6.25-50 mg/kg of E7386 with lenvatinib at 10 mg/kg. Next, we examined the effect on tumor angiogenesis. Each 50 mg/kg of E7386 and 10 mg/kg of lenvatinib decreased the microvessel density (MVD) in SNU398, reduction ratio of 37% and 79%, respectively. The combination treatment significantly decreased MVD more than each single treatment (reduction ratio of 91.4-95.1% at 12.5-50 mg/kg of E7386 with lenvatinib at 10 mg/kg). Similar combination effects were also observed in HepG2 and Huh-7 xenograft models.In conclusion, these results indicate that the combination of E7386 with lenvatinib exerted enhanced anti-angiogenic activity against tumor vessels and demonstrated the potent anti-tumor activity in HCC preclinical models.

#2928

**Bortezomib sensitizes glioblastoma with unmethylated** MGMT **promoter to temozolomide-chemotherapy through MGMT depletion and abrogated autophagy flux.**

Mohummad A. Rahman,1 Andrea G. Navarro,1 Jorunn Brekke,2 Christian Bindesbøll,3 Agnete Engelsen,1 Shahin Sarowar,1 Marzieh Bahador,1 Bjørn T. Gjertsen,1 Dorota Goplen,2 Per Ø. Enger,1 Frode Selheim,1 Anne Simonsen,3 Martha Chekenya1. 1 _University of Bergen, Bergen, Norway;_ 2 _Haukeland University Hospital, Bergen, Norway;_ 3 _University of Oslo, Oslo, Norway_.

Background: Glioblastoma with unmethylated O6-methyl guanine DNA methyltransferase (MGMT) promoter is highly resistant to temozolomide (TMZ) chemotherapy. Strategies that ameliorate drug resistance are sorely needed. Recent trials of the proteasome inhibitor bortezomib (BTZ) (Velcade) in combination with various drugs failed due to inappropriate schedule timing and dosing. We hypothesized that pretreatment with BTZ prior to TMZ administration may sensitize glioblastoma cell to TMZ chemotherapy.

Methods: We investigated treatment efficacy through DNA damage, apoptosis and autophagy flux by flow cytometry, western blotting, long-lived protein degradation assays, electron and fluorescence microscopy in cell lines (U87, T98G, HF66) and patient biopsy-derived cells (P3, 2012-18, BG5 and BG7). Treatment efficacy and tolerability was investigated in vivo in mice implanted orthotopically with patient-derived GBM xenografts and subsequently treated with human equivalent dose (HED) of BTZ 1.3 mg/m2 on days 1, 4, 8 and 11 for two cycles during TMZ 82 mg/m2 or 164 mg/m2 by oral gavage 5 days/week for 5 weeks. MRI, mouse survival times, tandem LC-MS/MS and clinical chemistry were used to monitor tumor growth and evaluate tissue and blood samples for biomarkers of treatment efficacy and toxicity.

Results: Patient-derived glioblastoma cells were universally more sensitive to BTZ than carfilzomib or MG-132 (P<0.0001), while their sensitivity to TMZ was strongly associated with MGMT promoter methylation status (P<0.0001). BTZ depleted MGMT protein (P<0.001) and mRNA (P<0.0001) in TMZ resistant tumor cells and sensitized to chemotherapy through induction of prominent DNA damage, G2-S phase cell cycle arrest and apoptosis with half the IC50 doses for both drugs. BTZ simultaneously abrogated TMZ-induced autophagic flux indicated by p62 accumulation, inhibition of utophagosome fusion and degradation of long-lived proteins. Human equivalent doses of BTZ 1.3 mg/m2 and TMZ 164 mg/m2 prolonged progression free and overall survival (P<0.0001), through diminished tumor growth (P<0.05), angiogenesis (P<0.01) and proliferation (P<0.05). Loss of structural and catalytic proteasomal subunits confirmed target inhibition at the therapeutically effective dose (P<0.01). Peroxiredoxin, thioredoxin and catalase antioxidant enzymes (P<0.01), alanine aminotransferase (P<0.05) and MGMT mRNA expression (P<0.01) were downregulated in combination treated animals, serving as accessible biomarkers of response.

Conclusion: Pretreatment with BTZ chemo-sensitized resistant glioblastoma through aborted autophagic flux and MGMT depletion to augment DNA damage, apoptosis and prolonged survival. Our findings warrant a controlled investigation of the treatment schedule in selected patients.

#2929

GC4419 enhances the response of non-small cell lung carcinoma cell lines to cisplatin and cisplatin plus radiation through a ROS-mediated pathway.

Ahneesh J. Mohanty,1 Brock J. Sishc,1 Kelly C. Falls,2 Collin D. Heer,2 Douglas R. Spitz,2 Michael D. Story1. 1 _The University of Texas Southwestern Medical Center, Dallas, TX;_ 2 _Holden Comprehensive Cancer Center, Carver College of Medicine, The University of Iowa, Iowa City, IA_.

Non-small cell lung carcinoma (NSCLC) is the primary cause of cancer-related death in the United States, with a five-year survival rate of only 8-10%. Current treatment methods for NSCLC aside from surgical resection, include platinum-based chemotherapy and chemo-radiation modalities. However, continued poor outcomes necessitate mechanisms to enhance the potency of current therapies but without increasing normal tissue morbidities. GC4419, a novel MnSOD mimetic that has shown promise in clinical trials as a radioprotective agent, may serve as a potential therapeutic adjuvant with these properties. Because MnSOD generates hydrogen peroxide, it can be hypothesized that supplementation of basal MnSOD levels using GC4419 could lead to lethal hydrogen peroxide formation in NSCLC cells and sub-lethal formation in normal cells following therapy-induced superoxide generation, due to differences in hydrogen peroxide metabolism. When H1299 and H460 cells were exposed to either cisplatin for 24h or cisplatin plus radiation, with or without GC4419, GC4419 synergistically decreased clonogenic survival in H460 and H1299 cells. Changes in ROS levels and cell death signatures were analyzed through flow cytometry and western blotting, respectively, following GC4419 treatment. GC4419 was found to reduce intracellular superoxide, increase intracellular hydrogen peroxide, and total cellular ROS in both H1299 and H460 cells, as well as induce early apoptosis. Specifically, across both cell lines, total cellular ROS and intracellular H2O2 was increased with GC4419 treatment by 46% and 54% following cisplatin and platinum chemo-radiation respectively, while intracellular superoxide was decreased by 48% and 42%, respectively. Early apoptosis was also increased with GC4419 across both cell lines on average by 26% following cisplatin, and 34% following chemo-radiation. Furthermore, GC4419 increased PARP cleavage 24h following cisplatin in H460 and H1299 by 83% and 146% respectively, and by 60% and 38% respectively, following chemo-radiation. However, GC4419 did not enhance cellular response to gemcitabine, which unlike cisplatin, does not induce intracellular superoxide formation. To test whether the production of increased cellular H2O2 via GC4419 superoxide dismutase activity was a potential mechanism for this enhanced toxicity, H1299 cells were engineered to express catalase on an inducible doxycycline promoter. In this cell line (H1299CAT), GC4419 enhancement of cytotoxicity was abrogated. Furthermore, in H1299CAT cells treated with GC4419 there was no change in PARP cleavage following either cisplatin or platinum chemo-radiation. As such, GC4419 has the potential to enhance the potency of platinum-based therapies without increasing normal tissue morbidities, leading to improved therapeutic outcomes and better quality of life for NSCLC patients.

#2930

Synergistic and additive anti-tumor effects of MIV-818 in combination with sorafenib in nonclinical hepatocellular carcinoma models.

Biljana Rizoska,1 Johan Bylund,1 Sveinn Briem,1 Alastair Kyle,2 Andrew Minchinton,2 Fredrik Öberg,1 Karin Göhlin,1 Mark Albertella1. 1 _Medivir AB, Huddinge, Sweden;_ 2 _BC Cancer, Vancouver, British Columbia, Canada_.

Background: MIV-818, a nucleotide prodrug of troxacitabine-monophosphate, has been designed to deliver high levels of the chain-terminating nucleotide troxacitabine-triphosphate (TRX-TP) to the liver after oral dosing while minimizing systemic exposure. Sorafenib is a multikinase inhibitor with antiangiogenic and antiproliferative effects that is approved for the treatment of advanced hepatocellular carcinoma (HCC). Hypoxia is induced as a result of the antiangiogenic effects of sorafenib. Since hypoxic conditions have been shown to increase cytotoxicity of TRX via increased conversion of TRX-diphosphate to TRX-TP, we investigated the effects of combining MIV-818 or TRX with sorafenib in cell lines and in xenograft mouse models of HCC.

Methods: Synergy of MIV-818 and sorafenib was evaluated in vitro using Bliss Independence combination analysis. In vivo effects were evaluated in nude mice with subcutaneous Hep3B or HepG2 xenografts. Due to instability of MIV-818 in mouse blood, treatment with TRX was also included. MIV-818 (30 or 100 mg/kg PO) or TRX (2.5 mg/kg IP) was given twice daily for 5 days alone or in combination with sorafenib (30 mg/kg PO) once daily for 21 days. Quantitative immuno-fluorescence was used to assess DNA damage (pH2AX), proliferation (BrdU), and hypoxia (pimonidazole) in the tumors. Results: MIV-818 shows strong synergistic anti-proliferative activity with sorafenib in several HCC cell lines in vitro. In the Hep3B xenograft model, treatment with TRX or sorafenib alone resulted in tumor growth inhibition (TGI) of 32% and 52%, respectively. Combination of TRX and sorafenib was substantially more active than either agent alone, reaching a TGI of 90%. Exposures of TRX and sorafenib in plasma and tumor were similar in the combination and single agent groups, suggesting no pharmacokinetic interactions. TRX treatment resulted in significant inhibition of proliferation (by 80%) and induction of DNA damage (15-fold) in vivo. Sorafenib treatment resulted in a small but significant inhibition of proliferation (by 27%) and 3-fold induction in hypoxia, but no induction of DNA damage. The data are consistent with the expected mechanisms of action of each agent. Combination of TRX and sorafenib resulted in 92% inhibition of proliferation and induction of DNA damage (15-fold). Notably, clear responses were seen even in hypoxic regions of the tumor, which can be resistant to therapy, indicating effective delivery of TRX-TP to regions far from blood vessels. Conclusions: Additive and synergistic anti-tumor effects were observed in nonclinical HCC models after combination of MIV-818 or TRX with sorafenib. The results suggest that add-on of MIV-818 to sorafenib may be beneficial for the treatment of HCC. MIV-818 is currently in nonclinical development in preparation for clinical trials in patients with advanced HCC and other liver cancers.

#2931

MTX-211, a dual and selective inhibitor of EGFR and PI3 kinase, shows promising activity in combination with MEK inhibition in preclinical models of pancreatic cancer.

Christy Frankowski-McGregor,1 Joel Maust,1 Elizabeth Ziemke,1 Rachel Mumby,2 Amy Delaney,1 Alnawaz Rehemtulla,1 Christopher Whitehead,2 Judith S. Sebolt-Leopold1. 1 _Univ. of Michigan, Ann Arbor, MI;_ 2 _Mekanistic Therapeutics, Ann Arbor, MI_.

Pancreatic cancer is one of the deadliest forms of cancer, with median 5-year survival rates less than 10%. This disease is recalcitrant to chemotherapeutic approaches and recently approved therapies afford only modest improvements in survival. KRAS is the most commonly mutated gene in pancreatic tumors with an incidence rate exceeding 90%. Despite intensive efforts, KRAS has remained undruggable. Kinases acting both upstream and downstream of RAS continue to be exploited for the development of novel agents to attenuate signaling through this critical oncogene. While EGFR and MEK inhibitor monotherapies have been evaluated in the clinic for the treatment of pancreatic cancer, efficacy has been modest despite their relevance to MAP kinase (MAPK) pathway-altered tumors. Novel therapies are urgently needed to address adaptive signaling mechanisms that diminish the effectiveness of kinase-targeted approaches. MTX-211 is a first-in-class dual inhibitor of PI3K and EGFR kinase with a promising pharmaceutical profile and proven ability to potentiate the effectiveness of MEK inhibitor therapy in KRAS mutant colorectal tumors. The present study was undertaken to extend our evaluation of MTX-211 to include pancreatic cancer model systems. We have found that MTX-211 exhibits low micromolar potency against an extensive panel of primary models of pancreatic cancer and is highly synergistic with the MEK inhibitor trametinib. Preliminary data suggest that this synergy is driven by the ability of MTX-211 to target compensatory transcriptional activation of HER3 that occurs in response to MEK inhibition. A genetically engineered KRAS and p53 mutant (KPC) mouse model was transduced with a lentiviral construct encoding a caspase reporter, which was used to show that the combination of MTX-211 and trametinib elicited a significant increase in apoptosis over single agent controls. Results from an animal study conducted with the KPC model further showed that the combination of MTX-211 and trametinib significantly slowed growth of these aggressive tumors. Employing both KPC and patient-derived xenograft models of pancreatic cancer, efforts are underway to optimize therapeutic outcome in response to MTX-211-based combination treatment regimens.

#2932

Evaluation of dual-acting PIM/PI3K inhibitor IBL-302 in preclinical breast cancer models.

Sean P. Kennedy,1 Michael O Neill,2 Darren Cunningham,2 Carmen Blanco-Aparicio,3 Sonia Martinez,3 Alex Eustace,4 Bryan Hennessy1. 1 _RCSI Royal College of Surgeons Ireland, Dublin, Ireland;_ 2 _Inflection Biosciences, Dublin, Ireland;_ 3 _CNIO Spainish National Cancer Research Centre, Madrid, Spain;_ 4 _National Institute for Cellular Biotechnology, Dublin, Ireland_.

Activating PI3K mutations have been identified in more than 30% of breast cancers (BC). These mutations have been associated with resistance (res) to trastuzumab (Tras) a HER2-binding monoclonal antibody. PIM kinase expression has been shown to be markedly elevated in PI3K treated BC samples suggesting that it could be a major res pathway for PI3K inhibitors in BC, potentially limiting their clinical utility. IBL-302 is a novel molecule that inhibits both PIM and PI3K signalling. This mechanism of action could afford significant benefit in the treatment of BC. We evaluated the preclinical activity of a IBL-302, in a range of BC models. IBL-302 was tested in the Sanger Institute GDSC screening panel of more than 700 different cancer cell lines in a CellTiter Glo anti-proliferation assay (72hr incubation). We subdivided a panel of 36 BC cell lines into their clinical subtype (HER2+ n=6, HR+ n=6 & TNBC n=24). IBL-302 had significantly lower IC50 in TNBC cell lines when compared to HR+ and HER2+ BC cell lines (r = 0.352, p = 0.038). Sensitivity to IBL-302 correlated significantly with PIM 1 expression (r= -0.45, p = 0.0045) and PIM 2 expression (r =- 0.3238, p = 0.0506) but no such relationship was observed for PIM 3.The HER2 gene or protein is amplified or overexpressed in ~25% of BC and promotes an aggressive phenotype, through the activation of the PI3K/AKT pathway. We utilised our HER2+ BC, Tras res cell lines which had previously been utilised in another PI3K / Tras refractory trial (NCT02705859). IBL-302 had anti-proliferative effects in HER2+ lines (SKBR3, HCC1954 and BT474 cells) including matched models of acquired Tras (SKBR-T, BT474-T) or lapatinib (SKBR-L, HCC1954-L) res. IBL-302 achieved IC50 ranging from (33.4 ± 2.9nM) in SKBR3-T cells to (136 ± 35.2nM) in SKBR3-L, but interestingly both acquired Tras res cell lines SKBR3-T and BT474-T cells were significantly more sensitive to IBL-302 than that observed in the matched SKBR3-P (p>0.01) and BT474-P (p>0.01) cells respectively. It was also observed that the combination of Tras and IBL-302 significantly increased the anti-proliferative effect in SKBR3-P (P<0.01) and SKBR3-T cells (P<0.01), relative to testing either drug alone. Treatment with IBL-302 (50mg/kg) PO for 21-days BT474 in xenograft model in BALB/c nude mice resulted in a significant decrease in tumour volume relative to the vehicle treated controls (P<0.01). IBL-302 had no significant impact on bodyweight at this dose. These results demonstrate in vitro efficacy of IBL-302 in a range of BC cell lines, including lines with acquired res to Tras. We have also shown the in vivo single agent efficacy of IBL-302 in the subcutaneous BT474 xenograft model in BALB/c nude mice. We believe IBL-302 represents an exciting new entity a dual-acting, PIM/PI3K inhibitor, with potential for the treatment of BC and given the potential to overcome Tras res in vitro in HER2+ BC cells that it should be clinically investigated in this setting.

#2933

Synergistic antitumor effect of ONC201 in combination with radiation therapy.

Lanlan Zhou, Jessica Wagner, Wafik S. El-Deiry. _Fox Chase Cancer Center, Philadelphia, PA_.

Radiation therapy is used as the main treatment when surgery is not feasible, before/after surgery, and to palliate symptoms in advanced stage. About 50% of cancer patients receive radiation therapy. 40% of the cure is achieved by radiation therapy. First-in-class small-molecule imipridone ONC201 can cause dual inactivation of ERK and AKT and induce an integrated stress response, proapoptotic TRAIL receptor DR5 activation, cancer stem cell depletion, and cell cycle arrest. ONC201 could synergize with radiation therapy because of its proapoptotic and antimetastasis effects. We tested this hypothesis in colorectal cancer, non-small cell lung cancer and glioblastoma cell lines (HCT116, RKO, LS513, H460, A549 and SNB19). Firstly, CellTiter-Glo® luminescent cell viability assays were performed with single treatment or combination of radiation therapy (0 to 4 Gy) and ONC201 (0 to 20 μM). For combination treatment, cancer cells were pretreated with ONC201 for 24 hours followed by radiation therapy. Next, colony formation assays with single treatment or combination were used to evaluate the effective dose range from cell viability assays. Flow cytometry and Western blot analysis were used for determination of apoptosis. We observed synergism between ONC201 and radiation therapy in several colorectal cancer, non-small cell lung cancer and glioblastoma cell lines. Combination indices are 0.53, 0.59 and 0.68 when non-small cell lung cancer cell line H460 was treated with ONC201 at 5 μM and radiation therapy at 1, 2 and 4 Gy. We are currently pursuing this promising combinational therapy in vivo, using the H460 xenograft model and potential syngeneic lung tumor models with wild-type p53. We plan to pursue the mechanism of this combination from in vivo samples. Our results indicate that the combination of radiation therapy and ONC201 could be an important approach in the application of ONC201 in clinical practice.

#2934

Cucurbitacin D enhances the therapeutic efficacy of docetaxel via targeting cancer stem cells and miR-145.

Mohammed Sikander,1 Shabnam Malik,1 Bilal Bin Hafeez,1 Hassan Mandil,1 Fathi T. Halaweish,2 Meena Jaggi,1 Subhash C. Chauhan1. 1 _Univ. of Tennessee Health Science Ctr., Memphis, TN;_ 2 _South Dakota State University, Brookings, SD_.

Background: Docetaxel (DTX) is an FDA approved drug for the treatment of advanced prostate cancer (PrCa). However, chemo-resistance and toxicity limits its use in clinic. Accumulating evidence suggests that activation of cancer stem cells (CSCs) is one of the mechanisms leading to developing chemo-resistance Therefore, identification of new pharmacophores that can target CSCs may improve the therapeutic efficacy of docetaxel for the treatment of advanced PrCa. Cucurbitacin D is one of the potent analogues of cucurbitacin and has shown several health benefits including anti-cancer activity. Herein, we report that Cucurbitacin D augments the therapeutic efficacy of DTX via modulating CSCs and miR-145.

Methods: Prostate cancer stem cells (pCSCs) were used as a model system to investigate the effect of Cucurbitacin D alone or in combination with docetaxel. Effect of Cucurbitacin D was evaluated on cell growth by MTS and xCELLigence assays and apoptosis induction was evaluated by flow cytometry. Colony formation assay was performed to investigate the effect of Cucurbitacin D on clonogenic potential of pCSCs cells. Effect of Cucurbitacin D on key oncogenic molecules was analyzed by Western blot and qRT-PCR analyses. To investigate the therapeutic and chemo-sensitization effect of Cucurbitacin D, we performed xenograft study using pCSCs.

Results: Our results demonstrated that Cucurbitacin D potentiates the therapeutic effect of docetaxel in pCSCs. Cucurbitacin D treatment significantly inhibited growth and clonogenic potential. Moreover, Cucurbitacin D effectively sensitized the effect of docetaxel as analyzed by significant (P<0.01) decrease in colony forming and proliferative potential of pCSCs compared to docetaxel treatment alone. To understand the underlying molecular mechanism of Cucurbitacin targeting CSCs, we analyzed the effect of Cucurbitacin D on CSCs markers such as CD133, OCT4, SOX2 and miR-145 in pCSCs. Cucurbitacin D effectively inhibited the expression of CD133, OCT4, and SOX2 and induced the expression of miR-145 in pCSCs. Cucurbiatcin D administration (1 mg/kg body weight intra-tumoral three times for five consecutive weeks) inhibited pCSCs derived xenograft tumors in athymic nude mice. This effect was additive, when combined with docetaxel treatment.

Conclusion: Our findings suggest that cucurbitacin D is a novel agent and has great promise to improve the therapeutic efficacy of docetaxel. Thus, cucurbitacin could be a novel therapeutic modality for the treatment of advanced prostate cancer.

#2935

Preclinical efficacy of the p70S6K/AKT dual inhibitor M2698 in combination with trastuzumab in models of gastric cancer.

Shota Fukuoka,1 Takashi Kojima,2 Yoshikatsu Koga,3 Mayumi Yamauchi,2 Masahiro Yasunaga,3 Yasuhiro Matsumura,3 Kohei Shitara,2 Toshihiko Doi,1 Takayuki Yoshino,2 Toshio Kuronita,4 Anderson Clark,5 Brian Elenbaas,5 Hong Zhang,5 Atsushi Ohtsu2. 1 _Experimental Therapeutics, National Cancer Center, Kashiwa, Japan;_ 2 _Division of Gastrointestinal Oncology, National Cancer Center Hospital East, Kashiwa, Japan;_ 3 _Division of Developmental Therapeutics, Research Center for Innovative Oncology, National Cancer Center Hospital East, Kashiwa, Japan;_ 4 _Merck Serono Co, Ltd (a business of Merck KGaA, Darmstadt, Germany), Tokyo, Japan;_ 5 _EMD Serono (a business of Merck KGaA, Darmstadt, Germany), Billierica, MA_.

M2698 is a potent and selective, ATP-competitive dual inhibitor of p70S6K and AKT1/3 that is being evaluated in a phase I clinical trial in cancer patients. Dual pathway inhibition by M2698 may result in improved clinical efficacy by inhibiting downstream pS6 and blocking the increased AKT activity that results from a compensatory feedback loop induced by PI3K/AKT/mTOR (PAM) pathway inhibition. In earlier studies, combination efficacy was observed with M2698 and trastuzumab in HER2+ breast cancer patient derived xenograft (PDX) models, including complete tumor regressions (Huck, BR. et.al. 105th AACR; 2014: Abstract 4516). To explore the potential combination effects in gastric cancer (GC), we evaluated the effect in in vitro and in vivo GC models. Ten human GC cell lines, which included two HER2+ lines, were treated with M2698 and trastuzumab over various concentrations and cellular proliferation was examined with a WST-8 assay. Combination effects were evaluated by Bliss independence scores and highest single agent (HSA) models. The OE-19 HER2+ cell line had the greatest Bliss and HSA sum values, whereas these measures in the other HER2+ cell line, NCI-N87, were among the lowest in the set of 10 lines, indicating that the combination is synergistic in some HER2+ GC cell lines, such as OE-19. The effects of this combination on downstream markers were analysed by Western blotting. Expression of pS6 was reduced in OE-19 cells by treatment with M2698, but pAKT and pERK were both increased. Upregulated pAKT is a known compensatory feedback mechanism that is inhibited by the dual nature of M2698. Upregulated pERK is a candidate resistance marker for PI3K pathway inhibition. However, the combination of trastuzumab with M2698 blocked this increase in pERK, suggesting that dual inhibition of the MAPK and PAM pathways may contribute to the synergistic anti-proliferative effects. The combination was then tested in the OE-19 xenograft model in vivo and significantly inhibited tumor growth compared to vehicle treatment and monotherapies (p<0.05), whereas both monotherapies were not different from vehicle (p>0.05). In an in vivo pharmacodynamic study, the combination of M2698 and trastuzumab inhibited pS6 and pERK showing effective PI3K and MAPK pathway inhibition by the combination. The agents were also tested alone and in combination (n=3/treatment group) in 27 PDX models of GC in mice (ChemPartner, Shanghai, China). The tumor control rate (tumor stasis or regression) was 11% (3/27) of models treated with M2698, 15% (4/27) with trastuzumab treatment and 22% (6/27) with the combination. The PDX models are currently being evaluated for HER2 status to determine the correlation with the efficacy of the treatments. Based on the data from the OE-19 model, pERK is a candidate marker of combination efficacy which can be further explored in additional GC models.

#2936

MTORC1/2 inhibition in combination with BCL-2/BCL-xL inhibition in APC and PIK3CA mutant colorectal cancer.

Stephanie L. Fricke, Susan N. Payne, Cheri A. Pasch, Demetra P. Korkos, Gioia Sha, Alex E. Yueh, Christopher Babiarz, Linda Clipson, Kristina A. Matkowskyj, Michael A. Newton, Dustin A. Deming. _University Of Wisconsin, Madison, WI_.

Background: Intrinsic resistance to agents targeting the PI3K/AKT/mTOR pathway has been commonly encountered in clinical trials of patients with PIK3CA mutant colorectal cancer (CRC). Upregulation of antiapoptotic signaling has been proposed as a mechanism of resistance to these therapies, including upregulation of BCL-2 and BCL-xL. To investigate if inhibition of BCL-2 family members would sensitize Pik3ca mutant cancers to MTORC1/2 inhibition, treatment studies were performed with TAK-228 (MTORC1/2 inhibitor), BEZ235 (dual PI3K/mTOR inhibitor), navitoclax (ABT-263; BCL-2, BCL-xL and BCL-w inhibitor) and the combination of navitoclax with either TAK-228 or BEZ235.

Methods: Therapeutic investigations with 200 nM TAK-228 or 200 nM BEZ235 and 250 nM navitoclax were performed in murine CRC spheroids with loss of APC and a constitutively active PI3K. Images were taken both pre- and post-treatment and changes in spheroid diameter were measured. Parallel treatment studies were performed on patient-derived organotypic CRC spheroids. Additionally, treatment studies were performed in vivo using a novel transgenic mouse model carrying Apc and Pik3ca mutations. The mice were treated with the combination of BEZ235 and navitoclax or with a single agent alone for 7 consecutive days.

Results: Treatment of CRC spheroids with TAK-228 resulted in a reduction of sphere size by 16% while control treated spheres increased by 77% of their size at day 0. No response was seen with navitoclax treatment alone. A profound synergistic treatment response was observed with the combination of TAK-228 and navitoclax (reduction of 26%, p<0.001), with most spheroids undergoing complete collapse. A similar treatment response was observed with BEZ235 and navitoclax. Human CRC spheroids treated with TAK-228 and/or navitoclax demonstrated a variable response with 3 of 5 lines having a greater than 15% reduction in sphere size when treated with the combination of TAK-228 and navitoclax (p<0.001). This enhanced treatment response correlated with an increase in apoptosis as measured by cleaved caspase 3. No enhanced activity was observed with the combination of BEZ235 and ABT-199 (selective BCL-2 inhibitor). In transgenic mice with Apc and Pik3ca mutant cancers, a median change in lumen occlusion of -42% was observed with the combination of BEZ235 and navitoclax compared to -15% with BEZ235 alone (one-sided p<0.03, n=34 mice).

Conclusion: Synergistic activity was seen with the combination of TAK-228 or BEZ235 and navitoclax. This combination deserves further study in future clinical trials.

#2937

Crizotinib inhibits growth, migration, and invasion of breast cancer cells in vitro and synergizes with chemotherapeutic agents.

Nehad M. Ayoub, Kamal M. Al-Shami, Mohammad A. Alqudah, Nizar M. Mhaidat. _Jordan Univ. of Science & Technology, Irbid, Jordan_.

MET is a receptor tyrosine kinase known to be associated with cancer development and progression. Dysregulations of MET expression and/or signaling have been reported in breast cancer patients and determined to be associated with inferior outcomes rendering MET an appealing target for therapeutic interventions. Crizotinib is a multi-targeted

small-molecule kinase inhibitor for MET, ALK, and ROS1 kinases. In this study, the anti- proliferative, cytotoxic, anti-migratory, and anti-invasive effects of crizotinib have been

evaluated in breast cancer cells in vitro. Cell viability was assessed by 3-(4,5-dimethyl-2- thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) colorimetric assay. In vitro wound-

healing assay was used to examine the effect of crizotinib on breast cancer cell migration. Anti-invasive effect of crizotinib treatment in MDA-MB-231 breast cancer cells was measured using Trevigen Cultrex® BME Cell Invasion Assay. The expressions of Ki-67, MET, and phospho-MET receptors were characterized using immunofluorescence staining. Results showed that crizotinib has significant anti-proliferative activity on all mammary tumor cells with IC50 values of 5.16, 1.5, and 3.85 μM in MDA-MB-231, MCF- 7, and SK-BR-3 cells, respectively. Crizotinib induced cytotoxic effects in all breast cancer cells examined after acute exposure in culture. Combined treatment of small dose of crizotinib with the chemotherapeutic agent paclitaxel or doxorubicin exhibited a highly synergistic inhibition of growth of MDA-MB-231 and MCF-7 cells with combination index values of less than 1. However, no significant effect was observed for the combined treatment of crizotinib with chemotherapy in SK-BR-3 cells. Treatment with crizotinib demonstrated a remarkable reduction in the total levels of the proliferation marker Ki-67 protein in all tested cell lines. Crizotinib inhibited migration and invasion of MDA-MB-231 cells in a dose-dependent fashion. Crizotinib reduced MET receptor activation in MDA-MB-231 cells when treated at effective concentrations. In conclusion, crizotinib suppressed proliferation, migration, and invasion of breast cancer cells in vitro. The results of this study demonstrated that combined treatment of crizotinib with chemotherapeutic agents resulted in a synergistic growth inhibition of specific breast cancer cell lines. Combination treatment of chemotherapy with novel targeted therapy could be a promising approach to overcome the limitations of conventional chemotherapeutic regimens in breast cancer management.

#2938

In vivo assessment of the combination of the JAK1 selective inhibitor itacitinib with first- and second-generation EGFR inhibitors in models of non-small cell lung cancer.

Matthew C. Stubbs, Xiaoming Wen, Chu-Biao Xue, Taisheng Huang, Wenqing Yao, Brian Metcalf, Reid Huber, Peggy Scherle, Bruce Ruggeri. _Incyte Corp., Wilmington, DE_.

Non-small cell lung cancers (NSCLC) make up the majority of lung cancers, and are predominantly driven by aberrant kinase pathway signaling. Oncogenic mutations leading to activation of the epidermal growth factor receptor (EGFR) have been identified in a substantial fraction of NSCLC patients, leading to EGFR-targeted therapies such as erlotinib that have improved patient outcome. However, inhibition of EGFR consistently leads to drug resistance through multiple pathways, creating a therapeutic need in NSCLC. One particular route to resistance of EGFR inhibitors is activation of pathways that can bypass the need for signaling through the EGFR, such as the JAK/STAT pathway. To explore the impact of JAK/STAT pathway modulation on EGFR inhibitor resistance, combination efficacy studies evaluating the JAK1 selective inhibitor itacitinib with either erlotinib or the EGFR T790M mutant inhibitor osimertinib, were conducted in xenograft models of activated and erlotinib resistant NSCLC. The HCC827 xenograft (EGFR-activating deletion in exon 19) model was very sensitive to both erlotinib and osimertinib, while the NCI-H1975 xenograft (EGFR T790M/L858R) model responded only to osimertinib. Itacitinib was efficacious in the HCC827 model, while only marginal tumor growth inhibition was observed with itacitinib in the NCI-H1975 model despite both models having detectable levels of pSTAT3. The combination of itacitinib with either erlotinib or osimertinib inhibited tumor growth to a greater degree than monotherapies in the HCC827 model. Despite marginal single agent efficacy from itacitinib in the NCI-H1975 model, itacitinib enhanced the efficacy of osimertinib at several dose levels in this model. Importantly, itacitinib and erlotinib administration had synergistic efficacy in this erlotinib-resistant model, indicating that JAK1 specific signaling may be a critical bypass mechanism for resistance to EGFR inhibitors. Downstream of EGFR, both erlotinib and osimertinib inhibited different signaling pathways when combined with itacitinib in the NCI-H1975 model: STAT signaling was regulated by erlotinib, while the AKT/S6 and ERK pathways were regulated by osimertinib. An analysis of possible upstream activators of signaling pathways relevant to NSCLC survival revealed that IL-6, MCP-1 and IL-8 levels were altered in H1975 tumors from mice treated with the combination of itacitinib and osimertinib, and to a lesser extent with the combination of itacitinib and erlotinib. These data demonstrate the potential utility of the JAK1 specific inhibitor itacitinib in EGFR activated NSCLC, or for patients with EGFR mutations who are no longer responsive to a first generation EGFR inhibitor such as erlotinib. The combination of itacitinib and osimertinib is currently in a Phase I/II study (NCT02917993).

#2939

ETC159, a porcupine inhibitor, exhibits synergism with PI3K inhibitors in 3-dimensional cell culture.

Sugunavathi Sepramaniam, Xin Hui Chew, Kao Chin Ngeow, May Ann Lee. _Experimental Therapeutics Centre, Singapore_.

Aberrations in WNT signaling, a pathway regulated by a family of secreted glycoproteins known as WNTs, is implicated as a cause for cancer. Post-translational modification of WNTs by porcupine is critical for their activity and eventual activation of the WNT pathway. ETC-159 is an orally available, potent porcupine inhibitor. Addition of ETC-159 suppresses cancer proliferation in several cell lines by blocking the secretion and activity of all WNTs. Nevertheless, tumor growth is driven by complex molecular interactions that dysregulate several pathways at any one time. Thus, early drug discovery studies have increasingly evolved to target multiple molecules or pathways. As therapeutic approaches using combinations of drugs directed at multiple targets can improve treatment response, in this study we explored the possibility of identifying compounds that are synergistic with ETC-159. Unlike conventional two-dimensional combination drug screening, we used the three-dimensional soft agar colony formation assays for the study. Dose-response curves were first independently established to obtain the EC50 ranges for each drug, before proceeding to combination treatments. Using the Chou-Talalay Drug Combination Software, CompuSyn, we identified several inhibitors, which target the PI3K pathway, to act synergistically with ETC-159 in pancreatic cell lines HPAF-II, ASPC-1 and CFPAC-I. Similar treatments in nonpancreatic cell lines indicated that the observed synergism was not cell-line specific, hence highlighting the applicability of such combination treatments for different cancers.

#2940

Chemosensitization of cholangiocarcinoma by guadecitabine.

Oliver Surriga, Gary K. Schwartz. _Columbia University Medical Center, New York, NY_.

Cholangiocarcinoma (CC) originates from biliary epithelial cells and is one of the most common hepatobiliary cancers second only to hepatocellular carcinoma. CC patients have poor long-term prognosis and have limited treatment options. Currently, a combination of gemcitabine and cisplatin is used for systemic treatment of CC patients. There are several tumor suppressor genes that are epigenetically inactivated in CC including p16INK4a, RASSF1A and SEMA3B. Guadecitabine is a next-generation hypomethylating agent that has never been tested in biliary cancer cells. It is a dinucleotide of decitabine and deoxyguanosine that is resistant to degradation by cytidine deaminase. Subcutaneous guadecitabine results in a longer window of exposure than IV decitabine in the clinic. The purpose of this study is to examine the effect of this agent alone and in combination with gemcitabine or cisplatin. We found that guadecitabine inhibits cell proliferation of CC cell lines, SK-ChA-1 and Mz-ChA-1, in a dose-dependent manner starting at 125 nM with concomitant inhibition of DNMT1 protein expression. Additionally, there is a dose-dependent induction of PARP cleavage and increase in expression of semaphorin 3B which is highly methylated in CC starting at doses of 125 nM. Sequential treatment with 250 nM guadecitabine for 48 hours and 20 nM gemcitabine or 1 µM cisplatin for 24 hours further inhibits cell proliferation of CC cells compared to either single agent treatments. Furthermore, western blot analysis of sequentially treated cell shows that DNMT1 is inhibited more by combining guadecitabine and gemcitabine than with either single agents or by combining guadecitabine with cisplatin. We also noted induction of PARP cleavage and increase in semaphorin 3B expression in the SGI-110 and gemcitabine combination treatment than in any other condition tested. In a xenograft model of SK-ChA-1 cells, sequential treatment with 2 mg/kg guadecitabine s.c. daily for 1 week and 15 mg/kg gemcitabine i.p. twice a week for 2 weeks significantly reduced tumor growth as compared to single agent therapy. In contrast to the other conditions, this effect continued for up to 18 days after the last treatment. In fact, the sequential treatment reduced tumor growth by 78% and 72% compared to single agent treatments of SGI-110 and gemcitabine, respectively. Furthermore the sequential combination also increased mRNA expression of p16INK4a, RASSF1A and SEMA3B compared to other treatments. However, guadecitabine did not sensitize SK-ChA-1 tumors when combined with 2 weeks of daily 2 mg/kg cisplatin s.c. treatments. In conclusion, guadecitabine is a potent demethylating agent for CC cells that can inhibit tumor growth by itself as well as sensitize the tumor to chemotherapy such as gemcitabine through epigenetic priming. This novel combination is a promising therapy for CC and may represent a significant improvement to current treatment strategies.

#2941

APE1/Ref-1 redox signaling regulates HIF1a-mediated CA9 expression in hypoxic pancreatic cancer cells: Combination treatment in patient-derived pancreatic tumor models.

Derek Logsdon,1 Fenil Shah,1 Fabrizio Carta,2 Claudiu Supuran,2 Melissa Fishel,1 Mark R. Kelley1. 1 _Indiana University, Indianapolis, IN;_ 2 _University of Florence, Florence, Italy_.

Pancreatic ductal adenocarcinoma (PDAC) is a deadly disease characterized by aggressive metastasis and therapeutic resistance. Reactive stroma in PDAC tumors leads to fibrosis, inflammation, and hypoxia. Hypoxia signaling creates a more aggressive phenotype with increased potential for metastasis and decreased therapeutic efficacy. Carbonic anhydrase IX (CA9) functions as part of the cellular response to hypoxia by regulating intracellular pH to promote cell survival. Apurinic/Apyrimidinic Endonuclease-1-Reduction/oxidation Effector Factor 1 (APE1/Ref-1) is a multifunctional protein with endonuclease activity in DNA base excision repair and redox signaling activity. This redox activity is responsible for reducing oxidized cysteines on specific transcription factors, including hypoxia inducible factor 1 alpha (HIF1α), enabling them to bind target sequences in DNA. We evaluated the mechanisms underlying PDAC cell responses to hypoxia and APE1/Ref-1 redox signaling control of HIF1α, a critical factor in hypoxia-induced CA9 transcription. We hypothesized that obstructing the HIF-CA9 axis at two points via APE1/Ref-1 inhibition (which results in a decrease in CA9 expression) and direct CA9 inhibition results in enhanced PDAC cell killing under hypoxic conditions. In our studies, HIF1α-mediated induction of CA9 is significantly attenuated following APE1/Ref-1 knock down or redox signaling inhibition in patient-derived PDAC cells and pancreatic cancer-associated fibroblast cells using the APE1/Ref-1 redox signaling inhibitor APX3330 (currently in clinical trials). Additionally, dual-targeting of APE1/Ref-1 redox signaling activity and CA9 activity results in additive-to-synergistic enhancement of acidification and cytotoxicity of PDAC cells under hypoxic conditions as well as decreased tumor growth in an ex vivo 3-dimensional tumor co-culture model. These studies are clinically relevant as we used the CA9 inhibitor SLC-0111 (phase I clinical trial completed), as well as APX3330 (Apexian Pharmaceuticals: IND 125360), for which a phase I clinical trial has opened. Further experiments characterized novel analogs of APX3330: APX2009 and APX2014, which demonstrated up to 50-fold improved potency as measured by pH reduction, cytotoxicity, and inhibition of hypoxia-induced CA9 expression. An SLC-0111 analog, FC12-531A, demonstrated up to 75-fold improved potency as measured by cytotoxicity. In combination, these analogs resulted in synergistic inhibition of 3D tumor spheroid growth at nanomolar-to-low-micromolar concentrations. These results underscore the concept that proper combination therapy has significant clinical utility of blocking APE1/Ref-1 and CA9 function for novel PDAC therapeutic treatment.

#2942

Enhanced cytotoxic activity of BRAF, MEK and PI3K inhibition with combination CDK4/6 inhibition in melanoma cells.

Yanping Zhang, Guangyong Peng, Eddy C. Hsueh. _St. Louis Univ., St. Louis, MO_.

Introduction: We have previously reported that CDK4/6 inhibition enhanced the anti-proliferation and anti-migration effects of BRAF, MEK and PI3K inhibitors in melanoma cells. (Zhang Y et al, AACR 2017) In this study, we investigated the effect of combination CDK4/6 inhibition and BRAF, MEK and PI3K inhibition on induction of cell cycle arrest and apoptosis in melanoma.

Methods: Five human melanoma cell lines were used. CDK4/6 inhibitor (PD0332991; PD), BRAF inhibitor (PLX4032; PLX), MEK inhibitor (AZD6244; AZD), and PI3K/AKT inhibitor (XL765; XL) were used alone or in combination. Cell apoptosis analysis was performed using the FITC Annexin V Apoptosis Detection Kit. Autophagy was evaluated by immunofluorescence using anti-LC3A/B and NECN-1 antibodies. Lysosomal morphology was evaluated using lysosomal associated membrane protein 1-green fluorescent protein fusion construct. Western blotting was performed for apoptosis markers (PARP and caspase-9) and autophagy markers (LC3A/B and Beclin1) using GAPDH as internal control. Data were presented as means ± SD for triplicate experiments. For comparison between groups, the student's t test was used and p< 0.05 was considered to be statically significant.

Results: Apoptosis was evaluated by Annexin V assay. Various levels of apoptosis were observed with single agent PD, PLX, XL and AZD in all 5 melanoma cell lines. Combination of PD enhanced the apoptosis effects of PLX, XL, and AZD in all 5 melanoma cell lines. Increased LC3 and BECN1 detection and increased density of autophagosomes on immunofluorescence was observed after combining CDK4 inhibition with BRAF, MEK, and PI3K/AKT inhibition. Inhibition of PARP and Beclin-1 expression and increase in caspase-9 expression were observed with combination of PD compared with single agent PLX, XL, and AZD.

Conclusion: CDK4 inhibition enhanced anti-melanoma activity of BRAF , PI3K/AKT and MEK inhibitions through increased induction of early autophagy and caspase-mediated apoptosis. This suggests that inhibition of CDK4/6 plus BRAF , MEK and PI3K/AKT inhibition may help understanding the interplay between autophagy and apoptosis and inform the development of future targeted agents.

#2943

In vivo combination of miransertib (ARQ 092) with anti-PD-1 antibody, trametinib, lapatinib, trastuzumab and paclitaxel.

Yi Yu, Terence Hall, Sudharshan Eathiraj, Ron E. Savage, Brian Schwartz. _ArQule, Inc., Burlington, MA_.

Dysregulation of the PI3K-AKT signaling pathway has been implicated as a key driver in cancer initiation and progression. AKT is a serine/threonine kinase and a critical component mediating the PI3K-AKT signaling axis. Although AKT inhibitors have been extensively studied, clinical outcome has not been impressive. Interestingly, it has been shown that PI3K/AKT pathway has been involved in resistance to conventional chemotherapy, and inhibition of AKT enhances targeted therapy and sensitizes radiation therapy. Miransertib is a potent and selective pan-AKT inhibitor and currently in early clinical studies. In this study, we assessed combined effect of Miransertib with immune checkpoint inhibitor, anti-MEK and anti-HER2 agents, and a chemotherapeutic agent in vivo. Miransertib at doses of range from 20mg/kg to 120mg/kg was tested in combination with anti-PD-1 antibody, trametinib, lapatinib, trastuzumab, or paclitaxel. Anti-tumor efficacy was assessed in syngeneic mouse CT-26 colon and 4T1 breast tumor models and xenografts models with endometrial (AN3CA) and breast (HCC1954, KPL-4, ZR-75-1) tumors, two patient-derived (PDX) models of endometrial cancer (with PIK3CAH1047R and R93W+D350G mutations) and in one vemurafenib-resistant melanoma PDX (with BRAFV600E and PIK3CA H1047R mutations). Combinability and efficacy of Miransertib and anti-PD-1 antibody was assessed in syngeneic mouse CT-26 colon tumor model. Miransertib at 60 mg/kg and anti-PD-1 antibody at 10mg/kg were combinable. Combined dosing of Miransertib with Anti-PD-1 antibody exerted superior anti-tumor activity in comparison to the single agents (TGI: 65% for combination, 50% for Miransertib and 55% for anti-PD-1 antibody after dosing for 9 days and more significant difference after 12 day dosing). In 4T1 breast tumor model, combination of Miransertib and anti-PD-1 antibody showed a very modest anti-tumor activity whereas there is no effect as single agents. In one of the endometrial PDX models, the combination of Miransertib with trametinib enhanced anti-tumor activity of each drug alone, reducing tumor growth by 67% compared to single-agent tumor reductions by 43% for either Miransertib or trametinib. In a vemurafenib-resistant melanoma PDX model, tumor growth was reduced by 73% when Miransertib was combined with trametinib, while trametinib alone only reduced tumor growth by 26% for trametinib and by 16% by Miransertib. Miransertib enhanced efficacy of in combination with trastuzumab, lapatinib, or paclitaxel, by reducing tumor growth as much as 92%, 73% and 85% respectively, while single agent comparators reduced tumors by less than 50%. Miransertib is combinable with anti-PD-1 antibody, trametinib, lapatinib, trastuzumab, and paclitaxel and exhibits enhanced anti-tumor activity. These results provide us rationale for the combination study of Miransertib in a clinical setting.

#2944

TGF-β inhibitor galunisertib combined with antiangiogenic therapies showed antitumor effects in vitro and in vivo in hepatocellular carcinoma (HCC).

Annemilaï Tijeras-Raballand,1 Christian Hobeika,2 Matthieu Martinet,1 Lucile Astorgues-Xerri,1 Elise Paven,2 Marie-Aude Le Bitoux,3 Anne Maillard,3 Clarisse Eveno,2 Marc Pocard,2 Philippe Bonnin,2 Alexandre Harari,3 Eric Raymond,4 Sandrine Faivre,5 Armand de Gramont1. 1 _AFR Oncology, Paris, France;_ 2 _Inserm U965- Lariboisière University Hospital, Paris, France;_ 3 _Immune Monitoring Core- CHUV, Lausanne, Switzerland;_ 4 _Paris Saint-Joseph Hospital, Paris, France;_ 5 _Beaujon University Hospital, Clichy, France_.

Introduction: TGF-β pathway, associated with hepatocellular carcinoma (HCC) progression, can be targeted by galunisertib, a selective ATP-mimetic TGF-β receptor (TβR)-I inhibitor in clinical trials for HCC patients. We investigated the antitumor effects of galunisertib (Galu) combined with antiangiogenic compounds (sorafenib or DC101, a fully human monoclonal antibody antagonizing VEGFR2, mimicking ramucirumab) in HCC.

Methods: In vitro, we assessed proliferation (MTT assay), migration (wound healing), and invasion (boyden chambers), in a panel of HCC cell lines. Transgenic mice developing stage-defined HCC were treated from weeks (W)8 to W16 with either vehicle, sorafenib (30mg/kg), Galu (100mg/kg), sorafenib plus Galu, DC101 (40mg/kg, twice weekly, IP), or DC101 plus galunisertib. Tumor growth was evaluated by ultrasound (liver size) and by the number of macronodules at sacrifice. Angiogenesis assessed by Doppler measuring the mean blood flow in the coeliac trunk (TCm) and CD31 staining.

Results: In vitro, we showed an inhibition of TGF-induced proliferation, migration and proliferation by Galu, further potentiated by sorafenib. Sorafenib-tolerant cell line SK-Sora was the most sensitive to Galu. In vivo, at W12 & W16, liver size and tumor macronodules number were significantly lower in all treatment arms compared to placebo. At W16, DC101 showed increased tumor control compared to sorafenib regarding liver weight (3,75g±0,39 vs 6,31 g±0,52, p<0.001) and macronodules number (75,7±12.0 vs 139±13,6, p<0.001). Galu/sorafenib and Galu/DC101 combinations showed increased tumor control at W16 as compared to monotherapies. Interestingly, at W16, Galu/DC101 combination showed greater effect on liver weight (3,09g±0,70 vs 3.75g±0,21, p<0.05, number of macronodules (55.1±14.8 vs 109.4±24.3, p<0.001), and micronodules surface (2.0 mm2±0.43 vs 1.43 mm2±0.33, p<0.05) as compared to Galu/sorafenib combination. Angiogenesis decreased in all treatment arms compared to placebo. At W16, we observed a TCm decrease over 50% in the combination arms compared to 24% with sorafenib, 26% with DC101 and 20% with Galu alone, respectively. These results were confirmed by CD31 staining for assessing number of vessels per field. In addition, Galu/sorafenib combination yielded an increased number of monocytes, a decreased number of neutrophils and Kuppfer cells, along with increased NK cells and decreased NKT cells. Dendritic cells as well as B and T cells population did not show particular variation except for CD4+CD25+ T cells that were strongly decreased in sorafenib-treated mice.

Conclusion: The combination of galunisertib with sorafenib or DC101 showed promising antitumor activities that were associated with decreased angiogenesis, DC101 displaying increased efficacy as compared to sorafenib.

#2945

Arsenic trioxide sensitizes glioma stem cells to brain penetrant PI3K and mTOR inhibitor GDC-0084.

Jianwen Dong,1 Emmanuel Martinez-Ledesma,1 Nghi Nguyen,2 Caroline Carrillo,1 Yuji Piao,1 Verlene Henry,1 Soon Young Park,1 Ningyi Tiao,1 Clifford Stephan,2 Roel Verhaak,3 Erik Sulman,1 Veerakumar Balasubramaniyan,1 John F. de Groot1. 1 _UT MD Anderson Cancer Center, Houston, TX;_ 2 _Texas A &M Health Science Center at Houston, Houston, TX; _3 _The Jackson Laboratory, Houston, TX_.

Glioblastoma is the most aggressive primary malignant brain tumor with few effective therapies. The current study evaluated arsenic trioxide (As2O3, ATO), a small-molecular agent that inhibits tumor growth via promoting promyelocytic leukemia protein (PML) degradation, in combination with multiple PI3K/mTOR inhibitors using high-throughput screening (HTS) to validate if ATO reverses glioblastoma resistance to PI3K/mTOR-targeted therapy. Quantitative single-agent and 2-drug combinations (5 drug doses, maximal concentration of 1 uM of each agent) were evaluated in 20 patient-derived glioma stem-like cells (GSCs). ATO was applied as an "anchor" drug and several mTOR and EGFR inhibitors as "probe" drugs to explore potential combination efficacy. Data from single-agent screening demonstrated that brain penetrant PI3K/mTOR inhibitor GDC-0084 potently inhibited cell viability with an IC50 ranging from 0.12μM to 5.78μM under normoxic conditions. Under hypoxic conditions, 10 of the 16 GSC cell lines remained sensitive, indicating less efficacy of GDC-0084 in the setting of a hypoxic microenvironment. Evaluation of drug combinations identified ATO and GDC-0084 as the most effective combination in vitro. ATO was synergistic with GDC-0084 in several GSCs resistant to GDC-0084 monotherapy. GSC sensitivity to GDC-0084 as single agent and in combination correlated with apoptosis, angiogenesis and PI3K/Akt pathways using gene set enrichment analyses (GSEA). In vitro combination treatment significantly inhibited PML, p-S6, p-AKT and p-mTOR expressions compared to single agent. In an orthotopic mouse model of glioma, targeting PI3K/mTOR with GDC-0084 prolonged the median survival to 81 days compared to 69 days in the control group. The efficacy of combining ATO and GDC-0084 in an orthotopic GSC mouse model is ongoing. Our studies confirm prior work demonstrating the efficacy of combining GDC-0084 with ATO, which now requires clinical validation.

#2946

Role of EphB3 receptor in mediating head and neck tumor growth, cell migration, and response to PI3K inhibitor.

Shilpa Bhatia, Anastacia Griego, Shelby Lennon, Ayman Oweida, Jaspreet Sharma, Christina Rohmer, Nomin Uyanga, Sanjana Bukkapatnam, Benjamin Van Court, David Raben, Christian Young, Lynn Heasley, Sana D. Karam. _University of Colorado Denver, Aurora, CO_.

Introduction: Eph proteins have emerged as critical drivers affecting tumor growth and progression in human malignancies. Dysregulated expression of EphB3, a member of the Eph receptor gene family, has been reported in different cancers. However, the precise role of EphB3 in head and neck squamous cell carcinoma (HNSCC) has not been explored. In the current study, we examined the effect of EphB3 downregulation on HNSCC tumor cell growth, migration, and response to PIK3CA inhibition both in vitro and in an orthotopic model of head and neck cancer.

Materials and methods: Alterations in EPHB3 across cancer types in the TCGA database were accessed via cBioportal. shRNA approach was used to knockdown the levels of EphB3 in human and murine HNSCC cell lines. The effect of EphB3 knockdown on cell viability and cell migration was determined by MTT/Trypan blue assay and by Boyden chamber assay respectively. We also generated a BKM120 resistant HNSCC cell line to elucidate the functional role of EphB3 in mediating sensitivity to PI3K inhibitor. The phospho-EphB3 levels were analyzed by an immunoprecipitation assay. For in vivo testing, we used an orthotopic model of HNSCC. Immunohistochemistry and Western blot analysis was performed on cultured HNSCC cells and on tumor tissues to determine key proteins that are altered in response to EphB3 knockdown in the presence and absence of PI3K inhibition.

Results: Our TCGA data analysis showed that EphB3 is frequently co-amplified with PIK3CA in HNSCC. We therefore hypothesized that EphB3 amplification plays a pro-tumorigenic role in HNSCC and that EphB3 and PIK3CA are co-operating oncogenes that contribute toward its pathogenesis. This hypothesis was not experimentally supported since EphB3 knockdown failed to alter HNSCC tumor cell growth in vitro or in vivo with an orthotopic model. However, responsiveness of EphB3 knockdown tumors to the PI3K inhibitor, BKM120, was significantly decreased in terms of both tumor growth delay and survival. This is associated with an increase in pro-survival proteins, S6 and BcL-XL in the EphB3 shRNA tumors treated with BKM120 compared to controls. We further observed that EphB3 knockdown resulted in increased migration in vitro and increased EMT gene signature in vivo. To explain these results, we examined EphB3 phosphorylation levels in HNSCC at baseline. While total EphB3 levels were high, we found low phospho-EphB3 levels in HNSCCs. Forced EphB3 phosphorylation with an ephrin-B2-Fc fusion protein resulted in decreased HNSCC migration and cell growth and enhanced response to BKM120 in vitro.

Conclusions: Our data collectively indicate that progression of HNSCC selects for low/inhibited EphB3 activity to enhance their survival and migratory abilities and decrease response to PI3K inhibitor. Therefore, strategies focused on activating EphB3 might be helpful to inhibit tumor growth and enhance sensitivity to PI3K inhibitors in HNSCC.

#2947

Apatinib and cytotoxic agents showed synergistic inhibitory effects in gastric cancer cells and fluorescent xenograft model.

Jiuhuan Feng,1 Shukui Qin2. 1 _Traditional Chinese Medical Hospital of Xinjiang Uygur Autonomous Region, Urumchi, China;_ 2 _Cancer Center of BaYi Hospital Affiliated to Nanjing University of Traditional Chinese Medicine, Nanjing, China_.

Background: Fluoropyrimidine and platinum-based combinations are the main treatment option for incurable advanced gastric cancer. While anti-angiogenic agents have been reported to bring survival benefit for gastric cancer patients after standard therapy. Apatinib is a small-molecular angiogenesis inhibitor that highly binds and selectively targets VEGFR-2. It has been approved by CFDA for treating pre-treated metastatic gastric cancer. The present study aimed to explore the anti-tumor effects of apatinib combined with traditional cytotoxic agents such as paclitaxel (TAX), oxaliplatin (L-OHP), 5- fluorouracil (5-FU) in vitro and in vivo.

Methods: Three human gastric adenocarcinoma cell lines (MKN-28, SGC-7901, BGC-823) were cultured for in vitro experiment. Apatinib with different dose (4 μg/mL, 8 μg/mL, and 16 μg/mL) combined with TAX, L-OHP or 5-FU were added to cells. MTT assay was performed to detect cell proliferation. The coefficient of drug interaction (CDI) value was calculated to analyze the synergistic inhibitory effects of drug combination. Tumor cell invasion and migration ability were detected by Transwell assay, and flow cytometry was performed to detect cell apoptosis. The in vivo anti-tumor effects of apatinib combined with cytotoxic agents were investigated in the green fluorescent protein-labeled gastric cancer-bearing mice.

Results: Compared with TAX, L-OHP or 5-FU alone, the combination of apatinib with these agents significantly inhibited cell proliferation in all three cell lines (P < 0.05). Based on the CDI values, the most obvious synergistic effect was obtained in the combination of apatinib with TAX. Additionally, apatinib at 4 μg/mL was selected for further analysis since apatinib at this dose showed more obvious and comprehensive synergistic effect in different combinations. Apatinib combined with TAX, L-OHP or 5-FU also significantly decreased the invasion and migration ability of tumor cells, and induced an obvious increase of cell apoptosis (P < 0.05). For in vivo assay, apatinib alone or cytotoxic agents alone could significantly inhibited tumor growth compared to control, while the combination of apatinib with above cytotoxic agents had more inhibitory effects (P < 0.05). Moreover, based on the changes in green fluorescence signal in mice, the tumor volume in mice treated with apatinib combined with cytotoxic agents, especially with TAX and 5-Fu, was markedly decreased than that in mice treated with cytotoxic agents alone. Furthermore, the combination of apatinib with TAX, L-OHP or 5-FU also significantly decreased the microvessel density when compared to these agents alone (P < 0.05).

Conclusion: Apatinib and cytotoxic agents have synergistic inhibitory effects in gastric cancer. The combination of apatinib with these agents may bring efficacy benefit for advanced gastric cancer patients in clinical practice.

#2948

Synergistic effect of combining Akt inhibitor SC66 with anticancer drugs cisplatin and paclitaxel in chemoresistant ovarian cancer cells.

Yi-Hui Wu, Cheng-Yang Chou. _National Cheng Kung University, Tainan, Taiwan_.

Background and aims Akt plays a central role in regulating cell growth and cell cycle progression and is regarded as a promising therapeutic target. We examined whether Akt inhibition by SC66 is therapeutically efficacious in the treatment of ovarian cancer as a single agent and in combination with chemotherapy drugs.

Methods Using eight human ovarian cancer cell lines, we determined the effect of SC66 by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, western blot, and apoptosis assays. We evaluated the association between phospho-Akt/mTOR, COL11A1 and SC66 sensitivity. We also determined the effect of SC66 on tumor growth using mice inoculated with human ovarian cancer cells.

Results Cell sensitivity to SC66 was inversely correlated with the expressions of phosphorylation of Akt and COL11A1, and cisplatin resistance. SC66 inhibited the phosphorylation of downstream 4E-BP1 and p70S6 kinase. SC66 also attenuated the expression of TWIST1 and Mcl-1, important factors in invasiveness and anti-apoptosis. SC66 sensitizes chemo-resistant cells to cisplatin and paclitaxel treatment, and enhanced apoptosis in the chemo-resistant cells. In addition, SC66 inhibited COL11A1 expression via decreasing the binding activity of c/EBPβ and NF-YA to the COL11A1 promoter, which was associated with chemoresistance and cell invasiveness respectively. In vivo, treatment with SC66 enhanced the therapeutic efficacy of cisplatin and paclitaxel in mouse xenograft.

Conclusions These results indicate the SC66 could have therapeutic efficacy in human ovarian cancers.

#2949

Synergistic effects of tyrosine kinase inhibitors and conventional chemotherapeutics on canine and human osteosarcoma cells.

Ya-Ting Yang, Vilma Yuzbasiyan-Gurkan. _Michigan State Univ., East Lansing, MI_.

Osteosarcoma (OS) is the most common bone tumor in both humans and dogs, and has a nearly ten fold higher incidence in dogs than humans. Despite advances in treatment of other cancers, the overall survival rates for OS have stagnated for the past decades. Therefore, there is a great need to identify novel and effective treatments. In the past few years, several classes of novel small molecules with anti-tumor activities have become available. Among them, we selected tyrosine kinase inhibitors (TKIs) with potential anti-tumor medications to evaluate the effects of treating OS. In our previous studies, two TKIs (sunitinib and sorafenib) showed good growth inhibition effects at low concentrations on three canine OS cell lines (D17, Abrams, and Gracie) and two human OS cell lines (SAOS2 and U2OS) in a series of drug screening assay. In addition to viability, migration, and invasion ability of OS cells were decreased by these two TKIs. Furthermore, we investigated the effect of combing one of the TKIs with current chemotherapeutics for OS. We determined the combination index to assess whether sorafenib and sunitinib have synergistic effects with conventional chemotherapeutics including cisplatin, carboplatin, or doxorubicin. Data from our cell lines point to synergistic effects of sorafenib combined with doxorubicin, but not when combined with cisplatin or carboplatin in both human and canine OS. Combination studies with sunitinib are ongoing. Based on current findings, clinical trials using combination of doxorubicin and sorafenib in proof of concept studies in dogs are warranted. These studies can be carried out relatively quickly in dogs where case load is high, and in turn, provide useful data for initiation of clinical trials in humans.

#2950

**Development of a 4-aminopyrazolo[3,4-** d **]pyrimidine-based dual IGF1R/Src inhibitor as a novel anticancer agent with minimal toxicity.**

Ho-Jin Lee, Hye-Young Min, Phuong Chi Pham, Byungyeob Baek, Byungjin Kim, Yunha Kim, Jeeyeon Lee, Ho-Young Lee. _Seoul National Univ., Seoul, Republic of Korea_.

Both the type I insulin-like growth factor receptor (IGF1R) and Src pathways are associated with the development and progression of numerous types of human cancer, and Src activation confers resistance to anti-IGF1R therapies. Hence, targeting both IGF1R and Src concurrently is one of the main challenges in combating resistance to the currently available anti-IGF1R-based anticancer therapies. However, the enhanced toxicity from this combinatorial treatment could be one of the main hurdle for this strategy, suggesting the necessity of developing a novel strategy for co-targeting IGF1R and Src to meet an urgent clinical need. In the present study, We synthesized a series of 4-aminopyrazolo[3,4-d]pyrimidine-based dual IGF1R/Src inhibitors, selected LL28 as an active compound, and evaluated its potential antitumor effects in vitro and in vivo. LL28 markedly suppressed the activation of IGF1R and Src and significantly inhibited the viability of several NSCLC cell lines in vitro by inducing apoptosis. Administration of mice with LL28 significantly suppressed the growth of H1299 NSCLC xenograft tumors without overt toxicity and substantially reduced the multiplicity, volume, and load of lung tumors in the KrasG12D/+-driven lung tumorigenesis model. These results suggest the potential of LL28 as a novel anticancer drug candidate targeting both IGF1R and Src, providing a new avenue to efficient anticancer therapies. Further investigation is warranted in advanced preclinical and clinical settings.

## BIOINFORMATICS AND SYSTEMS BIOLOGY:

### Systems and Computational Biology: Poster Discussion

#3296

SMMART: Serial measurements of molecular and architectural responses to therapy.

Brett Johnson, Jamie Keck, Max Morris, Kiara Siex, Annette Kolodzie, Swapnil Parmar, Jessica Riesterer, Koei Chin, Summer Gibbs, Laura Heiser, Paul Spellman, Kyle Ellrott, Ozgun Babur, Emek Demir, Adam Margolin, Jeremy Goecks, Lisa Coussens, Raymond Bergan, Joe Gray. _OHSU, Portland, OR_.

SMMART is a precision medicine research program focused on understanding the evolution of actionable biology and mechanisms of resistance in human tumors during therapy. This is accomplished through in depth functional, 'omic and multiscale image analysis of longitudinal samples acquired during treatment. Here we present a case report detailing the insights that can be gained from the comparative analysis of pre- and post-treatment biopsy specimens in a late-stage metastatic breast cancer patient. To understand the molecular evolution of cancer, we interrogated genomics with targeted and whole exome sequencing, transcriptomics with RNA and gene-fusion sequencing, and proteomics with reverse phase protein arrays. To understand cellular organization and architectural changes, we employed multi-scale imaging tools, including scanning electron microscopy (SEM), cyclic immunofluorescence, immune cell profiling with cyclic immunohistochemistry, and traditional pathological assessment. During the course of treatment, we monitored patient response to therapy with clinical imaging, circulating tumor DNA sequencing and cancer protein assessment.

Individual assays revealed key aspects of how this individual's cancer evolved under therapeutic pressure. For example, mutational profiling revealed the patterns of clonal evolution and the acquisition of new genetic driver events. 2D and 3D SEM showed changes in ECM organization, macropinocytosis, mitochondrion size, number and density and number and organization of filopodia-like protrusions. We used a 30-color cyclic immunofluorescence analysis to identify differences in cancer cell proliferation and differentiation state, as well as the composition and organization of infiltrating immune cells. In addition, integrative analyses of multiple data types provided insight into the evolution of actionable biology within this patient's disease. This included changes in the suitability of the patient for immune checkpoint inhibitors as well as specific tyrosine kinase inhibitors. The comprehensive molecular and architectural characterization of an individual patient's cancer at multiple time points provides biologically novel and clinically relevant insight into the ways in which cancers become resistant to treatment.

#3297

A tyrosine kinase interactome reveals network states that guide the use of targeted therapies in cancer.

Swati Kaushik, Gwendolyn Jang, Hsien-Ming Hu, Khyati Shah, Xin Zhao, John Jascur, John Von Dollen, Erik Verschueren, Jeffrey Johnson, Nevan Krogan, Sourav Bandyopadhyay. _University of California, San Francisco, San Francisco, CA_.

Characterization of the genomic landscapes of cancer patients has provided valuable insights into the key oncogenic drivers and revolutionized the concept of precision treatment of patients. However, a key limitation is that targetable alterations are only found in a small fraction of patients. This is due to the fact that the majority of cancer drugs are developed against specific oncogenes. However, oncogenes do not act in isolation but rather function as a part of complex protein interactions that can alter oncogene activity and dependence. We hypothesize that a systems approach to read the cellular activity of oncogenic proteins by mapping interaction network states of cancer cells can aid in patient stratification for targeted therapy. To identify interaction networks centered on the major class of cancer drug targets, we experimentally mapped protein-protein interaction (PPI) networks of all 90 human tyrosine kinases (TK) using proteomics approach of affinity purification and mass spectrometry. This analysis identified 1,458 high confidence interactors of TK in HEK293 cells. Detailed analyses of this interactome revealed the diverse cellular localizations and novel associations of TK with multiple protein complexes, suggesting a broader functional role in cellular signaling than previously appreciated. To map the cellular activity of TK in cancer patients, we developed a novel computational approach to integrate PPI networks with genomic data from cancer patients profiled in TCGA. Application to lung adenocarcinoma samples identified that activity of EGFR interactors could be used to define an EGFR network state that was highly predictive of the presence of EGFR mutation. Intriguingly, our analysis identified that 23% of EGFR wild-type samples were positive for the EGFR network state, suggesting a role for EGFR in lung cancer beyond EGFR mutant cases. Furthermore, this state was highly predictive of erlotinib sensitivity in EGFR wild-type lung PDX and cell lines. We identified that many KRAS and NF1 mutant NSCLC samples were EGFR network state positive and displayed evidence of EGFR activation identified by RPPA. These results indicate that a network state approach can precisely expand the pool of patients that may benefit from EGFR TK inhibitors (TKi). Finally, we tested whether components of TK networks were critical for their function by performing synthetic lethal RNAi screens in cell lines with mutation in key TK and identified many kinase interactors as drug sensitizers. Our results indicate that integration of high-throughput genomic datasets with the PPI networks provides an effective tool to understand complex oncogenic network states of cancer cells and provides a high-resolution readout of tumor cell dependence. This work provides the most complete interaction map for TK to date and is a valuable resource to probe mechanisms of oncogene addiction to improve the utility of TKi in cancer.

#3298

High-content phenotyping of somatic cancer mutations by functional variomics.

Nidhi Sahni, Patrick Kwok-Shing Ng, Kang Jin Jeong, Gordon B. Mills. _UT MD Anderson Cancer Ctr., Houston, TX_.

Cancer genomes are highly complex with numerous somatic mutations identified across patient populations. Previous studies on genomic mutations have shed light on new means for cancer therapeutic interventions. With rapid advances in next-generation sequencing, accumulating genotypic information in the absence of efficient and systematic functional analyses of genomic aberrations will create a bottleneck in understanding genotype-phenotype relationships in cancer. To address these challenges, here we report a systems-level functional variomics approach integrating high-throughput phenotyping with robust computational analyses to investigate mutation-specific effects. This systematic functional platform consists of massively parallel mutagenesis, sensitive survival assays using growth factor-dependent cell models, and functional network perturbation profiling of mutations on signaling effects. We profile several thousands of genomic aberrations, including point mutations, gene fusions and indels, and significantly expand the repertoire of characterized actionable mutations. This study represents a valuable resource and provides insights in prioritizing cancer-causing mutations, and uncovering patient-specific disease mechanisms at a high resolution, a critical step towards personalized precision medicine.

#3299

High-throughput combinatorial CRISPR-Cas9 gene knockout reveals most genetic interactions are context dependent.

John Paul Shen, Dongxin Zhao, Brenton Munson, Amanda Birmingham, Roman Sasik, Ana Bojorquez-Gomez, Katherine Licon, Kristin Klepper, Alex Beckett, Kyle Salinas Sanchez, Prashant Mali, Trey Ideker. _UCSD, San Diego, CA_.

Genetic interactions, in particular negative or "synthetic-lethal" interactions for which simultaneous disruption of two genes causes cell killing, have implications for therapeutic development as has been demonstrated by the clinical success of PARP inhibitors specifically for tumors with loss-of-function mutations in BRCA1/2. However, further applications of synthetic-lethal cancer therapy have been limited by poor understanding of the important genetic interactions in a cancer cell, and how these vary from one cancer type to another or from patient to patient. To enable systematic mapping of these genetic interaction networks, we recently developed a CRISPR-Cas9 screening methodology for knocking out single and pairs of genes in high throughput. Critical to this method is the precise determination of single-gene knockout effects, which is accomplished by serial measurement of the relative changes in gRNAs at days 3, 14, 21 and 28 post-transduction. To robustly quantify gene fitness and genetic interactions we developed a novel computational analysis framework that integrates all samples across the multiple days of the experiment; with said method we achieve Pearson correlation of 0.95 or greater between biologic replicates in the same cell line (p < 1x10-30). Additionally we demonstrate that our analysis method is robust to the compositional effects inherent in a pooled knockout experiment. To facilitate reproducibility of analyses and distribution to the scientific community, the code has been packaged into a modular series of python notebooks freely available on github. Evaluating all pairwise gene knockout combinations among a panel of 73 genes divided between tumor-suppressor genes (TSG) and cancer-relevant drug targets (DT) in a total of 5 cancer cell lines from diverse lineages (HeLa, A549, 293T, U2OS, LN229), we identified 226 synthetic lethal and 14 epistatic interactions at a Z-score cut-off of -3 (FDR ~0.3). Of the synthetic lethal interactions 203 (89.8%) were private to a single cell line, and no interaction was seen in more than 3 of 5 five cell lines. Thus far 10 (out of 16 tested) therapeutically relevant interactions have been replicated in low-throughput assays using either combinatorial drugs or CRISPR knockout of a TSG paired with a drug (71% precision or positive predictive value). The cell line specificity of interactions was also confirmed in low-throughput assays (75% negative predictive value). In summary, we have discovered many therapeutically relevant genetic interactions in cancer and identified the great importance of cellular context on the architecture of the genetic interaction network. Recognizing that there will be great diversity in genetic interaction between different tumors, it will be important to perform future studies across a large number of samples, which is enabled by the high-throughput method we have developed.

#3300

Network analysis of the human protein-protein interactome: Tumorigenic signaling mechanisms.

Diana Murray, Kamrun N. Begum, Andrea Califano, Barry Honig. _Columbia University Medical Center, New York, NY_.

The dysregulated signaling responsible for tumor cell state implementation and maintenance has been shown to be mediated by the concerted action of master regulator (MR) proteins. Highly connected MR protein modules serve as "tumor checkpoints" by translating upstream genetic alterations into aberrant protein activity that drives pathophysiological cellular phenotypes. Direct pathways for information flow among MR proteins, and between individual MR proteins and their upstream modulators and downstream effectors, are largely unknown. The Prediction of Protein-Protein Interactions (PrePPI) database contains 1.4M predictions for ~85% of the human proteome; of these, ~300K are predicted to be physical, i.e. direct one-to-one, interactions.We apply network analysis concepts, as implemented in the R package igraph, to the PrePPI interactome to predict structural mechanisms underlying tumorigenic signal transduction. Shortest path and random walk with restart algorithms elucidate 1) physical interactions between MR proteins, 2) physical interactions that connect MR proteins that do not directly bind to each other, 3) upstream signaling proteins with recurrent and patient-specific mutations that physically interact with MR proteins, and 4) downstream cofactors and transcription factors that may be involved in effecting a dysregulated phenotype.Our analysis predicts that physical interactions fully connect BACH2, BCL6, IRF8, and SPIB, established germinal center MR proteins. 1) BACH2 and BCL6 are predicted to interact directly through their BTB/POZ domains; BTB domains are known to mediate heterodimeric protein-protein interactions. 2) Additional proteins, including IBTK, ANFY1, PAX7, ZEB1, GABPB1/B2, connect SPIB to BACH2 and to BCL6, in some cases through ankyrin repeats, which are structural protein-protein interaction motifs. Several of the PrePPI-predicted proteins, e.g. IBTK, ZEB1, GABP, have previously been implicated in B-cell biology. The possible implications of our analyses for a wide range of MR proteins modules will be presented.The computational prediction of physical protein-protein interactions within the regulatory architecture of a tumor cell is a powerful approach to discovering novel tumorigenic signaling mechanisms, formulating specific experimentally testable hypotheses of function, including compound mechanisms of action, and prioritizing novel drug targets.

#3301

Using a novel single-cell lineage-tracing technique to uncover the mechanisms driving nongenetic cancer relapse.

Yaara Oren,1 Aviv Regev,2 Joan Brugge3. 1 _The Broad Institute and Harvard Medical School, Boston, MA;_ 2 _The Broad Institute, Cambridge, MA;_ 3 _Harvard Medical School, Boston, MA_.

Despite favorable initial response to platinum chemotherapy, the majority of patients with ovarian cancer will develop recurrent disease and succumb to it within 5 years of diagnosis. Initial recurrence is frequently platinum-sensitive and women can respond to multiple lines of platinum-based chemotherapy before eventually developing platinum-refractory disease. The observation that the tumor cells that survive the initial therapy give rise to a chemosensitive progeny is inconsistent with a simple Darwinian model of selection of genetically resistant clones. Tumor dormancy, stochastic cell state shifts and stem cell-like populations are among the mechanisms hypothesized to mediate re-emergence of platinum-sensitive ovarian cancer. However, given the lack of high-throughput methods to simultaneously track cell state and lineage, it is not currently feasible to distinguish the relative contribution of each of these factors. To address this need, we generated a new lentiviral barcode library, termed Watermelon. Unlike existing barcoding libraries that enable the tracking of multiple cancer cells solely at the DNA level, the Watermelon library encodes transcribed barcodes, which allows one to map the lineage to the transcriptional profile of each individual cell. This unique barcoding scheme, which is coupled with a genetic H2B retention system, enables simultaneous tracing of lineage as well as the transcriptional and proliferative states of each cell in the population during drug treatment. We have established and characterized an in vitro platinum-sensitive relapse model and are applying the Watermelon system together with time-lapse imaging to study the mechanisms underlying time-to-relapse variation and cell-fate decisions. By broadening our knowledge of the cellular and molecular pathways that affect noninherited drug resistance, our work will facilitate the future development of novel therapies that delay or even prevent the emergence of platinum-refractory ovarian cancer.

#3302

The molecular landscape of oncogenic signaling pathways in The Cancer Genome Atlas.

Francisco Sanchez-Vega,1 Marco Mina,2 Joshua Armenia,1 Walid K. Chatila,1 Augustin Luna,3 Konnor La,1 Sofia Dimitriadoy,4 David L. Liu,5 Havish S. Kantheti,6 Zachary Heins,1 Angelica Ochoa,1 Benjamin Gross,1 Jianjiong Gao,1 Hongxin Zhang,1 Ritika Kundra,1 Cyriac Kandoth,1 Istemi Bahceci,7 Leonard Dervishi,7 Ugur Dogrusoz,7 Wanding Zhou,8 Hui Shen,8 Peter W. Laird,8 Alice H. Berger,9 Trever G. Bivona,10 Alexander J. Lazar,11 Gary Hammer,12 Thomas Giordano,12 Lawrence Kwong,11 Grant McArthur,13 Chenfei Huang,14 Mitchell J. Frederick,14 Frank McCormick,10 Matthew Meyerson,3 The Cancer Genome Atlas Research Network, Eliezer Van Allen,5 Andrew D. Cherniack,5 Giovanni Ciriello,2 Chris Sander,3 Nikolaus Schultz1. 1 _Memorial Sloan Kettering, New York, NY;_ 2 _University of Lausanne, Lausanne, Switzerland;_ 3 _Dana-Farber Cancer Institute, Boston, MA;_ 4 _Princeton University, Princeton, NJ;_ 5 _Broad Institute of Harvard and MIT, Cambridge, MA;_ 6 _University of Texas at Dallas, Dallas, TX;_ 7 _Bilkent University, Ankara, Turkey;_ 8 _Van Andel Research Institute, Grand Rapids, MI;_ 9 _Fred Hutchinson Cancer Research Center, Seattle, WA;_ 10 _University of California San Francisco, CA;_ 11 _The University of Texas MD Anderson Cancer Center, Houston, TX;_ 12 _University of Michigan, Ann Arbor, MI;_ 13 _Peter MacCallum Cancer Centre, Melbourne, Australia;_ 14 _Baylor College of Medicine, Houston, TX_.

Over the past decade, The Cancer Genome Atlas (TCGA) has profiled more than 11,000 tumors spanning 33 distinct cancer types. The TCGA PanCanAtlas is a collaborative project by the TCGA Research Network that aims to address relevant overarching questions in oncology based on a cross-cancer analysis of the full, uniformly reprocessed TCGA data set. Here, we present results from our analysis of genetic alterations in mitogenic signaling pathways across cancer.

Genetic alterations in signaling pathways that control cell cycle progression, apoptosis, and cell growth are common hallmarks of cancer, but the extent, mechanisms, and co-occurrence of alterations in these pathways differ between individual tumors and tumor types. Using mutations and copy-number changes in 9,125 tumor samples profiled by TCGA, we analyzed the mechanisms and patterns of alterations in 10 canonical pathways: cell cycle, Hippo, Myc, Notch, beta-catenin / WNT, PI-3-Kinase / Akt, receptor-tyrosine kinase / RAS / MAP-kinase signaling, TP53, and TGF-beta signaling, as well as oxidative stress response. For each of these pathways, we propose an expert-curated description (or "template") that includes the relevant (altered) genes and the connections between them, as well as a detailed catalogue of the driver mutations and copy number changes with known oncogenic relevance. We provide a high-level map of pathway alteration frequencies across tissues and relevant cancer subtypes as well as detailed frequencies of alteration at the gene level for each individual pathway. We also investigate relationships of co-occurrence and mutual exclusivity across pathways and evaluate therapeutic implications, including drug combinations. Forty-nine percent of tumors had at least one potentially targetable alteration in the evaluated pathways, and 31% of tumors had multiple targetable alterations, making them candidates for combination therapy.

Our work delineates the full landscape of oncogenic alterations in mitogenic signaling pathways across cancer, and the pathway templates as well as the richly annotated data set that we provide will constitute an invaluable public resource for future use by the cancer genomics and precision oncology communities.

#3303

A comprehensive TCGA Pan-Cancer molecular study of gynecologic and breast cancers.

Ashton Berger,1 Anil Korkut,2 Rupa S. Kanchi,2 TCGA Pan-Gynecologic Group and Research Network, Gordon B. Mills,2 Douglas A. Levine,3 Rehan Akbani2. 1 _Broad Institute, Boston, MA;_ 2 _UT MD Anderson Cancer Ctr., Houston, TX;_ 3 _New York University, New York, NY_.

The Pan-Gynecologic (Pan-Gyn) molecular study analyzed data from 2,579 tumors representing five TCGA tumor-type projects: high-grade serous ovarian cystadenocarcinoma (OV), invasive breast carcinoma (BRCA), uterine corpus endometrial carcinoma (UCEC), cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC), and uterine carcinosarcoma (UCS). Principal aims were to identify shared and unique molecular features, clinically meaningful subtypes, and potential future therapeutic targets. We analyzed data of multiple types, including somatic copy number alterations (SCNAs), mutations, DNA methylation, mRNA expression (from mRNA-seq), microRNA (miRNA) expression (from miRNA-seq), long non-coding RNA (lncRNA) expression (from miRNA-seq), and protein expression (from reverse-phase protein arrays; RPPA).

We found multiple genomic and epigenomic features that were shared across the Pan-Gyn tumor types and distinguished them from other cancer types. Sixty-one somatic copy number alterations (SCNAs) and 46 significantly mutated genes (SMGs) were found in the Pan-Gyn cohort; 11 of the SCNAs and 11 of the SMGs had not been reported in previous TCGA single-disease papers on the same tumor types. Ten predominant mutation signatures were found that correlated with COSMIC signatures, whereas 10% of the samples had no SMGs. Most of the Pan-Gyn tumor types shared similar miRNA profiles. The exception, ovarian cancer, was extremely different from the rest, and, unexpectedly, the miRNA profiles of basal-like breast cancers closely resembled those of cervical cancers. Protein expression analysis (RPPA) revealed some OV and UCEC samples with the "reactive" signature previously identified in BRCA and shown to be prognostically relevant. It also revealed a subtype with low ER and AR expression levels (important markers for hormone therapy) that spanned all 5 tumor types. Novel lncRNA analysis showed several functionally significant ER-regulated lncRNAs and gene/lncRNA interaction networks. Pathway analysis identified subsets of tumor samples that had high levels of leukocyte infiltration, with potential implications for immunotherapy. Using 16 key molecular features across the Pan-Gyn cancers, we identified five novel cross-tumor and prognostic subtypes with possible clinical implications. Finally, we developed a decision tree that can classify patients into the five subtypes based on just 6 of the 16 molecular features. Those 6 features are potentially assessable in clinical laboratories.

## CLINICAL RESEARCH:

### Molecular Predictors of Response, Mediators of Resistance, Mechanisms of Action, Pharmacodynamic Markers, and Novel Disease Subsets

#2951

Gene expression profiling identifies new adult "triple-negative" acute lymphoblastic leukemia (ALL) subgroups.

Anna Ferrari,1 Silvia Vitali,1 Valentina Robustelli,1 Andrea Ghelli Luserna Di Rorà,1 Simona Righi,2 Cristina Papayannidis,1 Giovanni Marconi,1 Enrica Imbrogno,1 Alessandra Santoro,3 J M. Hernández-Rivas,4 Carmen Baldazzi,1 Maria Chiara Abbenante,1 Stefania Paolini,1 Nicoletta Testoni,1 Gastone Castellani,1 Elena Sabattini,2 Michele Cavo,1 Daniel Remondini,1 Giovanni Martinelli5. 1 _Università di Bologna, Bologna, Italy;_ 2 _University of Bologna, Bologna, Italy;_ 3 _Hematology department A.O. Ospedali Riuniti Villa Sofia-Cervello, Palermo, Italy;_ 4 _Fundación de Investigación del Cáncer de la Universidad de Salamanca, Salamanca, Spain;_ 5 _Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, Meldola, Italy_.

Background: Although there has been remarkable progress, there is a need to improve the molecular dissection of subtypes, identifying genetic alterations that predict the risk of treatment failure and developing novel and targeted therapies. B-ALL patients (pts) that do not have the most recurrent adult rearrangements (BCR-ABL1 t(9;22); TCF3-PBX1 t(1;19); MLL-AF4 t(4;11)) are collectively referred to as "triple negative" (Ph-/-/-) ALL.

Aims: Biologic characterization of Ph-/-/- ALL considering CRLF2 overexpression event (that represents near to 57% of B-ALL; Roberts KG, J Clin Oncol 2016), in in order to define and assess biomarkers in this subgroup to test new drugs.

Patients and Methods: Gene Expression Profiling (GEP; HTA 2.0 Affymetrix) was performed on 51 Ph-/-/- ALL, 25 B-ALL Ph+ at different time point of the disease and on 7 mononuclear cell of healthy donors. Data were normalized and analyzed with the Expression Console and the Transcriptome Analysis Console (TAC) Software (Affymetrix). Successively we cluster triple-negative GEP data with our validated pipeline, based in a top ten gene list.

Results: Comparing GEP of Ph-/-/- and Ph+ to donors we found some shared top upreg genes to focus on (e.g., EBF1, CD19, BLNK, PDLIM1, PXDN, NAV1, CTGF, LEF1, CD200, CRLF2). In triple-negative ALL GEP top upreg gene analysis we identify a well-defined 2-clusters-subdivision (Gr1 and Gr2). Furthermore, a third group , in the Gr1, can be identified by the algorithm without ambiguous assignments. The Gr2 is characterized by CTGF, CRLF2 and CD200 overexpression and it represents 11.3% of all B-ALL. Two groups t-test has been performed between Ph+ and the isolated subgroups of Ph- to determine the similarity of these two groups to Ph+. The Gr2 GEP is similar to Ph+ one.

Conclusions: We identified a new signature, related to CRLF2 high expression, to classify Ph-/-/- ALL B-based on 10 genes. Gr2 represents 11.3% of all B-ALL and it is characterized by high expression of three main genes: CRLF2, CTGF and CD200. Gr1 represents 46% of all B-ALL. Gr2 GEP similarity to Ph+ one, suggests that this Gr2 could contain Ph-like pts. This new Ph-/-/- subclassification identifies new potential therapeutic targets to test as a single agent or in combination.

ELN, AIL, AIRC, project Regione-Università 2010-12 (L. Bolondi), FP7 NGS-PTL project, HARMONY project, Fondazione del Monte BO e RA project.

#2952

Targeting CDK9 reactivates epigenetically silenced genes in cancer.

Hanghang Zhang,1 Somnath Pandey,1 Meghan Travers,2 Jittasak Khowsathit,3 George Morton,4 Hongxing Sum,1 Carlos A. Barrero,4 Carmen Merali,4 Yasuyuki Okamoto,1 Takahiro Sato,1 Judit Garriga,1 Natarajan V. Bhanu,5 Johayra Simithy,5 Bela Patel,1 Jozef Madzo,1 Noël Raynal,6 Benjamin A. Garcia,5 Marlene A. Jacobson,4 Salim Merali,4 Yi Zhang,1 Wayne Childers,4 Magid Abou-Gharbia,4 John Karanicolas,3 Stephen B. Baylin,2 Cynthia A. Zahnow,2 Jaroslav Jelinek,1 Xavier Graña,1 Jean-Pierre J. Issa1. 1 _Fels Institute for Cancer Research, Temple University School of Medicine, Philadelphia, PA;_ 2 _The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore, MD;_ 3 _Molecular Therapeutics Program, Fox Chase Cancer Center, Philadelphia, PA;_ 4 _Moulder Center for Drug Discovery Research, Temple University School of Pharmacy, Philadelphia, PA;_ 5 _Epigenetics Institute, Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA;_ 6 _Département de pharmacologie et physiologie, Université de Montréal, Quebec, Canada_.

In cancer, the epigenome is aberrantly reprogrammed leading to a wide range of heritable changes in gene expression, such as silencing of tumor suppressor genes (TSG). Altered epigenetic marks in cancer involve DNA methylation and histone post-translational modifications, and these come about as a result of aging and acquisition of genetic and epigenetic changes in readers/writers/editors of the epigenome. Given the reversible nature of epigenetic modifications, one goal of epigenetic therapy of cancer is to induce TSG reactivation, leading to cancer cell differentiation and cancer cell death.

To identify novel targets that can reactivate epigenetically silenced genes, we developed a phenotypic-based system, YB5. YB5 is a colon cancer cell line generated by stably transfecting SW48 cells with a vector containing GFP driven by a methylated and silenced CMV promoter. GFP re-expression can be achieved by known epigenetic drugs that lead to demethylation or induce active chromatin marks in the CMV promoter.

We screened a natural compound library for GFP activation in YB5 and identified a novel drug class that shares an aminothiazole core structure, and has epigenetic effects that are equivalent to DNA methyltransferase inhibitor (DNMTi). Target deconvolution identified CDK9 as the target of these drugs, which reactivate gene expression without affecting DNA methylation. It is well established that CDK9, the catalytic subunit of p-TEFb, is a transcriptional activator. CDK9 in complex with its regulatory subunit, Cyclin T1 or T2, is recruited by multiple mechanisms to promote RNAPII promoter-proximal pause release by phosphorylating negative elongation factors (DSIF and NELF). In addition, phosphorylation of the C-terminal domain (CTD) of RNAPII on Serine-2 allows recruitment of RNA processing factors, which work on the nascent RNA as it emerges from RNAPII. Our new data show that long-term CDK9 inhibition can reactivate epigenetically silenced genes with minimal downregulation effects, effects which are the opposite of the canonical role of CDK9-mediated transcriptional elongation. Mechanistically, we showed that CDK9 inhibition dephosphorylates the SWI/SNF protein SMARCA4 and represses HP1α expression, both of which contribute to gene reactivation. Based on gene activation, we developed the highly selective and potent CDK9 inhibitor MC180295 (IC50 =5nM) that has broad anti-cancer activity in-vitro and is effective in in-vivo cancer models. Additionally, CDK9 inhibition sensitizes with the immune checkpoint inhibitor α-PD-1 in vivo, making it an excellent target for epigenetic therapy of cancer. This is the first study that links CDK9 to maintaining gene silencing at epigenetically repressed loci in mammals. Excitingly, this is also the first example of kinase inhibitors as potential drugs that reverse epigenetic silencing.

#2953

Overall survival results of the single-institution molecular screening MOSCATO trial in hard-to-treat advanced cancers.

Yolla El Dakdouki, Adrien Allorant, Loic Verlingue, Ludovic Lacroix, Etienne Rouleau, Nathalie Auger, Stefan Michiels, Thierry Debaere, Fabrice Andre, Gilles Vassal, Cecile Jovelet, Lambros Tselikas, Jean-Charles Soria, Antoine Hollebecque, Christophe Massard. _Gustave Roussy Cancer Campus, Paris, France_.

Background: MOSCATO 01 (NCT01566019) is a non-randomized single institution prospective clinical trial evaluating clinical benefit of integrating comprehensive molecular profiling into clinical practice to guide treatment in advanced cancers relapsing after standard therapies. The primary outcome of this trial has been previously published. We hereby evaluate the effect of having an actionable molecular alteration on overall survival (OS) in the adult patients (pts) included in MOSCATO 01 trial, and, among those with an actionable alteration, the effect of receiving a targeted therapy matched to a molecular alteration on OS. Methods: Survival data of pts who gave written consent to be included in MOSCATO 01 trial were used for this analysis. OS was defined from date of inclusion to death, or last visit at the institution. The Inverse Probability-of-Censoring Weighting method with a propensity score was used to estimate causal effects in a COX MODEL. For actionable alteration effect, propensity score included age, sex, Royal Marsden Hospital prognostic score. For targeted therapy effect, the same variables were used for propensity score and an additional landmark time was set at 2 months to take lead guarantee-time bias into account. Actionable molecular alterations were defined by a weekly molecular tutor board. Results: Between December 2011 and March 2016, among 1035 pts included in MOSCATO trial, 948 had a tumor biopsy. Of 843 pts where molecular portrait was successful, an actionable molecular alteration has been identified in 411 pts. Of the 411 pts with actionable molecular alteration, 199 received targeted therapy matched to the identified genomic alteration. At the cut-off date of June 2017, survival data was obtained for a total of 906 pts (396 out of 411 pts with an actionable alteration and 493 out of 624 pts without). After median follow-up of 9.2 months (4.7-20.4 months), 749 deaths were observed and median OS was 7.4 months. In the MOSCATO 01 trial, pts with an actionable molecular alteration had a significantly worse OS than pts without (HR=1.51 [1.38,1.64], p<0.001). Among pts with an actionable alteration, pts who received a matched targeted therapy did not have a significantly improved OS (HR=0.81 [0.65,1.01], p=0.06). Conclusion: In MOSCATO 01 trial, pts with an actionable target had significantly worse OS, while pts with a targeted treatment did not have a significantly better OS than those without. Randomized controlled trials are needed to evaluate whether customizing targeted therapy to pts with advanced tumors harboring actionable molecular alterations could improve OS.

#2954

Immunomodulator maintenance post autologous stem cell transplant predicts better outcome in multiple myeloma patients with clonal hematopoiesis of indeterminate potential.

Tarek H. Mouhieddine, Jihye Park, Robert Redd, Christopher J. Gibson, Salomon Manier, Amin Nassar, Kalvis Hornburg, Marzia Capelletti, Daisy Huynh, Romanos Sklavenitits Pistofidis, Mark W. Bustoros, Saud H. AlDubayan, Brendan Reardon, Cody J. Boehner, Henry Dumke, Chia-Jen Lui, Darlys Schott, Eliezer M. Van Allen, Robert L. Schlossman, Nikhil C. Munshi, Kenneth C. Anderson, David P. Steensma, Jacob P. Laubach, Paul G. Richardson, Jerome Ritz, Benjamin L. Ebert, Robert J. Soiffer, Donna Neuberg, Irene M. Ghobrial. _Dana-Farber Cancer Institute, Boston, MA_.

Introduction: Multiple Myeloma (MM) is a clonal plasma cell malignancy, accounting for 10% of all hematological malignancies. Genetic analyses of large populations revealed that blood-specific somatic mutations in hematopoietic stem cells (HSCs) are commonly acquired during aging, a new entity labeled: clonal hematopoiesis of indeterminate potential (CHIP). We sought to determine the role of CHIP on survival of MM patients, specifically those receiving immunomodulators (IMiDs) maintenance (Lenalidomide or Thalidomide) post autologous stem cell transplant (ASCT).

Methods: We tested cryopreserved HSCs of 629 MM patients who underwent ASCT between 2003 and 2011 at the Dana-Farber Cancer Institute. We used a target bait panel of 224 genes and performed deep-targeted sequencing at 978x coverage and ultra-low pass whole-genome sequencing at 0.1x to account for tumor contamination. Sequencing data was analyzed using ichorCNA, MuTect, and Strelka and mutation annotations were based on reported mutations in the literature and databases (ClinVar, COSMIC, cBioPortal, TCGA, and ExAC).

Results: Our cohort had a median age of 58 years [24-83] at time of ASCT and median follow up post ASCT of 8 years [0.1-14.5]. 24% of patients had CHIP at time of ASCT, which is statistically similar to the 30% reported in non-Hodgkin's lymphoma (NHL), (Gibson et. al, JCO, 2017). The most commonly detected mutated genes were DNMT3A, TET2, TP53 and ASXL1. Acquiring mutations positively correlated with age (p=0.004). In contrast to NHL, PPM1D was not significantly mutated in MM (40% vs. 3.3%). 27 patients (4.3%) developed a second hematological malignancy at median of 4 years [1-10] post ASCT, of which 10 had CHIP. 22% received at least 3 years [0.06-12.8] of IMiD maintenance. Among those who did not receive IMiD maintenance, CHIP was associated with worse progression free survival (PFS) (p=0.047) where PFS at 3 years post ASCT was 31% (95%CI: 25-38) for those without CHIP vs. 15% with CHIP (95%CI: 7-25). In patients with IMiD maintenance, CHIP had no effect on PFS or overall survival (OS) (p=0.9). In patients with CHIP, receiving IMiD was associated with a better OS and PFS below the age of 58 and better PFS only in those above 58. In the overall cohort, CHIP was not associated with more adverse outcomes, which could be attributed to low OS and PFS in MM or the use of IMiD in 56% of this cohort. IMiD maintenance was associated with better OS (p<0.001) and PFS (p<0.001), consistent with prior studies, even in the presence of CHIP mutations.

Conclusion: CHIP is a common entity among MM patients that predicts a worse PFS in those who do not receive IMiD maintenance therapy post ASCT. The use of IMiDs abrogated the deleterious effect imposed by CHIP in this cohort. Larger cohorts with longer follow up are needed, especially in the era of novel agents and long-term use of Lenalidomide maintenance.

#2955

A radiomic-based MRI phenotype is uniquely associated with hypermutated genotype in gliomas.

Islam Hassan,1 Aikaterini Kotrotsou,1 Carlos Kamiya Matsuoka,1 Kristin D. Alfaro-Munoz,1 Nabil Elshafeey,1 Nancy El Shafei,2 Pascal O. Zinn,1 John F. de Groot,1 Rivka R. Colen1. 1 _UT MD Anderson Cancer Ctr., Houston, TX;_ 2 _National Research Centre, Cairo, Egypt_.

INTRODUCTION Hypermutation is the excessive accumulation of DNA mutation in cancer cells. This specific hypermutated genotype has been reported in low and high grade gliomas, specifically post-temozolomide treatment and is associated with treatment-resistance. Herein, we sought to identify an imaging-based signature for hypermutated gliomas using a radiomics-based approach.

MATERIALS AND METHODS In this IRB-approved retrospective study, we analyzed a total of 101 patients with primary gliomas from the University of Texas MD Anderson Cancer Center. Next generation sequencing (NGS) platforms (T200 and Foundation 1) were used to determine the Mutation burden status in post-biopsy (stereotactic/excisional). Patients were dichotomized based on their mutation burden; 77 hypomutated (<30 mutations) and 24 hypermutated (>=30 mutations or <30 with MMR gene or POLE/POLD gene mutations). Radiomic analysis was performed on the conventional MR images (FLAIR and T1 post-contrast) obtained prior to tumor tissue surgical sampling; and a total of 2480 rotation-invariant radiomic features were extracted using: (i) the first-order histogram and (ii) grey level co-occurrence matrix. The Maximum Relevance Minimum Redundancy technique was used to select the most relevant radiomic features. ROC analysis and leave-one-out cross-validation (LOOCV) were used to assess the performance of the Support Vector Machine (SVM) classifier as and AUC, Sensitivity, Specificity, and p-value were obtained.

RESULTS We found 100 radiomic features that can discriminate between hypermutated versus hypomutated gliomas, AUC 96.3% (CI: 90.2%-98.9%), Sensitivity 100%, Specificity 95%, p-value=3.769e-6.

CONCLUSION Hypermutated gliomas has a unique radiomic quantitative signature that can be used to predict mutation burden regardless of tumor grade or histopathology.

#2956

**Mechanisms of resistance for osimertinib for patients with** EGFR **-mutant lung cancer: MD Anderson Cancer Center single institution experience with osimertinib resistance.**

Xiuning Le, Marcelo Vailati Negrao, Monique Nilsson, Jacqulyne Robichaux, Emily Roarty, Waree Rinsurongkawong, Bonnie Glisson, Jianjun Zhang, John V. Heymach. _MD Anderson Cancer Center, Houston, TX_.

PURPOSE: Osimertinib was initially approved for treating epidermal growth factor receptor (EGFR) T790M positive non-small cell lung cancer (NSCLC), and recently is moving forward as the first-line treatment for EGFR sensitizing mutation positive NSCLC. However, the types and frequencies of resistance mechanisms to osimertinib have been incompletely described, and currently there are no guidelines for managing these patients. We evaluated clinical and genetic characteristics of patients who received osimertinib for EGFR-mutant NSCLC. We also provide treatment experience after progression on osimertinib.

PATIENTS AND METHODS: Using the MD Anderson Lung Cancer Moon Shot GEMINI database, we identified patients treated with osimertinib and performed clinical outcome analysis. Molecular profiling analysis was performed at the time of progression when available.

RESULTS: Eighty two patients were identified. Median PFS on osimertinib was 8.8 months (95% CI, 6.7 to 10.9). Fifty three of the 82 patients had disease progression on osimertinib. Among them, 30 had molecular profiling upon progression. Other than two cases with germline T790M, 11 cases preserved and 17 cases lost T790M mutation. In T790M preserved cases, tertiary mutations of C797S (5 cases) and L792H (2 cases) in EGFR gene, as well as MET amplification (4 cases), were the most common mechanisms of resistance. In T790M lost cases, 12 cases were without known resistance mechanisms. Upon progression, osimertinib was continued in 34 of 51 patients and 17 (50%) of them received local consolidation radiation. The osimertinib continued patients had longer PFS2 compared to the patients who discontinued on osimertinib upon first progression.

CONCLUSION: T790M loss is common (61%) in osimertinib resistance cases. In T790M-preserved cases, tertiary mutation within EGFR was the most common mechanism of resistance. MET amplification was observed in both T790M preserved and loss cases. In T790M loss cases, the mechanisms of resistance were mostly EGFR independent, highlighting the importance of understanding T790M-negative resistance mechanisms in the osimertinib era.

#2957

Sequential transcriptomic and phosphorylation landscape of acute myelogenous leukemia (AML) on the single-cell level.

Victoria E. Wang,1 Graham Heimberg,1 Gregory K. Behbehani,2 Chun Ye,1 Charalambos Babis Andreadis1. 1 _UCSF, San Francisco, CA;_ 2 _Ohio State University, Columbus, OH_.

Background: Patients with AML refractory to induction therapy or relapse within one year have poor outcomes. Elevated serum hepatocyte growth factor (HGF) level is an adverse prognostic factor in AML. Preclinical models have shown that myeloid blasts produce HGF in an autocrine fashion and pharmacologic blockade of the HGF/c-Met axis sensitizes blasts to cell death. We initiated a phase I study to assess the safety and tolerability of the HGF antibody ficlatuzumab (Fi) combined with high-dose cytarabine (Cy) in patients with AML who are refractory to 7+3 or have relapsed within one year of induction. Fi is given in escalated dosing of 10, 15, or 20 mg/kg for 4 doses every 2 weeks, starting on day 0, and Cy at a fixed dose of 2 g/m2 on days 2-7, using a 3x3 design. PBMCs, BM and serum are collected at defined times. Preliminary results demonstrating the safety of this combination with a promising response rate of 44% in nine evaluable patients were previously presented. Here we report the correlatives using single-cell RNA sequencing (scRNAseq) and mass cytometry (Cy-TOF) on PBMC specimens collected longitudinally during treatment for twelve patients.

Methods: scRNAseq was conducted using the 10x Genomics Chromium platform. Twelve genetically distinct samples were multiplexed per run and the resulting sequencing data were de-convoluted using individual SNP information from the OmniExpressExome54 Array and the novel DEMUXLET algorithm. The Cy-TOF antibody panel included markers for identification of myeloid blasts, T and B cells as well as intracellular phospho-signaling pathways downstream of the HGF/c-MET axis.

Results: Sixty samples from twelve patients corresponding to different treatment time points were analyzed using scRNAseq and thirty samples were run using Cy-TOF. On average, each scRNA run captured 17-23K cells and 19-24K reads per cell, corresponding to approximately 1,000 unique gene trasncripts/cell. Cy-TOF identified surface phospho-MET activation and downstream pathways.

Conclusions: scRNAseq combined with Cy-TOF identified changes in cellular populations corresponding to treatment. Effector signaling pathways were blunted with HGF blockade, suggesting on-target effect of the antibody. Each population also exhibited distinct transcription and phosphorylation profile in response to the drugs. Dose expansion is ongoing and future studies will apply these methods to analyze the bone marrow specimens to assess how the microenvironment evolves during the treatment course.

Clinical trial information: NCT02109627

### Use of Liquid Biopsies in Clinical Trials

#2958

Development of a superenhancer element-associated transcription factor signature in primary tumors for the identification of liver metastasis in colorectal cancer patients.

Roshni Roy,1 Jesper Grud Madsen,2 Yasuhide Yamada,3 Yoshinaga Okugawa,4 Yuji Toiyama,4 Susanne Mandrup,2 Ajay Goel1. 1 _Baylor Scott and White Research Institute, Dallas, TX;_ 2 _University of Southern Denmark, Denmark;_ 3 _National Cancer Center Hospital, Tokyo, Japan;_ 4 _Mie University, Mie, Japan_.

Background: More than 50% of patients with colorectal cancer (CRC) develop metastatic disease to liver, which ultimately results in death in more than two thirds of the cases. Hence, earlier detection, together with the identification of patients at high-risk for tumor recurrence could significantly improve patient outcomes. Current paradigm asserts that metastasis must at least in part be a reflection of mutational and transcriptional landscape present in the majority of cells which constitute the primary tumor mass. Among these genomic determinants, transcription factors (TFs) are of particular interest, as they regulate the expression of both protein coding and noncoding RNAs. Keeping this in mind, we undertook an effort to develop a TF-based gene expression signature in primary CRC tissues that can help identify patients with a metastatic disease.

Method: A comprehensive list of TFs was established from key relevant databases including TFdb, FANTOM and TFCAT. An in-silico discovery for differentially expressed TFs was performed in multiple, publicly available datasets. The final TF signature was examined in two independent cohorts of CRC patients (N=104 and 151 respectively). Statistical analyses included area under the receiver operating curve (AUROC) estimates, univariate and multivariate logistic regression,

and Kaplan Meier survival outcomes. Lastly, we analyzed associations between our selected TF and their binding to various superenhancer elements by analyzing a publicly available H3K27ac dataset on 21 colorectal cell lines.

Result: We developed a panel of 14 differentially expressed TFs by analyzing primary CRCs with and without liver metastasis. The AUROC values for liver metastasis-positive cases were 0.79 and 0.86 in the training and validation cohorts, respectively. The AUROC values improved to 0.93 in both cohorts on combining the signature with lymph node metastasis (LNM) status and the CEA levels. This signature was also associated with worse overall survival in the training (H.R (95%C.I) = 4.8(2.1-11.2), p<0.001) and validation cohorts (H.R (95%C.I) 8.9(3.1-25.6), p<0.001). In-silico analyses for superenhancer binding motifs for these TFs indicated that there exists a co-occurrence between many of the motif pairs, and 7 motifs (EHF, KLF7, MECP2, PURA, RARB, TCF4 and ZNF354C) were significantly more enriched within the superenhancers.

Conclusions: We present a TF gene expression signature that can identify liver metastasis and can predict poor survival by analyzing primary tumor tissues from CRC patients. Transcriptional cooperativity between 7 of these 14 TFs along with their enriched binding to key superenhancer elements underscores their functional role in metastasis.

#2959

Analysis of circulating tumor DNA reveals genomic alterations in metastatic prostate cancer patients treated with abiraterone acetate plus prednisone or enzalutamide.

Jelena Belic,1 Ricarda Graf,1 Thomas Bauernhofer,1 Yauheniya Cherkas,2 Ulz Peter,1 Julie Waldispuehl-Geigl,1 Samantha Perakis,1 Michael Gormley,2 Jaymala Patel,2 Weimin Li,2 Jochen B. Geigl,1 Denis Smirnov,2 Ellen Heitzer,1 Mitchell Gross,3 Michael R. Speicher1. 1 _Medical University of Graz, Graz, Austria;_ 2 _Pharmaceutical Companies of Johnson & Johnson, Spring House, PA; _3 _University of Southern California, Graz, CA_.

Background: Recent progress in the analysis of cell-free circulating tumor DNA (ctDNA) now allows for the monitoring of tumor genomes by noninvasive means. The introduction of abiraterone and enzalutamide for the treatment of metastatic castration-resistant prostate cancer (mCRPC) patients has led to a survival benefit. We analyzed genomic alterations as potential causes of disease progression using ctDNA in patients treated with abiraterone and enzalutamide. Furthermore, we investigated the correlation between cancer-associated clinical markers and the ctDNA detection in mCRPC patients.

Patients and Methods: In order to identify variables associated with ctDNA detection, we analyzed two cohorts including 94 plasma samples from 25 treatment courses (23 patients) and 334 plasma samples from 125 patients, respectively. We performed low-pass whole-genome sequencing (plasma-Seq) for genome-wide profiling of somatic copy number alterations (SCNAs) and targeted resequencing of 31 prostate cancer-associated genes.

Results: Applying plasma-Seq with targeted AR analyses we were able to identify significant genomic alterations in 16 of 25 (64%) cases in the first cohort. Our results indicated that AR amplification alone is not predictive for abiraterone and enzalutamide treatment outcome. In patients treated with abiraterone, low ctDNA levels at baseline were a significant determinant of progression-free survival. Next, we evaluated the determinants of ctDNA detection in an additional independent cohort of metastatic CRPC patients. Analysis of a total of 148 patients and 428 plasma samples from both cohorts revealed that increased levels of lactate dehydrogenase (LDH) highly correlated with the detection of ctDNA and could be used as the potential independent predictor of ctDNA release.

Conclusion: Our results showed that AR amplification alone is an insufficient marker for the prediction of therapy response in patients treated with abiraterone and enzalutamide. Analyses of ctDNA in two independent cohorts have provided insights into unique patterns of response and the emergence of resistance to abiraterone and enzalutamide. Noninvasive analysis of ctDNA plays an important role in treatment monitoring and the prediction of therapy outcome in mCRPC patients.

#2960

Concordance of genomic single-nucleotide variations (SNV) by next-generation sequencing (NGS) in paired tumor tissue and plasma in colorectal cancer (CRC).

Preeti Lal,1 John Lee,1 Hans-Peter Adams,1 Lijing Yao,1 Frederike Fuhlbrück1,1 Stephanie Yaung,1 Sylvie McNamara,1 Corinna Wöstmann,1 Sebastian Fröhler,1 LiTai Fang,1 Rainer Kube,2 Frank Marusch,3 Michael Heise,4 Thomas Steinmüller,5 Matthias Pross,5 René Mantke,6 John Palma,1 André Rosenthal11. 1 _Roche Sequencing Solutions, CA;_ 2 _Carl-Thiem-Klinikum, Germany;_ 3 _Ernst-von-Bergmann Klinikum, Germany;_ 4 _Sana Klinikum, Germany;_ 5 _DRK Klinikum, Germany;_ 6 _Klinikum Brandenburg, Germany_.

Introduction: Determining mutations in plasma (cfDNA) is a noninvasive method of profiling tumor genomic alterations. In this study, concordance of SNVs using NGS between matched plasma and formalin-fixed, paraffin-embedded (FFPE) tissue samples was investigated across all four stages of CRC. We focused primarily on stages I, II and III to determine the feasibility of using plasma for minimal residual disease (MRD) surveillance after surgical resection and for early detection of CRC.

Methods: From a CRC biobank of >9,000 subjects, 299 subjects (47 stage I, 131 stage II, 102 stage III, and 19 stage IV) were selected for whom paired treatment-naïve FFPE tumor tissue and 4mL of plasma was available. MSI was determined by PCR (Promega). DNA was isolated from plasma and FFPE using cobas® extraction kits. Sequencing was performed using the AVENIO ctDNA Surveillance Kit (Research Use Only) and AVENIO FFPET Surveillance kit (under development). The AVENIO kit detects four mutation classes. In this analysis only SNVs data is presented. Three different filtering methods for concordance were used for this analysis: (i) by adaptive call, (ii) by duplex support, and (iii) by single read. Concordance is defined as at least the presence of one identical SNV between matched tissue and plasma sample.

Results: The most frequently mutated COSMIC genes in the tumor tissues of this cohort are TP53, APC, KRAS, PIK3CA, and BRAF with frequencies of 59%, 53%, 20%, 19% and 17% respectively. Using the most sensitive method ("by single read"), overall concordance in this cohort was 79.26%. The concordance for various stages (Stage I to Stage IV) ranges from 50-100%. If a more stringent criterion of adaptive call is used, then the concordance ranges from 21-89%. The mean number of somatic variants for MSI-low tumors (n=247) was 5.2 compared to 15.5 in MSI-high tumors (n=52) p-value 3.3e-14. Multivariate analysis showed that in addition to the clinical stage, tumor size was the most important clinical variable associated with concordance of SNVs between the matched tissue and plasma.

Conclusions: This study demonstrated an overall concordance of 79.26%. Concordance was associated with the disease stage and most significantly with tumor size and T stage. High concordance for subjects with localized disease (stages IB; IIA-IIC, and IIIA-IIIC) suggests that cfDNA sequencing can be potentially used for surveillance monitoring of patients after surgery, in particular for the detection of MRD. High tissue-plasma concordance for localized CRC may allow the use of cfDNA sequencing for early detection. Prospective clinical studies are required for validation of this application.

#2961

First metastasis NGS analysis reveals new targets for personalized treatment in advanced breast cancer.

celine callens,1 anais boulai,1 virginie bernard,1 sylvain baulande,1 patricia legoix-ne,1 anne salomon,1 walid chemlali,1 vanessa benhamo,1 etienne brain,1 mario campone,2 thomas bachelot,3 sylvie giachetti,4 jacques bonneterre,5 francois-clement bidard,1 vincent servois,1 aurelie comte,1 frederique berger,1 ivan bieche,1 brigitte sigal,1 paul cottu1. 1 _institut curie, paris, France;_ 2 _ico, nantes, France;_ 3 _centre l berard, lyon, France;_ 4 _hôpital saint louis, paris, France;_ 5 _centre o lambret, paris, France_.

Background Genomic profile of breast cancer (BC) metastases (M) may differ from that of the primary tumor (PT). Based on a multicenter prospective study (ESOPE, NCT 01956552) including 130 patients with biopsies of the first metastatic site, we report here the results of a matched, comparative PT/M targeted next generation sequencing (NGS) mutational analysis.

Methods 117 paired PT/M FFPE samples obtained before any treatment for either early or advanced BC were available for analysis. Targeted sequencing was done using Illumina Hiseq2500 technology with a custom made 91 BC associated genes panel. Sequence data were aligned to the human reference genome (hg19) using Bowtie2 algorithm. High mapping quality and at least 70% coverage were achieved for 69 paired PT/M. Median depth was 723 and 98 % of targets achieved 100X depth. SNVs and indels were called using GATK. We defined the pathogenicity of variants using several public databases. We only retained confirmed pathogenic variant and variant of unknown pathogenicity observed at a frequency lower than 0,1% in population and reported at least once in cbioportal/tumorportal.

Results Patient characteristics are representative of patients with first line metastatic BC (Comte et al, ASCO 2016#550). In 69 paired PT/M we retained 256 somatic variants (188 shared, 28 only in PT and 40 only in M) distributed in 47 genes and including 62 indels and 194 SNV. There were 128 distinct variants (33 indels and 95 SNV) classified pathogenic for 98 and of unknown pathogenicity for 30 of them. TP53, PIK3CA, CDH1, GATA3, MAP3K1, BRCA2, ERBB2, ESR1, NF1, PTEN, ARID1A, BRCA1 and EGFR are genes with the more frequently PT/M shared variants (mutated at least in 2 patients). Private mutations are seen mostly in NF1, PIK3CA, MAP3K1, NCOR1, TSC1 genes for PT and in TP53, LAMA2, PIK3CA, ESR1, NF1, MAP2K4 genes for M. ESR1 D538G mutation was detected for 3 PT/M pairs and 1 M. Beyond highly shared PIK3CA and TP53 variants, overall crude PT/M discordance rate was 42%. PT/M shared variants could be considered for targeted treatment in 48% patients. For 13% patients, sequencing M revealed at least one additional druggable target in PIK3CA, ALK, PTEN, SF3B1, NOTCH4, KDR, TSC2, POLE, ESR1 and CDKN2A genes. Examination of M private variants to find a site-specific pattern of metastatic spread suggested enrichment in MAPK (p=0.02) and PI3K (p<0.0001) pathways mutations in liver M (n=30) when compared to other sites. Interestingly, all variants detected in bone M (n=8) were shared with PT.

Conclusion In this prospective multicenter study, the targeted mutational analysis showed that most metastatic samples at first relapse share majority of variants with their matched PT. Sequencing M showed additional druggable mutations in 13% of patients. Liver M had more variants in MAPK and PI3K pathways compared to other metastatic sites whereas bone M did not show a specific mutational pattern.

#2962

Clinical relevance of circulating cell-free DNA using amplicon-based next-generation sequencing panel in colorectal cancer patients with liver metastasis.

Hiroki Osumi,1 Eiji Shinozaki,1 Hitoshi Zembutsu,2 Yoshinori Takeda,1 Takeru Wakatsuki,1 Takashi Ichimura,1 Yumiko Ota,1 Izuma Nakayama,1 Mariko Ogura,1 Mitsukuni Suenaga,1 Daisuke Takahari,1 Keisho Chin,1 Akio Saiura,1 Kensei Yamaguchi1. 1 _Cancer Institute Hospital, Japanese Foundation for Cancer Research, Tokyo, Japan;_ 2 _Cancer Precision Medicine Center, Tokyo, Japan_.

Background: Recent advances in the next-generation sequencing technique (NGS) for the detection of cell-free DNA (cfDNA) will be possible to provide the utility as diagnostic and prognostic biomarkers of cancer. We conducted the study of feasibility to detect plasma cfDNA from patients with metastatic colorectal cancer (mCRC) using NGS panel and investigated their relationship with clinicopathological factors.

Methods: A total of 101 mCRC patients with liver metastasis, who had been treated with chemotherapy, were enrolled in this study from February to June in 2017. We investigated i) frequency of detectable mutations in plasma cfDNA, ii) concordance rate of RAS mutation between the DNA extracted from tissues and the plasma cfDNA, iii) relationship between the mutation allele frequencies (MAF) and clinicopathological factors including tumor location, metastatic site, number of metastatic organs, tumor marker (CEA and CA19-9), LDH level, D-dimer level and sum of the tumor diameter measured based on RECIST ver1.1 criteria. Sequencing of plasma cfDNA were performed using Ion Torrent™ Oncomine™ Colon cfDNA Assay.

Results: Mutations in plasma cfDNA were detected in 87.1% (88/101) of patients. The frequencies of plasma cfDNA mutation at TP53, KRAS, APC, and PIK3CA were 68.3%, 38.6%, 23.7%, and 14.8%, respectively. RAS mutational concordance rate between DNA extracted from tumor and cfDNA was 76.2% (77/101). MAF were significantly associated with CEA (P<0.0001), CA19-9 (P=0.006), LDH (P<0.0001) and number of metastatic organs (P<0.0001). Patients with liver or lymph node metastasis had significantly higher MAF compared with those without metastases (Liver: P<0.0001, Lymph node: P=0.008). Positive correlations between CEA level, CA19-9 level, LDH level, tumor diameter and MAF were observed. (CEA: r=0.52, CA19-9: r=0.34, LDH: r=0.55, tumor diameter: r=0.57)

Conclusions: Our results suggested that this cfDNA Assays could detect mutations at a high rate of mCRC patients undergoing chemotherapy and cfDNA analysis using NGS panel could be useful method of diagnostic biomarkers to monitor the change of RAS status and tumor burden. <!--EndFragment-->

#2963

Characterization of disease evolution in sequential sampled metastatic breast cancer using liquid biopsy.

Lisa Welter,1 Liya Xu,1 Dillon McKinley,1 Sara Restrepo-Vassalli,1 Angel Dago,2 Mariam Rodriguez Lee,1 Anand Kolatkar,1 James Hicks,1 Jorge Nieva,3 Peter Kuhn1. 1 _University of Southern California, Los Angeles, CA;_ 2 _The Scripps Research Institute, San Diego, CA;_ 3 _Keck School of Medicine USC, Los Angeles, CA_.

Background: It is established that tumors evolve over time due to treatment pressure or physiological constraints. Tissue biopsies give a one-time window into the characteristics of a primary tumor or metastatic nodule. Their main pitfall is procedure risk, pain, and cost, which is why they are rarely repeated. Liquid biopsies, however, are performed with minimal discomfort and risk, providing an easier way to monitor disease. Circulating tumor cells (CTCs) and cell-free DNA (cfDNA) have proven to be of high prognostic and diagnostic value. Here we evaluate 17 longitudinal blood draws collected from a metastatic breast cancer patient over 4 years and assess the power of single-cell analysis relative to analysis of tissue biopsies and cfDNA.

Methods: CTCs were enumerated using the high-definition single-cell assay (HD-SCA) and single CTCs were isolated by micromanipulation. DNA of single cells underwent whole genome amplification and Illumina library construction for copy number variation (CNV) analysis. Genomic DNA from microdissected FFPE tissue and cfDNA extracted from plasma were converted into Illumina sequencing libraries for CNV analysis. CA27.29 levels were reported by the clinic.

Results: Levels of CTCs and circulating tumor DNA (ctDNA) fractions were positively correlated with CA27.29. CNV analysis of single tumor cells at time of enrollment revealed 3 morphometrically indistinguishable clones that persisted during the 4-year follow-up. Genomic analysis of primary breast tissue and bone metastasis detected at time of diagnosis matched CNV data of the evolutionary first clone, while a later liver metastasis was comprised solely of clone 2. Remarkably, CTCs in clone 2 were identified years before discovery of the liver metastasis. To date, we have not detected a metastatic site harboring clone 3. We found that CNV profiles from cfDNA represented the aggregate of CTC clones.

Conclusion: Longitudinal blood sampling enabled tracing of tumor evolution robustly and less invasively than solid tumor biopsies. Metastasis associated clones were identified years before measureable disease, providing an opportunity for earlier interventions. While plasma-based assays detected the presence of tumor-derived DNA, the single-cell high-content resolution of the HD-SCA workflow was required to deconvolute 3 genomically distinct clones present in this patient. We propose that enumeration of CTCs and analysis of ctDNA could be used as additional clinical markers to monitor disease recurrence, adding valuable morphogenomic information about the primary tumor and metastatic sites.

Additionally, we are currently analyzing the results of single nucleotide variation analysis of single CTCs, cfDNA and FFPE tissue from this patient.

#2964

On-treatment changes in circulating tumor DNA (ctDNA) level as an early predictor of clinical outcome in the LOTUS randomized phase 2 trial of 1st-line ipatasertib (IPAT) + paclitaxel (PAC) for metastatic triple-negative breast cancer (mTNBC).

Matthew J. Wongchenko,1 Doron Lipson,2 Travis Clark,2 Mark Kennedy,2 Mandy Greene,2 Virginia Breese,2 Alyssa Tsiros,2 Sung-Bae Kim,3 Cristina Saura,4 Mafalda Oliveira,4 Jose Baselga,5 Amy V. Kapp,1 Wai Y. Chan,1 Stina M. Singel,1 Steven Gendreau,1 Rebecca Dent6. 1 _Genentech, Inc., South San Francisco, CA;_ 2 _Foundation Medicine, Inc., Cambridge, MA;_ 3 _Asan Medical Center, University of Ulsan College of Medicine, Seoul, Republic of Korea;_ 4 _Vall d'Hebron University Hospital, Vall d'Hebron Institute of Oncology, Barcelona, Spain;_ 5 _Memorial Sloan Kettering Cancer Center, New York, NY;_ 6 _National Cancer Centre, Singapore, Singapore_.

Purpose: There is high interest in longitudinal analysis of plasma ctDNA as an early predictor of response and long-term outcomes. We report a retrospective longitudinal analysis of ctDNA in the double-blind placebo (PBO)-controlled randomized phase 2 LOTUS (NCT02162719) trial of the oral Akt inhibitor IPAT + PAC for mTNBC.

Methods: Pre-treatment plasma samples were analyzed to identify and quantify genomic alterations in ctDNA using a hybrid-capture-based NGS assay that interrogates 62 genes (FoundationACT). A narrow ctDNA panel quantified the same mutations in on-treatment samples collected at 8 wks (d1, cycle 3). The ctDNA fraction (CTF) was defined as the highest mutant allele frequency in each sample. The ratio of CTF in on-treatment vs pre-treatment samples was compared with objective response (RECIST v1.1) and progression-free survival (PFS) in the experimental (IPAT + PAC) and control (PBO + PAC) arms.

Results: Paired on-treatment samples were evaluable from 66 of 88 patients with evaluable pre-treatment samples. The most common reason for no on-treatment sample was disease progression (PD) before cycle 3. In 81 baseline samples, 149 mutations were identified. Calculated CTF ratios ranged from 0% (not detected in on-treatment sample) to 648% (increase in on-treatment sample). In both arms and the pooled population, a greater CTF decrease was associated with objective response and longer PFS (Table).

Conclusions: On-treatment change in CTF shows a meaningful association with objective response and PFS in LOTUS. This effect was seen in the experimental and control arms, suggesting independence from the specific mechanism of therapy. Evaluation of samples from the time of PD is ongoing. Inclusion of additional timepoints will likely improve the predictive ability of CTF, possibly allowing its use as an early surrogate trial endpoint. | IPAT + PAC (n=32) | PBO + PAC (n=34) | Pooled arms (n=66)

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

Median CTF ratio, % (IQR)

Confirmed complete/partial response | (n=14) | (n=14) | (n=28)

0.4 (0-14.3) | 0.2 (0-46.9) | 0.3 (0-17.8)

Stable/progressive disease | (n=18) | (n=20) | (n=38)

8.2 (4.4-45.9) | 26.9 (8.4-91.4) | 20.7 (4.9-67.9)

Median PFS, months (95% CI)

CTF ratio ≤5% | (n=15) | (n=14) | (n=29)

7.2 (3.7-NE) | 6.3 (3.6-NE) | 7.2 (5.1-12.9)

CTF ratio >5% | (n=17) | (n=20) | (n=37)

3.7 (3.5-5.3) | 3.7 (2.8-5.4) | 3.7 (3.6-5.0)

PFS hazard ratio (95% CI) | 0.46 (0.19-1.09) | 0.43 (0.17-0.97) | 0.43 (0.23-0.78)

CI = confidence interval; IQR = interquartile range; NE = not estimable.

## EPIDEMIOLOGY:

### Genetic and Molecular Epidemiology of Cancer Risk and Prognosis

#2965

Functionally informed genome-wide interaction analysis of nonsteroidal anti-inflammatory drugs on colorectal cancer risk.

Xiaoliang Wang,1 Yu-Ru Su,1 Andrew T. Chan,2 Stephanie Bien,1 Sonja I. Bernt,3 Hermann Brenner,4 Graham Casey,5 Jenny Chang-Claude,6 Steven J. Gallinger,7 Robert W. Haile,8 Tabitha A. Harrison,1 Michael Hoffmeister,9 Mark A. Jenkins,10 Amit Joshi,11 Yi Lin,1 Noralane M. Lindor,12 Loic Le Marchand,13 Hongmei Nan,14 Polly A. Newcomb,1 John D. Potter,1 Martha L. Slattery,15 Steve N. Thibodeau,16 Emily White,1 Li Hsu,1 Ulrike Peters1. 1 _Fred Hutchinson Cancer Research Center, Seattle, WA;_ 2 _Massachusetts General Hospital, Boston, MA;_ 3 _National Cancer Institute, National Institutes of Health, Rockville, MD;_ 4 _German Cancer Research Center (DKFZ), Heidelberg, Germany;_ 5 _University of Virginia, Charlottesville, VA;_ 6 _German Cancer Research Center, Heidelberg, Germany;_ 7 _Mount Sinai Hospital, Toronto, Ontario, Canada;_ 8 _Cedars-Sinai, Los Angeles, CA;_ 9 _German Cancer Research Center, Germany;_ 10 _The University of Melbourne, Melbourne, Australia;_ 11 _Harvard School of Public Health, Boston, MA;_ 12 _Mayo Clinic, Scottsdale, AZ;_ 13 _University of Hawaii Cancer Center, Honolulu, HI;_ 14 _Richard M. Fairbanks School of Public Health, Indiana University, Indianapolis, IN;_ 15 _University of Utah Health Sciences Center, Salt Lake City, UT;_ 16 _Mayo Clinic, Rochester, MN_.

Background Regular use of aspirin and other non-steroidal anti-inflammatory drugs (NSAIDs) is associated with lower risk of colorectal cancer (CRC). Genome-wide interaction analysis (GxE) has identified a few variants that may modify the effects of NSAIDs on CRC risk. However, limited statistical power remains a concern. Restricting analyses by using functional genomic information to aggregate variants into biologically relevant sets can reduce the number of tests, thereby increasing statistical power. We tested the interactions between variant models of gene expression and NSAIDs use on CRC risk.

Methods Functional weights of each variant were estimated using PrediXcan based on jointly measured transcriptomes and genomes data of transverse colon tissues from the Genotype-Tissue Expression (GTEx) Project for all genes with sufficient heritability (≥1%). A mixed-effects model was used to assess the GxE effects in a gene among 9,917 incident CRC cases and 10,533 controls from 17 (nested) case-control studies from the Colon Cancer Family Registry (CCFR) and Genetics and Epidemiology of Colorectal Cancer Consortium (GECCO). GxE analysis was done by modeling the interaction between predicted gene expression and NSAID use (fixed effects), and residual variant-specific GxE effects that are not accounted for (random effects). Regular user of aspirin and/or non-aspirin NSAIDs was compared to non-regular users. False discovery rate (FDR) was used to account for multiple comparison and FDR≤0.2 was considered genome-wide significant. Secondary analysis was performed on significant genes and duration of use among users.

Results Regular use of aspirin was higher among controls (30.3%) compared to cases (24.3%). Among the 4,842 genes tested, SORD significantly modified the effect of regular use of aspirin on CRC risk (p-interaction = 1.45×10-5; FDR=0.07). The SORD gene encodes sorbitol dehydrogenase (SORD), which oxidizes sorbitol to fructose in the polyol pathway. Decreased SORD concentrations were previously observed to dramatically increase in colorectal adenoma cells when compared to normal mucosa cells, suggesting the involvement of dysregulated polyol metabolisms in colorectal tumorigenesis. However, the duration of aspirin use was not statistically significantly associated with SORD gene expression on CRC risk (p=0.149) among 5,560 aspirin users. No significant interactions were observed between genetically determined colon gene expression levels and any NSAID use or non-aspirin NSAID use at FDR<0.2.

Conclusions Incorporating functional information, we discovered a novel gene that may interact with aspirin use to confer CRC risk. These findings provide preliminary support for new biological insights that could help understand the chemopreventive mechanisms of aspirin on CRC. We aim to replicate these findings in additional studies.

#2966

A genome-wide scan identifies a new locus associated with pediatric rhabdomyosarcoma.

Philip J. Lupo,1 Libby Morimoto,2 Eric Karlins,3 Xiaorong Shao,2 Lindsay M. Morton,3 Michael E. Scheurer,1 Smita Bhatia,4 Leslie L. Robison,5 Gregory T. Armstrong,5 Simone Hettmer,6 Javed Khan,3 Stephen J. Chanock,3 Neal D. Freedman,3 Kathleen Wyatt,3 Belynda D. Hicks,3 Meredith Yeager,3 Casey L. Dagnall,3 Shengchao A. Li,3 Stephen X. Skapek,7 Douglas S. Hawkins,8 Catherine Metayer,2 Lisa Mirabello3. 1 _Baylor College of Medicine, Houston, TX;_ 2 _University of California, Berkeley, Berkeley, CA;_ 3 _National Cancer Institute, Bethesda, MD;_ 4 _UAB School of Medicine, Birmingham, AL;_ 5 _St. Jude Children's Research Hospital, Memphis, TN;_ 6 _University of Freiburg, Germany;_ 7 _UT Southwestern Medical Center, Dallas, TX;_ 8 _Seattle Children's, Seattle, WA_.

Background: Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children and has one of the poorest survival rates among all pediatric cancers. The two major histologic subtypes of RMS are embryonal (eRMS) and alveolar (aRMS), which display differences in terms of age-incidence patterns and somatic mutations. Approximately 10% of RMS cases are associated with germline mutations in known cancer predisposition genes (e.g., TP53, NF1), but very little is known about the genetic susceptibility to the ~90% of RMS cases that are sporadic. We conducted the first multi-institutional genome-wide association study (GWAS) of RMS in 727 cases and 3,384 controls.

Methods: Phase 1 of the GWAS included 421 RMS cases from Children's Oncology Group clinical trials, Texas Children's Hospital, and the Universidad de Navarra. Controls (n=2,763) were cancer-free individuals included in previous studies at the National Cancer Institute (NCI). Phase 2 included 306 cases from the Childhood Cancer Survivor Study and 621 independent controls from NCI. Genotypes were generated using the Illumina OmniExpress or the HumanOmni5Exome array and imputed based on the 1000 Genomes Project. Analyses were restricted to those of European (EUR) ancestry, and controls were matched to the cases based on principal components and genotype platform. Assuming an additive genetic model in SNPTEST, we used multivariable logistic regression models to estimate the odds ratio (OR), 95% confidence interval (CI), and P value for each variant on RMS overall and by two RMS subtypes: eRMS and aRMS.

Results: After quality control filtering and assessment of population substructure, there were 555 combined EUR RMS cases and 1,561 controls, which included: 1) 278 cases and 1,112 controls in phase 1; and 2) 277 cases and 449 controls in phase 2. In the combined set, we identified a new locus at chromosome 11p15.2 that was strongly associated with an increased risk of aRMS and significant at the genome-wide level (OR=2.3, P=2.2x10-8). Results were consistent across studies: phase 1 OR=2.3, 95% CI 1.7-3.2; and phase 2 OR=2.3, 95% CI 1.2-4.5. The top variant, rs12785926, mapped to an intron in the PSMA1 (proteasome subunit alpha 1) gene. Based on data from GTEx, rs12785926 is significantly associated with RRAS2 expression across multiple tissues. RRAS2 is involved in cell proliferation and is somatically mutated in several tumors. When evaluating eRMS and RMS overall in the combined set, there were no variants significant at the genome-wide level.

Conclusion: In the first GWAS of pediatric RMS, we identified a susceptibility locus associated with the more aggressive aRMS subtype that has a poorer prognosis. Additional replication analyses are underway using DNA obtained from archived newborn blood spots linked to population-based cancer registries, as well as other institutional cohorts. Further investigation will advance understanding of RMS etiology and biology.

#2967

Ethnic-specific risk of neurotoxocity and its impact on treatment outcomes among pediatric patients receiving acute lymphoblastic leukemia therapy.

Michael E. Scheurer,1 Olga A. Taylor,1 Austin L. Brown,1 Julienne Brackett,1 ZoAnne E. Dreyer,1 Ida (Ki) Moore,2 Pauline Mitby,3 Mary C. Hooke,4 Philip J. Lupo,1 Marilyn J. Hockenberry5. 1 _Baylor College of Medicine, Houston, TX;_ 2 _University of Arizona, Tucson, AZ;_ 3 _Children's Minnesota, Minneapolis, MN;_ 4 _University of Minnesota, Minneapolis, MN;_ 5 _Duke University, Durham, NC_.

Purpose: Because of the increasing awareness that Hispanics diagnosed with acute lymphoblastic leukemia (ALL) often experience worse outcomes, we sought to evaluate the risk of methotrexate (MTX)-related neurotoxicity (NT) by ethnic group and evaluate its effects on treatment outcomes in a multi-institutional prospective cohort of pediatric patients (2-17 years old).

Methods: Patients with ALL were prospectively recruited from 3 pediatric cancer centers in the United States for the period 2012-2017 and systematically followed for the development of treatment-related symptoms and toxicities. For this analysis, suspected NT cases were defined as patients with a neurologic event following intrathecal (IT) and/or intravenous (IV) MTX that led to a change in subsequent MTX therapy. Cumulative incidence of MTX NT was calculated by ethnic group. Multivariable linear regression models were generated to compare treatment differences between patients with and without MTX NT. The frequency of all-cause and central nervous system (CNS) relapse was compared between patients with and without MTX NT using the log-rank test and Cox regression models.

Results: Of the 280 patients enrolled, 39 (13.9%) experienced MTX NT (median follow-up = 22.6 months; range: 1.3 - 55.6 months). Cumulative incidence of MTX NT was 21.8% among Hispanic patients compared to 7.0% among non-Hispanic patients (p <0.001). After adjusting for relevant clinical characteristics, Hispanic patients were 2.74 times more likely to develop MTX NT compared to non-Hispanic white patients (95% CI: 1.21-6.19). Patients who experienced MTX NT received an average of 2.25 fewer doses of IT MTX (95% CI: 1.73-2.77), independent of treatment risk group, sex, or age. Six cases of MTX NT (15.4%) experienced all-cause relapse during the study period, compared to 13 (2.1%) patients without MTX NT (log-rank p = 0.0038). Similarly, CNS relapse was more frequent among patients with MTX NT (10.3%) than patients without NT (2.1%; log-rank p = 0.0014). In univariate Cox regression models, MTX NT was significantly associated with CNS relapse (HR: 3.80, 95% CI: 1.44-10.02), a trend which remained after individually accounting for treatment risk arm (HR: 2.92, 95% CI: 1.07-7.95), MRD status at day 29 (HR: 3.49, 95% CI: 1.32-9.24), race and ethnicity (HR: 3.15, 95% CI: 1.13-8.79), age at diagnosis (HR: 2.56, 95% CI: 0.91-7.21), and sex (HR: 3.82, 95% CI: 1.44-10.10).

Conclusion: We identified an increased risk of relapse, specifically in the CNS, among ALL patients following MTX NT, which was not fully explained by other clinical risk factors. Further, incidence of NT was higher among Hispanic patients in our clincs. This is particularly interesting given that Hispanic patients typically present with more favorable disease characterstics, yet often experience worse treatment outcomes.

#2968

Imputation of the prostate cancer transcriptome in over 230,000 men reveals novel germline-somatic interaction mechanism of cancer risk.

Nima C. Emami,1 Joshua Hoffman,2 Elad Ziv,1 John S. Witte1. 1 _University of California, San Francisco, San Francisco, CA;_ 2 _GlaxoSmithKline, Philadelphia, PA_.

Although genome-wide association studies of prostate cancer have revealed numerous genetic loci associated with disease risk, efforts to characterize the causal mechanisms of germline genetic variants have been unsystematic in their approach. While select studies have evaluated the effects of the most significantly associated variants on the expression of highly proximal genes, both the complex nature of gene expression regulation, as well as the majority fraction of prostate cancer risk unaccounted for by genome-wide association study loci, suggest that certain risk genes and variants remain undiscovered. In pursuit of such causal genetic factors, we analyzed germline genotype data paired with gene expression data from normal prostate tissue in 471 subjects to build regularized statistical models of how gene expression of the prostate is influenced by cis-genetic variation. By applying these models to genome-wide association study data, predictions of transcript expression levels were generated transcriptome-wide for over 230,000 male subjects (14,616 prostate cancer cases, 219,339 controls) from the UK Biobank, GERA Cohort, ProHealth Study, and California Men's Health Study. Finally, these transcript abundances were assessed in relation to prostate cancer case-control status to identify those genes with expression levels most predictive of prostate cancer diagnosis. Among the 12,014 genes for which models were successfully fit, 29 were transcriptome-wide significant via Bonferroni correction, while another 9 exhibited p-values that were suggestive. Moreover, 19 of the 38 significant or suggestive genes replicated in the validation cohort of Kaiser Permanente health plan members (GERA, ProHealth, CMHS). At previously implicated risk loci for prostate cancer, numerous genes were significantly associated with disease risk, including MSMB, NCOA4, and AGAP7 at 10q11.22, HNF1B at 17q12, as well as POU5F1B and PCAT1 at 8q24.21. Furthermore, several genes whose expression levels were not previously implicated were associated with prostate cancer risk. The most noteworthy of these was TMPRSS2, part of the TMPRSS2-ERG gene-fusion, which represents the most frequent somatic alteration discovered in prostate cancer tumors. When conditioned on a previously reported prostate cancer GWAS cis-variant at 21q22.3, TMPRSS2 retained transcriptome-wide significance. Further analysis of the genetic effects on TMPRSS2-ERG expression in prostate cancer tumors from The Cancer Genome Atlas (TCGA) suggested a novel mechanism by which germline and somatic factors cooperate to increase disease risk. By systematically characterizing gene expression of the prostate in this transcriptome-wide association study (TWAS) of prostate cancer, we identified prostate cancer risk genes and propose a novel germline-somatic interaction mechanism of cancer risk.

#2969

Genetically predicted blood protein biomarkers and prostate cancer risk: an analysis in over 140,000 European descendants.

Lang Wu,1 Xiang Shu,1 Jiandong Bao,1 Xingyi Guo,1 the PRACTICAL, CRUK, BPC3, CAPS, PEGASUS consortia, Zsofia Kote-Jarai,2 Christopher A. Haiman,3 Rosalind A. Eeles,2 Wei Zheng1. 1 _Vanderbilt Univ. School of Medicine, Nashville, TN;_ 2 _The Institute of Cancer Research, and The Royal Marsden NHS Foundation Trust, London, United Kingdom;_ 3 _University of Southern California, Los Angeles, CA_.

Prostate cancer (PrCa) is the second most frequently diagnosed malignancy among males in many countries. Several protein markers measured in blood have been found to be associated with PrCa risk. However, most previous studies assessed only a small number of biomarkers or included a small sample size. To search for novel protein biomarkers for PrCa risk, we performed a large study in 79,194 prostate cancer cases and 61,112 controls of European ancestry included in PRACTICAL/ELLIPSE consortia by using genetic instruments.

Protein quantitative trait loci (pQTLs) for 1,478 plasma proteins identified in a large study of 3,301 European descendants were used as instruments to evaluate associations between genetically predicted protein levels and PrCa risk. For proteins showing a significant association with PrCa risk, we further evaluated whether genetically predicted mRNA expression levels of the corresponding genes were associated with PrCa risk, by using mRNA expression prediction models for blood, prostate and cross tissue built using data of the Genotype-Tissue Expression Project.

We identified 31 proteins showing an association of their predicted levels with PrCa risk at a false discovery rate < 0.05, including 22 encoded by genes located more than 500 Kb away from any reported PrCa risk variants. These include proteins encoded by GSTP1, whose methylation was identified as a potential biomarker for PrCa detection, and MSMB, SPINT2, and CTSS, which were previously implicated as potential target genes of PrCa risk variants identified in genome-wide association studies. Eighteen of the proteins showed an inverse association and 13 showed a positive association. Among the 23 of the genes with mRNA expression prediction models built, seven showed an mRNA-PrCa association at p < 0.05 with a same direction of effect as the protein-PrCa association. For 28 of the identified genes, somatic changes of short indels, splice site, nonsense, or missense mutations were detected in PrCa patients in The Cancer Genome Atlas (enrichment p<0.0001). Pathway enrichment analysis showed that these genes were significantly enriched in cancer related pathways, including STAT3 Pathway, Glutathione Redox Reactions I, Glutathione-mediated Detoxification, Endoplasmic Reticulum Stress Pathway, and tRNA Splicing.

Our study has identified multiple proteins significantly associated with PrCa risk. Further research is needed to evaluate potential utility of the identified proteins in early detection of PrCa.

#2970

Multiple new susceptibility loci identified in genome-wide association study of Ewing sarcoma.

Mitchell J. Machiela,1 Thomas G. Grünewald,2 Didier Surdez,3 Stephanie Reynaud,3 Olivier Mirabeau,3 Eric Karlins,1 Rebeca Alba Rubio,2 Sakina Zaidi,3 Sandrine Grossetete-Lalami,3 Stelly Ballet,3 Eve Lapouble,3 Valérie Laurence,3 Jean Michon,3 Gaelle Pierron,3 Heinrich Kovar,4 Nathalie Gaspar,5 Udo Kontny,6 Anna González-Neira,7 Piero Picci,8 Javier Alonso,9 Ana Patino-Garcia,10 Nadège Corradini,11 Neal D. Freedman,1 Nathaniel Rothman,1 Casey L. Dagnall,1 Laurie Burdette,1 Kristine Jones,1 Michelle Manning,1 Kathleen Wyatt,1 Weiyin Zhou,1 Meredith Yeager,1 David G. Cox,12 Robert N. Hoover,1 Javed Khan,1 Gregory T. Armstrong,13 Wendy M. Leisenring,14 Smita Bhatia,15 Leslie L. Robison,13 Uta Dirksen,16 Markus Metzler,17 Wolfgang Hartmann,18 Konstantin Strauch,2 Thomas Kirchner,2 Andreas E. Kulozik,19 Lindsay M. Morton,1 Lisa Mirabello,1 Margaret A. Tucker,1 Franck Tirode,3 Stephen J. Chanock,1 Olivier Delattre3. 1 _National Cancer Institute, Bethesda, MD;_ 2 _LMU Munich, Germany;_ 3 _Institut Curie, France;_ 4 _Children's Cancer Research Institute, St. Anna Kinderkrebsforschung, Austria;_ 5 _Institut Gustave Roussy, France;_ 6 _RWTH Aachen University, Germany;_ 7 _Spanish National Cancer Research Centre, Spain;_ 8 _Istituto Ortopedico Rizzoli di Bologna, Italy;_ 9 _Instituto de Investigación de Enfermedades Raras, Spain;_ 10 _University of Navarra, Spain;_ 11 _IHOPe, Centre Léon Bérard, France;_ 12 _Centre Léon Bérard, INSERM U1052, France;_ 13 _St. Jude Children's Research Hospital, TN;_ 14 _Fred Hutchinson Cancer Research Center, WA;_ 15 _University of Alabama at Birmingham, AL;_ 16 _University Children's Hospital of Essen, Germany;_ 17 _University Children's Hospital of Erlangen, Germany;_ 18 _University Hospital of Münster, Germany;_ 19 _University Children's Hospital of Heidelberg, Germany_.

Ewing sarcoma (EWS) is a rare pediatric tumor predominantly occurring in children of European ancestry and is characterized by the pathognomonic EWSR1-FLI1 fusion oncogene. To identify germline susceptibility loci associated with EWS risk, we performed a genome-wide association study (GWAS) meta-analysis of 749 EWS cases and 1,378 unaffected individuals of European ancestry from sample collections within the Institut Curie, National Cancer Institute and the Childhood Cancer Survivor Study. Our study replicated previously reported susceptibility loci at 1p36.22, 10q21.3 and 15q15.1 as well as identified novel loci at 6p25.1, 8q24.23, 20p11.22 and 20p11.23 (P-values<5×10-8). These seven EWS susceptibility loci discovered in only 749 cases make EWS one of the most productive GWAS studied cancers when considering a locus to case discovery ratio. All estimated effect estimates were high for cancer GWAS with odds ratios in excess of 1.7 observed. These high per allele effects among relatively common germline variants are striking in light of the rarity of EWS cases and lack of evidence of EWS as part of a familial cancer syndrome and therefore suggest a distinctive genetic architecture for EWS. Interestingly, in silico bioinformatics analysis identified that most EWS susceptibility loci reside near GGAA nucleotide repeat sequences where binding of the EWSR1-FLI1 fusion protein occurs. ChIP-seq analyses confirmed in vivo binding of EWSR1-FLI1, suggesting germline variation in these regions could alter EWSR1-FLI1 binding and potentially deregulate neighboring genes. To identify genes with allele specific expression differences, we carried out expression quantitative trait locus (eQTL) analyses at each identified EWS susceptibility locus. We identified eQTLs for plausible candidate genes at 6p25.1 with RREB1, a RAS-responsive element, and at 20p11.23 with KIZ, a centrosomal stabilization protein. We also noted the 20p11.22 locus is near NKX2-2, a highly overexpressed gene in EWS, although no eQTL was observed in our expression data. Furthermore, knockdown of EWSR1-FLI1 in EWS cell lines indicated a more than 2-fold difference in expression of RREB1 and NKX2-2, further supporting the role of specific regulation of these genes by EWSR1-FLI1 and suggesting RREB1 and NKX2-2 may be transcription factors involved in core regulatory circuitries of EWS. Overall, our study suggests a distinctive underlying genetic architecture for EWS in which moderate risk common germline variants interact with EWSR1-FLI1 binding to alter expression of nearby target genes.

#2971

Digital image analysis based IHC4+C assay and prognosis in hormone receptor-positive breast cancer.

Mustapha Abubakar,1 Paul D. Pharoah,2 Mark E. Sherman,3 Montserrat Garcia-Closas,1 Mitch Dowsett4. 1 _National Cancer Institute, Rockville, MD;_ 2 _University of Cambridge, Cambridge, United Kingdom;_ 3 _Mayo Clinic, Jacksonville, FL;_ 4 _Institute of Cancer Research, London, United Kingdom_.

Background

The immunohistochemical (IHC) 4 + clinical treatment score (C) i.e. IHC4+C assay combines IHC assessment of four routinely measured markers in the clinical setting, including estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2) and KI67, with other clinical parameters for aiding decision-making on adjuvant chemotherapy in hormone positive (HR+) breast cancer. It is low cost, widely available and has been shown to provide similar prognostic information to the 21-gene (Oncotype DX) test. Clinical use of IHC4+C is limited by the need for standardized scoring of the IHC markers. Here, we examine the prognostic value of a digital image analysis (DIA) based IHC4+C assay in two study populations and compare this with pathologists' visual assay in a subset of patients. We also evaluated its potential clinical relevance in relation to the Nottingham prognostic index [NPI; i.e. low (<3.4), intermediate (3.4-5.4) and high (>5.4)] and IHC defined breast cancer subtypes (i.e. luminal A-like and B-like).

Methods

Using DIA data on ER, PR, HER2 and KI67, in addition to clinicopathological characteristics of tumors, we computed DIA based IHC4+C (i.e. DIAIHC4+C) assay for 2,034 women from study populations in Poland (PBCS) and the United Kingdom (SEARCH). A total of 246 deaths occurred over a median follow-up period of 6.7 years. A relevant threshold was determined using quartile analysis and the relationship between dichotomous categories (>75th vs ≤ 75th percentile) of DIAIHC4+C and 10-year breast cancer specific survival (BCSS) was investigated using Kaplan-Meier survival curves and in Cox proportional hazard regression models, overall and stratified by study, NPI and subtype.

Results

DIAIHC4+C score was associated with worse 10-year BCSS overall [HR (95% CI) >75th vs ≤ 75th percentile = 2.92 (1.77, 4.82); p-value <0.0001] and in both the PBCS [3.48 (1.40, 8.65); p-value = 0.007] and SEARCH [3.87 (2.24, 6.68); p-value <0.0001] study populations. DIAIHC4+C was associated with significantly worse survival outcomes in women with intermediate NPI [1.94 (1.31, 2.88); p-value = 0.001] and luminal A-like subtype of HR+ tumors [5.37 (3.50, 8.26); p-value <0.0001]. DIA and visual IHC4+C results were highly correlated (r = 0.95) and survival associations and model fit parameters were similar for DIA [3.82 (2.08, 7.00); p-value < 0.0001; AIC = 472.3] and visual [4.73 (2.55, 8.76); p-value < 0.0001; AIC = 466.1] IHC4+C scores amongst 444 patients with data from both sources.

Conclusion

These findings are in support of the prognostic value of DIAIHC4+C assay in HR+ breast cancer and demonstrate its potential clinical relevance as an adjunct to the NPI. Given the advantages of being high-throughput and highly reproducible, DIA based methods could find use in the standardization of the analytical processes for the IHC4+C assay.

## EXPERIMENTAL AND MOLECULAR THERAPEUTICS:

### Identification of Biomarkers and Molecular Drug Targets

#2972

Co-clinical trial of olaparib and temozolomide in SCLC PDX models uncovers new biomarkers of sensitivity.

Benjamin J. Drapkin,1 Julie George,2 Marcello Stanzione,1 Beow Y. Yeap,1 Mari Mino-Kenudson,1 Camilla L. Christensen,3 Ruben Dries,3 Sarah Phat,1 Jun Zhong,1 David T. Myers,1 Joseph A. Licausi,1 Tilak Sundaresan,1 Marina Kem,1 Nima Abedpour,2 Leica V. Sequist,1 Alice T. Shaw,1 Aaron N. Hata,1 Mehmet Toner,1 Shyamala Maheswaran,1 Daniel A. Haber,1 Martin Peifer,2 Roman K. Thomas,2 Anna F. Farago,1 Nicholas J. Dyson1. 1 _Massachusetts General Hospital, Boston, MA;_ 2 _University of Cologne, Cologne, Germany;_ 3 _Dana-Farber Cancer Institute, Boston, MA_.

Introduction: Small cell lung cancer (SCLC) is a common and rapidly fatal malignancy for which no biomarker-targeted therapies have been developed. Despite a critical need, progress suffers from (1) scarcity of cutting-edge laboratory models and (2) absence of promising targets. Patient-derived xenografts (PDX) may faithfully model the clinical disease, but because SCLC is rarely biopsied or resected, specimens for PDX generation are scarce. PARP inhibition has recently emerged as a compelling strategy for SCLC, and an ongoing phase 1/2 trial of combination olaparib tablets and temozolomide (O/T) has shown promising activity in patients. However, biomarkers for patient selection remain elusive.

Methods: We generated SCLC PDX models from circulating tumor cells (CTCs), biopsies and malignant effusions. CTCs were enriched with an automated microfluidic device, the CTC-iChip. To assess the genomic fidelity of the models, we performed comparative whole exome sequencing (WES) and RNA-seq on 6 sets of corresponding patient biopsies, founder (P0) PDX tumors, and early-passage PDXs. We then assessed the activity of combination O/T in a panel of PDX models, and compared PDX responses with molecular profiles to identify candidate biomarkers.

Results: 44 PDXs were generated from 32 patients, including 6 sets of serial models and 4 synchronous CTC- and biopsy-derived models. PDXs were derived with high efficiency from both CTCs (35% per blood draw) and biopsies/effusions (82% per implant). WES demonstrated that somatic alterations in tumor biopsies were stably maintained in both CTC and biopsy-derived models, without significant accumulation of new mutations, and transcriptional profiles remained consistent through early passages. Six models were derived from O/T trial patients, including two sets of serial models before and after durable responses. The serial models faithfully recapitulated patient responses to O/T: pre-trial models were highly sensitive and post-relapse were highly resistant. The co-clinical trial was expanded to 30 models, using the models derived from trial patients to delineate the margins of clinical sensitivity (6 models), intermediate sensitivity (6 models) and resistance (18 models). Among the molecular features evaluated, basal protein PARylation best distinguished the O/T-sensitive category from both intermediate (p < 0.001) and resistant models (p < 0.0001). In addition, PARylation decreased after relapse in serial models from O/T trial patients.

Conclusions: Both biopsy- and CTC-derived SCLC PDX models faithfully recapitulate the genomic and functional features of the donor patient tumor. O/T sensitivity in this panel correlated with basal PARylation. The value of the co-clinical trial is the potential to refine the clinical application of O/T in real time, to optimize follow-on clinical trials and to develop biomarker-directed therapy for SCLC.

#2973

A comprehensive panel of patient-derived xenografts representing the molecular heterogeneity and diversity of triple-negative breast cancer.

Florence Coussy,1 Virginie Bernard,1 Marion Lavigne,1 Anais Boulai,1 Sophie Chateau-Joubert,2 Ahmed Dahmani,1 Elodie Montaudon,1 Cécile Reyes,1 Rania El Botty,1 Gaëlle Pieron,1 Cécile Laurent,1 Samia Melaabi,1 Anne Vincent Salomon,1 Ivan Bièche,1 Elisabetta Marangoni1. 1 _Inst. Curie, Paris, France;_ 2 _Alfort Veterinary School, Maisons-Alfort, France_.

Purpose: Triple-negative breast cancer (TNBC) is a heterogeneous disease. Patients diagnosed with TNBC have a poor prognosis and identification of new biomarkers and therapeutic agents is a high priority. Patient-derived xenografts (PDX) are clinically relevant models that have emerged as important tool for the analysis of drug activity and predictive biomarker discovery. The purpose of this work was to analyze the molecular heterogeneity of a large panel of TNBC PDX with the perspective to test targeted therapy and to identify biomarkers of response.

Experimental Design: PDX of early-stage TNBC established from 2003 to 2016 (N=60) were analyzed by high-resolution array CGH and gene expression profiling (Cytoscan HD and Human Gene 1.1 ST arrays). Subtypes were identified with the tool TNBCtype (Chen et al., 2012) based on transcriptomic data. A targeted next-generation sequencing (NGS) of 100 genes (the top frequently mutated genes in breast cancer) was performed on Illumina HiSeq2500 sequencer. COSMIC, Tumorportal and cBioportal databases were used for the interpretation of genomic variants. Immunohistochemistry (IHC) and morphologic analysis of PDX were performed as compared to the corresponding patients' tumors. PDX carrying targetable genomic alterations in the PI3K/AKT/mTOR and MAPK signaling pathways were treated by specific inhibitors (selumetinib, BAY 80-6946 and PF-04691502).

Results: At the gene expression level, TNBC PDX represent all the different TNBC subtypes identified by the Lehmann classification. The frequency of the different TNBC subtypes was similar to the TGCA TNBC, except for the immunomodulatory subtype, underrepresented in PDX. Somatic pathologic mutations and copy number alterations were similar in PDX and TCGA TNBC patients. Among the top altered genes are TP53 and oncogenes and tumor suppressors of the PI3K/AKT/mTOR and MAPK pathways (including PIK3CA, AKT1, NF1 and NRAS/KRAS). At the histologic level, TNBC PDX were mainly composed of invasive ductal carcinoma of no special type, with some tumors being classified as apocrine or metaplastic carcinomas. Comparison with the original tumors show similar patterns (based on IHC analysis of CK5, CK8/18, CK14 and AR, FOXA1, EGFR and Ki67). In vivo efficacy experiments with PI3K and MAPK pathways inhibitors showed marked antitumor activity in PDX carrying genomic alterations of PIK3CA, AKT1 and NRAS, NF1 genes. Drug combination experiments are currently ongoing in PDX with simultaneous genomic alterations of PI3KCA and MAPK related genes.

Conclusions: TNBC PDX reproduce the molecular heterogeneity and diversity of TNBC patients. This large collection of PDX is a clinically relevant platform for drug testing, biomarker discovery and translational research.

#2974

Primary tumor data mining identifies a novel synthetic lethal partner of the BRCA1 mutation in breast cancer.

Yihui Shi, Xiaohe Liu, Jia Li, Lidia Sambucetti, Subarna Sinha. _SRI International, Menlo Park, CA_.

Identification of mutation-specific targeted therapies is a critical challenge in precision medicine, in part because many cancer mutations are loss-of-function and are not druggable. Even when a mutation-specific targeted therapy exists, resistance invariably develops necessitating alterative therapeutic options. Synthetic lethality can help identify new therapeutic targets for these mutations. In synthetic lethal (SL) interactions, an alteration in one gene leads to dependency on a second gene. Neither alteration by itself is essential for survival, but together these alterations lead to cancer cell death. We have recently developed a new computational method (Mining Synthetic Lethals, MiSL) that mines SL interactions from pan-cancer primary tumor data (such as TCGA). The underlying assumption of MiSL is that SL partners of a mutation will be selectively amplified or never deleted and overexpressed in primary tumor samples harbouring the mutation. Since MiSL mines relationships from primary human tumor data, it enables the identification of SL interactions in the native context of human tumors and is more likely to find relationships relevant to in vivo tumor biology than shRNA screens.

Here, we applied MiSL to identify SL partners of the BRCA1 mutation in triple-negative breast cancer (TNBC). BRCA1 is mutated in 15-20% of TNBC and is a known cancer susceptibility gene in breast cancer. MiSL identified 22 SL partners of the BRCA1 mutation in TNBC: these were enriched for DNA repair genes consistent with the known SL relationship between BRCA1 and the DNA repair gene PARP. Notably, XRCC6, a gene involved in non-homologous end joining, was predicted to be a SL partner of the BRCA1 mutation in TNBC. MiSL selected XRCC6 because XRCC6 deletions (which were associated with lowered expression of XRCC6) were mutually exclusive with BRCA1 mutations in pan-cancer analysis (p = 0.01), and XRCC6 was upregulated in BRCA1-mutated TNBC compared to BRCA1-WT TNBC (p = 0.02). To test the prediction, we examined the effect of XRCC6 knockdown (KD) in the BRCA1-mutated TNBC cell line SUM149. Consistent with our prediction, we found that XRCC6 KD significantly increased apoptosis (37.3%) and reduced viability (50% reduction, p < 0.0001) in SUM149 cells compared to controls. XRCC6 KD in a BRCA1-WT breast cancer cell line, T47D, resulted in significantly lower reduction in cell viability compared to the BRCA1-mutated cell line (p<0.0001). These data indicated that XRCC6 KD more effectively induced cell death in the presence of BRCA1 mutation than in cells WT for the gene. We are currently testing XRCC6 KD effects in TNBC cell lines harbouring germline BRCA1 mutations.

Thus, we have identified a novel SL partner of the BRCA1 mutation in TNBC that can lead to the development of new therapeutic options for BRCA1-mutated breast cancers and to new chemoprevention strategies for individuals carrying germline BRCA1 mutations.

#2975

Synthetic lethality in synovial sarcoma: SS18-SSX fusions and DNA damage response (DDR) inhibitors.

Emmy DG Fleuren, Sam Jones, Jessica Frankum, Chris Williamson, Malini Menon, Janet Shipley, Alan Ashworth, Winette van der Graaf, Christopher J. Lord. _The Institute of Cancer Research, London, United Kingdom_.

Aim: Synovial sarcoma (SS) is an aggressive soft-tissue malignancy predominantly affecting the young and characterised by pathognomonic SS18-SSX fusions. Treatment options are limited and therapeutic targeting of SS18-SSX oncoproteins has not yet been achieved. We carried out large-scale, multiplatform functional genomic screenings followed by extensive in vitro and in vivo validation studies to identify therapeutically actionable dependencies in SS with the highest translational potential.

Methods: High-throughput drug sensitivity and siRNA screens including 79 clinically relevant compounds (0.5-1000nM) and siRNAs targeting 1600 different genes, respectively, were carried out in SS cell lines (SYO-1, HS-SY-II, Aska-SS, Yamato-SS and CME-1) and >115 cancer cell lines of other histologies in triplicate in 5-day survival assays. SS drug and siRNA sensitivity data was compared to sensitivity profiles of non-SS tumour cell lines (Mann-Whitney test). Validation inhibitory and mechanistic assays were performed with selected drugs and siRNA in SS cells, non-SS cells ectopically expressing SS18-SSX fusion proteins, and relevant other controls. Presence of replication fork stress biomarkers was assessed by (fluorescent) immunohistochemistry and DNA fibre analysis.

Results: Both siRNA and drug assays identified a series of genes involved the DNA damage response (DDR) to be candidate genetic dependencies in SS. siRNA screen hits included ATR, the ATR-activating proteins RAD9A and RAD18, and the two tumour suppressor genes BRCA1 and BRCA2. The drug screen identified a dependency on two clinical PARP inhibitors (PARPi); talazoparib and rucaparib. ATR and PARP sensitivity in SS cells was comparable to that of tumour cells harbouring known molecular defects associated with sensitivity. A series of validation assays using clinical ATR (ATRi) and PARP inhibitors (PARPi) confirmed a synthetic lethal effect in SS tumour cell lines in vitro, which was confirmed for ATRi in patient-derived xenografts in vivo. Ectopic expression of SS18-SSX fusion proteins in non-SS cell lines elicited ATRi and PARPi sensitivity, which was mediated by a reduction in SS18 and SMARCB1 protein levels. ATRi sensitivity in SS was characterised by an increase in biomarkers of replication fork stress (increased yH2AX, decreased replication fork speed and increased R-loops), an apoptotic response and was found to be dependent upon Cyclin E expression. Finally, we found that combinations with cisplatin or PARP inhibitors enhanced the anti-tumour cell effect of ATRi, suggesting that either single agent ATRi or combination therapy involving ATRi might be further assessed as a candidate approach for SS treatment.

Conclusions: Our integrated analysis identified an unexpected dependency of SS tumour cells on DDR mediators. Importantly, sensitivity was synthetic lethal with SS18-SSX oncoproteins and is clinically actionable.

#2976

Confirmation of in-cell target engagement using the proteolysis targeting chimeras (PROTACs) against pirin.

Swee Y. Sharp,1 Nicola E. Chessum,1 John J. Caldwell,1 Marissa V. Powers,2 A Elisa Pasqua,1 Birgit Wilding,1 Ian Collins,1 Bugra Ozer,1 Martin Rowlands,1 Mark Stubbs,1 Rosemary Burke,1 Rob L. van Montfort,1 Matthew D. Cheeseman,1 Paul A. Clarke,1 Paul Workman,1 Keith Jones1. 1 _Institute of Cancer Research, London, United Kingdom;_ 2 _Inst. of Cancer Research, Sutton, United Kingdom_.

We recently reported the identification of the original bisamide lead compound CCT251236 as an inhibitor of the HSF1 stress pathway with a high affinity for the putative transcription factor co-regulator, pirin (SPR KD=44nM) (Cheeseman et al., J Med Chem, 60; 180-201, 2017). Pirin is a highly conserved non-heme iron-binding regulatory protein that is a member of the functionally diverse cupin superfamily, but has no known enzymatic function or biomarkers of activity. To understand further this poorly characterized protein and to confirm that CCT251236 binds to pirin within living cells, we conceived and optimized a heterobifunctional protein degradation probe using the proteolysis targeting chimeras (PROTACs; CCT367766) comprising a pirin-binding moiety linked to the cereblon-targeting ligand thalidomide. This PROTAC molecule was designed to recruit pirin to the E3 ubiquitin ligase cereblon resulting in the ubiquitylation and degradation of pirin. Negative control probes lacking binding to pirin (CCT367857) or cereblon (CCT367936) were also designed and synthesized. We demonstrated a concentration-dependent depletion of pirin protein from as low as 0.5nM and as early as 2 hr treatment of SKOV3 human ovarian cancer cells with the PROTAC. The negative controls CCT367857 and CCT367936 exhibited no pirin depletion at equimolar concentrations. At higher concentrations of the active probe, a hook effect is observed, consistent with the formation of a ternary complex. Degradation of pirin by the PROTAC was confirmed to be proteasome-dependent by rescue of depletion following pre-incubation with the proteasome inhibitor MG132. In addition, the PROTAC could not induce pirin degradation in CRISPR/cas9 cereblon knockout SKOV3 cells, confirming dependence on cereblon. Pre-treatment with the bisamide compound CCT251236 or free thalidomide abrogated the PROTAC-induced pirin degradation, consistent with pirin and cereblon engagement. Finally, to estimate the cellular selectivity of the PROTAC to pirin in an unbiased manner, we carried out whole proteome mass spectrometry in SKOV3 cells. From 8547 quantifiable proteins identified, only pirin (2.3-fold reduction) displayed a statistically significant (Padj<0.05) difference in protein expression, indicating impressive selectivity. In summary, we have designed a PROTAC as an intracellular probe against a poorly understood molecular target, pirin. This approach has allowed us to confirm in-cell target engagement of our bisamide lead CCT251236 with pirin and validates CCT367766 as a PROTAC tool to further study this largely unexplored protein. Our results also provide a systematic approach for the use of the powerful PROTAC technology to investigate potential and poorly understood cancer drug targets.

#2977

**PI3K/mTOR pathway inhibition induces Aurora B mediated cell death in** NOTCH1 **mutant head and neck squamous (HNSCC) cells.**

Vaishnavi Sambandam,1 Li Shen,1 Pan Tong,1 Shaohua Peng,1 Tuhina Mazumdar,1 Ratnakar Singh,1 Curtis R. Pickering,1 Jeffrey N. Myers,1 Jing Wang,1 Mitchell Frederick,2 Faye M. Johnson1. 1 _MD Anderson Cancer Center, Houston, TX;_ 2 _Baylor College of Medicine, Houston, TX_.

Genomic alterations in the PI3K/mTOR pathway occur in 54% of HNSCC patients. To identify novel biomarkers of response to PI3K/mTOR pathway inhibitors in HNSCC, we tested the efficacy of 7 PI3K/mTOR pathway inhibitors in 59 HNSCC cell lines and determined the association between drug sensitivity and genomic alterations. We identified that NOTCH1mut lines were significantly more sensitive to PI3K/mTOR pathway inhibitors than NOTCHWT lines: GSK2126458 (12/14 NOTCH1Mut lines), BYL719 (6/14), PQR309 (12/14), BKM120 (14/16), BEZ235 (12/16), BAY806942 (13/14) and GDC0980 (5/14 lines). In contrast to PIK3CAmut cell lines that experienced cell cycle arrest, after PI3K/mTOR pathway inhibition, NOTCH1mut lines underwent significant apoptosis in addition to G1/S cell cycle arrest. NOTCH1mut lines also showed reduced clonogenic growth in vitro and tumor growth inhibition in vivo in both oral orthotopic and subcutaneous xenograft mouse models. NOTCH1 knock out (KO) by CRISPR-Cas9 system in a NOTCH1WT line (PJ34) rendered it more sensitive to PI3K/mTOR inhibition.

After PI3K/mTOR inhibition, PJ34-NOTCH1 KO showed significant reduction in clonogenic growth (1.57-fold; P<0.05) and increased apoptosis (4.3-fold; P<0.05) compared to the parental line.

As no canonical pathways account for the underlying mechanism of sensitivity, we measured the level of 301 proteins by reverse phase protein array (RPPA) in 3 NOTCH1mut and 3 NOTCH1WT lines after GSK2126458 treatment. Several proteins related to cell cycle were differentially regulated in NOTCH1mutcells compared to wild type lines. Notably, both mRNA and protein levels of Aurora B were significantly decreased in NOTCH1mutcells but not in NOTCHwt cells following PI3K/mTOR inhibition. Aurora B is an important cell cycle regulator and deregulation of Aurora kinases leads to defective chromosomal segregation and mitotic catastrophe in numerous cancers. Aurora kinase inhibitors as single agent are highly effective in a panel of NOTCHwt cell lines as demonstrated by decreased colony formation ability and proliferation as well as G2/M arrest and apoptosis.

Inhibition of Aurora kinases in combination with PI3K inhibitors displayed synergy (Combination Index<1) in 64% of NOTCH1 wild type lines (26/44) and 66% of NOTCH1mutcell lines (8/12) also exhibited increased sensitivity as assessed by Cell-titer Glo assay. Aurora B knock down and over expression studies are underway to validate the finding.

This work is significant because inactivating NOTCH1 mutations, which occur in 18% of HNSCC patients and SCCs of the lung, esophagus, and other sites, may serve as a biomarker for response. Our present work may uncover potential combination therapies for HNSCC.

#2978

The utilization of a human MCL1 knockin mouse suggests that reductions in B-cells and monocytes may serve as clinically relevant pharmacodynamic markers of MCL1 inhibition.

Brian Belmontes, Sean R. Caenepeel, Jan Sun, Danny Chui, Angela Coxon, Gordon Moody, Jude Canon, Paul Hughes. _Amgen Inc., Thousand Oaks, CA_.

The development of small-molecule inhibitors targeting pro-survival BCL-2 family members, such as MCL1 and BCL-2, is an attractive approach for the treatment of cancer. AMG 176 is a potent and selective first-in-class MCL1 inhibitor currently in phase I clinical trials in multiple myeloma and AML. AMG 176 and its analogue AM-8621 exhibit picomolar affinity for human MCL1. However, binding affinity for murine MCL1 is reduced by ~200-fold. Examination of the AMG 176 binding pocket in human MCL1 suggests Leu248 contributes to the high affinity interaction with AMG 176. In mouse MCL1, the corresponding amino acid is a phenylalanine, reducing the affinity of AMG 176 for the mouse protein. This reduced affinity makes it challenging to assess the pharmacodynamic effect of AMG 176 on normal hematopoietic cell types at efficacious doses in human tumor xenograft models. To overcome this challenge we generated a human MCL1 knockin mouse in which the mouse genomic Mcl1 locus was replaced with its human counterpart. Human MCL1 knockin (KI) mice were created by targeting embryonic stem (ES) cells with a vector containing the human MCL1 genomic locus flanked by the homologous mouse sequences up- and downstream of mouse Mcl1. Targeted ES cells were confirmed by Southern blot and used to generate chimeric mice. Germline transmission of the human MCL1 KI allele and the removal of selectable markers was accomplished by breeding with cre-expressing transgenic mice. Homozygous human MCL1 KI mice were created by breeding heterozygous human MCL1 KI mice lacking the Cre transgene and selectable markers. We used a splenocyte viability assay to confirm the functional replacement of murine Mcl1 with its human ortholog. The in vitro treatment of splenocytes with AM-8621 for 6 hours resulted in dose-dependent Caspase 3 activation and reduced viability in B and T cells derived from human MCL1 knockin mice. Oral administration of AMG 176 at doses of 30 and 60 mg/kg resulted in the induction of apoptosis and the subsequent inhibition and regression of OPM-2 multiple myeloma tumor xenografts (54% tumor growth inhibition and 21% regression, respectively). The human MCL1 knockin mice were used to test the effect of AMG 176 at an equivalent dose and schedule on B cells, T cells and monocytes derived from peripheral blood, spleen and bone marrow. Samples treated with 30 and 60 mg/kg of AMG 176 were harvested 24 hours post-second dose of the first and second cycles. We observed a dose-dependent decrease in monocytes and B cells at both time points, with the most significant reductions observed in monocytes (70.1% and 94.5%, respectively) and B cells (81.5% and 94.0%, respectively) derived from peripheral blood after the first cycle. In conclusion, these data suggest that reductions in B cells and monocytes may serve as clinically relevant pharmacodynamic endpoints of MCL1 inhibition.

## IMMUNOLOGY:

### Defining New Immunotherapeutic Targets through Deep Molecular Characterization

#2979

A balance of genomic instability, tumor-immune contexture and TGF-β signaling contributing to exclusion of T cells governs response to PD-L1 checkpoint blockade.

Sanjeev Mariathasan,1 Shannon J. Turley,1 Dorothee Nickles,1 Alessandra Castiglioni,1 Kobe Yuen,1 Yulei Wang,1 Edward E. Kadel,1 Hartmut Koeppen,1 Jillian L. Astarita,1 Rafael Cubas,1 Suchit Jhunjhunwala,1 Yagai Yang,1 Yasin Şenbabaoğlu,1 Ira Mellman,1 Daniel S. Chen,1 Priti Hegde,1 Richard Bourgon,1 Thomas Powles2. 1 _Genentech, South San Francisco, CA;_ 2 _Barts Experimental Cancer Medicine Centre, London, CA_.

Checkpoint inhibitor blockade can result in robust and durable anti-tumor responses in various cancers, including metastatic urothelial cancer (mUC). However, these responses only occur in a subset of patients. Identifying determinants of response and resistance to cancer immunotherapy is critical for extending therapeutic benefit to more patients. Atezolizumab (anti-PD-L1) was approved in the US for the treatment of mUC based on the single-arm Phase II study IMvigor210 (NCT02108652; n= 429 patients). Here, we examined the biology underlying primary immune escape and responsiveness to anti-PD-L1 in tumor samples of patients from IMvigor210.

Methods: PD-L1 expression on tumor-infiltrating immune cells was assessed with SP142 IHC. Exploratory analyses in evaluable pre-treatment tissues included: (i) CD8 IHC analysis to define immune deserts, excluded and inflamed subtypes (ii) whole-transcriptome RNA sequencing to identify pathways associated with response and to perform tumor molecular subtyping, (iii) targeted mutational profiling (FoundationOne) to estimate tumor mutation burden, and (iv) whole-exome sequencing to predict putative neoantigens. EMT6-grafted BALB/c mice treated with anti-TGF-β and/or anti-PD-L1 antibodies were evaluated for tumor growth inhibition.

Results: Response was associated with CD8+ T-effector gene expression and, to an even greater extent, high neoantigen or tumor mutation burden (TMB). Gene expression pathways significantly associated with high TMB were those involved in cell cycle, DNA replication, DNA damage response (DDR). Tumors with mutations in DDR gene sets also had significantly high TMB and response rates. Lack of response was associated with a signature of transforming growth factor β (TGF-β) signaling in fibroblasts, particularly in patients with CD8+ T cells that were excluded from the tumor parenchyma and instead found in the fibroblast- and collagen-rich peritumoral stroma. Using a mouse model that recapitulates this immune excluded phenotype, we found that therapeutic administration of a TGF-β blocking antibody together with anti-PD-L1 reduced TGF-β signaling in stromal cells, facilitated T cell penetration into the centre of the tumor, and provoked vigorous anti-tumor immunity and tumor regression.

Conclusion: Pre-existing T-cell immunity and TMB are associated with response to atezolizumab in mUC, whereas TGF-β signaling in the stroma is a negative indicator of response, especially in immune-excluded tumors, a common phenotype of mUC. Integration of these three independent biological features provides a best basis for understanding clinical outcomes in this setting. Furthermore, the data suggests that TGF-β shapes the tumor microenvironment to restrain anti-tumor activity by restricting T cell infiltration.

#2980

Single cell RNA sequencing reveals AML immunoediting under pressure from engineered T cell therapy.

Kelly G. Paulson,1 Thomas M. Schmitt,1 Daniel Egan,2 Valentin Voillet,2 Miranda C. Lahman,3 Felecia D. Wagener,2 Daniel S. Hunter,2 Petri R. Muhlhauser,2 Paul C. Hendrie,4 Cecilia Yeung,2 Nathalie Vigneron,5 Benoit Van den Eynde,5 Jason H. Bielas,2 Merav Bar,3 Raphael Gottardo,2 Aude G. Chapuis,6 Philip D. Greenberg6. 1 _*Equal Contributions - Fred Hutchinson Cancer Research Center, Seattle, WA;_ 2 _Fred Hutchinson Cancer Research Center, Seattle, WA;_ 3 _University of Washington, Seattle, WA;_ 4 _Seattle Cancer Care Alliance, Seattle, WA;_ 5 _Ludwig Institute for Cancer Research, Brussels, Belgium;_ 6 _**Equal Contributions - Fred Hutchinson Cancer Research Center, Seattle, WA_.

Targeting relapsed acute myeloid leukemia (AML) with engineered T cells holds exceptional promise, but obstacles to efficacy exist. Understanding resistance mechanisms is essential to improve therapy and may broadly inform cancer immune escape. We and others have developed transgenic T cell receptor (TCR) therapies targeting the intracellular WT1 oncoprotein that promotes growth of most high-risk AMLs. TCR therapies generally target epitopes processed by the proteasome for Class I presentation. As mutation or loss of WT1 or loss of HLA expression is infrequent in AML, we hypothesized resistant clones may have defective antigen processing. A 25-year-old man with AML recurrent after a 2nd allogeneic stem cell transplant received reinduction chemotherapy and transgenic donor-derived T cells recognizing the HLA-A2 restricted WT1126-134 epitope (NCT01640301). The patient experienced a 12-month remission followed by relapse despite robust T cell persistence (>6% of CD8+ T cells). The relapsed leukemia expressed WT1 and HLA-A2 but was refractory to retreatment with additional WT1126-134-restricted T cells. Single cell RNA sequencing (scRNAseq) was performed on cryopreserved PBMCs from remission (n=4976 cells) and relapsed (n=2780) time points (10x Genomics platform). During apparent remission, transgenic T cells had a transcriptome distinct from native CD8s, expressing activation markers including HLA-DR, IL32, CD69 and CTLA4, suggesting recent TCR triggering from antigen encounter. However, at the time AML progression was observed, transgenic cells clustered with native quiescent CD8s, implying they were no longer encountering the targeted epitope. Transcriptome analysis of relapsed AML demonstrated downregulation of a single subunit of the immune proteasome (β1i), which we recently demonstrated in cell lines to be critical for processing the targeted WT1126-134 epitope. This patient's refractory AML was killed by T cells restricted to an alternate HLA-A2 epitope of WT1 processed independently of β1i, confirming the functional impact of immune proteasome downregulation. At an early responding timepoint, scRNAseq detected clinically unrecognized rare circulating AML cells already β1i-low, suggesting β1i loss represented immunoediting/outgrowth of a pre-existing clone as a consequence of T cell immunotherapy pressure. ScRNAseq is poised to become an important diagnostic in immunotherapy, with sufficient depth to profile both transgenic T cells and low burden/minimal residual AML, and, in our case, successfully identified a potentially targetable mechanism of immune escape. Our data suggest T cell therapy in AML and other tumors may be improved by in depth analysis of individual leukemic cells, and from selecting target epitopes processed by both standard and immunoproteasomes and/or early combination therapy combining TCRs recognizing differentially processed epitopes.

#2981

Clonal deletion of tumor-specific T cells by combination checkpoint blockade compromises antitumor efficacy in low tumor burden states.

Chien-Chun Steven Pai,1 Xiaoqing Lu,2 Donald Simons,3 Michel DuPage,4 Kole T. Roybal,1 Mingyi Chen,5 Serena Kwek,1 Amy-Jo Casbon,1 Gillian A. Kinsbury,3 Lawrence Fong1. 1 _UCSF, San Francisco, CA;_ 2 _Abbvie, MA;_ 3 _Former Abbvie Employee, MA;_ 4 _UC Berkeley, Berkeley, CA;_ 5 _UT Southwestern, Dallas, TX_.

A myriad of combination immunotherapies is being developed as cancer treatments with the goal of enhancing antitumor immunity. We found that depending on the disease setting, combined immune checkpoint blockade may not always lead to synergistic antitumor effects. While combined anti-CTLA-4 and anti-PD-1 improves control of established tumors, this combination can paradoxically compromise antitumor immunity in the low tumor burden state in preclinical models as well as in melanoma patients. This paradoxical outcome results from treatment-induced apoptosis of tumor-specific T cells. These changes further alter the overall T-cell receptor repertoire. Activated tumor-specific T cells express higher levels of IFN-γ receptor and are more susceptible to apoptosis. Deficiency of IFN-γ receptor on immune cells rescues this phenotype and restores antitumor activity. Additionally, tumor-specific T cells lacking the IFN-γ receptor demonstrate a significant survival advantage compared to their wild-type counterparts in tumor-bearing mice receiving combination therapy. Finally, combination therapy induces significantly higher levels of IFN-γ in the low versus high tumor burden state on a per cell basis, reflecting their less exhausted immune status. Thus, the optimal immunotherapy strategy may depend on disease context, and may not always favor more potent combination immunotherapies.

#2982

Somatic TP53 mutations alter the immune microenvironmentafter chemotherapy in breast cancer.

Mellissa J. Nixon,1 V. Monica Estrada,2 Susan R. Opalenki,1 Donna Hicks,1 Michael Korrer,1 Melinda E. Sanders,1 Roberto Salgado,3 Young Kim,1 Rebecca Cook,1 Carlos L. Arteaga,4 Justin M. Balko1. 1 _Vanderbilt University, Nashville, TN;_ 2 _UCSD, San Diego, CA;_ 3 _Institut Jules Bordet, Nashville, Belgium;_ 4 _UT Southwestern, Dallas, TX_.

Background: Neoadjuvant chemotherapy (NAC), followed by surgery, is the standard of care for triple-negative breast cancer (TNBC) patients. Unfortunately, only 30% achieve a pathologic complete response (pathCR, no evidence of invasive disease in the breast after NAC). In patients who do not achieve a pathCR, tumor-infiltrating lymphocytes (TILS) correlate with improved survival. This suggests there is an immune response to NAC in a subset of patients, which may be augmented by immune checkpoint inhibitors (ICI). Clinical trials are under way to examine the efficacy of ICI in breast cancer; therefore it is vital to identify biomarkers of response to better identify patients who may benefit from these modalities. Because many TNBC patients harbor somatic TP53 mutations and TP53 has been shown to be involved in immunity, we investigated the role of TP53 mutations on the immune microenvironment of breast cancer patients with residual disease.

Methods: We examined 34 matched pre- and post-NAC primary breast cancer. TILs were scored by a pathologist. RNA and DNA were extracted and analyzed using nanoString Pan-Cancer Immune panel (>700 genes) and ImmunoSeq T-cell receptor (TCR) sequencing, respectively. CRISPR technology was used in mouse tumor cell lines to generate Trp53 mutations commonly found in TNBC. Cells were treated with chemotherapy in vitro to determine tumor-specific changes in cytokines and PD-L1 expression. To determine alterations to the immune microenvironment, isogenic cell lines were injected into syngeneic mice and expression of immune recruiting cytokines and immune checkpoint molecules by the tumor as well as immune phenotyping of TILs after NAC was performed.

Results: TILs in the residual disease of TNBC patients correlated with improved survival after surgery, as expected. A third of patients had increased immune gene signatures after NAC, which correlated with increased TCR clonality, increased overall survival, and increased T cell-recruiting chemokine expression (CXCL9/10, etc.). In isogenic murine breast cancer cells, Trp53 mutant cells had higher levels of immune-recruiting chemokines after chemotherapy treatment in vitro, but demonstrated loss of Cdkn1a, a canonical p53 target gene. Trp53 mutant cells also exhibited increased PD-L1 expression after NAC compared to Trp53 wild-type cells. Mouse studies examining the immune infiltrate are ongoing.

Conclusions: Our work suggests that NAC induces immune gene signatures in a subset of TNBC that is correlated with a better prognosis. TP53 mutations in the tumor may contribute to an increased immune infiltrate after chemotherapy through upregulation of chemokine expression and promote T cell exhaustion through upregulation of the immune checkpoint PD-L1. Ongoing in vivo studies with chemotherapy and ICI will indicate if p53-mutant tumors have a better response to ICI after NAC.

#2983

CD25 enables oncogenic BCR- and TCR-signaling and represents a therapeutic target in lymphoblastic malignancies.

Jaewoong Lee,1 Huimin Geng,2 Zhengshan Chen,1 Gang Xiao,1 Kadriye Nehir Cosgun,1 Francesca Zammarchi,3 Patrick Van Berkel,4 Ari Melnick,5 Elisabeth Paietta,6 Markus Muschen1. 1 _Beckman Research Institute of City of Hope, Monrovia, CA;_ 2 _University of California San Francisco, CA;_ 3 _ADC Therapeutics (UK) Ltd, United Kingdom;_ 4 _ADC Therapeutics, NJ;_ 5 _Weill Cornell Medical College, NY;_ 6 _Montefiore Medical Ctr. North Div, Monrovia, NY_.

B- and T-cells critically depend on survival and proliferation signals from a functional B-cell (BCR) or T-cell receptor (TCR). In >50%, tumor clones are driven by oncogenic B/TCR-mimics, e.g. mutant B/TCR-signaling chains (Igβ, CD3ζ), BCR-ABL1, viral oncoproteins (e.g. EBV; KSHV, HTLV-I) and NF-κB-pathway lesions. Despite this heterogeneity, our correlative analyses showed that oncogenic B/TCR signaling in lymphoid malignancies is uniformly characterized by high surface levels of CD25.

CD25 is known as α-chain of the IL2 receptor. Our results revealed an unrecognized function as essential feedback regulator of oncogenic B/TCR-signaling. In adults (MDACC, ECOG) and children (COG; P9906) with Ph-like or Ph+ ALL, high surface levels of CD25 predict particularly poor outcome (n=416; P=0.005). While CD25 mediates IL2 signaling in T cells, CD25 on B- cells was not responsive to IL2. Our studies showed that oncogenic B/TCR-signaling rapidly induced surface translocation of CD25 by PKCδ-dependent phosphorylation (S268) and transcriptional activation by NF-κB. Engagement of CD25 induced recruitment of an inhibitory phosphatase complex (SHIP1, PP2A) at the cell membrane to limit kinase signaling (e.g. SYK, SRC and ERK) downstream of the B/TCR or its oncogenic mimics. Consistent with a role as negative feedback regulator, genetic deletion of CD25 in B cells caused uncoordinated Ca2+ oscillations, p53 checkpoint activation and cell cycle arrest.

In leukemia models, CD25-/- cells failed to initiate fatal disease in transplant recipients, which could be rescued by expression of the CD25 cytoplasmic tail. Depending on PKCδ-mediated phosphorylation at S268, CD25 shuttles between cytoplasm and cell membrane. Mirroring cellular activation states, only a fraction of tumor cells express CD25 on the surface at any given time, while pharmacological activation of PKCδ (I3A) forced continuous surface expression.

As strategy to eradicate CD25-expressing B- and T-cell malignancies, we combined PKCδ-activation by I3A with a CD25-antibody drug conjugate (ADCT-301) to treat NSG mice xenografted with patient-derived Ph+ ALL cells. While ADCT-301 induced durable remissions alone, combinations of I3A and ADCT-301 eradicated disease with NSG mice surviving for indefinite periods of time. In conclusion, we identified the IL2Rα chain CD25 as a previously unrecognized feedback regulator of oncogenic B/TCR-signaling and as a therapeutic target in refractory lymphoid malignancies.

#2984

Effects of anti-CTLA-4 and anti-PD-1 on memory T-cell differentiation and resistance to tumor relapse.

Stephen Mok, Colm R. Duffy, James P. Allison. _MD Anderson Cancer Center, Houston, TX_.

The FDA has begun to expand the approved uses of immune checkpoint blockade antibodies targeting CTLA-4 and PD-1. Blocking either checkpoint relieves the negative regulation of T-cells resulting in significant responses in patients with cancer. Data has now begun to emerge regarding differences between these two therapies. While αPD-1 therapy has a greater response rate (~30% vs. 11%) according to RECIST criteria, recent reports have suggested responses to αPD-1 may not be as durable as αCTLA-4. 25% of patients who initially responded to αPD-1 have tumor relapse within 24 months. In contrast, the 3-year survival rate of patients treated with αCTLA-4 is at least ~25% suggesting a durable response. Previous studies in bacterial or chronic LCMV infectious models have shown that αCTLA-4 can increase CD8+ memory T-cell formation, whereas genetic ablation of PD-1 on T-cells often promotes the terminally differentiated exhausted CD8+ T-cell phenotype, while attenuating memory T-cell formation. However, the mechanism which leads to relapse following αPD-1 treatment in tumor models is not clear. The goal of this project is to understand how immunotherapies shape memory T-cell formation and how that relates to the mechanism of tumor relapse.

To test whether αCTLA-4 or αPD-1 can induce a better memory T-cell response, mice were vaccinated with irradiated B16F10 murine melanoma cells and treated αCTLA-4 or αPD-1. Mice were re-challenged with B16F10 80 days after vaccination. Although both αCTLA-4 and αPD-1 improved tumor rejection compared with controls, αCTLA-4 treated mice exhibited superior tumor control compared to αPD-1 (p<0.0005) suggesting the memory T-cell response mediated by αCTLA-4 is more durable. In order to test whether this memory T-cell response is antigen-specific, mice were re-challenged with unrelated MC38 or 3LL cancer cells. The antibody treated groups did not show improved antitumor effect compared with vaccine control (p>0.5). To test whether the frequency of memory T-cells recruited to the re-challenged tumor could affect memory T-cell response, antigen-specific pmel-1 T-cells were infused to mice following vaccination with αCTLA-4 or αPD-1. Our result suggested that there were more tumor-infiltrating pmel-1 T-cells in the αCTLA-4 treated group compared to the αPD-1 treated group (p<0.05). In order to augment the durability of αPD-1 treatment, αPD-1 was combined with αCTLA-4 following vaccination. The combined treatment group has superior antitumor response compared to that with αPD-1 (p<0.000003) and overlapped with the αCTLA-4 treated group (P>0.05) during re-challenge, indicating that the effect of the combined treatment is dominated by αCTLA-4.

Collectively, our studies facilitate the design of combination immunotherapy treatments that enhance both response rates and generation of memory T-cells to prevent relapse.

#2985

Radiotherapy and αCD40 non-redundantly augment immunity to checkpoint blockade in refractory pancreatic ductal adenocarcinoma.

Hannah Dada, Andrew J. Rech, Christina Twyman-Saint Victor, Andy J. Minn, Robert H. Vonderheide. _University of Pennsylvania, Philadelphia, PA_.

Despite the success of cancer immunotherapy in many disease types, pancreatic ductal adenocarcinoma (PDA) is notably unresponsive to immune checkpoint blockade (ICB) with αPD1 and/or αCTLA4. The mechanism of resistance is poorly understood, but tumor epitopes and the microenvironment, which is immunosuppressive and excludes T cells, are thought to be contributory. To improve response to ICB, we used subcutaneous and orthotopic murine models of PDA to investigate the effect of combination therapy with ICB (CTLA-4 and PD-1 antagonist antibodies), CD40 agonist antibody and radiation therapy (RT). Combination therapy with CD40 agonist antibody, ICB and RT resulted in decrease tumor burden, increase overall survival, and generation of long-term immunity. Response is dependent on T and short-lived myeloid cells, while it is independent of innate activation pathways. Together, these results suggest a dual role for both the innate and adaptive immune response in treating PDA.

## MOLECULAR AND CELLULAR BIOLOGY / GENETICS:

### Drugging the Undruggable: Using Synthetic Lethality and Other Approaches to Develop New Treatment Strategies

#2986

E-cadherin/ROS1 inhibitor synthetic lethality in breast cancer.

Ilirjana Bajrami,1 Rebecca Marlow,2 Marieke van de Ven,3 Rachel Brough,1 Helen N. Pemberton,1 Jessica Frankum,1 Fei Fei Song,1 Rumana Rafiq,1 Asha Konde,1 Malini Menon,1 James Campbell,1 Aditi Gulati,1 Rahul Kumar,1 Stephen J. Pettitt,1 Mark D. Gurden,1 Marta Llorca Cardenosa,1 Irene Chong,1 Patrycja Gazinska,2 Fredrik Wallberg,1 Elinor J. Sawyer,2 Lesley-Ann Martin,1 Mitch Dowsett,1 Spiros Linardopoulos,1 Colm Ryan,4 Patrick W. Derksen,5 Jos Jonkers,3 Andrew N.J. Tutt,1 Alan Ashworth,6 Christopher J. Lord1. 1 _The Institute of Cancer Research, London, United Kingdom;_ 2 _Kings College London, London, United Kingdom;_ 3 _The Netherlands Cancer Institute, Amsterdam, Netherlands;_ 4 _University College Dublin, Dublin, Ireland;_ 5 _University Medical Center Utrecht, Utrecht, Netherlands;_ 6 _UCSF Helen Diller Family Comprehensive Cancer Centre, San Francisco, CA_.

The E-cadherin (CDH1) tumour suppressor gene encodes a calcium-dependent cell-cell adhesion glycoprotein, which has roles in maintaining cell polarity, differentiation, cell migration and survival. E-cadherin dysfunction is a feature common to many epithelial tumours, with the highest incidence occurring in diffuse gastric cancer (50%) and lobular breast cancer (63%) and can occur via CDH1 mutation, deletion or epigenetic silencing. Although E-cadherin dysfunction is relatively common, precision medicine approaches that exploit this pathogenic alteration are not yet available. Using genetic and small-molecule perturbation screens in breast tumour cells with CRISPR-Cas9 engineered CDH1 mutations, we identified a synthetic lethal interaction between E-cadherin deficiency and inhibition of the tyrosine kinase ROS1. Using data from large-scale genetic screens in molecularly diverse breast tumour cell lines, we found that the E-cadherin/ROS1 synthetic lethality was not only robust in the face of considerable molecular heterogeneity but was also elicited with clinical ROS1 inhibitors including foretinib and crizotinib. ROS1 inhibitors induced mitotic abnormalities and multinucleation in E-cadherin defective cells, phenotypes that were associated with a defect in cytokinesis and aberrant p120-catenin phosphorylation and localisation. In vivo, ROS1 inhibitors produced profound anti-tumour effects in multiple, distinct, models of E-cadherin defective breast cancer. This data therefore provides the pre-clinical rationale for assessing ROS1 inhibitors such as the licensed drug crizotinib in appropriately stratified patients.

#2987

Identification of new combination therapies for lung tumors harboring KRAS mutations.

Miriam Molina-Arcas,1 Christopher Moore,1 Sareena Rana,2 Febe van Maldegem,1 Stuart Horswell,1 David Hancock,1 Julian Downward1. 1 _The Francis Crick Institute, London, United Kingdom;_ 2 _Institute of Cancer Research, London, United Kingdom_.

Oncogenic mutations in KRAS are frequent in non-small cell lung cancer (NSCLC) and have been associated with poor prognosis and resistance to existing therapies. Thus, new therapeutic strategies are needed to treat these tumors. We have previously shown that NSCLC cells harboring KRAS mutations are more sensitive to inhibition of KRAS downstream effectors MEK and RAF and to treatment with IGF1R inhibitors than their wild-type counterparts. In order to identify complementary targets for the improvement of IGF1R and/or MEK targeting therapies, we performed a whole-genome shRNA screen in KRAS-mutant NSCLC cells. Interestingly, the list of sensitizers to IGF1R inhibitors included several genes encoding components of the mTOR pathway. Viability assays in a panel of lung cancer cell lines confirmed that combinations of IGF1R inhibitors with mTOR inhibitors, both rapalogs and kinase inhibitors, resulted in a synergistic anti-proliferative effect in KRAS-mutant NSCLC cells. Mechanistic investigations demonstrated some differences between the effects of rapalogs and mTOR kinase inhibitors although, in both cases, mTOR inhibitors increased IGF1R and AKT phosphorylation. Combination with IGF1R inhibitors blocked the reactivation of the PI3K pathway resulting in a robust suppression of PI3K and mTORC1 signaling. Notably, the inhibition of these signaling pathways was stronger in KRAS-mutant cells than in wild-type cells. Addition of a MEK inhibitor to the combination produced a more profound and durable suppression of cell proliferation and a stronger induction of apoptosis. Finally, we validated the different drug combinations in mouse models of KRAS-induced NSCLC. Results showed that the three-drug combination produced a marked tumor regression in a NSCLC mouse model driven by mutant Kras and p53 loss-of-function and also in urethane-induced lung tumors. These findings suggest potential novel therapeutic strategies for NSCLC tumors harboring KRAS mutations.

#2988

Loss of Rbm38 cooperates with mutant p53 to promote lymphomagenesis through downregulation of Pten.

Jin Zhang, Heejung yang, Yanhong Zhang, Mingyi Chen, Xinbin Chen. _UC Davis, Davis, CA_.

The tumor suppressor TP53 is the most commonly mutated gene in human cancers and mutant p53 has a gain of function by promoting tumor progression, metastasis, and resistance to anticancer therapy. Interestingly, the RNA binding protein Rbm38, a critical regulator of mutant p53 translation, is frequently lost in tumors with mutant p53. To determine the role of the Rbm38-mutant p53 axis in tumorigenesis, we showed that loss of Rbm38 significantly alters the cancer susceptibility of mutant p53 knockin mice by shortening the lifespan, altering the tumor incidence, and promoting T-cell lymphomagenesis. To understand how Rbm38 deficiency promotes mutant p53 gain of function, we showed that loss of Rbm38 not only enhances mutant p53 expression but also decreases expression of tumor suppressor Pten, a critical regulator of T-cell development. Furthermore, we showed that Rbm38 is required for Pten expression by stabilizing Pten mRNA through an AU-rich element in its 3'UTR. Thus, we have uncovered a novel mechanism by which Pten is regulated by Rbm38 via mRNA stability. Our data also suggest that Rbm38 controls T-cell lymphomagenesis by concomitantly modulating mutant p53 and Pten, which may be explored as a therapeutic strategy for the treatment of T-cell malignancies.

#2989

BET inhibitors induce Rac1-dependent MNK and eIF4E phosphorylation in cancer cells.

Thao Pham, Brian T. Decant, Krishan Kumar, Meng Shang, Maria Matsangou, Kazumi Ebine, Hidayatullah G. Munshi. _Northwestern University, Chicago, IL_.

Patients with recurrent differentiated thyroid cancers (DTCs) have poor prognosis and suffer from multiple complications from progressive symptomatic disease. Here we show that eukaryotic translation initiation factor 4E (eIF4E), an oncogene that is overexpressed in aggressive thyroid cancers, is required for growth of DTC cells in collagen microenvironment. We show that inhibitors targeting bromodomain and extra-terminal domain (BET) family of proteins also decrease growth of DTC cells in the collagen microenvironment. Unexpectedly, we have found that BET inhibitors induce MNK and eIF4E phosphorylation in cancer cells. Mechanistically, BET inhibitors induce Rac1-mediated cytoskeletal changes and targeting Rac1 blocks these changes as well as MNK and eIF4E phosphorylation. Functionally, we show that MNK inhibitors potentiate the effects of BET inhibitors at suppressing cancer cell proliferation and limiting sphere-forming ability. Together, these results demonstrate crosstalk between BET proteins and the MNK-eIF4E pathway, suggesting that combination therapy with BET and MNK inhibitors has the potential for synergistic inhibition of cancer cells.

#2990

Attenuation of RNA polymerase II pausing mitigates BRCA1-associated R-loop accumulation and tumorigenesis.

Xiaowen Zhang, Huai-Chin Chiang, Yao Wang, Chi Zhang, Victor X. Jin, Yanfen Hu, Rong Li. _UT Health Science Ctr. at San Antonio, San Antonio, TX_.

Most BRCA1-associated breast tumours are basal-like yet originate from luminal progenitors. BRCA1 is best known for its functions in double-strand break repair and resolution of DNA replication stress. However, it is unclear whether loss of these ubiquitously important functions fully explains the cell lineage-specific tumorigenesis. In vitro studies implicate BRCA1 in elimination of R-loops, DNA-RNA hybrid structures involved in transcription and genetic instability. Here we show that R-loops accumulate preferentially in breast luminal epithelial cells, not in basal epithelial or stromal cells, of BRCA1 mutation carriers. Furthermore, R-loops are enriched at the 5′ end of those genes with promoter-proximal RNA polymerase II (Pol II) pausing. Genetic ablation of Cobra1, which encodes a Pol II-pausing and BRCA1-binding protein, ameliorates R-loop accumulation and reduces tumorigenesis in Brca1-knockout mouse mammary epithelium. Our studies show that Pol II pausing is an important contributor to BRCA1-associated R-loop accumulation and breast cancer development.

#2991

Cytoplasmic mislocalization of CTCF by NPM1c in acute myeloid leukemia resulting in inhibited CTCF regulatory functions generating aberrant genetic and epigenetic profiles.

Atom Wang,1 Youqi Han,1 Peikun Chen,2 Nanyang Jia,2 Mark D. Minden1. 1 _Princess Margaret Cancer Center, TORONTO, Ontario, Canada;_ 2 _University of Toronto, TORONTO, Ontario, Canada_.

AML is not a single disease but rather a collection of diseases caused by a variety of mutations. About 30% of AML cases are characterized by a TCTG insertion in the exon 12 of Nucleophosmin 1 (NPM1). Due to this mutation, mutant NPM1 (NPM1c) is mislocalized and maintained predominantly in the cytoplasm of the cell. Previously it had been reported that NPM1 binds to CCCTC binding factor (CTCF). Based on this observation we hypothesized that NPM1c would mislocalize CTCF into the cytoplasm, preventing CTCF's access to binding sites in the nucleus, and allowing for epigenetic misregulation of gene expression.

To demonstrate that NPM1c could mislocalize CTCF to the cytoplasm we used confocal microscopy. While CTCF was completely nuclear in cells expressing NPM1, CTCF was present in the nucleus and the cytoplasm of cells expressing NPM1c. The cytoplasmic interaction of NPM1c and CTCF was confirmed by immunoprecipitation and western blotting. Using fluorescently tagged sub-fragments of NPM1c and CTCF we found that a.a. 1-264 of CTCF interacted with a.a. 244-298 of NPM1c.

CTCF has multiple effects on gene expression related to the level of CTCF in the cell/nucleus; overexpression of CTCF leads to cell senescence while reduced expression can lead to immortalization. Such effects are likely mediated by the effects of CTCF on gene expression and alternate splicing. Increased expression of HOXA9 is a characteristic of NPM1c AML and has been shown to be affected by CTCF binding between HOXA9 and HOXA7 in IMR90 cells. To determine if NPM1c would affect HOXA9 expression in IMR90 cells, we infected the cells with NPM1c or NPM1. Similar to CTCF knockdown in IMR90 cells, there was increased expression of HOXA9 in the NPM1c, but not NPM1 modified cells. Moreover, the IMR90 cells expressing NPM1c became immortal. Using chromatin immunoprecipitation we found that there was reduced binding of CTCF to the CTCF binding site 2kb downstream of HOXA9 in the IMR90 cells expressing NPM1c. Reduced CTCF binding to this site was observed in the NPM1c mutant human cell line OCI-AML-3 but not NB4 cells, an AML cell line, that lacks the mutation. Loss of CTCF binding in NPM1c expressing cells was further demonstrated by changes in CTCF mediated alternate splicing and enhancer blocking activity using reporter assays. Finally, interrogation of the TCGA AML data set showed increased methylation of CTCF binding sites in NPM1c AML. This is in keeping with de novo methylation that occurs when CTCF no longer binds to its cognate site.

Based on these studies we propose that the transforming effect of NPM1c is due, in large part, to its ability to mislocalize CTCF and suggests that targeting the interaction between these two proteins could be of potential therapeutic benefit.

### Cancer Epigenetics and Epigenetic Therapy

#2993

A switch in epigenetic silencing mechanisms of endogenous retroviruses during human genome evolution.

Hitoshi Ohtani,1 Minmin Liu,1 Wanding Zhou,1 Gangning Liang,2 Peter A. Jones1. 1 _Van Andel Institute, Grand Rapids, MI;_ 2 _University of Southern California, Los Angeles, CA_.

Approximately 9% of the human genome is derived from Endogenous Retroviruses (ERVs), which are a type of transposable element. ERVs started to become integrated into the ancestral human genome about 100 million years ago. Recent studies have reported that silencing mechanisms of long terminal repeats regions (LTRs), which are the putative promoters of ERVs, depend on DNA methylation, heterochromatin conformation (H3K9me3) and the PRC2 complex (H3K27me3). However, extensive maps of LTR distribution and the associated epigenetic marks have not yet been provided in human cancer cells. Here we report a switch in epigenetic silencing mechanisms of the approximately 450,000 LTR elements present in the non-genic regions of human DNA. Total RNA-seq in human colon cancer HCT116 cells following DNA methylation inhibitor treatment or knockdown of individual H3K9me2/3 histone methyltransferases (G9a, SETDB1, SETDB2, SUV39H1 and SUV39H2), H3K27me3 (EZH2) or a scaffold protein TRIM28 revealed that about 1,000 of evolutionary young LTRs were predominantly silenced by DNA methylation, whereas about 800 of intermediate age LTRs were silenced by histone methylation. They therefore have undergone an "epigenetic switch" in silencing mechanism during host genome evolution. Eventually, evolutionary old LTRs become inactive by accumulation of loss-of-function mutations. Consistent with these results, CpG sites in evolutionary young LTRs were methylated at higher levels than in intermediate age LTRs, whereas H3K9me2 and H3K9me3 marks were enriched on intermediate age LTRs. Interestingly, knockdowns of EZH2 showed limited upregulation of LTRs by itself, but multiple LTRs were upregulated by combination treatment with DNA methylation inhibitor. This is probably due to a process, in which removal of DNA methylation leads to the subsequent recruitment of EZH2 resulting in suppression by the polycomb repressive complex PRC2. Our earlier studies showed that LTR expressions lead to activation of viral defense pathways and apoptosis, however the species of LTRs was not characterized. We found that the activation of viral defense pathway was correlated with the upregulation of evolutionary young, but not intermediate age LTRs. Dual inhibition of DNA methylation and histone modifications by knockdown of G9a, SUV39H1 and EZH2 showed further upregulation of evolutionary young LTRs. Therefore, combination treatment targeting DNA methylation and the histone methyltransferases (G9a, SUV39H1 and EZH2) inhibitor may be useful in epigenetic therapy for cancer patients.

#2994

Discovery of selective, noncovalent small molecule inhibitors of DNMT1 as an alternative to traditional DNA hypomethylating agents.

Melissa B. Pappalardi,1 Mark Cockerill,2 Jessica L. Handler,1 Alexandra Stowell,2 Kathryn Keenan,1 Christian S. Sherk,1 Elisabeth A. Minthorn,1 Charles F. McHugh,1 Charlotte Burt,2 Kristen Wong,1 David T. Fosbenner,1 Mehul Patel,1 Jacques Briand,1 Helai Mohammad,1 Lourdes Rueda,1 Andrew Benowitz,1 Rab Prinjha,1 Dirk Heerding,1 Ryan G. Kruger,1 Ali Raoof,2 Allan Jordan,2 Bryan W. King,1 Michael T. McCabe1. 1 _GlaxoSmithKline, Collegeville, PA;_ 2 _Cancer Research UK Manchester Institute, Macclesfield, United Kingdom_.

Aberrant DNA hypermethylation within promoter regions and subsequent gene silencing are near universal hallmarks of human cancer. Reversal of DNA methylation by a hypomethylating agent, such as decitabine (Dacogen) or azacytidine (Vidaza), has shown clinical benefit for the treatment of heme malignancies. However, these agents have several limitations that preclude their full potential from being realized such as the requirement for IV administration, poor PK properties and a mechanism that requires incorporation into replicating DNA. This indirect, irreversible inhibition of the DNA methyltransferase (DNMT) family (DNMT1, 3a and 3b) and subsequent DNA damage induces significant dose-limiting toxicity thus preventing sufficient target engagement required for maximal demethylation. A drug discovery effort focused on DNMT1, the key family member responsible for maintaining the DNA methylation pattern, was initiated based on the compelling nature of the target coupled with the need for improved agents. A high-throughput screen identified a single dicyanopyridine (DCP) series of reversible, non-DNA incorporating, highly selective inhibitors for DNMT1 over DNMT3a or DNMT3b. Ensuing structure-activity relationship (SAR) optimization of the series led to the discovery of potent tool compounds that in cancer cells induced robust decreases in global DNA methylation, transcriptional activation of many silenced genes, and inhibition of cancer cell growth. In contrast to decitabine where most cells showed a cytotoxic response, our DNMT1 tool inhibitors primarily elicited a cytostatic response. Furthermore, studies in a mouse tumor model revealed decreased DNA methylation and a dose-dependent (1-45 mg/kg, BID) decrease in tumor growth with regression at the highest doses. In summary, a series of potent, selective DNMT1 inhibitors were discovered and refined delivering tool compounds capable of eliciting changes in DNA methylation, transcriptional activation, and tumor regression at well-tolerated doses. Thus demonstrating that selective, non-covalent inhibitors of DNMT1 may provide benefit over traditional DNA incorporating hypomethylating agents.

#2995

CDK9 phosphorylates BRG1 chromatin remodeler.

Somnath Pandey, Hanghang Zhang, Carlos A. Barrero, Salim Merali, Xavier Graña, Jean-Pierre Issa. _Temple University, Philadelphia, PA_.

Cyclin-dependent kinase 9 (CDK9) belongs to the class of CDKs involved in transcription regulation along with CDK7, 8, 10-13. Earlier works have established that CDK9 is the catalytic subunit of P-TEFb, and a transcriptional activator. CDK9 inhibition holds promise for patients belonging to Chronic Lymphocytic Leukemia (CLL) and BRD4-NUT-rearranged NUT midline carcinoma (NMC) groups. It is therefore fundamentally important to understand CDK9 mediated gene regulation. CDK9 in complex with its regulatory subunit, Cyclin T1 or T2, is known to promote RNAPII promoter-proximal pause release by phosphorylating negative elongation factors. Additionally, phosphorylation of the C-terminal domain (CTD) of RNAPII on Serine-2 allows recruitment of RNA processing factors, which work on the nascent RNA as it emerges from RNAPII. It is however not known whether CDK9 has any transcriptional repression activity. Earlier work from our lab demonstrated that long-term CDK9 inhibition leads to the activation of BRG1, an ATP-dependent nucleosome-remodeling complex from the SWI/SNF family of proteins. We therefore hypothesized that CDK9 directly phosphorylates BRG1 and regulates its activity. Using Co-immunoprecipitation (Co-IP) assay we discovered that CDK9 associates with BRG1 endogenously in colon cancer cells. We tested the association of these proteins more rigorously by transiently over-expressing either FLAG-tagged or GFP-tagged CDK9 in HEK293T cells and performed immunoprecipitation using BRG1 antibody. CDK9 was found to co-precipitate with BRG1. We validated this via reciprocal Co-IPs. We performed an in vitro kinase assay using purified CDK9 and BRG1 proteins to demonstrate that CDK9 directly phosphorylates BRG1 and that inhibition of CDK9 leads to reduced phosphorylation of BRG1. We validated these results in vivo in colon cancer cells. Furthermore, our LC-MS/MS data lead to the identification of novel phosphorylation sites in BRG1 following CDK9 inhibition. These findings provide a potential mechanism for the transcriptional repressor activity associated with CDK9 and may be relevant for developing therapies targeting this transcriptional regulator.

#2996

Insulator dysfunction and epigenetic oncogene activation in SDH-deficient gastrointestinal stromal tumor.

William A. Flavahan,1 Yotam Drier,1 Sarah E. Johnstone,1 Daniel R. Tarjan,1 Esmat Hegazi,1 Ewa T. Sicinska,2 Matthew L. Hemming,2 Chandrajit P. Raut,3 Jason L. Hornick,3 George D. Demetri,2 Bradley E. Bernstein1. 1 _Massachusetts General Hospital, Boston, MA;_ 2 _Dana Farber Cancer Institute, Boston, MA;_ 3 _Brigham and Women's Hospital, Boston, MA_.

Metabolic lesions with profound effects on epigenetic regulation are widely implicated in cancer, yet the mechanistic links between this epigenetic dysregulation and tumorigenesis remain unclear. Succinate dehydrogenase (SDH) deficiency, responsible for a subset of gastrointestinal stromal tumors (GISTs), causes accumulation of the metabolite succinate and DNA hypermethylation. We identified convergent mechanisms involving altered chromosomal conformation and pseudo-hypoxia that mediate the tumorigenic effects of SDH deficiency in GIST. To investigate epigenetic alterations in this disease, we created epigenetic maps of 14 clinical GIST specimens; including KIT and PDGFRA mutant, and SDH-deficient tumors. We characterized the landscapes of enhancers, genetic regulatory elements which can drive gene expression, through histone H3 lysine 27 acetylation chromatin immunoprecipitation sequencing (ChIP-seq). We characterized both the DNA methylation and CTCF occupancy of insulators, elements which help control chromatin conformation and restrict enhancer-gene interactions, through hybrid selection bisulfite sequencing and CTCF ChIP-seq, respectively. Analyzing these data, we uncovered thousands of putative insulators where DNA methylation replaced CTCF binding in SDH-deficient GISTs. One of the strongest disrupted insulators protected the receptor tyrosine kinase and known driver of GIST, c-KIT, from a nearby superenhancer. Chromatin conformation studies confirmed an SDH-deficient-specific interaction of this superenhancer with the KIT gene. CRISPR-mediated excision of the insulator in an SDH-intact GIST model resulted in enhancer interaction and KIT upregulation. Immunohistochemical studies confirm strong expression of c-KIT in SDH-deficient GIST clinical samples. SDH deficiency has also been reported to cause pseudohypoxia in tumors. We confirmed that the enhancer landscape of SDH-deficient tumors had a signature of pseudohypoxia. Additionally, following pseudohypoxia induction in a SDH-intact GIST model, the c-KIT ligand Stem Cell Factor (SCF/KITLG) was upregulated 12-fold. While activating KIT mutations drive the majority (~75%) of GIST tumors and are mutually exclusive with SDH deficiency, we show that a primary consequence of SDH loss is in fact induction of KIT signaling. Our findings demonstrate how metabolic lesions can provide alternate epigenetic mechanisms to activate classic tumorigenic pathways in the absence of canonical genetic mutations.

#2997

Epigenomic signatures of acquired platinum resistance in high grade serous ovarian cancer.

Fang Fang,1 Horacio Cardenas,2 Guanglong Jiang,3 Susan M. Perkins,3 Chi Zhang,3 Harold Keer,4 Yunlong Liu,3 Daniela Matei,2 Kenneth Nephew1. 1 _Indiana Univ., Bloomington, IN;_ 2 _Northwestern University, Chicago, IL;_ 3 _Indiana Univ., Indianapolis, IN;_ 4 _Axtex Pharmaceuticals, Inc., Pleasanton, CA_.

Epithelial ovarian cancer (OC) is the most lethal gynecologic malignancy. The majority of advanced stage patients develop uniformly fatal resistance to standard platinum-based treatments. Epigenetic changes, particularly DNA methylation aberrations have been implicated in acquired resistance to platinum in OC. The goal of the current study was to identify methylation changes associated with the development of acquired resistance to platinum-based chemotherapy. We hypothesized that OC tumors harboring an aberrant DNA methylome could be reversed by DNMTi and re-sensitized to platinum treatment. To achieve our objective, we generated and compared tumor DNA methylation profiles from patients with recurrent platinum-resistant OC treated on a clinical trial with a DNA methyl transferase inhibitor, guadecitabine, before (n=65) and after (n=19) treatment (NCT01696032), and patients with primary (platinum-naïve) OC (n=20). Human ovarian surface epithelial cells (HOSE) were used as controls (n=5). We examined methylation levels across the genome using the Infinium Human Methylation 450 Bead Chip (Illumina). By comparing the methylome of platinum naive and platinum resistant ovarian tumors, normalized to HOSE, we identified 444 CpG island-containing gene promoters that became hypermethylated in resistant tumors. Of those, 45 gene promoters were hypermethylated at an FDR<0.05 and 19 genes at an FDR<0.05 and Δβ>0.1. Among those significantly hypermethylated genes, acquired promoter CpG island methylation of Ras Association Domain Family 1A (RASSF1A; a tumor suppressor gene in multiple cancer types) was observed. Of the 444 hypermethylated promoters in tumor resistant samples, 93 CpG islands became hypomethylated after guadecitabine treatment, suggesting that these promoters may be associated with reversal of resistance. Of those were the IFNA8 promoter, a gene known to be associated with anti-tumor effects in ovarian and other solid malignancies, and DEFB124, which is involved in the innate immune response. Pathways associated with immune reactivation were highly enriched by guadecitabine treatment, including IL-12 signaling and production of macrophages and MAPK and apoptosis signaling. Together, these results support that aberrant DNA methylation affecting gene networks associate with immune response plays a role in platinum resistant development and could be a therapeutic target.

#2998

Dynamic 3d chromosomal landscapes in acute leukemia.

Palaniraja Thandapani,1 Andreas Kloetgen,1 Charalampos Lazaris,1 Xufeng Chen,1 Panagiotis Ntziachristos,2 Aristotelis Tsirigos,1 Iannis Aifantis1. 1 _New York Univ. School of Medicine, New York, NY;_ 2 _Northwestern University, New York, NY_.

T-cell acute lymphoblastic leukemia (T-ALL) accounts for approximately 10-15% of pediatric and 25% of adult ALL cases. Despite improved prognosis driven by the significant advances in the molecular understanding of T-ALL, the outcome of T-ALL patients with primary resistant and relapsed leukemia remains poor. Recent evidences have highlighted a role for changes in 3D chromatin architecture in cancer progression. Herein, for the first time we explored genomewide changes in chromatin organization in primary T-ALL samples relative to T cells from healthy donors. We show how the organization of topologically associated domains (TADs) influences key oncogenic and tumor-suppressive loci by modifying the promoter-enhancer landscape, deregulating the transcriptional program and ultimately causing disease progression.

To comprehensively characterize the chromatin landscape of clinically relevant loci in T-ALL, we sought to identify changes in intra-TAD activity and TAD disruptions on a genome-wide scale. We developed and implemented new computational approaches for Hi-C data analysis with a focus on Hi-C data integration with ChIP-Seq and RNA-Seq. We found significant correlations of TAD activity with CTCF occupancy, super-enhancer activity and gene expression of leukemia relevant loci. Furthermore, among the dozens of TAD disruptions we identified, we found a novel TAD "fusion" event around the MYC locus, which allows for chromatin interactions between a recently reported Notch-dependent MYC super-enhancer and promoter to drive overexpression of MYC. This particular TAD fusion is accompanied by a complete CTCF loss in the TAD boundary and increased MYC expression in T-ALL samples. Overall, this study sheds new light into how changes in chromatin architecture influence disease progression by restructuring oncogene and tumor suppressor landscapes.

#2999

Low-dose demethylation therapy for the treatment of cisplatin-resistant testicular cancer.

Andrea Corbet,1 Costantine Albany,2 Emmanuel Bikorimana,1 Ema Khan,1 Jennifer Rodriguez,1 Brock C. Christensen,3 George Sandusky,2 Lawrence H. Einhorn,2 Sarah J. Freemantle,1 Michael J. Spinella1. 1 _University of Illinois, Urbana, IL;_ 2 _Indiana University School of Medicine, Indianapolis, IN;_ 3 _Dartmouth Medical School, Hanover, NH_.

Testicular germ cell tumors (TGCTs) are the most common cancers of young males. A portion of TGCT patients are refractory to cisplatin. Only 30% of patients refractory to cisplatin respond to salvage therapies while to remainder die from progressive disease. Embryonal carcinoma (EC) are the stem cells of TGCTs. We have found that EC cells were highly sensitive to the DNA methyltransferase inhibitor, 5-aza deoxycytidine (5-aza). As an initial step in bringing demethylation therapy to the clinic for TGCT patients, we evaluated the effects of the clinically optimized, second generation demethylating agent guadecitabine (SGI-110) on EC cells in an animal model of cisplatin refractory testicular cancer. EC cells were exquisitely sensitive to guadecitabine and the hypersensitivity was dependent on high levels of DNA methyltransferase 3B. Guadecitabine mediated transcriptional reprogramming of EC cells included induction of p53 targets and repression of pluripotency genes. As a single agent, guadecitabine completely abolished progression and induced complete regression of cisplatin resistant EC xenografts even at doses well below those required to impact somatic solid tumors. Low dose guadecitabine also sensitized refractory EC cells to cisplatin in vivo. Genome-wide analysis indicated that in vivo antitumor activity was associated with activation of p53 and immune-related pathways and the antitumor effects of guadecitabine were dependent on p53, a gene rarely mutated in TGCTs. Together, these preclinical findings provided the rationale for our recently initiated and promising phase I clinical trial using SGI-110 to treat cisplatin refractory TGCT patients. We discuss our recent genome-wide molecular studies aimed to identify potential mechanism(s) to account for the hypersensitivity of TGCTs to 5-aza including promoter demethylation, p53 activation and dsRNA MDA5/MAVS/IRF7 viral mimicry. We also discuss preliminary findings from our ongoing trial. Our findings suggest that guadecitabine alone or in combination with cisplatin is a promising strategy to treat refractory TGCT patients.

### Sampling the Cancer Genome and the Epigenome: Opportunities and Exquisite Vulnerabilities

#3000

Pervasive intra-tumour heterogeneity and subclonal selection across cancer types.

Stefan Dentro,1 Ignaty Leshchiner,2 Kerstin Haase,3 Jeff Wintersinger,4 Amit Deshwar,4 Maxime Tarabichi,3 Yulia Rubanova,4 Kaixian Yu,5 Ignacio Vázquez García,1 Geoff Macintyre,6 Kortine Kleinheinz,7 Dimitri Livitz,2 Salem Malikic,8 Nilgun Donmez,8 Subhajit Sengupta,9 Yuan Ji,9 Jonas Demeulemeester,3 Pavana Anur,10 Clemency Jolly,3 Marek Cmero,6 Daniel Rosebrock,2 Steve Schumacher,2 Yu Fan,5 Matthew Fittall,3 Xiaotong Yao,11 Juhee Lee,12 Matthias Schlesner,7 Hongtu Zhu,5 David Adams,1 Gad Getz,2 Paul Boutros,4 Marcin Imielinski,11 Rameen Beroukhim,2 Cenk Sahinalp,8 Martin Peifer,13 Inigo Martincorena,1 Florian Markowetz,6 Ville Mustonen,14 Ke Yuan,15 Moritz Gerstung,16 Wenyi Wang,5 Paul Spellman,10 Quaid Morris,4 David Wedge,17 Peter Van Loo3. 1 _Wellcome Trust Sanger Inst., Cambridge, United Kingdom;_ 2 _Broad Institute, Boston, MA;_ 3 _Francis Crick Institute, London, United Kingdom;_ 4 _University of Toronto, Toronto, Ontario, Canada;_ 5 _MD Anderson, Houston, TX;_ 6 _Cancer Research UK Cambridge Institute, Cambridge, United Kingdom;_ 7 _German Cancer Research Centre, Heidelberg, Germany;_ 8 _Simon Fraser University, Burnaby, British Columbia, Canada;_ 9 _University of Chicago, Chicago, IL;_ 10 _Oregon Health & Science University, Portland, OR; _11 _Cornell University, New York, NY;_ 12 _University of California Santa Cruz, Santa Cruz, CA;_ 13 _University of Cologne, Cologne, Germany;_ 14 _University of Helsinki, Helsinki, Finland;_ 15 _University of Glasgow, Glasgow, United Kingdom;_ 16 _European Bioinformatics Institute, Cambridge, United Kingdom;_ 17 _Big Data Institute, Oxford, United Kingdom_.

We have characterised intra-tumour heterogeneity (ITH) across 2,778 whole genome sequences of tumours in the International Cancer Genome Consortium Pan-Cancer Analysis of Whole Genomes project, representing 36 distinct cancer types. We applied 6 copy number (CNA) callers and 11 subclonal reconstruction algorithms and developed approaches to integrate the results in robust, high-confidence CNA calls and subclonal architectures. The analysis reveals widespread ITH. We find at least one subclone in nearly all (96.7%) tumours with sufficient sequencing depth. Analysis using dN/dS ratios yields clear signs of positive selection in clonal and subclonal mutations and we find subclonal driver mutations in known driver genes. However, only 24% of subclones contain a driver mutation in a known driver gene, suggesting that a multitude of undiscovered late drivers exist and that tumours continue to undergo selection after tumourigenesis, at least until diagnosis. Consistent with other studies, we find that in 9% of tumours all clinically actionable mutations are subclonal, while 20% of tumours contain at least one subclonal actionable driver. These findings emphasise the relevance of ITH in treatment decision making. Distinct patterns of ITH emerge; for example, prostate, uterus and esophageal adenocarcinomas show high proportions of both subclonal single nucleotide variants (SNVs) and CNAs. Kidney chromophobe and pancreatic endocrine tumours also contain high proportions of subclonal SNVs, but few subclonal CNAs. On the other hand, hepatocellular carcinomas and head-and-neck and lung SCCs contain low proportions of subclonal SNVs and high proportions of subclonal CNAs. Mutational signature analysis reveals changes in signature activity. Exposures to UV light in melanomas and acid reflux in stomach and oesophageal cancers contribute more clonal mutations. While APOBEC and DNA damage repair response related signatures show increased activity in subclones. These findings highlight distinct evolutionary narratives between and within histologically distinct tumour types.

#3001

Broad/IBM Project: Discovery of treatment resistance mechanisms through use of liquid biopsy genomics services.

Gad Getz,1 Carrie Cibulskis,2 Ignaty Leshchiner,2 Megan Hanna,2 Dimitri Livitz,2 Kara Slowik,2 Chaya Levovitz,3 Filippo Utro,3 Kahn Rhrissorrakrai,3 Denisse Rotem,2 Gregory Gydush,2 Sarah C. Reed,2 Justin Rhoades,2 Gavin Ha,4 Samuel S. Freeman,5 Christopher Lo,2 Mark Fleharty,2 Justin Abreu,2 Katie Larkin,2 Michelle Cipicchio,2 Brendan Blumenstiel,2 Matt DeFelice,2 Jonna Grimsby,2 Susanna Hamilton,2 Niall Lennon,2 Viktor A. Adalsteinsson,2 Laxmi Parida3. 1 _MGH Cancer Center and Broad Institute, Charlestown, MA;_ 2 _Broad Institute, Cambridge, MA;_ 3 _IBM T. J. Watson Research, Yorktown Heights, NY;_ 4 _Broad Institute, Harvard University, Cambridge, MA;_ 5 _Broad Institute, Harvard Medical School, Cambridge, MA_.

The Broad/IBM Cancer Resistance Project has partnered with Broad Genomics to pilot the use of cutting edge sequencing technology for the analysis of cell free DNA in blood biopsies. Working closely with the Broad's Cancer Program, Broad Genomics has developed a suite of liquid biopsy sequencing products designed to provide optimal flexibility in conducting research studies with a broad range of applications including; biomarker discovery, treatment resistance monitoring, and detection of minimal residual disease (MRD) post-surgery. Cell-free DNA is extracted from the blood, and a dual unique-molecular-indexed library is created. From this library, low coverage whole genome (ultra-low-pass 0.1x coverage) data is generated to survey sample quality and evaluate the tumor fraction in the liquid specimen. Utilizing the same library, additional assays can be selected for processing based on the research aim (Targeted Panel Assays, MRD Detection or Whole Exomes). Since our approach utilizes the same genomic material for whole genome and targeted sequencing assays, it is possible to maximize the information learned from each valuable and limited liquid biopsy specimen. Our study design takes advantage of the discovery potential of combined tissue-based sequencing and serial liquid biopsy analysis to elucidate mechanisms of cancer resistance by tracking the evolution of clonal and subclonal populations in patients samples over time. This collaboration will utilize the ultra-low-pass sequencing and whole exome sequencing together with custom analysis pipelines to correlate the genomic events with patient clinical data. We aim to process 3,000 samples from 1,000 patients over the next 3 years. To date we have processed close to 500 samples through the ultra-low-pass pipeline and 100 samples through the whole exome sequencing pipeline (results to be provided).The ability to successfully investigate treatment resistant cancers from non-invasive liquid biopsies presents new opportunities for identifying markers, understanding dynamics and monitoring tumor dissemination and clonal evolution.

#3002

Genome engineering approaches to generate models of chromosome arm-level cancer aneuploidy.

Alison Marie Taylor,1 Xiaoyang Zhang,1 Juliann Shih,2 Gavin Ha,1 Galen F. Gao,2 Ashton C. Berger,2 Andrew D. Cherniack,2 Rameen Beroukhim,1 Matthew Meyerson1. 1 _Dana-Farber Cancer Inst., Boston, MA;_ 2 _Broad Institute, Cambridge, MA_.

Aneuploidy, whole chromosome or chromosome arm copy number imbalance, is a near-universal characteristic of human cancers. Although yeast and mammalian models of whole chromosome aneuploidies have been extensively investigated, chromosome arm-level aneuploidies have rarely been modeled. Cancer subtypes are often characterized by tumor-specific patterns of these arm-level copy number alterations; for example, squamous cell carcinomas (SCCs) from different tissues of origin (including lung, esophagus, and bladder) have a pattern of chromosome 3p loss and chromosome 3q gain. Our analysis of 495 lung SCCs found chromosome 3p deletion to be the most frequent genomic alteration, occurring in almost 80% of the tumors and covering the entire length of the chromosome arm. Over two-thirds of chromosome 3p genes showed significantly decreased expression in these samples. Without models of chromosome arm-level alterations, the phenotypic effects of specific aneuploidies in cancer, such as 3p deletion, remain unknown. However, recent advances in genome engineering and targeting of endonucleases allow new approaches to generate chromosomal alterations. Here, we used the CRISPR-Cas9 system to delete one copy of chromosome 3p in vitro. We successfully isolated almost 90 clones of immortalized lung epithelial cells with deletion of the 3p arm, with 8 validated by whole-genome sequencing. Consistent with patient data, expression of 3p genes was also decreased upon deletion. Phenotypic characterization revealed that cells with chromosome 3p deletion initially proliferated more slowly than their siblings. These chromosome 3p-deleted cells had increased G1 arrest, but did not undergo increased apoptosis or cell death. Interestingly, after several passages in culture, the proliferation defect was rescued in chromosome 3p-deleted cells; genome sequencing and karyotype analyses suggested that this was the result of chromosome 3 duplication. With our cellular model of chromosome arm-level aneuploidy, we uncovered a possible selection mechanism that allows aneuploidy tolerance in vitro. We used genome engineering to model chromosome arm-level deletions, providing a robust model that will address a gap in our understanding of aneuploidy in cancer.

#3003

Loss of heterozygosity of essential genes represents a novel class of cancer vulnerabilities.

Caitlin A. Nichols,1 Brenton R. Paolella,2 William J. Gibson,3 Meredith S. Brown,1 Laura M. Urbanski,1 Jack A. Kosmicki,4 John P. Busanovich,1 Ashton C. Berger,2 Galen F. Gao,2 Andrew D. Cherniack,2 Rameen Beroukhim1. 1 _Dana-Farber Cancer Institute, Boston, MA;_ 2 _Broad Institute of Harvard and MIT, Cambridge, MA;_ 3 _Brigham and Women's Hospital, Boston, MA;_ 4 _Massachusetts General Hospital and Harvard Medical School, Boston, MA_.

Despite progress in precision cancer drug discovery, few highly selective therapies exist in the clinic, creating the need for additional therapeutic targets. We have shown that copy number alterations (CNAs) in essential genes represent novel non-driver gene vulnerabilities in cancer. Here we interrogate loss of heterozygosity (LOH) of single nucleotide polymorphisms (SNPs) located in essential genes as a novel class of candidate therapeutic targets. We hypothesized that monoallelic inactivation of the single allele retained in tumors can selectively kill cancer cells, while somatic cells, which retain both alleles, will tolerate allele-specific knockout. We identified a list of over 1000 common missense SNPs in at least 1500 essential genes that undergo LOH in cancer and performed proof-of-concept allele-specific gene inactivation in two essential genes (PRIM1 and EXOSC8) using CRISPR-Cas9. We assessed the fidelity of allele-specific gene disruption and its cellular effects on gene expression, cell growth, and cell death in LOH and non-LOH genetic contexts. We determined that allele-specific knockout of PRIM1 and EXOSC8 selectively targets cells harboring only the single targeted allele of that gene. In cells retaining only the sensitive allele, we observed decreased target gene expression and cell viability that did not occur in cells retaining the resistant allele. We conclude that allele-selective inactivation of essential genes in regions of LOH (such as PRIM1 and EXOSC8) represents a novel candidate therapeutic strategy in cancer. The corresponding class of novel non-driver cancer vulnerabilities may provide a rich source of targets for future precision therapeutic development using gene editing, RNAi, or small-molecule approaches.

#3004

Single-cell mutational profiling of clonal evolution in myelodysplastic syndromes (MDS) during therapy and disease progression.

Alexey Aleshin,1 Peter L. Greenberg,1 Bruno C. Medeiros,1 Diane Heiser,2 Marianne Santaguida,2 Sacha Prashad,2 Robert Durruthy- Durruthy,3 Dennis J. Eastburn3. 1 _Stanford University, Stanford, CA;_ 2 _Notable Labs, San Francisco, CA;_ 3 _Mission Bio, South San Francisco, CA_.

Myelodysplastic syndromes (MDS) are a collection of clonal diseases with dysfunctional hematopoietic stem cells, characterized by ineffective hematopoiesis, cytopenias, and dysplasia. MDS is increasingly being recognized as a molecularly heterogeneous disease with variability in clinical phenotype, prognosis, and response to treatment. The clonal evolution in response to therapy remains a challenge in the management of MDS. However, resolution of complex clonal architectures is difficult with current bulk sequencing approaches. High-throughput single-cell genomic profiling enables the resolution of tumor heterogeneity, and may improve clinical diagnosis and treatment monitoring by allowing for characterization and early identification of resistant subclonal populations. To enable the characterization of genetic heterogeneity in tumor cell populations, we developed a novel microfluidic approach that barcodes amplified genomic DNA from thousands of individual cells confined to droplets. The barcodes were then used to reassemble the genetic profiles of cells from next-generation sequencing data. We applied this approach to sequential clinical MDS samples, genotyping the most clinically relevant loci across more than 15,000 individual cells. Additionally, to study effects of subclonal mutations on drug sensitivity, ex vivo functional testing was performed on red blood cell-lysed peripheral blood and/or bone marrow aspirate patient samples. Targeted single-cell sequencing was able to recapitulate bulk sequencing data from both peripheral blood and bone marrow aspirate samples. Furthermore, the single-cell nature of our approach enabled definitive determination of mutational co-occurrence within the same cell. For examples in one sample, bulk sequencing identified mutations in JAK2 and NRAS both at 3% variant allele frequency (VAF), where single-cell analysis suggested that these mutations were mutually exclusive, each defining a distinct subclone. Single-cell sequencing allowed for serial monitoring of clonal evolution, with analysis of sequential samples from this same patient showing increase of NRAS clone from 2.5% to 24.7% at time of disease progression after hypomethylating agent therapy. Furthermore, single-cell analysis was able to identify a distinct subclone characterized by a KRAS mutation (0.4% at initiation of therapy and 6.7% at relapse), missed by serial bulk sequencing. Taken together, our results suggest a greater degree of heterogeneity in MDS samples than suggested with bulk sequencing methods alone, and demonstrate utility of single-cell sequencing for sequential monitoring and identification of resistant clones prior to therapy initiation. We show here that this approach is a feasible, scalable, and effective way to identify and track heterogeneous populations of cells in MDS.

#3005

International Cancer Genome Consortium.

Andrew Biankin,1 Jennifer L. Jennings,2 Lincoln D. Stein3. 1 _University of Glasgow, United Kingdom;_ 2 _Ontario Institute for Cancer Research, Toronto, Ontario, Canada;_ 3 _Ontario Institute for Cancer Research, Ontario, Canada_.

The International Cancer Genome Consortium (ICGC) was established to bring together researchers from around the globe to comprehensively analyze the genomic, transcriptomic, and epigenomic changes in 50 different tumor types or subtypes that are of clinical and societal importance across the globe (International network of cancer genome projects. Nature 464, 993-998 (15 April 2010)). As of November 2017, the ICGC has received commitments from researchers and funding organizations in Asia, Australia, Europe, North America and South America for 90 project teams in 17 jurisdictions to study more than 25,000 tumor genomes. Processed data is available via the Data Coordination Centre (https://dcc.icgc.org/) based at the Ontario Institute for Cancer Research and is updated semi-annually. The November 2017 release (Version 26) in total comprises data from more than 17,000 cancer donors spanning 76 projects and 21 tumor sites. The Pan-Cancer Analysis of Whole Genomes (PCAWG) project of the ICGC and The Cancer Genome Atlas (TCGA) is coordinating analysis of more than 2,800 cancer genomes, with the extensive use of cloud computing. Because of the very large size of the pan-cancer dataset, PCAWG used distributed compute cloud environment spread across North America, Europe and Asia that meets the project's technical requirements and the bioethical framework of ICGC and its member projects. Each genome was characterized through a suite of standardized algorithms, including alignment to the reference genome, uniform quality assessment, and the calling of multiple classes of somatic mutations. Scientists participating in the research projects of PCAWG are now addressing a series of fundamental questions about cancer biology and evolution based on these data, and have gained new insights into the role of non-coding DNA in cancer. The first phase of ICGC, which is slated for completion in 2018, has focused on developing extensive catalogs of tumor genomic information. The proposed next phase of the consortium, ICGC-ARGO, will link genomic to extensive clinical information from clinical trials and community cohorts concerning lifestyle, environmental exposure, family history of disease, treatment and outcome data for a broad spectrum of cancers, including preneoplastic lesions. The goal will be to accelerate the translation of genomic information into the clinic to guide interventions including diagnosis, treatment, early detection and prevention. The ICGC develops policies and quality control criteria to help harmonize the work of member projects located in different jurisdictions. Data produced by ICGC projects are made rapidly and freely available to qualified researchers around the world via the data cloud and through the ICGC Data Coordination Center at (http://dcc.icgc.org). More information can be found on www.icgc.org.

#3006

Molecular characterization of baseline and serial multiple myeloma patients from the MMRF CoMMpass study.

Sheri Skerget,1 Austin Christofferson,1 Sara Nasser,1 Jessica Aldrich,1 Daniel Penaherrera,1 Christophe Legendre,1 Martin Boateng,1 Lori Cuyugan,1 Jonathan Adkins,1 Erica Tassone,1 The MMRF CoMMpass Network,2 Jen Yesil,2 Daniel Auclair,1 Winnie Liang,1 Jonathan J. Keats1. 1 _TGen (The Translational Genomics Research Institute), Phoenix, AZ;_ 2 _Multiple Myeloma Research Foundation, Norwalk, CT_.

Multiple myeloma (MM) is a hematological malignancy of plasma cells accounting for ~2% of new cancer cases each year in the United States. Our understanding of MM pathogenesis has improved dramatically with the development of whole genome analysis technologies, however, to date no study has comprehensively analyzed a large cohort of MM patients. The Multiple Myeloma Research Foundation CoMMpass Study (NCT01454297) is a fully accrued observational clinical trial with 1143 newly diagnosed MM patients from sites in the United States, Canada, Spain, and Italy. Clinical parameters are collected at baseline and every three months through the eight-year observation period. Tumor samples are collected and characterized using whole genome, exome, and RNA sequencing at diagnosis and each progression event. This unique prospective study design differentiates CoMMpass from other large cancer genomics studies performed to date.

This represents the first analysis of the CoMMpass interim analysis 12 dataset including 982 of whom are molecularly characterized at baseline. Median follow-up of the cohort exceeds 2 years, and while the median OS has still not been reached, median PFS of the cohort is 36 months. We identified a median of 153 non-immunoglobulin mutations, 29 structural events, and 133 copy number (CN) events per tumor at baseline. In order to identify a set of significantly mutated genes, we applied a consensus-based approach identifying 60 genes mutated in at least 1% of the baseline cohort. Consensus clustering of the CN and gene expression profiles identified 14 and 12 distinct MM subtypes, respectively. Integration of WGS and RNA sequencing data identified 1163 cross-validated fusion transcripts. An integrated analysis of all data sources identified a series of potential gain-of-function and loss-of-function genes, from which a pathway analysis highlighted alterations in the NF-kB, Ras, DNA repair, and cell-cycle pathways.

This dataset includes serial data for 121 patients (171 specimens), 21 of whom had multiple progression events. Mutational analyses revealed that ~ 24% of mutations identified at progression were not identified at the previous timepoint. Although rarely mutated at baseline, RRBP1 was frequently mutated at relapse and may represent a novel driver of disease progression or treatment resistance in MM. In progression samples we observed a greater proportion of patients with NRAS mutations, attributable to four patients who acquire NRAS mutations and five patients with baseline KRAS mutations that shift to NRAS mutations at relapse. All patients who exhibit a KRAS to NRAS shift were bortezomib treated, suggesting this shift may represent a mechanism of resistance. This comprehensive study has identified distinct genetic subgroups with variable clinical outcome and demonstrates the value of prospective collections to identify mechanisms of progression and resistance.

## MULTIDISCIPLINARY:

### Cancer Survivorship and Disparities

#3007

Monogenic and polygenic associations with subsequent breast cancer risk in survivors of childhood cancer: The St. Jude Lifetime Cohort Study (SJLIFE).

Zhaoming Wang,1 Carmen L. Wilson,1 Qi Liu,2 John Easton,1 Heather L. Mulder,1 Michael Rusch,1 Michael Edmonson,1 Shawn Levy,3 Aman Patel,1 Ying Shao,1 Ti-Cheng Chang,1 Stephen V. Rice,1 Yadav Sapkota,1 Russell J. Brooke,1 Wonjong Moon,1 Evadnie Rampersaud,1 Xiaotu Ma,1 Cynthia Pepper,1 Xin Zhou,1 Xiang Chen,1 Wenan Chen,1 Angela Jones,3 Braden Boone,3 Matthew J. Ehrhardt,1 Rebecca M. Howell,4 Nicholas Phillips,1 Courtney Lewis,1 Chimene A. Kesserwan,1 Gang Wu,1 Kim E. Nichols,1 James R. Downing,1 Melissa M. Hudson,1 Jinghui Zhang,1 Yutaka Yasui,1 Leslie L. Robison1. 1 _St. Jude Children's Research Hospital, Memphis, TN;_ 2 _University of Alberta, Edmonton, Alberta, Canada;_ 3 _HudsonAlpha, Huntsville, AL;_ 4 _University of Texas MD Anderson Cancer Center, Houston, TX_.

The allelic spectrum of the genetic architecture of breast cancer (BC) susceptibility includes at least 172 common variants with small effect sizes (per-allele odds ratio range: 1.03-1.31), plus rare variants with high (BRCA1, BRCA2, CHD1, PTEN, STK11, TP53) or moderate penetrance (ATM, CHEK2, NBN, NF1, PALB2). While these common variants confer modest risk individually, their combined effect in the form of a polygenic risk score (PRS) may be substantial. The SJLIFE whole-genome sequencing (WGS) data provide a unique opportunity to evaluate common and rare sets of genetic variants jointly, along with treatment exposures, for their contributions to subsequent BC risk in adult survivors of childhood cancer. This analysis utilized WGS data from 1131 females of European ancestry [median age at last follow-up: 34.9 years (range: 6.2-68.6)] of whom 47 were diagnosed with a subsequent BC (median age at BC 40.3 years, range: 25.5-53.0). The PRS (mean, 10.1; range, 8.3-12.2) was calculated using a weighted sum of the number of risk alleles and their log per-allele odds ratio from Michailidou et al. (Nature, Nov. 2017). A total of 34 (3.0%) survivors were carriers of pathogenic or likely pathogenic (P/LP) variants in the 11 BC predisposition genes (listed above). The standardized incidence ratio (SIR) for BC was 6.7 (95% CI, 5.0-8.9) for survivors relative to the SEER population. The SIR varied from 3.7 (95% CI, 1.4-8.1) for survivors with PRS in the 1st quintile to 3.6 (95% CI, 1.2-8.3), 7.3 (95% CI, 3.8-12.7), 7.6 (95% CI, 3.6-14.0), and 11.4 (95% CI, 6.8-18.1) in the 2nd, 3rd, 4th, and 5th quintiles, respectively. In the multivariable model adjusting for age at diagnosis, chest irradiation, alkylating agents, anthracyclines, attained age, and significant genotype eigenvectors, the relative rates (RR) of BC were 16.5 (95% CI, 6.4 - 42.6), 11.5 (95% CI, 4.4-29.9), and 47.8 (95% CI, 8.2-278.3) for carriers vs. non-carriers of the P/LP variants among all survivors, and survivors with and without chest irradiation, respectively. The RR per one standard deviation of PRS were 1.5 (95% CI, 1.1-1.9), 1.6 (95% CI, 1.2-2.0) and 1.3 (95% CI, 0.7-2.2), respectively, for the same three groups. Importantly, PRS was significantly associated with the rate of subsequent BC under the age of 45 (RR, 1.7; 95% CI, 1.3-2.2) but not over 45 (RR, 0.9; 95% CI, 0.6-1.5). To our knowledge, this is the first assessment of the joint effects of rare and common genetic variations implicated in the etiology of BC in the general population, among long-term survivors of childhood cancer. Clinically, we anticipate that an individual genetic profile utilizing common susceptibility loci in combination with rare P/LP variants will inform an improved strategy for personalized BC risk stratification and management for childhood cancer survivors. Further replication studies are warranted to confirm and refine our findings.

#3008

Effect of exercise on acute and late onset Doxorubicin-induced cardiotoxicity.

Fei Wang, Keri Schadler, Joya Chandra, Eugenie S. Kleinerman. _UT MD Anderson Cancer Ctr., Houston, TX_.

Introduction Doxorubicin (Dox)-induced cardiotoxicity interferes with QOL and longevity in childhood cancer survivors. We investigated whether exercise during or after Dox can reduce acute and late onset cardiotoxicity without inhibiting efficacy. Methods Exercise during Dox and acute cardiotoxicity: 4 week old nude mice were injected with A673 Ewing's sarcoma cells subcutaneously. When tumors reached 30-50mm mice were divided into 4 groups (8 mice/group): Control (saline, no exercise); Dox alone ( 2.5mg/kg 2x/week for 2 weeks); Exercise (treadmill running, 45min/day @ 12m/min 5days/week); Dox + Exercise. Tumor growth was quantified. Heart function was evaluated using echocardiography before and after Dox. We evaluated Ejection Fraction (EF), Fractional Shortening (FS), left ventricular posterior wall diameter (LVPW) in diastole and systole, and left ventricular internal diameter (LVID) in diastole and systole. Blood was collected to assess ROS levels in PBMCs and mice were euthanized 24 h after therapy. Heart Weight/Tibial Length (HW/TL) was determined. Heart sections were analyzed by WGA & H&E to assess histology and morphology; IHC to assess vessel morphology using CD31 and α-SMA; Masson's Trichrome to access deposition of collagen in heart tissues and transmission electron micrograph (TEM) to assess autophagy, vascular pericytes and endothelial cells. Exercise after Dox and long-term cardiotoxicity: 4 groups (8mice/group): Control (no Dox, no exercise); Dox alone-no exercise; Exercise; Dox then exercise after Dox for 14 weeks. For all groups heart function was evaluated by echo as above before and after Dox and then every 2 weeks for 14 weeks. Heart sections were analyzed as above. Results Exercise during therapy did not inhibit Dox efficacy. Dox induced a significant decrease in cardiac EF%, FS% and HW/TL ratio and increased ROS in PBMCs. Exercise during therapy inhibited these effects. Dox decreased cardiac vascular pericytes and endothelial cells and induced abnormal mitochondria, vacuolization and increased autophagosomes in the heart. These changes were not seen in the Dox+exercise mice. Exercise initiated after Dox also inhibited the late effects of Dox using the same echo and tissue evaluations. Additional there was a significant decrease in LVPW in diastole (but not in systole) 14 weeks after Dox. This was not seen when exercise was initiated after therapy. Conclusion Exercise prevented both the acute and late cardiotoxicity induced by Dox without inhibiting efficacy. Therefore, exercise interventions have the potential to decrease cardiac morbidity, improve cardiac health and the QOL for childhood cancer survivors. Diastolic function is also important in heart failure. While systolic function is typically monitored in children receiving Dox, our data suggest that Dox-induced diastolic dysfunction precedes systolic dysfunction and perhaps can be used as an early marker of cardiotoxicity.

#3009

Changes in recreational physical activity and prognosis in breast cancer survivors.

Audrey Y. Jung,1 Sabine Behrens,1 Kathrin Thoene,2 Martina Schmidt,1 Karen Steindorf,1 Jenny Chang-Claude1. 1 _German Cancer Research Center, Heidelberg, Germany;_ 2 _University Medical Center Hamburg-Eppendorf, Hamburg, Germany_.

Aim: Recreational physical activity (PA) both before and after a breast cancer diagnosis has been reported to be associated with lower total mortality. Whether there is benefit in changing PA levels between pre- and post-diagnosis is unclear and has not received much attention. This study aimed to examine changes in recreational PA and overall mortality (OM) and breast cancer-specific mortality (BCSM). Methods: We used data from the MARIE (Mamma Carcinoma Risk factor Investigation) study, a prospective population-based patient cohort study conducted in Germany, where 3813 breast cancer patients, aged 50-70 at diagnosis, were recruited from 2002-2005 and followed-up with re-interview in 2009 (FUP1) and for vital status until June 30, 2015. Based on median cut-points, women were categorized by their PA behavior pre- and post-diagnosis and combined to create four groups: pre-diagnosis low/post-diagnosis low, pre-diagnosis low/post-diagnosis high, pre-diagnosis high/post-diagnosis low, pre-diagnosis high/post-diagnosis high. Cox proportional hazards regression models with delayed-entry were used to assess the associations between change in recreational physical activity, and OM and BCSM, adjusting for age, menopausal status, and prognostic factors. Those in the pre-diagnosis low/post-diagnosis low group served as the reference. Results: A total of 2,066 patients were included in the analysis. There were 139 deaths (54 from breast cancer) over a median follow-up time of 11.6 years. Median recreational PA levels at pre-diagnosis and post-diagnosis were 3.6 MET-hr/week and 0 MET-hr/week, respectively. Compared to women who had low recreational PA levels at pre-diagnosis and post-diagnosis, women who had low recreational PA levels at pre-diagnosis and high PA levels at post-diagnosis had lower risk of OM (HR 0.6 (95%CI 0.4-1.0)), and women who had high PA levels at pre-diagnosis and maintained high PA levels at post-diagnosis also had lower risk of OM (HR 0.6 (0.8-0.9)). There was no association between changes in recreational PA and OM for women who had high PA levels at pre-diagnosis and low PA levels at post-diagnosis (HR 0.8 (0.5-1.3)). There were also no statistically significant associations between changes in recreational PA and BCSM (compared to low/low, HRs for low/high 0.8 (0.4-1.7), high/low 0.9 (0.4-1.9), and high/high 0.7 (0.3-1.3)). Conclusion: Our study suggests that there may be benefits in increasing recreational PA after diagnosis of a breast cancer even for women who were not particularly physically active pre-diagnosis.

#3010

Biobanking and genomic research: Understanding and acceptance of safety-net patients, primary care providers and minority groups.

Terry Davis,1 Connie Arnold,1 Lucio Miele,2 Glenn Mills1. 1 _LSUHSC-Shreveport, Shreveport, LA;_ 2 _LSUHSC-New Orleans, New Orleans, LA_.

Background. Disparities exists in recruitment in clinical trials and biorepositories among minority groups, rural residents and individuals with low SES.

Objective. Identify barriers and facilitators to awareness, understanding and acceptance of clinical trials and biobanking among safety-net patients and providers and support group participants and African American (AA) social and church groups.

Methods. We conducted 14 focus groups and 7 individual interviews January - May 2017 in urban and rural communities in Louisiana. Sites included safety-net oncology and primary care clinics, social service agencies, social and faith based groups. Themes were identified.

Results. 103 individuals participated: 78 clinic patient and community participants, 25 providers; 24% participants lived in rural areas. Patients and community members ranged in age from 45-88; 85% were female, 78% AA. Participants had an increasing awareness of clinical trials and personalized medicine due to ads on TV. Low income and minority patients were open to participating in genomic trials and biobanking even if it would not benefit them directly. Cancer patients and Alzheimer family caregivers were highly interested in clinical trials that might benefit them or their family. Community participants were less trusting of clinical trials than patients. All said information and recommendations about clinical trials would be most effective and actionable if it comes from a trusted physician. Terms such as clinical trials and biobanking were scary to some and not understood - medical studies and a bank to store blood and tissue were clearer. African American participants indicated that a recruitment message needs to be "ALL people are needed for studies to improve treatments" rather than "AA have been underrepresented and most treatments based on whites" which raises suspicion. Some participants were concerned with who would see data and if it would be a barrier to jobs or insurance - despite being told of protections in place for confidentiality. Rural and urban providers (n=25) were interested in being more involved in clinical trials. They lacked time to identify trials and explain them to patients. They wanted brief, plain language handouts with talking points and a card to give patients to call for more information. Transportation to academic centers is a barrier to patient participation in clinical trials. Very few patients, caregivers or providers had looked for clinical trials on the internet or social media.

Conclusions. African American and white adults were interested in clinical trials but not clear how to learn about them. Most were willing to participate but had never been asked. Recommendation to participate in a clinical trial is most effective coming from a trusted physician. Methods for getting community providers and the public useful information needs to be created with target audiences.

#3011

Determinants and prognostic value of quality of life in patients with pancreatic ductal adenocarcinoma.

Yang Deng,1 Huakang Tu,1 Jeanne A. Pierzynski,1 Ethan Miller,1 Maosheng Huang,1 Xiangjun Gu,2 David W. Chang,1 Yuanqing Ye,1 Michelle A. Hildebrandt,1 Alison P. Klein,3 Scott M. Lippman,4 Xifeng Wu1. 1 _MD Anderson Cancer Center, Houston, TX;_ 2 _Baylor College of Medicine Dan L Duncan Comprehensive Cancer Center, Houston, TX;_ 3 _The Sol Goldman Pancreatic Cancer Research Center, The Johns Hopkins University School of Medicine, Baltimore, MD;_ 4 _Moores Cancer Center, University of California San Diego, La Jolla, CA_.

Background Quality of life (QOL) is impaired in pancreatic cancer patients. Our aim was to investigate the determinants and prognostic value of QOL after diagnosis in a hospital-based cohort of racially/ethnically diverse patients with pancreatic ductal adenocarcinoma (PDAC). Patients and methods QOL was prospectively assessed using the Short Form-12 version 1 in 2478 PDAC patients. The Physical Component Summary (PCS) and Mental Component Summary (MCS) were categorized into tertiles based on their distribution. Ordered logistic regression was adopted to compare the risk of having lower PCS and MCS by patient sociodemographic and clinical characteristics. The association of PCS and MCS with mortality was assessed by Cox regression. Results Compared with non-Hispanic whites, Hispanics were at significantly higher risk of having lower PCS (odds ratio [95%CI], 1.69 [1.26-2.26]; P < .001) and lower MCS (1.66 [1.24-2.23]; P < .001). Patients diagnosed with stage III (1.80 [1.10-2.94]; P = .02) and stage IV (2.32 [1.50-3.59]; P < .001) were more likely to have lower PCS than were patients diagnosed with stage I. Other determinants included sex, age, drinking, smoking, education level, and comorbidities. The low tertile of PCS (hazard ratio [95%CI], 1.94 [1.72-2.18]; P < .001) and MCS (1.42 [1.26-1.59]; P < .001) were each related to poor prognosis. Similar results were found for non-Hispanic Whites as compared to African Americans/Hispanics/others. Conclusion QOL after diagnosis is a significant prognostic indicator for patients with PDAC, and multiple factors determine QOL, suggesting possible means of intervention to improve QOL and outcomes of PDAC patients.

#3012

Urban neighborhood and residential factors associated with breast cancer in African American women: A systematic review.

Brandi P. Smith, Zeynep Madak-Erdogan. _University of Illinois at Urbana-Champaign, Champaign, IL_.

Residential characteristics in urban neighborhoods impacts health and might be an important factor contributing to health disparities, especially in the African American population. The purpose of this systematic review is to understand the relationship between urban neighborhood and residential factors and breast cancer incidence and prognosis in African American women. Using PubMed and Web of Science, the existing literature was reviewed. Observational, cross-sectional, cohort, and prospective studies until February 2017 were examined. Studies including populations of African American women, setting in "urban" areas, and a measure of a neighborhood or residential factor were reviewed. Four parameters related to neighborhood or residential factors were extracted including: neighborhood socioeconomic status (nSES), residential segregation, spatial access to mammography, and residential pollution. Our analysis showed that African American women living in low nSES have greater odds of late stage diagnosis and mortality. Furthermore, African American women living in segregated areas (higher percentage of Blacks) have higher odds of late stage diagnosis and mortality compared to White and Hispanic women living in less segregated areas (lower percentage of Blacks). Late stage diagnosis was also shown to be significantly higher in areas with poor mammography access and areas with higher Black residential segregation. Lastly, residential pollution did not affect breast cancer risk in African American women. Overall, this systematic review provides a qualitative synthesis of major neighborhood and residential factors on breast cancer outcomes in African American women.

## TUMOR BIOLOGY:

### Deciphering Cancer Mechanisms in Animal Models

#3014

Location specificity in fusion-negative rhabdomyosarcoma driven by cell of origin.

Catherine J. Drummond, Jason A. Hanna, Matthew R. Garcia, Daniel J. Devine, Alana J. Heyrana, David Finkelstein, Mark E. Hatley. _St. Jude Childrens Research Hospital, Memphis, TN_.

Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma and despite aggressive treatment, clinical outcomes have not improved for three decades. There is a need to uncover the molecular underpinnings of RMS. Tumor location is a key prognostic indicator and although RMS occurs throughout the body, nearly 40% of all RMS occurs in the head and neck. It is unknown how the cell of origin affects location and therefore clinical outcome of RMS. Previously, we demonstrated that activation of Hedgehog signaling through expression of a conditional, constitutively active Smoothened allele, SmoM2, under the control of the adipose protein 2 (aP2)-Cre recombinase transgene gives rise to aggressive skeletal muscle tumors in mice that resemble human fusion negative RMS (FN-RMS). Interestingly, tumors were anatomically restricted to the neck in aP2-Cre;SmoM2 mice. In this study we leverage the aP2-Cre;SmoM2 model of FN-RMS to interrogate how cell of origin affects tumor localization.

By genetic fate mapping we determine that aP2-Cre labeled cells are non-myogenic and that aP2-Cre is not expressed in quiescent or activated muscle stem cells. Instead, we identify aP2-Cre expressing cells as endothelial cell progenitors within the muscle interstitium. Although aP2-Cre expressing endothelial cells were observed throughout the mouse, we observed that SmoM2 expression specifically drives embryonic expansion of aP2-Cre labeled cells only in the neck. SmoM2 expression reprograms endothelial progenitors resulting in a myogenic fate switch prior to terminal endothelial cell differentiation. We illustrate that endothelium and skeletal muscle within the head and neck arise from KDR expressing progenitors and that aP2-Cre is expressed after endothelial lineage commitment. Aberrant hedgehog activation in these aP2-Cre labeled endothelial progenitors results in TBX1 expression, a skeletal muscle specification factor in the head and neck, and subsequently MYOD1 expression, driving a partial myogenic program characteristic of FN-RMS. Further characterization of tumor cells isolated by flow cytometry revealed that FN-RMS in aP2-Cre;SmoM2 mice express TBX1, as well as PITX2, TCF21 and MSC, additional skeletal muscle specification factors in the head and neck. In contrast, PAX3, which specifies trunk and limb muscle, was not expressed in FN-RMS cells isolated from aP2-Cre;SmoM2 mice. Together, these results demonstrate that FN-RMS in the head and neck can arise from endothelial progenitor cells and suggest that aberrant activation of normal muscle development programs in non-myogenic cell types with developmental pliancy can drive location specific tumor formation.

#3015

Precise investigation of cancer stem cells in mouse glioblastoma.

Xuanhua P. Xie,1 Dan R. Laks,1 Daochun Sun,1 Asaf Poran,2 Ashley M. Laughney,1 German Belenguer,3 Joan Massague,1 Xiuping Zhou,4 Isabel Farinas,3 Olivier Elemento,2 Luis F. Parada1. 1 _Memorial Sloan Kettering Cancer Center, New York, NY;_ 2 _Weill Cornell Medicine, New York, NY;_ 3 _Universidad de Valencia, Valencia, Spain;_ 4 _Xuzhou Medical University, Jiangsu, China_.

In this study, we employ mouse models to investigate features and roles of cancer stem cells (CSCs) in glioblastoma (GBM). A nestin-TK-GFP transgene is firstly used to label CSCs in a fully penetrant mouse model of GBM (M7: hGFAP-Cre; Nf1fl/+; p53fl/fl; Ptenfl/+). Food-mediated ganciclovir (GCV) delivery kills proliferative transgene positive cells and significantly prolongs the lives of the transgene bearing mice. Isolation and transplantation of the tumor cells indicates the GFP+ cells are more tumorigenic than the GFP- cells. We then generate and characterize a novel transgene (CGD: nestin-CreERT2-H2BeGFP-hDTR) that labels all the neural stem/progenitor cells in the subventricular zone (SVZ). This transgene efficiently promotes brain tumors by eliminating tumor suppressor genes (CGD-M4: CGD; Nf1fl/+; p53fl/+; Ptenfl/+) in the corresponding cells. The CGD-GFP+ tumor cells are quiescent in vivo, yet form more spheres in vitro than the GFP- cells. The GFP+ cells are competent to develop tumors in a serial transplantation assay and constantly maintain a quiescent subpopulation pool in the newly formed tumors. Diphtheria toxin treatment ablates the CGD-GFP+ tumor cells and greatly reduces the tumor bulk. Temozolomide, the conventional chemotherapy for human GBM patients, benefits only mice transplanted with GFP- but not CGD-GFP+ tumor cells. Our study demonstrates the essential role of CSCs in GBM initiation, recurrence, and drug resistance. Further analysis of the CGD-GFP+ cells might delineate novel treatments for this deadly disease.

#3016

Ezh2 **is a dose-dependent mediator of prostate cancer aggressiveness and lineage transformation.**

Kristine M. Wadosky,1 Yanqing Wang,1 Leigh Ellis,2 David W. Goodrich1. 1 _Roswell Park Cancer Institute, Buffalo, NY;_ 2 _Dana Farber Cancer Institute, Boston, MA_.

Resistance to therapies for metastatic prostate cancer (PCa) causes patients to rapidly succumb to their disease, especially when resistance is associated with development of aggressive variant PCa. These PCa variants are found in 20-30% of patient autopsies relapsing from androgen deprivation therapy (ADT) and often lack androgen receptor (AR), aberrantly express alternative lineage markers, and have altered histology and clinical course. Of these PCa variants, neuroendocrine prostate cancer (NEPC) has been the most well-studied. Our recently published data show that Rb1 loss promotes NEPC transformation in the mouse and induces pervasive changes in gene expression characteristic of human NEPC. These changes include increased expression of histone methyltransferase Ezh2, a known PCa oncogene. In addition, treatment with Ezh2 inhibitors restores AR expression and ADT sensitivity in Rb1-deficient PCa. We hypothesize that Ezh2 is a driver of NEPC transformation in Rb1-deficient PCa. To test this hypothesis, we generated a new genetically engineered mouse model (GEMM) of Rb1-deficient PCa that also lacks Ezh2. Preliminary data suggest that overall survival of PBCre4:Ptenf/f:Rb1f/f:Ezh2f/+ (DKOE/+) mice with loss of one Ezh2 allele is increased compared to PBCre4:Ptenf/f:Rb1f/f (DKO). In prostate tumors from DKOE/+ mice, individual glands are apparent, characteristic of adenocarcinoma. Immunohistochemistry shows that expression of luminal markers, including AR, CK8, and CK18, are increased in DKOE/+ compared to DKO. NEPC markers synaptophysin and chromagranin A are decreased in DKOE/+ compared to DKO, suggesting that loss of one Ezh2 allele slows neuroendocrine transformation. To our surprise, overall survival of PBCre4:Ptenf/f:Rb1f/f:Ezh2f/f (DKOE/E) was decreased compared to DKO. Histological analysis of end-stage primary tumors from DKOE/E mice indicate they develop high grade lesions containing sheets of cells with minimal gland formation. These mice develop hemorrhagic ascites and metastases to the liver, lung, kidney, bone, thymus, and lymph node. AR expression in DKOE/E is decreased compared to both DKO and DKOE/+. When organoids were established from DKO, DKOE/+, and DKOE/E end-stage tumors, preliminary analysis suggest that DKOE/E have greater organoid renewal capability. Altogether, initial data from our new GEMMs show that loss of one allele of Ezh2 in DKOE/+ inhibits development of lethal NEPC transformation in Rb1-deficient PCa. Whereas, complete loss of Ezh2 in the context of Rb1-deficiency makes disease more aggressive. These data suggest that Ezh2's effect on PCa progression in the absence of Rb1 is dose-dependent. Future work is required to investigate of the exact mechanism of Ezh2's contribution to PCa aggressiveness and NEPC transformation.

#3017

Identifying drivers for advanced prostate cancer by a transposon-based genetic screen.

Min Zou, Alessandro Vasciaveo, Andrea Califano, Cory Abate-Shen. _Columbia Univ. Irving Comp. Cancer Ctr., New York, NY_.

Prostate cancer remains the second leading cause of cancer death in men in the United States. Androgen-deprivation therapy (ADT) often leads to progression to castration-resistant prostate cancer (CRPC), which is more aggressive and frequently associated with metastasis and poor prognosis. Despite the significance of CRPC in the clinic, the molecular mechanism driving the emergence of advanced prostate cancer is still not well understood. Identifying novel drivers is particularly important for developing therapeutic targets that lead to improved treatment outcome. We sought to explore drivers for advanced prostate cancer by utilizing the Sleeping Beauty (SB) system, a murine transposition-based forward mutagenesis screening approach. We generated mouse strain in which the SB transposase expression is under the control of a tamoxifen-inducible Cre allele driven by the prostate specific Nkx3.1 promoter. After crossing this strain to transgenic strain for the transposon element (T2/Onc2), as well as strain with floxed alleles for Pten and Trp53, we obtained a mouse model allowing mutagenesis screening in mouse prostate on a Pten and Trp53 null background (SB mice). Mice with only conditional deletion of Pten and Trp53 were generated as controls. Prostate tumor tissue from moribund mice were collected for sequencing and histopathologic analysis. Kaplan-Meier survival analysis was performed to examine the disease progression. Linker-mediated PCR was performed to amplify genomic loci containing SB transposon insertions. Barcoded sequencing library was generated and sequenced on an Illumina HiSeq4000. Sequencing reads and common insertion sites (CIS) were analyzed by published TAPDANCE pipeline. Survival analysis revealed that the SB mice (n=28) have a significantly shorter time to death than the controls (n=65) (log-rank p<0.0001). Interestingly, histologic analysis revealed frequent detection (11/28) of features associated with human neuroendocrine prostate cancer (NEPC), including presence of small round cells with prominent nuclei and scant cytoplasm that stain positively for neuroendocrine markers such as synaptophysin, but negatively for androgen receptor. Frequent detection of NEPC features in SB mice but not in control mice (1/53) suggests that SB-mediated mutagenesis drives NEPC development. CIS analysis revealed more than 200 insertion sites with statistical significance (integrated p<0.05), which require further validation in human tumors. In conclusion, we have implemented the SB genetic screen system in our existing CRPC mouse model. Our data show that SB mice present with accelerated disease progression and higher incidence of NEPC tumor features, consistent with a lethal and aggressive subtype of human CRPC. CIS analysis have identified hundreds of insertion sites, which are likely to include novel candidate drivers of aggressive prostate cancer.

#3018

Expression of oncogenic Nras and a MYC transgene in germinal center B cells induces a highly malignant multiple myeloma.

Zhi Wen,1 Adhithi Rajagopalan,2 Erik A. Ranheim,3 Remington Finn,4 Adam Pagenkopf,5 Grant Yun,4 Yun Zhou,1 Yidan Wang,1 Demin Wang,6 Marta Chesi,7 P. Leif Bergsagel,7 Fotis Asimakopoulos,5 Jing Zhang1. 1 _McArdle Laboratory for Cancer Research, University of Wisconsin-Madison, Madison, WI;_ 2 _Cellular and Molecular Biology Program, University of Wisconsin-Madison, Madison, WI;_ 3 _Department of Pathology & Laboratory Medicine, University of Wisconsin School of Medicine and Public Health, University of Wisconsin Carbone Cancer Center, Madison, WI; _4 _Department of Biochemistry, University of Wisconsin-Madison, Madison, WI;_ 5 _Division of Hematology/Oncology, Department of Medicine, University of Wisconsin-Madison School of Medicine and Public Health, University of Wisconsin Carbone Cancer Center, Madison, WI;_ 6 _Blood Research Institute, BloodCenter of Wisconsin, Milwaukee, WI;_ 7 _Department of Medicine, Mayo Clinic, Scottsdale, AZ_.

Multiple myeloma (MM) is characterized by clonal expansion of malignant plasma cells (PCs) and aberrant production of monoclonal immunoglobulin detected as an M spike using serum protein electrophoresis. In the United States, MM represents ~15% of hematologic malignancies and is one of the few cancers increasing in incidence (e.g., 14,400 in 1996 to 30,330 in 2016, from SEER). Previously, a Vk*MYC mouse model was described, in which AID-dependent activation of MYC transgene in germinal center (GC) B cells catalyzes a highly penetrant, indolent MM after a prolonged latency. However, the indolent MM developed in Vk*MYC mice rarely progresses to a malignant stage, suggesting that additional genetic mutations are required for the malignant MM progression. Recent sequencing of paired tumor/normal samples from advanced or refractory MM patients identified that constitutive activation of Ras signaling pathway (KRAS: 23%; NRAS:20%; BRAF: 8%) associates with MM progression and therapy resistance. To determine whether oncogenic Nras promotes the progression of Myc-induced indolent MM to a malignant stage, we generated NrasLSL Q61R/+; Vk*MYC; IgG1-Cre (VQ) mice along with single mutant mice Vk*MYC; IgG1-Cre (Vk) or NrasLSL Q61R/+; IgG1-Cre (Q61R). To boost NrasQ61R expression in GC B cells, 6-7 weeks old mice were immunized with NP-CGG. A significant fraction of VQ mice developed M-spike after immunization and subsequently died of a highly malignant MM. The MM developed in some mice were restricted in bone marrow and spleen and thus mimicked majority of MM patients, while MM developed in other mice was primarily located in lymph nodes and represented patients with extramedullary MM. Both types of MM cells displayed hyperproliferation and hyperactivation of AKT and ERK pathways. They could be cultured for several weeks with their original stromal cells and in the presence of Il-6. The cultured MM cells could be easily infected or transfected. Upon transplantation, both types of MM cells caused a rapid lethality (within 16 and 4 weeks respectively) in secondary and tertiary recipients and preserved the initial locations of donor MM cells. MM recipient mice developed bone lesions and/or fractures characteristic of MM patients. Preliminary preclinical studies indicated that both types of MM cells were resistant to Bortezomib, a proteasome inhibitor commonly used in treating MM patients, and AZD6244, a potent MEK inhibitor. Additional tests of combinatorial therapies are ongoing. Taken together, we generated a novel mouse model in which activation of Vk*MYC and oncogenic Nras in GC B cells results in a highly malignant, transplantable MM. This model can serve as a platform to investigate pathogenesis of highly malignant multiple myeloma and test the efficacy of novel therapeutic agents.

#3019

PRL3 contributes to the onset and progression of T-cell acute lymphoblastic leukemia in a zebrafish model.

Rachel E. Sieg, Elizabeth S. Hausman, Jessica Blackburn. _University of Kentucky, Lexington, KY_.

T-cell acute lymphoblastic leukemia (T-ALL) is a major clinical challenge, representing 15-25% of all acute lymphoblastic leukemias in children and adults. The survival rate for pediatric patients with relapsed T-ALL is a disheartening <30% in pediatric patients and <10% in adults. Current drugs against T-ALL include highly cytotoxic chemotherapies, which are generally ineffective against relapse and are particularly hard for young children to receive. These harsh therapies can result in a spectrum of developmental issues and potent side effects. Therefore, it is imperative to develop molecular therapies that can effectively inhibit T-ALL development without contributing to the adverse side effects of chemotherapy. We have found that the gene Protein Tyrosine Phosphatase 4A3 (PRL3) plays a role in T-ALL onset and development, making it a possible drug target for molecular therapies. Previously, PRL3 has not been associated with leukemia onset or progression, but here we show overexpression of PRL3 leads to increased T-ALL onset and aggressiveness, while PRL3 knockout leads to reduced T-ALL onset in a zebrafish T-ALL model. We identified PRL3 as an important driver of human T-ALL by performing a comparative genomic hybridization array on single-cell clones that evolved increased LPC frequency after serial transplantation from single leukemic cells in the zebrafish in a screen involving over 6,000 animals. We then overexpressed PRL3 via microinjection into 1-cell stage embryos, and tracked the rate of T-ALL onset and progression using fluorescent and confocal microscopy to generate a Kaplan-Meier graph. We found that overexpression increased the rate of leukemia onset and led to lethality quicker than in control zebrafish. We then knocked out PRL3 using a CRISPR-Cas9 strategy by microinjecting our constructs into 1-cell stage embryos and generating a similar Kaplan-Meier graph. By knocking out PRL3, we found that leukemia onset was delayed and progression to lethality was significantly slower than control zebrafish (p< 0.01). We then treated the T-ALL fish with two specific inhibitors, one that inhibited all PRLs and one that was specific to PRL3 (n= 60). We found that leukemia onset was significantly delayed when treated with the PRL inhibitor (p<0.001), but no significant difference was found when treated with the specific PRL3 inhibitor. We conclude that PRL3 is a novel gene important for T-ALL onset and progression. It has potential as a drug target to inhibit the growth and progression of leukemic cells. Future directions will be identifying and characterizing downstream substrates of this protein tyrosine phosphatase and doing combination drug screens to increase the inhibitory effects on T-ALL. We expect that these data will be directly relevant to future mammal studies and eventual clinical trials as alternatives to the traditional chemotherapy offered now.

#3020

**An essential role for** Argonaute 2 **in EGFR-KRAS signaling in pancreatic cancer development.**

Sunita Shankar, Jean Tien, Ronald F. Siebenaler, Vijaya L. Dommeti, Sylvia Zelenka-Wang, Kristin M. Juckette, Alice Xu, Malay Mody, Andrew Goodrum, Grace Tsaloff, Ingrid J. Apel, Lisha Wang, Javed Siddiqui, Jiaqi Shi, Chandan Kumar-Sinha, Arul Chinnaiyan. _University of Michigan, Ann Arbor, MI_.

KRAS and EGFR have been shown to function as essential mediators of pancreatic cancer development. In addition, KRAS and EGFR have been separately shown to interact with and perturb the function of Argonaute 2 (AGO2), a key mediator of RNA-mediated gene silencing. Here, we employed a genetically engineered mouse model of pancreatic cancer to define the effects of conditional loss of AGO2 in KRASG12D driven pancreatic cancer. Genetic ablation of AGO2 does not interfere with development of the normal pancreas or KRASG12D driven early precursor pancreatic intraepithelial (PanIN) lesions. However, AGO2 loss prevents progression from early to late PanIN lesions, development of pancreatic ductal adenocarcinoma (PDAC), and metastatic progression. This results in a dramatic increase in survival of KRASG12D mutant mice deficient in AGO2 expression. In both mouse and human pancreatic tissues, increased AGO2 expression at the plasma membrane is associated with PDAC progression. Mechanistically, within early precursor PanIN lesions, loss of AGO2 elevates phospho-EGFR levels and activates wild-type RAS, antagonizing KRASG12D activation and PDAC development. Furthermore, we observe that phosphorylation of AGO2Y393 by EGFR disrupts the interaction of wild-type RAS with AGO2, but does not affect the interaction of mutant KRAS with AGO2. Taken together, our study supports a biphasic model of pancreatic cancer development: an AGO2-independent early phase of PanIN formation reliant on EGFR and wild-type RAS signaling, and an AGO2-dependent phase wherein the KRAS-AGO2 interaction is critical to the progression from PanIN to PDAC. A recent study by Phillip Sharp and Tyler Jacks describes a requirement for the oncogenic KRAS-AGO2 interaction in the development of a transplant mouse model of plasmablastic lymphoma, and together these studies substantiate the role of the interaction in KRAS oncogenesis.

### Organ-Specific Metastasis

#3021

Plexin-B3 regulates cellular motility, invasiveness and metastasis in pancreatic cancer.

Sugandha Saxena, Yuri Hayashi, Satyanarayana Rachagani, Surinder Kumar Batra, Rakesh Kumar Singh. _Univ. of Nebraska Medical Ctr., Omaha, NE_.

Cancer metastasis significantly worsens the survival of pancreatic cancer (PC) patients. Currently, semaphorins and their high-affinity receptors plexins are considered versatile regulators of cancer cell migration, angiogenesis, invasion and metastasis. Our group identified semaphorin-5A (SEMA5A) as a putative cell adhesion molecule involved in organ-specific homing of PC cells and playing a significant role in PC angiogenesis and metastasis. Thus, SEMA5A and its receptor plexin-B3 represents an attractive targetable axis in PC metastasis. However, for a better understanding of the SEMA5A-initiated downstream signaling events and regulation of cellular phenotypes, it is necessary to delineate the function of its receptor plexin-B3 in PC as well. Thus, understanding the pathologic expression and functional role of plexin-B3 is essential for characterization of SEMA5A/plexin-B3 axis in PC. In the present study, we analyzed plexin-B3 expression in PC disease progression model of PDX-Cre-Kras(G12D) (KC) mice and evaluated the functional role by utilizing plexin-B3 knockdown T3M-4 and CD18/HPAF cells. We observed a reduction of plexin-B3 expression with disease progression in KC mice model and also the poor survival of pancreatic adenocarcinoma stage II patients (n = 24) with lower plexin-B3 expression. Functionally, knockdown of plexin-B3 enhanced in vitro cellular migration and invasiveness of PC cells. Furthermore, plexin-B3 knockdown cells showed a significant increase in cellular size with impaired colony formation in three-dimensional culture. Cell proliferation analysis demonstrated that plexin-B3 knockdown cells showed lower proliferation in comparison with the control cells. We also observed higher metastasis with lower tumor burden in nude mice injected with CD18/HPAF-sh plexin-B3 cells than CD18/HPAF-control cells. Furthermore, the tumors formed by CD18/HPAF-plexin-B3 cells showed higher fibrosis, a higher number of vimentin-positive cells, and lower number of proliferation marker Ki-67-positive cells in comparison with the CD18/HPAF-control tumors. In addition, we observed similar phenotypic changes in PC cells with loss of SEMA5A-ligand of plexin-B3. In summary, our data demonstrate that impairment in SEMA5A/plexin-B3 axis enhances cellular motility and invasiveness of PC cells, thereby resulting in higher metastasis.

#3022

Lymph node metastasis in solid tumors: A marker or driver of disease progression.

Ethel R. Pereira,1 Dmitriy Kedrin,1 Giorgio Seano,1 Olivia Gautier,2 Eelco F. Meijer,1 Dennis Jones,1 Shan-Min Chin,1 Shuji Kitahara,1 Echoe M. Bouta,1 Jonathan Chang,3 Elizabeth Beech,1 Han-Sin Jeong,4 Michael C. Carroll,3 Alphonse G. Taghian,1 Timothy P. Padera1. 1 _Massachusetts General Hospital/Harvard Medical School, Boston, MA;_ 2 _Massachusetts Institute of Technology, Cambridge, MA;_ 3 _Harvard Medical School, Boston, MA;_ 4 _Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Republic of Korea_.

Background: The presence of lymph node metastasis in patients with solid tumors is associated with tumor aggressiveness, poorer prognosis and the recommendation for systemic therapy. However, whether tumor cells exit the lymph node and contribute to distant metastases remains controversial. Methods: In this study, we used syngeneic murine cell lines representing breast and melanoma cancer that spontaneously metastasize to the lymph node. We engineered these cells to express Dendra2, a photoconvertable protein. Dendra2 is a green-fluorescent protein that can be converted to emit red light by exposure to 405nm light. Once tumor cells spontaneously metastasized to the lymph node from the primary site, a 405nm laser diode was used on 5 consecutive days to convert Dendra2-cancer cells from green to red fluorescence, restricting the light exposure only to the metastatic lymph node. This technology allowed us to specifically trace the fate of cancer cells in the lymph node and beyond to other organs. Results: We show that spontaneous lymph node metastasis from breast cancer and melanoma mouse models can leave the lymph node and enter the blood circulation. We identified micrometastatic disease in the lung that originated from the lymph node in both models. We hypothesized that cancer cells escape the lymph node by directly invading lymph node blood vessels, as opposed to draining through the efferent lymphatic vessel. Immunohistochemical analysis of metastatic lymph nodes revealed isolated cancer cells in close association with CD31+ blood vessels, within high endothelial venules (HEVs) and breaching the vascular basement membrane. Quantitative analysis showed that 23±2% of isolated cancer cells were within 5μm of a blood vessel, compared to only 11±1% using a predictive model of randomly distributed cells in the lymph node (p<0.05). Further, 6±2% of the cancer cells were inside blood vessels defined as cells within the lumen of blood vessels and having cell centroids more than 3μm from the blood vessel endothelium. To further confirm that metastatic cancer cells in a lymph node have affinity for lymph node blood vessels, we used multiphoton intravital microscopy to measure cancer cell migration in an optical lymph node window in mice. Dendra2 expressing metastatic cancer cells are first seen in the subcapsular sinus of the lymph node and later invade the cortex of the lymph node where they accumulate around rhodamine-dextran labeled blood vessels. Cancer cells can be observed in directed migration toward blood vessels as well as moving inside blood vessels. Finally, we analyzed lymph nodes with metastatic lesions from patients with head and neck cancer and identified cancer cells that were closely associated with and inside blood vessels. Conclusions: Together, our data show for the first time that in spontaneous breast and melanoma mouse models, tumor cells in the lymph node can invade blood vessels, exit the node and colonize the lung.

#3023

Dissecting mechanisms of breast cancer metastasis through patient-derived circulating tumor cells.

Remi Klotz,1 Amal Thomas,1 Sara Restrepo-Vassalli,1 Oihana Iriondo,1 Jane Han,1 Teng Teng,1 Matthew MacKay,1 Alan Wang,1 Negeen Izadian,1 Byoung-San Moon,1 Aditya Bardia,2 James Hicks,1 Andrew Smith,1 Min Yu1. 1 _USC, Los Angeles, CA;_ 2 _Massachusetts General Hospital, Boston, MA_.

During tumor progression, tumor cells invade the primary tumor microenvironment and intravasate into blood vessels, where they are referred to as circulating tumor cells (CTCs). These CTCs disseminate to other organs, and a subset of these cells will form metastasis. The growing interest for CTCs is confronted with the difficulty associated with their isolation and scarcity of material. To address this challenge, our established CTC lines from breast cancer patients offered a unique opportunity to investigate the signal pathways involved in the metastatic cascade. We assessed the metastatic potential of breast cancer patient-derived CTC lines by injecting them directly into the left ventricle of the heart in NSG mice. CTC lines are capable of generating metastases in most organs that are listed as common sites of metastases from breast cancer, such as the brain, lungs, bones and ovaries. Two patient-derived CTC lines harbored a high metastatic potential (over 80% of mice had metastases after 3 months) with generation of simultaneous metastases in the bones, lungs and ovaries. These mice remained brain-metastases free for up to 8 months. However, in 2 patient-derived CTC lines we demonstrated the brain as the preferential site of metastasis despite their low metastatic potential. Interestingly, in our mouse model, CTC lines recapitulated human cancer metastatic progression, as shown by the clinical data collected from patients where CTC lines were generated. We further investigated genetic and epigenetic determinants that regulate the organotropism of CTCs. To address this we isolated metastatic variants with a preferential tropism for the brain, lungs and bones. Investigating the gene expression signatures of metastatic variants lead us to identify the semaphorin 4D as a possible regulator of the brain metastasis. High SEMA4D expression at the primary site correlated with decreased brain metastasis-free survival in the breast cancer patients. Amplification of the chromosome 9 where the SEMA4D gene resides was also found in CTC-derived brain metastases. The SEMA4D overexpression promoted the ability of CTCs to transmigrate the blood-brain-barrier in vitro. We simultaneously investigated expression changes in stromal cells in organ-specific metastases, and among the pathways with the highest enrichment scores we found TNF and IFN signaling in the brain metastatic microenvironment. Next we used ATAC-seq to assay chromatin accessibility and identified potential regulatory factors mediating the brain metastasis formation. These data provides the evidence for the promising role of CTCs as an early prognostic factor for metastasis. Additionally, we expect to develop novel organ tropism associated markers, which can be considered for potential therapeutic targets in breast cancer.

#3024

Genomic characterization of organ-specific metastasis from prospective clinical sequencing of 20,000 cancer patients.

Francisco Sanchez-Vega, Walid K. Chatila, Karuna Ganesh, Alexander Penson, Niedzica Camacho, Philip Jonsson, Ed Reznik, Jianjiong Gao, Rona Yaeger, Michael F. Berger, Barry S. Taylor, Nikolaus Schultz. _Memorial Sloan Kettering, New York, NY_.

We investigated genomic differences in sequenced biopsies of patients with metastatic cancer to identify associations between somatic alterations and anatomic site of metastasis.

We analyzed data from 20,000 patients treated at Memorial Sloan Kettering Cancer Center, covering >50 cancer types and >20 metastatic sites. Tumors were sequenced using MSK-IMPACT, a targeted sequencing assay that identifies somatic mutations, copy number changes and structural rearrangements in 468 cancer genes using tumor and matched normal DNA. We identified tumor types such as prostate cancer, lung adenocarcinoma, uterine endometrioid carcinoma, head and neck cancer and gastrointestinal stromal tumors, for which the primary biopsies differed significantly from the metastatic biopsies at the genomic level, suggesting a need for sample stratification in clinical studies. We also identified site-specific enrichment of individual somatic alterations for tumors from the same cancer type, including therapeutically relevant targets. For example, when evaluating patients with esophagogastric cancer, we found an enrichment of HER2 amplified tumors, which are candidates for trastuzumab therapy, in sequenced lung metastases as opposed to biopsies sequenced at other metastatic sites (8/16, 50% vs. 20/145, 14%; p<0.001). Looking at sequenced metastases of lung adenocarcinoma (n=468), we found that brain metastases (n=34) exhibit increased aneuploidy (median fraction of genome altered 0.55 vs. 0.44; p<0.001, purity-controlled results) and adrenal gland metastases (n=21) exhibit increased mutational burden (13.3 mut/Mb vs. 6.1 mut/Mb; p<0.001) when compared to metastases sequenced at other anatomic sites. Other examples of novel findings include enrichment of amplifications in the MITF-ETV1 pathway in melanoma patients with brain metastatic disease (13/63, 21% vs. 10/234, 4%; p<0.001) and enrichment of 20q chromosomal amplifications in patients with microsatellite stable colorectal tumors that exhibit oligo-metastatic (as opposed to poly-metastatic) patterns at first time of metastasis diagnosis (158/223, 71% vs. 28/58, 48%; p=0.002).

We provide a detailed analysis of associations between genomic features and metastatic patterns within a clinical sequencing setting. Our work has the potential to directly influence the management of patients with metastatic cancer by enabling clinicians to (a) estimate the risk of future metastasis based on the genomic profile of early stage tumors, (b) choose the appropriate site for biopsy in cases where mutation identification will influence the selection of targeted therapy, (c) optimize the use and frequency of imaging modalities for metastasis surveillance and (d) investigate and functionally characterize novel genomic alterations that have a role in metastasis and can lead to improved therapies.

#3025

Ligand-independent EphA2 signaling drives an amoeboid phenotype that promotes melanoma brain metastasis development.

Chao Zhang,1 Inna Smalley,2 Ritin Sharma,2 Michael Emmons,2 Jane Messina,2 John Koomen,2 Keiran Smalley2. 1 _USF/Moffitt Cancer Center, Tampa, FL;_ 2 _Moffitt Cancer Center, Tampa, FL_.

The majority of melanoma patients treated with BRAF inhibitors (BRAFi) ultimately develop resistance and fail on therapy. It has previously been shown that BRAFi resistance is highly correlated with the adoption of a strongly invasive phenotype through ligand-independent EphA2 signaling.

In this study, we used comprehensive mass spectrometry-based proteomic approaches to delineate the global signaling changes associated with the aggressive phenotype driven by ligand-independent EphA2 signaling. Preliminary data have identified new signaling adaptations associated with a mesenchymal-to-amoeboid transition (MAT) phenotype, which was confirmed in 3D collagen cultures. Functional experiments demonstrated that the amoeboid phenotype promoted cell migration and invasion, which was mediated through the interaction of EphA2 and CDC42. These findings were confirmed by expressing a constitutively activated form of CDC42. BRAFi resistant cell lines also exhibited the MAT phenotype and were more invasive compared to their treatment-naïve counterparts, in line with their dependence upon ligand-independent EphA2 signaling following BRAFi selection pressure.

To further demonstrate the metastatic potential of these amoeboid cells driven by ligand-independent EpHA2 signaling in vivo, we performed intracardiac injections in mice with cell lines expressing either EpHA2 S897A (inactive) or S897E (constitutively active phosphomimetic). Interestingly, we found a preferential homing of EphA2 S897E cells to the brain, but no difference in metastasis to other organs including the lung and liver. An analysis of brain metastasis specimens from patients failing BRAF and BRAF/MEK inhibitor therapy showed strong staining for the amoeboid phenotype marker EphA2.

To investigate whether the amoeboid phenotype may confer a survival advantage in circulation, we carried out shear stress assays. These experiments demonstrated that the amoeboid phenotype driven by ligand-independent EphA2 signaling promoted the survival of melanoma cells under shear stress. Cell attachment assays, trans-endothelial invasion assays and vascular permeability assays show that these cells are better suited to attach to and permeate an endothelial monolayer. We further show that inhibiting PI3K reversed the amoeboid phenotype and limited the EphA2-driven invasive capacity.

In summary, we show for the first time that BRAFi resistance is associated with the adoption of an MAT phenotype that increases the metastatic seeding of melanoma cells to the brain, which can be reversed through inhibition of PI3K signaling.

#3026

Role of AnnexinA2, Sema3D and PlexinD1 in mediating perineural invasion as a mechanism of metastasis in pancreatic ductal adenocarcinoma.

Noelle R. Jurcak, Kenji Fujiwara, Agnieszka A. Rucki, Kelly Foley, Adrian Murphy, Stephen Muth, Abigail Brittingham, Elizabeth M. Jaffee, Lei Zheng. _Johns Hopkins School of Medicine, Baltimore, MD_.

Pancreatic ductal adenocarcinoma (PDA) is a devastating malignant disease. Upon diagnosis, eighty percent of patients present with metastatic disease. Perineural invasion, the spreading of cancer to the space surrounding or invading the nerve, is often found in surgically removed pancreatic tumors and is associated with a worse overall prognosis including high risk of recurrence and metastasis. Perineural invasion is thought to represent the initial steps of metastasis; however, the mechanism that mediates the role of neurons in precancerous inflammation and pancreatic cancer initiation is not known. We have previously found that Annexin A2 (AnxA2), is a metastasis-associated protein in PDA and is not required for primary PDA growth but is essential for the metastasis in genetically engineered spontaneous pancreatic tumor producing KPC mice. Knock-down of AnxA2 results in diminished metastasis and prolonged survival in mouse liver metastasis models of PDA. Moreover, the presence of perineural invasion is AnxA2 dependent in this model. By comparing tumor cells of AnxA2 wild type vs. knock-out KPC mice, we identified Semaphorin 3D (Sema3D) and PlexinD1 (PlxnD1) were among the most differentially expressed genes. We found that AnxA2 regulates the secretion of Sema3D. Originally characterized as axon guidance genes, ligand Sema3D along with its receptor PlxnD1 and co-receptor, Neuropilin 1 (NP1), are among the cellular pathways most frequently altered at the genetic level in PDA. Moreover, we found that knock down of Sema3D or PlxnD1 suppresses metastasis formation in mouse models of PDA (Foley et al. Science Signaling 2015). In the present study, our lab has found that PNI was present in 9/10 human PDA samples with abundant Sema3D expression compared to only 3/10 human PDA samples with low Sema3D expression. Using a Dorsal Root Ganglion (DRG) invasion assay, we found paracrine signaling between DRG and pancreatic cancer cells is important for increasing tumor cell invasion toward DRGs. Using small interfering RNA for AnxA2 in tumor cells, we find that decrease in AnxA2 significantly decreases tumor invasion. Furthermore, knock down of Sema3D from tumor cells, using shRNA, shows significant decrease in tumor cell invasion toward DRGs. We have also tested the role of the Sema3D receptor, PlexinD1, on DRGs in regulating the invasion of tumor cells towards nerves. A neutralizing antibody for PlexinD1 treated on DRGs caused significant decrease in tumor cell invasion in comparison to DRGs treated with IgG isotype control. These results demonstrate that AnxA2, Sema3D and PlexinD1 are involved in paracrine signaling between the tumor cells and nerves. Taken together, we conclude AnxA2/Sema3D/PlexinD1 signaling may aid in mediating perineural invasion of tumor cells towards nerves as a mechanism of metastasis in pancreatic ductal adenocarcinoma.

#3027

Single-cell RNA sequencing defines regulatory networks in ER+ breast cancer organ-specific metastases.

Nuria Padilla Just, Jessica Finlay-Schultz, Austin E. Gillen, Diana M. Cittelly, Elizabeth A. Wellberg, Kathryn B. Horwitz, Peter Kabos, Carol A. Sartorius. _University of Colorado Anschutz Medical Campus, Aurora, CO_.

Patients with estrogen receptor (ER)+ breast cancer are at chronic and prolonged risk of metastatic recurrences, which are usually fatal. The most frequent sites of ER+ breast cancer metastasis are bone, liver, lung, and brain, with individual tumors preferring one or more sites. The underlying mechanisms of site-specific metastasis are poorly understood and are critical for prevention and treatment of metastases to more lethal (brain, liver, lung) vs. static (bone) sites. The purpose of this study is to use single-cell RNA sequencing (sc RNA-seq) of ER+ breast cancer cells dispersed to breast tropic sites to define unique molecular determinants of organ-specific metastasis. Our group has developed ER+ patient-derived xenografts (PDX) at the University of Colorado Denver (UCD) labeled with constitutive luciferase-GFP. Our ER+ PDX fall into two general categories-pure "luminal" tumors with high ER plus/minus progesterone receptor (PR) (>50% of cells) and an absence of cytokeratin 5 (CK5)+ cells, and "luminobasal" tumors with a low percent of ER+PR+ cells (5-10%) and high CK5+ cells (15-90%). Using intracardiac (ic) dispersal as a measure of late-stage metastasis, we have defined the organ homing preference of several of these lines. Luminal PDX frequently colonize bone, with occasional secondary lesions to brain and liver. Luminobasal PDX ubiquitously colonize liver, with secondary lesions mostly to lung. We performed sc RNA-seq of luminal UCD65 brain metastatic and luminobasal UCD46 liver metastatic cells compared to their cognate primary tumors grown in the mammary fat pad. UCD65 brain metastatic vs. primary tumor cells have elevated expression of PR and carbonic anhydrase 2 (CA2), a cellular guard against intracellular pH changes, and a prospective PR-regulated gene. UCD46 liver metastatic cells have activated TGFbeta signaling, and increased cancer stem cell markers. We conclude that ER+ breast cancer PDX retain innate preference for specific breast-tropic organs, that this may relate to their ER+ subtype (luminal vs. luminobasal), and that sc RNA-seq may identify key molecular signatures of ER+ breast cancer metastases. We are profiling additional tumors and sites to make further comparisons and generate testable hypotheses. Furthermore, our models allow for functional studies to test innovative interventions and therapeutics that target site-specific metastases.

This work was supported by grants from the Breast Cancer Research Foundation (16-072, CAS, KBH, PK), the NIH (CA140985, CAS) and the UCD RNA Biosciences Initiative (AEG, CAS, PK). 
